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HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic radioresistance and monitoring via CA9 expression

HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic... Background: Hypoxia induces activation of the HIF-1 pathway and is an essential characteristic of malignant gliomas. Hypoxia has been linked to tumor progression, therapy resistance and poor prognosis. However, little is known about the impact of HIF-1a inhibition on radioresistance of malignant glioma. Methods: In this study, we investigated the effects of the inhibition of HIF-1a on cell survival and radiosensitivity in U251MG and U343MG glioma cells, using two different strategies. HIF-1a inhibition was achieved by siRNA targeting of HIF-1a or via chetomin, a disruptor of interactions between HIF-1a and p300. The inhibition of the HIF-1 pathway was monitored by quantitative real-time PCR and Western blot analyses of the expression levels of HIF-1a and CA9. CA9 expression was investigated as a potential indicator of the efficacy of HIF-1 inhibition and the resulting radiosensitivity of malignant glioma cell lines was determined by clonogenic assay after irradiation under normoxic (2-10 Gy) or hypoxic (2-15 Gy) conditions. Results: Although siRNA and chetomin show distinct modes of action, both attenuated the hypoxia-induced radioresistance of malignant glioma cell lines U251MG (DMF : 1.35 and 1.18) and U343MG (DMF : 1.78 and 1.48). 10 10 However, siRNA and chetomin showed diverse effects on radiosensitivity under normoxic conditions in U251MG (DMF : 0.86 and 1.35) and U343MG (DMF : 1.33 and 1.02) cells. 10 10 Conclusions: Results from this in vitro study suggest that inhibition of HIF-1a is a promising strategy to sensitize human malignant gliomas to radiotherapy and that CA9 could serve as an indicator of effective HIF-1-related radiosensitization. Background despite surgery, chemo- and radiotherapy [4]. With Malignant gliomas are tumors of the central nervous increasing malignancy, gliomas exhibit intratumoral system originating from glial cells or their progenitors. hypoxia [5], which has been associated with poor According to the WHO classification, malignant gliomas responses to radio- or chemotherapy [6,7]. The tran- are distributed in grade-III and grade-IV tumors [1,2]. scription factor hypoxia inducible factor-1 (HIF-1), a Histologically characterized as pleomorphic, infiltrative dimer of HIF-1a and HIF-1b, is a critical mediator of tumors with microvascular proliferation and high mito- the response to hypoxia. HIF-1 governs cellular adaption tic rates, these cells show poor response to treatment to oxygen deficiency by regulating tumor-relevant genes [3]. Hence, patients with gliomas have a mean life involved in energy metabolism, angiogenesis, cell prolif- expectancy of approximately one year in clinical trials, eration and apoptosis [8-10]. Overexpression of HIF-1a promotes tumor growth, whereas the loss of HIF-1a activity dramatically decreases tumor growth, vasculari- * Correspondence: [email protected] zation and energy metabolism [11]. Suppression of HIF- Department of Radiotherapy, Faculty of Medicine, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Straße 40, 06097 Halle (Saale), Germany © 2010 Kessler et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kessler et al. BMC Cancer 2010, 10:605 Page 2 of 11 http://www.biomedcentral.com/1471-2407/10/605 1a expression via antisense oligonucleotides was were transferred for 20h to hypoxic conditions or main- reported to reduce the survival of glioblastoma cells and tained in a normoxic environment. accelerate p53-independent apoptosis [12]. In addition, CTM (Alexis Biochemicals, Germany) was dissolved in knockdown of HIF-1a by RNA interference attenuates 0.1% dimethyl sulfoxide (DMSO) for a 100 mM stock 5 2 human glioma cell growth in vivo [13]. Furthermore, solution. Cells (3 × 10 ) were seeded in 25 cm flasks 24 downregulation of HIF-1a by siRNA increased the sen- hbeforetreatment with 75 nM CTM. At thetimeof sitivity of human brain glioma cells to doxorubicin and treatment, the confluency of the monolayer was 40-50%. etoposide [14]. Different concentrations and (pre-) incubation times of HIF-1 activity can also be inhibited by chetomin CTM were analyzed in pilot experiments. Cells were (CTM), an epidithiodiketopiperazine metabolite of the treated with CTM or DMSO for 4 h at 37°C under nor- fungal species Chaetomium [15]. Treatment with CTM moxic conditions. Then cells were transferred to attenuates hypoxia-induciblegeneexpression via reduc- hypoxic conditions or maintained in a normoxic envir- tion of the HIF-1a/p300 complex. At the molecular level, onment and incubated for 20 h. CTM disrupts the interactions of the C-terminal transac- tivation domain (TADC) of HIF-1a with the CH1 Hypoxia and Irradiation domain of p300, a transcriptional coactivator [15]. Pre- Hypoxia (<1% O ) was achieved using a gas generator vious studies have revealed that HIF-1a inhibition by system as previously described [17]. Four hours after CTM significantly reduced CA9 and VEGF mRNA siRNA transfection or incubation with CTM, the flasks expression and enhances radiation response under with the treated or untreated cells were transferred into severely hypoxic conditions in human HT 1080 cells [16]. gas-proof plastic bags and cultured for 20 h under In the present study, we analyzed the inhibitory effects hypoxic conditions. of two alternative HIF-1 targeting strategies, HIF-1a- Irradiation was delivered by a linear accelerator using siRNA and CTM, on HIF-1a expression and that of its 6 MV photons with adequate bolus material at a dose target gene carbonic anhydrase 9 (CA9) in human rate of 200 MU/min. When normoxic conditions were malignant glioma cells. Further, we investigated whether applied, the cells treated with siRNA or CTM were irra- targeting HIF-1a affects the hypoxia-induced radioresis- diated with a single dose of 2-10 Gy at room tempera- tance in these tumor cells. ture. Cells under hypoxia treated with siRNA or CTM were irradiated inside a plastic bag with a single dose of Methods 2-15 Gy at room temperature. After irradiation, the cells Cell Culture Conditions and Treatments were incubated under normoxia or hypoxia at 37°C for Early-passage human glioma cell lines U251MG and 1 h before harvest for the clonogenic assay, RNA isola- U343MG (American Type Culture Collection) were tion or protein isolation. grown in RPMI 1640 medium (Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum, 1% sodium Clonogenic Assay and Radiosensitivity pyruvate, 185 U/ml penicillin and 185 μg/ml streptomycin Clonogenic Assay was performed as previously described at 37°C in a humidified atmosphere containing 3% CO . [17]. The surviving fractions were determined as ratios Gene silencing by small interfering RNA (siRNA) was of the plating efficiencies (PE = counted colonies/seeded carried out by transfection using HIF-1a-directed or cells*100) of the irradiated cells to the non-irradiated control (Luciferase GL2) double-stranded RNA oligonu- cells. The dose-modifying factor at a 10% survival level cleotides. HIF-1a and Luciferase (Lu) siRNA were (DMF ) was established to analyze the effects of siRNA synthesized by Eurofins MWG Operon (Ebersberg, or CTM on the radiosensitivity of glioma cells (DMF Germany). The target sequences are depicted in addi- = radiation dose untreated cells/radiation dose treated tional file 1: “siRNA Target Sequences”. For siRNA cells). To fit the data a linear quadratic model lnS = 5 2 2 experiments, cells (1.5 × 10 ) were seeded in 12.5 cm -(aD + bD ) was applied, using origin 7.5. flasks 24 h before treatment with siRNA. At the time of transfection, the confluency of the monolayer was 40- Quantitative Real-Time PCR 50%. Different concentrations and (pre-) incubation Cells were washed twice with ice-cold phosphate buffered times of siRNA were analyzed in pilot experiments. saline (PBS) and harvested using a cell scraper. RNA was Transfection of 75 nM siRNA in U251MG and isolated by using the RNeasy Mini Kit according to U343MG cells was carried out using Interferin (poly- the manufacturer’s instructions (QIAGEN, Hilden, plus-transfection Inc., NY, USA) following the manufac- Germany). Reverse transcription was carried out as turer’s instructions. Cells were pre-treated with siRNA described in additional file 2: “Reverse Transcription”. for 4 h at 37°C under normoxic conditions. Then cells Conditions for the quantitative real-time PCR (qRT-PCR) Kessler et al. BMC Cancer 2010, 10:605 Page 3 of 11 http://www.biomedcentral.com/1471-2407/10/605 are depicted in additional file 3: “Conditions qRT-PCR”. (p = 0.005) under normoxic or hypoxic conditions, For normalization, hypoxanthine phosphoribosyltransfer- respectively (Figure 1A). In U343MG cells, HIF-1a- ase (HPRT) was used as housekeeping gene. The primers siRNA decreased the HIF-1a mRNA levels by 52% (p= for the amplification were synthesized by TIB MOLBIOL 0.17, not significant) or 67% (p = 0.01) under normoxic (Berlin, Germany), and their sequences are depicted in or hypoxic conditions, respectively (Figure 1B). additional file 4:” Primer Sequences”. Exposure to CTM had no effect on HIF-1a mRNA expression in U251MG cells (Figure 1C). However, Western Blot Analysis treatment of U343MG cells with CTM resulted in 67% Cells were washed twice with ice-cold phosphate buf- (p = 0.002) and 56% (p = 0.029) inhibition of HIF-1a fered saline (PBS), harvested using a cell scraper and mRNA under normoxic and hypoxic conditions lysed with RIPA-buffer (50 mM Tris-HCl, 200 mM (Figure 1D). NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, Inhibition of the HIF-1 activity was investigated by 0.25% deoxycholate, protease and phosphatase inhibi- monitoring the expression of the HIF-1a target gene car- tors) 1 h after irradiation. The lysates were incubated on bonic anhydrase 9 (Figure 2). In both U251MG and ice for 20 min and centrifuged for 10 min at 13,000 rpm U343MG cells, hypoxia induced an increase in CA9 at 4°C. The supernatants were collected and protein mRNA expression. Furthermore, neither control siRNA concentrations were determined using the Bradford nor DMSO had an effect on the CA9 mRNA expression. method. Equivalent amounts of protein were resolved by Twenty-four hours after treatment, the effect of the electrophoresis in 4-12% Bis-Tris Mini Gels (Invitrogen) siRNA or CTM on the CA9 mRNA levels was analyzed and then transferred to a PVDF membrane (Roth, under normoxia and hypoxia (Figure 2). Effects of the Karlsruhe, Germany) by tank-electroblotting (Bio-Rad, HIF-1a-siRNA and CTM on HIF-1a and CA9 mRNA München, Germany). Non-specific binding was blocked levels were compared to the respective control condition, by Tris buffered saline (TBS) containing 10% nonfat i.e., cells treated with control siRNA or the solvent powdered milk at room temperature for 1 h. The mem- DMSO. Knockdown of HIF-1a by siRNA in U251MG brane was incubated with the primary antibody at the cells caused a 67% (p= 0.14, not significant) or 68% (p = appropriate dilution in TBS-10% nonfat milk at 4°C 0.06, not significant) decrease in the CA9 mRNA expres- overnight. The membrane was then washed three times sion under normoxic or hypoxic conditions (Figure 2A). with TBS and incubated with the appropriate horserad- A reduction in the CA9 mRNA expression by 57% (p = ish peroxidise-conjugated secondary antibody at room 0.12, not significant) or 38% (p = 0.01), accomplished by temperature for 1 h. The protein-antibody complexes HIF-1a knockdown, was also observed under normoxia were detected by enhanced chemiluminescence (ECL- or hypoxia in U343MG cells (Figure 2B). kit; Amersham, Freiburg, Germany). Details of the In U251MG cells, inhibition of HIF-1a activity in utilized antibodies are described in additional file response to CTM caused a 92% (p= 0.002) or 51% (p= 5:"Antibodies”. 0.18, not significant) decrease in CA9 mRNA expression undernormoxicorhypoxic conditions, respectively Statistical Analyses and Figures (Figure 2C). In U343MG cells, treatment with CTM Results are presented as means ± SD of n = 3 indepen- caused 94% (p= 0.04) and 87% (p= 0.03) decrease of dent experiments. Statistical analyses were performed by the CA9 mRNA expression under normoxic or hypoxic Student’s t-test. A p-value < 0.05 was considered signifi- conditions (Figure 2D). cant. Results of figures (1;2;4) are expressed as a percen- tage of control (75 nM control siRNA/75 nM DMSO Effects of HIF-1a-siRNA or CTM on HIF-1a, CAIX and PARP under normoxic conditions) Protein Expression in U251MG and U343MG Cells When we analyzed glioma cell lines, we found that, Results under normoxic conditions, U251MG and U343MG Effects of HIF-1a-siRNA or CTM on HIF-1a and CA9 mRNA cells expressed small amounts of HIF-1a protein (Figure Expression in U251MG and U343MG Cells 3). Under hypoxia, HIF-1a protein levels were increased In both U251MG and U343MG cells, neither hypoxia in both U251MG and U343MG cells. Treatment with nor control siRNA or DMSO had an effect on HIF-1a control siRNA or DMSO did not affect HIF-1a protein mRNA expression. Twenty-four hours after treatment, expression (Figure 3). Treatment of U251MG and the effect of the HIF-1a-siRNA or CTM on the HIF-1a U343MG cells with the HIF-1a-siRNA strongly down- mRNA levels was analyzed under normoxic and hypoxic regulated HIF-1a protein expression under normoxic conditions (Figure 1). Treatment of U251MG cells with and hypoxic conditions (Figure 3A and 3B). the HIF-1a-siRNA resulted in an inhibition of HIF-1a Exposure to CTM resulted in an increase of HIF-1a mRNA by 58% (p= 0.07, not significant) or 63% protein expression under normoxic and hypoxic Kessler et al. BMC Cancer 2010, 10:605 Page 4 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 1 Effects of HIF-1a-siRNA and CTM in U251MG and U343MG Cells: qRT-PCR of HIF-1a mRNA Levels. A (U251MG) and B (U343MG), Silencing of normoxic and hypoxic HIF-1a mRNA levels using 75 nM HIF-1a-specific siRNA in comparison with 75 nM control siRNA treated and untreated cells. C (U251MG), CTM slightly enhanced the HIF-1a mRNA expression under normoxic and hypoxic conditions. 75 nM CTM in comparison with DMSO-treated and untreated cells. D (U343MG), CTM decreased the normoxic and hypoxic HIF-1a mRNA expression. 75 nM CTM in comparison with DMSO-treated and untreated cells. conditions in U251MG cells (Figure 3C). In contrast to both U251MG and U343MG cells, irrespective of U251MG cells, the U343MG cells showed decreased levels whether cells were grown under normoxic or hypoxic of HIF-1a protein in response to CTM (Figure 3D). conditions (Figure 3A and 3B). The CAIX protein is expressed in both U251MG and In U251MG cells, inhibition of the HIF-1a transcrip- U343MG cells under normoxic conditions. In response tional activity by CTM down-regulated the normoxic to hypoxia, CAIX protein levels were substantially and hypoxic CAIX protein expression (Figure 3C). increased in both cell lines (Figure 3). HIF-1a knock- Furthermore, CTM reduced the normoxic and hypoxic down strongly decreased CAIX protein expression in CAIX protein expression in U343MG cells (Figure 3D). Kessler et al. BMC Cancer 2010, 10:605 Page 5 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 2 Effects of HIF-1a-siRNA and CTM in U251MG and U343MG Cells: qRT-PCR of CA9 mRNA Levels. A (U251MG) and B (U343MG), Silencing of HIF-1a depleted the normoxic and hypoxic CA9 mRNA levels. CA9 mRNA levels using 75 nM HIF-1a-specific siRNA as compared to 75 nM control siRNA-treated and untreated cells. C (U251MG) and D (U343MG), CTM depleted the normoxic and hypoxic expression of CA9 mRNA. 75 nM CTM in comparison with DMSO-treated and untreated cells. The proteolytic cleavage of PARP via Western blot cells (Figure 3C). Furthermore, treatment with CTM analysis qualitatively assess induction of apoptosis. had no effect on apoptosis under normoxic conditions HIF-1a knockdown using siRNA did not affect the nor- but caused induction of apoptosis under hypoxic condi- moxic expression of the large fragment of PARP, but tions in U343MG cells (Figure 3D). did cause PARP cleavage under hypoxic conditions in U251MG cells (Figure 3A). In U343MG cells, HIF-1a Effects of HIF-1a-siRNA or CTM on Clonogenic Survival knockdown induced apoptosis under normoxic and and Radiosensitivity hypoxic conditions (Figure 3B). The clonogenic survival assay is a crucial endpoint read- Exposure to CTM caused induction of apoptosis out that we used to determine the fraction of cells sur- under normoxic and hypoxic conditions in U251MG viving the treatment with the HIF-1a-siRNA or CTM Kessler et al. BMC Cancer 2010, 10:605 Page 6 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 3 Effects of HIF-1a-siRNA and CTM in U251MG and U343MG Cells: Western Blot Analyses HIF-1a, CAIX and PARP protein Levels. HIF-1a/CAIX protein expression and PARP cleavage following treatment with 75 nM HIF-1a-siRNA/CTM or 75 nM control siRNA/DMSO in comparison with untreated cells. Specificity of siRNA on HIF-1a knockdown and effects of CTM on HIF-1 activity under normoxic and hypoxic conditions. A (U251MG), Treatment with HIF-1a-siRNA caused induction of apoptosis under hypoxic conditions. CAIX expression is inhibited by HIF-1a silencing under normoxia and hypoxia. B (U343MG), Transfection with HIF-1a-siRNA caused induction of apoptosis under normoxic and hypoxic conditions. CAIX expression is attenuated by HIF-1a silencing under normoxia and hypoxia. C (U251MG), CTM enhanced the normoxic and hypoxic HIF-1a protein expression. Treatment with CTM induced apoptosis under normoxic and hypoxic conditions. CTM attenuated the normoxic and hypoxic CAIX expression. D (U343MG), Treatment with CTM decreased the normoxic and hypoxic HIF-1a protein expression. Exposure to CTM caused induction of apoptosis under hypoxic conditions. CTM attenuated the normoxic and hypoxic expression of CAIX. b-Actin served as a loading control. (Figure4)and thecombinedtreatment of HIF-1a- with HIF-1a-siRNA decreased the plating efficiency siRNA or CTM and radiation (Figure 5). under normoxic (32%) (p = 0.10, not significant) and Treatment with control siRNA or DMSO did not hypoxic (36%) (p= 0.05) conditions as compared to that affect the plating efficiency of U251MG cells as com- of the control siRNA-treated cells (Figure 4B). pared to that of untreated cells (Figure 4A and 4C). In Treatment of U251MG cells with CTM reduced the U343MG cells, a toxic effect of control siRNA was plating efficiencies under normoxic (76%) (p = 0.02) and observed when compared to the untreated cells (reduc- hypoxic (75%) (p = 0.07, not significant) conditions as tion of PE by 40% under normoxia and by 60% under compared to those the DMSO-treated cells (Figure 4C). hypoxia), whereas DMSO did not affect the plating effi- Furthermore, treatment of U343MG cells with CTM ciency of U343MG cells (Figure 4B and 4D). decreased the plating efficiency under normoxic (45%) Treatment of U251MG cells with HIF-1a-siRNA (p = 0.0008) and hypoxic (65%) (p = 0.004) conditions as reduced the plating efficiency under normoxic (80%) compared to that of the DMSO-treated cells (Figure 4D). (p= 0.04) and hypoxic (59%) (p = 0.04) conditions as The clonogenic assay was also applied to determine compared to that of the control siRNA-treated cells whether HIF-1a-siRNA or CTM sensitized glioma cells (Figure 4A). Furthermore, treatment of U343MG cells U251MG and U343MG to radiation. The viability of Kessler et al. BMC Cancer 2010, 10:605 Page 7 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 4 Effects of HIF-1a-siRNA or CTM on Clonogenic Survival in U251MG and U343MG Cells (Cytotoxicity). A (U251MG) and B (U343MG), Effects of treatment with the indicated siRNA (75 nM) on the plating efficiency of non-irradiated cells. C (U251MG) and D (U343MG), Effects of treatment with 75 nM CTM on the plating efficiency of non-irradiated cells. non-irradiated cells was set as 100%. Survival of radiosensitivity of U343MG cells under normoxic U251MG and U343MG cells increased under hypoxic (DMF :1.33, p= 0.002) and hypoxic (DMF : 1.78, p= 10 10 conditions as compared to normoxia (Figure 5). Survival 0.001) conditions (Figure 5B), respectively. curves revealed that, although HIF-1a-siRNA only ThesurvivalcurvesindicatethatexposuretoCTM slightly diminished the cytotoxic effect of radiation on significantly enhanced the effect of radiation in U251MG cells under normoxic (DMF : 0.86, p = 0.04) U251MG cells under normoxic (DMF : 1.35, p = 0.001) 10 10 conditions, it significantly enhanced radiation sensitivity and hypoxic (DMF : 1.18, p = 0.04) conditions (Figure of U251MG cells under hypoxic conditions (DMF : 5C). Treatment with CTM did not affect radiosensitivity 1.35, p= 0.002) (Figure 5A). Further, treatment with of U343MG cells under normoxic (DMF :1.02, p = HIF-1a-siRNA resulted in a significantly enhanced 0.01) conditions, but resulted in significant enhanced Kessler et al. BMC Cancer 2010, 10:605 Page 8 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 5 Effects of HIF-1a-siRNA or CTM on Response to Radiation of U251MG and U343MG Cells (Radiosensitivity). A (U251MG) and B (U343MG), Clonogenic survival following treatment with the indicated siRNA (75 nM) and radiation under normoxia (0-10 Gy) and hypoxia (0- 15 Gy). HIF-1a-siRNA decreased the hypoxia-induced radioresistance of U251MG and U343MG cells. Under normoxia, HIF-1a-siRNA reduces the response to irradiation in U251MG cells and enhanced the effect of radiation in U343MG cells. C (U251MG) and D (U343MG), Clonogenic survival following exposure to 75 nM CTM and radiation under normoxia (0-10 Gy) and hypoxia (0-15 Gy). Exposure to CTM decreased hypoxia- induced radioresistance of U251MG and U343MG cells. CTM enhanced the effect of radiation in U251MG cells and reduced the response to irradiation in U343MG cells under normoxia. radiosensitivity under hypoxic (DMF :1.48, p =0.02) and disease progression [18-20]. Additionally, the malig- conditions (Figure 5D). nancy of brain tumors is associated with increased intra- tumoral hypoxia [5]. Discussion In the present study, we analyzed the effects of HIF- In most human cancers including malignant glioma, 1a inhibition by two alternative strategies, siRNA target- HIF-1a expression promotes tumor growth, angiogenesis ing of HIF-1a or CTM, on the radioresistance of two Kessler et al. BMC Cancer 2010, 10:605 Page 9 of 11 http://www.biomedcentral.com/1471-2407/10/605 malignant glioma cell lines. The glioma cell lines Results suggest that inhibition of HIF-1a decreased U251MG and U343MG displayed comparable HIF-1a tumor growth in malignant gliomas [24]. Additionally, mRNA expression levels under normoxic and hypoxic Dai et al. showed oxygen-independent cytotoxicity and conditions. U251MG and U343MG exhibited a weak p53-independent apoptosis by HIF-1a inhibition in glio- HIF-1a protein expression under normoxia with an blastoma cells [12]. In the present study, Western blot increase in HIF-1a protein, CA9 mRNA and CAIX pro- analyses of the cleavage of PARP confirm these findings. tein levels under hypoxia. Our previous studies using Furthermore, treatment of U251MG and U343MG cells the glioma cell line U87MG supported the potential use- with HIF-1a-siRNA or CTM reduced the plating effi- fulness of HIF-1a as an endogenous marker of tumor ciency under normoxic and hypoxic conditions as com- hypoxia and of resulting radioresistance of human pared to that of the control siRNA- or DMSO-treated tumor cells [21,22]. Furthermore, CAIX was found to be cells. a suitable marker of current or previous chronic hypoxia In the literature, hypoxia and HIF-1a-associated resis- andwepreviouslyproposedthatthe amount of CAIX- tance to radiotherapy and chemotherapy is discussed. positive cells in a tumor cell suspension may be related HIF-1a expression is a significant prognostic indicator to the radiosensitivity of this tumor in vivo [23]. Inhibi- in glial brain tumors treated by radiotherapy [18,25]. tion of the HIF-1 pathway by siRNA or CTM was per- Furthermore, HIF-1a exhibits an attractive target to formed by quantitative real-time PCR and Western blot overcome hypoxia-related radioresistance of tumors analyses. We showed that HIF-1 targeting via HIF-1a- in vitro and in vivo [26]. Silencing of HIF-1a has been siRNA effectively reduced the hypoxia-induced HIF-1a shown to inhibit hypoxia-mediated cell migration and protein levels of U251MG and U343MG cells. In addi- invasion in in vitro and in vivo malignant glioma mod- tion, exposure to CTM resulted in an increased HIF-1a els [13,27,28]. Further, silencing of HIF-1a by siRNA protein expression in U251MG cells and decreased sensitized glioma cells to the chemotherapeutic agents levels of HIF-1a proteininU343MGcells undernor- doxorubicin and etoposide [14]. This is the first study moxic and hypoxic conditions. CTM is an inhibitor of showing that inhibition of HIF-1a by siRNA or by the HIF-1a/p300 complex. CTM only interrupts the CTM under hypoxic conditions resulted in a signifi- transcriptional activity of HIF-1a. Thus, it diminishes cantly enhanced radiosensitivity of the human glioma the expression of HIF-1a target genes. U251MG cells cells. Recently, HIF-1a inhibition by CTM effectively responded as expected because CTM has no influence reduced hypoxia-dependent transcription and sensitized on the expression of HIF-1a mRNA. In U251MG cells, hypoxic HT 1080 human fibrosarcoma cells in vitro to treatment with CTM resulted in a slight increase of radiation [16]. HIF-1a protein stabilisation but attenuated the HIF-1a Although inhibition of HIF-1a under hypoxic condi- induced expression of CAIX. One possible explanation tions enhanced radiosensitivity of glioma cells U251MG for the slightly increased HIF-1a proteinexpressionin and U343MG, the effects of HIF-1a inhibition on radio- these cells might be that CTM attenuated the hypoxic sensitivity under normoxic conditions were not obvious. answer. So an auto regulatory feedback loop caused this In agreement with our results, Palayoor et al.showed stabilisation of HIF-1a. Moreover, another explanation that effects of HIF-1a inhibitor PX-478 on radiosensitity could be that CTM also inhibits the interaction of HIF- under normoxic conditions were less dramatic than 1a andVHL.Inthis, wayanunspecificsideeffectof under hypoxic conditions in prostate carcinoma cells CMT would inhibit the degradation of HIF-1a. [29]. Further studies have shown that HIF inhibitors are U343MG cell line didn’t respond as expected. In more potent in attenuation of HIF-dependent transcrip- U343MG cells CTM has an effect on the HIF-1a tional activity under hypoxia than under normoxia expression and on the target gene CAIX. One reason [30,31]. Further investigations are necessary to study the for that might have been that the applied dose of CTM normoxic expression of HIF-1a in malignant glioma cell in the lower grade 3 glioma cells line U343MG causes lines. side effects. Further, both cell lines differ in their Indeed, siRNA and CTM showed different impacts on genetic status and regulation of different pathways and the effects of radiation in the indicated cell lines under consequently in their disease aggressiveness and normoxia, as HIF-1a-siRNA reduces the response to response to treatment. Additionally, CTM is a small irradiation in U251MG cells, whereas in U343MG cells, molecule and the specificity for the target of small siRNA enhanced the effects of radiation. Furthermore, molecule is debatable and there might be another under normoxia, CTM enhanced the effects of radiation mechanism of CTM play a role. Furthermore, our data in U251MG cells, whereas in U343MG cells, CTM had indicate that monitoring CA9 mRNA and CAIX protein no effect on the response to irradiation under normoxia. expression is a means to verify the inhibition of HIF-1 The U343MG cell line originated from a de novo grade- activity. III anaplastic astrocytoma and U251MG was established Kessler et al. BMC Cancer 2010, 10:605 Page 10 of 11 http://www.biomedcentral.com/1471-2407/10/605 Authors’ contributions from a de novo grade-IV glioblastoma [1,2,32]. Thus, dif- JK designed the study, performed experimental procedures, analyzed the ferent genes involved in cellular pathways regulating data and drafted the manuscript. AH HW SR and MK aided in study design apoptosis, proliferation, and DNA repair could carry and reviewed the manuscript. MB analyzed the data and reviewed the manuscript. DV designed the study, performed experimental procedures, mutations or deletions in these cells. Both cell lines differ analyzed the data and drafted the manuscript. All authors read and in their genetic status of p53 and consequently in their approved the final manuscript. disease aggressiveness and response to treatment. Competing interests U343MG cells carry wild-type p53, whereas U251MG The authors declare that they have no competing interests. carry mutated p53 [32]. Hence, the ability to respond to radiation may differ in U251MG and U343MG cells. Received: 21 May 2010 Accepted: 4 November 2010 Published: 4 November 2010 Additionally, compared to HIF-1a-specific siRNA, CTM has a different mode of action. References However, the function of CTM is not fully understood. 1. Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, CTM is a member of the epidithiodiketopiperazine (ETP) Cavenee WK: The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002, 61:215-25, discussion 226-9. family. Previous studies have shown that ETPs react with 2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, p300 and cause zinc ion ejection. Additionally, the results Scheithauer BW, Kleihues P: The 2007 WHO classification of tumours of of Cook et al. suggest that ETPs also interact with other the central nervous system. Acta Neuropathol 2007, 114:97-109. 3. Burger PC, Vogel FS, Green SB, Strike TA: Glioblastoma multiforme and zinc ion binding proteins. In addition, it is likely that ETPs anaplastic astrocytoma. Pathologic criteria and prognostic implications. like CTM operate by more than one mechanism in cells Cancer 1985, 56:1106-11. and that CTM had an impact on transcription factors rele- 4. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, et al: Effects of radiotherapy vant to radiosensitivity [33]. with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year Conclusions analysis of the EORTC-NCIC trial. Lancet Oncol 2009, 10:459-66. 5. 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In conclusion, our results suggest that inhi- marker in premenopausal breast cancer patients with one to three bition of HIF-1a is a promising strategy to reduce cell positive lymph nodes and a putative marker of radiation resistance. Clin survival and to enhance the response of malignant glio- Cancer Res 2006, 12:6421-31. 8. Semenza GL: Involvement of hypoxia-inducible factor 1 in human cancer. mas to radiotherapy. Intern Med 2002, 41:79-83. 9. Bracken CP, Whitelaw ML, Peet DJ: The hypoxia-inducible factors: key Additional material transcriptional regulators of hypoxic responses. Cell Mol Life Sci 2003, 60:1376-93. 10. Goda N, Dozier SJ, Johnson RS: HIF-1 in cell cycle regulation, apoptosis, Additional file 1: siRNA Target Sequences. The file contains the target and tumor progression. Antioxid Redox Signal 2003, 5:467-73. sequences of the utilized siRNA. 11. Powis G, Kirkpatrick L: Hypoxia inducible factor-1alpha as a cancer drug Additional file 2: Reverse Transcription. The file contains the target. Mol Cancer Ther 2004, 3:647-54. conditions for the reverse transcription. 12. Dai S, Huang ML, Hsu CY, Chao KS: Inhibition of hypoxia inducible factor 1alpha causes oxygen-independent cytotoxicity and induces p53 Additional file 3: Conditions qRT-PCR. The file contains the conditions independent apoptosis in glioblastoma cells. Int J Radiat Oncol Biol Phys for the qRT-PCR. 2003, 55:1027-36. Additional file 4: Primer Sequences. The file contains the sequences of 13. Gillespie DL, Whang K, Ragel BT, Flynn JR, Kelly DA, Jensen RL: Silencing of the applied primer. hypoxia inducible factor-1alpha by RNA interference attenuates human Additional file 5: Antibodies. The file contains the details of the utilized glioma cell growth in vivo. Clin Cancer Res 2007, 13:2441-8. antibodies. 14. Chen L, Feng P, Li S, Long D, Cheng J, Lu Y, Zhou D: Effect of hypoxia- inducible factor-1alpha silencing on the sensitivity of human brain glioma cells to doxorubicin and etoposide. Neurochem Res 2009, 34:984-90. 15. Kung AL, Zabludoff SD, France DS, Freedman SJ, Tanner EA, Vieira A, Abbreviations Cornell-Kennon S, Lee J, Wang B, Wang J, et al: Small molecule blockade UT: untreated cells; control: control siRNA (luciferase) treated cells; HIF-1a: of transcriptional coactivation of the hypoxia-inducible factor pathway. HIF-1a-siRNA treated cells; DMSO: DMSO treated cells; CTM: CTM treated Cancer Cell 2004, 6:33-43. cells. 16. Staab A, Loeffler J, Said HM, Diehlmann D, Katzer A, Beyer M, Fleischer M, Schwab F, Baier K, Einsele H, et al: Effects of HIF-1 inhibition by chetomin Acknowledgements on hypoxia-related transcription and radiosensitivity in HT 1080 human This work was supported in part by a grant from the Deutsche fibrosarcoma cells. BMC Cancer 2007, 7:213. Forschungsgemeinschaft (DFG VO 871/2-3) to DV. We thank Dr. Thomas Greither for the helpful discussions. Kessler et al. BMC Cancer 2010, 10:605 Page 11 of 11 http://www.biomedcentral.com/1471-2407/10/605 17. Kappler M, Rot S, Taubert H, Greither T, Bartel F, Dellas K, Hansgen G, Pre-publication history Trott KR, Bache M: The effects of knockdown of wild-type survivin, The pre-publication history for this paper can be accessed here: survivin-2B or survivin-delta3 on the radiosensitization in a soft tissue http://www.biomedcentral.com/1471-2407/10/605/prepub sarcoma cells in vitro under different oxygen conditions. Cancer Gene doi:10.1186/1471-2407-10-605 Ther 2007, 14:994-1001. Cite this article as: Kessler et al.: HIF-1a inhibition by siRNA or chetomin 18. 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Int J Radiat Oncol Biol Phys 2003, 56:1184-93. 22. Vordermark D, Brown JM: Endogenous markers of tumor hypoxia predictors of clinical radiation resistance? Strahlenther Onkol 2003, 179:801-11. 23. Vordermark D, Kaffer A, Riedl S, Katzer A, Flentje M: Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells. Int J Radiat Oncol Biol Phys 2005, 61:1197-207. 24. Jensen RL, Ragel BT, Whang K, Gillespie D: Inhibition of hypoxia inducible factor-1alpha (HIF-1alpha) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas. J Neurooncol 2006, 78:233-47. 25. Korkolopoulou P, Patsouris E, Konstantinidou AE, Pavlopoulos PM, Kavantzas N, Boviatsis E, Thymara I, Perdiki M, Thomas-Tsagli E, Angelidakis D, et al: Hypoxia-inducible factor 1alpha/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis. Neuropathol Appl Neurobiol 2004, 30:267-78. 26. Bache M, Kappler M, Said HM, Staab A, Vordermark D: Detection and specific targeting of hypoxic regions within solid tumors: current preclinical and clinical strategies. Curr Med Chem 2008, 15:322-38. 27. Fujiwara S, Nakagawa K, Harada H, Nagato S, Furukawa K, Teraoka M, Seno T, Oka K, Iwata S, Ohnishi T: Silencing hypoxia-inducible factor- 1alpha inhibits cell migration and invasion under hypoxic environment in malignant gliomas. Int J Oncol 2007, 30:793-802. 28. Ali MA, Reis A, Ding LH, Story MD, Habib AA, Chattopadhyay A, Saha D: SNS-032 prevents hypoxia-mediated glioblastoma cell invasion by inhibiting hypoxia inducible factor-1alpha expression. Int J Oncol 2009, 34:1051-60. 29. Palayoor ST, Mitchell JB, Cerna D, Degraff W, John-Aryankalayil M, Coleman CN: PX-478, an inhibitor of hypoxia-inducible factor-1alpha, enhances radiosensitivity of prostate carcinoma cells. Int J Cancer 2008, 123:2430-7. 30. Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, Melillo G: Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 2002, 62:4316-24. 31. Zhong H, Willard M, Simons J: NS398 reduces hypoxia-inducible factor (HIF)-1alpha and HIF-1 activity: multiple-level effects involving cyclooxygenase-2 dependent and independent mechanisms. Int J Cancer 2004, 112:585-95. Submit your next manuscript to BioMed Central 32. Ishii N, Maier D, Merlo A, Tada M, Sawamura Y, Diserens AC, Van Meir EG: and take full advantage of: Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol 1999, 9:469-79. • Convenient online submission 33. Cook KM, Hilton ST, Mecinovic J, Motherwell WB, Figg WD, Schofield CJ: Epidithiodiketopiperazines block the interaction between hypoxia- • Thorough peer review inducible factor-1alpha (HIF-1alpha) and p300 by a zinc ejection • No space constraints or color figure charges mechanism. J Biol Chem 2009, 284:26831-8. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic radioresistance and monitoring via CA9 expression

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Springer Journals
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Copyright © 2010 by Kessler et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine general; Medicine/Public Health, general
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1471-2407
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10.1186/1471-2407-10-605
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21050481
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Abstract

Background: Hypoxia induces activation of the HIF-1 pathway and is an essential characteristic of malignant gliomas. Hypoxia has been linked to tumor progression, therapy resistance and poor prognosis. However, little is known about the impact of HIF-1a inhibition on radioresistance of malignant glioma. Methods: In this study, we investigated the effects of the inhibition of HIF-1a on cell survival and radiosensitivity in U251MG and U343MG glioma cells, using two different strategies. HIF-1a inhibition was achieved by siRNA targeting of HIF-1a or via chetomin, a disruptor of interactions between HIF-1a and p300. The inhibition of the HIF-1 pathway was monitored by quantitative real-time PCR and Western blot analyses of the expression levels of HIF-1a and CA9. CA9 expression was investigated as a potential indicator of the efficacy of HIF-1 inhibition and the resulting radiosensitivity of malignant glioma cell lines was determined by clonogenic assay after irradiation under normoxic (2-10 Gy) or hypoxic (2-15 Gy) conditions. Results: Although siRNA and chetomin show distinct modes of action, both attenuated the hypoxia-induced radioresistance of malignant glioma cell lines U251MG (DMF : 1.35 and 1.18) and U343MG (DMF : 1.78 and 1.48). 10 10 However, siRNA and chetomin showed diverse effects on radiosensitivity under normoxic conditions in U251MG (DMF : 0.86 and 1.35) and U343MG (DMF : 1.33 and 1.02) cells. 10 10 Conclusions: Results from this in vitro study suggest that inhibition of HIF-1a is a promising strategy to sensitize human malignant gliomas to radiotherapy and that CA9 could serve as an indicator of effective HIF-1-related radiosensitization. Background despite surgery, chemo- and radiotherapy [4]. With Malignant gliomas are tumors of the central nervous increasing malignancy, gliomas exhibit intratumoral system originating from glial cells or their progenitors. hypoxia [5], which has been associated with poor According to the WHO classification, malignant gliomas responses to radio- or chemotherapy [6,7]. The tran- are distributed in grade-III and grade-IV tumors [1,2]. scription factor hypoxia inducible factor-1 (HIF-1), a Histologically characterized as pleomorphic, infiltrative dimer of HIF-1a and HIF-1b, is a critical mediator of tumors with microvascular proliferation and high mito- the response to hypoxia. HIF-1 governs cellular adaption tic rates, these cells show poor response to treatment to oxygen deficiency by regulating tumor-relevant genes [3]. Hence, patients with gliomas have a mean life involved in energy metabolism, angiogenesis, cell prolif- expectancy of approximately one year in clinical trials, eration and apoptosis [8-10]. Overexpression of HIF-1a promotes tumor growth, whereas the loss of HIF-1a activity dramatically decreases tumor growth, vasculari- * Correspondence: [email protected] zation and energy metabolism [11]. Suppression of HIF- Department of Radiotherapy, Faculty of Medicine, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Straße 40, 06097 Halle (Saale), Germany © 2010 Kessler et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kessler et al. BMC Cancer 2010, 10:605 Page 2 of 11 http://www.biomedcentral.com/1471-2407/10/605 1a expression via antisense oligonucleotides was were transferred for 20h to hypoxic conditions or main- reported to reduce the survival of glioblastoma cells and tained in a normoxic environment. accelerate p53-independent apoptosis [12]. In addition, CTM (Alexis Biochemicals, Germany) was dissolved in knockdown of HIF-1a by RNA interference attenuates 0.1% dimethyl sulfoxide (DMSO) for a 100 mM stock 5 2 human glioma cell growth in vivo [13]. Furthermore, solution. Cells (3 × 10 ) were seeded in 25 cm flasks 24 downregulation of HIF-1a by siRNA increased the sen- hbeforetreatment with 75 nM CTM. At thetimeof sitivity of human brain glioma cells to doxorubicin and treatment, the confluency of the monolayer was 40-50%. etoposide [14]. Different concentrations and (pre-) incubation times of HIF-1 activity can also be inhibited by chetomin CTM were analyzed in pilot experiments. Cells were (CTM), an epidithiodiketopiperazine metabolite of the treated with CTM or DMSO for 4 h at 37°C under nor- fungal species Chaetomium [15]. Treatment with CTM moxic conditions. Then cells were transferred to attenuates hypoxia-induciblegeneexpression via reduc- hypoxic conditions or maintained in a normoxic envir- tion of the HIF-1a/p300 complex. At the molecular level, onment and incubated for 20 h. CTM disrupts the interactions of the C-terminal transac- tivation domain (TADC) of HIF-1a with the CH1 Hypoxia and Irradiation domain of p300, a transcriptional coactivator [15]. Pre- Hypoxia (<1% O ) was achieved using a gas generator vious studies have revealed that HIF-1a inhibition by system as previously described [17]. Four hours after CTM significantly reduced CA9 and VEGF mRNA siRNA transfection or incubation with CTM, the flasks expression and enhances radiation response under with the treated or untreated cells were transferred into severely hypoxic conditions in human HT 1080 cells [16]. gas-proof plastic bags and cultured for 20 h under In the present study, we analyzed the inhibitory effects hypoxic conditions. of two alternative HIF-1 targeting strategies, HIF-1a- Irradiation was delivered by a linear accelerator using siRNA and CTM, on HIF-1a expression and that of its 6 MV photons with adequate bolus material at a dose target gene carbonic anhydrase 9 (CA9) in human rate of 200 MU/min. When normoxic conditions were malignant glioma cells. Further, we investigated whether applied, the cells treated with siRNA or CTM were irra- targeting HIF-1a affects the hypoxia-induced radioresis- diated with a single dose of 2-10 Gy at room tempera- tance in these tumor cells. ture. Cells under hypoxia treated with siRNA or CTM were irradiated inside a plastic bag with a single dose of Methods 2-15 Gy at room temperature. After irradiation, the cells Cell Culture Conditions and Treatments were incubated under normoxia or hypoxia at 37°C for Early-passage human glioma cell lines U251MG and 1 h before harvest for the clonogenic assay, RNA isola- U343MG (American Type Culture Collection) were tion or protein isolation. grown in RPMI 1640 medium (Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum, 1% sodium Clonogenic Assay and Radiosensitivity pyruvate, 185 U/ml penicillin and 185 μg/ml streptomycin Clonogenic Assay was performed as previously described at 37°C in a humidified atmosphere containing 3% CO . [17]. The surviving fractions were determined as ratios Gene silencing by small interfering RNA (siRNA) was of the plating efficiencies (PE = counted colonies/seeded carried out by transfection using HIF-1a-directed or cells*100) of the irradiated cells to the non-irradiated control (Luciferase GL2) double-stranded RNA oligonu- cells. The dose-modifying factor at a 10% survival level cleotides. HIF-1a and Luciferase (Lu) siRNA were (DMF ) was established to analyze the effects of siRNA synthesized by Eurofins MWG Operon (Ebersberg, or CTM on the radiosensitivity of glioma cells (DMF Germany). The target sequences are depicted in addi- = radiation dose untreated cells/radiation dose treated tional file 1: “siRNA Target Sequences”. For siRNA cells). To fit the data a linear quadratic model lnS = 5 2 2 experiments, cells (1.5 × 10 ) were seeded in 12.5 cm -(aD + bD ) was applied, using origin 7.5. flasks 24 h before treatment with siRNA. At the time of transfection, the confluency of the monolayer was 40- Quantitative Real-Time PCR 50%. Different concentrations and (pre-) incubation Cells were washed twice with ice-cold phosphate buffered times of siRNA were analyzed in pilot experiments. saline (PBS) and harvested using a cell scraper. RNA was Transfection of 75 nM siRNA in U251MG and isolated by using the RNeasy Mini Kit according to U343MG cells was carried out using Interferin (poly- the manufacturer’s instructions (QIAGEN, Hilden, plus-transfection Inc., NY, USA) following the manufac- Germany). Reverse transcription was carried out as turer’s instructions. Cells were pre-treated with siRNA described in additional file 2: “Reverse Transcription”. for 4 h at 37°C under normoxic conditions. Then cells Conditions for the quantitative real-time PCR (qRT-PCR) Kessler et al. BMC Cancer 2010, 10:605 Page 3 of 11 http://www.biomedcentral.com/1471-2407/10/605 are depicted in additional file 3: “Conditions qRT-PCR”. (p = 0.005) under normoxic or hypoxic conditions, For normalization, hypoxanthine phosphoribosyltransfer- respectively (Figure 1A). In U343MG cells, HIF-1a- ase (HPRT) was used as housekeeping gene. The primers siRNA decreased the HIF-1a mRNA levels by 52% (p= for the amplification were synthesized by TIB MOLBIOL 0.17, not significant) or 67% (p = 0.01) under normoxic (Berlin, Germany), and their sequences are depicted in or hypoxic conditions, respectively (Figure 1B). additional file 4:” Primer Sequences”. Exposure to CTM had no effect on HIF-1a mRNA expression in U251MG cells (Figure 1C). However, Western Blot Analysis treatment of U343MG cells with CTM resulted in 67% Cells were washed twice with ice-cold phosphate buf- (p = 0.002) and 56% (p = 0.029) inhibition of HIF-1a fered saline (PBS), harvested using a cell scraper and mRNA under normoxic and hypoxic conditions lysed with RIPA-buffer (50 mM Tris-HCl, 200 mM (Figure 1D). NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, Inhibition of the HIF-1 activity was investigated by 0.25% deoxycholate, protease and phosphatase inhibi- monitoring the expression of the HIF-1a target gene car- tors) 1 h after irradiation. The lysates were incubated on bonic anhydrase 9 (Figure 2). In both U251MG and ice for 20 min and centrifuged for 10 min at 13,000 rpm U343MG cells, hypoxia induced an increase in CA9 at 4°C. The supernatants were collected and protein mRNA expression. Furthermore, neither control siRNA concentrations were determined using the Bradford nor DMSO had an effect on the CA9 mRNA expression. method. Equivalent amounts of protein were resolved by Twenty-four hours after treatment, the effect of the electrophoresis in 4-12% Bis-Tris Mini Gels (Invitrogen) siRNA or CTM on the CA9 mRNA levels was analyzed and then transferred to a PVDF membrane (Roth, under normoxia and hypoxia (Figure 2). Effects of the Karlsruhe, Germany) by tank-electroblotting (Bio-Rad, HIF-1a-siRNA and CTM on HIF-1a and CA9 mRNA München, Germany). Non-specific binding was blocked levels were compared to the respective control condition, by Tris buffered saline (TBS) containing 10% nonfat i.e., cells treated with control siRNA or the solvent powdered milk at room temperature for 1 h. The mem- DMSO. Knockdown of HIF-1a by siRNA in U251MG brane was incubated with the primary antibody at the cells caused a 67% (p= 0.14, not significant) or 68% (p = appropriate dilution in TBS-10% nonfat milk at 4°C 0.06, not significant) decrease in the CA9 mRNA expres- overnight. The membrane was then washed three times sion under normoxic or hypoxic conditions (Figure 2A). with TBS and incubated with the appropriate horserad- A reduction in the CA9 mRNA expression by 57% (p = ish peroxidise-conjugated secondary antibody at room 0.12, not significant) or 38% (p = 0.01), accomplished by temperature for 1 h. The protein-antibody complexes HIF-1a knockdown, was also observed under normoxia were detected by enhanced chemiluminescence (ECL- or hypoxia in U343MG cells (Figure 2B). kit; Amersham, Freiburg, Germany). Details of the In U251MG cells, inhibition of HIF-1a activity in utilized antibodies are described in additional file response to CTM caused a 92% (p= 0.002) or 51% (p= 5:"Antibodies”. 0.18, not significant) decrease in CA9 mRNA expression undernormoxicorhypoxic conditions, respectively Statistical Analyses and Figures (Figure 2C). In U343MG cells, treatment with CTM Results are presented as means ± SD of n = 3 indepen- caused 94% (p= 0.04) and 87% (p= 0.03) decrease of dent experiments. Statistical analyses were performed by the CA9 mRNA expression under normoxic or hypoxic Student’s t-test. A p-value < 0.05 was considered signifi- conditions (Figure 2D). cant. Results of figures (1;2;4) are expressed as a percen- tage of control (75 nM control siRNA/75 nM DMSO Effects of HIF-1a-siRNA or CTM on HIF-1a, CAIX and PARP under normoxic conditions) Protein Expression in U251MG and U343MG Cells When we analyzed glioma cell lines, we found that, Results under normoxic conditions, U251MG and U343MG Effects of HIF-1a-siRNA or CTM on HIF-1a and CA9 mRNA cells expressed small amounts of HIF-1a protein (Figure Expression in U251MG and U343MG Cells 3). Under hypoxia, HIF-1a protein levels were increased In both U251MG and U343MG cells, neither hypoxia in both U251MG and U343MG cells. Treatment with nor control siRNA or DMSO had an effect on HIF-1a control siRNA or DMSO did not affect HIF-1a protein mRNA expression. Twenty-four hours after treatment, expression (Figure 3). Treatment of U251MG and the effect of the HIF-1a-siRNA or CTM on the HIF-1a U343MG cells with the HIF-1a-siRNA strongly down- mRNA levels was analyzed under normoxic and hypoxic regulated HIF-1a protein expression under normoxic conditions (Figure 1). Treatment of U251MG cells with and hypoxic conditions (Figure 3A and 3B). the HIF-1a-siRNA resulted in an inhibition of HIF-1a Exposure to CTM resulted in an increase of HIF-1a mRNA by 58% (p= 0.07, not significant) or 63% protein expression under normoxic and hypoxic Kessler et al. BMC Cancer 2010, 10:605 Page 4 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 1 Effects of HIF-1a-siRNA and CTM in U251MG and U343MG Cells: qRT-PCR of HIF-1a mRNA Levels. A (U251MG) and B (U343MG), Silencing of normoxic and hypoxic HIF-1a mRNA levels using 75 nM HIF-1a-specific siRNA in comparison with 75 nM control siRNA treated and untreated cells. C (U251MG), CTM slightly enhanced the HIF-1a mRNA expression under normoxic and hypoxic conditions. 75 nM CTM in comparison with DMSO-treated and untreated cells. D (U343MG), CTM decreased the normoxic and hypoxic HIF-1a mRNA expression. 75 nM CTM in comparison with DMSO-treated and untreated cells. conditions in U251MG cells (Figure 3C). In contrast to both U251MG and U343MG cells, irrespective of U251MG cells, the U343MG cells showed decreased levels whether cells were grown under normoxic or hypoxic of HIF-1a protein in response to CTM (Figure 3D). conditions (Figure 3A and 3B). The CAIX protein is expressed in both U251MG and In U251MG cells, inhibition of the HIF-1a transcrip- U343MG cells under normoxic conditions. In response tional activity by CTM down-regulated the normoxic to hypoxia, CAIX protein levels were substantially and hypoxic CAIX protein expression (Figure 3C). increased in both cell lines (Figure 3). HIF-1a knock- Furthermore, CTM reduced the normoxic and hypoxic down strongly decreased CAIX protein expression in CAIX protein expression in U343MG cells (Figure 3D). Kessler et al. BMC Cancer 2010, 10:605 Page 5 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 2 Effects of HIF-1a-siRNA and CTM in U251MG and U343MG Cells: qRT-PCR of CA9 mRNA Levels. A (U251MG) and B (U343MG), Silencing of HIF-1a depleted the normoxic and hypoxic CA9 mRNA levels. CA9 mRNA levels using 75 nM HIF-1a-specific siRNA as compared to 75 nM control siRNA-treated and untreated cells. C (U251MG) and D (U343MG), CTM depleted the normoxic and hypoxic expression of CA9 mRNA. 75 nM CTM in comparison with DMSO-treated and untreated cells. The proteolytic cleavage of PARP via Western blot cells (Figure 3C). Furthermore, treatment with CTM analysis qualitatively assess induction of apoptosis. had no effect on apoptosis under normoxic conditions HIF-1a knockdown using siRNA did not affect the nor- but caused induction of apoptosis under hypoxic condi- moxic expression of the large fragment of PARP, but tions in U343MG cells (Figure 3D). did cause PARP cleavage under hypoxic conditions in U251MG cells (Figure 3A). In U343MG cells, HIF-1a Effects of HIF-1a-siRNA or CTM on Clonogenic Survival knockdown induced apoptosis under normoxic and and Radiosensitivity hypoxic conditions (Figure 3B). The clonogenic survival assay is a crucial endpoint read- Exposure to CTM caused induction of apoptosis out that we used to determine the fraction of cells sur- under normoxic and hypoxic conditions in U251MG viving the treatment with the HIF-1a-siRNA or CTM Kessler et al. BMC Cancer 2010, 10:605 Page 6 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 3 Effects of HIF-1a-siRNA and CTM in U251MG and U343MG Cells: Western Blot Analyses HIF-1a, CAIX and PARP protein Levels. HIF-1a/CAIX protein expression and PARP cleavage following treatment with 75 nM HIF-1a-siRNA/CTM or 75 nM control siRNA/DMSO in comparison with untreated cells. Specificity of siRNA on HIF-1a knockdown and effects of CTM on HIF-1 activity under normoxic and hypoxic conditions. A (U251MG), Treatment with HIF-1a-siRNA caused induction of apoptosis under hypoxic conditions. CAIX expression is inhibited by HIF-1a silencing under normoxia and hypoxia. B (U343MG), Transfection with HIF-1a-siRNA caused induction of apoptosis under normoxic and hypoxic conditions. CAIX expression is attenuated by HIF-1a silencing under normoxia and hypoxia. C (U251MG), CTM enhanced the normoxic and hypoxic HIF-1a protein expression. Treatment with CTM induced apoptosis under normoxic and hypoxic conditions. CTM attenuated the normoxic and hypoxic CAIX expression. D (U343MG), Treatment with CTM decreased the normoxic and hypoxic HIF-1a protein expression. Exposure to CTM caused induction of apoptosis under hypoxic conditions. CTM attenuated the normoxic and hypoxic expression of CAIX. b-Actin served as a loading control. (Figure4)and thecombinedtreatment of HIF-1a- with HIF-1a-siRNA decreased the plating efficiency siRNA or CTM and radiation (Figure 5). under normoxic (32%) (p = 0.10, not significant) and Treatment with control siRNA or DMSO did not hypoxic (36%) (p= 0.05) conditions as compared to that affect the plating efficiency of U251MG cells as com- of the control siRNA-treated cells (Figure 4B). pared to that of untreated cells (Figure 4A and 4C). In Treatment of U251MG cells with CTM reduced the U343MG cells, a toxic effect of control siRNA was plating efficiencies under normoxic (76%) (p = 0.02) and observed when compared to the untreated cells (reduc- hypoxic (75%) (p = 0.07, not significant) conditions as tion of PE by 40% under normoxia and by 60% under compared to those the DMSO-treated cells (Figure 4C). hypoxia), whereas DMSO did not affect the plating effi- Furthermore, treatment of U343MG cells with CTM ciency of U343MG cells (Figure 4B and 4D). decreased the plating efficiency under normoxic (45%) Treatment of U251MG cells with HIF-1a-siRNA (p = 0.0008) and hypoxic (65%) (p = 0.004) conditions as reduced the plating efficiency under normoxic (80%) compared to that of the DMSO-treated cells (Figure 4D). (p= 0.04) and hypoxic (59%) (p = 0.04) conditions as The clonogenic assay was also applied to determine compared to that of the control siRNA-treated cells whether HIF-1a-siRNA or CTM sensitized glioma cells (Figure 4A). Furthermore, treatment of U343MG cells U251MG and U343MG to radiation. The viability of Kessler et al. BMC Cancer 2010, 10:605 Page 7 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 4 Effects of HIF-1a-siRNA or CTM on Clonogenic Survival in U251MG and U343MG Cells (Cytotoxicity). A (U251MG) and B (U343MG), Effects of treatment with the indicated siRNA (75 nM) on the plating efficiency of non-irradiated cells. C (U251MG) and D (U343MG), Effects of treatment with 75 nM CTM on the plating efficiency of non-irradiated cells. non-irradiated cells was set as 100%. Survival of radiosensitivity of U343MG cells under normoxic U251MG and U343MG cells increased under hypoxic (DMF :1.33, p= 0.002) and hypoxic (DMF : 1.78, p= 10 10 conditions as compared to normoxia (Figure 5). Survival 0.001) conditions (Figure 5B), respectively. curves revealed that, although HIF-1a-siRNA only ThesurvivalcurvesindicatethatexposuretoCTM slightly diminished the cytotoxic effect of radiation on significantly enhanced the effect of radiation in U251MG cells under normoxic (DMF : 0.86, p = 0.04) U251MG cells under normoxic (DMF : 1.35, p = 0.001) 10 10 conditions, it significantly enhanced radiation sensitivity and hypoxic (DMF : 1.18, p = 0.04) conditions (Figure of U251MG cells under hypoxic conditions (DMF : 5C). Treatment with CTM did not affect radiosensitivity 1.35, p= 0.002) (Figure 5A). Further, treatment with of U343MG cells under normoxic (DMF :1.02, p = HIF-1a-siRNA resulted in a significantly enhanced 0.01) conditions, but resulted in significant enhanced Kessler et al. BMC Cancer 2010, 10:605 Page 8 of 11 http://www.biomedcentral.com/1471-2407/10/605 Figure 5 Effects of HIF-1a-siRNA or CTM on Response to Radiation of U251MG and U343MG Cells (Radiosensitivity). A (U251MG) and B (U343MG), Clonogenic survival following treatment with the indicated siRNA (75 nM) and radiation under normoxia (0-10 Gy) and hypoxia (0- 15 Gy). HIF-1a-siRNA decreased the hypoxia-induced radioresistance of U251MG and U343MG cells. Under normoxia, HIF-1a-siRNA reduces the response to irradiation in U251MG cells and enhanced the effect of radiation in U343MG cells. C (U251MG) and D (U343MG), Clonogenic survival following exposure to 75 nM CTM and radiation under normoxia (0-10 Gy) and hypoxia (0-15 Gy). Exposure to CTM decreased hypoxia- induced radioresistance of U251MG and U343MG cells. CTM enhanced the effect of radiation in U251MG cells and reduced the response to irradiation in U343MG cells under normoxia. radiosensitivity under hypoxic (DMF :1.48, p =0.02) and disease progression [18-20]. Additionally, the malig- conditions (Figure 5D). nancy of brain tumors is associated with increased intra- tumoral hypoxia [5]. Discussion In the present study, we analyzed the effects of HIF- In most human cancers including malignant glioma, 1a inhibition by two alternative strategies, siRNA target- HIF-1a expression promotes tumor growth, angiogenesis ing of HIF-1a or CTM, on the radioresistance of two Kessler et al. BMC Cancer 2010, 10:605 Page 9 of 11 http://www.biomedcentral.com/1471-2407/10/605 malignant glioma cell lines. The glioma cell lines Results suggest that inhibition of HIF-1a decreased U251MG and U343MG displayed comparable HIF-1a tumor growth in malignant gliomas [24]. Additionally, mRNA expression levels under normoxic and hypoxic Dai et al. showed oxygen-independent cytotoxicity and conditions. U251MG and U343MG exhibited a weak p53-independent apoptosis by HIF-1a inhibition in glio- HIF-1a protein expression under normoxia with an blastoma cells [12]. In the present study, Western blot increase in HIF-1a protein, CA9 mRNA and CAIX pro- analyses of the cleavage of PARP confirm these findings. tein levels under hypoxia. Our previous studies using Furthermore, treatment of U251MG and U343MG cells the glioma cell line U87MG supported the potential use- with HIF-1a-siRNA or CTM reduced the plating effi- fulness of HIF-1a as an endogenous marker of tumor ciency under normoxic and hypoxic conditions as com- hypoxia and of resulting radioresistance of human pared to that of the control siRNA- or DMSO-treated tumor cells [21,22]. Furthermore, CAIX was found to be cells. a suitable marker of current or previous chronic hypoxia In the literature, hypoxia and HIF-1a-associated resis- andwepreviouslyproposedthatthe amount of CAIX- tance to radiotherapy and chemotherapy is discussed. positive cells in a tumor cell suspension may be related HIF-1a expression is a significant prognostic indicator to the radiosensitivity of this tumor in vivo [23]. Inhibi- in glial brain tumors treated by radiotherapy [18,25]. tion of the HIF-1 pathway by siRNA or CTM was per- Furthermore, HIF-1a exhibits an attractive target to formed by quantitative real-time PCR and Western blot overcome hypoxia-related radioresistance of tumors analyses. We showed that HIF-1 targeting via HIF-1a- in vitro and in vivo [26]. Silencing of HIF-1a has been siRNA effectively reduced the hypoxia-induced HIF-1a shown to inhibit hypoxia-mediated cell migration and protein levels of U251MG and U343MG cells. In addi- invasion in in vitro and in vivo malignant glioma mod- tion, exposure to CTM resulted in an increased HIF-1a els [13,27,28]. Further, silencing of HIF-1a by siRNA protein expression in U251MG cells and decreased sensitized glioma cells to the chemotherapeutic agents levels of HIF-1a proteininU343MGcells undernor- doxorubicin and etoposide [14]. This is the first study moxic and hypoxic conditions. CTM is an inhibitor of showing that inhibition of HIF-1a by siRNA or by the HIF-1a/p300 complex. CTM only interrupts the CTM under hypoxic conditions resulted in a signifi- transcriptional activity of HIF-1a. Thus, it diminishes cantly enhanced radiosensitivity of the human glioma the expression of HIF-1a target genes. U251MG cells cells. Recently, HIF-1a inhibition by CTM effectively responded as expected because CTM has no influence reduced hypoxia-dependent transcription and sensitized on the expression of HIF-1a mRNA. In U251MG cells, hypoxic HT 1080 human fibrosarcoma cells in vitro to treatment with CTM resulted in a slight increase of radiation [16]. HIF-1a protein stabilisation but attenuated the HIF-1a Although inhibition of HIF-1a under hypoxic condi- induced expression of CAIX. One possible explanation tions enhanced radiosensitivity of glioma cells U251MG for the slightly increased HIF-1a proteinexpressionin and U343MG, the effects of HIF-1a inhibition on radio- these cells might be that CTM attenuated the hypoxic sensitivity under normoxic conditions were not obvious. answer. So an auto regulatory feedback loop caused this In agreement with our results, Palayoor et al.showed stabilisation of HIF-1a. Moreover, another explanation that effects of HIF-1a inhibitor PX-478 on radiosensitity could be that CTM also inhibits the interaction of HIF- under normoxic conditions were less dramatic than 1a andVHL.Inthis, wayanunspecificsideeffectof under hypoxic conditions in prostate carcinoma cells CMT would inhibit the degradation of HIF-1a. [29]. Further studies have shown that HIF inhibitors are U343MG cell line didn’t respond as expected. In more potent in attenuation of HIF-dependent transcrip- U343MG cells CTM has an effect on the HIF-1a tional activity under hypoxia than under normoxia expression and on the target gene CAIX. One reason [30,31]. Further investigations are necessary to study the for that might have been that the applied dose of CTM normoxic expression of HIF-1a in malignant glioma cell in the lower grade 3 glioma cells line U343MG causes lines. side effects. Further, both cell lines differ in their Indeed, siRNA and CTM showed different impacts on genetic status and regulation of different pathways and the effects of radiation in the indicated cell lines under consequently in their disease aggressiveness and normoxia, as HIF-1a-siRNA reduces the response to response to treatment. Additionally, CTM is a small irradiation in U251MG cells, whereas in U343MG cells, molecule and the specificity for the target of small siRNA enhanced the effects of radiation. Furthermore, molecule is debatable and there might be another under normoxia, CTM enhanced the effects of radiation mechanism of CTM play a role. Furthermore, our data in U251MG cells, whereas in U343MG cells, CTM had indicate that monitoring CA9 mRNA and CAIX protein no effect on the response to irradiation under normoxia. expression is a means to verify the inhibition of HIF-1 The U343MG cell line originated from a de novo grade- activity. III anaplastic astrocytoma and U251MG was established Kessler et al. BMC Cancer 2010, 10:605 Page 10 of 11 http://www.biomedcentral.com/1471-2407/10/605 Authors’ contributions from a de novo grade-IV glioblastoma [1,2,32]. Thus, dif- JK designed the study, performed experimental procedures, analyzed the ferent genes involved in cellular pathways regulating data and drafted the manuscript. AH HW SR and MK aided in study design apoptosis, proliferation, and DNA repair could carry and reviewed the manuscript. MB analyzed the data and reviewed the manuscript. DV designed the study, performed experimental procedures, mutations or deletions in these cells. Both cell lines differ analyzed the data and drafted the manuscript. All authors read and in their genetic status of p53 and consequently in their approved the final manuscript. disease aggressiveness and response to treatment. Competing interests U343MG cells carry wild-type p53, whereas U251MG The authors declare that they have no competing interests. carry mutated p53 [32]. Hence, the ability to respond to radiation may differ in U251MG and U343MG cells. Received: 21 May 2010 Accepted: 4 November 2010 Published: 4 November 2010 Additionally, compared to HIF-1a-specific siRNA, CTM has a different mode of action. References However, the function of CTM is not fully understood. 1. Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, CTM is a member of the epidithiodiketopiperazine (ETP) Cavenee WK: The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002, 61:215-25, discussion 226-9. family. Previous studies have shown that ETPs react with 2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, p300 and cause zinc ion ejection. 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Cook KM, Hilton ST, Mecinovic J, Motherwell WB, Figg WD, Schofield CJ: Epidithiodiketopiperazines block the interaction between hypoxia- • Thorough peer review inducible factor-1alpha (HIF-1alpha) and p300 by a zinc ejection • No space constraints or color figure charges mechanism. J Biol Chem 2009, 284:26831-8. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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BMC Cancer – Springer Journals

Published: Nov 4, 2010

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