Empirical Study Of Artesunate Anti-cancer Effect And Potentiating The Activity Of Erlotinib Against Non-small Cell Lung Cancer Cells

Background and objectiveLung cancer is one of the most prevalent malignancies and the leading cause ofcancer-related death worldwide in both men and women. Although multimodality therapiesand some molecular targeted therapies have been applied, the clinical responses to therapiesin patients with lung cancer are still unsatisfactory. The5-year over all survival in manycountries generally is less than15%. Despite aggressive therapies, resistance of manytumors to current treatment protocols still constitutes a major problem in cancer therapy. Topotentiate the efficacy and decrease the resistance and side effect of antitumor drugs, drugcombination is widely used clinically. Drug combination is an important progress in thetreatment of cancer in recent years and has become the trend of cancer treatment.Artesunate (ART), a major active ingredient of the Artemisia annua, displaysanticancer activity in vivo and has been successfully used to treat patients with metastaticmelanoma. It may rapidly convert into reactive oxygen species intracellularly, alterprincipal signaling pathways, inhibit metastasis, induce DNA damage, alter the cell cycleand reverse multidrug resistance.In this study we explored the effect of artesunate on potentiating anti-tumor activity oferlotinib in vivo and in vitro, and the molecular mechanism of the synergy with erlotinib inhuman non-small cell lung cancer A549cells. This research provides guidance forenhancing the anti-tumor effect and reversing the resistance of erlotinib clinically.Methods and results1. The Effects of artesunate on the proliferation and apoptosis of lung cancer A549cells.The effects of artesunate on A549cells proliferation were determined by MTT assay.AnnexinV-FITC and PI double staining was used to analyze apoptosis. ART inhibited Cell proliferation in A549cells in a concentration-dependent and time-dependent manner.Maximal proliferation inhibition was observed at96h with100μM ART, which inhibitedproliferation in93.5%. To further confirm the induction of apoptosis by ART, we stainedcells with AnnexinV-FITC/PI, The proportion of AnnexinV positive cells in ART treatedcells were inereased in a dose-dependent manner, which supports the finding that ARTinduced A549cells death by apoptosis.2. The effect of ART on enhancing anti-tumor activity of erlotinib in vivo and invitro.We explored the synergy of ART and erlotinib on A549cell lines. We found that ARTcombination with erlotinib had synergies on the tumor cell lines with CI value of0.58. Thenwe examined the synergic effect of ART combination with erlotinib on A549tumor growthinhibition in mice. The results showed that ART(60mg/kg) combination with erlotinib(50mg/kg) had the CI value of0.92, which means ART combination with erlotinib synergy isvery significant in vivo.3. ART potentiates the effect of erlotinib on apoptosis induction in A549cells.The results of Annexin V-FITC/PI stain and western blot showed ART enhanced theapoptosis induction activity of erlotinib in A549cell.4. The molecular mechanism of the synergy of ART combination with erlotinibWe further studied the mechanism of synergy. The results showed that ART potentiatethe inhibition effect of erlotinib in A549cells in EGFR/Akt dependent way and inhibition ofABCG2. It elevated levels of cell cycle G0/G1phase arrest. ART alone and combination witherlotinib can reduce the expression of pEGFR, pAkt and ABCG2level in A549and A549/ERcells. The data indicated that ART enhance apoptosis induction activity of erlotinib in A549and A549/ER cells associated with the inhibition of EGFR/Akt signal pathway.5. The molecular mechanism of the reverse of ART on erlotinib resistanceWe further studied the mechanism of reverse erlotinib resistance. The results showedthat ART have the reverse effect of erlotinib resistance in A549/ER and CD133C+D338~+A549cells by inhibiting ABCG2. It elevated levels of intracellular erlotinib and doxorubicinconcentration associated with reducing expression ABCG2.Conclusion Artesunate enhance apoptosis induction activity of erlotinib in A549and A549/ERcells associated with the inhibition of EGFR/Akt signal pathway. ABCG2involved in theerlotinib resistance and artesunate can reverse it by inhibiting ABCG2, increase intracellularerlotinib concentration.This study showed that artesunate as an anti-tumor sensitizer, has agreat potential value in clinical therapy.