Tumor cells generally proliferate rapidly and the demand for essential nutrients as well as oxygen always exceeds the supply due to the unregulated growth and the insufficient and inappropriate vascular supply. However, cancer cells show an inherent ability to tolerate extreme conditions, such as that characterized by low nutrient and oxygen supply, by modulating their energy metabolism. Thus, targeting nutrientdeprived cancer cells may be a novel strategy in anticancer drug development. Based on that, we established a novel screening method to discover anticancer agents that preferentially inhibit cancer cell viability under the nutrientdeprived condition. After screening 500 medicinal plant extracts used in Japanese Kampo medicine, we found that a CH 2 Cl 2 -soluble extract of Arctium lappa exhibited 100% preferential cytotoxicity under the nutrient-deprived condition at a concentration of 50 Mg/mL with virtually no cytotoxicity under nutrient-rich condition. Further bioassayguided fractionation and isolation led to the isolation of arctigenin as the primary compound responsible for such preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against nutrient-deprived cells at a concentration of 0.01 Mg/mL. Furthermore, arctigenin was also found to strongly suppress the PANC-1 tumor growth in nude mice, as well as the growth of several of the tested pancreatic cancer cell lines, suggesting the feasibility of this novel antiausterity approach in cancer therapy. Further investigation of the mechanism of action of arctigenin revealed that the compound blocked the activation of Akt induced by glucose starvation, which is a key process in the tolerance exhibited by cancer cells to glucose starvation.

Transcription of hypoxia-inducible genes is regulated by hypoxia response elements (HREs) located in either the promoter or enhancer regions. Analysis of these elements reveals the presence of one or more binding sites for hypoxia-inducible factor 1 (HIF-1). Hypoxia-inducible genes include vascular endothelial growth factor (VEGF), erythropoietin, and glycolytic enzyme genes. Site-directed mutational analysis of the VEGF gene promoter revealed that an HIF-1 binding site (HBS) and its downstream HIF-1 ancillary sequence (HAS) within the HRE are required as cis-elements for the transcriptional activation of VEGF by either hypoxia or nitric oxide (NO). The core sequences of the HBS and the HAS were determined as TACGTG and CAGGT, respectively. These elements form an imperfect inverted repeat, and the spacing between these motifs is crucial for activity of the promoter. Gel shift assays demonstrate that as yet unknown protein complexes constitutively bind to the HAS regardless of the presence of these stimuli in several cell lines, in contrast with hypoxia-or NO-induced activation of HIF-1 binding to the HBS. A common structure of the HRE, which consists of the HBS and the HAS, is seen among several hypoxia-inducible genes, suggesting the presence of a novel mechanism mediated by the HAS for the regulation of these genes.

An anthelminthic, pyrvinium pamoate (PP), 6-(dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-methyl-quinolinium pamoate salt, has been found to be extremely toxic to PANC-1 cells in glucose-free medium, but not to be toxic to the same cells cultured in ordinary medium, Dulbecco's modified Eagle's medium (DMEM). It showed the same preferential toxicity for various cancer cell lines during glucose starvation. When 0.1 µ µ µ µg/ml PP was added to the medium, spheroid growth of human colon cancer cell line WiDr was strongly inhibited to a diameter of 750 µ µ µ µm, and this finding is consistent with the concept of antiausterity. PP was also found to exert antitumor activity against human pancreatic cancer cell line PANC-1 in nude mice and SCID mice when it was administered subcutaneously or orally. Regarding the mechanism of PP action, inhibition of Akt phosphorylation, which has been found to be essential for the austerity mechanism, was observed in vitro and in vivo. These findings indicate that PP may be useful for anticancer therapy and that antiausterity therapy could be a novel strategy for anticancer therapy. (Cancer Sci 2004; 95: 685-690) uman cancers arise through many steps of carcinogenesis in which multiple genetic alterations, genetic or epigenetic, are often observed.

Nitric oxide (NO) regulates production of vascular endothelial growth factor (VEGF) by normal and transformed cells. We demonstrate that NO donors may up-regulate the activity of the human VEGF promoter in normoxic human glioblastoma and hepatoma cells independent of a cyclic guanosine monophosphate–mediated pathway. Deletion and mutation analysis of the VEGF promoter indicates that the NO-responsive cis-elements are the hypoxia-inducible factor-1 (HIF-1) binding site and an adjacent ancillary sequence that is located immediately downstream within the hypoxia-response element (HRE). This work demonstrates that the HRE of this promoter is the primary target of NO. In addition, VEGF gene regulation by NO, as well as by hypoxia, is potentiated by the AP-1 element of the gene. Our study also reveals that NO and hypoxia induce an increase in HIF-1 binding activity and HIF-1 protein levels, both in the nucleus and the whole cell. These results suggest that there are common features of the NO and hypoxic pathways of VEGF induction, while in part, NO mediates gene transcription by a mechanism distinct from hypoxia. This is demonstrated by a difference in sensitivity to guanylate cyclase inhibitors and a different pattern of HIF-1 binding. These results show that there is a primary role for NO in the control of VEGF synthesis and in cell adaptations to hypoxia. (Blood. 2000;95:189-197)

We evaluated the effect of nitric oxide (NO) on vascular endothelial growth factor (VEGF) gene expression in human A-172 glioblastoma cells and human HepG2 hepatocellular carcinoma cells. The mRNA level of VEGF increased in response to S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) in both cell lines, and increased in mRNA level well coincided with VEGF protein production in A-172 cells. SNAP at 0.5 mM induced maximal stimulation of 4.4 and 3.7 kb VEGF mRNA expression after 6 h about 11 and 8 fold increase, respectively above control level. Similar VEGF mRNA accumulation was observed also with NOR3, another chemical NO generator. To evaluate the effect of SNAP on VEGF mRNA stability, half-lives of VEGF mRNA were measured in A-172 cells cultured with or without 0.5 mM SNAP and treated with actinomycin D (25 microg/ml). Half-life for VEGF mRNA was found to be prolonged about 2.4 fold by SNAP. VEGF expression induced by SNAP was inhibited by guanylate cyclase inhibitors, methylene blue (10 microM) and LY-83583 (1 microM), and by the protein synthesis inhibitor, cycloheximide (25 microg/ml). These results suggest that induction of VEGF gene expression by NO is mediated through guanylate cyclase activity and requires on-going protein synthesis.

Hypoxia is a critical event for higher organisms, and cells and tissues react by increasing the oxygen supply by vasodilatation, angiogenesis, and erythropoiesis and maintaining cellular energy by increasing glycolysis and inhibiting anabolic pathways. Stimulation of glycolysis has been regarded as the main response that increases energy production during hypoxia; however, there is an obvious conflict during ischemia, because both the oxygen and glucose supply are insufficient. In this study, we found that exposure of HepG2 cells and normal fibroblasts to hypoxia induces cellular tolerance to glucose starvation. The tolerance induced by hypoxia is dependent on several amino acids, indicating a switch from glucose to amino acids as the energy source. When antisense RNA expression vector for 5-AMP-activated protein kinase or protein kinase B/Akt was transfected into HepG2 cells, the induction of tolerance to glucose was greatly inhibited, indicating that the tolerance was dependent on 5-AMP-activated protein kinase and protein kinase B/Akt. Similar tolerance was induced by nitric oxide exposure. The tolerance induced was observed in various cells and may represent a previously unknown physiological response related to hypoxia-preconditioning and tumor progression:austerity.

No abstract

Atypical adenomatous hyperplasia (AAH) has recently been implicated as a precursor to lung adenocarcinoma. We previously reported loss of heterozygosity (LOH) in tuberous sclerosis (TSC) geneassociated regions to frequently be observed in lung adenocarcinoma with multiple AAHs. In this study, we analyzed LOH in four microsatellite loci on 9q, including the TSC1 gene-associated region, and four loci on 16p, including the TSC2 gene-associated region, in both 18 AAHs and 17 concomitant lung adenocarcinomas from 11 patients. Seven of 18 (39%) AAHs and 9 of 17 (53%) adenocarcinomas displayed LOH on 9q. Carcinogenesis is a multistep process that results from an accumulation of genetic alterations in oncogenes and tumor suppressor genes. It is reasonable to regard each preneoplastic lesion as possibly having a characteristic genetic change and it is essential to investigate the biological features of preneoplastic lesions to elucidate the pathogenesis of carcinomas. In lung cancers, squamous dysplasia has long been recognized as a preneoplastic lesion of squamous cell carcinoma.