In addition, transcriptomic and metabolomic profiles, induced by Cur-SLN treatments, highlighted networks involved in this radiosensitization ability. The non tumorigenic MCF10A and the tumorigenic MCF7 and MDA-MB-231 BC cell lines were used. Curcumin-loaded solid nanoparticles were prepared using ethanolic precipitation and the loading capacity was evaluated by UV spectrophotometer analysis. Cell survival after treatments was evaluated by clonogenic assay. Dose–response curves were generated testing three concentrations of free-Cur and Cur-SLN in combination with increasing doses of IR (2–9 Gy). IC50 value and Dose Modifying Factor (DMF) was measured to quantify the sensitivity to curcumin and to combined treatments. A multi-“omic” approach was used to explain the Cur-SLN radiosensitizer effect by microarray and metobolomic analysis. We have shown the efficacy of the Cur-SLN formulation as radiosensitizer on three BC cell lines. The DMFs values, calculated at the isoeffect of SF = 50%, showed that the Luminal A MCF7 resulted sensitive to the combined treatments using increasing concentration of vehicled curcumin Cur-SLN  Instead, triple negative MDA-MB-231 cells were more sensitive to free-Cur, although these cells also receive a radiosensitization effect by combination with Cur-SLN (DMF: 1.38 with 10 µM Cur-SLN). The Cur-SLN radiosensitizing function, evaluated by transcriptomic and metabolomic approach, revealed anti-oxidant and anti-tumor effects. Curcumin loaded- SLN can be suggested in future preclinical and clinical studies to test its concomitant use during radiotherapy treatments with the double implications of being a radiosensitizing molecule against cancer cells, with a protective role against IR side effects.In breast cancer (BC) care, radiotherapy is considered an efficient treatment, prescribed both for controlling localized tumors or as a therapeutic option in case of inoperable, incompletely resected or recurrent tumors. However, approximately 90% of BC-related deaths are due to the metastatic tumor progression. Then, it is strongly desirable to improve tumor radiosensitivity using molecules with synergistic action. The main aim of this study is to develop curcumin-loaded solid nanoparticles (Cur-SLN) in order to increase curcumin bioavailability and to evaluate their radiosensitizing ability in comparison to free curcumin (free-Cur), by using an  For example, in Breast Cancer (BC) care, RT is considered an efficient treatment, prescribed as adjuvant therapy strategy combined with surgery for controlling localized tumors, as the last option for patients with inoperable cancers and also as the first choice in cases of incompletely resected or recurrent tumors after surgery2. Moreover, in some cases of high recurrence risk , a radiation boost of 10–20 Gy could be administered in order to improve local control. Nonetheless in a phase III study involving 2657 BC patients, the radiation boost after whole-breast irradiation had no effect on long-term overall survival, with the largest absolute benefit in young patients5. In addition, approximately 90% of BC-related deaths are due to the metastatic progression of the tumor6. Then, it is strongly desirable to improve the tumor radiosensitivity in order to further reduce cancer recurrence. In this sense, one successful strategy could be to combine RT schedules with targeted therapies by using molecules with synergistic and radiosensitizing action.Overall BC women candidates for conservative surgery were also treated with RT to consolidate surgical treatment and success rate. According to the international BC treatment guidelines, the recommended total dose to be administered to BC patients is 50–60, with a fractionation of 2 Gy/die for 5 days/weekCurcuma Longa plant for its potentially anti-cancer multitarget effect and its active compounds, which are represented by curcuminoids. The curcuminoids belong to the polyphenol family, and are known to play key roles in the control of inflammation and oxidative stress in many conditions7. In particular, among these compounds, curcumin is known to play multiple pleiotropic effects , thanks to its ability to interact and regulate multiple molecular targets such as transcription factors (TFs), growth factors, kinases, pro-inflammatory cytokines, adhesion molecules, etc. In turn, literature data highlight its potential therapeutic effects useful for the prevention and treatment of various diseases, including BC8.Among these molecules, characterized by specific anti-cancer actions, the nutraceutical compounds often available in medicinal plants, have recieved a recent strong interest by several authors. Among these, a prominent role is reserved to the 9. In particular, the radiosensitizing role is supposed to be related to curcumin-induced inhibition of NF-KB, AP-1 and STAT3 transcription factors (TFs), highly constitutively expressed in numerous cancers and also transiently induced after ionizing radiation (IR) exposure. Moreover, curcumin is described to induce radiosensitization through the inhibition of such genes involved in several processes such as survival , proliferation , angiogenesis (VEGF and IL-8), invasion (MM9) and metastasis . Overall, all these factors could contribute to the development of radioresistance in neoplastic cells11.On the other hand, thanks to its low toxicity and great anti-inflammatory effects, curcumin has recently been studied as a radiosensitizing molecule against tumor cells and radioprotective agent for healthy tissues12 or by enhancing the effect of RT to arrest cell cycle in G2/M13. In this phase, cells are about three times more radiosensitive because, once they pass the G2 checkpoint, they are unable to repair DNA damage. Interestingly, the effect of radiosensitization is also mediated by increasing the intrinsic and extrinsic apoptosis induced by ionizing radiation (IR), through an increase of p53 expression by the MDM2 down-regulation mediated by Akt.14. Regarding the intrinsic apoptosis process, curcumin has been found to increase the mitochondrial Ca2+ level and Reactive oxygen species (ROS) production, resulting in an increased permeability of the mitochondrial membrane, allowing cytochrome C to translocate into the cytosol and bind Apaf-1 to form the apoptosome complex which triggers the caspase cascade (by activation of caspase-9) and subsequent cell death15. On the other hand, curcumin induces extrinsic apoptosis of neoplastic cells by increasing the expression levels of the “death activators” Fas ligand, TRAIL and TNF-α and, consequently, causing the activation of caspases 8 and 316.On the other hand, literature data report that RT treatments combined with curcumin inhibit proliferation and induce apoptosis of neoplastic cells, significantly improving the effects of RT, thanks to the suppression of cyclin D1, implicated in the G1/S cell cycle transition17. Then, curcumin could play a key interesting role in radiosensitivity/radioresistance cell balance. However, considering its chemical characteristics, it is necessary to study new strategies to increase its absorption in cells. In particular, curcumin is a lipophilic and hydrophobic compound, and therefore insoluble in water and soluble in ethanol, dimethyl sulfoxide (DMSO) and acetone. It is poorly absorbed by the body, so much so that its serum concentrations after oral administration are particularly low. Curcumin stability is pH-dependent and in a basic environment it is degraded very quickly8. After oral administration, curcumin is metabolized by reduction and conjugation, producing metabolites vehicled by glucuronic acid and sulfate in plasma, whose biological activities are strongly reduced compared to that of curcumin. In this study, to facilitate its absorption, we have trapped curcumin into a Solid Lipid Nanoparticle carrier (SLN), in order to realize a molecule having a better in vivo biodistribution and an improved bioavailability and stability.Although tumor cell radiosensitization is considered a promising idea to fight cancer diseases, it is equally important to reduce IR toxic effects in healthy tissues surrounding tumors. So, since one of the most prominent cancer pathogenic factors are of an inflammatory nature, it follows that the curcumin anti-inflammatory powerful effects could down regulate inflammation and ROS production, through to the inhibition of NF-kB. This mechanism is also involved in the reduction of the RT induced fibrosis, driven by TGFβ which is also under the NF-kB transcription control, so that curcumin could even contribute in the reduction of normal tissue toxicities following RTin vitro approach on BC models. Here we used the MCF10A immortalized non tumorigenic mammary epithelial cell line and MCF7 (ER+/PR+/HER2−), MDA-MB-231 (ER−/PR−/HER2−) tumorigenic immortalized cell lines, characterized by different tumorigenic aggressive phenotypes18. In addition, we also reported in a descriptive way, transcriptomic and metabolomic profiles and a quantification of oxidative stress induced by Cur-SLN treatments, highlighting intracellular networks modulated sustaining the radiosensitizing Cur-SLN role in irradiated breast cell lines.Summarizing, the principal aim of this study was to evaluate the radiosensitizing ability of curcumin loaded SLN (Cur-SLN), in comparison to free curcumin (free-Cur) by using an We trust that all the data here reported may encourage the use of curcumin-loaded SLN in preclinical studies to test the combination with the radiotherapy treatment plans and its application in clinical trials.Empty and curcumin-loaded nanoparticles were prepared using an ethanolic precipitation technique followed by ultraturrax homogenization. Briefly, 118 mg of Precirol ATO was heated at 5–10 °C above its melting point (56 °C). In order to obtain drug-loaded nanoparticles, curcumin (20 mg) was added, under mechanical stirring, to the melted lipid phase. An ethanolic solution (2 ml) containing Pluronic F68 (200 mg) and then the cationic surfactant dimethyldioctadecyl-ammonium bromide (30 mg) was added to the melted lipid phase. Finally, the resulting warm organic solution was dispersed into the bidistilled water (100 ml) at 80 ± 1 °C and homogenized by Ultraturrax T25  at 17,500 rpm for 10 min. The obtained emulsion was quickly cooled using an ice bath for 30 min and then purified by exhaustive dialysis . After purification, to prevent any nanoparticle aggregation, a cryoprotector  to nanoparticle suspensions was added, using nanoparticles:cryoprotector ratio of 1:2 (w/w). Finally, curcumin nanoparticles were freeze-dried by using a lyophilizer  and stored at room temperature for successive characterization.19. The samples were diluted in bidistilled water until the appropriate concentration and then the measurements were carried out at 25 ± 1 °C, at a fixed angle of 173° (NIBS = non-invasive backscattering detection) in respect to the incident beam. Each sample was kept in a cuvette and analyzed in triplicate. Zeta potential  values were measured at 25 ± 1 °C using samples appropriately diluted in bidistilled water. The instrument setting conditions were equal to those described above for size measurements. Each sample was analyzed in triplicate.The hydrodynamic diameter (z-average) of empty and curcumin loaded nanoparticles was determined by Photon Correlation Spectroscopy (PCS) by using a Zetasizer Nano ZS , as reported in literatureCurcumin loading capacity was evaluated by UV spectrophotometer analysis. The nanoparticles were solubilized using organic mixtures until the appropriate concentrations [dichloromethane/acetonitrile/ethanol 4/4/2 (v/v/v)], filtered through 0.45 μm cellulose acetate membrane filters  and analyzed by UV spectrophotometer , at 423 nm. The peak areas were compared with the one obtained by analyzing standard solutions of curcumin at known concentrations. The straight-line equation was: y 132.8x + 0.0084 and the linear regression value was: r2 = 0.9992. The linearity of the method was studied for curcumin concentrations in the range of 1–20 μg/ml. DL% results were expressed as the weight percent ratio between the curcumin loaded and the total dried sample weight. Entrapment efficiency (EE%) was expressed as the weight percent ratio between the amount of curcumin entrapped into nanoparticles and the total amount of curcumin used to prepare the particles. Below, the equations used for DL% and EE% determination are reported.Wdrug represents the amount of curcumin entrapped into the nanoparticles and WNp represents the weight of the drug-loaded nanoparticles.Wf represents the amount of curcumin entrapped and Wi represents the amount of curcumin used to prepare the curcumin-loaded nanoparticles.2) and were routinely subcultured in 25-cm2 (T25) and 75-cm2 T75) standard tissue culture flasks. Cell lines were cultured in the presence and in the absence of 2.5 µM of free- Cur or Cur-SNL for 24 hrs. At the end of the stimulation, cells and supernatants were collected to assess the intracellular and extracellular content of curcumin by UV analysis. In particular, the supernatants were sucked from the wells and then collected by centrifugation at 1,300 rpm for 10 min. Cells were detached from the wells by tripsin, washed with PBS and stored as dry pellets at −80 °C.The human non-tumorigenic breast epithelial MCF10A cell line and the human breast adenocarcinoma MCF7 and MDA-MB-231 cell lines, were purchased from the American Type Culture Collection  and cultured according to the ATCC’s specifications. Cells were maintained in an exponentially growing culture condition in an incubator at 37 °C in a humidified atmosphere . As control , untreated cells were seeded in the same conditions in order to evaluate the plating efficiency (PE). All cell survival data post-treatment were normalized to the untreated sample. Colonies were allowed to grow under normal cell culture conditions for 10–15 days after treatments and then were fixed and stained with 50% methanol and 0.5% crystal violet . Colonies with more than 50 cells were counted manually under a Zeiss Axiovert phase-contrast microscope  and also automatically with a computer-assisted methodology22.Cell survival after treatments was evaluated by clonogenic assay performed as previously described50 value of free-Cur and Cur-SLN for each breast cell line, which represents the concentration required to obtain the 50% of in vitro inhibition, cells were treated in triplicates using a wide range of free-Cur and Cur-SLN concentrations  in 6-well plates. 24 hrs after treatments, the medium was replaced and the cells were maintained in standard growth conditions until processing according to the clonogenic assay protocol. The IC50 was calculated from the two drug dose–response curves obtained by the increase of concentrations of free-Cur and Cur-SLN, using a 2nd order polynomial fitting analysis. Moreover, dose-response curves were generated by means of the 2nd order polynomial fitting of FS% vs increasing IR dose for each concentration of free-Cur and Cur-SLN used.These curves were used to obtain the Dose Modifying Factor (DMF), arbitrarily calculated at the SF of 50% . In order to obtain the IC24. The irradiation setup and the dose distribution were studied using the Pinnacle Treatment Planning System . Cell irradiations were carried out using doses of 2, 4, 6 and 9 Gy to evaluate the radiation efficacy through a dose-response curve.A medical linear accelerator, Siemens Primus  emitting photon beams (X-rays) of 6 MV nominal energy, was used to perform cell irradiations. The Linac was calibrated under reference conditions defined by the International Atomic Energy Agency Technical Reports Series No. 398 “Adsorbed Dose Determination in External Beam Radiotherapy”5 cells were analyzed by fluorimeter for each determination performed in triplicate. The oxidation reaction was evaluated using the GloMax® Discover System (Promega) at the excitation wavelength of 475 nm and emission wavelength of 555 nm. The data shown are obtained from three independent experiments and are expressed as the mean ± standard error of the mean (SEM). The significance level respect to the control sample was set to *P < 0.05.A quantification of cellular oxidative stress has been performed measuring the intracellular reactive oxygen species (ROS) in the control (untreated), 2 Gy, 2 Gy + Cur-SLN, Cur-SLN samples of the three cell lines studied. The production of ROS was evaluated using 2′,7′-dichlorodiidrofluorescineacetate (DCFH-DA) . The DCFH-DA is de-esterified intracellularly and turns highly fluorescent 2′,7′-dichlorofluorescein upon oxidation, allowing a sensitive and rapid quantitation of oxygen reactive species in response to oxidative metabolism. 24 hours after treatments, cells were trypsinized, washed with PBS and incubated with 5 μM DCFH-DA reagent working solution according to manufacturing protocol. For consistency, 10We decided to evaluate only gene expression profiles (GEP) generated by the administration of Cur-SLN, as this formulation could have a clinic interest. Furthermore, we decided to evaluate the lowest dose/concentration combination (2 Gy/2.5 µM), a synergistic radiosensitizing treatment aims to obtain the maximum efficacy by administering the minimum doses of a drug and RI. Furthermore, it should be remembered that 2 Gy is the daily dose delivered in fractionated radiotherapy treatments.To analyze GEP induced by 2.5 µM Cur-SLN in MCF10A, MCF7 and MDA-MB-231 cell lines treated with 2 Gy of photon beams, we used whole human genome 4 × 44 K cDNA microarray expression analyses. In particular, we analyzed GEP of the following configurations: MCF10A Cur-SLN 2 Gy normalized with MCF10A 2 Gy; MCF7 Cur-SLN 2 Gy normalized with MCF7 2 Gy; MDA-MB-231 Cur-SLN 2 Gy normalized with MDA-MB-231 2 Gy.21. Microarray experiments were performed according to the Agilent Two-Color Microarray-Based Gene Expression Analysis protocol . In turn, 6 replicates for each configuration assayed were performed. Statistical data analysis, background correction, normalization and summary of expression measures were conducted with GeneSpring GX 10.0.2 software (Agilent Technologies). Finally, genes were identified as being differentially expressed if they showed a fold change (FC) of at least 1.5 with a p-value < 0.05 compared to irradiated cells of the same cell line analyzed, that were used as reference samples. The data discussed in this publication, in compliance with Minimum Information About a Microarray Experiment (MIAME) standards, have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO)25 and are accessible through the following GEO Series accession number: GSE118061.Total RNA extraction and its qualitative  and quantitative analyses were conducted as previously describedhttps://david.ncifcrf.gov/tools.jsp). The most representative significantly changed networks and pathways were selected and analyzed.In addition, the GEPs obtained in this work were also analyzed by pathway analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) network building tool that provides a comprehensive set of functional annotation tools for investigators to understand the biological meaning behind a long list of genes (26. Derivatization was performed using automated sample prep WorkBench instrument (Agilent Technologies). Dried polar metabolites were dissolved in 60 μl of 2% methoxyamine hydrochloride in pyridine (Pierce) and held at 40 °C for 6 hrs. After dissolution and reaction, 90 μl of MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamid) were added and samples were incubated at 60 °C for 1 h. GC/MS analysis was performed using 7200 accurate-mass Q-TOF GC/MS (Agilent Technologies) equipped with a 40-m DB-5MS capillary column operating under electron impact (EI) ionization at 70 eV. Samples (2 μl) were injected in a splitless mode at 250 °C, using helium as the carrier gas at a flow rate of 1 ml/min. The GC oven temperature was held at 100 °C for 2 min and increased to 325 °C at 10 °C/min.Metabolites from the same treatments described in the above transcriptomic section (2 Gy and 2.5 µM Cur-SLN) were extracted and analyzed by gas chromatography-mass spectrometry (GC-MS), as previously describedet al., 2016). Relative metabolites abundance was carried out after normalization to internal standard d27-myristic acid and cell number.GC/MS data processing was performed using Agilent Muss Hunter software and statistical analyses were performed using Mass Profile Professional (MPP) software .29. The oral bioavailability of curcuminoids in healthy humans is markedly enhanced by micellar solubilisation but not further improved by simultaneous ingestion of sesamin, ferulic acid, naringenin and xanthohumol30. To date, to increase the bioavailability of curcumin several methods were developed. In the present work, empty and curcumin-loaded solid lipid nanoparticles (SLNs) were successfully prepared by using the ethanolic precipitation technique and then, stored at room temperature for successive characterization in terms of mean size, polydispersity index (PDI) and ζ-potential in bidistilled water  and the Entrapment Efficiency (EE%), expressed as the weight percent ratio between the amount of curcumin entrapped into SLNs and the total amount of curcumin used to prepare the particles, were 1.64% and 68.62% respectively.50), MCF10A, MCF7 and MDA-MB-213 cell lines were treated with increasing doses of free-Cur and Cur-SLN, ranging from 2.5 to 100 μM for 24 hours and subjected to long-term clonogenic assay.In order to evaluate the cytotoxicity ability of free curcumin and curcumin-loaded SLN (Cur-SLN) in term of concentration that determined the 50% of growth inhibition  doses.23. These values show an overall increasing radiosensitization effect, rising with the concentration of both the two drugs. However, the three cell lines show a different level of sensitivity to free-Cur and Cur-SLN. Indeed, the normal epithelial MCF10A showed to be the most sensitive to all the radiosensitizer combined treatments, in comparison with the other two tumorigenic cell lines, being more sensitive to the vehicle curcumin Cur-SLN for each concentrations tested. On the other hand, although a lesser radiosensitizer effect can be observed for the two tumorigenic cell lines, MCF7 resulted more sensitive to the treatment with increasing concentration of vehicled curcumin Cur-SLN, whereas MDA-MB-231 have proved to be more sensitive to free-Cur, reaching the maximum of radiosensitizer effect using 10 µM of free-Cur.MCF10A, MCF7 and MDA-MB-231 cell lines were treated with only RT with increasing photon doses of 2, 4, 6, 9 Gy or were irradiated after a pretreatment with 2.5, 5, 10 µM of free-Cur and Cur-SLN for 24 hrs. The surviving fraction (SF) values, obtained by clonogenic assay after treatments, were used in order to generate dose-response curves in absence or presence of a certain concentration of free-Cur or Cur-SLN for the three BC cell lines . This, in turn, led to define the dose modifying factor (DMF), arbitrarily calculated at the SF of 50% by means of a 2nd order polynomial fitting from the dose response curves  , a synergistic radiosensitizing treatment aims to obtain the maximum efficacy by administering the minimum doses of a drug and RI. Furthermore, it should be well-known that 2 Gy is the daily dose delivered in fractionated radiotherapy treatments.Here, we reported GEP data obtained applying a Two-Color Microarray-Based Gene Expression Analysis (Agilent technologies) on MCF10A, MCF7 and MDA-MB-231 breast cell lines treated with 2.5 µM Cur-SLN and irradiated with photon beams using the IR dose of 2 Gy. In particular, comparative differential gene-expression analysis revealed that multiple deregulated genes (DEG) were significantly altered, by 1.5-fold or greater according to the specific experimental configuration. MCF10A cells treated with Cur-SLN changed the expression levels of 846 genes (189 down regulated and 657 up regulated). On the other hand, 1265 DEGs were selected in MCF7 BC cell line Cur-SLN treated and, among these, 752 were up regulated while 513 were down regulated. Finally, in MDA-MB-231 BC cell lines treated with Cur-SLN we selected 2301 DEGs (112 down regulated and 1179 up regulated).25 and the top molecular pathways of DEG datasets were selected and reported in Tables Moreover, up and down regulated transcripts were selected and grouped according to their involvement in specific biological networks using Integrated pathway enrichment analysis with the DAVID toolThe result of this mapping revealed the involvement of a set of factors controlling cellular processes described as follows.31, whereas transcriptional misregulation in cancer signaling contains several factors known to be deregulated in cancer cells because of their key roles in controlling cell survival/death balance.Summarizing, as displayed in Table 37.On the other hand, in MCF7 BC cell line Cur-SLN causes the deregulation of molecules belonging to apoptosis induction, inflammation signaling (sustained by cytokine and platelet activation pathways), tyrosine metabolism known to be involved in cellular stress response and in glucagon signaling pathway, recently described as modulated by curcumin to improve glucose tolerance Table 32–37.Finally, as displayed in Table Finally, in order to evaluate the unique and common DEG lists and cellular process modulated by these factors after Cur-SLN treatments in MCF10A, MCF7 and MDA-MB-231 cell lines, we performed Venn diagrams as shown in Fig. As displayed in Fig. Moreover, in the Supplementary Files These data highlight the wide range of curcumin bioactivities, that could help to obtain successful treatment plans in combination with RT.To further understand better the effects induced by combined treatment of 2.5 µM Curc-SLN and irradiation with 2 Gy of photon beams in MCF10A, MCF7 and MDA-MB-231 human breast cell lines, we performed metabolomic analysis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) systems.vs left side of the hierarchical clustering plot)  Fig. . In partot) Fig. . On the ot) Fig. . Moreoveot) Fig.  promptedmin Fig. . Similarcle Fig. . Finallyism Fig. .Figure 638.Therefore, taken together, these results showed that the effect of Cur-SNLB administration on breast cell lines exposed to irradiation is to exert a protective role against oxidative stress-induced by irradiation damage and to induce an antitumor effect through autophagy mechanism activationin vitro approach on three immortalized breast cell lines: the epithelial non-tumorigenic MCF10A cell line and the two tumorigenic MCF7 , and MDA-MB-231 (triple-negative: ER−/PR−/HER2−) cell lines. Specifically, these cell lines were subjected to single or combined treatment using four doses of IR  and 6 concentrations of free-Cur or Cur-SLN . Empty SLN has been also tested for cellular toxicity on the three cell lines under study using the same above reported concentrations, showing it not to be toxic. The IC50 evaluation showed that all the cell lines tested are sensitive to treatment with free and curcumin loaded-SLN. However, the MCF7 tumorigenic cell line showed a strong sensitivity to treatment with Cur-SLN and free-Cur, compared to the other two cell lines analysed, with IC50 values of 2.47 μM and 3.81 μM, respectively. On the other hand, the two cell lines MCF10A and MDA-MB-231 displayed lower levels of sensitivity to both the molecules. In particular, while in MDA-MB-231 cells the cytotoxic effect of Cur-SLN was very similar to that of free curcumin, with IC50 values of 7.62 μM and 6.62 μM, respectively, the MCF10A were proved to be more sensitive to Cur-SLN than free curcumin by finding IC50 values of 6.74 and 10.56 μM respectively.The main aim of this research study was to evaluate the radiosensitizing effects mediated by Cur-SLN in comparison to that generated by free-Cur, using an 50 values, we tested the radiosensitizing effect of free-Cur and Cur-SLN, used at concentrations of 2.5, 5, 10 μM, in combination with conventional radiotherapy treatment with increasing doses of 2, 4, 6, 9 Gy, in order to generate dose/response curves for all the treatment configurations tested.Based on the IC23.The radiosensitizing effect was determined by calculating the dose modifying factor (DMF), arbitrarily obtained at the SF of 50% , in order to highlight the capacity of enhancing tumor cells killing by the combined treatments in respect to irradiation onlyIn other terms, a DMF value of 2, means that the isoeffect could be obtained by the combined treatment with the half of IR dose, respect to the dose necessary using IR only.Overall, the DMFs value reported in Table in vivo, reducing the inflammation and the reactive oxygen species production, thanks to the down-regulation of NF-kB. This mechanism is also involved in the reduction of fibrosis induced by radiotherapy, in which a key role is played by TGFβ17. On these aspects, a preclinical study evaluated the adverse effects of cutaneous toxicity that occur following exposure to IR17, evaluating a possible protective role exerted by curcumin. In this case, curcumin was administered intragastric and intraperitoneally in C3H/HeN mice, 5 days before RT or 5 days after RT. Skin damage was assessed at 15–21 days (acute cutaneous toxicity) and at 90 days (chronic cutaneous toxicity) following a single dose of 50 Gy of radiation in the posterior leg of each mouse. The results showed that curcumin, administered before or after radiotherapy, markedly reduces acute and chronic cutaneous toxicity, significantly decreasing the expression of inflammatory IL-1, IL-6, IL-18, IL-1Ra and fibrogenic (TGF-β) cytokines in irradiated skin and muscles.Indeed, the normal epithelial MCF10A received a strong radiosensitizer effect by the combined treatments, although they were more sensitive to the vehicle curcumin Cur-SLN for each concentrations tested. This means that further studies are needed to understand if the use of curcumin as radiosensitizer could increase normal tissues toxicity post-irradiation. However, it should be noted that normal tissue complications (NTCP) are of inflammatory nature and the powerful anti-inflammatory effects of curcumin could reduce these toxicities On the other hand, although a lesser radiosensitizer effect can be observed for the two tumorigenic cell lines, MCF7 resulted more sensitive than MDA-MB-231 to the treatment with increasing concentration of vehicled curcumin Cur-SLN, reaching a DMF value of 1,78 using 10 µM of Cur-SLN. Instead, MDA-MB-231 were more sensitive to free-Cur, reaching the maximum of radiosensitizer effect with 10 µM of free-Cur . However, a clinical administration of curcumin is likely in the vehicle form, then the aggressive triple negative MDA-MB-231 BC cell line could receive a radiosensitization effect by Cur-SLN, as the DMF value is 1.38 with 10 µM of Cur-SLN. Thus, the possibility to use drug/IR combined treatments permits to increase the tumor control probability (TCP) even for radioresistant tumors such as the triple negative BC.Furthermore, in Figs Moreover, in order to highlight the action mechanism of Cur-SLN and to confirm the anti-oxidant properties of Cur-SLN, a quantification assay of oxidative stress induced by the treatments has been performed Fig. . On the vs 2 Gy + 2.5 μM Cur-SLN, for each cell line tested, analyzed with “whole genome microarray” approach and “two-color” staining method. The comparative gene expression analysis of MCF10A, MCF7 and MDA-MB-231 cells revealed that a large number of genes were deregulated (DEG) from treatment with Cur-SLN with a fold change (FC) ≥ 1.5. Below are the details of the genes deregulated in the three cell lines: 1) MCF10A: 846 DEG (189 down-regulated and 657 up-regulated); 2) MCF7: 1265 DEG (513 down-regulated and 752 up-regulated); 3) MDA-MB-231: 2301 DEG (112 down-regulated and 1179 up-regulated).In addition, we carried out a “omic” study (transcriptomic and metabolomic), in order to explore better this new formulation (Cur-SLN) action mechanism in producing a radiosensitivity effect on the three cell lines analyzed. This is thanks to its formulation which facilitates its transport in an aqueous solution, it could be administered in preclinical or clinical application. Moreover, we only compared the effects of lower IR doses/drug concentrations, since the combination with a radiosensitizer should, hopefully, allow the use of lower IR doses maintaining the isoeffect of therapeutic efficacy and reducing the side effects to the minimum. In addition, 2 Gy is the daily dose administered in fractionated treatments with external beams. Therefore, for the “omic” study the comparisons considered were: 2 Gy Figure 31.Thanks to the use of the DAVID tool, these deregulated genes have been grouped, based on their involvement in some biological networks and cellular processes. This approach has allowed to better characterize the unique and common molecular response among the analyzed cell lines. Specifically, in the non-tumorigenic MCF10A cell line, the specific molecular response to Cur-SLN treatment is characterized by the statistically significant deregulation of “Transcriptional misregulation in cancer”, which involves several key factors altered in neoplastic cells and “lysine degradation process” Table , the lat37. Finally, in MDA-MB-231 cells, deregulated genes involved in the cell cycle, in the TGF-β and in the TNF pathway, in the pathways of phosphatidylinositol and FoxO were identified , protein kinases and other oncogene molecules. In this context, the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway, usually activated in onset and neoplastic development, is inhibited by curcumin. This process could constitute a key molecular target for anti-cancer therapies as it represents a negative regulator of tumor progression processes and metastasis42.On the other hand, in MCF7 cell line the Cur-SLN treatment resulted in the deregulation of molecules involved in inflammatory processes, induction of apoptosis, tyrosine metabolism which is implicated in the response to cellular stress and glucagon signaling pathway, which is in turn regulated by curcumin in response to low glucose levels Table 31–37. Fied Table . Regardi48.Several studies have found that curcumin is also involved in the regulation of the FoxO pathway implicated in multiple cellular processes, such as cell cycle arrest, apoptosis, DNA repair, glucose metabolism and oxidative stress response. In particular, Fox TFs are negatively regulated by the AKT survival signal, as normally phospho-Akt prevents the nuclear localization of FOX and plays a key role in regulating its activity. Furthermore, tetrahydrocurcumin (THC), a metabolite of curcumin, regulates the oxidative stress response through FOXO, promoting its nuclear translocation and regulating, therefore, the expression of several genes involved in apoptosis, in cell cycle progression, in DNA repair, in oxidative stress, in the control of muscle growth, as well as in cell differentiation and glucose metabolism. Based on these findings, FOXO TF could be used as potential tumor suppressorsin vivo study which evaluated the radiosensitizing effect of curcumin on nude mice inoculated with colon cancer HCT 116 cells, performed by the group of Kunnumakkara49. This study evaluated the effect of the combined administration of IR  and curcumin administered orally 1 hour before IR exposure (1 g/kg). The effect was compared with a control group and two groups corresponding to the two treatments performed in single. The results showed that the tumor volume of mice that received combined treatment was significantly reduced compared to that of the control group of mice and those treated with curcumin alone or with radiation alone. Furthermore, it has been found that curcumin enhances the effect of RI, inhibiting the proliferation of neoplastic cells and generating a significant reduction of the Ki-67 cell proliferation marker and of CD31 (tumor density microvase marker) in tumor tissues, compared to the group that received only RIs. The same study showed a decrease in the expression levels of COX-2, VEGF, MMP-9, Cyclin D1, c-Myc, associated with angiogenesis, invasion, metastasis and proliferation and whose expression is generally increased following exposure to IR; this down-regulation was observed in the group treated with only curcumin and in the group subjected to the combined treatment. In general, since these factors are regulated by NF-kB, it can be concluded that curcumin exerts its powerful radiosensitizing effect through the down-regulation of NF-kB and its transcriptional targets, also allowing to reduce the toxic effects of inflammatory nature induced by radiation in healthy tissues.Similar molecular findings have been described in an Regarding the 51-gene signature composed by DEG common to three cell lines analyzed, the Gene Ontology analysis conducted using the DAVID tool, showed that the genes were involved in cell migration, in the calcium signaling pathways and in the activation of the chromatin modification process.Regarding the radiotherapy context, chromatin remodelling through histones acetylation and deacetylation represents attractive target for radiosensitization as this process plays a significant role in the repair mechanisms of the double strand breaks (DSB) generated by IR.51. Several authors have reported that curcumin limits the migration/proliferation process of neoplastic breast cancer cells by inducing crosstalk between the anchoring junctions and the Wnt pathway by means of the EPR-1 factor (Early Growth Response 1), and by inhibiting the expression of several genes involved in the epithelial-mesenchymal transition  , a process involved in cancer progression, thus suggesting its powerful anti-metastatic function53. The antitumor effect of curcumin is also related to the increase in intracellular calcium levels (Ca2+) inducing intrinsic apoptosis, as described above.In addition, preclinical studies have found that inhibitory molecules of HAT (histone acetyltransferase) and HDAC (histone deacetylase), such as curcumin, are able to radiosensitize neoplastic cells. In fact, it has been found that curcumin is able to significantly reduce the acetylation of H3/H4 histones and to increase the demethylation of histone H3K9 at the level of the cytokines promoter regions, thus inhibiting the expression of genes for proinflammatory chemokinesFinally, the 263 gene-signature constituted by deregulated genes common to the two tumorigenic MCF7 and MDA-MB-231 cell lines was found to consist of DEGs implicated in the cell cycle G2/M transition, in cell migration, in angiogenesis and in calcium signaling.54.Indeed, various studies have found that the curcumin anti-tumor effect is due to its ability to stop the cell cycle in G2/M phase and to trigger apoptosis of neoplastic cells by increasing the expression levels of FOX proteins, caspase 3 cleaved, Fas ligand (FasL) and the reduction of expression levels of cyclin-dependent kinasesThe metabolomic analysis allowed us to highlight the effects induced on metabolism by Cur-SLN in the combined treatment on the three cell lines.In particular, the two tumorigenic cell lines MCF7 and MDA-MB-231 showed greater similarities between them in the activated metabolic pathways, compared to the non-tumorigenic MCF10A cell line, highlighting a tumor specific metabolic response. Indeed, the hierarchical clustering reveals two defined area (the upper and the downer one) where it can be observed a tendency for which the up-regulated genes of tumorigenic cell lines are down-regulated in the MF10A, and on the contrary, the down-regulated genes of tumorigenic cell lines are up-regulated in the MCF10A Fig. . Specifivs 2 Gy + 2.5 µM Cur-SLN has been analysed. In MCF10A cells, a group of metabolites significantly involved in amino acid metabolism were found to be significantly deregulated, showing a significant increase in glucose and citrate  as radiosensitizer on three breast cell lines, deeply describing its molecular and metabolic effects in combined treatments.In conclusion, the data here described indicate that SLN-curcumin exerts a radiosensitizing effect, rising with its concentration increasing, particularly, Dose Modifying Factors (DMFs), calculated at the isoeffect of SF = 50%, showed that the Luminal A MCF7 resulted more sensitive to the combined treatments using increasing concentration of vehicled curcumin Cur-SLN, reaching a DMF value of 1,78 using 10 µM of Cur-SLN. Instead, the more aggressive triple negative MDA-MB-231 cells were more sensitive to free-Cur, reaching the maximum of radiosensitizer effect using 10 µM of free-Cur . However, a clinical administration of curcumin is likely in the vehicle form, then the aggressive triple negative MDA-MB-231 BC cell line could receive a radiosensitization effect by Cur-SLN, as the DMF value is 1.38 with 10 µM of Cur-SLN. In addition, the oxidative stress quantification and the “omic” study explained the radiosensitizing function of Cur-SLN, confirming at the transcriptomic level many action mechanisms already known for curcumin, which underline anti-oxidant and anti-tumor effects, thanks to its ability to regulate various cellular processes. Moreover, also the metabolomic study highlights, in synthesis, this double action of curcumin, which on the one hand activates an anti-oxidant metabolism with protective role against IR and, on the other hand, exerts an anti-tumor role, stimulating autophagy.Therefore, the use of curcumin loaded-SLN can be suggested in future preclinical studies and clinical trials to further test the clinical implications for the loaded curcumin concomitant use in the course of fractionated radiotherapy treatments, with the double implications of being a radiosensitizing molecule against cancer cells, with protective effects against the onset of normal tissue complications.Supplementary info51 gene signature.263 gene signature


Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum (SpeedVac) and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, 40–50 metabolites were identified as having statistically significant differences in abundance in redissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in the lyophilized samples, for example, n-methyl-α-aminoisobutyric acid, n-methylnicotimamide, sarcosine and 3-hydroxyisovaleric acid, whereas some metabolites were only present in SpeedVac dried samples, for example, trimethylamine. This research demonstrates that the choice of drying technique used during the preparation of samples of human cell lines or tissue extracts can significantly influence the observed metabolome, making it important to carefully consider the selection of a drying method prior to preparation of such samples for metabolic profiling. Biological fluids, cell line cultures and tissue extracts are common samples used in metabonomics analyses [Metabonomics is a metabolic profiling technique first described in 1999 ,3,4,5. Manalyses ,19,20,21analyses ,23,24. Manalyses . Mass spanalyses ,27.www.ncbi.nlm.nih.gov/pubmed) with the search terms “metabonomics OR metabolomics” AND “cell line” OR “cell culture” in the “Title/Abstract” field. Major research areas that employ metabonomics studies of human cell cultures include investigations of many human diseases, pharmacology, systems biology and toxicology [Cell lines have been used as models for studying human diseases and metabolic pathways since as early as 1956 . As withxicology ,30,31,32xicology ,34,35,36xicology ,40,41,42xicology ,43. Becaxicology ,36. Metabolic profiling of human cell cultures typically follows a five-step procedure post cell culture, including sample preparation and processing, data collection, data analysis, identification of metabolites, and determination and analysis of metabolic pathways to which identified metabolites belong . It has Here, we investigated how the observed metabolome of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, was influenced by application of drying either by SpeedVac or lyophilization. Ten replicate extract samples from each cell line were dried using each technique, and the metabolic profiles of the dried sample extracts were measured by NMR spectroscopy. The resulting metabolic profiles were compared to determine the influence of the choice of drying technique on the observed metabolic profiles. Representative 1D NMR spectra of samples prepared from redissolved speed vacuum dried and lyophilized cell extracts of MiaPaCa-2 cells used for metabolic profiling analysis are shown in A total of 407 manually defined spectral buckets were included in the analysis of MiaPaCa-2 cells. The PCA scores plot for MiaPaCa-2 samples showed significant cluster separation at 95% confidence interval based on an F-test analysis  A with 872 = 0.95) and good predictive power (Q2 = 0.75) using three PCs. In the PLS-DA, 120 buckets out of 407 had variable importance in projection (VIP) scores greater than 1.0, indicating that these buckets carried the most weight in being responsible for cluster separation.The PLS-DA scores plot for MiaPaCa-2 also exhibited statistically significant separation between data set clusters based on the F-test  B. PLS-DAp-value <0.05 or an AUC >0.70 [To be considered significant, a bucket had to have either a UC >0.70 . In MiaPRepresentative 1D NMR spectra of samples prepared from redissolved speed vacuum dried and lyophilized Panc-1 cell extracts used for metabolic profiling analysis are shown in A total of 359 manually defined spectral buckets were included in the analysis of Panc-1 cells. The PCA scores plot for Panc-1 exhibited significant cluster separation  C with 742 = 0.98) and high predictive capability of the model (Q2 = 0.90). In the PLS-DA, 160 buckets out of 359 had VIP scores greater than 1.0, indicating that these buckets carried the most weight in being responsible for cluster separation.The PLS-DA scores plot for Panc-1 showed statistically significant separation between data sets based on F-test analysis  D. Cross-In Panc-1 cells, 49 metabolites  out of aRepresentative 1D NMR spectra of samples prepared from redissolved speed vacuum dried and lyophilized AsPC-1 cell extracts used for metabolic profiling analysis are shown in A total of 307 manually defined spectral buckets were included in the analysis of Panc-1 cells. The PCA scores plot for AsPC-1 also displayed clear cluster separation at 95% confidence interval  E with 852 = 0.80 indicating excellent data agreement with the model, and Q2 = 0.94, indicating great model predictive power. In the PLS-DA, 113 buckets out of 307 had VIP scores greater than 1.0, indicating that these buckets carried the most weight in being responsible for cluster separation.The PLS-DA scores plot for AsPC-1 cell line samples showed separation of the two groups that was statistically significant based on F-test analysis  A. PLS-DAIn AsPC-1 cells, 44 metabolites  out of aThe data presented here indicate that the choice of drying technique used to prepare extracts from human cell lines for metabolic profiling has a significant impact on the observed metabolic profiles. 70–75% of all the buckets intensities were significantly different depending on the drying technique in the observed metabolome in each of the three cell lines. In the three human pancreatic cancer cell lines investigated in this study, the number of metabolites whose abundances were significantly different depending on drying technique ranged from 44 in AsPC-1 cell extracts to 53 in MiaPaCa-2 cell extracts. A prominent difference in the metabolic profiles depending on drying technique was that some metabolites were present in samples prepared by one drying technique but completely absent in the other . For insOn the other hand, one metabolite was present in speed-vacuumed samples but absent in the lyophilized samples , i.e., tThe majority of metabolites that were present at different abundances depending on drying technique were present in extracts prepared by both drying techniques . Forty-oAbout 10–15% of all significant buckets in each cell line could not be identified from the existing databases  therefore limiting our ability to draw specific conclusions from this missing fraction of data. While some metabolites were more abundant after SpeedVac drying, lyophilization seems to be the generally superior technique compared to centrifugal evaporation under vacuum as it seemed to preserve the original metabolome of the cell lines to a higher degree based on the general observation of including more metabolites, or the same metabolites at higher concentrations compared to SpeedVac prepared samples.g. The top layer (hydrophilic) and the bottom layer (lipophilic) were separated and transferred to new Eppendorf tubes. Hydrophilic extracts were dried and used for NMR spectroscopic analysis. MiaPaCa-2, Panc-1, and AsPC-1 were purchased from the American Type Culture Collection . MiaPaCa-2 and Panc-1 were grown in high glucose Dulbecco’s Modified Eagle Medium, (DMEM) and AsPC-1 cells were grown in Roswell Park Memorial Institute (RPMI) medium. Both media were supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Sixty 100 mm × 15 mm petri dishes per cell line were harvested and extracted as previously described by Watanabe et al. . BrieflyThirty samples of each cell line were dried in a speed vacuum centrifugal concentrator . Another thirty samples of each cell line were frozen in liquid nitrogen and dried using a lyophilizer . The drying time used for both SpeedVac and lyophilization were kept constant. MiaPaCa-2 and Panc-1 cells were dried for 21 h, and AsPC-1 for 20 h. For lyophilization, it was ensured that the samples remained frozen throughout the sublimation process and did not thaw into a liquid prior to complete lyophilization. Following resuspension in 150 µL of buffer , 0.01% sodium azide in 100% deuterium oxide ) , cell li1H Carr-Purcell-Meiboom-Gill (CPMG) NMR spectra were collected using a spectral width of 20.0 ppm and 32K points resulting in 1.21 s acquisition time, 512 scans, 4 dummy scans, 1.80 s recycle delay and 1 ms mixing time. Two-dimensional 1H-1H TOCSY NMR experiments were performed using a spectral width of 10.0 ppm and 2K points, 1.5 s recycle time and 60 ms mixing time [1H-13C HSQC NMR experiments were performed using non-uniform sampling in the indirect carbon dimension [All NMR experiments were recorded at 850.104 MHz at 298K on a Bruker Avance Spectrometer. 150 microliters of redissolved extracts were transferred to 3 mm NMR tubes for 1D experiments. Two hundred and fifty microliters of redissolved extracts were transferred to 5 mm Shigemi tubes for 2D experiments. One-dimensional ing time ,59,60,61imension ,63. www.metaboanalyst.ca) [NMR spectra were phase adjusted, baseline corrected, referenced to 0.0 ppm using the internal TSP standard manually using Top-Spin 3.6.1  and normalized to the total intensity prior to statistical analysis. Manual peak bucketing and principal components analyses (PCA) were performed using AMIX  as we have described previously ,58,60,61lyst.ca) ,58.https://www.chenomx.com/), the Biological Resonance Data Bank (BMRB) [https://ccic.ohio-state.edu/) to confirm metabolite assignments. The confidence in metabolite assignments was ranked using the RANCM scheme developed in our laboratory [One-dimensional NMR peaks that were statistically different between the groups were initially and putatively identified using ChenomX Profiler (k (BMRB) ,65 and tk (BMRB) ,67,68,69k (BMRB) ,71 (httpboratory . Sample preparation of cell line cultures for metabolomic analyses generally requires a drying step. In the case of metabolic profiling of human cell line cultures, a chloroform/methanol extraction procedure is normally used to isolate the hydrophobic and hydrophilic fractions that must be dried prior to reconstitution in a buffer appropriate for instrumental analysis. Considering absence of standardized procedures for sample preparation for metabolic profiling studies, we examined whether the choice of drying method had any influence on the observed metabolome of hydrophilic extracts measured by NMR of three different human pancreatic cell line cultures. We determined that the metabolic profiles of each cell line differed significantly depending on the drying technique used during sample preparation. Certain metabolites were only found in samples dried by a specific drying technique, suggesting either that some metabolites evaporated during drying by one method and not the other, or that some metabolites went through chemical transformation in one of the sample preparation methods compared to the other. Metabolites undetected in speed-vacuum-dried samples may have been lost due to more rapid evaporation over time due to their having higher vapor pressure near room temperatures compared to at the relatively low temperature of the sample maintained during lyophilization. Overall, the lyophilized samples contained more metabolites at higher concentrations in two out of the three cell lines tested, i.e., MiaPaCa-2 and Panc-1, appearing to make it the technique of choice for the sample drying step during sample preparation. However, the two cell lines that had more metabolites more abundant in the lyophilized samples, MiaPaCa-2 and Panc-1, were grown on the same DMEM media whereas the one cell line that had more metabolites abundant in the SpeedVac samples were cultured using a different media, i.e., RPMI. Therefore, future experiments should examine whether the choice of media used to culture human cell lines for metabolic profiling can influence the efficiency of metabolite recovery using different drying methods and potentially explain the difference in the abundances of metabolites following one drying method over the other. Another aspect of the research that warrants further investigation is simple replication of the reported experiments. For example, in the case of the MiaPaCa-2 samples, the PCA indicates that the lyophilized samples were widely scattered in the PCA eigenspace of the scores plot in comparison to the SpeedVac samples. In contrast, in the AsPC-1 cells, the lyophilized and SpeedVac samples were tightly clustered and well separated in the PCA scores plot. These disparate observations indicated that the experiments should be repeated to determine if these observations are reproducible. Future work could also include working with the database developers to generate more complete databases since approximately half of the observed NMR resonances could not be assigned to a metabolite. Finally, it would be beneficial to repeat the experiment using an MS-based analytical technique for data collection to achieve more comprehensive metabolic profiles. In conclusion, this research demonstrates that the drying technique used in the preparation of human cell culture samples for metabolic profiling can have a significant effect on the observed metabolomes and the selection of the drying technique used during sample preparation should be given careful consideration when designing the sample preparation strategy.


Cryptolepine, neocryptolepine and isocryptolepine are naturally occurring indoloquinoline alkaloids with various spectrum of biological properties. Structural modification is an extremely effective means to improve their bioactivities. This review enumerates several neocryptolepine and isocryptolepine analogues with potent antiproliferative activity against MV4-11 (leukemia), A549 (lung cancer), HCT116 (colon cancer) cell lines in vitro. Its activity towards normal mouse fibroblasts BALB/3T3 was also evaluated. Furthermore, structure activity relationships (SAR) are briefly discussed. The anticancer screening of neocryptolepine derivatives was performed in order to determine their cytotoxic and growth inhibitory activities across the JFCR39 cancer cell line panel. Compound 7d shows a notable cytotoxicity against the breast , CNS , colon , lung , and ovarian  cancer cell lines [For further studying cytotoxic and growth inhibitory activities of neocryptolepine derivatives, the anticancer screening was performed across the JFCR39 cancer cell line panel, evaluated at five concentration levels  . The resll lines .7d and 7k are more potent than compound 7q towards the cancer cell lines in the JFCR39 panel. Compounds 7d and 7k show a similar growth inhibitory activity (GI50 > 0.01 µM) against the 31 and 28 cell lines, respectively, of the JFCR39 panel. Compound 7k shows a notable cytotoxicity against the breast , CNS , colon , lung  and ovarian  cancer cell lines. Compound 7q shows a mentionable cytotoxicity for the CNS , colon , lung , and melanoma  cancer cell lines. Compound 7q displayed a strong growth inhibitory activity (GI50 < 1 µM) for 28 panels in the JFCR39 panel of the various cell lines. DMS273 (lung cancer) and NCI-H522 (lung cancer) are the most sensitive cell lines of the 39 cell lines (GI50 = 0.42 µM for both and LC50 = 4.7 µM and 5.5 µM respectively). Compound 7q also showed a strong antitumor effect on LOX-IMVI .11a, 11b and the artemisinin-isocryptolepine hybrids 12 showed the potent antiproliferative activity, based on the data in In our group, we achieved the synthesis of the artemisinin-indoloquinoline hybrids and studied their antiproliferative activity . The art13 was a highly potent hAChEI having IC50 = 0.95 ± 0.04 nM, and a highly potent hBuChE having IC50 = 2.29 ± 0.14 nM [In 2017, Li Wang synthesized the novel tacrine-neocryptolepine hybrids, and evaluated as inhibitors of human AChE (hAChE) and human BuChE (hBuChE) . Compoun 0.14 nM .The COMPARE analysis assesses the correlation coefficient between the fingerprints of the test compounds and those of the various reference compounds . This sy7d and 7k have a very good match to actinomycin D (r = 0.7 for both). Similarly, compound 7k has a slight similarity to paclitaxel (r = 0.64). Compound 7q shows some resemblance to vindesine sulfate (r = 0.58) and aclarubicinHCl (r = 0.57). The COMPARE analysis revealed that compounds 7d and 7k have a mode of action similar to actinomycin D. It also suggested that 7l has a mode of action similar to vindesine sulfate or aclarubicin HCl. However, the new compounds may have other unique modes of action since the correlation coefficients (r) were at relatively low levels, which present an interesting possibility to examine in further studies.Indoloquinoline alkaloids are important scaffolds for antitumoral drug development. This review discussed the SAR of neocryptolepine and isocryptolepine, and presents the useful methods for improving the antitumoral activity of neocryptolepine and isocryptolepine analogues. The amino substituent effect and methyl localization effect are now available strategies, but the substituent group effect at the benzene ring of the quinoline moiety has no effect on regular SAR. Thus, the antitumoral activity is highly related to the activity of interacting with DNA. The computer-assisted database analysis, COMPARE, suggested that


All-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied.We used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and –resistant cell-lines. Bio-computing approaches were used to analyse the high-throughput lipidomic and transcriptomic data.ATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative-phosphorylation. ATRA reduces the amounts of cardiolipins and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells.The observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria. All-trans-retinoic acid (ATRA) is a non-conventional and promising therapeutic agent acting on different types of solid/hematologic malignancies –5 and it+ mammary tumors are characterized by sensitivity to the anti-proliferative action of ATRA, while the triple-negative counterparts tend to be resistant [Breast-cancer is a heterogeneous disease , althougesistant , 12. Theesistant , 13. ATResistant .There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression –18. In aCLs) as the main lipid class modulated by ATRA in retinoid-sensitive breast-cancer cell-lines. Mechanistic and RNA-sequencing studies show that the ATRA-dependent down-regulation of CLs in sensitive cell-lines is accompanied by a reduction in the amounts and activity of mitochondria.Here, we evaluate the constitutive lipidomic profiles of breast-cancer cell-lines recapitulating the heterogeneity of the disease , 12 and RARA-C5) and relative control (Vect-C1) clones from MDA-MB-453 cells as well as the RARα silenced (RARsA-sh18) and relative control (Vect-C6) clones from SK-BR-3 cells have been described [The source and the characteristics of the 16 breast-cancer cell-lines used are available in Additional file escribed . The groescribed .Single-cell motility assays were performed on BSA-coated culture wells by time-lapse microscopy, using the Imaging Station CellRˆ  and the software Image J .Untargeted lipidomics studies were performed with Lipostar, a high-throughput software supporting targeted and untargeted liquid-chromatography/mass-spectrometry (LC-MS) lipidomics , 28. FurSK-BR-3, HCC-1419, MDA-MB-361 and HCC-202 lines, mitotracker stained cells were also subjected to quantitative microscopic analysis using the ImageJ software. Cells were counterstained with Hoechst 3342 (Thermofisher) for the determination of cell nuclei. For each experimental point, a minimum of 200 cells/field in at least 4 fields/experimental triplicate were quantitated. Mitochondria were isolated using a described protocol [Mitochondria were stained with MitoTarcker Deep Red FM (Invitrogen) according to the manufacturer instructions. Following staining, cells were fixed with 2% formalin and subjected to quantitative FACS analysis, using a fluorescence activated cell sorter . In the case of the experiments performed on the protocol . The enzprotocol , 31. Theprotocol .− 6 M) for another 24 h. RNA was extracted with the mRNeasy Mini Kit (QIAGEN). RNA sequencing was performed using the Illumina TruSeq RNA library-preparation kit and sequenced on the Illumina NextSeq500 with paired-end, 150 base pair long reads. The overall quality of sequencing reads was evaluated using FastQC [Gencode [v27 release of the Gene Transfer File (GTF). Raw-counts were further processed in the R Statistical environment and downstream differential expression analysis was performed using the DESeq2 pipeline. All the RNA-sequencing data relevant for this study were deposited in the EMBL-EBI Arrayexpress database (Accession No: E-MTAB-8408). Genes characterized by low mean normalized counts were filtered out by the Independent Filtering feature embedded in DESeq2 . DESeq2-computed statistics were used as input for gene-set enrichment testing performed with the pre-ranked version of Camera. Statistical enrichments were determined for gene-sets obtained from the Hallmark (H), which are curated by the Molecular Signature DataBase (MSigDB).Three paired biological replicates of each breast-cancer cell-line were grown in DMEMF12 medium containing 5% charcolated FBS  for 24 h. Cells were treated with vehicle (DMSO) or ATRA  for 10 min. Cells were scraped from the dishes, centrifuged at 13,000 rpm and kept in 0.12 M phosphate buffer containing 4% paraformaldehyde and 2% glutaraldehyde for 2 h. After post-fixation in 0.12 M cacodylate buffer containing 1% OsO4 for 2 h and subsequent dehydration in graded series of ethanol, samples were cleared in propylene oxide, embedded in Epoxy medium (Epon 812 Fluka) and polymerized at 60 °C for 72 h. Ultrathin sections (70 nm thickness) were obtained with a Leica EM UC6 ultramicrotome, counterstained with uranyl acetate and lead citrate and examined with an Energy Filter Transmission Electron Microscope  equipped with a yttrium aluminium garnet (YAG) scintillator slow-scan charge-coupled device (CCD) camera . The numerical density of mitochondria  was estimated by morphometrical analysis using 30 digitized electron microscope fields of cells for each group acquired by the iTem software  and digitally superimposing an orthogonal grid with ImageJ (1.52a version). Briefly, the mitochondrial profile area density (NA) was estimated by the ratio between the number of mitochondria and the cytoplasmic area. Mitochondrial volume density (VV) was determined by the ratio of grid points falling over mitochondria divided by the total number of points of the grid contained in the cytoplasm. The numerical density of mitochondria (NV) was then estimated for each cell using the formula: NV = (1/β) (NA3/2 / VV1/2), where β is the shape coefficient for ellipsoidal mitochondria, calculated from the ratio of the armonic mean of major and minor axis of mitochondria sections measured on digital images. The mean mitochondrial volume was calculated for each cell as the ratio of mitochondrial volume density VV and numerical density NV.ATRA-sensitive − 6 M) for 30 min at 37° and the fluorescence polarization values were determined using a fluorescence detector . The fluorescence polarization (FP) value is a function of the emission value (emission = 420 nm), which was detected through analyzers oriented in parallel (FP1) and perpendicular (FP2) to the direction of polarization of the excitation beam (excitation = 365 nm), according to the eq. FP = (FP2 - FP1 / FP2 + FP1).The microviscosity of mitochondrial membranes was measured as described by M. Salmona et al. following staining with 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe [TN) breast-cancers, while the other 8 cell-lines derive from luminal tumors characterized for HER2 and ER/PR expression. Consistent with the tumor origin, the constitutive gene-expression profiles determined by RNA-sequencing classify the cell-lines in two distinct basal and luminal groups  and 4 luminal  cell-lines are classified as highly sensitive to ATRA. Three luminal  and 3 basal  cell-lines are endowed with intermediate sensitivity. One luminal (HCC-1419) and 4 basal  cell-lines show low sensitivity/resistance to ATRA.To conduct the study, we used 16 cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA has been determined , 12. EigPCs) consist of 203 features, while 40 cardiolipins (CLs) are identified. PCA of the constitutive lipid profiles separates luminal from basal cell-lines  and PCs, some diacylglycerophosphoinositols or phosphatidylinositols (PIs), neutral glycosphingolipids (N-glyco-SPs), CLs, lysophosphatidylcholines (LPCs), sphingomyelins (SMs) and 1-alkyl-2-acylglycerophosphocholines/1-alkenyl-2-acylglycerophosphocholines (o-PCs/ p-PCs), as well as all the identified steryl-esters (SEs), monoacylglycerophosphoethanolamines  and 1-alkenyl-phosphatidylethanolamine (p-PE) , 2 PCs, 1 p-PC, 1 ceramide (CER) and 1 diacylglycerophosphoethanolamine  are higher in luminal than basal cell-lines. Although luminal and basal cell-lines show different complements of individual features, the average constitutive levels of the 23 classes of lipids do not vary in the two groups  for 48 h. The selection of the retinoid concentration and exposure time is based on pilot studies conducted in SK-BR-3 cells. These studies demonstrate that maximal alterations in the lipidomic profiles are observed with 10− 6 M ATRA, while the 48-h exposure time precedes any significant effect on the number of cells [We compared the mass-spectrometry data obtained in all the cell-lines exposed to DMSO or ATRA  which results in the conversion of all-trans retinol into all-trans retinyl ester, a storage form of Vitamin A [d groups . SimilarTRA Fig. , left. Tore Fig. , right. itamin A .PSs are cell membrane glycerophospholipids differing in their fatty acid composition and they are involved in cell-cycle signaling and apoptosis [SK-BR-3, HCC-1500, CAMA1 and MDA-MB-361 cell-lines, ATRA causes a reproducible reduction in the total amounts of the PSs identified  or vehicle (DMSO) for 16 h and subjected to a random-motility assay for another 24 h . To obtain insights into the mechanism underlying the ATRA-dependent down-regulation of CLs, we evaluated the RNA-sequencing data obtained in all the 16 cell-lines exposed to ATRA (10− 6 M) or vehicle for 24 h . Using these data, we determined the effects of ATRA on the expression of the 77 genes involved in the glycerophospholipid metabolic pathway  between the ATRA/DMSO expression ratio of any of these genes and the ATRA-score values. The results support the idea that the ATRA-dependent CLs decrease observed in retinoid sensitive cell-lines cannot be explained by up-regulation of specific genes controlling the catabolism of CLs or down-regulation of genes involved in their biosynthesis. CLs are predominantly located in the inner membrane of mitochondria, where they are involved in oxidative phosphorylation [Oxidative Phosphorylation” lays in third position among the 4 top Hallmark gene-sets enriched for genes down-regulated by ATRA in sensitive luminal and basal cell-lines  situated in the inner mitochondrial membrane. ATRA down-regulates approximately half of the “Oxidative Phosphorylation” genes in the luminal and basal cell-lines characterized by high/intermediate sensitivity to the retinoid  for 8 h . Indeed, PCA demonstrates that the constitutive lipidomic profiles of the ATRA-sensitive SK-BR-3 and the ATRA-insensitive HCC-1419 cell-lines are very similar  for 24, 48 and 72 h, stained with Mitotracker and subjected to quantitative fluorescence microscopy  for 48 h  for 48 h. We measured the enzymatic activity of respiratory complexes I-V following normalization for the levels of citrate synthase activity  or the corresponding void vector (Vect-C1) [RARA-C5 cells are characterized by higher RARα expression levels than the Vect-C1 counterparts and the nuclear retinoid receptor is functionally active as indicated by its ATRA-dependent degradation  for 48 h does not alter the overall levels of CLs in Vect-C1 cells, while it causes a reproducible decrease in RARA-C5 cells  . As expeion Fig. a, right,53 cells , which aoid Fig. a, left. y action . The liplls Fig. d. In addSK-BR-3 cells stably infected with a retroviral construct containing a RARα-targeting shRNA (RARA-sh18) and the corresponding void vector (Vect-C6) [RARA-sh18 are characterized by lower levels of functionally active RARα than the corresponding Vect-C6 control cell-line  for 48 h decreases CL levels  . As expeine Fig. b, right.A RARA-sh8 and theion Fig. b, left. lls Fig. a, exposu to ATRA − 6 M for to ATRA − 6 M for to ATRA − 6 M for to ATRA − 6 M forCLs and the associated reduction in mitochondrial membrane microviscosity are dependent on RARα, which is a crucial determinant of ATRA anti-tumor activity in breast cancer [All these results indicate that the ATRA-induced decrease in t cancer .+ breast cancer patients > 65 years of age undergoing neo-adjuvant treatment with aromatase inhibitors . The present report provides new insights into the mechanisms underlying the anti-tumor action of ATRA, a non-conventional therapeutic agent [ATRA is a promising anti-tumor agent and it is the object of an approved window-of-opportunity clinical trial we are conducting in the context of the personalized treatment of ERic agent , 12, 26.ic agent . In partic agent .CLs, a class of lipids predominantly located in the inner mitochondrial membrane and playing an important role in oxidative phosphorylation [CLs and this effect is correlated to the growth inhibitory activity of the retinoid. Down-regulation of CLs is accompanied by a significant reduction in the number and functional activity of mitochondria, two phenomena which are likely to contribute to the growth inhibitory action of ATRA in sensitive cell-lines. Our transcriptomic data indicate that ATRA-dependent down-regulation of CLs is consequent to a decrease in the assembly of mitochondria. In fact, ATRA does not exert direct effects on the expression of genes involved in the biosynthesis/catabolism of CLs, while the retinoid down-regulates numerous genes coding for mitochondrial proteins via direct or indirect transcriptional events mediated by RARα. In addition, the ATRA-dependent down-regulation of nuclear genes encoding mitochondrial proteins is a relatively early event, preceding the decrease in CL levels. Although CLs down-regulation is unlikely to be a determinant of the reduction in the amounts of mitochondria, the process may contribute to the functional deficiency of these organelles induced by ATRA in sensitive cells. In fact, the decrease in the enzymatic activity of mitochondrial complexes I, III IV and V observed in SK-BR-3 cells may be explained by the fact that ATRA induces a relative deficit of CLs in the mitochondrion. This, in turn, reduces the microviscosity of mitochondrial membranes, a phenomenon which is potentially at the basis of the ATRA induced decrease in mitochondrial complexes activity [The most interesting effects of ATRA are observed on rylation . ATRA reactivity –52.CLs caused by ATRA must be further investigated. Nevertheless, we recently observed that exposure of retinoid-sensitive luminal and basal breast cancer cell lines to ATRA induces the expression of various endogenous retroviral RNAs, resulting in an unusual type of “viral-mimicry” [viral-mimicry” causes the induction of the transcriptional factor, IRF1 (Interferon Responsive Factor 1), via RARα activation and it is associated with the up-regulation of numerous other interferon-responsive genes. Interestingly, viruses [viral-mimicry” and the consequent cascade of transcriptional effects involving interferon-dependent genes are at the basis of the reduction in the number and functional activity of mitochondria observed in sensitive breast cancer cell lines exposed to ATRA.The molecular mechanisms underlying the reduction in the number of mitochondria and the consequent decrease in the levels of mimicry”  response viruses , IRF1 [5 viruses  and otheCLs depletion, which is at the basis of the induced michondrion-dependent apoptotic response [CLs.The involvement of mitochondria in the anti-proliferative action exerted by ATRA on the neoplastic cell is of relevance for the development of innovative approaches in the treatment of breast-cancer. In fact, cancer cell mitochondria are novel therapeutic targets, given their structural and functional differences from the normal counterparts , 57. Theresponse , 60. TheOverall, the study provides new insights into the mechanisms underlying the anti-tumor activity of ATRA in breast cancer. To the best of our knowledge, this is the first study which demonstrates that the anti-proliferative effects of the retinoid are, at least partially, the consequence of a decrease in the amounts of mitochondria which leads to deficits in the respiration and energy balance of the neoplastic cells. From a basic point of view, our integrated approach defines the constitutive lipidomic profiles of luminal and basal breast-cancer cells. In addition, it demonstrates that the anti-proliferative effects of ATRA are, at least partially, the consequence of a decrease in the amounts of mitochondria which leads to deficits in the energy balance of the breast-cancer cell. As for the applicative aspect, the study opens the way to therapeutic strategies based on rational combinations involving ATRA and organic molecules targeting the mitochondria, an emerging group of anti-tumor agents.Additional file 1: Figures S1-S12. Supplementary Information, Supplementary Methods, Supplementary References.Additional file 2: Table S1. Lipidomics raw data, Highthroughput lipid ID.Additional file 3: Table S2. Effects of ATRA on cardiolipin amounts in SK-BR-3 cells.Additional file 4: Table S3. Effects of ATRA on cardiolipin amounts in genetically engineered breast cancer MDA-MB-453 and SK-BR-3 cells.


Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, and new therapeutic strategies are still required. Here we screened a synthetic cannabinoid library to identify compounds that uniformly reduce the viability of seven CRC cell lines.Material and Methods: Seven distinct CRC cell lines were treated with 10 μM cannabinoid compounds (from a library of 370 molecules) for 48 h, and cell viability was subsequently measured with MTS assay. Dose–response curves were conducted for compounds that were found to reproducibly reduce cell viability of one or more cell lines.Results: We identified 10 compounds from the library that were able to reduce cell viability of CRC cell lines (with an IC50 ≤ 30 μM). Of these compounds, seven were specific for CRC cells, and six were effective in all CRC cell lines tested. Treatment with traditional phytocannabinoids (THC or CBD) was either ineffective or much less potent and only partially efficacious. Treatment with antagonists for the known cannabinoid receptors  failed to block the activity of the most potent of identified compounds.Conclusion: We identified three families of cannabinoid compounds that reduce CRC cell viability through a noncanonical receptor mechanism. Future modification of these compounds may lead to the development of novel therapies to treat this disease. Despite this knowledge, attempts to target the Wnt/β-catenin pathway to inhibit cancer growth have been largely unsuccessful. In light of these facts, new therapeutic approaches need to be undertaken.With an estimated 97,220 new cases and over 50,000 deaths each year, colorectal cancer (CRC) is the third most common cancer and the third most common cause of cancer death.5–7; however, a number of studies have proposed the use of cannabinoid compounds as anti-tumor agents.8–11 These plant-derived cannabinoids interact with the endocannabinoid system and have a much higher affinity for the receptors than do endogenous ligands. The expression of cannabinoid receptors 1 and 2 (CB1 and CB2) and GPR55 has been reported to have increased in CRC, and this is associated with a poorer prognosis and more advanced disease.12–17Over the past decade, the number of countries and states that have legalized medical cannabis has grown rapidly, and cannabinoid compounds may serve as a novel therapeutic agent to combat a number of diseases. With regard to cancer, medical cannabis has largely been utilized for palliative purposesAPC, the most common activating mutation of the Wnt/β-catenin pathway in CRCs; in contrast, HCT116 and LS174 have direct activating mutations in CTNNB1, the gene that encodes β-catenin, while RKO cells have intact Wnt/β-catenin signaling. SW480 and SW620 cells were isolated from the same individual, with SW480 cells representing a primary tumor and SW620 a lymph node metastatic tumor. Our approach of analyzing CRC cell lines with distinct Wnt signaling mutations allowed us to determine whether the genetic lesion or tumor subtype could influence sensitivity to cannabinoid treatment. Here we identified several cannabinoid compounds that show potential as novel therapies to combat CRC. Intriguingly, these agents do not appear to act through traditional cannabinoid receptor signaling.The objective of our study was to identify compounds from a synthetic cannabinoid library that universally reduces cell viability of seven distinct CRC cell lines . Importantly, these cell lines represent a diversity of tumor types: the first four cell lines contain mutations in 2. CCD 841 CoTr cells were cultured as described above, but grown at the permissive temperature of 33°C.The human CRC cell lines SW480, SW620, HT29, DLD-1, HCT115, LS174, RKO; the temperature-sensitive t-antigen-transformed normal human colonic epithelial cell line CCD 841 CoTr; and the human embryonic kidney cell line HEK 293 were obtained from the American Type Culture Collection (ATCC). Each line, with the exception of the RKO line that was cultured in RPMI, was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM Glutamax, 10 U/mL penicillin, 10 μg/mL streptomycin, and 0.25 μg/mL Amphotericin B at 37°C in 5% CO19 Target gene expression was measured by quantitative real-time polymerase chain reaction using TaqMan™ probes for CNR1 (Hs01038522_s1), CNR2 (Hs00275635_m1), GPR55 (Hs00271662_s1), TRPV1 (Hs00218912_m1), and GAPDH (Hs99999905_m1) . Relative levels were determined using the 2−ΔΔCT method.20RNA isolation and cDNA synthesis were performed as previously described.Cells were seeded in 96-well plates at a density of 20,000 cells per well, incubated for 8 h, and then treated with members of a synthetic cannabinoid library  at 10 μM. This library contains 370 distinct molecules consisting of parent compounds along with positional isomers, analogs, and homologs. Cells were treated with the compounds for 48 h, and then cell viability was measured using the MTS assay following the manufacturer's protocol . Cells were incubated with MTS for 1.25 h , after which absorbance at 590 nm was measured using a FlexStation 3 spectrophotometer . Cell viability for DMSO-treated controls was set to 100%. A z-score was calculated for individual plates, and compounds that displayed a z-score of −1.5 or greater were selected for repeat screening . Importantly, any compounds identified that reduced viability of one of the cell lines tested were rescreened against all cell lines, to reduce the possibility that potential compounds would be overlooked. These experiments were supplemented with the traditional phytocannabinoids  at the same concentration. For select compounds, MTS results were confirmed with trypan blue staining. Cells were plated and treated as described, and after 48 h, adherent and nonadherent cells were collected, stained with 0.2% trypan blue, and counted on a hemocytometer.Any compounds that reduced viability upon rescreening in either the original cell line or in a second cell line were pursued, and dose–response curves were performed on these compounds for all cell lines. Cells were seeded as described above, incubated for 8 h, and treated with select cannabinoid compounds at concentrations of 100 nM, 333 nM, 1 μM, 3.3 μM, 5.6 μM, 10 μM, 33 μM, and 100 μM. Viability was measured as described in the To explore the receptors mediating cell death, experiments were conducted using selective antagonists to inhibit each of the four primary cannabinoid receptors. SW480 cells were seeded as described in the n=3) dose–effect curves in all of the cell lines. Statistical significance was determined using the Student's t-test .Each compound identified in the primary screen that reduced viability was rescreened two additional times; therefore, each potential compound identified from the original screen was tested three times at 10 μM in all seven cell lines. Moreover, 30 compounds were subjected to replicate . Levels of cannabinoid receptor 1 (CNR1) were similar between most of the cell lines tested; however, mRNA expression was markedly higher in HT29 and LS174 cells compared with other cell lines (CNR2) was undetected in each of the cell lines tested (using multiple dilutions of the prepared cDNA). The expression of G protein-coupled receptor 55 (GPR55), sometimes referred to as CB3,22 was similar to that observed for CNR1, with the highest expression in HT29 and LS174 cells (TRPV1) was similar between all colon cell lines (To determine if cannabinoid receptor messenger RNA (mRNA) expression levels vary in our cell line panel , we conducted reverse transcribed-quantitative polymerase chain reaction (RT-qPCR) analysis of the four most common cannabinoid receptors , PTI-2, and PTI-1 were not selective and inhibited normal epithelial cells and/or HEK 293 cells (data not shown).Dose–response curves were performed for each of the 30 compounds that reduced viability of the rescreening process . Of these compounds, only 10 were identified that had an ICTHC, while not effective at reducing cell viability at 10 μM, did reduce cell viability at higher concentrations. THC was effective in SW480  and HCT1We next sought to identify which cannabinoid receptor was mediating the action of our identified cannabinoids. We repeated the cannabinoid treatment with our most potent cannabinoid  and antagonists to each of the four major receptors using SW480 cells. Treatment with these antagonists  failed to prevent the reduction in cell viability observed with (±)-5-epi CP 55,940 .24–28 However, a large-scale screening of these compounds to assess the potency and efficacies of different synthetic cannabinoids has not previously been performed. Here, we demonstrated that 10 synthetic compounds are highly efficacious and moderately potent for reducing the viability of seven CRC cell lines. The structures of these compounds are illustrated in Previous work has demonstrated the ability of cannabinoid compounds to reduce the viability of CRC cell lines.50 of 16.3–23.1 μM and efficacy in only three cell lines. Additionally, for CP 55,940 and CP 47,497, we found that certain isomers were more potent than others. In particular, of the four CP 55,940 compounds in the library, only one  failed to reduce CRC cell viability, suggesting that the (+)-stereoisomer is an active form as this was present in the other three derivatives. With regard to CP 47,497, the library contains nine derivatives of which only three reduced viability by >50% . Of the three compounds that were identified, two were stereoisomers of the more potent C-8 homolog and one was a stereoisomer of the parental C-7 isomer, (−)-CP 47,497.29 Mixtures containing the (+)-stereoisomer of the parental compound or the (+)-stereoisomer alone were unable to reduce CRC cell viability. Additionally, neither the para-quinone analog nor the C-6 or C-9 homologs were able to reduce CRC cell viability.Interestingly, these compounds can be placed into three families: CP 55,940, CP 47,497, and PTI. Additionally, both CBD and the synthetic derivative of CBD, HU-331, exhibited the ability to reduce CRC cell viability, although CBD displayed a weak activity with an IC30–32 The ability of CP 55,940 to antagonize GPR55 is consistent with our finding that CBD, but not THC, was able to reduce the viability of CRC cells. THC is an agonist of GPR55, whereas CBD has been shown to be an antagonist of GPR55.33 There are no published data on the pharmacological activity of the identified synthetic cannabinoids against GPR55. While this is an attractive idea, we did not detect any effect on cell viability by GPR55 antagonist ML-193. This suggests that the cannabinoids may be signaling through GPR55 in a manner different from ML-193, or that antagonism of GPR55 is not the mechanism of action of the identified cannabinoids.Despite having a similar binding affinity for CB1 and CB2 as CP 55,940 and CP 47,497, WIN 55,212-2 was much less potent and less efficacious than these two compounds -CP 47,497 C-8 homolog while of low potency was efficacious in five of the cell lines tested, but was ineffective in both SW480 and SW620 cells that were derived from the same patient. We also failed to observe a complete reduction of viability of any cell line. Our most potent and efficacious compounds only reduced viability to between 10% and 15% even at the highest dose (100 μM) tested. This could be due to new cell divisions occurring during the 48 h treatment period, and the daughter cells not being exposed to the synthetic cannabinoid for a long enough time period. This finding could also be due to the degradation of cannabinoid or reduced concentration of the cannabinoid over time. It is also possible that, given the limited solubility of these agents, we were simply unable to deliver high enough concentrations.In the majority of CRC tumors, there is aberrant activation of the Wnt/β-catenin pathway. Of the cell lines tested, six of the seven had mutations that activated this pathway; however, we did not observe any difference in susceptibility to cannabinoids based on the activation of this pathway. Interestingly, we did find that only tumor cell lines isolated from primary tumors, with mutations in the APC gene , were more susceptible to CBD, and cells that contained mutations in the β-catenin gene (HCT116 and LS174) were only moderately impacted by CBD where viability did not typically drop below 50% even at the highest concentrations tested. The treatment-resistant metastatic cell line SW620 was also not susceptible to CBD. This might suggest that CBD is able to reduce the activity of Wnt/β-catenin pathway before the translocation of β-catenin into the nucleus.In summary, we identified 10 synthetic cannabinoids demonstrating reproducible activity against human cells, with seven compounds reducing viability only in CRC cells. These compounds fall into three families of molecules, CP 55,940, CP 47,497, and PTI. Interestingly, CBD had only incomplete activity against a subset of CRC lines, and THC was without activity at 10 μM. We also identified potential isomers and modifications that may contribute to increasing the potency and efficacy of CP 55,940 and CP 47,497 that may lead to novel drugs to treat this deadly disease.


EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM).EGFR  and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 μM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 μM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment.The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours.MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR mutations.Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with The online version of this article (10.1186/s12890-018-0760-z) contains supplementary material, which is available to authorized users. EGFR can drive tumour growth -2,5-diphényltétrazolium bromide test (MTT) . Cell viability was assessed at increased concentration of tracer and genuine erlotinib. As our tracer had a 3.5-fold higher molar mass than erlotinib, cell viability assay was also performed at equimolar erlotinib concentrations. For each cell line, we calculated the concentration that resulted in 50% cell death (IC50). MTT assay was chosen because of its lack of interference with the emission wavelength of our tracer.6 cells were re-suspended in a non-fluorescent culture container using 0.5 ml of PBS containing 1 μmol/l of tracer, 1 μmol/l of erlotinib or PBS alone during an hour at 37 °C, in the dark, with 5% CO2 atmosphere. Cells were then washed twice using PBS. Each washing was followed by 3 min centrifugation at 2000 rpm. FCFM imaging was then immediately performed by application of the probe on the cell pellet. Experiments and imaging were performed six times for each cell line. Results of imaging are expressed as median fluorescence intensity (MFI), in arbitrary units (A.U).In vitro imaging was performed on centrifuged cell pellet. Cells were removed from the plates at confluence by trypsination (1 ml of Trypsin in 0.05% EDTA), suspended in 8 ml of culture medium, centrifuged twice at 2000 rpm for 3 min and finally re-suspended in PBS at room temperature. 1 × 10For ex vivo imaging, xenografts were excised under general anaesthesia after intra-peritoneal injection of 100 mg/kg of ketamine and 10 mg/kg of xylazine. After xenograft excision, and under general anaesthesia, mice were sacrificed by cervical dislocation. The xenograft was then immerged in a 2 ml solution containing 1 μmol/l of tracer at room temperature and kept out of light. After an hour, each tumour was washed by consecutive immersion in 3 separate wells containing 2 ml of 0.9% saline serum. FCFM imaging was then immediately performed by application of the optical probe on the surface of each xenograft. Experiments and imaging were performed at least eight times for each cell line. Results of imaging are expressed as MFI.Results are expressed as headcounts and percentages, medians, and first and third quartiles (IQR) or means and standard deviation (SD). Comparisons were performed using Student T-test and ANOVA test. Correlations were assessed using the Spearman correlation coefficient. ROC-curve was used to establish sensitivity and specificity and to determine optimal cut-off value. All tests were two-sided, the type I error rate was set at 0.05. Analyses were performed using GraphPad Prism 6® for Mac OS X® .p = 0.1) . The tracer was able to distinguish cell lines with a mutated EFGR from wild-type EGFR cell lines, and to distinguish cell lines with a sensitive phenotype (HCC827) from those with a resistant phenotype (H1975 and H1650) (p = 0.002) . Using a cut-off value of 354 A.U, MFI had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours. MFI did not differ significantly between xenografts derived from the three cell lines with a mutation of EGFR . Therefore, ex vivo imaging could not distinguish tumours with a sensitive phenotype from those with a resistant phenotype to erlotinib