In Vivo Reversal of P-Glycoprotein-Mediated Drug Resistance in a Breast Cancer Xenograft and in Leukemia Models Using a Novel, Potent, and Nontoxic Epicatechin EC31.

The modulation of P-glycoprotein (P-gp, ABCB1) can reverse multidrug resistance (MDR) and potentiate the efficacy of anticancer drugs. Tea polyphenols, such as epigallocatechin gallate (EGCG), have low P-gp-modulating activity, with an EC50 over 10 µM. In this study, we optimized a series of tea polyphenol derivatives and demonstrated that epicatechin EC31 was a potent and nontoxic P-gp inhibitor. Its EC50 for reversing paclitaxel, doxorubicin, and vincristine resistance in three P-gp-overexpressing cell lines ranged from 37 to 249 nM. Mechanistic studies revealed that EC31 restored intracellular drug accumulation by inhibiting P-gp-mediated drug efflux. It did not downregulate the plasma membrane P-gp level nor inhibit P-gp ATPase. It was not a transport substrate of P-gp. A pharmacokinetic study revealed that the intraperitoneal administration of 30 mg/kg of EC31 could achieve a plasma concentration above its in vitro EC50 (94 nM) for more than 18 h. It did not affect the pharmacokinetic profile of coadministered paclitaxel. In the xenograft model of the P-gp-overexpressing LCC6MDR cell line, EC31 reversed P-gp-mediated paclitaxel resistance and inhibited tumor growth by 27.4 to 36.1% (p < 0.001). Moreover, it also increased the intratumor paclitaxel level in the LCC6MDR xenograft by 6 fold (p < 0.001). In both murine leukemia P388ADR and human leukemia K562/P-gp mice models, the cotreatment of EC31 and doxorubicin significantly prolonged the survival of the mice (p < 0.001 and p < 0.01) as compared to the doxorubicin alone group, respectively. Our results suggested that EC31 was a promising candidate for further investigation on combination therapy for treating P-gp-overexpressing cancers.

Curcumin synergizes with 5-fluorouracil by impairing AMPK/ULK1-dependent autophagy, AKT activity and enhancing apoptosis in colon cancer cells with tumor growth inhibition in xenograft mice.

BACKGROUND: Chemoresistance is a major obstacle that limits the benefits of 5-Fluorouracil (5-Fu)-based chemotherapy for colon cancer patients. Autophagy is an important cellular mechanism underlying chemoresistance. Recent research advances have given new insights into the use of natural bioactive compounds to overcome chemoresistance in colon cancer chemotherapy. As one of the multitargeted and safer phytomedicines, curcumin has been reported to work as cancer-specific chemosensitizer, presumably via induction of autophagic signaling pathways. The precise therapeutic effect of curcumin on autophagy in determining tumorous cells' fate, however, remains unclear. This study was conducted to investigate the differential modulations of the treatments either with 5-Fu alone or 5-Fu combined with curcumin on cellular autophagic responses and viabilities in the human colon cancer cells HCT116 and HT29, and explore molecular signaling transductions underlying the curcumin-mediated autophagic changes and potentiation of 5-Fu's cytotoxicity in vitro and in vivo. METHODS: Cell proliferation assay and morphology observation were used to identify the cytotoxicity of different combinations of curcumin and 5-Fu in HCT116 and HT29 cells. Cell immunofluorescence assay, Flow cytometry and Western blot were employed to detect changes of autophagy and the autophagy-related signaling pathways in the colon cancer cells and/or xenograft mice. RESULTS: Curcumin could significantly augment the cytotoxicity of 5-Fu to the tumorous cells, and the pre-treatment with curcumin followed by 5-Fu (pre-Cur) proved to be the most effective one compared to other two combinations. The chemosensitizing role of curcumin might attribute to the autophagy turnover from being activated in 5-Fu mono-treatment to being inhibited in the pre-Cur treatment as indicated by the changes in expression of beclin-1, p62 and LC3II/LC3I and the intensity of Cyto-ID Green staining. The autophagic alterations appeared to be contributed by down-regulation of not only the phospho-Akt and phospho-mTOR expressions but the phospho-AMPK and phospho-ULK1 levels as well. The cellular activation of AMPK by addition of A-769662 to the pre-Cur combination resulted in reversed changes in expressions of the autophagy protein markers and apoptotic status compared to those of the pre-Cur combination treatment. The findings were validated in the xenograft mice, in which the tumor growth was significantly suppressed in the mice with 25-day combination treatment, and meanwhile expressions of the autophagy markers, P-AMPK and P-ULK1 were all reversely altered in line with those observed in HCT116 cells. CONCLUSION: Pre-treatment with curcumin followed by 5-Fu may mediate autophagy turnover both in vitro and in vivo via AMPK/ULK1-dependent autophagy inhibition and AKT modulation, which may account for the increased susceptibility of the colon cancer cells/xenograft to the cytotoxicity of 5-Fu.