Cellular transformation by cigarette smoke extract involves alteration of glycolysis and mitochondrial function in esophageal epithelial cells

Cigarette‐smoking increases the risk of developing various types of human cancers including esophageal cancers. To test the effects of chronic cigarette smoke exposure directly on esophageal epithelium, cellular resistance to mainstream extract (MSE), or sidestream smoke extract (SSE) was developed in chronically exposed nonmalignant Het‐1A cells. Anchorage‐independent growth, in vitro invasion capacity and proliferation of the resistant cells increased compared with the unexposed, sensitive cells. An epithelial marker E‐cadherin was down‐regulated and mesenchymal markers N‐cadherin and vimentin were up‐regulated in the resistant cells. Het‐1A cells resistant to MSE or SSE consumed more glucose, and produced more lactate than the sensitive cells. The increased anchorage‐independent cell growth of the resistant cells was suppressed by a glycolysis inhibitor, 2‐deoxy‐D ‐glucose, indicating that these cells are highly dependent on the glycolytic pathway for survival. Decreased mitochondrial membrane potential and ATP production in the resistant cells indicate the presence of mitochondrial dysfunction induced by chronic exposure of cigarette smoke extract. Increased expression of nuclear genes in the glycolytic pathway and decreased levels of mitochondrial genes in the resistant cells support the notion that cigarette smoking significantly contributes to the transformation of nonmalignant esophageal epithelial cells into a tumorigenic phenotype.     

Chronic exposure to chewing tobacco selects for overexpression of stearoyl-CoA desaturase in normal oral keratinocytes

Chewing tobacco is a common practice in certain socio-economic sections of southern Asia, particularly in the Indian subcontinent and has been well associated with head and neck squamous cell carcinoma. The molecular mechanisms of chewing tobacco which leads to malignancy remains unclear. In large majority of studies, short-term exposure to tobacco has been evaluated. From a biological perspective, however, long-term (chronic) exposure to tobacco mimics the pathogenesis of oral cancer more closely. We developed a cell line model to investigate the chronic effects of chewing tobacco. Chronic exposure to tobacco resulted in higher cellular proliferation and invasive ability of the normal oral keratinocytes (OKF6/TERT1). We carried out quantitative proteomic analysis of OKF6/TERT1 cells chronically treated with chewing tobacco compared to the untreated cells. We identified a total of 3,636 proteins among which expression of 408 proteins were found to be significantly altered. Among the overexpressed proteins, stearoyl-CoA desaturase (SCD) was found to be 2.6-fold overexpressed in the tobacco treated cells. Silencing/inhibition of SCD using its specific siRNA or inhibitor led to a decrease in cellular proliferation, invasion and colony forming ability of not only the tobacco treated cells but also in a panel of head and neck cancer cell lines. These findings suggest that chronic exposure to chewing tobacco induced carcinogenesis in non-malignant oral epithelial cells and SCD plays an essential role in this process. The current study provides evidence that SCD can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients who are users of tobacco.     

Autophagy in fibroblasts induced by cigarette smoke extract promotes invasion in lung cancer cells

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 β and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.     

Enhanced expression of anti-apoptotic proteins in human papillomavirus–immortalized and cigarette smoke condensate–transformed human endocervical cells: Correlation with resistance to apoptosis induced by DNA damage

Apoptosis plays an important role in various biological processes including embryogenesis, differentiation, homeostasis, and oncogenesis. We have developed a system composed of primary human endocervical cells (HEN), HEN immortalized by human papillomavirus (HPV) type 16, and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). To understand the role of apoptosis in the multistep oncogenesis of human cervical cells, we examined the expression of apoptosis-associated proteins in our in vitro model system. The results showed no significant difference in the levels of apoptosis-inducing proteins bak and bax among all the cell types examined. On the other hand, the levels of apoptosis-inhibiting proteins bcl-2, bcl-x_L and BAG-1 increased progressively after immortalization and transformation. The p53 protein level decreased in the HPV16-immortalized HEN and increased in one of two lines of the CSC-transformed HEN. Further, the increased levels of apoptosis-inhibiting proteins in the HPV16-immortalized and the CSC-transformed HEN correlated with progressively increased resistance of these cells to apoptosis induced by staurosporine or cisplatin. This study provided the first evidence that overexpression of apoptosis-inhibiting proteins is important for both multistep oncogenesis and resistance of human endocervical cells to apoptosis induced by DNA-damaging reagents. Mol. Carcinog. 22:95–101, 1998. © 1998 Wiley-Liss, Inc.     

Induction of osteopontin expression by nicotine and cigarette smoke in the pancreas and pancreatic ductal adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with etiological association with cigarette smoking. Nicotine, an important component of cigarettes, exists at high concentrations in the bloodstream of smokers. Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival, proliferation, invasion, and metastasis. Here, we investigated the potential molecular basis of nicotine's role in PDA through studying its effect on OPN. Nicotine significantly (p < 0.02) increased OPN mRNA and protein secretion in PDA cells through activation of the OPN gene promoter. The OPN mRNA induction was inhibited by the nicotinic acetylcholine receptor antagonist, mechamylamine. Further, the tyrosine kinase inhibitor genistein inhibited the nicotine‐mediated induction of OPN, suggesting that mitogen activated protein kinase signaling mechanism is involved. Nicotine activated the phosphorylation of ERK1/2, but not p38 or c‐Jun NH2‐terminal MAP kinases. Inhibition of ERK1/2 activation reduced the nicotine‐induced OPN synthesis. Rats exposed to cigarette smoke showed a dose‐dependent increase in pancreatic OPN that paralleled the rise of pancreatic and plasma nicotine levels. Analysis of cancer tissue from invasive PDA patients, the majority of whom were smokers, showed the presence of significant amounts of OPN in the malignant ducts and the surrounding pancreatic acini. Our data suggest that nicotine may contribute to PDA pathogenesis through upregulation of OPN. They provide the first insight into a nicotine‐initiated signal transduction pathway that regulates OPN as a possible tumorigenic mechanism in PDA. © 2009 UICC     

香烟烟雾与慢性应激联合暴露对大鼠学习记忆能力的影响

目的：研究香烟烟雾与慢性应激联合暴露对大鼠学习记忆能力的影响。方法：40只SPF级SD大鼠,每组10只,雌雄各半,随机分为对照组、香烟烟雾组、慢性应激组、联合干预组。对照组不采取任何处理;香烟烟雾组大鼠采用呼吸道静式染毒,每天1次,每次10支香烟,每次1 h,连续12周;慢性应激组从5种慢性应激方案中每日随机给予1种慢性应激,每种慢性应激方式不连续应用,进行12周;联合干预组每日进行慢性应激处理的同时吸烟10支,持续12周。在染毒第2、4、8、10、12周时,各组大鼠进行Morris水迷宫实验,检测大鼠逃避潜伏期,测试各组大鼠学习记忆能力。染毒12周后,腹主动脉采血,剥取大脑,观察大鼠体质量、脑质量、脑脏器系数、脑组织病理学变化,测定血清中Cort浓度、ACH、CHAT含量及ACHE活力。结果：与对照组比较,慢性应激组大鼠Cort浓度显著增加（P < 0.05）,提示慢性应激组大鼠处于紧张状态;与慢性应激组比较,第2周联合干预组逃避潜伏期潜伏期减少,呈拮抗作用（P < 0.05）;与对照组比较,香烟烟雾、慢性应激单独及联合干预组大鼠第10周逃避潜伏期增加（P < 0.05）;病理学观察结果显示香烟烟雾组大鼠神经元轻度核固缩,脑膜血管中度充血及轻度出血,胶质细胞增生,慢性应激组大鼠神经元可见轻度核固缩及淤血,联合干预组变化最明显,联合干预组大鼠神经元中度核固缩,脑膜轻度淤血及水肿,神经元周围空泡形成。与对照组比较,二者单独及联合暴露组大鼠ACH含量下降（P < 0.05）,与对照组比较,应激组大鼠CHAT含量下降、ACHE活力升高（P < 0.05）。结论：香烟烟雾和慢性应激联合暴露对大鼠学习记忆能力的影响存在交互作用;香烟烟雾和慢性应激单独及联合暴露均会导致大鼠学习和记忆能力下降;二者单独及联合暴露主要通过造成ACH合成受损,导致大鼠学习和记忆能力降低;联合暴露对ACH、CHAT含量及ACHE活力未产生交互作用;其联合作用具体机制需进一步深入研究。     

Chronic exposure to cigarette smoke increases matrix metalloproteinases and Filaggrin mRNA expression in oral keratinocytes: role of nicotine stimulation

The vegetal alkaloid nicotine has been proved to modify the expression of many keratinocyte markers. In this study, the basal expression of MMP-2, MMP-9, MMP-28, and Filaggrin has been evaluated in oral keratinocytes, in order to collect information about the ability of cigarette smoke to modify the basal expression pattern of these key enzymes in the absence of evident clinical signs in the oral epithelium. MMP-2, MMP-9, MMP-28, and Filaggrin basal expression was investigated by RT-PCR in oral keratinocytes derived from smokers (n = 11), non-smokers (n = 11), and ex-smokers (n = 6) healthy volunteers. Moreover keratinocytes from non-smokers volunteers were stimulated in vitro by a single dose administration of nicotine (10 μM) in order to estimate the effect of nicotinic receptors activation on the basal expression of the studied markers. RT-PCR analysis showed that all the markers studied were overexpressed in keratinocytes from smoker donors compared to control keratinocytes, while a single dose of nicotine was able to induce only Filaggrin expression in keratinocytes from non-smoking donors. Markers expression in ex-smoker donors was similar to that observed in normal non-smoker donors. These data indicate for the first time that cigarette smoking affects basal expression of some important markers in oral mucosa keratinocytes in vivo in the absence of clinical signs and that smoke quitting restores basal expression levels of these markers.     

Preneoplastic and neoplastic lesions in the lung, liver and urinary tract of mice exposed to environmental cigarette smoke and UV light since birth

It is difficult to reproduce the carcinogenicity of cigarette smoke (CS) in animal models. Recently, we showed that exposure of mice to mainstream CS (MCS) for 120 days, starting immediately after birth, resulted in an early and potent carcinogenic response. In parallel, we implemented studies evaluating intermediate biomarkers and tumors in mice exposed to environmental CS (ECS). To this purpose, we used 263 newborn CD-1 mice born from 27 dams. The whole-body exposure to ECS for 120 days, starting within 12 hr after birth, resulted in an early appearance of preneoplastic lesions in lung, which however tended to attenuate after discontinuing exposure. When the experiment was stopped, after 330 days, the number of lung adenomas was higher in ECS-exposed mice as compared to sham-exposed mice, but such increase was statistically significant only in mice co-exposed to smoke and halogen light mimicking solar irradiation. Moreover, exposure to ECS produced extensive histopathological changes, mainly parenchymatous degeneration, in liver. The alterations produced in both lung and liver require that exposure to ECS starts immediately after birth, no effect being observed when exposure started 8 days later. In contrast, induction by ECS of alterations in the urinary tract, such as microadenomas and adenomas in renal pelvis and kidney, papillary hyperplasia of urothelium, and urinary bladder papillomas, were unrelated to the exposure time after birth. The results obtained with ECS cannot be directly compared to those previously obtained with MCS, since the latter involved shorter daily exposures to more massive CS doses.     

Molecular alterations associated with chronic exposure to cigarette smoke and chewing tobacco in normal oral keratinocytes

Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.     

LDOC1 silenced by cigarette exposure and involved in oral neoplastic transformation

Previously, we identified global epigenetic aberrations in smoking-associated oral squamous cell carcinoma (OSCC). We hypothesized that cigarette exposure triggers OSCC through alteration of the methylome of oral cells. Here we report that cigarette smoke condensate (CSC) significantly changes the genomic 5-methyldeoxycytidine content and nuclear accumulation of DNA methyltransferase 1 (DNMT1) and DNMT3A in human untransformed oral cells. By using integrated analysis of cDNA and methylation arrays of the smoking-associated dysplastic oral cell line and OSCC tumors, respectively, we identified four epigenetic targets—UCHL1, GPX3, LXN, and LDOC1—which may be silenced by cigarette. Results of quantitative methylation-specific PCR showed that among these four genes, LDOC1 promoter was the most sensitive to CSC. LDOC1 promoter hypermethylation and gene silencing followed 3 weeks of CSC treatment. LDOC1 knockdown led to a proliferative response and acquired clonogenicity of untransformed oral cells. Immunohistochemistry showed that LDOC1 was downregulated in 53.3% (8/15) and 57.1% (20/35) of premalignant oral tissues and early stage OSCCs, respectively, whereas 76.5% (13/17) of normal oral tissues showed high LDOC1 expression. Furthermore, the microarray data showed that LDOC1 expression had decreased in the lung tissues of current smokers compared with that in those of never smokers and had significantly decreased in the lung tumors of smokers compared with that in normal lung tissues. Our data suggest that CSC-induced promoter methylation may contribute to LDOC1 downregulation, thereby conferring oncogenic features to oral cells. These findings also imply a tumor suppressor role of LDOC1 in smoking-related malignancies such as OSCC and lung cancer.     

Lycorine hydrochloride induces reactive oxygen species-mediated apoptosis via the mitochondrial apoptotic pathway and the JNK signaling pathway in the oral squamous cell carcinoma HSC-3 cell line

Poor drug efficacy is a prominent cause of oral squamous cell carcinoma (OSCC) treatment failure. Although increased efforts in developing OSCC therapeutic strategies have been achieved in recent decades, the 5-year survival rate of patients with OSCC remains poor and effective drugs to treat OSCC are lacking. The aim of the present study was to investigate the apoptotic effect caused by lycorine hydrochloride (LH) and to identify its mechanism in the OSCC HSC-3 cell line. The findings demonstrated that LH effectively induced HSC-3 cell apoptosis and cell cycle arrest at the G₀/G₁ phase, resulting in the inhibition of cell proliferation. Furthermore, it was found that LH increased reactive oxygen species (ROS) production, triggered mitochondrial membrane potential (MMP) disorder, enhanced the protein expression levels of Bax, Bim, cleaved caspase-9, caspase-3 and poly(ADP-ribose) polymerase 1 and decreased Mcl-1 expression. The protein expression levels of important members of the JNK signaling pathway, including phosphorylated (p)-JNK, p-mitogen-activated protein kinase kinase 4 and p-c-Jun, were significantly increased in LH-treated cells, accompanied by an increase in ROS. However, N-acetyl cysteine (NAC), a potent antioxidant, reversed the upregulated mRNA expression of c-Jun, as well as the enhanced ROS production, the disorder of MMP and the apoptosis of HSC-3 cells induced by LH. These results suggested that LH may induce HSC-3 cell apoptosis via the ROS-mediated mitochondrial apoptotic pathway and the JNK signaling pathway, which indicated that LH may be a potential drug candidate for anti-OSCC therapy.