Down-regulation of autophagy-associated protein increased acquired radio-resistance bladder cancer cells sensitivity to taxol
Down-regulation of autophagy-associated protein increased acquired radio-resistance bladder cancer cells sensitivity to taxol
复制标题
自噬相关蛋白的下调增加了获得性放射抗性膀胱癌细胞对紫杉醇的敏感性
DOI：
10.1080/09553002.2021.1872812
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发表时间：
2021-01-16
影响因子：
2.6
通讯作者：
Kong, Zhaolu
中科院分区：
文献类型：
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作者：
Ma, Xiangli;Mao, Guangmin;Kong, Zhaolu
背景放射治疗作为一种保膀胱的治疗方法，在膀胱癌（BCa）的治疗中得到了广泛的应用，并在过去的几十年里取得了很大的进展。但部分BCa患者的RT反应性较低，RT后局部复发率可达50%。获得性放射耐药（Acquired radio-resistance， ARR）是放疗失败的重要原因之一。然而，这些ARR细胞对化疗缺乏敏感性，导致肿瘤复发和转移。目的建立ARR表型BCa细胞模型，探讨ARR可能的分子机制，寻找克服ARR的有效靶分子。材料与方法将56,637个细胞经2 Gy γ射线照射30次，存活细胞命名为5637R。采用集落形成法和MTT法分别评价细胞对电离辐射（IR）和抗肿瘤药物的敏感性。用transwell法测定细胞的迁移和侵袭能力。采用实时荧光定量聚合酶链反应（RT-qPCR）和免疫印迹（WB）技术比较5637和5637R细胞基因和蛋白表达的差异。分子抑制剂和小干扰RNA （siRNA）系统分别用于降低靶蛋白的表达。结果分次辐照（FI）后存活的BCa细胞对IR和化疗药物均表现出耐受性。这些ARR细胞（5637R）具有较高的迁移和侵袭能力，并伴有上皮间充质转化（EMT）相关转录因子（ZEB1/Snail/Twist）的表达增加。此外，5637R细胞表现出增强的癌症干细胞（CSC）样特征，激活KMT1A-GATA3-STAT3回路，这是一种新报道的人类膀胱癌干细胞（BCSC）自我更新途径。结合Kaplan-Meier分析，我们推测GATA3/MMP9/STAT3可能是预测BCa不良预后的有效分子面板。为了增强耐药细胞对辐射的敏感性，我们引入了ERK抑制剂（FR 180204）和STAT3抑制剂（sgi -201）。但两者均不能增强ARR细胞对IR的应答。另一方面，分别实施sirna抑制BCa细胞中内源性Beclin1和Atg5这两个重要自噬相关基因的表达，显著增加了紫杉醇暴露后5637R细胞的死亡。同样，经典的自噬抑制剂氯喹（chloroquine， CQ）仅对5637R细胞增强紫杉醇的细胞毒性。结论长期FI治疗是建立arr表型BCa细胞模型的有效方法，通过丰富BCSCs，增强细胞迁移和侵袭能力。抑制自噬相关蛋白的表达和使用自噬抑制剂均可增加ARR细胞对紫杉醇的敏感性，提示自噬可能在ARR细胞的化学耐受性中起重要作用。
Abstract Background As a bladder-preserving therapy, radiation therapy (RT) has been widely used in the treatment of bladder cancer (BCa) and made great progress in the past few decades. However, some BCa patients have low RT responsiveness and local recurrence rate after RT could reach 50%. Acquired radio-resistance (ARR) is one of the important reasons for the failure of RT. Unfortunately, these ARR cells also lack sensitivity to chemotherapy and cause tumor recurrence and metastasis. Purpose To build ARR-phenotype BCa cell model, discuss the possible molecular mechanism of ARR and find effective target molecules to overcome ARR. Materials and methods Five thousand six hundred and thirty-seven cells were subjected 30 times to 2 Gy of γ-rays and the surviving cells were called 5637R. Colony formation and MTT assay were applied to evaluate cells sensitivity to ionizing radiation (IR) and anti-neoplastic agents, respectively. Cells abilities of migration and invasion were determined using transwell method. Quantitative real-time polymerase chain reaction (RT-qPCR) and western blot (WB) were respectively utilized to compare the difference of gene and protein expression between 5637 and 5637R cells. Molecule inhibitors and small interfering RNA (siRNA) systems were employed to decrease the expression of target proteins, respectively. Results BCa cells survived from fractionated irradiation (FI) exhibited tolerance to both IR and chemotherapy drugs. These ARR cells (5637R) had elevated migration and invasion abilities, accompanied by increased expression of epithelial mesenchymal transition (EMT)-related transcription factors (ZEB1/Snail/Twist). Moreover, 5637R cells showed enhanced cancer stem cell (CSC)-like characteristics with activated KMT1A–GATA3–STAT3 circuit, a newly reported self-renewal pathway of human bladder cancer stem cell (BCSC). Combined with Kaplan–Meier’s analysis, we speculated that GATA3/MMP9/STAT3 could be an effective molecular panel predicting poor prognosis of BCa. In order to enhance the sensitivity of resistant cells to radiation, we introduced ERK inhibitor (FR 180204) and STAT3 inhibitor (S3I-201). However, both of them could not enhance ARR cells response to IR. On the other hand, siRNAs were respectively implemented to inhibit the expression of endogenous Beclin1 and Atg5, two important autophagy-related genes, in BCa cells, which significantly increased 5637R cells death upon taxol exposing. Similarly, chloroquine (CQ), a classic autophagy inhibitor, enhanced the cytotoxicity of taxol only on 5637R cells. Conclusions Long-term FI treatment is an effective method to establish the ARR-phenotype BCa cell model, by enriching BCSCs and enhancing cells migration and invasion. Both inhibiting the expression of autophagy-related proteins and using autophagy inhibitor can increase the sensitivity of ARR cells to taxol, suggesting that autophagy may play an important role in ARR cells chemical tolerance.