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.
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 humanbladder 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.