Anti-proliferation Effect And Mechanism Of Chidamide Alone Or Combined With Bortezomib On Multiple Myeloma Cells

Multiple myeloma(MM)is a kind of malignant plasma cell tumors.Although given a comprehensive treatment-of traditional chemotherapy drugs,protease inhibitors(bortezomib),immune modulators(thalidomide,lenalidomide)and autologous stem cell transplantation,most patients still can not avoid disease recurrence or progression.Therefore,the search for more effective drugs or drug combination has been a research hotspot in the MM treatment field.Histone epigenetics is a research hotspot in genetics.Histone acetylation,methylation,ubiquitination and phosphorylation modify gene transcription by changing the mutual affinity between histone and DNA,in which histone acetylation is of particular concem.Many studies have shown that the occurrence and development of·tumors are closely related to the acetylation status of histones.Histone acetylation enzyme inhibitors(HDACi),as a new anticancer drug targeting histone deacetylase(HDAC),have attracted more and more attentions.So far,the FDA have approved vorinostat(SAHA),belinostat and and romidepsin for the treatment of recurrent or refractory peripheral T-cell lymphoma(PTCL),have approved panobinostat for the treatment of recurrent or refractory MM.Chidamide is the first self-developed selective HDACi'.in China,which has achieved good therapeutic effects in the treatment of recurrent or refractory PTCL.Chidamide was approved by CFDA for the treatment of recurrent or refractory PTCL.In recent years,researchers have applied Chidamide to diffeirent types of tumor cell lines,and a large number of studies have shown that Chidamide has strong anti-tumor activities against breast cancer,prostate cancer,colon cancer,lung cancer,liver cancer,lymphoma and other tumors.HD AC is overexpressed in multiple myeloma,and the prognosis of patients with HDAC overexpression is poor,and what's more,HDACi(vorinostat and panobinostat)alone or in combination with bortezomib has a good anti-myeloma effect.Therefore,we speculated that Chidamide may be an effective drug in the treatment of multiple myeloma and have synergistic anti-myeloma effect with bortezomib too.Thus,we selected multiple myeloma cell lines RPMI8226 and U266 as the study subjects to observe the anti-myeloma effect of Chidamide alone or combined with bortezomib and to explore the possible mechanisms.This research was divided into three parts:Part ?:Anti-proliferation effect and mechanism of Chidamide on multiple myeloma cellsPart ?:Chidamide sensitizes multiple myeloma cells to bortezomib and its mechanisms.Part ?:Whole transcriptome sequencing revealing the possible mechanisms of anti-myeloma effect of Chidamide.Part ? Anti-proliferation effect and' mechanism of Chidamide on multiple myeloma cellsObjective:We sought to investigate the effect of Chidamide on.the growth inhibiton and apoptosis induction of human multiple myeloma cells and to explore the relevant mechanisms.Methods:1.CCK-8 assay was used to detect the inhibitory effect of Chidamide on human myeloma cell lines RPMI8226 and U266.2.Flow cytometry with PI staining was performed to characterize cell cycle profile after Chidamide treatment.3.Wright-Giemsa staining was used to observe the morphological changes of apotosis upon Chidamide treatment.Flow cytometry with AnnexinV/PI double staining was used to detect the ratio of apoptosis after Chidamide treatment.4.Western Blot was used to measure the expression of histone acetylases,cell cycle-related and apoptosis-related proteins after Chidamide treatment.Results:1.Concentration of 0.5-8?mol/l Chidamide inhibited the proliferation of RPMI8226 and U266 cells in a dose-and time-dependent manner.The IC50 values of Chidamide on RPMI8226 cell lines at 24h,48h and 72h were 9.09±0.58?mol/l,1.19±0.36?mol/l and 0.77±0.21?mol/l(p<0.001),respectively.The IC50 values of Chidamide on U266 cell lines at 24h,48h and 72h were 16.16±2.51?mnol/l,8.06±1.02?mol/l and 2.86±0.58?mol/l(p<0.001),respectively.2.After treatment with 2?mol/l Chidamide for 48h,the percentage of RPMI8226 cells in the G1 phase increased from 28.12±1.50%to 42.42±4.80%(p<0.05),and the percentage of U266 cells in the G1 phase increased from 42.31±1.43%to 71.25±4.65%(p<0.05)after treatment with 8 ?mol/1 Chidamide for 48h.3.The total apoptosis rate of RPMI8226 cells increased from 7.12±2.50%to 31.51±4.52%(p<0.05)after treatment with 2 ?mol/l Chidamide for 48h.The total apoptosis rate of U266 cells increased from 6.82±2.41%to 18.21±3.51%(p<0.05)after treatment with 8 ?mnol/l Chidamide for 48h.Moreover,morphological changes of apoptotic characteristics were observed under light microscope in both cell lines afterChidamide treatment.4.Chidamide can significantly down-regulate the expression of HDAC1,HDAC2 and HDAC3,thereby up-regulating the acetylation level of H3 and H4,and can activate caspase-3,caspase-8,caspase-9 and PAPR.5.Following the treatment of Chidamide,On one hand,p53 phosphorylation was increased,c-myc and cyclinDl were significantly down-regulated,and p21 expression was significantly up-regulated;On the other hand,Bax was significantly up-regulated,Bcl-2 and mcl-1 were significantly down-regulated,leading to the increased Bax/Bcl-2 ratio.Conclusion:Chidamide inhibited the proliferation of MM cells by caspase dependent apoptosis and p53-dependent G0/G1 cell cycle arrest after increasing acetylation levels of H3 and H4.Chidemide is a promising drug against myeloma.Part ? Chidamide sensitizes multiple myeloma cells to bortezomib and its mechanisms.Objective:To investigate the combination effects of Chidamide and bortezomib on myeloma cells and to explore the relevant mechanisms.Methods:1.CCK-8 assay was used to detect the inhibitory effect of the combination of bortezomib and Chidamide on human myeloma cell lines RPMI8226 and U266.2.Flow cytometry with PI staining was performed to characterize cell cycle profile upon combinedtreatment.3.Flow cytometry with AnnexinV/PI double staining method was used to detect the ratio of apoptosis after combined treatment.4.Western Blot was used to measure the expression of histone acetylases,cell cycle related and apoptosis related proteins after combined treatment.Results:1.Combining bortezomib with Chidamide for 48h has a remarkable effect on proliferation inhibition of RPMI8226 and U266 cells compared with the control group.The inhibitory rate of 0.5?mol/1 single Chidamide on RPMI8226 cells for 48h was 18.02±3.74%.In RPMI8226 cells,combined with 0.5?mol/l Chidamide for 48h,the inhibition rate of bortezomib(1.25ng/ml,2.5ng/ml,5ng/ml)increased from(31.35±5.94%,85,71±4.33%,96.88±2.77%)to(65.40±3.35%,96.70±2.65%,98.35±1.14%)(p<0.05),respectively,with the combined index<1.The inhibitory rate of 2?mol/l single Chidamide on U266 cells for 48h was 21.91±2.45%.In U266 cells,combined with 2?mol/l Chidamide for 48h,the inhibition rates of bortezomib(1.25ng/ml,2.5ng/ml,5ng/ml)increased from(3.87±1.79%,28.48±5.63%,55.26±4.97%)to(35.51±3.14%,69.34±8.61%,89.21±1.35%)(p<0.05),respectively,with the combined index<1.2.The cell cycle distribution of bortezomib combined with Chidamide was not obviously changed when compared with that of bortezomib alone.In RPMI8226 cells,the percentage of cells in the G2 phase increased from 12.83±1.10%to 42.09±19.81%after treatment with 1.25ng/ml bortezomib for 48h(p<0.05),and the percentage of cells in the G2 phase was 51.900±14.12%after combination of 0.5p?mol/l Chidamide for 48h,which was not different from that of bortezomib alone(p>0.05).In U266 cells,the percentage of cells in the G2 phase increased from 19.70±5.49%to 46.86±11.65%(p<0.05)after treatment with 2.5ng/ml bortezomib for 48h,and the percentage of cells in the G2 phase was 42.98±5.57%after combination of 2?mol/l Chidamide for 48h,which was not different from that of bortezomib alone(p>0.05).3.The apoptosis rate of bortezomib combined with Chidamide was significantly increased.The total apoptosis rate of RPMI8226 cells treated with the combined group for 48h was 52.14±5.23%,which was higher than that of Chidamide or bortezomibalone with total apoptosis rate of 18.34±2.23%and 36.23±2.45%(p<0.05),respectively.The total apoptosis rate of U266 cells treated with the combined group for 48h was 35.42±4.54%,which was higher than that of Chidamide or bortezomib alone with the apoptosis rate of 8.94±3.16%and 23.28±3.57%(p<0.05),respectively.4.Chidamide can sensitizes bortezomib down-regulateing HDAC expression,maintaining H3 and H4 high acetylation levels,and activating caspase-3,caspase-8,caspase-9 and PAPR.5.After treatment with the combination of bortezomib and Chidamide for 48h,on one hand,the expression of Bax was significantly up-regulated,while the expression of bcl-2 and McL-1 was significantly down-regulated,which increased the ratio of Bax/bcl-2.On the other hand,the expression of cyclin B was down-regulated and the expression of p21 was up-regulated.Conclusion:Chidamide sensitizes bortezomib proliferation inhibition and apoptosis induction on RPMI8226 and U266 cells.This study provides experimental basis for the clinical apply of the combination of Chidamide and bortezomib for the treatment ofMM.Part III Whole transcriptome sequencing revealing the possible mechanisms of anti-myeloma effect of Chidamide.Objective:To reveal the possible mechanisms of anti-myeloma effect of Chidamide by whole transcriptome sequencingMethods:RPMI8226 cell lines were.treated with 1?mol/l Chidamide or equal volume cell culture medium for 24h.RNA was extracted by TRIzol method and then the samples were sent to Novogene Company to perform the whole transcriptome sequencing.Illumina sequencing was used to detect differentially expressed genes.ClusterProfiler software was used for the Gene Ontology(GO)function enrichment analysis,KEGG pathway enrichment analysis,and Reactome pathway enrichment analysis.Cytoscape'software was used to analyze the protein-protein interation.Ten genes with obvious expression differences were selected and verified by qRT-PCR.Results:1.With padj<0.05,RNA-seq revealed that a total of 463 differentially expressed genes were detected in RPMI8226 cell lines after treatment with 1?mol/1 Chidamide for 24h,when compared with the control,including 457 up-regulated genes and 6 down-regulated genes.The qRT-PCR results of the 10 genes were basically consistent with whole transcriptome sequencing.2.Enrichment analysis with Gene Ontology(GO)showed that chemical signal transmission,synaptic formation,cell morphology,cell growth signal regulation,and intercellular interaction were the most important aspects.According to KEGG Enrichment analysis,there were 8 signaling pathways enriched,including axon guidance,PI3K/AKT signaling pathway,extracellular matrix receptor interaction,cell adhesion molecules,cancer-related proteoglycan and so on.Enrichment analysis with Reactome showde extracellular matrix metabolism,axon guidance signal,nervous system diseases,and activation of matrix metalloproteinase were the most important aspects.3.There were 23 differentially expressed genes related to the PI3K/AKT signaling pathway,all of which were up-regulated genes.There were 16 differentially expressed genes related to axon guidance,all of which were up-regulated genes.4.Seven important genes were found through protein-protein interaction analysis,namely SRC,MMP9,MMP2,NOTCH3,MYH14,GNAO1 and RHOC.Conclusion:Chidamide changes multiple signaling pathways arid multiple biological functions in multiple myeloma cells,and we should pay special attention to PI3K/AKT signaling pathway and axon guiding pathway.Conclusions1.Chidamide inhibites the proliferation and induces apoptosis of MM cells.2.Chidamide induces caspase dependent apoptosis and p53-dependent G0/G1 cell cycle arrest after increasing acetylation levels of H3 and H4.3.Chidamide sensitizes bortezomib proliferation inhibition and apoptosis induction on RPMI8226 and U266 cells.4.Chidamide sensitizes bortezomib to induce caspase dependent apoptosis.5.Chidamide changes multiple signaling pathways and multiple biological functions in multiple myeloma cells,and we should pay special attention to PI3K/AKT signaling pathway and axon guiding pathway.6.Chidemide is a promising drug against myeloma.