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Journal of Dental Research
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Areca-treated Fibroblasts Enhance Tumorigenesis of Oral Epithelial Cells

H.-H. Lu1, C.-J. Liu1,2, T.-Y. Liu1,3, S.-Y. Kao1,4, S.-C. Lin1,* and K.-W. Chang1,4,*

1 Institute of Oral Biology, School of Dentistry, National Yang-Ming University, No. 155, Li-Nong St., Sec.2, Taipei, Taiwan 112;
2 Oral and Maxillofacial Surgery, Taipei Mackay Memorial Hospital, Taipei, Taiwan; and
3 Department of Medical Education and Research and
4 Department of Dentistry, Taipei Veterans General Hospital, Taipei, Taiwan


Figure 1
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  		Figure 1. Areca nut extract up-regulated MMP-2 activity in oral fibroblasts. RT-PCR analysis, ELISA assay, and zymography were performed. Representative illustrations are in Appendix Fig. 5. (a) Quantitation of mRNA expression of KGF-1, MMP-2, TIMP-1, and u-PA. (b) Quantitation of MMP-2 production in the supernatants from equal numbers of cells. For each cell, triplicate analyses were performed. (c) Quantitation of active MMP-2 in zymography. Values are means ± SE from 3 distinctive oral fibroblast cultures. A, areca nut extract treatment; numbers before A, concentration (µ g/mL) for treatment. Number in the bottom of each Fig., population-doubling number. *p < 0.05; **p < 0.01; ***p < 0.001.

 

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  		Figure 2. Supernatant of arrested oral fibroblasts enhanced the proliferation and migration of oral squamous carcinoma cells through MMP-2 activity. (a,b) Growth curves for SAS and OC3, respectively. (c,d) Migration for SAS and OC3, respectively. SAS cells were cultured in media containing 2% and 0.5% fetal bovine serum in (a) and (c), respectively. Values are expressed as means ± SE from 3 individual analyses. *p < 0.05; **p < 0.01; in comparison with controls. #p < 0.05; ##p < 0.01; in comparison with cells without OA-Hy treatment.

 

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  		Figure 3. Supernatant of arrested oral fibroblasts enhanced the anchorage-independent growth of SAS cells. Representative illustrations of colonies after various treatments are shown in Appendix Fig. 7a. (a) Representative colonies after treatment with MMP-2 and supernatants. (b,c) Quantitation of total colonies after various treatments. (d) Quantitation of colonies after treatment with MMP-2 or supernatant of arrested oral fibroblasts and various doses of OA-Hy or LY294002. The enhancement of anchorage-independent growth was gradually reversed by a progressive increase in the doses of OA-Hy and LY294002. Each triangle represents a gradient dose of 0, 2.5, 5, and 10 µ M OA-Hy or LY294002. *p < 0.05; **p < 0.01; ***p < 0.001; in comparison with controls. ##p < 0.01; ###p < 0.001; in comparison with cells without OA-Hy or LY294002 treatment. {Delta} , p < 0.05; in comparison with cells with pre-immune IgG treatment. Bars, 500 µ m. Quantitation of large colonies is shown in Appendix Figs. 7b, 7c.

 

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  		Figure 4. Co-injection of arrested oral fibroblasts enhanced the tumorigenesis of SAS cells. (a) Co-injection of SAS cells with arrested oral fibroblasts induced larger tumors compared with control oral fibroblast co-injection and SAS alone injection. Injection of arrested oral fibroblasts alone did not induce tumors. (b) Co-injection of OECM-1 cells with arrested oral fibroblasts significantly induced tumorigenesis of the OECM-1 cells, but from days 15 to 23. Injection of OECM-1 alone or OECM-1 and control oral fibroblast co-injection did not induce tumorigenesis. SAS alone injection in this experiment was used as a control (3 mice). Except for SAS injection in (b), values are expressed as means ± SE from 6 mice in each group. The experiments were performed reproducibly. *p < 0.05; **p < 0.01; ***p < 0.001.

 

Journal of Dental Research, Vol. 87, No. 11, 1069-1074 (2008)
DOI: 10.1177/154405910808701111
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