as a common malignant tumor in the digestive tract , esophagus carcinoma derives from a cascade process which is dependent on multiple factors . cells in the precancerous lesion are able to re - differentiate into normal cells or transform into primary carcinoma . therefore , elucidation of the molecular mechanism of esophagus carcinoma pathogenesis and the establishment of drug targets are critically important for the prevention and treatment of disease . both the occurrence and progression of malignant tumors are correlated with cell proliferation / apoptosis , which is mainly regulated by tumor suppressor gene b - cell lymphoma-2 ( bcl-2 ) . the over - expression of bcl-2 proteins in tumor cells may promote the growth of lesions , suggesting the correlation of bcl-2 and biological behavior of esophagus cancer cells . another tumor suppressor gene , tp53 , may induce oncogenesis when it has been mutated . vascular epithelial growth factor ( vegf ) studies have revealed the expression of tp53 , bcl-2 , and vegfa genes in both esophagus tumor and adjacent tissues , suggesting the biological significance of those 3 factors in esophagus cancer . this study therefore investigated the effect of tp53 , bcl-2 , and vegfa genes on the proliferation and apoptosis of esophagus carcinoma cells , via a methyl - benzyl - nitrosamine ( mbna)-induced rat esophagus cancer model , in an attempt to investigate the potential molecular mechanism underlying cancer pathogenesis . a cohort of 60 male wistar rats ( 7~8 weeks old , body weight between 180 and 200 grams ) were provided by the animal center of the institute of oncology , shandong university . animals were kept in a specific pathogen - free ( spf ) grade facility with food and water provided ad libitum . after acclimation , all animals were randomly divided into control and model groups ( n=30 each ) . rats in the model group received bi - weekly subcutaneous injection of 0.15% mbna ( at 3.5 mg / kg , sigma , usa ) in 0.9% saline . general condition of animals was daily observed during the whole experiment , which lasted 30 consecutive weeks . animals from both groups were sacrificed at week 4 , 10 , 20 , and 30 ( n=7 each ) . tissue samples were then fixed in 10% neutral buffered formalin ( nbf ) solution and stained by he method . we also quantified the condition of tumor cell apoptosis using a tunel kit ( boster biotech , china ) following the manual instruction . a total of 100 nuclei were observed from each of 5 randomly selected fields from 1 slide . the apoptosis index ( ai ) after dewaxing and rehydration , mouse anti - p53 monoclonal antibody ( 1:100 , boster biotech , china ) , mouse anti - bcl-2 monoclonal antibody ( 1:60 , boster biotech , china ) , or mouse anti - vegf monoclonal antibody ( 1:100 , boster biotech , china ) were added for overnight incubation . goat anti - mouse igg secondary antibody conjugated with horseradish peroxidase ( boster biotech , china ) was applied , followed by color development using dab substrate . positive signals were deduced as yellow / brown granules in the cytoplasm ( for vegf and bcl-2 ) or the nucleus ( for p53 ) . the relative staining intensity was calculated by the averaged optical density ( od ) value . the spss 19.0 software package was used to analyze all collected data , of which measurement data with normal distribution are presented as mean  standard deviation ( sd ) . between - group comparison was done by t - test or one - way analysis of variance ( anova ) . a cohort of 60 male wistar rats ( 7~8 weeks old , body weight between 180 and 200 grams ) were provided by the animal center of the institute of oncology , shandong university . animals were kept in a specific pathogen - free ( spf ) grade facility with food and water provided ad libitum . after acclimation , all animals were randomly divided into control and model groups ( n=30 each ) . rats in the model group received bi - weekly subcutaneous injection of 0.15% mbna ( at 3.5 mg / kg , sigma , usa ) in 0.9% saline . general condition of animals was daily observed during the whole experiment , which lasted 30 consecutive weeks . animals from both groups were sacrificed at week 4 , 10 , 20 , and 30 ( n=7 each ) . tissue samples were then fixed in 10% neutral buffered formalin ( nbf ) solution and stained by he method . we also quantified the condition of tumor cell apoptosis using a tunel kit ( boster biotech , china ) following the manual instruction . a total of 100 nuclei were observed from each of 5 randomly selected fields from 1 slide . the apoptosis index ( ai ) after dewaxing and rehydration , mouse anti - p53 monoclonal antibody ( 1:100 , boster biotech , china ) , mouse anti - bcl-2 monoclonal antibody ( 1:60 , boster biotech , china ) , or mouse anti - vegf monoclonal antibody ( 1:100 , boster biotech , china ) were added for overnight incubation . goat anti - mouse igg secondary antibody conjugated with horseradish peroxidase ( boster biotech , china ) was applied , followed by color development using dab substrate . positive signals were deduced as yellow / brown granules in the cytoplasm ( for vegf and bcl-2 ) or the nucleus ( for p53 ) . the relative staining intensity was calculated by the averaged optical density ( od ) value . the spss 19.0 software package was used to analyze all collected data , of which measurement data with normal distribution are presented as mean  standard deviation ( sd ) . between - group comparison was done by t - test or one - way analysis of variance ( anova ) . at each time point , control rats had smooth mucosa with no significant abnormality in the esophagus ( figure 1a ) . after 4 weeks , small granules occurred in the rough mucosa of rat esophagus in the model group ( figure 1b ) . papillomas were observed from esophagus mucosa from 10 weeks , and were aggravated at 20 and 30 weeks . control rats had a basal cell layer in a regular arrangement , with evenly distributed epithelial cells ( figure 2a ) . model rats , however , had inflammation from 4 weeks , as shown by irregular arranged epithelial cells , hyperplasia nucleus containing granular chromatin , and unclear cell border ( figure 2b ) . esophagus tissue at 10 weeks had atypical proliferation , loosely arranged cells , and enlarged nuclei containing granular chromatin ( figure 2c ) . at 20 weeks , atypical proliferation was aggravated , as shown by cells with discrete sizes , enlarged and darkened nuclei , and abnormal nucleus / cytoplasm ratio ( figure 2d ) . after 30 weeks , features of squamous carcinoma could be identified by irregular cell arrangement , abnormal cell shape , elevated chromatin , and nuclei with dark color and abnormal shape ( figure 2e ) . ihc staining results showed that bcl-2 proteins were mainly identified in the cell membrane and cytoplasm , vegf proteins existed in the cytoplasm , and p53 proteins occurred in the nucleus . in the control group , all those 3 proteins had lower expression levels ( figures 3a , 4a , and 5a ) . in the model group , after 4 weeks of mbna - induction , expression levels of p53 , bcl-2 , and vegf proteins were all elevated ( figures 3b , 4b , and 5b ) . with the progression of the disease , expression levels of those 3 proteins were further elevated ( figures 3c3e , 4c4e , and 5c5e ) in a time - dependent manner . with the elongation of induction , model rats had decreased levels of esophagus carcinoma cells , as supported by the significantly suppressed ai compared to the control group ( p<0.05 , figure 6 ) . we analyzed the correlation among p53 , bcl-2 , and vegf proteins in esophagus carcinoma tissues . results showed a significant positive correlation between p53 and bcl-2 or vegf proteins ( r=0.753 and 0.762 , p<0.05 in both cases ) . the correlation between bcl-2 and vegf proteins was not significant ( r=0.109 , p>0.05 ) . at each time point , control rats had smooth mucosa with no significant abnormality in the esophagus ( figure 1a ) . after 4 weeks , small granules occurred in the rough mucosa of rat esophagus in the model group ( figure 1b ) . papillomas were observed from esophagus mucosa from 10 weeks , and were aggravated at 20 and 30 weeks . control rats had a basal cell layer in a regular arrangement , with evenly distributed epithelial cells ( figure 2a ) . model rats , however , had inflammation from 4 weeks , as shown by irregular arranged epithelial cells , hyperplasia nucleus containing granular chromatin , and unclear cell border ( figure 2b ) . esophagus tissue at 10 weeks had atypical proliferation , loosely arranged cells , and enlarged nuclei containing granular chromatin ( figure 2c ) . at 20 weeks , atypical proliferation was aggravated , as shown by cells with discrete sizes , enlarged and darkened nuclei , and abnormal nucleus / cytoplasm ratio ( figure 2d ) . after 30 weeks , features of squamous carcinoma could be identified by irregular cell arrangement , abnormal cell shape , elevated chromatin , and nuclei with dark color and abnormal shape ( figure 2e ) . ihc staining results showed that bcl-2 proteins were mainly identified in the cell membrane and cytoplasm , vegf proteins existed in the cytoplasm , and p53 proteins occurred in the nucleus . in the control group , all those 3 proteins had lower expression levels ( figures 3a , 4a , and 5a ) . in the model group , after 4 weeks of mbna - induction , expression levels of p53 , bcl-2 , and vegf proteins were all elevated ( figures 3b , 4b , and 5b ) . with the progression of the disease , expression levels of those 3 proteins were further elevated ( figures 3c3e , 4c4e , and 5c5e ) in a time - dependent manner . with the elongation of induction , model rats had decreased levels of esophagus carcinoma cells , as supported by the significantly suppressed ai compared to the control group ( p<0.05 , figure 6 ) . we analyzed the correlation among p53 , bcl-2 , and vegf proteins in esophagus carcinoma tissues . results showed a significant positive correlation between p53 and bcl-2 or vegf proteins ( r=0.753 and 0.762 , p<0.05 in both cases ) . the correlation between bcl-2 and vegf proteins was not significant ( r=0.109 , p>0.05 ) . the pathogenesis of esophagus carcinoma is closely related with the imbalance of cell apoptosis . under normal physiological conditions the dysregulation of apoptosis , therefore , may lead to various diseases , including malignant tumors . actually , it has been well established that the inactivation of tumor - suppressing genes and activation of oncogenes are molecular mechanisms underlying the immortality of cancer cells . tumor - suppressor genes inhibit the formation of tumors via its participation in cell cycle - related signal transduction , cell apoptosis , and differentiation regulation . it has been shown that inhibition of cell apoptosis play an important role in the pathogenesis of esophagus carcinoma . the most widely studied tumor - related genes in esophagus cancer are tp53 and bcl-2 genes . as an anti - apoptotic gene , the bcl-2 gene locates at chromosome 14q18 and encodes a membrane protein to prolong the cell survival period . the over - expression of bcl-2 gene in tumor cells can cause resistance to chemotherapy . the elevated expression of bcl-2 gene in esophagus carcinoma cells may sustain the cell cycle via its inhibition on intracellular ca , blocking nucleus transportation and anti - oxidation . the wild - type tp53 gene encodes p53 protein , which can suppress tumor formation by its surveillance on cell growth and genome , along with the induction of apoptosis of cells with mutated dna . bcl-2 protein expression can be down - regulated by wild - type tp53 gene to facilitate cell apoptosis , accompanied with elevated expression of bax proteins . vegf , on the other hand , directly induces the tumor angiogenesis by the elevated permeability of micro - vessels , thereby providing a favorable environment for endothelial cell proliferation . the highly specific induction of vascular endothelial cell mitosis by vegf directly facilitates de novo formation of tumor vessels . it has been reported that both tumor proliferation and invasion are correlated with elevated vegf expression , which can be regulated by the tp53 gene . various oncology studies all suggested the correlation between tp53 and bcl-2/vegfa expression and the existence of a possibly shared transcriptional activation pathway . this study thus investigated the role and correlation of p53 , bcl-2 , and vegf in the apoptosis of esophagus carcinoma . our results showed significantly lowered apoptosis level in the esophagus model group compared to control animals . with the disease progression , esophagus cell apoptosis decreased gradually , as shown by lowered ai , suggesting the potential involvement of apoptosis in esophagus cancer . compared to those in the control group , model rats had elevated expression of bcl-2 , p53 , and vegf proteins in their esophagus tissues , in addition to the existence of inflammatory lesions and atypical hyperplasia . expression of these proteins also increased with the progression of disease , in a time - dependent manner . all those results suggest the involvement of these proteins in the dysregulation of cell apoptosis and further tumor progression . in esophagus carcinoma tissues , tp53 gene expression level was positively correlated with bcl-2 or vegf proteins , suggesting the role of p53 in the directing apoptotic pathway in the occurrence of esophagus cancer . because elevated bcl-2 may keep cells in the active cycle and decrease apoptotic level , it can increase tumor cell proliferation . our results thus suggest the synergistic effect of tp53 , bcl-2 , and vegfa genes in esophagus cancer occurrence . the possible cellular pathway among those proteins has been reported previously : bcl-2 altered the redox status of mitochondrial and membrane potential , thus releasing apoptotic protein precursor apaf-1 , cytochrome c and activating caspase-9 ; p53 induced the death receptor via cascade activation of fas , dr5 and dr4 to facilitate cell apoptosis . p53 also regulated the endogenous apoptotic pathway as an upstream factor of the bax / bak pathway to mediate bcl-2 protein expression . as the downstream effector of p53 gene , both bax and bcl-2 can be selectively regulated by p53 . the imbalance between bcl-2 and bax gene expression may favor the occurrence of esophagus cancer . further studies supported the role of p53 in tumor angiogenesis via its regulation of vegf expression . our study provided further evidence , as the expression of p53 was closely related with bcl-2 in esophagus carcinoma tissues , suggesting potential synergistic effects . previous study of genomic screening in esophagus cancer patients revealed the loss of chromosome 17p , where tp53 gene localizes in cancer patients , suggesting the genetic grounds of oncogenesis . moreover , the gene polymorphism of the tp53 gene may also reside in the tumor pathogenesis as the so - called unique tumor principle , in which each individual tumor may have various tumor suppressor gene polymorphisms , in addition to certain epigenetic regulations , such as methylation patterns . therefore , the expressional regulation of tp53 gene is of crucial importance in the development of esophagus tumor . our study provides further evidence regarding the role of tp53 in bcl-2 gene expression regulation . such selective modulation of tp53 on bax and bcl-2 gene expression may be affected by environmental factors , leaving the detailed cellular pathway unclear . it is commonly believed that tp53 gene mutation and protein deposition occur early in the oncogenesis of esophagus cancer . the wild - type tp53 gene may inhibit tumor angiogenesis via its down - regulation of vegf proteins ; however , mutant p53 can facilitate the proliferation and metastasis of tumor cells . these phenomena suggest the existence of a common pathway that is regulated by both p53 and vegf . the illustration of a synergistic effect between vegf and p53 proteins requires further knowledge about the role of p53 in mediating tumor angiogenesis . it has been postulated that p53 may regulate tumor vascular formation via the increase of protein kinase c activity and modulating vegf expression . wild - type tp53 gene may inhibit vegf expression via negative feedback in its transcription , thereby inhibiting tumor vessel formation . mutant tp53 gene , however , loses its ability to inhibit vegf transcription , as does the inhibition of vascular formation and tumor invasiveness . our results revealed that a time - dependent relationship between p53 and bcl-2 or vegf protein levels , suggesting their close relationship with tumor cell proliferation and apoptosis . the abnormally elevated expression of tp53 , bcl-2 , and vegfa genes in esophagus carcinoma suggest potential synergistic effects between these proteins , which may all participate in the proliferation and apoptosis of tumor cells .
backgroundthe pathogenesis of esophagus carcinoma involves a cascade process consisting of multiple factors and accumulation of gene mutations . 
 it is known that vascular endothelial growth factor ( vegf ) mainly regulates de novo vascular formation while b - cell lymphoma-2 ( bcl-2 ) gene exerts a tumor - suppressing effect . 
 the prominent expression of vegfa and bcl-2 genes , along with the most famous tumor - suppressor gene , tp53 , raise the possibly of gene interaction . 
 this study therefore investigated the effect and correlation of tp53 , bcl-2 , and vegfa genes on cell proliferation and apoptosis of esophagus carcinoma.material/methodsa total of 30 male rats were prepared by subcutaneous injection of methyl - benzyl - nitrosamine ( mbna ) to induce esophagus cancer , along with 30 controlled rats which received saline instead . 
 after 4 , 10 , 20 , or 30 weeks , rats were sacrificed to observe the morphological changes of esophageal mucosa . 
 cell apoptosis was quantified by terminal deoxynucleotidyl transferase - mediated dutp nick - end - labeling ( tunel ) assay . 
 immunohistochemical ( ihc ) staining was employed to examine the expression of tp53 , bcl-2 and vegfa genes.resultswith the progression of cancer , pathological damages of esophageal tissue aggravated while the cancer cell apoptosis gradually decreased compared to controlled animals . 
 protein levels of p53 , bcl-2 , and vegf in the model group were significantly elevated at each time point . 
 positive correlations existed between p53 and bcl-2 or vegf.conclusionsabnormally elevated expression of tp53 , bcl-2 , and vegfa genes may participate in the proliferation of esophagus cancer cells in a synergistic manner .