| Cutaneous T cell lymphoma(CTCL)was a heterogeneous group of T cell derived lymphomas,and it belonged to extranodal non-hodgkin’s lymphoma(NHL).Mycosis fungoides(MF)and sezary syndrome(SS)were the two most common subtypes.The latest epidemiological survey showed that the incidence rate of CTCL was increasing year by year,and it had become an important problem threatening public health in many countries.The exact mechanism of its pathogenesis was not clear,and it is related to many factors.Recently,more and more evidences showed that apoptosis resistance was closely related to the development of MF and SS.The treatment of CTCL was mainly based on its stages.For the early limitations,local or physical therapy was often used,while patients who were in extension or refractory need to adopt systemic treatment or combined treatment.There was no complete cure method at present.Histone deacetylase inhibitors(HDACi)was a new type of non-cytotoxic targeted therapy drug for tumor,which had a strong anti-tumor effect in vitro and vivo.Currently,two kinds of HDACi,vorinostat and romidepsin,had been approved by the food and drug administration(FDA)for refractory or recurrent CTCL.But because of its low response rate and drug resistance,clinical application had been seriously restricted,so it was very important to find a combination of treatment methods or drugs that can help to enhance the response rate,improve the efficacy,delay or reverse its resistance.Anti-tumor traditional Chinese medicine(TCM)had gradually become a hot spot in the research and development of anti-tumor drugs in recent years because of its multi-link and multi-target influence on the occurrence,invasion and metastasis of tumor,and it is often used as an adjuvant drug for anti-tumor treatment in clinic.TCM monomer referred to the active components with clear chemical structure in TCM.Some of them have obvious anti-tumor activities,such as curcumin,quercetin,emodin,icaritin,resveratrol and ursolic acid.In addition,some monomers could be used in combination with existing anti-tumor drugs to exert synergistic anti-proliferation and pro-apoptosis effects.This research includes the following five parts:Part 1 Screening of traditional Chinese medicine monomer against cutaneous T-cell lymphomaObjective:Cell proliferation was used as the screening model to investigate the effects of six kinds of anti-tumor TCM monomers(quercetin,emodin,icariin,resveratrol,curcumin,ursolic acid)on the proliferation of three kinds of CTCL cells(Myla,Hut-78,HH),and to screen the best TCM monomer,which would be the target monomer in the follow-up study of vorinostat combination.Methods:After three kinds of CTCL cells were treated with six kinds of T CM monomers at concentrations of 10μM,20μM and 40μM for 72h,the optical density(OD)was detected by CCK-8 cell proliferation detection kit,and the percentage of cell viability was converted to evaluate the effect of six kinds of TCM monomers on the proliferation of CTCL cells.Results:For Myla cells,the cell viability of resveratrol and ursolic acid at 20μM was less than 50%,and the cell viability of the other four drugs at 40μM was also less than 50%.For Hut-78 cells,the cell viability of curcumin,resveratrol,icaritin and ursolic acid at 20μM was lower than 50%,and that of quercetin at 40μM was lower than 50%,but that of emodin at 40μM was still higher than 50%.In HH cells,the cell viability of curcumin,resveratrol and ursolic acid at 20μM was lower than 50%,while the cell viability of the other three drugs at 40μM was still higher than 50%.The cell viability of the three kinds of CTCL cells decreased the most after treatment with ursolic acid,and the decreased proportion was greater than that of the other five kinds of TCM monomers.Conclusion:At the same concentration,ursolic acid had a stronger inhibitory effect on the proliferation of CTCL cells than the other five monomers,and Ursolic acid was the target TCM monomer of the combination of vorinostat.Part 2 Effect of ursolic acid combined with vorinostatObjective:To evaluate the effect of ursolic acid combined with vorinostat on the proliferation and apoptosis of CTCL cells.Methods:(1)Myla and Hut-78 cells were treated with ursolic acid with gradient concentrations(100μM,40μM,20μM,10μM,5μM,1μM,0.1μM)for 24h,48h and 72h respectively.The OD was measured,and the inhibition rate and IC50 were calculated to find out the appropriate concentration of the combined drug.After 48h of combined treatment of ursolic acid and vorinostat on Myla and Hut-78 cells,the percentage of cell viability was converted and CI value was calculated to determine the combined effects of the two drugs(additive,synergistic or antagonistic).(2)The apoptosis rate of Myla and Hut-78 cells were detected by flow cytometry after 24h treatment with 10μM,15μM and 20μM respectively.Then,Myla and Hut-78 cells were treated with 10μM ursolic acid and 2/μM vorinostat for 48h.The apoptosis rate and expression of apoptosis related proteins were detected by flow cytometry and western blotting.Results:(1)The IC50 of Myla cells treated with ursolic acid for 24h,48h and 72h were 34.67±7.56μM,16.22±4.09μM,14.45±1.71μM,respectively.The IC50 of Hut-78 cells treated with ursolic acid for 24h,48h and 72h were 22.91±3.24μM,16.22±1.59μM,7.76±0.99μM.The cell viability of CTCL cells treated with ursolic acid and vorinolta was lower than that of the two single drug groups.The CI values of the two drugs in Myla and Hut-78 cells were 0.334 and 0.498(0.3 ≤CI<0.7 was synergistic effect).(2)The total apoptosis rates of Myla cells of control,10μM,15μM and 20μM group were 2.78%,3.30%,44.76%and 69.74 respectively,and the total apoptosis rate of Hut-78 cells was 11.29%,20.39%,65.01%and 85.29%.The total apoptosis rate of the combined group was higher than single drug groups,and the total apoptosis rate of the experimental group was reduced respecti vely from the total apoptosis rate of the corresponding control group,and the total apoptosis rate of the combination group was higher than single drug groups.Western blotting showed that Bcl-2 in the combination group was significantly down regulated,lower than that of the single drug groups,while Bax,Cleaved Caspase-3 and Cleaved PARP were up regulated in the combination group,which was higher than single drug groups,but the above changes were not significant in the Hut-78 cells.Conclusion:Ursolic acid and vorinostat have synergistic effects on the proliferation inhibition and apoptosis promotion of CTCL cells.Part 3 Transcriptome sequencing of cutaneous T cell lymphoma treated with ursolic acid and vorinostatObjective:To explore the molecular mechanism of ursolic acid and vorinostat in inhibiting proliferation and promoting apoptosis by transcriptome sequencing.Methods:(1)Myla cells treated with 10μM ursolic acid for 48 h and control cells were sequenced and analyzed.RT-qPCR and western blotting were used to verify the expression of the key genes and proteins.(2)After Myla cells were treated with 10μM ursolic acid and 2μM vorinostat for 48h,the transcriptome sequencing and data analysis were performed.The expression levels of the key genes and proteins were verified by RT-qPCR and western blotting.Results:(1)2466 differentially expressed genes were obtained by transcriptome sequencing.After further analysis,many signaling pathways related to cell proliferation and apoptosis(TNF-α signaling pathway,NOD-like receptor signaling pathway,Toll-like receptor signaling pathway,MAPK signaling pathway and RIG-I receptor signaling pathway)and some key node proteins(NF-κB family related proteins,proteasome,TNF family proteins)were found.Through the verification of gene and protein levels,the expression of key molecules in these pathways and key node proteins in protein interaction were basically consistent with the sequencing results.(2)Through transcriptome sequencing and data analysis,24 genes and 5 proteins were selected for gene and protein level verification,and the results were basically consistent with the sequencing results.Vorinostat can increase the expression of CKMT1A,while ursolic acid can decrease the expression of CKMT1A.Conclusion:Ursolic acid could significantly inhibit the proliferation and promote apoptosis of CTCL cells,which might be the result of the interaction of TNF-α,NLRP1,JNK,MDA5 and other molecular proteins and signaling pathways.Besides,vorinostat could enhance the expression of CKMT1A in CTCL cells,while ursolic acid could down regulate the expression of CKMT1A,suggesting that CKMT1 A might be a potential target for both of them to exert synergistic anti-CTCL effect.Part 4 Role of CKMT1A in cutaneous T cell lymphoma treated with ursolic acid and vorinostatObjective:To explore the role of CKMT1A in ursolic acid combined with vorinostat in the treatment of CTCL.Methods:(1)Western blotting was used to further investigate the changes of CKMT1A protein expression in Myla cells treated with vorinostat for different time(0h,24h,48h,72h)and different concentrations of vorinostat(0μM,0.5μM,1μM,2μM),the effects of vorinostat on CKMT1A protein expression in different CTCL cells(Myla,Hut-78,HH)and the expression of CKMT1A protein in Myla cells treated with different HDACi(vorinostat,romidepsin,belinostat,panobinostat).(2)CKMT1A substrate(creatine)and substrate analogue(cyclocreatine)were used to investigate the effect of CKMT1A on cell proliferation(cell proliferation experiment).Objective to construct and identify lentivirus mediated CKMTIA gene silencing cell line,and to investigate the effect of vorinostat on apoptosis(flow cytometry)of CTCL cells after CKMTIA gene silencing.(3)The direct interaction between ursolic acid and CKMTIA was analyzed by molecular docking technique.Results:(1)The protein expression level of CKMTIA in Myla cells was increased after treatment with vorinostat of different concentrations and time,and the up-regulation expression of CKMTIA protein was reflected in other HDACi.Except HH cells,vorinostat could increase the expression of CKMTIA protein in Myla and Hut-78 cells.(2)The expression change of CKMT1A could affect the content of its substrate phosphocreatine.Increasing the content of its substrate creatine or its analogue cyclocreatine did not affect the content of its substrate product,so it had no effect on cell proliferation.In the stable transfected CKMT1A silenced cell line,only the silencing of CKMT1A had no effect on cell apoptosis,but the apoptosis rate was significantly increased after stimulation with vorinostat compared with the control group.(3)Three hydrogen bonds were formed between CKMT1A and ursolic acid,and the molecular docking score(binding energy)was-7kcal/mol.Ursolic acid has strong binding activity with CKMT1A.Conclusion:The high expression of CKMT1A induced by vorinostat had a certain effect on the anti-tumor effect of vorinostat in CTCL cells,and the down-regulation of ursolic acid on CKMT1A may be one of the molecular mechanisms of its synergistic anti-CTCL with vorinostat.The binding activity of ursolic acid and CKMT1A is strong,which suggested that ursolic acid can interact with CKMT1A directly,which may affect the expression or function of CKMT1A.CKMT1A might be one of the targets of ursolic acid in coordination with vorinostat in anti-CTCL.Part 5 Extension of molecular docking technology in broad-spectrum anti-proliferative drugObjective:To explore the direct interaction between broad-spectrum anti-proliferative drugs and CKMT1 A,so as to pave the way for systematic research on whether CKMT1A can be selected as a synergistic target of anti-proliferative drugs in the future.Methods:(1)After treated with adapalene for 72h,the OD of the representative cells of various abnormal proliferative diseases(melanoma,gastric cancer,squamous cell carcinoma,tissue cell lymphoma,CTCL,psoriasis)was measured using CCK-8 cell proliferation detection kit,and inhibition rate was converted.(2)The direct interaction between adapalene and CKMT1A was analyzed by molecular docking technique.Results:(1)The proliferation of these cells was inhibited in a dose-dependent manner.The IC50 of different cells were 3.95±0.19μM(A375),1.89±0.22μM(M14),9.6±1,53μM(SGC-7901),7.62±0.72μM(SCC-9),7.17±1.37μM(U937),19.48±4.55μM(Myla),0.72±0.26μM(Hut-78),2.21±0.35μM(HaCat).(2)Molecular docking analysis showed that four hydrogen bonds were formed between adapalene and CKMT1A,and the molecular docking score(binding energy)was-7.3kcal/mol.Conclusion:Adapalene had extensive inhibitory effect on the proliferation of representative cells(including CTCL cells)derived from abnormal proliferative diseases,and had strong binding activity with CKMT1 A,suggesting that CKMT1A might be a target for some anti-proliferative drugs to exert synergistic effect. |
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