Down-regulation of autophagy-associated protein increased acquired radio-resistance bladder cancer cells sensitivity to taxol

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.

Keywords:

• Acquired radio-resistance
• radiosensitivity
• autophagy
• chemoresistance

Author contributions

ZK designed the experiments. XM, GM, RC and FW performed the experiments. ZK analyzed the data. ZK and XM wrote the manuscript. All authors reviewed the manuscript.

Disclosure statement

The authors declare that they have no competing interests.

Data availability statement

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Additional information

Funding

The present study was supported by National Natural Science Foundation of China [Grant Nos. 31870846 and 31270896] and Natural Science Foundation of Shanghai [Grant No. 18ZR1403600].

Notes on contributors

Xiangli Ma

Xiangli Ma is a postgraduate at the Department of Radiobiology, Institute of Radiation Medicine, Fudan University, Shanghai, PR China.

Guangmin Mao

Guangmin Mao, MD, is a doctor of Radiotherapy, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, PR China.

Rulve Chang

Rulve Chang, PhD, is a senior researcher at Department of Information Project, Yangtze River Pharmaceutical Group, Shanghai, PR China.

Fang Wang

Fang Wang, MD, is a senior researcher at Shanghai Medicilon Biopharmaceutical Co., Ltd., Shanghai, PR China.

Xiangyan Zhang

Xiangyan Zhang, is a postgraduate at the Department of Radiobiology, Institute of Radiation Medicine, Fudan University, Shanghai, PR China.

Zhaolu Kong

Zhaolu Kong, PhD, is an associate researcher at the Department of Radiobiology, Institute of Radiation Medicine, Fudan University, Shanghai, PR China.