Inhibitory Effect And Molecular Mechanism Of Antisense RNA Technology By Downregulating Critical Enzymes Of Polyamine Metabolism On Colorectal Cancer

Colorectal cancer is the most frequently diagnosed cancer in China. It is also the third leading cause of cancer mortality and has becoming higher and younger in its attack rate around the world. According to the unknown etiopathogenesis, it is difficult for the early diagnosis. Frequently, it has been into the advanced stage when the patient comes to visit the doctor. Now the first-line therapy is radical surgery with adjuvant chemotherapy. But overall 5 year survival rate for this disease is around 40%. The combination of chemotherapy and radiotherapy has some kind of therapeutic effect, but is not satisfactory well enough. Therefore, it is important to explore its etiopathogenesis and find a novel non-surgical intervention for treating colorectal cancer.The polyamines putrescine, spermidine and spermine are organic cations shown to participate in a bewildering number of cellular reactions. Their positive charges enable polyamines to interact electrostatically with polyanionic macromolecules within the cell, such as DNA, RNA and proteins. Polyamines are essential for normal cell growth, and aberrant polyamine metabolism is known to play an important role in the development of tumors. Cancer cells always have higher intracellular polyamine content than the equivalent normal tissue. Polyamine content of colorectal cancers is high and show strong positive correlations with prognostic factors and tumor recurrence, which acting like oncological markers in early detection of colon cancer. There are two critical enzymes controlling the polyamine metabolic pathway in eukaryotic cells. The first one is ornithine decarboxylase (ODC), which catalyzes the decarboxylation of ornithine to produce putrescine. The second rate-limiting enzyme is S-adenosylmethionine decarboxylase (AdoMetDC), which catalyzes the formation of decarboxylated S-adenosylmethionine (dcSAM). DcSAM donates its propylamine moiety for the biosynthesis of the spermidine and spermine by spermidine synthase and spermine synthase, respectively. In colorectal cancer, the activities of ODC and AdoMetDC are increased by 3- to 4-fold over those found in the equivalent normal tissue. ODC and AdoMetDC have been extensively regarded as anticancer target. The rivalrous inhibitor of them have some effect on animal experiment about anticancer, but the side effects restrict its clinic research. To investigate the pathogenesis and gene therapy of tumor, a previously constructed replication-deficient recombinant adenovirus (Ad-ODC-AdoMetDCas) to transfer antisense ODC and AdoMetDC gene to cells was utilized on colorectal cancer cell HT-29. The results suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas downregulated the expression of ODC and AdoMetDC, significantly inhibited the cell growth, and induced G1 arrest in HT-29 cells. And in the present study, the underlying regulatory responses of Ad-ODC-AdoMetDCas on cell cycle distribution were investigated. The results showed that recombinant adenovirus Ad-ODC-AdoMetDCas significantly decreased levels of cyclin D1 protein and mRNA level and suppressed the promoter activity. Ad-ODC-AdoMetDCas also inhibited nuclear translocation of β-catenin which is a regulated upstream molecular of cyclin D1. These findings indicated a close association between G1 arrest induced by Ad-ODC-AdoMetDCas and the suppression of cyclin D1.The antibody of ODC and AdoMetDC were required in this study. The monoclonal antibody of ODC has been prepared previously, but no antibody of AdoMetDC could obtain then. In favor of accomplishing the study, the prokaryotic expression plasmid of human AdoMetDC α subunit was constructed. The recombined AdoMetDC-α protein was induced and purified, which was used as a good immunogenic agent to immunize the Balb/c mouse, then got polyclonal antibody of AdoMetDC. ELISA and Western blotting showed that the prepared polyclonal anti-AdoMetDC antibody could combine the AdoMetDC protein specially. Immunohistochemical detection using the polyclonal antibody showed significant high concentration of AdoMetDC in tumor tissues than that in normal tissues. The preparation of polyclonal anti-AdoMetDC antibody not only facilitated the following study, but also established a method of AdoMetDC examine, consequently would favor diagnosis of colorectal cancer.Part 1 Prokaryotic expression of human S-Adenosylmethionine Decarboxylase gene α subunit and preparation of polyclonalanti-AdoMetDC antibodyObjective:To construct the prokaryotic expression plasmid of human AdoMetDC α subunit, and purified the fusion protein to prepare polyclonal antibody of AdoMetDC.Methods:1. Domain of human AdoMetDC α subunit cDNA was amplified by RT-PCR from colorectal cancer cells and it was cloned into prokaryotic expression vector pTriEx-4 by TA clone.2. The positive recombinant pTriEx-AdoMetDC-α was transformed into an expression strain of E.coli JM109(DE3) and the bacteria was induced to express AdoMetDC-α protein by IPTG The protein product was identified by SDS-PAGE and Western blotting with anti His·Tag monoclonal antibody.3. The protein was purified by Ni-NTA affinity chromatography and tested by SDS-PAGE.4. Purified AdoMetDC-α fusion protein was used to immunize Balb/c mice and collected murine antiserum. The antiserum was evaluated by ELISA and Western blotting.5. Detect the expression of AdoMetDC in human colorectal cancer tissues by immunohistochemical technique using the obtained polyclonal antibody.Results: 1. The complete encoding sequence of AdoMetDC α subunit cDNA was amplified from human AdoMetDC total RNA. The construction of the expression recombinant plasmid pTriEx-AdoMetDC-α was confirmed through restriction enzyme mapping analysis and DNA sequencing.2. SDS-PAGE and Western blotting showed that pTriEx-AdoMetDC-α in E.coli JM109(DE3) can express a specific protein (32kD) which was conform to the expectancy and including 6×His·Tag.3. Purified AdoMetDC-α were obtained by the HisTrap^TMprotein purification kit.4. The polyclonal anti-AdoMetDC antibody was prepared successfully. ELISA and Western blotting showed that the antibody could combine the AdoMetDC protein.5. Immunohistochemical detection showed that there was high concentration of AdoMetDC in colorectal cancer tissues.Conclusions:The polyclonal anti-AdoMetDC antibody was prepared successfully which facilitates the following study and establishes a measure of colorectal cancer diagnosis.Part 2 Adenovirus-mediated expression of both antisense ODC andAdoMetDC inhibits colorectal cancer cell growthObjective:To evaluate the inhibitory effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ODC and AdoMetDC on colorectal cancer cells growth and cell cycle distribution.Methods:1. Human colorectal cancer cells HT-29 were cultured in RPMI 1640 medium and were infected with Ad-ODC-AdoMetDCas. MTS was used to assess adenovirus-mediated gene transduction efficiency.2. Cell viable curve was protracted to assess inhibitory effect of Ad-ODC-AdoMetDCas on HT-29. 3. Western blotting was used to detect the regulation of Ad-ODC-AdoMetDCas on ODC and AdoMetDC protein levels in cells.4. Cell cycle progression was detected by flow cytometry analysis. Results:1. Adenovirus-mediated gene transduction efficiency was 50MOI.2. Ad-ODC-AdoMetDCas could significantly inhibit the growth of HT-29 cells and invasive ability is 50MOI without cell toxicity.3. Western blotting showed that more than 45% of ODC and AdoMetDC protein expression were inhibited in Ad-ODC-AdoMetDCas infected HT-29 cells.4. Cell flow cytometry analysis showed that Ad-ODC-AdoMetDCas could lead the infected cell to arrest at G1 phase.Conclusions:Downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection can inhibit cell growth and Induces G1 arrest in HT-29 cells. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.Part 3 Underlying regulatory responses of Adenovirus-mediatedexpression of both antisense ODC and AdoMetDC induces G1 arrestObjective:To investigate underlying regulatory responses of Adenovirus-mediated expression of both antisense ODC and AdoMetDC induces G1 arrest in HT-29 cells.Methods:1. The effect of Ad-ODC-AdoMetDCas on protein levels of cell cycle regulated proteins was measured by Western blotting analysis.2. The effect of Ad-ODC-AdoMetDCas on mRNA levels of cell cycle regulated proteins was measured by RT-PCR.3. A luciferase reporter plasmid of cyclin D1 promoter was constructed to observe the effect of Ad-ODC-AdoMetDCas on cyclin D1 promoter activity. 4. The effect of Ad-ODC-AdoMetDCas on expression level of β-catenin wasmeasured by Western blotting analysis. Results:1. Ad-ODC-AdoMetDCas could decrease protein expression of cyclin D1, but there were no obvious changes in CDK4 protein level in HT-29 cells.2. The mRNA level of cyclin D1 decreased significantly in Ad-ODC-AdoMetDCas-treated cells but no changes in CDK4 mRNA level.3. The cyclin D1 promoter luciferase reporter plasmid (pGL3-Dl) was constructed successfully and its activity decreased significantly by Ad-ODC-AdoMetDCas infection.4. Ad-ODC-AdoMetDCas had no effect on total β-catenin level but inhibited the translocation from cytoplasm to nucleus of β-catenin.Conclusions:Downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection induces G1 arrest in HT-29 cells and the arrest was associated with suppression of cyclin D1 expression and inhibition of β-catenin nuclear translocation.SummaryIn this study, the prokaryotic expression recombinant plasmid pTriEx--AdoMetDC-α was constructed. Purified AdoMetDC-α fusion protein was used to prepare polyclonal anti-AdoMetDC antibody. Immunohistochemical detection showed that there was high concentration of AdoMetDC in colorectal cancer tissues with the obtained polyclonal antibody. The antibody facilitates the following study and establishes a measure of colorectal cancer diagnosis. The recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ODC and AdoMetDC downregulated ODC and AdoMetDC, inhibited cell growth and induced G1 arrest in HT-29 cells. And the arrest was associated with suppression of cyclin D1 expression and inhibition of β-catenin nuclear translocation. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.