The Investigation Of The Mechanisms Of Celastrol Sensitizing Prostate Cancer Cells To Tumor Necrosis Factor-α

Celastrol is a kind of triterpenoid isolated from the traditional Chinese medicine,Thunder of God Vine that has been used in the treatment of autoimmune disease,asthma, chronic inflammation. Recently, celastrol was found to exhibit significantanticancer activities, including the induction of apoptosis in different cancer cells,synergistically enhancing the cytotoxicity of other chemotherapeutic agents, andinhibiting the growth of glioma, melanoma and prostate cancer in nude mice. However,the target and mechanism of celastrol are not completely clear. Celastrol has beenidentified as a novel inhibitor of heat shock protein90(HSP90). In addition, celastrolhas been reported to be a potent inhibitor of proteasomes and induce cytotoxicity inglioma and prostate cancer models in vivo and in vitro. Several other molecular targetshave been proposed to explain the anticancer effects of celastrol, including NF-B, andxc-Cystine/Glutamate antiporter. As celastrol is moved into clinical studies, it isimportant to gain a better understanding of its target and mechanism.Tumor necrosis factor-.(TNF-.), is a pleiotropic cytokine inducing a variety ofcellular responses, ranging from inflammatory cytokine production, cell survival, cellproliferation, and cell death.After binding and activating its receptor TNFR1, the deathdomain at the cytoplasmic tail of TNFRI rapidly recruits an adaptorprotein.Subsequently,many other signal proteins including TNF receptor associatedprotein2(TRAF2),RIPK1and Ciap1and cIAP2,will be further recruited to form a signalcomplex refered as complex1.Complex I then recruits I B kinase (IKK) complex,leading to the activation of NF-B. In a process that is still not well characterized,complex I internalizes and converts into a Caspase-8activating, death inducing complex,complex II, with additional recruitment of FADD and proCaspase-8. ActivatedCaspase-8induces Caspase-3activity. Moreover, it is known that poly (ADP-ribose)polymerase (PARP) is cleaved by Caspase-3, a downstream Caspase of Caspase-8, and -9, and causes cell apoptosis. Caspase-8activates apoptotic signal through anothermechanism involving BID cleavage to truncated Bid (t-Bid). T-Bid translocates to themitochondria, increasing its outer membrane permeability. That results in cytochrome Crelease and activation of other Caspases ultimately leading to apoptosis. However theintegrity of mitochondria permeability also protect by a series of anti-apoptotic proteins,mainly Bcl-2family members, such as Bcl-2and Bcl-xL. The down-regulation of theseproteins would be accelerates the process of apoptosis.In addition to its cytotoxicity to various tumor cells, Celastrol also introduceschemo-sensitization to TRAIL and Bortezomib in various tumor cells. In order toinvestigate the impact of celastrol on the cell death signalling pathway mediated byTNF-; è a serious of experiments have been conducted in different prostate cancer celllines. Firstly, we compared the cytotoxicity of celastrol and TNF-., and found that thethree prostate cell line è LNCaP and PC-3and DU145è are all resistant to the eachtreatment. However when treated the cells with the combination of celastrol and TNF-.,the cell survival decrease obviously. Through the staining of Annexin-V and detectionof Caspases and PARP proteins, the tumor cells were introduced to go apoptosis bytreatment of celastrol and TNF-..More interestingly, the pretreatment of pan Caspaseinhibitor zVAD-fmk could be almost reverts the injury of celastrol and TNF-., furtherindicated that after the combined treatment, there is activation of capasaes.In the exploration of the molecular mechanisms of the apoptosis caused by thecombined treatment, several signaling pathway have been detected, such as JNK, p38and NF-B. Compared with the single treatment, there was a noticeable increase ofphosphorylated JNK proteins in combinatuin group. The pretreatment JNK specificinhibitor SP600125could block the apoptosis induced by celastrol and TNF-.,indicating that the apoptosis can attribute to the activation of JNK signaling. UnlikeJNK protein, the phosphorylation of p38protein all decreased after different treatmentand the inactivation of p38caused by the treatment of p38inhibitor SB could acceleratethe injury of celastrol and TNF-.. At the same time celastrol also inhibited theactivation of NF-B mediated by the TNF-.. The anti-apoptotic proteins, such as FLIP, Bcl-2, and Bcl-xL decreased obviously in the combined treatment. Finally, thecombination treatment of celastrol of TNF-. could also increased obviously reactiveoxygen species (ROS) and decreased mitochondrial membrane potential (MMP).In conclusion, our finding revealed that celastrol can sensitize prostate cancer toTNF-. and introduce the cancer cells to apoptosis accompanied by the activation ofCaspases. The underlying mechanism involves the activation of JNK signaling, theinactivation of p38and NF-B pathways. The down-regulation of anti-apoptoticproteins, such FLIP, Bcl-2, and Bcl-xL, promotes the process of apoptosis. In addition,the ROS production and MMP injury accelerate the apoptosis.