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BACKGROUND Long-term chemotherapy reduces the sensitivity of colon cancer cells to chemotherapeutics like vincristine (VCR) and lead to drug resistance, which has become a major barrier for colon cancer treatment. Calcium antagonists are used as clinical tumor multidrug resistance reversal agents to regulate the P-glycoprotein (P-gp) level and block efflux pump function now, but they have significant side effects. Hyperin as active component with low toxicity in traditional Chinese medicine has calcium antagonistic effect. Thus, the purpose of this study was to evaluate the inhibitory effect of hyperin on the growth of HCT8/VCR colon cancer cell line (vincristine-resistant) and analyze the enhancing effect of hyperin on the sensitivity of cancer cells to VCR and its relationship to the expression and function of P-gp. METHODS Using the MTT method, we investigated the influence of hyperin, VCR alone, and hyperin plus VCR on the growth of HCT8/VCR cells. Western blot analysis was employed to detect the expression of P-gp, and flow cytometry was used to evaluate P-gp function by detecting the fluorescence intensity of intracellular Rho123. RESULTS The inhibitory effect of hyperin at the dose of 12.5 μM on HCT8/VCR cell growth was not enhanced as time progressed and no significant inhibitory effect was found for VCR-treated cells at the dose of 2 μM. But the inhibition of cell growth was observed after the combined treatment of hyperin (12.5 μM) and VCR (2 μM). P-gp expression levels in HCT8/VCR cells treated with hyperin plus VCR were markedly lower than the levels in control cells and those treated with VCR. In addition, the intensity of Rho123 fluorescence of HCT8/VCR cells treated with hyperin plus VCR or hyperin alone was significantly higher than intensity observed in control cells and those treated with VCR alone. CONCLUSIONS Hyperin synergistically augments the growth inhibitory effect of vincristine. The underlying mechanism most probably involves down-regulation of P-gp expression and inhibiting the function of the P-gp pump in HCT8/VCR cells.