Digoxin Induces Lung Cancer Cell Death By Increasing Generation Of Reactive Oxygen Species

Objective: The digitalis drugs have been widely used for the treatment of cardiac arrhythmias and heart failure for more than two hundrend years. Digoxin is one of the commonly used digitalis drugs in clinic. Epidemiological Studies have confirmed that the recurrence rate and mortality rate were lower in patients treated with digitalis drugs. Many laboratory studies also confirmed that digitalis drugs inhibited the growth of various tumor cells. Oxidative stress plays an important role in cancer treatment. Reactive oxygen species(ROS) caused cell damage by oxidizing nucleic acids, proteins and other macromolecules of tumor cells. ROS can regulate cell proliferation as a signaling molecule involved in intracellular signal transduction. Many anti-tumor drugs kill tumor cells by increasing ROS level. In our study, we intend to study the effect of oxidative stress in anti-tumor effect of digoxin, and further explore the source of reactive oxygen species.Methods:MTT assay was used to test the effect of digoxin and other drugs on cell proliferation of cancer cells. The level of reactive oxygen was estimated using the luorescent dye DCFH-DA and observed under confocal laser scanning microscopy. The level of superoxide anion was analyzed by staining with dihydroethidium probe and observed under confocal laser scanning microscopy. The mitochondrial membrane potential of cancer cells was detected with JC-1 kit. The levels of malondialdehyde(MDA), GSH/GSSG and the activity of NADPH oxidase were detected with corresponding kits. The effect of digoxin on necrosis was detected by flow cytometry(FCM), electron microscopy, and PI staining. The formation of autophagy lysosome was detected with AO staining. Digoxin-induced DNA damage was detected by comet assay. The expression of H2 A.X protein was detected by western analysis.Results:1 The growth inhibition effect of digoxin on different cancer cellsWe examined the growth inhibition effect of digoxin on different cell. A549 cells and HCC827 cells were the most sensitive cells. The IC50 of A549 and HCC827 cells for 24 hours were 0.21 μmol/L and 0.26 μmol/L. The IC50 of TE-1 cells, MCF-7 cells, BGC-823 cells for 24 hours were 1.12 μmol / L, 9.18μmol/L, and 37.14μmol/L.2 Digoxin inhibited the growth of A549 cells by ROS generationDigoxin significantly increased the level of reactive oxygen and superoxide anion in A549 cells(P<0.01). The level of reactive oxygen and superoxide anion gradually increased with the extension of time. The level of mitochondrial membrane potential was decreased with the increase of reactive oxygen species(P<0.01). NAC completely antagonized the increase of reactive oxygen species and superoxide anion, and the anti-cancer effect of digoxin(P<0.01). GSH also partially antagonized the anti-cancer effect of digoxin(P<0.01). Equal concentrations of digoxin caused only a slight increase of ROS in TE-1 cells, and had no influence on the level of ROS in MCF-7 cells and BGC-823 cellsThe increase of ROS induced by digoxin occured earlier than intracellular calcium, which indicated that the increase of ROS caused by digoxin was not due to intracellular calcium. Digoxin at 0.2μmol/L inhibited the growth of A549 cells and had no effect on the activity of Na+/ k+-ATPase, which indicated that the anti-cancer effect of digoxin may be not related to Na +/ k+-ATPase.3 Digoxin induced the increase of ROS from two ways: mitochondria and NADPH oxidaseDigoxin significantly increased the level of MDA content in A549 cells(P<0.01), decreased the ratio of GSH/GSSG(P<0.01), and increased the activity of NADPH oxidase(P<0.01). The anti-cancer effect of digoxin can beantagonized by NADPH oxidase inhibitor diphenylene iodonium hloride(DPI), mitochondrial inhibitor rotenone and the electron transport hain complex I inhibitor cyclosporine A(Cs A)(P<0.01).4 Necrosis and autophagy were involved in the digoxin-induced cell death in A549 cellsFlow cytometry and PI staining experiments showed that digoxin induced cell necrosis(P<0.01). Hoechest 33342 staining experiments showed that the condensed nucleus does not appear in digoxin group. The cells in digoxin group showed typical features of necrosis under the electron microscope: nuclear membrane rupture, organelles dissolution. The effect of digoxin was significantly antagonized by necrosis inhibitor necrostatin-1(P<0.05 or 0.01). The apoptosis inhibitor Z-VAD-FMK and Ac-DEVD-CHO have no influence on the effect of digoxin. Acridine orange staining experiments showed digoxin induced autophagy lysosome in A549 cells. The effect of digoxin was antagonized by autophagy inhibitor 3-MA(P<0.05 or 0.01).5 Digoxin caused DNA damage in A549 cells by ROS generationComet assay results showed that digoxin induced tailing DNA cells in A549 cells(P<0.01). Immunohistochemistry and western blotting results showed that digoxin induced the expression of p-H2 A.X(P<0.01). NAC antagonized the effect of digoxin(P<0.01).6 Digoxin induced HCC827 cells necrosis and autophagy by ROS generationDigoxin significantly increased the level of ROS in HCC827 cells, which could be antagonized by NAC(P<0.01). NAC also antagonized the anti cancet effect of digoxin(P<0.01). These data indicated that digoxin-induced ROS was involved in cell death in HCC827 cells. Digoxin also increased the activity of NADPH oxidase in HCC827 cells(P<0.01). The anti-cancer effect of digoxin was antagonized by DPI, Cs A and rotenone(P<0.01). These data indicated that ROS induced by digoxin was derived from mitochondria and NADPH oxidase.FCM and PI Staining experiments showed that digoxin increased the necrosis rate in HCC827 cells(P<0.01).The necrosis induced by digoxin was antagonized by NAC and necrostatin-1(P<0.01). Digoxin induced autophagy lysosome in HCC827 cells, and autophagy inhibitor 3-MA partially antagonized the effect of digoxin.Conclusions:1 Digoxin has selective killing activity on different cancer cell. A549 cells and HCC827 cells are the two most sensitive cells among all the cells we tested.2 Digoxin increases the level of intracellular ROS from two ways: mitochondria and NADPH oxidase.3 Digoxin induces DNA damage, necrosis and autophagy of lung cancer cells by increasing the level of ROS.