title: Effects of hexahydrocurcumin in combination with 5-fluorouracil on dimethylhydrazine-induced colon cancer in rats.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  The combined effects of HHC with 5-FU exhibit a synergistic inhibition by decreasing ACF formation mediated by down-regulation of COX-2 expression.
authors: Department of Anatomy, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand.
authors: Khanitta
authors: K
authors: Srimuangwong
authors: Chainarong
authors: C
authors: Tocharus
authors: Jiraporn
authors: J
authors: Tocharus
authors: Apichart
authors: A
authors: Suksamrarn
authors: Pornphorm Yoysungnoen
authors: PY
authors: Chintana
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols; D017209:Apoptosis; D003110:Colonic Neoplasms / Q000139:chemically induced / Q000188:drug therapy*; D003474:Curcumin / Q000008:administration & dosage / Q000031:analogs & derivatives*; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2; D004127:Dimethylhydrazines / Q000009:adverse effects*; D005472:Fluorouracil / Q000008:administration & dosage*; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004127:Dimethylhydrazines; D008565:Membrane Proteins; C569902:hexahydrocurcumin; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496513:Ptgs1 protein, rat; C489691:Ptgs2 protein, rat; D003474:Curcumin; D005472:Fluorouracil
keywords: Apoptosis; Colon cancer; Combination treatment; Curcumin analog; Cyclooxygenase-2; Hexahydrocurcumin