Sulforaphane inhibited expression of hypoxia-inducible factor-1alpha in human tongue squamous cancer cells and prostate cancer cells

Previous studies show that a number of natural compounds from our diet have anticancer effects. Sulforaphane is the most characterized isothiocyanates (ITCs), which are identified in cruciferous vegetables. Sulforaphane is viewed as a conceptually promising agent in cancer prevention. Because of its ability to induce cancer cell apoptosis, it inhibits progression of benign tumors to malignant tumors and interrupts metastasis. However, the effect of sulforaphane on tongue cancer cell proliferation has not yet been reported, and the mechanisms that sulforaphane inhibits cancer development are still unclear. Hypoxia-inducible factor 1 (HIF-1) expression is associated with tumorigenesis and angiogenesis. It regulates the expression of many genes including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase, and lactate dehydrogenase A. In our study, we investigated the effects of sulforaphane on expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was overexpressed in many human malignant tumors, human tongue squamous cell carcinoma and prostate cancer DU145 cells. Sulforaphane inhibited hypoxia induced expression of HIF-1alpha via inhibiting synthesis of HIF-1alpha. Sulforaphane was also found to inhibit hypoxia induced HIF-1alpha expression through activating JNK and ERK signaling pathways, but not AKT pathway. Inhibition of HIF-1alpha by sulforaphane resulted in decreasing expression of VEGF. Taken together, these results suggest that sulforaphane is an effective chemopreventive compound against tongue cancers and prostate cell angiogenesis in vitro, and that the HIF-1alpha target provides a new sight into the mechanisms of sulforaphane's inhibition against tumor cell proliferation.     

Glucose requirement for hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha)

Hypoxia-inducible factor-1alpha (HIF-1alpha) is both a potential endogenous marker of tumor hypoxia and therapeutic target, and elevated HIF-1alpha protein levels have been shown to be associated with increased hypoxic radiation resistance in FaDu human pharyngeal carcinoma cells in vitro. Here, we found that in FaDu xenografts, no significant HIF-1alpha protein accumulation was detectable by either flow cytometry or Western blot, despite the presence of hypoxic (pimonidazole-positive, radiation resistant) cells. To investigate the effect of different tumor microenvironment conditions on hypoxic HIF-1alpha accumulation, we performed in vitro hypoxia experiments (0.1% O2, 24 h) with manipulation of pH (7.4 vs. 6.7), glucose (0-5.5 mM) and serum (0 or 10%) availability in FaDu and HT 1080 human fibrosarcoma cells. Hypoxic induction of HIF-1alpha protein was strongly dependent on glucose availability and largely abolished at 0.55 mM glucose or less in both cell lines. This glucose effect was confirmed in a hypoxia-responsive-element (HRE)/enhanced-green-fluorescent-protein (EGFP) reporter assay in transfected HT 1080 cells and possibly explains a lack of HIF-1alpha protein in hypoxic tumor cells.     

Prognostic impact of hypoxia-inducible factors 1alpha and 2alpha in colorectal cancer patients: correlation with tumor angiogenesis and cyclooxygenase-2 expression.

PURPOSE: Angiogenesis plays an important role in a multitude of biological processes including those of tumorigenesis and cancer progression. Hypoxia is the prime driving factor for tumor angiogenesis and the family of hypoxia-inducible factors (HIFs) plays a pivotal role in this process. The role of HIF in tumor angiogenesis has been underscored in different carcinomas but yet to be reported for colorectal carcinomas. EXPERIMENTAL DESIGN: In this study, we examined HIF [HIF-1alpha (HIF1) and HIF-2alpha (HIF2)] expression in 87 curatively resected colorectal carcinoma samples, and the results were correlated with clinicopathological factors, microvessel density, cyclooxygenase 2 expression, and patient prognosis. RESULTS: HIF1 (44.8%) was more frequently expressed than HIF2 (29.9%). Most of the clinicopathological factors representing the tumor aggressiveness were significantly correlated with overexpression of HIF2 but not with HIF1 expression. HIF2 expression had direct correlation with microvessel density and cyclooxygenase 2 expression. and, in contrast, HIF1 expression had a weak but significant inverse correlation in T1 and T2 tumors only. HIF2 expression alone and the combined expression of HIF1 and HIF2 had significant impact on patient survival. In the multivariate analysis, however, only the combined expression of HIF1 and HIF2 remained independently significant. CONCLUSIONS: Taken together, our results suggest that HIF2 expression may play an important role in angiogenesis and that the combined expression of HIF1 and HIF2 may play an important role in tumor progression and prognosis of colorectal carcinomas. Therefore, HIF expression could be a useful target for therapeutic intervention.     

Differential gene up-regulation by hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha in HEK293T cells

Cells exposed to hypoxia respond by increasing the level of hypoxia-inducible factor-1 (HIF-1). This factor then activates a number of genes by binding to hypoxia response elements in their promoter regions. A second hypoxia-responsive factor, HIF-2, can activate many of the same genes as HIF-1. Overexpression of HIFs accompanies the pathogenesis of many tumors. It is unclear, however, as to the respective role of these factors in responsiveness to hypoxia and other stresses. To address this issue, we used microarray technology to study the genes activated in HEK293T cells by hypoxia or transfection with the alpha chain of HIF-1 (or mutant HIF-1 resistant to degradation) or HIF-2. Fifty-six genes were found to be up-regulated at least 3-fold by either hypoxia or transfection. Of these, 21 were elevated both by transfection with HIF-1alpha and with HIF-2alpha, and 14 were preferentially activated by HIF-1alpha including several involved in glycolysis. Ten genes were preferentially activated by HIF-2alpha, including two (CACNA1A and PTPRZ1) implicated in neurologic diseases. Interestingly, most HIF-2alpha-responsive genes were not substantially activated by hypoxia. An additional 10 genes were up-regulated by hypoxia but minimally activated by HIF-1alpha or HIF-2alpha transfection. Ten of the genes were studied by quantitative real-time PCR and/or by Northern blot and the results paralleled those found with microarray technology. Although confirmation in other systems will be necessary, these results indicate that whereas some genes are robustly activated by both HIF-1 and HIF-2, others can be preferentially activated by one or the other factor.     

PDGFRbeta and HIF-1alpha inhibition with imatinib and radioimmunotherapy of experimental prostate cancer

The clinical application of radioimmunotherapy (RIT) as a single modality in the treatment of prostate cancer is held back because of poor tumor responses to RIT and unacceptable normal tissue toxicities. The purpose of reported here studies was to develop a multimodality approach to RIT of prostate cancer that includes imatinib, a potent PDGFRbeta inhibitor, and in the course of these studies to define the mechanism of imatinib effects on RIT. Hypothesized interactions between these two modalities depend on the reduction of tumor interstitial fluid pressure with the subsequent increase of (131)ICC49 uptake into the tumor, and the inhibition of HIF-1alpha resulting in the improved tumor radiosensitivity. Levels of HIF-1alpha, IGF-1, PDGF-BB, phospho-PDGFRbeta and VEGF in response to imatinib were examined in PC-3 human prostate adenocarcinoma cells in vitro and in xenografts. RIT was based on (131)ICC49 and it was augmented with imatinib. Although PDGFRbeta appears to be functional in PC-3 tumors, the effect of imatinib on the tumor interstitial fluid pressure was insignificant. PC-3 cells and PC-3 xenografts express constitutive HIF-1alpha, which was significantly inhibited by imatinib. Reduced levels of HIF-1alpha were accompanied by the notable suppression of IGF-1. Simultaneously the increase in tumor levels of mouse and human PDGF-BB was observed. Improved PC-3 responses to RIT+imatinib treatment were significant and lasted approximately two weeks. Tumor doubling times in mice treated with (131)ICC49+imatinib were 21.6 +/- 0.7 days compared to 17.2 +/- 0.5 days in (131)ICC49+PBS-treated control mice. Imatinib alone had no effect on the tumor growth. In conclusion, imatinib inhibits HIF-1alpha expression in PC-3 tumors and improves RIT, but it has no effect on VEGF indicating absence of anti-angiogenic effects. There is a significant time- and dose-dependent reduction in the expression of IGF-1 suggesting an alternative pathway of imatinib-regulated HIF-1alpha expression leading to the improved PC-3 responses to RIT.     

The relation between hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression with anemia and outcome in surgically treated head and neck cancer

BACKGROUND: Hypoxia promotes tumorigenesis through the hypoxia-inducible factor (HIF) pathway. There are 2 main homologues of the regulatory proteins, HIF-1alpha and HIF-2alpha, which have different effects in genetic knock-out experiments. Anemia may contribute to hypoxia by reducing oxygen delivery, but it is not known whether this influences HIF-alpha expression in tumors. METHODS: The expression of HIF-1alpha, HIF-2alpha, carbonic anhydrase-9 (CA-9), and peripheral hemoglobin (Hb) levels in 151 patients who underwent surgery for head and neck squamous cell carcinoma (HNSCC) were analyzed and related to outcome. RESULTS: High HIF-1alpha was expressed in 45 of 140 tumors (30%), HIF-2alpha was expressed in 21 of 139 tumors (14%), and CA-9 was expressed in 56 of 149 tumors (62%). There was a positive correlation between HIF-1alpha expression and HIF-2alpha expression (P =.0001). HIF-1alpha alone was associated with a worse disease-specific survival (DSS) (P =.05) and disease-free survival (DFS) (P = .03) in multivariate analyses. Nine percent of tumors expressed both high HIF-1alpha and high HIF-2alpha. High HIF-1alpha/high HIF-2alpha expression was an independent prognostic factors in DSS (P = .04) and DFS (P =.005) in multivariate analyses. There was no correlation noted between Hb and HIF-1alpha, HIF-2alpha, or CA-9. CONCLUSIONS: HIF-1alpha alone was correlated with DSS and DFS. The additive effect of HIF-2alpha on poor prognosis suggested that different pathways may be regulated by HIF-2alph. Anemia that was not related to HIF-alpha expression suggests that tumor intrinsic factors regulate HIF-alpha therefore, anemia may be a surrogate marker for other factors that affect outcome.     

Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype

In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.     

Expression of HIF-1alpha and Glut-1 in human bladder cancer

HIF-1 is a heterodimer consisting of the HIF-1alpha and HIF-1beta subunits, and HIF-1alpha is the unique oxygen regulated subunit that determines HIF-1 activity. HIF-1alpha upgrades many gene products which include the glucose transporter protein 1 (Glut-1). Immunohistochemical studies using a monoclonal antibody specific for HIF-1alpha indicate that the overexpression of HIF-1alpha occurs in the most common forms of human cancer, including bladder cancer. The expression of Glut-1 in human bladder cancer is associated with poor prognosis and a low survival rate. To our knowledge, this is the first study to compare the expression of both HIF-1alpha and Glut-1 with clinicopathological characteristics in superficial and invasive human bladder cancer (all invasive bladder cancer patients received radical radiotherapy). The Kaplan-Meier survival analysis curve shows a significant association of HIF-1alpha expression with recurrence and survival in superficial bladder cancer and shows a significant association of Glut-1 with survival in invasive bladder cancer [chi2 (4)=10.52; Pr >chi2 =0.0012].     

Relation of hypoxia-inducible factor-2 alpha (HIF-2 alpha) expression in tumor-infiltrative macrophages to tumor angiogenesis and the oxidative thymidine phosphorylase pathway in Human breast cancer

Tumor-associated macrophages (TAMs) produce angiogenic factors and in breast cancer are associated with high vascular grade and poor survival. TAMs preferentially migrate to hypoxic areas within tumors and strongly express hypoxia-inducible factor (HIF)-2 alpha. This study examined whether HIF-2 alpha was involved in TAM angiogenic activation by correlating its expression with tumor microvessel density as a marker of angiogenesis, and other tumor variables, in a series of human primary invasive breast carcinomas. A correlation was found between high TAM HIF-2 alpha and high tumor vascularity (P < 0.0001), as well as high tumor grade (P = 0.007). The relation of HIF-2 alpha expression to a recently described oxygen-dependent pathway of angiogenesis was also studied, and an inverse relationship was found between TAM HIF-2 alpha and tumor thymidine phosphorylase expression (P = 0.02). These results suggest that TAM HIF-2 signaling may be a useful target for future antiangiogenic strategies but show that tumors use both oxygen-dependent and oxygen deficiency-regulated pathways for angiogenesis. Thus, combined blockade of pathways and careful assessment of these pathways in trials are necessary.