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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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.[7] 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,[12] 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.[13] 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.[11] 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.[15] 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cell culture

Human HCC cell lines, Hep3B and HepG2, and the human embryonic kidney cell line, HEK293 were obtained from ATCC (Rockville, MD, USA). The cells were cultured in DMEM (Wako, Tokyo, Japan), supplemented with 10% FCS and penicillin-streptomycin (Wako). HEK293 cells were transfected with expression vectors with Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA), and then stable clones were established by treatment with G418 (Sigma-Aldrich, St. Louis, MO, USA). For normoxic cell cultures, the cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and 21% O2. For hypoxic cell cultures, the cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and 1% O2 at a hypoxia workstation (Ruskinn Technology, Pencoed, UK). The cells were treated with a growth medium containing FeAC, LS081 (TimTec, Neward, DE, USA) and MG132 (Enzo Life Sciences, Farmingdale, NY, USA) as indicated for each of the experiments.

Iron uptake measurements by atomic absorption spectrophotometry

Hep3B and HepG2 cells were incubated with LS081 at concentrations of 0–30 μM in the presence of 10-μM FeAC for 1 h. The cells were removed from the plates with trypsin, washed extensively with HEPES-buffered saline, and enumerated; after lysis with 0.1% SDS, the iron content (fmol/cell) was measured by atomic absorption spectrophotometry with an Hitachi Z8100 Atomic Absorption Spectrophotometer (Hitachi, Tokyo, Japan).

Western blot analysis

Cell and tissue samples were lysed in Radio-Immunoprecipitation Assay (RIPA) Buffer, separated with polyacrylamide gel and electro-transferred to nitrocellulose membranes. After the membranes were blocked with 5% nonfat dry milk in PBST buffer (PBS containing 0.05% Tween-20), the membranes were probed with anti-HIF-1α antibody (Novus Biological, Littleton, CO, USA), anti-FLAG antibody (Sigma-Aldrich), anti-actin antibody (BD Biosciences, Franklin Lakes, NJ, USA), and anti-histone H1 antibody (Santa Cruz, Santa Cruz, CA, USA). They were then incubated in HRP-conjugated anti-mouse IgG secondary antibody (R&D Systems, Minneapolis, MN, USA). Antibody binding was then visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Real-time RT-PCR

Total RNA was isolated from cells with RNA purification system PureLink RNA Mini Kit (Invitrogen) and reverse transcribed with a high-capacity complementary DNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). Real-time RT-PCR was then performed using ABI 7300 system (Applied Biosystems) with TaqMan probes for human HIF-1α mRNA (Applied Biosystems). 18S ribosomal RNA was analyzed as an internal control, and the ratio of HIF-1α to 18S ribosomal RNA was calculated.

Immunohistocytometry

Hep3B cells were cultured under hypoxic conditions for 18 h and fixed with 4% paraformaldehyde solution. The cells were then incubated first with the anti-HIF-1α monoclonal antibody (Novus) and then with the Alexa Fluora 488 conjugated anti-mouse IgG antibody (Invitrogen); this was followed by nuclear staining with DAPI.

Cell growth assay

Hep3B and HepG2 cells were cultured both in normoxic and hypoxic conditions with LS081 and/or FeAC for 24 and 48 h. Cell numbers were enumerated by the MTT assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. Absorbance was measured at 490 nm with a Powerscan HT (DS Pharma, Osaka, Japan), and the ratio against the control at 24 h was calculated.

Expression vectors

To establish an expression vector that expresses N-terminal FLAG-tagged, wild-type HIF-1α (pCI-neo-3 × FLAG HIF-1α), complementary DNA of human HIF-1α was subcloned into pCI-neo-3 × FLAG, which was constructed by inserting oligonucleotides encoding the 3 × FLAG epitope into the pCI-neo mammalian expression vector (Promega). Additionally, the DNA sequences corresponding to the proline residues at amino acids 402 and 564 of HIF-1α were replaced by site-directed mutagenesis, and a vector expressing N-terminal FLAG-tagged HIF-1α, with proline residues replaced by alanine residues (pCI-neo-3 × FLAG HIF-1α P402/564A), was established.

Xenografts

Hep3B cells were inoculated into the subcutaneous tissue of the back of Balb/c nude mice (Clea Japan, Tokyo, Japan), and when the tumor volume reached approximately 400 mm3, daily intraperitoneal injections with LS081 (5 and 25 mg/kg/day) were started. The controls were treated with vehicle alone (30% polyethyleneglycol in PBS). Tumor diameters were measured with calipers, and the tumor volume was calculated using the formula: inline image. These mice were killed on day 7 of treatment with the collection of serum and tissues. All experimental procedures performed were approved by the animal experiments committee of Asahikawa Medical University (Hokkaido, Japan) based on the guidelines for the protection of animals.

Serum assays for hepatic function, transferrin, and iron content

Serum analysis for GOT, GPT, serum iron and UIBC was performed with the automatic serum analyzer LABOSPECT 008 (Hitachi). Assay reagents used were as follows: L-type wako GOT-J2 (Wako) for GOT, L-type wako GPT-J2 (Wako) for GPT, quick auto neo Fe (Sino Test, Tokyo, Japan) for serum iron, and quick auto neo UIBC (Sino Test) for UIBC. Analysis for NTBI was performed using HPLC, with a minor modification as previously reported by our laboratory.[16]

Mass spectrophotometry

Liquid chromatography-mass spectrometry was performed using NanoFrontier eLD (Hitachi). LS081 and/or FeAC were first dissolved in ultrapure water and then further diluted in ultrapure methanol. Infusion analysis for these samples was accomplished with a micro syringe at 3 μL/min flow speed with negative electrospray ionization. The spectra of samples were compared with the virtual spectra simulated by system software.

Statistics

The Student paired t-test was used. < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

LS081 and iron inhibits HIF-1α protein but not mRNA expression in HCC cell lines

The iron facilitating activity of LS081 in Hep3B and HepG2 cells was verified by atomic absorption spectrophotometry. Figure 1(a) shows that in the presence of 10-μM FeAC, iron uptake was facilitated by LS081 treatment at a dose of 5–30 μM in both cell lines. To determine whether iron facilitation by LS081 inhibits HIF-1α expression in HCC cells, Hep3B and HepG2 cells were cultured both under normoxic and hypoxic conditions with FeAC and/or LS081. The data showed that a combination of LS081 and FeAC significantly inhibited HIF-1α protein expression (Fig. 1b). Nuclear HIF-1α protein also disappeared when FeAC and LS081 were added to the culture medium under hypoxic conditions (Fig. 1c). The presence of HIF-1α protein as detected by immunohistocytometry also showed that the expression of HIF-1α protein both in the cytosol and nucleus was decreased when the cells were treated with LS081 and FeAC (Fig. 1d). In contrast, HIF-1α mRNA expression was not significantly affected by FeAC and/or LS081 under normoxic or hypoxic conditions (Fig. 1e). These results indicate that LS081-dependent iron facilitation post-transcriptionally inhibits HIF-1α expression.

image

Figure 1. (a) Dose response curve of LS081 on iron facilitation in Hep3B and HepG2 cells analyzed by atomic absorption spectrometry. The mean ± SD of three independent experiments are shown with each point in triplicate. (b) Western blot analysis for HIF-1α and actin (loading control) in whole cell lysates of Hep3B and HepG2 cells treated with LS081 and/or FeAC both under normoxia and hypoxia for 24 h. (c) Western blot analysis for HIF-1α and histone H1 (loading control) in nuclear lysates of Hep3B and HepG2 cells treated with LS081 and/or FeAC under hypoxic conditions for 24 h. (d) Immunohistocytometry detection of HIF-1α in Hep3B cells treated with no addition (control), LS081, FeAC, or LS081 and FeAC under hypoxic conditions for 24 h. Shown is a single optical slice through the level of the nuclei. (e) Real-time RT-PCR analysis for HIF-1α mRNA in Hep3B (left) and HepG2 (right) cells treated with LS081 or FeAC under both normoxic and hypoxic conditions for 24 h. The columns represent the mean ± SD of three independent experiments. Cont., control; FeAC, ferric ammonium citrate; HIF, hypoxia inducible factor.

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Iron facilitation by LS081 enhances HIF-1α prolyl-hydroxylation

We hypothesized that the reduction in HIF-1α protein expression was caused by increased HIF-prolyl-hydroxylases activity with subsequent increased ubiquination and HIF-1α proteosomal degradation. Figure 2(a) shows that in the presence of the proteasome inhibitor MG132, HIF-1α protein reduction was no longer observed. To determine whether the iron-facilitating activity of LS081 decreases HIF-1α protein by enhancing HIF-prolyl-hydroxylation, we established HEK293 cell lines that stably express FLAG-tagged, wild-type HIF-1α (293 HIF-1α WT) and FLAG-tagged HIF-1α with proline residues that were replaced with alanine (293 HIF-1α P402/564A) (Fig. 2b). These cell lines were then treated with LS081 and FeAC for 24 h. As seen in Figure 2(c), culture in the presence of the combination of LS081 and FeAC decreased wild-type HIF-1α protein but had no effect on HIF-1α P402/564A expression. Interestingly, the mutated-HIF-1α that lost proline residues did not decrease. These data are consistent with iron facilitation mediated by LS081, thus increasing PHD activity and prolyl-hydroxylation of HIF-1α.

image

Figure 2. (a) Western blot analysis for HIF-1α and actin (loading control) in Hep3B cell treated with 10-μM LS081, 10-μM FeAC and 10-μM MG132 under hypoxic conditions. (b) A schematic illustration of the expression vectors used in this study showing that with the loss of the proline residues FLAG-HIF-1α, P402/564A could not be hydroxylated by PHD. (c) Left panel: Western blot analysis for FLAG-tagged HIF-1α and actin (loading control) in HEK293 cells treated with LS081/FeAC. Right panel: The ratio of FLAG-HIF-1α to the actin loading control by densitometric analysis. The columns represent the mean ± SD of three independent experiments. *P < 0.05, compared with control (HIF-1α WT without treatment). FeAC, ferric ammonium citrate; HIF, hypoxia inducible factor; PHD, prolyl-hydroxylases; WT, wild type.

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Treatment with LS081 and iron inhibits cell growth in HCC

Because HIF-1α transcriptional activity upregulates many kinds of growth factors and anti-apoptotic factors, inhibition of HIF-1α by LS081 might cause both growth arrest and apoptosis. To investigate the effect of LS081 on cell growth, Hep3B and HepG2 cells were cultured with LS081 and/or FeAC in normoxia and hypoxia for 24 and 48 h, and the cell numbers were enumerated (Fig. 3). Growth of Hep3B cells was not affected by LS081, slightly inhibited by FeAC alone, and markedly inhibited by the combination of LS081 and FeAC both under normoxia and hypoxia (Fig. 3a). The combination of LS081 and FeAC inhibited cell growth only under hypoxic conditions in HepG2 cells (Fig. 3b). These observations correspond to the results that HIF-1α protein expression was detectable even under normoxia in Hep3B cells while HIF-1α protein was detectable only in hypoxia in HepG2 cells (Fig. 1a). As the fold changes of the combination of LS081 and FeAC were lower than the controls and time dependently reduced, this treatment may have induced both growth arrest and apoptosis. The data further suggest that LS081 itself does not have a cytotoxic effect on cell growth while a combination of LS081 and iron inhibits cell growth corresponding to HIF-1α expression.

image

Figure 3. MTT assay in (a) Hep3B and (b) HepG2 cells cultured with 10-μM LS081, 10-μM FeAC, or 10-μM LS081 and FeAC under normoxic and hypoxic conditions for 24 and 48 h. Left panel: normoxia, Right panel: hypoxia. The columns represent the mean ± SD of three independent experiments. *P < 0.05, compared with control. Cont., control; FeAC, ferric ammonium citrate.

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LS081 treatment inhibits tumor growth of Hep3B xenograft in nude mice

Hep3B xenografts were initiated, and when the tumors reached about 400 mm3, LS081 was given intraperitoneally in the absence of any supplemental iron. Growth curves of the xenografts show that LS081 significantly inhibited tumor growth (Fig. 4a). Western blot analysis after treatment showed that LS081 reduced HIF-1α expression in the tumor tissue (Fig. 4b). LS081 treatment caused hemorrhagic necrosis in the tumor tissue, indicating that the treatment induced destruction of intratumoral vessels (Fig. 4c). LS081-treated mice showed neither significant body weight change, hepatic damage nor aberrant iron overload in histological analysis (data not shown). Furthermore, the levels of serum hepatic enzymes were not affected by LS081 treatment (Fig. 4d), which is consistent with a lack of hepatic damage by LS081. These results suggest that LS081 might play a role as an anticancer agent without hepatic toxicity, despite being active against hepatomas.

image

Figure 4. (a) The change in tumor volume during the treatments. Each point represents the mean ± SD for four mice. (b) Western blot analysis for HIF-1α and actin (loading control) in the tumor lysates. (c) H&E staining of tumor tissue after the treatment (original magnification × 100). (d) The levels of serum hepatic enzymes after the treatment. The columns represent the mean ± SD for four mice. Cont., control; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvate transaminase; HIF, hypoxia inducible factor.

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LS081 mainly affects NTBI

In the animals bearing the xenografts, LS081 treatment did not affect either serum iron levels (i.e. primarily ferri-transferrin) or UIBC (i.e. apo or unsaturated transferrin), while LS081 reduced NTBI, which exists as a minor component of body iron (Fig. 5). These results indicate that LS081 may primarily increase serum NTBI into tissues without affecting transferrin bound iron.

image

Figure 5. Levels of serum iron, UIBC and NTBI in Hep3B xenograft models. The columns represent the mean ± SD for four mice. *< 0.05, compared with control. NTBI, non-transferrin-bound iron; UIBC, unsaturated iron binding capacity.

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LS081 directly binds to free iron

Based on serum analysis, we speculated that LS081 binds to free iron and transports the chelated iron into the cell, but it has a lesser effect on uptake of iron from transferrin. LS081 chelates were analyzed by mass spectrometry of in vitro mixtures of LS081 and FeAC (Fig. 6). The spectrum of LS081 shows LS081 to have predominant isotopes with a mass of 363.0725 and 365.0713 daltons (Fig. 6a). However, in the mixture of LS081 and FeAC, the spectrum shifted to a larger mass of about 781 daltons (Fig. 6b). The observed spectra were quite similar to that predicted by computer simulation and are consistent with two molecules of LS081 binding one molecule of iron.

image

Figure 6. (a) Left panel: The spectrum of LS081 (overall view). Right panel: Predicted spectrum pattern by computer simulation (upper) and enlarged view of left panel (lower). (b) Left panel: The spectrum of the mixture of LS081 and FeAC (overall view). Right panel: Predicted spectrum pattern by computer simulation (upper) and enlarged view of left panel (lower).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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.[17] 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.[20] 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.[16] 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,[15] 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.[15] The side-chain composition of LS081 differs from that of the hydrazone derivatives that inhibit iron uptake.[15] 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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Grant support: We received grant support from the Japanese Ministry of Education, Culture, Sports, Science and Technology and the Japanese Ministry of Health, Welfare and Labour. The authors would like to thank Ms Kotoe Shibusa, Ms Akemi Kita and Mr.Hiroaki Konishi for their technical assistance in our experiments.

Abbreviations
FeAC

ferric ammonium citrate

GOT

glutamic oxaloacetic transaminase

GPT

glutamic pyruvate transaminase

HCC

hepatocellular carcinoma

HIF

hypoxia inducible factor

NTBI

non-transferrin-bound iron

PHD

prolyl-hydroxylases

UIBC

unsaturated iron binding capacity

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References