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 combination treatment,low doses of 5-FU were used combined with various concentrations of HHC to minimize the toxicity 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:MTT 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 observation 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 μmol/L in combination with HHC at the concentration of 25 μmol/L significantly 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.

Phonopheresis Associated with Nanoparticle Gel from Phyllanthus amarus Relieves Pain by Reducing Oxidative Stress and Proinflammatory Markers in Adults with Knee Osteoarthritis

Objective: To determine the changes in serum levels of inflammatory biomarkers and antioxidant levels among the knee osteoarthritis(OA) patients after treatment with Phyllanthus amarus(PP) by nanoparticle gel phonophoresis. Methods: This study was a randomized, double-blind, placebo-control, parallel-group, clinical trial involving 30 subjects with mild-to-moderate degree of knee OA. The patients were allocated to two groups using a computer-generated random numbers, and received conventional ultrasound therapy(control group, 15 cases) and PP(treatment group, 15 cases) once daily for 10 sessions. The pain was evaluated by visual analogue scale(VAS). Serum levels of tumor necrosis factor-α(TNF-α) were determined by enzyme-linked immunosorbnent assay(ELISA). Nitric oxide(NO) was determined by modified Griess reagent. The antioxidant effects, including superoxide dismutase(SOD) and total antioxidant capacity(TAC), were also measured by ELISA assay. Results: The VAS score was significantly decreased in the treatment group compared with the control group after treatment(P<0.01). The serum concentrations of TNF-α and NO were significantly reduced in the treatment group compared with the control group(P<0.01) after treatment. However, the serum concentrations of SOD and TAC in the treatment group were significantly higher after treatment compared with the control group(P<0.01). Conclusion: PP could alleviate knee pain and significantly reduce systemic antiinflammatory effects in knee OA patients.