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HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells
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Research Article
HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells Junye Liu a,b , Jing Zhang b , Xiaowu Wang a , Yan Li b , Yongbin Chen a , Kangchu Li a , Jian Zhang b , Libo Yao b,⁎, Guozhen Guo a,⁎ a b
Department of Radiation Medicine, Fourth Military Medical University, Xi'an, China Department of Biochemistry and Molecular Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, China
A R T I C L E I N F O R M A T I O N
AB S TR AC T
Article Chronology:
Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been
Received 1 December 2009
shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are
Revised version received
unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride
19 February 2010
could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic
Accepted 24 February 2010
expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-
Available online 3 March 2010
down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. NMyc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was
Keywords:
found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression
HIF-1
of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to
NDRG2
radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced
Cervical cancer
apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest
Hypoxia
that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2
Radioresistance
acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy. © 2010 Elsevier Inc. All rights reserved.
Introduction Hypoxia is an important characteristic feature of solid tumors, such as cervical cancer. Evidence has accumulated showing that up to 50–60% of locally advanced solid tumors contain hypoxic regions that have considerably lower oxygen tension than the normal tissues [1]. A large body of clinical data has been published demonstrating that hypoxia negatively influence radiotherapy. By utilizing the Eppendorf probe, a polarographic microelectrode which can accurately measure microregional tissue oxygen
pressures, researchers have been able to evaluate the correlation of oxygen tension inside solid tumors with local control, diseasefree survival and overall survival after radiotherapy. As reviewed in [2], the poor prognostic value of hypoxia has been proved for irradiated cervical carcinomas, head and neck carcinomas as well as soft tissue sarcomas, which strongly indicate that tumor hypoxia could induce radioresistance. Oxygen enhancement effect has been proposed as one of the mechanisms underlying hypoxia-induced radioresistance [2]. Oxygen can react with the radiation-created broken ends of
⁎ Corresponding authors. L. Yao is to be contacted at fax: +86 29 84774513. G. Guo, fax: +86 29 84774873. E-mail addresses: [email protected] (L. Yao), [email protected] (G. Guo). Abbreviations: HIF-1, hypoxia inducible factor 1; HPV, human papillomaviruse; HRE, hypoxia responsive element; NDRG2, N-Myc downstreamregulated gene 2; OER, oxygen enhancement ratio; REF, resistance enhancement factor; SEF, sensitivity enhancement factor 0014-4827/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2010.02.028
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DNA, impair its repair and thus facilitate radiation-induced cell death. Hypoxia can abolish oxygen enhancement effect and lead to decreased radiosensitivity. However, there are increasing reports indicating that hypoxia may also modulate tumor radiosensitivity through biological effects. Hypoxia is a powerful stimulus of many critical tumor phenotypes and regulates various important cellular processes. Hypoxia inducible factor 1 (HIF-1) is one the key mediators of hypoxia signaling pathways [3]. HIF-1 is a heterodimeric transcription factor consisting of two subunits, HIF-1α and HIF-1β. While HIF-1β is constitutively expressed, HIF-1α is rapidly degraded in normoxic conditions and can be stabilized by low oxygen tensions. Therefore, hypoxia can induce HIF-1 expression, which in turn increases the expression of more than 100 gene products that take parts in the regulation of angiogenesis, metabolism, cell cycle progression and apoptotic process [3]. HIF-1 has been implicated in regulating tumor radiosensitivity [1,4]. As revealed by a multicentre study, HIF-1α expression in uterine cervical cancer tissues negatively correlates with 5-year survival and positively correlates with the adverse effects of radiotherapy [5]. In another study, HIF-1α expression in patients with definitive radiotherapy for cervical cancer indicates an increased risk of tumorrelated death [6]. These data suggests that HIF-1 might play a role in regulating tumor radiosensitivity. Human N-Myc downstream-regulated gene 2 (NDRG2), also named SYLD/KIAA1248, was first cloned in our laboratory in 1999 (GenBank accession no. AF159092). NDRG2 belongs to the NDRG gene family, a new class of Myc-repressed genes which also consist of NDRG1, NDRG3 and NDRG4 [7]. The NDRGs share 57–65% amino acid identity and are highly conserved in plants, invertebrates and mammals, suggesting that this gene family may have important cellular functions [8]. Similar to NDRG1, NDRG2 is also a differentiation-related gene and could be repressed by c-myc [9]. A line of evidence suggests that NDRG2 is deregulated in human malignant tumors, including glioblastoma [10,11], meningioma [12], colorectal carcinoma [13–16], gastric cancer [17], breast cancer [18], thyroid cancer [19], clear cell renal cell carcinoma [20], liver cancer and pancreatic cancer [21]. Interestingly, NDRG2 has been identified as a target gene of HIF-1 and can be upregulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein levels [22]. This inspired us to conduct the present study to explore whether NDRG2 has a role in regulation of hypoxiainduced tumor radioresistance.
the presence or absence of 100 μM CoCl2 for a specified time period.
Constructs and transfection Expression constructs were derived by subcloning cDNAs encoding full-length HIF-1α or NDRG2 into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The recombinant pSilencer 3.1 (Ambion, Austin, TX) expressing a scramble control siRNA or siRNA specific to HIF-1α or NDRG2 has been described elsewhere [22,23]. All construct sequences were directly confirmed by DNA sequencing. Hela cells were transfected with the corresponding constructs using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction.
Irradiation Irradiation was performed using a 60Co γ-ray therapeutic machine, RCR-120 (Toshiba, Tokyo, Japan), at a dose rate of 1.6 Gy/min.
Colony-forming assay
Materials and methods
Exponentially growing cells were trypsinized into single cell suspension and exposed to indicated dosage of irradiation. Following exposure, cells (200–2000) were plated in 60 mm dishes cells and incubated at 37 °C, 5% CO2 for colony formation. After 10–14 days of growth, the colonies were fixed with 10% (v/ v) methanol for 15 min and stained with 5% (g/v) Giemsa solution (Sigma) for 20 min. Colonies that consisted of more than 50 cells were scored. Colony plating efficiency was calculated to be the number of viable plated cells, and expressed as a percentage of inoculated cells. In each irradiation dose group, survival fraction of cells was calculated as plating efficiency of the irradiated cells divided by the plating efficiency of the untreated control. Survival curves were plotted as the log of survival fraction versus radiation dose. The ID50 (radiation dose producing a surviving fraction of 50%) was determined by SPSS software (version 10.0; SPSS, Chicago, IL). The oxygen enhancement ratio (OER) was determined by dividing the ID50 under hypoxic conditions by the ID50 under aerobic conditions. The radioresistance-promoting effects of CoCl2, HIF-1 and NDRG2 were represented by the resistance enhancement factor (REF), calculated as the ID50 for treated cells divided by the ID50 for control cells. The radiosensitivitypromoting effects of HIF-1α siRNA and NDRG2 siRNA were represented by the sensitivity enhancement factor (SEF), calculated as the ID50 for control cells divided by the ID50 for the treated cells.
Cell culture and hypoxia induction
Western blot analysis
The human cervical cancer cell line Hela was obtained from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sijiqing Biological Engineering Materials Co., Hangzhou, China) at 37 °C in the presence of 5% CO2-balanced air. To induce hypoxia, cells were rendered in a chamber with a gas mixture of 1% O2 and 5% CO2balanced N2 at 37 °C. The level of oxygen in the chamber was verified using a gas monitor (SKC, Inc., Eighty Four, PA). To mimic hypoxia using chemicals, cells were cultured under 20% oxygen in
The proteins extracted from Hela cells with lysis buffer (50 mM Tris–HCl, pH 7.2, 1% Triton X-100, 10% Glycerol, 5 mM EDTA, 150 mM NaCl, 5 mM CaCl2, 10 mM MgCl2 with 10 μg/ml leupeptin, 10 μg/ml aprotinin, 5 μg/ml pepstatin and 1 mM PMSF) were resolved over 12% SDS-PAGE and transferred to nitrocellulose membranes (0.22 μm, Millipore, Bedford, MA). The blots were probed with antibodies against HIF-1α (BD Biosciences, San Diego, CA), NDRG2 [24], Bax and Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA). Equal loading of all lanes was confirmed by reprobing the membranes with anti-β-actin antibody (Sigma, St. Louis, MO).
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Real-time PCR The total RNA of Hela cells was isolated using TRIzol (Invitrogen, Carlsbad, CA) and then quantified. One microgram of total RNA was reverse transcribed with Superscript II RNase H reverse transcriptase using oligo (dT) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Primers for NDRG2 expression have been described previously [25]. NDRG2 mRNA expression levels were determined by real-time PCR using the ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster City, CA). Fluorescent data were converted into cycle threshold measurements using the SDS system software and exported to Microsoft Excel. Fold expression changes relative to untreated control were calculated with the ΔΔCT method [26] using ARF1 (ADP-ribosylation factor 1) as the reference transcript [25].
Detection of apoptosis Apoptosis was detected using flow cytometry analysis. In brief, cells were harvested following mild trypsinization, washed in phosphate-buffered saline and stained with fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science, Basel, Switzerland) and propidium iodide (PI). Apoptotic cells were measured using a Becton Dickinson fluorescence-activated cell sorter (FACS) apparatus.
Statistical analysis Data are expressed as the mean ± SD. Statistical analyses were performed with the SPSS software (version 10.0; SPSS, Chicago, IL) by using one-way ANOVA followed by the t-test for independent groups. Statistical significance was based on a value of p < 0.05.
Results Hypoxia and its mimetic induced radioresistance of Hela cells Many studies linking hypoxia to tumor radioresistance were based on clinical data or transplanted tumor xenograft in nude mice [1,2]. To exclude the influence of tumor stromal cells and endothelial cells and better understand the effects of hypoxia on cancer cell radiosensitivity, the cultured monolayer of human cervical cancer Hela cells were used in this study. As indicated in Fig. 1A, hypoxia treatment significantly increased the survival fraction of irradiated Hela cells in a dosage-dependent manner. As revealed by calculation, the OER was 2.31 ± 0.36. Cobalt chloride (CoCl2), one kind of the hypoxia mimetic, was also employed to test hypoxia impact on radiosensitivity. It was clearly demonstrated that pre-treatment with CoCl2 could increase radioresistance of Hela cells (Fig. 1B) with REF as 1.93 ± 0.27. Taken together, these data indicated that both hypoxia and its mimetic agent could induce resistance of Hela cells to radiation.
HIF-1 promoted radioresistance of Hela cells HIF-1 expression in Hela cells was genetically modulated by transient transfection with constructs expressing HIF-1α or HIF1α-specific siRNA, which resulted in overexpression or inhibition
Fig. 1 – Hypoxia enhances resistance of Hela cells to radiation. Cervical cancer Hela cells pre-treated (A) in hypoxic conditions or (B) with hypoxia mimetic CoCl2 (100 μM) for 12 h were subjected to radiation at different dosage as indicated. The survival fractions were determined by colony-forming assay as described in “Materials and methods”. Data was expressed as mean ± SD of triplicates in one experiment. Shown was representative of at least 3 experiments.
of HIF-1α in Hela cells (Fig. 2A). Under normoxic condition, elevated expression of HIF-1α dramatically increased the survival fraction of irradiated Hela cells (Fig. 2B) with REF as 1.71 ± 0.19. Meanwhile, knocking-down of HIF-1α expression could sensitize Hela cells pre-treated in hypoxic condition to radiation (Fig. 2C) with SEF as 1.48 ± 0.13. These data indicated that HIF-1 could promote the radioresistance of Hela cells.
NDRG2 expression was induced in Hela cells by both hypoxia and radiation in a HIF-1-dependent manner Our previous work has revealed NDRG2 as a new target gene of HIF-1 and demonstrated that NDRG2 can be induced by hypoxia in human lung cancer cell line A549, hepatocellular carcinoma cell line HepG2 and breast carcinoma cell line SKBR-3 [22]. In the present study, we tested whether hypoxia could regulate NDRG2 expression in human cervical cancer Hela cells. It was shown that hypoxia treatment dramatically upregulated NDRG2 expression in Hela cells in a time-dependent manner (Fig. 3A). NDRG2 expression in Hela cells apparently increased after 1 h of exposure to hypoxia, steadily increased and reached to its peak after 12– 18 h of hypoxia treatment. Knock-down of HIF-1α did not change NDRG2 expression but did abolish hypoxia-induced upregulation of NDRG2 in Hela cells (Fig. 3B). Interestingly, NDRG2 could be also induced by radiation in time-dependent and dose-dependent manner (Fig. 4). Hela cells were irradiated at 6 Gy and NDRG2 expression was monitored by
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real-time PCR and Western blot analysis. It was evidenced that both the mRNA and protein expression level of NDRG2 apparently elevated 8 h after radiation, increased to its peak at 12 h, begun to decrease at 16 h and came back to basal level at 24 h (Figs. 4A,B). In another set of experiment, Hela cells were irradiated at different dosage and NDRG2 expression was followed 12 h after radiation. While 2 Gy of radiation has no significant influence on NDRG2, 4– 10 Gy of radiation upregulated NDRG2 mRNA and protein expression with the profound effect at 6 Gy (Figs. 4C,D). It has been reported that radiation could cause HIF-1 expression levels to increase in tumors [27] and NDRG2 promoter contains hypoxia responsive element (HRE) which is under the control of HIF-1 [22]. We therefore asked whether HIF-1 mediates radiation-induced NDRG2 expression. The present study revealed that HIF-1α expression significantly increased 2 h after radiation, reached to its peak at 8 h, gradually decreased at 12 h and came back to its basal level at 24 h (Fig. 5A). It was clear that HIF-1α responded to radiation about 6 h earlier than NDRG2 in Hela cells, which indicated HIF-1 activation might be an event upstream of NDRG2 upregulation. HIF-1α expression was silenced in Hela cells with HIF-1α-specific siRNA construct and the effect of radiation on NDRG2 expression was revisited. As shown in Fig. 5B, knock-down of HIF-1α completely inhibited radiation-induced upregulation of NDRG2 in Hela cells. These data suggested that radiation-induced NDRG2 expression was HIF-1-dependent.
NDRG2 promoted radioresistance of Hela cells
Fig. 2 – HIF-1 mediates hypoxia-induced radioresistance of Hela cells. Hela cells were transfected with cDNAs of pcDNA3.1 construct expressing HIF-1α, pcDNA3.1 (vector), pSliencer3.1 constructs expressing HIF-1α-specific siRNA (siHIF-1α) or scramble siRNA (control). Twenty four hours later, cells were harvested and subjected to Western blot to detect HIF-1α expression (A. Exposure time: 2 min), or irradiated (B), or treated in hypoxic condition for 12 h and then irradiated at indicated dosage (C). The dose–survival curves (B, C) were determined as described in Fig. 1.
The fact that NDRG2 was a responsive gene to radiation led us to evaluate the role of NDRG2 in radioresistance of Hela cells. NDRG2 expression in Hela cells was upregulated or downregulated by transfection with constructs expressing NDRG2 or NDRG2-specific siRNA, respectively (Fig. 6A). Under normoxic condition, overexpression of NDRG2 dramatically increased the survival fraction of irradiated Hela cells (Fig. 6B) with REF as 1.94 ± 0.28, indicating that NDRG2 could promote the radioresistance of Hela cells. Meanwhile, silence of NDRG2 expression significantly decreased the survival fraction of irradiated Hela cells (Fig. 6C) with SEF as 1.81 ± 0.23, which suggested that inhibition of NDRG2 could reverse hypoxia-induced radioresistance of Hela cells.
NDRG2 protected Hela cells from radiation-induced apoptosis The effects of NDRG2 on radiation-induced apoptosis were further investigated. It was observed that ectopic expression of NDRG2 inhibited radiation-induced apoptosis (Fig. 7A) whereas knockdown of NDRG2 increased radiation-induced apoptosis of Hela cells (Fig. 7B), which indicated that NDRG2 could promote the resistance of Hela cells to radiation-induced apoptosis.
NDRG2 inhibited radiation-induced upregulation of Bax Fig. 3 – HIF-1 mediates hypoxia-induced NDRG2 expression in Hela cells. A. Hela cells were incubated in hypoxic condition for the indicated time. B. Hela cells transfected with cDNAs of pSliencer3.1 constructs expressing HIF-1α-specific siRNA (siHIF-1α) or scramble siRNA (control) were incubated in normoxic or hypoxic condition for 12 h. NDRG2 expression in Hela cells was determined by Western blot analysis. β-actin was detected as loading control.
To further illustrate the role of NDRG2 in radiation-induced apoptosis, Bax and Bcl-2 expression was monitored in irradiated Hela cells. As shown in Fig. 8, radiation induced no changes of Bcl-2 but elevated expression of Bax, thus increased the Bax/Bcl-2 ratio, which would transduce signals to downstream molecules and initiate apoptotic process. Surprisingly, overexpression of NDRG2 almost completely abolished upregulation of Bax as well as Bax/ Bcl-2 ratio induced by radiation (Fig. 8A). Meanwhile, silence of
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Fig. 4 – Radiation up-regulates NDRG2 expression in Hela cells. A and B. Hela cells were irradiated at 6 Gy and harvested at indicated time points. C and D. Hela cells were irradiated at indicated dosage and harvested 12 h later. A and C. NDRG2 mRNA expression level was evaluated by real-time PCR as described in “Materials and methods”. B and D. NDRG2 protein was detected by Western blot analysis (upper panel). β-actin was also detected as loading control. NDRG2 expression was quantitated by densitometry and plotted as fold change (lower panel). Values were mean ± SD of 3 independent experiments.
NDRG2 facilitated radiation-induced Bax expression and promoted increase of Bax/Bcl-2 ratio (Fig. 8B). These strongly suggested that NDRG2 could promote resistance of Hela cells to radiation-induced apoptosis through inhibiting radiation-induced upregulation of Bax.
Discussion It is widely accepted that Oxygen can increase the cytotoxicity of radiation by modifying radiation-induced DNA damage and
Fig. 5 – HIF-1 mediates radiation-induced NDRG2 expression in Hela cells. A. Hela cells were irradiated at 6 Gy and harvested indicated time points. HIF-1α expression was analyzed by Western blot analysis (exposure time: 15 s). B. Hela cells transfected with cDNAs of pSliencer3.1 constructs expressing HIF-1α-specific siRNA (siHIF-1α) or scramble siRNA (control) were irradiated at 6 Gy and harvested 12 h later. NDRG2 expression was detected by Western blot analysis. β-actin was detected as loading control.
impairing DNA repair. This phenomenon is termed oxygen enhancement effect, which could make oxygenated cells 3-times more radiosensitive than hypoxic cells [2]. Depriving cells of oxygen would abolish oxygen enhancement effect and thus leads to decreased sensitivity of cells to irradiation. As revealed by the present study, pre-treatment of Hela cells in hypoxic condition resulted in increased radioresistance and the calculated OER was 2.31 ± 0.36, which was close to the previous report for Hela cells [28]. Interestingly, the hypoxia mimetic cobalt chloride could enhance the radioresistance of Hela cells in the presence of normal oxygen tension. Cobalt chloride does not change the oxygenation of cells but activate the hypoxia signaling transduction pathway, especially the HIF-1 pathway [3]. Our data supports the idea that, in addition to repressed oxygen enhancement effect by hypoxia, there must be some biological mechanisms mediating hypoxiainduced radioresistance [2]. HIF-1 is the most important mediator of hypoxia signal transduction, which controls the expression of greater than 100 genes and thus exerts diverse and even controversial impacts on tumor radiosensitivity [3]. HIF-1 can induce cell cycle arrest [29], inhibit apoptosis signal [30] and promote angiogenesis [31,32], which would result in decreased radiosensitivity. HIF-1 can also reinforce apoptosis signal [31] and enhance glycolysis [33], which would lead to increased radiosensitivity. Therefore, the net effect of HIF-1 is determined by the balance of those two aspects. Studies have shown that HIF-1-deficiency promotes tumor radiosensitivity in vivo [27,34–36], suggesting that the net result of HIF-1 activity is to promote radioresistance in tumors. It has been proposed that
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Fig. 6 – NDRG2 contributes to hypoxia-induced radioresistance of Hela cells. Hela cells were transfected with cDNAs of pcDNA3.1 construct expressing NDRG2, pcDNA3.1 (vector), pSliencer3.1 constructs expressing NDRG2-specific siRNA (siNDRG2) or scramble siRNA (control). Twenty four hours later, cells were harvested and subjected to Western blot to detect NDRG2 expression (A), or irradiated (B), or treated in hypoxic condition for 12 h and then irradiated at indicated dosage (C). The dose–survival curves (B, C) were determined as described in Fig. 1.
HIF-1 in cancer cells would induce the expression of a variety of proangiogenic cytokines which protects stromal endothelial cells from radiation-induced cell death and eventually enhances radioresistance of tumor cells [4,27,35]. From this view point, that is the indirect protective effect of HIF-1 on tumor cells. The present study demonstrated that overexpression of HIF-1α could promote radioresistance while silence of HIF-1α could increase radiosensitivity of Hela cells in vitro. In line with this finding, a recent study revealed that radioresistant lung cancer cell lines exhibited higher expression level of HIF-1α than radiosensitive lung cancer cell lines and that inhibition of HIF-1α expression sensitized lung cancer cells to radiation [37]. These data indicate that HIF-1 can directly enhance radioresistance of cancer cells. Taken together, there are at least two mechanisms mediating the effect of HIF-1 on tumor radioresistance. Firstly HIF-1 could directly protect cancer cells from radiation-induced cell death. Secondly, HIF-1 increases the expression of proangiogenic cytokines and promotes the survival of stromal endothelial cells which in turn provide support
Fig. 7 – NDRG2 regulates radiation-induced apoptosis of Hela cells. Hela cells were transfected with cDNAs of (A) pcDNA3.1 construct expressing NDRG2 or pcDNA3.1 (vector), (B) pSliencer3.1 constructs expressing NDRG2-specific siRNA (siNDRG2) or scramble siRNA (control). Twenty four hours later, cells were irradiated at indicated dosage and then cultured for another 24 h. The apoptotic cells were stained with FITC-labeled annexin V and propidium iodide and analyzed by flow cytometry. The percentage of double positive cells was calculated and data was expressed as mean ± SD of triplicates in one experiment. Shown was representative of at least 3 experiments. *p < 0.01 vs. vector or control.
to cancer cells. However, how does HIF-1 directly induce radioresistance of cancer cells needs further investigation. HIF-1 can be induced and activated not only by hypoxia and its mimetics but also by irradiation. Moeller et al. [27] found that HIF1 activity in tumors growing in skinfold window chambers began to increase steadily approximately 12–24 h after radiation, peaking 48 h following treatment. However, they also reported that there was no detectable increase in HIF-1 expression and activation at 24, 48 and 72 h after irradiation of cancer cells in vitro [27]. They therefore inferred that radiation induces HIF-1 activation only in vivo. We argue here that HIF-1 can be induced also in vitro. The present study demonstrated that HIF-1α expression in monolayer of Hela cells increased 2 h after radiation, reached to its peak at 8 h, gradually decreased at 12 h and came back to its basal level at 24 h. Recently, there are two studies that report similar results. Kim et al. [37] examined a series of human lung cancer cell lines and found that HIF-1α expression was induced in H1299, H182 and H226B cells as early as 30 min to 3 h after administering a single dose of radiation. Singh-Gupta et al. [38] observed that radiation-induced upregulation of HIF-1α in PC-3 cells occurred at 3–5 h after radiation. These data suggest that HIF-1α responses to radiation very quickly and that it will be very late to check HIF-1α more than 24 h after radiation. Moeller et al. [27] didn't examine the change of HIF-1 activity within 24 h of
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Fig. 8 – NDRG2 regulates radiation-induced apoptosis of Hela cells. Hela cells were transfected with cDNAs of (A) pcDNA3.1 construct expressing NDRG2 or pcDNA3.1 (vector), (B) pSliencer3.1 constructs expressing NDRG2-specific siRNA (siNDRG2) or scramble siRNA (control). Twenty four hours later, cells were irradiated at 6 Gy and then cultured for another 12 h. The cellular extracts were analyzed by Western blot to determine Bax and Bcl-2 expression level (upper panel). β-actin was detected as loading control. Bax and Bcl-2 expression were quantitated by densitometry and plotted as Bax/Bcl-2 ratio (lower panel). Values were mean ± SD of 3 independent experiments. *p < 0.01 vs. vector or control.
radiation and might miss the opportunity to uncover radiationinduced increase of HIF-1 activity in vitro. Altogether, radiation can induce the expression and activation of HIF-1 in cancer cells both in vitro and in vivo. In addition, it has been discovered that human papillomaviruse (HPV) E6 and E7 were able independently to enhance induction of HIF-1α upon hypoxic treatment [39]. Considering HPVs are the causative agents of cervical cancer, it would be possible for HIF-1 to be induced and activated by hypoxia, radiation and E6/E7 proteins in HPV + cervical cancer. So, HIF-1 blockade would be much promise and beneficial to the patients with HPV + cervical cancer who underwent radiotherapy. The present study revealed that NDRG2 was upregulated in cervical cancer Hela cells not only by hypoxia but also by radiation. Silence of HIF-1α could suppress hypoxia- and radiation-induced NDRG2 expression, indicating response of NDRG2 to hypoxia and radiation is HIF-1-dependent. It was also demonstrated that enforcing the expression of NDRG2 led to increased radioresistance of Hela cells whereas silencing NDRG2 strongly enhanced radiosensitivity of hypoxic Hela cells. Furthermore, NDRG2 could inhibit radiation-induced upregulation of Bax and prevent Hela cells from radiation-induced apoptosis. It thus can be established that NDRG2 responses to HIF-1 activation and promotes radioresistance of cancer cells, which represents one of the mechanisms underlying HIF-1mediated radioresistance. However, it should be noted that radiation could induce mitotic catastrophe as well as apoptosis. Thus, further studies are needed to explore the effects of NDRG2 on radiationinduced mitotic catastrophe. Moreover, the mechanisms depicting the effect of NDRG2 on Bax expression remain unclear. It has been found that NDRG2 could translocate to nucleus upon stimulation with hypoxia [22] and radiation (data not shown). A physical interaction of NDRG2 with transcription factor MSP58 has been
demonstrated and their colocalization in nucleus of Hela cells during cell stress has been confirmed [40]. It can be speculated that NDRG2 might interact with transcription factors and thus regulate gene expression profile during response to radiation. Further investigation is ongoing to validate this speculation. In conclusion, the present study demonstrated that NDRG2 can be induced in Hela cells by both hypoxia and radiation in a HIF-1dependent manner and that both NDRG2 and HIF-1 contribute to hypoxia-induced tumor radioresistance. These data can help us to better understand the molecular mechanisms underlying hypoxiainduced tumor radioresistance and the precise role of NDRG2 in tumor development. It is also suggested that blockage of HIF-1 and/ or NDRG2 may sensitize tumors to radiotherapy. Further studies are encouraged to evaluate the clinical application potential of HIF1 and NDRG2 blockade as radiosensitizer for tumor therapy.
Acknowledgments This work was supported by National Natural Science Foundation of China (nos. 30600161, 60871068 and 30970862), National Basic Research Program of China (no. 2006CB504100), Natural Science Foundation of Shaanxi Province (no. 2006C202) and Postdoctoral Science Foundation of China (no. 200503826).
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Research Article
HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells Junye Liu a,b , Jing Zhang b , Xiaowu Wang a , Yan Li b , Yongbin Chen a , Kangchu Li a , Jian Zhang b , Libo Yao b,⁎, Guozhen Guo a,⁎ a b
Department of Radiation Medicine, Fourth Military Medical University, Xi'an, China Department of Biochemistry and Molecular Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, China
A R T I C L E I N F O R M A T I O N
AB S TR AC T
Article Chronology:
Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been
Received 1 December 2009
shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are
Revised version received
unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride
19 February 2010
could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic
Accepted 24 February 2010
expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-
Available online 3 March 2010
down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. NMyc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was
Keywords:
found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression
HIF-1
of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to
NDRG2
radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced
Cervical cancer
apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest
Hypoxia
that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2
Radioresistance
acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy. © 2010 Elsevier Inc. All rights reserved.
Introduction Hypoxia is an important characteristic feature of solid tumors, such as cervical cancer. Evidence has accumulated showing that up to 50–60% of locally advanced solid tumors contain hypoxic regions that have considerably lower oxygen tension than the normal tissues [1]. A large body of clinical data has been published demonstrating that hypoxia negatively influence radiotherapy. By utilizing the Eppendorf probe, a polarographic microelectrode which can accurately measure microregional tissue oxygen
pressures, researchers have been able to evaluate the correlation of oxygen tension inside solid tumors with local control, diseasefree survival and overall survival after radiotherapy. As reviewed in [2], the poor prognostic value of hypoxia has been proved for irradiated cervical carcinomas, head and neck carcinomas as well as soft tissue sarcomas, which strongly indicate that tumor hypoxia could induce radioresistance. Oxygen enhancement effect has been proposed as one of the mechanisms underlying hypoxia-induced radioresistance [2]. Oxygen can react with the radiation-created broken ends of
⁎ Corresponding authors. L. Yao is to be contacted at fax: +86 29 84774513. G. Guo, fax: +86 29 84774873. E-mail addresses: [email protected] (L. Yao), [email protected] (G. Guo). Abbreviations: HIF-1, hypoxia inducible factor 1; HPV, human papillomaviruse; HRE, hypoxia responsive element; NDRG2, N-Myc downstreamregulated gene 2; OER, oxygen enhancement ratio; REF, resistance enhancement factor; SEF, sensitivity enhancement factor 0014-4827/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2010.02.028
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DNA, impair its repair and thus facilitate radiation-induced cell death. Hypoxia can abolish oxygen enhancement effect and lead to decreased radiosensitivity. However, there are increasing reports indicating that hypoxia may also modulate tumor radiosensitivity through biological effects. Hypoxia is a powerful stimulus of many critical tumor phenotypes and regulates various important cellular processes. Hypoxia inducible factor 1 (HIF-1) is one the key mediators of hypoxia signaling pathways [3]. HIF-1 is a heterodimeric transcription factor consisting of two subunits, HIF-1α and HIF-1β. While HIF-1β is constitutively expressed, HIF-1α is rapidly degraded in normoxic conditions and can be stabilized by low oxygen tensions. Therefore, hypoxia can induce HIF-1 expression, which in turn increases the expression of more than 100 gene products that take parts in the regulation of angiogenesis, metabolism, cell cycle progression and apoptotic process [3]. HIF-1 has been implicated in regulating tumor radiosensitivity [1,4]. As revealed by a multicentre study, HIF-1α expression in uterine cervical cancer tissues negatively correlates with 5-year survival and positively correlates with the adverse effects of radiotherapy [5]. In another study, HIF-1α expression in patients with definitive radiotherapy for cervical cancer indicates an increased risk of tumorrelated death [6]. These data suggests that HIF-1 might play a role in regulating tumor radiosensitivity. Human N-Myc downstream-regulated gene 2 (NDRG2), also named SYLD/KIAA1248, was first cloned in our laboratory in 1999 (GenBank accession no. AF159092). NDRG2 belongs to the NDRG gene family, a new class of Myc-repressed genes which also consist of NDRG1, NDRG3 and NDRG4 [7]. The NDRGs share 57–65% amino acid identity and are highly conserved in plants, invertebrates and mammals, suggesting that this gene family may have important cellular functions [8]. Similar to NDRG1, NDRG2 is also a differentiation-related gene and could be repressed by c-myc [9]. A line of evidence suggests that NDRG2 is deregulated in human malignant tumors, including glioblastoma [10,11], meningioma [12], colorectal carcinoma [13–16], gastric cancer [17], breast cancer [18], thyroid cancer [19], clear cell renal cell carcinoma [20], liver cancer and pancreatic cancer [21]. Interestingly, NDRG2 has been identified as a target gene of HIF-1 and can be upregulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein levels [22]. This inspired us to conduct the present study to explore whether NDRG2 has a role in regulation of hypoxiainduced tumor radioresistance.
the presence or absence of 100 μM CoCl2 for a specified time period.
Constructs and transfection Expression constructs were derived by subcloning cDNAs encoding full-length HIF-1α or NDRG2 into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The recombinant pSilencer 3.1 (Ambion, Austin, TX) expressing a scramble control siRNA or siRNA specific to HIF-1α or NDRG2 has been described elsewhere [22,23]. All construct sequences were directly confirmed by DNA sequencing. Hela cells were transfected with the corresponding constructs using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction.
Irradiation Irradiation was performed using a 60Co γ-ray therapeutic machine, RCR-120 (Toshiba, Tokyo, Japan), at a dose rate of 1.6 Gy/min.
Colony-forming assay
Materials and methods
Exponentially growing cells were trypsinized into single cell suspension and exposed to indicated dosage of irradiation. Following exposure, cells (200–2000) were plated in 60 mm dishes cells and incubated at 37 °C, 5% CO2 for colony formation. After 10–14 days of growth, the colonies were fixed with 10% (v/ v) methanol for 15 min and stained with 5% (g/v) Giemsa solution (Sigma) for 20 min. Colonies that consisted of more than 50 cells were scored. Colony plating efficiency was calculated to be the number of viable plated cells, and expressed as a percentage of inoculated cells. In each irradiation dose group, survival fraction of cells was calculated as plating efficiency of the irradiated cells divided by the plating efficiency of the untreated control. Survival curves were plotted as the log of survival fraction versus radiation dose. The ID50 (radiation dose producing a surviving fraction of 50%) was determined by SPSS software (version 10.0; SPSS, Chicago, IL). The oxygen enhancement ratio (OER) was determined by dividing the ID50 under hypoxic conditions by the ID50 under aerobic conditions. The radioresistance-promoting effects of CoCl2, HIF-1 and NDRG2 were represented by the resistance enhancement factor (REF), calculated as the ID50 for treated cells divided by the ID50 for control cells. The radiosensitivitypromoting effects of HIF-1α siRNA and NDRG2 siRNA were represented by the sensitivity enhancement factor (SEF), calculated as the ID50 for control cells divided by the ID50 for the treated cells.
Cell culture and hypoxia induction
Western blot analysis
The human cervical cancer cell line Hela was obtained from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sijiqing Biological Engineering Materials Co., Hangzhou, China) at 37 °C in the presence of 5% CO2-balanced air. To induce hypoxia, cells were rendered in a chamber with a gas mixture of 1% O2 and 5% CO2balanced N2 at 37 °C. The level of oxygen in the chamber was verified using a gas monitor (SKC, Inc., Eighty Four, PA). To mimic hypoxia using chemicals, cells were cultured under 20% oxygen in
The proteins extracted from Hela cells with lysis buffer (50 mM Tris–HCl, pH 7.2, 1% Triton X-100, 10% Glycerol, 5 mM EDTA, 150 mM NaCl, 5 mM CaCl2, 10 mM MgCl2 with 10 μg/ml leupeptin, 10 μg/ml aprotinin, 5 μg/ml pepstatin and 1 mM PMSF) were resolved over 12% SDS-PAGE and transferred to nitrocellulose membranes (0.22 μm, Millipore, Bedford, MA). The blots were probed with antibodies against HIF-1α (BD Biosciences, San Diego, CA), NDRG2 [24], Bax and Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA). Equal loading of all lanes was confirmed by reprobing the membranes with anti-β-actin antibody (Sigma, St. Louis, MO).
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Real-time PCR The total RNA of Hela cells was isolated using TRIzol (Invitrogen, Carlsbad, CA) and then quantified. One microgram of total RNA was reverse transcribed with Superscript II RNase H reverse transcriptase using oligo (dT) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Primers for NDRG2 expression have been described previously [25]. NDRG2 mRNA expression levels were determined by real-time PCR using the ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster City, CA). Fluorescent data were converted into cycle threshold measurements using the SDS system software and exported to Microsoft Excel. Fold expression changes relative to untreated control were calculated with the ΔΔCT method [26] using ARF1 (ADP-ribosylation factor 1) as the reference transcript [25].
Detection of apoptosis Apoptosis was detected using flow cytometry analysis. In brief, cells were harvested following mild trypsinization, washed in phosphate-buffered saline and stained with fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science, Basel, Switzerland) and propidium iodide (PI). Apoptotic cells were measured using a Becton Dickinson fluorescence-activated cell sorter (FACS) apparatus.
Statistical analysis Data are expressed as the mean ± SD. Statistical analyses were performed with the SPSS software (version 10.0; SPSS, Chicago, IL) by using one-way ANOVA followed by the t-test for independent groups. Statistical significance was based on a value of p < 0.05.
Results Hypoxia and its mimetic induced radioresistance of Hela cells Many studies linking hypoxia to tumor radioresistance were based on clinical data or transplanted tumor xenograft in nude mice [1,2]. To exclude the influence of tumor stromal cells and endothelial cells and better understand the effects of hypoxia on cancer cell radiosensitivity, the cultured monolayer of human cervical cancer Hela cells were used in this study. As indicated in Fig. 1A, hypoxia treatment significantly increased the survival fraction of irradiated Hela cells in a dosage-dependent manner. As revealed by calculation, the OER was 2.31 ± 0.36. Cobalt chloride (CoCl2), one kind of the hypoxia mimetic, was also employed to test hypoxia impact on radiosensitivity. It was clearly demonstrated that pre-treatment with CoCl2 could increase radioresistance of Hela cells (Fig. 1B) with REF as 1.93 ± 0.27. Taken together, these data indicated that both hypoxia and its mimetic agent could induce resistance of Hela cells to radiation.
HIF-1 promoted radioresistance of Hela cells HIF-1 expression in Hela cells was genetically modulated by transient transfection with constructs expressing HIF-1α or HIF1α-specific siRNA, which resulted in overexpression or inhibition
Fig. 1 – Hypoxia enhances resistance of Hela cells to radiation. Cervical cancer Hela cells pre-treated (A) in hypoxic conditions or (B) with hypoxia mimetic CoCl2 (100 μM) for 12 h were subjected to radiation at different dosage as indicated. The survival fractions were determined by colony-forming assay as described in “Materials and methods”. Data was expressed as mean ± SD of triplicates in one experiment. Shown was representative of at least 3 experiments.
of HIF-1α in Hela cells (Fig. 2A). Under normoxic condition, elevated expression of HIF-1α dramatically increased the survival fraction of irradiated Hela cells (Fig. 2B) with REF as 1.71 ± 0.19. Meanwhile, knocking-down of HIF-1α expression could sensitize Hela cells pre-treated in hypoxic condition to radiation (Fig. 2C) with SEF as 1.48 ± 0.13. These data indicated that HIF-1 could promote the radioresistance of Hela cells.
NDRG2 expression was induced in Hela cells by both hypoxia and radiation in a HIF-1-dependent manner Our previous work has revealed NDRG2 as a new target gene of HIF-1 and demonstrated that NDRG2 can be induced by hypoxia in human lung cancer cell line A549, hepatocellular carcinoma cell line HepG2 and breast carcinoma cell line SKBR-3 [22]. In the present study, we tested whether hypoxia could regulate NDRG2 expression in human cervical cancer Hela cells. It was shown that hypoxia treatment dramatically upregulated NDRG2 expression in Hela cells in a time-dependent manner (Fig. 3A). NDRG2 expression in Hela cells apparently increased after 1 h of exposure to hypoxia, steadily increased and reached to its peak after 12– 18 h of hypoxia treatment. Knock-down of HIF-1α did not change NDRG2 expression but did abolish hypoxia-induced upregulation of NDRG2 in Hela cells (Fig. 3B). Interestingly, NDRG2 could be also induced by radiation in time-dependent and dose-dependent manner (Fig. 4). Hela cells were irradiated at 6 Gy and NDRG2 expression was monitored by
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real-time PCR and Western blot analysis. It was evidenced that both the mRNA and protein expression level of NDRG2 apparently elevated 8 h after radiation, increased to its peak at 12 h, begun to decrease at 16 h and came back to basal level at 24 h (Figs. 4A,B). In another set of experiment, Hela cells were irradiated at different dosage and NDRG2 expression was followed 12 h after radiation. While 2 Gy of radiation has no significant influence on NDRG2, 4– 10 Gy of radiation upregulated NDRG2 mRNA and protein expression with the profound effect at 6 Gy (Figs. 4C,D). It has been reported that radiation could cause HIF-1 expression levels to increase in tumors [27] and NDRG2 promoter contains hypoxia responsive element (HRE) which is under the control of HIF-1 [22]. We therefore asked whether HIF-1 mediates radiation-induced NDRG2 expression. The present study revealed that HIF-1α expression significantly increased 2 h after radiation, reached to its peak at 8 h, gradually decreased at 12 h and came back to its basal level at 24 h (Fig. 5A). It was clear that HIF-1α responded to radiation about 6 h earlier than NDRG2 in Hela cells, which indicated HIF-1 activation might be an event upstream of NDRG2 upregulation. HIF-1α expression was silenced in Hela cells with HIF-1α-specific siRNA construct and the effect of radiation on NDRG2 expression was revisited. As shown in Fig. 5B, knock-down of HIF-1α completely inhibited radiation-induced upregulation of NDRG2 in Hela cells. These data suggested that radiation-induced NDRG2 expression was HIF-1-dependent.
NDRG2 promoted radioresistance of Hela cells
Fig. 2 – HIF-1 mediates hypoxia-induced radioresistance of Hela cells. Hela cells were transfected with cDNAs of pcDNA3.1 construct expressing HIF-1α, pcDNA3.1 (vector), pSliencer3.1 constructs expressing HIF-1α-specific siRNA (siHIF-1α) or scramble siRNA (control). Twenty four hours later, cells were harvested and subjected to Western blot to detect HIF-1α expression (A. Exposure time: 2 min), or irradiated (B), or treated in hypoxic condition for 12 h and then irradiated at indicated dosage (C). The dose–survival curves (B, C) were determined as described in Fig. 1.
The fact that NDRG2 was a responsive gene to radiation led us to evaluate the role of NDRG2 in radioresistance of Hela cells. NDRG2 expression in Hela cells was upregulated or downregulated by transfection with constructs expressing NDRG2 or NDRG2-specific siRNA, respectively (Fig. 6A). Under normoxic condition, overexpression of NDRG2 dramatically increased the survival fraction of irradiated Hela cells (Fig. 6B) with REF as 1.94 ± 0.28, indicating that NDRG2 could promote the radioresistance of Hela cells. Meanwhile, silence of NDRG2 expression significantly decreased the survival fraction of irradiated Hela cells (Fig. 6C) with SEF as 1.81 ± 0.23, which suggested that inhibition of NDRG2 could reverse hypoxia-induced radioresistance of Hela cells.
NDRG2 protected Hela cells from radiation-induced apoptosis The effects of NDRG2 on radiation-induced apoptosis were further investigated. It was observed that ectopic expression of NDRG2 inhibited radiation-induced apoptosis (Fig. 7A) whereas knockdown of NDRG2 increased radiation-induced apoptosis of Hela cells (Fig. 7B), which indicated that NDRG2 could promote the resistance of Hela cells to radiation-induced apoptosis.
NDRG2 inhibited radiation-induced upregulation of Bax Fig. 3 – HIF-1 mediates hypoxia-induced NDRG2 expression in Hela cells. A. Hela cells were incubated in hypoxic condition for the indicated time. B. Hela cells transfected with cDNAs of pSliencer3.1 constructs expressing HIF-1α-specific siRNA (siHIF-1α) or scramble siRNA (control) were incubated in normoxic or hypoxic condition for 12 h. NDRG2 expression in Hela cells was determined by Western blot analysis. β-actin was detected as loading control.
To further illustrate the role of NDRG2 in radiation-induced apoptosis, Bax and Bcl-2 expression was monitored in irradiated Hela cells. As shown in Fig. 8, radiation induced no changes of Bcl-2 but elevated expression of Bax, thus increased the Bax/Bcl-2 ratio, which would transduce signals to downstream molecules and initiate apoptotic process. Surprisingly, overexpression of NDRG2 almost completely abolished upregulation of Bax as well as Bax/ Bcl-2 ratio induced by radiation (Fig. 8A). Meanwhile, silence of
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Fig. 4 – Radiation up-regulates NDRG2 expression in Hela cells. A and B. Hela cells were irradiated at 6 Gy and harvested at indicated time points. C and D. Hela cells were irradiated at indicated dosage and harvested 12 h later. A and C. NDRG2 mRNA expression level was evaluated by real-time PCR as described in “Materials and methods”. B and D. NDRG2 protein was detected by Western blot analysis (upper panel). β-actin was also detected as loading control. NDRG2 expression was quantitated by densitometry and plotted as fold change (lower panel). Values were mean ± SD of 3 independent experiments.
NDRG2 facilitated radiation-induced Bax expression and promoted increase of Bax/Bcl-2 ratio (Fig. 8B). These strongly suggested that NDRG2 could promote resistance of Hela cells to radiation-induced apoptosis through inhibiting radiation-induced upregulation of Bax.
Discussion It is widely accepted that Oxygen can increase the cytotoxicity of radiation by modifying radiation-induced DNA damage and
Fig. 5 – HIF-1 mediates radiation-induced NDRG2 expression in Hela cells. A. Hela cells were irradiated at 6 Gy and harvested indicated time points. HIF-1α expression was analyzed by Western blot analysis (exposure time: 15 s). B. Hela cells transfected with cDNAs of pSliencer3.1 constructs expressing HIF-1α-specific siRNA (siHIF-1α) or scramble siRNA (control) were irradiated at 6 Gy and harvested 12 h later. NDRG2 expression was detected by Western blot analysis. β-actin was detected as loading control.
impairing DNA repair. This phenomenon is termed oxygen enhancement effect, which could make oxygenated cells 3-times more radiosensitive than hypoxic cells [2]. Depriving cells of oxygen would abolish oxygen enhancement effect and thus leads to decreased sensitivity of cells to irradiation. As revealed by the present study, pre-treatment of Hela cells in hypoxic condition resulted in increased radioresistance and the calculated OER was 2.31 ± 0.36, which was close to the previous report for Hela cells [28]. Interestingly, the hypoxia mimetic cobalt chloride could enhance the radioresistance of Hela cells in the presence of normal oxygen tension. Cobalt chloride does not change the oxygenation of cells but activate the hypoxia signaling transduction pathway, especially the HIF-1 pathway [3]. Our data supports the idea that, in addition to repressed oxygen enhancement effect by hypoxia, there must be some biological mechanisms mediating hypoxiainduced radioresistance [2]. HIF-1 is the most important mediator of hypoxia signal transduction, which controls the expression of greater than 100 genes and thus exerts diverse and even controversial impacts on tumor radiosensitivity [3]. HIF-1 can induce cell cycle arrest [29], inhibit apoptosis signal [30] and promote angiogenesis [31,32], which would result in decreased radiosensitivity. HIF-1 can also reinforce apoptosis signal [31] and enhance glycolysis [33], which would lead to increased radiosensitivity. Therefore, the net effect of HIF-1 is determined by the balance of those two aspects. Studies have shown that HIF-1-deficiency promotes tumor radiosensitivity in vivo [27,34–36], suggesting that the net result of HIF-1 activity is to promote radioresistance in tumors. It has been proposed that
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Fig. 6 – NDRG2 contributes to hypoxia-induced radioresistance of Hela cells. Hela cells were transfected with cDNAs of pcDNA3.1 construct expressing NDRG2, pcDNA3.1 (vector), pSliencer3.1 constructs expressing NDRG2-specific siRNA (siNDRG2) or scramble siRNA (control). Twenty four hours later, cells were harvested and subjected to Western blot to detect NDRG2 expression (A), or irradiated (B), or treated in hypoxic condition for 12 h and then irradiated at indicated dosage (C). The dose–survival curves (B, C) were determined as described in Fig. 1.
HIF-1 in cancer cells would induce the expression of a variety of proangiogenic cytokines which protects stromal endothelial cells from radiation-induced cell death and eventually enhances radioresistance of tumor cells [4,27,35]. From this view point, that is the indirect protective effect of HIF-1 on tumor cells. The present study demonstrated that overexpression of HIF-1α could promote radioresistance while silence of HIF-1α could increase radiosensitivity of Hela cells in vitro. In line with this finding, a recent study revealed that radioresistant lung cancer cell lines exhibited higher expression level of HIF-1α than radiosensitive lung cancer cell lines and that inhibition of HIF-1α expression sensitized lung cancer cells to radiation [37]. These data indicate that HIF-1 can directly enhance radioresistance of cancer cells. Taken together, there are at least two mechanisms mediating the effect of HIF-1 on tumor radioresistance. Firstly HIF-1 could directly protect cancer cells from radiation-induced cell death. Secondly, HIF-1 increases the expression of proangiogenic cytokines and promotes the survival of stromal endothelial cells which in turn provide support
Fig. 7 – NDRG2 regulates radiation-induced apoptosis of Hela cells. Hela cells were transfected with cDNAs of (A) pcDNA3.1 construct expressing NDRG2 or pcDNA3.1 (vector), (B) pSliencer3.1 constructs expressing NDRG2-specific siRNA (siNDRG2) or scramble siRNA (control). Twenty four hours later, cells were irradiated at indicated dosage and then cultured for another 24 h. The apoptotic cells were stained with FITC-labeled annexin V and propidium iodide and analyzed by flow cytometry. The percentage of double positive cells was calculated and data was expressed as mean ± SD of triplicates in one experiment. Shown was representative of at least 3 experiments. *p < 0.01 vs. vector or control.
to cancer cells. However, how does HIF-1 directly induce radioresistance of cancer cells needs further investigation. HIF-1 can be induced and activated not only by hypoxia and its mimetics but also by irradiation. Moeller et al. [27] found that HIF1 activity in tumors growing in skinfold window chambers began to increase steadily approximately 12–24 h after radiation, peaking 48 h following treatment. However, they also reported that there was no detectable increase in HIF-1 expression and activation at 24, 48 and 72 h after irradiation of cancer cells in vitro [27]. They therefore inferred that radiation induces HIF-1 activation only in vivo. We argue here that HIF-1 can be induced also in vitro. The present study demonstrated that HIF-1α expression in monolayer of Hela cells increased 2 h after radiation, reached to its peak at 8 h, gradually decreased at 12 h and came back to its basal level at 24 h. Recently, there are two studies that report similar results. Kim et al. [37] examined a series of human lung cancer cell lines and found that HIF-1α expression was induced in H1299, H182 and H226B cells as early as 30 min to 3 h after administering a single dose of radiation. Singh-Gupta et al. [38] observed that radiation-induced upregulation of HIF-1α in PC-3 cells occurred at 3–5 h after radiation. These data suggest that HIF-1α responses to radiation very quickly and that it will be very late to check HIF-1α more than 24 h after radiation. Moeller et al. [27] didn't examine the change of HIF-1 activity within 24 h of
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Fig. 8 – NDRG2 regulates radiation-induced apoptosis of Hela cells. Hela cells were transfected with cDNAs of (A) pcDNA3.1 construct expressing NDRG2 or pcDNA3.1 (vector), (B) pSliencer3.1 constructs expressing NDRG2-specific siRNA (siNDRG2) or scramble siRNA (control). Twenty four hours later, cells were irradiated at 6 Gy and then cultured for another 12 h. The cellular extracts were analyzed by Western blot to determine Bax and Bcl-2 expression level (upper panel). β-actin was detected as loading control. Bax and Bcl-2 expression were quantitated by densitometry and plotted as Bax/Bcl-2 ratio (lower panel). Values were mean ± SD of 3 independent experiments. *p < 0.01 vs. vector or control.
radiation and might miss the opportunity to uncover radiationinduced increase of HIF-1 activity in vitro. Altogether, radiation can induce the expression and activation of HIF-1 in cancer cells both in vitro and in vivo. In addition, it has been discovered that human papillomaviruse (HPV) E6 and E7 were able independently to enhance induction of HIF-1α upon hypoxic treatment [39]. Considering HPVs are the causative agents of cervical cancer, it would be possible for HIF-1 to be induced and activated by hypoxia, radiation and E6/E7 proteins in HPV + cervical cancer. So, HIF-1 blockade would be much promise and beneficial to the patients with HPV + cervical cancer who underwent radiotherapy. The present study revealed that NDRG2 was upregulated in cervical cancer Hela cells not only by hypoxia but also by radiation. Silence of HIF-1α could suppress hypoxia- and radiation-induced NDRG2 expression, indicating response of NDRG2 to hypoxia and radiation is HIF-1-dependent. It was also demonstrated that enforcing the expression of NDRG2 led to increased radioresistance of Hela cells whereas silencing NDRG2 strongly enhanced radiosensitivity of hypoxic Hela cells. Furthermore, NDRG2 could inhibit radiation-induced upregulation of Bax and prevent Hela cells from radiation-induced apoptosis. It thus can be established that NDRG2 responses to HIF-1 activation and promotes radioresistance of cancer cells, which represents one of the mechanisms underlying HIF-1mediated radioresistance. However, it should be noted that radiation could induce mitotic catastrophe as well as apoptosis. Thus, further studies are needed to explore the effects of NDRG2 on radiationinduced mitotic catastrophe. Moreover, the mechanisms depicting the effect of NDRG2 on Bax expression remain unclear. It has been found that NDRG2 could translocate to nucleus upon stimulation with hypoxia [22] and radiation (data not shown). A physical interaction of NDRG2 with transcription factor MSP58 has been
demonstrated and their colocalization in nucleus of Hela cells during cell stress has been confirmed [40]. It can be speculated that NDRG2 might interact with transcription factors and thus regulate gene expression profile during response to radiation. Further investigation is ongoing to validate this speculation. In conclusion, the present study demonstrated that NDRG2 can be induced in Hela cells by both hypoxia and radiation in a HIF-1dependent manner and that both NDRG2 and HIF-1 contribute to hypoxia-induced tumor radioresistance. These data can help us to better understand the molecular mechanisms underlying hypoxiainduced tumor radioresistance and the precise role of NDRG2 in tumor development. It is also suggested that blockage of HIF-1 and/ or NDRG2 may sensitize tumors to radiotherapy. Further studies are encouraged to evaluate the clinical application potential of HIF1 and NDRG2 blockade as radiosensitizer for tumor therapy.
Acknowledgments This work was supported by National Natural Science Foundation of China (nos. 30600161, 60871068 and 30970862), National Basic Research Program of China (no. 2006CB504100), Natural Science Foundation of Shaanxi Province (no. 2006C202) and Postdoctoral Science Foundation of China (no. 200503826).
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