Chloroform extract of cigarette smoke induces proliferation of human esophageal squamous-cell carcinoma cells: Modulation by β-adrenoceptors

Abstract

The present study aimed to delineate the actions of cigarette smoke extracts on esophageal squamous- cell carcinoma cell growth in vitro. Both chloroform- and ethanol-extracts from cigarette smoke stimu- lated human esophageal squamous carcinoma EC109 cell proliferation. Chloroform- and ethanol-extracts also upregulated β-adrenoceptors expression in EC109 cells.

Cyclo-oxygenase-2 (COX-2) expression was increased by chloroform-extract. The stimulatory actions of chloroform-extract on cell proliferation and COX-2 expression were abolished by β1- and β2-adrenoceptor selective antagonists, implicating that COX-2 was downstream to the β-adrenoceptors.

Collectively, the promoting action of chloroform-extract from cigarette smoke on esophageal squamous-cell carcinoma cell proliferation is β-adrenoceptor- and COX- 2-dependent.

Introduction

Cigarette smoking is a significant public health concern and is responsible for millions of deaths worldwide. It is a major risk factor for both squamous cell carcinoma and adenocarcinoma of the esophagus. The ingestion of tobacco condensates is believed to expose the esophageal epithelium to tobacco carcinogens, particularly nitrosamines, which contribute to cancer development.

The risk of esophageal cancer is strongly associated with both the number of cigarettes smoked per day and the duration of smoking. Esophageal cancer is the sixth leading cause of cancer-related deaths globally, accounting for more than 335,000 fatalities each year. Among its subtypes, esophageal squamous cell carcinoma represents approximately 70% of all esophageal cancer cases, and its prognosis remains poor.

Despite the well-established link between smoking and esophageal squamous cell carcinoma, the molecular mechanisms underlying its pathogenesis are still not fully understood. Further research is needed to elucidate the specific pathways involved in smoking-induced esophageal cancer development.

The β-adrenoceptor has recently been implicated in the carcinogenesis of various types of cancers. Studies have shown that β-adrenoceptor activation can stimulate the growth of pulmonary, pancreatic, and colon carcinoma cells. Additionally, polymorphisms in β-adrenoceptor genes have been associated with an increased risk of breast, colon, and endometrial cancers.

Furthermore, β-adrenoceptor blockers have been found to inhibit the migration of colon cancer cells induced by norepinephrine. Recent clinical studies also suggest that the use of β-blockers is negatively associated with cancer risk, further supporting the idea that β-adrenoceptors play a crucial role in cancer development.

Similarly, recent clinical data indicate that cyclooxygenase-2 (COX-2) is upregulated in squamous dysplasia and squamous cell carcinoma of the esophagus. Functional genetic variants in COX-2 have also been linked to an increased risk of esophageal cancer. In support of this, COX-2 inhibitors have demonstrated the ability to suppress the proliferation of esophageal cancer cells both in vitro and in vivo.

Moreover, chronic use of COX-2 inhibitors appears to be associated with a reduced incidence of esophageal cancer. These findings strongly suggest that COX-2 plays a significant role in the development of esophageal squamous cell carcinoma, highlighting the potential therapeutic benefits of targeting COX-2 in cancer prevention and treatment.

Several studies have reported that cigarette smoking upregulates the arachidonic acid cascade, thereby promoting cancer growth. However, the specific involvement of COX-2 and its relationship with β-adrenoceptor activation in the pathogenesis of smoking-related esophageal squamous cell carcinoma remains unclear.

In the present study, we investigated the effects of cigarette smoke extracts on the growth of esophageal squamous carcinoma (EC109) cells. Additionally, we examined the roles of β-adrenoceptors and COX-2 in this process to better understand their contributions to smoking-induced esophageal cancer progression.

Materials and methods

Reagents and drugs

Atenolol (β1-selective antagonists), ICI 118,551 (β2- selective antagonists), nimesulide (COX-2 inhibitor), and antibody for β-actin were purchased from Sigma (St. Louis, Missouri, USA). Antibody for COX-2 was obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA).

Preparation of Chloroform and Ethanol Extracts from Cigarette Smoke

Cigarette smoke extracts were prepared following previously established methods with slight modifications. Commercial non-filtered Camel cigarettes (R.J. Reynolds, Winston-Salem, North Carolina, USA) were used for this study.

Briefly, smoke from burning cigarettes was sequentially bubbled into chloroform and subsequently into 95% ethanol. The substances dissolved in ethanol were concentrated using a rotary evaporator (RE 47; Yamato Scientific Co., Tokyo, Japan) connected to a cooling system (F10; Julabo Labortechnik, Seelbach, Germany) to remove excess ethanol. After evaporation, chloroform was added to this fraction to extract the chloroform-soluble substances. The remaining insoluble portion was designated as the ethanol extract.

The chloroform-soluble fractions were then combined and further concentrated using the same procedure to produce the final chloroform extract. Both the chloroform and ethanol extracts were prepared in a 0.1% dimethyl sulfoxide (DMSO) solution at a concentration of 25 mg/mL before use.

A control group was processed following the same extraction procedure but without exposure to cigarette smoke. The control extract was also prepared in 0.1% DMSO and served as the experimental control. A single batch of both chloroform and ethanol extracts was used for the entire study.

Cell culture and viability assays

The human esophageal squamous carcinoma cell line, EC109, was established by National Laboratory of Molecular Oncology. Chinese Academy of Medical Sciences. EC109 cells were maintained in RPMI-1640 medium, supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) reduction method, as previously described (Shin et al., 2004).

Briefly, after incubation with various agents, cells were incubated with 2.5% MTT solution (5 mg/mL) for another 4 hours at 37°C. Then, 0.04 M of HCl- isopropanol was added to the solution and mixed thoroughly. The color change was determined by an MRX microplate reader (Dynex Technologies, Chantilly, Virginia, USA) at 570 nm.

Western blot analysis

Cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer containing 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L sodium chloride, 0.5% α-cholic acid, 0.1% sodium dodecyl sulfate (SDS), 2 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, and 10% glycerol. Additionally, 1.0 mmol/L phenylmethylsulfonyl fluoride and 1 μg/mL aprotinin were added to the buffer to prevent protein degradation.

Following lysis, the samples were sonicated on ice for 30 seconds and centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was then collected, and protein concentrations were determined using a protein assay kit (Bio-Rad), with bovine serum albumin (BSA) serving as the standard.

For Western blot analysis, 50 micrograms of protein from each sample were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto Hybond C nitrocellulose membranes (Amersham Corporation, Arlington Heights, Illinois, USA). The membranes were incubated overnight at 4°C with primary antibodies specific for COX-2 or β-actin. After primary antibody incubation, the membranes were exposed to peroxidase-conjugated secondary antibodies for 1 hour at room temperature.

β-actin was used as an internal loading control. The protein signals were visualized using an enhanced chemiluminescence system (Amersham Corporation) and detected on X-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan). The quantification of protein bands was performed using video densitometry (Scan Marker III; Microtek, Carson, California, USA).

Statistical analysis

Results were expressed as the mean ± standard error (SE) for at least three independent experiments, and statistical comparisons were based on the Student’s t-test or analysis of variance (ANOVA), followed by Tukey’s t-test. P < 0.05 was considered to be significant.

Results

Both chloroform- and ethanol-extracts from cigarette smoke stimulated EC109 cell proliferation in a dose- dependent manner (Figure 1). At a dose of 100 μg/ mL, chloroform- and ethanol-extract increased cell proliferation of EC109 cells by 44 and 35%, respec- tively, when compared to the control group.

Discussion

Cigarette smoking is widely recognized as the most consistent environmental risk factor for squamous-cell carcinoma of the esophagus. However, the precise molecular and cellular mechanisms underlying this epidemiological association have remained unclear (Enzinger and Mayer, 2003).

In the present study, we demonstrated that both chloroform- and ethanol-extracts at a concentration of 100 μg/mL significantly stimulated the proliferation of esophageal squamous carcinoma EC109 cells. Additionally, exposure to these extracts led to an increase in the expression of β1- and β2-adrenoceptors.

Interestingly, while treatment with the chloroform-extract also elevated COX-2 expression in EC109 cells, the ethanol-extract did not produce the same effect. Furthermore, the proliferation induced by the chloroform-extract was effectively inhibited by β1- and β2-adrenoceptor antagonists, as well as by a COX-2 inhibitor.

These findings strongly suggest that the proliferative effect of the chloroform-extract from cigarette smoke operates through a β-adrenoceptor- and COX-2-dependent pathway. However, the exact mechanism by which the ethanol-extract stimulates EC109 cell growth remains unclear and requires further investigation.

Previous studies have demonstrated that COX-2 expression is upregulated during the progression of esophageal cancer (Kase et al., 2004a, 2004b; Oyama et al., 2005; Zhang et al., 2005). In this study, we present novel findings showing that the chloroform-extract from cigarette smoke increases COX-2 expression in EC109 cells. This effect was completely abolished by β-adrenoceptor antagonists, suggesting that COX-2 acts downstream of β-adrenoceptors in esophageal squamous carcinoma cells.

In this context, previous research has indicated that the recruitment of β-arrestin by activated β-adrenoceptors can lead to the phosphorylation of phospholipase Cγ. This process subsequently generates the second messenger diacylglycerol, which activates protein kinase Cα (Cook and Wakelam, 1992; Luttrell et al., 1999; Kim et al., 2002). Notably, activation of protein kinase Cα has been reported to upregulate COX-2 expression (Wang et al., 2001; Kim and Chun, 2003). However, whether the ethanol-extract also activates protein kinase Cα to induce COX-2 expression in cancer cells remains an open question that requires further investigation.

Alternatively, recent studies have suggested that β-adrenoceptor stimulation can transactivate the epidermal growth factor receptor (EGFR), which subsequently leads to the phosphorylation of extracellular signal-regulated kinases 1 and 2 (Erk1/2) (Askari et al., 2005; Yeh et al., 2005). In this regard, Erk1/2 activation has also been shown to enhance COX-2 expression (Brambilla et al., 2002; Chen et al., 2004). These potential pathways warrant further exploration to fully understand the molecular mechanisms underlying cigarette smoke-induced COX-2 upregulation in esophageal squamous carcinoma cells.

Conclusion

The findings of this study clearly highlight the significant role of β-adrenoceptors in the progression of smoking-related esophageal cancer. Specifically, the chloroform-extract from cigarette smoke has been shown to stimulate the growth of esophageal squamous-cell carcinoma through β-adrenoceptor activation.

Furthermore, the activation of β-adrenoceptors and the subsequent elevation of COX-2 appear to be a crucial mechanistic cascade in the promotion of esophageal cancer growth. These insights not only deepen our understanding of the molecular mechanisms underlying smoking-related malignancies but may also contribute to the development of novel therapeutic and chemoprophylactic strategies for the prevention and treatment of esophageal cancer. ICI-118551