Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells

Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells

Research Article Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells Eunyoung Tak1, S...

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Research Article

Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells Eunyoung Tak1, Seonmin Lee1, Jisun Lee1, M.A. Rashid1, Youn Wha Kim2, Jae-Hoon Park2, Won Sang Park3, Kevan M. Shokat4, Joohun Ha1, Sung Soo Kim1,⇑ 1 Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea; 2Department of Pathology, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea; 3Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea; 4Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA

Background & Aims: Human carbonyl reductase1 (CBR1) has been reported to protect cells against lipid peroxidation. Since human hepatocellular carcinoma (HCC) cells are under oxidative stress in hypoxic conditions, we tested if CBR1 is upregulated by hypoxia inducible factor (HIF)-1a, helps tumor growth under hypoxia, and renders chemoresistance to cisplatin and doxorubicin in HCC. Methods: Luciferase, EMSA, and chromatin immunoprecipitation (ChIP) assays were performed to analyze whether HIF-1a transactivates CBR1 promoter. CBR1 overexpression, siRNA, and inhibitors were used to study the role of CBR1 in tumor survival under hypoxia and chemoresistance to cisplatin and doxorubicin in HCC. FACS and Western blot analysis for oxidative stress markers were performed to measure ROS. Immunohistochemistry (IHC) was performed to analyze CBR1 expression in 78 cases of HCC and 123 cases of colon cancer tissues. Results: The CBR1 promoter was activated by HIF-1a. CBR1 overexpression enhanced cell survival by decreasing oxidative stress under hypoxia, cisplatin, and doxorubicin treatment. CBR1-siRNA increased apoptosis via increasing oxidative stress. Combinational therapy of CBR1 inhibitors with cisplatin or doxorubicin enhanced cell death in HCC cells. IHC showed CBR1 overexpression in 56 (72%) out of 78 HCC and 88 (72%) out of 123 colon cancer cases.

Keywords: CBR1; HCC; HIF-1a; Lipid peroxidation; ROS. Received 24 December 2009; received in revised form 21 June 2010; accepted 22 June 2010; available online 17 September 2010 ⇑Corresponding author. Address: Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, #1, Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 0524; fax: +82 2 959 8168. E-mail address: [email protected] (S.S. Kim). Abbreviations: CBR1, carbonyl reductase 1; HCC, hepatocellular carcinoma cell; HRE, hypoxia-responsive element; SDR, short chain dehydrogenase/reductase; ONE, 4-oxonon-2-enal; HIF-1a, hypoxia inducible factor-1a; TACE, transarterial chemoembolization; HO-1, heme oxygenase-1; TRXr-1, thioredoxin reductase-1; ALDH, aldehyde dehydrogenase; AR, aldose reductase; HNE, hydroxynonenal; MDA, malondialdehyde; NOS2, nitric oxide synthase 2; Hydroxy-PP, 3-(1-tertbutyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol; Hydroxy-PP-Me, 3-(7isopropyl-4-(methylamino)-7H-pyrrolo[2,3-d]pyrimidin-5yl)phenol; ROS, reactive oxygen species.

Conclusions: Overexpressed CBR1 by HIF-1a plays important roles in tumor growth under hypoxia and chemoresistance to anticancer drugs. The inhibition of CBR1 by specific inhibitors enhances anticancer drug efficacy in HCC. Therefore, we concluded that CBR1 is a good molecular target for the development of anticancer drugs for HCC patients. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Human carbonyl reductase 1 (CBR1) is a ubiquitous NADPH dependent enzyme belonging to the short chain dehydrogenase/reductase (SDR) family. It consists of 277 amino acid residues and catalyzes a large number of biologically and pharmacologically active substrates, including a variety of endogenous and xenobiotic carbonyl compounds [1]. CBR1 inactivates highly reactive lipid aldehydes, such as 4-oxonon-2-enal (ONE), which are able to modify protein and DNA [2]. Hypoxia induces a number of cellular responses such as angiogenesis, erythropoiesis, glycolysis, etc., through both gene regulation and post-transcriptional modification of certain proteins [3]. The homeostatic responses to hypoxia are predominantly mediated by hypoxia-inducible factor (HIF)-1. It is a heterodimer composed of HIF-1a and HIF-1b subunits [4,5]. In contrast to a constitutively expressed HIF-1b, HIF-1a is sensitively regulated within cells in response to hypoxia and growth factor stimulation. Under normoxic conditions, HIF-1a is rapidly degraded by ubiquitination and proteasome pathways, after hydroxylation of two proline residues (Pro402 and Pro504) [6]. Therefore, HIF-1a usually remains undetectable in most cells. When O2 concentration declines, HIF-1a protein level is markedly increased due to a blockade of prolyl hydroxylation. The increased HIF-1a transactivates many genes related to hypoxia by binding to the hypoxiaresponse elements (HREs; 50 -A/CGTG-30 ) in hypoxia-inducible promoters. Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer [7,8]. Although there are several treatment options for HCC patients, the long-term prognosis is generally poor.

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JOURNAL OF HEPATOLOGY Surgery is usually considered a curative treatment option. However, most patients can receive only palliative treatments including transarterial chemoembolization (TACE) [9]. The overall recurrence rate of HCC patients with initial remission following TACE is very high. Therefore, anticancer drugs with high efficacy should be developed to overcome HCC [10,11]. HCC cells are frequently under hypoxic conditions that subsequently leads to cell death. However, HCC cells also develop cellular defense mechanisms, mostly regulated by HIF-1, and thus evade cell death. Here, we show, for the first time, the possible role of CBR1 in tumor growth under hypoxia and resistance to anticancer drugs such as cisplatin and doxorubicin.

Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco-BRL (Grand Island, NY, USA). Antibody against CBR1 was purchased from Imgenex (San Diego, CA). Antibodies against HIF-1a, heme oxygenase-1 (HO-1), thioredoxin reductase-1 (TRXr-1), nitric oxide synthase 2 (NOS2), aldehyde dehydrogenase (ALDH), aldose reductase (AR), PARP, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Hsp72, Hsp60, hydroxynonenal (HNE), malondialdehyde (MDA), and acrolein antibodies were acquired from Abcam (Cambridge, MA). Antibody against caspase-3 was purchased from Assay Designs (Ann Arbor, MI). Cisplatin, CoCl2, 3-(4,5-dimethylthiasol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin, Hoechst 33342, H2O2, doxorubicin, and actinomycin D were acquired from Sigma Aldrich (St. Louis, MO). The pGL3-Basic and pSV-b-gal vector and restriction enzymes such as KpnI and HindIII were purchased from Promega (Madison, WI). [c-32P] ATP was purchased from Amersham Biosciences (Little Chalfont, UK).

Cell culture and hypoxia

NJ). Calculations were based on the ‘‘Delta–Delta method’’ using the following equation: R (ratio) = 2[DCT sample–DCT control] [13]. The data were expressed as fold change of the treatment groups relative to the control. GAPDH was amplified as a control for real-time RT-PCR.

Promoter analysis and luciferase assay The CBR1 promoter sequence was analyzed using Genomatix MatInspector (http://www.genomatix.de). For the construction of the luciferase reporter plasmids, 1000 base pairs of the CBR1 promoter sequence was amplified via PCR. The amplified fragments were cloned into the pGL3 basic vector (Promega, Madison, WI) with KpnI and HindIII restriction enzymes. For mutational analysis, the HRE site was mutated by PCR-based site-directed mutagenesis. Primers used for constructing luciferase reporters containing various regions of the CBR1 promoter or mutated HRE were as follows: pGL3-CBR1–1000 forward, 50 -CGGGTACCCACATCTGGA-30 ; pGL3-CBR1–1000 reverse, 50 -ATAAGCTTGCCGCTGAGCGCGCAGGC-30 ; pGL3-CBR1–350 forward, 50 -CCGGTACCGAATGGTTCTTCACT-30 ; pGL3-CBR1–350 reverse, 50 -ATAAGCTTGCCGCTGAGCGCGCAGGC-30 ; pGL3-CBR1–350 M forward, 50 -CTTCACTGCCACGGGGGCAGCCAA-30 ; pGL3-CBR1–350 M reverse, 50 -TTGGCTGCCCACGGGGCAGTGAAG-30 . Chang cells were transfected with 0.2 lg of the pGL3 basic-derived plasmids together with the internal control plasmid, pCMV-Lac (Promega, Madison, WI). Luciferase and b-gal activities (not shown) were measured using 50 ll cell lysates in a microplate reader (BIO-RAD), and the luciferase activity was normalized to bgal activity, as previously reported [14].

Electrophoretic mobility shift assay (EMSA) EMSA was performed as described previously [15] using the following oligonucleotides, CBR1 wild type promoter, 50 -CTTCACTGCCACGTGGGCAGCCAA-30 ; CBR1-M, 50 -CTTCACTGCCAAAAGGGCAGCCAA-30 . The probes were labeled with [c-32P] ATP. For a competition study, a 100-fold molar excess of unlabeled oligonucleotides was added to the reaction mixture before the addition of the radiolabeled probe.

Chromatin immunoprecipitation (ChIP)

Human Chang liver and the two human HCC cell lines, HepG2, and Hep3B cells (1  106/ml) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in DMEM supplemented with 10% fetal bovine serum, since these cell lines have been used for in vitro experiments for the investigations on hepatocellular carcinoma cells [12]. Hypoxic conditions were generated in the hypoxia chamber (0.1% O2).

Conventional ChIP was conducted as described previously [16]. The cross-linked chromatin was subjected to immunoprecipitation with antibodies against HIF-1a. CBR-HRE was amplified using the following primers 50 -GAAGAAAACCCCTCAGAGAACC-30 and 50 -CTCGCCGGGGTGCGGAGCAGGCGG-30 .

Establishment of stable cell lines

RNA interference

Chang and HepG2 cells were transfected with pcDNA3-CBR1 wild type (CBR1/WT, 3 lg), or pcDNA3 (3 lg) using an OmniPOTER (QBIOgene, OH). Cells transfected with pcDNA plasmid alone without the CBR1 gene (mock) were used as a control. For stable transfection, cells were cultured in the selective medium with 600 mg/ ml G418 for 1 month. Then, drug resistant individual clones were isolated and incubated for further amplification in the presence of a selective medium.

Small interfering RNAs (siRNA) specific to either CBR1 (CBR1-siRNA) or scrambled sequence (scrambled-siRNA) were prepared by Sigma Aldrich (St. Louis, MO). si-RNA (0.5 lg) was used for transfection using OmniPOTER transfection reagent (QBIOgene, OH) and the siRNA target sequences were as follows: CBR1-siRNA sense, 50 -rCrArCrArGArAUUrArCUrCrCrCUrCUrCUrATT-30 ; anti-sense, 50 -UrArGrArGrGrArGUrArAUUrCUrGUrGTT-30 , scrambled-siRNA 50 -UCCCAGAUAGAGACUUCAATT-30 ; anti-sense, 50 -UUGAAGUCUCUAUCUGGGATT-30 . Efficiency of siRNA-based interference for CBR1 production was monitored by Western blot analysis.

RNA isolation and reverse transcription-polymerase chain reaction For analysis of CBR1 mRNA, total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA) and amplified by a two step protocol using AMV reverse-transcriptase (Promega, Madison, WI) and Taq polymerase. Primers for amplifying CBR1 transcripts were as follows; forward, 50 -GAATTCATGTCGTCCGGCATCCAT30 ; reverse, 50 -AAGCTTTCACCACTGTTCAACTCTCTT-30 .

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Real-time quantitative RT-PCR

Reactive oxygen species (ROS) analysis

The following primer sets were used: GAPDH control forward, 50 -CATCGAGCACGGCATCGTCAC-30 and reverse, 50 -TCGAAGTCCAGGGCGACATAG-30 ; CBR1 forward 50 -AACAAGTTTGTGGAAGGATACAAAGAAGGGA-30 , and reverse 50 TGTTCAACTCCTTCTCTGAAACAAATTGTC-30 . Real-time PCR amplification was conducted using TaqMan Universal Mastermix (PE Applied Biosystems, Branchburg,

ROS were measured using 20 -70 -dichlorodihydrofluorescein diacetate (DCF-DA). The cells were incubated with 10 lM DCF-DA at 37 °C for 30 min and re-suspended in 1 ml of PBS. Fluorescence was measured by a flow cytometer. The mean DCF fluorescence intensity was measured with excitation at 488 nm and emission at 525 nm.

MTT assays were executed in 12-well plates. Optical density was assessed at 550 nm using a microplate reader (Bio-Rad). Cell survival was expressed as the percentage of absorbance relative to that of untreated cells.

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Cells were incubated for 30 min with Hoechst 33342 (Molecular Probes) loading dye and fixed for 20 min in 4% formaldehyde. Stained cells were monitored using a confocal laser microscope (META 510, Zeiss).

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Tissue samples The formalin-fixed and paraffin-embedded 78 HCC and 123 colon cancer tissues collected between 2003 and 2004 were used in this study. No patients received any form of treatment prior to surgery. One 6 lm section stained with haematoxylin and eosin was independently reviewed by three pathologists. To construct the tissue microarray block, two pathologists screened the histologic sections and selected areas representative of the tumor cells. Two and one tissue core samples from each cancer and non-cancerous area, respectively, were taken and placed in a new recipient paraffin block using a commercially available microarray instrument (Beecher Instruments, Microarray Technologies, Silver Spring, MD, USA).

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The deparaffinized and rehydrated specimens were incubated overnight at 4 °C with a monoclonal antibody against CBR1 (Imgenex, San Diego, CA) at a 1:1000 dilution. The immunostained section was visualized using the Dako EnVision Detection Kit (Dako, Denmark). Routine haematoxylin and eosin stained sections were examined to ensure the structural integrity of the tissues. Three pathologists, who were blinded to the specific diagnosis and prognosis for each case, interpreted results. Staining intensities were interpreted as negative when immunostaining was weak. Immunostaining results were considered to be positive when >30% of the cancer cells showed distinct immunoreactivity. Immunoreactivity was assessed using a three positive grading system; +, minimal staining; ++, uniform or intense staining, and +++, more intense staining. Only specimens exhibiting above ++ immunoreactivity were considered positive.

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CBR1 is transcriptionally up-regulated in response to hypoxia To investigate whether CBR1 protein is induced by hypoxia, we performed Western blot analysis using Chang and HepG2 cells subjected to hypoxic conditions for the specified periods of time. As shown in Fig. 1A, CBR1 protein level began to be detected after a 3 h exposure to hypoxia, and kept continuously increasing up to 48 h. The HIF-1a protein level also increased dramatically during the periods of experiments. To test the induction of CBR1 mRNA under hypoxia, we performed RT-PCR. The results showed that the level of CBR1 mRNA is highly increased under hypoxia (Fig. 1B). CoCl2, a hypoxia mimicking agent, showed the same result as hypoxia. To rule out the possibility of mRNA stabilization, we kept the cells under hypoxia for 9 h, and then treated with 5 lg/ml actinomycin D, followed by incubation under hypoxic or normoxic condition for another 12 h. Actinomycin D was used to inhibit the de novo synthesis of the CBR1 transcript. As shown in Fig. 1C, the half life of CBR1 mRNA turned out to be

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Fig. 1. Transcriptional upregulation of CBR1 in response to hypoxia. (A) Western blot analysis. Total cell lysates (10 lg) extracted after exposure to hypoxia for the indicated periods of time were subjected to Western blot analysis. (B) RT-PCR analysis. Total RNA was extracted from Chang cells and HepG2 cells exposed to hypoxia or treated with 200 lM CoCl2 for the indicated periods of time and subjected to RT-PCR analysis. GAPDH was used as a loading control. (C) Decay rate of Chang and HepG2 CBR1 mRNA. Cells were exposed to hypoxia for 9 h and then incubated with 5 lg/ml actinomycin D for the indicated periods of time under normoxic or hypoxic conditions. (D) Real-time quantitative RT-PCR. Cells were incubated under hypoxia for 12 h, and expression levels of CBR1 mRNA were quantified. Data represent mean ± SE, n = 3. *p <0.01 vs untreated Chang cells in normoxia. #p <0.01 vs untreated HepG2 cells in normoxia.

similar in both conditions, indicating that CBR1 mRNA is really regulated at the transcriptional level under hypoxia. The realtime quantitative RT-PCR also showed a significant increase in the CBR1 mRNA level in hypoxia (Fig. 1D).

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JOURNAL OF HEPATOLOGY CBR1 promoter is activated by the HIF-1a transcription factor HIF-1 is a major transcription factor in hypoxia. Upregulation of the CBR1 mRNA level by hypoxia led us to explore whether the CBR1 promoter contains a hypoxia-responsive element (HRE), HIF-1 binding site. First, bioinformatic analysis of the promoter of the CBR1 gene was performed to search for a consensus HRE sequence. Since HRE sequences contained the HIF-1 binding site (HBS), 50 -A/CGTG-30 , and the HIF-1 ancillary sequence (HAS), GGCAGCCAA, 8–9 nucleotides down- or upstream of HBS, we tried to find both sequences in CBR1 promoter. As a result, we identified one typical HRE containing both HBS and HAS located at 313 bp upstream to the CBR1 start codon (Fig. 2A). To test the reactivity of HIF-1 to HRE under hypoxia, we constructed several luciferase reporter constructs, and performed luciferase activity assay under hypoxia (Fig. 2A). Neither under normoxic nor hypoxic conditions was any luciferase activity observed for the empty pGL3-basic plasmid. In contrast, pGL3-CBR1–1000 and pGL3-CBR1–350 showed significant increase in luciferase activity under hypoxia, while pGL3-CBR1–350 M containing 50 AAAG-30 , instead of 50 -CGTG-30 , at HRE showed quite less activity than normal both in Chang and HepG2 cells. The co-transfection study with HIF-1a also showed the same results (Fig. 2B). Next, we performed EMSA experiments using nuclear extracts from Chang and HepG2 cells incubated for 12 h under hypoxia. Oligonucleotides containing wild type or mutated version of HRE were used in the experiments. Only the wild type oligonucleotide incubated with nuclear extracts from HepG2 cells showed strong mobility shifted bands. The binding was abolished by both the mutated oligonucleotide and competitive inhibition with cold oligonucleotide (Fig. 2C). The nuclear extracts from Chang cells showed the same results (data not shown). In addition, ChIP assays also showed that CBR1 HRE readily associated with HIF-1a (Fig. 2D). To further investigate the effect of HIF-1a on CBR1 induction, we treated Chang and HepG2 cells with HIF-1a inhibitors, deguelin, and 17-AAG, for 12 h under hypoxia and then observed if they suppress CBR1 protein levels (Fig. 2E). Results showed a decrease in both CBR1 and HIF-1a protein levels. The knockdown of HIF-1a using specific siRNA also showed the same results as the inhibitors (Fig. 2F). Both of the HIF-1a inhibitors and specific siRNAs decreased mRNA levels of HIF-1a and CBR1 (data not shown). Based on all these results, we concluded that HIF-1a really regulates CBR1 expression at the transcriptional level in response to hypoxia.

Overexpression of CBR1 protects cells from hypoxia, H2O2, cisplatin, and doxorubicin-induced apoptotic cell death HIF-1 has been known to play an important role in the growth and survival of solid tumor cells [17,18]. We have demonstrated that CBR1 is induced by hypoxia via HIF-1a activation. Therefore, we hypothesized that CBR1 might play an important role in growth and survival of HCC cells especially in response to cellular stresses including hypoxia, CoCl2, cisplatin, doxorubicin, and oxidative stress such as H2O2 treatment. Cisplatin and doxorubicin are known to induce apoptosis partly through the generation of ROS [19,20]. We made stably transfected cell lines with CBR1, using Chang and HepG2 cells. CBR1 was overexpressed about 3fold higher in stably transfected cells than mock transfectants

in both cell lines (Fig. 3A). When these cells were exposed to hypoxia for up to 72 h, cells overexpressing CBR1 survived much more than mock-transfected cells (Fig. 3B). Hoechst 33342 staining showed similar results (Fig. 3C). To see the effect of CBR1 on other stresses including CoCl2, H2O2, cisplatin, and doxorubicin, we performed a MTT assay (Fig. 3D) as well as a Western blot analysis for the detection of apoptotic markers such as cleavages of PARP and caspase-3 (Fig. 3E). For these experiments, we performed dose- and timedependent experiments and found that 200 lM CoCl2, 100 lM cisplatin, or 0.1 lM doxorubicin is optimal for our experiments at 72 h (data not shown). Therefore, cells were treated with each condition for 72 h for the final experiments. All results showed that overexpression of CBR1 protected cells from apoptotic cell death induced by these stressors.

Knockdown of CBR1 enhances cell death under treatment of hypoxia, H2O2, cisplatin, and doxorubicin stress To further explore the role of CBR1 in cell death in response to hypoxia, H2O2, cisplatin, and doxorubicin treatment, we conducted RNA interference experiments using small interfering RNA (siRNA) specific to CBR1 in Chang and HepG2 cells. As shown by Western blot analysis, the expression of CBR1 was almost completely suppressed by CBR1 specific siRNA. In order to observe the distinct difference in cell death rate, we treated cells with each stress for 36 h, rather than 76 h as was used for the overexpression of CBR1. First, hypoxia-induced cell death was monitored, by microscopy, in scrambled-siRNA and CBR1-siRNA transfected cells by microscopy (Fig. 4B). It was also monitored after Hoechst 33342 staining (Fig. 4C). Compared to scrambled-siRNA, CBR1 specific siRNA decreased cell survival quite significantly. The same results were observed with other stressors using the MTT assay (Fig. 4D), and by Western blot analysis for the detection of apoptotic markers (Fig. 4E).

CBR1 protects cells by attenuating oxidative stress To find the molecular mechanism by which CBR1 protects cells from the diverse stresses described above, we first measured ROS level in mock, wild-type CBR1, scrambled-siRNA, and CBR1 specific-siRNA transfected Chang and HepG2 cells after 24 h exposure to hypoxia. ROS levels were measured after DCF-DA loading for 30 min. As expected, results showed that the highest and lowest ROS levels were detected in CBR1 specific-siRNA and wild-type CBR1 transfected cells, respectively (Fig. 5A). To further confirm these observations, we measured, by Western blotting, the expression levels of diverse stress-responsive proteins including heme oxygenase-1 (HO-1), thioredoxin reductase-1 (TRXr-1), nitric oxide synthase 2 (NOS2), Hsp72, Hsp60, and lipid peroxidation products including HNE, MDA, and acrolein. Results showed that all the oxidative stress markers were decreased in wild-type CBR1 transfected cells, but increased in CBR1 specific-siRNA transfected cells. Moreover, the aldehyde dehydrogenase (ALDH), and aldose reductase (AR) enzymes, which are known to detoxify HNE [21], were also decreased in CBR1 overexpressed cells, but increased in CBR1 knockdown cells (Fig. 5B).

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Fig. 2. Functional analysis of human CBR1 promoter. (A) Luciferase reporter constructs of the CBR1 promoter. (B) Luciferase reporter assay. Chang and HepG2 cells transfected with each luciferase reporter construct shown in (A) were exposed to hypoxia or cotransfected with pcDNA3-HIF-1a. Relative luciferase activities were expressed in comparison with the activity of pGL3-Basic. Data represent mean ± SE, n = 3. *p <0.01 vs pGL3-CBR1/1000 and pGL3-CBR1/350 under normoxia. (C) EMSA. Nuclear extracts were prepared from HepG2 cells exposed to hypoxia for 12 h, and then 10 lg of the nuclear extracts was incubated with 32P-labeled wild type or mutant oligonucleotide probes. For a competition study, a 100-fold molar excess of unlabeled wild-type probe (Cold) was added to the reaction mixture. (D) ChIP assay. Input, amplified CBR1 from a 1:100 dilution of total input chromatin: IgG, immunoprecipitation with non-specific IgG. (E) Western blot analysis. Effect of HIF-1a suppression by deguelin and 17-AAG on CBR1 expression. Cells were treated with 100 lM deguelin and 100 lM 17-AAG for 12 h under hypoxia. Actin was utilized as a loading control. (F) Effect of HIF-1a siRNA on CBR1 expression. Cells were transfected with HIF-1a siRNA or scrambled siRNA. Cells were then allowed to recover in regular culture medium for 20 h after transfection, followed by exposure to hypoxia for 12 h. SC, scrambled siRNA.

Combined treatment of hydroxy-PP and hydroxy-PP-Me with cisplatin and doxorubicin enhances anticancer drug effects in hepatocelluar carcinoma cell lines Recently, it was reported that 3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (hydroxy-PP) and 3-(7-isopropyl-4-(methylamino)-7H-pyrrolo[2,3-d]pyrimidin-5yl)phenol (hydroxy-PP-Me) are selective inhibitors of CBR1 [22]. In 332

addition, it was reported that these two inhibitors enhance cell death, when combined with doxorubicin [22]. To further examine the role of CBR1 in the protection against cell death induced by hypoxia and anticancer drugs in HCC, we treated Chang, HepG2, and Hep3B cells with hydroxy-PP and hydroxy-PP-Me alone or in combination with cisplatin and doxorubicin. We first performed dose-dependent experiments and then chose 25 lM concentrations of hydroxy-PP and hydroxy-PP-Me, since those concentrations

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Fig. 3. Effects of CBR1 overexpression on apoptotic cell death. (A) Western blot analysis. Expression level of CBR1 was monitored in Chang cells stably transfected and HepG2 cells transiently transfected with mock and wild-type CBR1. (B) Microscopic observation of hypoxia-induced cell death. (C) Apoptosis. Cells under normoxia (None) or hypoxia were stained with Hoechst 33342 and visualized under confocal microscopy. The arrows represent chromosomal DNA fragmentation. (D) MTT assay. Each transfectant was exposed to hypoxia, 200 lM CoCl2, 100 lM H2O2, 40 lM cisplatin or 0.1 lM doxorubicin for 72 h for the assay. Data represent mean ± SE, n = 3. (E) PARP cleavage and caspase-3. CBR1/WT, wild-type CBR1.

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ox ia yp ox ia C oC l 2 H 2 O C is 2 pl D ox atin or ub ic in

N

H

or m

ox ia yp ox ia C oC l 2 H 2 O C is 2 pl D ox atin or ub ic in

0

0

N

SC-siRNA CBR1-siRNA

PARP Cleaved Cleaved Caspase-3 Actin

H

Relative cell survival (%)

120

116 kDa 87 kDa

PARP Cleaved Cleaved Caspase-3

Normoxia Hypoxia

CoCl2

H2O2

Cisplatin Doxorubicin

Normoxia Hypoxia

CoCl2

H2O2

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CBR1

CBR1

SC

CBR1

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CBR1

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CBR1

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siRNA

CBR1

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CBR1

SC

CBR1

SC

CBR1

SC

CBR1

CBR1

SC

SC

Actin

siRNA

CBR1

E

SC

None

Cisplatin Doxorubicin

Fig. 4. Effect of CBR1 knockdown on apoptotic cell death. (A) Western blot analysis. CBR1 protein expression level was monitored in Chang and HepG2 cells transfected with scrambled or CBR1 specific siRNA. (B) Microscopic observation of hypoxia-induced cell death. (C) Hoechst 33342 staining. (D) MTT assay. Cells were exposed to each condition for 36 h for the assay. Data represent mean ± SE, n = 3. Drug concentrations were same as those in Fig. 3D. (E) Cleavage of PARP and caspase-3. SC, scrambled siRNA.

334

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JOURNAL OF HEPATOLOGY Chang

HepG2

8 7

7

6

6

5 4 CBR1/WT

3 2

*

1

**

R N A si

W T

C BR 1-

C BR 1/

si

C BR 1-

T

T

siRNA

1 SC

BR

k oc M

C

1 BR C

SC

HO-1

1/

W

W 1/ BR C

k oc M

2

HepG2 siRNA

HO-1

TRXr-1

Oxidative stress markers

NOS-2 HSP72 HSP60

CBR1 down stream enzymes

3

M oc k

R N A

W T C BR 1/

CBR1-siRNA

Chang

Lipid peroxidation products

4

0 M oc k

0

Oxidative stress markers

5

1

CBR1-siRNA

B

##

BR

CBR1/WT

8

Mock

#

Mean fold of ROS

Mean fold of ROS

Mock

C

A

TRXr-1 NOS-2 HSP72 HSP60

HNE

Lipid peroxidation products

MDA Acrolein AR

CBR1 down stream enzymes

ALDH Actin

HNE MDA Acrolein AR ALDH Actin

Fig. 5. Antioxidant effect of CBR1. (A) ROS measurement. Each transfectant was treated with or without hypoxia for 24 h. Then, ROS level was measured by flow cytometry after loading with DCF-DA and shown as a histogram. Data represent mean ± SE, n = 3. FACS analytic data (right) and their quantifications (left) are shown for both cell lines. *p and #p <0.01 vs Chang mock transfectants, **p and ##p <0.01 vs HepG2 mock transfectants. (B) Western blot analysis. Samples were prepared from cells under hypoxia for 24 h. CBR1/WT, wild-type CBR1; SC, scrambled siRNA; TRXr-1, thioredoxin reductase-1; ALDH, aldehyde dehydrogenase; AR, aldose reductase.

did not show any toxicity, but showed remarkable effects on cell death, when combined with cisplatin and doxorubicin. Our MTT assay, performed after exposure to 40 lM cisplatin and 0.2 lM doxorubicin alone, or in combination with 25 lM hydroxy-PP and hydroxyl-PP-Me for 72 h, revealed that both inhibitors remarkably enhanced cell death by cisplatin or doxorubicin both in HepG2 and Hep3B cells (Fig. 6A). When the two conditions were combined, most of the cells died. Western blot analysis also revealed increased cleavages of PARP and caspase-3, when hydroxy-PP and hydroxy-PP-Me were used in combination with cisplatin and doxorubicin. Interestingly, inhibition of CBR1 enhanced the anticancer drug effects even in p53-defective HCC

cells. We think this finding is important because p53 inactivation, by mutation or binding to HBX, has been known to be one of the main causes in the emergence of resistance to anticancer drugs in HCC and because inhibition of CBR1 may pave the new way to overcome the chemoresistance often observed in HCC patients. CBR1 is overexpressed in human hepatocellular carcinoma and colon cancer tissues To assess the clinical relevance of CBR1, its expression level was measured in human HCC. Pathologically confirmed HCC and corresponding non-cancerous tissue specimens were obtained and

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Research Article Table 1. Histopathological features and CBR1 expression in HCC and colon cancer taken from patients.

140

A

Chang HepG2 Hep3B

Relative cell survival (%)

120

CBR1 expression in HCC (n = 78)

100

Gender

Grade 1 80 Grade 2 60

0 Cisplatin Doxorubicin Hydroxy-PP Hydroxy-PP-Me

Grade 3

***

40

**

*** **

+ -

+ -

+ -

+

+ + -

## #

*

-

### ## ###

*

20

Grade 4

#

+ +

+ + -

+ +

Chang

116 87

Cleaved Caspase-3 Actin

116 87

PARP Cleaved Caspase-3 Actin

Hep3B 116 87

PARP Cleaved Caspase-3 Actin

Cisplatin Doxorubicin Hydroxy-PP Hydroxy-PP-Me

-

+ -

+ -

+ -

+

+ + -

+ +

+ + -

+ +

Fig. 6. Reduced cell survival by combined treatment of CBR1 inhibitors and cisplatin in HepG2 and Hep3B cells. (A) MTT assay. Cells were exposed to 40 lM cisplatin and 0.2 lM doxorubicin, 25 lM hydroxy-PP, and 25 lM hydroxy-PP-Me alone or in combination for 72 h for the assay. Data represent mean ± SE, n = 3. *p <0.01 vs HepG2 cells under cisplatin alone, **p <0.01 vs Chang cells under cisplatin alone, ***p <0.01 vs Hep3B cells under cisplatin alone, ##p <0.01 vs Chang cells under doxorubicin alone, #p <0.01 vs HepG2 cells under doxorubicin alone, ###p <0.01 vs Hep3B cells under doxorubicin alone. (B) Cleavage of PARP and caspase-3. Actin was used as the loading control.

CBR1 expression was evaluated via immunohistochemical analysis. In 56 (72%) out of the 78 HCC cases, a moderate to strong immunopositivity was observed (Table 1). The corresponding non-cancerous hepatocytes were negative or weakly positive

++

+++

M

0

0

7

0

F

0

1

2

0

M

4

4

9

2

F

1

1

4

0

M

3

3

16

7

F

2

2

6

1

M

0

1

2

0

F

0

0

0

0

Gender

Grade 1

Grade 3 Grade 4

HepG2

+

Grading

kDa

PARP

-

CBR1 expression in Colon cancer (n = 123)

Grade 2

B

336

Grading

-

+

++

+++

M

0

1

9

0

F

0

1

4

0

M

4

6

14

3

F

1

2

6

1

M

3

7

25

11

F

2

3

8

4

M

0

1

2

1

F

0

1

0

0

Note: The multivariate model was adjusted for tumor grade, gender, and pathologic stage.

for CBR1. Typical overexpression of CBR1 is shown in Fig. 7A. Interestingly, the CBR1 expression level was not associated with tumor grades and cancer development. Western blot analysis showed CBR1 upregulation in seven out of 10 HCC (Fig. 7B). To further confirm the overexpression of CBR1 during tumor development, we studied 123 human colon cancer tissues via immunohistochemical analysis, and found similar results (Table 1, Fig. 7). Western blot analysis showed the same results in 10 colon cancer tissues (Fig. 7B).

Relation between CBR1 expression and access to blood supply in human hepatocellular carcinoma and colon cancer tissues To finally confirm that CBR1 expression is really regulated by hypoxia in human cancer tissues, we investigated whether CBR1 expression is not increased in oxygenated cancer tissues. Cancer tissues surrounding blood vessels in HCC and tumor surface areas in colon cancers were considered oxygenated areas, because oxygen would more likely be delivered in those tissues than tissues without blood vessels around necrotic or central areas. It is well known that necrotic areas in cancer tissues are deficient in blood supply and are not well supplied with oxygen. As expected, CBR1 was weakly overexpressed in oxygenated cancer tissues, while cancer tissues surrounding necrotic debris, caused by non-blood supply, showed intense immunoreactivity for CBR1 in both HCC and colon cancers (Fig. 8).

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Fig. 7. CBR1 overexpression in human HCC and colon cancer tissues. (A) Immunohistochemical analysis. Typical immunoreactivity photomicrographs from  to +++ staining are shown, using normal liver and grade 3 HCC and colon cancer tissues. These criteria were used for Table 1. The cancer cells have a strong cytoplasmic staining of CBR1 compared to corresponding normal liver and colon tissues. (B) Western blot analysis. CBR1 is overexpressed in seven of the 10 HCC cases and seven of the 10 colon cancer cases, compared with the matched normal surrounding liver and colon tissues.

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Research Article

Fig. 8. Relationship between oxygenation and CBR1 overexpression in HCC and colon cancer tissues. Non-cancerous regions (left) do not show any positive CBR1 immunostaining. Cancer tissues surrounding blood vessels in HCC and tumor surface areas in colon cancer (middle) show weak to mild positive CBR1 immunostaining. Cancer tissues without blood supply (right) show strong immunostaining. Both liver and colon tissues show similar immunostaining patterns. THV, terminal hepatic venule; S, sinusoid; A, artery; ND, and necrotic debris. Original magnification, 100.

Discussion Here, we first showed that CBR1 expression is upregulated at the transcriptional level by HIF-1a under hypoxic conditions in both Chang and HepG2 cells. Also, we showed that overexpressed CBR1 protects cells against hypoxia and anticancer drugs such as cisplatin and doxorubicin, by reducing oxidative stress within cells. In addition, we showed that CBR1 inhibitors can remarkably enhance cell death under hypoxia or when combined with anticancer drugs. Finally, overexpression of CBR1 was observed by immunohistochemistry, in 56 (72%) out of the 78 HCC cases. Additionally, CBR1 was also observed to be overexpressed in human colon cancer tissues. The transcriptional regulatory mechanisms for the induction of CBR1 have rarely been studied, though microarray studies in mouse identified CBR1 as one of the responsive proteins to several inducers, including phenethyl isothiocyanate [23], D3T [24], and sulphorophane [25,26]. The transcription factor Nrf2 and the aryl hydrocarbon receptor ligand have been suggested to induce CBR1 [27]. Here, we argue that HIF-1a is another transcription factor for CBR1 induction under hypoxic conditions. HIF-1a regulates CBR1 by binding to the HRE sequence, 50 -A/ CGTG-30 , contained within CBR1’s promoter. The human HRE sequence containing the HIF-1 binding site (HBS), 50 -A/CGTG-30 , and the HIF-1 ancillary sequence (HAS), 50 -GGCAGCCAA-30 , nine nucleotides downstream of HBS, was found to be located at 313 bp upstream of the CBR1 start codon. Since mouse, rat, and monkey CBR1 promoters also contain typical consensus HRE sequences, we believe transcriptional regulation of CBR1 by HIF-1a occurs in diverse mammalian systems. Hypoxia generates a toxic outburst of ROS from the mitochondrial electron transport chain (mETC) complex III [28,29], and thus induces hypoxic cell death [30–32]. The rapidly growing HCC cells are frequently exposed to hypoxic conditions especially in the central regions. Growth under hypoxic conditions subsequently leads to accumulation of oxidative stress including lipid peroxidation and ultimately tumor cell death. However, hypoxia also stabilizes HIF-1a, and prevents hypoxic cell death by inducing a wide variety of anti-apoptotic molecules, including Bcl-2, Bax, and IAP-2 [33]. Furthermore, HIF-1a increases blood supply 338

by regulating the transcription of several genes involved in angiogenesis [34]. As a result, HCC cells are able to evade cell death and continue to grow, even under hypoxic conditions. Indeed, it was suggested that hypoxia enhances cell proliferation, suppresses apoptotic cell death, and consequently leads to enhanced tumor malignancy in HCC. Our results show that HIF-1a upregulates CBR1, and thereby attenuates cell death under hypoxic conditions. The attenuated cell death is due to the ability of CBR1 to reduce oxidative stresses, including lipid peroxidation. Therefore, we propose that CBR1 is one of the anti-apoptotic molecules regulated by HIF-1a. HCC is very resistant to diverse therapeutic modalities, including combination therapy with TACE, anticancer drugs, and antiangiogenic agents. Therefore, the development of more effective treatment modalities and new anticancer drugs is urgent. Our results indicate that suppression of either CBR1 expression or enzymatic activity increases sensitivity to cisplatin and doxorubicin through increased oxidative stress in HCC cells. Conversely, overexpression of CBR1 renders resistance to those agents. Therefore, therapeutic targeting of CBR1 expression or enzymatic activity may be effective in HCC treatment. Indeed, our results showed that CBR1 inhibitors, hydroxy-PP, and hydroxy-PP-Me, remarkably increase cell death in both HepG2 and Hep3B cell lines. We believe that increased drug sensitivity in Hep3B cells by these inhibitors is especially important, because Hep3B cells do not have the p53 tumor suppressor gene. During the molecular pathogenesis of HCC, p53 mutations occur frequently. Both hepatitis B and C viruses, aflatoxin B1, and oxyradicals are implicated in p53 mutations. An important consequence to attenuated p53 activity in HCC tumors is increased chemoresistance. Therefore, the enhanced chemotherapeutic effect of anticancer drugs, in combination with CBR1 enzymatic inhibitors in Hep3B cells, demonstrates the effectiveness of CBR1 targeting in more resistant disease, and will lead to the development of more effective anticancer modalities in HCC patients. In our immunohistochemical analyses, CBR1 was overexpressed in 56 (72%) out of the 78 HCC cases and 88 (72%) out of the 123 colon cancer cases, indicating that CBR1 may really play important roles in tumorigenesis and resistance to anticancer therapies not only in HCC but also other cancers. However, we could not find any correlation of CBR1 overexpression with tumor grades or development. This is highly possible because CBR1 expression is mainly regulated by hypoxia rather than tumor invasiveness or metastasis. The more convincing evidence on the role of CBR1 overexpression in tumor malignancy will come out after massive studies with more HCC samples and other cancer cases. In summary, we have demonstrated that CBR1 is upregulated by hypoxia and confers resistance to apoptotic cell death induced by hypoxia, which in turn stimulates tumor growth. Also, we have demonstrated that CBR1 overexpression is a major cause of resistance to anticancer drugs, and inhibition of CBR1 enzymatic activity enhances anticancer drug efficacy in HCC. Therefore, we propose that CBR1 will be a valuable target for future drug development in patients with HCC.

Financial support This work has been supported by a grant from the Korea Science & Engineering Foundation (No. 20090091346) to S.S. Kim.

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