The Mechanism Research Of Calcium Channel Alpha1C Associated With The Efficacy Of Immunochemotherpy On DLBCL

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common form of theaggressive lymphoma, the treatment and outcome of them has made tremendousprogress in the last decades. Although DLBCL has kinds of subtypes, one thing incommon is that they highly expressed CD20antigenin tumor cells. Rituximab, whichis the anti-CD20monoclonal antibody, is considered as a major breakthrough in thetreatment of B-cell lymphomas.A large number of clinical trials confirmed that thecombination of rituximab with CHOP has significantly improved the response rateand survival rate of DLBCL. the NCCN treatment guidelines set the R-CHOP as thestandard first-line treatment for DLBCL in2003. However, in some patients, theapplication of rituximab did not increase the efficacy of CHOP, these patients showedbe resistance to the regimen or relapse after complete remission in a short time. Earlystudies have shown that Calcium channels and pumps are closely related totheoccurrence and development of tumor, calcium signaling pathways also play a vitalrole in rituximab-related apoptosis:we found that the intracellular calcium was higherin cells which were killed by rituximab.Microarray analysis found that among the patients with different responses to immunochemotherpy, there are differences in theexpression of the calcium channel protein CACNA1C, so the relationship betweencalcium channel protein and the efficacy of immunochemotherpy deserves furtherstudy.ObjectiveThis study aimed to define the relationship between CACNA1C and the efficacyof immunochemotherpy, then find ways to improve its efficacy.Materials and MethodsSelected the111cases of DLBCL patients in affiliated Cancer Hospital ofZhengzhou University from2007-2012, collected the clinical data and pathologicaltissue, all patients were initial treated with R-CHOP as first-line therapy, then dividedinto sensitive group and resistant group accord to the efficacy. Selected two cell linesof DLBCL, OCI-Ly7and OCI-Ly3, OCI-Ly7is sensitive for rituximab whileOCI-Ly3is resistant for rituximab.1. Clinical researchSelect the48cases of DLBCL patients for gene chip, analysis of gene expressionprofiles of the48paitients, there are differences in the expression of the calciumchannel gene between the sensitive group and resistant group, There are obviouslydifference in CACNA1C but no significant difference in CD20expression. Select theleft63cases of DLBCL patients,41cases of sensitive group,22cases of resistantgroup.RNA was extracted from paraffin-embedded tissues, qRT-PCR was used todetect the expression of CACNA1C gene. Immunohistochemical method was used todetect the expression of CACNA1C and CD20in patients.2. experimental research2.1Application of CCK-8detected rituximab andepirubicin acting onproliferation in both cell lines. Filter out the optimal concentration of drugs, provide the basis for post-drug testing.2.2qRT-PCR and Western Blot were applied to detect the expression ofCACNA1C genes and proteins in two cell lines.2.3use Indo1-AM by flow cytometry to test the calcium influx of cell dealedwith rituximab with or without calcium channel blocker nimodipine.2.4Annexin V-FITC flow cytometry detect the apoptosis rateof two cell linesdealed with rituximab and epirubicin, and the role of nimodipine to apoptosis.3. Statistical MethodsSPSS13.0statistical software to analyze the results. Immunohistochemical alldata are mean±standard deviation that were to test its normality, homogeneity ofvariance was used to compare the mean between the t test.Results1． CACNA1C gene expression in the sensitive group of patients was higher thanresistant group, the difference was statistically significant (p=0.025). thepositive rate of CACNA1C protein in resistant group is lower than the sensitivegroup (10/22vs31/41, p=0.021), CD20expression was no significantdifference in the two groups.2． Rituximab and epirubicin act on two cell lines severally, the cell proliferationinhibition rate showed a concentration-dependent, and is not time-dependent,the optimal concentration of rituximab is50μg/ml, epirubicin is50ng/ml.3． Gene and protein expression levels of CACNA1C were significantly higher inOCI-Ly7than OCI-Ly3(19.617±1.627vs5.230±0.120, p=0.001).4． Rituximab joint Fab antibody can induce calcium influx in both cell lines, andthe calcium influx can significantly inhibited by nimodipine, Inhibited intensityof calcium influx was significantly stronger in OCI-Ly7than OCI-Ly3.5． Treated both cell lines with rituximab after48h, OCI-Ly7cells have a higherapoptosis rate than OCI-Ly3cells; treatment by rituximab in combination withepirubicin, nimodipine can significantly reduce both cell lines apoptosis: OCI-Ly7(31.96±5.12vs19.53±5.48, p=0.031) and OCI-Ly3(11.27±5.04vs5.30±2.87, p=0.045).Conclusion1. CACNA1C is positively correlated with the immunochemotherpy of DLBCL,which low expression lead to drug-resistance.2. Rituximab mediates the calcium influx,which results in cell apoptosis, thenimproved the efficacy of chemotherapy; The specific blocker of CACNA1Creduces the apoptosis induced by rituximab, which result in drug-resistance.