The Circadian Clock Protein CRY1 Inhibits Paclitaxel-induced Senescence Of Bladder Cancer Cells

BackgroundBladder cancer(BC)is a common cancer worldwide with high morbidity and mortality whose incidence has increased in recent years.The first-line treatment is cisplatin-containing combination chemotherapy such as gemcitabine plus cisplatin or MVAC(methotrexate,vinblastine,doxorubicin,and cisplatin).Clinical trials prove that paclitaxel combined with radiation or gemcitabine are effective treatment strategies for BC patients,making paclitaxel a promising second-line treatment option for patients with metastatic UC.Unfortunately,a considerable number of patients will become resistant to the above treatment options and then relapse.Therefore,enhancing the sensitivity of bladder cancer cells to drugs and reversing drug resistance are of great clinical significance for bladder cancer patientsCellular senescence refers to irreversible cell cycle arrest of cellular stress response.Cellular senescence is often seen as a tumor suppressor mechanism that inhibits cell proliferation and thus prevents cancer.According to different inducing factors,cellular senescence can be divided into replicative senescence,oncogene-induced senescence,and treatment-induced senescence(Therapy-induced Senescence,TIS).Among them,TIS can inhibit the occurrence and development of tumors more effectively and safely than apoptosis,and its role in tumor treatment has gradually attracted attention.However,there are few studies on TIS in the treatment of bladder cancer,and studies on drug-resistant bladder cancer have not been reportedCircadian rhythm is an internal mechanism produced by the body's long-term adaptation to changes in the external environment,regulating many important physiological and biochemical processes,such as cell proliferation and senescence.The disruption of the circadian clock increases the risk of cancer in humans,but the impact of each of the four core circadian clock genes on tumors is still not consensus,In particular,the relationship between circadian rhythm and drug resistance remains elusive.Objectives1.To clarify whether drug-resistant bladder cancer cells resist therapy-induced senescence.2.To define the characteristics of circadian rhythm in drug-resistant bladder cancer cells.3.To explore the mechanism between circadian rhythm genes and cellular anti-senescence.Methods and Results1.Establish of Cisplatin-resistant bladder cancer cell line(CRBC).Long-term of Cisplatin(4?g/ml)treatment was used to obtain the Cisplatin-resistant bladder cancer UMUC3 and EJ cells.Real-time PCR test results showed increased expression of resistance genes and MTS confirmed the resistance of these cells to cytotocity of not only Cisplatin,but also PTX,indicating muti-drugs resistant ability2.The resistant cells enter quiescence to resist senescence under PTX treatment.After PTX treatment of bladder cancer cells,the cells were arrested in G2/M phase,and the nucleus showed polyploid phenotype,which was consistent with the inhibitory effect on mitosis by PTX.However,in CRBC,the cell cycle accumulated in the G0/G1 phase,the cell nucleus did not show polyploidy after PTX treatment.Moreover,the proliferative ability of CRBC was reduced,and the expression of proliferation-related proteins including Cyclin D1,Cyclin E1 and MCM7 were reduced,indicating CRBC enter quiescence under PTX stressAfter PTX treatment of bladder cancer cells,SA-?-gal staining cells increased significantly and the protein levels of P53 and P21 increased,indicating that PTX could induce bladder cancer cell aging.But in CRBC,the above-mentioned senescence-related phenotypes were not significant,which means the CRBC resisted paclitaxel-induced senescence3.Circadian rhythm is prolonged and CRY1 accumulated in CRBC after PTX treatmentAfter PTX treatment of CRBC,the cells entered the quiescent phase and resisted senescence.The results of Real-time PCR and Western blot showed that the circadian rhythm cycle was prolonged and the amplitude increased.Western blot results showed that the levels of circadian rhythm proteins BMAL1,CLOCK and PER2 decreased,but CRY1 increased.In addition,serum-free-induced quiescent UMUC3 and EJ cells also showed prolonged circadian rhythm and accumulated CRY1 protein level4.CRY1 mediates resistance of CRBC to PTX-induced senescence by promoting p53 degradationWhen RNAi was used to inhibit cryl,SA-?-gal staining cells in CRBC increased significantly and p53 protein level increased under the effect of PTX.But there was no significant change in p53 mRNA level,indicating thatf CRY1 maycontribute tothe resistance of CRBC to PTX-induced senescence by promoting p53 degradation.The results of CO-IP showed that MDM2 and CRY1 were detected in the immunoprecipitation complex by anti-p53 antibaody,while inhibition of CRY1 significantly reduced the binding of p53 and MDM2.This suggests that CRY1 can promote the combination of p53 and MDM2 to promote the degradation of p53.5.The activation of AMPK promotes the senescence of CRBC.It hasbeen shown that AMPK can phosphorylate CRY1 and hence promote its degradation.After treatment of CRBC with AMPK activator,the level of CRY1 decreased significantly,the level of P53 protein increased significantly,and the number of senescent cells increased.However,after treating CRBC with AMPK inhibitor,the expression of P53 was significantly reduced,and the cells were not senescent.Conclusions1.CRBC enter the quiescece and resist senescence under PTX stress.2.The circadian rhythm is prolonged and the expression of CRY1 is increased inquiescent CRBC.3.The clock protein CRY1 mediates the resistance of CRBC to PTX-induced senescence by promoting p53 degradation.4.The activation of AMPK down-regulates CRY1,leading to increase of P53protein 1,which in turn promotes senescence.