No abstract

Adaptive cellular responses resulting from multiple microenvironmental stresses, such as hypoxia and nutrient deprivation, are potential novel drug targets for cancer treatment. Accordingly, we focused on developing anticancer agents targeting the tumor microenvironment (TME). In this study, to search for selective antitumor agents blocking adaptive responses in the TME, thirteen new compounds, designed and synthesized on the basis of the arylmethylbiguanide scaffold of phenformin, were used in structure activity relationship studies of inhibition of hypoxia inducible factor (HIF)-1 and unfolded protein response (UPR) activation and of selective cytotoxicity under glucose-deprived stress conditions, using HT29 cells. We conducted luciferase reporter assays using stable cell lines expressing either an HIF-1-responsive reporter gene or a glucose-regulated protein 78 promoter-reporter gene, which were induced by hypoxia and glucose deprivation stress, respectively, to screen for TME-targeting antitumor drugs. The guanidine analog (compound 2), obtained by bioisosteric replacement of the biguanide group, had activities comparable with those of phenformin (compound 1). Introduction of various substituents on the phenyl ring significantly affected the activities. In particular, the o-methylphenyl analog compound 7 and the o-chlorophenyl analog compound 12 showed considerably more potent inhibitory effects on HIF-1 and UPR activation than did phenformin, and excellent selective cytotoxicity under glucose deprivation. These compounds, therefore, represent an improvement over phenformin. They also suppressed HIF-1- and UPR-related protein expression and secretion of vascular endothelial growth factor-A. Moreover, these compounds exhibited significant antiangiogenic effects in the chick chorioallantoic membrane assay. Our structural development studies of biguanide derivatives provided promising candidates for a novel anticancer agent targeting the TME for selective cancer therapy, to be subjected to further in vivo study.

The tumor microenvironment, characterized by regions of hypoxia, low nutrition, and acidosis due to incomplete blood vessel networks, has been recognized as a major factor that influences not only the response to conventional anti-cancer therapies but also malignant progression and metastasis. However, exploiting such a cumbersome tumor microenvironment for cancer treatment could provide tumor-specific therapeutic approaches. In particular, hypoxia is now considered a fundamentally important characteristic of the tumor microenvironment in which HIF-1 mediated gene regulation being essential for angiogenesis and tumor development. This has led to the current extensive interest in the drug discovery for tumor microenvironment including tumor specific potential molecular targets for cancer therapeutics. Propolis has pleiotropic effects and contains a variety of bioactive polyphenolic compounds. Some flavonoids of them have been reported to inhibit HIF-1α. We therefore evaluated the effects of composition of Brazilian propolis on cellular responses to hypoxia or nutrition deprivation stress for the development of novel anti-cancer drug targeting the tumor microenvironment. Materials and Method: The ethanol extract of Brazilian propolis was subjected to a series of chromatographic separations to examine HIF-1 inhibition and cytotoxicity under hypoxic and/or glucose deprived condition. The HIF-1α trans-activation effect was evaluated by HIF-1 reporter assay using stable cell lines expressing p2.1. The effect on unfolded protein response (UPR) activation in tumor cell induced by glucose deprivation or 2-deoxyglucose was also examined by GRP78 promoter reporter assay. We performed Immunoblot analysis of the HIF-1α and UPR target genes. The effects on HIF-1α dependent gene expression were investigated by RT-PCR using HCT116 cells. Result: The propolis whole extract increased HRE-dependent luciferase activity in a dose-dependent manner. From a series of chromatographic separations, we obtained fractions 3, 5 and 8 that inhibited HIF-1-dependent induction of luciferase, while fractions 6 and 10-13 increased the luciferase activity. Cinnamic acid derivative, GPU-215 was isolated from Fr.6 reduced HIF1 transactivation activity, expression of HIF-1α protein and GLUT1 mRNA under hypoxia, while flavonoid, GPU-216 from Fr.8 increased HIF-1α transactivation activity and UPR activation. Conclusion: We discovered promising compounds from Brazilian propolis for novel anti-cancer drug targeting tumor microenvironment. GPU-215 has inhibitory effect on HIF-1α expression and function, while GPU-216 increased HIF-1α transactivation and UPR activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 765.