| Background:Previous epidemiological studies have revealed deficiency of vitamin E (Ve) and selenium (Se) as a risk factor of ESCC in Linxian, a county in the Henan province in northern China, which has the highest incidence rate worldwide. The General Population Nutrition Intervention Trial conducted in this area demonstrated that supplementation with Ve/Se was preventive against esophageal cancer in subjects who entered in the trial at ages younger than 55 years old, but not in older subjects. On the other hand, a parallel trial with antioxidants and other nutrients failed to demonstrate a protective effect against esophageal and gastric cardia cancer among adults with esophageal dysplasia. Taken together, these results suggest that supplementation with antioxidant nutrients was effective in decreasing the incidence and mortality of esophageal cancer, however, the preventive effect probably occurred in early stage rather than late stage of cancer process.Among a variety of nutritional intervention trials, the results were inconsistent or controversial, because some interventions on high caner risk population showed no chemopreventive effect of nutrition supplementation, and some even raise concern that certain micronutrients could promote growth of preexisting tumors or precancerous lesions. Therefore, a novel concept was proposed by scientists that the efficacy of cancer chemoprevention by nutrients may be time-selective during the multistages of carcinogenesis—intervention at different stages of caner might generate diverse effects, i.e. effective at early stage while noneffective at late stage. This concept is consistent with the above mentioned Linxian intervention results, but it is unclear whether the "timing" of the intervention period can account for the conflicting results from chemoprevention trials with nutrients. However, time-selective effect of nutrients intervention in cancer chemoprevention has been given great attention over recent years.Considering results from Linxian trials of VE/Se intervention on esophageal cancer, as well as the great significance of these two micronutrients in human nutrition and cancer prevention, if the preventive effect of VE/Se supplementation at early and late stage of esophageal cancer could be tested and verified in animal model studies, and the efficacy of intervention time explained with the underlying mechanism, it would be of great significance to provide further evidence for the preventive effect of VE/Se and the scientific viewpoint of time-selective effect of cancer chemoprevention, which could be valuable in directing the future research and the practice of human cancer prevention with nutrients.Objective:(1)To prove that VE/Se deficiency could promote esophageal carcinogenesis, while supplementation with nutrients could inhibit cancer generation, with which providing further evidence to the chemoprevention effect of nutrient on cancer; (2)To explicit the time-selective effect of VE/Se supplementation on esophageal cancer, by which proving the conclusion from Linxian trial.Methods:(1) Explore the time progress of of N-Nitrosomethylbenzylamine (NMBzA)-induced tumorigenesis in rat esophageal cancer model, get the basis for early/late stage intervention in the next experiment.150 nine-week-old male F344 rats were randomized into three experimental groups:Model group A, Model group B and Control group. Rats in Model groups were treated with NMBzA, solubilized in 20% DMSO/water, at 0.35 mg/kg body weight, s.c.,3 times per week for 5 weeks. Control group was given the vehicle as negative control. Body weight and food intake were measured once a week during the experiment. Ten rats from each groups were sacrificed at Weeks 10 and 15 to assess the development of esophageal lesions, the number and size of visible tumors in esophagus were recorded, and fixation with 10% Neutral formaldehyde was performed for pathological examination. At week 25, all the remained animals were sacrificed and inspected and fixed in the same way. Histopathological lesions were defined in four grades:hyperplasia, dysplasia, papilloma and carcinoma, and they were respectively analyzed in each esophageal tissue section.(2) Mimic the low VE/Se nutritional status in the human populations such as that in Linxian using rat esophageal cancer model, study the promotive effect of VE/Se deficiency on esophageal cancer and probe into the time-selective effect of intervention.190 F344 rats were randomized into five experimental groups:rats in Groups A, B, C and D were treated with NMBzA, solubilized in 20% DMSO/water, at 0.35 mg/kg body weight, s.c.,3 times per week for 5 weeks. Group E was given the vehicle as negative control. Body weight and food intake were measured once a week during the experiment. Two different diets were prepared, normal diet and low Ve/Se diet. The normal diet was produced according to AIN-93M formula which contained 80 IU/kg a-tocopherol and 0.15 mg/kg Se. The low Ve/Se diet was made with the same formula but contained 46 IU a-tocopherol and 0.05 mg Se per kg diet. Diet intervention:rats were maintained on the low VE/Se diet as the control group(group A) and other groups were put on the normal AIN-93M diet at the early stage (Week 0-10, group B), late stage (Week 11-25, group C), or throughout the entire experiment (Week 0-25, group D). At Weeks 0,5,15 and 25, blood samples were collected from the retroorbital vein after anesthesia with ether. Plasma was stored at-80℃for VE/Se analysis. a-Tocopherol levels were determined by HPLC. Se in plasma was determined by fluorimetry with 2,3-diaminonaphthalene. At week 25, rats from each groups were sacrificed, the number and size of visible tumors in esophagus were quickly recorded by photograph and the esophagus was cut longitudinally, with one-half fixed in 10% neutral buffered formalin for pathological analysis. The other half of the esophagus was stripped of muscle, quickly frozen in liquid nitrogen and then stored at-80℃for Western blotting and enzyme immunoassay (EIA). Cell proliferation in esophageal mucosa was detected by BrdU immunohistochemistry(IHC). Microvessels were detected by immunofluorescence staining for von Willebrand factor, a marker for vascular endothelial cells. COX2,5LOX proteins expression were determined by Western blotting. EIA was used to determine the PGE2/LTB4 levels and 8-OhdG IHC was used to monitor DNA oxidative damage. Antioxidative enzymes activity was respectively detected with GPx,GST EIA assay kits.(3) According to the VE/Se supplementation levels in Linxian trial, intervention at different cancer stage was performed using rat esophageal cancer model to study the time-selective effect of VE/Se supplementation and its mechanism.240 F344 rats were randomized into six experimental groups:rats in Groups A, B, C, D and E were treated with NMBzA, solubilized in 20% DMSO/water, at 0.35 mg/kg body weight, s.c.,3 times per week for 5 weeks. Group F was given the vehicle as negative control. Body weight and food intake were measured once a week during the experiment. Three different diets were prepared, normal diet and low Ve/Se diet. The normal diet contained 80 IU/kg a-tocopherol and 0.15 mg/kg Se. The low Ve/Se diet contained 46 IU/kg a-tocopherol and 0.05 mg/kg Se. The high Ve/Se diet contained 162IU/kg a-tocopherol and 0.30mg/kg Se. Diet intervention:rats were maintained on the low VE/Se diet as the control group(group A) and normal diet as the normal control(group B), other groups were put on the high VE/Se diet at the early stage (Week 0-10, group C), late stage (Week 11-25, group D), or throughout the entire experiment (Week 0-25, group E). At Weeks 0,5,15 and 25, blood samples were collected from the retroorbital vein after anesthesia with ether. Plasma was stored at-80℃for VE/Se analysis. a-Tocopherol levels were determined by HPLC. Se in plasma was determined by atomic fluorescence spectrometry. At week 25, rats from each groups were sacrificed, the number and size of visible tumors in esophagus were quickly recorded by photograph and the esophagus was cut longitudinally, with one-half fixed in 10% neutral buffered formalin for pathological analysis. The other half of the esophagus was stripped of muscle, quickly frozen in liquid nitrogen and then stored at-80℃for Western blotting and EIA. Cell proliferation in esophageal mucosa was detected by BrdU IHC. NFκB p65 expression were determined by Western blotting. COX2,5LOX mRNA levels were determined by RT-PCR. EIA was used to determine the PGE2/LTB4 and 8-OhdG levels. Antioxidative enzymes activity in tissue and plasma was respectively detected with GPx,GST and SOD assay kits.Results:(1)Visible tumors:At Week 10, visible tumor incidences of Model group A and B were both 50%, and respectively increased to 70% and 80% at Week 15. At Week 25, tumor incidence of Model group B was 100%, significantly higher than Model group A, and also higher than that at Week 10 and 15. From Week 10 to 15, tumor volume in Model group A rapidly increased, and got closed to group B at Week 15 and higher than Week 10. From Week 15 to 25, Tumor multiplicity and volume in Model group A and B both obviously increased, significantly higher than that of Week 10 and 15. Histopathological examination:Rats in control group showed a few hyperplasia and no other pathologic lesions. At Week 10, the incidence and number of papilloma in both Model groups were low, no squamous cell carcinoma was observed. At Week 15, one rat in each group(10%) developed carcinoma; dyplasia number in Model group B significantly increased when comparing with that at Week 10, and also higher than group A. At Week 25, papilloma and carcinoma numbers in Model group B were significantly higher than that at Week 10.(2) General observations:The general appearance and activity level of animals were not affected by NMBzA treatments and low VE/Se diet intervention. Nutritional status:Both plasma a-tocopherol and Se levels in NMBzA-treated groups decreased after carcinogen treatment at Week 5. The low VE/Se diet had a profound effect in lowering the plasmaα-tocopherol and Se levels in rats; Switching to normal diet during the late stage increasedα-tocopherol and significantly Se levels in group C. Visible tumors and histological analysis:In comparison to group A which had a tumor incidence of 100%, early, late or continuous supplementation of Ve/Se (group B, C, D) significantly reduced tumor incidence to 82.9%,81.8%or 71.8%, respectively. Early supplementation (group B) and continuous supplementation (group D) both significantly decreased the numbers of visible esophageal tumors, dysplasia, papilloma and carcinoma, but late supplementation (group C) had similar numbers of visible tumors, dysplasia, papilloma, and also the incidences of papilloma and carcinoma, when comparing with low VE/Se group (group A). Cell proliferation:Compared with Low VE/Se group (group A), continuous supplementation (group D) significantly reduced cell proliferation in hyperplasia, papilloma and carcinoma. Early and late supplementation also lowed cell proliferation but the effect was significantly weaker than continuous supplementation (group D). In dysplasia and papilloma, the proliferation index of early supplementation (group B) was lower than that of late supplementation (Goup C), but not statistically significant. Angiogenesis:Compared with Low VE/Se group (group A), continuous supplementation (group D) significantly inbibited angiogenesis in dysplasia and papilloma, early and late supplementation (group B and C) also exhibited suppressing effect without significant difference. COX2,5LOX proteins expression:COX2/5LOX levels in NMBzA-treated groups (groupA-D) were significantly higher than that of Control (group E). Early and late supplementation (group B and C) both decreased COX2 and 5LOX expression that in Low VE/Se group (group A), but they were not statistically different from that of continuous supplementation (group D). PGE2/LTB4 levels:Low VE/Se group (group A) and late supplementation (goup C) resulted much higher PGE2/LTB4 levels than control group (group E), but early and continuous supplementation (group B and D) did not. Early supplementation seemed more effective in decreasing PGE2/LTB4 levels than late supplementation but there was no statistical significance between two groups. DNA oxidative damage:Compared with Low VE/Se group (group A), continuous supplementation (group D) significantly decreased the 8-OHdG positive cells in dysplasia, papilloma and carcinoma. As for dysplasia, the potive cell numbers of early supplementation group (group B) were significantly less than that of late supplementation group (goup C). In papilloma and carcinoma, less DNA damage was also observed in early supplementation group (group B) but not significantly different from late supplementation (goup C). Antioxidative enzymes activity:Esophagus GPx,GST and plasma GPx activity were significantly lower in Low VE/Se group (group A) than those in continuous supplementation (group D) and control group (group E). There was no difference between group C and D, but group B had lower esophagus GST activity and plasma GPx activity.(3) General observations:The general appearance and activity level of animals were not affected by NMBzA treatments and low or high VE/Se diet intervention. Nutritional status:Both plasma a-tocopherol and Se levels in early and continuous intervention group (group C and E) increased after changing to high VE/Se supplementation during the first 10 weeks. Since Week 11, switching to low VE/Se diet during the late stage significantly lowered a-tocopherol and Se levels in group C. On the contrary, late intervention group (group D) elevated plasma VE and Se levels after changed to high VE/Se diet. Visible tumors:Group A had the highest tumor incidence of 100%; When compared with group B (70.3%), early and continuous supplementation (group C and E) reduced tumor incidence to 64.1% and 65.8%, respectively, which were significantly lower than that of late supplementation (group D, 86.5%). Comparing with low VE/Se group (group A), early and continuous supplementation (group C and E) both significantly decreased the numbers of visible esophageal tumors, but late supplementation (group C) did not. Histological analysis: Early and continuous supplementation group (group C and E) had less ESCC incidence and number than low VE/Se group and late supplementation group (group A and D). Early supplementation group (group C) also had lower papilloma incidence and multiplicity than late supplementation group (group D), but without statistical significance. Late supplementation (group D) had similar dysplasia number with low VE/Se group (group A), both significantly more than early and continuous supplementation (group C and E). Cell proliferation:Late VE/Se supplementation (group D) did not inhibited cell proliferation in hyperplasia, dysplasia and papilloma, which were significantly higher than that of normal diet group (group B), early supplementation (group C) and continuous supplementation (group E). Cell proliferation in dyplasia and papilloma of group B was not statistically different with that in group E. NFκB expression:NFκB expression level in early supplementation group (group C) was significantly lower than that in late supplementation group (group D) which was not statistically different from that of Low VE/Se group (group A). When comparing with normal diet (group B), both early and continuous supplementation (group C and E) significantly decreased NFκB expression. COX2/5LOX mRNA levels:RT-PCT results demonstrated that COX2/5LOX mRNA levels in low VE/Se group (group A) were approximately 5～14 fold of those in control group, significantly higher than normal diet and other supplementation groups. Early supplementation group (group C) had lower COX2 mRNA levels than late and continuous supplementation group (group D and E). PGE2/LTB4 levels:PGE2/LTB4 levels of early supplementation group (goup C) were not different with continuous supplementation (group E), but significantly higher than that of normal VE/Se group (group B) and late supplementation group (group D) DNA oxidative damage:Low VE/Se group (group A) and early supplementation group (group C) resulted in similar 8-OHdG level in esophagus and plasma, which was higher than other NMBzA-treated groups. Early supplementation group (group C) had significantly higher plasma 8-OHdG level than late supplementation group (goup D). Antioxidative enzymes activity:Esophagus GPx,SOD and plasma GPx activity were significantly lower in Low VE/Se group (group A) than late and continuous supplementation (group D and E), but were not different from those in early supplementation group (group C). Early supplementation group (group C) had lower esophagus and plasma GPx, SOD activity than late supplementation group (group D).Conclusions:(1)VE/Se deficiency could significantly promote NMBzA-induced esophageal carcinogenesis in rat; supplementation with VE/Se at normal diet level did not show obvious time-selective effect on esophageal cancer prevention. (2)VE/Se supplementation at higher level demonstrated significant time-selective effect on cancer prevention:early supplementation was more effective than late supplementation which showed no effect in suppressing tumorigenesis. (3)Oxidative stress and aberrant metabolism of Arachidonic acid may play important role in NMBzA-induced esophageal carcinogenesis. Suppression of oxidative stress and NFκB-mediated inflammatory response at the early stage of carcinogenesis might be the underlying mechanism of time-selective chemoprevention of VE/Se supplementation on esophageal cancer. |
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