NADPH Oxidase Subunit 4 Mediates Cycling
Hypoxia-promoted Radiation Resistance in
Glioblastoma Multiforme
Chia-Hung Hsieh a,b,*, Chung-Pu Wu c,Hsu-Tung Lee d, Ji-An Liang e,
Chun-Yen Yu e, Yu-Jung Lin a
a Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan b Department of Medical Research, China Medical University Hospital, Taichung, Taiwan
c Department of Physiology and Pharmacology, Chang Gung University, Tao-Yuan,
Taiwan
d Department of Neurosurgery, Taichung Veterans General Hospital, Taichung, Taiwan
e Department of Radiation Therapy and Oncology, China Medical University Hospital,
Taichung, Taiwan
*Corresponding authors:
Chia-Hung Hsieh, PhD
Mailing address: No. 91, Hsueh-Shih Road, Taichung 404, Taiwan
E-mail: [email protected] .edu.tw
Tel: 886-4-22052121 ext. 7712 Fax: 886-4-22333641
Words: 7061 Figures: 5
Running title: Nox4 mediates cycling hypoxia-promoted radiation resistance
Conflict of Interest Notification
None 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Abstract
Cycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It mediates tumor progression and radiotherapy resistance through mechanisms that involve the reactive oxygen species (ROS) production. However, the details of the mechanism underlying cycling hypoxia-mediated radioresistance remain obscure. We have previously shown that in glioblastoma, NADPH oxidase subunit 4 (Nox4) is a critical mediator involved in cycling hypoxia-mediated ROS production and tumor progression. Here, we examined the impact of in vivo tumor microenvironment on Nox4 expression pattern and its impact on radiosensitivity in GBM8401 and U251, two glioblastoma cell lines stably transfected with a dual hypoxia-inducible factor-1 (HIF-1) signaling reporter construct. Furthermore, in order to isolate hypoxic tumor cell subpopulations from human glioblastoma xenografts based on the physiological and molecular characteristics of tumor hypoxia, several techniques were utilized. In this study, the perfusion marker Hoechst 33342 staining and HIF-1 activation labeling were used together with immunofluorescence imaging and fluorescence activated cell sorting (FACS). Our results revealed that Nox4 was predominantly highly expressed in the endogenous cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. Moreover, when compared to the normoxic or chronic hypoxic cells, the cycling hypoxic tumor cells derived from glioblastoma xenografts have much higher Nox4 expression, ROS levels and radioresistance. Nox4 suppression in intracerebral glioblastoma-bearing mice suppressed tumor microenvironment-mediated radioresistance and enhanced the efficiency of radiotherapy. In summary, our findings indicated that cycling hypoxia-induced Nox4 plays an important role in tumor microenvironment-promoted radioresistance in glioblastoma, hence targeting Nox4 may be an attractive therapeutic strategy to block cycling hypoxia-mediated radioresistance.
Key Words: glioblastoma, cycling hypoxia, NADPH oxidase subunit 4 (Nox4),
hypoxia-inducible factor-1, radioresistance 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Introduction
Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor in humans. Current therapy consists of surgical resection followed by radiation therapy and concomitant chemotherapy . Despite these treatments, the median survival for GBM patients is usually less than 1 year from the time of diagnosis . Most patients receiving radiation therapy following GBM resection still develop tumor recurrence in the proximity of the primary site, emphasizing the radioresistant nature of GBM . As such, understanding the mechanism of radioresistance is essential for developing more effective radiotherapy treatment regimens for GBM.
The intrinsic radiosensitivity of GBM is a pivotal factor that determines the effectiveness of radiotherapy . Many factors, including genetic alterations and microenvironments, affect tumor response to ionizing radiation . One such factor is tumor hypoxia that exists in various forms. Chronic hypoxia is the consequence of increased oxygen diffusion distance due to tumor expansion, which is typically found in tissue with low microvascular density or tissue adjacent to necroses. Cycling or acute hypoxia on the other hand, is due to the abnormal structure or function of the blood vessels within a tumor, causing inadequate blood flow. The cells in the cycling hypoxia areas often experience several short-term periods of hypoxia during their lifetime .
Recent evidence shows that both cycling hypoxia and chronic hypoxia play roles in many aspects of tumor progression and therapy resistance . Elevation of reactive oxygen species (ROS) production during cycling hypoxia is implicated in radioresistance through several mechanisms, which includes the possibility of death-resistant cell selection by the repetition of hypoxia episodes and activation of anti-apoptotic signaling pathways . Consequently, antioxidant supplementation during radiation therapy poses a conundrum for the radiation oncologists because antioxidants may provide the protection effect to tumor cells and reduce the efficacy of treatment . Therefore, it is important to explore other new targets for blocking cycling hypoxia-induced radioresistance during radiotherapy.
ROS in a solid tumor may emanate from several sources, including mitochondria, NADPH oxidases of the Nox family, uncoupled nitric oxide (NO) synthases, and xanthine oxidases, but the functional roles of individual sources remain unclear . It is recognized, however, that different sources may modulate distinct signaling pathways 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
through regulated, spatially restricted ROS production. Recent studies have demonstrated that NADPH oxidase subunit 4 (Nox4) is expressed in several tumor types such as hepatoma, breast cancer, ovarian cancer, melanoma, prostate cancer and various neuroepithelial neoplasms. These studies also demonstrated the involvement of Nox4 in cellular senescence, resistance to apoptosis, tumorigenic transformation, cell proliferation, cell survival and radiation resistance . Moreover, strong evidence suggests that these processes were up-regulated through Nox4 generation of ROS. While the over-expression of Nox4 in glioblastoma cells or tissue samples has been reported previously , the expression pattern of Nox4 in solid tumor and its impact on tumor microenvironment-mediated radiosensitivity remain unknown.
In the present study, we determined the tumor microenvironment-mediated expression pattern of Nox4 and its impact on radiosensitivity in glioblastoma. We showed that Nox4 tends to be overexpressed in endogenous cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor. Furthermore, the cycling hypoxic tumor cells derived from glioblastoma xenografts have much higher Nox4 expression, NADPH oxidase activity, ROS levels and radioresistance than chronic hypoxic cells or normoxic cells. Though same phenomena were observed in glioblastoma cells receiving in vitro cycling hypoxic stress, the knockdown of Nox4 inhibited this effect. Nox4 blockade in intracerebral glioblastoma-bearing mice decreased tumor microenvironment-induced Nox4 expression and radioresistance, and further increased overall therapeutic efficiency of radiotherapy.
Methods and materials
Viral transduction and stable cell lines
The pGreenFire1-SFFV and dxHRE-NESTKGFP:dMODC-CMV-Red2XPRT were used to generate glioblastoma reporter cells bearing SFFV promoter-driven a dual optical reporter gene encoding both green fluorescence protein (GFP) and luciferase (Luc) or dual reporter cassette which has HIF-1–inducible reporter gene (NESTKGFP:dMODC) and a constitutively expressed reporter gene (Red2XPRT). The lentiviral vector pLVCT-tTR-KRAB (Addgene) was used to express Nox4 short hairpin RNA (shRNA) (Sigma) following the manufacturer’s protocol. Lentivirus or retrovirus production and cell transduction were carried out according to protocols described elsewhere GBM8401 and U251 cells bearing the spleen focus-forming virus (SFFV) promoter-driven a dual optical reporter gene and tetracycline (Tet)-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
inducible Nox4 shRNA were termed GBM8401/SFFV-LucGFP-Nox4 shRNA and U251/SFFV-LucGFP-Nox4 shRNA cells, respectively. Moreover, GBM8401 cells bearing the dual reporter gene cassette were termed GBM8401/hif-1-r cells.
Cell culture and in vitro hypoxic treatments
GBM8401/SFFV-LucGFP-Nox4 shRNA, U251/SFFV-LucGFP-Nox4 shRNA,
GBM8401/hif-1-r and their parental cells were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and 1% penicillin-streptomycin. Cells were treated with or without in vitro non-interrupted hypoxic or cycling hypoxic stress as previously described . Briefly, the cells were treated in a Biospherix C-Chamber (Biospherix) inside a standard culture
chamber by means of exhausting and gassing with 95% N2 and 5% CO2 to produce
oxygen concentrations of 0.5 to 1% for 4 h at 37°C to achieve non-interrupted hypoxic conditions. For the cycling hypoxic treatment, cell cultures were exposed to
12 cycles of 0.5 to 1% O2 for 10 min interrupted by 5% CO2 and air for 10 min at
37°C in a hypoxia chamber with a timer-controlled regulator. In vitro medium oxygen during cycling hypoxia was determined using the Oxford Oxylite fiberoptic probe
(Oxford) and this condition resulted in the medium pO2 of 0.8–1.5 mmHg during
hypoxic phase.
Cellular assays
Several cellular assays were used in this study. These included the following: ROS
levels analysis to determine the ROS levels; real-time quantitative PCR (Q-PCR) and Western blot analysis to assess the Nox4 expression in mRNA and protein levels; and clonogenic survival assay to evaluate the cell survival of irradiated tumor cells. ROS levels were assessed by using carboxy-2′7′-dihydrodichlorofluorescein diacetate (H2DCFDA, Molecular Probes). The detailed information of ROS levels analysis, Q-PCR and Western blot analysis is available as our previous report . In the cellular assays, GBM8401/SFFV-LucGFP-Nox4 shRNA and U251/SFFV-LucGFP-Nox4 shRNA cells were treated with 0.04 μg/mL doxycycline (Dox) for 48 h to induce Nox4 knockdown and then, exposed to in vitro cycling hypoxic stress. After cycling hypoxic stress, cells were collected for various cellular assays.
NADPH oxidase assay.
NADPH oxidase activity was measured by superoxide production from NADPH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
using the lucigenin-enhanced luminescence assay . Briefly, cells were homogenized in lysis buffer and 100-μl aliquots of cell homogenates were added to 900 μl of 50 mM phosphate buffer, pH 7.0, containing 1 mM EGTA, 150 mM sucrose, 5 μM lucigenin, and 100 μM NADPH. Enhanced lucigenin luminescence indicative of superoxide concentration was measured by a SpectraMax M2/M2e Microplate Readers Reader (Molecular Devices) and normalized to the protein amount. Protein content was measured using the Bio-Rad protein assay reagent. Superoxide production was expressed as relative chemiluminescence (light) units (RLU)/mg protein. Data were represented as relative units of superoxide production fold change from normoxic group using paired experiments.
Cells and animals irradiation
Cells were irradiated (2.85 Gy/min) with single doses of X-ray (0–10 Gy) using a high-energy X-ray linear accelerator (Varian), harvested, and assayed for clonogenic survival. For animal irradiation, the tumors in living mice were locally irradiated (4.25 Gy/min) at a dose of 10 Gy under anesthesia. After irradiation, tumors were excised for cell sorting and clonogenic survival assay. The clonogenic survival assay was carried out as previously described .
Animal model
Eight-week-old male Nude mice (Balb/c nu/nu) were purchased from the Animal Facility of the National Science Counsel (NSC) and were used to establish the orthotopic glioblastoma xenograft model according to the published methods .
Briefly, 2 × 105 GBM8401/SFFV-LucGFP-Nox4-shRNA and GBM8401/hif-1-r were
harvested by trypsinization and injected into the left basal ganglia of anesthetized mice. The tumors developed at 18 days after tumor implantation for immunofluorescence imaging, flow cytometry analysis and cell sorting studies and at 14 days after tumor implantation for evaluating the efficiency of radiotherapy studies. All animal experiments were conducted according to Institutional Guidelines of China Medical University after obtaining permission from the local Ethical Committee for Animal Experimentation.
Immunofluorescence imaging
A hypoxia marker, pimonidazole (70 mg/kg, intraperitoneal; HPI ), and a perfusion
marker, Hoechst 33342 (1 mg/mouse, intravenous; Sigma), were administered 3 hr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
and 30 min prior to tumor excision in the immunofluorescence imaging of glioblastoma xenografts. The immunofluorescence imaging was performed as previously described . For the immunofluorescence imaging of GBM specimens, fresh GBM operative specimens were obtained from patients undergoing a craniotomy at the China Medical University Hospital with ethical approval from the Research Ethics Board. Frozen primary tumor sections (10 μm) were incubated with primary antibodies, pimonidazole (1:500; HPI), Nox4 (1:500; Novus), HIF-1α(1:100; Novus) or CD31(1:100; Novus) overnight at 4°C and secondary antibodies, DyLight 649-conjugated goat anti-rabbit antibody (1:100; Molecular Probes) or DyLight 488-conjugated goat-anti-mouse antibody (1:100; Abcam). Tissue fluorescence was visualized with the Axio Observer A1 digital fluorescence microscope system (ZEISS).
Flow cytometry
Tumor tissues were disaggregated with an enzyme cocktail containing collagenase type III (Sigma), hyaluronidase (Sigma), and collagenase type IV (Sigma), washed several times, and resuspended in phosphate-buffered saline (PBS) to produce a single cell suspension. The total time for tumor digestion to single cell suspension was 20 min. Prior to flow cytometry, cells were incubated with rabbit polyclonal Nox4 antibody in cold fluorescence-activated cell sorting (FACS) buffer (PBS, 0.5% BSA) on ice for 30 min. After washing in FACS buffer, cells were incubated with DyLght 649-conjugated goat anti-rabbit antibody. After the final washing step, fluorescence was measured using a FACScalibur instrument and FACSDiva 6.0 software (BD Bioscience). Tumor cells were gated according to DsRed expression and side scatter (SSC). Nox4 expression was further evaluated after Hoechst 3342 and GFP gating on
cycling hypoxic tumor cells (DsRed+, Hoechst 3342+, and GFP+), chronic hypoxic
tumor cells (DsRed+, Hoechst 3342- and GFP+), or normoxic tumor cells (DsRed+,
Hoechst 3342+, and GFP-).
Chemiluminescent probe L-012 assay
The cell suspensions were incubated in 0.5 ml of 0.9% NaCl solution containing 10
mM phosphate buffer (pH 7.4), 6 mM KCl and 6 mM MgCl2 (KRP) in the presence of
either 400 μM L-012. After incubation of the mixture for 3 min at 37°C, the chemiluminescence intensity was recorded continuously for 20–40 min using SpectraMax M2/M2e Microplate Readers Reader (Molecular Devices). Data were presented as relative units of chemiluminescence intensity change from normoxic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
group.
Animal survival assay
Intracerebral GBM8401/SFFV-LucGFP-Nox4 shRNA-bearing mice were randomly
assigned to four different therapeutic groups: control (drinking water containing 5% sucrose only), Nox4 knockdown (5% sucrose plus 2 mg/mL Dox), 10 Gy irradiation or Nox4 knockdown + 10 Gy irradiation. Nox4 knockdown was performed for 2 days starting day 14 after tumor cell injection. Tumor irradiation was carried out at day 16 after tumor cell implantation. Tumor progression was monitored by bioluminescence imaging and mice were monitored daily for survival. Animals were killed at the onset of neurologic signs or any type of distress.
Bioluminescent imaging (BLI)
Mice were imaged with the IVIS Imaging System 200 Series (Caliper) to record bioluminescent signal emitted from the engrafted tumors. Mice were anesthetized with isoflurane and received intraperitoneal injection of D-Luciferin (Caliper) at a dose of 270 µg/g body weight. Imaging acquisition was performed at 15 min after intraperitoneal injection of luciferin. For BLI analysis, regions of interest encompassing the intracranial area of signal were defined using Living Image software, and the total number of photons per second per steradian per square centimeter were recorded. To facilitate comparison of growth rates, each mouse’s luminescence readings were normalized against its own luminescence reading at day 14, thereby allowing each mouse to serve as its own control.
Statistical analysis
For multiple comparisons of nonparametric variables, Kruskal-Wallis ANOVA was
used. For parametric variables, ANOVA was used along with Fisher’s least-significant-difference (LSD). For survival analysis, statistical software for Kaplan-Meier Survival Analysis with Tarone-Ware statistics (SPSS Inc) was used. P < 0.05 was considered significant. All analyses were two-tailed.
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Results
Nox4 is highly expressed in the endogenous cycling hypoxic areas in glioblastoma xenografts and human glioblastoma specimens
Growing evidence has suggested that cycling hypoxia tended to occur in highly vascular regions with relatively high permeability and therefore, cycling hypoxic areas still have blood perfusion after transient occlusion or narrowing of the vasculature. Moreover, cells exposed to cycling hypoxia exhibit more robust HIF-1 activation than cells that are chronically hypoxic. Therefore, the combination of the perfusion marker Hoechst 33342 staining and HIF-1 activation labeling approach could be utilized to distinguish the different hypoxic subtypes in vivo. The GBM8401/hif-1-r tumors showed highly heterogeneous Hoechst 33342 staining and
HIF-1 activation on immunofluorescence images (Fig. 1A). Regions that are positive
for Hoechst 33342 staining and GFP expression (HIF-1 activation) are predicted cycling hypoxic areas. In contrast, GFP-positive but Hoechst 33342-negative regions are mostly chronic hypoxic areas. In addition, pimonidazole, a probe known for selectively staining chronically hypoxic cells, was used to verify chronic hypoxic areas. The staining pattern of pimonidazole was consistent with staining of chronically hypoxic cells using the Hoechst 33342 staining and HIF-1 activation labeling approach. Next, we investigated the hypoxic microenvironment-mediated Nox4 expression pattern in solid tumors. The expression of Nox4 tended to be confined in the cycling hypoxic regions, and not in the chronic hypoxic regions (Fig.
1B). Moreover, the immunofluorescence staining of Nox4, HIF-1α and CD31 human
glioblastoma specimens also demonstrated that the Nox4 expressing cells were frequently positioned in perivascular or vascular areas (Fig. 1C). The fact that the expression of Nox4 is colocalized with HIF-1α overexpression, suggests that endogenous cycling hypoxia can lead to the induction of Nox4 in glioblastoma.
Exogenous cycling hypoxia induces Nox4 expression, NADPH oxidase activity and ROS production in glioblastoma cells
In order to validate whether cycling hypoxia can trigger Nox4 activation and further promote ROS production, we examined the effect of glioblastoma cells exposing to in vitro cycling hypoxic or non-interrupted hypoxic stress on Nox4 expression, NADPH oxidase activity and ROS production. Our results demonstrated 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
that the levels of Nox4 mRNA and protein, NADPH oxidase activity and ROS were all elevated significantly in cycling hypoxia-treated cells (Fig. 2A, B, C and D). In contrast, no significant changes were observed in normoxic or non-interrupted hypoxic cells. Next, we investigated whether Nox4 is a critical mediator involved in cycling hypoxia-induced NADPH oxidase activity and ROS generation. Tet-regulatable lentiviral vectors encoding shRNAs were used to stably and specifically knockdown Nox4 induction in GBM8401 and U251 cells under cycling hypoxia. RT-PCR and western blot analysis showed that this shRNA successfully knocked down
the cycling hypoxia-mediated Nox4 expression level in both cell lines (Fig. 2A and
B). Moreover, cycling hypoxia-induced ROS were inhibited by Nox4 knockdown and
were cleared from the cell by the action of polyethylene glycol (PEG)-superoxide dismutase (SOD) and catalase (Fig. 2D). These results indicate that Nox4 is the major isoform responsible for the increased ROS that is induced by cycling hypoxia in glioblastoma cells.
Nox4 knockdown inhibits exogenous cycling hypoxia-induced radioresistance in glioblastoma cells
To investigate whether the cycling hypoxia-mediated Nox4 up-regulation is responsible for radiation resistance in glioblastoma cells, we first looked at the effect of cycling hypoxia on radiosensitivity using the clonogenic survival assay. Our results showed that cycling hypoxia pretreatment significantly increased cell resistance to ionizing radiation compared with normoxic controls in GBM8401 and U251 cells
(Fig. 3A and B). But, loss of Nox4 significantly inhibits cycling hypoxia-induced radioresistance and increases the radiosensitivity of glioblastoma cells to cycling hypoxia or normoxia exposure. We next determined the effect of ROS scavengers, PEG-SOD, and PEG-catalase, on cycling hypoxia-mediated radiosensitivity. We found that treatment of SOD or catalase provides a protection rather than sensitization to irradiation with 6 Gy, indicating that these ROS scavengers contribute to the quenching of free radicals produced by the radiation and thus provide some degree of protection (Fig. 3C).
Cycling hypoxic cells isolated from glioblastoma xenografts have much more Nox4 expression, ROS levels and radioresistance than other tumor subpopulation cells
To further determine the Nox4 expression, NADPH oxidase activity, ROS levels and radioresistance of hypoxic tumor cell subpopulations in glioblastoma xenografts, we utilized fluorescence activated cell sorting to isolate these subpopulations derived 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
from disaggregated the orthotopic GBM8401/hif-1-r xenografts. These tumor cells were further analyzed their Nox4 expression, NADPH oxidase activity, ROS levels and radiosensitivity using FACS, lucigenin-enhanced chemiluminescence assay, chemiluminescent probe L-012 assay and clonogenic survival assay, respectively. Flow cytometry analysis of tumor cells (DsRed positive cells) after Hoechst 3342 and
GFP gating showed approximately 28 ± 4 % of cycling hypoxic cells (Hoechst 3342+
and GFP+), 10 ± 2 % of chronic hypoxic cells (Hoechst 3342- and GFP+) and 58 ± 6
% of normoxic cells (Hoechst 3342+ and GFP-) were present in tumor suspension
cells (Fig. 4A). We discovered that Nox4 expression was up-regulated significantly in
cycling hypoxic cells (Hoechst 3342+ and GFP+) and chronic hypoxic cells (Hoechst
3342- and GFP+) when compared to normoxic cells (Hoechst 3342+ and GFP-) (Fig.
4B, C and D). Likewise, Nox4 expression, NADPH oxidase activity and ROS levels
in cycling hypoxic cells were higher than in chronic hypoxic cells (Fig. 4E and F). Moreover, tumors of intracerebral glioblastoma-bearing mice received local tumor irradiation of 10 Gy were excised for cell sorting, and performed clonogenic survival assays immediately after irradiation. Our results showed that cycling hypoxic tumor cells possessed stronger radioresistance than chronic hypoxic tumor cells and
normoxic tumor cells (Fig. 4G). Taken together, these data indicate that endogenous
cycling hypoxia enhances Nox4 expression and NADPH oxidase activity and further promotes radioresistance in glioblastoma xenografts.
Nox4 blockage enhances the efficiency of radiotherapy in glioblastoma xenografts
Finally, we investigated whether Nox4 is a potential therapeutic target of tumor microenvironment-mediated radioresistance in GBM. To noninvasively monitor tumor treatment by following xenograft glioblastoma with or without Nox4 knockdown responses to radiotherapy in vivo, bioluminescent imaging (BLI) was utilized to assess intracranial tumor growth in the orthotopic GBM8401/SFFV-LucGFP-Nox4 shRNA xenograft model. This imaging technique can detect photon emissions associate with an ATP-dependent process catalyzed by metabolically active cells that express a luciferase reporter gene and its substrate, luciferin, in living subjects. It has been shown to allow noninvasive, quantitative, and real-time monitoring of tumor growth and response to therapy in many small-animal models . Prior to radiotherapy, the mice received two-day treatment with doxycycline to inhibit Nox4 expression in glioblastoma xenografts, which is confirmed by Western blot and
immunohistochemical analysis (Fig. 5A and B). Results clearly showed that mice that
had two-day treatment with doxycycline prior to initiation of radiotherapy had significantly better tumor growth delay and survival rate than those not pretreated 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
with doxycycline (Fig. 5C, D and E). Overall, these results indicate that Nox4 blockage suppresses tumor microenvironment-mediated radioresistance and enhances the efficiency of radiotherapy in glioblastoma xenografts.
Discussion
Resistance to radiotherapy is a primary cause for treatment failure in the vast majority of patients with advanced and inoperable cancer . Though the precise mechanisms of radioresistance in tumors are complex and multifactorial , it is now widely accepted that the particularities of tumor microenvironment contribute significantly to radiotherapy failure and radioresistance . Hypoxia is a hallmark of the tumor microenvironment and a modifier of physical effects of radiation on cell DNA integrality. Both chronic and acute hypoxia can alter the expression of a myriad of genes, including transcription factors, growth factors, cytokines, and DNA repair enzymes contributing to both tumor progression and therapy resistance. Of all the factors, HIF-1α is one of the master regulators that orchestrate the hypoxia-mediated cellular responses . Recent data suggest that cycling hypoxia can induce ischemia-reperfusion and reoxygenation-dependent HIF-1 signaling, which prompts prosurvival or proangiogenic signaling events and enhances malignant progression and resistance to radiotherapy . However, this mechanism remains unconfirmed in endogenous tumor microenvironment. Here, our data highlighted that both cycling hypoxia and chronic hypoxia can induce HIF-1 signal transduction within the endogenous tumor microenvironment. Moreover, the majority of HIF-1 signal transduction activity occurs in areas with relatively high perfusion. In keeping with the pathophysiologic consequences, cycling hypoxia tends to occur in highly vascular regions with relatively high permeability and therefore, cycling hypoxic areas still have blood perfusion after transient occlusion or narrowing of the vasculature . Although one cannot distinguish between normoxia and cycling hypoxia via perfusion marker, HIF-1 signal transduction activity usually does not exist in normoxic areas. We also confirmed that the reporter cells exposed to normoxic condition have no significant HIF-1 activation (data not shown). In contrast, chronic hypoxic areas do not have blood perfusion, even when the blood perfusion of the areas proximal to the blood vessels has been restored. Therefore, tumor cells with positive Hoechst 33342 staining and HIF-1 activation are potential cycling hypoxic cells. Moreover, results from our FACS analysis showing approximately 30 % of the cycling hypoxic tumor cells in glioblastoma xenografts further support the notion that cycling hypoxia is a common feature in solid tumors.
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A key observation of our study is that Nox4 is highly expressed in the cycling hypoxic areas of glioblastoma xenografts. We showed that glioblastoma cells received several cycles of hypoxia/reoxygenation resulted in significant increased levels of Nox4 mRNA and protein, NADPH oxidase activity and ROS, indicating that Nox4 expression and function can be induced by cycling hypoxia. In addition to our findings, induction of Nox4 mRNA expression is observed under endoplasmic reticulum stress, shear stress, carotid artery injury, hypoxia and ischemia, as well as transforming growth factor (TGF)-1 and tumor necrosis factor (TNF)-α stimulation . Increasing evidence suggests that Nox4 plays a pivotal role in tumor hypoxia-promoted cancer progression , chronic intermittent hypoxia-induced pulmonary vascular remodeling and hypertension and ischemia-mediated endothelial angiogenesis . Although the precise mechanism involved in these biological events is not fully understood, Nox4-derived ROS are thought to be the essential signaling mediators involved in the regulation of relative molecular events. In general, Nox proteins produce superoxide anion via a single electron reduction. Depending on the microenvironment or cellular compartment wherein it is produced, spontaneous or SOD catalyzed reduction of superoxide anion into hydrogen peroxide may occur in association with the generation of other ROS . Nevertheless, a specific feature of Nox4 activity is its capacity for constitutive generation of extracellular hydrogen peroxide . Nox4 is constitutively associated with p22phox, which is active in an agonist-independent manner, and produces mostly hydrogen peroxide and less detectable superoxide . Interestingly, NADPH oxidase activity in cell homogenates measured by superoxide production via lucigenin-enhanced luminescence assay is increased in cycling hypoxia, but Nox4 knockdown inhibits this effect. Similar effects can be found in IGF-I or TNF-α-induced NADPH-dependent production of superoxide . Therefore, these findings suggest the possibility that cycling hypoxia or other stresses disturbs the relative production of superoxide versus hydrogen peroxide by Nox4.
ROS play an important role in the regulation of HIF-1 under normoxia and hypoxia. We recently reported that ROS contribute to cycling hypoxia-induced HIF-1 or NF-κB activation and their downstream biological processes . While the role of ROS in oxygen sensing and activation of HIF-1 is complex, earlier discoveries indicate that increased ROS levels in response to many agonists or stresses appear to serve as signaling molecules to up-regulate HIF-1 activation via attenuation of its degradation and activation of its transcriptional activity in an adenosine monophosphate-activated protein kinase (AMPK)-dependent manner . Moreover, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
accumulating evidence also shows that ROS inhibited prolyl 4-hydroxylases (PHD) activity and further stabilized HIF-1α through modulation of its cofactors iron and 2-oxo-glutarate availability . In addition, ROS can also up-regulate HIF-1α transcription by activating NF-κB, because the promoter of HIF-1α contains a functional NF-κB binding site . Therefore, Nox4-derived ROS may induce HIF-1 activation via these potential molecular pathways in glioblastoma under cycling hypoxia. On the other hand, ROS may further enhance Nox4 expression and function to maintain ROS levels as a positive feedback loop via ROS-induced signaling molecules. Recently, HIF-1α , NF-κB and AP-1 sites were reported to be located in Nox4 promoter, and these transcription factors regulate Nox4 expression and Nox activity. Therefore, induction of Nox4 by HIF-1α may contribute to maintain ROS levels during cycling hypoxia and cycling hypoxia-induced malignant progression or transformation in glioblastoma.
Although we have investigated ROS and the related signaling pathways involved in cycling hypoxia-mediated glioma cell radioresistance previously , the mechanism of cycling hypoxia-induced ROS generation remained elusive. In this study, we revealed the source of ROS in cycling hypoxia-induced radioresistance. We showed that the cycling hypoxia-induced ROS production is significantly decreased by Nox4 knockdown, suggesting that Nox plays an important role in cycling hypoxia-mediated ROS generation. The Nox family has seven homologs (Nox1, Nox3, Nox4, Nox5, Duox1, Duox2, and the gp91phox protein, known as Nox2), that contributes to normal physiological processes ranging from bactericidal activity to remodeling of the extracellular matrix . Consequently, imbalance of Nox activities can be the potential cause of acute or chronic diseases as well as cancer development. On top of a recent report showing that Nox4 mRNA and protein expression levels in Nox family were increased in human glioma cell lines or tumor specimens and contributed to the malignant phenotype of gliomas , our results further indicates the association between tumor microenvironment and Nox4 induction. We found that Nox4 expression tends to occur in probable cycling hypoxic areas with high HIF-1 activation and blood perfusion within the tumor microenvironment in glioblastoma xenografts and human glioblastoma specimens. Moreover, exposing glioblastoma cells to cycling hypoxic stress also induces Nox4 expression, ROS production and radioresistance, which can be reduced significantly by RNA interference. These results indicate that Nox4 is a critical mediator for tumor microenvironment-induced ROS production and radioresistance in glioblastoma.
In a recent study, Lu et al demonstrate that Nox4 is involved in androgens-induced 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
oxidative stress and radiation resistance in prostate cancer cells . This androgens induce ROS production and radioresistance in prostate cancer cells are mediated via Nox2 and Nox4, and the inhibition of Nox by apocynin can sensitize in vitro tumor cells to radiation. Although in vivo studies have not yet been established, these findings further prompt us to examine whether Nox4 is a critical mediator involved in tumor microenvironment-induced radioresistance. Our results show that cycling hypoxia causes a substantial elevation in Nox4 expression and further increases ROS production and radioresistance in glioblastoma. Even though we cannot rule out the possibility that the procedure of tumor digestion and cell sorting of hypoxic tumor cell subpopulations may affect the original behaviors of isolated cells in vivo. Considering the data derived from these cells are consistent with in vitro results obtained using exogenous hypoxic stresses, it is unlikely that this factor had significant effect on the observed phenotypes or molecular events in the isolated cell subpopulations. Moreover, similar methodology have been employed in previous studies to isolate hypoxic cell subpopulations for radiation sensitivity studies, and these isolated hypoxic cells maintained the radiation resistance phenotypes after tumor digestion and cell sorting .
In summary, the findings derived from the current study are highly significant. Not only do we provide the direct evidence that Nox contributes to tumor microenvironment-mediated radioresistance, but we also show that Nox4 is a therapeutic targeting of cycling hypoxia-promoted radioresistance in GBM. As expected, our results confirmed that intracerebral glioblastoma-bearing mice received Nox4 knockdown result in an improved overall therapeutic efficiency in radiotherapy. Although we did not investigate the effect of this therapeutic approach on normal tissues, recent studies highlight that radiation induces chronic oxidative stress in rat brain microvascular endothelial cells , murine hematopoietic stem cells and human lung fibroblast cells , at least in part via up-regulation of Nox4 that leads to the induction of brain and bone marrow injury or fibrosis. Therefore, treatment via Nox4 inhibition may be a good clinical practice to block cycling hypoxia-mediated tumor radioresistance and prevent radiation-induced normal tissue injury in the radiotherapy of GBM.
Acknowledgments
Grant support was provided by grant NSC97-2314-B-039-033-MY3 from the National Science Council, Taipei, Taiwan, and grants CMU97-221 and CMU98-BC-02 from China Medical University, Taichung, Taiwan (C-H. Hsieh). The National 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Science Council of ROC (grant no. NSC100-2320-B-182-002) and the Chang Gung Medical Research Program CMRPD190652 (C-P. Wu)
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Figure legends
Figure 1. The majority of Nox4 expression occurred in endogenous cycling hypoxic
areas in glioblastoma A. Hypoxic subtypes in GBM8401/hif-1-r xenografts. Top left, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
fluorescence image of DsRed reporter (red), indicating tumor cell localization within the brain. Top right, fluorescence image of GFP reporter (green), demonstrating HIF-1 transcriptional activity in tumor cells. Middle left, fluorescence image of Hoechst 33342 (blue) showing perfusion within tumor tissue. Middle right, fluorescence image of pimonidazole for chronic hypoxia staining (red). Bottom left, fluorescence overlay image of Hoechst 33342 (blue) and GFP reporter (green), indicating potential chronic hypoxic areas (Hoechst 3342-, GFP+) and cycling hypoxic areas (Hoechst 3342+,
GFP+). Bottom right, fluorescence overlay image of Hoechst 33342 (blue), GFP
reporter (green), and pimonidazole (red). B. Nox4 staining colocalized with the potential chronic hypoxic areas. Upper left, fluorescence image of DsRed reporter (red), indicating tumor cell localization within the brain. Upper right, fluorescence overlay image of Hoechst 33342 (blue) and GFP reporter (green). Lower left, fluorescence image of Nox4 (red). Lower right, fluorescence overlay image of Hoechst 33342 (blue), GFP reporter (green), and Nox4 (red) C. Expression pattern of Nox4 in primary GBMs. Upper panels, Nox4 (red) was expressed in some perivascular GBM cells and in some endothelial cells (green) around the blood vessels. Lower panels, Nox4 (red) expression colocalized with high expression of HIF-1α (green).
Figure 2. Exogenous cycling hypoxia induces Nox4 expression, NADPH oxidase
activity and ROS production in glioblastoma cells. GBM8401/SFFV-LucGFP-Nox4 shRNA and U251/SFFV-LucGFP-Nox4 shRNA cells were treated with non-interrupted hypoxic stress or cycling hypoxic stress for 4 h in the absence or presence of Dox (0.04 μg/mL), PEG-SOD (100 units/mL), PEG-catalase (100 units/mL) or PEG (18 μmol/L) and the levels of Nox4 mRNA (A), Nox4 protein (B), NADPH oxidase activity (C) and intracellular ROS (D) were evaluated by Q-PCR, western blotting, Lucigenin-enhanced chemiluminescence assay, and H2DCFDA assay, respectively. Each bar represents the mean ± standard deviation of triplicate measurements. * p < 0.01 compared to normoxia. # p < 0.01 compared to cycling hypoxia.
Figure 3. Nox4 knockdown inhibits exogenous cycling hypoxia-induced
radioresistance in glioblastoma cells. A. Radiation cell survival curves for GBM8401/SFFV-LucGFP-Nox4 shRNA cells. Cells were pre-treated for 48 h with Dox and exposed to in vitro cycling hypoxic stress before irradiation. B. Surviving fraction for GBM8401/SFFV-LucGFP-Nox4 shRNA and U251/SFFV-LucGFP-Nox4 shRNA cells with or without Dox treatment and received in vitro cycling hypoxic stress followed by 4-Gy irradiation. C. Surviving fraction for GBM8401/SFFV-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
LucGFP-Nox4 shRNA and U251/SFFV-LucGFP-Nox4 shRNA cells treated either with PEG-SOD (100 units/mL), PEG-catalase (100 units/mL) or PEG (18 μmol/L) during in vitro cycling hypoxic stress and 4-Gy irradiation. Each bar represents the mean ± standard deviation of triplicate measurements. * p < 0.01 compared to normoxia. # p < 0.01 compared to cycling hypoxia.
Figure 4. Cycling hypoxic cells isolated from glioblastoma xenografts have much
more Nox4 expression, NADPH oxidase activity, ROS levels and radioresistance than other tumor subpopulation cells. Normoxic tumor cells, cycling hypoxic tumor cells, and chronic hypoxic tumor cells were isolated from disaggregated GBM8401/hif-1-r xenografts via cell sorting (A) and the levels of Nox4 expression (B, C and D), NADPH oxidase activity (E) , intracellular ROS (F) and radiosensitivity (G) were evaluated by FACS, Western blot analysis, lucigenin-enhanced chemiluminescence assay, chemiluminescent probe L-012 assay and clonogenic survival assay, respectively. Each bar represents the mean ± standard deviation of triplicate measurements. * p < 0.01 compared to normoxia. # p < 0.01 compared to cycling hypoxia.
Figure 5. Nox4 blockage enhances the efficiency of radiotherapy in glioblastoma
xenografts. Western blot (A) and immunohistochemical analysis (B) of Nox4 in GBM8401/SFFV-LucGFP-Nox4 shRNA xenografts with or without Nox4 knockdown. Original magnification, ×200. C. The mean normalized BLI values associated with longitudinal monitoring of intracranial tumor growth for each treatment group. D. Bioluminescence imaging of intracranial tumor growth for each treatment group on day days 14 and 20 after tumor implantation. E. The corresponding survival curves of GBM8401/SFFV-LucGFP-Nox4 shRNA xenograft-bearing mice for each treatment group. Bars report the mean ± standard deviation of measurements in 6 mice. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
