Thesis:Research On The Inhibition Effect Of Cardiac Glycosides Of Human Burkitt’s Lymphoma Cell Line (Raji)

Objective:(a) To study the suppressing effect of cardiac glycosides (digoxin, digitoxin, ouabain) on cell viability of human Burkitt’s lymphoma cell line (Raji) in vitro and in vivo.(b) To explore the mechanisms of the anti-tumor effect of cardiac glycosides.(c) To study the side effect of cardiac glycosides (digoxin, digitoxin, ouabain) on human bone marrow mononuclear cells.(d) To find the possible synergistic effect of digitoxin and vinblastine in treating Raji cells.Methods:(a) The human Burkitt’s lymphoma cell line (Raji) was cultured in RPMI1640supplemented with10%FCS. The human bone marrow mononuclear cells were cultured in RPMI1640supplemented with20%FCS.(b) The overall viability of cells treated with cardiac glycosides or vinblastine was assessed with the Cell Counting Kit-8(CCK-8) assay. IC50values as well as the combination index were determined.(c) The apoptosis induction effect of cardiac glycosides on Raji cells was detected by Annexin-V and PI flow Cytometry Assay Kits, and the IC50values of cell apoptosis was determined as well.(d) The expression of c-myc protein in cardiac glycosides treated cells was analysed by western blotting.(e) The c-myc mRNA expression level in cardiac glycosides treated cells was measured by realtime-PCR.(f) To carry out in vivo studies, the human Burkitt’s lymphoma in xenograft mice models were built up and then cardiac glycosides were used to treat them.Results:(a) The IC50values of Raji cells for digoxin, digitoxin and ouabain were82.45±13.14nM,42.62±4.26nM and59.99±9.17nM, respectively. The IC50values of human bone marrow mononuclear cells for digoxin, digitoxin and ouabain were between50nM and500nM.(b) The IC50values of Raji cells apoptosis for digoxin, digitoxin and ouabain were159.12±115.07nM,112.64±52.05nM and109.61±34.70nM, respectively. The IC50values of human bone marrow mononuclear cells for digoxin, digitoxin and ouabain were between800nM and2000nM.(c) Compared with the untreated control group of Burkitt’s lymphoma xenografts mice, the animals treated with digitoxin1mg/kg i.p. injection showed a life-extension (p<0.05), with a median survival time of19days, while the median survival time of control group was11days.(d) Western blotting analysis showed the expression of c-myc protein was significantly reduced after48h cardiac glycosides treatment (low or high concentrations) with dose-dependent manner in Raji cells(p<0.05). However, the protein level of c-myc in human bone marrow mononuclear cells was not markedly changed (p>0.05).(e) Realtime-PCR analysis in Raji cells revealed that mRNA levels of c-myc had no significant change after48h cardiac glycosides treatment (digoxin50ng/ml, digitoxin50ng/ml, ouabain50nM,ouabain100nM)(p>0.05).(f) Digitoxin and vinblastine combination treatment had an additive effect or a moderate synergy effect on Raji cells.Conclusion:(a) Cardiac glycosides at low concentrations can suppress the cell viability of human Burkitt’s lymphoma cell line (Raji) in vitro, both repress cell proliferation and induce cell apoptosis.(b) Digitoxin (i.p. injection) can prolong the life span of Burkitt’s lymphoma xenografts mice.(c) Cardiac glycosides can reduce the c-myc protein level in Raji cells in a dose-dependent way, but have no impact on the level of c-myc mRNA.(d) Cardiac glycosides at low concentrations induced no significantly changes in cell proliferation, apoptosis or expression of c-myc protein in human bone marrow mononuclear cells.(e) Digitoxin combined with vinblastine can exert an synergistic effect on Raji cells.