Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells

Abstract

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase.

Oleandrin sensitizes human osteosarcoma cells to cisplatin by preventing degradation of the copper transporter 1

“…Our mRNA microarray assays showed that OLE inhibited the expression of multiple proteasome‐related genes, which is consistent with the results that OLE suppressed the proteasome activity detected by proteasome activity assays. In line with our observation, Arenobufagin, another kind of cardiac glycoside, inhibited the proteasome activity of Hela cervical carcinoma cells (Yue et al, ). Previous studies demonstrated that proteasome inhibitors lactacystin and AllnL increase the content of Pt‐DNA adducts in A2780/CP70 DDP‐resistant human ovarian carcinoma cells (Mimnaugh et al, ).…”

“…So proteasome activity suppression may be a novel way to increase DDP uptake. Arenobufagin, another kind of cardiac glycoside, was found to inhibit the function of the proteasome in Hela cervical carcinoma cells (Yue et al, ). Whether OLE affects the proteasome activity to regulate CTR1 function deserves to be clarified.…”

Arenobufagin Induces Apoptotic Cell Death in Human Non-Small-Cell Lung Cancer Cells via the Noxa-Related Pathway

“…Toad venom has been widely used in China as a local anesthetic, cardiotonic, antimicrobial, and antineoplastic agent for many years [ 26 , 27 ]. Arenobufagin, as one of the main biologically active compounds extracted from toad venom, has been showed to exhibit potential anti-cancer effects in human hepatocellular carcinoma (HCC), esophageal squamous cell carcinoma and cervical carcinoma cells [ 19 , 21 , 22 ]. However, the anti-NSCLC effects of arenobufagin are largely unknown.…”

“…Deng et al found that arenobufagin could intercalate with DNA to induce G2 cell cycle arrest through the ATM/ATR pathway in HCC cells [ 20 ]. Except in HCC, arenobufagin has also been shown to inhibit growth and induce apoptosis in human esophageal squamous cell carcinoma cells and cervical carcinoma cells [ 21 , 22 ]. However, the effects and mechanisms of arenobufagin on lung cancer are still not clear.…”

Arenobufagin Promoted Oxidative Stress‐Associated Mitochondrial Pathway Apoptosis in A549 Non‐Small‐Cell Lung Cancer Cell Line

“…Anyway, a lot of studies have demonstrated its broadspectrum antitumor activities in cancers such as breast cancer, pancreatic carcinoma, and liver cancer [12][13][14]. We previously found that ARE can induce liver cancer cell apoptosis and autophagy through PI3K/Akt/mTOR signal routing [14]; induce cell cycle arrest and apoptosis in human cervical cancer HeLa cells [15]; have anticancer effect on human esophageal squamous cell carcinoma (its mechanism of exerting anticancer efficacy may be activation of cysteinecontaining aspartate proteolytic enzyme (caspase) by endogenous and exogenous pathways); promote apoptosis of esophageal cancer cells by enhancing caspase phosphorylation and activating p53 signaling [16]; promote apoptosis of human glioblastoma U-87 cells by inhibiting p38MAPK signaling pathway [17]; and inhibit epithelial-mesenchymal conversion by going down the β-catenin pathway, consequently repressing motility and invasiveness of prostate cancer PC3 cells [18]. Nevertheless, the role and mechanisms of ARE on NSCLC remain unclear.…”