STAT3 inhibitor NSC74859 radiosensitizes esophageal cancer via the downregulation of HIF-1α
Abstract
Radiotherapy is the main therapy for inoperable and locally advanced esophageal squamous cell carcinoma (ESCC). However, radioresistance in ESCC remains a chal- lenge. The aim of this study is to investigate the radiosensitizing effects of STAT3 inhibitor NSC74859 on ESCC and explore the underlying mechanisms. ECA109 and TE13 cells were exposed to hypoxia, and treated with NSC74859 or radiation, alone or in combination. Cell prolif- eration, survival, apoptosis, and double-stranded DNA breaks (DSBs) were examined. Nude mice model of ECA109 xeno- graft was treated with radiation and/or NSC74859. The levels of STAT3, p-STAT3, HIF-1α, and VEGF were detected by Western blot analysis. NSC74859 efficiently radiosensitized ESCC cells and xenografts in nude mice, and inhibited hypoxia-/radiation-induced activation of STAT3 and upregu- lation of HIF-1α and VEGF expression. NSC74859 confers radiosensitivity in ESCC via the inhibition of STAT3 activa- tion and the downregulation of HIF-1α and VEGF expression.
Keywords : Esophageal squamous cell carcinoma . NSC74859 . STAT3 . HIF-1α . Radiotherapy
Introduction
Esophageal cancer is one of the most common cancers in the world, consisting of esophageal adenocarcinoma and esopha- geal squamous cell carcinoma (ESCC). Radiotherapy is a common therapy for patients with inoperable and locally advanced ESCC. However, a large proportion of ESCC tu- mors develop resistance to radiotherapy, indicating the neces- sity to enhance radiosensitivity of ESCC.
During radiotherapy, the oxygen generates free oxygen radicals that induce DNA damage and kill tumor cells [1]. Hypoxic environments contribute to some malignant behav- iors, particularly the acquisition of resistance to radiotherapy. Hypoxia-inducible factor 1α (HIF-1α) plays a vital role in the response to hypoxia and transcriptionally activates genes such as vascular endothelial growth factor (VEGF) to promote the resistance of cancer cells to radiotherapy [2–4].
Signal transducer and activator of transcription 3 (STAT3) acts as a signal transducer as well as transcription factor, and is constitutively activated in numerous human tumors. Interest- ingly, hypoxia has been shown to activate STAT3 in renal cell carcinoma, contributing to both stability and synthesis of HIF- 1α protein. In hypoxia, STAT3 protein has been shown to bind to the HIF-1α promoter both in vitro and in vivo [5]. Further- more, activated STAT3 behaves as an angiogenesis inductor to promote VEGF expression, enhance HIF-1α stability, and acts as a co-activator under hypoxia [6]. STAT3 inhibitor NSC74859, also known as S3I-201, is an inhibitor of the dimerization and phosphorylation of STAT3, and has been shown to selectively suppress the viability of human breast, pancreatic and glioblastoma cancer cell lines, and potentiate the antiproliferative activity of cetuximab [7, 8]. The antitu- mor effects of NSC74859 were also observed in mouse models of human breast cancer and liver cancer [9–11]. Inter- estingly, blocking phospho-STAT3 (p-STAT3) with NSC74859 inhibited the upregulation of HIF-1α and VEGF in hypoxic human umbilical vein endothelial cells, indicating the existence of an autocrine loop involving STAT3, HIF-1, and VEGF [12].
Given the crucial role of HIF-1 and VEGF in cancer radioresistance, we wondered whether STAT3 inhibitor could enhance the effects of radiotherapy in ESCC. The present study aimed to investigate the radiosensitizing effects of STAT3 inhibitor NSC74859 on ESCC and explore the under- lying mechanisms.
Materials and methods
Reagents and cell lines
NSC74859 (Selleck, USA) was formulated at 1,000x stock solution in DMSO for in vitro experiments and used at 10 mg/ml in salad oil for in vivo experiments. Two human malignant esophageal cancer cell lines ECA109 and TE13 were obtained from Shanghai Institutes for Biological Sci- ence, China. Cells were propagated in RPMI-1640 medium supplemented with 10 % fetal bovine serum (Gibco, CA, USA), 100 units/ml penicillin, and 100 mg/ml streptomycin (Sigma, St. Louis, USA).
Hypoxia and irradiation protocols
Hypoxia was induced by incubating cells in a hypoxia cham- ber (glass pot maintaining 0.5 % O2). Irradiation was per- formed at 566 cGy/min using an X-ray irradiator (Elekta, Sweden). Cells were irradiated at a single dose. Animals received 6 Gy (2.47 cGy/min) for tumor radiotherapy (RS- 200 Pro Biological Irradiator).
Cell proliferation assay
CCK8 assay (Beyotime, China) was to evaluate the prolifer- ation of esophageal cancer cells. Then 96-well plates were seeded with cells at a density of 4×104 cells/well. After 24 h, cells were treated with agents at indicated concentrations for 22 h and incubated with fresh medium containing 10 % CCK8 for another 2 h. Viable cells were detected by measuring absorbance at 450 nm by a microplate reader (BioTek ELx800, USA).
Clonogenic survival assay
Cells were seeded into 6-well plates overnight and then treated with S3I-201 (100 μM) or cisplatin (10 μM) as positive controls under normoxic or hypoxic conditions for 24 h. Next, the cells were subjected to X-rays of 2, 4, 6, and 8 Gy, and cultured at 37 °C for 10 days, fixed with methanol and stained with Giemsa. Finally, the numbers of the colonies containing at least 50 cells were counted under microscope.
Immunocytofluorescence
Cells were incubated on glass coverslips for 24 h. Cells were fixed with 4 % paraformaldehyde at 0.5, 4, and 24 h after irradiation, permeabilized with 0.1 % Triton X-100, stained with anti-phospho-histone γ-H2AX (Millipore, France, dilut- ed 1: 250), and incubated with 0.25 mg/mL DAPI (Beyotime) for 5 min. Slides were observed under a laser scanning con- focal microscope.
Western blot analysis
Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Kaygen). Amount of protein in the lysates was quantified by BCA kit (Beyotime). Equal amounts of proteins were separated by SDS-PAGE and trans- ferred to PVDF membranes (Millipore, France). The mem- branes were blocked with 5 % skim milk, incubated with primary antibodies against STAT3, p-STAT3 and HIF-1α (Cell Signaling Technology, France), VEGF, and β-actin (Santa Cruz, USA) at 4 °C overnight, and incubated with HRP-conjugated secondary antibodies (BioWorld, USA) for 1 h at room temperature. Immunoblotted proteins were visu- alized by ECL reagents and the signals were detected by ChemiDoc XRS imaging system (Quantity One Quantitation software, Bio-Rad Laboratories, Hercules, CA, USA).
Flow cytometry
Cells were plated in 6-well plates at a specific density. The cells were treated in normoxia or hypoxia for 24 h, exposed to X-rays (6 Gy). After 24 h, the cells were collected and ana- lyzed by using AnnexinV-FITC Apoptosis Detection kit (BD Bioscience, Oxford, UK). The apoptotic cells were detected by flow cytometry.
Xenografts
ECA109 cells (1×106) were injected subcutaneously in the fossa axillaris of 6-week old male nude mice (Engene, China). When the tumors grew to 100 mm3, the animals were fed with 10 mg/kg NSC or vehicle (PBS) every other day. Tumor volume was calculated. When the average volumes increased to 300 mm3, tumors were irradiated by RS-2000 biological irradiator at a dose of 6 Gy (2 Gy/min) 2 h after injection. The tumor doubling time (DT) was calculated as follows: DT=d× lg2/lg(Vd/V0), where d was the length of time between two measurements, Vd was the volume of the tumor treated with X-ray, and V0 was the volume of the tumor before the X-ray. The mice were sacrificed when the average tumor volume reached 1,500–2,000 mm3, and tumor tissues were dissected, weighed, and then stored in 4 % buffered formaldehyde.
Statistical analysis
Statistical analyses were conducted by t tests or one-way ANOVA, with 95 % confidence estimations (GraphPad Prism 5.0, CA, USA). A P value <0.05 was consider statistically significant. Results NSC74859 promotes the radiosensitivity of ESCC cells NSC74859 inhibited the proliferation of ESCC cells in a dose- dependent manner (Fig. 1a). At 24 h, the IC50 for ECA109 and TE13 cells were 172.8 and 184.6 μM, respectively. Thus, a low cytotoxic concentration (100 μM) was selected for further in vitro experiments. NSC74859 inhibited clonogenic survival of ECA109 cells similarly under conditions of hyp- oxia (SER=1.34) and normoxia (SER=1.32) (Table 1). How- ever, compared with hypoxic cells, cells in normoxia exhibit- ed a reduction in colony formation after irradiation, indicating hypoxia-induced radioresistance (normoxia: SER= 1.32, normoxia+NSC: SER=1.75). The reduction was observed in cisplatin-treated cells as positive control (SER= 1.95; Fig. 1b). Similar results were observed in TE13 cells (Table 1; Fig. 1c). NSC74859 induces apoptosis and DNA breaks in ESCC cells We wondered whether NSC74859-induced radiosensitization resulted from increased apoptosis. Treatment with NSC74859 (100 μM) alone for 24 h did not significantly induce apoptosis under either normoxia or hypoxia. Interestingly, the propor- tion of apoptotic cells was remarkably increased in irradiation- treated groups compared with control group (normoxia: p= 0.021, p=0.006; hypoxia: p=0.049, p=0.009), and apoptotic cells in combined treatment group were significantly more than in irradiation alone group in both normoxic and hypoxic conditions (p=0.010 and p=0.013, respectively; Fig. 1d–f). To better understand the anti-clongenic effect of NSC74859, we detected DNA double-strand breaks (DSBs) by immunofluorescence staining of γ-H2AX foci in ESCC cells at different time points after irradiation. We observed an experiments. Notably, hypoxia-stimulated accumulation of phospho-STAT3, HIF-1α, and VEGF levels was significantly attenuated by NSC74859 (Fig. 2c, e). Fig. 1 NSC74859 increases radiosensitivity of ESCC cells. a NSC74859 inhibited the proliferation of ESCC cells in a dose-dependent manner; the IC50 for ESCC cells ECA109 and TE13 were 172.8 and 184.6 μM, respectively. b, c Clonogenic survival assay of ECA109 and TE13 cells exposed to 0–8 Gy and/or NSC74859 (100 μΜ) in normoxia and hypoxia conditions. d–f NSC74859 (100 μΜ) significantly enhanced irradiation- induced apoptosis of ECA109 cells under hypoxia and normoxia, *p<0.05. Fig. 2 NSC74859 leads to the inhibition of STAT3 activation and the downregulation of HIF-1α and VEGF expression in ESCC cells. a, b Determination of phospho-S139 cH2AX foci in ECA109 and TE13 cells 30 min, 4 h, and 24 h after treatment with 1 Gy and/or 100 μM NSC74859 (mean±SEM, n=3),*p<0.05, **p<0.01. c, e Western blot analysis showing that NSC74859 reduced the levels of p-STAT3, HIF- 1α, and VEGF in ECA109 and TE13 cells. d, f Western blot analysis showing that hypoxia increased the levels of p-STAT3, HIF-1α, and VEGF in ECA109 and TE13 cells. NSC74859 promotes the radiosensitivity of ESCC xenografts in nude mice To confirm radiosensitization effect of NSC74859 on ESCC xenograft in vivo, ECA109 tumor-bearing mice were treated with a single fraction of 6 Gy irradiation, and received intra- peritoneal injection of NSC74859 every other day for 1 week before irradiation. Compared to control group, either irradia- tion or combined treatment effectively delayed tumor growth and reduced tumor weight (p=0.038 for irradiation and p= 0.008 for NSC74859+IR on the 22th day; Fig. 3a, b, d). Moreover, we calculated the doubling time for ECA109 tumor. In control group and NSC74859 alone group, the doubling time was 7.5±0.9 and 8.3±0.8 days, respectively. The doubling time was significantly extended to 12.5 ± 0.7 days in combination treatment group (p=0.002), while it was 10.5± 1.5 days in irradiation alone group (p =0.034). Taken together, these results demonstrate that NSC74859 enhances the radiosensitivity of ESCC xenografts. To investigate whether NSC74859 inhibits STAT3 activa- tion and HIF-1α and VEGF expression in hypoxic ESCC xenografts, we temporarily blocked the tumor blood supply for 5 min before irradiation for a hypoxic model. After treat- ment with NSC74859 and/or irradiation, tumor xenografts were sectioned for immunohistochemistry. The results showed that NSC74859 significantly decreased the levels of phospho-STAT3, HIF-1α, and VEGF compared to control group (Fig. 3c). Discussion Hypoxic environments contribute to some malignant behav- iors, particularly the acquisition of resistance to radiotherapy. Hypoxia-inducible factor 1α (HIF-1α) plays a vital role in the response to hypoxia and transcriptionally activates genes such as vascular endothelial growth factor (VEGF) to promote the resistance of cancer cells to radiotherapy. Previous studies have shown that it could increase the radiosensitivity in esophageal, prostate, and nasopharyngeal cancers by down- regulating the level of HIF-1α and VEGF protein [13–15]. In several epithelial cancers, STAT3 signaling is involved in cell proliferation, metastasis, angiogenesis, host immune evasion, and resistance to apoptosis [16, 17]. Activated STAT3 behaves as an angiogenesis inductor to promote VEGF expression, enhance HIF-1α stability, and acts as a co-activator under hypoxia. STAT3 knockdown reduced the proliferation and migration of ESCC cells [18, 19]. The inhi- bition of STAT3 displayed antitumor effect, and promoted chemosensitivity and radiosensitivity in NPC and HNSCC via modulating HIF-1α expression [20, 21]. Fig. 3 NSC74859 enhances the radiosensitivity of ESCC xenografts. a The volume of ECA109 xenografts treated by 6 Gy on day 6 and/or 5 mg/kg NSC74859 on day 0, 2, 4, and 6 (mean±SEM, n=6). b, d Tumor weight on the 22th day after first administration in four groups (n=6) (compared with control group: p>0.05 for NSC74859 alone, p=0.038 for irradiation, p=0.008 for combination group). c Immunohistochemical staining for p-STAT3, HIF-1α, and VEGF in ECA109 xenografts dis- sected from nude mice.
In this study, by using NSC74859 as a STAT3 inhibitor, we demonstrated that NSC74859 increased the radiosensitivity of ESCC cells by inhibiting cell proliferation and colony forma- tion. Next, we determined whether NSC74859-induced radiosensitization is related to increased apoptosis. While NSC74859 alone did not significantly induce apoptosis of ESCC cells under normoxia or hypoxia condition, the combi- nation of NSC74859 and irradiation significantly increased apoptosis of ESCC cells compared to irradiation treatment alone group. These data suggest that NSC74859 increases the radiosensitivity of ESCC cells by enhancing irradiation- induced cancer cell apoptosis.
To explore the signaling mechanism by which NSC74859 increases radiosensitization effects in ESCC cells, we investi- gated the effects of NSC74859 and/or irradiation on the acti- vation of STAT3 and HIF-1α and VEGF expression in ECSS cells. We found that hypoxia stimulated the activation of STAT3 and the expression of HIF-1α and VEGF in ESCC cells, confirming that hypoxia contributes to cancer radioresistance. However, NSC74859 significantly attenuated hypoxia-induced activation of STAT3 and the upregulation of HIF-1α and VEGF expression. These data suggest that NSC74859 increases the radiosensitivity of ESCC cells by interfering with the activation of STAT3/HIF-1α/VEGF signaling.
Furthermore, we used xenograft nude mouse model to provide in vivo evidence that NSC74859 enhances the radio- sensitivity of ESCC xenografts. NSC74859 significantly en- hanced irradiation-induced tumor growth inhibition. Interest- ingly, by immunohistochemistry, we found that NSC74859 significantly decreased the levels of phospho-STAT3, HIF- 1α, and VEGF in dissected tumor xenografts, confirming that NSC74859 interferes with the activation of STAT3/HIF-1α/ VEGF signaling in vivo.
In conclusion, NSC74859 radiosensitizes normoxic and hypoxic ESCC cells and xenografts, mainly due to the inhi- bition of STAT3 activation and the downregulation of HIF-1α and VEGF expression. To overcome the radioresistance of ESCC, especially in hypoxic condition, a hypoxic radiosensitizer is needed to be used in combination with radiotherapy. Our results provide support for the utility of NSC74859 as an antitumor agent in different cancers and expand our understanding of the mechanisms of NSC74859 activity. NSC74859 appears to be a promising radiosensitizer TC-S 7009 for radiotherapy of ESCC.