HIF-1αSilencing Inhibits the Growth of Osteosarcoma Cells by Inducing Apoptosis

140 Available online at www.annclinlabsci.org HIF-1αSilencing Inhibits the Growth of Osteosarcoma Cells by Inducing Apoptosis Fei Lv 1, Rui Du 2, Wenqiang Shang 1, Sai Suo 1, Dangen Yu 1, and Jianhe Zhang 1 1 Department of Orthopedics, Jizhong Energy Fengfeng Group Hospital, Handan, Hebei and 2 Postgraduate School, Hebei Medical University, Shijiazhuang Hebei, China Abstract. Background. Osteosarcoma (OS) is a malignant tumor of mesenchymal origin, which is generally locally aggressive and tends to produce early systemic metastases. Therefore, an identification of a novel therapeutic target is required. Methods. We investigated the effect of small interfering RNA (sirna) targeting hypoxia-inducible factor 1α (HIF-1α) on the growth of Osteosarcoma MG-63 and U2-OS cells. Two cell-lines were transfected with various concentrations of HIF-1α or control sirna, and the effect on HIF-1α expression was analyzed by using quantitative polymerase chain reaction and western blot analysis. The effects of HIF-1α sirna on growth inhibition and apoptosis were then evaluated by standard methods. Result. HIF-1α sirna treatment reduced HIF-1α mrna and protein expression in MG-63 and U2-OS cells significantly. The downregulation of HIF-1α expression upregulated caspase-3 expression subsequently inhibited the growth of MG-63 and U2-OS cells and induced apoptosis of the two cells. Our in vitro data indicate that the downregulation of HIF-1α is capable of suppressing OS cells growth, through the induction of apoptosis. HIF-1α inhibition may be a new strategy for the treatment of Osteosarcoma. Key words: RNA interference, HIF-1α, target therapy, Osteosarcoma Introduction Osteosarcoma (OS) is a locally aggressive malignant tumor of mesenchymal origin, and patients with OS are susceptible to early systemic metastases [1]. Although the long-term outcome for patients who undergo surgery for high-grade OS has improved with the addition of systemic chemotherapy, prognosis remains unsatisfactory [2]. OS largely occurs in the young but about one-third of OS occurs in patients over 40 years of age [3], which is characterized by local invasion and early lung metastasis, resulting in a 5-year survival rate of 20% in patients with metastases [4,5]. Therefore, a novel and effective therapeutic targets is urgently required for patients with refractory OS. Hypoxia is a typical feature of solid tumors. HIF- 1α is a major survival factor for tumor cells growing in a low oxygen environment. Moreover, it plays a critical role in inducing hypoxia-related gene expression and cellular Address correspondence to Dr. Jianhe Zhang, Department of Orthopedics, Jizhong Energy Fengfeng Group Hospital, Handan, Hebei 056200, P. R. China; e mail: gukezhangjianhe@163.com responses [6]. HIF1-α can transcribe more than one hundred genes. HIF-1α influences a number of genes that in part play roles in tumor progression, including insulin-like growth factor binding protein-3, endothelin-1, inducible nitric oxide synthetase, hemeoxygenase 1, vascular endothelial growth factor (VEGF), and different glucose transporters and glycolytic enzymes [7,8]. While upregulation or activation of HIF-1α promotes tumor growth, downregulation or loss of HIF-1α activity has been demonstrated to markedly decrease tumor growth, vascularization, and energy metabolism [9]. The suppression of HIF-1α expression by RNA interference (RNAi) has been shown to be an effective method of cancer therapy [10]. Evidence suggests that overexpression of HIF1-α was implicated with the poor prognosis of ovarian cancer, pancreatic carcinoma, breast cancer, nasopharyngeal carcinoma, cervix cancer, and so on. However, none of the presently literatures exclusively target HIF-1α to address the potential of small interfering RNA (sirna) targeting HIF1-α for the treatment of OS. The present study was undertaken with the purpose of investigating sirna 0091-7370/16/0200-140. 2016 by the Association of Clinical Scientists, Inc.

HIF-1α Silencing Inhibits the Growth of Osteosarcoma Cells 141 Figure 1. MG-63 and U2-OS cells transfection with the HIF-1α sirna (25, 50 and 100 nm) and control sequences. sirna, small interfering RNA. Figure 2. HIF-1α sirna downregulates HIF-1α mrna in osteosarcoma MG-63 and U2-OS cells. (A and C). HIF-1α mrna expression in MG-63 and U2-OS cells under normoxia and hypoxia. The two cell-lines were exposed to hypoxia for 24, 48 and 72 h, respectively and HIF-1α mrna was assayed using quantitative-pcr. GAPDH served as an internal control. (B and D). HIF-1α sirna suppresses expression of HIF-1α mrna in MG-63 and U2-OS cells. Relative HIF-1α mrna expression was calculated to assess the ability of indicated agents to inhibit HIF-1α mrna expression. Data are mean±sd. *P<0.05 vs. normal; # P<0.05 vs. control. HIF-1α, hypoxia-inducible factor 1α; sirna, small interfering RNA; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

142 targeting HIF-1α on the growth of OS MG-63 and U2-OS cells. We found that sirna targeting HIF- 1α significantly inhibited the expression of HIF-1α in the two cell-lines. The results also indicated that silencing of HIF-1α may be a new strategy in the treatment of OS through induction of apoptosis. Materials and Methods Cell culture. Human OS cell line MG-63 and U2-OS was cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen Gibco Cell Culture Products, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 50 units of penicillin 50 μg/ml gentamicin, 2.5 μg/ml amphotericin B, 1% glutamine, 2% HEPES at natural temperature (37 C) in atmosphere containing 5% CO 2. For hypoxic treatment, cells were cultured in a modular incubator chamber (Forma, Thermo Fisher Scientific, Waltham, MA, USA) that was infused with 2% O 2, 5% CO 2, and 93% N 2. RNAi treatment. HIF1-α interference RNA (sirna) was designed, constructed, and purified by Boya Bioengineering Company (Shanghai, China). The antisense sequence of sirna targeting HIF-1α was 5'-AGTTCACCTGAGCCTAATA-3', and the control sequence was 5'-CTTAGCCTTCGAATGATCT-3'. Gene silencing was performed via transfection with the HIF-1α sirna (25, 50, and 100 nm) and control sequences. sirna was transfected into MG-63 cells and U2-OS using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA, USA) (Figure 1). After incubation for 2h, medium was replaced with fresh medium. According to the indicated time, the cells were harvested for subsequent studies. Quantitative polymerase chain reaction (qpcr) analysis. Total RNA was extracted from the cells using the Total RNA Kit (TIANGEN Co., Beijing, China) and 3 mg of RNA was converted into cdna using the High Capacity cdna Archive Kit (Applied Biosystems, Foster City, CA, USA). The primers were synthesized based on the published sequence [11]. The master mix of each PCR reaction included SYBR GREEN master mix (Solarbio Co., Beijing, China), forward and reverse primers, and 10 ng of template cdna. The PCR reaction was carried out at 95 1C for 5 min followed by 40 cycles of 95 1C for 30 s, 60 1C for 30 s, and 72 1C for 30 s. Data were analyzed using the comparative Δ Ct method (ABPrism software, Applied Biosystems, Foster City, CA) using GAPDH as the internal normalization control. Western blot analysis. Whole-cell extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors (BD Pharmingen, San Diego, CA). Protein samples (60 mg) were separated in 10% SDS polyacrylamide gels (Sigma- Aldrich, St. Louis, MO) and transferred to polyvinylidene difluoride (PVDF) membranes (BD Pharmingen, San Diego, CA). The membranes were blocked with nonfat milk in tris buffered saline with tween, and probed with primary antibodies against HIF-1α or caspase-3 (Invitrogen Life Technologies) at 4 C overnight. Membranes were then probed with an alkaline phosphatase-linked secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at 37 C. Signals were detected using a chemiluminescence Phototope -horseradish peroxidase (HRP) kit, according to the manufacturer's instructions (Cell Signaling Technology Inc., Beverly, MA, USA). Where necessary, β-actin (Invitrogen Life Technologies) was used as an internal control. MTT assay. Cell proliferation was measured by MTT assay. Cells were grown in monolayer culture to 60% confluency, harvested using Trypsin and plated at a density of 4 10 4 cells/well into separate wells of a 96-well plate (Costar; Corning Inc., Corning, NY, USA). Incubation was continued in hypoxia conditions. Following incubation for the indicated times, 20 μl 5 mg/ml MTT (Sigma, St. Louis, MO, USA) solution in PBS was added to each well for an additional 4 h. Subsequently, the supernatant was discarded and 150 μl dimethylsulfoxide (DMSO) was added to each well. Following MTT incubation, the absorbance of the samples was determined using a microplate reader at 490 nm (SunriseTM; Tecan Group Ltd., Männedorf, Switzerland). Annexin-V assay. Effect of HIF-1α sirna on cell apoptotic measured by Annexin-V Apoptosis Detection Kit I (BD). As follows, 2 10 5 cells were collected, washed twice with cold PBS, suspended in 100 μl binding buffer, and incubated with Annexin V-FITC at room temperature for 10 min. Subsequently, 5 μl propidium iodide (PI, 20 μg/ml) was added and cells were incubated away from the light at room temperature for 5 min. Cells were then directly analyzed by FAC ScanTM (Becton Dickinson) and evaluated using the Cell Quest program (Becton Dickinson). Statistical analysis. All the experiments were repeated in triplicate. Analyses were carried out using SPSS 21.0 statistical software. One-way analysis of variance (ANOVA) followed by post hoc least significant difference (LSD) or Dunnett's tests was performed to determine the significance of the differences in multiple comparisons. Differences were considered significant at P<0.05.

HIF-1α Silencing Inhibits the Growth of Osteosarcoma Cells 143 Figure 3. HIF-1α sirna downregulates the expression of HIF-1α protein in osteosarcoma MG-63 and U2-OS cells. The protein level of HIF-1 awas detected by Western blot analysis. (A and C) HIF-1α protein expression of MG-63 and U2- OS cells under normoxia and hypoxia (24, 48 and 72 h). (B and D) Various concentrations of HIF-1α sirna downregulated levels of HIF-1α protein expression under hypoxic conditions. Data are mean±sd (n=3). *P<0.05 vs. normal; # P<0.05 vs. control. HIF-1α, hypoxia-inducible factor 1α; sirna, small interfering RNA. Figure 4. The expression of caspase-3 protein was increased after transfection with 100 nm HIF-1α sirna. sirna, small interfering RNA. This study was approved by the Human Ethics and Research Ethics committees of Jizhong Energy Fengfeng Group Hospital. Results HIF-1α sirna downregulates HIF-1α mrna expression. To assess the potential effect of HIF-1α sirna, we evaluated HIF-1α mrna expression levels in MG-63 and U2-OS cells by qpcr. Under normoxic conditions, HIF-1α mrna was expressed at low levels; however, under hypoxic conditions, HIF-1α mrna expression was significantly increased (Figure 2A and C). HIF-1α sirna significantly suppressed the expression of HIF-1α mrna in HIF1α sirna-treated cells, (P<0.05), particularly at concentrations of 50 and 100 nm, compared to control cells (Figure 2B and D).

144 Figure 5. Effect of HIF-1α sirna on the proliferation of osteosarcoma cells in vitro. Following exposure to the indicated hypoxic conditions (24,48,72 h), the inhibition of proliferation by HIF-1α was assessed using MTT assay. Data are mean±sd (n=3). *P<0.05 vs. normal; # P<0.05 vs. control. HIF-1α, hypoxia-inducible factor1α; sirna, small interfering RNA. HIF-1α sirna reduces HIF-1α protein expression. Western blot analysis showed the effect of HIF-1α sirna on HIF-1α protein expression. Under normoxic conditions, HIF-1α protein expression in MG-63 and U2-OS cells was low; however, expression was markedly induced under hypoxia (Figure 3A and C). Treatment with control sirna did not affect HIF-1α protein expression; however, HIF-1α sirna significantly downregulated HIF-1α protein expression under hypoxic conditions (Figure 3B and D). In addition, the decrease of HIF-1α expression upregulated the expression of caspase-3 (Figure 4). HIF-1α sirna suppresses the growth of MG-63 and U2-OS cells. The effect of HIF-1α silencing on the two cell-lines growth was estimated in vitro. As shown in Figure 5, under hypoxia, HIF-1α sir- NA significantly inhibited the growth of MG-63 and U2-OS cells, particularly at concentrations of 50 and 100 nm (Figure 6), compared to control sirna. Moreover, the suppression of growth in the HIF-1α sirna-transfected cells was more efficient under 72 h hypoxia, compared to 24 h. Discussion There have been lots of previous literature proved that HIF-1α plays a vital role in invasion, proliferation, metastasis and apoptosis of malignant tumors [9,12]. Therefore, therapeutic agents that inhibit HIF-1α function have the potential to improve the outcome of patients with OS. In this study, we found hypoxia markedly increased HIF-1α mrna and protein expression in MG-63 and U2-OS cells. In comparison, silencing HIF-1α with sir- NA significantly inhibited them. Moreover, HIF- 1α sirna significantly inhibited the two cell-lines growth under hypoxia, and promoted apoptosis through upregulation of caspase-3 expression. Tumor cells and tissues adapt to hypoxic microenvironment via the activation of numerous hypoxia-related molecules, among which hypoxia-inducible factors (HIFs) are the predominant ones [13].Therefore, understanding the central role of HIF-1α in the adaptive response to hypoxia is essential for an effective strategy to the clinic [14]. As previously described [9,15], our study results demonstrated that in MG-63 and U2-OS cells, HIF-1α mrna and protein were expressed at low levels under normoxic conditions; however, mrna and protein levels increased notably under hypoxia. Published literature has confirmed that HIF-1α is important in the regulation of cell growth, development and invasion of OS [16]. Thus, we hypothesized that the downregulation of HIF-1α might represent a novel therapeutic target in OS. It s reported that 17-AAG, temsirolimus and rapamycin have been demonstrated to suppress the growth of HIF-1α expressing tumors; however, none of these drugs have been shown to directly and specifically target HIF-1α [14]. Here we observed that HIF-1α silencing by sirna not only significantly inhibited the expression of HIF- 1α mrna and protein, but also effectively suppressed the proliferation of OS cells under hypoxic conditions.

HIF-1α Silencing Inhibits the Growth of Osteosarcoma Cells 145 Figure 6. The growth of MG-63 and U2-OS cells was inhibited after transfection with 100 nm HIF-1α sirna. sirna, small interfering RNA. Apoptosis plays an important role in the regulation of tumor cell growth, metastasis, and invasion. Plenty of studies have demonstrated that HIF-1α possesses dual functions in mediating apoptosis of tumor cells, i.e, anti- and pro-apoptotic function [17-20]. Unfortunately, the complete effect of HIF- 1α on cell apoptosis remains unknown. As Theodoropoulos et al reported, HIF-1α may depend on tumor microenvironment, particularly hypoxia, to regulate cell apoptosis [21]. As we all know, cancer cells, especially in these malignant tumors, always grow faster than the blood vessels that nourish it, which makes the tumor environment have low oxygen availability, driving tumor cells to adapt to hunger. Evidence suggests that HIF-1α is widely expressed in OS tissue [15]. Our study demonstrated that, under hypoxia, HIF-1α sirna induced MG-63 caspase-dependent cell apoptosis in vitro. In the program of cell death (apoptosis), caspase act as a crucial mediator. This occurs because caspase-3 represents a frequently activated death protease that catalyzes the specific cleavage of key cellular proteins, and is required for certain hallmarks of apoptosis [22]. In the present study, it was observed that HIF-1α sirna significantly upregulated the level of caspase-3 expression of OS cells under hypoxic conditions, in accord with the apoptosis induced by HIF-1α sirna. In conclusion, our study showed that sirna technology may be used to specifically inhibit HIF-1α expression in OS cell lines. Silencing of HIF-1α not only induced apoptosis in OS MG-63 and U2- OS cells, but also suppressed growth of OS. However, achieving the specific efficiency of HIF- 1α sirna in vivo remains a challenge. This study indicates that further insights into the role of HIF- 1α sirna would contribute to the development of therapeutic strategies to prevent OS. References 1. Luetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment-where do we stand? A state of the art review. Cancer Treat Rev. 2014. 40(4): 523-32. 2. K. Dobrenkov, N.K. Cheung, GD2-targeted immunotherapy and radioimmunotherapy, Semin. Oncol.. 2014. 41(2014)589-612. 3. Sergi C, Zwerschke W. Osteogenic sarcoma (osteosarcoma) in the elderly: tumor delineation and predisposing conditions. Exp Gerontol. 2008. 43(12): 1039-43.

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