Since our data suggest that promotion of autophagy by Rg3 can sensitize doxorubicin-induced cell death, we carried the subsequent study on the therapeutic efficacy of combined treatment of Rg3 and using a xenograft mouse model. When the tumors were measureable, mice were matched for tumor volumes and assigned to control, Rg3, doxorubicin, or combination of Rg3 and doxorubicin. As shown in Fig. 10. A, Rg3 or doxorubicin treatment alone had minimal effect on the growth of tumors, with the tumor size similar to the control mice whereas combined treatment with Rg3 and doxorubicin led to significant reduction of the tumor volume. Furthermore, no significant weight loss was observed during these periods of combination treatments (Fig. 10. B). Consistent with the data of tumor volume, the tumor weight was also significantly decreased in combination of Rg3 and doxorubicin (Fig. 10. C). Taken together, these findings suggest that Rg3 potently enhances the therapeutic activity of doxorubicin c in vivo. We next investigate the effect of the drug combination on in vivo using H&E staining of paraffin-embedded sections of xenografted tumors. As shown in Fig. 10. D, Cell density of the combination of Rg3 and Doxorubicin group was greatly lower than Rg3 or Doxorubicin treated groups and control group shows much higher cell density.
A. B.
B. D.
Fig. 10. Combination of Rg3 plus Doxorubicin inhibits hepatoma growth in mouse xenograft model. A & B. Effect of Rg3 plus Doxorubicin on Huh-7 hepatocellular carcinoma cell xenograft in athymic BALB/c nude mice. Huh-7 tumors were established subcutaneously and treated with Rg3, Doxorubicin or Rg3 plus Doxorubicin for 21 days. Tumor growth (tumor volume in A and body weight in B) was monitored. C. Tumor weight was also measured after 21 days treatment. At the end of treatment mice were killed and measured tumor weight and
IV. Discussion
At present, it has been well established that autophagy possesses important biological functions and has been also implicated closely in oncogenesis and cancer development (Mathew et al., 2007; Levine and Kroemer, 2008; Apel et al., 2009; Chen and Debnath, 2010). One particularly important and yet highly controversial issue in autophagy study is the role of autophagy in cell death (Gozuacik and Kimchi, 2007; Kroemer et al., 2008;
Scarlatti et al., 2008). In general, autophagy serves as pro-survival function under starvation and several other cell stress condition and suppression of autophagy has been accepted as a novel therapeutic strategy in combination cancer therapy (Kourtis and ginseng's efficacy. Because ginsenosides appear to affect multiple signaling pathways, their effects are complex and it is unclear their action of mechanisms. In the present study, we provide convincing evidence suggesting that suppression effect of Rg3 on autophagy is functionally related to its sensitization effect on a DNA-damage agent, doxorubicin and such a combination could serve as a novel strategy to improve cancer therapeutic efficiency, based on the following observation: First, we found that that Rg3
enhances autophagy markers by suppressing autophagic flux. Suppressing autophagic flux was analyzed by the tfLC3. Essentially, this method is based on the difference in the nature of the two fluorescent proteins (GFP and mRFP), i.e. mRFP is much more resistant to lysosomal quenching than GFP in the acidic environment. Therefore, if the autolysosome maturation proceeds normally, it would give rise to more red-only puncta.
Reversely, if the autophagosome does not fuse with lysosome or the lysosome function
is blocked, most of the puncta should exhibit both red and green signals and appear to be yellow (Kimura et al., 2007). Based on this, our results provided evidence that Rg3 is likely to inhibit autophagosome maturation and autolysosome formation. Although the underlying molecular mechanisms for Rg3’s effects on inhibition of late stage of autophagic flux remain to be further investigated.
Secondly, Rg3 suppressed autophagy can contribute synergic effects on doxorubicin-induced cell death. DNA damage induces autophagy and autophagy delays apoptotic cell death induced by the DNA-damaging agents. Doxorubicin has been recently shown to be paralleled by autophagy (Kobayashi et al., 2010) and low doses of doxorubicin induced autophagy and apoptosis simultaneously. For instance, in multiple myeloma cell lines, pharmacologic or genetic inhibition by knockdown of Beclin 1 or Atg5, markedly augmented doxorubicin-induced cell death (Pan et al., 2011). Consistent with this report, our results revealed that inhibition of autophagy by Rg3 treatment markedly augmented doxorubicin-induced cell death in hepatocellular carcinoma cells in vitro, which was further evidenced by using mouse xenograft model. Therefore, suppression of autophagy becomes a potent strategy in combination therapy to overcome the resistance and enhance the chemotherapeutic efficacy. Although the role of autophagy in cancer cell fate is still controversial, there is growing evidence that autophagy plays a pro-survival mechanism in cancer cells and there is an urgent need to develop specific autophagy inhibitors that are clinically viable. At present, few inhibitors of autophagy are used in autophagy research and CQ is the only autophagy inhibitor in clinical trial (Rubinsztein et al., 2007). Based on the above, Rg3 could be further developed as important adjunct to enhance antihepatoma efficacy of existing DNA-damaging agents.
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- 국문요약 - 핵심어 : Autophagy, Hepatocellular carcinoma, Ginsenoside Rg3, DNA damage agent