|Year : 2016 | Volume
| Issue : 2 | Page : 640-644
Involvement of syk and VEGF-C in invasion of lung adenocarcinoma A549 cells
Qifeng Sun, Chuanliang Peng, Bo Cong, Yingtao Hao, Jiazhong Guo, Yunpeng Zhao, Xiaogang Zhao
Department of Thoracic Surgery, Second Hospital of Shandong University, Jinan, Shandong, China
|Date of Web Publication||25-Jul-2016|
Department of Thoracic Surgery, Second Hospital of Shandong University, 247 Beiyuan Street, Jinan, Shandong 250 033
Source of Support: This study was supported by a grant from the natural
science fund of Shandong Province (No:ZR2013HQ061)., Conflict of Interest: None
Background and Aims: Lung cancer has become one of the most dangerous malignant tumors in the world nowadays, whose pathogenesis is complex involving multi-genes and multi-elements. This study aims to investigate the values of spleen tyrosine kinase (Syk) and vascular endothelial growth factor-C (VEGF-C) in lymphangiogenesis and metastasis of lung adenocarcinoma A549 cells.
Materials and Methods: The pcDNA3.1-VEGF-C and pLNCX-syk were constructed and transfected into A549 cells. After cells with stable expression were sorted, the level of VEGF-C was tested by RT-PCR and immunohistochemistry and the mRNA of syk was tested by RT-PCR. The cell invasion assay was investigated by transwell chamber in vitro. Restriction enzyme digestion and gel electrophoresis demonstrated successful construction of the pcDNA3.1-VEGF-C.
Results: RT-PCR and immunohistochemistry revealed higher expression of VEGF-C in VEGFC-construct-transfected A549 cells than that in controls (P < 0.05). Successful construction of the pLNCX-syk was demonstrated by restriction enzyme electrophoresis and sequencing. RT-PCR revealed Syk expression higher in syk-construct-transfected cells than in controls (P < 0.05).
Conclusions: The results indicate a potential link between the upregulation of Syk and VEGF-C expression and lung adenocarcinoma.
Keywords: Lung adenocarcinoma, lymph node, metastasis, Syk, VEGF-C
|How to cite this article:|
Sun Q, Peng C, Cong B, Hao Y, Guo J, Zhao Y, Zhao X. Involvement of syk and VEGF-C in invasion of lung adenocarcinoma A549 cells. J Can Res Ther 2016;12:640-4
| > Introduction|| |
Lung cancer is one of the most common malignant tumor in the worldwide, and its morbidity and mortality are both on the high rank of all malignant tumors. Spleen tyrosine kinase (Syk) is widely expressed in hematopoietic cells, and is one of two members of the Syk family (Syk and ZAP-70). Syk plays an essential role in lymphocyte development and activation of immune cells.,,, As a non-receptor protein tyrosine kinase, Syk has recently been recognized as a new candidate tumor suppressor. Decrease or loss of Syk expression has been associated with a malignant phenotype and poor prognosis in a variety of cancers.,, Vascular endothelial growth factor (VEGF), now termed VEGF-A, belongs to the platelet-derived growth factor family and is the most potent inducer of angiogenesis and vessel permeability. VEGF-C is a ligand at the VEGF receptor VEGF-R3. This is a tyrosine kinase receptor that is expressed predominantly in the endothelium of lymphatic vessels. It has been shown in experimental studies that VEGF-C is the only factor known to cause lymphangiogenesis. In recent investigations, VEGF-C has been detected in several different cancers, and its levels in some studies seem to correlate with lymphangiogenesis and metastasis and patient prognosis.,,
This study aims to determine the precise role of Syk and VEGF-C in invasion of lung adenocarcinoma A549 cells.
| > Materials and Methods|| |
Recombinant pcDNA3.1-VEGF-C and pLNCX-Syk construct
Eukaryotic expression vector pcDNA3.1 and retroviral vector pLNCX2 were purchased from Clontech. E. coli DH5α was stored by the laboratory. pCR II-VEGF-C recombinant plasmid, restriction endonuclease EcoR I and Xho Iwere purchased from Shanghai Biological Engineering. Reverse transcription PCR Amplification Kit, DNA connection kit and restriction enzymes Cla I and Hind III were purchased from TaKaRa Biotechnology Dalian Co., Ltd.
PCR II-VEGF-C recombinant plasmid was transformed into competent E. coli DH5α cells. monoclonal was picked out and cultured overnight at 37o C in Luria-Bertani (LB) medium containing ampicillin. The plasmid was extracted according to the plasmid extraction kit manufacturer's instructions and double digested by EcoR I and Xho I. DNA fragments were confirmed by agarose gel electrophoresis containing 1% ethidium bromide (EtBr). The emergence of 1.1 kb band was believed to be the specific band of VEGF-C. The recovery of DNA was performed from gel containing VEGF-C gene.
Eukaryotic expression vector pcDNA3.1 was double digested by EcoR I and Xho Iat 37o C. The connection system was carried out in a final volume of 10 μl containing 5μl of the purpose of gene, 1 μl of recovered pcDNA3.1 double digested fragment, 1μl of T4 DNA ligase, 1 μl 10X T4 Buffer, and sterilized deionizated H2O was added to a final volume of 10 μl. The reaction mixture was incubated for 18 h in 16o C water bath. pLNCX-Syk was reconstructed following the same protocol [Figure 1].
|Figure 1: Construction of pcDNA3.1-VEGF-C and pLNCX-syk. Schematic representation of the construction of the recombinant adenovirus, VEGF-C and Syk gene, using AdMax system|
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Cell culture and transfection experiments
Non-small cell lung cancer A549 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Beijing) and cultured under recommended conditions. Briefly, cells were incubated in a humidified atmosphere of 5% CO2 in air at 37o C, and fed with the Dulbecco's Modified Eagle Medium (DMEM) (Sigma) containing 10% fetal bovine serum (FBS, Gibco-Invitrogen) and 1% of a stock solution containing 10 000 IU/mL penicillin–streptomycin solution. For routine passages, cultures were split 1:4 when they reached 80% to 90% confluence, generally every 3N4 days. All experiments were performed on exponentially growing cells.
A549 cells at 50–60% confluence were transfected with pcDNA3.1-VEGF-C in six-well culture plates according to the Lipofectamine ™ 2000 manufacturer's instructions (Gibco-Invitrogen, USA). Cells transfected with plasmid vector pcDNA3.1 were designed as controls. After G418 (400 g/ml) (Gibco-Invitrogen, USA) screening for 2 weeks, cells were transferred to a flask containing G418 (200 g/ml) for further amplification.
The pLNCX-Syk plasmid was transfected into A549 cells by Lipofectamine in the same way. 36 hours after transfection, Genectin (200 g/ml) (Gibco-Invitrogen, USA) was added into the culture medium for cell sorting. The positive clone selected by neomycin was named pLNCX-Syk and picked out for further culture 7 days later. A549 cells were continued to culture for 5 weeks in the medium containing Genecitin (100 g/ml). Cells transfected with plasmid vector were designed as controls and named pLNCX.
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis for Syk and VEGF-C
Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. The RNA concentration was measured at absorbance of 260 and 280 nm using UV4501S (Shanghai eGuide Analytical Instrument CO. China) by calculating OD260/OD280 ratio. To synthesize first-strand cDNA, 1 μg RNA was reverse transcribed with use of 100 U PrimerScript Reverse Transcriptase (TaKaRa Biotechnology) and 50 μM Oligo dt Primer (Takara Biotechnology) according to the manufacturer's protocol. GAPDH was used as an internal control to ensure that cDNA was complete and Taq was deactivated in each reaction. Primers used for Syk and GAPDH and primers for VEGF-C and GAPDH gene amplification were synthesized by Shanghai Biological Engineering [Table 1].
|Table 1: Summary of primer sequences and PCR product sizes used for RT-PCR|
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PCR was carried out in a final volume of 10 μl containing 10X LA-PCR buffer (TaKaRa Biotechnology), 2μM of each primer, 500μM of each dNTP, 50 ng of template cDNA, and 0.1 U of TaKaRa LA Taq (TaKaRa Biotechnology). The PCR conditions for amplification of Syk and hGAPDH were as follows: Initial denaturation at 95o C for 1 min, followed by 30 cycles at 55o C for 30 s, 72o C for 1 min, and 72o C for 5 min. The PCR conditions for amplification of VEGF-C were as follows: Initial denaturation at 95°C for 1 min, followed by 28 cycles at 55o C for 45 s, 72o C for 10 s, and 72o C for 5 min. The PCR conditions for GAPDH were initial denaturation at 95o C for 1 min, followed by 26 cycles at 53o C for 30 s, 72o C for 10 s, and 72o C for 5 min. The RT-PCR products were electrophoresed on a 2% agarose gel and visualized by staining with EtBr.
Immunohistochemical analysis for VEGF-C
The immunohistochemistry kit was from Zhongshan GoldenBridge Biotechnology Co. (China). VEGF-C expression was evaluated by immuno-histochemistry with rabbit polyclonal antibody against VEGF-C (R and D Systems, USA). All cell crawling slides were fixed in cold acetone for 20 min. After slides were washed in 0.5% Triton X-100 for 20 min, endogenous peroxidase was blocked by immersing the sections in 3% H2O2 for 30 min at room temperature. Sections were blocked in 10% goat serum and incubated with the primary antibody (anti-VEGF-C; 1:50) at 4°C overnight. The peroxidase-labeled polymer conjugated to goat anti-rabbit antibody was used to detect antigen-antibody reaction. After three additional washes, sections were incubated with polyvalent biotinylated goat anti-rabbit antibody for 1 hour at 37o C. Sections were then visualized with 3, 3-diaminobenzidine as a chromogen for 5 min and counterstained with Mayer's hematoxylin for 7 min. Immunoreactivity was visualized with use of a standard ABC method (DAB kit, Boster Biological Technology).
The number of stained cells per 200 cells was determined under an Olympus microscope (Olympus Optical Co., Ltd., Tokyo, Japan) in five visual fields at a magnification of 200X. The assessment was performed independently by two observers who were blinded to the cells' information, based on the same positive slides, and the final result was obtained by consensus in case of a discrepancy.
Transwell invasion assays
Matrigel was thawed at 4o C overnight and diluted to 1 mg/ml in serum free-cold cell culture media DMEM. 100 µl of the diluted matrigel was put into upper chamber of 24-well transwell and incubated at 37o C at least 4–5 hours for gelling. Transfected A549 cells were harvested by Trypsin/EDTA. After washing and and centrifugation, the cells were resuspended in media containing 1% FBS at a density of 2 × 106 cells/ml. 100 µl of the cell suspension were put onto the matrigel, and lower chamber of the transwell was filled with 600 µl DMEM containing 1% FBS as an adhesive subtrate. Then the cells were incubated at 37o C, 5% CO2 for 24 hours. After transwells from 24-well plates were removed, noninvaded cells on the top of the transwell were scraped off with a cotton swab. The invasive cells were fixed with 95% ethanol, stained with hematoxylin - eosin (HE) and counted under a light microscope.
All statistical analyses were performed using the SPSS13 software package. Students t - test and χ2 test were used to compare data from two groups of. Differences associated with a P value of 0.05 were considered statistically significant.
| > Results|| |
The eukaryotic expressing vector for VEGF-C and Syk were constructed successfully and transfected into A549 cells by lipofectamine TM2000 protocols. The result of agarose gel electrophoresis showed two fragments of 5.4 kb and 1.1 kb after the pcDNA3.1-VEGF-C were double digested by EcoRI and XhoI, which were consistent with the size of pcDNA3.1 and VEGF- C [Figure 2]a. After the pLNCX-syk were double digested by Cla I and Hind III, the image of 1.5% agarose gel electrophoresis showed that the size of bands matched with the size of designed [Figure 2]b.
|Figure 2: Restriction enzyme digestion and gel electrophoresis for pcDNA3.1 (a) VEGF-C and (b) pLNCX-Syk|
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After G418 selection, the transfected cells expressing VEGF-C stably were obtained. RT-PCR and immunohistochemistry revealed higher expression of VEGF-C in VEGFC-construct–transfected A549 cells than that in controls [Figure 3] and [Figure 4], P < 0.05 and P < 0.01 respectively]. Positive expression of Syk was conformed in A549 cells transfected with pLNCX-Syk by RT-PCR [Figure 5]. The results of Gene sequencing were consistent with the information from Gene Bank (AF015950) and there was no occurrence of mutation.
|Figure 3: Expression of VEGF-C in cells of different groups by RT-PCR. Expression of VEGF-C (412 bp) in pcDNA3.1-VEGF-C transfected A549 cells was lower than that in pcDNA3.1 transfected cells. GAPDH was 268 bp|
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|Figure 4: Expression of VEGF-C in A549 cells by immunohistochemistry. Immunohistochemistry realed higher expression of VEGF-C in pcDNA3.1-VEGF-C transfected A549 cells (A-2) than that in control group (A-1). (×200)|
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|Figure 5: Expression of Syk in A549 cells by RT-PCR. The result of RT-PCR confirmed positive expression of Syk (1909 bp) in the pLNCX-Syk A549 cells and negative in pLNCX transfected cells|
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After cultured in Transwell chamber for 24 hours, the number of invasive cells in pcDNA3.1-VEGF-C group was significantly higher than that in pcDNA 3.1 groups (50.29 ± 7.56 vs 20.14 ± 1.51, P < 0.01). The number of invasive cells in pLNCX-Syk group was 5 ± 1.73, which was significantly lower than that in the pLNCX control group (12.29 ± 2.75, P < 0.01) [Figure 6].
|Figure 6: Verification of invasive cells using Transwell chambers. Transwell assays verified more invasive cells in pcDNA3.1-VEGF-C transfected A549 cells and lower invasive cells in pLNCX transfected group, (×200)|
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| > Discussion|| |
Increasing evidence suggests that gene inactivation, particularly of tumor suppressors genes, plays a crucial role in the initiation, progression, and metastasis of tumors. Likewise, more data indicate that alterations in Syk expression and/or function factor in the development and behavior of various malignancies.,,,, Our data add to this trend, strongly suggesting that upregulation of Syk expression is associated with the invasion of A549 cells.
Experimental murine tumor models have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastasis. It is also an important prognostic factor because cancer spread via the lymphatics is a characteristic in the early stages of some tumors. In the present study, our results suggest VEGF-C is a potent enhancer of tumor invasion, leading to increased invasive lung carcinoma A549 cells. The secretion of VEGF-C and VEGF-D by some tumors could induce the activation of their receptor, VEGFR-3 on the vascular endothelium and thereby inducing the formation of new lymphatic vessels. VEGFR-3 is the primary receptor for VEGF-C and is largely restricted to lymphatic endothelial cells. However, increasing numbers of clinical and experimental findings strongly suggest that VEGFR-3 is also expressed in many cancer cells,, and, therefore, an autocrine mechanism in human tumors has been proposed. However, little is currently known about the factors that make tumors secret these lymphangiogenic factors.
Tyrosine kinase-targeting anticancer agents of the EGFR family are currently under investigation. A highlight is the development of quinazoline-derived agents that are specific ATP-competitors of EGFR tyrosine kinase, one representative of which is ZD1839 ('Iressa'). ZD1839 shows antiproliferative activity in various human cancer cell types in vitro. Tumor growth inhibition in vivo by ZD1839 is potentiated by combination with a variety of cytotoxic anticancer agents., However, Syk tyrosine kinase has no cross sensitivity for these kinds of agents because they selectively inhibit only EGFR activity.
Matrigel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans) but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumor cells into Matrigel has been used to characterize involvement of extracellular matrix (ECM) receptors and matrix degrading enzymes which play roles in tumor progression. In this study, Transwell and Matrigel were used to investigated the invasive ability of A549 cells. However, further studies with large numbers of clinical samples are needed to determine with more certainty the influence of Syk and VEGF-C expression on non-small cell lung carcinoma development and/or prognosis. And the association between the two factors is still indefinite. These findings may provide an important new therapeutic target for the treatment of lung carcinoma.
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