Synergism Of Ginsenoside Rh2 And Betulinic Acid To Induce Apoptosis In Tumor Cells

Induction of apoptosis in cancer cells is a key killing mechanism for most antitumor therapies including chemotherapy,γ?irradiation, immunotherapy or cytokines. Thus, defects in apoptosis may cause resistance. Combination treatment of anticancer reagents has been shown to be advantageous in malignancies that still partially respond to either treatment alone, because they may help to amplify weaker death signals. The old paradigm involved combining drugs to reduce toxicity and maximize pharmacokinetics, whereas the new paradigm is for targeting pathways. Several recent studies have shown a mechanism-based combination strategy for cancer treatment.In this study, we used two plant-derived drugs, G-Rh2 and Bet A, to examine the synergistic anticancer effects in human cancer cells of different origin. The two reagents were chosen for the following reasons. First, G-Rh2 can induce acute and extensive apoptotic cell death in various human cancer cells. The caspase-8-mediated caspase activation cascade plays an important role in G-Rh2-induced apoptosis. More importantly, overexpression of Bcl-2 and/or Bcl-XL cannot suppress G-Rh2-triggered apoptosis. Second, Bet A induces apoptosis in tumor cells mainly through the mitochondrial pathway. Also, they do not sensitize normal human cells that show some tumor specificity. Therefore, we speculated that combined treatment with G-Rh2 and Bet A may synergistically trigger apoptosis in cancer cells, through targeting of the death receptor pathway by G-Rh2 and the mitochondrial pathway by Bet A. In this paper, we have described four major observations: (i) cotreatment with G-Rh2 and Bet A synergistically induced apoptotic cell death in HeLa, A549, and HepG2 cells. The CI values were 0.567, 0.670 and 0.716, respectively.(ii) co-treatment with G-Rh2 and Bet A synergistically activated caspase-8 and consequently enhanced Bid cleavage; (iii) co-treatment with G-Rh2 and Bet A synergistically induced translocation of Bax to the mitochondria, and thus increased cytochrome c release from the mitochondria; and (iv) specific inhibition of caspase-8 by the caspase-8-targeting siRNA effectively reduced caspase-9 processing, PARP cleavage, and apoptosis in HeLa cells.When apoptosis is induced in HeLa cells by co-treatment with G-Rh2 and Bet A, we observed that proteolytic activation of caspase-8, down-regulation of Bid, Bax translocation, cytochrome c release, and PARP cleavage all occurred within the same time period. In contrast, these apoptotic features were not observed in cells treated with either drug alone. In this study, we observed that another multidomain pro-apoptosis member, Bak, also translocated to the mitochondria under the same apoptotic conditions. However, this translocation occurred in cells treated with Bet A alone or G-Rh2 and Bet A. Thus, the translocation of Bak may not correlate with enhanced caspase-8 activation and cytochrome c release. This is in agreement with a recent study that showed that TRAIL triggers caspase-8 processing and Bid cleavage, and sensitizes tumor cells by a Bax-dependent mechanism, but not by Bak. Our results suggest that caspase-8-mediated Bid cleavage may trigger the translocation of Bax to the mitochondria and consequent release of cytochrome c in apoptotic cells induced by co-treatment with G-Rh2 and Bet A.We produced further evidence to support this notion. Specific inhibition of caspase-8 by siRNA effectively reduced the level of proteolytic activation of caspase-9, PARP cleavage, caspase-3 activation, and apoptosis in G-Rh2/Bet A co-treated cells to that in control cells treated with Bet A, which expressed normal levels of caspase-8. Cells treated with G-Rh2 alone displayed only minimal caspase-3 activation and apoptotic morphology. At the concentrations used in this study, we did not observe any alteration in Smac release and mitochondria-associated Bcl-2 in differently treated cells, which suggests that Smac and Bcl-2 are not involved in this synergistic process.Previous studies have shown that G-Rh2 induces cancer cell apoptosis in a Bcl-2- and/or Bcl-XL-independent manner, and Bet A induces apoptosis mainly through the mitochondrial pathway, with some tumor specificity. Since the anti-apoptotic Bcl-2 and Bcl-XL are frequently overexpressed in human cancer cells, combination treatment with G-Rh2 and Bet A may be a novel strategy to enhance efficacy of anticancer therapy.In conclusion, co-treatment with G-Rh2 and Bet A triggers caspase-8 processing and Bid cleavage, and sensitizes tumor cells by a Bax-dependent mechanism, followed by caspase-9 and -3 processing, and apoptosis. Thus, co-treatment with G-Rh2 and Bet A may be a novel strategy to enhance the efficacy of Bet A-based therapy, which warrants further investigation.