A Study Of Sodium Butyrate On It's Anti-Carcinogenesis And Molecule Mechanism

Background Colorectal Cancer is one of the most common malignant tumors, which incidence prove to be ascending in recent years. Recent progress in molecular biology and genetics has improved the understanding of it's carcinogenesis mechanisms. However, there are few effective methods for prevention or therapies against cancer based on such elucidated molecular mechanisms of carcinogenesis.The exclusive radical cure method of the disease is to resect the tumor as early as possible. But the principal diagnostic approach at present mainly depend on pathological diagnosis through colonoscope, and most patients are already in the advanced stage when their disease are final diagnosed, and they have no chance to be cured by operation. Meanwhile the colon carcinoma is not sensitive to chemotherapeutics, and radiotherapy is merely a perioperative adjunctive therapy (AT). All of these acutely affect and restrict the prognosis of the disease, and the five- year survival rates are less than 50%. Therefore to study its pathogenesy and to explore effective chemopreventive agents become the tumor researchers' important and urgent mission.The tumor-suppressor gene p53 is the most important anti-oncogene, and it is mutated in about 50% of human malignancies, including colorectal cancer. It is known that p53 stimulates the promoter activities of p21wafl, gadd45 and bax genes to enhance their expression as a transcriptional factor, resulting in cell cycle arrest, DNA repair and apoptosis, respectively. Therefore, chemical compounds or food factors that can stimulate these genes might compensate for part of the p53 function. We therefore hypothesized that a strategy for up-regulating p53-target genes such as p21waf1, gadd45 and bax might be useful for cancer prevention or therapy, and we proposed that "Gene-regulating chemoprevention" and"Gene-regulating chemotherapy" may be new promising strategies for cancer prevention or therapy, and histone deacetylase inhibitors, such as butyrate and TSA are good candidates for these strategies.The cause of colorectal cancer is still not fully understood at present. The major reason is considered to be the result of synthetic action of environmental and genetic factors. Among the environmental agent, the relationship between colorectal cancer and dietary factor is paid more and more attention. According to many reports, the role of dietary factor is about 50% , but genetic factor is approximately 15%.Data of epidemiology and animal experiments have proved that high fat diet and /or insufficient dietary fiber are the major correlation factors, and epidemiological evidence suggests that a high intake of resistant starch and dietary fiber protects against colorectal cancer. The mechanisms underlying this protection are thought to be mediated by the short-chain fatty acid butyrate.With the development of epigenetics, butyrate, the ferment of dietary fiber in the colon bacterium, draws the people's interest again. Sodium butyrate is a very important intestinal mucosa nutritional agent, and it has been knownto be active on many kinds of cultured mammal cells in vitro, including inhibiting cellular proliferation, inducing apoptosis and so on. Being a very potential anticancer drug it is increasingly paid close attention.In recent years ,a serial studies on the anti-tumor effects of sodium butyrate have been carried out all over the world. But there are few studies about of regulations of sodium butyrate on expression of some p53's target genes, such as p21wafl, Bax and Gadd45 gene, and there is no systematic study both in vitro and in vivo . The aim of our study is to approach the molecule mechanisms of sodium butyrate in inhibiting cellular proliferation and inducing apoptosis by analyzing its regulations on the expressions of three main target genes of p53, including p21wafl, Bax and Gadd45 gene.In this research, a thorough investigation of the regulations of sodium butyrate on expression of the three main target genes of p53 both in vitro and in vivo was processed.This study will enrich the anticancer mechanism of butyrate and provide data for applying sodium butyrate in clinical therapy of colon cancer. Meanwhile it will also prompt that high butyrate dietary will be helpful to reduce or even prevent the incidence of colorectal cancer.Objectives Experiment 1:Is To test if there is any difference in inhibiting proliferation and prompting apoptosis among human colon cell line Lovo with wild p53, HT-29 with mutant p53,and the human embryo kidney 293 cell.2> To observe the regulation of sodium butyrate on the three main target genes of p53 including p21wafl,Bax and Gadd45,and further explore the molecule mechanism of its inhibiting proliferation and inducing apoptosis of colon cancer cells.Experiment 2:1, To observe the carcinogenesis of colorectal cancer on male mice of KUNMING species by subcutaneous injection of DMH;2> To explore the relationship between the status of proliferation and apoptosis of the large bowel mucosa during the mice colon carcinogenesis induced by DMH and the carcinogenesis of colorectal cancer;3n To analyze the relationship between the expressions of p21wafl,Bax and Gadd45 and the carcinogenesis;Experiment 3:K To observe the inhibitory actions of sodium butyrate (50mM and lOOmM) given by intra- rectal infuse on colon carcinogenesis of the experimental mice;2^ To investigate the regulations of sodium butyrate on status of the proliferationand apoptosis of the large bowel mucosa ,as well as expressions of the p21wafl,Bax and Gadd45 genes. To probe the anti-tumor roles of sodium butyrate in vivo furthermore;Materials and MethodsThe colon cancer cell line Lovo (with wild-type p53 gene ) , HT-29 (with mutant-type p53 gene),and human embryo kidney 293 cell line(diploid) were cultivated in RPMI1640 medium, which contain 10% calf blood serum and were placed in a constant temperature oven of 37°C and 5% CC>2.Then NaBT of different concentrations were used to dispose these cells. The final concentrations of experiment groups were 1.25mMs 2.5mlVk 5.0mML 1 OmM respectively , and PBS was used in control group. We applied respectively (1) MTT to detect the cellular proliferation and draw the growth curve; (2 ) flow cytometry to examine the apoptosis and periodic changes of these cells;(3)inverted phase contrast microscope, Bradford Variable Projective Microscope(BVPM),and scanning electron microscope (SEM) ,as well as transmission electron microscope (TEM) to observe morphologic changes of these cells, and the changes of cells' ultra-microstructure and apoptotic body;(4) Annexin V-FITC/PI double tagging and TUNEL staining to survey apoptosis;(5) RT-PCR and Western blot to study the expression of p21wafl,Bax,and Gadd45 messenger ribonuclcic acid(mRNA) and that of the protein.Colorectal cancer of male mice of KUNMING species were induced by subcutaneous injection of DMH weekly for 11 weeks .The dose of DMH given to each mouse was SOmg.kg"1 wt.w"1.Simultaneously butyrate was intrarectal administration daily for 24 weeks, and the dose of butyrate given to each mouse was 0.5ml.d"'.The mice were killed in batches in the 12th, 18th and 24lh weeks of carcinoma induction separately. We studied (l)the incidence of colon tumors and burden with macroscopic observation;(2)the state of proliferation and apoptosis of the mucous membrane of colon during the carcinogenesis and the regulation of sodium butyrate with PCNA and TUNEL respectively;(3)the mRNAexpressions of these three genes,p21wafl,Bax and Gadd45 by RT-PCR and these protein expressions by both immunohistochemistry and Western blot. We also observed the relationship between these three genes and the carcinogenesis of the mice's colon,as well as the regulation to them by sodium butyrate. Meanwhile the general state, the body weight increasing, the liver and renal function of lOOmM butyrate group mice, as well as the pathological changes of its liver, kidney, lungs, and pancreas were also observed .The difference of all items between control group and 100 mM butyrate enema group were analyzed.Methods of Statistics The data were statistically processed analysis of variance(ANOVA), x2test and / test by the software SPSS11.0,and with the value of P smaller than 0.05 being considered statistically significant.Results Experiment 1:1 n Cancer cells' growth apoptosis examination: Different concentration of sodium butyratewere used to dispose cultured cells independently. With the increase in drug's concentration,the apoptosis rate of cancer cells kept increasing,the differences are significant.2^ Morphologic change of cells: In the control group, cancer cells showed oval-shape, some of them showed fusiform due to extension. After sodium butyrate disposing, the appearance of Lovo and HT-29 cell became round, its volume became smaller and they abscised from the inner surface of the bottle. With the prolonged of time, there were more and more cells detached and floated up, some nuclear fragments and apoptotic bodies were observed. The number and appearance of human embryo kidney 293 cell had no change after sodium butyrate disposing.3n Sodium butyrate inhibited proliferation and induced apoptosis of Lovo and HT-29 cell in a time and dose-dependant manner.4> Sodium butyrate blocked HT-29 cell mainly in Gl phase of the cell cycle while Lovo cell were stopped mainly in G2 phase.5^ Sodium butyrate stimulated p21wafl and Bax expression of HT-29 cell both at mRNA and protein levels, but it had little effect on transcription of Gadd45 in HT-29 cell. In Lovo cell, these three genes' expression were all enhanced after being treated with sodium butyrate, and the expression of Gadd45 was the most obviousof all.6> Sodium butyrate had little effect on human embryo kidney 293 cell, and these three genes' expression of the cellhad no obvious change after being treated with sodium butyrate.Experiment 2:1 x In the 18th and 24th weeks of carcinoma induction,the incidence of carcinoma in model group was 65% (13/20) and 94.12% (16/17) respectively, and no tumor was found in the control group.2^ There were 29 mice bore cancer in the study and 187 nodus were induced. Among all the 187 nodus, 178 nodus(95.19%) were located distal colon,7 nodus (3.74)were located proximated end ,and only 2 nodus (1.07)were situated small intestine .No tumor was found in liver ,lungs,kidney,and pancrease.3^ During the carcinoma induction, the mucosas of the model mice appeared different atypital hyperplasia, adenoma, andcarcinoma in turns. Among the inducing cancer both intramucosal carcinoma and infiltrating carcinoma were found, and the ratio of infiltrating carcinoma was raised with the prolonged of experimental time.4> Different types of colon mucosa in the model group mice had different levels of PCNA expression. The strongest PCNA expression was found in carcinoma, and following by the adenoma and atypital hyperplasia .It was very low in normal colon mucosa in the model group mice. The differences were verysignificant (p< 0.01) .It suggested that colon mucosa of the mice showed a very high proliferation activity during colon carcinogenesis by DMH.5^ Different types of colon mucosa in the model group mice had different levels of TUNEL expression. Contrary to that of PCNA, the results of TUNEL showed that it was the lowest in carcinoma, and the ratio of proliferation and apoptosis of the colon mucosa increased gradually from atypital hyperplasia to adenoma .It was suggested that there were presented disequilibrium between proliferation and apoptosis in the large bowel mucosa during the mice colon carcinogenesis induced by DMH.6^ The results of RT-PCR, Western blot and immunohistochemistry showed that p21wafl,Bax and Gadd45 gene had different expressions in different categories of the large bowel mucosa of the mice both at mRNA and protein levels during the mice colon carcinogenesis induced by DMH. All the three genes were expressed lowestly in carcinoma.Experiment 3:K The mice in model group showed anorexy and lazy from the 11th weeks of carcinoma induction,and its body weight lighten obviously at the 18th and 24th weeks.lt had no difference between the mice of 100 mM butyrate enema + NS group and control group.2^ The incidence of carcinoma in model group, 50mM butyrate group, and 100 mM butyrate group were 65%( 26/40 ), 30%( 12/40 ) and 7.5%( 3/40 Respectively, and the differences were very significant (p<0.01) .No tumor was found in the mice NS (normal saline, NS) enema group (control group) and 100 mM butyrate enema + NS group .3^ Although the PCNA expression of the DMH +100mM butyrate enema mice was stronger than that in control group mice, it was obviously lower than that of model group ,and there was no difference between 100 mM butyrate enema+ NS group and control group. All the results suggested that 100 mM butyrate enema may inhibit the proliferation of large bowel mucosa during the mice colon carcinogenesis induced by DMH.4^ The apoptosis extent of DMH +100mM butyrate enema mice was obviously higher than that of model group,and this suggested that 100 mM butyrate enema may promote the apoptosis of large bowel mucosa during the mice colon carcinogenesis induced by DMH (p<0.001) . The ratio of proliferation and apoptosis of DMH +100mM butyrate enema group was lower than that of model group,and this suggested that 100 mM butyrate enema may adjust the proliferation and apoptosis of the mice large bowel mucosa.5^ The expressions of p21wafl, Bax and Gadd45 gene of lOOmM butyrate enema group mice were obviously higher then that of control and model group mice. Among of them , p21wafl had a significant change.6n There were no difference in the general condition of mice, the body weight increasing, the liver and renal function ,as well as the pathological changes of liver, kidney ,and pancreas between control group and 100 mM butyrate enema + NS group (P>0.05) .Conclusions Experiment 1:1% Sodium butyrate above 2.5mM may inhibit proliferation and induce apoptosis of Lovo and HT-29 cell selectively in a time and dose-dependant manner. Maybe the effect associated with its up-regulating the expressions of p21wafl,Bax and Gadd45 within the two cell lines.2n The up-regulation of butyrate on the expressions of p21wafl,Bax and Gadd45 within the two cell lines is p53 gene independent.3% The biological effects of sodium butyrate on HT-29 and Lovo are not identical, and some differences of its regulation on p21wafl,Bax and Gadd45 genes'expression are also observed. Experiment 2:1% The carcinogenesis , histopathology and tumor location of the experimental large bowel carcinoma of KUNMING mice induced by DMH are similar to that of human's. This method not only have a high tumor incidence but also is simple. It may be very helpful to the study of carcinogenesis of human large bowel cancer.2n There are disequilibrium between proliferation and apoptosis in the large bowel mucosa during the mice colon carcinogenesis induced by DMH.3 > The colon carcinogenesis induced by DMH is concerned with three antioncogenes, p21wafl as well as Bax and Gadd45.Experiment 3:K Intrarectal application of sodium butyrate(lOOmM) significantly decreased the incidence of malignancies in the colon and postponed the carcinogenesis.2n The anti-tumor effects of sodium butyrate in vivo are at least through its regulation on the disequilibrium between proliferation and apoptosis in the large bowel mucosa during the mice colon carcinogenesis induced by dimethylhydrazine, and the role come true by its regulation on the main three target genes of p53 ,such as p21wafl,Bax and Gadd45.3n Intrarectal application of sodium butyrate(lOOmM) inhibites the carcinoma development of colorectal cancer induced by DMH,and no toxic effects are found in our reserch.