Abstract：
BACKGROUND: Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown.
METHODS: Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry.
RESULTS: Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells.
CONCLUSIONS: Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.
METHODS: Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry.
RESULTS: Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells.
CONCLUSIONS: Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.
摘要：
背景：硼替佐米（BTZ）是一种强大的蛋白酶体抑制剂，已被批准用于血液系统恶性肿瘤的治疗。已针对不同类型的实体瘤评估了其有效性。由于耐药性，BTZ在大多数实体瘤中无效，包括胆管癌,这与蛋白酶体反弹效应有关。然而,蛋白酶体抑制剂诱导蛋白酶体反弹效应的机制在很大程度上是未知的。
方法：用BTZ处理胆管癌细胞，顺铂,或两者的组合。使用定量实时聚合酶链反应（qPCR）确定Nfe2l1和蛋白酶体亚基基因（PSMA1，PSMB7，PSMD1，PSMD11，PSMD14和PSME4）的mRNA水平。使用蛋白质印迹和蛋白酶体活性测定评估核因子-红细胞2相关因子1（Nfe2l1）的蛋白质水平和蛋白酶体酶活性，分别。进行转录组测序以筛选调节Nfe2l1表达的潜在转录因子。锌指E盒结合同源异型盒1(ZEB1)对Nfe2l1和蛋白酶体亚基基因表达的影响,以及蛋白酶体酶活性，在用BTZ处理之前用siRNA敲除ZEB1表达后进行评估。使用双荧光素酶报告基因和染色质免疫沉淀测定法检测ZEB1在Nfe2l1启动子上的转录活性。使用细胞计数试剂盒-8(CCK-8)测定测量细胞活力,并使用蛋白质印迹和流式细胞术评估细胞凋亡。
结果：顺铂治疗BTZ处理的人胆管癌细胞系（RBE）抑制蛋白酶体亚基基因表达（蛋白酶体反弹）和蛋白酶体酶活性。这种作用是通过降低Nfe2lmRNA和蛋白质的水平来实现的。我们的研究利用转录组测序将ZEB1鉴定为Nfe2l1的上游转录因子，这通过双荧光素酶报告基因和染色质免疫沉淀测定得到证实。值得注意的是,在基础和BTZ诱导的条件下，使用siRNA（si-ZEB1）的ZEB1敲低抑制蛋白酶体亚基基因的表达，导致蛋白酶体酶活性的抑制。此外，与BTZ联合治疗，顺铂,和si-ZEB1显著降低RBE细胞的活力。
结论：我们的研究揭示了一种新的机制，顺铂通过抑制胆管癌中ZEB1/Nfe2l1轴破坏BTZ诱导的蛋白酶体反弹效应。这一发现为开发基于蛋白酶体抑制剂的胆管癌和其他肿瘤的临床治疗策略提供了理论基础。
方法：用BTZ处理胆管癌细胞，顺铂,或两者的组合。使用定量实时聚合酶链反应（qPCR）确定Nfe2l1和蛋白酶体亚基基因（PSMA1，PSMB7，PSMD1，PSMD11，PSMD14和PSME4）的mRNA水平。使用蛋白质印迹和蛋白酶体活性测定评估核因子-红细胞2相关因子1（Nfe2l1）的蛋白质水平和蛋白酶体酶活性，分别。进行转录组测序以筛选调节Nfe2l1表达的潜在转录因子。锌指E盒结合同源异型盒1(ZEB1)对Nfe2l1和蛋白酶体亚基基因表达的影响,以及蛋白酶体酶活性，在用BTZ处理之前用siRNA敲除ZEB1表达后进行评估。使用双荧光素酶报告基因和染色质免疫沉淀测定法检测ZEB1在Nfe2l1启动子上的转录活性。使用细胞计数试剂盒-8(CCK-8)测定测量细胞活力,并使用蛋白质印迹和流式细胞术评估细胞凋亡。
结果：顺铂治疗BTZ处理的人胆管癌细胞系（RBE）抑制蛋白酶体亚基基因表达（蛋白酶体反弹）和蛋白酶体酶活性。这种作用是通过降低Nfe2lmRNA和蛋白质的水平来实现的。我们的研究利用转录组测序将ZEB1鉴定为Nfe2l1的上游转录因子，这通过双荧光素酶报告基因和染色质免疫沉淀测定得到证实。值得注意的是,在基础和BTZ诱导的条件下，使用siRNA（si-ZEB1）的ZEB1敲低抑制蛋白酶体亚基基因的表达，导致蛋白酶体酶活性的抑制。此外，与BTZ联合治疗，顺铂,和si-ZEB1显著降低RBE细胞的活力。
结论：我们的研究揭示了一种新的机制，顺铂通过抑制胆管癌中ZEB1/Nfe2l1轴破坏BTZ诱导的蛋白酶体反弹效应。这一发现为开发基于蛋白酶体抑制剂的胆管癌和其他肿瘤的临床治疗策略提供了理论基础。
