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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)
Polycyclic 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 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.
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We quantified the effect of PAH treatment on liver cancer cell migration to assess whether the effects of B[a]A, B[k]F, and IND are due to ROS-mediated p38 MAPK signal transduction. We hypothesized that the mechanism by which B[a]A, B[k]F, and IND stimulate migration of HepG2 cells is by augmentation of the ROS-mediated p38 MAPK activation pathway. Our data support this hypothesis: cell migration stimulated by B[a]A, B[k]F, and IND was inhibited by pretreatment with the ROS scavenger NAC as well as with the p38 MAPK inhibitor SB203580, and expression of the migration-related genes AP-1, SNAI1, ETS-1, ITGA2, and MMPs was decreased by NAC and SB203580. Our results show that PAHs, acting through ROS-mediated p38 MAPK, enhance the migration of HepG2 cells.
As stated previously, PAHs include pollutants of high human and ecotoxicological relevance, due to their ubiquitous occurrence and potentially toxic, mutagenic, and carcinogenic effects.(22,23) Many researchers have undertaken risk assessments and toxicological studies of PAHs with human cell lines or other organisms by means of physical and chemical measurements, but such physical–chemical analyses might not be sufficient to provide detailed information on how the PAHs affect cells on a molecular level.(24) Therefore, a toxicological study looking at the effects of PAHs on the molecular level is required, with gene expression analysis being an appropriate method. We have investigated the mechanism(s) of toxicity of PAHs and other environmental toxicants in human cell lines, based on alteration of gene expression.(25,26) In this study, we identified gene expression profiles induced by three PAHs in HepG2 cells using a 44K whole human oligonucleotide microarray. With a statistical cut-off of ≥1.5-fold change, the expression levels of 532 genes were commonly upregulated and those of 742 genes were commonly downregulated upon exposure to B[a]A, B[k]F, and IND (Fig. 2B,C). As shown in Table 2, 24 of these genes were involved in the process of migration. Therefore, we focused on the mechanism of HCC migration induced by exposure to PAHs.
Hepatocellular carcinoma is a major malignant neoplasm, especially in Asia.(27) Previous studies have indicated that HCC is an environmentally related cancer, with both viral and chemical carcinogens involved in the multistage process. Exposure to several PAHs or to manufactured gas plant residues that contain known carcinogens, including benzene and PAHs, induces liver tumors.(28,29) Hepatocellular carcinoma must acquire the capacity to migrate to become invasive and metastatic. In spite of recent advances in diagnostic and therapeutic methods, the prognosis of HCC patients with intrahepatic metastasis is poor.(5) Accordingly, analysis of the mechanism of migration in cancer cells might lead to new strategies for preventing the progression of HCC. The molecular and cellular mechanisms of intrahepatic metastasis are not fully understood, although much evidence has pointed to signal transduction, exerted by the microenvironment around the primary tumor locus, as the trigger for tumor metastasis, especially at the migration stage.(30,31) Hazardous chemicals present in cigarette smoke, including PAHs, increase cellular invasion and metastasis of tumors, but the mechanisms underlying these effects are unknown. From the results of this study, we propose that the molecular mechanism is the cross-talk between major signaling cascades, leading to MAPK activation through ROS generation, followed by cell migration.
First, we focused on ROS, which have recently been proposed to be involved in the key intracellular signal transduction pathway for a variety of cellular processes.(32) Elevated oxidative status has been found in many types of cancer cells and is believed to contribute to carcinogenesis.(33) Recent studies have pointed to the involvement of ROS-mediated signaling in tumor metastasis, which is a complicated process including migration, invasion by the tumor cells, and angiogenesis around the tumor lesion.(34) Several important signal transduction pathways, including the MAPK, PI3K, Rho-GTPase, and Smads cascades, are known to mediate the transcriptional regulation of metastasis-related genes. Importantly, ROS are closely associated with these signal cascades, which suggest their involvement in tumor progression.(35,36)
Polycyclic aromatic hydrocarbons require metabolic activation to electrophiles that react with DNA leading to mutation. The aldo-keto reductase superfamily can activate PAHs by forming reactive and redox active o-quinones that are the ultimate carcinogenic forms of PAHs. These PAH o-quinones are reactive Michael acceptors and produce significant amounts of ROS(37,38) and can give rise to covalent DNA adducts as well as oxidative DNA lesions. For example, BP-7,8-dione produces N2 or N6 stable adducts with 2′-dGuo and 2′-dAdo by Michael addition which can hydrate and cyclize, respectively.(39,40) In addition, BP-7,8-dione reacts with guanine to form N7-depurinating guanine adducts in vitro.(41) The PAH o-quinones can also generate ROS to form 8-oxo-dGuo in vitro.(42–44) In this study, the levels of ROS increased in a dose-dependent manner following treatment with B[a]A, B[k]F, and IND at three different dose levels (IC20, IC30, and IC50; Fig. 3). Furthermore, the ROS scavenger NAC prevented cell migration induced by B[a]A, B[k]F, and IND (Fig. 6).
An important signaling cascade, MAPK is involved in tumor invasion and includes ERK, JNK, and p38.(9) It is a major signaling cascade for cell migration in diverse systems.(8) Several studies have suggested that, of all MAPKs, p38 is specifically involved in the migration of diverse cell types, and that SB203580 and SB202190 inhibit the migration of smooth muscle cells.(45,46) In this study, we confirmed that B[a]A, B[k]F, and IND phosphorylated p38 MAPK in a dose-dependent manner (Fig. 4), and that this activation was inhibited by SB203580 (data not shown). How do PAHs activate p38 MAPK? We used SB203580, which can inhibit the p38 MAPK autophosphorylation pathway but does not usually inhibit the classic pathway through MKK3/6 to activate/phosphorylate p38 MAPK.(47,48) A previous report showed that SB203580 did not change the phosphorylated MKK3/6 but decreased the phosphorylated p38 MAPK and MAPKAP2 after isoflurane exposure.(49) Therefore, we suggest that PAHs might increase the phosphorylated p38 MAPK at least partly through the autophosphorylation pathway.
Furthermore, Gupta et al.(50) established that ROS generation activated MAPK in the progression of malignancy in mouse keratinocyte cell lines. Based on previous research, we hypothesized that p38 MAPK could play an important role in B[a]A-, B[k]F-, and IND-mediated migration through ROS generation. In the current study, we confirmed that B[a]A, B[k]F, and IND-induced phosphorylation of p38 MAPK was inhibited by the ROS scavenger NAC (Fig. 4).
These results suggest that B[a]A, B[k]F, and IND induce p38 MAPK activation through ROS generation. Furthermore, the p38 MAPK inhibitor SB203580 prevented B[a]A-, B[k]F-, and IND-induced cell migration (Fig. 6).
It has been shown that MAPK may regulate cellular motility by promoting specific gene transcription, including early responsive genes, such as AP-1, SNAI1, and ETS-1.(51) These genes could play important roles in tumor progression. The transcription factor Ap-1 is an important mediator of several cellular processes, including migration of various cancer cells.(52) Snail is a transcriptional repressor that plays a major role in triggering epithelial–mesenchymal transition.(53) More importantly, Snail is expressed at the invasive front of epidermoid carcinomas and is associated with lymph node status and/or invasiveness of ductal breast carcinomas and hepatocarcinomas.(54,55) Ets-1 expression is also upregulated in tumor progression and its activation is intimately linked with cellular migration, invasion, and tumor metastasis. Ets-1 expression is significantly increased in adenomas, carcinomas in situ, and colonic adenocarcinoma, but is absent from normal or hyperplastic polyps.(56) In other cancer models, Ets-1 and Ets-2 expression is directly linked to lymph node metastasis and depth of invasion.(57) These transcription factors may regulate the expression of other migration-related genes, such as MMPs and integrins.(15,58) In the present study, we found that the expressions of Ap-1, Snail, and Ets-1 were increased in a dose-dependent manner following B[a]A, B[k]F, and IND exposure. Furthermore, NAC and SB203580 prevented this increase (Fig. 5).
Invasive tumor cells must be able to cross the basement membrane to extend from the primary site. Matrix dissolution, which is mediated in part by MMPs, is required for this process.(59) Matrix metalloproteinases are a group of zinc-containing metallopeptidases that play an important role in the invasion of cancer cells by degrading basement membranes. The MMP family plays a key role in tumor invasion in various human cancers, including HCC. Increased expression of MMP-2, MMP-7, and MT1-MMP is strongly associated with dedifferentiation, portal invasion, intrahepatic metastasis, and recurrence in HCC.(60) As cancer cells become metastatic, they develop an altered affinity and avidity for the ECM. This phenotypic change is initially mediated by changes in the expression of cell surface molecules known as integrins, by the release of proteases that remodel the ECM, and by the deposition of new ECM molecules. These events activate signaling cascades that regulate gene expression, cytoskeletal organization, cell adhesion, and cell survival. As a result, cancer cells become more invasive, migratory, and better able to survive in different microenvironments. In the present study, we found that the expression of several MMPs increased in a dose-dependent manner following B[a]A, B[k]F, and IND exposure. Furthermore, NAC and SB203580 prevented this increase (Fig. 5). However, further mechanistic studies are warranted for assessing the role of MMPs and integrins in the processes of migration in HCC. These results suggest that ROS and p38 MAPK may play important roles in the expression of migration-related genes.
In summary, the results of this study show that ROS-mediated p38 MAPK signaling may play an important role in B[a]A-, B[k]F-, and IND-induced migration of HepG2 cells. Figure 7 is a schematic representation of the possible mechanism for the effect of PAHs on the migration of HepG2 cells. Further analyses and a better understanding of this mechanism could lead to developments in the management and treatment of patients with HCC.
Figure 7. Predictive mechanism of reactive oxygen species (ROS)-mediated p38 MAPK-dependent migration of HepG2 hepatocellular carcinoma cells induced by benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F), and indeno[1,2,3-c,d]pyrene (IND). AP-1, activator protein 1; Ets-1, v-ets erythroblastosis virus E26 oncogene homolog 1; HCC, hepatocellular carcinoma; PAH, polycyclic aromatic hydrocarbon.
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