背景:顺铂(DDP)耐药性仍然是改善卵巢癌(OC)患者临床预后的关键挑战。Gli2过表达可导致OC细胞的DDP耐药,但具体的潜在监管机制仍不清楚。膜转运蛋白编码基因MDR1正调节各种癌症类型的化疗抗性。我们评估了MDR1作为潜在的Gli2下游靶标以及Gli2/MDR1轴在促进OC细胞DDP抗性中的贡献。
方法:为了产生耐药的SKOV3/DDP细胞,SKOV3细胞在连续诱导下生长6个月,其中DDP浓度稳定增加。使用蛋白质印迹检测具有不同DDP敏感性的OC细胞中的Gli2表达。细胞计数试剂盒-8测定用于评估SKOV3,SKOV3/DDP的DDP敏感性,A2780和A2780/DDP细胞和逆转SKOV3/DDP和A2780/DDP细胞中的DDP抗性。使用5-乙炔基-2'-脱氧尿苷(EdU)掺入测定法分析细胞增殖。使用荧光素酶报告基因测定确定Gli2对MDR1的转录调节。最后,异种移植OC肿瘤在裸鼠中产生,然后用腹膜内DDP或磷酸盐缓冲盐水(PBS)注射处理,以研究Gli2是否影响体内OC中的DDP抗性。
结果:DDP耐药的SKOV3/DDP和A2780/DDP细胞与DDP敏感的OC细胞相比,Gli2和MDR1的表达更高。SKOV3/DDP细胞中Gli2敲低显著降低MDR1表达,虽然它增加了DNA损伤,从而使OC细胞对DDP敏感。在SKOV3/DDP和A2780/DDP细胞中用Gli拮抗剂61抑制剂(GANT61)靶向Gli2表达后获得类似结果。在稳定过表达Gli2的细胞中,用梯度浓度的维拉帕米处理,MDR1抑制剂,显著抑制MDR1表达。我们的发现表明,MDR1表达的下调可能会逆转OC细胞对DDP的抗性。此外,双荧光素酶报告基因测定证实,MDR1是Gli2的直接下游靶标,Gli2正调控MDR1的表达。最后,裸鼠皮下异种移植证明Gli2在调节OC耐药中起关键作用。
结论:我们确定了Hedgehog-Gli信号通过调节MDR1表达调节OC化学抗性的机制。因此,Gli2和MDR1是化疗耐药型OC患者的潜在生物标志物和治疗靶点。
BACKGROUND: Cisplatin (DDP) resistance remains a key challenge in improving the clinical outcome of patients with ovarian cancer (OC). Gli2 overexpression can lead to DDP resistance in OC cells, but the specific underlying regulatory mechanism remains unclear. The membrane transporter encoding gene
MDR1 positively regulates chemotherapy resistance in various cancer types. We evaluated
MDR1 as a potential Gli2 downstream target and the contribution of the Gli2/
MDR1 axis in promoting DDP resistance in OC cells.
METHODS: To generate drug-resistant SKOV3/DDP cells, SKOV3 cells were grown for six months under continuous induction wherein the DDP concentration was steadily increased. Gli2 expression in OC cells with varying DDP sensitivities was detected using western blot. Cell counting kit-8 assays were used to assess the DDP sensitivity of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells and reversal of DDP resistance in SKOV3/DDP and A2780/DDP cells. Cell proliferation was analyzed using 5-ethynyl-2\'-deoxyuridine (EdU) incorporation assays. The transcriptional regulation of
MDR1 by Gli2 was determined using luciferase reporter assays. Finally, xenograft OC tumors were generated in nude mice, which were then treated with intraperitoneal DDP or phosphate-buffered saline (PBS) injections to investigate if Gli2 affected DDP resistance in OC in vivo.
RESULTS: DDP-resistant SKOV3/DDP and A2780/DDP cells showed higher expression of Gli2 and MDR1 as compared with that in DDP-sensitive OC cells. Gli2 knockdown in SKOV3/DDP cells significantly reduced MDR1 expression, whereas it increased DNA damage, thereby sensitizing OC cells to DDP. Similar results were obtained after targeting Gli2 expression with the Gli-antagonist 61 inhibitor (GANT61) in SKOV3/DDP and A2780/DDP cells. In cells stably overexpressing Gli2, treatment with gradient concentrations of verapamil, an MDR1 inhibitor, significantly inhibited
MDR1 expression. Our findings indicate that downregulation of MDR1 expression may reverse OC cell resistance to DDP. Moreover, dual-luciferase reporter gene assays confirmed that MDR1 is a direct downstream target of Gli2, with Gli2 positively regulating
MDR1 expression. Finally, subcutaneous xenotransplantation in nude mice demonstrated that Gli2 plays a key role in regulating OC drug resistance.
CONCLUSIONS: We identified a mechanism by which Hedgehog-Gli signaling regulates OC chemoresistance by modulating MDR1 expression. Hence, Gli2 and MDR1 are potential biomarkers and therapeutic targets in patients with chemoresistant OC.