|Year : 2016 | Volume
| Issue : 4 | Page : 542-547
Vascular endothelial growth factor: Evidence for autocrine signaling in hepatocellular carcinoma cell lines affecting invasion
BK Sharma1, R Srinivasan2, YK Chawla1, A Chakraborti3
1 Department of Hepatology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
2 Department of Cytology and Gynecological Pathology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
3 Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
|Date of Web Publication||21-Apr-2017|
Department of Cytology and Gynecological Pathology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh
Source of Support: None, Conflict of Interest: None
BACKGROUND AND AIM: Vascular endothelial growth factor (VEGF) is a well-known pivotal regulator of tumor angiogenesis. Apart from endothelial cells, it is also expressed in nonendothelial cells, including tumor cells themselves. Hence the aim of this study was to investigate the autocrine effects of VEGF in hepatocellular carcinoma (HCC) -derived cell lines. MATERIALS AND METHODS: Two hepatocellular carcinoma cell lines (Hep3B and HepG2) were screened for expression of VEGF by quantitative real-time polymerase chain reaction (PCR) and its receptors VEGF-R1, VEGF-R2, and neuropilin-1 expression by reverse transcriptase-PCR, respectively. Furthermore, VEGF transcript was silenced by siRNA and the effects on cell migration, viability, and proliferation were determined by the wound healing assay, MTT assay, and propidium iodide staining, respectively. RESULTS: Both Hep3B and HepG2 cell lines expressed VEGF and all the three receptors at high levels. VEGF siRNA inhibited VEGF expression significantly in both Hep3B and HepG2 cell lines. Silencing of VEGF showed decreased migration in the Hep3B cell line. In both cell lines tested, there was decreased cell viability but no effect on cellular proliferation. CONCLUSION: Our data indicates that autocrine signaling of VEGF through its receptors exists in HCC cell lines, which has important implications for tumor invasion, metastasis, and for designing interventional strategies.
Keywords: Autocrine, hepatocellular carcinoma, small interfering RNA, Vascular endothelial growth factor
|How to cite this article:|
Sharma B, Srinivasan R, Chawla Y, Chakraborti A. Vascular endothelial growth factor: Evidence for autocrine signaling in hepatocellular carcinoma cell lines affecting invasion. Indian J Cancer 2016;53:542-7
|How to cite this URL:|
Sharma B, Srinivasan R, Chawla Y, Chakraborti A. Vascular endothelial growth factor: Evidence for autocrine signaling in hepatocellular carcinoma cell lines affecting invasion. Indian J Cancer [serial online] 2016 [cited 2020 Nov 29];53:542-7. Available from: https://www.indianjcancer.com/text.asp?2016/53/4/542/204765
| » Introduction|| |
Hepatocarcinogenesis is a complex multistep process involving the interaction of numerous factors, including growth factors., Vascular endothelial growth factor (VEGF) is a well-studied growth factor in hepatocellular carcinoma (HCC) and is established as a key regulator of angiogenesis.,, VEGF is a soluble, dimeric 46-kDa glycoprotein that binds to VEGF receptor 1 (VEGF-R1), VEGF-R2, and a third new VEGFR-labeled neuropilin-1 (NRP-1) present on the endothelial cells. Binding of VEGF secreted by tumor cells to its receptor present on endothelial cells results in subsequent activation of the signal transduction pathway, which promotes endothelial cell migration, proliferation, and prolongation of cell survival, ultimately leading to angiogenesis., Thus angiogenesis mediated by VEGF is essentially paracrine in nature., On the other hand, the autocrine effects of VEGF are less well known and have not been reported in HCC-derived cell lines. A few studies on breast cancer-derived cell lines have shown that VEGF functions not only in a paracrine manner on endothelial cells but also affects the neoplastic cells themselves in an autocrine manner, which ultimately leads to the invasion, migration, and survival of the cancerous cells in the hypoxic or hostile conditions., Hypoxia is a potent inducer of VEGF expression and is mediated by hypoxic inducible factor-1α (HIF-1α) in turn VEGF stimulates angiogenesis. However, the fact that most tumors contain hypoxic pockets implies that angiogenesis induced by VEGF in tumors is not sufficient to mitigate hypoxia.,,
Although hypoxia kills most normal cells and some tumor cells, it also provides a strong selective pressure for the survival of the cancerous cells. An understanding of the mechanisms that enable tumor cells to survive in hypoxia, therefore, is essential for deciphering the biology of cancer progression and for designing therapeutic interventions.,,, The possibility that VEGF also functions in an autocrine fashion on hepatocellular carcinoma cells themselves to stimulate signaling pathways that maintain their survival and its effect on cell proliferation and migration has not been studied previously. Thus the aim of this study was to explore the autocrine function of VEGF on HCC-derived (Hep3B and HepG2) cell lines.
| » Materials and Methods|| |
Human hepatocellular carcinoma cell lines (Hep3B and HepG2) were obtained from National Centre for Cell Science (NCCS), Pune, India. Hep3B and HepG2 cells were maintained in continuous culture at 37°C temperature and 5% CO2 in high glucose containing Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.5 μg/ml fungizone (Life Technologies, New York, USA). After every 48 h, depleted medium from the culture flask was replaced with fresh medium. When required for the assays confluent monolayer of Hep3B and HepG2 cells was washed twice with sterile phosphate-buffered saline (PBS). Thereafter, Trypsin –EDTA solution (250 mg Tripsin, 30 mg EDTA in 100 mL PBS) was added to deadhere the cells from the culture flask. Cells were deadhered, collected, and centrifuged at 200 ×g for 5 min. Pellet was washed with DMEM medium and resuspended in fresh medium with 10% FBS for experiments.
VEGF siRNA and scrambled siRNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were transfected into hepatic Hep3B and HepG2 cell lines using transfection reagent (Santa Cruz Biotechnology Inc.) and following manufacturer's instructions.
The expression of VEGF as well as its receptors VEGF-R1, VEGF-R2, and NRP-1 was evaluated by reverse transcriptase-polymerase chain reaction (PCR) using the primers and PCR conditions detailed in [Table 1]. The total RNA was extracted using the Trizol reagent (Ambion Inc., Life Technologies, New York, USA) and 1μg of this was converted to cDNA using a commercially available kit (Fermentas Life Sciences, Maryland, USA). These transcripts were evaluated in VEGF-specific siRNA-treated cells, control (scrambled) siRNA-treated cells, and from the control cells (not treated with anything).
|Table 1: Specific primer sequences and PCR conditions used for different genes|
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Quantitative real-time reverse transcriptase-PCR (qRT-PCR) was carried out for quantitating VEGF transcript expression using SYBR green master mix (Roche Diagnostic, Penzberg, Germany). VEGF standard curve was derived based on the method of Leong et al., 2007. β-actin transcript expression was used as an internal control. The number of copies of VEGF transcript in a test sample was calculated from the VEGF standard curve as described previously.
The effect of VEGF siRNA on cell migration was studied by the method of Liang et al., 2007. The basic steps involve creating a “wound” in a cultured cell monolayer with the help of pipette tip and capturing the images at the beginning and at regular intervals during cell migration to close the wound, and comparing the images to quantify the migration rate of the cells.
Effect of siRNA on cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) dye uptake method. This assay is based on the reduction of yellow MTT dye into insoluble purple formazan crystals by dehydrogenase activity in mitochondria, a conversion that occurs only in living cells. Briefly, 2 × 103 cells were seeded in 96 well plates and allowed to grow overnight. After 24 h of priming, cells were treated with VEGF siRNA for 24 h. Before treatment, medium were replaced with fresh medium. Each experiment was performed in triplicate. Four hours before the end of desired time interval, 20 μL of MTT solution (2.5 mg/mL) was added to each well. After 4 h, resulting formazan crystals were dissolved in 40 μL of lysis buffer (20% sodium dodecyl sulphate (SDS) dissolved in 50% each of dimethylformamide and double distilled water (ddH2O)). The developed color was read at 540 nm on an enzyme-linked immunosorbent assay reader. The relative viability was calculated as follows:
To determine the effect of VEGF siRNA on the cell cycle, cells (Hep3B and HepG2) were first treated with VEGF siRNA and incubated for 24 h. After 24 h, cells were washed, trypsinized, and harvested. Cells were pelleted by centrifugation at 800 ×g for 5 min at 4°C, washed with ice-chilled PBS, and then fixed with ice-chilled 70% ethanol (70 mL of absolute ethanol in 30 mL PBS). Cells were kept at 20°C overnight. After fixation, cells were centrifuged at 3000 rpm for 5 min, washed with PBS, and centrifuged again. Cell pellet was resuspended in 40 μL of phosphate –citrate buffer, consisting of 192 parts of 0.2 M Na2 HPO4 and 8 parts of 0.1 M citric acid (pH 7.8) at room temperature for at least 30 min. After 30 min, cells were pelleted by centrifugation at 1000 ×g for 5 min. The pellets were resuspended in 1 mL of PBS containing propidium iodide (PI) (50 μg/mL) and RNase (200 μg/mL) in polypropylene tubes and incubated at room temperature for 30 min. The tubes were then placed at 4°C in the dark before the flow cytometric analysis using a FACscan flow cytometer (Becton–Dickinson, New Jersey, USA). The PI signals were detected by the FL-2 photomultiplier tube. The percentage of cells in G1, S, and G2/M was evaluated.
All experiments were performed in triplicate. Significant differences were analyzed using Student's t test. Data were considered statistically significant if P< 0.05. Data are expressed as mean ± standard deviation.
| » Results|| |
The Hep3B and HepG2 cell lines expressed VEGF as well as all the 3 receptors of VEGF, namely, VEGFR1, VEGFR2, and NRP-1 [Figure 1].
|Figure 1: VEGF and VEGF receptors expression in Hep3B and HepG2 cell lines Lane 1: Molecular marker 100 bp; Lane 2: Hep3B cells; Lane 3: HepG2 cells. (VEGF: Vascular endothelial growth factor)|
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Treatment with VEGF-siRNA revealed a marked decrease in VEGF copy number as evaluated by qRT-PCR in the Hep3B cell line (25210.6 ± 129.6 copies/mL in control cells vs 121.6 ± 40.1 copies/mL in VEGF-siRNA–treated cells; P = 0.0001) and in the HepG2 cell line (19742.5 ± 219.9 copies/mL in control cells vs 601.7 ± 72.9 copies/mL; P =0.0001) [Figure 2]a and [Figure 2]b.
|Figure 2: (a and b) qRT-PCR analysis of VEGF in Hep3B and HepG2 cell *P < 0.05 vs control cells. (qRT-PCR: Quantitative real-time reverse transcriptase–polymerase chain reaction; VEGF: Vascular endothelial growth factor)|
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The wound healing assay could be performed only in the Hep3B cell line, which grows as a monolayer; it could not be performed on the HepG2 cell line as the cells grow in clumps as well as sheets. We observed that cell migration was retarded by VEGF-siRNA treatment to a significant extent [Figure 3]. At 0 h the mean wound width was 22.15 ± 2.01 mm, 23.92 ± 1.27 mm, and 24.73 ± 3.51 mm in control cells, control-siRNA-treated cells, and VEGF-siRNA–treated cells, respectively. At 72 h, the gap was fully covered by the cells in the control cells and control-siRNA–treated cells, whereas the wound width was 11 mm in VEGF-siRNA–treated cells [Figure 4].
|Figure 3: Effect of VEGF-siRNA on cell migration in Hep3B cell line-control cells (a, d, g, j);control siRNA cells (b, e, h, k); VEGF siRNA treated cells (c, f, i, l). Note the decreased migration in the VEGF-siRNA treated cells at 48 and 72 h. (VEGF: Vascular endothelial growth factor)|
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|Figure 4: Effect of VEGF-siRNA on cell migration of Hep3B cells at different time intervals|
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The MTT assay for cell viability showed reduced cell viability in both Hep3B and HepG2 cell lines by 30% and 27%, respectively [Figure 5]. On the other hand cell proliferation assessed by propidium iodide staining did not reveal any significant effect of VEGF-siRNA on cell proliferation [Figure 6].
|Figure 5: Effect of VEGF-siRNA on cell viability in Hep3B and HepG2 cells.*P < 0.05 vs control cells. (VEGF: Vascular endothelial growth factor)|
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|Figure 6: The analysis of propidium iodide staining in Hep3B cell line (a, b, c) and HepG2 cell line (d, e, f) M1: G0/G1 cells; M2:G2 cells; M3: S cells; M4: Apoptotic cells. Note that there is no effect of VEGF silencing on cell proliferation|
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| » Discussion|| |
Hepatocellular carcinoma has characteristics of invasiveness, such as recurrence, intrahepatic spread, and systemic metastasis, which has made it one of the most fatal malignancies., Tumor angiogenesis plays a role in the invasiveness and progression of HCC and is mainly driven by VEGF and its isoforms.,,
Overexpression and elevated serum levels of VEGF are known to correlate with different characteristics of HCC, such as tumor size, venous invasion, and poor patient prognosis.,, VEGF-mediated angiogenesis results chiefly from its paracrine effects on endothelial cells. However, in addition to endothelial cells, VEGF and VEGFRs are expressed on numerous nonendothelial cells, including tumor cells. This prompted us to evaluate the interaction of VEGF on tumor cells themselves. There are only a few studies on the role of VEGF as an autocrine growth factor in the different cancers, including breast,, malignant mesothelioma, and pancreatic cancers., In the present study the autocrine effect of VEGF was evaluated in vitro in two HCC-derived cell lines (Hep3B and HepG2). We hypothesised that knockdown of VEGF in these cell lines can affect cell invasion, migration, proliferation, and eventually the survival of the HCC cells.
Initially, we screened Hep3B and HepG2 cell lines for the expression of VEGF and VEGFRs and observed that both these cell lines express VEGF and its receptors VEGF-R1, VEGF-R2, as well as NRP-1. This made it a good system for evaluating the VEGF autocrine function. Furthermore, several studies reported the increased tissue expression of VEGF and VEGFRs (VEGFR1, VEGFR-2) in HCC patients and highlighted their prognostic roles for the disease.,,, We also found upregulated expression of VEGF and its receptors in our cohort of 67 HCC patients (unpublished data) when compared with chronic hepatitis and cirrhosis patients. Thus in HCC, VEGF operates in both paracrine and autocrine fashion. The existence of VEGF autocrine mitogenic loop in pancreatic cancer was provided by the study of von Marschall et al., who showed the expression of VEGF and its receptors in pancreatic cancer tissues but not in the ductal epithelial cells of normal pancreas and chronic pancreatitis. Further they demonstrated the mitogenic effect of VEGF signaling in Dan-G and AsPc-1 pancreatic cancer cell lines was blocked in dominant-negative KDR/flt-1 constructs.
We employed siRNA technology to inhibit VEGF in the two HCC cell lines. The present study showed significant inhibition of VEGF expression by VEGF-siRNA in both cell lines. A near-total VEGF inhibition was demonstrated by the qRT-PCR assay in both Hep3B and HepG2 cells. Our results are in accordance with Bachelder et al., who have shown that transfection of antisense oligonucleotide in two breast cancer cell lines (MDA-MB-231 and MDA-MB-435) resulted in 50% reduction of steady state levels of VEGF mRNA compared with the sense oligonucleotide transfected cells, as measured by qRT-PCR.
The most significant observation of this study was the inhibition of migration of the Hep3B cell line upon VEGF silencing as shown by wound healing assay. This inhibiting effect of VEGF on cell migration and invasion is supported by a previous study by Bachelder et al., wherein authors had shown a 70% decrease in matrigel invasion by the breast cancer cells upon VEGF silencing. They further demonstrated that this effect of VEGF was through NRP-1 and not the other receptors. It is therefore conceivable that VEGF silencing can halt tumor progression by inhibiting migration and invasion of tumor cells in addition to the inhibition of endothelial cells and angiogenesis, a key requisition for invasion. Furthermore, VEGF silencing leads to a reduction in cell viability. Masood et al. studied the effect of VEGF antisense oligos on VEGF expression and cell viability in different human cancer cell lines (KSY1, MS2, A375, 526, Hey, Hoc-7, and PanC3) and found that VEGF inhibition reduced cell viability significantly, which could be reversed by exogenously supplemented VEGF. Bachelder et al. have also reported 3-fold increase in cell death in VEGF antisense transfected cells relative to the sense transfected cells and finally concluded that VEGF autocrine function promotes survival of these cells.
The effects of VEGF silencing on cell proliferation is also interesting. There was no effect of VEGF silencing on cellular proliferation in both the cell lines studied. Our observation is in contrast to observation of von Marschall et al. and Strizzi et al. who have shown VEGF stimulation can induce cellular proliferation. On the other hand, several studies support our observation that VEGF does not affect cellular proliferation, although it inhibits both cell migration and cell viability. Morelli et al. treated different gastrointestinal cancer cell lines with cediranib (an inhibitor of VEGFR) and found that at a clinically relevant dose, it failed to have any effect on cell proliferation, whereas cell migration and cell viability were profoundly affected. These findings are in agreement with another report, which showed that VEGF-A and VEGF-B did not increase proliferation in colorectal cancer cell lines. In a differently designed study on pancreatic cell lines, Wey et al. have shown that activation of VEGFR-1 by VEGF promoted cell migration and cell invasion but not cellular proliferation. Thus the differential effects of VEGF on cellular proliferation and cell migration may be tissue dependent.
The clinical implications of this experimental work are that targeting VEGF in HCC will inhibit tumor cell invasion and migration in addition to angiogenic inhibition. However, targeting VEGF alone seems to be insufficient for controlling the growth of HCC as evidenced by this study. Hence VEGF inhibition as, for example, by sorafenib, has been used in addition to conventional chemotherapy in clinical trials with some success.,
In conclusion, our results suggest an even greater role for VEGF in liver cancer, which is not restricted merely to paracrine induction of angiogenesis through endothelial cells but also as an autocrine stimulator, which may facilitate migration and cell survival effecting tumor invasion.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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