The Apoptotic Effect Of Zoledronic Acid On The Nasopharyngeal Carcinoma Cells Via ROS Mediated Chloride Channel Activation

Objectives:Recently,the clinical anti-cancer effect of zoledronic acid(ZA),the third generation of bisphosphonates,has been widely concerned.Studies have found that ZA could exert its anti-tumor effect by inducing apoptosis of tumor cells,but its mechanism has not been fully elucidated.Our previous studies have found that chloride channels were involved in the regulation of tumor cell proliferation,apoptosis,and migration.The relationship between chloride channel and ZA-induced apoptosis was investigated in this study,to find whether chloride channels worked as the target of ZA-induced apoptosis,and its related mechanism was investigated.This study provides a new method to investigate the anti-tumor effect of ZA.Method:All experiments were performed on the poorly differentiated human nasopharyngeal carcinoma(CNE-2Z)cells.(1)Using MTT colorimetric assay to analyze the anti-proliferation effect of ZA on CNE-2Z cells;(2)Appling Flow Cytometry(FCM)to measure the apoptotic rate of CNE-2Z cells;(3)Using IPP software to processes cell images,and using formula to calculate the standardized cell volume;(4)Appling whole cell patch method to record the activation of chloride current under different conditions;(5)Using FCM to detect the transfection efficiency of si RNA(Cl C-3 si RNA and NC si RNA);(6)Using si RNA interference technology to down-regulate Cl C-3 expression;(7)Appling western blot method to detect the expression of related protein;(8)Using FCM to measure the level of intracellular reactive oxygen species(ROS).Results:(1)ZA exerted anti-proliferative effect on CNE-2Z cells with a dose-dependent and time-dependent manner.Compared with the anti-proliferative effect for 24 h,ZA exerted an obvious anti-proliferation effect for 48 h or 72 h.And the IC50 of ZA for 48 h and 72 h were 41.58 ?M and 23.89 ?M on CNE-2Z cells respectively.(2)To further clarify the mechanism of ZA against CNE-2Z cells in vitro,we found that ZA exerted a time-dependent and dose-dependent apoptotic effect on CNE-2Z cells.(3)Apoptotic volume decrease(AVD)often occurs in the early stage of apoptosis.The experimental results showed that ZA(50 ?M)could induce AVD of CNE-2Z cells,and ZA-induced AVD could be inhibited by the chloride channel blocker NPPB,indicating that the chloride channels may be involved in ZA-induced apoptosis;(4)To further clarify whether the chloride channel is involved in ZA-induced apoptosis,we found that the chloride channel blocker NPPB significantly inhibited the apoptosis of CNE-2Z cells induced by ZA(50 ?M),indicating that the chloride channel did involved in ZA-induced apoptosis;(5)When cells were bathed in the isotonic solution containing 50 ?M ZA,a current with a specific outward rectification property,and its reversed potential was close to the Cl-equilibrium potential;(6)ZA activated current had a property of the anionic selectivity,the sequence of anion permeability was I-> Br-> Cl-> gluconate-.And ZA activated current could be inhibited by NPPB(100 ?M),DIDS(100 ?M)or Tamoxifen(20 ?M).These results indicated that ZA-activated currents belong to chloride currents.(7)To determine whether the Cl C-3 chloride channel is the main chloride channel of ZA-acted on CNE-2Z cells,we found that after down-regulated Cl C-3,ZA-activated chloride currents and ZA-induced apoptosis were significantly inhibited,indicating that Cl C-3 chloride channel may be the potential target of ZA.(8)50 ?M ZA could significantly increase ROS content in CNE-2Z cells for 24 h,and inhibiting ROS generation by the antioxidant L-NAC(1 m M),ZA-induced apoptosis was significantly inhibited,suggesting that ZA may induce apoptosis by generating ROS;(9)In order to further explore the relationship between ROS and chloride channels,we found that ZA could hardly activate chloride currents when the generation of ROS was inhibited by L-NAC,suggesting that ROS may mediate in ZA-activated chloride current;(10)To clarify the relationship between ROS and chloride channels,we used Cl C-3 si RNA to silence Cl C-3 chloride channel in CNE-2Z cells,and found that ZA-generated ROS was not significantly inhibited,suggesting that ROS may act as the upstream of chloride channels.Conclusion:(1)Chloride channel is involved in ZA-induced apoptosis of CNE-2Z cells,and Cl C-3 chloride channel is its main molecular basis;(2)Reactive oxygen species(ROS)participated in ZA-induced apoptosis and ZA-activated chloride current in CNE-2Z cells;(3)ZA induced apoptosis of CNE-2Z cells through promoting ROS production,which subsequently activates Cl C-3 chloride channel.