The Mechanism Of Chemotherapy Drug Resistance Of Bladder Cancer Reversed By CYLD Regulated Autophagy

Part 1 Effect of Cell Autophagy on chemotherapy drug resistance of bladder cancerBackgroundBladder cancer is the most common malignancy of the urinary system in China. Approximately 25 percent of newly diagnosed patients is muscle invasive bladder cancer. Another 75 percent of patients is non-muscle invasive bladder cancer, but most of them will relapse or infringe muscle. Therefore, early surgery along with conventional chemotherapy is the key to the treatment of bladder cancer, which can reduce the recurrence rate of bladder cancer, improve the prognosis of patients and improve their survival. Nevertherless,2/3 of patients still appears relapse and 15% to 20% patients with recurrence will progress to a higher pathological stage and/or associate with elevated grade. Most of the deaths were due to local invasion or distant metastasis caused by muscle invasive bladder cancer. Higher mortality requires urologists to find out new and more effective measures to treat bladder cancer. In recent years, although as the first-line chemotherapy, the new GC joint treatment programs of Gemcitabine and Cisplatin has had a significant effect on advanced bladder cancer, but drug resistance limits the long-term efficacy of chemotherapy drugs greatly.Gemcitabine is widely used in the treatment of pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, non-small cell lung cancer and other tumors. However, more and more urologists are aware of disadvantages caused by Gemcitabine resistant. Despite initial curative effect of bladder cancer chemotherapy is notable, but 60 to 70 percent effective patients relapse within 5 years, with an average survival of only 12 to 14 months, which can be attributed to the emergence of drug resistance during treatment. Many studies have shown that chemotherapy drugs can induce apoptosis of tumor cells to play a therapeutic role, but chemotherapy can also induce autophagy in tumor cells. Autophagy induced under such conditions is often used as a protective mechanism to inhibit cell apoptosis, and this protective mechanism may make tumor cells against various chemotherapeutic drugs, which make tumor cells chemotherapy resistant and is disadvantage to the treatment of tumors.Studies have shown that application of RNAi on disturbing autophagy related gene expression can accelerate tumor cell death after receiving chemotherapy. In addition, some of autophagy inhibitors, such as 3-methyl adenine, chloroquine, and anti-tumor chemotherapy drugs have a synergistic effect in inducing tumor cell apoptosis aspect. Thus we should start the apoptosis signaling pathways to promote apoptosis of tumor cells and improve chemotherapy efficacy, as long as we can inhibit the chemotherapy resistance induced by tumor cells autophagy by some way in the anti-cancer treatment process.ObjectiveTo investigate the effects of autophagy (cell autophagy) on Gemcitabine resistance of bladder cancer influence.Materials and methodsWe repeated screening concentration gradient dosing of Gemcitabine, and constructed Gemcitabine-resistant strains (RT112-GR) by using bladder cancer cell RT112, and detected the drug resistance of bladder cancer cell RT112 by MTT cytotoxicity assay. We studied differentially expressed mRNA levels and protein levels of autophagy marker protein (LC-3) in two cell lines RT112 and RT112-GR by RT-PCR and Western boltting methods. We detected differentially expressed autophagy body levels in two cell lines RT112 and RT112-GR by electron microscopy. We detected the changes of the Gemcitabine resistance in RT112-GR cell lines before and after autophagy levels inhibited by chloroquine (CQ) by using WST-1 cell viability experiments and LDH cytotoxicity assay.ResultsMTT results showed that, Gemcitabine half inhibitory concentration (IC50) in RT112-GR cell line was 4.2 umol/L, which was 350 times in its parent cell RT112’s (RT112 IC50 value was 12 nmol/L), and Gemcitabine resistance RT112-GR cell lines were no cross-resistance to doxorubicin and Cisplatin. After the addition of autophagy inhibitor CQ to treatment resistant cell lines, MTT results showed that the IC50 value was 10.9 nmol/L, and had no statistically significant difference (NS) when compared with the conventional bladder cancer cells. We used Gemcitabine of 45 nM to treatment RT112 and RT112-GR two cell lines. RT-PCR results showed that mRNA expression levels of LC-3 in RT112-GR cell line was significantly higher than ordinary RT112 cells line (P=0.023). Western boltting results showed that the protein levels of LC3 in RT112-GR bladder cancer cell lines was significantly higher than ordinary RT112 cells group (P< 0.05). Electron micrograph showed that autophagic bodies in RT112-GR cytoplasm increased significantly(P<0.05) after Gemcitabine of 45 nM treated the two cell lines. MTT cytotoxicity assay showed that Gemcitabine toxicity to resistant group was recovered after adding CQ.ConclusionWe successfully constructed Gemcitabine-resistant bladder cancer cell strains (RT112-GR) in our study. After equal treatment dose of 45 nM Gemcitabine treated the two cell lines RT112 and RT112-GR, autophagy level in RT112-GR cells was significantly higher than the parental RT112 cells, which suggesting that the protective autophagy was produced. However, when we added with autophagy inhibitor CQ to treat RT112-GR cells, the level of autophagy was reduced and the resistance is missing.Part 2 The construction of CYLD transfected cells and related functional experimentsBackgroundTumor suppressor CYLD (Cylindromatosis, cylindrical tumor gene) encodes a deubiquitination enzymes. Mutation or deletion in CYLD gene may induce cylindromatosis. This gene is also closely related to the occurrence and development of a variety of tumors. CYLD regulates a plurality of signal pathways including NF-κB, JNK and AKT by removing 63-linked ubiquitin chains from several specific substrates through its own deubiquitination enzymes activity. So far, there has been no study about the relationship between CYLD and autophagy in tumors at home and abroad. Studies have suggested that CYLD may affect cell autophagy by influencing associated ubiquitination of lysine48 and lysine6 in adult rat brain post-synapses. It has been cleared that autophagy is related to many types of chemotherapy drug resistance. Whether or not CYLD affects the growth, apoptosis and chemosensitivity capacity of bladder cancer by adjusting autophagy needs our further study to confirm. Our preliminary findings indicated that the signal pathways activation level of NF-κB, JNK and Akt could be increased by reducing CYLD expression level in rat vascular smooth muscle cell through the process of adenovirus infection, which is the key pathway activated by autophagy. Li Dan-dan et al confirmed that chemotherapy upregulated Beclin 1 gene expression and mediated tumor cell Hep3B, CNE2 autophagy death through activating JNK and then phosphorylating downstream substrate c-Jun. Nevertherless, Gao Junling et al verified that effectively inhibiting neurons autophagy after traumatic brain by blocking the JNK pathway could have cytoprotection. Wang W et al found that regulation of NF-κB might affect the protective effect of autophagy on cells. In addition, our recent findings showed that upregulated CYLD expression level of vascular smooth muscle cells in rats could significantly reduce the expression of autophagy protein LC3, and downregulated CYLD expression level of vascular smooth muscle cells in rats by the use of adenovirus infection could significantly increase the expression of autophagy protein LC3, which suggested that CYLD expression level were closely related to autophagy. In summary, previous studies have verified that autophagy can affect tumor growth, apoptosis and resistance to chemotherapy. And cell autophagy may be regulated by CYLD, of which regulatory mechanism may be likely related to the activation levels of NF-κB, JNK, Akt conduction pathways. Synthesizing results of our previous study, we propose a hypothesis:upregulated expression level of CYLD in bladder cancer cells might affect downregulated activation levels of signal transduction pathways including key NF-κB, JNK and Akt, which inhibiting autophagy, thereby inducing autophagic cell death or apoptosis, reversing chemosensitivity of bladder cancer.ObjectiveTo investigate the relationship of cylindrical tumor gene (CYLD) and autophagy of bladder cancer.Materilas and methodsThe results were detected by immunohistochemistry staining.55 cases of bladder cancer patients in our hospital library were collected and the carcinoma tissue of patients were collected by surgery,10 cases of benign bladder tissue as control tissues, and constructed tissue chip. These tissue chips were performed CYLD immunohistochemical staining and LC-3 staining by Immunohistochemical staining method. Application of Pearson correlation analysis to study whether there is a correlation between LC-3 and CYLD expression. We constructed CYLD knock-down bladder cancer cells. CYLD protein and mRNA expression levels in the experimental group (knockout CYLD, Ad-Cyld shRNA silencing gland virus), CYLD overexpression group (Ad-Cyld upregulated adenovirus), empty viral transfection group and control group were measured by Western Blot and RT-PCR technology. LC-3 protein and mRNA expression levels in the CYLD knockouted bladder cancer cells, CYLD overexpression group, empty viral transfection group and normal cells group were measured by Western Blot and RT-PCR technology.ResultsImmunohistochemical staining results suggested that CYLD expression levels were significantly reduced in the bladder cancer tissues (P<0.05), compared with the control group (normal bladder tissue), the difference was statistically significant. LC-3 expression was negatively correlated with CYLD expression in bladder cancer tissues, and Pearson correlation coefficient was -0.511 (P<0.05).Real-time quantitative PCR results showed that:LC-3 gene expression level in the experimental cells (CYLD knockouted RT112) was increased while CYLD gene expression level was significantly decreased, compared with control cells (RT112) and empty viral transfection group, and the difference was statistically significant (P <0.05).ConclusionWe successfully constructed CYLD knockouted in common bladder cancer. The role of CYLD in bladder cancer may be associated with autophagy.Part 3 The relationship of the expression of cylindrical tumor gene (CYLD) and chemotherapy resistance of bladder cancerBackgroundCYLD is a recognized tumor suppressor gene. CYLD gene mutations or deletions may affect tumor occurrence and development. Microtubule-associated protein light chain-3 (Microtubule associated protein light chain3, LC-3) is homologous genes of a yeast cell autophagy-related genes ATG8. LC-3 expression level in various tumor diseases was abnormal. Studies have found that expression level of LC-3 is negatively correlated with the depth of tumor invasion, lymph node metastasis, lymph node invasion and poor prognosis. Compared with the low expression of LC-3, the overall survival of cancer patients with high LC-3 expression is better. LC-3 protein is a good marker correlated with autophagy. Studies have shown that autophagy may be regulated by CYLD, of which regulatory mechanism may be related to activation levels of NF-κB, JNK, Akt conduction pathways. During the chemotherapy process of bladder cancer, whether CYLD interact with LC-3 need our further studies. The regulation and mechanism of CYLD to bladder cancer autophagy are significantly meaningful for us to know the precise molecular biological mechanism of autophagy regulation, and to design novel and rational targeted chemotherapy methods, and to further improve diagnosis and treatment levels of bladder cancer in our country.ObjectiveTo investigate the relationship between the expression of CYLD and Gemcitabine resistance in bladder cancer.Material and methodsCYLD gene expression in experimental group (knockout CYLD:RT112-CYLD shRNA), drug resistance group (RT112-GR) and ordinary cell group (RT112) was measured by real-time quantitative PCR technology. IC50 values and differences of the experimental group (knockout CYLD) and the other two cell groups (RT112-GR and RT112) were measured and compared by using CCK8 assay. IC50 values difference of pre-three groups was measured after autophagy inhibitor CQ treated the experimental group (knockout CYLD). After the same treatment amount of Gemcitabine treated three groups of cells, LC-3 mRNA expression of three groups was measured by using real-time quantitative PCR technology and cytotoxicity testing of each group was detected by CCK8 assay. The experimental group was treated with CQ, and LC-3 mRNA levels were detected by RT-PCR technique and cytotoxicity of CQ was detected by CCK8 assay.ResultsReal-time quantitative PCR results showed that CYLD in bladder cancer drug resistant group exhibited low expression similar to the knockout experimental group, with statistical significance difference (P< 0.05) compared with ordinary cells. LC3 expression in CYLD knockouted experimental group showed high expression similar to drug resistance group. CYLD knockouted experimental group showed Gemcitabine resistance similar to drug resistance group. IC50 of Gemcitabine in drug resistance was 4.2μmol/L, while IC50 in CYLD knockouted experimental group was about 4.12μmol/L and both of them had no significant difference, namely IC50 and drug resistance group had no significant difference (P> 0.05). After adding autophagy inhibitor CQ, drug resistance was reversed, namely difference of reduced IC50 had no statistically significance compared with ordinary cell group (P> 0.05).ConclusionProtective autophagy induced by low expression of CYLD in drug-resistant cell lines may be reasons for drug resistance. While applying autophagy inhibitor CQ on the cells, resistant strains restore the sensitivity to Gemcitabine.