Down-regulation Of Survivin Contributes To Resistance Of Melanoma Cells To ER Stress-induced Apoptosis

Background&Objective The human menaloma is the deadliest form skincancer, the harm of the human melanoma is constantly increasing, and the meansurvival time of the patients have the shift with melanoma only six to10months. Theshift melanoma cells have very powerful proliferation ability, and resist to mostcancer treatment, so the drug has obvious effect, therefore the treatment effect is verylow. Study shows that the melanoma drug resistance due to its own a variety of cellapoptosis pathways deactivation have happened. The treatments include a variety ofhas been used in the three phase of clinical test methods, such as immune therapy, cellfactor treatment, radiation and chemotherapy. Even chemical treatment reagentcombined use of therapy in the past20years did not change the survival rates ofclinical trials.Melanoma cells are highly resistant to chemotherapeutic treatments due to it canwell adapt to pharmacological ER stress and successly escape the apoptosis inducedby it. So it is critical to understand mechanisms of the adaptability. Traditionaltreatment can not improve survival rates of melanoma, its basic reason is the lack of agood understanding of the mechanisms for melanoma cell to apoptosis. A lot ofspecific survival mechanisms lead to conventional treatments completely lost theeffect. At the same time, melanoma has the very strong adaptability. In recent years,the report also proved that more and more survival mechanisms involved in themelanoma drug resistance, The research of this aspect is also more and more notedby people. On this basis, this work aim to study the survival mechanism and specificsignaling pathways for melanoma cells in endoplasmic reticulum stress conditions, We expect to find some proteins to be effective biomarkers of melanoma duringtreatment, in order to provide the new ideas and drug targets to the clinical treatmentof melanoma.Methods (1) Melanoma cell line were treated with tunicamycin for differentperiods, we performed mRNA microarray analysis of melanoma cell line,thatresistive to ER stress,Mel-RM under pharmacological ER stress that induced bytunicamycin (TM). Among the changes, an down-regulation of survivin was the mostnoticeable and sustained. The QRT-PCR and the Western Bloting used to validate chipresults, at the same time, detection the survivin expression under the endoplasmicreticulum stress situations in different melanoma cell lines and other types of tumorcell.(2) we stably overexpressed pBABE and pBABE-survivin in melanoma cell lineMel-RM by lentivirus, examined whether down-regulation of survivin was able toprotect the melanoma cells from apoptosis induced by TM via hoechst33342dyenuclear, the cell vitalitydetected by MTT, Western Bloting detection molecularmarkers of cell apoptosis.(3) The cell cycle of melanoma cells block in the G1phaseunder endoplasmic reticulum stress, after the survivin overexpression the cell cyclechanges were tested by flow cytometry, Western Bloting testing the cell cycle relatedproteins changes;(4) Examination of the flanking genomic DNA region identified aputative E2F1binding site located in the promoter region of survivin viabiology-information methods. Through the fluorescent enzyme experiments,Survivinpromoter activity in the endoplasmic reticulum stress are suppressed, but exogenousexperiments shows it can be activated by E2F1.Use RNA interference method toknockdown E2F1, the changes of survivin disappeared.Results (1) Survivin is down-regulated in melanoma cells undergoing ER stressand this effect of ER stress on survivin is specific for melanoam cells.(2)Downregulation of survivin protects melanoma cells from ER Stress-inducedapoptosis and forced expression with survivin can effectively enhanced TM induced apoptosis in melanoma cells;(3) Protection against ER Stress-induced apoptosis bydownregulation of survivin is associated with its inhibitory effect on cell cycleprogression;(4) E2F1transcrip-tionally regulates survivin in melanoma cells underER stress. Exogenous expressed E2F1also reduced the endoplasmic reticulum stressresistance of melanoma cells.Conclusion We found that down-regulation of survivin protects melanoma cellsfrom ER stress-induced apoptosis, overexpression of survivin enhances sensitivity ofmelanoma cells to ER stress. Mechanistically, the protection of down-regulationsurvivin via its effect on G1cell cycle arrest. Furthermore, survivin is transcrptionalregulated by E2F1under ER stress, stablely overexpressed wildtype E2F1much moreenhanced the sensibility of melanoma cell to ER stress. Our findings indicated theE2F1-survivin-G1cell cycle arrest pathway as a mechanism involved in the resistanceof melanoma cells to ER stress induced apoptosis.