Study On The Mechanism Of G-Rh2 And Etoposide Synergistic Activity In HeLa Cell Apoptosis

Tumor is a leading cause of human death in the world. The standard treatment for most tumors continues to be surgery,radiation and chemotherapy. Chemotherapy - the use of medications to treat cancer - has played a major role in cancer treatment for half a century. Years of research and testing have proved chemotherapy to be an effective method for cancer therapy. However, chemoresistance, and the damage for normal cells are the major causes of cancer treatment failure. To overcome tumor resistant and enhance sensitivity of anticancer reagents to tumor cells are the key step for the tumor treatment.Combination therapy has been the basis for most success stories in cancer treatment, which is understandable that components of a combination affect different molecular targets, and have a favorable pharmacological interaction (same target, but different large-organ toxicities) to circumvent tumor resistance. A barrier to major advancements in combination therapy, however, has been a lack of understanding about the intersection of critical signaling pathways.Etoposide, a semisynthetic derivative of the podophyllotoxins, is used in the current tumor treatment protocols, such as lung cancer (especially small cell lung cancer), acute lymphoblastic leukemias (ALL) and acute myeloblastic leukemia (AML). However, tumor resistance, non-specific tissue cytotoxicity are the limitations for successful development of this drug as one of the primary therapeutic agents for the treatment of other tumors.Ginsenoside Rh2(G-Rh2),with a dammarane skeleton is reported to have remarkable anti-cancer potential based on growth inhibitory activity, and induction of apoptosis. Previous studies have demonstrated that G-Rh2 can induce apoptotic cell death in MCF-7 human breast cancer, human hepatoma SK-HEP-1, human leukemia THP-1 and human lung adenocarcinoma A549 cells. Notably, several observations have shown that G-Rh2 induces apoptosis in a Bcl-2- and/or Bcl-XL-independent manner. Disruption of mitochondria apoptosis signailing and concomitant development of resistance to anticancer therapy occurs as a consequence of overexpression of antiapoptotic members of the Bcl-2 family such as Bcl-2 or Bcl-xL, or through the loss of the proapoptotic Bcl-2 homologs such as Bax or Bak. In this vein, we hypothesize the combination of G-Rh2 and etoposide may cause synergistic effect to overcome tumor chemotherapeutic resistance.In this thesis we draw some conclusions as follows:1. Etoposide and G-Rh2 act synergistically to induce apoptotic cell death in three tumor cell lines with different origin, HeLa, SGC-7901, HepG2), and the combination index (CI) were 0.78, 0.88, 0.41 respectively.2. We selected HeLa cells, which is not sensitive to etoposide, to carry out further study. Our results showed the apoptotic effect can be markedly enhanced by treatment with G-Rh2.3. Etoposide and G-Rh2-induced synergistic apoptosis in HeLa cells involves the participation of the mitochondria/caspase-9 activation-mediated pathway, but not death receptor–mediated pathway. G-Rh2 enhanced the release of cytochrome c and Smac from the mitochondria to the cytosol induced by etoposide. The enhancement release of cytochrome c and Smac may resulte from the up-regulated translocation of Bak and Bax to mitochondria.4. In our study, we observed that insufficient Smac release was the reason of resistance to apoptosis induced by etoposide, while G-Rh2 could markedly enhance the release of Smac in HeLa cells and it may be one of the key steps in the synergistic effect.Smac was identified as a protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing the inhibitor of apoptosis proteins (IAPs). IAPs are highly expressed in many tumors and they have been associated with refractory disease and poor prognosis. The strategies targeting Smac may prove to be especially effective to overcome tumor resistance. In this study we found that etoposide alone induced the depolarization of mitochondrial membrane potential and a detectable amount of cytochrome c release, while the apoptotic characteristic changes were not detected. In same experimental condition, the release of Smac was not detected. The Smac release was observed when cells were cotreated with G-Rh2 and etoposide. It suggested that G-Rh2 augments the release, and this effect may be in large part responsible for the sensitization of etoposide-mediated apoptosis in HeLa cells. To provide further evidence for this notion, we silenced Smac expression using Smac specific siRNA and found that ablation of Smac markedly reduced the level of apoptosis induced by G-Rh2 in combination with etoposide in HeLa cells. Those results provide an useful information for the key role of Smac in the apoptosis program.Bax and Bak are major regulators of mitochondrial integrity among multiple apoptotic signals release cytochrome c and Smac from the mitochondrial intermembrane space to activate caspase. The loss of Bax or Bak which cause the disruption of mitochondria apoptosis signailing, is one of the reasons to develop into the resistance of anticancer therapy. In this study, the translocation of Bak and Bax to the mitochondria remarkably enhanced in etoposide and G-Rh2 cotreated cells, which resulted in the upregulated release of cytochrome c and Smac. The function of increasing the translocation of Bak and Bax of G-Rh2 provide some new insight to widen the application of G-Rh2 in tumor therapy. In precent study we proposed a new therapeutic strategy to overcome the chemoresistance which target topoisomerase II, and provided a novel method for the understanding about the intersection of critical signaling pathways in combination therapy.