Alkaloids from Stephania venosa as Chemo-Sensitizers in SKOV3 Ovarian Cancer Cells via Akt/NF-κB Signaling

Crebanine (CN), tetrahydropalmatine (THP), O-methylbulbocapnine (OMBC) and N-methyl tetrahydropalmatine (NMTHP) are isoquinoline derived natural alkaloids isolated from tubers of Stephania venosa. We investigated chemo-sensitizing effects of these alkaloids in ovarian cancer cells and evaluated underlying molecular mechanisms involved in chemo-sensitivity. Detection of cell apoptosis was evaluated by using flow cytometry. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chou-Talalay median effect principle was used to evaluate potential drug interactions. Protein analyses were performed on ovarian carcinoma cells using Western blotting upon treatment with anticancer drug and alkaloids. Aporphine alkaloids, such as CN and OMBC, enhanced cisplatin sensitivity in intrinsic cisplatin resistant SKOV3 cells, but not in cisplatin sensitive A2780 cells. Protoberberine alkaloids, such as THP and NMTHP, had no synergistic effect on cisplatin sensitivity in either cell line. Chemo-sensitizing effects of CN and OMBC in SKOV3 cells were mediated via activating apoptosis-induced cell death through caspase-3, -8 and cleaved poly ADP-ribose polymerase (PARP) and via inhibiting anti-apopotic and survival protein expression, such as Bcl-xL, Baculoviral IAP repeat-containing protein 3 (cIAP-2), survivin and interleukin (IL) -6. Cisplatin stimulated protein kinase B (Akt) and nuclear factor-kappaB (NF-κB) signaling pathways, but not mitogen-activated protein kinase (MAPK), activator protein 1 (AP-1) and signal transducer and activator of transcription 3 (STAT3) in SKOV3 cells. Akt/NF-κB signaling was blocked by CN and OMBC leading to increased sensitization to cisplatin. These findings demonstrate that CN and OMBC sensitizes SKOV3 cells to cisplatin via inhibition of Akt/NF-κB signaling and the down regulation of NF-κB mediated gene products. Our results suggest that alkaloids obtained from S. venosa could be used as chemo-sensitizers in ovarian cancer to sensitize and minimize the dose related toxicity of platinum-based chemotherapeutic drugs.