Combination Therapy With Protein Kinase Inhibitor H89 And Tetrandrine Elicits Enhanced Synergistic Antitumor Efficacy

Cancer is a multigenic disease caused by the abnormal proliferation and differentiation of cells governed by tumorigenic factors.Cancer continues to be a worldwide killer and becomes the second most common cause of death after heart disease despite the enormous amount of research and rapid developments seen during the past decade.The treatment of cancer is very complicated in clinical.Chemotherapy is one of the major cancer treatment strategies,especially for advanced malignant tumors or leukemia.Natural products have garnered increased attention in the chemotherapy drug discovery field.Remarkably,among the FDA approved anticancer drugs,more than 75%are from natural sources and are being used either in their actual form or as simple modifications from actual form.Tetrandrine(Tet),a bisbenzylisoquinoline alkaloid that was isolated from the medicinal plant Stephania tetrandrine S.Moore,has been widely used as an effective agent to treat patients with hypertension,arrhythmia,arthritis,inflammation,and even silicosis in traditional Chinese medicine.Of note,tetrandrine has been recently identified as a potential leading compound among anticancer agents with a variety of pharmacological effects including regulating cell viability,migration,invasion,angiogenesis and multidrug resistance of tumors.Due to the complexity of cancer with the involvement of multiple signaling pathways,it is difficult for a single compound to combat cancer.Even though,if a compound exhibit potent anticancer effect,there is a chance for the development of resistance against that compound by tumor cells making the drug ineffective.Thus,combination therapy could be an available strategy to improve the treatment efficiency.Increasing studies have shown that tetrandrine can bring about synergistic activity to enhance cytotoxicity when combined with molecularly targeted drugs.H89,is a potent protein kinase A(PKA)inhibitor with the ability to readily cross the cell membrane,with preclinical activity demonstrated in vitro and in vivo.Recently,H89 has garnered increased attention in the other functions such as anti-tumor activity.To examine whether a cooperative effect exists between H89 and tetrandrine in tumor chemotherapy,we treated cancer cells with a series of concentrations of H89 and tetrandrine alone or in combination.The results showed that tetrandrine plus H89 had a clear effect on most human cancer cells.In contrast,normal cells,such as HBL-100,L02 and HEK-293T,were substantially less sensitive to H89/tetrandrine,which suggests that the H89/tetrandrine combined treatment is relatively selective towards cancer cells.The use of the Chou-Talalay method to calculate the H89/tetrandrine interaction showed combination indexes<1,which indicated their synergistic effects under a number of the treatment conditions.Flow cytometry with Annexin V/PI showed that H89/tetrandrine combination treatment increased apoptotic cell death compared with each agent alone.Western blot and GFP-LC3 fluorescence assays showed that the H89/tetrandrine combination treatment synergistically increased autophagy.In addition,we used an mRFP-EGFP-LC3 tandem-tagged fluorescent protein to determine the dynamic process of autophagic flux.The results suggested that the combination of H89/tetrandrine promotes functional autophagy in cancer cells.Mechanistically,H89/tetrandrine combination treatment substantially increased intracellular ROS compared with either agent alone in cancer cells.Pretreatment of cells with the ROS scavenger NAC significantly rescued them from cell death induced by combination treatment.H89 is a strong inhibitor of cyclic AMP-dependent protein kinase A(PKA).H89 alone or combined with tetrandrine significantly decreased the phosphorylation of the transcription factor CREB in cancer cells.H89/tetrandrine transiently activated MEK/ERK1/2 signaling,and these effects mainly originated from tetrandrine.Inhibition of ERK1/2 with PD98059 could partially rescue H89/tetrandrine induced cell apoptosis,whereas it had no effect on autophagy.However,concomitant pretreatment of cells with FSK and PD98059 almost completely restored cell viability,which implies that PKA and ERK signaling are simultaneously involved in H89/tetrandrine induced cell death.Notably,in this study,we determined that H89/tetrandrine sensitive cells showed higher levels of c-Myc expression than resistant cells.The overexpression of c-Myc enhanced the effects of combination treatment induced cell death and apoptosis We also demonstrated that c-Myc mplification downregulates Mcl-1 expression and increases intracellular ROS,which contributes to H89/tetrandrine sensitivity.In summary,our findings demonstrate that the combination of tetrandrine and H89 exhibits an enhanced therapeutic effect and may become a promising therapeutic strategy for cancer patients.It also provides a significant clinical application of tetrandrine in the treatment of human cancer.Moreover,the combination of H89/tetrandrine offers new selectively targeted therapy strategies for patients with c-Myc amplification.