ABSTRACT
BACKGROUND: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Polyamine concentrations are elevated in colorectal cancer. Depletion of polyamine content in colorectal cancer by chemotherapy is related to tumor regression and impaired tumorigenicity. The current study evaluates the therapeutic effects of antisense ODC and AdoMetDC sequences on colorectal cancer in vitro and in vivo. METHODS: Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). The human colon cancer cell lines, HT-29 and Caco-2, were infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore, the antitumor effects of Ad-ODC-AdoMetDCas were also evaluated in vivo in a nude mouse tumor model. RESULTS: Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. The adenovirus also induced tumor regression in established tumors in nude mice. CONCLUSIONS: Our study suggests that Ad-ODC-AdoMetDCas has antitumor activity and therapeutic potential for the treatment of colorectal cancer.
Subject(s)
Adenosylmethionine Decarboxylase/metabolism , Colorectal Neoplasms/genetics , Genetic Vectors/genetics , Oligonucleotides, Antisense/pharmacology , Ornithine Decarboxylase/metabolism , Adenosylmethionine Decarboxylase/chemistry , Adenosylmethionine Decarboxylase/genetics , Animals , Animals, Newborn , Cell Proliferation , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Cytomegalovirus/genetics , Dependovirus/classification , Dependovirus/genetics , Evaluation Studies as Topic , Factor IX/genetics , Female , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors/administration & dosage , Genetic Vectors/chemistry , Humans , In Vitro Techniques , Injections, Intraperitoneal , Injections, Intravenous , Luciferases/genetics , Luminescent Measurements , Male , Mice , Mice, Inbred C57BL , Neoplasm Invasiveness , Oligonucleotides, Antisense/therapeutic use , Ornithine Decarboxylase/chemistry , Ornithine Decarboxylase/genetics , Polyamines/chemistry , Polyamines/metabolism , Serotyping , Sex Factors , Time Factors , Tissue Distribution , Transgenes , beta-Galactosidase/metabolismABSTRACT
AIM: To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase ( AdoMetDC ) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth. METHODS: A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by PacI. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine. RESULTS: Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine. CONCLUSION: The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.
Subject(s)
Adenosylmethionine Decarboxylase/metabolism , HT29 Cells/pathology , Oligodeoxyribonucleotides, Antisense , Ornithine Decarboxylase/metabolism , Polyamines/metabolism , Adenosylmethionine Decarboxylase/genetics , Adenoviridae/genetics , Cell Line , Cell Proliferation , Embryo, Mammalian , Genetic Vectors , HT29 Cells/metabolism , Humans , Kidney/cytology , Ornithine Decarboxylase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
OBJECTIVE: To investigate the effect of arterial elongation on the arteries and to set up a new method for vessel repair. METHODS: This study was performed in 24 arteries that underwent different traumatic deficit and acute intra-operative elongation repair. The vessels were harvested and studied by histology, electron microscopy, and immunohistochemistry on day 1, 7 and 28 after operation. RESULTS: The total unobstructed rate was 100% for deficit group as well as control experimental group. The content of collagen and elastin at various intervals showed no significant difference, the ratio of them also had no significant difference. Following anastomosis, the compliance of arteries was decreased, the peak value of proliferative cell nuclear antigen (PCNA) positive cell percentage was less than 20%, the internal diameter of elongation and anastomosis edge had no significant difference between deficit group and control group. There were no marked differences in pathological changes between deficit group and control group. CONCLUSION: Acute intraoperative artery elongation after balloon dilatation is a simple, safe, and effective alternative to vein interposition in the repair of traumatic arterial injuries. It appears to be a useful method in the emergency treatment.
Subject(s)
Anastomosis, Surgical/methods , Arteries/surgery , Tissue Expansion , Animals , Arteries/injuries , Disease Models, Animal , Dogs , Proliferating Cell Nuclear Antigen/metabolism , Random Allocation , Wound HealingABSTRACT
OBJECTIVE: To investigate fluid resuscitation affecting the result of treatment of patients with traumatic shock. METHODS: Two hundred and fifty-six cases of patients with traumatic shock treated in our hospital between January 1989 and December 2002 were analysed retrospectively, and the volume-effect relationship between fluid resuscitation during the first hour and future of traumatic shock was summarised. RESULTS: The successful rate of resuscitation was 73.05 percent (187 cases), and traumatic mortality was 26.95 percent (69 cases). After 1-hour trauma, the survival rate was 52.27 percent in patients with 1 liter of resuscitation fluid, 75.90 percent in patients with 15-20 liter of resuscitation fluid, and 78.29 percent of more than 20 liter resuscitation fluid, 86.79 percent in mild shock, 80.34 percent in middle shock and 54.65 percent in severe shock. CONCLUSION: Severe trauma and shock, hypotension and incorrect resuscitation manipulation are the main factors affecting the result of resuscitation and treatment of traumatic shock. It is important to early and correctly expand, especially at the first hour. The aim of resuscitation of traumatic shock is to maintain the hemodynamics and correct O2 defect.
