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Nicotine prevents the apoptosis induced by menadione in human lung cancer cells
BBRC Biochemical and Biophysical Research Communications 342 (2006) 928–934 www.elsevier.com/locate/ybbrc
Nicotine prevents the apoptosis induced by menadione in human lung cancer cells Tao Zhang a, Heng Lu a, Xuan Shang a, Yihao Tian a, Congyi Zheng b, Shiwen Wang c, Hanhua Cheng a,*, Rongjia Zhou a,* a
Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China b Centre for Type Culture Collection, College of Life Sciences, Wuhan University, Wuhan 430072, PR China c Institute of Geriatric Cardiology, General Hospital of PLA, Beijing 100853, PR China Received 9 February 2006 Available online 20 February 2006
Abstract Approximately 50% of long-term cigarette smokers die prematurely from the adverse effects of smoking, including on lung cancer and other illnesses. Nicotine is a main component in tobacco and has been implicated as a potential factor in the pathogenesis of human lung cancer. However, the mechanism of nicotine action in the development of lung cancer remains largely unknown. In the present study, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making lung cancer cell A549 to be in a physiological nicotine environment, and observed that nicotine promoted cell proliferation and prevented the menadione-induced apoptosis, and exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage, in which NF-jB was up-regulated. Interference analysis of NF-jB in A549 cells showed that knock down of NF-jB resulted in apoptosis promotion and counteracted the protective effect of nicotine. The findings suggest that nicotine has potential effect in lung cancer genesis, especially in patients with undetectable early tumor development and development of specific NF-jB inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer. 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Lung cancer; NF-jB; Tobacco
The median adult smoking prevalence was 20.9% (61.2 million) in the United States [1], 26.9% (350million) in China (http://www.wpro.who.int), and even around 30% in the European Region [2]. Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction [3]. Cigarette smoking causes approximately 440,000 deaths annually in the United States or 18.1% of all deaths nationwide. With only 15% of the world’s population, the European Region faces nearly one-third of the worldwide burden of tobacco-related diseases. Cigarette smokers have two times the mortality of non-smokers in middle age (35 to 69 years). In men, *
Corresponding authors. Fax: +86 27 68756253. E-mail addresses: [email protected] (H. Cheng), rjzhou@whu. edu.cn (R. Zhou). 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.02.047
approximately 90% of lung cancer deaths are attributed to smoking, 60% in females, respectively. Furthermore, about 40,000 nonsmokers die each year in the United States as a consequence of their involuntary exposure to environmental smoke. Nicotine is a major component in tobacco. The typical pack-per-day smoker absorbs 20–40 mg of nicotine each day, achieving plasma concentrations of 23–35 ng/ml by the afternoon. This level of nicotine delivery provides sufficient nicotine intake to produce a cascade of physiological and pathological effects via nicotinic acetylcholine receptors, which were found in many types of cells including human lung cancer cells of all histologic types and normal lung tissue, in addition to the central nervous system [4]. Nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer. It has been shown that
T. Zhang et al. / Biochemical and Biophysical Research Communications 342 (2006) 928–934
nicotine may play roles in lung tumorigenesis by stimulating the growth of lung cancer cells, for example Akt-dependent proliferation and survival [5], and by prevention of apoptosis by diverse stimuli such as administration of opioids, tumor necrosis factor, UV light or c-radiation [6]. Nicotine might exert a regulatory effect on Bcl-2 because nicotine induces extensive Bcl-2 phosphorylation, or activates the MAPK kinase ERK2, which results in increased
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expression of the Bcl-2 oncoprotein and suppression of apoptosis [7], or inactivates the proapoptotic function of Bax through phosphorylation [8]. However, in addition to the inhibitory effects of nicotine on apoptosis, stimulatory effect on apoptosis is also suggested for nicotine in some experimental systems. It was shown that nicotine stimulates the apoptotic pathway possibly through the Hsp 90a expression [9]. Therefore, it remains a challenge to explain
Fig. 1. Menadione induces A549 Cell Apoptosis. (A–D) Morphological profiles of menadione-induced apoptosis using Hoechst33258 staining. (A–D) A549 cells treated with 0, 50, 100, and 200 lM menadione for 1 h, respectively. The cells were then changed with fresh medium and further cultured for 12 h. Magnification 400·. (E–H) FITC-labeled TUNEL assay of menadione-treated A549 cells. (E–H) A549 cells treated with 0, 50, 100, and 200 lM menadione for 1 h, respectively. Magnification 100 ·. (I–L) Apoptosis analysis using propidium iodide staining and flow cytometry. (I–L) A549 cells treated with 0, 50, 100, and 200 lM menadione for 1 h, respectively. (M) Percentage of cell death based on the analysis of (I–L). (N) The mitochondrial enzymatic activities of menadione-induced apoptosis determined by MTT assay. Two asterisks, P < 0.001, a significant difference.
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the controversial actions of nicotine. Furthermore, although nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer, the mechanism of its action in the development of this cancer remains largely unknown. More studies will still be needed to clarify the biological effects of nicotine in different experimental systems. Here we showed protection of nicotine from apoptosis induced by menadione and found that NF-jB is involved in the process, suggesting that nicotine plays potential role in lung cancer genesis through inhibition of apoptosis, and development of NF-jB inhibitors would be a promising pharmacotherapeutical approach for tobacco-related lung cancer. Materials and methods Cell culture. Human lung adenocarcinoma cell line A549 was obtained from Centre for Type Culture Collection (Wuhan, China). The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37 C, with 5% CO2. Medium was replaced every other day. MTT assay. Cell viability was determined using the MTT assay kit according to the manufacturer’s protocol (Amresco, Solon, OH, USA). After the treatments with nicotine hydrogen tartrate salt (Sigma, St. Louis, MO, USA) or menadione sodium bisulfite (Sigma, St. Louis, MO, USA) (cultures of the control group were left untreated), 5 lL of the MTT labeling reagent (5 mg/mL in PBS) was added and further incubated at the same condition above for 2 h. 50 lL of DMSO was then added and the cells were disrupted by pipetting up and down. The spectrophotometric absorbance of the sample was measured using an ELIASA (Biotek, Winooski, VT, USA). The wavelength to measure absorbance was 570 nm, and the reference wavelength was 650 nm. Optical density (O.D.) was calculated as the difference between the absorbance at the test wavelength and that at the reference wavelength. Percent viability was calculated as (O.D. of drug-treated sample/control O.D.) · 100. Morphological profiles of apoptosis cell using Hoechst stain. Cell samples were fixed with 3.7% fresh formaldehyde for 15 min and then were washed once with PBS. The samples were dyed by Hoecchst33258
Fig. 2. Nicotine prevents the apoptosis of A549 cells induced by menadione. (A) Mitochondrial enzymatic activities of both nicotine- and menadionetreated A549 cells determined by MTT assay. A549 cells were pre-treated with 0, 50, 100, and 200 lM nicotine for 6 h, respectively, then changed with fresh medium and treated with or without 100 lM menadione for 1 h. The cells were changed with fresh medium and further cultured for 12 h. (B–E) FITC-labeled TUNEL assay of both nicotine- and menadione-treated A549 cells. The cells were pre-treated with (C,E) or without (B,D) 100 lM nicotine for 6 h, then changed with fresh medium and treated with (D,E) or without (B,C) 100 lM menadione for 1 h. The cells were changed with fresh medium and further cultured for 12 h. Magnification 100 ·. (F) Assessment of apoptosis by Annexin V. The cells were pre-treated with (panels II and IV) or without (panels I and III) 100 lM nicotine for 6 h, then changed with fresh medium and treated with (panels III and IV) or without (panels I and II) 100 lM menadione for 1 h. The cells were then changed with fresh medium and further cultured for 12 h. The necrotic cells lost cell membrane integrity that permits PI entry. Viable cells exhibit Annexin V/PI (symbol 3 in the plot); early apoptotic cells exhibit Annexin V+/PI (symbol 4 in the plot); late apoptotic cells or necrotic cells exhibit Annexin V+/PI+ (symbol 2 in the plot). (G) Percentage of cell death based on the assessment of apoptosis by Annexin V in (F). Single asterisk, P < 0.05; two asterisks, P < 0.001, a significant difference.
T. Zhang et al. / Biochemical and Biophysical Research Communications 342 (2006) 928–934 (Amresco, Ohio, USA) for 15 min at 37 C, and washed one time with PBS. They were analyzed by fluorescence microscopy (Leica, Germany) and images were captured with a color imaging system. TdT-mediated dUTP nick end labeling. Cell samples were fixed with 3.7% fresh formaldehyde for 15 min and then bound to glass slides. The slides were rinsed with PBS, incubated in permeabilization solution for 2 min on ice, and rinsed twice with PBS. They were then incubated with 50 lL terminal deoxynucleotidyl transferase reaction mixture (Roche, Basel, Switzerland) for 60 min at 37 C, rinsed three times with PBS, and analyzed by fluorescence microscopy (Leica, German) and images were captured with a color imaging system. Apoptosis analysis using propidium iodide staining and flow cytometry. Cell samples were suspended in the solution A (Amresco, Solon, OH, USA) containing 50 mg/mL propidium iodide, 50 mg/mL RNAse, and 0.05% (v/v) Nonidet P40 for 30 min at 4 C. The DNA was analyzed by FACScan Flow Cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA). The numbers of cells with sub-G1 DNA content were determined as indicators of apoptotic population. Assessment of apoptosis by Annexin V. Apoptotic cell death was measured using FITC-conjugated Annexin V/propidium iodide assay (Biovision, Palo Alto, CA, USA) by flow cytometry (Becton–Dickinson, Franklin Lakes, NJ, USA). Briefly, 5 · 104cells were washed with ice-cold PBS, resuspended in 0.1 mL binding buffer, and stained with 10 lL of FITC-conjugated Annexin V (10 mg/mL) and 10 lL of PI (50 mg/mL). The cells were incubated for 15 min at room temperature in the dark, 400 lL of binding buffer was added, and analyzed by a FACScan flow cytometer (Annexin V excitation 488 nm, emission 515 nm; PI excitation 488 nm, emission 580 nm). Real time fluorescent quantitative RT-PCR. Total RNAs were prepared by the Trizol kit (Invitrogen, Carlsbad, CA, USA) according to the
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manufacturer’s instructions. All the RNAs were digested by Rnase-free DNase I and purified. About 3 lg RNAs were used as template for reverse transcription using 0.5 lg poly(T)20 primer and 200 U MMLV reverse transcriptase (Promega, Madison, WI, USA). Real time RT-PCR was used for the quantification of the NF-jB expression using the multichannel RotorGene 3000 (Corbett Research, Australia), according to the supplied protocol. PCR cycling conditions were: 5 min at 95 C; 40 cycles of 30 s at 95 C, 30 s at 65 C, and 30 s at 72 C in a 25 lL reaction mix containing 1· Sybr Green I. The primers used were 5 0 -AGAGCAACC TAAACAGAGAG-3 0 and 5 0 -TTGCTGGTCCCACATAGTTG-3 0 for NF-jB1 (NM_003998) and 5 0 -TCCAAAATCAAGTGGGGCGA-3 0 and 5 0 -AGTAGAGGCAGGGATGATGT-3 0 for GAPD (NM_002046). Simultaneous detection of GAPD gene was used to normalize NF-jB1 expression. Amplification procedure for GAPD was same as that of NF-jB1. For robustness issues, each sample was performed in triplicate at least. Data were analyzed by the software Rotor-gene version 4.6 and then plotted in Microsoft Excel. RNAi analysis. The siRNA included in the analysis was determined using Ambion online siRNA design tool (www.ambion.com/techlib/misc/ siRNA_design.html, Ambion, Austin, TX, USA). Hairpin DNA sequences were synthesized as two complementary oligonucleotides, annealed, and ligated between the BbsI and XbaI sites, and replaced EGFP coding sequence of pmU6pro vector (a gift from David Turner) to generate interference vector NF-RNAi. The sequences are sense: 5 0 -TTTGGGGCTATAATCC TGGACTATGAGTCCAGGATTATAGCCCCTTTTT-3 0 , and antisense: 3 0 -CCCGATATTAGGACCTGATACTCAGGTCCTAATATC GGGGAAAAAGATC-5 0 , which located in amino acid 163–168 of NF-jB (p50). Transfection and luciferase assay. The pNF-jB-Luc vector (Stratagene, La Jolla, CA, USA) containing the Photinus pyralis luciferase reporter
Fig. 3. Relative mRNA expression of NF-jB to GAPD [(A) real time fluorescent quantitative RT-PCR analysis] and NF-jB protein activity [(B) luciferase assay] are up-regulated by nicotine and the up-regulated effect was repressed by menadione. The cells were pre-treated with or without 100 lM nicotine for 6 h and then changed with fresh medium and treated with or without 100 lM menadione for 1 h. The cells were changed with fresh medium and further cultured for 12 h. The cells were then analyzed for real time fluorescent quantitative RT-PCR and luciferase assay. (C) Schematic structure of luciferase reporter. Single asterisk, P < 0.05; two asterisks, P < 0.001, a significant difference.
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gene driven by the basic promoter element (TATA box) plus five repeats of NF-jB cis-enhancer element (TGGGGACTTTCCGC) was used as a reporter for NF-jB activity. The pNF-jB-Luc was transfected or cotransfected with NF-RNAi into A549 cells with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The ratio of DNA (lg)/Lipofectamine 2000 (ll) was 0.8/2.5. The pcDNA3.0 was used to adjust the transfected DNAs to same DNA quantity. The pcDNA3.0 vector was used as the negative control. After the treatments with nicotine hydrogen tartrate salt or menadione sodium bisulfite (cultures of the control group were left untreated), the transfected cells were harvested and lysed in the reporter lysis buffer (Promega, Madison, WI, USA). The luciferase activity was measured by a luminometer (Turner BioSystems, Sunnyvale, CA, USA) and normalized using the protein concentration of the cell lysates according to the directions provided by the manufacturer.
Results Nicotine promoted cell proliferation and prevented the apoptosis To study roles of nicotine in apoptosis, menadione was first used to set up apoptosis system in lung cancer cells
A549. The cells were treated with concentration gradient of menadione as 1–50–100–200 lM. The apoptotic cells turned round and their nucleus broke into pieces, forming apoptotic bodies (Figs. 1A–D). Apoptotic cells were obviously observed at concentrations of 100 lM menadione by FITC-labeled TdT-mediated dUTP nick end labeling (TUNEL) assay (Figs. 1E–H). Quantitative analysis using propidium iodide staining and flow cytometry showed the proportion of apoptotic cells in the cells from 19% to 56% after treated with 50 to 200 lM menadione, respectively (Figs. 1I–M). To further characterize the apoptosis effects of menadione on A549 cells, the mitochondrial enzymatic activity was analyzed with the MTT assay. The MTT reaction was attributed mainly to mitochondrial succinate dehydrogenase and closely correlated with overall cell viability. As the menadione concentrations increased, the MTT values decreased (Fig. 1N). The average IC50 (inhibitory concentration 50%) for menadione was 100 lM, which was selected for use in all subsequent experiments. After the cells were pre-treated with different concentrations of nicotine, whenever 100 lM menadione was added,
Fig. 4. NF-jB RNA interference analysis. (A) Relative mRNA expression of NF-jB to GAPD using real time fluorescent quantitative RT-PCR analysis, after NF-RNAi was transfected, the cells were further treated with both nicotine and menadione in the same way as shown in Fig. 3. (B) Luciferase assay after the same treatment as (A). (C) Assessment of apoptosis by Annexin V after the same treatment as (A). Symbols 1–4 in the plots represented the same as those in Fig. 2. (D) Percentage of cell death based on the assessment of apoptosis by Annexin V in (C). I–VIII represented different combinations of treatments. Structure of luciferase reporter was the same as shown in Fig. 3C. Single asterisk, P < 0.05; two asterisks, P < 0.001, a significant difference. The difference between V and VI is 0.05 < P < 0.1.
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the MTT values increased with nicotine concentrations up, and then the value was stable (82%) at 100 lM nicotine, which suggested nicotine’s role in promoting proliferation of the cells. However, the DNA fragmentation in the menadione-induced apoptotic cells decreased when the nicotine was pre-treated at 100 lM (Fig. 2E). Quantitative analysis using Annexin V/PI assay further showed that the proportion of early stage apoptotic cells (Annexin V+/PI) increased significantly from 1.7% to 7.8%, while the proportion of late stage apoptotic cells (Annexin V+/PI+) decreased significantly from 52% to 30% in the group of nicotine+/menadione+, compared with the group of nicotine/menadione+ (Figs. 2F and G). These results suggested that nicotine protected cells from menadione-induced apoptosis. Anti-apoptotic effect of nicotine involved in NF-jB When the cells were pre-treated with nicotine, obvious increase of NF-jB mRNA expression (12-fold) was observed by real-time PCR, and further protein activity by luciferase assay increased 4-fold (Figs. 3A and B). However, after menadione was treated afterwards, both NF-jB mRNA and protein activity decreased 2.8-fold and 1.4-fold, respectively (Figs. 3A and B). These results showed that the NF-jB may play a role in nicotine effects on the apoptosis. To further investigate the role of NFjB, RNA interference analysis was used. In the treatment of nicotine+/menadione+, the NF-jB mRNA expression level of the transfected cells with NF-jB RNAi vector decreased 2.8-fold in contrast with that of the control vector transfected cells, and protein activity by luciferase assay decreased 1.8-fold. To further investigate characters of the apoptosis afterNF-jB RNA interference, Annexin V/PI assay was used. In the treatment of nicotine/menadione+ (Figs. 4C V,VI), the proportion of late stage apoptotic cells (Annexin V+/PI+) in the transfected cells with NF-jB RNAi vector increased from 48.2% to 58.1%, while early stage apoptotic cells (Annexin V+/PI) decreased from 2.76% to 1.65%, comparing with that of the control vector transfected cells. Moreover, in the treatment of nicotine+/ menadione+ (Figs. 4C VII,VIII), the same apoptotic trend was observed. The proportion of late stage apoptotic cells increased from 27.9% to 40.4% and early stage apoptotic cells decreased from 6.52% to 3.03%. These results suggested that NF-jB played an important role in the anti-apoptotic effect of nicotine against menadione and its effect might act by delaying the process of apoptosis. Discussion Both hereditary and environmental factors contributed to the genesis of lung cancer, which involved many genes including those in processes of DNA reparation, signal transduction, and cell cycle regulation [10]. Defects of these processes will result in activation of oncogenes,
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the inactivation of anticancer genes, and genomic unstabilization, and thus lung cancer genesis [11–13]. Although some factors were elucidated to play a role in lung cancer genesis, the molecular mechanisms are complicated and still remain largely unknown. As a main biological component of tobacco, nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer. To investigate whether nicotine contributes to the genesis of human lung cancer through apoptosis, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making cells to be in a physiological environment, which mimics in vivo a certain extent physiological status of long-term smoker. When the cells in nicotine environment were induced to be apoptotic, we observed protection of nicotine from apoptosis induced by menadione, and nicotine exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage. It suggested that nicotine would be an inducer for lung oncogenesis by preventing or postponing their apoptosis. Inhibitory and stimulatory effects of nicotine on apoptosis involve several pathways including ERK (extracellular signal-regulated protein kinase) [14], JNK (c-jun N-terminal kinase) [15], p38 MAPK [16] and AKT (v-akt murine thymoma viral oncogene homolog) [17], and PKC (protein kinase C) [18]. Here we observed anti-apoptosis effect of nicotine, which is involved in NF-jB action. We found that pre-treating of nicotine could up-regulate the NF-jB expression both mRNA and protein in the A549 cells, and the effect could be down-regulated by menadione. Furthermore in the NF-jB RNA interference experiments, the interference of NF-jB in A549 cells promoted the apoptosis induced by menadione and counteracted the protective effect of nicotine. However, the relations between NFjB and apoptosis were very complicated, for example, the NF-jB could activate expression of apoptotic genes including p53, TNF, Fas, IL-2B, and c-myc to induce apoptosis [19], in addition to activation of expression of anti-apoptotic genes including TRAF1/2, c-IAP1/2, Bcl-xl, Mn SOD and Bcl-2 to inhibit apoptosis [20]. Activation of NF-jB may result from different signaling pathways triggered by a variety of cytokines, growth factors, tyrosine kinases, epidermal growth factor receptor, insulin growth factor receptor, and tumor necrosis factor receptor, Ras/MAPK and PI3K/Akt [21]. Nicotine might also regulate NF-jB by nicotinic acetylcholine receptors mainly via three signal pathways including ERK [22], AKT [23], and PKC [24]. Our results supported that nicotine would exert its role of anti-apoptosis through ERK or AKT via NF-jB upregulation. Thus, further development of specific NF-jB inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer. However, we should keep in mind that NF-jB exerts functions that we might not want to inhibit and thus appropriate caution should be taken.
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Acknowledgments The work was supported by the National Natural Science Foundation of China, the National Key Basic Research project (G2000057004), the Program for New Century Excellent Talents in University and the Key Project of Chinese Ministry of Education (No. 2004.28). There is not any financial conflict of interest. References [1] A.M.N Kuiper, J. Bombard, E. Maurice, K. Jackson, State-specific prevalence of cigarette smoking and quitting among adults—United States, 2004, MMWR Morb. Mortal. Wkly. Rep. 54 (2005) 1124– 1127. [2] WHO, in: The WHO Informal Meeting on Health Professionals and Tobacco Control, 2004, Geneva, Switzerland. [3] R. Peto, A.D. Lopez, J. Boreham, M. Thun, C. Heath Jr., R. Doll, Mortality from smoking worldwide, Br. Med. Bull. 52 (1996) 12–21. [4] K.D. Macklin, A.D. Maus, E.F. Pereira, E.X. Albuquerque, B.M. Conti-Fine, Human vascular endothelial cells express functional nicotinic acetylcholine receptors, J. Pharmacol. Exp. Ther. 287 (1998) 435–439. [5] H. Nakayama, T. Numakawa, T. Ikeuchi, Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells, J. Neurochem. 83 (2002) 1372–1379. [6] S.C. Wright, J. Zhong, H. Zheng, J.W. Larrick, Nicotine inhibition of apoptosis suggests a role in tumor promotion, FASEB J. 7 (1993) 1045–1051. [7] W.L. Heusch, R. Maneckjee, Signalling pathways involved in nicotine regulation of apoptosis of human lung cancer cells, Carcinogenesis 19 (1998) 551–556. [8] M. Xin, X. Deng, Nicotine inactivation of the proapoptotic function of Bax through phosphorylation, J. Biol. Chem. 280 (2005) 10781– 10789. [9] Y.P. Wu, K. Kita, N. Suzuki, Involvement of human heat shock protein 90 alpha in nicotine-induced apoptosis, Int. J. Cancer 100 (2002) 37–42. [10] L.H. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science 266 (1994) 1821–1828. [11] S.J. Weintraub, Inactivation of tumor suppressor proteins in lung cancer, Am. J. Respir. Cell. Mol. Biol. 15 (1996) 150–155. [12] J.D. Hasday, K.A. McCrea, Inherited predisposition to lung cancer, Occup. Med. 7 (1992) 227–240. [13] F.J. Kaye, R.A. Kratzke, J.L. Gerster, P.S. Lin, Recessive oncogenes in lung cancer, Am. Rev. Respir. Dis. 142 (1990) S44–S47.
[14] C.Y. Huang, J.H. Chen, C.H. Tsai, W.W. Kuo, J.Y. Liu, Y.C. Chang, Regulation of extracellular signal-regulated protein kinase signaling in human osteosarcoma cells stimulated with nicotine, J. Periodontal. Res. 40 (2005) 176–181. [15] N. Onoda, A. Nehmi, D. Weiner, S. Mujumdar, R. Christen, G. Los, Nicotine affects the signaling of the death pathway, reducing the response of head and neck cancer cell lines to DNA damaging agents, Head Neck 23 (2001) 860–870. [16] A.K. Murashov, I. Ul Haq, C. Hill, E. Park, M. Smith, X. Wang, X. Wang, D.J. Goldberg, D.J. Wolgemuth, Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury, Brain Res. Mol. Brain Res. 93 (2001) 199–208. [17] K.A. West, J. Brognard, A.S. Clark, I.R. Linnoila, X. Yang, S.M. Swain, C. Harris, S. Belinsky, P.A. Dennis, Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells, J. Clin. Invest. 111 (2003) 81–90. [18] L. Xu, X. Deng, Protein kinase Ciota promotes nicotine-induced migration and invasion of cancer cells via phosphorylation of mu and m-calpains, J. Biol. Chem. 281 (2006) 4457–4466. [19] S. Shishodia, B.B. Aggarwal, Nuclear factor-kappaB activation: a question of life or death, J. Biochem. Mol. Biol. 35 (2002) 28–40. [20] M.J. Binnicker, R.D. Williams, M.A. Apicella, Gonococcal porin IB activates NF-kappaB in human urethral epithelium and increases the expression of host antiapoptotic factors, Infect. Immun. 72 (2004) 6408–6417. [21] X. Dolcet, D. Llobet, J. Pallares, X. Matias-Guiu, NF-kB in development and progression of human cancer, Virchows Arch. 446 (2005) 475–482. [22] Y.S. Ho, C.H. Chen, Y.J. Wang, R.G. Pestell, C. Albanese, R.J. Chen, M.C. Chang, J.H. Jeng, S.Y. Lin, Y.C. Liang, H. Tseng, W.S. Lee, J.K. Lin, J.S. Chu, L.C. Chen, C.H. Lee, W.L. Tso, Y.C. Lai, C.H. Wu, Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK) induces cell proliferation in normal human bronchial epithelial cells through NFkappaB activation and cyclin D1 up-regulation, Toxicol. Appl. Pharmacol. 205 (2005) 133–148. [23] J. Tsurutani, S.S. Castillo, J. Brognard, C.A. Granville, C. Zhang, J.J. Gills, J. Sayyah, P.A. Dennis, Tobacco components stimulate Aktdependent proliferation and NFkappaB-dependent survival in lung cancer cells, Carcinogenesis 26 (2005) 1182–1195. [24] J.K. Min, Y.M. Kim, S.W. Kim, M.C. Kwon, Y.Y. Kong, I.K. Hwang, M.H. Won, J. Rho, Y.G. Kwon, TNF-related activationinduced cytokine enhances leukocyte adhesiveness: induction of ICAM-1 and VCAM-1 via TNF receptor-associated factor and protein kinase C-dependent NF-kappaB activation in endothelial cells, J. Immunol. 175 (2005) 531–540.
Nicotine prevents the apoptosis induced by menadione in human lung cancer cells Tao Zhang a, Heng Lu a, Xuan Shang a, Yihao Tian a, Congyi Zheng b, Shiwen Wang c, Hanhua Cheng a,*, Rongjia Zhou a,* a
Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China b Centre for Type Culture Collection, College of Life Sciences, Wuhan University, Wuhan 430072, PR China c Institute of Geriatric Cardiology, General Hospital of PLA, Beijing 100853, PR China Received 9 February 2006 Available online 20 February 2006
Abstract Approximately 50% of long-term cigarette smokers die prematurely from the adverse effects of smoking, including on lung cancer and other illnesses. Nicotine is a main component in tobacco and has been implicated as a potential factor in the pathogenesis of human lung cancer. However, the mechanism of nicotine action in the development of lung cancer remains largely unknown. In the present study, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making lung cancer cell A549 to be in a physiological nicotine environment, and observed that nicotine promoted cell proliferation and prevented the menadione-induced apoptosis, and exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage, in which NF-jB was up-regulated. Interference analysis of NF-jB in A549 cells showed that knock down of NF-jB resulted in apoptosis promotion and counteracted the protective effect of nicotine. The findings suggest that nicotine has potential effect in lung cancer genesis, especially in patients with undetectable early tumor development and development of specific NF-jB inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer. 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Lung cancer; NF-jB; Tobacco
The median adult smoking prevalence was 20.9% (61.2 million) in the United States [1], 26.9% (350million) in China (http://www.wpro.who.int), and even around 30% in the European Region [2]. Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction [3]. Cigarette smoking causes approximately 440,000 deaths annually in the United States or 18.1% of all deaths nationwide. With only 15% of the world’s population, the European Region faces nearly one-third of the worldwide burden of tobacco-related diseases. Cigarette smokers have two times the mortality of non-smokers in middle age (35 to 69 years). In men, *
Corresponding authors. Fax: +86 27 68756253. E-mail addresses: [email protected] (H. Cheng), rjzhou@whu. edu.cn (R. Zhou). 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.02.047
approximately 90% of lung cancer deaths are attributed to smoking, 60% in females, respectively. Furthermore, about 40,000 nonsmokers die each year in the United States as a consequence of their involuntary exposure to environmental smoke. Nicotine is a major component in tobacco. The typical pack-per-day smoker absorbs 20–40 mg of nicotine each day, achieving plasma concentrations of 23–35 ng/ml by the afternoon. This level of nicotine delivery provides sufficient nicotine intake to produce a cascade of physiological and pathological effects via nicotinic acetylcholine receptors, which were found in many types of cells including human lung cancer cells of all histologic types and normal lung tissue, in addition to the central nervous system [4]. Nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer. It has been shown that
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nicotine may play roles in lung tumorigenesis by stimulating the growth of lung cancer cells, for example Akt-dependent proliferation and survival [5], and by prevention of apoptosis by diverse stimuli such as administration of opioids, tumor necrosis factor, UV light or c-radiation [6]. Nicotine might exert a regulatory effect on Bcl-2 because nicotine induces extensive Bcl-2 phosphorylation, or activates the MAPK kinase ERK2, which results in increased
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expression of the Bcl-2 oncoprotein and suppression of apoptosis [7], or inactivates the proapoptotic function of Bax through phosphorylation [8]. However, in addition to the inhibitory effects of nicotine on apoptosis, stimulatory effect on apoptosis is also suggested for nicotine in some experimental systems. It was shown that nicotine stimulates the apoptotic pathway possibly through the Hsp 90a expression [9]. Therefore, it remains a challenge to explain
Fig. 1. Menadione induces A549 Cell Apoptosis. (A–D) Morphological profiles of menadione-induced apoptosis using Hoechst33258 staining. (A–D) A549 cells treated with 0, 50, 100, and 200 lM menadione for 1 h, respectively. The cells were then changed with fresh medium and further cultured for 12 h. Magnification 400·. (E–H) FITC-labeled TUNEL assay of menadione-treated A549 cells. (E–H) A549 cells treated with 0, 50, 100, and 200 lM menadione for 1 h, respectively. Magnification 100 ·. (I–L) Apoptosis analysis using propidium iodide staining and flow cytometry. (I–L) A549 cells treated with 0, 50, 100, and 200 lM menadione for 1 h, respectively. (M) Percentage of cell death based on the analysis of (I–L). (N) The mitochondrial enzymatic activities of menadione-induced apoptosis determined by MTT assay. Two asterisks, P < 0.001, a significant difference.
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the controversial actions of nicotine. Furthermore, although nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer, the mechanism of its action in the development of this cancer remains largely unknown. More studies will still be needed to clarify the biological effects of nicotine in different experimental systems. Here we showed protection of nicotine from apoptosis induced by menadione and found that NF-jB is involved in the process, suggesting that nicotine plays potential role in lung cancer genesis through inhibition of apoptosis, and development of NF-jB inhibitors would be a promising pharmacotherapeutical approach for tobacco-related lung cancer. Materials and methods Cell culture. Human lung adenocarcinoma cell line A549 was obtained from Centre for Type Culture Collection (Wuhan, China). The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37 C, with 5% CO2. Medium was replaced every other day. MTT assay. Cell viability was determined using the MTT assay kit according to the manufacturer’s protocol (Amresco, Solon, OH, USA). After the treatments with nicotine hydrogen tartrate salt (Sigma, St. Louis, MO, USA) or menadione sodium bisulfite (Sigma, St. Louis, MO, USA) (cultures of the control group were left untreated), 5 lL of the MTT labeling reagent (5 mg/mL in PBS) was added and further incubated at the same condition above for 2 h. 50 lL of DMSO was then added and the cells were disrupted by pipetting up and down. The spectrophotometric absorbance of the sample was measured using an ELIASA (Biotek, Winooski, VT, USA). The wavelength to measure absorbance was 570 nm, and the reference wavelength was 650 nm. Optical density (O.D.) was calculated as the difference between the absorbance at the test wavelength and that at the reference wavelength. Percent viability was calculated as (O.D. of drug-treated sample/control O.D.) · 100. Morphological profiles of apoptosis cell using Hoechst stain. Cell samples were fixed with 3.7% fresh formaldehyde for 15 min and then were washed once with PBS. The samples were dyed by Hoecchst33258
Fig. 2. Nicotine prevents the apoptosis of A549 cells induced by menadione. (A) Mitochondrial enzymatic activities of both nicotine- and menadionetreated A549 cells determined by MTT assay. A549 cells were pre-treated with 0, 50, 100, and 200 lM nicotine for 6 h, respectively, then changed with fresh medium and treated with or without 100 lM menadione for 1 h. The cells were changed with fresh medium and further cultured for 12 h. (B–E) FITC-labeled TUNEL assay of both nicotine- and menadione-treated A549 cells. The cells were pre-treated with (C,E) or without (B,D) 100 lM nicotine for 6 h, then changed with fresh medium and treated with (D,E) or without (B,C) 100 lM menadione for 1 h. The cells were changed with fresh medium and further cultured for 12 h. Magnification 100 ·. (F) Assessment of apoptosis by Annexin V. The cells were pre-treated with (panels II and IV) or without (panels I and III) 100 lM nicotine for 6 h, then changed with fresh medium and treated with (panels III and IV) or without (panels I and II) 100 lM menadione for 1 h. The cells were then changed with fresh medium and further cultured for 12 h. The necrotic cells lost cell membrane integrity that permits PI entry. Viable cells exhibit Annexin V/PI (symbol 3 in the plot); early apoptotic cells exhibit Annexin V+/PI (symbol 4 in the plot); late apoptotic cells or necrotic cells exhibit Annexin V+/PI+ (symbol 2 in the plot). (G) Percentage of cell death based on the assessment of apoptosis by Annexin V in (F). Single asterisk, P < 0.05; two asterisks, P < 0.001, a significant difference.
T. Zhang et al. / Biochemical and Biophysical Research Communications 342 (2006) 928–934 (Amresco, Ohio, USA) for 15 min at 37 C, and washed one time with PBS. They were analyzed by fluorescence microscopy (Leica, Germany) and images were captured with a color imaging system. TdT-mediated dUTP nick end labeling. Cell samples were fixed with 3.7% fresh formaldehyde for 15 min and then bound to glass slides. The slides were rinsed with PBS, incubated in permeabilization solution for 2 min on ice, and rinsed twice with PBS. They were then incubated with 50 lL terminal deoxynucleotidyl transferase reaction mixture (Roche, Basel, Switzerland) for 60 min at 37 C, rinsed three times with PBS, and analyzed by fluorescence microscopy (Leica, German) and images were captured with a color imaging system. Apoptosis analysis using propidium iodide staining and flow cytometry. Cell samples were suspended in the solution A (Amresco, Solon, OH, USA) containing 50 mg/mL propidium iodide, 50 mg/mL RNAse, and 0.05% (v/v) Nonidet P40 for 30 min at 4 C. The DNA was analyzed by FACScan Flow Cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA). The numbers of cells with sub-G1 DNA content were determined as indicators of apoptotic population. Assessment of apoptosis by Annexin V. Apoptotic cell death was measured using FITC-conjugated Annexin V/propidium iodide assay (Biovision, Palo Alto, CA, USA) by flow cytometry (Becton–Dickinson, Franklin Lakes, NJ, USA). Briefly, 5 · 104cells were washed with ice-cold PBS, resuspended in 0.1 mL binding buffer, and stained with 10 lL of FITC-conjugated Annexin V (10 mg/mL) and 10 lL of PI (50 mg/mL). The cells were incubated for 15 min at room temperature in the dark, 400 lL of binding buffer was added, and analyzed by a FACScan flow cytometer (Annexin V excitation 488 nm, emission 515 nm; PI excitation 488 nm, emission 580 nm). Real time fluorescent quantitative RT-PCR. Total RNAs were prepared by the Trizol kit (Invitrogen, Carlsbad, CA, USA) according to the
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manufacturer’s instructions. All the RNAs were digested by Rnase-free DNase I and purified. About 3 lg RNAs were used as template for reverse transcription using 0.5 lg poly(T)20 primer and 200 U MMLV reverse transcriptase (Promega, Madison, WI, USA). Real time RT-PCR was used for the quantification of the NF-jB expression using the multichannel RotorGene 3000 (Corbett Research, Australia), according to the supplied protocol. PCR cycling conditions were: 5 min at 95 C; 40 cycles of 30 s at 95 C, 30 s at 65 C, and 30 s at 72 C in a 25 lL reaction mix containing 1· Sybr Green I. The primers used were 5 0 -AGAGCAACC TAAACAGAGAG-3 0 and 5 0 -TTGCTGGTCCCACATAGTTG-3 0 for NF-jB1 (NM_003998) and 5 0 -TCCAAAATCAAGTGGGGCGA-3 0 and 5 0 -AGTAGAGGCAGGGATGATGT-3 0 for GAPD (NM_002046). Simultaneous detection of GAPD gene was used to normalize NF-jB1 expression. Amplification procedure for GAPD was same as that of NF-jB1. For robustness issues, each sample was performed in triplicate at least. Data were analyzed by the software Rotor-gene version 4.6 and then plotted in Microsoft Excel. RNAi analysis. The siRNA included in the analysis was determined using Ambion online siRNA design tool (www.ambion.com/techlib/misc/ siRNA_design.html, Ambion, Austin, TX, USA). Hairpin DNA sequences were synthesized as two complementary oligonucleotides, annealed, and ligated between the BbsI and XbaI sites, and replaced EGFP coding sequence of pmU6pro vector (a gift from David Turner) to generate interference vector NF-RNAi. The sequences are sense: 5 0 -TTTGGGGCTATAATCC TGGACTATGAGTCCAGGATTATAGCCCCTTTTT-3 0 , and antisense: 3 0 -CCCGATATTAGGACCTGATACTCAGGTCCTAATATC GGGGAAAAAGATC-5 0 , which located in amino acid 163–168 of NF-jB (p50). Transfection and luciferase assay. The pNF-jB-Luc vector (Stratagene, La Jolla, CA, USA) containing the Photinus pyralis luciferase reporter
Fig. 3. Relative mRNA expression of NF-jB to GAPD [(A) real time fluorescent quantitative RT-PCR analysis] and NF-jB protein activity [(B) luciferase assay] are up-regulated by nicotine and the up-regulated effect was repressed by menadione. The cells were pre-treated with or without 100 lM nicotine for 6 h and then changed with fresh medium and treated with or without 100 lM menadione for 1 h. The cells were changed with fresh medium and further cultured for 12 h. The cells were then analyzed for real time fluorescent quantitative RT-PCR and luciferase assay. (C) Schematic structure of luciferase reporter. Single asterisk, P < 0.05; two asterisks, P < 0.001, a significant difference.
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gene driven by the basic promoter element (TATA box) plus five repeats of NF-jB cis-enhancer element (TGGGGACTTTCCGC) was used as a reporter for NF-jB activity. The pNF-jB-Luc was transfected or cotransfected with NF-RNAi into A549 cells with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The ratio of DNA (lg)/Lipofectamine 2000 (ll) was 0.8/2.5. The pcDNA3.0 was used to adjust the transfected DNAs to same DNA quantity. The pcDNA3.0 vector was used as the negative control. After the treatments with nicotine hydrogen tartrate salt or menadione sodium bisulfite (cultures of the control group were left untreated), the transfected cells were harvested and lysed in the reporter lysis buffer (Promega, Madison, WI, USA). The luciferase activity was measured by a luminometer (Turner BioSystems, Sunnyvale, CA, USA) and normalized using the protein concentration of the cell lysates according to the directions provided by the manufacturer.
Results Nicotine promoted cell proliferation and prevented the apoptosis To study roles of nicotine in apoptosis, menadione was first used to set up apoptosis system in lung cancer cells
A549. The cells were treated with concentration gradient of menadione as 1–50–100–200 lM. The apoptotic cells turned round and their nucleus broke into pieces, forming apoptotic bodies (Figs. 1A–D). Apoptotic cells were obviously observed at concentrations of 100 lM menadione by FITC-labeled TdT-mediated dUTP nick end labeling (TUNEL) assay (Figs. 1E–H). Quantitative analysis using propidium iodide staining and flow cytometry showed the proportion of apoptotic cells in the cells from 19% to 56% after treated with 50 to 200 lM menadione, respectively (Figs. 1I–M). To further characterize the apoptosis effects of menadione on A549 cells, the mitochondrial enzymatic activity was analyzed with the MTT assay. The MTT reaction was attributed mainly to mitochondrial succinate dehydrogenase and closely correlated with overall cell viability. As the menadione concentrations increased, the MTT values decreased (Fig. 1N). The average IC50 (inhibitory concentration 50%) for menadione was 100 lM, which was selected for use in all subsequent experiments. After the cells were pre-treated with different concentrations of nicotine, whenever 100 lM menadione was added,
Fig. 4. NF-jB RNA interference analysis. (A) Relative mRNA expression of NF-jB to GAPD using real time fluorescent quantitative RT-PCR analysis, after NF-RNAi was transfected, the cells were further treated with both nicotine and menadione in the same way as shown in Fig. 3. (B) Luciferase assay after the same treatment as (A). (C) Assessment of apoptosis by Annexin V after the same treatment as (A). Symbols 1–4 in the plots represented the same as those in Fig. 2. (D) Percentage of cell death based on the assessment of apoptosis by Annexin V in (C). I–VIII represented different combinations of treatments. Structure of luciferase reporter was the same as shown in Fig. 3C. Single asterisk, P < 0.05; two asterisks, P < 0.001, a significant difference. The difference between V and VI is 0.05 < P < 0.1.
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the MTT values increased with nicotine concentrations up, and then the value was stable (82%) at 100 lM nicotine, which suggested nicotine’s role in promoting proliferation of the cells. However, the DNA fragmentation in the menadione-induced apoptotic cells decreased when the nicotine was pre-treated at 100 lM (Fig. 2E). Quantitative analysis using Annexin V/PI assay further showed that the proportion of early stage apoptotic cells (Annexin V+/PI) increased significantly from 1.7% to 7.8%, while the proportion of late stage apoptotic cells (Annexin V+/PI+) decreased significantly from 52% to 30% in the group of nicotine+/menadione+, compared with the group of nicotine/menadione+ (Figs. 2F and G). These results suggested that nicotine protected cells from menadione-induced apoptosis. Anti-apoptotic effect of nicotine involved in NF-jB When the cells were pre-treated with nicotine, obvious increase of NF-jB mRNA expression (12-fold) was observed by real-time PCR, and further protein activity by luciferase assay increased 4-fold (Figs. 3A and B). However, after menadione was treated afterwards, both NF-jB mRNA and protein activity decreased 2.8-fold and 1.4-fold, respectively (Figs. 3A and B). These results showed that the NF-jB may play a role in nicotine effects on the apoptosis. To further investigate the role of NFjB, RNA interference analysis was used. In the treatment of nicotine+/menadione+, the NF-jB mRNA expression level of the transfected cells with NF-jB RNAi vector decreased 2.8-fold in contrast with that of the control vector transfected cells, and protein activity by luciferase assay decreased 1.8-fold. To further investigate characters of the apoptosis afterNF-jB RNA interference, Annexin V/PI assay was used. In the treatment of nicotine/menadione+ (Figs. 4C V,VI), the proportion of late stage apoptotic cells (Annexin V+/PI+) in the transfected cells with NF-jB RNAi vector increased from 48.2% to 58.1%, while early stage apoptotic cells (Annexin V+/PI) decreased from 2.76% to 1.65%, comparing with that of the control vector transfected cells. Moreover, in the treatment of nicotine+/ menadione+ (Figs. 4C VII,VIII), the same apoptotic trend was observed. The proportion of late stage apoptotic cells increased from 27.9% to 40.4% and early stage apoptotic cells decreased from 6.52% to 3.03%. These results suggested that NF-jB played an important role in the anti-apoptotic effect of nicotine against menadione and its effect might act by delaying the process of apoptosis. Discussion Both hereditary and environmental factors contributed to the genesis of lung cancer, which involved many genes including those in processes of DNA reparation, signal transduction, and cell cycle regulation [10]. Defects of these processes will result in activation of oncogenes,
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the inactivation of anticancer genes, and genomic unstabilization, and thus lung cancer genesis [11–13]. Although some factors were elucidated to play a role in lung cancer genesis, the molecular mechanisms are complicated and still remain largely unknown. As a main biological component of tobacco, nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer. To investigate whether nicotine contributes to the genesis of human lung cancer through apoptosis, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making cells to be in a physiological environment, which mimics in vivo a certain extent physiological status of long-term smoker. When the cells in nicotine environment were induced to be apoptotic, we observed protection of nicotine from apoptosis induced by menadione, and nicotine exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage. It suggested that nicotine would be an inducer for lung oncogenesis by preventing or postponing their apoptosis. Inhibitory and stimulatory effects of nicotine on apoptosis involve several pathways including ERK (extracellular signal-regulated protein kinase) [14], JNK (c-jun N-terminal kinase) [15], p38 MAPK [16] and AKT (v-akt murine thymoma viral oncogene homolog) [17], and PKC (protein kinase C) [18]. Here we observed anti-apoptosis effect of nicotine, which is involved in NF-jB action. We found that pre-treating of nicotine could up-regulate the NF-jB expression both mRNA and protein in the A549 cells, and the effect could be down-regulated by menadione. Furthermore in the NF-jB RNA interference experiments, the interference of NF-jB in A549 cells promoted the apoptosis induced by menadione and counteracted the protective effect of nicotine. However, the relations between NFjB and apoptosis were very complicated, for example, the NF-jB could activate expression of apoptotic genes including p53, TNF, Fas, IL-2B, and c-myc to induce apoptosis [19], in addition to activation of expression of anti-apoptotic genes including TRAF1/2, c-IAP1/2, Bcl-xl, Mn SOD and Bcl-2 to inhibit apoptosis [20]. Activation of NF-jB may result from different signaling pathways triggered by a variety of cytokines, growth factors, tyrosine kinases, epidermal growth factor receptor, insulin growth factor receptor, and tumor necrosis factor receptor, Ras/MAPK and PI3K/Akt [21]. Nicotine might also regulate NF-jB by nicotinic acetylcholine receptors mainly via three signal pathways including ERK [22], AKT [23], and PKC [24]. Our results supported that nicotine would exert its role of anti-apoptosis through ERK or AKT via NF-jB upregulation. Thus, further development of specific NF-jB inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer. However, we should keep in mind that NF-jB exerts functions that we might not want to inhibit and thus appropriate caution should be taken.
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Acknowledgments The work was supported by the National Natural Science Foundation of China, the National Key Basic Research project (G2000057004), the Program for New Century Excellent Talents in University and the Key Project of Chinese Ministry of Education (No. 2004.28). There is not any financial conflict of interest. References [1] A.M.N Kuiper, J. Bombard, E. Maurice, K. Jackson, State-specific prevalence of cigarette smoking and quitting among adults—United States, 2004, MMWR Morb. Mortal. Wkly. Rep. 54 (2005) 1124– 1127. [2] WHO, in: The WHO Informal Meeting on Health Professionals and Tobacco Control, 2004, Geneva, Switzerland. [3] R. Peto, A.D. Lopez, J. Boreham, M. Thun, C. Heath Jr., R. Doll, Mortality from smoking worldwide, Br. Med. Bull. 52 (1996) 12–21. [4] K.D. Macklin, A.D. Maus, E.F. Pereira, E.X. Albuquerque, B.M. Conti-Fine, Human vascular endothelial cells express functional nicotinic acetylcholine receptors, J. Pharmacol. Exp. Ther. 287 (1998) 435–439. [5] H. Nakayama, T. Numakawa, T. Ikeuchi, Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells, J. Neurochem. 83 (2002) 1372–1379. [6] S.C. Wright, J. Zhong, H. Zheng, J.W. Larrick, Nicotine inhibition of apoptosis suggests a role in tumor promotion, FASEB J. 7 (1993) 1045–1051. [7] W.L. Heusch, R. Maneckjee, Signalling pathways involved in nicotine regulation of apoptosis of human lung cancer cells, Carcinogenesis 19 (1998) 551–556. [8] M. Xin, X. Deng, Nicotine inactivation of the proapoptotic function of Bax through phosphorylation, J. Biol. Chem. 280 (2005) 10781– 10789. [9] Y.P. Wu, K. Kita, N. Suzuki, Involvement of human heat shock protein 90 alpha in nicotine-induced apoptosis, Int. J. Cancer 100 (2002) 37–42. [10] L.H. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science 266 (1994) 1821–1828. [11] S.J. Weintraub, Inactivation of tumor suppressor proteins in lung cancer, Am. J. Respir. Cell. Mol. Biol. 15 (1996) 150–155. [12] J.D. Hasday, K.A. McCrea, Inherited predisposition to lung cancer, Occup. Med. 7 (1992) 227–240. [13] F.J. Kaye, R.A. Kratzke, J.L. Gerster, P.S. Lin, Recessive oncogenes in lung cancer, Am. Rev. Respir. Dis. 142 (1990) S44–S47.
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