Effects Of Digoxin Exerts In Raji Cells And Detection Of The Possible Mechanism

Background:Burkitt’s lymphoma is a highly malignant B-cell non-Hodgkin’s Lymphoma.Digoxin,a cardiac glycoside,has recently been shown to induce an inhibitory action on human tumor cell lines,but it’s still not known on Burkitt’s lymphoma.In the present study we intended to evaluate the antitumor properties of digoxin on Burkitt’s lymphoma Raji cells and the possible mechanisms.Methods:In vitro:After the co-culture of Raji cells with digoxin,we(1.)observedcultured-Raji cells directly with an inverted microscope to evaluate the cell mass in the digoxin group and the control;(2)investigated cell viability using the Cell Counting Kit-8(CCK-8)assay;(3)analyzed the DNA synthesis by Click-iT(?)EdU assay;(4)detected the cell cycle population by Cycle TESTM PLUS DNA Reagent Kit;(5)evaluated the morphological changes of apoptotic cells by Giemsa staining;(6)measured the apoptosis by Annexin V-FITC/PI doubling detection method;(7)analyzed protein levels of p21cip1,cyclin D1,c-myc,and caspase-3,-8,-9 and their active forms,as well as protein Bcl-2,Bax,and protein p65,p-p65,IκBα,p-IκBαusing western blot.In vivo:We established a BALB/c-nu mouse model of Burkitt’s lymphoma by injecting Raji cells subcutaneously.To study the effects of digoxin on xenograft tumors,digoxin was injected into the abdominal cavity daily since the size of the xenograft reached the tumor criteria.Mice weight and volume of xenograft tumors were measured twice a week.The mice sacrificed 21 days later.The peripheral blood smears,xenograft tumors and important organs of mice were retained for further studies:(1)Wright Giemsa staining for the peripheral blood smears;(2)HE staining;(3)detect human CD20 of important organs using immunohistochemical staining;(4)immunohistochemical staining was used to detect CD20,Ki-67 and c-myc protein levels of xenograft tumors.Results:In vitro:We found that(1)digoxin reduced the size of Raji cells mass;(2)cell viability decreased obviously in a dose-and time-dependent manner;(3)DNA synthesis capacity decreased;(4)the cell cycle assay showed the G0/G1 phase arrest;(5)Giemsa staining showed that nuclear membrane was ruptured and the condensation and margination of chromatin was found in the apoptotic Raji cells treated with digoxin;(6)cell populations of the digoxin group in both early and late apoptosis stage increased,and most apoptotic cells were in the early apoptosis stage;(7)digoxin decreased cyclin D1 and c-myc protein levels,on the contrary,increased the p21cip1 protein level.Besides,digoxin increased the cleavage of caspase-9 and caspase-3,but not caspase-8.Also,the Bcl-2/Bax ratio decreased.Digoxin suppressed the protein distribution of p65 in the nucleus and the cytoplasm,reduced p65,p-p65 and p-IκBα levels,however,increased IκBα level,while the expression of RelB remained stable.In vivo:We found that(1)digoxin suppressed the growth of subcutaneous tumors,but exerted no effect on mice weight;(2)there was no existence of CD20+ Raji cells in the peripheral blood smears and the tissue slice of important organs;(3)digoxin reduced the protein expression of CD20,Ki-67 and c-myc in tumor tissues.Conclusions:These findings suggest digoxin has an antitumor effect in Raji cells,including the anti-proliferation and pro-apoptosis aspects,and this effect may be achieved partly through G0/G1 phase arrest and the alteration of cell cycle regulatory proteins p21cip1,cyclin D1 and c-myc.The activation of caspase-3,-9,the reduction of Bcl-2/Bax ratio,as well as the down-regulation of the NF-κB pathway may also contribute to the mechanism.Besides,digoxin inhibits the growth of Raji cells in nude mice and reduces the levels of Ki-67 and c-myc in tumor xenografts.