Suppression of Chloride Channel 3 Expression Facilitates Sensitivity of Human Glioma U251 Cells to Cisplatin Through Concomitant Inhibition of Akt and Autophagy

Abstract

Cisplatin resistance is a difficult problem in clinical chemotherapy, and the mechanisms involved in cisplatin resistance require further study. In this study, we investigated the role of chloride channel-3 (ClC-3) in cisplatin resistance. Autophagy was demonstrated by accumulation of LC3-II, beclin 1 and Atg12-Atg5. The ultrastructure changes were observed under electron microscope. Chemical staining with acridine orange or MDC was used to detect acidic vesicular organelles. Quantification of apoptosis was detected by PI and Annexin V staining. The mechanisms involved in the Akt pathway and autophagy were studied by western blot analysis. Our results showed that Akt phosphorylation and autophagy were induced by cisplatin in human glioma U251 cells. Specific inhibition of ClC-3 by ClC-3 siRNA sensitized the apoptosis-resistant U251 cells to cisplatin-mediated cell death and downregulated phosphorylated Akt. Interestingly, ClC-3 suppression also inhibited induction of autophagy by cisplatin although the Akt/mTOR pathway was deregulated. Counteracting the autophagic process by 3-methylademine enhanced cytotoxicity of cisplatin, revealing that autophagy plays a key role in chemoresistance. Suppressing the Akt/mTOR pathway by the NADPH oxidase inhibitor diphenyl iodonium (DPI) indicated that cisplatin-induced activation of Akt/mTOR pathway requires generation of reactive oxygen species (ROS) through NADPH oxidase. Collectively, our results suggest that ClC-3 suppression causes the inhibition of Akt and autophagy, which can enhance the therapeutic benefit of cisplatin in U251 cells. Anat Rec,

CLC-3 channels in cancer (Review)

“…Thus, CLC-3 may be related to multidrug resistance in cancer cells via increased cell proliferation. Consistent with this idea, suppression of CLC-3 was found to increase the sensitivity of human glioma u251 cells to cisplatin via inhibition of Akt and autophagy (37). CLC-3 is expressed in late endosomes and lysosomes and contributes to their acidity.…”

“…Overexpression of CLC-3 is also associated with apoptosis (88,89) due to inhibition of the PI3K/Akt/mTOR signaling pathway (89). In addition, as a Cl -/h + transporter, CLC-3 contributes to the acidity of the late endosome and lysosomes, promoting sequestration of basic chemotherapeutic drugs and leading to drug resistance (35,37). CLC-3 inhibitors can be used for the treatment of cancer by inhibiting cell proliferation and preventing drug resistance, whereas CLC-3 activators exhibit antitumor activity through promotion of apoptosis.…”

p62 participates in the inhibition of NF-κB signaling and apoptosis induced by sulfasalazine in human glioma U251 cells

“…3-MA as a classic autophagy inhibitor has been widely used in related research (29,30). Previous research used 3-MA to inhibit autophagy and prove that autophagy is involved in the growth inhibition of hepatoma cells induced by SAS (31).…”

Ion channels in the regulation of autophagy

“…192 Similarly, the CLCN3 channel also resides mainly on endosomal compartments 193 and positively regulates autophagy. 194 It should be noted, however, that both CLCN3 and CLCN7 function as Cl ¡ /H + exchangers rather than voltage-dependent anion channels. 186 Taken together, the available data suggest that chloride channels can regulate autophagy at both early and late stages.…”