Amendment Of Chemoresistence In Hypoxic Ovarian Cancer By Noscapine Through Inhibition Of HIF-1α

Amendment of chemoresistence in hypoxic ovarian cancer by noscapine through inhibition of HIF-1αMany studies have shown that hypoxic microenvironment are frequently found in solid tumors and the transcriptional factor HIF-1 is the key regulator in that condition. HIF-1 is closely related with tumors' malignant behaviours through regulation of more than 60 target genes. The subunit HIF-1αis highly expressed in more than 70% solid tumors and is closely related with vascular formation, tumor progression and chemoresistance. Ovarian cancer is one of the most common female genital malignancy and it is hard to be diagnosed in the early stage. Almost all ovarian cancers relapsed and became multi-drug-resistant to the traditional chemotherapeutics such as cisplatin and paclitaxel. Although cytoreductive surgery plus radiotherapy and chemotherapy based on paclitaxel and cisplatin have prolonged the patients' survival, the five year survival rate of the patients maintained at about 30%. It is reported that HIF-1αis up-regulated in ovarian cancer and we suppose it an important regulator in ovarian cancer chemoresistance. Human ovarian cancer cell line C13K as the model, we detected the impact of the small molecule compound noscapine on C13K chemosensitivity in both normoxic and hypoxic conditions, investigated the regulatory mechanism of HIF-1αby noscapine in levels of mRNA and protein, and studied the transcriptional activity of HIF-1 through inspections of HIF-1αlocation, fluorescent reporter plasmids' activity and target genes' mRNA expression. This study provides an experimental basis for the possibility taking noscapine in adjuvant chemotherapy of ovarian cancer with the target gene HIF-1α.Part 1 The effects of hypoxia on chemosensitivity of human ovarian cancer cellsObjective To examine the effects of hypoxia on sensitivity of ovarian cancer cell line C13K to cisplatin. Methods C13K cells were treated with CoCl₂, then typan blue exclusion test was used to detect cell survival and western blotting was used to detect expression of HIF-1α. After "effective hypoxia" was built, C13K cells were established with gradient concentrations of cisplatin under both hypoxia and normoxia, then MTT assay was used to detect proliferation of each group. Results HIF-1αwas significantly up-regulated after exposure of CoCl₂ without obvious cytotoxicity. Cisplatin inhibited C13K proliferation in a time and concentration dependent manner under both hypoxic and normoxic conditions (P<0.05). However, cells in hypoxia group showed less sensitivity than those in normoxia to cisplatin (P<0.05). Conclusions We established "effective hypoxia" model with C13K successfully. Hypoxia C13K cells showed less sensitivity to cisplatin than those in normoxia.Part 2 The effects of noscapine on chemosensitivity of hypoxic ovarian cancer cellsObjective To explore the impacts of noscapine on chemosensitivity of ovarian cancer cell line C13K. Methods C13K cells were divided into Normoxia Group (control, noscapine, cisplatin, noscapine & cisplatin group) and Hypoxia Group (control, noscapine, cisplatin, noscapine & cisplatin group). Trypan blue exclusion test was used to detect cell survival and flow cytometer was used to measure apoptosis rate. Results In Group Hypoxia, the percentage of dead cells and cell apoptosis rate in group noscapine & cisplatin was significantly higher than that in group noscapine(P<0.05). However, this effect was not obvious in Group Normoxia. Conclusion Noscapine reverses C13K chemoresistance to cisplatin induced by hypoxia.Part 3 The inhibition effects of noscapine on HIF-1αObjective To investigate the impacts of noscapine on HIF-1αand the transcriptional activity of HIF-1 in C13K. Methods C13K cells were divided into Group Normoxia (control, noscapine) and Group Hypoxia (control, noscapine). Then, RT-PCR was used to detect mRNA levels of HIF-1α, MDR1 and Bax, western blotting was used to detect HIF-1αprotein, immunofluorescence detection was used to detect location of HIF-1αin C13K cells. Hypoxia C13K cells were divided into Group Control and Group Noscapine and after incubation with CHX for gradient durations, western blotting was used to detect protein levels and half-life periods of HIF-1α. Hypoxic C13K cells were divided into 4 groups: group control, group noscapine, group MG132, group noscapine & MG132, then western blotting was used to detect HIF-1αprotein levels. HIF-1αfluorescent reporter plasmid pGL3-VEGF-HRE was transfected into C13K cells and treated with noscapine in normoxia or hypoxia, then luciferase activity in each group was inspected. Results Noscapine had no significant effect on HIF-1αmRNA but the half-life period of HIF-1αand MG132 blocked the inhibitory effect of noscapine on HIF-1αprotein. Cell responses induced by CoCl₂ such as HIF-1αnuclear translocation, enhanced luciferase activity, elevated MDR1 mRNA level are all inhibited by noscapine. Noscapine elevated Bax mRNA level in hypoxia. Conclusion Noscapine inhibites HIF-1αprotein expression by promoting its proteasome degradation pathway. Noscapine inhibites transcriptional activity of HIF-1.