Effect And Mechanism Of Adenovirus SiMDM2 And NDRG2 Gene Therapy On Proliferation And Apoptosis Of SCC-13

Background: Skin cancer is one of the most common malignant tumors. It can generally be divided into malignant melanoma and non melanoma skin cancer(NMSC), NMSC mainly including squamous cell carcinoma (SCC) and basal cell carcinoma. In recent years, the incidence of skin cancer is on the rise, which has become a global public health problem. SCC mainly occurs in the elderly, 50 to 60 years old for the peak incidence, men more than women, tend to occur in the head and neck, face and back of the hand and other exposed partsSCC is a malignant tumor which originates from the epidermis and skin appendages keratinocyte and often occurs on the basis of some, or by some precancerosis has evolved SCC cells malignant etiology is not clear, but clearly related to the following factors:(1) Ultraviolet irradiation.2 Chemical carcinogens.(3)Precancerosis.(4) Trauma scar and other chronic skin disease.(5) Immunosuppression.Ultraviolet radiation is the main cause of skin cancer. It tend to take several years or decades accumulate excessive exposure to the sun's ultraviolet rays before development to the malignant tumor by the Epidemiological data .The most important defense mechanism to protect the skin from ultraviolet radiation is involved in the synthesis of melanin, and actively repair mechanism.SCC on histopathology of invasive growth, since the skin break through basal layer the dermis, a cord like and irregular mass, composed of normal squamous cells and atypical squamous cells. Differentiation of the former is good, some can form keratinocytes, the latter differentiation is not good, that is, the so-called cancer cells.Common keratin pearl and dyskeratosis cells of cancer cell differentiation in the direction of keratinocyte. The well-differentiated squamous cells can be seen in the intercellular bridge. Undifferentiated and poorly differentiated squamous cells volume is small, there are no intercellular bridges and dyskeratosis cells. Broders divided SCC into 4 levels according to the percentage of undifferentiated cancer cells,combined with the atypical degree of cancer cells and the degree of invasion of the damage . ISCC (Well differentiated squamous cell carcinoma) , containing less than 25% of atypical squamous cells, invasion to the dermis downward, but no more than the level of sweat glands, in the SCC mass, keratin pear can be seen more,keratinization of the center can completely or partly, discontinuous edges visible basal layer cells, no obvious clear boundaries with the surrounding stroma and around the SCC mass in the dermis have obvious inflammation, generally is not metastatic. II SCC: containing 25%?50% atypical squamous cells, invasion to the deep dermis .in the SCC mass, rare keratin pear can be seen, keratinization of the center is light and see more parakeratosis, and the surrounding stroma no obvious boundary, surrounding dermal inflammation is light compared to ? SCC. ? SCC:containing 50%?70% atypical squamous cells, in the SCC mass, Invisible keratin pearl , keratinization of the center is not obvious and see remarkable mitotic figure, the nucleus is atypical, SCC inflammation in dermis around is not obvious. SCC IV(Poorly differentiated squamous cell carcinoma), almost all are atypical squamous cells in SCC, there are more mitotic figure but no intercellular bridge in SCC mass,without keratinization of the central, when the fusiform cancer cells were in spiral arrangement, the SCC was difficult to differentiate from the sarcoma.The standard SCC treatment including surgery, radiotherapy and chemical therapy.However, patients who have been treated for 2 years are still likely to relapse and distant metastasis. In view of the rapid progress of gene therapy and the low stability and effectiveness of single gene therapy, And previous studies have shown that the recombinant adenovirus carrying two or more genes has cytotoxic effects on multiple myeloma, as well as laryngeal cancer and other tumors, It needs a kind of combined gene high stability and effectiveness of treatment to improve the prognosis of SCC patients. In this paper we focus on the study of human recombinant N-myc wild type p53 gene related downstream regulated gene 2 (NDRG2) and 2(MDM2) in the double mutant effect alone and combined with the application of SCCP53 is a well-known tumor-suppressor gene and the p53 signaling pathway is the most common mutations pathway in the tumor.P53 can induce cell cycle arrest and apoptosis when DNA damage and cytotoxicity are occurred. It not only plays an important role in tumor suppression but also an extensive transcription factor which regulates more than 2500 genes and most of the genes related to the occurrence,development and invasion of the tumor. NDRG2 is the target gene of p53 gene, and it can be independent of p53 gene.NDRG2 belongs to the NDRG family. It has significant structural similarity with ABH super family. It contains two subtypes. The main amino acid sequence information indicates that both NDRG2 protein showed significantly structural similarity with ABH super family[].Through regulation of different cytokines, NDRG2 plays roles in many biological processes including cell stress response, cell differentiation,proliferation, and apoptosis. The expression of NDRG2 is decreased or absent in various tumor cells. Moreover, the prognosis of various malignant tumors is negatively correlated with the tumorous expression of NDRG2. In recent years, a number of in vitro and in vivo studies have shown that NDRG2 has anti-proliferation and anti-invasion activities, and induces apoptosis in many tumor cells. In the present study, we investigated the anti-tumor effect of the recombinant adenovirus carrying siMDM2 and NDRG2 gene on tumor volume regression both in vitro and in a SCC nude mice model.Murine double minute gene 2 (MDM2) is a proto oncogene. Mutation and amplification of MDM2 have been found in a variety of tumors, and the amplification of MDM2 is closely related to the metastasis of tumor. MDM2 is a highly amplification gene originally cloned from a spontaneous BALB/3T3DM cells containing double minute chromosomes (murine double minute MDM). This gene in mice on chromosome 10 CI-C3 District, Momand et al. for the first time in 1992 separate and proves that the rat mdm2 gene product is a is a 90000Da protein molecular. Its expression is induced by wild-type p53 and in turn, it forms a negative feedback loop through promoting degradation and inhibiting the transcriptional activity of wild-type p53. MDM2 involves in the development of many human cancers through negative regulation of p53 transcription activation, mediation of cell arrest and apoptosis, and promotion of tumorigenesis. Overexpression of MDM2 was reported to act against p53 gene therapy in tumors. Thus, interference of the p53-MDM2 feedback loop offers a new anti-tumor strategy to restore the p53 tumor suppressor activity. MDM2 is an E3 ubiquitin ligase, and is an important regulator of p53 pathway. Its negative regulating p53 in two ways: one is through direct binding to inhibit transcription; Another is by its E3 ligase activity to degradation of p53 protein.ARF can directly combined MDM2 and then inhibit the activity of E3 ubiquitin enzyme activity to regulate the transcriptional activity of p53.In tumor cells,Ad-p53 gene therapy to increase the expression of wild-type p53, which is a possible therapeutic method to overcome the mutation of the gene. In clinical trials, Lang et al. Found that the transcription of p53 gene and functional exogenous p53 expression which was caused by the injection of adenovirus vector containing p53 in the tumor. Therefore, direct down-regulation of MDM2 expression by specific siRNA may be an effective method for the treatment of skin cancer that has the expression of exogenous p53 gene.Objective: To research the therapeutic effects of recombinant adenovirus Ad-GFP,Ad-siMDM2, Ad-NDRG2 and Ad-siMDM2-NDRG2 gene therapy on SCC cell line and tumor bearing mice model, so as to find a more effective method for the treatment of SCC.Method: (1) We entrust Hanbio Biotechnology to Construct recombinant adenovirus Ad-GFP, Ad-siMDM2, Ad-NDRG2, Ad-siMDM2-NDRG2, using 293A cells for virus propagation, purification, and getting enough dose and concentration of the virus for gene therapy trials.(2) The SCC-13 cell line was recovered, cultured, passaged in vitro and recombinant adenovirus Ad-GFP, Ad-siMDM2, Ad-NDRG2 and Ad-siMDM2-NDRG2 transfection experiments.(3) Establishing the tumor-bearing mice model and subcutaneous injection of 0.9%NaCl and recombinant adenovirus Ad-GFP, Ad-siMDM2, Ad-NDRG2,Ad-siMDM2-NDRG2 treatment, observing the tumor size changes in each group.(4) Using of CCK 8 kits to detect the proliferation potential of SCC - 13 cell which infected by the recombinant adenovirus Ad-GFP, Ad-siMDM2, Ad-NDRG2, and Ad-siMDM2-NDRG2.(5) Using RT-PCR and Western blot from the level of gene and protein respectively research the changes of mRNA expression and the corresponding protein products of gene MDM2 and NDRG2 in SCC-13 cell infection by recombinant adenovirus Ad-GFP,Ad-siMDM2, Ad-NDRG2, Ad-siMDM2-NDRG2 . Especially by virtue of Caspase3 protein expression level in SCC-13 cell to further study the SCC-13 cell apoptosis mechanism induced by gene therapy with recombinant adenovirus Ad-GFP,Ad-siMDM2,Ad-NDRG2,Ad-siMDM2-NDRG2.(6) Annexin and V/PI double staining and flow cytometry were used to evaluate the apoptosis rate of SCC-13 cells in tumor bearing mice.(7) Using MTT to study the cytotoxic effect after the recombinant adenovirus Ad-GFP,Ad-siMDM2, Ad-NDRG2, Ad-siMDM2-NDRG2 transfected SCC-13 cells at different time points.Results: (1) Sufficient doses and concentrations of experimental SCC-13 cells and recombinant adenovirus were obtained.(2) The SCC - 13 cells can be highly effective transfect by recombinant adenovirus Ad-GFP, an optimal expression of GFP at 48 hours post-infection .And the efficiency of infection is best when MOI for 100.(3) Subcutaneous injection of 0.9%NaCl and recombinant adenovirus Ad-GFP,Ad-siMDM2, Ad-NDRG2, Ad-siMDM2-NDRG2 mice tumor volume comparison showed that 0.9%NaCl and Ad-GFP group the average tumor volume the biggest,Ad-siMDM2, Ad-NDRG2 group the average tumor volume is similar,Ad-siMDM2-NDRG2 group the average tumor volume minimum.(4) Compared with the Ad-GFP group, in the Ad-GFP, Ad-siMDM2, Ad-NDRG2,Ad-siMDM2-NDRG2 SCC-13 cell transfection, Ad-siMDM2, Ad-NDRG2,Ad-siMDM2-NDRG2 group on SCC-13 cell proliferation have obvious inhibiting.(5) In gene level analysis by RT-PCR results showed that: ?compared with control group, MDM2 gene mRNA expression significantly reduced in SCC-13 cells which transfect by the recombinant adenovirus Ad-siMDM2, Ad-siMDM2-NDRG2;?scc-13 cells within itself NDRG2 gene expression level is low, but in the SCC-13 cell transfect by recombinant adenovirus Ad - NDRG2, Ad-siMDM2-NDRG2,overexpression of NDRG2 corresponding increase its mRNA expression. Recombinant adenovirus Ad-siMDM2, Ad-NDRG2, Ad-siMDM2-NDRG2 gene therapy can promote the Caspase3 protein expression in SCC-13. Western blot studies at the protein level confirmed the results of the RT-PCR gene study.(6) Annexin V/PI double staining and flow cytometry analysis of tumor bearing mouse tumor cells showed that MDM2 gene silencing and overexpression of NDRG2 gene significantly induced apoptosis in mouse tumor cells(7) MTT studied the cytotoxic effects of SCC-13, and the results showed that the Ad-siMDM2-NDRG2 group compared with the Ad-GFP group, P<0.05; the same Ad-siMDM2-NDRG2 group compared with the Ad-siMDM2 group and the Ad-NDRG2 group, P<0.05.Conclusion : Recombinant adenovirus Ad-siMDM2-NDRG2 gene therapy can infect SCC more effectively than single Ad-siMDM2 or Ad-NDRG2 gene therapy, and can provide a potentially effective means for clinical treatment of SCC.