In Vivo And In Vitro Evaluation On Anti-tumor Effects Of An Artemisinin Sensitizing Compound And Mechanistic Investigation Of Artemisinin Targeting Tumor Cells

The malignant tumor is one of the most major diseases that severely threatens the human health. Artemisinin is not only the most effective anti-malarial agent, but also exhibits an anti-tumor activity. However, the peroxidized bridge structure within the artemisinin molecule that is deeply implicated in tumor cell killing is prone to be destroyed by antioxidant agents and antioxidant enzymes present in the human body, which can significantly affect the efficacy of artemisinin in anti-tumors. Additionally, due to the molecular target and the lethal mechanism of artemisinin acting on tumor cells being unclear, the application of artemisinin in the clinic tumor therapy has been largely delayed. To this end, an exhauster of the antioxidant glutathione (GSH), diethyl maleate, an inhibitor of glutathione peroxidase (GSH-PH), mercaptosuccinic acid, and an inhibitor of catalase, aminotriazole, were chosen to combine with artesunate as a sensitizing compound for further study. The in vivo and in vitro evaluations regarding the efficiency of artesunate and the artesunate-containing compound in prohibiting the propagation of hepatoma HepG2 cells and postponing the growth of transplated tumor cells on tumor-bearing nude mice were conducted by comparison between artesunate and the artesunate-containing compound. Furthermore, the sensitizing nature of selected prooxidant agents (inhibitors of antioxidants) on artesunate was also elucidated preliminarily. In consequence, the growth inhibition rate of tumor cells was increased to 21.29% in the artesunate-containing compound as compared with treatment by the identical titer of artesunate alone, while the tumor inhibition rate for the artesunate-contaning compound in tumor-bearing nude mice was increased up to 16.75%-22.91% as compared with treatment by artesunate alone. Upon the artesunate-containing compound treatment, the content of GSH and activities of GSH-PX and CAT in HepG2 cells were all declined when compared with the artesunate treatment, in which the GSH-PX activity was significantly different from the control after 24h (P<0.05), whereas the CAT activity was very significantly different from the control after 24h and 48h (P<0.01).Moreover, we have found for the first time that the molecular target of artesunate attacking a tumor cell is likely the prothsetic heme of heme-containing enzymes such as nitric oxide synthase (NOS) and catalase (CAT), which was evident by that the shift of absorbance is directly detected from the heme (A415) to the artesunate-heme conjugate (A476). On the other hand, the generation of NO can be dramatically affected by the sumplementation of artesunate, and it also closely correlates with the growth inhibition rate available from HepG2 cells. Subsequently, a low concentration of artesunate (50μmol/L) induces NO production and decreases H2O2 content to protect tumor cells from growth inhibition. In contrast, high titers of artesunate (100-200 u mol/L) suppress NO production and increase H2O2 content, thereby promoting tumor cell death. Furthermore, it was observed that a combination of the chemical therapeutic agent,5-fluorouracil (5-FU), with artesunate demonstrates a dominant synergistic effect that enables the inhibition rate of HepG2 elevation from 25% to 40% by a mechanism underlying abrogating the protection of tumor cells from the excess NO, thereby suggesting a possibility of 5-FU and other chemotherapeutics serving as a synergist to artesunate.The anti-tumor effect of the artesunate sensitizing compound has been evaluated using the HepG2 tumor cell model and the tumor-bearing naked mice experimental animal model, and the sensitizing property of prooxidants that ihibit antioxidation, alter the cellular redox state, and creat a microenvironment beneficial to artesulate killing tumors was primarily clarified in the present study. We report here for the first time that NOS is likely one of the artesunate-conjugated molecular targets, and propose a feasibility of using conventional chemotherapeutics as sensitizers of artesunate. These results should pave an avenue towards the eventual application of artemisinin-derived drugs in anti-tumor clinic trials.