Anticancer Gene PTEN On Human Colon Cancer

Background:Colorectal cancer are the most common malignant tumors in humanbeing, At present, therapies for Colorectal cancer includes surgical therapy, chemotherapy, radiotherapy and endocrine therapy.Following with the application and study of medical molecular biology, Gene therapy is becoming an important component of biotherapy for tumors and possessesing a good applied perspective of Colorectal cancer therapeutics. After p53 and Rb gene,PTEN gene is hitherto the first tumor suppressor gene encoding a dual specificity protein–lipid phosphatase that plays an important role in cell apoptosis, blockage of cell cycle ,cell migration and tumorigenesis. In recent years,the studies on PTEN gene mostly have been focusing on its various kinds of functional mechanisms in tumorigenesis and tumor growth ,which seldom involved in its effects on tumor cells,solid tumors and related pharmacal research and development. Therefore,we constructed eukaryotic cell expression vector containing human PTEN gene and green fluorescence protein gene,observed its inhibition on human Colorectal cancer in vitro and in vivo,made a primary approach to mechanisms in its inhibition on tumors,which may provide theoretical and experimental basis of feasibility of PTEN gene treatment for Colorectal cancer, lay a foundation for gene therapy in clinical application.Methods:1.Expression of PTEN on the cells of colorectal carcinomaTotal 58 patients pathological diagnosed with colorectal Cancer surgery in The China Japan union Hospital of jilin university from January 2007 to of January 2008 were collected . Methods including nomal Expression of PTEN was detected in 58 samples mucosa membrane and carcinoma tissue by immunohistochemistry ABC method. PTEN positive signal was localized in cytoplasm and nucleus. 2.Construction of eukaryotic cell expression vector containing human PTEN gene and green fluorescence protein geneThe recombinant plasmid was constructed by using nested polymerase chain reaction. The total RNA that extracted from normal adult female placental tissue was taken as template to synthesize the first chain,The product of nested PCR fragments including PTEN gene were amplified and directly cloned into the pMD18-T Vector, the recombinant plasmid was digested by double-endonuclease and the positive plasmids obtained through screening. Both pMD18-T Vector including PTEN gene and pEGFP-C1 Vector were digested by double-endonuclease again , PTEN gene was directly ligated into enhanced green fluorescent protein expression vector pEGFP-C1, the positive recombinant plasmids obtained through screening, which were identified by double-endonuclease, sequencing and sequence alignment.3.The inhibition on human colon cancer cells by PTEN geneThe recombinant pEGFP-PTEN was transfected into Colorectal cancer cell line LoVo by lipofection. Positive cell clones LoVo-vect,LoVo-PTEN were obtained by G418 screening.Fluorescence microscopy,PTEN gene DNA assay, RNA assay, SDS-PAGE, Western-blot assay and immunocytochemistry assay were used to identify if the PTEN gene be integrated into Colorectal cancer cell line LoVo or stably expressed. Through DNA agarose gel electrophoresis, FCM Analysis, MTT assay, morphological observation by transmission electron microscope, we observed the inhibition on human Colorectal cancer cells by PTEN gene.By detecting PTEN protein and P-Akt protein using Western-blot assay, detecting PTEN protein and mutant P53 protein using immunocytochemistry assay,we made a primary approach to mechanisms in human wild type PTEN gene`s inhibition on Colorectal cancer cells in vitro.Results:1. The positive rate of PTEN protein expression in the tumor tissues and normal mucosa were 77.58 % (45/58)and 100 %(20/20 ) respectively and their difference is statistically (P<0.05).The immunostaining of PTEN was strong positive in normal colon mucosa. The positive cells distributed in the whole colon mucosa, and weakly positive or negative in poorly differentiated adenocarcinoma and metastatic focus. The high level expression of PTEN protein in tumor tissue was relative to the earlier tumor stage (P<0.05 );.Thereis no relationship between PTEN protein and gender, age,tumor location (P>0.05). The expression of PTEN was associated with tumor stage, tumor differentiation degree and metastasis.2. The recombinant plasmid was identified by double-endonuclease, sequencing and sequence alignment, the sequence of which were fully consistent with that announced in Gene Bank. the results showed that the recombinant plasmid was successfully constructed.3. Fluorescence microscopy showed LoVo-vect and LoVo-PTEN cell lines sent out green fluorescence,whereas the LoVo cell line did not . The target gene band emerged in PTEN gene DNA assay and RNA assay. Western-blot assay showed that the contoll group cells did not express PTEN gene,but the cells transfected with PTEN gene expressed it.Furthermore, the expression of Phospho-Akt (Ser473) in transfected cells decreased.The results of MTT showed proliferation velocity of LoVo-PTEN is significantly slower than that of controll group(P<0.05).FCM analysis showed that, compared with the control group, the group (LoVo-PTEN)were significantly increased(P<0.05), the proportion of Lovo and Lovo-vect did not changed, (P>0.05). These results demonstrate that PTEN gene can inhibit proliferation of carcinoma of,Colorectal cancer, induce apoptosis, cause cell arrest at G1 phase.The results of detecting PTEN protein protein by immunocytochemistry assay showed that the staining of immunocytochemistry pEGFP-C1-PTEN group was only positive,whereas other groups were negtive, which demonstrated that the PTEN gene was integrated into Colorectal cancer cell line LoVo and stably expressed. The results of transmission electron microscope showed that ultrastructure of LoVo-vect cells included uniform nucleus which was composed of regular dispersed chromatin and nucleoli. on the contrary, ultrastructure of LoVo-PTEN cells appeared typical morphological features of apoptosis: irregular nucleus with condensed chromatin along the nuclear envelope and condensed cytoplasm (feature of apoptosis), In the cytoplasm, double-membraned giant autophagic vacuoles (ultrastructural feature of autophagy) contain a large part of the cytoplasm with some preserved organelles like endocytoplasmic reticulum, mitochondria, ribosomes,apoptotic body was unseen.These results demonstrated that the PTEN gene was sucsessfully transfected into Colorectal cancer cell line LoVo by lipofection which was integrated into Colorectal cancer cell line LoVo and stably expressed, PTEN gene can induce cell apoptosis , arrest cell cycle can down-regulate the expression of mutant Phospholated Akt.Conclusion:These results indicate that we successfully constructed the eukaryotic cell expression vector containing human wild type PTEN gene and green fluorescence protein gene, the PTEN gene was successfully transfected into Colorectal cancer cell line LoVo and stably expressed, PTEN gene can inhibit growth of tumor in vitro and in vivo. These results show the prospects for PTEN gene therapy in Colorectal cancer treatment, also provide theoretical and experimental basis for PTEN gene in clinical application, but specific mechanisms need to be further discussed.