Experimental Study On Bortezomib Combined With Pirarubicin Induced Apoptosis Of T Cell Lymphoma/leukemia In Vitro

Objectives: T cell lymphoma (TCL) is a malignant diseasederived from T lymphocytes, its clinical manifestations are aggressive or highlyaggressive, rapidly progressive and short survival time, poor response to thegenerally antitumor drugs, poor prognosis, and therefore need to find theeffective treatment measures, but there are still no substantive progress so far.Bortezomib，the proteasome inhibitor,can inhibit the expression of genes relatedto cell proliferation and thereby cause the apoptosis of tumor cell by affectingthe intracellular regulatory protein degradation and controlling the NF-κBactivity. Pirarubicin can interfere with the transcription process and mRNAsynthesis by rapidly embedding between DNA nucleic acid base pairs whenenter into cells,, inhibit the growth of tumor cells by affecting the activity ofDNA polymerase which can prevent DNA syntheis. It is confirmed in vitro andmulticenter studies that bortezomib (BTZ), pirarubicin (THP) can inductapoptosis in many solid tumors, but whether bortezomib can induct apoptosison T cell lymphoma and its mechanism is not fully understood, and whetherthere is a synergetic effect in bortezomib and pirarubicin remains to be studiedfurther. This study will explore bortezomib induced apoptosis on lymphomacell line Jurkat cells and primary T-cell leukemia cells, and observe thesynergetic effect in bortezomib plus pirarubicin, to provide the experimentalbasis for the clinical treatment of T cell lymphoma/leukemia. Methods:1.Cell culture and group: Jurkat cells are cultured in vitro and primary T lymphocyticleukemic cells are isolated and cultured with different concentration of BTZ,THP alone and in combination on apoptosis in cells stimulated by24h and48h.2. Test the inhibitory ratio of the cell proliferation by using cell counting Kit(Cell Counting Kit-8, CCK-8), observe the cell morphology by invertedmicroscope, detect the apoptosis of cells by flow cytometry (flow cytometry,FCM),analysis the apoptosis of cells by immune fluorescence, then observeand analysis whether there in synergistic effect between the drugs.3. Statisticalanalysis: use SPSS17.0statistical software for statistical analysis of dataprocessing, data are presented as mean±standard deviation (x’s±s), one-wayANOVA used among the groups,t-test used in pairwise comparisons,inspectionlevel of α=0.05, statistical analysis the probability of P<0.05was statisticallysignificant representation.Results:1.The morphology of the cells whichstimulated by drugs has changed significantly under the inverted phase contrastmicroscope.Cells become dim and dark, shrinkage,their membranes areincomplete and their nuclear are fragmentation.In addition, as the drugconcentration or the role of time increases,the apoptosis rate increases.2.Cellcounting kit were detected and the results show that, there is obvious inhibitedeffect on Jurkat cells and primary T leukemia cell when Bortezomib andPirarubicin used alone, and the inhibition rate was time and concentrationdependent. When treated with different concentrations of bortezomib incombination with small dose of Pirarubicin on Jurkat cells interfere with24h, each increased significantly (P<0.05) compared with bortezomib monotherapygroup.It indicates that there was a good synergistic action when BTZ combinedwith THP; and also perfect synergistic action to primary T leukemia cells. Butthere is no significant difference of inhibited effect of BTZ alone or incombination with THP on Jurkat cells and primary T leukemia cells.3.By flowcytometry the results of apoptosis detection with drug on cells display that:with the concentration increasing, the apoptosis rate was obviously more higherwhile treat Jurkat cells and primary T cells with different concentrations ofbortezomib after24h than the normal control group; and when using differentconcentrations of bortezomib plus pirarubicin for induction of apoptosis in cells,flow cytometry analysis revealed that apoptosis efficiency two drugcombination is higher than that of bortezomib, pirarubicin single drug group(P<0.01).But there is no significant difference between the rate of BTZ-inducedapoptosis of Jurkat cells and primary T leukemia cells.4.Immunofluorescencetechnique showes that after treating with drug, cells apoptosis,then themembrane became incompletement, propidium iodide through the cellmembrane defect and make nuclear coloring.And with the increasing of drugconcentration, the nuclei were stained red phenomenon more obviously whichmeans more apoptosis. Conclusion:1. Bortezomib can be uesd to induceapoptosis in human T cell leukemia cell line Jurkat cells and primary T cellleukemia cells, and the apoptosis rate depends on concentration and time.2.There is a good synergistic action when bortezomib combined with pirarubicin, they can enhance bortezomib-induced apoptosis of Jurkat cells and primary Tcell leukemia cells.3.There are no significant differences between bortezomibmonotherapy and combination used in Jurkat cells and primary T-cell leukemiacells.4.The results provide an experimental basis for bortezomib in the clinicaltreatment of T cell-derived hematopoietic and lymphoid malignancies.