Up-regulation of vascular endothelial growth factor-C by nicotine in cervical cancer cell lines.
OBJECTIVE
Smoking and infection with human papilloma virus (HPV) are major risk factors for cervical cancer. Our earlier work shows that nicotine enhances cellular proliferation of cervical cancer cell lines by up-regulating epidermal growth factor (EGF) and its receptor EGF-R, which leads to increased insulin-like growth factor II in vitro. We found that the vascular endothelial growth factor (VEGF)-C, one of the five isoforms of VEGF, may be specifically involved in lymphogenic metastasis of cervical cancer. This has prompted us to study if in vitro nicotine treatment will up-regulate VEGF-C alongside EGF-R levels, while down regulating the anti-proliferative transforming growth factor (TGF)-beta levels in HPV positive cervical cancer cell lines.


METHODS
Cervical cancer cell lines CaSki, HeLa and ME-180, were cultured in serum free DMEM medium for 24-hr, and treated with 10 ng/mL nicotine in the medium supplemented with 10% fetal bovine serum. A group of untreated cells served as controls. The cells were cultured in chamber slides (for immunofluorescent antibody assay) as well as microtiter plate wells (for BrdU cell proliferation assay). The cellular levels of VEGF-C, TGF-beta, EGF-R and HPV-E6 (early protein 6) were measured by a semi-quantitative immunofluorescent antibody assay. The cell proliferation and immunofluorescent assay data were analyzed by a Student's t-test.


RESULTS
Cell proliferation was significantly increased after nicotine treatment in all the cell lines. The VEGF-C levels were significantly increased, while TGF-beta levels were decreased by nicotine in all the cell lines (P < 0.00001). EGF-R levels were also significantly increased after nicotine treatment in HeLa and ME-180, while HPV-E6 levels remained unchanged in all three.


CONCLUSIONS
Nicotine up regulates expression of cell proliferative VEGF-C and EGF-R, while down-regulating anti-proliferative TGF-beta. Our data suggest that nicotine in circulation and in cervical squamous epithelial cells may promote not only rapid tumor growth but its lympho-angiogenic spread (VEGF-C) as well. It appears that nicotine does not promote HPV spread in the cervical cancer cells.
Lane
Derrick
D
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, SC 29425, USA.
Gray
Elizabeth A
EA
Mathur
Rajesh S
RS
Mathur
Subbi P
SP
D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D006367:HeLa Cells; D006801:Humans; D009538:Nicotine / Q000378:metabolism* / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D027383:Papillomaviridae / Q000187:drug effects / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D015854:Up-Regulation; D002583:Uterine Cervical Neoplasms / Q000378:metabolism* / Q000821:virology; D042582:Vascular Endothelial Growth Factor C / Q000378:metabolism*
C068651:E6 protein, Human papillomavirus type 16; D018722:Nicotinic Agonists; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D016212:Transforming Growth Factor beta; C467486:VEGFC protein, human; D042582:Vascular Endothelial Growth Factor C; D009538:Nicotine