INVESTIGATIVE TREATMENT DIVISION

24.INVESTIGATIVE TREATMENT DIVISION

    The main goal of the research in this division is to develop innovative strategies for cancer prevention, diagnosis and treatment based on the better understanding of the biology of cancer tissues and the interaction between cancer and host. Improvement of preexistent modalities of cancer diagnosis and treatment is also within the scope of the research activity of the division.
Drug Delivery Systems in Cancer Chemotherapy

    An objective of DDS in cancer chemotherapy is to find methods by which anticancer agents selectively target solid tumors. Two main concepts constitute selective tumor targeting, active targeting and passive targeting. The former involves monoclonal antibodies or ligands to tumor related receptors which can target the tumor by utilizing the specific binding ability between antibody and antigen or between the ligand and its receptor. The latter system can be achieved by the so-called "EPR effect, the enhanced permeability and retention effect". The EPR effect in solid tumor tissue was named according to the pathophysiological characteristics: (a) hypervasculature; (b) incomplete vascular architecture; (c) several vascular permeability factors stimulating extravasation within the cancer; and (d) little drainage of macromolecules and particulates. Macromolecular anticancer agents such as liposomal or micellar drugs have long plasma half-lives because they are too large to pass through the normal vessel walls unless they are trapped by the reticuloendothelial system in various organs. Such macromolecular agents can diffuse out of tumor blood vessels, reach the solid tumor tissue effectively and be retained for a long period due to the EPR effect ^(298,²⁹⁹⁾.
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. The activation of HIF-1 is known to cause both an increase in oxygen supply as a result of vasodilatation, angiogenesis, and erythropoiesis and a switch in energy metabolism by stimulation of glycolysis. Induction of tolerance to glucose starvation was discovered, and it was found to be induced in both normal and cancer cell lines by exposing cells 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 ⁽³⁰⁰⁾, but HIF-1 was found not to be involved in the induction of the tolerance. Instead, 5'-AMP-activated protein kinase was involved in the tolerance ⁽³⁰¹⁾. When HepG2 cells were stably transected with the AMPK antisense expression vector, cell survival stimulated by AICAR, an AMPK activator ⁽³⁰²⁾, during hypoxia was largely abolished in the condition of glucose starvation ⁽³⁰³⁾, indicating that the role of AMPK is important for the tolerance, and the 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 the phosphorylation of p53 at ser 15 and 20. Involvement of the ATM gene in this process is also suspected. Recently it was found that both Akt and AMPK are necessary for tumor cell survival under nutrient starvation, and a novel AMPK family member ARK5, which was newly identified in our division, is unique in that its activation is directly regulated by Akt and the activated ARK5 phosphorylates ATM during nutrient starvation. The precise biochemical mechanisms of energy production without glucose in the canfidion of hypoxia, in which the TCA cycle and oxidative-phosphorylation are almost completely inactive, remain to be elucidated.
Construction of a New Strategy of Gene Therapy

    A new expression vector for gene therapy was constructed based on the human U1 snRNA transcription cassette. U1 snRNA forms a complex with pre-mRNA between the 5’terminus and the splice donor sites and regulates mRNA splicing, and is characterized by its extremely long half life. When mutations were introduced into the 5’end of U1 snRNA using the human K-ras gene, tumorigenesis of pancreatic cancer cells having K-ras mutation was dramatically reduced in nude mice ⁽³⁰⁴⁾. Thus, this vector could be applicable to a wide variety of gene systems.
Role of Nitric Oxide (NO) in Bile Duct Injury

    The role of nitric oxide synthase (NOS) in autoimmune disease is gaining increased attention because of the relationships between NOS activity and T-lymphocyte subpopulations and, in particular, the influence of NO on cytokine production by Th1 versus Th2 cells. In addition, there is evidence that both the liver and infiltrating hepatic T cells have inducible NOS-2 activity. Our study suggests that NO produced through NOS-2 may play a role in the pathogenesis of bile duct injury in some PBC patients ⁽³⁰⁵⁾.
Research for the Mechanism of the "Field Cancerization" Phenomenon in the Upper Aerodigestive Tract

    Patients with squamous-cell carcinoma in the head and neck (HNSCC) often develop second primary esophageal squamous-cell carcinomas (ESCC). In addition, widespread epithelial oncogenic alterations are also frequently observed in the esophagus and can be made visible as multiple lugol-voiding lesions (multiple LVL) by Lugol chromoendoscopy. Multiple occurrences of neoplastic change in the upper aerodigestive tract have been explained by the concept of "field cancerization", usually associated with repeated exposure to carcinogens such as alcohol and cigarette smoke. We found strong evidence for a gene-environmental interaction between the ALDH2-2 allele and alcohol drinking, for the risk of developing multiple LVL, resulting in the development of second ESCC in patients with HNSCC. Ultimately, increased local acetaldehyde exposure thus appears to be a critical determinant of the phenomenon of "field cancerization" ⁽³⁰⁶⁾.




24.INVESTIGATIVE TREATMENT DIVISION



	The main goal of the research in this division is to develop innovative strategies for cancer prevention, diagnosis and treatment based on the better understanding of the biology of cancer tissues and the interaction between cancer and host. Improvement of preexistent modalities of cancer diagnosis and treatment is also within the scope of the research activity of the division. Drug Delivery Systems in Cancer Chemotherapy An objective of DDS in cancer chemotherapy is to find methods by which anticancer agents selectively target solid tumors. Two main concepts constitute selective tumor targeting, active targeting and passive targeting. The former involves monoclonal antibodies or ligands to tumor related receptors which can target the tumor by utilizing the specific binding ability between antibody and antigen or between the ligand and its receptor. The latter system can be achieved by the so-called "EPR effect, the enhanced permeability and retention effect". The EPR effect in solid tumor tissue was named according to the pathophysiological characteristics: (a) hypervasculature; (b) incomplete vascular architecture; (c) several vascular permeability factors stimulating extravasation within the cancer; and (d) little drainage of macromolecules and particulates. Macromolecular anticancer agents such as liposomal or micellar drugs have long plasma half-lives because they are too large to pass through the normal vessel walls unless they are trapped by the reticuloendothelial system in various organs. Such macromolecular agents can diffuse out of tumor blood vessels, reach the solid tumor tissue effectively and be retained for a long period due to the EPR effect ^(298,²⁹⁹⁾. 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. The activation of HIF-1 is known to cause both an increase in oxygen supply as a result of vasodilatation, angiogenesis, and erythropoiesis and a switch in energy metabolism by stimulation of glycolysis. Induction of tolerance to glucose starvation was discovered, and it was found to be induced in both normal and cancer cell lines by exposing cells 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 ⁽³⁰⁰⁾, but HIF-1 was found not to be involved in the induction of the tolerance. Instead, 5'-AMP-activated protein kinase was involved in the tolerance ⁽³⁰¹⁾. When HepG2 cells were stably transected with the AMPK antisense expression vector, cell survival stimulated by AICAR, an AMPK activator ⁽³⁰²⁾, during hypoxia was largely abolished in the condition of glucose starvation ⁽³⁰³⁾, indicating that the role of AMPK is important for the tolerance, and the 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 the phosphorylation of p53 at ser 15 and 20. Involvement of the ATM gene in this process is also suspected. Recently it was found that both Akt and AMPK are necessary for tumor cell survival under nutrient starvation, and a novel AMPK family member ARK5, which was newly identified in our division, is unique in that its activation is directly regulated by Akt and the activated ARK5 phosphorylates ATM during nutrient starvation. The precise biochemical mechanisms of energy production without glucose in the canfidion of hypoxia, in which the TCA cycle and oxidative-phosphorylation are almost completely inactive, remain to be elucidated. Construction of a New Strategy of Gene Therapy A new expression vector for gene therapy was constructed based on the human U1 snRNA transcription cassette. U1 snRNA forms a complex with pre-mRNA between the 5’terminus and the splice donor sites and regulates mRNA splicing, and is characterized by its extremely long half life. When mutations were introduced into the 5’end of U1 snRNA using the human K-ras gene, tumorigenesis of pancreatic cancer cells having K-ras mutation was dramatically reduced in nude mice ⁽³⁰⁴⁾. Thus, this vector could be applicable to a wide variety of gene systems. Role of Nitric Oxide (NO) in Bile Duct Injury The role of nitric oxide synthase (NOS) in autoimmune disease is gaining increased attention because of the relationships between NOS activity and T-lymphocyte subpopulations and, in particular, the influence of NO on cytokine production by Th1 versus Th2 cells. In addition, there is evidence that both the liver and infiltrating hepatic T cells have inducible NOS-2 activity. Our study suggests that NO produced through NOS-2 may play a role in the pathogenesis of bile duct injury in some PBC patients ⁽³⁰⁵⁾. Research for the Mechanism of the "Field Cancerization" Phenomenon in the Upper Aerodigestive Tract Patients with squamous-cell carcinoma in the head and neck (HNSCC) often develop second primary esophageal squamous-cell carcinomas (ESCC). In addition, widespread epithelial oncogenic alterations are also frequently observed in the esophagus and can be made visible as multiple lugol-voiding lesions (multiple LVL) by Lugol chromoendoscopy. Multiple occurrences of neoplastic change in the upper aerodigestive tract have been explained by the concept of "field cancerization", usually associated with repeated exposure to carcinogens such as alcohol and cigarette smoke. We found strong evidence for a gene-environmental interaction between the ALDH2-2 allele and alcohol drinking, for the risk of developing multiple LVL, resulting in the development of second ESCC in patients with HNSCC. Ultimately, increased local acetaldehyde exposure thus appears to be a critical determinant of the phenomenon of "field cancerization" ⁽³⁰⁶⁾.