- Email: [email protected]
Hypoxia inducing factor-1α regulates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in tumor cells exposed to hypoxia
Biochemical and Biophysical Research Communications 399 (2010) 379–383
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hypoxia inducing factor-1a regulates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in tumor cells exposed to hypoxia Jae-Kyo Jeong, Myung-Hee Moon, Jae-Suk Seo, Jae-Won Seol, Sang-Youel Park *, You-Jin Lee Center for Healthcare Technology Development, Bio-Safety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea
a r t i c l e
i n f o
Article history: Received 22 July 2010 Available online 24 July 2010 Keywords: Hypoxia HIF-1a TRAIL
a b s t r a c t Hypoxia is a common environmental stress. Particularly, the center of rapidly-growing solid tumors is easily exposed to hypoxic conditions. Hypoxia is well known to attenuate the therapeutic response to radio and chemotherapies including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein. HIF-1a is a critical mediator of the hypoxic response. However, little is known about the function of hypoxia-inducible factor-1a (HIF-1a) on hypoxic inhibition of TRAIL-mediated apoptosis. In this study, we investigated whether hypoxic inhibition of TRAIL-mediated apoptosis can be regulated by modulating HIF-1a protein. Hypoxia- and DEF-induced HIF-1a activation inhibited the TRAIL-mediated apoptosis in SK-N-SH, HeLa, A549 and SNU-638 cells. And also, HIF-1a inactivating reagents including DOX increased the sensitivity to TRAIL protein in tumor cells exposed to hypoxia. Furthermore, knock-down of HIF-1a using lentiviral RNA interference sensitized tumor cells to TRAIL-mediated cell death under hypoxic condition. Taken together, these results indicate that HIF-1a inactivation increased TRAIL sensitivity in hypoxia-induced TRAIL-resistant tumor cells and also suggest that HIF-1a inhibitors may have benefits in combination therapy with TRAIL against hypoxic tumor cells. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Programmed cell death, also known as apoptosis, can be triggered in tumor cells by anti-cancer agents. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, a member of the TNF family) is stimuli stimulus that can induce cancer cell death [1]. TRAIL has been the subject of interest in oncology as it displays specific anti-cancer activity against a wide range of cancer cells without significant side effects [2]. TRAIL interacts with four major receptors: death receptor 4 and 5 (DR4 and DR5) can induce apoptosis, while decoy receptor 1 and 2(DcR1 and DcR2) can attenuate TRAIL-mediated cell death by binding to TRAIL [3–4]. TRAIL-induced apoptosis via the p53-mediated increase of DR5 has been reported [5]. In addition, one early study found that tumor cells expressing wild-type p53 are more sensitive to TRAIL compared to cells with mutations in p53 genes [6]. Thus, TRAIL-mediated apoptosis is related to p53 tumor suppressor functions during cancer therapy [7]. Hypoxia is a common stress found within tumors. In particular, rapidly-growing solid tumor cells are often hypoxic at their center. Hypoxia exhibits an anti-apoptotic effect via regulation of many
* Corresponding author. Address: College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea. Fax: +82 63 270 3780. E-mail address: [email protected] (S.-Y. Park). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.082
apoptosis pathways [8], and decreases the effects of anti-cancer drugs, including TRAIL, in solid tumor cells [9]. Hypoxia-inducible factor-1(HIF-1), a transcription factor composed of a- and b- subunits, is a key regulator of metabolic adaptation to hypoxia [10]. Targets of HIF-1a include genes coding for cytokines and growth factors, angiogenesis, cell cycle progression, glucose uptake and metabolism, and cell survival. HIF-1a has been implicated as an oncogene that is over-expressed in human cancer cells. Blockade of HIF-1a as a therapeutic target has been explored alone or in combination with other chemotherapeutic reagents [11,12]. It has been suggested that HIF-1a acts with p53 as a co-regulator of transcription regulate apoptosis [13]. While moderate and mild hypoxia maintains cell viability by activating survival genes, prolonged and severe hypoxia provokes cell death, which is associated with stability of p53 [14–17]. Indeed, the apoptotic function of p53 can be regulated by the status of HIF-1a in cells, and blocking HIF-1a expression can drive p53-mediated tumor cell death in mild hypoxia [13,18]. This study focuses on the influence of HIF-1a on regulation of TRAIL apoptotic activity in hypoxic tumor cells. We show that inhibition of HIF-1a increased the apoptosis-inducing potential of TRAIL even in mild hypoxia (1% O2). These results support the view that suppression of HIF-1a is a viable therapeutic strategy for solid tumor cells that are resistant to anti-cancer drugs, including TRAIL, in a hypoxic environment.
380
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383
2. Materials and methods 2.1. Cell culture and hypoxic treatment A human neuroblastoma cell line (SK-N-SH), a human uterine cervical cancer cell line (HeLa), a human lung cancer cell line (A549) and a human gastric cancer cell line (SNU-638) were obtained from the American Type Culture collection (ATCC; Manassas, VA, USA). A hypoxia chamber was used to create a low oxygen (O2) environment; a gas mixture of 1% O2, 5% CO2 and 94% N2 was flowed into the sealed chamber. 2.2. Crystal violet assay Cell viability was determined by crystal violet staining, as previously described [19]. Cell viability was calculated based on the relative dye intensity compared to controls. 2.3. Lactate dehydrogenase (LDH) assay Cytotoxicity was assessed by LDH assay in the supernatant using a LDH Cytotoxicity Detection kit (Takara Bio, Tokyo, Japan) according to the protocol of the manufacturer. LDH activity was determined by measuring the absorbance at 490 nm. 2.4. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay TUNEL analysis was performed to measure the degree of cellular apoptosis using an in situ ApoBrdU DNA fragmentation assay kit (BioVision, San Francisco, CA, USA) following the manufacturer’s instructions. 2.5. DNA fragmentation assay Cell pellets were suspended in 0.5 ml of 50 mM Tris–HCl buffer (pH 8.0) and digested with 500 lg/ml proteinase K for 4 h at 65 °C. After digestion, the DNA was extracted with phenol/chloroform (1:1, v/v). Purified DNA was electrophoresed on an agarose gel and visualized by ultraviolet trans-illumination. 2.6. Western blots Cell lines were lysed in a lysis buffer. Equal amounts of proteins were electrophoretically resolved by 8–15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting to a nitrocellulose membrane was performed as previously described [20]. Antibodies used for immunoblotting were directed towards p53, HIF-1a, and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2.7. HIF-1a short hairpin RNA (shRNA) lentivirus The sh-RNA plasmid constructs for HIF-1a were a gift from Dr. Y.N. Kim, National Cancer Center, Seoul, Korea. Lentiviruses were produced as described previously [21]. Briefly, SK-N-SH cells were transfected with either sh-HIF-1a or a control sh-RNA plasmid (shMock). When necessary, drug selection of cells was done with 1 lg/ml puromycin after a 24 h recovery in standard growth medium. 2.8. Statistical analyses All data are expressed as means ± standard deviation (SD), and the data were compared using the Student’s t-test and the ANOVA
Duncan test with the SAS statistical package (SAS Institute, Cary, NC, USA). The results were considered significant at P < 0.05 (*) or P < 0.01 (**). 3. Results 3.1. Hypoxia decreases TRAIL-induced apoptosis in cancer cell lines The influence of mild hypoxia (1% O2) on TRAIL-induced apoptosis in SK-N-SH, SNU-638, HeLa, and A549 cells was estimated by crystal violet staining. All cancer cells were exposed to hypoxic conditions alone or in combination with TRAIL. All cells were responsive to TRAIL treatment (30–50% reduction in viability of all cell populations), and hypoxia had no effect on cell viability. However, hypoxia inhibited TRAIL-induced tumor cell apoptosis (Fig. 1A). These results were confirmed by morphological examination of the cell population (Fig. 1B). Consistent with these results, the TUNEL assay also showed that hypoxia completely inhibited TRAIL-induced apoptosis, as demonstrated by the decrease in green color (Fig. 1C). Collectively, these results are consistent with the idea that hypoxia decreases TRAIL-mediated apoptosis. 3.2. HIF-1a activation regulates TRAIL-mediated apoptosis in tumor cells While the role of HIF-1a as an anti-apoptotic factor in hypoxic conditions is well known [22,23], we sought to better understand the effect of mild hypoxia (1% O2) on sensitivity to TRAIL-induced apoptosis. Western blot analysis was performed to evaluate the HIF-1a protein level in SK-N-SH neuroblastoma cells exposed to hypoxia with or without TRAIL (Fig. 2A). Cells displayed increased HIF-1a protein levels under hypoxic conditions independent of TRAIL treatment. To investigate the influence of HIF-1a activity on TRAIL-induced cell death, we utilized the HIF-1a activator, deferoxamine (DEF), and the HIF-1a inhibitor, doxorubicin (DOX), under hypoxic/normoxic conditions. Cells receiving DEF in combination with TRAIL had inhibition of TRAIL-induced apoptosis under normoxic conditions. Hypoxia-induced TRAIL-resistance cells treated with DOX followed by exposure to TRAIL were strongly sensitized to TRAIL activity (Fig. 2B). These data were further corroborated by morphological examination of cells (Fig. 2C). Western blot analysis was performed to detect expression of HIF-1a in cells exposed to TRAIL, DEF, and DOX in hypoxic/normoxic conditions (Fig. 2D). The hypoxic mimicking compound DEF increased the stability of HIF-1a and decreased p53 expression under normoxia independent of TRAIL treatment. In contrast, DOX decreased the expression of HIF-1a protein levels, and increased p53 protein levels under hypoxia, independent of TRAIL treatment. These results are consistent with the idea that HIF-1a regulates TRAIL-induced apoptosis in low oxygen conditions.
3.3. Maintenance of TRAIL-induced apoptosis in cancer cells by HIF-1a instability under hypoxic conditions To examine the protective role of HIF-1a in TRAIL-induced apoptosis, a HIF-1a RNAi lentivirus was utilized to knock-down HIF-1a expression. Hypoxia (1% O2) inhibited TRAIL-induced apoptosis in sh-Mock transfected cells. In contrast, treatment with HIF1a RNAi lentivirus blocked hypoxic inhibition of TRAIL-induced tumor cell death (Fig. 3A and C). LDH and DNA fragmentation results confirmed that hypoxia-mediated induction of HIF-1a protected the hypoxic tumor cells from TRAIL-mediated cell death (Figs. 3B and E). These data demonstrate that hypoxic inhibition of TRAILinduced cancer cell death can be blocked by HIF-1a inactivation using natural products or RNAi tools.
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383
381
Fig. 1. Hypoxia inhibits TRAIL-mediated apoptosis in cancer cell lines. (A) All cancer cells were preincubated in hypoxic conditions (1% O2, 12 h) and were then exposed to 400 ng/ml TRAIL for 6 h. The treated cell viability was measured by crystal violet staining. Viability of control cells was set at 100%, and the viability relative to the control is presented. Magnification 100, scale bar = 50 lm. Bar graph indicates the mean ± SEM (n = 2). (B) Cell morphology was photographed with a light microscope (200). (C) Representative immunofluorescence images of TUNEL-positive (green) SK-N-SH cells at 6 h after exposure to 400 ng/ml of TRAIL in the absence and presence of hypoxia. The cells were counterstained with propidium iodide (red) to show all cell nuclei. Magnification 400, scale bar = 10 lm. Bar graph indicates the mean ± SEM (n = 2). **P < 0.05, significant differences between control and each treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. HIF-1a inhibits TRAIL-induced apoptosis in cancer cell lines. (A) For the Western blot analysis of HIF-1a from SK-N-SH cells, cells were preincubated in hypoxic conditions (1% O2, 12 h) and then exposed to 400 ng/ml TRAIL for 6 h. b-actin was used as a loading control. (B) SK-N-SH cells were treated with 2.5 lM DEF in the absence or presence of TRAIL (400 ng/ml) under normoxia and were treated with 100 nM DOX in the absence or presence of TRAIL (400 ng/ml) under hypoxia (1% O2, 12 h). (C) Cell morphology was photographed with a light microscope (200) and scale bar = 20 lm. Bar graph indicates the mean ± SEM (n = 2). (D) Western blot analysis of HIF-1a from SK-N-SH cells treated as described in (B), b-actin was used as a loading control. *P < 0.01, **P < 0.05, significant differences between control and each treatment group.
382
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383
Fig. 3. Lentiviral shRNA knock-down of HIF-1a sensitizes SK-N-SH cells to TRAIL-induced apoptosis. (A) HIF-1a-shRNA or mock transfected SH-SY5Y cells were cultured in 21% or 1% oxygen tension for 12 h, and then treated with TRAIL for 6 h. Cell viability was measured by crystal violet staining. Viability of control cells was set at 100%, and viability relative to the control is presented. (B) Cells were treated with 400 ng/ml TRAIL for 6 h after exposure to hypoxia for 12 h, and release of LDH into the cell culture supernatant was measured. (C) The treated cells were photographed with a light microscope (100) and scale bar = 200 lm. Bar graph indicates the means ± SEM (n = 2). (D) Western blot analysis of HIF-1a from HIF-1a-shRNA or mock transfected SH-SY5Y cells incubated with or without hypoxia for 12 h. b-actin was used as a loading control. *P < 0.05, significant differences between control and each treatment group. #P < 0.05, significant differences between the cells transfected with HIF-1a-shRNA and with mock. (E) Analysis of genomic DNA isolated from SK-N-SH cells treated 400 ng/ml TRAIL under hypoxia or under normoxia. Marker: 100 bp DNA ladder.
4. Discussion The purpose of this study was to investigate the role HIF-1a plays in hypoxic inhibition of TRAIL-induced cell death. This study suggests therapeutic potential for HIF-1a suppressors including DOX in chemotherapeutic intervention strategies that involve the use of TRAIL in hypoxic solid tumors. TRAIL, a member of the TNF family, is a stimulus that can induce cancer cell death via death receptors [4,7]. TRAIL-mediated apoptosis is stimulated by DR4 and DR5 and suppressed by Dcr1 and Dcr2. A link between DR5 and the status of the p53 tumor suppressor has been extensively studied [5]. Early studies found that a number of tumor cell lines expressing wild-type p53 are sensitive to TRAIL compared to lines with mutations in p53 genes [5,6]. Consistent with this, TRAIL treatment of SK-N-SH cells under normoxia (21% O2), but not hypoxia (1% O2), increased p53 protein levels and activated the apoptotic signal pathway (Fig. 2). In response to low oxygen conditions, cells switch their metabolism and gene expression to adapt to hypoxia. HIF-1a, as a transcription factor, is known to play a central role in the cellular response to hypoxia [10,22]. Hypoxia and HIF-1a have been reported to confer protection against drug-induced apoptosis in many cancer cell lines [13]. A recent study suggested that HIF-1a increases the expression of decoy receptor 2, which has an important role in suppressing TRAIL-induced apoptosis [24]. Our data showed that TRAIL-mediated apoptosis can be regulated by HIF1a-dependent regulation of p53 protein levels under hypoxic conditions (Fig. 2). Taken together, this data suggests that inactivation of HIF-1a could enhance the TRAIL-induced therapeutic effect in hypoxic tumors.
Some reports have shown that severe hypoxia leads to p53 activation, while mild hypoxia has no effect in many cancer cell lines [14,15,18]. Mayes et al. demonstrated that HIF-1a independent expression of p53 is a determinant of cancer cell sensitivity to TRAIL-induced apoptosis during severe hypoxia (0.2% O2) [16]. Our data has shown that the expression of p53 is regulated by the stability of HIf-1a in SK-N-SH cells under mild hypoxic conditions (1% O2) (Fig. 2D). Furthermore, our results show that increased HIF-1a stability during DEF treatment may decrease p53 proteins levels in TRAIL-treated cancer cells under normoxic conditions, while decrease of HIF-1a during DOX treatment increased p53 expression under hypoxic conditions (Fig. 2D). These observations support the hypothesis that HIF-1a inhibition increases the expression of p53 and enhances hypoxia-exposed TRAIL-resistant tumor cell death. Taken together, these results demonstrate that HIF-1a inactivation increased TRAIL-induced apoptosis in hypoxic TRAIL-resistant tumor cells. These results also suggest that HIF-1a inhibitors may have clinical benefits when used in combination therapy with TRAIL for the treatment of hypoxia-exposed solid tumor cells. Acknowledgments This work was supported by National Research Foundation of Korea Grant (KRF-2009-0065822) and Regional Research Universities Program and the Center for Healthcare Technology Development. References [1] R.K. Srivastava, Intracellular mechanisms of TRAIL and its role in cancer therapy, Mol. Cell Biol. Res. Commun. 4 (2000) 67–75.
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383 [2] A. Almasan, A. Ashkenazi, Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy, Cytokine Growth Factor Rev. 14 (2003) 337–348. [3] V. Pavet, J. Beyrath, C. Pardin, A. Morizot, M.C. Lechner, J.P. Briand, M. Wendland, W. Maison, S. Fournel, O. Micheau, G. Guichard, H. Gronemeyer, Multivalent DR5 peptides activate the TRAIL death pathway and exert tumoricidal activity, Cancer Res. 70 (2010) 1101–1110. [4] G. Mellier, S. Huang, K. Shenoy, S. Pervaiz, TRAILing death in cancer, Mol. Aspects Med. 31 (2010) 93–112. [5] B.Z. Carter, D.H. Mak, W.D. Schober, M.F. Dietrich, C. Pinilla, L.T. Vassilev, J.C. Reed, M. Andreeff, Triptolide sensitizes AML cells to TRAIL-induced apoptosis via decrease of XIAP and p53-mediated increase of DR5, Blood 111 (2008) 3742–3750. [6] L. Weinmann, J. Wischhusen, M.J. Demma, U. Naumann, P. Roth, B. Dasmahapatra, M. Weller, A novel p53 rescue compound induces p53dependent growth arrest and sensitises glioma cells to Apo2L/TRAIL-induced apoptosis, Cell Death Differ. 15 (2008) 718–729. [7] D. Mahalingam, E. Szegezdi, M. Keane, S. Jong, A. Samali, TRAIL receptor signalling and modulation: are we on the right TRAIL?, Cancer Treat Rev. 35 (2009) 280–288. [8] K. Selvendiran, A. Bratasz, M.L. Kuppusamy, M.F. Tazi, B.K. Rivera, P. Kuppusamy, Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3, Int. J. Cancer 125 (2009) 2198–2204. [9] S.Y. Park, T.R. Billiar, D.W. Seol, Hypoxia inhibition of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, TRAIL, Biochem. Biophys. Res. Commun. 291 (2002) 150–153. [10] J.M. Adams, L.T. Difazio, R.H. Rolandelli, J.J. Lujan, G. Hasko, B. Csoka, Z. Selmeczy, Z.H. Nemeth, HIF-1: a key mediator in hypoxia, Acta Physiol. Hung. 96 (2009) 19–28. [11] Y. Hu, K. Kirito, K. Yoshida, T. Mitsumori, K. Nakajima, Y. Nozaki, S. Hamanaka, T. Nagashima, M. Kunitama, K. Sakoe, N. Komatsu, Inhibition of hypoxiainducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan, Mol. Cancer Ther. 8 (2009) 2329–2338. [12] W.G. Kaelin Jr., Treatment of kidney cancer: insights provided by the VHL tumor-suppressor protein, Cancer 115 (2009) 2262–2272. [13] E. Poon, A.L. Harris, M. Ashcroft, Targeting the hypoxia-inducible factor (HIF) pathway in cancer, Expert. Rev. Mol. Med. 11 (2009) e26. [14] N.V. Iyer, L.E. Kotch, F. Agani, S.W. Leung, E. Laughner, R.H. Wenger, M. Gassmann, J.D. Gearhart, A.M. Lawler, A.Y. Yu, G.L. Semenza, Cellular and
[15]
[16]
[17] [18]
[19]
[20]
[21]
[22] [23]
[24]
383
developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha, Genes Dev. 12 (1998) 149–162. C. Koumenis, R. Alarcon, E. Hammond, P. Sutphin, W. Hoffman, M. Murphy, J. Derr, Y. Taya, S.W. Lowe, M. Kastan, A. Giaccia, Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation, Mol. Cell Biol. 21 (2001) 1297–1310. P.A. Mayes, L. Campbell, M.S. Ricci, J.P. Plastaras, D.T. Dicker, W.S. El-Deiry, Modulation of TRAIL-induced tumor cell apoptosis in a hypoxic environment, Cancer Biol. Ther. 4 (2005) 1068–1074. Y. Zhao, X.Q. Chen, J.Z. Du, Cellular adaptation to hypoxia and p53 transcription regulation, J. Zhejiang Univ. Sci. B 10 (2009) 404–410. J. Yang, A. Ahmed, E. Poon, N. Perusinghe, A. de Haven Brandon, G. Box, M. Valenti, S. Eccles, K. Rouschop, B. Wouters, M. Ashcroft, Small-molecule activation of p53 blocks hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in vivo and leads to tumor cell apoptosis in normoxia and hypoxia, Mol. Cell Biol. 29 (2009) 2243–2253. J.S. Seo, J.W. Seol, M.H. Moon, J.K. Jeong, Y.J. Lee, S.Y. Park, Hypoxia protects neuronal cells from human prion protein fragment-induced apoptosis, J. Neurochem. 112 (2010) 715–722. J.W. Seol, A.A. Chaudhari, Y.J. Lee, H.S. Kang, I.S. Kim, N.S. Kim, J.H. Park, T.H. Kim, D.W. Seol, S.Y. Park, Regulation of DR-5 protein and mitochondrial transmembrane potential by gemcitabine, a possible mechanism of gemcitabine-enhanced TRAIL-induced apoptosis, Oncol. Rep. 18 (2007) 523– 529. S.H. Lee, K.H. Koo, J.W. Park, H.J. Kim, S.K. Ye, J.B. Park, B.K. Park, Y.N. Kim, HIF-1 is induced via EGFR activation and mediates resistance to anoikis-like cell death under lipid rafts/caveolae-disrupting stress, Carcinogenesis 30 (2009) 1997–2004. H.E. Ryan, J. Lo, R.S. Johnson, HIF-1 alpha is required for solid tumor formation and embryonic vascularization, EMBO J. 17 (1998) 3005–3015. H. Yasuda, Solid tumor physiology and hypoxia-induced chemo/radioresistance. Novel strategy for cancer therapy: nitric oxide donor as a therapeutic enhancer, Nitric oxide 19 (2008) 205–216. G.T. Pei, C.W. Wu, W.W. Lin, Hypoxia-induced decoy receptor 2 gene expression is regulated via a hypoxia-inducible factor 1alpha-mediated mechanism, Biochem. Biophys. Res. Commun. 391 (2010) 1274–1279.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hypoxia inducing factor-1a regulates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in tumor cells exposed to hypoxia Jae-Kyo Jeong, Myung-Hee Moon, Jae-Suk Seo, Jae-Won Seol, Sang-Youel Park *, You-Jin Lee Center for Healthcare Technology Development, Bio-Safety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea
a r t i c l e
i n f o
Article history: Received 22 July 2010 Available online 24 July 2010 Keywords: Hypoxia HIF-1a TRAIL
a b s t r a c t Hypoxia is a common environmental stress. Particularly, the center of rapidly-growing solid tumors is easily exposed to hypoxic conditions. Hypoxia is well known to attenuate the therapeutic response to radio and chemotherapies including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein. HIF-1a is a critical mediator of the hypoxic response. However, little is known about the function of hypoxia-inducible factor-1a (HIF-1a) on hypoxic inhibition of TRAIL-mediated apoptosis. In this study, we investigated whether hypoxic inhibition of TRAIL-mediated apoptosis can be regulated by modulating HIF-1a protein. Hypoxia- and DEF-induced HIF-1a activation inhibited the TRAIL-mediated apoptosis in SK-N-SH, HeLa, A549 and SNU-638 cells. And also, HIF-1a inactivating reagents including DOX increased the sensitivity to TRAIL protein in tumor cells exposed to hypoxia. Furthermore, knock-down of HIF-1a using lentiviral RNA interference sensitized tumor cells to TRAIL-mediated cell death under hypoxic condition. Taken together, these results indicate that HIF-1a inactivation increased TRAIL sensitivity in hypoxia-induced TRAIL-resistant tumor cells and also suggest that HIF-1a inhibitors may have benefits in combination therapy with TRAIL against hypoxic tumor cells. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Programmed cell death, also known as apoptosis, can be triggered in tumor cells by anti-cancer agents. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, a member of the TNF family) is stimuli stimulus that can induce cancer cell death [1]. TRAIL has been the subject of interest in oncology as it displays specific anti-cancer activity against a wide range of cancer cells without significant side effects [2]. TRAIL interacts with four major receptors: death receptor 4 and 5 (DR4 and DR5) can induce apoptosis, while decoy receptor 1 and 2(DcR1 and DcR2) can attenuate TRAIL-mediated cell death by binding to TRAIL [3–4]. TRAIL-induced apoptosis via the p53-mediated increase of DR5 has been reported [5]. In addition, one early study found that tumor cells expressing wild-type p53 are more sensitive to TRAIL compared to cells with mutations in p53 genes [6]. Thus, TRAIL-mediated apoptosis is related to p53 tumor suppressor functions during cancer therapy [7]. Hypoxia is a common stress found within tumors. In particular, rapidly-growing solid tumor cells are often hypoxic at their center. Hypoxia exhibits an anti-apoptotic effect via regulation of many
* Corresponding author. Address: College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea. Fax: +82 63 270 3780. E-mail address: [email protected] (S.-Y. Park). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.082
apoptosis pathways [8], and decreases the effects of anti-cancer drugs, including TRAIL, in solid tumor cells [9]. Hypoxia-inducible factor-1(HIF-1), a transcription factor composed of a- and b- subunits, is a key regulator of metabolic adaptation to hypoxia [10]. Targets of HIF-1a include genes coding for cytokines and growth factors, angiogenesis, cell cycle progression, glucose uptake and metabolism, and cell survival. HIF-1a has been implicated as an oncogene that is over-expressed in human cancer cells. Blockade of HIF-1a as a therapeutic target has been explored alone or in combination with other chemotherapeutic reagents [11,12]. It has been suggested that HIF-1a acts with p53 as a co-regulator of transcription regulate apoptosis [13]. While moderate and mild hypoxia maintains cell viability by activating survival genes, prolonged and severe hypoxia provokes cell death, which is associated with stability of p53 [14–17]. Indeed, the apoptotic function of p53 can be regulated by the status of HIF-1a in cells, and blocking HIF-1a expression can drive p53-mediated tumor cell death in mild hypoxia [13,18]. This study focuses on the influence of HIF-1a on regulation of TRAIL apoptotic activity in hypoxic tumor cells. We show that inhibition of HIF-1a increased the apoptosis-inducing potential of TRAIL even in mild hypoxia (1% O2). These results support the view that suppression of HIF-1a is a viable therapeutic strategy for solid tumor cells that are resistant to anti-cancer drugs, including TRAIL, in a hypoxic environment.
380
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383
2. Materials and methods 2.1. Cell culture and hypoxic treatment A human neuroblastoma cell line (SK-N-SH), a human uterine cervical cancer cell line (HeLa), a human lung cancer cell line (A549) and a human gastric cancer cell line (SNU-638) were obtained from the American Type Culture collection (ATCC; Manassas, VA, USA). A hypoxia chamber was used to create a low oxygen (O2) environment; a gas mixture of 1% O2, 5% CO2 and 94% N2 was flowed into the sealed chamber. 2.2. Crystal violet assay Cell viability was determined by crystal violet staining, as previously described [19]. Cell viability was calculated based on the relative dye intensity compared to controls. 2.3. Lactate dehydrogenase (LDH) assay Cytotoxicity was assessed by LDH assay in the supernatant using a LDH Cytotoxicity Detection kit (Takara Bio, Tokyo, Japan) according to the protocol of the manufacturer. LDH activity was determined by measuring the absorbance at 490 nm. 2.4. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay TUNEL analysis was performed to measure the degree of cellular apoptosis using an in situ ApoBrdU DNA fragmentation assay kit (BioVision, San Francisco, CA, USA) following the manufacturer’s instructions. 2.5. DNA fragmentation assay Cell pellets were suspended in 0.5 ml of 50 mM Tris–HCl buffer (pH 8.0) and digested with 500 lg/ml proteinase K for 4 h at 65 °C. After digestion, the DNA was extracted with phenol/chloroform (1:1, v/v). Purified DNA was electrophoresed on an agarose gel and visualized by ultraviolet trans-illumination. 2.6. Western blots Cell lines were lysed in a lysis buffer. Equal amounts of proteins were electrophoretically resolved by 8–15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting to a nitrocellulose membrane was performed as previously described [20]. Antibodies used for immunoblotting were directed towards p53, HIF-1a, and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2.7. HIF-1a short hairpin RNA (shRNA) lentivirus The sh-RNA plasmid constructs for HIF-1a were a gift from Dr. Y.N. Kim, National Cancer Center, Seoul, Korea. Lentiviruses were produced as described previously [21]. Briefly, SK-N-SH cells were transfected with either sh-HIF-1a or a control sh-RNA plasmid (shMock). When necessary, drug selection of cells was done with 1 lg/ml puromycin after a 24 h recovery in standard growth medium. 2.8. Statistical analyses All data are expressed as means ± standard deviation (SD), and the data were compared using the Student’s t-test and the ANOVA
Duncan test with the SAS statistical package (SAS Institute, Cary, NC, USA). The results were considered significant at P < 0.05 (*) or P < 0.01 (**). 3. Results 3.1. Hypoxia decreases TRAIL-induced apoptosis in cancer cell lines The influence of mild hypoxia (1% O2) on TRAIL-induced apoptosis in SK-N-SH, SNU-638, HeLa, and A549 cells was estimated by crystal violet staining. All cancer cells were exposed to hypoxic conditions alone or in combination with TRAIL. All cells were responsive to TRAIL treatment (30–50% reduction in viability of all cell populations), and hypoxia had no effect on cell viability. However, hypoxia inhibited TRAIL-induced tumor cell apoptosis (Fig. 1A). These results were confirmed by morphological examination of the cell population (Fig. 1B). Consistent with these results, the TUNEL assay also showed that hypoxia completely inhibited TRAIL-induced apoptosis, as demonstrated by the decrease in green color (Fig. 1C). Collectively, these results are consistent with the idea that hypoxia decreases TRAIL-mediated apoptosis. 3.2. HIF-1a activation regulates TRAIL-mediated apoptosis in tumor cells While the role of HIF-1a as an anti-apoptotic factor in hypoxic conditions is well known [22,23], we sought to better understand the effect of mild hypoxia (1% O2) on sensitivity to TRAIL-induced apoptosis. Western blot analysis was performed to evaluate the HIF-1a protein level in SK-N-SH neuroblastoma cells exposed to hypoxia with or without TRAIL (Fig. 2A). Cells displayed increased HIF-1a protein levels under hypoxic conditions independent of TRAIL treatment. To investigate the influence of HIF-1a activity on TRAIL-induced cell death, we utilized the HIF-1a activator, deferoxamine (DEF), and the HIF-1a inhibitor, doxorubicin (DOX), under hypoxic/normoxic conditions. Cells receiving DEF in combination with TRAIL had inhibition of TRAIL-induced apoptosis under normoxic conditions. Hypoxia-induced TRAIL-resistance cells treated with DOX followed by exposure to TRAIL were strongly sensitized to TRAIL activity (Fig. 2B). These data were further corroborated by morphological examination of cells (Fig. 2C). Western blot analysis was performed to detect expression of HIF-1a in cells exposed to TRAIL, DEF, and DOX in hypoxic/normoxic conditions (Fig. 2D). The hypoxic mimicking compound DEF increased the stability of HIF-1a and decreased p53 expression under normoxia independent of TRAIL treatment. In contrast, DOX decreased the expression of HIF-1a protein levels, and increased p53 protein levels under hypoxia, independent of TRAIL treatment. These results are consistent with the idea that HIF-1a regulates TRAIL-induced apoptosis in low oxygen conditions.
3.3. Maintenance of TRAIL-induced apoptosis in cancer cells by HIF-1a instability under hypoxic conditions To examine the protective role of HIF-1a in TRAIL-induced apoptosis, a HIF-1a RNAi lentivirus was utilized to knock-down HIF-1a expression. Hypoxia (1% O2) inhibited TRAIL-induced apoptosis in sh-Mock transfected cells. In contrast, treatment with HIF1a RNAi lentivirus blocked hypoxic inhibition of TRAIL-induced tumor cell death (Fig. 3A and C). LDH and DNA fragmentation results confirmed that hypoxia-mediated induction of HIF-1a protected the hypoxic tumor cells from TRAIL-mediated cell death (Figs. 3B and E). These data demonstrate that hypoxic inhibition of TRAILinduced cancer cell death can be blocked by HIF-1a inactivation using natural products or RNAi tools.
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383
381
Fig. 1. Hypoxia inhibits TRAIL-mediated apoptosis in cancer cell lines. (A) All cancer cells were preincubated in hypoxic conditions (1% O2, 12 h) and were then exposed to 400 ng/ml TRAIL for 6 h. The treated cell viability was measured by crystal violet staining. Viability of control cells was set at 100%, and the viability relative to the control is presented. Magnification 100, scale bar = 50 lm. Bar graph indicates the mean ± SEM (n = 2). (B) Cell morphology was photographed with a light microscope (200). (C) Representative immunofluorescence images of TUNEL-positive (green) SK-N-SH cells at 6 h after exposure to 400 ng/ml of TRAIL in the absence and presence of hypoxia. The cells were counterstained with propidium iodide (red) to show all cell nuclei. Magnification 400, scale bar = 10 lm. Bar graph indicates the mean ± SEM (n = 2). **P < 0.05, significant differences between control and each treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. HIF-1a inhibits TRAIL-induced apoptosis in cancer cell lines. (A) For the Western blot analysis of HIF-1a from SK-N-SH cells, cells were preincubated in hypoxic conditions (1% O2, 12 h) and then exposed to 400 ng/ml TRAIL for 6 h. b-actin was used as a loading control. (B) SK-N-SH cells were treated with 2.5 lM DEF in the absence or presence of TRAIL (400 ng/ml) under normoxia and were treated with 100 nM DOX in the absence or presence of TRAIL (400 ng/ml) under hypoxia (1% O2, 12 h). (C) Cell morphology was photographed with a light microscope (200) and scale bar = 20 lm. Bar graph indicates the mean ± SEM (n = 2). (D) Western blot analysis of HIF-1a from SK-N-SH cells treated as described in (B), b-actin was used as a loading control. *P < 0.01, **P < 0.05, significant differences between control and each treatment group.
382
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383
Fig. 3. Lentiviral shRNA knock-down of HIF-1a sensitizes SK-N-SH cells to TRAIL-induced apoptosis. (A) HIF-1a-shRNA or mock transfected SH-SY5Y cells were cultured in 21% or 1% oxygen tension for 12 h, and then treated with TRAIL for 6 h. Cell viability was measured by crystal violet staining. Viability of control cells was set at 100%, and viability relative to the control is presented. (B) Cells were treated with 400 ng/ml TRAIL for 6 h after exposure to hypoxia for 12 h, and release of LDH into the cell culture supernatant was measured. (C) The treated cells were photographed with a light microscope (100) and scale bar = 200 lm. Bar graph indicates the means ± SEM (n = 2). (D) Western blot analysis of HIF-1a from HIF-1a-shRNA or mock transfected SH-SY5Y cells incubated with or without hypoxia for 12 h. b-actin was used as a loading control. *P < 0.05, significant differences between control and each treatment group. #P < 0.05, significant differences between the cells transfected with HIF-1a-shRNA and with mock. (E) Analysis of genomic DNA isolated from SK-N-SH cells treated 400 ng/ml TRAIL under hypoxia or under normoxia. Marker: 100 bp DNA ladder.
4. Discussion The purpose of this study was to investigate the role HIF-1a plays in hypoxic inhibition of TRAIL-induced cell death. This study suggests therapeutic potential for HIF-1a suppressors including DOX in chemotherapeutic intervention strategies that involve the use of TRAIL in hypoxic solid tumors. TRAIL, a member of the TNF family, is a stimulus that can induce cancer cell death via death receptors [4,7]. TRAIL-mediated apoptosis is stimulated by DR4 and DR5 and suppressed by Dcr1 and Dcr2. A link between DR5 and the status of the p53 tumor suppressor has been extensively studied [5]. Early studies found that a number of tumor cell lines expressing wild-type p53 are sensitive to TRAIL compared to lines with mutations in p53 genes [5,6]. Consistent with this, TRAIL treatment of SK-N-SH cells under normoxia (21% O2), but not hypoxia (1% O2), increased p53 protein levels and activated the apoptotic signal pathway (Fig. 2). In response to low oxygen conditions, cells switch their metabolism and gene expression to adapt to hypoxia. HIF-1a, as a transcription factor, is known to play a central role in the cellular response to hypoxia [10,22]. Hypoxia and HIF-1a have been reported to confer protection against drug-induced apoptosis in many cancer cell lines [13]. A recent study suggested that HIF-1a increases the expression of decoy receptor 2, which has an important role in suppressing TRAIL-induced apoptosis [24]. Our data showed that TRAIL-mediated apoptosis can be regulated by HIF1a-dependent regulation of p53 protein levels under hypoxic conditions (Fig. 2). Taken together, this data suggests that inactivation of HIF-1a could enhance the TRAIL-induced therapeutic effect in hypoxic tumors.
Some reports have shown that severe hypoxia leads to p53 activation, while mild hypoxia has no effect in many cancer cell lines [14,15,18]. Mayes et al. demonstrated that HIF-1a independent expression of p53 is a determinant of cancer cell sensitivity to TRAIL-induced apoptosis during severe hypoxia (0.2% O2) [16]. Our data has shown that the expression of p53 is regulated by the stability of HIf-1a in SK-N-SH cells under mild hypoxic conditions (1% O2) (Fig. 2D). Furthermore, our results show that increased HIF-1a stability during DEF treatment may decrease p53 proteins levels in TRAIL-treated cancer cells under normoxic conditions, while decrease of HIF-1a during DOX treatment increased p53 expression under hypoxic conditions (Fig. 2D). These observations support the hypothesis that HIF-1a inhibition increases the expression of p53 and enhances hypoxia-exposed TRAIL-resistant tumor cell death. Taken together, these results demonstrate that HIF-1a inactivation increased TRAIL-induced apoptosis in hypoxic TRAIL-resistant tumor cells. These results also suggest that HIF-1a inhibitors may have clinical benefits when used in combination therapy with TRAIL for the treatment of hypoxia-exposed solid tumor cells. Acknowledgments This work was supported by National Research Foundation of Korea Grant (KRF-2009-0065822) and Regional Research Universities Program and the Center for Healthcare Technology Development. References [1] R.K. Srivastava, Intracellular mechanisms of TRAIL and its role in cancer therapy, Mol. Cell Biol. Res. Commun. 4 (2000) 67–75.
J.-K. Jeong et al. / Biochemical and Biophysical Research Communications 399 (2010) 379–383 [2] A. Almasan, A. Ashkenazi, Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy, Cytokine Growth Factor Rev. 14 (2003) 337–348. [3] V. Pavet, J. Beyrath, C. Pardin, A. Morizot, M.C. Lechner, J.P. Briand, M. Wendland, W. Maison, S. Fournel, O. Micheau, G. Guichard, H. Gronemeyer, Multivalent DR5 peptides activate the TRAIL death pathway and exert tumoricidal activity, Cancer Res. 70 (2010) 1101–1110. [4] G. Mellier, S. Huang, K. Shenoy, S. Pervaiz, TRAILing death in cancer, Mol. Aspects Med. 31 (2010) 93–112. [5] B.Z. Carter, D.H. Mak, W.D. Schober, M.F. Dietrich, C. Pinilla, L.T. Vassilev, J.C. Reed, M. Andreeff, Triptolide sensitizes AML cells to TRAIL-induced apoptosis via decrease of XIAP and p53-mediated increase of DR5, Blood 111 (2008) 3742–3750. [6] L. Weinmann, J. Wischhusen, M.J. Demma, U. Naumann, P. Roth, B. Dasmahapatra, M. Weller, A novel p53 rescue compound induces p53dependent growth arrest and sensitises glioma cells to Apo2L/TRAIL-induced apoptosis, Cell Death Differ. 15 (2008) 718–729. [7] D. Mahalingam, E. Szegezdi, M. Keane, S. Jong, A. Samali, TRAIL receptor signalling and modulation: are we on the right TRAIL?, Cancer Treat Rev. 35 (2009) 280–288. [8] K. Selvendiran, A. Bratasz, M.L. Kuppusamy, M.F. Tazi, B.K. Rivera, P. Kuppusamy, Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3, Int. J. Cancer 125 (2009) 2198–2204. [9] S.Y. Park, T.R. Billiar, D.W. Seol, Hypoxia inhibition of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, TRAIL, Biochem. Biophys. Res. Commun. 291 (2002) 150–153. [10] J.M. Adams, L.T. Difazio, R.H. Rolandelli, J.J. Lujan, G. Hasko, B. Csoka, Z. Selmeczy, Z.H. Nemeth, HIF-1: a key mediator in hypoxia, Acta Physiol. Hung. 96 (2009) 19–28. [11] Y. Hu, K. Kirito, K. Yoshida, T. Mitsumori, K. Nakajima, Y. Nozaki, S. Hamanaka, T. Nagashima, M. Kunitama, K. Sakoe, N. Komatsu, Inhibition of hypoxiainducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan, Mol. Cancer Ther. 8 (2009) 2329–2338. [12] W.G. Kaelin Jr., Treatment of kidney cancer: insights provided by the VHL tumor-suppressor protein, Cancer 115 (2009) 2262–2272. [13] E. Poon, A.L. Harris, M. Ashcroft, Targeting the hypoxia-inducible factor (HIF) pathway in cancer, Expert. Rev. Mol. Med. 11 (2009) e26. [14] N.V. Iyer, L.E. Kotch, F. Agani, S.W. Leung, E. Laughner, R.H. Wenger, M. Gassmann, J.D. Gearhart, A.M. Lawler, A.Y. Yu, G.L. Semenza, Cellular and
[15]
[16]
[17] [18]
[19]
[20]
[21]
[22] [23]
[24]
383
developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha, Genes Dev. 12 (1998) 149–162. C. Koumenis, R. Alarcon, E. Hammond, P. Sutphin, W. Hoffman, M. Murphy, J. Derr, Y. Taya, S.W. Lowe, M. Kastan, A. Giaccia, Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation, Mol. Cell Biol. 21 (2001) 1297–1310. P.A. Mayes, L. Campbell, M.S. Ricci, J.P. Plastaras, D.T. Dicker, W.S. El-Deiry, Modulation of TRAIL-induced tumor cell apoptosis in a hypoxic environment, Cancer Biol. Ther. 4 (2005) 1068–1074. Y. Zhao, X.Q. Chen, J.Z. Du, Cellular adaptation to hypoxia and p53 transcription regulation, J. Zhejiang Univ. Sci. B 10 (2009) 404–410. J. Yang, A. Ahmed, E. Poon, N. Perusinghe, A. de Haven Brandon, G. Box, M. Valenti, S. Eccles, K. Rouschop, B. Wouters, M. Ashcroft, Small-molecule activation of p53 blocks hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in vivo and leads to tumor cell apoptosis in normoxia and hypoxia, Mol. Cell Biol. 29 (2009) 2243–2253. J.S. Seo, J.W. Seol, M.H. Moon, J.K. Jeong, Y.J. Lee, S.Y. Park, Hypoxia protects neuronal cells from human prion protein fragment-induced apoptosis, J. Neurochem. 112 (2010) 715–722. J.W. Seol, A.A. Chaudhari, Y.J. Lee, H.S. Kang, I.S. Kim, N.S. Kim, J.H. Park, T.H. Kim, D.W. Seol, S.Y. Park, Regulation of DR-5 protein and mitochondrial transmembrane potential by gemcitabine, a possible mechanism of gemcitabine-enhanced TRAIL-induced apoptosis, Oncol. Rep. 18 (2007) 523– 529. S.H. Lee, K.H. Koo, J.W. Park, H.J. Kim, S.K. Ye, J.B. Park, B.K. Park, Y.N. Kim, HIF-1 is induced via EGFR activation and mediates resistance to anoikis-like cell death under lipid rafts/caveolae-disrupting stress, Carcinogenesis 30 (2009) 1997–2004. H.E. Ryan, J. Lo, R.S. Johnson, HIF-1 alpha is required for solid tumor formation and embryonic vascularization, EMBO J. 17 (1998) 3005–3015. H. Yasuda, Solid tumor physiology and hypoxia-induced chemo/radioresistance. Novel strategy for cancer therapy: nitric oxide donor as a therapeutic enhancer, Nitric oxide 19 (2008) 205–216. G.T. Pei, C.W. Wu, W.W. Lin, Hypoxia-induced decoy receptor 2 gene expression is regulated via a hypoxia-inducible factor 1alpha-mediated mechanism, Biochem. Biophys. Res. Commun. 391 (2010) 1274–1279.