The Clinical Efficacy And Biological Activity Of Decitabine In The Treatment Of Diffuse Large B-cell Lymphoma

BackgroundLymphoma is a malignant tumor originating from the lymphatic hematopoietic system.The clinical manifestations mainly include painless lymphadenopathy,hepatosplenomegaly.Almost all the organs of body can be involved by lymphoma cells,and patients are often accompanied by systemic symptoms such as fever,night sweats,weight loss,and itchy skin.There are two subtypes of Hodgkin’s lymphoma（HL）and Non-Hodgkin’s lymphoma（NHL）.NHL can be divided into B-cell lymphoma and T/NK cell lymphoma according to biology source of the cell.The most common pathological type is Diffuse Large B Cell Lymphoma（DLBCL）.The standard treatment regimen is rituximab,one type of CD20 monoclonal antibody,combined with CHOP（cyclophosphamide,doxorubicin,vincristine,prednisone）.Lymphoma is one of malignant disease that may be cured.What’s more,many new treatments emerging prominently in recent years,such as Chimeric Antigen Receptor（CAR-T）therapy and target drugs,are potentially effective,but there are still many refractory and relapsed lymphoma patients who have no response to available treatments in the clinic.So it is necessary to find new anti-tumor drugs and treatments for refractory and relapsed patients.Decitabine（DAC）is a nucleotide analogue which can be directly incorporated into DNA by phosphorylation of deoxycytidine kinase inside tumor cells,and reverse DNA methylation by inhibiting DNA methyltransferase.DAC can not only induce the differentiation or apoptosis of tumor cells,but also exhibit various anti-tumor activities.It has been applied to the treatment of more and more tumors recently.At present,domestic and foreign literatures document that DAC may has good application potential in lymphoma treatment,but most of these researches had limitedly focused on biological studies such as cytotoxicity and pro-apoptosis.So far,there are few basic and clinical studies,although some studies have confirmed the methylation level of some genes may be involved in the mechanism of action of DAC in lymphoma cells.But in DLBCL patients,especially relapsed and refractory ones,it is still unclear which genes or epigenetic indicators are involved in the mechanism of action of DAC.Studies have shown that low concentrations of DACs can lead to demethylation,and high concentrations of DACs lead to cytotoxicity without a clear-cut mechanism.Literature documented that microRNAs may be involved in the demethylation process of DAC.MicroRNAs are types of endogenous,small-sized,non-coding single-stranded RNA that can be involved in the regulation of various biological processes such as cell cycle,apoptosis,differentiation,proliferation,and immune response.Among these microRNAs,miR-196b has dual roles of tumor suppressor and carcinogenesis.In laryngeal squamous cell carcinoma,the high expression of miR-196b promotes the proliferation,migration and invasion of laryngeal squamous cell carcinoma.On the contrary,over-expression miR-196b in breast cancer could significantly inhibit the proliferation and migration of breast cancer cells MDA-MB-231 and MDA-MB-468.It also induced cell cycle arrest and apoptosis in acute B-cell lymphobalstic leukemia.The c-myc gene plays an important role in cell proliferation,differentiation and apoptosis.Studies have shown that c-myc might be a target gene of miR-196b,and miR-196b can inhibit the expression of c-myc gene in acute B-cell lymphobalstic leukemia.Other studies have found that the down-expression of miR-196b by hypermethylation of CpG islands in endometriosis might promote the expression of c-myc.Combined with these above studies,we speculate that the high expression of c-myc might lead to silencing of miR-196b hypermethylation in DLBCL cells.Hereby,this study is divided into clinical and basic experiments:10 cases of DAC combined with ESHAP（etoposide 60mg/m²,cytarabine 100mg/m²,cisplatin20mg/m²,dexamethasone 20mg）or DHAP（cytarabine 2g/m²,cisplatin 20mg/m²,dexamethasone 20mg）regimen were collected to evaluate clinical efficacy.In our experiment,different concentrations of DAC were selected to coculture with DLBCL cell line LY8 to observe the effects of proliferation,apoptosis and expression of c-myc protein and miR-196b,investigate whether the high expression of c-myc is related to the hypermethylation of CpG islands in promoter regin of miR-196b,observe the synergy effect of DAC with traditional chemotherapy drugs,and detect the methylation level of CpG islands promoter regin of miR-196b in LY8 cells,which provided a theoretical basis for usage of DAC in treatment of DLBCL.Objects1.A retrospective analysis of 10 patients with relapsed and refractory DLBCL treated with DAC combined with ESHAP/DHAP regimen was performed to evaluate the short-term clinical efficacy of DAC in the treatment of relapsed and refractory DLBCL.2.To analyze the effects of DAC on the proliferation,apoptosis,c-myc protein,expression of miR-196b and the methylation of CpG islands in promoter regin of miR-196b in DLBCL cell line LY8,explore the biological activity and mechanism of DAC in DLBCL,and observe DAC and traditional chemotherapy drugs,including etoposide,cytarabine,cisplatin and dexamethasone,have a synergistic effect with DAC,providing a theoretical basis for the clinical application of DAC and the above drugs.Methods1.The clinical data of 10 patients with relapsed and refractory DLBCL who underwent DAC combined with ESHAP/DHAP regimen were collected from the lymphoma clinic of the First Affiliated Hospital of Zhengzhou University.Retrospective analysis was performed to evaluate the clinical efficacy.2.The CCK-8 method was used to measure the effect of different concentrations（0.1,1,10,100μmmol/L）of DAC on the proliferation of DLBCL cell line LY8.The IC50 value of DAC was calculated by SPSS 21.0 statistical software.Carry out subsequent experiments.3.Flow cytometry was used to measure the effect of DAC on apoptosis of LY8cells.4.Western Blot was used to detect the expression of c-myc protein before and after DAC treatment of LY8 cells.5.The expression of miR-196b in LY8 cell line before and after DAC was detected by qRT-PCR.6.MSP（methylation specific PCR）method was used to detect the methylation level of CpG islands in promoter regin of miR-196b before and after DAC treatment;7.CCK-8 method was used to detect the IC₅₀ value of traditional chemotherapy drugs etoposide,cytarabine,cisplatin and dexamethasone on LY8,and the inhibition rate of LY8 cells combined with DAC,calculate DAC and traditional chemotherapy synergy index between drugs.Results1.A total of 10 patients with relapsed refractory DLBCL were enrolled,and 8patients were included in the analysis.The total response rate（ORR）was 62.5%,and2 patients（25%）achieved complete response（Complete Response,CR）.Two patients（25%）achieved progressive response（PR）,one patient（12.5%）had stable disease（Stable Disease,SD）,and only one patient was terminated due to heavier bone marrow suppression.2.The inhibitory rates of 0.1,1,10,100μmol/L DAC on LY8 cells at 24h,48h and 72h were（4.92±0.21）%,（15.15±0.66）%,（20.82±0.39）%;（14.88±4.3）%,（35.4±5.14）%,（55.99±3.18）%;（28.47±1.93）%,（48.49±3.18）%,（62.66±2.66）%;（28.48±1.94）%,（52.86±2.54）%,（63.39±1.92）%.The IC50 value of the DAC on LY8was 10μmol/L,and subsequent experiments were carried out with 10μmol/L DAC.The apoptotic rates of the blank control group,24h,48h and 72h were 1.90±0.60)%、（21.29±2.10）%、（46.00±1.45）%、（49.63±2.23）%,respectively.The expression of c-myc protein in LY8 cell line after 10μmol/L DAC was significantly lower than that before treatment,p<0.05 between the two groups;the relative expression of miR-196b was significantly increased after treatment.The p<0.05 between the groups;the methylation level of the CpG islands in promoter regin of miR-196b was significantly lower than that before the action.The IC50 values of traditional chemotherapy drugs etoposide,cytarabine,cisplatin and dexamethasone in LY8 cell lines were 283 ng/mL,12.15 ng/mL,65 ng/mL,216.1μg/mL,respectively.The synergy index between drug and DAC was 0.88,0.92,0.78 and 0.83,respectively.Conclusion1.DAC has effective clinical therapeutic activity and application potential in the treatment of relapsed and refractory DLBCL.2.DAC inhibited proliferation and induced apoptosis of DLBCL cell line LY8 in a dose-and time-dependent manner.3.DAC can inhibit the expression of c-myc protein by decreasing the methylation level of CpG islands in promoter regin of miR-196b,and has a good synergistic effect with traditional chemotherapy drugs.