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Hypoxia inducible factor-1α (HIF-1α) has a central role in cellular oxygen-sensing, and its overexpression in many types of cancer is considered important in tumor progression. Thus, targeting HIF-1α production and activity has been of great therapeutic interest. In normoxic conditions, HIF-1α is hydroxylated by oxygen-dependent prolyl-hydroxylases, which require ferrous iron for its activity. The tumor suppressor protein von Hippel Lindau binds to the hydroxylated HIF-1α, which is then ubiquitinated and degraded by proteasomes. We focused on the physiological degradation machinery of HIF-1α mediated by prolyl hydroxylases. Previously, we identified a small molecule, LS081, that is capable of stimulating iron uptake into cells. In the present study, we aimed to inhibit the expression of HIF-1α protein and growth of hepatocellular carcinoma by using the iron-facilitating activity of LS081. In the human hepatocellular carcinoma cell lines Hep3B and HepG2, a combination of LS081 and ferric ammonium citrate (LS081/FeAC) inhibited HIF-1α protein expression but did not inhibit HIF-1α mRNA expression. A mutated HIF-1α protein, which has proline residues that were replaced with alanine and transfected into HEK293 cells, was not affected by the combination of LS081 and FeAC. Furthermore, the iron-facilitating activity of LS081 resulted in Hep3B and HepG2 growth inhibition in vitro and in vivo. These results indicate that the iron-facilitating activity of LS081 inhibits HIF-1α expression through prolyl-hydroxylation of HIF-1α and might have a therapeutic effect in the treatment of hepatocellular carcinoma. (Cancer Sci 2012; 103: 767–774)
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that enhances the expression of many genes, including those involved in angiogenesis, cell proliferation, glucose metabolism, erythropoiesis and cell survival. HIF-1 is composed of α and β subunits, where the β subunit is constitutively expressed and the α subunit is degraded under normoxic conditions despite the fact that it is continuously synthesized.[1, 2] In the presence of oxygen, HIF-PHD1, 2 and 3 catalyze the iron-dependent hydroxylation of specific prolyl-residues on HIF-1α. Once hydroxylated, HIF-1α binds to von Hippel Lindau tumor suppressor protein, is ubiquitinated and then degraded by proteasomes. However, under hypoxic conditions, HIF-1α is hydroxylated to a lesser extent and imported into the nucleus, where it binds to HIF-1β and other transcription factors and co-activators to transactivate a variety of genes containing the hypoxia response element.[3-6] In most cancer cells, HIF-1α is overexpressed via either hypoxia-dependent or independent mechanisms, resulting in increased HIF transcriptional activity,[7-11] which helps the cancer cells to survive and grow by enhancing angiogenesis, motility and glycolysis. HIF activities are also involved in resistance to chemotherapy and radiation therapy. Therefore, inhibition of HIF activities should be of importance in cancer treatment.
The iron-chelator deferoxamine deprives cells of iron and upregulates the expression of HIF-1α protein, indicating that cellular iron content has an essential role in regulating HIF-1α protein degradation. In fact, FeCl3 alone or ferri-transferrin reduces HIF-1α expression in hypoxic conditions. Based on these reports, we hypothesized that the facilitation of iron uptake in cancer cells might downregulate the expression of HIF-1α protein by enhancing the activity of PHD. Although HIF-1 inhibitors have been identified,[7, 14] there have been no reports to date on increasing HIF-1α protein degradation by stimulating iron uptake.
In a previous study, we reported that in mouse HCC models, HIF-1α was overexpressed and inhibition of HIF-1α mRNA expression resulted in remarkable growth reduction. Furthermore, tumor vascularization, which is significantly observed in typical HCC tissues compared to tumors in other organs, proves that the inhibition of HIF-1α could greatly affect the treatment of HCC. We also reported that a novel iron facilitator, LS081, inhibited HIF-1α expression in prostate cancer cell lines and the growth of these cells in cell culture. However, neither the mechanism of HIF-1α inhibition nor the effect of LS081 on tumor xenografts was determined. In this study, we present data that LS081 leads to increased activity of PHD, a resulting increase in hydroxylation of HIF-1 and consequent decrease of HIF-1 protein expression, and that LS081 markedly affects the growth of HCC xenografts.
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Although there have been considerable efforts to identify inhibitors of HIF-1 transcriptional activity, there have been no reports that directly focus on modifying the physiological degradation machinery of HIF-1α as mediated by PHD or that suggest that increased intracellular iron could be manipulated to increase PHD activity and, hence, decrease HIF-1 expression. Our previous report and current studies were undertaken to determine if compounds that stimulated iron uptake would, in fact, affect HIF expression and alter cancer cell proliferation. In fact, iron facilitation by LS081 significantly decreased HIF-1α protein in a prolyl-hydroxylation–dependent manner and, presumably as a consequence, inhibited growth of HCC. In some cancer cells, hypoxia-independent overexpression of HIF-1α has been observed.[9-11] This phenomenon is thought to be caused by abnormal activation of HIF-1α translation mediated by oncogenic signaling through the intracellular signal pathway mediated by phosphoinositide 3-kinase (PI3 kinase)/Akt/mammalian target of Rapamycin (mTOR) pathway. Furthermore, the status of a well-known tumor suppressor, p53, may influence HIF-1α downregulation. In our study, detectable levels of HIF-1α protein were observed in Hep3B cells (p53 null) grown under normoxic conditions, whereas it was undetectable in HepG2 (p53 wild). These levels were significantly decreased by treatment with LS081/FeAC, suggesting that iron facilitation might have a therapeutic advantage in targeting HIF-1 in some cancers even when normoxic conditions prevail.
Iron content is lower in human hepatomas than in normal tissue. In animal models, liver tumors contain reduced amounts of iron and resist iron accumulation.[18-20] Furthermore, a key regulator of iron uptake, transferrin receptor 1, which is also known as a transcriptional target of HIF-1, is increased in HCC. In many other cancers, a similar elevation of transferrin receptor 1 expression and decreased cellular iron content is present, which is consistent with an iron deficiency phenotype. This decrease could then result in reduced activity of PHD with HIF-1α protein upregulation.[21, 22] Iron depletion observed in HCC may therefore be a cause of HIF-1α upregulation. Oxygen- and iron-dependent PHD, which are key factors in HIF-1α degradation, are also known to be transcriptional targets of HIF-1.[23-25] Thus, HIF-1–dependent upregulation of PHD might contribute to a negative feedback control of HIF-1α upregulation. Even with sufficient oxygen levels in tumor tissues, limited iron content would interfere with the negative feedback control mediated by PHD. Iron facilitation in this situation may help to reduce HIF-1α expression by enhancing the activity of PHD. In our present study, the LS081 succeeded in reducing HIF-1α protein levels of HCC both in cell culture and animal models. We provided iron facilitation as a novel approach for HIF-1–targeting treatment in HCC.
Iron is an essential metal for hemoglobin synthesis in erythrocytes, oxidation–reduction reactions, and cellular proliferation, whereas iron-overload causes organ dysfunction through the production of reactive oxygen species production.[26, 27] Most serum-circulating iron binds to transferrin while NTBI exists as a minor component of body iron. In our study, the treatment with LS081 reduced serum NTBI levels whereas no significant changes were observed in serum iron and UIBC levels. Additionally, mass spectrometry analysis suggested that two molecules of LS081 directly bind one molecule of free iron. These data suggest that LS081 mainly binds NTBI and transports it into cells through unknown transporters or receptors without an effect on transferrin bound iron. This selective effect can be explained by the hypothesis that the affinity of transferrin for iron is stronger than LS081 affinity for free iron. Although iron facilitation by LS081 increased ferritin levels in cell culture as previously described, we did not observe a change in ferritin levels in the Hep3B xenografts in our present study (data not shown). As NTBI levels are extremely low under physiological conditions in vivo, LS081 would passively facilitate NTBI uptake into tissues without resulting in ferritin upregulation and iron overload.
Ponka and his colleagues pioneered the use of hydrazone derivatives, many of which are iron chelators that inhibit iron uptake into reticulocytes as well as various cell lines, to study cellular iron metabolism.[28, 29] Our results suggest that LS081, which does chelate iron, facilitates the uptake of iron. The side-chain composition of LS081 differs from that of the hydrazone derivatives that inhibit iron uptake. In the initial screen of the chemical library that identified LS081 as an iron facilitator, other hydrazone derivatives were identified both as facilitators and as iron uptake inhibitors. A detailed structure-activity analysis is being undertaken to determine the side-chain composition that dictates the effects on iron uptake. The hemorrhagic necrosis in the xenografts suggests that the effects of LS081 may not be limited to the cancer cells alone. However, it remains to be seen if LS081 has a direct effect on endothelium or if increased iron content in the xenografts leads to the generation of reactive oxygen species and disruption of the neovascular tissue.
In conclusion, we presented a unique property of a novel iron facilitator, LS081, which enhanced HIF-1α degradation by modulation of prolyl-hydroxylation activity. We also succeeded in inhibiting cell growth of HCC both under normoxic and hypoxic conditions in cell culture and in xenograft models. Furthermore, LS081 itself did not show cytotoxic effects on cell growth in vitro, and no hepatic toxicity was observed in the xenografts. Therefore, treatment with LS081 might be a novel approach for HIF-1–targeting treatment in cancer.