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Ectopic hCGβ may induce epithelial-mensenchymal transition on human keratinocytes in vitro and this could promote tumour progression and invasion Background: Ectopic expression of the free beta subunit of human chorionic gonadotropin (hCGβ) is associated with poor prognosis in various cancers, including colon, cervical, prostate and bladder carcinomas. Earlier studies have indicated that the measurement of serum and urine hCGβ is a prognostic indicator of primary cervical carcinoma that will rapidly progress to metastasis (1). Our more recent work on cervical cancer showed that there was a positive correlation between hCGβ expression and tumour progression, invasion and metastasis (2). However, the mechanism of how hCGβ participates in these malignant aspects of oncogenesis is unknown; furthermore structural studies are beginning to suggest that hCGβ expressed by tumours is different from that derived during pregnancy. Aim: This study aims to examine the effect of tumour cell conditioned culture media containing ectopically expressed hCGβ on human keratinocyte cell line. Methods: Conditioned medium (CM) from ScaBER cell line (HTB-3, ATCC, USA), a stable bladder cancer cell line that is known to ectopically express hCGβ, was used to incubate normal Human Keratinocytes (HKScienCell, USA) in culture. HK were also incubated with either conditioned media from TCL-1 (a non-malignant derived trophoblast cell line) or 3T3 cells (mouse fibroblast cell line) or by adding recombinant hCGβ to untreated control medium. HK cells were cultured for 72 hours and cellular morphological changes were observed. The expressions of Cytokeratin, Vimentin and E-cadherin proteins were studied by immunocytochemistry. The percentage and intensity of positive stained cells were evaluated by averaging of six individual fields under light microscope (200 x magnifications). Results: Exposure to SCaBER CM resulted in a significant spindled shape change in HK morphology while those treated with TCL-1, 3T3 or recombinant hCGβ showed no significant changes. Immunohistochemistry staining analysis showed dramatically reduced expression of Cytokeratin (from 100% strong staining to 30% weak staining), significantly increased expression of Vimentin (from negative to 95% moderate staining) and down regulation of E-cadherin (100% moderate to 20% weak staining) in HK cells when they were incubated with SCaBER conditioned media. Conclusion: The data showed that factors present in the SCaBER cell CM transformed HK cells from an epithelial to mesenchymal phenotype. Although hCGβ is secreted in to the culture media the effects maybe due to other factors produced by SCaBER cells and hCGβ blocking experiments and receptor studies are underway. Nevertheless, the result could indicate that the changes are due to ectopic hCGβ secreted by cancer cells. The cellular morphological changes and the down-regulation of epithelial marker and up-regulation of the mensenchymal marker combined with the down regulation of E-cadherin suggested that SCaBER CM (ectopically expressed hCGβ) induce EMT changes in normal HK cells which is a feature linked to tumour progression and invasion.
1. Crawford RA et al. 1998. J Clin pathol. 51:685-8. |
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