Tellurium is a rare element that has been regarded as a toxic, nonessential element, and its biological role is not clearly established. In addition, the biological effects of elemental tellurium and some of its organic and inorganic derivatives have been studied, leading to a set of interesting and promising applications. Diphenyl ditelluride (DPDT), an organic tellurium derivate, showed antioxidant, antigenotoxic, antimutagenic, and anticancer properties. The antioxidant and prooxidant properties of DPDT are complex and depend on experimental conditions, which may explain the contradictory reports of these properties. In addition, DPDT may exert its effects through different pathways, including distinct ones to those responsible for chemotherapy resistance phenotypes: transcription factors, membrane receptors, adhesion, structural molecules, cell cycle regulatory components, and apoptosis pathways. This review aims to present recent advances in our understanding of the biological effects, therapeutic potential, and safety of DPDT treatment. Moreover, original results demonstrating the cytotoxic effects of DPDT in different mammalian cell lines and systems biology analysis are included, and emerging approaches for possible future applications are inferred. Tellurium (Te) is a stable and solid element that pertains to chalcogens (group 16 in the periodic table), which is the same group that includes sulfur, selenium, and polonium. Te is classified as a metalloid because of its features between metals and nonmetals . It was The industrial applications of inorganic Te compounds include production of nanoparticulate semiconductors and metal-oxidizing solutions , 7. FurtThe risk of human environmental exposure to Te is unpredictable due to its elevated usage . The useAlthough there was limited use of synthetic organotellurium (OT) compounds in the past, they have turned a promising alternative for various applications, as evidenced by the increase in reports on OT compounds in the literature , 19. SynIn the present review, we emphasize the biological activities of an OT compound, diphenyl ditelluride (DPDT) , aiming The antioxidant effects of certain molecules are based on their ability to retard or inhibit oxidative damage. Their antioxidant role includes blocking oxidative reactions induced by highly reactive oxidant molecules—the so-called free radicals or reactive oxygen species (ROS)—that damage other molecules. The antioxidant properties of substances such as OT compounds can protect the biomolecules and cell components against oxidative damage –26. OT cin vitro studies and rodent models could be attributed to their ROS scavenging and glutathione peroxidase mimetic properties . Th. ThμM DP5 μg/mL) .μg/mL DPDT for all cell lines. A DPDT concentration of 0.28 μg/mL induced a greater reduction in cell viability, about 40–50%, for all cell lines  was evaluated by clonogenic assay. The cells were treated for 72 h at 0.028, 0.28, and 2.8 ll lines . These rThe administration of traditional chemotherapeutic agents inhibits the cell division inducing not only cell cycle alterations and apoptosis but other forms of nonapoptotic cell death such as necrosis, autophagy, and mitotic catastrophe. Since the most effective cancer treatment method used after surgery is chemotherapy, the search for new drugs with antiproliferative properties is currently ongoing , 55.μM DPDT. This was the first study showing the effects of DPDT on the cell cycle (μM DPDT resulted in the accumulation of S-phaseV79 cells . In. InμM DP79 cells . Moreovell cycle . We alsoll lines . FurtherC6 cells .μM) (μM) in V79 cells . In. InμM)  showed pronounced tolerance in the top1Δ strain to DPDT exposure. The same study also reported DPDT-induced inhibition of human TopoI activity in vitro using DNA relaxation assays [The results reported in DPDT studies lead us to infer the possible mechanisms of action of this drug and suggest its application as an antiproliferative agent in cancer therapy. As previously mentioned, results from our group demonstrated that DPDT can induce frameshift mutations in bacterial DNA and induce the formation of micronuclei in V79 cells , 22. On 79 cells . In thisn assays . These rRegarding the possibility of TopoI inhibition, the search for a noncamptothecin (CPT) TopoI inhibitor has been the target of several studies because of the limitation of CPT and its derivatives –62. The The intertwining of DNA strands and helices, produced during the essential cellular processes of replication, transcription, recombination, and chromosome segregation, must be resolved in order to maintain genome stability and cell viability. DNA topoisomerases supply an important solution for resolving such topological DNA problems . Howeverp value ≤ 0.01. After this, we obtained initial C6 protein-protein interaction (PPI) networks based on physical interactions from the GeneMANIA prediction server [As described in previous sections, DPDT promotes different cellular phenomena. However, no molecular target associated with DPDT has yet been identified. We used a systems biology approach to predict putative cell signaling pathways or specific protein targets for DPDT. To this end, we prospected networks based on C6 cell line gene expression data. Contrast analysis was applied, and differentially expressed genes before simulation of DPDT treatment were selected using the rank product method, a technique based on calculating rank products (RP) from replicate experiments. We used three sample replicates of the C6 rat glioma cell line (untreated) from the GEO database (GSE1139 accession number) under culture conditions similar to those of our experimental model. For each sample, the average of the signal between the same probes was calculated and applied to the normalized microarray data using the limma package in the R/Bioconductor software , 72. Then server  which aln server . It is an server –77.In addition, to define Akt interconnectivity and relevance in the C6 CP-PPI network, we analyzed some topological parameters. Firstly, we applied CentiScaPe software to explore node degree and betweenness centralities . Based o2+ influx activated protein kinase A and protein kinase C in astrocytes, causing the hyperphosphorylation of glial fibrillary acidic protein and vimentin. These disrupt the organization of actin stress fibres formed by endogenous RhoA activation and led to altered cell morphology. In neurons, the overexpression Ca2+ levels activated Erk and p38MAPK, beyond the protein kinase A and protein kinase C, provoking hyperphosphorylation of neurofilament subunits  was observed at a similar concentration range to that of the antitumor agent DOX (IC50: 0.02 μg/mL). This concentration is more than an order of magnitude lower compared to the toxic DPDT concentration in normal CMSPH cells (0.4 μg/mL). Furthermore, the hemolytic potential in erythrocytes was observed at high concentration, and the cytotoxicity of DPDT cannot be attributed to unspecific damage to cell membranes. Albeit the conventional prejudice concerning organotellurium compounds, DPDT use should be considered with caution because of its high reactivity and toxicity at relatively low concentrations. The exact nature of DPDT cytotoxic effects remains unclear, although some mechanisms have been proposed that differ depending on the system studied. New trials regarding toxicity, checkpoint activation, and mechanisms of cell death induction by DPDT should be explored in a greater number of cell lines. Moreover, its topoisomerase inhibition potential should be further investigated, keeping in mind that this compound probably presents several distinct mechanisms of action. DPDT have particular chemistry with the thiol which is related to many of the biological effects observed so far. The DPDT depletes GSH because of oxidation and/or as a possible substrate for GSH conjugation and could modulate cellular antioxidant defenses inducing GSH synthesis. The dual action of DPDT (protective and toxic) opens possibilities for distinct applications in cancer treatment (2+ levels activated Erk and p38MAPK, beyond the PKA and PKC, provoking hyperphosphorylation of neurofilament subunits, and as a consequence must have caused cellular redox imbalance, increasing ROS and inducing cell death. ROS-generating drugs induce cancer-specific cytotoxicity by elevated endogenous ROS production in preclinical studies [In the past decade, several organometallic compounds have entered clinical trial owing to their unique redox-modulating features and great potential in cancer therapy , 81. Thereatment . In neur studies , 83. Thu


The results showed that after 24 h of lectin exposure, the cells lines showed a dose-dependent cytotoxic effect, the effect being higher on MCF-7, while C33-A showed the highest resistance. Cell proliferation studies showed that the toxic effect induced by lectins is higher even when lectins are removed, and in fact, the inhibition of proliferation continues after 48 h. Due to the use of two techniques to analyze the cytotoxic and antiproliferative effect, differences were observed in the results, which can be explained by the fact that one technique is based on metabolic reactions, while the other is based on the 3[H]-thymidine incorporated in DNA by cells under division. These results allow concluding that lectins exert a cytotoxic effect after 24 h of exposure, exhibiting a dose-dependent effect. In some cases, the cytotoxic effect is higher even when the lectins are eliminated, however, in other cases, the cells showed a proliferative effect.For many years, several studies have been employing lectin from vegetables in order to prove its toxic effect on various cell lines. In this work, we analyzed the cytotoxic, antiproliferative, and post-incubatory effect of pure tepary bean lectins on four lines of malignant cells: C33-A; MCF-7; SKNSH, and SW480. The tests were carried out employing MTT and  Worldwide cancer-related mortality has increased considerably, thus the need to search for novel drugs and alternative treatments to solve this health problem. One of these alternatives has suggested the use of diverse alternative treatments such as herbal medicines, something that has been known for many decades by humans ,2,3.in vitro and in vivo, inhibit growth when incorporated into the diet, and are toxic when injected into animals; therefore, it has been suggested that lectins be utilized as an alternative treatment for some diseases, such as cancer -thymidine assays.Tepary bean -2,5 diphenyltetrazolium bromide (MTT) assay. The MTT assay is a colorimetric assay for assessing cell viability, however, tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferative to resting status) of potential medicinal agents and toxic materials.The cytotoxicity of the TBL to SKNSH, SW480, MCF-7, and C33-A cells after 24 h of exposure is depicted in Studies carried out employing tritium-labeled thymidine shown in  indicate3[H]-thymidine assays.To establish whether the effect of lectin toxicity continues when this substance has been eliminated from the cell culture, we carried out another assay in which, after a 24-h lectin exposure, these proteins were removed and cytotoxicity and proliferation was again determined after 24 and 48 h of incubation with only culture medium, employing MTT and The results obtained by MTT assay are depicted in These results indicated that once the lectin was removed from the cells, the cytotoxic effect of these proteins continued, causing the cells to inhibit their proliferation after 24 h of incubation, which at this point indicated that the lectins were attached to the cell membranes. This prevents the cells from being able to metabolize the MTT, which is reflected in the decreased production of the formazan compound. However, at this point, we are unable to know with certainty whether the cells are able to maintain this effect, thereby continuing cell incubation without lectin for an additional 24 h, up to 48 h.When the cell proliferations were analyzed after 48 h of incubation without lectin, the results illustrated in As can be observed, some of the cell lines continued to decrease in viability; SKNSH cells, however, were able to recover and increase their cell viability. These results may indicate that although the lectin caused damage, these cells are able to repair this damage and to continue to perform certain metabolic functions, or that the level of damage caused by the lectins was not permanent and that cell proliferation could be recovered at some time after the lectins were removed from the culture medium. However, on the other hand, in the remaining cell lines, damage was more severe, perhaps because the lectins were more strongly bound to the cells and were unable to recover.The post-incubatory assay carried out employing is shown in The results when proliferation was measured after 48 h without lectins are illustrated in These results indicated that at low concentrations, the cytotoxic effect of lectins was maintained, even if these were not still present in the culture medium. However, in lectins that is bound strongly the damage continue although the lectin solution was retired, but at higher concentrations of 100 µg/mL, recovery cells exhibited activity, thus an increase in the ability to divide by increasing the amount of thymidine incorporated into the DNA.Many studies have reported the purification of the lectins of several vegetable sources, including ion exchange and affinity ,52,53,54During lectin purification by affinity chromatography, it was determined that the fraction not retained by the matrix continued to demonstrate considerably high agglutination activity. This is probably due to that the fetuin matrix does not possess the ability to retain some of the lectin isoforms contained in the beans, retaining in this manner only a fraction of these. There are reports that indicate that lectins of different bean varieties consist of several isoforms and that in order to purify these independently, it is necessary to employ certain chromatography techniques, either independently or in combination, such as ion exchange or affinity, because each isoform possesses a different purification pattern ,56,57.When we analyzed the pure lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (data not shown), this exhibited only one band with a molecular weight of approximately 31 KDa, which led us to think that only one protein was purified. However, previously published studies in which this same affinity matrix was employed showed that such a matrix not only retains a single isoform, but also receives a mixture of all three of these, which differ little in their isoelectric points and molecular weights . AnotherThe MTT technique is known as an assay that is based on the ability of living cells to transform 3--2,5-diphenyltetrazolium bromide (MTT) by means of the action of the mitochondria into a colorful formazan product, which is a direct indicator of the amount of viable cells, and also can be used to measure cytotoxicity or cytostatic activity (shift from proliferative to resting status); however, there was a degree of error, due to that there may be cells that do not exhibit immediate metabolic activity or even that their activity is decreased due to the action of the lectin bonding to the cells. Although this methodology is well accepted for carrying out feasibility and cell proliferation studies, there are other methodologies that can yield better results concerning the cells’ biological activity, such as the technique that includes DNA synthesis, as well as the use of tritiated thymidine.The results observed in the study with the MTT assay indicate that there is great variability in the cytotoxic effect of lectins on the four cell lines studied. It is noteworthy that these cell lines derive from different cancer origins, but it has been documented that there are significant changes in the pattern of the glycosylation of cell membranes in the carcinogenic process. The binding of lectins to cell membranes depends on the type of carbohydrate to which the lectin is related, and also to that found in the cell membrane and, on the other hand, to the strength of the binding of these lectins. As we can observe, although in all cell lines the lectins were most likely bound by the union pattern, this was considerably variable and the quantity of lectins bound was also different, all of this due to the differences of each cell line, as well as to that the damage caused by the lectin in each cell line is varied, despite that statistically, not a great difference has been noted among the lines. As mentioned previously, the cell line that exhibited the greatest effect was SW480; this could have been due to that lectin bound in a greater quantity and exerted a greater toxic effect on these cells, in contrast with the remaining cell lines studied.With the use of the tritiated thymidine technique, we are very well aware that this is a much more accurate technique than the MTT one for directly measuring the amount of thymidine incorporated by living cells and their capability of dividing, which renders it a better technique for measuring lectin-associated cellular damage. Thus, this technique more clearly shows the level of damage caused by the lectins in each of the cell lines.in vivo, causing damage to several cells in organs -Thymidine Assay4.3.2. 4 cells/well and incubated at different TBL concentrations  in DMEM for 24 h. After a 24 h exposure to the lectin, the solution was removed, and DMEM supplemented with FBS was added. Incubation was continued for 24 and 48 h and cell proliferation was measured at each period by detection of the incorporation of tritium-labeled thymidine (3 µCi/well) during a 30 min incubation period. To stop the uptake of radioactivity, the medium was removed and the cells were washed twice with phosphate buffer pH 7.6 containing 150 mM NaCl; then, we added 500 µL of SDS 0.1% containing 10 mM ethylenediaminetetraacetic acid  to each well. After 30 min at room temperature, the lysate was added with 500 µL of 10% cold trichloroacetic acid (TCA) and the precipitate was collected on a nitrocellulose paper, washed three times with TCA 5%, dried, and counted in a liquid scintillation counter . All determinations were performed in triplicate.The cells were cultured in 24-well flat-bottomed plates at a concentration of 5 × 10p < 0.05), followed by the Tukey post hoc test for multiple comparisons. IC50 values were calculated from linear regression analysis.Experimental results are expressed as mean ± SEM. All measurements were replicated three times. The data were analyzed by an analysis of variance (Purification of lectins from tepary beans using the fetuin affinity column was a good choice for purification, however, purification could be improved by employing another affinity matrix with higher affinity for tepary lectins, or some combination with other chromatographic techniques, to improve lectin purification and separate the lectin isoforms. We observed a marked effect of the lectins on the inhibition of cell proliferation in the SW480 cell line, with the effect not being so marked in the remaining three cell lines studied.The post-incubation results obtained 24 and 48 h after the lectin was eliminated from the cell culture showed that by the MTT technique, the four cell lines studied possessed some recovery capacity, displayed by increasing cell proliferation. On the other hand, post-incubation proliferation results obtained by tritium-labeled thymidine after 24 and 48 h prior to the lectin solution being eliminated from the cell culture showed that SW480 cells were not able to recover their proliferation activity: the C33-A and MCF-7 cell lines did not present recovery at low lectin concentrations, while at high concentrations, both cell lines presented recovery. In the case of SKNSH cells, these demonstrated proliferation activity after elimination of the lectin. Recovery in cell proliferation 24 and 48 h after the lectin was eliminated, as observed in SKNSH, C33-A, and MCF-7 cell lines, was highest at the 100-µg/mL concentration.


Aeschynomene fascicularis (Fabaceae) has been used in Mayan traditional medicine in the Yucatan peninsula. However, the compounds present in the plant responsible for its curative properties have not yet been investigated. Aeschynomene fascicularis root bark was extracted with 100% methanol to obtain a crude extract. The methanol extract was partitioned successively with solvents with increasing polarity to obtain the corresponding hexane (Hx), dichloromethane (DCM) and ethyl acetate fractions (EtOAc), as well as a residual water-alcoholic fraction. These fractions were tested for their cytotoxic activities using an MTT assay against Hep-2 cancer cell lines. The Hx fraction led to the isolation of spinochalcone C (1), spinochalcone A (2), isocordoin (3) and secundiflorol G (4). Their structures were identified based on spectroscopic evidence and chemical properties. All compounds were subjected to cytotoxicity and antiproliferative assays against a panel of seven cell lines, including one normal-type cell line. Spinochalcone A (2) exhibited cytotoxic activity against DU-145 cell line and antiproliferative activity against the KB cell line. Secundiflorol G (4) showed strong cytotoxic activity towards KB and Hep-2 cell lines. In addition, isocordoin (3) showed moderate activity on KB, Hep-2 and DU-145 cell lines. The active Compounds 2, 3 and 4 are potential therapeutic entities against cancer.The plant  The flora of the Yucatan peninsula is rich in vascular plants, including 2600–3000 species . Some ofAeschynomene fascicularis root bark has a pronounced cytotoxic activity on cancer cell lines.In the Yucatan, medicinal plants have been used in urban and rural communities as a common practice for the control of many types of diseases, including cancer. Moreover, they are used in the treatment of conditions consistent with the symptoms of cancer: abscesses, calluses, corns, hard lumps, polyps, tumors or warts. In a preliminary screening for cytotoxic constituents from plants used in Mayan traditional medicine to treat cancer-like symptoms, 21 species were collected, and the crude methanol extracts of various plant parts were subjected to a cytotoxicity assay against a panel of cancer cell lines . The resAeschynomene fascicularis Cham. and Schltdl. (Fabaceae) is a shrub extensively distributed from Mexico to Colombia. This plant is commonly known as kabal pich by the ancient inhabitants of the Yucatan Peninsula, and it has been used in Mayan traditional medicine to treat superficial tumors [Aeschynomene genus have been done [A. fascicularis [A. fascicularis.l tumors . Few cheeen done . Recentlicularis . TherefoA. fascicularis.Continuing our effort to search for novel anti-cancer agents from Mayan medicinal plants of the Yucatan peninsula, we performed a guided fractionation of the root bark extract to isolate and identify the cytotoxic compound(s) from this plant. Thus, the aim of this study was to isolate and identify the active cytotoxic compounds from A. fascicularis root bark was subjected to chromatographic purification, resulting in the isolation of four compounds. The chemical structures of all compounds were determined by the analysis of their NMR spectra  and mass spectra and by comparison of these data with those published in the literature.The active hexane fraction obtained from the methanol extract of 1: yellow oil. UV λmax (CHCl3) nm: 239, 308; FTIR (film) νmax cm−1: 3385, 1649, 1600, 1450, 1332, 1122; EI-MS 70 eV, m/z (relative intensity %): 374 , 359 (100), 303 (25), 255 (20), 131 (15), 103 (17). For 1H-NMR  and 13C-NMR  data, see 1 showed a molecular mass of 374 a.m.u. Its FTIR spectrum showed bands at 3385 and 1649 cm−1, which in conjunction with the 1H-NMR signal of a chelated proton at δ 13.74 , signals due to two trans-olefinic protons at δ 7.57  and δ 7.86 , and an α,β-unsaturated carbonyl signal at δ 191.9 in the 13C-NMR spectrum, confirmed the structure of a chalcone. Additionally, the 1H-NMR spectrum showed signals in the aromatic region belonging to a singlet at δ 7.40 and two sets of multiplets at δ 7.43 (3H) and δ 7.65 (2H) characteristic of protons in penta-substituted and mono-substituted rings, respectively. The presence of two cis-olefinic protons at δ 5.59  and δ 6.32  along with two signals for two methyls (δ 25.9 and 18.0) and a signal at δ 77.8 due to a quaternary carbon in the 13C-NMR spectrum are consistent with the occurrence of a 2,2-dimethyl-pyran ring. Furthermore, signals at δ 1.46 (6H), δ 3.36  and a multiplet at δ 5.26 (1H) indicate the presence of an isoprenyl group. The position of all protons and carbons was established by combining the two-dimensional HSQC and HMBC experiments. The assigned structure (Tephrosia spinosa [Compound tructure  correspo2: yellow amorphous solid. UV λmax (CHCl3) nm: 238, 322; FTIR (film) νmax cm−1: 3390, 1634, 1608, 1567, 1362, 1147; 1H-  and 13C-  NMR: see m/z (relative intensity %): 376 , 333 (40), 305 (45), 265 (30), 201 (40), 161 (60), 131 (40), 103 (35). Compound 2 has a molecular mass of 376 a.m.u. A set of signals in the 1H, 13C-NMR and FTIR spectra indicated the presence of a chalcone skeleton similar to Compound 1. However, 2 presents two isoprenyl groups confirmed by the presence of methyl signals at δ 1.76, 1.79, 1.80 and 1.84 (3H each), two signals at δ 3.33 and 3.46  and a multiplet at δ 5.30 (2H). The location of all protons and carbons was established by combining the two-dimensional HSQC and HMBC experiments. The assigned structure (Tephrosia spinosa [1 and 2 are reported here for the first time.Compound tructure  correspo3: yellow amorphous solid. FTIR (film) νmax cm−1: 3231, 1629, 1613, 1480, 1444, 1362, 1234; EI-MS 70 eV, m/z (relative intensity %): 308 , 265 (90), 231 (12), 149 (100), 131 (37), 103 (40), 77 (20). 1H-NMR : δ 1.78 , 1.85 , 3.49 , 5.31 , 6.44 , 7.43 , 7.60 , 7.74 , 7.89 , 13.76 . 13C-NMR : δ 18.2 (3′′-Me), 22.0 (C-1′′), 26.0 (3′′-Me), 108.1 (C-5′), 114.3 (C-1′), 114.4 (C-3′), 120.8 (C-α), 121.3 (C-2′′), 128.7 (C-2/C-6), 129.2 (C-3/C-5), 129.6 (C-6′), 130.8 (C-4), 135.1 (C-1) 136.1 (C-3′′), 144.5 (C-β), 162.0 (C-4′), 164.2 (C-2′), 192.3 (C=O). Compound 3 gave identical spectral features with those described for isocordoin, which has previously been isolated from Cordoa piaca [Lonchocarpus xuul [Compound 4: brown-red amorphous solid. c 0.02, CHCl3); UV λmax (CHCl3) nm: 238, 283; FTIR (film) νmax cm−1: 3385, 1618, 1598, 1506, 1454, 1275, 1157, 1116. In the 1H-NMR spectrum, a set of mutually-coupled five protons , 3.02 , 3.52 , 4.09  and 4.36 ) suggested the presence of an isoflavan skeleton. The 1H-NMR spectrum further exhibited the presence of a methoxyl at δ 3.74 (s), and signals of a typical α,α-dimethyl-allyl group were observed , 1.51 , 4.97 , 5.02 , and 6.13 . An isolated aromatic proton at δ 6.56  and three protons in an ABC system , 6.38  and 6.92 ) indicate that an aromatic ring is penta-substituted, and another ring is tri-substituted. Characterization of Compound 4 was made by comparing the 1H-NMR spectrum to that reported for secundiflorol G, a flavan previously described as isolated from Sophora secundiflora [Aeschynomene and, particularly, on the contribution to the phytochemistry of the species A. fascicularis.Compound A. fascicularis. Our guided fractionation study suggests that flavan 4 and chalcones 2 and 3 are the main compounds responsible for the observed biological activity in the original extract and the active hexane fraction. The results of the bioassays towards six human cancer cell lines and their selectivity indices (SI) compared with a normal cell line are presented in This study was conducted towards the isolation of active compounds from the root bark of 1 did not show any activity on tested cell lines to the maximum concentration (100 µM).Compound 2 showed good cytotoxic activity against the DU-145 and PC-3 cell line; moreover, this compound showed high selectivity compared with a normal cell line. Interestingly, only this compound showed good antiproliferative activity toward the KB cell line  towards all tested cell lines. Cytotoxic activity of isocordoin (3) on ovarian, prostate and leukemia human cancer cell lines has been documented [Leishmania mexicana and Trypanosoma cruzi [Compound cumented . Further4) showed potent activity and moderate selectivity towards oral-laryngeal cancer cell lines Hep-2 and KB. Previously, the cytotoxic activity of various isoflavans had been reported against mouth cancer cell lines [4) towards KB and Hep-2 lines. Therefore, we propose the potential use of secundiflorol G in oral-laryngeal cancer chemotherapy.Secundiflorol G  is the most abundant compound present, as much as 30% of the hexane fraction of A. fascicularis root bark (measured by GC-MS), and this could be exploited to develop 2 as a potential marker molecule (together with 3 and 4) to standardize the hexane fraction of A. fascicularis as a phytomedicine. In addition to this, it is worth highlighting the antiproliferative effect that 2 presented against the KB cell line, providing an added value to the development of A. fascicularis as a phytomedicine for the treatment of oral-laryngeal cancer. At present, cancer therapies are using multiple cytotoxic drugs acting synergistically, in order to enhance the curative potency of all substances together. This emphasizes the effectiveness of a phytomedicine prepared from the hexane fraction of A. fascicularis.Finally, spinochalcone A . The FTIR spectra were measured with a Nicolet Protégé spectrophotometer . NMR spectra  were acquired on a Bruker Avance  400 Ultra Shield spectrometer and TMS was used as an internal standard. Low-resolution mass spectra were taken on an Agilent Technologies  6890N gas chromatograph coupled to a 5975B mass spectrometer (GC-MS). Column chromatography was carried out on silica gel 60 (70–230 mesh) from Merck  and Sephadex LH-20 from Sigma . TLC was performed on precoated silica gel 60 F254 (0.25 mm) (Merck). Spots on TLC were visualized under UV light and by spraying phosphomolybdic acid reagent, followed by heating. The optical density of the developed plates with the 3--2,5-diphenyl tetrazolium bromide  and sulforhodamine B  was read in a ThermoSpectronic spectrophotometer from Bio-Rad .UV spectra were taken in CHA. fascicularis was collected in May 2009 from Yaxcabá, Yucatan  (Mexico). The plant material was identified and authenticated by a taxonomist from the Natural Resources Unit of the Centro de Investigación Científica de Yucatán (CICY). Specimens under the voucher number P. Simá 2997 were deposited at CICY’s U Najil Tikin Xiw herbarium.Root bark of 2O (1:3) and extracted using solvents of increasing polarity (3 × 1000 mL): hexane, CH2Cl2 and EtOAc. The solvents were removed using a rotary evaporator (40 °C) to obtain the hexane, dichloromethane, ethyl acetate and aqueous fractions. Only the hexane fraction showed cytotoxic activity against the Hep-2 cancer cell line (CC50 = 18 µg/mL).Dried powdered root bark (594 g) was extracted by maceration at room temperature with MeOH (3 × 500 mL) for 24 h. The supernatants were filtered and evaporated under vacuum by means of a rotary evaporator to obtain a dried extract. The MeOH extract (84 g) was suspended in 500 mL MeOH/H1, 60 mg) and spinochalcone A , respectively. Fraction A4 was explored using a Sephadex LH-20 CC to obtained four fractions (B1–B4). Fraction B2 was eluted with MeOH (100%) and was further subjected to CC silica gel using the isocratic system Hx/An (8:2) to obtain Compound 3 (5 mg). Finally, from Fraction A6, Compound 4 (20 mg) was isolated by means of a CC silica gel using the solvent system CH2Cl2/MeOH (9:1).The active hexane fraction (6.4 g) was submitted to a vacuum liquid chromatography (VLC) on silica gel using mixtures of hexane (Hx) and acetone (An) of ascending polarity to yield 11 fractions, which were pooled into six final fractions (A1–A6), according to their similarity on TLC. Fractions A2 and A3 were each purified by silica gel column chromatography (CC) under isocratic elution with Hx/An (9:1) to yield spinochalcone C (v/v), 100 U/mL penicillin G, and 100 mg/mL streptomycin at 37 °C in an atmosphere of 5% CO2 (95% humidity). At 70%–80% confluence, cells were detached from the cultured flask by treatment with 0.05% trypsin-EDTA (Gibco), and a suspension of 5 × 104 cells/mL of viable cells was seeded in a 96-well microtiter plate  and incubated.Three types of cancer cell lines obtained from the American Type Culture Collection (ATCC) were cultured: two oral-laryngeal cancer cell lines, nasopharynx carcinoma  and laryngeal carcinoma ; two cervix-uterine cancer cell lines, cervix adenocarcinoma  and cervix squamous carcinoma ; and two prostate cancer cell lines, prostate carcinoma  and prostate adenocarcinoma ; along with one normal cell line . The cells were cultured in sterile Costar T75 flasks containing DMEM , supplemented with fetal bovine serum . The amount of MTT-formazan is directly proportional to the number of living cells and was determined by measuring the optical density (OD) at 590 nm using a bioassay reader (Bio-Rad). All determinations were performed in triplicate.Cytotoxicity was assessed by the MTT assay as previously described by Rahman . When ceThe growth inhibition of cancer cells was evaluated by the sulforhodamine B method . When cecontrol − ODsample/ODcontrol) × 100. Results are expressed as the concentration of compound that killed 50% of the cells (CC50) and that reduced cell growth by 50% (IC50), respectively, and they were calculated using the software GraphPad Prism 4. In addition, the level of cytotoxicity on normal cells was evaluated by determining the selectivity index (SI) of each compound, which is calculated as the ratio of cytotoxicity or reduced cell growth on normal cells to cancer cells  [The cytotoxic and antiproliferative activities of tested compounds were calculated as the percentage of cells killed or the percentage of inhibition of cell proliferation, respectively, using the equation: growth inhibition (%) = (ODr cells) . It is gr cells) .2 and 3 and flavan 4 are the main compounds responsible for the observed biological activity in the original extract of A. fascicularis and are potential candidates for drug development based on their effective cytotoxic and antiproliferative activities.The present study suggests that chalcones


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