The Mechanisms Of Apoptosis In Cutaneous Squamous Cell Carcinoma Cells Induced By Retinoids

IntroductionCutaneous squamous cell carcinoma(SCC)is one of the most common skin carcinoma,and the incidence rate of SCC has been alarmingly increasing worldwide, representing a major medical and economic problem in terms of cancer management.Although surgery is still the major therapeutic modality of SCC of skin,its use may be limited in treating multiple lesions.So the search for effective and appropriate antitumor medicines is in of great importance.Retinoids are structural and functional analogs of vitamin A that display a wide range of biologic activity.As retinoids are critical for the maintenance of epithelial growth and differentiation,they are effective therapeutic and chemopreventive agents for some skin diseases and epithelial cancers.In some clinical trails,the first generation of retinoids was proved to be effective in treating SCC or reducing cutaneous squamous cell carcinoma risk in high risk population.Systemic retinoid,specifically acitretin,have shown to be effective in preventing the development of SCC in solid organ transplant recipients and in patients with psoriasis treated with psoralen-UVA.A single regimen of acitretin was also successfully used to treat a patient who had multiple,extensive verrucous carcinomas. Tazarotene was demonstrated to significantly reduce proliferation and increased apoptosis in basal cell carcinomas in vitro.In addition,in order to improve the sensitivity of retinoids on cancer,retinoids combined with interferon have obtained satisfying outcome in clinic study.To date,retinol and the retinoids are the only compounds proved chemopreventive for -skin carcinogenesis,but the mechanisms have not been fully clarified.In this study,we evaluated the apoptotic effects and the molecular transduction mechanisms in cutaneous squamous cell carcinoma cell line SCL-1 induced by retinoids or retinoids in combination with IFNα-2a,offering new thought for finding more effective treatment measure of SCC.Methods1.Cell cultureSCL-1 cells and HaCaT cells were cultured in DMEM medium supplemented with 10%heat-inactivated fetal bovine serum,100U/ml penicillin and 100U/ml streptomycin and in a humidified atmosphere of 5%CO₂ at 37℃.2.Assay for cell proliferation by MTT(1)Effects of acitretin on the cell proliferation of cell line SCL-1;(2)Effects of all-trans retinoic acid(ATRA),tazarotene on the cell proliferation of cell line SCL-1;(3)Effects of acitretin on the cell proliferation of HaCaT;(4)Effects of acitretin with IFNα-2a on the cell proliferation of cell line SCL-1;3.Detection of apoptosis with Cell Death Detection ELISA(1)Detection of apoptosis in SCL-1 cells induced by acitretin;(2)Detection of apoptosis in SCL-1 cells induced by ATRA,tazarotene;(3)Detection of apoptosis in HaCaT cells induced by acitretin;(4)Detection of apoptosis in SCL-1 cells induced by acitretin with IFNα-2a;4.Annexin V/PI assay by flow cytometry(1)The early apoptotic cells induced by ATRA,tazarotene,acitretin were quantified by Annexin V/PI assay;(2)The early apoptotic cells induced by acitretin with IFNα-2a were quantified by Annexin V/PI assay;5.The cell cycle was assessed by flow cytometry6.Apoptotic morphological changes(1)Ultrastructural changes in SCL-1 cells treated with acitretin were determined by transmission electron microscopy. (2)Apoptotic SCL-lcells treated with acitretin with IFNα-2a were detected by acridine orange staining.7.Detection of proteins by Western blotting(1)Effects of acitretin on protein expressions in Fas apoptotic signaling pathway;(2)Effects of acitretin with IFNα-2a treatment on caspase activation;8.Detection of Fas protein on cell surface by flow cytometry9.Real-time polymerase chain reaction(PCR)was performed to assess Fas and FasL expression in SCL-1 cells10.Blocking assay with ELISA(1)Effects of caspase inhibitor on apoptosis in SCL-1 cells induced by acitretin;(2)Effects of Neutralizing ZB4 anti-Fas antibody on apoptosis in SCL-1 cells induced by acitretin;Results1.MTT assayAcitretin inhibited growth of SCL-1 cells in a dose- and time-dependent manner. ATRA,acitretin and tazarotene all inhibited growth of SCL-lcells in a time-dependent manner,acitretin played the most prominent role among them.In contrast,acitretin exhibited a few inhibitory effects on the proliferation of HaCaT cells.The combination of acitretin with IFNα-2a inhibited growth of SCL-1 cells,inhibitory rate of combination has remarkable difference compared with ATRA,IFN-αand control group(P＜0.05).2.Detection of apoptosis with Cell Death Detection ELISAAcitretin induced apoptosis of SCL-1 cells in a dose- and time-dependent manner. ATRA,acitretin and tazarotene all induced apoptosis in a time-dependent manner,acitretin played the most prominent role among them.However,the pro-apoptotic effect of acitretin on HaCaT was much less prominent than that on SCL-1.The combination of acitretin with IFNα-2a enhanced the apoptosis induced by acitretin or IFNα-2a alone in SCL-1 cells. 3.Detection using flow cytometryATRA,acitretin and tazarotene all induced early apoptosis in SCL-1 cells,increased G₁-phase population,acitretin played the most prominent role among them.The combination of acitretin with IFNα-2a enhanced the early apoptosis in SCL-1 cells.4.Apoptotic morphological changesUnder transmission electron microscopy,nucleus of SCL-1 cells treated with acitretin for 5 days showed features of apoptosis,including disappeared villi on cell membrane and dense aggregation of chromatin.In control group,SCL-1 cells displayed normal morphology with rich villi on the cell membrane,normal shape of nucleus,and nuclear chromatin which evenly distributed.Observation with fluorescence microscope at 490nm, the typical apoptosis of SCL-1 cell treated with acitretin with IFNα-2a for 72 h is obvious. Cells treated with acitretin or IFN alone didn't appear apoptosis phenomenon and control group displayed normal morphology.5.Western blotThe levels of Fas,FasL,and Fas-associated death domain(FADD)increased significantly after 12 hours exposing to acitretin,with activation of caspase-8,whereas the corresponding caspase-9,caspase-3 and poly(ADP-ribose)polymerase(PARP)active fragments occurred 24 hours after acitretin treatment.All of the above proteins changed in a time-dependent manner.Caspase-8,caspase-3 protein in SCL-1 cells treatmed with acitretin/IFNαwere activated significantly compared with control and acitretin,IFNαalone.6.Detection of Fas protein on cell surface by flow cytometryThe level of the cell surface Fas receptor was significantly increased in acitretin-treated cells compared with the untreated controls.7.RT-PCRThe results with relative quantification analysis also demonstrated that both Fas and FasL mRNAs increased at 12 hour after the addition of acitretin and continued to increase substantially in a time-dependent manner. 8.Blocking assayCaspases-8 inhibitor effectively suppressed acitretin-induced apoptosis while caspase-9 inhibitor did not.Neutralizing ZB4 anti-Fas antibody significantly inhibited the apoptosis in SCL-1 cells induced by acitretin.Conclusions1.ATRA,acitretin and tazarotene can inhibit SCL-1 cell proliferation and induce SCL-1 cell apoptosis;acitretin played the most prominent role among them.2.Acitretin is a potent inducer of apoptosis in human cutaneous squamous cell carcinoma cell line SCL-1 through Fas death receptor pathway,and that SCL-1 cells undergoes a typeâ… apoptotic pathway induced by acitretin.3.The combination of acitretin with IFNα-2a can inhibit proliferation and induce apoptosis of SCL-1 cells,which is related to the activation of caspase-8 and caspase-9.