Experimental Study Of ES2, A Jatrophane Diterpenoid Ester From Euphorbia Sororia, Reverses Tumor Multidrug Resistance

Background:Multidrug resistance (MDR) in cancer is a major complication in clinical oncology and constitutes one of the most common treatment limitations in cancer patients. Although several Pgp inhibitors have been tested in controlled clinical trials, no satisfactory results have been obtained so far. Therefore, to search for novel MDR modulators with higher efficacy and lower toxicity is still an urgent problem to be solved. Natural products, especially traditional Chinese herbal medicines, are investigated by many researchers as MDR modulators for their low cost, lower toxicity, good efficacy and a greater number of targets.9 new jatrophane-type diterpenoids in Euphorbia sororia A. Schrenk were found for the first time by Huang Y. and the screening results showed that the natural compounds had no appreciable cytotoxic effect against tumor cell-lines, but exhibited potential MDR reversal activity.Purpose:In this study, we will screen and evaluate the biological activity of 54 natural compounds isolated from the Euphorbia sororia, Euphorbia macrorrhiza and Euphorbia soongarica Boiss and find the most potential MDR reversal compound. In addition, we will focused on the deep investigation of the compound’s MDR reversal activity and an attempt at elucidating its underlying mechanism to provide solid molecular evidence in supporting the development of ES2 to be a novel MDR modulator in clinical cancer therapy in the future.Methods:1. The biological activity of 54 natural compounds isolated from the Euphorbia sororia, Euphorbia macrorrhiza were screened and evaluated and the most potential MDR reversal compound was found by MTT assay and flow cytometric analysis.2. Cytotoxicity effect and modulation of MDR of ES2 on MDR cells and their corresponding parental cells were tested by MTT assay.3. The effect of ES2 on the accumulation and efflux of DOX or Rhodamine123 in MDR cells and their corresponding parental cells were measured by fluorescence microscope and flow cytometry assay.4. The effect of ES2 on the ATPase activity of Pgp was assessed by Pgp-GloTM Assay Systems.5. Molecular docking model of ES2 binding to Pgp was performed by a virtual docking experiment.6. The effect of ES2 on Pgp expression at protein levels and its underlying mechanism were assessed by Western blot analysis.7. The effect of ES2 on Pgp expression at mRNA levels were tested by real-time fluorescent quantitative PCR.8. The duration of ES2 reversal activity using MTT assay was studied.Result:1. ES2 was the most potential tumor MDR reversal compound in the 54 natural compounds isolated from the Euphorbia sororia, Euphorbia macrorrhiza and Euphorbia soongarica Boiss.2. ES2, at up to 10 μM, had no appreciable cytotoxic effect to all cell lines and profoundly sensitized Pgp-overexpressing cells to chemotherapeutic drugs but not in parental cells. Moreover, its potency was greater than that of Verapamil.3. ES2 modulated Pgp-mediated transport by enhancing the accumulation of DOX or Rho123 and inhibiting the efflux of Rho123 in MDR cells overexpressing Pgp.4. ES2 stimulated the ATPase activity of Pgp and had no effect on verapamil-stimulated ATPase activity.5. ES2 was able to bind to Pgp at the same site as verapamil but did not completely overlap.6. ES2 significantly down-regulated Pgp expression at protein levels, but did not affect its expression at mRNA levels in MDR cells.7. The proteasome may be involved in the degradation of P-gp.8. ES2 had partially persistent reversal activity.Conclusion:ES2, a a jatrophane diterpenoid ester from Euphorbia sororia, reversed Pgp-mediated MDR by stimulating the ATPase activity of Pgp, down-regulating Pgp expression, inhibition of its drug efflux function, increasing the intracellular accumulation of anticancer drugs in Pgp-overexpressing cells. These results provide solid molecular evidence in supporting the development of ES2 to be a novel MDR modulator in clinical cancer therapy in the future.