Login to Access Video or Poster Abstract: MP48-08
Sources of Funding: Grants from the National Natural Science Foundation of China (NSFC 81101936 to JZ;NSFC 81172436 to YS; NSFC 81302227 to YC)

Introduction

We previously identified intravesical silibinin, a natural flavonoid, as a novel and effective chemopreventivetherapy against bladder cancer (BCa), which were accompanied with its proapoptotic effects. However, the exact mechanisms are not thoroughly understood.Heat shock proteins (Hsps) have been identified as key determinants of cancer cell survival, which can modulate apoptosis by directly interacting with components of the apoptotic machinery. Manipulation of Hsps by chemical agents represents a viable strategy against cancer. Therefore, the purpose of this study was to examine the role of Hsps in regulating silibinin-induced apoptosis in BCa.

Methods

Human BCa cell line RT4 and T24 served as the model system in vitro and in vivo. The expression of Hsps, heat shock factor-1(HSF1) and_x000D_ apoptosis related molecules after silibinin treatment were examined by western blot. Co-immunoprecipitation was used to determine the interaction between apoptotic protease activating factor-1 (Apaf-1) and Hsp70 or pro-caspase 9. Both nude mice xenografts and orthotopic rat bladder cancer tissues were analyzed for apoptosis molecular alterations after oral or intravesical silibinin treatment in vivo.

Results

Exposure of BCa cells to silibinin resulted in significant downregulation of Hsp70 both in mRNA and protein levels. No change of Hsp90,Hsp60, Hsp40 and Hsp27 were observed. Either heat shock pretreatment-induced expression of endogenous Hsp70 or overexpression of exogenous Hsp70 attenuated silibinin-induced cell apoptosis, while knocking down Hsp70 sensitized cells to apoptosis by silibinin. Silibinin-induced disruption of mitochondrial membrane potential and release of cytochrome c from mitochondria were inhibited by overexpression of Hsp70. Furthermore, silibinin inhibited the formation of complexes containing Apaf-1 and Hsp70 and increased the interaction between Apaf-1 and pro caspase-9. Additionally, the downregulation of Hsp70 by silibinin was correlated with a diminished presence of HSF1 in the nucleus, and the inhibition of transcriptional activity of HSF1. More importantly, silibinin competed with_x000D_ ATP for binding to the ATPase domain of Hsp70 as determined by silibinin-conjugated Sepharose pull-down assay. Consistently, either oral silibinin or intravesical instillation of silibinin suppressed the growth of xenografts in nude mice or orthotopic bladder cancer in rats respectively, which were accompanied with_x000D_ downregulation of Hsp70 and HSF1.

Conclusions

These findings firstly identify the role of Hsp70 in mediating mitochondrial apoptotic pathway induced by silibinin, and suggest selective targeting HSF1/Hsp70 signaling could produce synthetic lethality in the management of BCa.

Funding

Grants from the National Natural Science Foundation of China (NSFC 81101936 to JZ;NSFC 81172436 to YS; NSFC 81302227 to YC)