Effect Of Chidamide On NK/T Cell Lymphoma Cell Lines

Background:NK/T cell lymphoma(NKTCL),a rare type of non-Hodgkin’s lymphoma(NHL).It is characterized by a poor prognosis and undesirable survival rates.It occurs more common in East Asia and always associated with EBV and rhinitis.The EBV-DNA quantitation can predict the prognosis of this highly aggressive disease.Although multiple systemic chemotherapy and radiotherapy are applied,the overall survival(OS)rate remains low.Recently,PEG-Asp-based DDGP(cisplatin,dexamethasone,gemcitabine,and pegaspargase)regimen explicated impressive clinical outcome due to its tolerable toxicities and high efficacy.New therapeutic method has become the focus of current research.Histone acetylation status is frequently altered in cancer cells.Histone acetylation level is controlled by histone acetyltransferases(HATs)and histone deacetylases(HDACs).Histone deacetylases inhibitors(HDACIs)upregulate acetylation levels of both histone and nonhistone protein,disrupt the histone–DNA affinity and induce a less compacted chromatin leading to increased transcriptional activity and gene expression.Chidamide,the structural analog of MS-275 and a benzamide type HDAC inhibitor targeting HDAC 1,2,3 and10,which is just approved in China for the treatment of recurrent or refractory peripheral T-cell lymphoma(PTCL)in December 2014.Many preclinical studies have demonstrated that Chidamide showed anticancer effect in lymphoma,coloncancer,pancreatic cancer and leukemia.Gemcitabine,a pyrimidine nucleoside antimetabolite agent and a type of specific cell cycle nucleosides chemotherapeutics drugs,mainly inhibits cell DNA synthesis and is clinically applied to a variety of human malignancies,particularly NSCLC.Gemcitabine has frequently been used in combinational treatments with other anticancer agents.Gemcitabine alone or gemcitabine-containing combination is widely used as part of salvage treatment in lymphoma.In recent years,gemcitabine-containing combination chemotherapies are widely used in patients with relapsed or refractory ENKL.Gemcitabine-containing combination chemotherapy DDGP(comprising gemcitabine,pegaspargase,cisplatin,and dexamethasone)showed dramatic effectiveness in NKTCL patients.Gemcitabine has become one of the effective drugs in clinical treatment of NKTCL.In our study,Chidamide increased acetylation of histone H3,caused cell cycle shift and suppressed cell proliferation in a dose-dependent manner.Besides,downregulated Bcl-2 expression decreased mitochondrial membrane potential and caspase-dependent apoptosis were examined,which may contribute to the underlying mechanism.The results also showed another mechanism for the cytotoxicity of Chidamide which is blocking multiple survival signaling pathways.Rhodamine 123 staining was used to detect cell transmembrane potential,flow cytometry was used to detect cell cycle and apoptosis.The results showed that,the cell growth of YTS and NKL cells can be inhibited by chidamide alone,and chidamide joint gemcitabine has a synergistic inhibitory effect on the YTS and NKL cells.After stained by Rhodamine 123,the fluorescence intensity in the combined administration group was dramatically decreased compared to the monotherapy group.And the apoptosis rate of YTS and NKL cells can be significantly increased by chidamide combined with gemcitabine.Western blotting analysis results show gemcitabine synergistically augments chidamide–induced expressions of γH2AX,cleaved Caspase-3 Caspase-9,and cleaved PARP.Herein,we present evidence that Chidamide showing promising activity in treating NK/T cell lymphoma.Our results also demonstrate the significant efficacy and safety of a new paradigm in the treatment of NK/T cell lymphoma,and indicatethe potential of this regimen as a rational therapy against this disease.Objective:To study the effects of chidamide and gemcitabine on human NK/T cell lymphoma cell lines and to investigate its possible mechanisms.Materials and methods:1.Drug:Chidamide(CS055)was supplied by Chipscreen Biosciences.For stock solution,it was prepared in dimethyl sulfoxide(DMSO)(Sigma,USA)at 100 mM and stored at-20°C.Then dissolved in physiological saline for consequent experimental concentrations.Gemcitabine was purchased from Selleck Chemicals(Houston,TX)and dissolved in physiological saline for consequent experimental concentrations.2.Cell lines:NK/T cell lymphoma cell lines(YTS and NKL)was a gift from Professor Scott Kaufmann(Mayo Medical Center,Rochester,MN,USA).YTS were cultured in RPMI-1640(Beijing Solarbio Science and Technology Co.,Ltd.,Beijing,China)and 10% fetal calf serum.NKL were cultured in RPMI-1640 supplemented with10% fetal calf serum and 1,000 U/ml interleukin(IL)-2(Beijing SL Pharmaceutical Co.,Ltd.,Beijing,China).YTS and NKL cell lines were cultured in constant temperature incubator.3.CCK8 Assays: For both two cell lines,16×10 4 /ml cells per well were seeded in 96-well plates for treated with dose-escalation of Chidamide.For combination treatment,both drugs were simultaneously added together in the culture medium.cck8 was then added to each well,the antitumor inter actions between the two agents were determined by calculating combination index(CI)using the CalcuSyn software(Biosoft,Cambridge,UK),CI<1,CI=1,and CI>1 indicate synergistic,additive,and antagonistic effects,respectively.After incubating for 48 h,10ul CCK8 solution was added into each well,the cells were then incubated for2 h.The 96-well plate was read on a multiwell scanning spectrophotometer(Thermo,USA)at a wavelength of 450 nm.The rate of cell proliferation was determined by theequation(1-OD treated /OD control)*100.Results were calculated from three independent experiments.The consequent data were calculated using GraphPad Prism software.4.Cell Cycle and Apoptosis Analyses:Cells were seeded in 6-well plates and were treated with Chidamide or combined with gemcitabine at indicated concentrations for 48 h.Cells were harvested,fixed with cold 70% ethanol,after PI/RNase staining,all the cells were subjected to flow cytometry analysis.Apoptosis Analyses was performed by Annexin V-FITC apoptosis detection kit(KeyGEN BioTECH),as the manufacturer’s recommendations described,then subjected to flow cytometry analysis.5.Measurement of MMP :Cells were treated with chidamide or gemcitabine alone or combined,then washed with PBS and resuspended in RPMI 1640 containing1 μM Rhodamine 123(Beijing Solarbio Science and Technology Co.,Ltd.,Beijing,China)and incubated at 37?C for 30 min in the dark,then observed under fluorescent microscope.6.Western Blot Analysis : After treated with variable concentrations of Chidamide,gemcitabine(individually or combined)for 48 h,the cells were collected and washed with PBS,then lysed in RIPA buffer on ice for 30 min.the protein lysates were centrifuged at 13200 g for 10 min at 4?C to remove cell debris.Then,the protein concentration was determined.After this,equal amounts of protein were separated by SDS/PAGE and then electrotransferred onto a PVDF membrane.The membrane was immunoblotted with anti-acetylated histone H3(ac-H3),-p21,-Bcl-2,cleaved-caspase3,cleaved-caspase9,-parp,?-H2 AX,-GAPDH(Cell Signaling Technology,Danvers,MA,USA).7.Comet Assay:Alkaline Comet Assay was performed using the Trevigen Comet Assay kit(Trevigen,Gaithersburg,MD).Briefly,combine cells at 1 x 10 5 /ml with molten LMAgarose(at 37° C)at a ratio of 1:10(v/v)and immediately pipette 50μl spread onto CometSlide then lysed at 4°C for 1 h.Immerse Comet Slide in Alkaline Unwinding Solution for 20 minutes at room temperature or 1 hour at 4° C,in the dark.The slides were then subjected to electrophoresis(set power supply to 21 volts and apply voltage for 30 minutes at 4°C),gently drain excess electrophoresis solution,immerse twice in dH2 O for 5 minutes each,then in 70% ethanol for 5 minutes.Dry samples at 37° C for 10-15 minutes.Place 10 μl of diluted BD onto each circle of dried agarose and stain 1 minute(room temperature)in the dark.Gently rinse the slide briefly in water.Allow slides to dry completely at 37° C.Images were captured using a Leica fluorescent microscope.8.Statistical Analysis : We use one-way ANOVA to analysis data for the experiments with more than two groups.P < 0.05 was considered statistically significant.Results:1.Chidamide resulted in increased acetylation of histone H3 and showed anti-proliferative effects in NK/T cell lymphoma cell lines.Chidamide,in a dose-and time-dependent manner,strongly inhibited proliferation in both two NK/T cell lymphoma cell lines.The combination group dramatically induced growth arrest.2.Chidamide induced caspase-dependent apoptosis in NK/T cell lymphoma cells.The efficacy of drug-induced cell apoptosis was augmented at elevated concentrations.Increased expression of the proapoptotic markers cleaved PARP,cleaved caspase 3,and cleaved caspase 9 was observed in both cell lines in a concentration-dependent manner.when chidamide and gemcitabine were combined together,an amplifying apoptosis rate and a noteworthy expression of cleaved caspase 9,cleaved caspase 3 and cleaved PARP were observed.3.Chidamide induced cell cycle arrest in NK/T cell lymphoma cells: Chidamide induced G1 arrest and up-regulated p21 expression,whereas the CDK1 protein expression was down-regulated after Chidamide treatment.When chidamide and gemcitabine were combined together,the proportion of both cell lines in S phase increased compared to gemcitabine group.4.Chidamide treatment could caused MMP(membrane potential of mitochondria)loss in NK/T cell lymphoma cells.The decreased fluorescence intensity was accompanied by downregulation of Bcl-2,while the MMP change in combination group was more dramatic.Chidamide and gemcitabine cooperativelyinduced DNA damage as reflected by the abundant expression of γH2AX in NK/T cell lymphoma cells,the comet assay showed the proportion of cells with tail DNA increased following exposure to gemcitabine and chidamide alone,but the tails were not significantly longer than those of control cells.The proportion of cells with tail DNA and tail lengths were even greater in the combination group,whereas the effect was less pronounced in the monotherapy group.5.Chidamide exerted its antitumor effect by regulating multiple signaling pathways.A dose-dependent decrease in the level of p-stat3,P-Erk1/2,p-Akt was observed in both cell lines.In contrast to the decreased phosphorylated protein levels,the total STAT3,Akt,Erk1/2 levels were not notably changed.Conclusion:1.Chidamide inhibits the proliferation of YTS and NKL cell lines;2.Chidamide inhibits malignant NK/T cell growth by inducing cell cycle arrest,MMP loss,caspase-dependent apoptosis and blocking multiple signaling pathways.3.Combined treatment of chidamide with gemcitabine establishes remarkable antitumor effect.