Hedgehog-Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1.
Hedgehog-Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1.
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DOI：
10.3389/fonc.2021.794959
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发表时间：
2021
影响因子：
4.7
通讯作者：
Chen Q
中科院分区：
文献类型：
--
作者：
Wang Q;Wei X;Hu L;Zhuang L;Zhang H;Chen Q
顺铂（DDP）耐药仍然是改善卵巢癌（OC）患者临床结局的关键挑战。Gli 2过表达可导致OC细胞对DDP耐药，但具体的调控机制尚不清楚。膜转运蛋白编码基因MDR 1积极调节各种癌症类型的化疗耐药性。我们评估了MDR 1作为潜在的Gli 2下游靶点以及Gli 2/MDR 1轴在促进OC细胞DDP耐药中的作用。为了产生耐药性SKOV 3/DDP细胞，SKOV 3细胞在连续诱导下生长6个月，其中DDP浓度稳定增加。Western blot检测不同顺铂敏感性OC细胞中Gli 2的表达。采用细胞计数试剂盒-8法检测SKOV 3、SKOV 3/DDP、A2780、A2780/DDP细胞对顺铂的敏感性及SKOV 3/DDP、A2780/DDP细胞对顺铂耐药性的逆转情况。采用5-乙炔基-2 ′-脱氧尿苷（EdU）掺入法分析细胞增殖。使用荧光素酶报告基因测定确定Gli 2对MDR 1的转录调节。最后，在裸鼠中产生异种移植OC肿瘤，然后用腹膜内DDP或磷酸盐缓冲盐水（PBS）注射处理以研究Gli 2是否影响体内OC中的DDP抗性。顺铂耐药的SKOV 3/DDP和A2780/DDP细胞Gli 2和MDR 1的表达均高于顺铂敏感的OC细胞。SKOV 3/DDP细胞中Gli 2的敲低显著降低了MDR 1的表达，但增加了DNA损伤，从而使OC细胞对DDP敏感。在SKOV 3/DDP和A2780/DDP细胞中，用Gli拮抗剂61抑制剂（GANT 61）靶向Gli 2表达后获得了类似的结果。在稳定过表达Gli 2的细胞中，用梯度浓度的维拉帕米（一种MDR 1抑制剂）处理可显著抑制MDR 1表达。我们的研究结果表明，MDR 1表达下调可能会逆转OC细胞对DDP的耐药性。此外，双荧光素酶报告基因测定证实，MDR 1是Gli 2的直接下游靶点，Gli 2正调控MDR 1表达。最后，裸鼠皮下异种移植实验表明，Gli 2在调节OC耐药性中起关键作用。我们确定了Hedgehog-Gli信号通过调节MDR 1表达调节OC化疗耐药性的机制。因此，Gli 2和MDR 1是化疗耐药OC患者的潜在生物标志物和治疗靶点。
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. 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. 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. 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.
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影响因子：
8
作者：
Min Q;Wang Y;Wu Q;Li X;Teng H;Fan J;Cao Y;Fan P;Zhan Q
通讯作者：
Zhan Q
影响因子：
14.5
作者：
Jiang J
通讯作者：
Jiang J
影响因子：
11.2
作者：
Regl, G;Kasper, M;Aberger, F
通讯作者：
Aberger, F
影响因子：
11.2
作者：
Kubo, M;Nakamura, M;Katano, M
通讯作者：
Katano, M
影响因子：
11.1
作者：
Hoppe MM;Jaynes P;Wardyn JD;Upadhyayula SS;Tan TZ;Lie S;Lim DGZ;Pang BNK;Lim S;P S Yeong J;Karnezis A;Chiu DS;Leung S;Huntsman DG;Sedukhina AS;Sato K;Topp MD;Scott CL;Choi H;Patel NR;Brown R;Kaye SB;Pitt JJ;Tan DSP;Jeyasekharan AD
通讯作者：
Jeyasekharan AD