The tobacco carcinogen NNK disturbs mitotic chromosome alignment by interrupting p53 targeting to the centrosome

Park, Ji Eun,Jang, Yu Lim,Jang, Chang-Young Elsevier/North-Holland 2017 Toxicology letters Vol.281 No.-

<P><B>Abstract</B></P> <P>The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent risk factor among tobacco-related carcinogens in lung cancer progression and outcomes. Although genetic mutations and chromosome instability have been detected in NNK-induced lung tumors, the oncogenic mechanisms of NNK are not fully understood. Here, we show that NNK increases chromosomal instability by disrupting spindle microtubule (MT) attachment to the kinetochore (KT) and spindle dynamics. Mechanistically, NNK blocks the targeting of p53 to the centrosome during mitosis, leading to chromosome alignment defects in metaphase. Therefore, lung cancer cells with wild-type p53, such as A594 and H226B, are more resistant to the NNK treatment than p53-mutant lung cancer cells, such as A1299 and H226Br. Although NNK does not affect the levels or transcriptional activity of p53, the reduction of the p53 level at the centrosome exacerbates the NNK-induced chromosome alignment defect in A549 and H226B cells. Therefore, p53 protects against NNK-induced chromosome instability by modulating the function of centrosome-localized p53 and not by modulating transcriptional activity. We conclude that NNK may increase the risk of lung cancer progression and poorer outcomes in patients with p53 mutations by perturbing proper mitotic progression and chromosome integrity.</P> <P><B>Highlights</B></P> <P> <UL> <LI> NNK induces chromosome alignment defects in mitosis. </LI> <LI> NNK interrupts the targeting of p53 to the centrosome. </LI> <LI> p53 Protects nnk-induced chromosome alignment defects. </LI> <LI> p53-Mutant lung cancer cells are sensitive to the nnk treatment. </LI> </UL> </P>

인체 세포 모델을 이용한 HPV-16과 NNK의 발암 잠재력에 관한 연구

양재호,이세영 한국독성학회 1996 Toxicological Research Vol.12 No.2

Carcinogenic potential of HPV-16 DNA and NNK in a human keratinocyte cell line was assessed to study effects of viral-chemical interaction. Human cells were transfected with HPV-16 DNA and 6 clonal cell lines were subsequently obtained. Clonal line-3 and 6 at passage 7 showed characteristics of tumor cells such as increases of saturation density, soft-agar colony formation, cell aggregation and foci appearance. Among cells treated with 1$\mu M$, 10$\mu M$, 100$\mu M$ or 1 mM of NNK for 4 weeks, 100$\mu M$ treatment showed most tumorigenic characteristics at passage 7. These results indicate that either HPV-16 or NNK alone is tumorigenic in this in human in vitro model. When cells transfected with HPV-16 were subsequently exposed by 100 uM NNK for 4 weeks, all the clonal cells except clone-1 showed higher levels of tumor cell characteristics than HPV-16 DNA or NNK exposure alone. Clonal line-6, the most tumorigenic cells, showed higher transcriptional level of fibronectin and lower level of TGF-$\beta_1$, as compared to control cells, suggesting that alteration of growth factor or extracellular matrix may play a role in carcinogenesis process induced by HPV-16 and NNK. Taken together, the present study indicates that viral-chemical interactions between HPV-16 DNA and NNK enhance carcinogenic potentials of human cells and implies that smoking among people infected with human papillomavirus may pose an additional risk of causing cancer.

흡연자와 비흡연자의 치은섬유아세포에서 니코틴과 NNK가 부착과 성장에 미치는 영향

김일영,박미영,최성호,조규성,김종관,채중규,Kim, Il-Young,Park, Mi-Young,Choi, Seong-Ho,Cho, Kyoo-Sung,Kim, Chong-Kwan,Chai, Jung-Kiu 대한치주과학회 1998 Journal of Periodontal & Implant Science Vol.28 No.4

In order to study the effects of cigarette smoking on periodontal tissue, gingival fibroblast from the smoking and nonsmoking groups were cultured and each group were treated with nicotine(50ng/ml,100ng/ml) and NNK(50ng/ml, 100ng/ml) to test their attachment ability at time intervals of 30minutes, 60minutes, 90minutes, 120minutes, and 240minutes. Using the same method, the growth each group treated with nicotine and NNK in order to compare their attachment ability and growth rate was done. The Results are as follows. 1. In comparing the attachment ability and growth rate between the smoking and non-smoking group were significantly higher in all time intervals. 2. When the attachment ability was com-pared among these two groups after treatment with nicotine and NNK, the non-smoking group showed decrease in attachment ability while the smoking group was not affected. 3. The growth rate of these two groups were compared after treating with nicotine and NNK. The growth rate of fibroblast from the non-smoking group decreased while fibroblast from the smoking group was not affected. These results suggest that fibroblast from the non-smoking group showed higher attachment ability, growth rate, and sensitivity to nicotine and NNK. This implies that fibroblast from the non-smoking group is a more reliable source in testing the cytotoxicity of nicotine and NNK. Also it could be reasonable to think that nicotine and NNK is a probable cause for problems in attachment and repair mechanism.

Nicotine 및 Tobacco-specific nitrosamine이 발암과정에 미치는 영향

강호일,황명실,김은정,김윤정,이국경,정자영,원도희,김옥희 식품의약품안전청 1998 식품의약품안전청 연보 Vol.2 No.-

Nicotine은 그동안 계암발생파정에 관여되어 있는 것으로 추정되어지고 있으나 현재 이에 대한 기전.은 밝혀진 것이 거의 없다.본 연구에서는 폐암발생과정에서 있어서 nicotine의 역할을 조사하기 위해 첫번째 실험군으로 nicotine을 Sprague-Dawley 랫드에 연속적으로 10ㅇ리간 투여한 후 암유전자 및 암억제유전자의 발현변화를 단백질과 mRNA레벨에서 검토하엿다. 그 결과 5종류의 암유전자인 ras,raf,myc,fos, jun,및 2종류의 암억제유전자인 p53,Rb 단백질의 발현변화는 거의 없는것으로 나타났다.그리고 2종류의 암유전자인 myc,fos및 암억제유전자 p53 mRNA발현변화 역시 거의 없어 단백질 발현 실험결과와 일치하는 것으로 나타났다. 두번째 실험군으로 nicotine 및 sodium nitrite그리고 NNK를 Fischer344 랫드에 투여하여 발암물질인 NNK의 생성여부를 검토한 결과 NNK를 단독투여한 실험군의 경우 8-OHdG 레벨이 1.8배에서 2.3배까진 현저하게 증가하였으나 nicotine 및 sodium nitrite를 단독 혹은 병용 투여한 경우 8-OHdG 레벨의 유의한 차이가 관찰되지 않아 NNK 생성을 확인할수 없었다. Nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer, however its mechanism of action in the development of lllng cancer remains largely unknown.To explore the role of Bicotine in the developmene of lung cancer, lue first investigated the effects of nic-otine on the expression of tumor associated genes by treating Spragve-Barley rats with nirotine (10 mf/kg) by gavage once daily for 10 days. We determined the expression of proteins and rnRNAs of the raf,raf, mrt JHn, foa oncogenes and p53, ab tumor suppressor genes by Western and Northern blotting,respectively. We did not detect any changes on t31e levels of prote·ins aBO mRNAs of these tumor associ-ated genes in the lung of Sprague-Dawley rats from 3 days to 12 ·weeks after the last treatment of nico-tine, indicating that nicotine appears to have no effect on the exE)ression of these oncogenes aBd turnersuppressor genes at an early stage in multistage chemical carcinoifenesis. In a second experiment, we in-vestigated the possibility that 4-(methylnitrosamino)- t- (3-p!rridyl)-1-butaaone (NNK) could beformed eBdogenously bf treating with nicotine and sodium nitrite. We treated groups of Fischer 344 ratswith nicetiBe (60 rrnol/kg) and sodium nitrite (180 rmol/kg), nicotiae, sodiu:n-nitrite and NNK (120 #mol/kg) flfne by gavage once daily to, 7 dafs, .espectively and dete,mined the 8-hydroxy-deoByguanosine (8-OHdG), as an indicator of NNK formation, in the lungs of rats 24 hours and 48hours after the last treatmeilt by HPLC/ECD method. We detect 3ncreased level of 8-OHdG in the tungsof rats treated with NNK, but in the case of nicotine plus sodiLlra nitrite, nicotine and sodium nitritealone we could not detected any changes of 8-OHdG, respectively.

4-(N-Methyl-N-nitrosamino)-1(3-pyridyl)-1-butanone(NNK) Restored the Cap-dependent Protein Translation Blocked by Rapamycin

Kim Jun-Sung,Park Jin Hong,Park Sung-Jin,Kim Hyun Woo,Hua Jin,Cho Hyun Sun,Hwang Soon Kyung,Chang Seung Hee,Tehrani Arash Minai,Cho Myung Haing Korean Society of ToxicologyKorea Environmental Mu 2005 Toxicological Research Vol.21 No.4

Eukaryotic initiation factor 4E (elF4E) is a key element for cap-dependent protein translation controlled by affinity between elF4E and 4E-binding protein 1 (4E-BP1). Rapamycin can also affect protein translation by regulating 4E-BP1 phosphorylation. Tobacco-specific nitrosamine, 4(N-methyl-N-nitrosamino )-1-(3-pyridyl)-1-butanone (NNK) is a strong lung carcinogen, but its precise lung cancer induction mechanism remains unknown. Relative roles of cap-dependent and -independent protein translation in terms of NNK-induced lung carcinogenesis were elucidated using normal human bronchial epithelial cells. NNK concentrations applied in this study did not decrease cell viability. Addition of NNK restored rapamycin-induced decrease of protein synthesis and rapamycin-induced phosphorylation of 4E-BP1, and increased expression levels of mTOR, ERK1/2, p70S6K, and Raf-1 in a concentration-dependent manner. NNK also caused perturbation of normal cell cycle progression. Taken together, NNK might cause toxicity through the combination of restoration of 4E-BP1 phosphorylation and increase of elF4E as well as mTOR protein expression, interruption of Raf1/ERK as well as the cyclin G-associated p53 network. Our data could be applied towards elucidation of the molecular basis for lung cancer treatment.

Binding Pattern Elucidation of NNK and NNAL Cigarette Smoke Carcinogens with NER Pathway Enzymes: an Onco-Informatics Study

Jamal, Qazi Mohammad Sajid,Dhasmana, Anupam,Lohani, Mohtashim,Firdaus, Sumbul,Ansari, Md Yousuf,Sahoo, Ganesh Chandra,Haque, Shafiul Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.13

Cigarette smoke derivatives like NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol) are well-known carcinogens. We analyzed the interaction of enzymes involved in the NER (nucleotide excision repair) pathway with ligands (NNK and NNAL). Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. The highest obtained docking energy between NNK and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.13 kcal/mol, -7.27 kcal/mol, -8.05 kcal/mol and -7.58 kcal/mol respectively. Similarly the highest obtained docking energy between NNAL and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.46 kcal/mol, -7.94 kcal/mol, -7.83 kcal/mol and -7.67 kcal/mol respectively. In order to find out the effect of NNK and NNAL on enzymes involved in the NER pathway applying protein-protein interaction and protein-complex (i.e. enzymes docked with NNK/NNAL) interaction analysis. It was found that carcinogens are well capable to reduce the normal functioning of genes like RAD23A (HR23A), CCNH, CDK7 and CETN2. In silico analysis indicated loss of functions of these genes and their corresponding enzymes, which possibly might be a cause for alteration of DNA repair pathways leading to damage buildup and finally contributing to cancer formation.

Development of a method for the determination of 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone in dust using liquld chromatography tandem mass spectrometry

이원경,강수진,오지은,황상현,이도훈 한국분석과학회 2015 분석과학 Vol.28 No.1

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco specific nitrosamine found only intobacco products. The ability to monitor biomarker concentrations is very important in understanding environmentaltobacco smoke (ETS). In this study, an efficient and sensitive method for the analysis of NNK in dust wasdeveloped and validated using liquid chromatography tandem mass spectrometry. Dust was collected with filterpaper soaked in methanol. The standard solution and dust sample were diluted with 100 mM ammonium acetateand extracted using dichloromethane. Our calibration curves ranged from 25 to 104 pg/mL. Excellent linearitywas obtained with correlation coefficient values between 0.9996 and 1.0000. The limit of detection (LOD) was5 pg/mL (S/N ≥ 3) and the retention time was 10 min. The limit of quantification (LOQ) was 25 pg/mL, andthe acceptance criteria was the rate of 98-103% (80-120% at levels up to 3×LOQ). The coefficient of variations(CV) was 2.8%. Accuracies determined from dust samples spiked with four different levels of NNK racurvesranged that from 25 to 104 pg/mL. Excellent linearity was obtained between 92.1% and 114%. The precisionof the method was acceptable (5% of CV). The recovery rates of the whole analytical procedure at low, medium,and high levels were 105.7-116.5% for NNK. The carry-over effects during LC-MS/MS analysis were notobserved for NNK. This manuscript summarizes the scientific evidence on the use of markers to measure ETS.

Effects of Physalis peruviana L on Toxicity and Lung Cancer Induction by Nicotine Derived Nitrosamine Ketone in Rats

El-Meghawry El-Kenawy, Ayman,Elshama, Said Said,Osman, Hosam-Eldin Hussein Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.14

Nicotine-derived nitrosamine ketone (NNK) is considered a key tobacco smoke carcinogen inducing lung tumors. Physalis peruviana L (harankash) is considered one plant with marked health benefits. This study aimed to evaluate Physalis peruviana L effect on the toxic effect of NNK induced lung cancer in the rats by using pulmonary histopathological, immunohistochemical and DNA flow cytometric analyses. Sixty adult male rats were divided into four groups, each consisting of fifteen animals. The first group received saline, the second received two successive toxic doses of NNK only while the third received two successive toxic doses of NNK with a single daily dose of Physalis peruviana L. The fourth group received a single daily dose of Physalis peruviana L only. Toxic doses of NNK induced hyperplasia and adenocarcinoma in the lung and positive immunoreactivity for Ki-67 and p53 staining with disturbance of the lung DNA content. Administration of Physalis peruviana L with NNK led to a mild pulmonary hyperplasia and weak expression of Ki-67 and p53 with an improvement in the lung DNA content. Physalis peruviana L may protect against NNK induced lung carcinogenesis due to its antioxidant and anti-proliferative effects.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Induces Retinoic Acid Receptor β Hypermethylation through DNA Methyltransferase 1 Accumulation in Esophageal Squamous Epithelial Cells

Wang, Jing,Zhao, Shu-Lei,Li, Yan,Meng, Mei,Qin, Cheng-Yong Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.5

Overexpression of DNA methyltransferase 1 (DNMT1) has been detected in many cancers. Tobacco exposure is known to induce genetic and epigenetic changes in the pathogenesis of malignancy. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen present in tobacco smoke; however the detailed molecular mechanism of how NNK induces esophageal carcinogenesis is still unclear. We found that DNMT1 was overexpressed in ESCC tissues compared with paired non-cancerous tissues, the overexpression being correlated with smoking status and low expression of $RAR{\beta}$. The latter could be upregulated by NNK treatment in Het-1A cells, and the increased DNMT1 expression level reflected promoter hypermethylation and downregulation of retinoic acid receptor ${\beta}$($RAR{\beta}$). RNA interference mediated knockdown of DNMT1 resulted in promoter demethylation and upregulation of $RAR{\beta}$ in KYSE30 and TE-1 cells. 3-(4,5-Dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometric analysis demonstrated that NNK treatment in Het-1A cells could enhance cell proliferation and inhibit cell apoptosis in a dose-dependent manner. In conclusion, DNMT1 overexpression is correlated with smoking status and low expression of $RAR{\beta}$ in esophageal SCC patients. NNK could induce $RAR{\beta}$ promoter hypermethylation through upregulation of DNMT1 in esophageal squamous epithelial cells, finally leading to enhancement of cell proliferation and inhibition of apoptosis.

흡연과 금연 나이트클럽의 간접흡연 노출의 차이에 대한 탐색연구

곽수영,이보람,이기영,이도훈,Guak, Sooyoung,Lee, Boram,Xu, Siyu,Lee, Kiyoung,Lee, Dohoon 한국환경보건학회 2014 한국환경보건학회지 Vol.40 No.1

Objectives: This pilot study assessed secondhand smoke (SHS) exposure in smoking and non-smoking nightclubs in Seoul, Korea by measuring the concentration of particulate matter smaller than $2.5{\mu}m$ ($PM_{2.5}$). Methods: This comparative study was conducted in three nightclubs in Seoul. While one non-smoking nightclub was measured on weekdays and weekends, different smoking nightclubs were measured on weekdays and weekends. The concentration of $PM_{2.5}$ was observed using a real-time monitor over an average of three hours. The number of people in the clubs was also estimated. Settled dust was collected in a smoking and a non-smoking nightclub and analyzed for NNK concentration. Results: The $PM_{2.5}$ concentration in the smoking nightclubs was higher than those found in the non-smoking nightclub by 26 times on weekdays and three times on weekends. Indoor $PM_{2.5}$ concentration was correlated with the number of people in the smoking nightclubs. Relatively high $PM_{2.5}$ concentration was observed in the non-smoking nightclub on weekends. NNK concentration in the smoking nightclub was 7 times higer than in the non-smoking nightclub. Conclusion: Smoking in nightclubs caused high $PM_{2.5}$ concentration. Although the non-smoking nightclub had a lower $PM_{2.5}$ concentration, $PM_{2.5}$ concentration on weekends was higher due to the smoking room. Complete prohibition of smoking in nightclubs can protect patrons from secondhand smoke exposure.