Trans, trans-2,4-decadienal induced cell proliferation via p27 pathway in human bronchial epithelial cells

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Abstract

Lung cancer is the leading cause of cancer deaths worldwide. Epidemiological studies have shown that exposure to cooking oil fumes (COF) is a risk factor for lung cancer. Trans, trans-2,4-decadienal (tt-DDE), a dienaldehyde, is abundant in heated oils and COF. Previously, we found that long-term exposure (45 days) to a sub-lethal dose (1 μM) of tt-DDE significantly increased growth of human bronchial epithelial cells (BEAS-2B). Aims of this study are to understand the mechanism of tt-DDE-induced cell proliferation and possible protective effects of antioxidant, vitamin C and N-acetylcysteine (NAC) in BEAS-2B cells. Utilizing the real-time RT-PCR and Western immunoblotting, we found that p27 mRNA and protein levels were significantly increased by 1 μM tt-DDE treatment. Co-treatment with vitamin C or NAC partially prevented tt-DDE-induced cell proliferation. In addition, the downstream targets of p27, including CDK4, cyclin D1 and phosphorylated-Rb proteins, increased in 1 μM tt-DDE-treated cells and these changes were prevented by NAC co-treatment. Therefore, these results suggest that tt-DDE increased cell proliferation via inhibition of p27 expression, increase in CDK4/cyclin D1 protein accumulation and enhancement of Rb phosphorylation. Increased cell proliferation is considered as the early stages of lung carcinogenesis. Administration of antioxidants may prevent COF-associated lung carcinogenesis.

Introduction

Trans, trans-2,4-decadienal (tt-DDE), a specific type of dienaldehydes, is a by-product of peroxidation of polyunsaturated lipids during storage (National Toxicology Program, 1993), heated oils during cooking (Wu et al., 2001) and it is also used as a food additive/flavoring agent. While tt-DDE appeared to be inactive in most mutagenicity tests (National Toxicology Program, 1993), it is known to form DNA adducts with calf thymus DNA (Loureiro et al., 2000, Carvalho et al., 2001). Because of a potential role of tt-DDE in human carcinogenesis and its widespread presence in food products, there are increasing concerns for a potential link between dienaldehydes exposure and development of cancers in humans (Hageman et al., 1991, National Toxicology Program, P. H. S., National Institutes of Health, US Department of Health and Human Services, 1993).

Exposure to cooking oil fumes (COF) is a risk factor for lung cancer among Chinese women (Zhong et al., 1999a, Zhong et al., 1999b, Ko et al., 2000). The potential risk of inhaled COF for personnel who operate deep frying facilities is also of great concern. tt-DDE is the most abundant dienaldehyde in COF. Wu and Yen (2004) reported that tt-DDE increased oxidative stress and genotoxicity in human lung carcinoma A549 cells. Our laboratory also demonstrated that long-term exposure (up to 45 days) to tt-DDE increased oxidative tress, secretion of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1 β), and cell proliferation in human bronchial epithelial cells BEAS-2B (Chang et al., 2005). Increased cell proliferation is the earliest step of tumor promotion and our results suggested that tt-DDE might be a tumor promoter in human lung epithelial cells.

Cellular proliferation is divided into four distinct phases; G1, S (DNA synthesis), G2 and M (mitosis). Transition from one phase to the next is tightly controlled by specific cell cycle regulatory proteins in response to various extracellular signals (Sherr, 1994). Progression of cell cycle is mediated by a series of positive regulators (cyclin-dependent kinases (CDKs) and cyclins) and negative regulators (cyclin-dependent kinase inhibitors (CKIs) (Reed et al., 1994, Pines, 1997). The CDKs are a family of serine/threonine protein kinases which are activated by binding to their partner cyclins (MacLachlan et al., 1995). In mammalian cells, there are two main classes of CDKs that are involved in G1/S phase transition. Activities of CDK4 and CDK2 play a critical role in the progression through the G1 restriction point and in transition to S phase, respectively (Pines, 1995, Sherr, 1996). Cyclin D1 and cyclin E are the partners of CDK4 and CDK2, respectively. A downstream target of the CDK/cyclin complexes is retinoblastoma protein (Rb), a tumor suppressor gene. Phosphorylation of Rb modulates its activity, which is regulated by CDK/cyclin. Additionally, phosphorylation of Rb releases transcription factors such as E2F, allowing entry of cells into S phase (Weinberg, 1995).

Recent studies indicated that the activity of CDK/cyclin complexes is negatively regulated by CKIs. The CKIs consist of two distinct families: (i) the INK4 family includes p16INK4a, p15INK4b, p18INK4c and p19INK4d, which specifically inhibit CDK4/cyclin D and CDK6/cyclin D complexes; (ii) the KIP/CIP family includes p21 (also named Cip1, Waf1, Sdi1, Cap20, Pic1), p27Kip1 and p57Kip2, which broadly inhibit activities of CDK2/cyclin E, CDK4/cyclin D, CDK6/cyclin D, CDK2/cyclin A and CDC2/cyclin B complexes and influence throughout cell cycle (Sherr and Roberts, 1995, Sherr and Roberts, 1999, Sherr, 2000). During the growth factors induced transition from G1 to S phase of cell cycle, p27 and p21 were down-regulated to increase CDK activity and phosphorylation of Rb. The purpose of the present study was to characterize the mechanisms by which tt-DDE impacts cell cycle regulatory events to induce BEAS-2B cell proliferation. The role of ROS in this mechanism was also investigated.

Section snippets

Materials

tt-DDE was purchased from Acros (Geel, Belgium). Dimethylsulfoxide (DMSO), dimethylthiazol-diphenyltetrazolium, 2′-7′-dichlorofluorescein diacetate, Tris, NaCl, dithiothreitol, glycerol, leupeptin, aprotinin, pepstatin A, sodium dodecyl sulfate (SDS), phenylmethylsulfonyl fluoride, vitamin C, N-acetylcysteine (NAC) and antibody against β-actin were purchased from Sigma (St. Louis, MO). PhosphoPlus® Rb (Ser780, Ser795, Ser807/811) antibody kit was purchased from Cell Signaling Technology

Prevention of tt-DDE-induced cell growth by antioxidants

Previously, we demonstrated that co-treatment with antioxidant NAC prevented 1 μM tt-DDE-induced cell proliferation in BEAS-2B cells 45 days later (Chang et al., 2005). Because of NAC-induced cytotoxicity, co-treatment was done for the first 7 days. However, after 45 days, NAC significantly reduced cell counts by 72.94% compared to control (Table 2). To confirm the preventive effect of antioxidants, we co-treated cells with another antioxidant vitamin C (2 mM) for 45 days, which showed no

Discussion

tt-DDE is a toxic aldehyde, abundant in restaurant exhausts (Yang et al., 2007) and COF (Wu et al., 2001). Exposure to COF is an important risk factor for lung cancer among Chinese females (Ko et al., 2000). Previously we showed that tt-DDE induced cell proliferation via increasing ROS production and oxidative stress in human lung cells BEAS-2B by a yet unknown mechanism (Chang et al., 2005). The results of the present study show that continuous tt-DDE (1 μM) treatment for 45 days might

Acknowledgments

This work was supported by research grant, 96A1-EOSP01-005, from the National Research Program for Genomic Medicine and Department of Health. The scientific content of this manuscript does not necessarily signify the views and policies of the Department of Health, or condemn, endorse or recommend for use.

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