http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, 2014; 30(6): 461–465 ! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2014.898054
VEGF AND CERVICAL CANCER
Vascular endothelial growth factor C enhances cervical cancer cell invasiveness via upregulation of galectin-3 protein Junxiu Liu1, Yang Cheng2, Mian He1, and Shuzhong Yao1 1
Department of Gynecology and Obstetrics, The First Affiliated Hospital of Sun Yat-Sen University, Guangdong, Guangzhou, China and Department of Gynecology and Obstetrics, Guangzhou First Municipal People’s Hospital, Guangdong, Guangzhou, China
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Abstract
Keywords
Vascular endothelial growth factor C (VEGF-C) promotes cervical cancer metastasis, while the detailed mechanism remains obscure. Recent evidence shows that galectin-3 (Gal-3), a glycan binding protein, interacts with the VEGF receptors and reinforces their signal transduction. In this study, we investigated the role of Gal-3 in VEGF-C-induced cervical cancer cell invasion. On cervical carcinoma cell line SiHa cells, silencing of Gal-3 expression with specific siRNA largely impaired VEGF-C-enhanced cell invasion. Treatment with VEGF-C for 12–48 h enhanced Gal-3 protein expression, which was inhibited by the addition of NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC). Moreover, the silencing of NF-kB subunit p65 expression with specific siRNA attenuated VEGF-C-enhanced Gal-3 expression, suggesting that NF-kB is the key intermediate. Under VEGF-C stimulation, an enhanced interaction between VEGF receptor-3 (VEGF-R3) and Gal-3 was found, which may possibly lead to VEGF-R3 activation since exogenous Gal-3 induced VEGF-R3 phosphorylation in a dose- and time-dependent manner. In conclusion, our findings implied that VEGF-C enhanced cervical cancer invasiveness via upregulation of Gal-3 protein through NF-kB pathway, which may shed light on potential therapeutic strategies for cervical cancer therapy.
Cervical cancer, galectin-3, invasiveness, VEGF-C
Introduction Cervical cancer is the third most common malignancy in women worldwide and it remains the leading cause of cancer-related death, especially for women in developing countries [1]. The root reason for cervical cancer mortality is the spread of cancer cells [2]. Previously we have demonstrated that vascular endothelial growth factor C (VEGF-C) directly drives cervical cancer cell migration and invasion [3]. However, the molecular mechanism remains largely unknown. VEGF-C exerts biological effects mainly through VEGF receptor-3 (VEGF-R3) [4,5]. When it binds to VEGF-C, VEGF-R3 is activated to recruit intracellular signaling cascades. Subsequently the receptor internalizes from plasma membrane to endosome, resulting in the attenuation of the signaling [6]. Recently it has been reported that galectin-3 (Gal-3), a glycan binding protein, interacts with the VEGF-R2 and reinforces its signal transduction in endothelial cells [7]. Moreover, Gal-3 is found to be positively related to cervical cancer progression [8]. These works hinted the possibility that Gal-3 may be implicated in VEGF-R3 signaling in cervical cancer. To test this hypothesis, we investigated the role of Gal-3 in VEGF-C-promoted cer-
Address for correspondence: Prof. Shuzhong Yao, Department of Gynecology and Obstetrics, The First Affiliated Hospital of Sun Yat-Sen University, Guangdong, Guangzhou 510089, China. E-mail: [email protected]
History Received 6 November 2013 Revised 21 January 2014 Accepted 21 February 2014 Published online 20 March 2014
vical cancer SiHa cells invasion and explored the underlying mechanism.
Materials and methods Cell cultures and treatments Cervical carcinoma cell line SiHa cells were obtained from China Center for Type Culture Collection [3] and were incubated in RPMI 1640 medium containing 10% fetal calf serum (FCS), L-glutamine and penicillin streptomycin under a 5% CO2 atmosphere at 37 C. Whenever an inhibitor was used, the compound was added 1 h before starting the treatment. Recombinant human VEGF-C wild type (2179-VC-025) was purchased from R&D Systems (Minneapolis, MN). Pyrrolidine dithiocarbamate (PDTC) and mithramycin A (MA) were purchased from Sigma-Aldrich (Saint-Louis, MO). SR 11302 [(E,E,Z,E)3-Methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1yl)-2,4,6,8-nonatetraenoic acid] was obtained from Tocris Bioscience (Bristol, UK). Immunoblottings Cell lysates were separated by SDS-PAGE. Antibodies used were: Gal-3 (sc-20157, Santa Cruz, CA), Phoshop-VEGFR3 (CB5793, Cell Applications, Inc., Wiltshire, UK), VEGF-R3 (CB5792, Cell Applications, Inc.). Primary and secondary antibodies were incubated with the membranes with a standard technique. Immunodetection was accomplished using enhanced chemiluminescence. Densitometry values were adjusted to b-actin or VEGFR3 intensity, then normalized to expression from the control sample.
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Immunoprecipitation assay SiHa cells were harvested in 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 1 mM Na3VO4, 1 mM NaF and 1 mM PMSF. Equal amounts (700 mg) of cell lysates were incubated with 1 mg of precipitating anti-Gal-3 antibody (sc-20157, Santa Cruz, CA) for 1 hour at 4 C under gentle agitation. 25 mL of protein A-agarose slurry (Cell signaling) were added, and the samples were rolled at 4 C for another hour. The samples were then pelleted, washed and resuspended in 50 mL of 2X Laemmli buffer for immunoblotting by using anti-VEGF-R3 antibody or anti-phosphorylated VEGF-R3 antibody.
SiHa cells (40% confluent) were serum-starved for 1 h followed by incubation with 100 nM target siRNA or control siRNA for 6 h in serum-free media. Specific siRNA used are: Gal-3 siRNA (sc-155994, Santa Cruz, CA), NF-kB p65 siRNA (sc-29410, Santa Cruz, CA) and scrambled siRNA (sc-37007, Santa Cruz, CA). The serum-containing media was then added (10% serum final concentration) for 42 h before experiments and/or functional assays were conducted. Target protein silencing was assessed through protein analysis up to 48 h after transfection. Cell invasion assays As we previously described [3], cell invasion were assayed following the standard method using the BD BioCoatÔ Growth Factor Reduced (GFR) MatrigelÔ Invasion Chamber (BD Bioscience, San Jose, CA, USA). The invading cells were observed under the microscope at 100 magnification. Cells were counted in the central field of triplicate membranes. The invasion index was calculated as the % invasion test cell/% invasion control cell. Statistical analysis All values are expressed as mean ± SD. Statistical differences between mean values were determined by ANOVA, followed Figure 1. Silencing of Gal-3 expression impairs VEGF-C-induced SiHa cell invasion. (A) and (B) Cells were treated with VEGF-C (100 ng/mL) for 48 h, with the transfection of scrambled or specific Gal-3 siRNA. Invading cells were counted in three different central fields of triplicate membranes and invasion indexes are shown. **p50.01 versus control in scrambled siRNA group, #p50.01 versus VEGF-C in scrambles siRNA group. (C) SiHa cells were transfected with scrambled siRNA or Gal-3 targeted siRNA for 48 h. After that the level of Gal-3 expression was detected by Western blot as indicated. b-Actin was used as the loading control.
Results Silencing of Gal-3 expression impairs VEGF-C-induced SiHa cell invasion Previously we have found that VEGF-C promoted SiHa cell invasion [3]. In agreement with this, stimulation with VEGF-C (100 ng/mL) for 48 h increased invading cell number into the matrix (Figure 1A and B). However, when Gal-3 expression was silenced by specific siRNA (Figure 1C), VEGF-C largely lost its ability to promote SiHa cell invasion, as indicated by the value of invasion indexes (Figure 1A and B). VEGF-C enhances Gal-3 protein expression via NF-kB pathway Next we investigated the effect of VEGF-C on Gal-3 expression. Indeed, VEGF-C (100 ng/mL) significantly enhanced Gal-3 protein expression from 12–48 h (Figure 2A). This effect was inhibited by NF-kB inhibitor PDTC (20 mM), but not by AP-1(activator protein 1) inhibitor SR 11302 (SR – 1 mM) or Sp1 binding inhibitor mithramycin A (MA – 100 nM) (Figure 2B), suggesting a crucial role of NF-kB in this effect. To confirm the exact role of NF-kB, we silenced NF-kB subunit p65 expression by using specific siRNA (Figure 2C). As a result, the silencing of p65 markedly impaired the effect of VEGF-C on Gal-3 expression (Figure 2D). VEGF-C increases the interaction between VEGF-R3 and Gal-3 protein Immunoprecipitation assay was performed and it was found that in the resting state, Gal-3 interacted with VEGF-R3. After stimulation with VEGF-C (100 ng/mL) for 10 min, an enhanced interaction between these two proteins was observed (Figure 3A). Moreover, VEGF-C (100 ng/mL) resulted in the enhanced interaction between Gal-3 and phosphorylated VEGF-R3 (Figure 3B).
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Figure 2. VEGF-C enhances Gal-3 protein expression via NF-kB pathway. (A) Cells were treated with VEGF-C (100 ng/mL) for indicated time and the level of Gal-3 expression was detected. *p50.05 versus control, **p50.01 versus control. (B) Cells were treated with VEGF-C (100 ng/mL) for 48 h, in the presence or absence of AP-1 inhibitor SR 11302 (SR – 1 mM) or Sp1 binding inhibitor mithramycin A (MA – 100 nM) or NF-kB inhibitor PDTC (20 mM). The level of Gal-3 expression was detected. **p50.01 versus control, #p50.01 versus VEGF-C. (C) SiHa cells were transfected with scrambled siRNA or p65 targeted siRNA for 48 h. After that the level of p65 expression was detected. (D) Cells were treated with VEGF-C (100 ng/mL) for 48 h, with the transfection of scrambled siRNA or specific p65 siRNA. Gal-3 expression was detected. **p50.01 versus control in scrambled group, #p50.01 versus VEGF-C in scrambled group. All these experiments were performed in triplicates and representative images are shown.
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Figure 3. VEGF-C increases the interaction between VEGF-R3 and Gal-3 protein and Gal-3 induces VEGF-R3 phosphorylation in a dose- and timedependent manner. (A–B) SiHa cells were treated for 10 min with VEGF-C (100 ng/mL) and protein extracts were immunoprecipitated with an antibody versus Gal-3 and the IPs were assayed for co-immunoprecipitation of VEGF-R3 (A) or phosphorylated VEGF-R3 (P-VEGF-R3) (B) Gal-3 as indicated. Cell extract (30 mg) was used as input. Normal mouse IgG was used as the control antibodies, respectively. (C-D) Cells were treated with different doses of Gal-3 (C) or treated with Gal-3 for different time (D) as indicated and the level of phosphorylated VEGF-R3 (P-VEGF-R3) expression was detected by western blot as indicated. VEGF-R3 was used as the loading control. *p50.05 versus control, **p50.01 versus control. All these experiments were performed in triplicates and representative images are shown.
Gal-3 induces VEGF-R3 phosphorylation in a dose- and time-dependent manner To examine the role of Gal-3 on VEGF-R3 activation, we treated SiHa cells with exogenous Gal-3 and observed its effect on VEGF-R3 phosphorylation. Indeed, incubation with Gal-3 (10 mg/ mL) resulted in time-dependent phosphorylation of VEGF-R3 (Figure 3C). Moreover, treatment with different doses of Gal-3 (2, 5, 10 mg/mL) for 10 min all increased VEGF-R3 phosphorylation (Figure 3D).
Discussion Tumor metastasis is the leading cause of death in patients with cervical cancer, while the molecular mechanism remains unclear. VEGF-C plays critical role in a most of aggressive tumors, which is mainly attributed to its ability to induce lymphangiogenesis. However, VEGF-R3, the specific receptor of VEGF-C, is also expressed in a variety of human tumor cells [4], indicating that
VEGF-C may have direct impact on tumor cells. In support of this, we found that VEGF-C drove cervical cancer cell invasion into matrices, consistent with effects of VEGF-C in other types of cancer cells [9–11]. VEGF-C exerts its biological effects mainly through VEGFR3. It has been reported that Gal-3 interacts with the VEGF-C receptor and reinforces its signal transduction in endothelial cells [7]. In our study, we found that VEGF-C largely lost its ability to promote SiHa cell invasion when Gal-3 expression was silenced, suggesting a crucial role of Gal-3 in VEGF-C-promoted cervical cancer cell invasiveness. This is consistent with other studies showing that Gal-3 overexpression is associated with increased invasiveness of melanoma, ovarian, thyroid and colorectal cancer cells [12–15]. Gal-3 expression is modulated by a variety of extra- and intracellular stimulators [16–18]. In this work, we found that VEGF-C increased Gal-3 protein expression, indicating that Gal-3 is the target protein of VEGF-C. In the human Gal-3 gene promoter
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DOI: 10.3109/09513590.2014.898054
area, there are binding sites for transcriptional factors including Sp1, AP-1 and NF-kB [18–20], which are well-known as the intermediates of VEGF-C signaling [21, 22]. Therefore, we investigated the role of these transcription factors and found that the NF-kB inhibitor, while not the inhibitor of Sp1 or AP-1, largely inhibited VEGF-C-enhanced Gal-3 expression, implying that NF-kB is the key pathway. This is further confirmed by the fact that silencing of p65 protein, the functional subunit of NF-kB, markedly impaired VEGF-C-enhanced Gal-3 expression. In this study, we found that at resting state, there is an interaction between VEGF-R3 and Gal-3 in cervical SiHa cells, while treatment with VEGF-C enhanced the interaction not only between VEGF-R3, but also between phosphorylated VEGF-R3 with Gal-3, implying that Gal-3 may be important in retaining active VEGF-R3 at the plasma membrane. Moreover, we found that exogenous Gal-3 activated VEGF-R3 in a timeand dose-dependent manner, implying that Gal-3 functions not only as the detainer for VEGF-R3 in the plasma membrane, but also as the activator of VEGF-R3. Therefore, a positive feedback loop may exist between VEGF-C/VEGF-R3 signaling and Gal-3. Galectins are mammalian b-galactoside binding proteins characterized by highly conserved carbohydrate recognition domains (CRDs). Currently 15 galectin members have been identified [23]. Although we found that Gal-3 plays important role in cervical cancer metastasis, we do not exclude the roles of other galectin members in this effect. For example, it was recently reported that high galectin 1 expression was significantly correlated with depth of invasion in cervical lesions and lymph node metastasis of cervical cancer [24]. Therefore, further efforts are needed to elucidate their exact role in tumor metastasis. In conclusion, our work implied that VEGF-C enhances Gal-3 protein expression through NF-kB pathway. The increased Gal-3 protein interacts and activates VEGF-R3, leading to increased invasiveness of cervical cancer SiHa cells. These findings may shed light on potential therapeutic strategies for cervical cancer therapy.
Declaration of interest The authors of this manuscript have nothing to disclose. The Project is supported by Guangdong Natural Science Foundation (To Yang Cheng, NO:S2013010016548).
References 1. Sherman SM, Moss E, Redman CW. The invasive cervical cancer review: psychological issues surrounding disclosure. Cytopathology 2013;24:77–80. 2. Pierce Campbell CM, Menezes LJ, Paskett ED, Giuliano AR. Prevention of invasive cervical cancer in the United States: past, present, and future. Cancer Epidemiol Biomarkers Prev 2012;21: 1402–8. 3. He M, Cheng Y, Li W, et al. Vascular endothelial growth factor C promotes cervical cancer metastasis via up-regulation and activation of RhoA/ROCK-2/moesin cascade. BMC Cancer 2010;10: 170–9.
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4. Su JL, Yen CJ, Chen PS, et al. The role of the VEGF-C/VEGFR-3 axis in cancer progression. Br J Cancer 2007;96:541–5. 5. Van Trappen PO, Steele D, Lowe DG, et al. Expression of vascular endothelial growth factor (VEGF)-C and VEGF-D, and their receptor VEGFR-3, during different stages of cervical carcinogenesis. J Pathol 2003;201:544–54. 6. Karpanen T, Heckman CA, Keskitalo S, et al. Functional interaction of VEGF-C and VEGF-D with neuropilin receptors. FASEB J 2006; 20:1462–72. 7. Markowska AI, Jefferies KC, Panjwani N. Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells. J Biol Chem 2011;286:29913–21. 8. Balasubramanian K, Vasudevamurthy R, Venkateshaiah SU, et al. Galectin-3 in urine of cancer patients: stage and tissue specificity. J Cancer Res Clin Oncol 2009;135:355–63. 9. Su JL, Yang PC, Shih JY, et al. The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells. Cancer Cell 2006;9:209–23. 10. Marchio S, Primo L, Pagano M, et al. Vascular endothelial growth factor-C stimulates the migration and proliferation of Kaposi’s sarcoma cells. J Biol Chem 1999;274:27617–22. 11. Zhao R, Liu XQ, Wu XP, et al. Vascular endothelial growth factor (VEGF) enhances gastric carcinoma invasiveness via integrin alpha(v)beta6. Cancer Lett 2010;287:150–6. 12. Levy R, Grafi-Cohen M, Kraiem Z, Kloog Y. Galectin-3 promotes chronic activation of K-Ras and differentiation block in malignant thyroid carcinomas. Mol Cancer Ther 2010;9:2208–19. 13. Kim MK, Sung CO, Do IG, et al. Overexpression of Galectin-3 and its clinical significance in ovarian carcinoma. Int J Clin Oncol 2011; 16:352–8. 14. Braeuer RR, Zigler M, Kamiya T, et al. Galectin-3 contributes to melanoma growth and metastasis via regulation of NFAT1 and autotaxin. Cancer Res 2012;72:5757–66. 15. Barrow H, Rhodes JM, Yu LG. The role of galectins in colorectal cancer progression. Int J Cancer 2011;129:1–8. 16. Kim K, Mayer EP, Nachtigal M. Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins. Biochim Biophys Acta 2003;1641:13–23. 17. Lavra L, Rinaldo C, Ulivieri A, et al. The loss of the p53 activator HIPK2 is responsible for galectin-3 overexpression in well differentiated thyroid carcinomas. PLoS One 2011;6:e20665. 18. Fogel S, Guittaut M, Legrand A, et al. The tat protein of HIV-1 induces galectin-3 expression. Glycobiology 1999;9:383–7. 19. Wang YG, Kim SJ, Baek JH, et al. Galectin-3 increases the motility of mouse melanoma cells by regulating matrix metalloproteinase-1 expression. Exp Mol Med 2012;44:387–93. 20. Noma N, Simizu S, Kambayashi Y, et al. Involvement of NF-kappaB-mediated expression of galectin-3-binding protein in TNF-alpha-induced breast cancer cell adhesion. Oncol Rep 2012;27: 2080–4. 21. Chien MH, Ku CC, Johansson G, et al. Vascular endothelial growth factor-C (VEGF-C) promotes angiogenesis by induction of COX-2 in leukemic cells via the VEGF-R3/JNK/AP-1 pathway. Carcinogenesis 2009;30:2005–13. 22. Tsuka N, Motokawa M, Kaku M, et al. Fms-like tyrosine kinase (Flt)-4 signaling participates in osteoclast differentiation in osteopetrotic (op/op) mice. Biomed Res 2009;30:31–7. 23. Nakahara S, Raz A. Regulation of cancer-related gene expression by galectin-3 and the molecular mechanism of its nuclear import pathway. Cancer Metastasis Rev 2007;26:605–10. 24. Kim HJ, Do IG, Jeon HK, et al. Galectin 1 expression is associated with tumor invasion and metastasis in stage IB to IIA cervical cancer. Hum Pathol 2013;44:62–8.
VEGF AND CERVICAL CANCER
Vascular endothelial growth factor C enhances cervical cancer cell invasiveness via upregulation of galectin-3 protein Junxiu Liu1, Yang Cheng2, Mian He1, and Shuzhong Yao1 1
Department of Gynecology and Obstetrics, The First Affiliated Hospital of Sun Yat-Sen University, Guangdong, Guangzhou, China and Department of Gynecology and Obstetrics, Guangzhou First Municipal People’s Hospital, Guangdong, Guangzhou, China
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 02/08/15 For personal use only.
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Abstract
Keywords
Vascular endothelial growth factor C (VEGF-C) promotes cervical cancer metastasis, while the detailed mechanism remains obscure. Recent evidence shows that galectin-3 (Gal-3), a glycan binding protein, interacts with the VEGF receptors and reinforces their signal transduction. In this study, we investigated the role of Gal-3 in VEGF-C-induced cervical cancer cell invasion. On cervical carcinoma cell line SiHa cells, silencing of Gal-3 expression with specific siRNA largely impaired VEGF-C-enhanced cell invasion. Treatment with VEGF-C for 12–48 h enhanced Gal-3 protein expression, which was inhibited by the addition of NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC). Moreover, the silencing of NF-kB subunit p65 expression with specific siRNA attenuated VEGF-C-enhanced Gal-3 expression, suggesting that NF-kB is the key intermediate. Under VEGF-C stimulation, an enhanced interaction between VEGF receptor-3 (VEGF-R3) and Gal-3 was found, which may possibly lead to VEGF-R3 activation since exogenous Gal-3 induced VEGF-R3 phosphorylation in a dose- and time-dependent manner. In conclusion, our findings implied that VEGF-C enhanced cervical cancer invasiveness via upregulation of Gal-3 protein through NF-kB pathway, which may shed light on potential therapeutic strategies for cervical cancer therapy.
Cervical cancer, galectin-3, invasiveness, VEGF-C
Introduction Cervical cancer is the third most common malignancy in women worldwide and it remains the leading cause of cancer-related death, especially for women in developing countries [1]. The root reason for cervical cancer mortality is the spread of cancer cells [2]. Previously we have demonstrated that vascular endothelial growth factor C (VEGF-C) directly drives cervical cancer cell migration and invasion [3]. However, the molecular mechanism remains largely unknown. VEGF-C exerts biological effects mainly through VEGF receptor-3 (VEGF-R3) [4,5]. When it binds to VEGF-C, VEGF-R3 is activated to recruit intracellular signaling cascades. Subsequently the receptor internalizes from plasma membrane to endosome, resulting in the attenuation of the signaling [6]. Recently it has been reported that galectin-3 (Gal-3), a glycan binding protein, interacts with the VEGF-R2 and reinforces its signal transduction in endothelial cells [7]. Moreover, Gal-3 is found to be positively related to cervical cancer progression [8]. These works hinted the possibility that Gal-3 may be implicated in VEGF-R3 signaling in cervical cancer. To test this hypothesis, we investigated the role of Gal-3 in VEGF-C-promoted cer-
Address for correspondence: Prof. Shuzhong Yao, Department of Gynecology and Obstetrics, The First Affiliated Hospital of Sun Yat-Sen University, Guangdong, Guangzhou 510089, China. E-mail: [email protected]
History Received 6 November 2013 Revised 21 January 2014 Accepted 21 February 2014 Published online 20 March 2014
vical cancer SiHa cells invasion and explored the underlying mechanism.
Materials and methods Cell cultures and treatments Cervical carcinoma cell line SiHa cells were obtained from China Center for Type Culture Collection [3] and were incubated in RPMI 1640 medium containing 10% fetal calf serum (FCS), L-glutamine and penicillin streptomycin under a 5% CO2 atmosphere at 37 C. Whenever an inhibitor was used, the compound was added 1 h before starting the treatment. Recombinant human VEGF-C wild type (2179-VC-025) was purchased from R&D Systems (Minneapolis, MN). Pyrrolidine dithiocarbamate (PDTC) and mithramycin A (MA) were purchased from Sigma-Aldrich (Saint-Louis, MO). SR 11302 [(E,E,Z,E)3-Methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1yl)-2,4,6,8-nonatetraenoic acid] was obtained from Tocris Bioscience (Bristol, UK). Immunoblottings Cell lysates were separated by SDS-PAGE. Antibodies used were: Gal-3 (sc-20157, Santa Cruz, CA), Phoshop-VEGFR3 (CB5793, Cell Applications, Inc., Wiltshire, UK), VEGF-R3 (CB5792, Cell Applications, Inc.). Primary and secondary antibodies were incubated with the membranes with a standard technique. Immunodetection was accomplished using enhanced chemiluminescence. Densitometry values were adjusted to b-actin or VEGFR3 intensity, then normalized to expression from the control sample.
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Immunoprecipitation assay SiHa cells were harvested in 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 1 mM Na3VO4, 1 mM NaF and 1 mM PMSF. Equal amounts (700 mg) of cell lysates were incubated with 1 mg of precipitating anti-Gal-3 antibody (sc-20157, Santa Cruz, CA) for 1 hour at 4 C under gentle agitation. 25 mL of protein A-agarose slurry (Cell signaling) were added, and the samples were rolled at 4 C for another hour. The samples were then pelleted, washed and resuspended in 50 mL of 2X Laemmli buffer for immunoblotting by using anti-VEGF-R3 antibody or anti-phosphorylated VEGF-R3 antibody.
SiHa cells (40% confluent) were serum-starved for 1 h followed by incubation with 100 nM target siRNA or control siRNA for 6 h in serum-free media. Specific siRNA used are: Gal-3 siRNA (sc-155994, Santa Cruz, CA), NF-kB p65 siRNA (sc-29410, Santa Cruz, CA) and scrambled siRNA (sc-37007, Santa Cruz, CA). The serum-containing media was then added (10% serum final concentration) for 42 h before experiments and/or functional assays were conducted. Target protein silencing was assessed through protein analysis up to 48 h after transfection. Cell invasion assays As we previously described [3], cell invasion were assayed following the standard method using the BD BioCoatÔ Growth Factor Reduced (GFR) MatrigelÔ Invasion Chamber (BD Bioscience, San Jose, CA, USA). The invading cells were observed under the microscope at 100 magnification. Cells were counted in the central field of triplicate membranes. The invasion index was calculated as the % invasion test cell/% invasion control cell. Statistical analysis All values are expressed as mean ± SD. Statistical differences between mean values were determined by ANOVA, followed Figure 1. Silencing of Gal-3 expression impairs VEGF-C-induced SiHa cell invasion. (A) and (B) Cells were treated with VEGF-C (100 ng/mL) for 48 h, with the transfection of scrambled or specific Gal-3 siRNA. Invading cells were counted in three different central fields of triplicate membranes and invasion indexes are shown. **p50.01 versus control in scrambled siRNA group, #p50.01 versus VEGF-C in scrambles siRNA group. (C) SiHa cells were transfected with scrambled siRNA or Gal-3 targeted siRNA for 48 h. After that the level of Gal-3 expression was detected by Western blot as indicated. b-Actin was used as the loading control.
Results Silencing of Gal-3 expression impairs VEGF-C-induced SiHa cell invasion Previously we have found that VEGF-C promoted SiHa cell invasion [3]. In agreement with this, stimulation with VEGF-C (100 ng/mL) for 48 h increased invading cell number into the matrix (Figure 1A and B). However, when Gal-3 expression was silenced by specific siRNA (Figure 1C), VEGF-C largely lost its ability to promote SiHa cell invasion, as indicated by the value of invasion indexes (Figure 1A and B). VEGF-C enhances Gal-3 protein expression via NF-kB pathway Next we investigated the effect of VEGF-C on Gal-3 expression. Indeed, VEGF-C (100 ng/mL) significantly enhanced Gal-3 protein expression from 12–48 h (Figure 2A). This effect was inhibited by NF-kB inhibitor PDTC (20 mM), but not by AP-1(activator protein 1) inhibitor SR 11302 (SR – 1 mM) or Sp1 binding inhibitor mithramycin A (MA – 100 nM) (Figure 2B), suggesting a crucial role of NF-kB in this effect. To confirm the exact role of NF-kB, we silenced NF-kB subunit p65 expression by using specific siRNA (Figure 2C). As a result, the silencing of p65 markedly impaired the effect of VEGF-C on Gal-3 expression (Figure 2D). VEGF-C increases the interaction between VEGF-R3 and Gal-3 protein Immunoprecipitation assay was performed and it was found that in the resting state, Gal-3 interacted with VEGF-R3. After stimulation with VEGF-C (100 ng/mL) for 10 min, an enhanced interaction between these two proteins was observed (Figure 3A). Moreover, VEGF-C (100 ng/mL) resulted in the enhanced interaction between Gal-3 and phosphorylated VEGF-R3 (Figure 3B).
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VEGF-C and cervical cancer
DOI: 10.3109/09513590.2014.898054
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Figure 2. VEGF-C enhances Gal-3 protein expression via NF-kB pathway. (A) Cells were treated with VEGF-C (100 ng/mL) for indicated time and the level of Gal-3 expression was detected. *p50.05 versus control, **p50.01 versus control. (B) Cells were treated with VEGF-C (100 ng/mL) for 48 h, in the presence or absence of AP-1 inhibitor SR 11302 (SR – 1 mM) or Sp1 binding inhibitor mithramycin A (MA – 100 nM) or NF-kB inhibitor PDTC (20 mM). The level of Gal-3 expression was detected. **p50.01 versus control, #p50.01 versus VEGF-C. (C) SiHa cells were transfected with scrambled siRNA or p65 targeted siRNA for 48 h. After that the level of p65 expression was detected. (D) Cells were treated with VEGF-C (100 ng/mL) for 48 h, with the transfection of scrambled siRNA or specific p65 siRNA. Gal-3 expression was detected. **p50.01 versus control in scrambled group, #p50.01 versus VEGF-C in scrambled group. All these experiments were performed in triplicates and representative images are shown.
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IP: Gal-3 IB: P-VEGF-R3
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Figure 3. VEGF-C increases the interaction between VEGF-R3 and Gal-3 protein and Gal-3 induces VEGF-R3 phosphorylation in a dose- and timedependent manner. (A–B) SiHa cells were treated for 10 min with VEGF-C (100 ng/mL) and protein extracts were immunoprecipitated with an antibody versus Gal-3 and the IPs were assayed for co-immunoprecipitation of VEGF-R3 (A) or phosphorylated VEGF-R3 (P-VEGF-R3) (B) Gal-3 as indicated. Cell extract (30 mg) was used as input. Normal mouse IgG was used as the control antibodies, respectively. (C-D) Cells were treated with different doses of Gal-3 (C) or treated with Gal-3 for different time (D) as indicated and the level of phosphorylated VEGF-R3 (P-VEGF-R3) expression was detected by western blot as indicated. VEGF-R3 was used as the loading control. *p50.05 versus control, **p50.01 versus control. All these experiments were performed in triplicates and representative images are shown.
Gal-3 induces VEGF-R3 phosphorylation in a dose- and time-dependent manner To examine the role of Gal-3 on VEGF-R3 activation, we treated SiHa cells with exogenous Gal-3 and observed its effect on VEGF-R3 phosphorylation. Indeed, incubation with Gal-3 (10 mg/ mL) resulted in time-dependent phosphorylation of VEGF-R3 (Figure 3C). Moreover, treatment with different doses of Gal-3 (2, 5, 10 mg/mL) for 10 min all increased VEGF-R3 phosphorylation (Figure 3D).
Discussion Tumor metastasis is the leading cause of death in patients with cervical cancer, while the molecular mechanism remains unclear. VEGF-C plays critical role in a most of aggressive tumors, which is mainly attributed to its ability to induce lymphangiogenesis. However, VEGF-R3, the specific receptor of VEGF-C, is also expressed in a variety of human tumor cells [4], indicating that
VEGF-C may have direct impact on tumor cells. In support of this, we found that VEGF-C drove cervical cancer cell invasion into matrices, consistent with effects of VEGF-C in other types of cancer cells [9–11]. VEGF-C exerts its biological effects mainly through VEGFR3. It has been reported that Gal-3 interacts with the VEGF-C receptor and reinforces its signal transduction in endothelial cells [7]. In our study, we found that VEGF-C largely lost its ability to promote SiHa cell invasion when Gal-3 expression was silenced, suggesting a crucial role of Gal-3 in VEGF-C-promoted cervical cancer cell invasiveness. This is consistent with other studies showing that Gal-3 overexpression is associated with increased invasiveness of melanoma, ovarian, thyroid and colorectal cancer cells [12–15]. Gal-3 expression is modulated by a variety of extra- and intracellular stimulators [16–18]. In this work, we found that VEGF-C increased Gal-3 protein expression, indicating that Gal-3 is the target protein of VEGF-C. In the human Gal-3 gene promoter
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 02/08/15 For personal use only.
DOI: 10.3109/09513590.2014.898054
area, there are binding sites for transcriptional factors including Sp1, AP-1 and NF-kB [18–20], which are well-known as the intermediates of VEGF-C signaling [21, 22]. Therefore, we investigated the role of these transcription factors and found that the NF-kB inhibitor, while not the inhibitor of Sp1 or AP-1, largely inhibited VEGF-C-enhanced Gal-3 expression, implying that NF-kB is the key pathway. This is further confirmed by the fact that silencing of p65 protein, the functional subunit of NF-kB, markedly impaired VEGF-C-enhanced Gal-3 expression. In this study, we found that at resting state, there is an interaction between VEGF-R3 and Gal-3 in cervical SiHa cells, while treatment with VEGF-C enhanced the interaction not only between VEGF-R3, but also between phosphorylated VEGF-R3 with Gal-3, implying that Gal-3 may be important in retaining active VEGF-R3 at the plasma membrane. Moreover, we found that exogenous Gal-3 activated VEGF-R3 in a timeand dose-dependent manner, implying that Gal-3 functions not only as the detainer for VEGF-R3 in the plasma membrane, but also as the activator of VEGF-R3. Therefore, a positive feedback loop may exist between VEGF-C/VEGF-R3 signaling and Gal-3. Galectins are mammalian b-galactoside binding proteins characterized by highly conserved carbohydrate recognition domains (CRDs). Currently 15 galectin members have been identified [23]. Although we found that Gal-3 plays important role in cervical cancer metastasis, we do not exclude the roles of other galectin members in this effect. For example, it was recently reported that high galectin 1 expression was significantly correlated with depth of invasion in cervical lesions and lymph node metastasis of cervical cancer [24]. Therefore, further efforts are needed to elucidate their exact role in tumor metastasis. In conclusion, our work implied that VEGF-C enhances Gal-3 protein expression through NF-kB pathway. The increased Gal-3 protein interacts and activates VEGF-R3, leading to increased invasiveness of cervical cancer SiHa cells. These findings may shed light on potential therapeutic strategies for cervical cancer therapy.
Declaration of interest The authors of this manuscript have nothing to disclose. The Project is supported by Guangdong Natural Science Foundation (To Yang Cheng, NO:S2013010016548).
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