Objective: To investigate the potential mechanisms that curcumin reverses 5-fluorouracil(5-FU) multidrug resistance(MDR). Methods: Cell growth and the inhibitory rate of curcumin(2–25 μg/mL) and/or5-FU(0.05–1000 μ...Objective: To investigate the potential mechanisms that curcumin reverses 5-fluorouracil(5-FU) multidrug resistance(MDR). Methods: Cell growth and the inhibitory rate of curcumin(2–25 μg/mL) and/or5-FU(0.05–1000 μg/mL) on human colon cancer HCT-8 and HCT-8/5-FU(5-FU-resistant cel line) were determined using cel counting kit-8(CCK-8) assay. Apoptosis and cel cycle after 5-FU and/or curcumin treatment were detected by ?ow cytometry(FCM) and transmission electron microscopy(TEM). The expression of the multidrug resistance related factors p-glycoprotein(P-gp) and heat shock protein 27(HSP-27) genes and proteins were analyzed by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting(WB), respectively. Results: The inhibitory rate of curcumin or 5-FU on HCT-8 and HCT-8/5-FU cells proliferation at exponential phase were in a dosedependent manner, HCT-8 cell line was more sensitive to curcumin or 5-FU when compared the inhibitory rate of HCT-8/5-FU. The 50% inhibitory concentration(IC50) of combination 5-FU and curcumin(4.0 μg/mL)in HCT-8/5-FU was calculated as 179.26 μg/mL, with reversal fold of 1.85. Another IC50 of combination 5-FU and curcumin(5.5 μg/mL) in HCT-8/5-FU was calculated as 89.25 μg/mL, with reversal fold of 3.71. Synergistic effect of 5-FU and curcumin on HCT-8 and HCT-8/5-FU cells were found. The cell cycle analysis performed by FCM showed that HCT-8 and HCT-8/5-FU cel s mostly accumulated at G0/G1 phase, which suggested a synergistic effect of curcumin and 5-FU to induce apoptosis. FCM analysis found that the percentage of apoptosis of cel s treated with curcumin, 5-FU and their combination were signi?cantly increased compared to the control group(P<0.05), and the percentage of apoptosis of the combination groups were slightly higher than other groups(P<0.05). The m RNA levels of P-gp(0.28±0.02) and HSP-27(0.28±0.09) in HCT-8/5-FU cel s treated with combination drugs were lower than cel s treated with 5-FU alone(P-gp, 0.48±0.07, P=0.009;HSP-27, 0.57±0.10, P=0.007). The protein levels of P-gp(0.25±0.06) and HSP-27(0.09±0.02) in HCT-8/5-FU cells treated with combination drugs were decreased when compared to 5-FU alone(P-gp, 0.46±0.02, P=0.005;HSP-27, 0.43±0.01, P=0.000). Conclusions: Curcumin can inhibit the proliferation of human colon cancer cells. Curcumin has the ability of reversal effects on the multidrug resistance of human colon cancer cells lines HCT-8/5-FU. Down-regulation of P-gp and HSP-27 may be the mechanism of curcumin reversing the drug resistance of HCT-8/5-FU to 5-FU.展开更多
基金Supported by China Science Fund of Clinical Oncology(No.Y-L2014-002)
文摘Objective: To investigate the potential mechanisms that curcumin reverses 5-fluorouracil(5-FU) multidrug resistance(MDR). Methods: Cell growth and the inhibitory rate of curcumin(2–25 μg/mL) and/or5-FU(0.05–1000 μg/mL) on human colon cancer HCT-8 and HCT-8/5-FU(5-FU-resistant cel line) were determined using cel counting kit-8(CCK-8) assay. Apoptosis and cel cycle after 5-FU and/or curcumin treatment were detected by ?ow cytometry(FCM) and transmission electron microscopy(TEM). The expression of the multidrug resistance related factors p-glycoprotein(P-gp) and heat shock protein 27(HSP-27) genes and proteins were analyzed by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting(WB), respectively. Results: The inhibitory rate of curcumin or 5-FU on HCT-8 and HCT-8/5-FU cells proliferation at exponential phase were in a dosedependent manner, HCT-8 cell line was more sensitive to curcumin or 5-FU when compared the inhibitory rate of HCT-8/5-FU. The 50% inhibitory concentration(IC50) of combination 5-FU and curcumin(4.0 μg/mL)in HCT-8/5-FU was calculated as 179.26 μg/mL, with reversal fold of 1.85. Another IC50 of combination 5-FU and curcumin(5.5 μg/mL) in HCT-8/5-FU was calculated as 89.25 μg/mL, with reversal fold of 3.71. Synergistic effect of 5-FU and curcumin on HCT-8 and HCT-8/5-FU cells were found. The cell cycle analysis performed by FCM showed that HCT-8 and HCT-8/5-FU cel s mostly accumulated at G0/G1 phase, which suggested a synergistic effect of curcumin and 5-FU to induce apoptosis. FCM analysis found that the percentage of apoptosis of cel s treated with curcumin, 5-FU and their combination were signi?cantly increased compared to the control group(P<0.05), and the percentage of apoptosis of the combination groups were slightly higher than other groups(P<0.05). The m RNA levels of P-gp(0.28±0.02) and HSP-27(0.28±0.09) in HCT-8/5-FU cel s treated with combination drugs were lower than cel s treated with 5-FU alone(P-gp, 0.48±0.07, P=0.009;HSP-27, 0.57±0.10, P=0.007). The protein levels of P-gp(0.25±0.06) and HSP-27(0.09±0.02) in HCT-8/5-FU cells treated with combination drugs were decreased when compared to 5-FU alone(P-gp, 0.46±0.02, P=0.005;HSP-27, 0.43±0.01, P=0.000). Conclusions: Curcumin can inhibit the proliferation of human colon cancer cells. Curcumin has the ability of reversal effects on the multidrug resistance of human colon cancer cells lines HCT-8/5-FU. Down-regulation of P-gp and HSP-27 may be the mechanism of curcumin reversing the drug resistance of HCT-8/5-FU to 5-FU.
