An activator of PHD2, KRH102140, decreases angiogenesis via inhibition of HIF-1α

cell biochemistry and function

Cell Biochem Funct 2011; 29: 126–134.

Published online 1 February 2011 in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/cbf.1732

An activator of PHD2, KRH102140, decreases angiogenesisvia inhibition of HIF-1a

Manoj Nepal 1, Young-Dae Gong 2, Young Ran Park 1 and Yunjo Soh 1*

1Department of Dental Pharmacology, School of Dentistry and Institute of Oral Bioscience, Brain Korea 21 Project, Chonbuk NationalUniversity, Jeon-Ju, Republic of Korea2Innovative Drug Library Research Center, Science College, Dongguk Univeristy, Jung-gu, Seoul, Republic of Korea

Hypoxia-inducible transcription factors (HIFs) play a pivotal role in the response of cells to hypoxia. HIFs are dimers of an oxygen-sensitivea-subunit (HIF-1a or HIF-2a), and a constitutively expressed b-subunit. In normoxia, HIF-1a is destabilized by post-translationalhydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. Prolyl hydroxylation leads to von Hippel–Lindauprotein-dependent ubiquitination and rapid degradation of HIF-1a. We previously reported that KRH102053, an activator of PHD2, rapidlydecreased HIF-1a and eventually inhibited angiogenesis. Here, we report a potent activator of PHD2, KRH102140, which has a structuresimilar to KRH102053. KRH102140 more efficiently suppressed HIF-1a than KRH102053 in human osteosarcoma cells under hypoxia.Furthermore, KRH102140 decreased the mRNA levels of HIF-regulated downstream target genes associated with angiogenesis and energymetabolism such as vascular endothelial growth factor, adrenomedullin, Glut1, aldolase A, enolase 1 and monocarboxylate transporter 4.KRH102140 also inhibited tube formation in human umbilical vein endothelium cells. The results suggest that KRH102140 has potentialtherapeutic effects in alleviating various diseases associated with HIFs. Copyright # 2011 John Wiley & Sons, Ltd.

key words — hypoxia-inducible factors; KRH102140 [(2-Fluorobenzyl)-(2-methyl-2-phenyl-2H-chromen-6-yl_-amine]; prolyl hydro-xylation; von Hippel–Lindau protein; vascular endothelial growth factor; angiogenesis

INTRODUCTION

Hypoxia-inducible factor (HIF)-1 is a heterodimeric, basichelix–loop–helix, PAS-domain transcription factor consist-ing of HIF-1a and HIF-1bsubunits.1 HIF-1 is regulatedby O2-dependent prolyl hydroxylation in the ODD region,which is required for ubiquitinylation by E3 ubiquitin–protein ligases, including von Hippel–Lindau tumour-suppressor protein (VHL), and subsequent proteasomaldegradation.2,3 Under low oxygen, hydroxylation does notoccur because of the low oxygen affinity of prolylhydroxylase preventing the interaction of HIF-1a withVHL.4 As a result, HIF-1a is stabilized and promotes theexpression of target genes. HIF-1a expression increases inresponse to hypoxia in tissues, and functional HIF-1aupregulates target genes by binding to hypoxia-responsiveelements in gene regulatory regions. HIF-1 is considered acentral regulator of the adaptation responses of cancer cellsto hypoxia and is responsible for gene expression thatinfluences angiogenesis, and modulates glucose metab-olism, cell proliferation, survival and invasion in solidtumours during tumour progression and metastasis.5 Several

* Correspondence to: Y. Soh, Department of Dental Pharmacology, Schoolof Dentistry, Chonbuk National University, Duck-Jin Dong, Duck-Jin Ku,Jeon-Ju 561-756, Republic of Korea. E-mail: [email protected]

Copyright # 2011 John Wiley & Sons, Ltd.

clinical studies indicate that HIF-1a protein expressioncorrelates with advance disease stage, metastasis, treatmentresistance, and poor prognosis in cancer patients.6

HIF-1 is a major molecular target for hypoxia-selectiveanticancer drug discovery.7 In animal models, HIF-1aoverexpression is associated with increased tumour growth,vascularization and metastasis, while loss-of-function hasthe opposite effect.8 When HIF-1a inhibition is combinedwith chemotherapy and radiation, efficient treatment wasobserved in preclinical models.9 Thus, small molecule HIF-1a inhibitors are attractive lead drugs for suppressingtumour growth and enhancing chemotherapy and radiationby downregulating hypoxia-inducible gene expression.Recently, considerable effort has been directed to thediscovery of HIF-1 inhibitors from chemical libraries andnatural products. These inhibitors reportedly regulate theHIF-1 signalling pathway through a variety of molecularmechanisms, including transcriptional regulation, folding,stabilization, nuclear translocation, degradation and trans-activation.

In a previous study, we demonstrated the potential ofsmall molecule KRH102053, which targets HIF-1a inhypoxic conditions in human osteosarcoma (HOS), HepG2hepatoma cells and rat PC12 phaeochromocytoma cells.10 Inaddition to this HIF-1a inhibitor, we identified anothersmall molecule, KRH102140, with a structure similar to

Received 24 September 2010Revised 18 December 2010

Accepted 18 December 2010

Table 1. Primer sequences and conditions for RT-PCR

Targetgenes

Incubationtime in

hypoxia(h)� Primers (forward, reverse)

AnnealingTm

(8C)PCR

cycles

HIF-1a 4 5’-gctggccccagccgctggag-3’ 53 305’-gagtgcagggtcagcactac-3’

VEGF 8 5’-acccatggcagaaggaggag-3’ 55 305’-acgcgagtctgtgtttttgc-3’

MCT4 8 5’-cctgggcttcattgacatct-3’ 53 305’-agcaaaatcagggaggaggt-3’

ADM 24 5’-ggatgccgcccgcatccgag-3’ 60 305’-gacaccagagtccgacccgg-3’

Aldolase A 24 5’-tgtgggcatcaaggtagaca-3’ 55 255’-aaggtgatcccagtgacagc-3’

Enolase 1 24 5’-tgatcgagatggatggaaca-3’ 55 255’-atgccgatgaccaccttatc-3’

Glut1 24 5’-gcaacggcttagacttcgac-3’ 55 255’-ccaaatcggcatcttctcat-3’

b-actin 4, 8, 24 5’-agaaaatctggcaccacacc-3’ 55 255’-ccatctcttgctcgaagtcc-3’

�Incubation time in hypoxia was determined according to the expressionpattern of each gene.

phd activator decreases angiogenesis 127

KRH102053, which efficiently inhibited the elevated proteinand mRNA levels of hypoxia-regulated genes in in vitroexperiments (Figure 1). We suggest that KRH102140 mayhave therapeutic potential for the treatment of variousdiseases associated with elevated levels of hypoxia.

MATERIALS AND METHODS

Cell culture and treatment

HOS and HepG2 cells were grown in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% heatinactivated foetal bovine serum (FBS), penicillin(100 U ml�1) and streptomycin (100 mg ml�1). All cellswere maintained in a humidified atmosphere containing 5%CO2 at 378C. For incubation of cells under hypoxic states,cells were treated with serum-free media for 18 h andincubated in an airtight chamber (Thermo Forma Co.,Marietta, OH, USA), flushed with a gas mixture containing1% O2, 5% CO2 and 94% N2 for indicated times at 378C.

MTT assay

Cells (5� 103) were seeded onto 96-well plates withmedium supplemented with 10% FBS and incubated for24 h. Cells were exposed to various concentrations ofKRH102140 for an additional 24 h (HOS and HepG2 cells)or 48 h (HUVECs). Cells were washed twice withphosphate-buffered saline (PBS) and dead cells wereassayed by treating with 100 mg ml�1 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]for 2 h at 378C. After washing with PBS, the resultingpurple formazan was dissolved in 200 ml of dimethylsulphoxide and absorbance read at 540 nm to quantify celldeath rates.

Reverse transcription-PCR

Total RNAwas isolated from cultured cells using Trizol, andcDNA was synthesized with SuperScript II reversetranscriptase according to the manufacturer’s protocol.Hypoxia time, PCR conditions and primer sequences arepresented in Table 1. After 1 min at 958C, PCR was usingTaq polymerase for various cycles (30 s at 948C, 1 min atannealing temperature and 2 min at 728C). Reactionproducts (10 ml) were separated on 0�8% agarose gels andstained with ethidium bromide. DNA band intensity wasanalysed by densitometry using a Phosphoimager andQuantity One software (Version 4�3�1) (Bio-Rad, Hercules,CA, USA).

Cytoplasmic and nuclear extract preparation

Cells were harvested and resolved in lysis buffer (20 mMTris-HCl pH 7�5, 137 mM NaCl, 10% glycerol (v v�1), 1%Triton X-100 (v v�1), 1 mM phenylmethylsulphonylfluoride,1 mM Na3VO4 and 1� protease inhibitor cocktail). Aftercentrifugation at 16 000� g for 15 min, supernatants wereused as cytoplasmic extracts. To detect HIF-1a, cells were


resuspended in 150 ml of buffer A (10 mM HEPES pH 7�9,1�5 mM MgCl2, 10 mM KCl, 0�5 mM dithiotreitol, 0�5 mMphenylmethylsulphonylfluoride, 0�4% Nonidet P-40 (v v�1)and 1� protease inhibitor cocktail) for 20 min on ice andcentrifuged at 2300� g for 5 min, as described previously.11

Pellets were dissolved in 100 ml of buffer C (20 mMHEPES pH 7�9, 420 mM NaCl, 1�5 mM MgCl2, 0�2 mMEDTA, 0�5 mM dithiotreitol, 0�5 mM phenylmethylsulpho-nylfluoride and 1� protease inhibitor cocktail) for 30 minon ice. After centrifugation at 16 000� g for 15 min,supernatants were used as nuclear extracts for HIF-1aimmunoblot.

Western blot analysis

The concentration of glucose transporter proteins wasdetermined from cell extracts by western blot. Cell extractswere prepared using standard techniques. Briefly, cells werewashed twice in cold PBS (pH 7�4) and lysed for 30 min at48C in 20 mM Tris-HCl (pH 7�4), 2 mM EDTA, 1 mMsodium orthovanadate, 0�1 mM PMSF and a cocktail ofprotease inhibitors. Lysates were centrifuged at 15 000� gfor 30 min at 48C, and supernatants were separated intoaliquots and stored at �808C. Aliquots of cellular fractions(approximately 30 mg) were run on SDS-PAGE gels underdenaturing and reducing conditions, and transblotted intoPVDF membranes. Membranes were blocked in 5% non-fatdried milk for 1 h and incubated overnight with primaryrabbit polyclonal IgG antibody mAbs against HIF-1a, HIF-1b, VEGF and actin, followed by 1 h with goat anti-rabbitIgG HRP-conjugated secondary antibody. Immunoreactivitywas detected using an enhanced chemiluminescencedetection kit (Pierce), and blot densities were analysedusing a luminescent image analyser (LAS-3000, Fuji Film,Japan).


Figure 1. Chemical structure of KRH102140 [(2-Fluorobenzyl)-(2-methyl-2-phenyl-2H-chromen-6-yl_-amine]

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Tube formation and proliferation assays

For reconstitution of basement membranes, growth factor-reduced Matrigel was added to 48-well tissue culture plates(150 mg per well) on ice. Plates were incubated for 1 h toallow Matrigel to solidify. HUVECs were trypsinized,resuspended in medium containing the indicated concen-trations of KRH102140 and added on top of thereconstructed basement membrane (3� 104 cells per well).Cells were incubated in a hypoxic chamber for 40 h toinduce growth factors such as VEGF and to form capillary-like tubular structures.

Statistical analysis

All the experimental data shown are expressed as mean-s� S.E.M. and all experiments were repeated at least three

Figure 2. Effect of KRH102140 on protein and mRNA level of HIF-1a in HOS cKRH102140 for 4 h under hypoxia. The protein levels of HIF-1a and HIF-1b were dhistogram represents the level of HIF-1a protein in different conditions compare(p< 0�05) compared with the control (#). Each histogram represents the mean�S.Eby RT-PCR after treating with 2 or 20 mM KRH102140. (C) HOS cells were treatrespectively. The protein levels of HIF-1a and HIF-1b were determined by imm


times, unless otherwise indicated. Statistical analyses wereperformed by Dunnett’s multiple comparison test usingSPSS ver. 12�0 software and P-values less than 0�05 wereconsidered significant.

RESULTS

KRH102140 inhibits HIF-1a protein without affectingits mRNA level

Under normoxic conditions, HIF-1a is degraded byubiquition-dependent proteasomes, while under hypoxiait is stabilized.12 To investigate whether KRH102140[(2-Fluorobenzyl)-(2-methyl-2-phenyl-2H-chromen-6-yl_-amine] could affect HIF-1a protein and mRNA levels inHOS and HepG2 cells under hypoxic conditions, weanalysed HIF-1a protein and mRNA levels by immunoblot-ting and RT-PCR. To ensure that downregulation of HIF-1alevels by KRH102140 was not because of non-specificcytotoxicity on the target cells, we performed MTT assays todetermine cell death rates at different KRH102140concentrations over 24 h. Although KRH102140 did notexhibit cytotoxic effects up to 100 mM (data not shown), weused a maximum of 20 mM for all experiments, and foundefficient reductions in HIF-1a protein level at thisconcentration. An IC50 of KRH102140 in inhibition ofHIF-1a was estimated to 12�2 mM, which was lower thanthat of KRH102053 with 17�1 mM (Figure 2A). In responseto hypoxia, the level of HIF-1a increased sharply within 4 h,compared to HIF-1b, which was used as a negative control.

ells. (A) Exponentially growing HOS cells were treated with 0�2, 2 or 20 mMetermined by immunoblot using anti-HIF-1a or anti-HIF-1b antibodies. Thed to hypoxia (as % control). Asterisk (�) indicates a significant difference.M. of three separate determinations. (B) HIF-1a mRNA level was analysed

ed with KRH102140 or KRH102053 (0�2 or 20 mM) for 4 h under hypoxia,unoblot using anti-HIF-1a or anti-HIF-1b antibodies, respectively



Under these conditions, KRH102140 significantly reducedHIF-1a levels in a concentration-dependent manner.The effect of KRH102140 was specific to HIF-1a, andHIF-1b remained unchanged. Furthermore, the effect ofKRH102140 in HIF-1a mRNA level, normalized to b-actinwas not observed even with an adequate concentration andtime (Figure 2B). The results suggested that KRH102140could be a strong inhibitor of HIF-1a. Previously, wereported that KRH102053 significantly activated PHD2, anupstream regulator of HIF-1a, by 26�4%. However,KRH102140 increased PHD2 activity by 15�4% in HPLCassay system (data not shown). To compare the activities ofKRH102140 with KRH102053 in inhibition of HIF-1a, bothchemicals with 0�2 and 20 mM were treated and the levels ofHIF-1a were measured by western blot analysis (Figure 2C).The result showed that KRH102140 inhibited HIF-1a morepotently than KRH102053.

KRH102140 stimulates interaction between VHL andHIF-1a

To investigate whether KRH102140 could modulateinteraction between HIF-1a and VHL in HOS cells, wedetermined the level of VHL-HIF-1a interaction byimmunoprecipitation with anti-HIF-1a antibody followedby immunoblot analysis with anti-VHL antibody using HOScells treated with KRH102140 (Figure 3). Under hypoxia,

Figure 3. The level of VHL protein interacting with HIF-1a was analysedafter immunoprecipitation of HIF-1a in the absence or presence ofKRH102140 under hypoxia. The histogram represents the level of VHLprotein in different conditions compared to hypoxia (as % control). Asterisk(�) indicates a significant difference (p< 0�05) compared with the control (#)


the level of VHL bound to HIF-1a was reduced in HOS cellscompared to the normoxia control group. Treatment with20 mM KRH102140 increased the binding of VHL toendogenous HIF-1a. This result suggested that KRH102140enhanced VHL interaction in hypoxia, possibly leading toincreased prolyl hydroxylase domain 2 (PHD2) activity.

Downregulation of angiogenesis by KRH102140

Hypoxia can occur in both physiological and pathophysio-logical conditions, and HIF1a is important in both cases.Successful foetal development is highly dependent onestablishing an efficient haematopoietic and vascularsystem, and both are controlled by HIF-1a targets such aserythropoietin5 vascular endothelial growth factor(VEGF).13 VEGF is also a downstream target and ismarkedly induced by HIF-1a.14 So, we evaluated whetherKRH102140 affected the VEGF expression and angiogen-esis regulated by HIF-1a. Under hypoxic conditions, theVEGF protein level was significantly increased by more thantwo-fold compared to actin. Upon treatment withKRH102140, the elevation in VEGF protein level wassignificantly inhibited in a concentration-dependent manner(Figure 4A). Furthermore, at 20 mM KRH102140, therelative expression of VEGF was more strongly inhibitedthan 2 mM-treated and non-treated cells. We also analysedthe mRNA level by RT-PCR. As expected, mRNAexpression was also inhibited by KRH102140, dependenton concentration. KRH102140 did not affect the level of theb-actin negative control (Figure 4B). These results demon-strated that KRH102140 is a potent inhibitor of angiogenesismediated by hypoxia.

KRH102140 inhibits hypoxia-induced in vitro tubeformation in endothelial cells

Tube formation is widely used as an in vitro assay forangiogenesis and hypoxia is reported to induce tubeformation in human umbilical vein endothelium cells(HUVECs).15 We examined the effect of KRH102140 onthe angiogenesis rate by measuring the degree of tubeformation in HUVEC using matrigel. Previous studiessuggested that VEGF or fibroblast growth factors alone or incombination with serum induce tube formation.15,16 Weused hypoxia as the sole inducer to evaluate effects ofKRH102140 on tube formation through inhibition of HIF-1a. To ensure that tube formation was not inhibited byKRH102140 cytotoxic effects, we tested for cell viability inHUVECs (Figure 5A). No cytotoxicity was observed, evenafter treatment with 100 mM of KRH102140. We used amaximum of 20 mM KRH102140 for the rest of the assays.Under normoxic conditions, tube formation was clearlyobserved starting at 2 h, and the formation of microvesselswas neither inhibited nor stimulated by treatment withKRH102140 (Figure 5B, upper panel). Conversely, underhypoxic conditions, HUVECs displayed greater motility anddifferentiated into a network structure that was betterdefined than the normoxic control groups (Figure 5B,


Figure 4. Concentration-dependent suppression of VEGF protein and mRNA by KRH102140. (A) HOS cells were treated with KRH102140 at 2 or 20 mM for8 h under hypoxia. VEGF protein level was determined by western blotting. The same amount of protein was loaded in each lane, as demonstrated by b-actin.The histogram represents the level of VEGF protein in different conditions, compared to hypoxia as a percentage of the control. Asterisk (�) indicates asignificant difference ( p< 0�05) compared with the control (#). (B) VEGF mRNA in HOS cells evaluated by RT-PCR and compared to b-actin

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bottom first panel). KRH102140 treatment significantlydecreased the rate of tube formation in the hypoxia group ina concentration-dependant manner (Figure 5B, lower, sec-ond and third panel), as visualized by the reduced width andlength of the endothelial network-like structure. Wequantified tube formation by counting the number of visiblevessels. In response to hypoxia, microvessels increased bymore than two-fold compared to the correspondingnormoxic groups, while in normoxia, formation of micro-vessels was unaffected, even after treatment with 20 mMKRH102140 for 18 h. The number of microvessels wasdistinctly reduced by KRH 102140 treatment in hypoxicconditions, with significant differences and concentrationdependence (Figure 5C). From this finding we hypothesizedthat the suppression of microvessel formation byKRH102140 was mediated through the HIF-1a.

Downregulation of adrenomedullin mRNA byKRH102140

Adrenomedullin (ADM) is a secreted protein that is avasodilatory agent with proangiogenic effects. It stimulatesproliferation of HUVECs and blood vessel development 17

and upregulated by hypoxia in several cancer cell lines,through three HIF-1a-binding sites.18 Expression of ADMcorrelates with vascularity in renal cells, and in breast andendometrial carcinomas, and has been demonstrated to


stimulate tumour angiogenesis.19 We evaluated whetherKRH102140 also downregulated ADM mRNA by analysingmRNA levels by RT-PCR. As shown in Figure 6A, the ADMmRNA level was significantly raised in hypoxic conditions.Interestingly, this elevation in mRNA under hypoxia wasefficiently downregulated by KRH102140 treatment, andwas significantly different from corresponding untreatedcells that were used as negative controls. The mRNA levelwas normalized to b-actin, which was unchanged, even aftertreatment with the maximum concentration of KRH102140.Figure 6B shows the increased mRNA level of ADM as ahistogram. Under hypoxic conditions, mRNA expressionincreased significantly compared to normoxic conditions.This increased in expression in mRNA was clearly down-regulated upon treatment with KRH102140 in a concen-tration-dependent manner. This result suggested thatKRH102140 had an inhibitory effect ADM mRNA incancer cells by downregulating the level of HIF-1a. Fromthis finding we concluded that KRH102140 had aninhibitory effect on angiogenesis.

Inhibition of mRNA of glucose-metabolizing genes

Hypoxia upregulates several genes involved in cellularenergy metabolism to adapt to changes in oxygenconcentration.20 Aldolase A, aldolase C, enolase 1, glut 1,glut-421 and monocarboxylate transporter-4 (MCT4) are


Figure 5. Concentration-dependent effect of KRH102140 on tube formation in HUVECs under normoxia and hypoxia. Culture 48-well plates were coatedwith 200 ml of Matrigel per well, for 12 h. HUVECs (3� 104) were grown overnight and exposed to 2 or 20 mM of KRH102140 under normoxia and hypoxia for18 h. (A) Cell viability of HUVECs treated with KRH102140 by MTT assay. (B) Photographs of tube formation (�40). (C) Histogram represents themean� S.E.M. number of tubes per microscopic area (�100) from separate experiments. Asterisk (�) indicates a significant difference (p< 0�05) comparedwith the control (#). Each column represents the mean�S.E.M. of three or four independent experiments


pivotal genes involved in glucose metabolism, and aremediated in part by the transcriptional complex of HIF-1a.Therefore, we examined the effect of KRH102140 on themRNA level of these genes in HOS cells in hypoxic

Figure 6. Concentration-dependent effect of KRH102140 on ADM mRNA. (A) Efor 24 h under hypoxia. ADM mRNA in HOS cells was evaluated by RT-PCR and cb-actin. Asterisk (�) indicates a significant difference (p< 0�05) compared with


conditions. We analysed the mRNA levels of glut1, aldolaseA, enolase 1 and MCT4 by RT-PCR in both hypoxic andnormoxic conditions (Figure 7A). Interestingly, underhypoxia, mRNA expression was significantly increased

xponentially growing HOS cells were treated with 2 or 20 mM KRH102140ompared to b-actin. (B) The histogram represents ADM mRNA compared tothe control (#)


Figure 7. Concentration-dependent effect of KRH102140 on expression of genes involved in glucose metabolism under hypoxia. (A) HOS cells weremaintained for various times (Table 1) under hypoxia in the presence and absence of 2 or 20 mM KRH102140. RT-PCR determined the mRNA level of glut1,aldolase A, enolase 1 and MCT4 with b-actin as a negative control. (B) The histogram depicts the mRNA of target genes compared to b-actin. Asterisk (�)indicates a significant difference ( p< 0�05) compared with the control (#). Each column represents the mean�S.E.M. of three or four separate experiments

Copyright # 2011 John Wiley & Sons, Ltd. Cell Biochem Funct 2011; 29: 126–134.

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compared to normoxic conditions. Furthermore, upontreatment with KRH102140, mRNA levels of these geneswere significantly inhibited in a concentration-dependentmanner. KRH102140 at 20 mM almost abolished expression.The effect of KRH102140 on the mRNA of hypoxiainducible genes is shown in the histogram in Figure 7B. Therelative mRNA expression was significantly greater inhypoxic conditions compared to the corresponding controlgroup, suggesting that KRH102140 had a potentialinhibitory effect that blocked the energy level and glucosemetabolism modulated by hypoxia.

DUSCUSSION

Drugs that can modulate hypoxia will clearly be useful intreating various hypoxia-related diseases. Several smallmolecules have been reported to inhibit HIF-1a activity bydirectly affecting diverse molecular pathways. We pre-viously studied the effect of the small molecule KRH102053in HOS, HepG2 and PC12 cell lines, and found that itactivates or inhibits HIF-PHD activities. KRH102053efficiently decreased the protein level of HIF-1a, as wellas the mRNA levels of HIF-regulated downstream targetgenes such as VEGF, aldolase A, enolase 1 and MCT4 in cellmodels. Here, we studied the effect of KRH102140, whichmimics the structure of KRH102053, in HOS and HepG2cells. KRH102140 was selected among 2000 analogues of 6-amino-2,2-dimethyl-3,4,6-trisubstituted 2H-1-benzopyrenwith a significant antioxidant activity. KRH102140 effi-ciently activated PHD activity and inhibited HIF-1a in aconcentration-dependent manner, although not by affectingits mRNA level. In addition, KRH102140 also inhibited theincrease in the protein level of VEGF, and the mRNA levelsof VEGF, ADM, glut1, aldolase A, enolase1 and MCT4under hypoxic conditions.

Of the three members of the HIF-PHD genes, PHD2 isthought to be more important than other PHD isoenzymesfor controlling HIF-1a levels during normoxia.12,22 Therelative expression of PHD2 is higher than the expression ofother PHD isoenzymes in human osteosarcoma U-2 OS,hepatocellular carcinoma and Hep3B cell lines.23 Severalstudies suggest that PHD2, but not PHD1 and PHD3, is amajor negative regulator of vascular growth.24 Moreover,PHD2 enzymes are expressed in many proliferating tumourcells.25 Under hypoxia, PHD2 can be induced to levels ashigh as PHD3.23 In a recent study, PHD3 and PHD2 werefound to be efficiently hypoxia-induced in human glio-blastomas.10,26 In our previous study, we showed that PHD2had a major function in HOS and HepG2 cells. Higher levelsof PHD2 transcripts were observed than PHD1 and PHD3under normoxic conditions. In this study, we hypothesizedthat KRH102140 is likely to affect other PHD2 isoforms,based on their similar structures and kinetic parameters.22,23

From the results and hypotheses presented here, the use ofPHD2 as a target enzyme for identifying activators of PHDisoenzymes might be justified, although inclusion of otherPHD enzymes would be highly desirable.


HIF-1a expression promotes cell survival in a hypoxicmicroenvironment by assisting the expression of genesinvolved in the regulation of angiogenesis, metabolicadaptation, invasion and metastastis.27,28 In addition, over-expression of HIF-1a ultimately leads to cancer pro-gression.5 In this study, downregulation of the elevatedlevels of HIF-1a by KRH102140 positively correlated withdecreased levels of downstream HIF-1a genes. Thus, wehave demonstrated that expression of VEGF under hypoxia,which is a hallmark of angiogenesis, was inhibited bytreatment with KRH102140. Moreover, KRH102140 alsoinhibited tube formation in HUVECs, and hypoxiastimulates tube formation in HUVECs.29 Another importantangiogenic factor is ADM, which is a novel growth factor forendothelial cells, stimulates the growth of HUVECs andpromotes angiogenesis in vivo.17 Furthermore, ADMstimulates migration, proliferation, invasion and theformation of capillary tubes in HUVECs.30 KRH102140potently downregulated the mRNA level of ADM in aconcentration-dependent manner. The potential ofKRH102140 for inhibiting transcription of ADM andVEGF, and capillary tube formation in HUVECs providesstrong evidence of its antiangiogenic capacity.

The proliferation and development of tumour cellsrequires copious amounts of glucose, and recent workindicates that glucose transport and metabolism are essentialfor post-treatment survival of tumour cells. Transport ofglucose across the cell membrane is maintained by thefacilitative glucose transporter GLUT family, with the mostimportant member considered to be Glut1.31 Aldolase A,enolase1 and Glut3 are also involved in glucose transport totumour cells.21 On the basis of these findings, the inhibitoryaction of KRH102140 on glut1, aldolase A, enolase1 andmonocarboxylate transporter probably suppresses thetransport of glucose into tumour cells, eventually prohibitingcellular adaptation to a hypoxic microenvironment throughimpaired glycolytic rates.

In summary, KRH102140 inhibited in vitro tubeformation in HUVECs and suppressed gene expressiondownstream of HIF-1a related to angiogenesis. However,whether KRH102140 could be useful for suppressingtumour angiogenesis and growth in animal models is notclear, and further in vivo experiments are important toresolve this question. Nevertheless, from the in vitro findingswe conclude that KRH102140 has more potent inhibitoryeffect of HIF-1a than KRH102053. By targeting multipleprocesses that are essential for tumour cells to adopt, surviveand induce angiogenesis, small compounds such asKRH102140 may offer an improved therapeutic outcomeagainst angiogenesis, against the effects of genes down-stream of HIF-1a. The simple structure of KRH102140might provide an attractive pharmacological target againstvarious diseases associated with an elevated HIF-1a level.

CONFLICT OF INTEREST

No conflict of interest.


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ACKNOWLEDGEMENTS

This work was supported by the Korea Research FoundationGrant funded by the Korean Government (MOEHRD, BasicResearch Promotion Fund) (2009-0074645).

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An activator of PHD2, KRH102140, decreases angiogenesis via inhibition of HIF-1α