We analyzed whether HaCaT cells treated with PLD can
We analyzed whether HaCaT Calcium Colorimetric Assay Kit treated with PLD can alter the expression of genes involved in DNA damage response. Key DNA damage cues activate the sensory DNA-PK/ATM/ATR kinases, which relay two parallel cascades that ultimately serve to inactivate the Cyclin B-cdc2 complex. The protein kinases Chk1 and Chk2 together with ATM and ATR, act to reduce cyclin-dependent kinase (CDK) activity by various mechanisms, some of which are mediated by activation of the p53 transcription factor (Smits and Gillespie, 2015, Massey et al., 2015, Köpper et al., 2013). Inhibition of CDKs slowed down or arrested cell-cycle progression at the G1-S, intra-S and G2/M ‘cell-cycle checkpoints’, which were thought to increase the time available for DNA repair before replication or mitosis ensued. The proteins of ATM, Chk and P53 phosphorylation levels were all changed. In addition, as we showed that PLD caused early activation of p53 in HaCaT cells which was followed by p21 and Bax activation for the first 24h. In the cells treated at 0.125μM for 48h, or cultured in fresh medium for another 72h and 96h, the expression of p53 active form, p21 active form, and Bax/Bcl-2 protein remained increased. Moreover, cdc2 which is a key regulator in G2/M transition (Taylor and Stark, 2001) began to decline at the 24h time point and lasted until the wash period. At the same time, cdk2, cdk6 and cyclin E2, which contribute to G1 phase arrest were decreased as well. As mentioned above, many CDKs declined in response to PLD treatment, but it is not yet known why the PDL-treated-HaCaT cells continue to arrest at the G2/M phase, partly because the ATM/ATR signaling regulation network is so complex. Finally, the G2/M phase arrested cells have no effective DNA repair mechanisms and the damage cannot be removed.
Conflict of interest
Introduction Arsenic, one of the most prevalent environmental toxicants, is considered to be a potent human carcinogen as well as a tumor-promoting agent. Inorganic arsenic, particularly arsenite, causes cancers of the lung, skin, kidney, urinary bladder, and liver (Bagla and Kaiser, 1996). Although skin is thought to be the most sensitive site for arsenite toxicity, lung tissue is now recognized as a target (Chen et al., 2004). Arsenite is mutagenic in cultured cells and in mice (Basu et al., 2001, Noda et al., 2002). Various hypotheses have been proposed to explain the carcinogenicity of arsenite. Oxidative stress, chromosomal abnormality, and altered growth factors are possible modes of action (Huang et al., 2004). Benzo(a)pyrene (BaP), a carcinogen present in cigarette smoke and lampblack, is a ubiquitous environmental pollutant found in the air, water, and soil; it is mainly produced by incomplete combustion of organic materials (An and Li, 2009). BaP has been classified as human carcinogen by the International Agency for Research on Cancer (Smith et al., 2000). In cultured cells exposed to BaP or BaP diolepoxide, various genes and proteins involved in regulation of transcription, cell proliferation, energy metabolism, DNA synthesis, cell structure and motility, apoptosis, tumor suppression, and other biological processes are altered (Uno et al., 2001, Wester et al., 2012). BaP is a procarcinogen requiring metabolism and metabolic activation to form the ultimate carcinogen (Chen et al., 2003). Indeed, both arsenite and BaP are known human carcinogens. Studies on the mode of action of arsenite indicate that it acts as a co-carcinogen or a promoter and that it facilitates progression of carcinogenesis (Chen et al., 2004). Although arsenite is not a strong mutagen, it potentiates the genotoxicity induced by carcinogens, such as BaP (Evans et al., 2004, Fischer et al., 2005, Li et al., 2008a, Li et al., 2008b) and ultraviolet radiation (Wiencke et al., 1997). These studies suggest that arsenite acts as a co-carcinogen to promote tumor formation. In development of lung cancers, there may be a synergism between arsenite exposure and cigarette smoking, for epidemiological studies have suggested that cigarette smoking acts as a co-carcinogen with arsenite (Chen et al., 2004). This synergistic effect in lung cancer development is particularly notable, because, among arsenite-induced cancers, lung cancer may be the main cause of arsenite-related deaths (Chen et al., 2004). BaP exposure from smoking is significantly correlated with cancer occurrence (Perera et al., 2007, Pfeifer et al., 2002, Veglia et al., 2007). Cigarette smokers may be also exposed to arsenic directly from cigarette smoke (Chang et al., 2005, Torrence et al., 2002) and/or indirectly from contaminated drinking water (Khan et al., 2003, Yoshida et al., 2004). Recent epidemiologic studies have shown that arsenic exposure via drinking water is associated with an increased risk of lung cancer. A meta-analysis of studies on occupational arsenic exposure via inhalation found a synergistic effect of cigarette smoking and arsenic on lung cancer, and 30–54% of lung cancer cases were attributable to both exposures (Miyamoto et al., 2007). Co-exposure to arsenic may influence the BaP-induced biological consequences.