The Study of Low-dose Olaparib Inhibiting Ovarian Tumor Growth Based on the Mechanism of Cellular Senescence

概要

Backgroud: Ovarian cancer is one of the most serious female cancers. According to the data collected by The Global Cancer Observatory, there are millions of new ovarian cancer cases and death cases every year. The incidence rate and mortality rates is 9.3 and 3.1 in Japan, 5.3 and 3.3 in China per 100,000 people. Due to the mild early symptoms and the lack of effective early diagnosis measures, 75% of patients are already in the advanced stage when discovered. Cytoreductive surgery plus platinum-based chemotherapy will bring a big damage to patients and poor prognosis. High dose chemotherapy may cause many side effects. Olaparib as a novel oral Poly (ADP-ribose) polymerase (PARP) inhibitor have been verified in many clinical trials to have good anti-tumor effects and are well tolerated. It is indicated in maintenance treatment of breast cancer susceptibility gene (BRCA) mutated advanced ovarian cancer or recurrent ovarian cancer. However, roles of olaparib in many aspects remain to be studied.
Objective: 1)The efficiency of olaparib on non-BRCA mutant ovarian cancer cells in short term. 2)The inhibitory effect of olaparib combined with cisplatin on non-BRAC mutant ovarian cancer cells in short term. 3) The effect and mechanism of continuous lowdose olaparib in vivo and in vitro. 4)After adding rosiglitazone, the inhibitory effect of olaparib on ovarian cancer cells and the improvement of side effect caused by cellular senescence.

Methods: Cell counting kit-8 (CCK-8) assay, crystal violet staining and fluorescence activated cell sorter (FACS) methods were used to detect the number and viability of ovarian cancer cells. The dose synergistic effect was analyzed by Chou's Compusyn Software. The ratio of side population (SP) cells, apoptosis and cell cycle were detected by FACS. Xenografted mice (n=6 or 12 per group) were treated with continuous low-dose (10 mg/kg/day) olaparib to study the changes of tumor volume and verify the effect of continuous lowdose olaparib combined with rosiglitazone (10 mg/kg/day). Senescence-associated 6-galactosidase (SA-6-Gal) staining and senescence-associated heterochromatin aggregation (SAHF) were used to determine whether the cells were senescent. Senescence-associated secretory phenotype (SASP) was detected by RT-qPCR, and the main cell cycle signaling pathway was studied by Western bolt. Differences between multiple groups were analyzed by one-way AN OVA (Tukey). The studenfs t-test was adopted in the comparison of two groups. When p< 0.05, the difference was statistically significant.

Results: Through the results of Chapter Two and Three, we found that low-dose(0.25x IC50) olaparib has an inhibitory effect on non-BRAC 1/2 mutated ovarian cancer cells and when combined with cisplatin, it can significantly enhance its proliferation inhibitory effect with a strong synergistic effect (p< 0.05, p< 0.01). In Chapter Four, we have confirmed that ovarian cancer cell growth is inhibited, cell cycle (G0/G1 phase) is blocked, and SA-6-Gal staining and SAHF positive cells significantly increased by the administration of low­ dose (5pM) olaparib. At the same time, 5pM olaparib increased expression levels of P16/P53 and Rb protein in SKOV3 and A2780 ovarian cancer cells, which suggests that olaparib can depend on the P16-Rb /P53-Rb signing pathway to induce the ovarian cancer cell senescence, thereby inhibiting tumor cell proliferation. In Chapter Five, In vivo experiment, the final volume of ovarian tumors was significantly decreased in the olaparib + rosiglitazone group compared with the control group (p<0.05). In vitro experiment, the olaparib (5pM) + rosiglitazone(5pM) group showed a significant down-regulation of cell proliferation compared with the control group (p<0.05). According to the results of RT-PCR, rosiglitazone can down-regulate expression of SASP caused by olaparib-induced cellular senescence. Olaparib combined with rosiglitazone can promote the apoptosis of ovarian cancer cells. And its possible inhibitory mechanism is that the rosiglitazone inactivated olaparib-induced senescence of ovarian cancer cells through the SIRT1/P53 signaling pathway, decrease the SASP and promoted olaparib to inhibit tumor cell proliferation.

Conclusion: 1)low-dose olaparib has an inhibitory effect of on non-BRAC mutant ovarian cancer cells. 2) Olaparib combined with cisplatin can effectively enhance its inhibitory effect with a synergistic dose effect. 3) Continuous lowdose olaparib can induce cellular senescence under P16 or P53 dependent manner in ovarian cancer. 4)After rosiglitazone is added, continuous lowdose olaparib effectively improve its inhibitory effect on ovarian tumor and cells. Rosiglitazone ameliorates cellular senescence and promotes apoptosis in ovarian cancer induced by olaparib.