Study On The Mechanism Of Valinomycin-induced Apoptosis In HeLa Cells

Valinomycin,a K⁺ selective ionophore,serves to dissipate the membrane potential of respiring mitochondria.It has been reported that loss of mitochondrial membrane potential is observed in the early stages of apoptosis induced by various agents.Its effect as an apoptosis inducer was reported in the late 1980s to early 1990s. It has been shown to induce apoptosis in several cell types including neurons, thymocytes,ascites hepatoma cells,pulmonary artery myocytes.In this study,we attempted to evaluate in detail the signaling cascades resulting in Valinomycin -induced HeLa cells apoptosis,focusing particularly on mitochondrial changes and the role of p38 mitogen-activated protein kinase activation.We first testified the antitumor activity of Valinomycin against HeLa,HepG2, Raji,MG-63 and KB cell lines,and the IC₅₀ values were 4.2μg/mL,1.67μg/mL,0.8 ng/mL,3.1μg/mL and 1.5μg/mL,separately.We found that Valinomycin induced HeLa apoptosis was correlated with the depolarization of mitochondria and the regulation of Bcl-2 family proteins(Bax and Bcl-2),involved the generation of reactive oxygen species,the release of Cytochrome C from mitochondria,and the activation of caspase and p38 MAPK pathway,but no obvious activation of JNK MAPK.Caspase inhibitor(Z-VAD-FMK) induced a partially decrease of Valinomycin-induced apoptosis,p38 MAPK inhibitor, SB203580,also reduced an attenuation of Valinomycin-induced apoptosis and the activation of caspase-3.Collectively,our findings indicate that p38 MAPK and caspase signaling cascade are involved in the Valinomycin-induced HeLa cells apoptosis.Moreover,we examined the cross-talk between p38 MAPK and mitochondria -mediated caspase pathway.After treated with SB203580,caspase-9 activation was block with the decrease of p38 MAPK activation,while the expression of Bcl-2 family proteins(Bcl-2 and Bax) were not affected and the release of Cytochome C were not blocked.It illuminated that the activation of p38 MAPK affects caspase-3 activation,but not Bcl-2 family proteins and the release of Cytochome C. Overall,we demonstrated that the caspase and p38 MAPK were involved in Valinomycin-induced HeLa cells apoptosis.These results indicated that Valinomycin activates two parallel caspase activation pathways,i.e.,Valinomycin induces the disruption of mitochondrial potential,followed by the release of Cytochorme C relased.Meantime,Valinomycin activates p38 pathway which crosstalks to the mitochondrial pathway,both affect the activation of downstream caspase-9 and caspase-3 simultaneously.In addition,we studied the apoptosis of HepG2 cells induced by Valinomycin. We found that Valinomycin activated the expression of p38 and JNK,but the block of p38 and JNK pathways by SB203580 and SP600125 did not attenuate Valinomycin -induced apoptosis,indicating that p38 and JNK pathways did not involve in this apoptosis.However,more researches are needed to elucidate the apoptotic pathways induced by Valinomycin in HepG2 cells.