Natural Product Apigenin Exertes Anti-tumor Effect Via Inhibiting Hsp90 Chaperone Function

Apigenin, a common flavonoid, is found in a variety of fruits and vegetables, including parsley, onions, apple and certain seasonings. Apigenin has gained increasing attention due to its anti-tumor benefits, and has been shown to suppress proliferation in a wide variety of tumor cells, including those of the prostate, breast, and leukemia. Several mechanisms have been elucidated for its anti-tumor effect, such as deregulating cell cycle proteins, inducing apoptotic proteins, inhibiting fatty acid synthesis, producing reactive oxygen species, and inactiving survival signaling pathways. Apigeninn exhibits multiple effects on tumor cells, such as proliferative inhibition, cell cycle arrest and apoptosis induction, however, the underlying mechamisms are not fully elucidated.Hsp90 (Heat shock protein 90), one of the most prominent chaperone, promotes the maturation and maintains the stability of numerous client proteins, most of which involving in the signal transduction, cell cycle and apoptosis. The interaction of Hsp90 and its client proteins also require several co-chaperones to maintain the chaperone complex, such as Hsp70, cdc37, Hop, p23, et al. Among the co-chaperones, Cdc37 is special because it is in charge to recruit a variety of proteins (such as Raf-1, Src, Akt and Cdk4 et al) to Hsp90 and maintains their stability and activities. Previous studies have shown that the Ser13 phosphorylation of Cdc37 induced by CK2 is essential for maintaining the function of Hsp90-Cdc37 complex, which suggestes that targeting CK2 may influence pleiotropic cellular functions by disturbing Cdc37 phosphorylation.Apigenin can competitively binds to the ATP-binding site in CK2αand blocks its kinase activity. In order to explore wehter apigenin can influence the Hsp90 chaperone function by inhibition of Cdc37 phosphorylation, and promote the degradation of a variety of client proteins; we firstly choose the prostate cancer cells as the model to investigate the antitumor effect of apigenin.In prostate cancer cells, apigenin showed the effects to inhibit cell proliferation, arrest cell cycle, and induce apoptosis. In androgen-dependent LNCaP cells, apigenin time and dose-dependetly induced the degradation of androgen receptor (AR) protein, an important Hsp90 client, and blocked its transactivation on PSA. Western blot analysis also showed that apigenin promoted the degradation of several other Hsp90 client proteins through ubiquitin-proteasome pathway, which is independent of apoptosis, indicating that apigenin could inhibit Hsp90 chaprone function. The results of ATP-Resin pull-down and immunoprecipitation plus western blotting showed that unlike GA or FK228, apigenin neither bindes with Hsp90 nor enhances its acetylation, suggesting that apigenin may influence Hsp90 function through other mechanims.As a selective CK2 inhibitor, apigenin can down-regulate CK2 protein levels and inhibit its kinase activity. We noticed that accompany the decline of phosphorylation of Cdc37 protein, the interaction of Cdc37 with Hsp90 and its clients were also decreased, indicating that the molecular chaperone complex was disaaociated, which directly promoted the degradation of Hsp90 client proeins. The important role of Cdc37 on maintaining Hsp90 chapreon function was also comfirmed by Cdc37 siRNA. Additionally, apigenin showed synergic effect on depleting Hsp90 clients with either Hsp90 inhibitor GA or Hsp70 inhibitor quercetin, which indicating that Hsp90 complex has multiple targets.Since CK2 protein is highly expresses in multiple myeloma (MM) cells, which plays a crucial role in controlling survival and sensitivity to chemotherapeutics of MM cells. We further detected the effect of apigenin on MM cells. The experiments revealed that apigenin inhibited proliferation of different multiple myeloma cell lines, inhibited CK2 kinase and promoted degradation of a variety of Hsp90 client proteins. The experiments also confirmed that the inhibitory effect of apigenin was correlated with the down-regulation of CK2 protein. Apigenin inactivated the intracellular and the extracellular cytokines-induced survival signaling pathways, and resulted in the activation of caspase-8/caspase-3 apoptosis pathways, finally induced cell apoptosis. Apigenin selectively inhibited the proliferation of purified primary MM cells, but had no effect on the normal PBMC cells. In the purified malignant plasma cells, apigenin also down-regulated CK2 protein and inhibited its kinase activity, promoted degradation of various Hsp90 client proteins, and induced cells to go apoptosis through casapase-dependent pathways.In conclusion, our studies show for the first time that apigenin exerts its antitumor effect via indirectly inhibiting Hsp90 chaperone function. The mechanism involves inhibiting CK2-mediated Cdc37 phosphorylation, which resulting in Hsp90-Cdc37-clients complex disassociation. This study also supportes that targeting the Hsp90/Cdc37 chaperone complex is a new stratagem for finding new functional Hsp90 inhibitors.