| Sign In to gain access to subscriptions and/or personal tools. |
Areca-treated Fibroblasts Enhance Tumorigenesis of Oral Epithelial Cells
1 Institute of Oral Biology, School of Dentistry, National Yang-Ming University, No. 155, Li-Nong St., Sec.2, Taipei, Taiwan 112; Correspondence: * corresponding authors, ckcw{at}ym.edu.tw and sclin{at}ym.edu.tw
Several hundred million Asians chew areca nut, which is strongly associated with oral carcinogenesis in people of this region. The impacts of areca nut extract on oral target cells are largely unclear. This study hypothesized an inductive role for areca-nut-exposed stromal cells in the progression of oral carcinomas in an at-risk population. Oral fibroblasts with chronic subtoxic areca nut extract treatment exhibited growth arrest and MMP-2 activation. The supernatant of arrested oral fibroblasts activated the AKT signaling pathway in oral carcinoma cells. The enhancement of proliferation, migration, and anchorage-independent growth of oral carcinoma cells elicited by such supernatant could be abrogated by blockers against MMP-2 or AKT. Subcutaneous co-injection of arrested oral fibroblasts into nude mice significantly enhanced the tumorigenicity of xenographic oral carcinoma cells. This study concludes that areca nut extract may impair oral fibroblasts and then modulate the progression of oral epithelial oncogenesis via their secreted molecules.
Key Words: areca • betel • carcinoma • fibroblast • MMP-2
A round 200–600 million people worldwide, predominantly in Southern and Southeastern Asia, chew areca (betel), the major components of which are areca nut and other ingredients (Chang et al., 2002; Gupta and Warnakulasuriya, 2002; Lin et al., 2005a). Areca nut chewing is related to a higher risk of oral carcinoma and oral submucosal fibrosis (Jeng et al., 2001; Tilakaratne et al., 2006; Shieh et al., 2007). In a monograph published by the International Agency of Research on Cancer (IARC) for the evaluation of cancer risks (IARC, 2004), areca nut was ranked as a group I carcinogen to humans. Previous studies have shown that areca nut extract activates MAPKs, AKT, and NF-  B in normal oral keratinocytes (Lin et al., 2004a, 2005b; Lu et al., 2006; Tseng et al., 2007). Matrix metalloproteinases-2 (MMP-2) primarily hydrolyze type IV collagen and laminin, which are key elements of the basement membrane and also play significant roles in the progression of cancers, including oral carcinoma (Oku et al., 2006). Functional MMP-2 polymorphism is a risk factor for oral carcinogenesis in areca users (Lin et al., 2004b). Areca ingredients seem to modulate MMPs pathologically, since MMP-2 is increased in the saliva of areca chewers (Liu et al., 2005). Recent studies have shown that senescent dermal fibroblasts can affect their adjacent epithelial cells through the provision of various factors (Campisi, 2005; Parrinello et al., 2005; Coppe et al., 2006). Areca nut extract induces senescence of oral epithelial cells (Lu et al., 2006). This study hypothesized that areca nut extract could impair the growth of oral fibroblasts, and that such impaired fibroblasts might provide factors facilitating epithelial oncogenesis.
Cell Culture, Growth Curve, and Reagents Sampling of normal human gingival tissues was approved by an institutional review board. After informed consent was obtained from the participants, oral fibroblasts were cultivated from excised gingival tissues (Ko et al., 2003). The analysis of the growth curve was carried out by trypan blue exclusion assay. For serial culture, the population doubling was calculated at each passage according to the following equation: population doubling = Ln/Ln 2 (number of collected vital cells/number of plated cells). SAS, OECM-1, and OC3 oral squamous carcinoma cells were cultured in media containing 0, 2, or 3.3% fetal bovine serum (Lin et al., 2005b). The preparation of areca nut extract followed protocols previously described (Ko et al., 2003). Cells treated with areca nut extract were washed and cultured with serum-free medium for 24 hrs for collection of supernatants. Active recombinant MMP-2 and the MMP-2 inhibitor I, OA-Hy (Berton et al., 2001), were purchased from Calbiochem (San Diego, CA, USA). A MMP-2 neutralizing antibody (Shen et al., 2006) and unrelated pre-immune rabbit IgG, which served as a negative control, were purchased from Chemicon (Temecula, CA, USA). An AKT blocker, LY294002, was purchased from Sigma-Aldrich (St. Louis, MO, USA) (Tseng et al., 2007).
Statistics The methodologies for other analyses are described in the APPENDIX.
Areca Nut Extract Regulated MMP-2, TIMP-1, and u-PA mRNA Expression and Up-regulated MMP-2 Activity Serially passaged oral fibroblasts treated with 10 µ g/mL areca nut extract to reach premature growth arrest were defined as ‘arrested oral fibroblasts’ (Appendix Fig. 1  ). Untreated cells grown to the same population-doubling were designated ‘control oral fibroblasts’. An increase in p16, Rb, and p21 expression was found in serially passaged oral fibroblasts at later passages compared with earlier passages. There was an early or sustained increase in p16 expression, conspicuous down-regulation of p21, and slight down-regulation in Rb in oral fibroblasts treated with areca nut extract (Appendix Fig. 2). The supernatant of arrested oral fibroblasts enhanced the proliferation and migration of oral carcinoma cells, but the supernatant from control oral fibroblasts did not exert such an effect (Appendix Figs. 1, 3, 4).
The changes of secreted factors during the gradual progression of arrested oral fibroblasts were investigated. Initial screening by RT-PCR analysis excluded changes in mRNA expression of MMP-3, -7, -9, -13, and -15 in the areca-nut-extract-treated oral fibroblasts, which were reported or assumed to be regulated by areca in other types of cells (detailed analysis not shown) (Lin et al., 2005b; Lu et al., 2006). A marked down-regulation of KGF-1 and TIMP-1 mRNA expression and a slight up-regulation of MMP-2 and u-PA mRNA expression were noted in the oral fibroblasts following areca nut extract treatment (Fig. 1a  , Appendix Fig. 5a). The notable down-regulation in KGF-1 in this study agrees with a previous study and supports the validity of our analysis (Ko et al., 2003). The gelatin lysis activities of MMP-2 (72-kDa protein as the proform and 62-kDa protein as the activated form) in the supernatant of areca-nut-extract-treated oral fibroblasts were much higher than those in the controls during various population-doublings (Appendix Figs. 5b, 5c). Relative to control oral fibroblasts, the arrested oral fibroblasts had increased MMP-2 mRNA expression and ~ three-fold increase in MMP-2 secretion (Figs. 1a, 1b). There was also a very marked increase in MMP-2 activation in arrested oral fibroblasts relative to controls (Fig. 1c).
Supernatant of Arrested Oral Fibroblasts Enhanced the Proliferation and Migration of Carcinoma Cells through MMP-2 Activity
Supernatant of Arrested Oral Fibroblasts Enhanced Anchorage-independent Growth of Carcinoma Cells
Arrested Oral Fibroblasts Enhanced the Tumorigenicity of SAS Cells A series of inoculates composed of 2.5 x 105 control SAS cells (SAS alone), the same number of SAS cells mixed with 1 x 106 arrested oral fibroblasts (SAS + arrested oral fibroblasts), the same number of SAS cells mixed with 1 x 106 control oral fibroblasts (SAS + control oral fibroblasts), and 1 x 106 arrested oral fibroblasts alone was injected subcutaneously into several nude mice. The SAS + arrested oral fibroblast mice showed a significantly faster tumor induction and an increase in final tumor volume compared with the other groups at the end of 5+ wks (Fig. 4a ). No tumor mass was noted in mice injected with arrested oral fibroblasts. OECM-1 was non-tumorigenic following subcutaneous injection of 5 x 106 cells over 8 wks. However, co-injection with 1 x 106 arrested oral fibroblasts dramatically induced the genesis of small xenographic OECM-1 tumors over a period of between 7 and 28 days (Fig. 4b). It was intriguing that the tumors shrank spontaneously after they reached about 200 mm3. Attempts to rescue the tumors by the injection of an additional 1 x 106 arrested oral fibroblasts into the area surrounding the regressing tumors at days 14 and 21 temporarily maintained the tumor size, but did not sustain tumor growth (not shown). The characteristics of xenographic tumors (Appendix Fig. 9) and the cultivated cells from xenographs are described in the APPENDIX.
Our previous study has shown that areca nut extract can induce senescence in oral keratinocytes. p16 up-regulation is a key event during the senescence process of oral keratinocytes (Lu et al., 2006). Evidence from the present study demonstrated that similar treatment also resulted in the growth arrest of oral fibroblasts. p16, Rb, and p21 expression could be inhibitory to cell growth. During the premature growth arrest process of oral fibroblasts induced by areca nut extract, the impact of the key p16 up-regulation seemed to surpass the down-regulation of p21 and Rb induced by the treatment for growth inhibition. The findings may be in agreement with the existence of a p16-mediated irreversible growth arrest event that is refractory to inactivation involving p21 (Beausejour et al., 2003). A complete discussion of growth arrest can be found in the APPENDIX. Senescent dermal fibroblasts promoted epithelial cell growth and tumorigenesis (Krtolica et al., 2001). Senescent fibroblasts affected the morphogenesis of breast epithelium by producing MMP-3 (Campisi, 2005; Parrinello et al., 2005). Senescent fibroblasts could secrete VEGF, which might promote angiogenesis (Coppe et al., 2006). By intensive screening, we excluded MMP-3, -7, -9, -13, and -15 as potential factors regulated by areca in oral fibroblasts, whereas an increase of MMP-2 mRNA expression and a change of mRNA expression for TIMP-1 and u-PA were identified. In addition, a further significant increase in MMP-2 activity was noted in the supernatant of arrested oral fibroblasts. Thus, it is likely that up-regulation in MMP-2 transcription and the homeostatic disturbance of the known or unknown MMP-2 regulators could underlie MMP-2 activation in arrested oral fibroblasts (Tilakaratne et al., 2006). SAS, OECM-1,and OC3 cells showed little MMP-2 activity (not shown). On treatment with supernatant from arrested oral fibroblasts, the proliferation and migration of oral carcinoma cells were enhanced to different levels. Since a specific blocker or a nullifying antibody of MMP-2 reversed such enhancement, together with the effects of the added MMP-2, this suggests that MMP-2 may be a crucial secreted molecule, produced by arrested oral fibroblasts, which caused the enhancement. The prominent enhancement in migration suggests a novel mechanism whereby the arrested oral fibroblasts may affect adjacent epithelial cells by influencing their migration or invasion resulting from the secreted factors (Campisi, 2005). In SAS cells, the intense activation of IGFR and AKT by the supernatant of arrested oral fibroblasts may be mechanistically involved in phenotypic enhancement (Tseng et al., 2007). MMP-2 can cleave IGF-binding proteins to release IGF, which results in the activation of IGFR (Fowlkes et al., 1994). It is likely that by secreting MMP-2, the arrested oral fibroblasts activate IGFR in SAS cells. The subsequent activation of the AKT cascade may finally lead to phenotypic changes. It is also clear that blockage of MMP-2 and AKT impaired the anchorage-independent growth of SAS and OECM-1 cells that had been enhanced by the supernatant of arrested oral fibroblasts. These findings support the hypothesis that arrested oral fibroblasts enhance the transformation of oral carcinoma cells, probably through MMP-2 secretion and the subsequent AKT activation. This is in agreement with previous findings where AKT signaling was crucial for the resistance to anoikis in transformed cells (Martin et al., 2006). Since the supernatant of arrested oral fibroblasts appreciably increased the fraction of large colonies compared with MMP-2 in carcinoma cells, presumably other factors in addition to MMP-2 in supernatants are also able to stimulate transformation. Proteomic analysis is being carried out at present to elucidate these novel factors. Areca nut has been proven to be a promoter of oral chemical carcinogenesis in a rodent model (Lin et al., 1997). To evaluate the enhancing effects of arrested oral fibroblasts on the growth of oral carcinoma, we used both non-tumorigenic OC3 and OECM-1 cells and high-grade tumorigenic SAS cells in a series of in vivo tumor induction experiments. The significant enhancing effects of arrested oral fibroblasts on xenographic SAS growth in nude mice support the notion that the stromal cell changes elicited by areca might support neoplastic growth of oral epithelium in an advantageous microenvironment. Since the cultured cells from this tumor exhibited significantly greater transformation-phenotypic changes compared with control cells, we can speculate that such phenotypic changes become persistent in SAS cells. Anchorage-independence is a measure of autonomous cell growth and an important hallmark of cancer. In agreement with the in vitro studies involving the arrested oral fibroblast supernatant, which was able to enhance the transformation of OECM-1 but not OC3, in vivo experiments also revealed that co-injection of arrested oral fibroblasts was sufficient to induce transient tumorigenesis of OECM-1, but not of OC3. Whether the limitations in the proliferative enhancement provided by the arrested oral fibroblasts or a lack of specific mutations in OC3 underlies the discrepancies deserves further elucidation. It is likely that the regression of the OECM-1 xenographic tumor was secondary to the mortality of the arrested oral fibroblasts in the transplanted environment that originally sustained OECM-1. The findings suggest that factors produced by the arrested oral fibroblasts may also be important to the initiation of tumor growth in vivo. Chronic subtoxic ANE treatment results in the genesis of tetraploid OC3 (Lu et al., 2006), which could be an antecedent of aneupoidity or advanced neoplastic cells (Fujiwara et al., 2005). The co-culture model of OC3 and oral fibroblast might be useful as a way of addressing the interactive roles between keratinocytes and fibroblasts for epithelial oncogenesis modulated by areca. In the present study, we have identified the induction of MMP-2 activation in oral fibroblasts by areca nut extract treatment. Not only may this event may play a role in submucosal pathogenesis and impaired wound healing, but also it provides an advantageous environment for the activation of growth factors that may contribute significantly to the progression of oral carcinomas. Although the areca nut ingredients responsible for the induction require further investigation, clues to this interactive role in oral carcinoma progression substantiate the view that areca nut per se might promote tumor formation by neoplastic cells. An anti-MMP-2 therapeutic regimen might prove to be valuable as a means of intervention.
This study is supported by the following grants: V95ER2-011 from the Veterans General Hospital, Taipei; NSC95-2314-B010-043 from National Science Council; Mackay-Yang-Ming Research Grant MMHY3-N-010-014; and Aim for the Top University Plan from the Department of Education, Taiwan.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/11/1069/DC1. Received for publication November 10, 2007. Revision received July 19, 2008. Accepted for publication August 1, 2008.
Journal of Dental Research, Vol. 87, No. 11,
1069-1074 (2008)
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
, 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.