Cigarette smoke condensate promotes cell proliferation through disturbance in cellular redox homeostasis of transformed lung epithelial type-II cells

Abstract

Present study was initiated to evaluate the effects of cigarette smoke condensate (CSC) on the cellular changes at molecular levels in non-small lung carcinoma cells (A549). Cigarette smoke condensate at low concentration (0.1 μg/ml) induced cancer cell proliferation, DNA synthesis, reduced glutathione (GSH) levels and intercellular adhesion molecule-1 (ICAM-1) expression without any significant change in reactive oxygen species (ROS) and superoxide radicals (SOR) production. The increased levels of GSH and ICAM-1 due to increased γ-glutamylcysteine synthetase (γ-GCS) activity and transcriptional activation of ICAM-1 gene respectively might be via activation of p38 mitogen activated protein kinase (p38 MAPK). The induction of ICAM-1 expression and cell proliferation reflect the tumor promoting activity of low CSC concentration. On the other hand, high CSC concentration (50 μg/ml), which is doubtful to be achieved in the lungs even in the chain smokers, induced killing effects on cancer cells by increasing apoptosis, ROS and SOR production, inducing cell cycle arrest, and increased ICAM-1 levels. These changes were found to be associated with altered GSH/GSSG ratio which shifted the redox balance towards more oxidizing equivalent followed by activation of p38 MAPK and stress-activated protein kinase (SAPK) involved in signaling cascade and finally transcriptional activation of γ-GCS and ICAM-1 genes. These changes were found to be p38 and SAPK dependent.

Introduction

Lung cancer is the most common tobacco related cause of cancer mortality, with one case being produced for every 3 million cigarettes smoked [1], [2]. Tissue damage due to the inhalation of cigarette smoke induces a complex sequence of events collectively known as the inflammatory response. The release of oxidants from activated neutrophils in the pulmonary microcirculation has been implicated as one of the contributors to the inflammatory responses in lung diseases. Oxidants and chemicals in cigarette smoke are highly reactive and, when generated close to cell membranes, oxidize membrane phospholipids (lipid peroxidation) and proteins, which may continue in a chain reaction. This may severely disrupts cell functions and lead to cell death, or to damage of DNA in alveolar cells [3]. Therefore, mutations or oxidant-mediated modifications in cancer-related genes or post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation-by free radicals or lipid peroxidation byproducts are some of the key events that can increase the inflammation and cancer risks [4]. ROS generated during smoking may induce cell proliferation during the tumor promotion stage of carcinogenesis [5], [6]. Evidences are emerging which link ROS with altered expression of growth regulatory genes. Recently there has been an increasing recognition that the effects of oxidants are not solely mediated through gross damage of the cellular constituents but also by their more discriminating role as a redox regulator of signal transduction, mitosis, energy metabolism, the GSH/GSSG ratio and the glutathione cycle, cellular redox homeostasis, gene transcriptional regulation and post-translational modification of proteins [7], [8]. ROS therefore are important determinants of the redox state and constitute a regulatory mechanism in the cell through modification of protein conformation and function and regulation of signal transduction [9], [10]. These species have been implicated as second messengers involved in activation of various redox sensitive transcription factors. Oxidative changes may amplify the receptor-mediated signal, and can function either to activate protein kinases [e.g., MAPK] and inhibit or activate transcription factors, such as AP-1 and NF-κB [11]. Through regulation of gene transcription factors, and disruption of signal transduction pathways, ROS are intimately involved in the maintenance of concerted networks of gene expression that may interrelate with neoplastic development [5]. Therefore, the decision to commit to cell death or cell survival or cell proliferation will in part depend on the strength and duration of oxidant exposure and on the cell type involved. During multistep process, i.e., initiation, promotion and progression, of carcinogenesis, the genomes of incipient cancer cells acquire mutant alleles of proto-oncogenes, tumor-suppressor genes, and other genes that directly or indirectly control cell proliferation, apoptosis and differentiation. Earlier report from our laboratory has shown that cigarette smoke acts on the initiation and promotion phases of lung carcinogenesis-induced by the most prominent carcinogen, 3,4-benzo(a)pyrene, present in the cigarette smoke [12]. However, more studies are warranted to understand the mechanistic aspects of CSC action on the growth of tumor cells. This aspect is quite relevant as the smokers keep their smoking habits intact till the disease becomes aggressive and is detectable by the modern diagnostic modalities. In the present study, we have found lung cancer cell proliferating activities with low concentrations of cigarette smoke condensate in A549 cells.