Effects Of The Diet With Vitamins On Proliferation Of Human Esophageal Cancer Cell Line And Its Mechanisms

Objectives:Esophageal cancer (EC) is prevalent in China where Yanting (YT) country is one of the regions with the highest incidence, and EC has been the leading cause of cancer deaths in YT region for more than thirty years. It is suggested that diet has a profound effect on susceptibility to EC. Studies on the etiology of esophageal cancer have been conducted in Yanting region since the 1970s. Little evidence, however, is available on the potential role of the local diet in Yanting region (YT diet) on the progression of esophageal cancer.The relationship between low intakes of vitamins and EC has attracted great interest worldwide; especially in those antioxidants that protect epithelial tissue. Vitamin A, riboflavin and folic acid play crucial roles in maintaining epithelial tissue, otherwise carcinogens could attack the damaged epithelial cells more easily. As indispensable anti-oxidants, vitamin E and vitamin C cooperate with each other in inhibiting carcinogenesis. It is confirmed that riboflavin and vitamin C are capable of lowering the risk of EC. Other vitamins such as vitamin A, E and folic acid have uncertain roles in the pathogenesis of EC.Recent research on diet and cancers has mainly focused on epidemiological studies, in vivo and in vitro experiments. Combining these methods together, could provide greater insight into the etiology of EC in Yanting region. Therefore, in the present study, a sero-physiology method, similar to the sero-pharmacology, was applied to avoid the shortcomings of each of the above methods when used independently.Nowadays, the roles of cell-cycle-regulating-gene on the etiology of EC have attracted increasing attentions, while few studies conducted in Yanting region focused on this field.In the present study, to assess the feasibility of feeding the rat with human adult diet, we observed effects of the human adult diet in Chengdu region on growth of the rats, which lay foundation for further study on the relationship between the Yanting diet and EC. A sero-physiology method was used to examine for possible effects of the sera of rats fed the Yanting diet and the Yanting diet supplemented with vitamins ( Mix.1, vit.A, E and folic acid; Mix.2 was Mix.1 plus riboflavin and vit.C ) at two doses on the proliferation of the human esophageal cancer cell line Eca-109. That will not only offer a new way to study the roles of the diet and nutrients on the development of cancer, but also provide evidence for regulation of the Yanitng diet and chemoprevention of EC in future human trials in the Yanting region. The third objective was to explore effects of the sera of rats fed the Yanting diet and the Yanting diet supplemented with vitamins on expression of cell-cycle-regulating-gene in human esophageal cancer cell line Eca-109, by means of real-time polymerase chain reaction (RT-PCR) and immunofluorescence methods, which will give insight into the mechanisms of the Yanting diet and vitamins on the progression of EC and maybe provide evidence for gene-therapy in clinic.Methods:(1) To assess the feasibility of feeding the rat with human adult diet, we observed effects of the healthy human adult diet in Chengdu region (Chengdu diet) on diet intakes and body weights of the rats, and a standard diet for rat was set as the control.(2) MTT cell growth assay was used to select the optimal conditions of rat's serum, such as different concentrations and deactivated or not, for cultivating the human esophageal cancer cell line Eca-109. In addition to this, 10% FBS (fetal bovine serum) and human normal liver epithelial cell line HL7702 were set as serum control and normal cell line control, respectively, which made the sero-physiology method more perfect.(3) The effects of sera of rats fed the Chengdu diet, the Yanting diet and the Yanting diet supplemented with vitamins (Mix. 1, vit. A, E and folic acid; Mix.2 was Mix.1 plus riboflavin and vit.C) at two doses on proliferation of human esophageal cancer cell line Eca-109 were assessed by means of MTT assay, doubling time, DNA synthesis, and flow cytometry assays.(4) Real-time polymerase chain reaction (RT-PCR) and immunofluorescence methods were adopted to explore effects of the rats' sera fed the Yanting diet and the Yanting diet supplemented with vitamins on expression of cell-cycle-regulating-gene at mRNA and protein level of human esophageal cancer cell line.Results:(1) In the animal trial, the mean daily food intakes were significantly lower in the Chengdu diet group than the standard diet group. However, no obvious differences of body weight gains were found between the two groups.(2) Non-deactivated rat serum (serum of rats fed the standard diet) at a concentration of 5% provided the optimal conditions for cultivating the Eca-109 cell, and such condition had exactly the same effect on the proliferation of human normal liver epithelial cell line HL7702 as that of FBS.(3) MTT assay, DNA synthesis, and flow cytometry assays showed that rats' sera fed the YT diet significantly accelerated Eca-109 cell proliferation, while inhibited the proliferation of HL7702 cell in contrast with the control (FBS). These changes, however, were reversed by supplementation with two vitamin mixtures, and especially in the groups with high doses. Rats' sera fed the Chengdu diet had similar effects on the proliferation of two cell lines as those of the control.(4) Rats' sera fed the Yanting diet abnormally increased the expression of oncogenes CyclinD1 and CDK4 at mRNA and protein level of human esophageal cancer cell line, whereas the sera had the contrary effects on those of anti-oncogene P16. The sera showed the tendency to lower the expression of anti-oncogene Rb, but there were no obvious differences between it and the control. These changes, however, were reversed by supplementation with two vitamin mixtures, and especially in the groups with high doses.Conclusions:(1) It is feasible to feed the rat with human adult diet without affecting its' growth.(2) It is feasible to cultivate the human esophageal cancer cell line Eca-109 with rat serum instead of the conventional fetal bovine serum (FBS).(3) Compared to sera of rats fed the Chengdu diet, sera of rats fed the Yanting diet significantly accelerated Eca-109 cell proliferation, while inhibited the proliferation of HL7702 cell, which indicates that there maybe exist cancer-promoting factors in the Yanting diet.(4) The effects of sera of rats fed the Yanting diet on the proliferation of two cell lines were reversed by sera of rats fed the Yanting diet supplemented with vitamin mixture, which shows that an abundance of dietary vitamins may protect the epithelial cell against carcinogens, while vitamin deficiency has the opposite effect. (5) Sera of rats fed the Yanting diet made the oncogenes abnormally highly expression, while lowered the expression of anti-oncogenes in human esophageal cancer cell line, which maybe one of the mechanisms that the sera could promote esophageal cancer cell proliferation. However, vitamin mixtures having opposite effects on those genes maybe contribute to the inhibitory effects on the proliferation of the cancer cells.