Nerium oleander L., termed Breastin. The phytochemical characterization by nuclear magnetic resonance spectroscopy (NMR) and low- and high-resolution mass spectrometry revealed several monoglycosidic cardenolides as major constituents . Breastin inhibited the growth of 14 cell lines from hematopoietic tumors and 5 of 6 carcinomas. Remarkably, the cellular responsiveness of odoroside H and neritaloside was not correlated with all other classical drug resistance mechanisms, i.e., ATP-binding cassette transporters , oncogenes , tumor suppressors , and others , in 59 tumor cell lines of the National Cancer Institute , indicating that Breastin may indeed bypass drug resistance. COMPARE analyses with 153 anticancer agents in 74 tumor cell lines of the Oncotest panel revealed frequent correlations of Breastin with mitosis-inhibiting drugs. Using tubulin-GFP-transfected U2OS cells and confocal microscopy, it was found that the microtubule-disturbing effect of Breastin was comparable to that of the tubulin-depolymerizing drug paclitaxel. This result was verified by a tubulin polymerization assay in vitro and molecular docking in silico. Proteome profiling of 3171 proteins in the NCI panel revealed protein subsets whose expression significantly correlated with cellular responsiveness to odoroside H and neritaloside, indicating that protein expression profiles can be identified to predict the sensitivity or resistance of tumor cells to Breastin constituents. Breastin moderately inhibited breast cancer xenograft tumors in vivo. Remarkably, in contrast to what was observed with paclitaxel monotherapy, the combination of paclitaxel and Breastin prevented tumor relapse, indicating Breastin’s potential for drug combination regimens.Cancer drug resistance remains a major obstacle in clinical oncology. As most anticancer drugs are of natural origin, we investigated the anticancer potential of a standardized cold-water leaf extract from  Nerium oleander L. (Family: Apocynaceae) is a shrub growing in subtropical regions . It is the only species belonging to the genus Nerium. It is an ornamental plant in parks and gardens and has a long history in ancient Europe. In the Near East and Southern Asia, it has also been used in traditional medicine as anti-inflammatory, antidiabetic, and anticancer herbal medicine as well as an herbal drug against indigestion, malaria, leprosy, mental diseases, etc. [es, etc. ,2,3,4. Ies, etc. .N. oleander are cardiac glycosides such as oleandrin, which are toxic upon ingestion and have a narrow therapeutic window. In academic Western medicine, cardiac glycosides have been used to treat heart insufficiency [+/K+-ATPase . The ade  in being a cold-water extract [COMPARE analyses using the National Cancer Institute (USA) and Oncotest panels of cell lines and Breastin as well as two of Breastin’s constituents, odoroside H and neritaloside. We have chosen these two compounds as data were not available for the other phytochemicals of Breastin. The inhibition of the microtubule network was identified as a possible mode of action of Breastin and verified in subsequent experiments. Furthermore, proteomic profiling identified subsets of proteins that predicted the sensitivity or resistance to odoroside H and neritaloside.The present study aimed to investigate the anticancer activity of a cold-water extract of  extract . Then, wm/z 593, N. oleander leaves with water at room temperature [The chemical composition of the Breastin preparation was investigated by nuclear magnetic resonance spectroscopy (NMR) and low- and high-resolution mass spectrometry. The intension was to confirm the previously published chemoprofile  as a meaperature .−3 to 102 µg/mL (50). For the multiple myeloma cell lines, they ranged from 0.28 to 0.72 µg/mL . Based on the IC50 values, the multidrug-resistant CEM/ADR5000 subline of CCRF-CEM was not resistant or was only minimally resistant (1.4-fold) to Breastin compared to the parental drug-sensitive cells. This was remarkable since CEM/ADR5000 were characterized as expressing a phenotype of full-blown high-degree multidrug resistance to a broad array of clinically established anticancer drugs, including anthracyclines, Vinca alkaloids, epipodophyllotoxines, taxanes, and others [The effect of Breastin on the viability of multiple myeloma and leukemia cell lines was analyzed by generating dose–response curves with concentrations ranging from 1002 µg/mL . The dosd others . 50 values ranging from 1.01 to 5.54 µg/mL. Another cell line (LNCaP) was not responsive to Breastin and an IC50 value could not be calculated, even up to the highest concentration of 50 µg/mL. Furthermore, we compared the effects of incubation with Breastin for 24 h and 48 h. We did not observe an increased growth inhibition after 48 h compared to 24 h, indicating that shorter incubation might be sufficient to exert Breastin’s effect.For comparison, we also exemplarily tested cell lines derived from solid tumors. The results of six carcinoma cell lines treated with Breastin are shown in https://dtp.cancer.gov; accessed on 8 February 2023). In the case of oleandrin, its cytotoxicity was beyond the concentration range tested in the NCI panel; therefore, it could not be used further.The fact that Breastin inhibited multidrug-resistant CEM/ADR5000 cells with nearly the same efficacy as drug-sensitive CCRF-CEM cells piqued our interest, and we wanted to analyze the activity of Breastin in a panel of classical drug resistance mechanisms in greater detail. Odoroside H and neritaloside as two of the main compounds of Breastin were investigated in the NCI panel of 59 tumor cell lines. The data deposited in the NCI database were used for their evaluation. Both compounds are cardenolides and have the same scaffold, and neritaloside has an additional acetoxy group at position C16 A. The otWe first analyzed whether both compounds are involved in drug resistance phenotypes caused by diverse mechanisms, such as ATP-binding cassette transporters , oncogenes and tumor suppressors , and others  . Importa10IC50 values of odoroside H and neritaloside were first correlated with each other. We found a significant relationship  (The log× 10−14) B. The cy× 10−14) C–E. Ther50 and IC70 values for Breastin of 74 cell lines with 153 reference anticancer agents with known modes of action.In addition to the two isolated compounds, odoroside H and neritaloside, we were also interested in the N. oleander extract itself. We performed COMPARE analyses with Breastin in another panel of cell lines. For this reason, we used the tumor cell line panel of Oncotest GmbH  and correlated the IC50 and IC70 of the top-ranked compounds  are listed in 70 values have been compiled in addition to the usually used IC50 values to have additional information at drug concentrations that are noncytotoxic and which therefore might better reflect the mode of action without the involvement of cytotoxic effects. It was a striking result that the COMPARE profile of Breastin was associated with many anticancer agents affecting mitosis. This can be taken as an indication that mitosis and/or DNA damage-related mechanisms may contribute to the cytotoxic effect of Breastin on cancer cells. Therefore, we independently performed in vitro experiments to prove these biostatistical correlation analyses.The ICU2OS human osteosarcoma cells expressing α-tubulin-GFP were treated with Breastin (13.2 µg/mL) for 24 h to study the impact of Breastin on microtubules. Paclitaxel (10 µM) and nocodazole (10 µM) were taken as positive controls. As shown in An in vitro tubulin polymerization assay was performed to validate the results obtained by confocal microscopy. Paclitaxel, a microtubule-stabilizing agent, was taken as a positive control. As depicted in Our phytochemical analyses  identifiTherefore, we performed defined docking by laying the grid boxes over the main drug-binding sites of tubulin . In the defined docking approach, the LBE and predicted inhibition constant (pKi) values were generally lower at the Vinca alkaloid- and taxane-binding sites than at the colchicine-binding site. Vinorelbine and paclitaxel showed the lowest LBE and pKi values at their corresponding binding sites, indicating that the molecular docking approach likely was correct. The results of defined molecular docking revealed that not only odoroside H and neritaloside but also the other cardenolides of Breastin were bound to tubulin, supporting the hypothesis that Breastin inhibits cancer growth, at least in part, by affecting tubulin. The interaction of adynerin, neritaloside, odoroside A, odoroside H, oleandrin, and vanderoside with β-tubulin, illustrated using Discovery Studio Visualizer, is shown in We used two different docking protocols: Using a blind docking approach, we scanned the whole surface of the target proteins to identify the docking pose of the compounds on the target. These results were subsequently verified by a defined docking approach. Here, we focused only on the specific docking pose identified by blind docking and found consistent results. These two in silico approaches were experimentally verified by two in vitro techniques, i.e., immunofluorescence-based confocal microscopy and a biochemical tubulin binding assay. Therefore, using two in silico and two in vitro assays provided sufficient confidence in the validity of our results.10IC50 values of 59 tumor cell lines with the cellular expression of 3171 proteins as measured by mass spectrometry deposited at the database of the Developmental Therapeutics Program of NCI (USA) . We selected 40 out of 3171 proteins consisting of the top 20 directly and the top 20 inversely correlating proteins with the log10IC50 values of these two compounds. The proteins and their biological functions identified for odoroside H are shown in To investigate the influence of protein expression on cellular resistance and sensitivity to odoroside H and neritaloside, we correlated the log10IC50 values for the two compounds in the second dimension led to two-dimensional color-coded heat maps. For odoroside H, four major clusters were obtained for the 59 tumor cell lines (clusters 1–4), and seven clusters were obtained for the 40 proteins (clusters A–G) .We analyzed the protein expression profiles using hierarchical cluster analysis (Ward method). The cellular expression of these 40 proteins was assembled in the first dimension, and the logers A–G) . As a nep = 8.27 × 10−5, χ2 test).For neritaloside, we found three clusters for the cell lines (clusters 1–3) and another four clusters for the proteins . The disThe results in Finally, we were interested in whether Breastin can inhibit cancer cells not only in vitro but also in animals. For this reason, we used a tumor xenograft model. MAXF 401 cells were transplanted into nude mice. After an initial induction growth phase of MAXF 401 cells for 24 days, the mice were treated with Breastin , paclitaxel , Breastin plus paclitaxel , or vehicle control (group 1). The results are shown in Identification of the constituents by HPLC in the current batch of Breastin revealed the same compounds that have been identified in a previous investigation of another batch, i.e., oleandrin, oleandrigenin sarmentoside, neritaloside, odoroside H, and odoroside A . This me50 values of 36 tumor cell lines in a preliminary investigation were reported in cell lines originating from solid tumors but not in hematopoietic tumor cell lines [Previously, the ICll lines . InspireA COMPARE analysis of the Oncotest cell line panel also indicated that a majority of the top 10 most closely correlated reference anticancer compounds were related to disturbing the mitotic spindle. Therefore, we hypothesized that the mitotic spindle and tubulin, as the most predominant protein in mitosis, might be disturbed by Breastin. Indeed, we were able to verify this assumption by independent in vitro experiments regarding microtubule stabilization . From the literature, there are no hints that adynerin, odoroside A and H, vanderoside, and neritaloside might inhibit or stabilize microtubules. Oleandrin has been reported to arrest the cell cycle of cancer cells in the G2/M phase, which can be taken as a hint that mitotic arrest might indicate an interaction with microtubules . The ethThe isolated compounds were only used to obtain insights into the molecular modes of action of the selected constituents of Breastin. In this sense, the isolated compounds were only tools on our way to explain the activity of Breastin and to bring this extract closer to clinical application. The final goal is to develop Breastin as a herbal preparation for clinical application but not to perform classical drug development with chemical compounds such as odoroside and neritaloside.+/K+-ATPase [Other targets have also been discussed in the literature. Cardenolides (including oleandrin) are well characterized for their inhibitory activity towards Na+-ATPase ,36,37,38+-ATPase , and N. +-ATPase ,41,42,43+-ATPase ,45,46,47+-ATPase , increas+-ATPase .The cytotoxic activity of N. oleander is not only due to oleandrin but also due to other compounds in N. oleander such as odoroside A, nerigoside, and oleandrigenin ,50,51. SDuring the past two decades, it became more and more clear that the sensitivity and resistance of tumor cells to cytotoxic drugs are determined not only by single factors but by transcriptome-wide gene expression  and may explain the growth-inhibitory activity of odoroside H. Moreover, the expression of several proteins involved in RNA function and processing  correlated with the anticancer effect of odoroside H. This possibly points to another mechanism of action of odoroside H that is worth investigating further in the future. Interestingly, some potential resistance mechanisms were also found, such as DNA repair (APEX1), chaperone , and cytoskeletal elements (SYNM).The dataset of proteins that were significantly associated with the response of tumor cells to odoroside H usefully complemented our previous mechanism-of-action studies. Several proteins have functions in primary and energy metabolism , which could be an effect of the NaThe cellular response to neritaloside was associated with mechanism classes similar to those of odoroside H, e.g., primary and energy metabolism , proliferation , and proteins related to RNA function and processing . This indicates that odoroside H and neritaloside may complement each other in their action against cancer cells and possibly enhance each other. The expression of 14 proteins was significantly correlated with sensitivity or resistance to both compounds . Future experiments should clarify whether combination therapy with both substances has additive or synergistic effects on cancer cells. The anticancer activity in vivo indicates that Breastin might also be active in human patients. In our experiments, Breastin significantly reduced tumor growth, indicating that Breastin has the potential to prevent both tumor development and progression. Oleandrin indeed inhibited tumor promotion in a murine chemically induced skin carcinogenesis model . For tumFurthermore, it was encouraging for us to see that Breastin exerts a strong synergistic effect if combined with the standard anticancer drug paclitaxel. This is a remarkable result. Clearly, Breastin was a dose modifier for paclitaxel. If we assume that Breastin has a mode of action similar to that of paclitaxel , then a dose intensification of paclitaxel alone should lead to a similar effect. High-dose chemotherapy with paclitaxel is, however, associated with significant toxicities ,66,67 an2 extract of N. oleander in glioblastoma xenograft tumors [If Breastin were ever to be established in clinical oncology, then it would probably not be used as monotherapy, but as part of combination regimens. Therefore, Breastin must not generate antagonisms with other drugs but synergisms or at least additive effects. The fact that the relapse of paclitaxel-treated tumors was prevented by the addition of Breastin may be taken as a strong argument for the clinical promise of Breastin. Recently, another study described the synergistic effects of radiotherapy and a supercritical COt tumors . Other at tumors ,70,71,72In general, it is advisable to be careful with cardiac glycosides because they are known for their toxicity. We found some toxic reactions, but they were rather mild and tolerable in our mouse experiments. Another aqueous N. oleander extract (Anvirzel) also only exerted mild toxicities in a clinical phase 1 trial in 18 patients with refractory solid tumors. The side effects included fatigue, nausea, vomiting, dyspnea, and mild injection site pain . A revie50 of Breastin in Albino Balb/C mice was 0.39 mL/25 g i.m. (=15.6 mg/kg), 0.30 mL/25 g i.p., (=12.0 mg/kg), and 0.33 mL/25 g s.c. (=13.2 mg/kg). Tachycardia, myorelaxation, and motoric incoordination were found at high doses, but single or repeated doses i.m., i.p., or s.c. up to 0.15 mL/25 g (6 mL/kg) did not provoke side effects. Animals generally gained weight , indicating that Breastin was not anorexic at the doses used. Breastin did not decrease blood counts or change hemoglobin contents or blood indices during the treatment applied for 8 weeks in mice. In contrast, Breastin increased the leukocyte counts in the experimental groups. Liver and renal function abnormalities were not observed. Breastin caused neither erythema nor any cutaneous sensitization in the skin of guinea pigs nor any inflammatory changes in the eyes of the tested rabbits as compared to the untreated controls. Given chronically for 6 months in rats, Breastin did not cause significant differences in the kidney profiles but did cause a significant increase in aspartate and alanine aminotransferases. The histopathological studies performed on biopsies obtained from the kidneys and the livers of rats also revealed normal results. However, the microscopic anatomy performed on biopsies taken from the liver of the dead animals showed some changes such as infiltration of inflammatory cells and passive congestion, but no central necrosis was detected. In summary, Breastin did not show severe or life-threatening (grade 3 and 4) toxicities in rodents. However, caution is advised as it is known that rodents have a much higher acceptance (100×) of cardiac glycosides than humans and oleandrin can pass the blood–brain barrier [The toxicities of Breastin were already investigated in detail . Briefly barrier ,79.Breastin is a cold-water extract from leaves of N. oleander. The preparation of the extract has been described . The onl2O as solvent. Chemical shifts in ppm were referenced to the internal standard TMS (δ = 0). The high-resolution ESI mass spectra were obtained using a Bruker Apex III 70 eV Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a 7.0 Tesla superconducting magnet, an RF-only hexapole ion guide, and an external electrospray ion source. Nitrogen was used as drying gas at 150 °C. The sample solutions were introduced continuously via a syringe pump with a flow rate of 120 µL/h. The data were evaluated using the Bruker XMASS 7.0.8 software.Proton NMR spectra were recorded using a Varian Unity 500 spectrometer at 499.83 MHz using D2O:CH3CN gradient system containing 0.2% HOAc was used for HPLC measurements.Low-resolution negative LCMS spectra were obtained from a Finnigan MAT TSQ 7000 instrument  coupled with a Surveyor MicroLC system equipped with an RP18-column . An HThe multiple myeloma cell lines KMS11, KMS12BM, NCI-H929, MolP8, JJN3, OPM2, AMO1, and L363 were provided by Dr. Ellen Leich and Manik Chatterjee, University of Würzburg, Würzburg, Germany. RPMI8226 cells were purchased from the American Type Cell Culture Collection . Their maintenance has been described .Human NB4 acute promyelocytic leukemia cells were provided by Dr. Gabriele Greve . Human HL60 acute promyelocytic leukemia cells were provided by Dr. Andreas Schwarting and Dr. Julia Weinmann-Menke . Human MOLT-4 acute lymphoblastic leukemia cells were obtained from the tumor bank of the German Cancer Research Center . Drug-sensitive CCRF-CEM acute lymphoblastic leukemia cells and P-glycoprotein-overexpressing CEM/ADR5000 cells were cultured as described . The mai2 atmosphere at 37 °C. Cells were passaged twice weekly. All experiments were conducted with the cells in their logarithmic growth phase. Multiple myeloma and leukemia cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin (100 U/mL)–streptomycin (100 µG/mL) (PIS) antibiotic (Invitrogen) and incubated in humidified 5% COSeventy-four cell lines were established from patient-derived xenograft tumors (PDX) subcutaneously passaged in nude mice by Oncotest GmbH . Other cell lines were obtained from ATCC , DSMZ , or the National Cancer Institute  as described ,85.Several other tumor cell lines of the Research Genetic Cancer Centre S.A.  have been tested for their sensitivity toward Breastin. We used the following human carcinoma cell lines from human carcinoma types: KRAS-mutated HCT-116 colorectal carcinoma, estrogen-receptor-positive MCF-7 breast carcinoma, triple-negative MDA-MB-231 breast adenocarcinoma, MOR lung adenocarcinoma, PANC-1 ductal cell pancreatic carcinoma, and androgen-responsive LNCaP prostate carcinoma. A GFP fusion construct of α-tubulin was used to generate stably transfected U2OS-GFP-α-tubulin cells. The cell line was a generous gift from Joachim Hehl, Light Microscopy Centre, ETH Zürich. Wild-type U2OS cells were obtained from Dr. Wynand Roos .10IC50 values obtained by a sulforhodamine 123 assay) and transcriptomic and proteomic expression data were deposited on the NCI website (https://dtp.cancer.gov (accessed on 30 August 2022)).The Developmental Therapeutics Program of the National Cancer Institute  uses a panel of 55 human tumor cell lines  for drug screening . The dru50) were calculated by nonlinear regression using Microsoft Excel.The resazurin reduction assay was used to investigate the cytotoxicity of Breastin to tumor cells. The assay is based on the reduction of the indicator dye, resazurin, to the highly fluorescent resorufin by viable but not drug-treated dead cells . NonviabA modified propidium iodide assay  was usedp values < 0.05. Results were calculated using Microsoft Excel 2016. The measurements from the proliferation assays were used to determine the IC50 values upon 48 h incubation by using Microsoft Excel 2016.Cell lines of the RGCC panel were tested with the MTT assay as described  with a c4 U2OS-GFP-α-tubulin cells were seeded in each well of a sterile ibiTreat μ-slide , and cells were allowed to attach overnight. Cells were treated with 13.2 μg/mL Breastin or DMSO (solvent control) and incubated at 37 °C for 2 h. After rinsing with PBS and staining for 15 min with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) , the cells were washed with PBS and mounted. Fluorescence imaging was performed by using 470 nm excitation and 525 nm emission for GFP and 360 nm excitation and 447 nm emission for DAPI with an EVOS digital inverted microscope (Life Technologies). Each experiment was repeated at least three times and representative images were selected.The method has been described . AliquotBreastin was investigated by using the In Vitro Tubulin Polymerization Assay Kit  following the manufacturer’s instructions. The analyses were accomplished using a FlexStation 3 Multi-Mode Microplate Reader . The readings were obtained by measuring the turbidity variation (light scattering) every 30 s for 3 h  at 350 nm .The COMPARE algorithm ,93 was a10C50 values for odoroside H and neritaloside were deposited in the database of the NCI, USA  and correlated with the corresponding log10C50 values of 91 standard anticancer agents determined against the NCI tumor cell line panel. These standard agents represent the main molecular mechanisms of action for established anticancer drugs. Similarities between the sensitivity pattern of a test compound and the sensitivity pattern of standard drugs were expressed quantitatively as Pearson correlation coefficients. High-level correlations between the sensitivity patterns of two compounds are strongly suggestive of a similar mode of action.The logCOMPARE analyses with the cell line panel of Oncotest GmbH . Here, the IC50 and IC70 values of Breastin were correlated with the corresponding IC50/IC70 values of 153 standard agents with known modes of action determined against the same cell line panel. These standard agents represent the main molecular mechanisms of action for established anticancer drugs. Similarities between the sensitivity pattern of a test compound and the sensitivity pattern of standard drugs are expressed quantitatively as Spearman correlation coefficients [As a second independent approach, we performed ficients . High-lehttp://www.PubChem.nih.gov; accessed on 30 August 2022). The crystal structure of tubulin (PDB ID: 5N5N) was obtained from the Protein Data Bank . AutoDockTools 1.5.6 was used to convert all six compounds, tubulin microtubule inhibitors , and tubulin to Protein Data Bank Partial Charge and Atom Type (PDBQT) format. Four different grid boxes were prepared around the whole tubulin protein, the Vinca alkaloid-binding site, the taxol-binding site, and the colchicine-binding site to study the tubulin-binding behavior of Breastin compounds in silico. In AutoDock4.2.6. software, Lamarckian Algorithm was set with 10 runs and 2,500,000 energy evaluations to generate the docking log file. Binding energy and predicted Ki were collected from the RMSD cluster analysis while the interacting amino acids were identified with AutoDockTools [The three-dimensional structures of six phytochemicals from Breastin  were downloaded from PubChem  [10IC50 values of the cell lines. Heat maps based on agglomerative hierarchical clustering according to the Ward method were generated by using the CIM miner software  [2 .The Pearson correlation test and hierarchical cluster analyses were performed by using mass-spectrometry-based proteomic expression data of 59 NCI cell lines (st 2022)  to generst 2022) . The linIn-house outbred NMRI nude mice were used . The animals were maintained in Macrolon cages under natural daylight cycles at an ambient temperature of 24 ± 1 °C. Mice were fed with standard chow  and water ad libitum. All animal experiments were approved by the Regierungspräsidium Freiburg, and studies were conducted following the UKCCR guidelines for the use of animals in experimental neoplasia .7 tumor cells resuspended in 100% Matrigel. The antitumor activity was monitored for Breastin or paclitaxel monotherapy and the combination of Breastin with paclitaxel. Mice were randomized into four treatment groups  if tumors reached 50–250 mm3 . The drugs were administered intraperitoneally at 40 mg/kg of BW three times a week for a duration of three to four weeks. Tumor size was measured twice weekly via calipers. Antitumor activity was evaluated as maximum tumor volume inhibition compared with the vehicle control group . Before euthanasia, tumors were carefully excised and immediately snap-frozen in liquid nitrogen for further analysis.To study the antitumor efficacy of Breastin in human MAXF 401 xenograft breast cancer, mice were subcutaneously injected with 2 × 10Breastin inhibits cancer in vitro and in vivo in hematopoietic cell lines  and epidermal cell lines (diverse carcinomas). Phytochemical profiling revealed the monoglycosidic cardenolides adynerin, neritaloside, odoroside A, odoroside H, oleandrin, and vanderoside as major bioactive secondary metabolites of Breastin. Major molecular mechanisms of drug resistance were not correlated with the activity of odoroside H and neritaloside. In silico and in vitro mode-of-action analyses revealed that Breastin is a mitotic spindle poison that inhibits the depolymerization of microtubules. The inhibition of human xenograft tumors and the observed prevention of tumor relapse by Breastin after treatment with paclitaxel suggest Breastin as a promising cotreatment. These findings may justify clinical investigations in human cancer patients in the future. Special attention has to be paid to the dosing and toxicity profile in patients.


Four of these complexes featured a ruthenium(II) core, where p‐cymene, toluene, benzene and indane were used as arenes. Furthermore, the osmium(II) p‐cymene dimer, as well as the Cp* dimers of rhodium(III) and its heavier analogue iridium(III) were included in this work . While the cytotoxic potencies of the ruthenium(II) and osmium(II) dimers are very low (or not even detectable at applicable concentrations), surprising activity, especially in cells from ovarian malignancies , have been found for the rhodium(III) and iridium(III) representatives. This publication is aimed at all researchers using synthetic procedures based on functionalization of these dimeric starting materials to rationalize changes in biological properties, especially cytotoxicity in cancer cells.The cytotoxicities of seven dimeric metal species of the general formula [M(arene)Cl Dimeric metal precursors are commonly employed in organometallic chemistry; however, comprehensive evaluation of their cytotoxic potency has been neglected so far. While organoruthenium dimers did not show appreciable cytotoxicity against seven commonly investigated human cancer cell lines, rhodium(III) and iridium(III) Cp* dimers exhibited notable activity in in vitro experiments. So far, most publications are based on the assumption that the dimeric metal precursors show no relevant anticancer activity; however, there are also examples where remarkable IC50 values were observed .50 values were determined, are scarce, and finding the respective values is laborious Cl2]2 have been synthesized, and their IC50 values have been determined in a panel of seven different human cancer cell lines by means of the MTT assay.We wanted to provide the scientific community with a summary of IC1–7 were synthesized according to literature (syntheses and minor modifications are described in the Supporting Information).1–4, 6, 7) were synthesized by the treatment of the corresponding metal chlorides  with dienes , which provided good to excellent yields (52–98 %)  precursor (5) in good yield (75 %) over two steps , others can only be given as >100 μm (1–3). These differences arise due to biophysical factors, mainly limited solubility.To give the broadest overview in literature yet, the antiproliferative activity of all seven described dimeric precursors has been determined in exponentially growing monolayer cultures of seven adherent human cancer cell lines by means of the colorimetric MTT assay with 96 h exposure of cells to the compounds (plus 24 h tests to complement those settings which were studied further in the apoptosis/necrosis assay) (Table 1–4), the results constitute a proof of principle, as these complexes show IC50 values higher than 100 μm in almost all of the investigated cell lines. The only exceptions to this trend were observed in A2780 cells where ruthenium benzene and indane dimers 2 and 4 showed IC50 values of 65 and 33 μm after 96 h, respectively. A similar pattern was observed for osmium p‐cymene complex 5, which can be considered inactive in most of the cell lines, except for CH1/PA‐1 teratocarcinoma cells, where activity was observed in concentrations around 50 μm.For the dimeric ruthenium compounds (6) and iridium (7) Cp* dimers, unexpected observations can be reported, as they showed moderate cytotoxicity in A549 and CH1/PA‐1 cells and turned out to be highly active in A2780 cells at an exposure time of 96 h , rhodium compound 6 was more active, whereas this trend was reversed in the more sensitive cells. In other words, iridium dimer 7 was more active in A549, CH1/PA‐1, and A2780 cells compared to rhodium 6. Their surprisingly high activity may be attributed to the slower ligand exchange rates,1–5 and 7 after 24 h of treatment. Only compound 6 induced necrosis in up to 9 % of the cells, depending on the concentration .In the four cell lines where these two dimers showed low potency  were synthesized and their purity was confirmed by standard analytical methods . The half maximal inhibitory activity of all compounds was determined in seven frequently investigated human cancer cell lines. The p‐cymene, toluene, and benzene organoruthenium dimers (1–3) were almost inactive in all tested cancer cell lines, while organorhodium and its heavier homologic iridium compound  revealed noteworthy cytotoxicity in several cell lines, especially in A2780. These findings showed that rhodium(III) and iridium(III) Cp* dimer exhibited notable activity in in vitro experiments and should be considered in future studies when the cytotoxicity of organorhodium and organoiridium metallodrugs are evaluated.Seven commonly used dimeric metal precursors  should be addressed to the authors.Supporting InformationClick here for additional data file.


Only low RAD51 (p = 0.041), TopBP1 (p = 0.026) and APOBEC3B (p = 0.015) protein expression were associated with increased VE-821 sensitivity. HGS cells had increased levels of RS (pRPASer4/8 and γH2AX nuclear immunofluorescence), and elevated RS predicted sensitivity to VE-821 independently of the cell line subtype. These data suggest that functional assessment of RS biomarkers may be a better predictive biomarker of ATRi response than any single aberrant gene in ovarian cancer and potentially other cancers.Ataxia telangiectasia and Rad-3 related kinase (ATR) signals DNA lesions and replication stress (RS) to the S and G2/M checkpoints and DNA repair pathways making it a promising target to exploit the dysregulated DNA damage response in cancer. ATR inhibitors (ATRi) are under clinical investigation as monotherapy and in combination with other anticancer agents. Molecular determinants of sensitivity to ATRi are common in ovarian cancer, suggesting the therapeutic potential of ATRi. We investigated the cytotoxicity of the ATRi, VE-821, in a panel of human ovarian cancer cell lines. High grade serous (HGS) cell lines were significantly more sensitive to VE-821 than non-HGS ( Ovarian cancer is the seventh most common cancer among women accounting for ~300,000 cancer cases globally in 2018 and is the leading cause of death from gynecological malignancy . There iThe exploitation of DDR defects is a promising approach to cancer therapy, exemplified by the success of the synthetic lethality of poly-ADP ribose polymerase (PARP) inhibitors (PARPi) in cancers with HRD. The introduction of PARPi in the treatment of platinum sensitive HRD HGSOC has resulted in significant clinical benefits with prolongation of progression-free survival (PFS) ,7,8,9. HTP53 mutations  amplification, which drives S-phase entry is present in ~20% of HGSOC [Ataxia telangiectasia and Rad-3 related kinase (ATR) is a key DDR kinase, responsible for signaling single stranded DNA (ssDNA), principally due to replication stress (RS), to the S and G2/M checkpoints and DNA repair . In cancof HGSOC ,16,17,18of HGSOC ,20,21,22of HGSOC ,24,25, Tof HGSOC , and CHKof HGSOC . Of noteof HGSOC , and BRCof HGSOC ,29. Defeof HGSOC , and linof HGSOC  and APOBof HGSOC ,33 overeof HGSOC ,35, havePutative biomarkers of response need to be sensitive and easy to measure for successful translation into clinical practice. However, unless a single gene/protein loss accounts for the response, clinical utility often fails due to the complexity of molecular characteristics of human cancer. This study aimed to investigate whether previously identified determinants of sensitivity to ATR inhibition in non-ovarian cancers would act as predictive biomarkers in in vitro models of ovarian cancer, and to investigate novel determinants of sensitivity. Sensitivity to the predecessor of the clinical ATRi berzosertib , VE-821 , was inv2. Cell lines were tested for mycoplasma every 2 months. Derivatives of the MMR deficient A2780-CP70 cell line, CP70 A2 and CP70 B1 were established by transfection with chromosome 3 containing; wild-type hMLH1 (CP70 B1), or mutant hMLH1 and were grown in media supplemented with 200 μg/mL Hygromycin B. The BRCA1 mutant cell line, UWB1.289, and its BRCA1 corrected derivative, UWB1.289 + Br1, were grown in 50% RPMI, 50% MEBM media supplemented with 20% FBS and 200 μg/mL G418S for UWB1.289 + Br1 cells.Human cancer cell lines were obtained from ATCC or ECACC, excluding the NUCOLL43 cell line which was derived de novo from a sample of malignant ascites that spontaneously immortalized , and theTP53 mutation, high CNA frequency, and a low frequency of mutations overall . As well as features of non-HGS subtypes  including wild-type TP53, closer to normal gene copy numbers, mutations in PIK3CA, PTEN, KRAS, BRAF, CTNNB1 and ARID1A, and CNAs in ERBB2. Cell lines not included in the assessment by Domcke et al. [Classification of the panel of cell lines into “HGS” or “non-HGS” was made according to the characterization by Domcke et al. , which ae et al. , UWB1.28V/V) and stained in 0.4% crystal violet (W/V). Colonies were counted and percentage survival was calculated relative to the plating efficiency of treated vs. untreated DMSO controls. Interpolation of the survival curves was used to calculate the LC50, the concentration causing a 50% decrease in cell survival, of VE-821.Low densities of cells  were seeded into 6-well plates and allowed to adhere for 24 h before being treated with 0–30 μM VE-821 for 48 h. The DMSO concentration was kept consistent at 0.5% in treated and untreated control cells. After 48 h, drug was removed and replaced with fresh media before cells were left to form colonies. After 9–21 days, cells were fixed in methanol: acetic acid , and blocked in 5% nonfat milk in TBS-T. Primary antibodies were incubated overnight at 4 °C in either 5% nonfat milk or 5% BSA in TBS-T. The following primary antibodies were used: ATM , ATR , ARID1A , CHK1 , Cyclin E , DNA-PKcs , Ku70 , Ku80 , RAD51 , PARP1 , TopBP1  and XRCC1 . HRP conjugated secondary antibodies  were incubated in 5% nonfat milk for 1 h at room temperature. For detection, Clarity Western enhanced chemiluminescence substrate (BioRad) was added and bands were visualized using the G-box gel documentation system , quantified using ImageJ software Version 2.3.0/1.53q , and normalized to Ponceau S staining , following manufacturer instructions. Protein content was estimated by Pierce BCA assay (Thermo Fisher) following manufacturers guidelines, then samples were diluted to equal concentrations between 0.8–1 mg/mL in XT sample buffer and XT reducing agent , and boiled at 95 °C for 10 min. Lysates were separated by SDS-PAGE using 3–8% Tris-Acetate gels (BioRad), transferred to nitrocellulose Hybondstaining . PonceauSer4/8  and γH2AXSer139 . After primary antibody incubation Cells were washed once in 0.2% BSA 0.3% Triton-X/PBS before being washed three times in 0.2% BSA/PBS, then incubated with Alexa Fluor conjugated secondary antibodies  for 1 h at room temperature. Cells were washed once in 0.2% BSA/PBS before being incubated with DAPI diluted 1:1000 for 30 min at room temperature. After DAPI, cells were washed 3 times in 0.2% BSA/PBS then mounted onto slides with Prolong Glass Antifade mountant (Thermo Fisher) and imaged using a DM6 widefield microscope. Multiple images per cell line were captured in each independent experiment and analyzed using ImageJ software, Version 2.3.0/1.53q (NIH)).Cells were seeded onto coverslips and allowed to establish exponential growth in drug-free medium for 24 h before the coverslips were washed once in ice cold PBS, permeabilized in 0.2% triton-X/PBS for one minute, and then fixed in 2% paraformaldehyde. After fixing cells were washed once in PBS before being washed 3 times in 0.2% BSA 0.3% triton-X/PBS. Cells were then blocked in 5% BSA + 1:25 goat serum/PBS for one hour at room temperature. After blocking, primary antibodies were incubated overnight at 4 °C in 0.2% BSA 0.3% triton-X/PBS. The following primary antibodies were used: pRPA50 values between the CP70 B1 cell line (LC50 = 4.13 µM) and the UWB1.289 cell line (LC50 = 0.64 µM)  . To demo0.64 µM) . The CAOp-value = 0.009) . However, VE-821 was not particularly cytotoxic in the homozygous BRCA1 mutant COV362 cell line (The data clearly shows that that HGS cell lines were significantly more sensitive to single-agent VE-821 than non-HGS cell lines (= 0.009) . Howevertoxicity ,17, and ell line d despiteTP53 and BRCA1 were not predictive of sensitivity to VE-821, we investigated whether expression of 13 key proteins  (Since mutations in and ATM) a, previop-value = 0.041), TopBP1  and APOBEC3B  protein expression  or low (below the median) protein expression for each of the 13 proteins analyzed. The percentage cell survival at 10 µM VE-821 of the cell lines with high or low protein expression was then assessed and compared for each individual protein. Of the 13 proteins analyzed, cells with low RAD51  tended to have a lower percentage cell survival at 10 µM VE-821 (p = 0.065), and it may be worth investigating in a larger panel of cell lines.ATRi and PARPi combinations have been shown to have synergistic cytotoxic activity in a variety of preclinical models ,43,44 anPrevious studies have shown that ATRi are more cytotoxic in cancer cell lines with increased RS ,47,48,49Ser4/8 and γH2AX were measured across the panel of cell lines by immunofluorescence (IF) microscopy      .Ser4/8 and γH2AX measure slightly different aspects of RS. Whilst pRPASer4/8 measures the accumulation of ssDNA, an early event in RS onset, γH2AX measures the development of DSBs, a probable consequence of persistent RS. Despite this, levels of pRPASer4/8 and γH2AX correlated well with each other across the panel of cell lines .pRPAMultiple ATRi are currently in clinical trial as monotherapy and in combination with chemotherapeutics, radiotherapy, PARPi or immune checkpoint inhibitors. The development of these molecularly targeted drugs has driven the search for predictive biomarkers for patient treatment stratification. Whilst preclinical data are essential to identify and develop potential predictive biomarkers, much of it is based on studies using genetic modification of a single gene, which does not reflect the clinical situation where multiple determinants of resistance and sensitivity may be expressed in the same tumor.TP53 mutant. p53 has been described as a determinant of sensitivity to ATR inhibition, although data has been conflicting with some studies reporting that p53 dysfunction is a determinant of sensitivity to ATRi [TP53 mutation did not correlate with sensitivity to VE-821. Therefore p53 status may be a better determinant of chemo- or radio-sensitization to ATR inhibition, than ATRi monotherapy [We discovered that HGSOC cell lines were more sensitive to single agent VE-821 and focused our efforts on establishing why. Almost all HGSOCs are  to ATRi ,16,17 wh to ATRi ,50. Despotherapy .ATM mutations or loss of ATM protein expression [ATM mutations are common in hematological malignancies, it has been predicted only 1–5% ovarian cancers contain somatic ATM mutations [ATM; therefore, ATM mutation was not investigated as a determinant of sensitivity to VE-821. Despite this, ATM protein expression varied considerably across the panel of cell lines with an 18-fold difference in expression between the cells with the lowest ATM expression (CAOV3) and the highest expression (A2780). However, there was not a significant association between ATM expression levels and sensitivity to VE-821 in the panel of cell lines. This is in keeping with a recent study which found no association between ATM mutations and sensitivity to ATRi AZD6738, whilst ATM function did predict sensitivity to the drug [The most powerful determinant of sensitivity to ATR inhibition described pre-clinically and clinically is ATM dysfunction ,22,42,51pression ,53. Howeutations . In thisutations  containethe drug . Furtherthe drug ,57. TherBRCA1 was also found to confer sensitivity to VE-821 but only in the context of an isogenic cell line pair, suggesting that whilst HRR defects may be predictive of sensitivity to ATRi, BRCA genetic testing alone may not be sufficient for patient stratification.RAD51 inhibition by the small molecular inhibitor BO2 was previously shown to significantly sensitize cells to the ATR inhibitor VE-821 , and HRRSer4/8 and γH2AX . Preclinical data suggesting APOBEC as a determinant of sensitivity to ATR inhibition has led to investigation of APOBEC in clinical trials with a subset of patients with mutations in APOBEC genes being investigated in the OLAPCO clinical trial (NCT02576444) investigating olaparib in combination with ATRi AZD6738, the results of which may further elucidate the significance of APOBEC as a predictive biomarker.Additional proteins identified as potential determinants of sensitivity to VE-821 in this panel of cell lines were APOBEC3B and TopBP1. APOBEC3B, belonging to a subclass of cytidine deaminases responsible for converting cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, have previously been linked to increased levels of RS and mutagenesis , and oveSer4/8 and γH2AX.Previous studies reporting RS as a determinant of sensitivity to ATRi have assessed oncogene-induced RS by MYC ,49, or oSer4/8 or γH2AX) were significantly more sensitive to VE-821. Cells lines representative of HGSOC were found to have significantly higher levels of RS, which could underlie their increased sensitivity to VE-821. Whilst pRPASer4/8 and γH2AX independently predicted sensitivity to VE-821, there was a more significant association between γH2AX FI and sensitivity to VE-821. γH2AX is the primary response to DSBs and is phosphorylated by ATR in S-phase in response to RS and collapsed replication forks [Ser33 in triple negative breast cancer [Ser4/8 and γH2AX as well as high-throughput quantitative imaging analysis should be assessed and validated.Given the difficulty in translating single previously identified determinants of sensitivity to VE-821, we decided to assess the level of intrinsic RS by IF microscopy. Previous functional assessment of replication fork stability in short-term patient derived HGSOC organoids, indicated that replication fork instability is associated with increased sensitivity to berzosertib . Here, won forks , as wellon forks , therefot cancer , and γH2t cancer . TherefoTP53, ATM, BRCA1 may be more easily translated to the clinic, this study demonstrates that this approach may not be reliable when faced with complex cancer phenotypes. In conclusion, we propose RS may be a better predictive biomarker of ATRi monotherapy response than any single gene or protein in ovarian cancers.Whilst assessment of a single predictive biomarker, e.g.,


Phellinus linteus. Thus far, HME’s anticancer activity and mechanisms remain largely unknown. We herein report HME was cytotoxic, more potent than cisplatin, and proapoptotic to various human bladder transitional carcinoma cell lines. Of note, HME blocked STAT3 activation, evidenced by HME-elicited reduction in tyrosine 705-phosphorylated STAT3 levels constitutively expressed or induced by interleukin-6. Significantly, HME-induced cytotoxicity was abrogated in cells expressing a dominant-active STAT3 mutant (STAT3-C), confirming STAT3 blockage as a pivotal mechanism of HME’s cytotoxic action. We further revealed that survivin was downregulated by HME, while its levels were rescued in STAT3-C-expressing cells. Moreover, survivin overexpression abolished HME-induced cytotoxicity, illustrating survivin as a central downstream mediator of STAT3 targeted by HME. Lastly, HME was shown to lower tyrosine 416-phosphorylated SRC levels, suggesting that HME inhibits STAT3 by repressing the activation of SRC, a STAT3 upstream kinase. In conclusion, we present the first evidence of HME’s anti-bladder cancer effect, likely proceeding by evoking apoptosis through suppression of the antiapoptotic SRC/STAT3/survivin signaling axis.Bladder cancer is a leading human malignancy worldwide. Signal transducer and activator of transcription (STAT) 3 is an oncogenic transcription factor commonly hyperactivated in most human cancers, including bladder cancer. Notably, preclinical evidence has validated STAT3 blockade as a promising therapeutic strategy for bladder cancer. Hispolon Methyl Ether (HME) is a structural analog of hispolon, an anticancer component of the medicinal mushroom  Bladder cancer overwhelms over 430,000 people worldwide per year and remains a significant malignancy globally . The risSignal transducer and activator of transcription (STAT) 3 is a member of the STAT family of transcription factors. Physiologically, STAT3 regulates immunity along with cell proliferation, survival, and apoptosis . Under n12H12O4) is a natural polyphenolic compound widely present in Phellinus genus mushrooms, including the traditional medicinal mushroom Phellinus linteus commonly used in East Asia [P. linteus, including antidiabetic, antiinflammatory, antioxidant, and anticancer [Hispolon -4-hydroxyhexa-3,5-dien-2-one; Cast Asia . Hispoloticancer ,24,25. Oticancer ,27. In tticancer . On the Herein, we present the first report demonstrating HME’s anti-bladder cancer effect and its mechanisms of anticancer action. In brief, we validated HME’s cytotoxic effect, more potent than that of cisplatin, on a panel of human bladder cancer cell lines. This cytotoxic action is attributed to the induction of bladder cancer cell apoptosis by thwarting the antiapoptotic SRC/STAT3/survivin signaling axis. Our discovery thereby implicates HME as a potential agent for bladder cancer chemotherapy.To evaluate HME’s potential as an anti-bladder cancer agent, we began by testing whether HME induces cytotoxicity against human bladder cancer cells using an MTS assay. Cisplatin, one of the first-line chemotherapeutic agents for bladder cancer treatment , was incThe nature of HME-induced cytotoxicity was next explored. Immunoblot analysis indicated that HME treatment led to an increase in the levels of cleaved poly-ADP-ribose polymerase (PARP) (c-PARP) in all three bladder TCC cell lines, illustrating that HME elicited caspases activation and hence the induction of apoptosis A. FurtheAccumulating evidence has underscored the link between aberrant STAT3 activation and the malignant progression of diverse human cancers, including bladder cancer . NotablyGiven that HME suppresses STAT3 activation, we aimed to define the role of STAT3 inhibition in HME-mediated bladder cancer cell death. To address this, we generated a stable TCCSUP cell clone that expresses a dominant-active STAT3 mutant (STAT3-C) to withstand HME-mediated blockage of STAT3 activation . It is nAfter establishing a pivotal role of STAT3 blockage in HME’s cytotoxic action, we further explored the candidate effectors downstream of STAT3 to mediate HME-induced bladder cancer cytotoxicity. Along this line, we found earlier that HME downregulates survivin, a vital pro-survival protein known as a STAT3 downstream target A. IndeedHow HME suppresses STAT3 activation in bladder cancer cells was next addressed. To this end, we began by testing the effect of HME on the activation status of JAK2 and SRC, two eminent STAT3 upstream kinases that phosphorylate STAT3′s tyrosine 705 to induce STAT3 activation . ImmunobGiven that SRC activation was inhibited in all HME-treated bladder cancer cell lines, we figured SRC is the primary target of HME for blocking STAT3 activation, in turn promoting bladder cancer cytotoxicity. To address this, we observed that HME failed to downregulate both p-STAT3 (Y705) and survivin nor upregulate c-PARP in TCCSUP cells stably expressing v-src, a dominant-active SRC mutant B. As a rIn this study, we established the cytotoxic effect of HME, an analog of hispolon, on human bladder cancer cells and the underlying mechanism of action. Specifically, we demonstrated that HME is cytotoxic and proapoptotic to a panel of human bladder TCC cell lines and exhibits more potent cytotoxicity than Cisplatin . FurtherP. linteus, exerts a marked anticancer effect on diverse human cancer cell lines, including bladder cancer [CIP1 as a result of ERK-mediated downregulation of MDM2 [Recent studies have demonstrated that hispolon, a main pharmacological constituent of r cancer ,25. Thisr cancer ,27. Alonr cancer . In this of MDM2 . In viewMounting evidence has substantiated the notion that blockage of STAT3 activation is a promising therapeutic strategy for multiple human malignancies, including bladder cancer ,19,20,21Data presented here underscored that survivin downregulation due to STAT3 blockage by HME is a vital mechanism of HME’s cytotoxic action on bladder cancer cells . SurviviOur inquiry about how HME suppresses STAT3 activation revealed that SRC appears as the primary STAT3 upstream kinase targeted by HME, as the levels of tyrosine 416-phosphorylated SRC were markedly lowered in all HME-treated bladder cancer cell lines . In thisIn conclusion, we first report HME’s cytotoxic effect on human bladder cancer cells via suppression of the antiapoptotic SRC/STAT3/survivin signaling axis . Conside® Bioscience . Recombinant human IL-6 was acquired from PeproTech .Hispolon methyl ether was chemically synthesized according to the published procedure . HispoloBIRC5 (Genebank number: NM_001168) was PCR-amplified from the cDNA pool of human colorectal cancer cell line HCT 116 (ATCC CCL-247™) using the following primer pair: 5′-GCCATTAACCGCCAGATTTG-3′ (forward) and 5′-CTAAGACATTGCTAAGGGGC-3′ (reverse). The PCR-amplified BIRC5 ORF was TA-cloned into the pGEM-Teasy vector  for sequence verification. The sequence-verified BIRC5 ORF was then retrieved by EcoR I digestion and subcloned to the pBabe.puro vector at the EcoR I site, followed by validation of in-frame orientation. For generating pBabe-HA-v-src, the v-src-overexpressing vector, the v-src ORF was PCR-amplified using pLNCX chick src Y527F (Addgene plasmid #13660) as the template by the following primer pair: 5′-ACCGGTGGGAGCAGCAAGAGCAAG-3′ (forward) and 5′-CTATAGGTTCTCTCCAGGCTG-3′ (reverse). Then, the PCR-amplified product was TA-cloned, sequence verified, digested by Age I and Eco RI, and then directionally subcloned to the pBabe-HA vector established in our laboratory [http://n2t.net/addgene:13660; RRID:Addgene_13660 accessed on 28 November 2016).pBabe-HA-STAT3-C, the pBabe plasmid-based expression vector for ectopic expression of the dominant-active STAT3 mutant (STAT3 (A661C/N663C); STAT3-C) tagged with an N-terminal hemagglutinin (HA) epitope, has been described previously . To consboratory . pLNCX c2 in their respective culture media supplemented with non-essential amino acids, 1 mM sodium pyruvate, 10% fetal bovine serum, and 1% penicillin–streptomycin. All chemicals involved in the cell culture were purchased from Gibco Life Technologies .Human urinary bladder transitional cell carcinoma cell lines J82 (ATCC HTB-1™) and TCCSUP (ATCC HTB-5™) were both cultured in Eagle’s Minimum Essential Medium, while the T24 cell line (ATCC HTB-4™) in McCoy’s 5a medium in accordance with the recommendation of the American Type Culture Collection (ATCC) . Cells were grown at 37 °C in a humidified environment with 5% CO® AQueous One Solution Cell Proliferation Assay (MTS) assay in accordance with the manufacturer’s protocol . Human bladder TCC cells were grown in 96-well culture plates (7 × 103 cells/well) for 24 h before drug treatment, and the cell viability after drug treatment for 24 h and 48 h was measured thereafter. Long-term in vitro cytotoxicity was assessed by the effect of drugs on cells’ ability to form colonies (clonogenicity) after culture for 10~14 days. Clonogenicity assay was performed as described previously [Short-term in vitro cytotoxicity was determined by the effect of drugs on cell viability using the CellTiter 96eviously ,50.® Annexin V & Dead Cell Assay Kit  as previously reported [5 cells grown on each well of a 6-well plate were subjected to HME treatment  for 24 h, followed by resuspension using trypsinization, two-times washed with phosphate-buffered saline (PBS), and 20 min-incubation with 100 μL of Annexin V & Dead Cell reagent at room temperature in the dark. Thereafter, the annexin V-positive (apoptotic) cell population levels were gauged using flow cytometry analysis on the Muse® Cell Analyzer .HME-induced human bladder TCC cell apoptosis was scored quantitatively using the Musereported ,50. In sTCCSUP cell clones stably expressing HA-STAT3-C, survivin, or HA-v-src were established by infecting TCCSUP cells with pBabe vector-derived retroviral particles carrying the ORFs of HA-STAT3-C, survivin, or v-src, followed by positive selection for 2 days with puromycin for the cells with successful infection. The puromycin-selected cell clones were then subjected to immunoblotting to confirm the ectopic expression of HA-STAT3, survivin, or HA-v-src proteins. The pBabe vector-derived retroviral particles were produced by following the established protocols reported previously ,50.Immunoblot analysis was conducted in accordance with our established procedures ,50. Prim


Merkel cell carcinoma (MCC) is an aggressive skin cancer with limited treatment options for the advanced disease once immunotherapy has failed. MCC carcinogenesis has been attributed to the integration of an oncogenic virus or chronic UV radiation. The antineoplastic effects of dimethyl fumarate (DMF) have recently been shown in melanoma. Three virus-negative UV-associated MCC cell lines were treated with different doses of DMF. The cytotoxic effects and cell proliferation were assessed by the MTT cytotoxicity assay and BrdU proliferation assay for four different treatment periods. The reductions in cell viability and proliferation were demonstrated for all the cell lines used, with DMF proving to be significantly effective.Merkel cell carcinoma (MCC) is a rare, difficult-to-treat skin cancer once immunotherapy has failed. MCC is associated either with the clonal integration of the Merkel cell polyomavirus (MCPyV) or mutagenic UV-radiation. Fumaric acid esters, including dimethyl fumarate (DMF), have been shown to inhibit cell growth in cutaneous melanoma and lymphoma. We aimed to explore the effects of DMF on MCPyV-negative MCC cell lines. Three MCC cell lines  were treated with different doses of DMF. The cytotoxic effects and cell proliferation were assessed by the MTT cytotoxicity assay and BrdU proliferation assay at different time points. A significant reduction in cell viability and proliferation were demonstrated for all the cell lines used, with DMF proving to be effective. Merkel cell carcinoma (MCC) is an uncommon cutaneous malignancy with epithelial and neuroendocrine differentiation. MCC cells share morphological, immunohistological, and ultrastructural features with Merkel cells. However, there is no obvious direct histogenetic link between MCC and Merkel cells. Importantly, Merkel cell polyomavirus (MCPyV) is clonally integrated into about 80% of the MCC in patients of the Northern hemisphere. By contrast, MCPyV-negative MCC is characterized by a high mutational burden similar to other ultraviolet (UV)-associated skin tumors such as cutaneous melanoma. Nevertheless, the distinct molecular pathogenesis of MCC and its link to MCPyV is not yet fully understood. The biological behavior of MCC is highly aggressive with high rates of local recurrences, regional lymph node, and distant metastasis. When compared to the chemotherapy era, the management of advanced MCC has significantly been improved since the introduction of immune checkpoint inhibitors. Nevertheless, only about 50% of patients with MCC benefit from immunotherapy ,4,5,6,7.™), dimethyl fumarate , diroximel fumarate (Vumerity™), and zinc, magnesium, and calcium salts of monoethyl fumarate (Fumaderm®). DMF was first shown to possess cytoprotective and antioxidant effects in non-cancer models, which appeared to be related to the induction of the nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) pathway which also crosstalks with the NF-κB pathway [Fumaric acid esters (FAE) are successfully used for the treatment of psoriasis and multiple sclerosis. Their antioxidative, immunomodulatory, and neuroprotective properties cause FAE to be attractive therapeutic candidates for other conditions as well. Currently, four FAE formulations are approved as human drugs: monomethyl fumarate , MCC14.2 (source: CBA-1340), and MCC26 (source: CBA1341). On the basis of a material transfer agreement, all the cell lines have previously been provided to J.C. Becker by J.H. Leonard . The cells were cultured in RPMI 1640 with 10% fetal calf serum, 2 mM L-glutamine, and a penicillin (100 U/mL)/streptomycin (100 U/mL) mixture. All the substances were purchased from PAN-Biotech, Germany. The cells were grown at 37 °C, 95% humidity, and 5% COThe DMF was provided by Almirall Hermal GmbH . The DMF with high purity was dissolved in DMSO and sterile filtered. All three cell lines were treated with the prepared substance in different concentrations for different treatment periods . After the treatment time, the assay-specific procedures and measurements were performed. To analyze the antineoplastic effects of the substance DMF on cell viability and to determine its dose–response relationship, colorimetric MTT cytotoxicity assays were performed on all three cell lines. The cells were treated with increasing concentrations of DMF in RPMI 1640 medium for 2 h, 6 h, 24 h, or 48 h. After the end of the incubation period, the yellow-colored MTT reagent 3--2,5-diphenyltetrazolium bromide (5 mg/mL) was administered to the cells. The MTT is reduced via mitochondrial dehydrogenase by metabolically viable cells to violet-colored formazan crystals that can be measured using photometry. The cell viability was analyzed using a microplate absorbance reader  measuring the optical density. The amount of purple formazan crystals is directly proportional to the number of viable cells. All the MTT assays were performed with eight replicates. In order to analyze and quantify the antiproliferative effect of the substance DMF, a BrdU (5-bromo-2-deoxyuridine) proliferation assay  was carried out with all cell lines according to the manufacturer’s instructions. This proliferation assay is a non-isotopic colorimetric immunoassay to quantify BrdU incorporation into newly synthesized DNA from actively proliferating cells. All the experiments were set up according to the same scheme as in the MMT assay. The extent of the cell proliferation was determined via optical density using a microplate absorbance reader . All the BrdU assays were performed with eight replicates.The statistical analyses were performed using the software package GraphPad Prism Version 9.4.1. Based on the assumption of a normal distribution for naturally measured values, an ordinary one-way ANOVA was used to calculate the significance. For a comparison between the individual measured values, a Holm-Šídák test was connected. If possible, the measured values were used to estimate an IC50.Based on previously published papers ,20,21,22p < 0.0001) was achieved (p = 0.0111) at the highest concentration used. The reduction was dependent on the dosage used as well as the duration of the treatment time. The strongest reductions were visible for the 24 h and 48 h treatment of each cell line. In this regard, the SR was reduced to 2.6% (MCC13), 0.8% (MCC14.2), and 2.0% (MCC26) after 48 h of treatment with 200 µmol/L. However, similarly low levels below 5% SR were also seen in all three cell lines after only 24 h. Notably, the SR was already reduced below 5% in cell line MCC26 at a concentration of 100 µmol/L compared to 150 µmol/L (MCC14.2) and 200 µmol/L (MCC13). Additionally, when comparing the IC5048 h values of the three different cell lines it could be observed that MCC26 (31.60 µmol/L) was more sensitive than MCC14.2 (67.99 µmol/L) and MCC13 (97.80 µmol/L). For all three cell lines, a very significant reduction in cell viability (achieved . StrikinThe concentrations of DMF used were derived from the previously performed MTTs (0 µmol/L–200 µmol/L). The three cell lines were seeded at different previously identified concentrations. MCC13 was seeded at 30.000 cells/well. MCC14.2 was seeded at 20.000 cells/well and MCC26 at 50.000 cells/well. To cause the results to be more descriptive and comparable, a proliferation rate (PR) in percent was calculated. For this purpose, the negative control was set as 100% and the other values were related to it. p ≤ 0.0001) in PR for all the treatment durations  and 50.53 µmol/L (MCC26).The proliferation assays showed a very significant reduction , mutational patterns are strongly associated with UV-induced DNA damage, and many coding mutations of cancer-related genes do exist . Inspiren extent . A signin extent .p ≤ 0.0001) in proliferation for all cell lines and treatment periods, with all three showing very significant reductions (p ≤ 0.0001) below 10%. For the 24 h and 48 h treatment periods, strong reductions to under 5% were evident at relatively low concentrations starting at 100 µmol/L  or 150 µmol/L (MCC13). Similar to our data, where one could see a tendency toward time dependent PR reduction and a dosage-dependent PR reduction, some data suggest that DMF induces a concentration-dependent reduction in proliferation in melanoma cells as well as in human colorectal adenocarcinoma cell lines, similar to our results [The three BrdUs performed with DMF all showed similar trends regarding the results. There were very significant reductions  and functional experiments with respect to the NF-κB signaling should be included. Regarding the issue of drug resistance development, several rounds of DMF treatments could be performed on the eventual surviving MCC cells. If drug resistance is consistently and easily occurring, DMF might be combined with additional drugs. NF-κB signaling is known to be regulated by the p38 stress-signaling pathway. It would be worth seeing whether NF-κB as well as p38 signaling is downregulated by DMF. If yes, then p38 inhibitors could possibly potentiate or even act synergistically with the DMF treatment, resulting in a better outcome and a decrease in the emergence of drug resistant cancer cells. Moreover, in vitro cell invasion assays are recommendable for future studies to exclude that DMF induce a paradoxical increase in cell invasion in the MCC cell lines used.Despite current immunotherapy regimens, there is still a need for other treatment approaches for MCC patients. Using different MCPyV-negative cell lines, we have investigated the in vitro effect of DMF, a substance for which an antineoplastic effect in combination with other tumors has been shown. A reduction in cell viability and an inhibition of proliferation were observed, with DMF proving effectiveness at doses starting from 10 µmol/L or 50 µmol/L depending on the assay and cell line. For the treatment duration of the cell lines with the substrate, a dependent effect was clearly shown in the MTT-assays, as well as a tendency in the BrdU-assays. Repeating the tests and extending them to include supplementary procedures could provide further insights. Even with the existing data, it is becoming apparent that FAE such as DMF should be investigated further as a promising therapeutic agent for MCPyV-positive MCC as well.


The yeast molecular chaperone Hsp104 is a disaggregase that has been shown to fragment amyloidogenic aggregates associated with certain yeast prions and reduce protein aggregation associated with human neurogenerative diseases. In yeast, we found that TTR forms both SDS-resistant oligomers and SDS-sensitive large molecular weight complexes. In actively dividing cultures, Hsp104 has no impact on oligomeric or large aggregate populations, yet overexpression of Hsp104 is loosely associated with an increase in overall aggregate size. Interestingly, a potentiating mutation in the middle domain of Hsp104 consistently results in an increase in overall TTR aggregate size. These data suggest a novel approach to aggregate management, where the Hsp104 variant shifts aggregate populations away from toxic oligomeric species to more inert larger aggregates. In aged cultures Hsp104 overexpression has no impact on TTR aggregation profiles suggesting that these chaperone approaches to shift aggregate populations are not effective with age, possibly due to proteostasis decline.Patients with the fatal disorder Transthyretin Amyloidosis (ATTR) experience polyneuropathy through the progressive destruction of peripheral nervous tissue. In these patients, the transthyretin (TTR) protein dissociates from its functional tetrameric structure, misfolds, and aggregates into extracellular amyloid deposits that are associated with disease progression. These aggregates form large fibrillar structures as well as shorter oligomeric aggregates that are suspected to be cytotoxic. Several studies have shown that these extracellular TTR aggregates enter the cell and accumulate intracellularly, which is associated with increased proteostasis response. However, there are limited experimental models to study how proteostasis influences internalized TTR aggregates. Here, we use a humanized yeast system to recapitulate intracellular TTR aggregating protein  In vitro evidence suggests that these small oligomeric TTR aggregates are cytotoxic  is associated with the amyloidogenic aggregation of the human transthyretin (TTR) protein. As a functional tetramer, TTR shuttles hormone and proteins within the bloodstream and CNS. However, the tetramer can dissociate with age, leading to the misfolding and subsequent aggregation of the TTR monomer. It is the tetramer dissociation that is considered the rate limiting step of the aggregation process . The misytotoxic . Over tiytotoxic . Interesin vivo molecular chaperone response.Transthyretin aggregates have been observed within cells, detected as intracellular cytoplasmic deposits . Cells wMolecular chaperones play an important role in maintaining protein homeostasis by facilitating proper polypeptide folding while managing the accumulation of excess aberrantly folded proteins . Elevatein vitro studies show that wildtype-Hsp104 can disassemble human amyloidogenic aggregating proteins  were also co-transformed with plasmids containing an Hsp104 promoter (HSE) driving either empty vector , wildtype Hsp104 , Hsp104 with a A503V mutation , or Hsp104 with a A503S mutation .All yeast strains are derivatives of the 74-D694 background and listed in 600 = 0.4–0.8; approximately 24 h), or until saturation .Yeast strains were grown using standard media and cultivation procedures . NutrienOE (p3169), Hsp104A503V (p3170), or Hsp104A503S (p3171). Strains were grown to log or saturation, and 3D images of independent transformants were captured and quantified for the number of cells that contained GFP puncta compared to all cells expressing GFP.For co-localization experiments, strains were transformed with TTR-eGFP (p3146) and mCherry (p3197) or Hsp104-mCherry (p3172). 3D images were captured with a 63X  objective on a DMI600 Leica inverted microscope using Leica K5 camera and LasX software. For print, images were subjected to 3D deconvolution, using AutoQuant deconvolution software and shown as maximum projection images. For quantifying the number of cells with TTR-eGFP puncta, strains were transformed with TTR-eGFP (p3146) and either empty vector (p3168), Hsp1042•6H20, 2 mM EDTA, 10% glycerol; pH 7.5), protease inhibitor cocktail (Sigma) and PMSF (Sigma), while kept on ice to prevent protease degradation, as described in Cell lysates were prepared from 50 ml cultures by glass bead lysis with 1x lysis buffer  growth after 24 h. Wildtype strains with an endogenous copy of Hsp104 (WT) exhibited on average less than 0.5% of cells with puncta, while strains lacking HSP104 (hsp104Δ) had almost three times as many cells with puncta , we found that cells did not have any detectable TTR-GFP puncta after 24 h of growth  can trap protein complexes and aggregates of different sizes within the interface between different sucrose concentrations  shows TTR preferentially fractionating to either the 10% or 60% fractions  and the National Institutes of Health (NIH) grant (GM131365) to AM, and the Schmitt Leadership Fellowship to AK.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cell Death and Disease 10.1038/s41419-018-1002-2, published online 11 September 2018Retraction to: Figure panels 1A, B, and C, labeled as cell line MDA-MB-468, appeared to be falsified and/or fabricated, and were also used in multiple NIH grant applications labeled as breast cancer cell line 4T1 or brain cancer cell line U118.Photos in figure panels 2A and 2C, labeled as breast cancer cell line MDA-MB-468, appeared to be falsified, and were also used in multiple NIH grant applications labeled as cell lines 4T1 or BT20.Figure panel 2B, labeled as breast cancer cell line MDA-MB-468, appeared to be falsified and/or fabricated, and was also labeled as brain cancer cell line GBM U118 in another publication by the same authors [Figure 4C, labeled as iFLs from breast cancer cell line MDA-MB-468 in the publication, appeared to be falsified and/or fabricated, and was also used in  labeled An additional concern identified as part of the investigation is the extensive overlap between this study and another article by the same authors .The Editor-in-Chief has retracted this article. An investigation by the Office of the Vice Chancellor for Research at Washington University in St. Louis concluded that:The authors have not responded to any correspondence from the editor or publisher about this retraction.


Metabolomics represent the set of small organic molecules generally called metabolites, which are located within cells, tissues or organisms. This new “omic” technology, together with other similar technologies  is becoming a widely used tool in cancer research, aiming at the understanding of global biology systems in their physiologic or altered conditions. Cancer is among the most alarming human diseases and it causes a considerable number of deaths each year. Cancer research is one of the most important fields in life sciences. In fact, several scientific advances have been made in recent years, aiming to illuminate the metabolism of cancer cells, which is different from that of healthy cells, as suggested by Otto Warburg in the 1950s. Studies on sponges and algae revealed that these organisms are the main sources of the marine bioactive compounds involved in drug discovery for cancer treatment and prevention. In this review, we analyzed these two promising groups of marine organisms to focus on new metabolomics approaches for the study of metabolic changes in cancer cell lines treated with chemical extracts from sponges and algae, and for the classification of the chemical structures of bioactive compounds that may potentially prove useful for specific biotechnological applications. Metabolomics (or metabonomics ) is usedMetabolomics represents a powerful approach, because metabolites and their concentrations directly reflect the underlying biochemical activity and state of cells and/or tissues, producing the molecular phenotype. The set of these small molecules, such as peptides, amino acids, nucleic acids, carbohydrates, organic acids, vitamins, polyphenols and alkaloids, represents the metabolome, through which the final or intermediate products of a biochemical process can be studied in order to build a metabolic pathway . This arThe concept of the metabolic profile first appeared in literature in the 1950s, but only in the following three decades was this area of research completely developed. Despite this observation, metabolomics has only recently aroused the interest of researchers, thanks to the development of advanced technologies for the quantification of metabolites, such as gas chromatography (GC) and mass spectrometry (MS) . InitialDifferent methods can be applied to prepare, to extract and to analyse samples. In this paragraph, we explain the most used techniques to treat marine specimens in order to avoid rapid alteration of their metabolic profile and to stop metabolic reactions.t-test, analysis of variance, principal component analysis (PCA), and partial least squares (PLS) and orthogonal-PLS (OPLS) analyses are the most used statistical methods in metabolomics studies [Obtaining a broad coverage of the metabolome is difficult, due to a wide range of physico-chemical properties exhibited by small molecules. For this reason, various techniques are used to evaluate the set of metabolites . Once sa studies ,39. In tSeveral separation techniques are routinely applied according to the characteristics of the putative compounds to be identified. Among them, Gas Chromatography (GC) is an excellent separation technique that was considerably improved with the introduction in 1979 of fused-silica capillary columns that resulted in higher resolution, higher efficiency, better reproducibility and smaller sample size . In addi13C, 15N) or biologically reactive groups, including phosphate atoms (31P) [1H-NMR for the analyses of marine samples, but other nuclides  may provide more information about this set of metabolites. The main limits of this technique concern the spectral resolution and sensitivity, which can be improved by high intensity magnetic field [1H-NMR spectroscopy is almost 100 times less sensitive. This means that a typical 1H-NMR-based metabolomics study only gives back information on 50–200 identified metabolites with concentrations >1 μM, while a typical LC-MS study can render information on more than 1000 identified metabolites with concentrations of >10 to 100 nM [MS and NMR are the most widely used analytical techniques in metabolomics . MS provms (31P) ,58,59,60ms (31P) . Jeremy ms (31P) . Therefoms (31P) ,64. Our ic field ,65. Furto 100 nM .Cancer is one of the deadliest human diseases, able to alter the metabolism of a cell . It is wTethya crypta [Halichondria okadai [Dunaliella salina [According to an analysis of literature, in the last years, the marine environment has shown to be the most promising source of bioactive compounds against cancer. In particular, sponges and algae are likely to be the marine organisms from which the largest number of natural compounds with antiproliferative activity could be isolated. During the past 50 years, sponges are considered a gold mine for the discovery of bioactive natural products ,75. In fa crypta . In 2010a okadai , developa okadai . Algae, a okadai . Potent a okadai ,81,82,83a salina ,86,87,88In the frame of evolutive processes, several marine organisms, such as macro- and microalgae, sponges and fishes, developed appropriate defence mechanisms. They are based on the use of a variety of natural weapons, i.e., molecules that allow them to survive in a hostile environment characterized by stressful conditions. This is due to variable salinity, pressure, temperature and light, as well as to the need to avoid microbial and/or viral attacks . These cThe first approach involves the use of bioassay-guided fractionation of chemical extracts from the marine organisms under analysis on different cancer cell lines, and, once an active fraction has been identified, metabolomics approaches are applied to elucidate the chemical structure of the potentially bioactive compounds. Otherwise, in the second approach, the bioassay-guided fractionation of chemical extracts is also performed. However, once a cancer cell line on which the active fractions have antiproliferative effects has been identified, metabolomics approaches are applied to define the changes in metabolites of treated cells. To date, the data reported in literature (see below) demonstrated that the first approach is the most used and fewer applications have been reported on the second approach, because, at the moment, the second approach represents a novelty in the pharmacological field.Stylissa carteri sampled in the Red Sea, were extracted and analysed using LC-MS and 1H-NMR. Further, these compounds were tested on two cancer cell lines. The IC50 values for Stylissamide A on MCF7 (breast cancer) and on HepG2 (liver cancer) were of 21.1 µM and 36.8 µM, respectively, while the IC50 values for Stylissoside A were of 27.5 µM on MCF7 and 30.5 µM on HepG2 [Callyspongia siphonella. The molecules were characterized by using 1H-NMR and named 5-bromotrisindoline and 6-bromotrisindoline  responsible for the reduction of the 70–90% of cell viability of SKBR3 breast cancer cell line [Coscinoderma sp. on cancer cells. Crude extracts alone showed moderate activity, but were enhanced when the extract was encapsulated in liposomes. In this case, the IC50 values were notably lower compared to the positive control (Doxorubicin). The tested cell lines were HepG2 (IC50 = 2.2 µg/mL), MCF7 (IC50 = 4.1 µg/mL) and CaCo2  [Geodia macandrewii, thanks to a complex combination of two techniques for the generation of metabolomics data: UHPLC and MS. The cytotoxic activity of this compound was tested against a melanoma cancer cell line (A2058) and showed weak cytotoxic activity (IC50 = 8.5 μM) [Latrunculia biformis caught the attention of Li et al. [1H-NMR and MS analyses, the presence of known and unknown compounds. However, only three of them showed cytotoxic activity on the HCT-116 colon cancer cell line. The first tested compound was already known: (−)-discorhabdin L, and it showed IC50 equal to 0.33 µg/mL. The other two interesting compounds were new. They are two new discorhabdin analogs, i.e., (−)-1-acetyl-discorhabdin L and (+)-1-octacosatrienoyl-discorhabdin L, exhibiting IC50 of 1.1 µg/mL and 25.6 µg/mL, respectively [Theonella swinhoei performed by MS and 1H-NMR showed the presence of theonellamides. In particular, Theopalauamide was an already known compound whose cytotoxic activity was evaluated on the HTC-116 colon carcinoma cell line (IC50 = 2.8 µM), while two new compounds  have been largely demonstrated to be one of the richest sources of exclusive secondary metabolites with relevant bioactivity, by means of bioassays ,90. Manyon HepG2 . Similarectively . Some spell line  at the cell line . LC-MS l7 µg/mL) . A new c 8.5 μM) . Similari et al. . The autectively . Metabolectively .Petrosia ficiformis sampled in the Mediterranean Sea , a bacterial strain of Streptomyces sp. (SBT348) has been isolated. Thanks to a metabolomic approach performed through LC-MS analyses, it was possible to isolate a known compound, namely Petrocidin A , a new compound whose structure has been elucidated  has been isolated, from which Nocardiotide A has been detected and characterized through MS analyses. Surprisingly, this compound showed activity against several different cancer cell lines, such as the CT26 , HeLa  and MM.1S (IC50 = 8 µM/mL) cancer cell lines [Nocardiopsis sp. (UR 92) were isolated from another sponge belonging to Amphimedon sp., coming from the Red Sea. These bacterial strains were cultured in different culture conditions and their crude extracts were analysed with an MS approach. The extract of Nocardia sp. (UR86) caused cellular inhibition of several cancer cell lines, such as HepG2, MCF7 and CaCo2, with IC50 values of 3.1 µg/mL, 3.9 µg/mL and 14.4 µg/mL, respectively. Similarly, extracts of Nocardiopsis sp. (UR92) were active against the same cancer cell lines with IC50 values of 3.7 µg/mL, 14.7 µg/mL and 14.3 µg/mL, respectively [Micromonospora sp. was isolated from Callyspongia sp., while Actinokineospora sp. was isolated from Spheciospongia vagabunda. Several compounds were detected using LC–MS metabolomics analyses, but only one was noteworthy in terms of its cytotoxic effect: the N-(2-hydroxyphenyl)-acetamide  against HepG2, MCF7 and Hela cancer cell lines. They also assessed the value of IC50 on the above three cell lines, being 29.7 μg/mL, 25.1 μg/mL and 36.3 μg/mL, respectively. In conclusion, ulvan showed a significant cytotoxic activity in a dose-dependent manner and it can be developed as a promising cancer-fighting compound. In the same work, the researchers determined the fine structure of the ulvan, using 1H-NMR and MS methods. Similarly, Mofeed et al. [U. lactuca and Ulva fasciata on MCF7 and HTC-116  cell lines at different concentrations , highlighting a significant dose-dependent response after 48 h of exposure. They also tested the extracts of three additional species of seaweeds, which, similarly, exhibited cytotoxic activity: two red algae, namely Amphiroa anceps and Corallina mediterranea, and the fucales Sargassum filipendula. The extracts were analysed by means of GC-MS. Moreover, various researches have been carried out on seaweed belonging to order Fucales, such as Fucus vesiculosus. Geisen et al. [F. vesiculosus, Zenthoefer et al. [1H-NMR spectroscopy techniques, revealing a characteristic fingerprint that was significantly correlated with the activity. In particular, the acetonic crude extract (FvT_A) showed the strongest activity against Panc89 and PancTu1, with an inhibitory rate of 80.3% and 82.6%, respectively. It is worth observing that the particular attention paid to some brown algae is due to the production and storage of a sulphated polysaccharide called Fucoidan, which is well known as a promising compound to be applied for cancer treatment. Among various species of brown algae, one of the best known is Cladosiphon okamuranus, an edible alga that is commonly cultured in Japan. This alga largely produces an accessory pigment named fucoxanthin, which is mainly metabolised and transformed into fucoxanthinol by the digestive enzymes of the gastrointestinal tracts of consumers. Both compounds are well known and studied because they exert an anti-proliferative effect [C. okamuranus using HPLC and MS. The results showed potentially anti-osteosarcoma properties, which appear to be at least partially attributable to the inhibition of Akt and AP-1 signal pathways in human and mouse osteosarcoma cancer cell lines . In addition, various sulphated compounds extracted from algal biomasses have aroused interest for their biotechnological applications. In particular, Shao et al. [U. fasciata (UFP), Gloiopeltis furcata (GFP) and Sargassum henslouianum (SHP). The polysaccharides were extracted after ultrasonic disruption applying Radial Flow Chromatography (RFC) separation and then tested on MKN45 (gastric cancer) and DLD  cell lines. After incubating with those three extracts for 24 h at different concentrations (from 0.125 to 1.00 mg/mL), the inhibitive effects on MKN45 cancer cells were observed. In particular, SHP exhibited the strongest cytotoxic effect, with a growth inhibition percentage of almost 50% at the concentration of 1.00 mg/mL. In contrast, all samples showed low percentages of growth inhibition on DLD cancer cells, at all concentrations.Algae are a complex and heterogeneous group of photosynthetic organisms, showing an extraordinary biological diversity represented by more than 166,000 species . It is cd et al.  tested ar et al.  analysede effect . Rokkakue effect  extracteo et al.  demonstr1H-NMR and MS metabolomic approaches to record the variation of metabolites naturally present in the dinoflagellate Amphidinium carterae in a long-term culture. Specifically, among other compounds of interest, as fatty acids and carotenoids, they focused on the amphidinol family and their content variation in relationship to different levels of daily irradiance and nutrients in anf/2 medium. Three concentrations of methanolic extract  were tested on four cancer cell lines: namely A549, HT29, MDA-MB-231 and PSN-1, showing a high antiproliferative activity against all four tumor cell lines (80%). Similarly, other studies investigated the chemical composition of the extracts of various microalgae along with their anticancer effects. Arslan et al. [Isochrysis galbana through 1H-NMR and GC-MS and tested its cytotoxic effect on four cell lines: chronic myelogenous leukemia K562, human acute T lymphoblastic leukaemia MOLT-4, human Caucasian histiocytic lymphoma U937, Caucasian promyelocytic leukemia HL60 and human Burkitt′s lymphoma Raji cancer cells, showing the highest cytotoxicity (about 24.07%) at a concentration of 500 μg/mL against Raji cells. An interesting approach to the assay of natural products against cancer cell lines was reported by Karakas et al. [Chlorella protothecoides and Nannochloropsis oculata) against A172 (brain glioblastoma) and HCT116 cell lines. Assays on cells were carried out, testing not only the crude extract at increasing concentrations , but also three micro- and nano-particles loaded with the extracts, represented by PVA: Chitosan solution (PCH) and PVA: NaAlg solution (PNA) (produced through electrospraying techniques), and NPAL (obtained using the microemulsion method). The particles PCH and PNA were prepared by loading with concentrations of 70, 35 and 17.5 μg/mL, while NPAL particles obtained by the microemulsion method were loaded with 50, 25 and 12.5 μg/mL. Each particle was preliminarily tested on HUVEC cell lines (non-cancerous cells) in order to ensure absence of cytotoxicity against normal cells. Crude extracts were analysed with GC-MS. The results of the assay showed cytotoxic effects of both microalgal extracts and encapsulated microalgal extracts on two cancer cell lines while they did not have cytotoxic effects on healthy cells. This study showed that microalgal extracts have cytotoxic effects on cancer cells and did not lose their cytotoxic effects after encapsulation. In a similar study, Hussein et al. [Tetraselmis suecica in conjunction with an innovative compound, through the application of silver nanoparticles adopted as a carrier, against MCF7, mammary carcinoma 4 T1 and normal Vero cell-lines. The cytotoxicity assays were carried out by separately testing the crude extract of the microalgae, the silver nanoparticles AgNPs. In conclusion, the co-application of the two. T. suecica single application only showed the IC50 of 46.77 μg/mL on MCF7 and 83.17 μg/mL on 4 T1 cells. The AgNPs single application displayed the highest cytotoxicity according to a dose-dependent pathway after 72 h treatments with an IC50 of 5.3 μg/mL on MCF7, 17.78 μg/mL on 4 T1, and 25.11 μg/mL on Vero cells. Besides, the AgNPs-T. suecica co-application reached the IC50 of 6.60 μg/mL and 53.7 μg/mL, respectively, while the T. suecica-CHL single application only showed the IC50 of 46.77 μg/mL and 83.17 μg/mL against MCF7 and 4 T1, respectively. Moreover, they analysed the crude extract of T. suecica through GC-MS. In another study, Hussein et al. [Nannochloropsis oculata’s water extract with IC50 of 15.8 μg/mL, TMX-T. suecica’s ethanolic extract with IC50 of 16.9 μg/mL, TMX-Chlorella sp.’s chloroform extract with IC50 of 13.4 μg/mL, while TMX-N. oculata’s chloroform extract, TMX-T. suecica’s ethanolic extract and TMX-Chrolella sp.’s ethanolic extract showed cytotoxic effect against 4 T1 cells with IC50 of 15.4, 13.8 and 16.9 μg/mL, respectively. Moreover, the synergistic application of TMX-algae’s extracts maintains an antiproliferative effect on cancer cell lines and reduced the toxicity on normal Vero cells. In addition, after 1H-NMR analyses, isoleucine was found only in the ethanolic extract of Chlorella sp., glycerol only in the ethanolic extract of T. suecica and in the chloroform extract of Chlorella sp., while xanthine was found only in the chloroform extract of Chlorella sp. These metabolites can help reduce the toxicity of non-cancerous Vero cells. Fayyad et al. [Spirulina platensis to identify the most active chemical compounds and also to check the cytotoxic effect on cancer cell lines L20 B (mice intestine carcinoma) and MCF7 (breast cancer) after 24 h and 48 h exposure. GC mass analysis showed that the active chemical compounds in the extracts contained alkaloids, terpenes, phenols, resins, saponines, flavones, steroids, proteins and amino acids. Moreover, this extract exhibited the highest growth inhibition by testing the 12.5 mg/mL concentration against L20 B (32.5%) and against MCF7 (71.5%). These percentages increased after 48 h application (35.5% against L20 B and 78% against MCF7). Other studies, rather than testing the entire extract from microalgae, worked on different fractions obtained with distinct methods. El-Baz et al. [Haematococcus pluvialis and Dunaliella salina and tested them on HePG2, MCF7, HCT116, and A549 cancer cell lines. Moreover, carotenoids of H. pluvialis identified using LC-MS showed high cytotoxic activity against the HCT116 (100% inhibition at 0.1 mg/mL) and mild activity against both MCF7 and HePG2 lines, while the carotenoid-rich fraction of D. salina showed moderate cytotoxic activity on the MCF7 and HePG2 cancer cell lines.After the aforementioned works, dealing with various macroalgae, hereafter we explore studies focused on the use of microalgae as a source of anti-cancer compounds. The study of Abreu et al.  investign et al.  analyseds et al. . They ten et al.  analyzedn et al.  demonstrd et al.  tested vz et al.  extractePhaeodactylum tricornutum and Staurosirella pinnata that were analysed with 1H-NMR. They performed a bioactivity assay on human immortalised keratinocytes HaCaT and human melanoma CHL-1 cell lines in a 24 h dose-response test with different concentrations . The most important result showed that S. pinnata extract had an important dose-dependent effect on CHL-1. Extracts and compounds from the macro- and micro-algae analysed in this section are shown in Savio et al.  obtainedGeodia cydonium and it was tested on three breast cancer cell lines, MCF7, MDA-MB-231 and MDA-MB-468. An organic extract of this sponge had a cytotoxic effect with IC50 of 37, 44 and 70 μg/mL, respectively, on three cancer cell lines analysed, whereas it was inactive on normal breast cells (MCF-10A). After the treatment, the metabolomic profile was studied through 1H-NMR spectra. Interestingly, the active fraction was able to interfere with the glycolysis, lipid and amino acid metabolism of the tumor cells, enabling them to support their bioenergetic and macromolecular synthesis. In particular, the proton resonances related to the metabolites identified in three breast cancer cells were studied. The spectral region from 0.5 to 3 ppm showed the presence of signals from alanine, arginine, aspartate, glutamate, glutamine, isoleucine, lactate, leucine, lipids, lysine, proline, threonine and valine. The spectral region from 3 to 5.5 ppm was mainly composed of signals corresponding to choline, -glucose, -glucose, glycine, glycero-phosphocholine and phosphocholine. The 5.5–8.5 ppm region contained the resonances of histidine, phenylalanine and tyrosine. They also showed an increasing level of lactate after treatment in all three cell lines, and a decrease of α-glucose, β-glucose, choline, glycerophosphocholine, glutamine, glutamate and lipids. Other metabolites were also reduced: proline in MCF-7 cells, threonine in MDA-MB231 and asparagine and lysine in MDA-MB468 cells, whereas glycine was reduced in both MDA-MB231 and MDA-MB468 cells.As mentioned above, a few studies report analyses of metabolite changes after treatment of cancer cells with extracts/molecules isolated from sponges or algae. An interesting method to study cytotoxic activity is to provide a metabolic profiling of the treated cells to understand at which level the compound is interacting. This was the aim of the research conducted by Costantini et al. . The stuA decrease of pro-inflammatory cytokines  levels was detected, as well, along with an increase of anti-inflammatory cytokines levels (IL-4 and IL-10). Finally, the chemical entities present in this fraction were analyzed by liquid chromatography high-resolution mass spectrometry combined with molecular networking.F. vesiculosus was performed by Heavisides et al. [F. vesiculosus due to phlorotannins [An additional interesting approach to the brown macroalga s et al. , who invotannins , fucoidaotannins  and fucootannins . This stAmong the “omics” platforms, metabolomics has a great power to aid cancer research, thanks to the possibility for rapid analysis of tissue or biofluid samples. In fact, by coupling metabolite profiling and organism biology, it is possible to provide significant impacts for the discovery of new compounds and define their possible biotechnological applications for blocking the progression of cancer. Metabolic profiling is usually referred to a quantitative study of a group of metabolites that is associated with a given pathway. As demonstrated by the data reported in this review, metabolomics has been extensively used in the last years, with the aim to isolate bioactive compounds from marine organisms  against cancer, defining their chemical structures. A few examples, in contrast, may be reported on the use of a metabolomics approach to study how treatment with chemical extracts from sponges and algae could induce metabolite changes in treated cancer cell lines. For this reason, we think that this is a strength of our review, pushing the scientific world to invest in research projects aimed to test marine natural products in metabolomic studies. In fact, we think that this represents a very important application for cancer research, helping in the understanding of changes in the metabolic pathways induced by natural compounds from the sea. This is possible because of a high diversity of marine natural products, which represent promising opportunities for drug discovery and the development of marine biotechnologies. In addition, high-throughput techniques, such as metabolomics, are extremely useful for rapidly exploring the chemical diversity of marine environments with respect to the classical approaches, being also able to detect metabolites when present at low concentrations. Metabolite identification remains the main metabolomics bottleneck, and together with bioinformatic tools, such as molecular networks, it can lead to the annotation of unknown metabolites, leading to the discovery of new compounds. Furthermore, understanding the ecological and biological factors that contribute to the production of a certain metabolite can be extremely useful for selecting and optimizing the extraction of bioactive compounds, enhancing their yields and elucidating gene clusters associated with the biosynthetic pathways to which they belong.