Study On The Effect And Mechanisms Of Novel Retinoids ECPIRM To Induce Apoptosis And Immunomodulatory Of Cutaneous T Cell Lymphoma Cell HUT78

Cutaneous T cell lymphomas(CTCL)is a heterogenous group of extranodal non-Hodgkin lymphomas,which is T-cell derived.Mycosis fungoides(MF)and Sezary Syndrome(SZS)are the most common forms,comprising 67%of all CTCL.The pathogenesis of CTCL is multiple.On the one hand,there are immune suppressor in CTCL.The tumor microenvironment with unbalance of Thl/Th2 cytokines contribute to the growth and survival of malignant T cells.On the other hand,malignant cell is observed resistance to apoptosis.Organism reduce ability about eliminating the abnormal cells to tumor.Treatment of CTCL has encompassed a variety of modalities.Many researches report effectiveness of retinoid in the treatment of CTCL.And retinoid has been used in CTCL in every stage for over.Retinoids,which are natural and synthetic analogs and derivatives of vitamin A,exert a broad spectrum of biological activities by modulating embryonic development,vision,reproduction,bone formation,cell growth,apoptosis,differentiation and malignant transformation.Retinoids is an immunomodulator against tumor,especially Hematologic tumor.ATRA treatment for APL resulted in effective rate from 40%to 90%..Retinoids inhibite proliferation,induce apoptosis by binding to nuclear retinoid receptors.And side effects of retinoids depend also on the modulation of the retinoid receptors.Its application is limited usually by side effects by retinoid receptors.ECPIRM,a 13-cis RA derivative,was synthesized in our laboratory,and was shown to inhibit proliferation and induce apoptosis through RAR/RXR-independent pathway in cutaneous squamous carcinoma cell(SCC)SCL-1.In addition,skin irritation reaction such as erythema and desquamation was not demonstrated in mice treated with ECPIRM.We have proved that ECPIRM does not activate the transcription of retinoic acid receptors,suggesting that the novel synthesized compound was superior to the other retinoids and it is a potential therapeutic drug.Retinoids is widespread use in the treatment of blood malignancies.T cell express all retinoid receptors excepect RXR?.SCL-1 belong to keratinocytes which express RARa and RAR?.As advantages of ECPIRM in SCL-1,we investigate the effects of ECPIRM in hematological tumor cell,the underpinning mechanism and the relationship with retinoid receptors based on HUT78 cells originating from patient with SZS.The current study include three sections:1)To investigate the effect and mechanisms of proliferation and apoptosis;2)To illuminate the effect and mechanisms of immunomoderator;3)To demonstrate the effect of retinoid receptors.Part1 The effect and mechanisms of proliferation and apoptosis of Cutaneous T Cell Lymphoma Cell HUT78Objective To investigate the effect and mechanisms of ECPIRM on proliferation and apoptosis in CTCL cell lines.Section 1 the effect of ECPIRM on proliferation and apoptosis in CTCL cell lines.Method 1.The anti-proliferative activity of ECPIRM,ATAT,13-cis RA and bexarotene(1?M,5?M,10?M,20pM,40pM)for 24,48,72 and 96 hours respectively were investigated by MTT assay and Trypan blue exclusion assay in CTCL cell and human Lymphocytes2.Morphological changes in cell apoptosis were determined by fluorescence microscope.The pro-apoptotic activity of ECPIRM was detected by Annexin-V/PI staining flow cytometry after HUT78 cells were treated with ECPIRM(5?M,10?M,20?M)for 24,48,72h.3.Cell cycle progression was analysed by flow cytometry after HUT78 cells were treated with ECPIRM(5?M,10?M,20?M)for 24,48,72h.4.CDK2?CDK4?CyclinD1?CyclinE?p21 and p27 were detected by western blot analysis after HUT78 cells were treated with ECPIRM(5?M,10?M,20pM)for 24,48,72h.Results:(1)ECPIRM inhibit proliferation of HUT78 cell.Cell growth was inhibited by 40pM ECPIRM at 24h.10?M and up 10?M ECPIRM also exhibited inhibitory effects on the proliferation of HUT78 cell at 48h(p<0.01)and the max to inhibit rate was observed at 96h.The anti-proliferative effect of ECPIRM is stronger than ARTA,13-cis RA and bexarotene when the concentration was greater than or equal to 20 ?M(p<0.01).Anti-proliferation activity of ECPIRM in lymphocyte was much less than that of HUT78.(2)ECPIRM induced apoptosis of HUT78 cell:1)Apoptotic morphological alterations were observed by microscope.There were no noticeable changes in cellular morphological changes after treatment with ECPIRM for 24h.Typical apoptotic morphology including condensed nuclei,apoptotic body and fragmented DNA was shown in HUT78 cells treated with ECPIRM for 48h and 72h.2)The apoptosis rate of HUT78 was detected by Annexin-V/PI staining flow cytometry treatment with ECPIRM for 24,48,72h.The result shows that the apoptosis of HUT78 cells treated with ECPIRM for 24h was not obvious.The rate of apoptosis increased at 48h(p<0.01).The apoptosis rate was 75.04%treatment with ECPIRM.The apoptosis of lymphocyte treated with ECPIRM was not obvious.(3)ECPIRM induces G0/G1 arrest in HUT78 cells:1)The cell cycle of HUT78 was detected by flow cytometry.The result show that the cell cycle of HUT78 cells treated with ECPIRM for 24h was not obvious.As the ECPIRM concentration increases,the rate of increase G0/G1 phase(p<0.01)at 48h.The G0/G1 phase rate was 71.70±2.48 treatment with ECPIRM at 72h.The rate of S and G2/M was reduced.2)The cell cycle related genes were detected by westbolt.The expression levels of cyclinD1,cyclin E,CDK2 and CDK4 markedly decreased following treatment with ECPIRM(p<0.01).In contrast,the expression level of p21 and p27 increased(p<0.01).Conclutions ECPIRM can inhibit the proliferation of HUT78,exhibiting better inhibitory effect than 13-cis RA,ATRA,bexarotene(p<0.01).ECPIRM induces apoptosis of HUT78 and induces G0/G1 cell cycle arrest.The proliferation and apoptosis of lymphocyte treated with ECPIRM was not obvious.Section 2 The mechanisms of ECPIRM on proliferation and apoptosis in CTCL cell lines.Methods:The JAK/STAT pathway genes(JAK1?pJAK1?STAT3?STAT5?pSTAT3?pSTAT5,BCL-XL?c-Myc)were detected by western blot analysis after HUT78 cells were treated with ECPIRM for 72h.Result ECPIRM suppression JAK/STAT signaling pathway and it may contribute to the inhibition of proliferation.Part 2 The effect and mechanisms of ECPIRM on immune regulationObjective To investigate the effect and mechanisms of ECPIRM on immune regulation in CTCL cell lines.Section 1 The effect of ECPIRM on immune regulationMethods 1.The anti-proliferative activity of ECPIRM,(0.01?M,1?M,10?M)for 24,48,72 and 96 hours respectively were investigated by MTT assay in CTCL cell 2.The L-2,IL-12,IFGy,IL-4,IL-5,IL-10,TGF-?was detected by flow cytometry and real-time qPCR after HUT78 cells were treated with ECPIRM(0.01?m,0.1?m,1?m)for 48h.Results:1)The proliferation of HUT78 treated with ECPIRM(0.01?m,0.1?m,1?m)was not obvious.10?M ECPIRM inhibited proliferation of HUT78 cell at 48h2)The results of RT-qPCR showed that IL-2,IL-12,IFN-y,IL-4 and IL-5 was lowly expressed in HUT78.ECPIRM increase the express of IL-2,IL-12,IFN-?and decrease the expression of IL-10,TGF-?.The change of IL-4and IL-5 was not obvision.3)The results of western blot showed the effect of ECPIRM on IL-2,IL-12,IFN-y,IL-4,IL-5,IL-10 and TGF-?consistent with RT-qPCR.Conclution ECPIRM regulate immune of CTCL cell by decease IL-10 and TGF-?Section 2 The mechanisms of ECPIRM on immune regulationMethods The P65 and IkBawas detected by real-time qPCR and P65,IkBaand pIkBaby flow cytometry and after HUT78 cells were treated with ECPIRM(0.01?m,0.1?m,1?m)for 96h.Results The results of RT-qPCR showed that P65 and IkBawere decreased after HUT78 cells were treated with 1?m ECPIRM.The change of P65 and IkBain protein levels consistent with mRNA level.The expression of pIkB? was deceased.Conclution ECPIRM suppression NF-KB signaling pathway and it may contribute to the inhibition of IL10 and TGF-?.Part 3 The effect of ECPIRM on the expression and transcriptional activation of retinoic acid receptor in HUT78 cellsObjective To investigate the effect of ECPIRM on the expression and transcriptional activation of retinoic acid receptors in HUT78 cell.Section 1 The effect of ECPIRM on RAR/RXR in HUT78 cellMethods The RAR,RXR,TIGI and ST AT1 was detected by real-time qPCR after HUT78 cells were treated with ECPIRM(0.01?m,0.1?m,1?m)for 96h.The RAR,RXR,TIGI,STAT1 and pSTAT1 were detected by real-time qPCR after HUT78 cells were treated with ECPIRM(0.01?m,0.1?m,1?m)for 96hResults:ATRA significantly increased both of the protein expression of RARa,RAR?,CYP26A1 ? STAT1 and their mRNA expression respectively.However,ECPIRM did not upregulate any retinoic acid receptors in HUT78 cellsSection 2 The effection of ECPIRM,ATRA1?Compared effect of ECPIRM on proliferation in HUT78 cell with ATRA and bezxratene.Methods 1)The anti-proliferative activity of ECPIRM,ATRA(0.01?M,1?M,10?M)for 24,48,72 and 96 hours respectively were investigated by MTT assay in CTCL cell and the anti-proliferative activity of ECPIRM,ATRA(0.01?M,1?M,10?M)combined with AGN193109 for 24,48,72 and 96 hours respectively were investigated by MTT assay in CTCL cell2)The anti-proliferative activity of ECPIRM,bexarotene(0.01?M,1?M,10?M)for 24,48,72 and 96 hours respectively were investigated by MTT assay in CTCL cell and the anti-proliferative activity of ECPIRM,bexarotene(0.01?M,1?M,10?M)combined with danthron for 24,48,72 and 96 hours respectively were investigated by MTT assay in CTCL cellResults:The effect of combination group on proliferation consistent with ECPIRM on proliferation.The anti-proliferative effect of ATRA is stronger than ARTA combination with AGN193109.The anti-proliferative effect of bexarotene is stronger than bexarotene combination with danthronConclusions ECPIRM could neither induce the expression of RAR?,RAR?,RAR?and RXRa,RXRP nor activate the transcription of retinoic acid receptors,suggesting that the novel synthesized compound will have less side-effect in chemotherapy.Summary1.ECPIRM can inhibite proliferation,induce the apoptosis and moderate Th1/Th2 cyctekine.The biological action of ECPIRM prior to ATRA,13-cis RA and bexarotene.There are no biological action by ECPIRM on lymphocyte,2.Meanwhile,ECPIRM induces G1 cell cycle arrest in HUT78 cells,via the downregulation of CDK2,CDK4,cycling D1 and cyclin E expression and upregulation of p21 and p27.3.ECPIRM suppression JAK/STAT signaling pathway and it may contribute to the inhibition of proliferation.4.ECPIRM suppression NF-KB signaling pathway and it may contribute to the inhibition of IL10 and TGF-?.5.ECPIRM could neither induce the expression of RARa,RAR?,RAR?,RXR? and RAR?,nor activate the transcription of retinoic acid receptors,suggesting that the novel synthesized compound will have less side-effect in chemotherapy.