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Hypoxia and growth factor stimulation induce hypoxia-inducible factor-1α (HIF-1α), conferring upon cancer cells the ability to adapt to microenvironments and enhance proliferation, angiogenesis and metastasis. Hemin, an iron-binding porphyrin, has been used to treat porphyria attacks, particularly in acute intermittent porphyria. Although the anti-inflammatory and antitumor effects of hemin were reported, no information is available regarding its effect on HIF-1α. Our study investigated whether hemin and other protoporphyrin compounds have the ability to inhibit HIF-1α activity, and if so, what is the molecular basis of inhibition. Hemin treatment prevented CoCl2-induced HIF-1α expression. HIF-1α inhibition by hemin resulted from an increase in its facilitated ubiquitination and degradation, as shown by the experimental results using cychloheximide treatment and ubiquitination assays. Consistently, hemin repressed HIF-1α-dependent gene transactivation. Intriguingly, hemin directly impeded the binding between heat shock protein 90 (HSP90) and HIF-1α, which was reversed by the addition of an excess amount of ATP required for HSP90 activity. In addition, hemin decreased the expression of client proteins of HSP90. Thus, the inhibition of HIF-1α activity by hemin might result from its interaction with HSP90. Moreover, treatment of protoporphyrin IX, ZnPP or Co(III)PP, but not Mn(III)PP, inhibited HIF-1α induction, indicating that protoporphyrin ring in association with the nature of binding metal leads to HSP90 inhibition. In an in vivo model, hemin treatment inhibited not only the formation of new vessels but also cancer cell proliferation and migration/invasion, supporting the notion that hemin may be applied to the prevention and/or treatment of angiogenesis and/or cancer metastasis.
Hemin (ferriprotoporphrin IX), which belongs to the porphyrin family containing an iron, is the oxidative form of the heme molecules. It has been used to treat porphyria attacks, especially in acute intermittent porphyria.1 Hemin administration had a therapeutic effect in β°-thalassemic patients by enhancing HbF (α2γ2) synthesis, eventually resulting in a threefold increase in hemoglobin content.1 In recent studies, it has been shown that the chemicals containing protoporphyrin ring such as hemin possess substantial antigenotoxic and anti-inflammatory effects against diverse mutagens.2–4 When heme is oxidized, its binding with hemoglobin is less tight, and consequently, it interacts more easily with alternative acceptor proteins and the lipid membranes of cells.5 Hence, hemin may promote oxidative stress, which would facilitate the antioxidant adaptive response in cells. Hemin induced heme oxygenase-1 (HO-1), which enhanced the oxidative degradation of heme molecules to release free iron, biliverdin and carbon monoxide,1 and this, in turn, raised the possibility that hemin has an anticancer effect.1
Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric complex composed of the inducible subunit of HIF-1α and the constitutively expressed HIF-1β (also known as “aryl hydrocarbon nuclear translocator”).6, 7 HIF-1α can be controlled by either an oxygen-dependent or -independent mechanism.8, 9 Hypoxic conditions and hypoxia-mimetic agents (e.g., CoCl2) increase the accumulation of HIF-1α via the enhanced protein stability of HIF-1α. Consequently, activation of HIF-1α promotes cancer progression by inducing its target genes, whose protein products stimulate angiogenesis, glycolysis and tumor proliferation.9, 10 Thus, activated HIF-1α controls the expression of genes involved in the adaptation of cancer cells to unfavorable tumor microenvironments where the supply of oxygen and nutrients are limited.7, 10 Consequently, cancer cells with augmented HIF-1α expression levels become aggressive and acquire chemotherapy resistance.11
Heat shock protein 90 (HSP90) is a ubiquitously expressed molecular chaperone that plays a role in cell signaling, proliferation and survival.12, 13 Moreover, HSP90 is upregulated in cancer cells to adapt to environmental stresses, including nutrient deprivation and hypoxia.12, 13 Under these conditions, HSP90 prevents its client proteins [i.e., HIF-1α, epidermal growth factor receptor (EGFR), endothelial nitric oxide synthase (eNOS), glucocorticoid receptor (GR), p53, Bcr-Abl, cyclin-dependent kinase 4 (CDK4), Raf1, Akt and human epidermal growth factor receptor 2 (HER2)] from degradation and malfunction against cellular stress through its ATPase activity.12–14 The ability of HSP90 to regulate HIF-1α is independent of canonical O2/proline hydroxylase-domain proteins (PHD)/von Hippel-Lindau (VHL) pathway.14–16 Certain chemicals bound to the ATP-binding pocket of HSP90 promote the disruption of HSP90 function, causing ubiquitin-mediated degradation of HIF-1α.15, 17, 18 Therefore, an increasing number of studies have described a series of compounds, such as geldanamycin, 17-AAG, radicicol and deguelin, that inhibit HIF-1α in cell and animal models.7, 17, 18
In view of the possibility that hemin affects the pathways responsible for oxidative degradation of heme molecules, our study explored the effects of hemin and other protoporphyrin compounds on HIF-1α expression and its activity and target gene induction. We determined whether hemin prevents the ability of CoCl2 or hypoxia to increase HIF-1α activity and studied the underlying molecular basis. Here, we report that hemin treatment accelerates HIF-1α ubiquitination and its degradation. As hemin activated various signaling pathways known to affect HIF-1α, our study examined the possible causal relationship between the activated signaling pathways and a decrease in HIF-1α stability by hemin. Finally, the results of the chick chorioallantoic membrane (CAM) model, and cancer cell proliferation and migration/invasion assays confirmed the efficacy of hemin in the repression of HIF-1α and angiogenesis. Our findings indicate that hemin directly interferes with the interaction between HSP90 and HIF-1α. This approach enabled us to identify hemin and other protoporphyrins as novel compounds that inhibit HIF-1α-mediated angiogenesis.
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HIF-1α belongs to the bHLH and PAS families and forms a heterodimer complex with HIF-1β.6, 32 This complex binds to HIF-1-specific DNA sequences in the mammalian HRE (5′-B(A/G)CGTGVBBB-3′; B represents to all bases except A, and V represents to all bases except T) of target genes.6, 32 HIF-1α contains two transactivation domains, N-terminal and C-terminal. The C-terminal domain especially attributes to the binding with transcription coactivators, such as CBP/p300.9 Activation of HIF-1α by hypoxia or growth factors causes cancer progression by initiating angiogenic gene transcription. Therefore, HIF-1α is considered as a promising pharmacological target against cancer cell development and growth independent of normal cells. A series of anticancer agents have been reported to inhibit HIF-1α.33 Moreover, additional new chemicals are being investigated for HIF-1α inhibition.
The oxygen-dependent degradation domain (ODDD) of HIF-1α is essential for its regulation by oxygen.7 This domain has two proline residues hydroxylated by PHD, which recruits the E3 ubiquitin ligase complex.7 Thus, HIF-1α constantly undergoes the ubiquitin-proteasome degradation under normoxic conditions.7 In our study, we found that hemin increased HIF-1α ubiquitination in cancer cells exposed to CoCl2. Our data showing a substantial increase in HIF-1α ubiquitination by hemin correlate well with a decrease in total HIF-1α level. The hypothesis that hemin enhances proteosomal degradation of HIF-1α is supported by our data showing that hemin accelerates HIF-1α ubiquitination, decreases nuclear HIF-1α content and inhibits HRE target gene transactivation. Hypoxic conditions impede PHD from hydroxylating proline residues due to oxygen deficiency.34, 35 We found that hemin also inhibited HIF-1α activation stimulated by hypoxia. Therefore, it was predicted that the target site of hemin, which led to accelerated HIF-1α degradation, might not depend on the ubiquitination step.
Our observation that hemin, ZnPP, protoporphyrin IX and Co(III)PP, but not Mn(III)PP, had the ability to inhibit HIF-1α induction indicates that metal-binding protoporphyrins have differential inhibitory effects. In addition, we found that cyanocobalamin and hydroxocobalamin, which belong to protoporphyrin IX families, failed to inhibit HIF-1α activation (data not shown), suggesting that the structure of aliphatic chains of protoporphyrin is important. Hence, it is highly likely that the inhibitory effect of hemin on HIF-1α results from protoporphyrin ring in its chemical structure in association with the chemical nature of binding metal. The exact role and chemical properties of metals comprised in protoporphyrin ring remain to be established.
An intriguing finding of our study is that hemin has an inhibitory effect on HSP90 for the repression of HIF-1α activity. This hypothesis is supported by the result of the immunoprecipitation assays showing that hemin attenuated the interaction between HSP90 and HIF-1α. This finding, in turn, led to 26S proteasome-mediated degradation of HIF-1α. In addition, the ability of hemin to suppress the mRNA levels of HSP90′s client proteins strongly supports our hypothesis. Moreover, the result of cell-free in vitro assay strengthens the concept that the inhibitory effect of hemin on the interaction of HSP90 (ATP-dependent chaperone) and HIF-1α may result from the competition of hemin with ATP for the ATP-binding site of HSP90. This contention was strengthened by the reversal of hemin's inhibitory effect on the binding of HSP90 and HIF-1α by the addition of excess amount of ATP. Studies have shown that geldanamycin, radicicol and deguelin impede the interaction of HSP90 and oncogenic proteins, thereby facilitating their protein degradation.17, 36 These chemicals possess a high affinity to the ATP-binding site of HSP90, which contributes to HSP90 dimerization via its conformational change.36 Oh et al. displayed the docked conformation of geldanamycin (a model structure) or deguelin attached to the ATP-binding site of HSP90.17 These structural analyses suggest the possibility that hemin also binds to the same region of HSP90 and competes with ATP.
Reactive oxygen species, in particular H2O2, inactivates PHD enzymes, thereby stabilizing HIF-1α.34, 35 Thus, antioxidants exert inhibitory effects against tumorigenesis and tumor proliferation that rely on HIF-1α activation.37 The roles of hemin on oxidative stress in the cell remain controversial. It has been reported that hemin transcriptionally induced thioredoxin, which acts as an antioxidant and redox regulator.38 In addition, phase II enzymes induced by hemin may help protect cells from oxidative stress-related condition through its radical scavenging activity.38, 39 Hemin induces activation of NF-E2-related factor 2 (Nrf2) and its target antioxidative genes.38 These results raised the possibility that hemin may inhibit HIF-1α through its increased expression of phase II enzymes including hemeoxygenase-1 (HO-1). However, we found that ZnPP, a well-known HO-1 inhibitor, also suppressed CoCl2- or hypoxia-inducible HIF-1α activation in HCT116 cells, which rules out the possibility that Nrf2-mediated HO-1 induction by hemin contributes to its inhibitory action on HIF-1α. Moreover, hemin treatment alone substantially increased H2O2 production and further enhanced CoCl2-induced H2O2 production (Supporting Information Fig. S1b). This result agreed to another report, which stated that high concentration of hemin would cause oxidative stress, leading to metabolic stress, tissue damage and cell death.40 Therefore, potential antioxidant effect of hemin might not be associated with its inhibition of HIF-1α by hemin.
In view of the known signaling pathways controlling HIF-1α, our study further examined changes in the phosphorylation status of major kinases that regulate HIF-1α activity in cells treated with hemin. S6K1 is involved in the regulation of HIF-1α: (i) insulin promotes HIF-1A mRNA translation through S6K1 and thus increases de novo synthesis of the protein, and (ii) S6K1 enhances translation of HIF-1α mRNA via 5′ terminal oligopolypyrimidine (TOP) sequences.41, 42 In HCT116 cells, hemin treatment alone enhanced phosphorylations of Akt and S6K1 rapidly, which did not match the inhibitory action of hemin on HIF-1α (Supporting Information Fig. S2a). Consistently, chemical inhibitors such as Ly294002 and rapamycin (respectively, a phosphatidylinositol 3-kinase/Akt inhibitor and an S6K1 inhibitor) failed to restore the ability of hemin to inhibit HIF-1α induction by CoCl2 (Supporting Information Fig. S2b, top).
AMP-activated protein kinase (AMPK) is also implicated with HIF-1α activity.43, 44 AMPK activation by either 5-aminoimidazole-4-carboxamide ribonucleotide or metformin antagonized HIF-1α induction by insulin or insulin-like growth factor, indicating that chemical activation of AMPK might be associated with HIF-1α inhibition.45 In the subsequent experiment, we examined the possible role of AMPK activation in hemin's inhibition of HIF-1α in cells. Although hemin enhanced the phosphorylation of AMPK at 3–6 hr, compound C (an AMPK inhibitor) treatment did not show reversal of hemin's inhibitory effect on HIF-1α induction (Supporting Information Fig. S2b, upper middle). Recently, it has been known that glycogen synthase kinase-3β (GSK3β) phosphorylates three residues (i.e., S551, T555 and S589) of HIF-1α and provokes its proteasomal degradation.46 Hemin increased the inhibitory phosphorylation of GSK3β at 10–60 min. Nonetheless, the inhibitory effect of hemin on CoCl2 induction of HIF-1α was not antagonized by SB21676, a well-known GSK3β inhibitor (Supporting Information Fig. S2b, upper middle). Therefore, neither AMPK nor GSK3β might be engaged in the inhibition of HIF-1α by hemin.
Protein kinase C δ (PKCδ) is phosphorylated and activated under hypoxia.47 The phosphorylation of PKCδ contributes to stabilization of HIF-1α protein.47 Constitutively active c-Jun N-terminal kinase 1 (JNK1) is also implicated in HIF-1α stabilization in hypoxia-mimicked conditions.48 Therefore, PKCδ and JNK are involved in the regulation of HIF-1α. Unexpectedly, hemin activates both PKCδ and JNK (Supporting Information Fig. S2a), and the inhibitory action of hemin on HIF-1α was not reversed by treatment with either a PKCδ inhibitor (rottlerin) or a JNK inhibitor (SP600125) (Supporting Information Fig. S2b, lower middle and bottom), suggesting that the inhibitory effect of hemin on HIF-1α activity might not result from the activation of PKCδ or JNK. Collectively, our results indicate that the inhibitory effect of hemin on HSP90 interaction with HIF-1α, which leads to 26S proteasome-mediated HIF-1α degradation, may not depend on the signaling pathways examined.
After heme synthesis in mitochondria, it would be subsequently incorporated as a prosthetic group into cellular proteins (i.e., hemoglobin and myoglobin).49 Heme incorporated into apoprotein is unlikely to affect HIF-1α activity because the side chains and metal of protoporphyrin would interact with amino acid residues of apoproteins. Therefore, free heme might have rare chance to encounter and inhibit HSP90. Pharmacokinetic studies showed that the plasma concentration of hemin was calculated to be ∼46.1 μM with the half-life time of 10.8 hr after intravenous administration of hemin arginate (3 mg/kg) in healthy men and porphyric patients.50 During pathological conditions such as ischemia reperfusion or malaria, the level of free heme may be raised up to 20 μM because of severe hemolysis.40 Considering the fact that heme in neutral solutions and in the presence of oxygen is quickly converted to hemin, the estimated half maximal inhibitory concentration (IC50) (∼10 μM) of hemin required for HIF-1α inhibition in our study seems to be physiologically relevant.
Our in vivo study confirmed a decrease in new vessel formation in a CAM assay, strengthening the concept that hemin inhibits HIF-1α activity and thereby diminishes angiogenesis. Moreover, the findings that hemin repressed DNA synthesis and invasion/migration of HCT116 cells support the concept that hemin may have inhibitory effects on angiogenesis, and tumor proliferation and invasion/migration presumably at least in part through HIF-1α inhibition. In summary, the results of our study demonstrate that hemin inhibits HIF-1α activity and HIF-1α-dependent functions (i.e., angiogenesis, cancer cell growth, invasion and migration), and which may result from a decrease in HIF-1α stability via the inhibition of HSP90 responsible for HIF-1α protection. Our finding showing hemin and other protoporphyrins as new HIF-1α inhibitors implies their potential applications not only for the prevention and/or treatment of diseases associated with angiogenesis but also for the inhibition of tumor growth and metastasis.