Antitumor Effect Of MS-275 Combined With Cisplatin On Esophageal Squamous Cell Carcinoma

Background and objectiveEsophageal squamous cell carcinoma(ESCC)is a common malignant tumor in the world.Despite significant advances in the understanding,prevention,diagnosis,and treatment of ESCC,most ESCC patients have a poor prognosis and a low five-year survival rate.Chemotherapy is an important method to treat ESCC,but it is easy to cause tumor cell tolerance and normal cell damage,which will affect the therapeutic effect.Cisplatin(DDP)is a first-line chemotherapy drug for the treatment of ESCC.A number of studies have demonstrated that platinum-based chemotherapy regimens have good therapeutic effect in ESCC patients,but the nephrotoxicity and drug resistance are the major obstacles for their clinical use.Therefore,it is necessary and urgent to search safe and effective new drugs to improve the efficacy of DDP and reduce the toxicity and drug resistance of DDP for the clinical treatment of ESCC.Histone acetylation modification is a common epigenetic modification,which is mainly regulated by histone deacetylases(HDAC)and histone acetyltransferases(HAT).A growing number of studies have shown that the high expression of HDAC1 and HDAC2 could lead to tumorigenesis and is associated with poor prognosis.MS-275 is an HDAC inhibitor,which significantly inhibits the expression of HDA1 and HDAC2.Previous studies demonstrate that MS-275 exhibits anti-tumor activity and low toxicity to various tumors,such as liver cancer,breast cancer and colon cancer.So,MS-275 is an antitumor drug with broad application prospects,but its research on ESCC remains unclear.Other studies have shown that DDP could induce cytoprotective autophagy in the treatment of cancer,and the activation of autophagy may be one of the mechanisms for DDP resistance.However,the effect of MS-275 on tumor cell autophagy has not been reported.Here we have five questions:(1)Is there a correlation between abnormal expression of HDAC1 or HDAC2 and clinical pathological parameters of ESCC?(2)Could MS-275 kill the ESCC cells?(3)What about the synergistic anti-tumor effect of MS-275 combined with DDP on ESCC cells?(4)Could MS-275 enhance DDP-induced autophagy?(5)How about the role of autophagy in this combination?In view of the above problems,our study aims to explore the anti-tumor effect and related mechanism of MS-275 alone or combined with DDP on ESCC,which will provide certain scientific bases and strategies for the treatment of ESCC with MS-275 and DDP.MethodsPart ?: Correlation between HDAC1 and HDAC2 expression and clinical pathological parameters of ESCCImmunohistochemical technique was used to detect the localization and expression of HDAC1 and HDAC2 in 64 cases of ESCC patients and their associated adjacent atypical hyperplasia tissues and non-cancerous tissues.The relationship and correlation between protein(HDAC1 and HDAC2)expression and clinical pathological parameters(sex,age,depth of invasion,histological grade,clinical stage,and lymph node metastasis)were statistically analyzed.Part ?: MS-275 induces apoptosis in ESCC cells KYSE-70 and EC9706 cellsWestern blot was used to detect the expression of HDAC1 and HDAC2 in Het-1A,KYSE-70 and EC9706 cells.The effect of MS-275 on the survival,colony formation,apoptosis or cycle,migration and stemness of KYSE-70 and EC9706 cells was respectively detected by CCK-8 assay,plate cloning assay,flow cytometry,Transwell and cell scratch assay,and sphere formation assay.Western blot was used to detect the expression of HDAC1,HDAC2,Cyclin D1,Cyclin A,Cleaved-caspase-3,Bax,Bcl-2,E-cadherin,Vimentin,Fibronectin,CD44 and PI3K/Akt/m TOR signaling pathway-related protein(Akt1,p-Akt1,m TOR,and p-m TOR)after MS-275 treatment.Part ?: Antitumor effect of MS-275 combined with DDP on EC9706 cells and xenograftsCCK-8 assay was used to detect the effect of DDP on the survival of EC9706 cells.In addition,CCK-8 assay,plate cloning assay,flow cytometry,AO-EB staining,mitochondrial membrane potential kit,oxidative stress' kits,transwell and cell scratch test,and sphere formation assay were respectively used to detect the effect of MS-275 combined with DDP on cell inhibition rate,colony formation,apoptosis,cell membrane damage,mitochondrial membrane potential,oxidative stress level,migration and stemness of EC9706.Western blot was used to detect the expression of Bax,Bcl-2,PRDX1,Trx,E-cadherin,MMP-7,c-Myc,Snail,Oct-4 and Sox2.Finally,a nude mouse model of human EC9706 cell xenograft was established,and then treated with MS-275 and DDP;the weight and volume of tumor were respectively measured;necrosis of tumor cells was detected by HE staining;apoptosis of tumor cells was detected by Tunel and immunohistochemistry;the expression of Ki67 in tumor tissues was detected by immunofluorescence.Part ?: Inhibition of autophagy enhances the sensitivity of EC9706 cells to MS-275 combined with DDPWestern blot was used to analyze the expression of autophagy-associated protein(P62 and LC3)in EC9706 cells after treated by MS-275 combined with DDP.The effect of 3-MA and Rapamycin on the survival of EC9706 was respectively detected by CCK-8 assay.In addition,the effect of autophagy on cell survival,apoptosis,mitochondrial membrane potential and migration mediated by MS-275 and DDP was individually examined by CCK-8 assay,flow cytometry,mitochondrial membrane potential kit and transwell assay.Finally,Western blot was used to detect the expresson of P62,LC3,Cyt-C,E-cadherin,Beclin-1,Atg5 and Atg7.ResultsPart ?: Correlation between HDAC1 and HDAC2 expression and clinical pathological parameters of ESCC1.HDAC1 and HDAC2 were mainly localized in the nucleus and highly expressed in ESCC tissues.The expression of HDAC1 and HDAC2 in ESCC tissues were 67.19%(43/64)and 76.56%(49/64)respectively,compared with atypical hyperplasia tissues(HDAC1,43.75%,28/64;HDAC2,48.44%,31/64)and non-cancerous tissues(HDAC1,17.19%,11/64;HDAC2,21.88%,14/64).2.There was no correlation between HDAC1 and HDAC2 expression and age and gender of ESCC patients(both P>0.05).HDAC1 expression was associated with tumor invasion and histologic grade(both P<0.05).HDAC2 expression was associated with clinical stage and Lymph node metastases(both P<0.05).Part ?: MS-275 induces apoptosis in ESCC cells KYSE-70 and EC9706 cells1.Compared with Het-1A cells,the protein expression levels of HDAC1 and HDAC2 were significantly increased in KYSE-70 and EC9706 cells(all P<0.05).2.After MS-275 treatment for 48 h,with the increase of MS-275,the survival and clone formation rate of KYSE-70 and EC9706 was decreased(P<0.05).MS-275 could significantly arrest cell cycle in G0/G1 phase,promote apoptosis and inhibit migration in KYSE-70 and EC9706 cells(all P<0.05).In addition,MS-275 also inhibited the EMT process(all P<0.05)in KYSE-70 and EC9706 cells.3.Compared with CON group,with the increase of MS-275,the expression of HDAC1 and HDAC2 was significantly decreased,while Ac H3 and Ac H2 B expression was significantly increased.In addition,the expression of p-Akt1 and p-m TOR were decreased,while the total expression of Akt1 and m TOR was not significantly changed.Part ?: Antitumor effect of MS-275 combined with DDP on EC9706 cells and xenografts1.There was synergistic anti-tumor effect between MS-275 and DDP.2.Compared with CON,MS-275 and DDP group,MS-275 combined with DDP could significantly inhibit the proliferation of EC9706 cells,induce apoptosis and oxidative damage(all P<0.05),and inhibit cell migration and EMT process(all P <0.05).3.Compared with CON,MS-275 and DDP group,MS-275 combined with DDP could significantly inhibit the growth of EC9706 xenografts,promote tumor necrosis and apoptosis,and inhibit cell proliferation(all P<0.05).Part ?: Inhibition of autophagy enhances the sensitivity of EC9706 cells to MS-275 combined with DDP1.Compared with CON,MS-275 and DDP group,MS-275 combined with DDP could significantly inhibit the protein expression of P62 and increase the ratio of LC3-II/LC3-I(all P<0.05).2.Rapamycin and 3-MA affected the survival of EC9706 cells in a time-and dose-dependent manner(all P<0.05).In addition,compared with MS-275+DDP group,50 n M Rapamycin could significantly inhibit P62 expression and increase the ratio of LC3-II/LC3-I(P<0.05),but 2m M 3-MA could significantly promote the expression of P62 and decrease the ratio of LC3-II/LC3-I(P<0.05).3.Compared with MS-275+DDP group,activation of autophagy by Rapamycin reversed the inhibitory effect of MS-275 in combination with DDP on the survival and migration of EC9706,and resists apoptosis induced by MS-275 and DDP.Inhibition of autophagy by 3-MA could significantly promote the inhibitory effect of MS-275 combined with DDP on the survival and migration of EC9706 cells,and promote cell apoptosis(all P<0.05).Conclution1.HDAC1 and HDAC2 are highly expressed in ESCC and associated with clinical pathological parameters of ESCC.2.MS-275 exerts anti-tumor effect on KYSE-70 and EC9706 cells.3.MS-275 combined with DDP exert synergistic anti-tumor effect on EC9706 cells and xenografts.4.Activation of autophagy to a certain extent can reduce the anti-tumor effect of MS-275 combined with DDP to EC9706 cells;inhibition of autophagy can enhance the sensitivity of EC9706 cells to MS-275 combined with DDP.This results suggest that inhibition of autophagy can be used as an adjuvant measure against MS-275 in combination with DDP.