Reduction of hypoxia-induced angiogenesis in ovarian cancer cells by inhibition of HIF-1 alpha gene expression
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- Bryant, C.S., Munkarah, A.R., Kumar, S. et al. Arch Gynecol Obstet (2010) 282: 677. doi:10.1007/s00404-010-1381-9
The goal of this study was to investigate the effects of silencing HIF-1 alpha gene expression with specific small interfering RNA (siRNA) on VEGF production and angiogenesis in epithelial ovarian cancer (EOC) cells.
Two EOC cell lines, MDAH-2774 and SKOV-3, were cultured under normoxic (20% O2) and hypoxic (2% O2) conditions using standard techniques. After EOC cells were transfected with siRNA, HIF-1 alpha and VEGF mRNA levels were measured by real-time RT–PCR. Angiogenesis was evaluated utilizing an in vitro assay model consisting of human umbilical vein endothelial cells (HUVEC) and polymerized ECM Matrix.
Both EOC cell lines evaluated constitutively expressed HIF-1 alpha and VEGF mRNA. HIF-1 alpha and VEGF mRNA levels were significantly increased in response to hypoxia (P < 0.05). Under hypoxic conditions, inhibition of HIF-1 alpha gene expression by a specific siRNA resulted in a significant reduction in HIF-1 alpha and VEGF mRNA levels (P < 0.05). In the in vitro angiogenesis model, supernatant from the hypoxic EOC cells induced the HUVEC to form a complex tubular network, a hallmark of angiogenesis. Semi-quantitative analysis of the angiogenesis assay revealed a significant reduction in tube formation when supernatant from HIF-1 alpha siRNA-treated hypoxic EOC cell was used (P < 0.05).
Inhibition of HIF-1 alpha expression by specific siRNA resulted in a significant decrease in VEGF production and angiogenesis. Further investigation of HIF-1 alpha inhibition for anti-tumor activity is warranted and may potentially prove HIF-1 alpha as a therapeutic target in the management ovarian cancer.
KeywordsOvarian cancerAngiogenesissiRNAGene silencingHIF-1 alphaVEGF
Angiogenesis is the development of new blood vessels from the preexisting vasculature. This process is a key factor in the progression of cancer and has been demonstrated to strongly correlate with risk of invasion and metastasis [1, 2]. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor that promotes neovascularization . Elevated expression of VEGF has been reported in ovarian cancer and is associated with a poor prognosis .
The balance of pro- and anti-angiogenic factors governs angiogenesis. During tumorigenesis, tumor progression, and metastasis, there is a disruption in the net balance of angiogenic factors, favoring angiogenesis . Hypoxia is believed to be a key signal for activation of the ‘angiogenic switch’ and the resultant increase in expression of VEGF [5, 6]. Hypoxia-induced VEGF expression is regulated by multiple mechanisms, one of which is transcriptional activation .
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor composed of a constitutively-expressed HIF-1 beta subunit and an inducibly-expressed HIF-1 alpha subunit . In response to hypoxia, HIF-1 alpha protein accumulates in the cytosol and translocates to the nucleus, where it activates hypoxia-sensitive genes, such as VEGF, by binding to their promoter regions [8, 9]. We have previously shown, when epithelial ovarian cancer (EOC) cell lines MDAH-2774 and SkOV-3 were cultured under hypoxic conditions, a significant increase in the expression of HIF-1 alpha and VEGF mRNA occurs .
The goal of this study was to investigate the effect of silencing HIF-1 alpha gene expression with specific small interfering RNA (siRNA) on VEGF mRNA production and angiogenesis of ovarian cancer cells.
Cell line and cell culture
The human EOC cell lines, MDAH-2774 and SkOV-3, were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Cell lines were cultured in 100 × 20 mm cell culture dish (Corning Inc., NY) with McCoy’s 5A medium (Invitrogen, CA) supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin, 0.1 μg of streptomycin (ATCC). Cells were maintained at confluency at 37°C incubation with 5% carbon dioxide. Culture medium was replaced every 2 days. Culture medium for hypoxic conditions was preequilibrated to the experimental oxygen conditions overnight. For each experiment, cells were plated in 24-well culture dish (Corning Inc., NY) at a cell density of approximately 4.5 × 104 cells per well and cultured for 24 h under normoxic (20% O2, 5% CO2, and 75% N2) or hypoxic conditions (2% O2, 2% CO2, balanced of nitrogen) provided by a gas mixture tank (Wilson Medical Gases Inc., MI).
HIF-1 alpha siRNA design and transfection
siRNAs were designed after determination of target sequences by aligning target (HIF-1 alpha) sequence to an Ambion® web-based algorithm (Ambion, TX). The 21-nucleotide duplex siRNA molecules with 3-dTdT overhangs were resuspended in nuclease-free water according to the instructions of the manufacturer (Ambion, TX). To ensure stringent controls, both a 2A-based mutated control with two nucleotide mismatches (siRNA-2Amut) and a scrambled control sequence (siRNA-SCR) obtained from Ambion® (Silencer Negative Control No.1 siRNA, Ambion, TX, catalog No. 4610) were used. After optimizing transfection condition according to the manufacturer (Ambion, TX), the reverse transfection-, or neofection method was performed, in which cells are transfected as they adhere to the plate after trypsinization. siRNA transfection procedure in the cell lines was performed in accordance with our previously reported works . For siRNA transfection, cells were transferred to a 24-well cell culture plate (Corning Inc., NY) at a concentration of 4.5 × 104 cells per well (cell volume of 450 μL) and transfected with the use of 1.5 μL siPORT™ NeoFX™ reagent and 1 μL of 20 μM siRNA (Silencer™ siRNA Transfection II Kit, Ambion, TX, catalog No. 1631), and 25 μL OptiMEM® medium (Invitrogen, CA, catalog No. 31985–047), up to a final volume of 500 μL. NeoFX™ reagent and siRNA were incubated at room temperature for 10 min and then applied onto 4.5 × 104 cell per well. Transfection mixtures were incubated with cells for 8 h before washing with media and incubated for additional time period to equal total incubation time of 24 h.
Semi-quantitative analysis of angiogenesis
Individual cells, well separated
Cells begin to migrate and align themselves
Capillary tubes visible, no sprouting
Sprouting of new capillary tubes visible
Closed polygons begin to form
Complex mesh like structures develop
Measurement of HIF-1 alpha and VEGF mRNA levels by real-time reverse transcriptase polymerase chain reaction
Preparation of complimentary DNA (cDNA): using the QuantiTect® reverse transcription (RT) kit (Qiagen, CA, catalog No. 205311), the genomic DNA elimination reaction was prepared on ice, 2 μL of gDNA wipeout buffer, 1 μg of template RNA, and 2 μL of RNase-free water was incubated at 42°C for 2 min. The RT reaction components were prepared by adding 1 μL of Quantiscript® reverse transcriptase, 4 μL of Quantiscript® RT Buffer, and 1 μL of RT primer mix to the genomic DNA elimination reaction mixture and were incubated for 15 min at 42°C. The Quantiscript® reverse transcriptase mixture was then incubated for 3 min at 95°C to inactivate the enzyme.
VEGF: An initial cycle was performed at 95°C for 8 min, followed by 35 cycles of 95°C for 15 s, 55°C for 30 s, 72°C for 30 s, and then a final cycle at 72°C for 7 min to allow completion of product synthesis
HIF-1 alpha: An initial cycle was performed at 95°C for 15 min, followed by 35 cycles of 95°Cfor 15 s, 56°C for 30 s, 72°C for 30 s, and then a final cycle at 72°C for 7 min to allow completion of product synthesis
Each treatment was performed in three independent experiments. The data are presented as the means ± SD. Student’s t test was conducted using SPSS to evaluate the difference of the means between groups. Pearson’s correlation was used to assess associations between mRNA levels and treatments (v15.0 for Windows, SPSS, IL). Differences were considered to be significant if P < 0.05.
Silencing HIF-1 alpha gene expression by specific siRNA reduced HIF- alpha and VEGF mRNA levels in ovarian cancer cells exposed to hypoxia
Correlation between HIF-1 alpha and VEGF mRNA levels
A significant positive correlation was observed between HIF-1 alpha and VEGF mRNA levels in both EOC cell lines when cultured under hypoxic conditions (MDAH-2774 r = 0.971, P = 0.001; SkOV-3 r = 0.974, P = 0.001). There was no correlation noted between HIF-1 alpha and VEGF mRNA levels when cultured under normoxic conditions. There was a significant positive correlation observed between HIF-1 alpha and VEGF mRNA levels when both cell lines were treated with HIF-1 alpha-specific siRNA under hypoxic conditions (MDAH-2774 r = 0.941, P = 0.005; SkOV-3 r = 0.982, P < 0.001).
Silencing HIF-1 alpha gene expression by specific siRNA significantly reduced angiogenesis
In 1971, Folkman  proposed that tumor growth and metastasis are angiogenesis-dependent, and hence blocking angiogenesis could be a strategy to arrest tumor growth. Based on successful preclinical data, several anti-angiogenic agents alone or in combination with conventional therapies are now in clinical trials . Clinical and pre-clinical research have identified the following strategies for targeting angiogenesis: (1) interference with angiogenic ligands, their receptors or downstream signaling, (2) up-regulation or delivery of endogenous inhibitors, (3) directly targeting the tumor vasculature, or recently (4) the use of RNA interference to silence genes specific for angiogenesis [4, 11, 14–16].
Angiogenesis is a critical step in the process of cancer progression. VEGF, one of the most potent angiogenic cytokines, is over expressed in a large number of malignancies, including epithelial ovarian carcinomas [17, 18]. Angiogenesis commonly occurs in response to conditions of low oxygen concentration . We have previously reported a positive correlation between HIF-1 alpha and VEGF mRNA levels after ovarian cancer cells were cultured under hypoxic conditions . We have also previously reported on the pharmacologic inhibition and siRNA gene silencing of alternate angiogenic pathways that resulted in decreased VEGF mRNA levels and angiogenesis [11, 19].
VEGF production is regulated through a number of pathways, one of which is transcriptional activation via HIF-1 alpha. HIF-1 alpha functions as a master regulator of oxygen homeostasis and is commonly overexpressed in human cancers and their metastases [20, 21]. HIF-1 alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis, and angiogenesis [5, 7].
Carmeliet et al. reported that when embryonic stem (ES) cells with inactivated HIF-1 alpha genes were exposed to hypoxic conditions, the cellular responses included reduced expression of VEGF, impaired formation of large vessels in ES-derived tumors, and impaired vascular function. Their findings suggest that tumor vascularization is largely controlled by HIF-1 alpha, in part as a result of VEGF production . Wang et al., found that HIF-1 alpha protein plays a bimodal role in cellular hypoxic response in cancer cells including opposite effects of hypoxia, simultaneously promoting cell death and activating cellular antiapoptotic resistance. They went onto describe that downregulation of HIF-1 alpha promoted cell death and prevented activation of cellular defenses by hypoxia .
According to our previous  and current studies, HIF- alpha and VEGF mRNA levels are constitutively expressed in MDAH-2774 and SkOV-3 cells. Treatment with hypoxia was associated with a significant increase in HIF-1 alpha and VEGF mRNA levels. VEGF was not significantly altered by HIF-1 alpha siRNA under normoxic conditions. This can be explained by the regulator mechanism of HIF-alpha activity. A decrease in cellular O2 tension leads to elevation of HIF-1 alpha activity via stabilization of the HIF-1 alpha protein. Conversely, ubiquitin-mediated proteolysis of HIF-1 alpha protein on exposure to normoxic environment results in a rapid decay of HIF-1 alpha activity .
Surprisingly, HIF-1a mRNA was not significantly reduced by HIF-1 alpha siRNA in both cell lines when cultured under normoxic conditions. Optimization of the siRNA transfection process resulted in a lesser reduction in HIF-1 alpha mRNA in the MDAH-2774 cells as compared with the SkOV-3 cells. The less-than-expected decrease in HIF- alpha mRNA level in the MDAH-2774 cells might be explained by a less effective transfection process during our experiment or by similar mechanisms reported in the literature describing target-specific and off-target effects of RNA interference .
The findings in the current study with regard to hypoxia induction of HIF-1 alpha and VEGF mRNA production and angiogenesis are consistent with previous reports in the literature. Treatment with HIF-1 alpha-specific siRNA resulted in a significant decrease in HIF-1 alpha and VEGF mRNA in both ovarian cancer cell lines when cultured under hypoxic conditions. The in vitro angiogenesis model revealed that treatment of the ovarian cancer cells with HIF-1 alpha-specific siRNA resulted in a significant reduction and inhibition of tube formation by the HUVEC endothelial cells.
In this study, we have confirmed a role of HIF-1 alpha in ovarian cancer angiogenesis with the use of an in vitro model. In addition, current preclinical and clinical data with selective angiogenesis inhibitors are yielding very encouraging results. Further investigation of HIF-1 alpha inhibition for anti-tumor activity is warranted and may potentially prove HIF-1 alpha as a therapeutic target in the management ovarian cancer.
Conflict of interest statement