INVESTIGATIVE TREATMENT DIVISION

23.INVESTIGATIVE TREATMENT DIVISION

    The main goal of the research in this division is the design of new strategies for cancer prevention, diagnosis, and treatment based on a better understanding of the biology of cancer tissues and interaction between cancers and their host. Improvement of existing modalities of cancer diagnosis and treatment also falls within the scope of the division's research activity.
Biochemical and Molecular Basis of Cellular Responses to Insufficient Blood Supply

    Glucose has long been thought to be the major source of energy production during hypoxia. HIF-1 is the key transcription factor in the hypoxia response, in which both an increase in oxygen supply as a result of vasodilatation, angiogenesis, and erythropoiesis and a switch in energy metabolism by stimulation glycolysis are known to occur. Induction of tolerance to glucose starvation was discovered, and it was found to be induced in both normal and cancer cell lines by exposure to either hypoxia or nitric oxide or both. The tolerance to glucose starvation during hypoxia was totally unexpected, because glycolysis had been regarded as the mechanism of energy production during hypoxia. Stimulation of the synthesis of a tumor angiogenesis factor, VEGF, through activation of HIF-1 was discovered in this division,⁽²⁵⁸⁾ but HIF-1 was found not to be involved in the induction of tolerance. Instead, 5'-AMP-activated protein kinase was involved in the tolerance. When antisense RNA expression vector was stably transfected into HrpG2 cells, cell survival stimulated by AICAR, an AMPK activator, during hypoxia without glucose was largely abolished, indicating the role of AMPK in the tolerance, and cell cycle progression was also altered under these conditions. When HepG2 cells were treated with AICAR, the cell cycle stopped at the G1 phase as a result of induction of p21waf-1 by phosphorylation of p53 at ser 15 and 20.⁽²⁵⁹⁾ Involvement of the AMT gene in this process is also suspected. The precise biochemical mechanisms of energy production without glucose during hypoxia, in which the TCA cycle and oxidative-phosphorylation are almost completely inactive, remains to be elucidated.
Development of new cancer therapies

    Based on the above findings and hypothesis, a new strategy for screening new cancer therapies was developed in which drugs capable of abolishing the tolerance to nutrient starvation that is typically observed constitutively in pancreatic cancer cell lines. Several hundred Actinomyces culture supernatant were newly examined for the above activity, and four of them were found to be active, meaning that the supernatants were toxic to pancreatic cancer cells only when nutrients were absent. Some of them were purified to homogeneity and found to be previously unknown compounds. Interestingly, all 8 active compounds and fractions identified thus far have exhibited the same ability to inhibit PKB/Akt activation by glucose starvation, indicating the importance of the PKB/Akt pathway in the tolerance mechanism.
Studies on tumor progression

    Intestinal metaplasia is the one of representative preneoplastic lesions of human cancer, and expression of CYP 1A1, 1A2 had been discovered in intestinal metaplasia. High expression of p450 reductase activity was also found in intestinal metaplasia, and a new method of localizing lesions was invented based on its activity.⁽²⁶⁰⁾ Preneoplastic lesions of lung cancer and genetic changes in these lesions were analyzed in a similar manner. ^(261,²⁶²⁾ In addition to these analyses of precancerous conditions, cell biology analyses were performed in connection with nitric oxide and nutrient starvation. When a variety of tumor cells were exposed to nitric oxide or glucose and/or amino acid starvation, various matrix metalloproteinases were found to be overproduced at the transcription level, with MMP1, MMP3, MMP10 and MMP13 being the most significantly induced. AICAR treatment was also found to cause similar effects. These findings prompted us to hypothesize that tumor cells might undergo progression as a result of intrinsic physiological reactions and inflammatory processes in response to the selective pressure of oxygen and nutrient deprivation. This possibility was examined in iNOS-gene-deficient mice and by chemical carcinogenesis with B(a)P. Tumor incidence and time to carcinogenesis were not different from in wild-type mice, but the degree of invasion was higher in the wild-type than in the knockout mice. The metastatic ability of the tumors induced in both strains of mice was remarkable only in wild type mice, indicating that the presence of iNOS gene greatly influences the outcome of metastasis. This research was conducted as a part of project concerning the implications of inflammation and nitric oxide in cancer.^(258,²⁶³⁾




23.INVESTIGATIVE TREATMENT DIVISION



	The main goal of the research in this division is the design of new strategies for cancer prevention, diagnosis, and treatment based on a better understanding of the biology of cancer tissues and interaction between cancers and their host. Improvement of existing modalities of cancer diagnosis and treatment also falls within the scope of the division's research activity. Biochemical and Molecular Basis of Cellular Responses to Insufficient Blood Supply Glucose has long been thought to be the major source of energy production during hypoxia. HIF-1 is the key transcription factor in the hypoxia response, in which both an increase in oxygen supply as a result of vasodilatation, angiogenesis, and erythropoiesis and a switch in energy metabolism by stimulation glycolysis are known to occur. Induction of tolerance to glucose starvation was discovered, and it was found to be induced in both normal and cancer cell lines by exposure to either hypoxia or nitric oxide or both. The tolerance to glucose starvation during hypoxia was totally unexpected, because glycolysis had been regarded as the mechanism of energy production during hypoxia. Stimulation of the synthesis of a tumor angiogenesis factor, VEGF, through activation of HIF-1 was discovered in this division,⁽²⁵⁸⁾ but HIF-1 was found not to be involved in the induction of tolerance. Instead, 5'-AMP-activated protein kinase was involved in the tolerance. When antisense RNA expression vector was stably transfected into HrpG2 cells, cell survival stimulated by AICAR, an AMPK activator, during hypoxia without glucose was largely abolished, indicating the role of AMPK in the tolerance, and cell cycle progression was also altered under these conditions. When HepG2 cells were treated with AICAR, the cell cycle stopped at the G1 phase as a result of induction of p21waf-1 by phosphorylation of p53 at ser 15 and 20.⁽²⁵⁹⁾ Involvement of the AMT gene in this process is also suspected. The precise biochemical mechanisms of energy production without glucose during hypoxia, in which the TCA cycle and oxidative-phosphorylation are almost completely inactive, remains to be elucidated. Development of new cancer therapies Based on the above findings and hypothesis, a new strategy for screening new cancer therapies was developed in which drugs capable of abolishing the tolerance to nutrient starvation that is typically observed constitutively in pancreatic cancer cell lines. Several hundred Actinomyces culture supernatant were newly examined for the above activity, and four of them were found to be active, meaning that the supernatants were toxic to pancreatic cancer cells only when nutrients were absent. Some of them were purified to homogeneity and found to be previously unknown compounds. Interestingly, all 8 active compounds and fractions identified thus far have exhibited the same ability to inhibit PKB/Akt activation by glucose starvation, indicating the importance of the PKB/Akt pathway in the tolerance mechanism. Studies on tumor progression Intestinal metaplasia is the one of representative preneoplastic lesions of human cancer, and expression of CYP 1A1, 1A2 had been discovered in intestinal metaplasia. High expression of p450 reductase activity was also found in intestinal metaplasia, and a new method of localizing lesions was invented based on its activity.⁽²⁶⁰⁾ Preneoplastic lesions of lung cancer and genetic changes in these lesions were analyzed in a similar manner. ^(261,²⁶²⁾ In addition to these analyses of precancerous conditions, cell biology analyses were performed in connection with nitric oxide and nutrient starvation. When a variety of tumor cells were exposed to nitric oxide or glucose and/or amino acid starvation, various matrix metalloproteinases were found to be overproduced at the transcription level, with MMP1, MMP3, MMP10 and MMP13 being the most significantly induced. AICAR treatment was also found to cause similar effects. These findings prompted us to hypothesize that tumor cells might undergo progression as a result of intrinsic physiological reactions and inflammatory processes in response to the selective pressure of oxygen and nutrient deprivation. This possibility was examined in iNOS-gene-deficient mice and by chemical carcinogenesis with B(a)P. Tumor incidence and time to carcinogenesis were not different from in wild-type mice, but the degree of invasion was higher in the wild-type than in the knockout mice. The metastatic ability of the tumors induced in both strains of mice was remarkable only in wild type mice, indicating that the presence of iNOS gene greatly influences the outcome of metastasis. This research was conducted as a part of project concerning the implications of inflammation and nitric oxide in cancer.^(258,²⁶³⁾