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 radiotherapy responsiveness and local recurrence rate after radiotherapy 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

5637 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 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 couldn’t 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.

中文翻译：

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自噬相关蛋白的下调增加获得性抗癌膀胱癌细胞对紫杉醇的敏感性。

摘要

背景

作为保存膀胱的疗法，放射疗法（RT）已经广泛用于治疗膀胱癌（BCa），并且在过去的几十年中取得了长足的进步。然而，一些BCa患者放疗反应性低，放疗后局部复发率可达到50％。获得性抗射频干扰（ARR）是RT失败的重要原因之一。不幸的是，这些ARR细胞也缺乏对化学疗法的敏感性，并导致肿瘤复发和转移。

目的

建立ARR表型BCa细胞模型，探讨ARR的可能分子机制，寻找克服ARR的有效靶分子。

材料和方法

5637细胞接受了2 Gy的γ射线照射30次，存活的细胞称为5637R。集落形成和MTT测定分别用于评估细胞对电离辐射（IR）和抗肿瘤剂的敏感性。使用transwell方法确定细胞的迁移和侵袭能力。分别通过实时定量聚合酶链反应（RT-qPCR）和蛋白质印迹（WB）来比较5637和5637R细胞之间基因和蛋白质表达的差异。分别使用分子抑制剂和小干扰RNA（siRNA）系统来降低靶蛋白的表达。

结果

从分次照射（FI）中幸存的BCa细胞对IR和化疗药物均表现出耐受性。这些ARR细胞（5637R）具有提高的迁移和侵袭能力，并伴随着上皮间质转化（EMT）相关转录因子（ZEB1 / Snail / Twist）的表达增加。此外，5637R细胞通过激活的KMT1A-GATA3-STAT3回路显示了增强的癌干细胞（CSC）样特征，这是新报道的人膀胱癌干细胞（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预后不良的有效分子检测方法。为了增强耐药细胞对放射线的敏感性，我们引入了ERK抑制剂（FR 180204）和STAT3抑制剂（S3I-201）。然而，它们都不能增强ARR细胞对IR的反应。另一方面，siRNA分别用于抑制BCa细胞中两个重要的自噬相关基因内源性Beclin1和Atg5的表达，这显着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（CQ）仅在5637R细胞上增强了紫杉酚的细胞毒性。

结论

长期FI治疗是通过丰富BCSCs和增强细胞迁移和侵袭能力来建立ARR表型BCa细胞模型的有效方法。抑制自噬相关蛋白的表达和使用自噬抑制剂均可以提高ARR细胞对紫杉醇的敏感性，这表明自噬可能在ARR细胞的化学耐受性中起重要作用。