The Anti-tumor And Radio-Sensitizing Effect Of Atovaquone In Non-Hodgkin’s Lymphoma

Objective:To explore the effect of Atovaquone(ATO)on the cell proliferation,cycle and apoptosis of non-Hodgkin’s lymphoma Raji cells,and to clarify the related mechanisms.Moreover,to explore whether Atorvaquone could enhance the radiosensitivity of Raji cells,and to provide a new research strategy for the treatment of non-Hodgkin’s lymphoma.Methods:(1)MTT assay,trypan blue dye and cell colone formation assay were used to evaluate the effect of atorvaquone on the proliferation of Raji cells.(2)Cell cycle distribution of Raji cells was determined using PI staining by flow cytometry.(3)Apoptosis was analyzed using Annexin V/PI double binding assay.(4)The intracellular alterations of reactive oxygen species(ROS)were detected by using DCFH-DA.(5)Western blot was used to detect the protein expression of cell cycle and apoptosis related molecules.(6)Establish the model of non-Hodgkin’s lymphoma cell transplantation in mice to evaluate atovaquone with radiotherapy sensitization effect of non-Hodgkin’s lymphoma.Results:(1)Atorvaquone inhibits the proliferation of Raji cellsVarious concentrations of atovaquone(5-40μmol/L)inhibited the growth of Raji cells in a concentration-dependent manner(r=0.951),and its IC50 value was about23.5μmol/L.The proliferation of Raji cells was significantly inhibited by the treatment with atovaquone(20 and 30μmol/L)for 72 h,which was statistically different with that in the control group(P<0.01,P<0.001).The number of clones in Raji cells treated with atorvaquone(10,20 and 30μmol/L)was significantly lower than that in the control group.That was further proved that atorvaquone could inhibit the proliferation of Raji cells.(2)Atorvaquone induced G1 phase arrest and apoptosis in Raji cellsSignificant G1 phase arrest(P<0.01,P<0.001)and apoptosis(P<0.01)was induced in Raji cells treated with atovaquone(20 and 30μmol/L)for 24 h and 48 h,respectively.(3)Atorvaquone decreased ROS level in Raji cellsThe fluorescence intensity of intracellular DCF probes in Raji cells treated with atorvaquone for 6h decreased in a concentration-dependent manner.(4)Atorvaquone inhibited STAT3 activation in Raji cellsAfter treatment with atovaquone(20 and 30μmol/L)for 24 h in Raji cells,the protein expression of p-JAK2 and p-STAT3(Y705)was significantly decreased(P<0.001,P<0.05).(5)Atorvaquone modulates Raji cell cycle and apoptosis modulates molecular protein expressionAtovaquone(20 and 30μmol/L)treatment induced an apparent decrease in the levels of antiapoptotic protein Mcl-1,Bcl-2,and Bcl-xl(P<0.05)but increase in the level of cleaved-caspase 3 protein expression.In addition,atovaquone induced down-regulation of c-Myc and cell cycle related molecules Cyclin D1,CDK4,and CDK6(P<0.01,P<0.05)protein expression.(6)Atovaquone combined with X-ray inhibited the proliferation of Raji cellsCompared with the 4Gy irradiation group,the 4Gy combined with atorvaquone(20μmol/L)treatment group significantly inhibited the proliferation of Raji cells(P<0.001),suggesting that atorvaquone combined with X ray had synergistically influence on the proliferation of Raji cells.(7)Atorvaquone combined with X-ray increased the apoptosis of Raji cellsCompared with the 4Gy irradiation group,the 4Gy combined with atorvaquone(20μmol/L)treatment group significantly increased the apoptosis of Raji cells(P<0.001),suggesting that atorvaquone combined with X ray had synergistically induced apoptosis in Raji cells.(8)Atovaquone combined with radiation inhibited tumor growth of non-Hodgkin’s lymphoma cell transplantation in mice.Establish the model of non-Hodgkin’s lymphoma cell transplantation in mice to evaluate atovaquone with radiotherapy sensitization effect of non-Hodgkin’s lymphoma.Compared with the radiation group,the group of atorvaquone combined with radiation obviously inhibited tumor growth,suggesting atovaquone has radiotherapy sensitization effect of non-Hodgkin’s lymphoma(P<0.05).Conclusion:(1)Atovaquone effectively inhibits cell proliferation and induces cell cycle of G1 arrest and apoptosis by suppression of STAT3 signaling pathway in Raji cells.It could be a potent therapeutic agent against non-Hodgkin’s lymphoma.(2)Atorvaquone combined with X-ray inhibited the proliferation of Raji cells and induced cell apoptosis.It is suggested atorvaquone may increase radiosensitivity of Raji cells.(3)Atorvaquone combined with radiation inhibited tumor growth in non-Hodgkin’s lymphoma cell bearing mice,suggesting that atorvaquone has radiotherapy sensitization effect on non-Hodgkin’s lymphoma.