Polycyclic aromatic hydrocarbons induce migration in human hepatocellular carcinoma cells (HepG2) through reactive oxygen species-mediated p38 MAPK signal transduction Mi-Kyung Song,1,2 Youn-Jung Kim,3 Mee Song,1 Han-Seam Choi,1 Yong-Keun Park2 and Jae-Chun Ryu1,4 1Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology, Seoul; 2School of Life Sciences and Biotechnology, Korea University, Anam-Dong, Seoungbuk-Gu, Seoul; 3Department of Applied Chemistry, Kyung Hee University, Yongin, Korea
(Received December 14, 2010 ⁄ Revised May 5, 2011 ⁄ Accepted May 25, 2011 ⁄ Accepted manuscript online June 2, 2011 ⁄ Article first published online July 8, 2011)
Although polycyclic aromatic hydrocarbons (PAHs) are carcinogenic and have been extensively studied with regard to tumor formation, few studies have investigated the involvement of these environmental chemicals in tumor migration and invasion. Polycyclic aromatic hydrocarbons induce reactive oxygen species (ROS) and activate MAPK signal transduction. The p38 signaling transduction pathway, one of the most typical MAPK pathways, plays an essential role in regulating cell migration. Therefore, we investigated whether three PAHs, benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F), and indeno[1,2,3-c,d]pyrene (IND), induce migration in human hepatocellular carcinoma cell line HepG2 through ROS-mediated p38 MAPK signal transduction. Reactive oxygen species generation and p38 MAPK activity both increased in a dose-dependent manner and were prevented by SB203580, an inhibitor of p38 MAPK, and N-acetylcysteine (NAC), a ROS scavenger. Expression of migration-related genes was also increased by B[a]A, B[k]F, and IND in a dose-dependent manner and was inhibited by SB203580 and NAC. The migration of HepG2 cells, observed using the Transwell migration assay, also increased in a dose-dependent manner and was prevented by SB203580 and NAC. Our results indicate that the ROS-mediated p38 MAPK signaling pathway plays an essential role in the PAH-induced migration of HepG2 cells. (Cancer Sci 2011; 102: 1636–1644)
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olycyclic aromatic hydrocarbons, which contain two or more fused benzene rings, are a type of organic pollutant.(1) Humans are systemically exposed to PAHs through combustion generated by cigarette smoking, automobile exhaust, municipal waste, and chemical and paper processing. The contamination of the environment by PAHs and their derivatives is detrimental to human health, because they may be mutagenic and carcinogenic.(2,3) Numerous epidemiologic studies have linked PAHs to cancer development;(4) however, few investigations have addressed the effects of PAH exposure on the characteristics of invasion or metastasis in developed cancer cells. Because most patient deaths from solid malignancies result not from growth of the primary tumor but rather from metastasis to secondary organs, such investigations are warranted. Hepatocellular carcinoma is one of the most common cancers worldwide, especially in Asia.(5) Most patients with HCC die within 1 year of diagnosis, largely because of frequent tumor recurrence and metastasis. It frequently shows early invasion into blood vessels, together with intrahepatic extensions and, later on, extrahepatic metastasis.(6) A better understanding of the processes involved in the development of metastasis might improve future prognosis by facilitating effective treatment strategies. The migration of tumor cells and the loss of cell contacts between tumor cells are prerequisites for tumor cell invasion and metastasis.(7) The Cancer Sci | September 2011 | vol. 102 | no. 9 | 1636–1644
candidate signaling molecule associated with migration is MAPK. The MAPK signaling cascade, including ERK, c-Jun NH2-terminal kinase, and p38, has been implicated in the migration of numerous cell types.(8) In addition to participating in cell transformation and tumorigenesis, p38 MAPK activation is associated with invasive or metastatic potential in some human cancer cells.(9,10) Many genes with various functions are also associated with cell migration. Among these genes, transcription factors, such as Ap-1, Ets-1, and the snail homolog family, are the most important genes for cell migration.(11–13) Importantly, p38 MAPK may influence these transcription factors by regulating their gene expression. In addition, these transcription factors regulate the expression of the MMP family, which is thought to accelerate cancer invasion, and integrins, which participate in a number of pathological conditions, such as chronic inflammation, invasion by cancer cells, and metastasis.(14,15) Furthermore, ROS are key candidate messengers involved in MAPK signaling.(16) Reactive oxygen species may mediate many cellular effects, including migration,(17) and ROS-triggered cellular signaling may be induced by several PAHs.(18) The objective of this study was to determine whether the PAHs B[a]A, B[k]F, and IND may alter migration through ROS-mediated p38 MAPK signal transduction in human HCC cells. Materials and Methods Chemicals and reagents. Both B[a]A and B[k]F were purchased from Acros Chimica (Geel, Belgium), and IND was purchased from Wako (Osaka, Japan). Dimethylsulphoxide and SB203580 were obtained from Sigma-Aldrich (St. Louis, MO, USA) and NAC was purchased from Fluka (St Louis, MO, USA). Dulbecco’s PBS and FBS were the products of Gibco (Grand Island, NY, USA). All other chemicals used were of analytical grade or the highest grade available. Cell culture. The human HCC cell line, HepG2, was purchased from Korean Cell Line Bank (Seoul, Korea) and was maintained under a humidified atmosphere of 5% CO2 and 95% air at 37C. The culture medium was 90% DMEM supplemented with 10% FBS, sodium bicarbonate (Sigma-Aldrich), sodium pyruvate (Gibco), and penicillin and streptomycin (Gibco). The medium was refreshed every 2–3 days. Determination of cell viability. To determine the cytotoxicity and effects on cell growth, the MTT cell proliferation assay was carried out using the modifications described by Mosmann.(19) Linear equation was calculated from Sigma Plot (Systat Software, San Jose, CA, USA).
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doi: 10.1111/j.1349-7006.2011.02000.x ª 2011 Japanese Cancer Association
Microarray analysis. Gene expression analysis was carried out using a 44K whole human genome microarray (Agilent Technologies, Palo Alto, CA, USA). Total RNA was extracted from the PAHs-treated HepG2 cells using TRIzol (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy mini kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. Labeling and hybridization were carried out using a FairPlay microarray labeling kit (Stratagene, Glenville, VA, USA), followed by the coupling of Cy3 (controls) or Cy5 (treated samples) dye. The hybridized slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA, USA) and the images were analyzed using GenePix 4.1 software (Axon Instruments) to obtain gene expression ratios. The fluorescence intensity of each spot was calculated by local median background subtraction. We then used the robust scatter-plot smoother LOWESS function to carry out intensitydependent normalization of gene expression. Scatter-plot analysis was carried out using Microsoft Excel 2000 (Microsoft, Redmond, WA, USA). A significance analysis of microarray was carried out for genes with significant changes in expression. To determine whether changes in expression were statistically significant, a q-value was calculated for each gene, using the permutation procedure. For each permutation, two-sample t statistics were computed for each gene. Genes were considered to be differentially expressed when the logarithmic gene expression ratios in three independent hybridizations were >0.5 or
