A mycobacterial iron chelator, desferri-exochelin, induces hypoxia-inducible factors 1 and 2, NIP3, and vascular endothelial growth factor in cancer cell lines.

Hypoxia is a key phenomenon in tumor behavior, selecting for resistance to apoptosis, conferring resistance to radiotherapy and chemotherapy, and also inducing angiogenic factors such as vascular endothelial growth factor (VEGF). Exochelins are naturally evolved iron chelators produced by Mycobacterium tuberculosis. Because iron chelation has been reported to activate the hypoxia-inducible factor (HIF), we investigated the effects of an exochelin [desferri-exochelin (DFE) 772SM] on this hypoxia-inducible pathway and downstream target genes. DFE induced HIF-1alpha and HIF-2alpha transcription factors regulating the hypoxic response in the breast tumor cell line MDA468. DFE was 10 times more potent and more rapid in onset of effect than the clinically used iron chelator deferoxamine. The expression of downstream hypoxia-responsive target genes VEGF and the proapoptotic protein NIP3 was activated by transcription. MDA468 proliferation was inhibited via HIF-independent pathways, related to other effects of iron chelation. DFE inhibited effects of VEGF on endothelial cell proliferation. DFE potentially could be useful in cancer therapy by inducing apoptosis via NIP3 in conjunction with other non-HIF-related growth inhibitory pathways and blocking endothelial proliferation despite the presence of VEGF.

Chong TW ，Horwitz LD ，Moore JW ，Sowter HM ，Harris AL ... -  《CANCER RESEARCH》

Association of hypoxia-inducible factors 1alpha and 2alpha with activated angiogenic pathways and prognosis in patients with endometrial carcinoma.

Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha are essential regulatory proteins for the adaptation of tumor cells to hypoxia, and they stimulate angiogenesis through activation of the vascular endothelial growth factor (VEGF) gene. HIF-1alpha and HIF-2alpha proteins were studied immunohistochemically in a group of 81 patients with Stage I endometrial adenocarcinoma of the endometrioid cell type. The results were correlated with intratumoral angiogenesis, the expression of the angiogenic factors VEGF and thymidine phosphorylase (TP), and the VEGF/receptor (VEGF/KDR) complex. Relations also were sought with estrogen receptor (ER) and progesterone receptor (PR), with the apoptosis-related proteins bcl-2 and p53, with several histopathologic parameters, and with patient prognosis. In addition, a sample of 25 normal endometria at various phases of the menstrual cycle was studied for the presence of HIF-1alpha and HIF-2alpha. HIF-1alpha expression was detected in 49% of endometrial carcinomas. The expression was cytoplasmic or mixed nuclear/cytoplasmic. HIF-1alpha expression was associated with up-regulation of the VEGF pathway and with increased standard microvessel density (sMVD) and activated VEGF/KDR microvessel density (aMVD). It also was associated with a poor prognosis in both univariate and multivariate analyses. HIF-2alpha protein showed a pattern of expression similar to the pattern seen in HIF-1alpha, but expression of HIF-2alpha protein occurred in only 17% of endometrial carcinomas, and it was associated with increased TP reactivity. There also was a relation of HIF-1alpha expression with well-differentiated endometrial neoplasms, and there was a marginal association of HIF-1alpha and HIF-2alpha with ER expression. With reference to normally cycling tissues, HIF-1alpha nuclear/cytoplasmic expression was particularly strong in the samples of early proliferative phase endometrium compared with HIF-2alpha protein expression, which showed a constant reaction throughout the menstrual cycle. The up-regulation of HIF-1alpha and, to a lesser extent, of HIF-2alpha is a common event in Stage I endometrial adenocarcinomas. In these tumors, HIF-1alpha expression is related to increased angiogenesis, through activation of the VEGF angiogenic pathway, and to an unfavorable prognosis. HIF-2alpha accumulation is associated with increased expression of the angiogenic factor TP.

Sivridis E ，Giatromanolaki A ，Gatter KC ，Harris AL ，Koukourakis MI ，Tumor and Angiogenesis Research Group ... -  《CANCER》

Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia-inducible factor-2 alpha in human bladder tumors and cell lines.

Hypoxia is an important regulator of vascular endothelial growth factor (VEGF) expression, and VEGF is associated with poor prognosis in bladder cancer. To investigate further the mechanisms of VEGF regulation, we examined VEGF expression by mRNA and protein analysis in four human bladder cancer cell lines, showing a progression from well to poorly differentiated phenotypes under varying conditions of confluence and hypoxia (0.1% O(2)) and with chemical mimics of hypoxia. Hypoxia significantly increased VEGF protein expression in all cell lines, although this effect was dependent on the degree of confluence. The superficial bladder cancer cell line RT4 lost hypoxia inducibility at confluence, whereas inducibility was maintained in the invasive cell lines 253J and EJ28. This pattern of VEGF expression in the invasive cell lines correlated with the expression of the transcription factor hypoxia inducible factor-1 alpha (HIF-1 alpha) and with hypoxia-inducible factor-2 alpha (HIF-2 alpha) and in RT4 correlated with a marked reduction in HIF-1 alpha inducibility at confluence. Using the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY 294002, we show that this VEGF hypoxia-inducible pathway regulated by HIF-1 alpha is distinct from a PI 3-kinase-dependent pathway, which regulates basal amounts of VEGF, but does not affect inducibility. Both HIF-1 alpha and HIF-2 alpha protein and mRNA were up-regulated in primary human bladder tumors (n = 12) compared with normal bladder specimens (n = 4), with significant intertumor variation. These results suggest that components of the hypoxia response pathway, including HIF-1 alpha and HIF-2 alpha, are important cofactors in the regulation of VEGF in bladder cancer and are therapeutic targets in this disease.

Jones A ，Fujiyama C ，Blanche C ，Moore JW ，Fuggle S ，Cranston D ，Bicknell R ，Harris AL ... -  《CLINICAL CANCER RESEARCH》

Hypoxia-induced changes in the expression of VEGF, HIF-1 alpha and cell cycle-related molecules in ovarian cancer cells.

Hypoxia is important in cancer progression, and at the stage of detachment of the cancer cells from the primary lesion. This study was undertaken to analyze the effect of hypoxia on angiogenesis and cell proliferation in ovarian cancer. We first used immunohistochemistry to examine the expression of VEGF in 42 cases of ovarian carcinoma, with relevance to the p53 expression. Then, the expression of VEGF, HIF-1 alpha, cell cycle-related molecules and cell numbers were examined in 4 ovarian cancer cell lines with various p53 gene status. Immunohistochemistry showed that there was no significant correlation between VEGF and p53 expression. Moreover, hypoxia increased the expression of VEGF via up-regulation of HIF-1 alpha irrespective of p53 gene status. However hypoxia did not change the cell numbers, but influenced the expression of cell cycle-related molecules (increased p27 and decreased cyclin D1 and Rb). Hypoxia increased VEGF expression in ovarian cancer cells irrespective of p53 gene status.

Horiuchi A ，Imai T ，Shimizu M ，Oka K ，Wang C ，Nikaido T ，Konishi I ... -  《ANTICANCER RESEARCH》

V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression.

Adaptation to hypoxia represents an important aspect of tumor progression. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates essential homeostatic responses to cellular and systemic hypoxia by activating transcription of multiple genes including those encoding glycolytic enzymes and vascular endothelial growth factor (VEGF). In this report, we demonstrate that whereas C-SRC expression is not required for expression of HIF-1 or transcriptional activation of genes encoding VEGF and enolase 1 (ENO1), cells expressing the v-Src oncogene have increased expression of HIF-1, VEGF, and ENO1 under both hypoxic and nonhypoxic conditions. Expression of V-SRC was associated with increased transcription of reporter genes containing cis-acting hypoxia-response elements from the VEGF and ENO1 genes, and this transcriptional activation required an intact HIF-1 binding site. When three rat hepatoma subclones that differed with respect to the level of HIF-1 expression were injected into nude mice, tumor growth correlated with HIF-1 expression, suggesting that HIF-1 may be generally involved in tumor progression. These studies link an oncogene to the induction of HIF-1 expression, thus providing a mechanism for hypoxic adaptation by tumor cells.

Jiang BH ，Agani F ，Passaniti A ，Semenza GL ... -  《CANCER RESEARCH》