Effects of areca nut and other oral risk factors-polarized macrophages on the activation of oral fibroblasts

• Main Authors: Po-Ju Hsiao, 蕭伯儒
• Other Authors: Lien-Yu Chang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/99361058427741478333

碩士 === 國立陽明大學 === 口腔生物研究所 === 105 === Oral cancer, one of the leading causes of cancer death in Taiwan, is closely related to risk factors such as areca nut chewing habit, smoking and microbial infection. Activation of fibroblasts showing higher expression of collagen, α-smooth muscle actin (α-SMA) and CXCL12 is observed in carcinogenesis. Macrophage polarization can be classified into two types: M1 and M2. M1 type markers like CD86, IL-12 and TNF- may enhance inflammation, while M2 type markers like arginase-1, CD163, IL-10 and TGF- may promote tumor growth. M2 macrophages could promote the stroma expression of -SMA in colorectal cancer, however, little is known about the influence of macrophage polarization on fibroblast activation during oral carcinogenesis. Previous studies showed that areca nut extract (ANE) may polarize macrophages into M2-like phenotype. The study further examined the effects of these M2-like macrophages on the activation of oral fibroblasts. Phenotypes of macrophages and fibroblasts in oral cancer specimens were also evaluated. Specific protein expression was detected by western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). Macrophages differentiated from THP-1 cells by phorbol 12-myrsitate 13-acetate (PMA) were further treated with Porphyromonas gingivalis LPS, nicotine (NT) and/or areca nut extract (ANE) for 24 hours. As compared to control group, expression of M1 markers: CD86 and IL-12 showed no significant change, while expression of M2 markers: Arginase-1 and CD163 was higher in macrophages treated with ANE with/without LPS and NT. However, the expression of TGF-β showed no significant change between groups. Production of IL-10 and TNF-α was significantly increased by LPS, but not by NT and ANE. The supernatants collected from treated macrophages were used as conditioned media (CM-Mac) for the treatment of fibroblasts. Significantly increased expression of collagen and IL-6 by fibroblasts was found in cells treated with CM-Mac from ANE-related groups, and α-SMA was also increased in ANE-related groups though without statistically significance. CM-Mac from LPS-related groups also increased the production of IL-6 by fibroblasts. However, both CM-Mac from LPS-related and ANE-related groups decreased the production of CXCL12 by fibroblasts. On the other hand, oral fibroblasts directly treated with ANE, NT and/or LPS, also increased the production of collagen and α-SMA. Tissue specimens were obtained from routine dental surgery at the department of stomatology in Taipei Veterans General Hospital after IRB approval and informed consent. Using IHC, levels of positive staining for CD68、CD163、arginase-1 and α-SMA, but not CD86, in stromal connective tissues were significantly higher in oral cancer than in adjacent normal tissues. Taken together, the results showed that the expression of M2 macrophages and activated fibroblasts were higher in connective tissue stroma of OSCC. In in vitro cell experiments, ANE with/without NT and LPS might polarize macrophages into M2-like phenotype. CM-Mac from these ANE-related groups in turn could increase collagen production and IL-6 secretion, but not CXCL12 secretion, by fibroblasts. In conclusion, besides the direct effects of oral risk factors, the immune cells altered by risk factors, the M2 or M2-like macrophages, could in turn modify the resident cell function resulting in abnormal collagen production by fibroblasts, which might play an important role in oral carcinogenesis.