The Anticancer Activity Of Histone Deacetylase Inhibitor(IBH589) In Rituximab-resistant Raji Cell

Objective:Since anti-CD20monoclonal antibody-rituximab (Rituximabwas) has been used in the treatment of CD20+B cell lymphoma, no matter asingle agent or with traditional chemotherapy drugs in combination, greatlyimproved efficacy,complete remission rate (CR), overall efficiency rate (OER)and overall survival (OS) of patients.But there are still nearly half of patientswith relapsed or refractory. How to treat rituximab-resistant lymphoma hasbecoming an urgent problem to solve.The histone deacetylase inhibitors (HDACIs) is a kind of novelanticancer drugs by inhibiting histone deacetylase (HDAC) activity.It inducesacetylating histones of target cells, changes binding stasus between histoneand DNA chains and triggers expression of some specific genes. It can inhibitproliferation, induce differentiation and promote apoptosis on a variety oftumor cells including bladder cancer, stomach cancer and other solid tumorsand hematological malignancies such as lymphoma in in vitro experiments[3-5].However, there is no report whether rituximab resistance cell line susceptibleto HDACIs and the mechanisms over the world.This study was designed to investigate the role and possible mechanismsof histone deacetylase inhibitors-LBH589on rituximab resistant humanBurkitt lymphoma cell lines (RRCL).Methods:1Culturing of tumor cells: human Burkitt lymphoma cell lines Raji and RRCLwere cultured in RPMI-1640medium containing10%fetal bovine serum(FBS) at37°C,5%CO2, passaged or changed medium at saturated humidityconditions, and used in the experiments at logarithmic growth phase.2MTT method: The spectrophotometric values (OD) were measured byspectrophotometer testing after being exposed by LBH589on both tumor cells: The logarithmic growth phase Raji and RRCL cells were seeded in96-well plates, five concentrations,(10nM,500nM,5000nM,10000nM,25000nM) which screened by the pre-experiments of LBH589were dilutedand added to96well plates (5wells per concentration). Culturing medium wasused as experimental control. The OD values, and growth inhibition rate oftwo types of cells were tested after24,48and72hours exposion to LBH589with tetrazolium blue (MTT) colorimetric assay.3Apoptosis rate, cell cycle and Bcl-2, Fas proteins' expression levels weretested at three concentrations of LBH589(10nM,500nM,5000nM).4After screening effective concentration with pre-experiments, LBH589werediluted to0.5nM,1nM,2nM, respectively. The two types of cells wereexposed for24-48hours by LBH589. Test the expression of CD20protein byflow cytometry.Result：1MTT assay to test effect of LBH589on Raji cells and RRCLRaji cells and RRCL were exposed by LBH589on different concentrations(10nM,500nM,5000nM10000nM,25000nM) after24h-72h, OD valuesreduced and the inhibition rate increased gradually with MTT method.Different concentrations of LBH589handle the same time, graduallyincreasing the inhibition rates of the two cell lines; the same concentration,processing time, within a certain time frame, with its gradually extended, theinhibition rate of Raji cells and RRCL was increased to varying degrees. Rajicells in the LBH589of five concentration groups, except for500nM and5000nM,5000nM and10000nM, the rest of either of the two groups werestatistically significant (P <0.05), in groups of three different processing time,only between24h and72h groups were statistically significant (P<0.05),LBH589inhibits Raji cells can reach the highest rate (64.03±5.33)%in72h,25000nM condition. For RRCL in different concentrations and time groups,the differencies between any two groups were statistically significant (P<0.05). Under the conditions of72h,5000nM, the LBH589inhibits RRCLcan reach80%. LBH589has anti-proliferative activity on the two cell lines and there is a significant time-dose-dependent relationship.2The expression levels of Bcl-2, Fas and CD20proteins, cell apoptosis andcell cycles were tested by Flow cytometric in two tumors2.1With the increasing of the LBH589concentrations (10nM,500nM,5000nM), apoptosis peak and apoptosis rate were increased in Raji cells, andRRCL cell. Comparing between the blank control group and the experimentalgroup, the difference was statistically significant (P <0.05).2.2With different concentrations (10nM,500nM,5000nM) of LBH589incresed, the G0/G1phase cells increased gradually on Raji cells and RRCLafter24hours, while the proportion of S phase and G2/M phase cells graduallydecreased. LBH589may arrest cell cycle at G0/G1phase.2.3With different concentrations (10nM,500nM,5000nM) of LBH589incresed, the expression of Bcl-2protein gradually decreased, suggesting thatthe LBH589may induce apoptosis by down-regulation of Bcl-2proteinexpression. LBH589, respectively, deal with two kinds of tumor cells6-48hours after Raji cells Fas protein fluorescence channel value graduallyincreased the most significant changes at48h, the control group andexperimental group, the difference was statistically significant (P <0.05); butFas protein on RRCL did not change, suggesting that LBH589inducingapoptosis of Raji cells may be associated with the expression of Fas proteinupregulation, but Fas protein has nothing to do with apoptosis on RRCL.2.4Low concentrations (0.5nM,1nM,2nM) LBH589can increase theexpression of CD20protein after exposed by LBH589during24h to48h.Thecontrol group and experimental group difference was statistically significant(P <0.05).Conclusions:1LBH589can inhibit the in vitro proliferation of Raji cells and RRCL. Thisantiproliferative effect was positively related to time-and dose–dependenceto RRCL;2LBH589can induce apoptosis and arrest cell cycle at G0/G1phase on Rajicells and RRCL; 3LBH589can induce apoptosis of Raji cells, which maybe related to theexpression of Bcl-2protein down-regulation and expression of Fas proteinupregulation. But maybe there is no relationship to Fas on RRCL;4LBH589inhibits RRCL more stronger;5Low-dose LBH589can induce CD20+B-cell lymphoma cell lines CD20protein upregulation, may play a role to overcome rituximab resistanceaspects.