Supplementary MaterialsSupplementary Information. normal lung fibroblasts (TIG-3) and three cancer cell types, osteosarcoma (U2OS; wild type p53), breast adenocarcinoma (MCF-7; wild type but functionally inactive p53) and HT1080 fibrosarcoma (mutant p53). In comparative cytotoxicity analysis, cells were treated with 5 M of all the compounds for 24C48?h. We found that Phytochemical (PH)-11 (Soyaspogenol-A; Snol-A) was significantly cytotoxic (50C70%) to U2OS, HT1080 and MCF-7 cells; normal human fibroblast cells showed milder (20C30%) in several independent experiments (Fig.?S1). Furthermore, PH-10 (Soyasaponin-I; Snin-I) with similar structure was not toxic to any of the cancer cell types (Fig.?S1). In order to confirm such differential effect of Snol-A and Snin-I, we investigated dose-dependent response using more human cancer cells including osteosarcoma (U2OS; wild type p53 and Saos-2; null p53), ovarian adenocarcinoma (SKOV3; null p53), and breast adenocarcinoma (MDA-MB-231; mutant p53). Captopril disulfide As shown in Fig.?1A,B, whereas Snol-A caused dose-dependent inhibition of cell proliferation in all the four cancer cell types, Snin-I was ineffective. Microscopic observations of U2OS and SKOV-3 cancer cells treated with Snol-A (2C10 M) for 48?h showed stressed phenotype (irregular and flattened cell shapes) and restricted growth as compared to control cells (Fig.?1C) and was further confirmed by long-term clonogenicity assay (Fig.?1D). Snin-I caused no effect in these assays (Figs.?1B,C and S2ACC). Of note, Snol-A treated cancer cells that lacked wild type p53 function (SKOV-3 and Saos-2) also showed considerable dose-dependent cytotoxicity. Open in a separate window Figure 1 Snol-A, but not Snin-I, caused potent cytotoxicity to cancer cells. (A,B) Viability of control, Snol-A (A) and Snin-I (B) treated (48?h) cancer cells (SKOV-3, U2OS, Saos-2 and MDA-MB-231). Snol-A, but not Snin-I, showed dose-dependent toxicity. (C) Phase contrast images of control and treated SKOV-3 and U2OS cells; Snol-A, not Snin-I, treated cells showed stressed morphology marked by flat, irregular and branched phenotypes. (D) Colony-forming assay showing crystal violet stained colonies in control and Snol-A treated U2OS, SKOV-3, Saos-2 and MDA-MB-231 cancer cells. Quantitation of colony forming assay is shown on Rabbit Polyclonal to MCL1 the right. Snin-I structurally owns one hydroxyl group at C-22 and three sugars at C-3, while Snol-A was found to have no sugar chains on the C-3, and possessed two hydroxyl groups at C-21 and C-2238 (Fig.?S3ACC). ADMET predictions on pharmacodynamic activity (distribution, metabolism, excretion and toxicity) revealed better human intestinal absorption (HIA) score for Snol-A than Snin-I (Fig.?S3D,E) suggesting that Snol-A will be better absorbed from the intestinal tract upon oral administration. The score for penetration through the Blood-Brain Barrier (BBB) was also higher for Snol-A than Snin-I (Fig.?S3D,E). In terms of metabolism, both compounds showed similar characteristics as a substrate for CYP450 enzyme (Fig.?S3D,E). Caco-2 permeability, predicts assimilation of drugs into human intestinal39, showed better score for Snol-A than Snin-I Captopril disulfide (Fig.?S3D,E). Toxicity predictions (LD50) revealed40 higher toxicity of Snol-A than Snin-I (Fig.?S3D,E). These predictions matched with our results for several cancer cells lines. Snol-A caused growth arrest that was mediated by upregulation of p21WAF1 In order to investigate the molecular mechanism of Snol-A induced toxicity, we first analyzed the cell cycle profiles in control and treated {sub-toxic (IC30) and moderately toxic doses (IC50) of Snol-A: SKOV-3 (6 M and 10 M), and MDA-MB-231 and Saos-2 (10 M & 20 M) cells. As shown in Fig.?2A, there was an increase in S phase population, suggesting cell cycle arrest, in all the three cell lines in response to Snol-A treatment. Cells treated with higher dose (10C20 M) showed a distinct apoptotic sub-population, clearly evident in SKOV-3. Analyses of cell cycle progression key proteins by immunoblotting revealed decrease in the levels of CDK2, Cyclin A, Cyclin D1 and CDK4 in SKOV-3 cells (Fig.?2B). Of note, p21WAF1 showed a significant increase in Snol-A treated cells (Fig.?2B). Analysis of its transcript levels reaffirmed increased p21WAF1 levels in Snol-A treated SKOV-3 cells (Fig.?2C). To analyze the effect of Snol-A on p21WAF1 transcript, we enrolled p21WAF1 promoter (pWWP)-Luciferase reporter system that affirmed Captopril disulfide a significant increase in p21WAF1 promoter activity in Snol-A treated.