19.INVESTIGATIVE TREATMENT DIVISION
Implication of NO in Cancer
We have reported that NO stimulates synthesis of the tumor angiogenesis factor VEGF.(260,261) The mechanisms of this up- regulation has been extensively analyzed. Site- directed mutagenesis of the human VEGF gene promoter revealed that both hypoxia and NO activate the VEGF gene through a hypoxia response element (HRE). Gel retardation analysis of HIF- 1 binding to the promoter revealed that (T)GCGTG is the core sequence of the HIF- 1 binding site. Extensive analysis of HRE indicated the existence of an additional cis- element in HRE. Mutation analysis indicated that HIF- 1 binding is not influenced by this novel cis- element and supershift experiments identified an unknown nuclear factor which binds to this element. We named this element the HIF- 1 binding site ancillary sequence. When human cancer cell lines, Hep3B and A172 were treated with various NO- generators other than sodium nitroprusside, HIF- 1 was found to be markedly activated and this activation was completely blocked by the NO scavenger, PTIO. Similar activation of HIF- 1 was observed by hypoxic treatment of cells at 1%O2 and HIF- 1 was synergistically activated by hypoxia and NO treatment. Pharmacological experiments using various inhibitors indicated that the NO- induced pathway which activates HIF- 1 is distinct from that induced by hypoxia. NO induces not only VEGF expression but also confers cellular resistance against nutrient- deficiency. In addition, it confers resistance against apotosis. The mechanisms of apoptosis resistance were analyzed and found to be at least due to inhibition of caspase 3 activation, and caspase 9 activation.
Tolerance of Cancer Cells for Nutrient-starvation
We discovered that NO treatment and/or hypoxia treatment of HepG2 cells induced tolerance to glucose- starvation and this tolerance includes not only a switch of the substrate from glucose to amino acids but also drastic changes in energy metabolism. The molecular mechanisms of this induction are not clear yet, but may involve PKB/ Akt and AMP kinase. This tolerance is similar to the stringent control of E. coli and dauer formation of nematoda in biology. Because cancer progresses through many steps of selection under stringent conditions, some cancers might acquire this tolerance. This possibility was examined directly by culturing cancer cell lines in various deficient media and was found to be true. Some undifferentiated cancer cell lines including pancreatic cancer were extremely resistant to nutrient deficiency. This resistance might be a new target for cancer therapy.
High alcohol Dehydrogenase Activity of the Normal Oral Microflora Neisseria
An association between high alcohol consumption and head and neck cancer has been firmly proven by epidemiological studies. Recently it was demonstrated that not only alcohol itself but also the activity of alcohol metabolizing enzymes, especially aldehyde dehydrogenase, strongly influence the risk for esophageal and head and neck cancers. This raised the possibility that acetaldehyde concentration might be a determinant of risk. The possibility of local production of aldehyde was examined and the normal oral microflora Neisseria was found to have extremely high alcohol dehydrogenase activity. The proportion of oral microflora was examined in volunteers with various lifestyles in terms of alcohol consumption and smoking, and those people frequently consuming alcohol were found to have high Neisseria levels. A case- control study and ecological studies are ongoing.
CYP Expression in Intestinal Metaplasia of Stomach
During a study of intestinal metaplasia by staining with methylene blue, we happened to find methlene blue- unstained lesions which were subsequently found to be intestinal metaplasia. By this method, even single gland metaplasia could be detected. The mechanisms by which the intestinal metaplasia turned white was found to be dependent on p450 reductase activity which was discovered to be expressed abundantly in intestinal metaplasia tissue. (262) Because p450 reductase couples with CYP, aberrantly expressed molecular species were analyzed and found to include 1A1, 1A2 which are responsible for the mutagenic activation of various environmental mutagens including heterocyclic amines. The mutagenic activation of heterocyclic amines was directly proven using the microsome fraction of intestinal metaplasia. Genetic changes of other preneoplastic lesions are also studied.(263)