Synergistic Anti-tumor Activity Of ABT-737 And Celastrol

Purpose:To confirm synergistic effects of Celastrol in combination with ABT-737 and study on their cellular mechanism.Methords:1. MTT cell viability assay was used to evaluate proliferation of human liver cancer Bel-7402 and HepG2 cells after treatment of ABT-737, Celastrol and the combination. Then calculate the CI values by the CalcuSyn software; 2. DAPI staining was utilized to observe apoptotic bodies in Bel-7402 and HepG2 cells after treatment of ABT-737, Celastrol and the combination; 3. Propidium iodide staining (PI) staining and flow cytometry was used to analyze cell apoptosis after treatment of ABT-737, Celastrol and the combination; 4. JC-1 staining was used to determine the mitochondrial membrane depolarization after treatment of ABT-737, Celastrol and the combination; 5. Western blotting analysis was used to detect the expression of protein such as apoptosis-related proteins, HSP90 client proteins, NOXA and so on; 6. real-time RT-PCR was adopted to measure NOXA mRNA after treatment of Celastrol; 7. ATF4 gene was silenced by small interfering RNA technique. Then measure NOXA mRNA after treatment of Celastrol; 8. NOXA gene was silenced by small interfering RNA technique. Then detect apoptosis-related protein expressions and cell proliferation after treatment of ABT-737, Celastrol and the combination; 9. Immunoprecipitation was used to detect the interaction between NOXA protein to Mcl-1 protein after treatment of ABT-737, Celastrol and the combination.Results:1. The combination of ABT-737 and Celastrol exhibited synergistic cytotoxicity in human liver cancer Bel-7402 and HepG2 cells. CI values were mostly less than 0.3, which was regarded as strong synergism; Few apoptosis bodies were observed after treatment of 10μM ABT-737 alone and 1.25μM Celastrol alone for 72 h, while many apoptosis bodies were observed after treatment of the combination for 72 h.; ABT-737 in combination with Celastrol could synergistically induce cell apoptosis after treating cells for 24 h,48 h and 72 h. The cell apoptosis was time-dependently increased;2. Compared to the corresponding control,10μM ABT-737 alone and 1.25μM Celastrol alone caused slight decrease ofΔΨm, while the combination of them ledΔΨm in Bel-7402 and HepG2 cells to obviously time-dependently diminish; ABT-737 in combination with Celastrol could result in evident release of cytochrome c in HepG2 cells; The combination of ABT-737 and Celastrol contributed to caspases-3 activation and PARP cleavage in Bel-7402 and HepG2 cells; 3. Celastrol could inhibit HSP activity in Bel-7402 and HepG2 cells, which was indicated by increase of Ub, degradation of HSP90 client proteins p-ERK, CDK4 and accumulation of HSP70; Celastrol could activate ER Stress response increasing p- eIF2a and ATF4, followed by up-regulation of NOXA mRNA levels; Celastrol induced less NOXA mRNA when ATF4 gene was knocked down than control group; 4. Either Celastrol alone or in combination with ABT-737 could increase NOXA protein expressions, which were earlier than cell apoptosis; The combination of ABT-737 and Celastrol caused less caspases-3 activation, PARP cleavage as well as inhibition of cell proliferation when NOXA gene was silenced, compared with control group; 5. Either Celastrol alone or in combination with ABT-737 could increase interaction between NOXA and Mcl-1.Conclusion:Celastrol in combination with ABT-737 could induce strong synergistic killing of liver cancer cells; through inhibition of Hsp90 and induction of ER stress in liver cancer cells, Celastrol could promote the up-regulation of Noxa and its binding to Mcl-1, then released Bak from Mcl-1 to activate the mitochondrial apoptosis pathway, and finally sensitized liver cancer cells to ABT-737.