| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 97 === Human basal cell carcinoma (BCC) is the most common human cancer. BCC affects millions of people worldwide annually. Although BCC never metastasis, but local invasion and destruction are very common. Cyclooxygenase-2 (COX-2) and tumor-associated macrophages (TAM) are known to increase tumorigenesis, angiogenesis, and metastasis in many human cancers. However, the role of COX-2 and TAM remain elusive in human basal cell carcinoma. We found that both COX-2 expression in BCC epithelial cells and number of TAM associate with increased invasion depth and microvessel density (MVD) by immunohistochemistry (IHC). Multivariate linear regression analysis showed that both grades of COX-2 expression in BCC epithelial cells as well as number of TAM are independent predictors for depth of invasion and MVD in human BCC.
In the first part of the study, we focused first on the effects of COX-2 expression in human BCC cells. Overexpression of COX-2 in human BCC cell line conferred cells resistance to ultraviolet B (UVB) induced apoptosis by up-regulation of anti-apoptotic proteins Mcl-1 and Bcl-2. COX-2 overexpression also increased secretion of vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) and increased in vitro and in vivo angiogenesis. Blocking COX-2 fuction by COX-2 specific RNA interference (siRNA) or specific inhibitor NS-398 abrogated increased VEGF-A and bFGF secretion by BCC cells. When COX-2 overexpressing BCC cells were inoculated into SCID subcutaneously, they developed xenograft larger than vector control counterparts. Significant increased neovascularization was observed in COX-2 overexpressing xenograft. We confirmed COX-2 promote BCC progression by enhancing anti-apoptotic ability, angiogenesis and tumorigenesis.
Prominent TAM aggregated adjacent to COX-2 overexpressing tumor nests in BCC. In addition, increased number of TAM significantly correlated grading of COX-2 expression in BCC cancer cells. Taken the special adjacency and statistics together, we hypothesized that TAM might induce COX-2 expression in BCC cells and subsequently increase invasion and angiogenesis in BCC.
TAM is a kind of M2 macrophages, which is activated by Th2 cytokines. Human THP-1 cell line differentiated to macrophages after treatment with phorbol myristate acetate (PMA). We demonstrated PMA-treated THP-1 macrophages were a kind of M2 macrophages because they express M2 surface markers (i.e. CD204 & CD206) and a M2 cytokine profile. Non-contact coculture with PMA-treated THP-1 macrophages induced COX-2 expression, incrased invasiveness, and in vitro angiogenesis in human BCC cells. Once COX-2 activity was abrogated by COX-2 specific siRNA or specific inhibitor celecoxib, the increased invasiveness and angiogenesis induced by coculture were attenuated. Coculture with macrophages induced COX-2 expression in BCC by activating the p38 MAPK/NF-κB signaling pathway. Coculture with macrophages induced MMP-9 activation by p38 MAPK/ NF-κB /COX-2 cascades and increased invasion of BCC cells. Increased angiogenesis induced by coculture resulted from increased secretion of VEGF-A and bFGF by p38 MAPK/ NF-κB /COX-2 cascades. To verify our findings, we generate another kind of M2 macrophages from human peripheral monocytes. Coculture with monocyte-derived M2 macrophages also induced COX-2 dependent invasion and angiogenesis, as well as increased expression of MMP-9, VEGF-A and bFGF. We conclude that TAM might induce BCC invasion and angiogenesis via a COX-2 dependent manner in BCC cells.
Our study highlights the importance of COX-2 and TAM in the pathogenesis and progression of human BCC. COX-2 and TAM might be used as therapeutic targets for the non-surgical treatments of human basal cell carcinoma in the near future.
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