Effects of hexahydrocurcumin in combination with 5-fluorouracil on dimethylhydrazine-induced colon cancer in rats

AIM:To investigate the effects of hexahydrocurcumin(HHC),and its combination with 5-fluorouracil(5-FU) on dimethylhydrazine(DMH)-induced colon cancer in rats.METHODS:Male Wistar rats weighing 100-120 g were used as subject models.Aberrant crypt foci(ACF),early preneoplastic lesions of colon cancer,were induced by subcutaneous injection of DHM(40 mg/kg) twice a week for two weeks.After the first DMH injection,rats were treated daily with vehicle(n = 12),curcumin(CUR)(50 mg/kg)(n = 12),HHC(50 mg/kg) orally(n = 12),and treated weekly with an intraperitoneal injection of 5-FU(50 mg/kg)(n = 12),or a combination of 5-FU plus CUR(n = 12) and HHC(n = 12) at the same dosage(s) for 16 wk.The total number of ACF and large ACF were assessed.Cyclooxygenase(COX)-1 and COX-2 expression were detected by immunohistochemistry in colon tissues.The quantitative data of both COX-1 and COX-2 expression were presented as the percentage of number of positive-stained cells to the total number of cells counted.Apoptotic cells in colon tissues were also visualized using the dUTP-biotin nick end labeling method.Apoptotic index(AI) was determined as the percentage of labeled nuclei with respect to the total number of nuclei counted.RESULTS:The total number of ACF was highest in the DMH-vehicle group(1558.20 ± 17.37),however,the number of ACF was significantly reduced by all treatments,5-FU(1231.20 ± 25.62 vs 1558.20 ± 17.37,P < 0.001),CUR(1284.20 ± 25.47 vs 1558.20 ± 17.37,P < 0.001),HHC(1086.80 ± 53.47 vs 1558.20 ± 17.37,P < 0.001),DMH-5-FU + CUR(880.20 ± 13.67 vs 1558.20 ± 17.37,P < 0.001) and DMH-5-FU + HHC(665.80 ± 16.64 vs 1558.20 ± 17.37,P < 0.001).Interestingly,the total number of ACF in the combined treatment groups,the DMH-5-FU + CUR group(880.20 ± 13.67 vs 1231.20 ± 25.62,P < 0.001;880.20 ± 13.67 vs 1284.20 ± 25.47,P < 0.001) and the DMH-5-FU + HHC group(665.80 ± 16.64 vs 1231.20 ± 25.62,P < 0.001;665.80 ± 16.64 vs 1086.80 ± 53.47,P < 0.001) were significantly reduced as compared to 5-FU or each treatment alone.Large ACF were also significantly reduced in all treatment groups,5-FU(111.00 ± 7.88 vs 262.20 ± 10.18,P < 0.001),CUR(178.00 ± 7.33 vs 262.20 ± 10.18,P < 0.001),HHC(186.60 ± 21.51 vs 262.20 ± 10.18,P < 0.001),DMH-5-FU + CUR(122.00 ± 5.94 vs 262.20 ± 10.18,P < 0.001) and DMH-5-FU + HHC(119.00 ± 17.92 vs 262.20 ± 10.18,P < 0.001) when compared to the vehicle group.Furthermore,in the DMH-5-FU + CUR and DMH-5-FU + HHC groups the formation of large ACF was significantly reduced when compared to CUR(122.00 ± 5.94 vs 178.00 ± 7.33,P < 0.005) or HHC treatment alone(119.00 ± 17.92 vs 186.60 ± 21.51,P < 0.001),however,this reduction was not statistically different to 5-FU monotherapy(122.00 ± 5.94 vs 111.00 ± 7.88,P = 0.217;119.00 ± 17.92 vs 111.00 ± 7.88,P = 0.619,respectively).The levels of COX-1 protein after all treatments were not different from normal rats.A marked increase in the expression of COX-2 protein was observed in the DMH-vehicle group.Over-expression of COX-2 was not significantly decreased by 5-FU treatment alone(95.79 ± 1.60 vs 100 ± 0.00,P = 0.198).However,over-expression of COX-2 was significantly suppressed by CUR(77.52 ± 1.68 vs 100 ± 0.00,P < 0.001),HHC(71.33 ± 3.01 vs 100 ± 0.00,P < 0.001),5-FU + CUR(76.25 ± 3.32 vs 100 ± 0.00,P < 0.001) and 5-FU + HHC(68.48 ± 2.24 vs 100 ± 0.00,P < 0.001) in the treated groups compared to the vehicle group.Moreover,CUR(77.52 ± 1.68 vs 95.79 ± 1.60,P < 0.001),HHC(71.33 ± 3.01 vs 95.79 ± 1.60,P < 0.001),5-FU + CUR treatments(76.25 ± 3.32 vs 95.79 ± 1.60,P < 0.001) and 5-FU + HHC(68.48 ± 2.24 vs 95.79 ± 1.60,P < 0.001) markedly decreased COX-2 protein expression more than 5-FU alone.Furthermore,the AI in all treated groups,5-FU(38.86 ± 4.73 vs 23.56 ± 2.12,P = 0.038),CUR(41.78 ± 6.92 vs 23.56 ± 2.12,P < 0.001),HHC(41.06 ± 4.81 vs 23.56 ± 2.12,P < 0.001),5-FU + CUR(49.05 ± 6.75 vs 23.56 ± 2.12,P < 0.001) and 5-FU + HHC(53.69 ± 8.59 vs 23.56 ± 2.12,P < 0.001) significantly increased when compared to the DMH-vehicle group.However,the AI in the combination treatments,5-FU + CUR(49.05 ± 6.75 vs 41.78 ± 6.92,P = 0.192;49.05 ± 6.75 vs 38.86 ± 4.73,P = 0.771) and 5-FU + HHC(53.69 ± 8.59 vs 41.06 ± 4.81,P = 0.379;53.69 ± 8.59 vs 38.86 ± 4.73,P = 0.245) did not reach significant levels as compared with each treatment alone and 5-FU monotherapy,respectively.CONCLUSION:The combined effects of HHC with 5-FU exhibit a synergistic inhibition by decreasing ACF formation mediated by down-regulation of COX-2 expression.

Hexahydrocurcumin enhances inhibitory effect of 5-fluorouracil on HT-29 human colon cancer cells

AIM： To investigate the ability of hexahydrocurcumin （HHC） to enhance 5-fluorouracil （5-FU） in inhibiting the growth of HT-29 cells by focusing on cyclooxygenase （COX）-2 expression.METHODS： Antiproliferative effects of HHC and 5-FU, alone and in combination, on growth of HT-29 human colon cancer cells were assessed using 5-diphenyltetrazolium bromide （MTT） reduction assay. In combinationtreatment, low doses of 5-FU were used combined with various concentrations of HHC to minimize the toxic- ity and side effects of 5-FU. The therapeutic effects of these drugs on down-regulation of COX-2 mRNA and protein expression were examined using semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting analysis.RESULTS： Ml-I- reduction assay indicated that HHC alone markedly decreased the viability of HT-29 human colon cancer cells compared to control. Semi-quantitative RT-PCR analysis indicated that HHC is a selective COX-2 inhibitor. This finding was supported by the ob- servation that HHC significantly down-regulates COX-2 mRNA expression compared to the control （control： 100.05% ± 0.03% vs HHC： 61.01% ± 0.35%, P 〈 0.05） but does not alter COX-1 mRNA. In combined treatment, addition of HHC to a low dose of 5-FU exerts a synergistic effect against the growth of HT-29 cells by markedly reducing cell viability to a greater degree than monotherapy. Semi-quantitative RT-PCR indicated that 5-FU at the concentration of 5 pmol/L in combina- tion with HHC at the concentration of 25 pmol/L signifi- cantly down-regulates COX-2 mRNA expression when compared with values in cells treated with 5-FU or HHC alone （HHC ＋ 5-FU： 31.93% ± 5.69%, 5-FU： 100.66% ± 4.52% vs HHC： 61.01% ±0.35%, P 〈 0.05）.CONCLUSION： HHC together with 5-FU exerts a synergistic effect and may prove chemotherapeutically useful in treating human colon cancer.