The Synergistic Inhibitory Effects Of A PLK1 Inhibitor And An HSP90 Inhibitor Against Proliferation Of Gastric Cancer Cells

Objective:To investigate the inhibitory effect of PLK1 inhibitor Volasertib and HSP90 inhibitor PU-H71 on the proliferation of gastric cancer cells,and to study the possible mechanism of the synergistic apoptotic induction effects of the combination of these two chemicals.Methods:(1)MTT assay was used to assess the proliferation inhibitory effects of Volasertib and PU-H71 in gastric cancer cells;(2)The method of colony formation was used to evaluate the influence of the chemicals on colony formation ability of MGC-803 cells;(3)Annexin V-FITC/PI staining was used to detect the apoptosis-induction effects of Volasertib and PU-H71 on MGC-803 cells;(4)Annexin V-FITC/PI staining was used to evaluate the contribution of the caspases to the apoptosis-induction effects of Volasertib;(5)JC-1 staining was used to evaluate the influence of Volasertib on mitochondrial membrane potential;(6)Western blot Analysis was used to detect the influence of Volasertib and PU-H71 on apoptotic signal pathways;(7)High Content Analysis(HCA)and Western blot methods were used to measure the influence of Volasertib and PU-H71 on DNA damage response;(8)Western blot Analysis was used to evaluate the contribution of reactive oxygen species to the effects of Volasertib;(9)Colorimetry method was used to assess the production of superoxide anion radicals triggered by Volasertib and PU-H71.Results:(1)Volasertib and PU-H71 obviously inhibited proliferation of gastric cells,the combination of the two chemicals caused synergistic effects;(2)MGC-803 cells were mainly arrested in G2/M phase by Volasertib;(3)Volasertib dose dependently decreased the mitochondrial membrane potential and induced apoptosis in MGC-803 cells;(4)Volasertib modulated the expression of pro-apoptotic and pro-survival members of Bcl-2 family and upregulated DR5,FADD,Bid etc,thus activated mitochondria and death receptor mediated apoptosis pathways;(5)Volasertib induced DNA damage response in a time and dose-dependent manner,combination of Volasertib and PU-H71 resulted in significantly strengthened DNA damage;(6)N-acetylcysteine(NAC)can block the apoptosis-inducing effects of Volasertib.the production of superoxide anion free radicals was increased by the addition of PU-H71 to Volasertib;(7)Compared to treatment of Volasertib alone,combined treatment of Volasertib and PU-H71 caused even stronger inhibition of PLK1 and stronger activation of mitochondria and death receptor mediated apoptosis signal transduction;(8)Volasertib does dependently activated PI3K-AKT pathway,combination of Volasertib and PU-H71 blocked the activation of these pathways.Conclusion: Both PLK1 inhibitor Volasertib and HSP90 inhibitor PU-H71 can inhibit the proliferation of gastric cancer cells,combination of the two chemicals can produce synergistic effects.Volasertib induced DNA damage and triggered apoptosis via mitochondria and death receptor signal pathways.Production of ROS played a key role in Volasertib-induced apoptosis,but also caused enhanced activities of several pro-survival pathways.Combination of Volasertib and PU-H71 augmented Volasertib induced DNA damage and production of superoxide anion free radicals,blocked the activation of AKT,ERK,STAT signal transduction,and thus caused higher apoptosis rates.These enhanced anti-tumor effects are possibly related to the HSP90-inhibition effects of PU-H71,which caused blockade of diverse pro-survival pathways.