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Phosphoproteomic analysis of gossypol-induced apoptosis in ovarian cancer cell line, HOC1a.

ABSTRACT: Ovarian cancer is a major cause for death of gynecological cancer patients. The efficacy of traditional surgery and chemotherapy is rather compromised and platinum-resistant cancer recurs. Finding new therapeutic targets is urgently needed to increase the survival rate and to improve life quality of patients with ovarian cancer. In the present work, phosphoproteomic analysis was carried out on untreated and gossypol-treated ovarian cancer cell line, HOC1a. We identified approximately 9750 phosphopeptides from 3030 phosphoproteins, which are involved in diverse cellular processes including cytoskeletal organization, RNA and nucleotide binding, and cell cycle regulation. Upon gossypol treatment, changes in phosphorylation of twenty-nine proteins including YAP1 and AKAP12 were characterized. Western blotting and qPCR analysis were used to determine expression levels of proteins in YAP1-related Hippo pathway showing that gossypol induced upregulation of LATS1, which phosphorylates YAP1 at Ser 61. Furthermore, our data showed that gossypol targets the actin cytoskeletal organization through mediating phosphorylation states of actin-binding proteins. Taken together, our data provide valuable information to understand effects of gossypol on protein phosphorylation and apoptosis of ovarian cancer cells.

SUBMITTER: Jin L

PROVIDER: S-EPMC4144078 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Steroidal saponins are a group of compounds with complex structures and biological activities. They have anti-inflammatory, antimicrobial, fungicidal, and antitumor properties. Yamogenin is one of the spirostane saponins and occurs in Trigonella foenum-graecum, Asparagus officinalis, and Dioscorea collettii. It is a stereoisomer of diosgenin-a well-known compound whose activity and mechanisms of action in cancer cells are determined. However, the antitumor effect of yamogenin is still little known, and the mechanism of action has not been determined. In this study, we evaluated the effect of yamogenin on human ovarian cancer SKOV-3 cells in vitro by determining the cellular factors that trigger cell death. The viability of the cells was assessed with a Real-Time xCELLigence system and the cell cycle arrest with flow cytometry. The activity of initiator and executioner caspases (-8, -9, and -3/7) was estimated with luminometry and flow cytometry, respectively. The mitochondrial membrane depolarization, the level of oxidative stress, and DNA damage in the yamogenin-treated cells were also evaluated by flow cytometry. Genes expression analysis at the mRNA level was conducted with Real-Time PCR. Bid activation and chromatin condensation were estimated with fluorescent microscopy. The obtained results indicate that yamogenin has cytotoxic activity in SKOV-3 cells with an IC50 value of 23.90 ± 1.48 µg/mL and strongly inhibits the cell cycle in the sub-G1 phase. The compound also triggers cell death with a significant decrease in mitochondrial membrane potential, an increase in the level of oxidative stress (over two times higher in comparison to the control), and activation of caspase-8, -9, -3/7, as well as Bid. The results of genes expression indicate that the Tumor Necrosis Factor (TNF) Receptor Superfamily Members (TNF, TNFRSF10, TNFRSF10B, TNFRSF1B, and TNFRSF25), Fas Associated via Death Domain (FADD), and Death Effector Domain Containing 2 (DEDD2) were significantly upregulated and their relative expression was at least two times higher than in the control. Our work shows that yamogenin induces apoptosis in ovarian cancer cells, and both the extrinsic and mitochondrial-intrinsic pathways are involved in this process.

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Phosphoproteomic analysis of gossypol-induced apoptosis in ovarian cancer cell line, HOC1a.

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