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Aim: Men are more susceptible to liver fibrosis (LF) than women. However, the underlying molecular mechanism, especially the role of estrogen/estrogen receptor (ER) activation in this sexual dimorphism is unclear. Therefore, the aim of the current study was to investigate the impact and the underlying molecular mechanisms of estrogen/ER activation on diethyl nitrosamine (DEN)-induced LF., Main Methods: Thirty ovariectomized (OVX) female rats were randomly allocated into five groups (n = 6), and received no treatment, diethyl nitrosamine (DEN), DEN/fulvestrant, DEN/silymarin or DEN/estradiol benzoate (EB). In addition, three sham groups received no treatment, DEN or DEN/fulvestrant, and one control group that neither ovariectomized nor treated. Directly after treatment, liver injury biomarkers were measured. In addition, hepatic tissue hydroxyproline, TNF- α, TGF- β, and IL-10 were evaluated. Expression of NF-kβ, CD68 (a marker for macrophage infiltration), ER-β and TLR-4 were measured. Finally, liver tissue histopathology was assessed., Key Findings: Ovariectomy aggravates DEN-induced LF, as it significantly elevated all liver tissue injury biomarkers. This effect has become even worse after blocking ER by fulvestrant, indicating a protective role of estrogen/ER activation against DEN-induced LF. Inhibition of TLR-4/NF-kβ signaling pathway contributed to this protective effect, as estrogen deprivation or blocking of ER significantly activates this pathway during the onset of LF. While administration of EB or silymarin (selective ER-β activator) improved LF indices and deactivated this pathway., Significance: These results provide new insight into the pivotal role of estrogen/ER activation via modulation of TLR-4/NF-kβ, in the alleviation of LF pathogenesis., Competing Interests: Declaration of competing interest The authors report no declaration of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this study, a visible-light-induced intermolecular [2+2] photocycloaddition reaction based on flavonoids was constructed to address the problems of low yield, poor physicochemical properties, and lack of target definition in total synthesis of (±)-millpuline A whose bioactivity remains unknown. As a result, 20 derivatives were synthesized for bioactivity evaluation. Consequently, lung cell protective effects of (±)-millpuline A and compound B13 a were revealed for the first time and the crucial role of stereoconfiguration of the cyclobutane moiety in their protective effects against NNK in normal lung cells was demonstrated. Moreover, through target prediction and experimental verification in MLE-12 cells, SRC was determined to be the target of (±)-millpuline A regarding its protective effect in NNK-induced lung cell injury. Results from RT-Q-PCR and HTRF experiments verified that (±)-millpuline A could repress SRC activity through a transcriptional mechanism but not acting as an inhibitor to directly bind to and thereby inhibit SRC protein. The results in this paper are informative for the further development of visible light-catalyzed cycloaddition of flavonoids and lay a scientific foundation for understanding the bioactivity and underlying mechanism of (±)-millpuline A and other structurally similar natural skeletons., (© 2023 Wiley-VCH GmbH.)

Low responsiveness to chemotherapy is an important cause of poor prognosis in pancreatic cancer. Smoking is a high-risk factor for pancreatic cancer and cancer resistance to gemcitabine; however, the underlying mechanisms remain unclear. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the main metabolite of tobacco burning and has been shown to be associated with cancer development and chemoresistance. However, in pancreatic cancer, its mechanism remains poorly understood. In this study, we found that NNK promoted stemness and gemcitabine resistance in pancreatic cancer cell lines. Moreover, NNK increased autophagy and elevated the expression levels of the autophagy-related markers autophagy-related gene 5 (ATG5), autophagy-related gene 7 (ATG7), and Beclin1. Furthermore, the results showed that NNK-promoted stemness and gemcitabine resistance was partially dependent on the role of NNK in cell autophagy, which is mediated by the β2-adrenergic receptor (β2AR)-Akt axis. Finally, we proved that NNK intervention could not only activate β2AR, but also increase its expression, making β2AR and Akt form a feedback loop. Overall, these findings show that the NNK-induced β2AR-Akt feedback loop promotes stemness and gemcitabine resistance in pancreatic cancer cells., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Aims: Lung cancer is the leading cause of cancer death worldwide, and tobacco smoking is a recognized major risk factor for lung tumor development. We analyzed the effect of tobacco-specific nitrosamines (TSNAs) on human lung adenocarcinoma metabolic reprogramming, an emergent hallmark of carcinogenesis. Results: A series of in vitro and in vivo bioenergetic, proteomic, metabolomic, and tumor biology studies were performed to analyze changes in lung cancer cell metabolism and the consequences for hallmarks of cancer, including tumor growth, cancer cell invasion, and redox signaling. The findings revealed that nicotine-derived nitrosamine ketone (NNK) stimulates mitochondrial function and promotes lung tumor growth in vivo . These malignant properties were acquired from the induction of mitochondrial biogenesis induced by the upregulation and activation of the beta-2 adrenergic receptors (β2-AR)-cholinergic receptor nicotinic alpha 7 subunit (CHRNAα7)-dependent nitrosamine canonical signaling pathway. The observed NNK metabolic effects were mediated by TFAM overexpression and revealed a key role for mitochondrial reactive oxygen species and Annexin A1 in tumor growth promotion. Conversely, ectopic expression of the mitochondrial antioxidant enzyme manganese superoxide dismutase rescued the reprogramming and malignant metabolic effects of exposure to NNK and overexpression of TFAM, underlining the link between NNK and mitochondrial redox signaling in lung cancer. Innovation: Our findings describe the metabolic changes caused by NNK in a mechanistic framework for understanding how cigarette smoking causes lung cancer. Conclusion: Mitochondria play a role in the promotion of lung cancer induced by tobacco-specific nitrosamines. Antioxid. Redox Signal. 36, 525-549.

Cigarette smoking greatly promotes the progression of kidney renal clear cell carcinoma (KIRC), however, the underlying molecular events has not been fully established. In this study, RCC cells were exposed to the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine) for 120 days (40 passages), and then the soft agar colony formation, wound healing and transwell assays were used to explore characteristics of RCC cells. RNA-seq was used to explore differentially expressed genes. We found that NNK promoted RCC cell growth and migration in a dose-dependent manner, and RNA-seq explored 14 differentially expressed genes. In TCGA-KIRC cohort, Lasso regression and multivariate COX regression models screened and constructed a five-gene signature containing ANKRD1, CYB5A, ECHDC3, MT1E, and AKT1S1. This novel gene signature significantly associated with TNM stage, invasion depth, metastasis, and tumor grade. Moreover, when compared with individual genes, the gene signature contained a higher hazard ratio and therefore had a more powerful value for the prognosis of KIRC. A nomogram was also developed based on clinical features and the gene signature, which showed good application. Finally, AKT1S1, the most crucial component of the gene signature, was significantly induced after NNK exposure and its related AKT/mTOR signaling pathway was dramatically activated. Our findings supported that NNK exposure would promote the KIRC progression, and the novel cigarette smoke-related five-gene signature might serve as a highly efficient biomarker to identify progression of KIRC patients, AKT1S1 might play an important role in cigarette smoke exposure-induced KIRC progression., (© 2022. The Author(s).)

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a Group 1 human carcinogen, as classified by the International Agency for Research of Cancer (IARC), and plays a significant role in lung carcinogenesis. However, its carcinogenic mechanism has not yet been fully elucidated. In this study, we performed colony formation assays, soft-agar assays, and tumor growth in nude mice to show that 100 mg/L NNK facilitates the malignant transformation of human bronchial epithelial Beas-2B cells. Transcriptome sequencing showed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a post-transcriptional regulator, was differentially expressed in NNK-induced malignant transformed Beas-2B cells (2B-NNK cells). Small interfering RNA (SiRNA) was used to downregulate the expression of the IGF2BP1 gene. The reduction in protein expression, cell proliferation rate, and colony-forming ability and the increase in the apoptosis rate of Beas-2B cells transfected with the SiRNA indicated a role for IGF2BP1 in NNK-induced malignant transformation. IGF2BP1 is an N6-methyladenosine (m6A) regulatory factor, but it is not known whether its association with m6A mediates the malignant transformation of cells. Therefore, we measured the overall levels of m6A in Beas-2B cells. We found that the overall m6A level was lower in 2B-NNK cells, and knocking down IGF2BP1, the overall level of m6A was restored. Hence, we concluded that IGF2BP1 is involved in the NNK-induced malignant transformation of Beas-2B cells, possibly via m6A modification. This study therefore contributes novel insights into the environmental pathogenesis of lung cancer and the gene regulatory mechanisms of chemical carcinogenesis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Iron oxide nanoparticles (magnetite) have been widely used in industry and medicine. However, the safety assessment of magnetite has not been fully completed. The present study was conducted to assess effects of magnetite on carcinogenic activity, using a medium-term bioassay protocol. A total of 100 male Fischer 344 rats, 6 weeks old, were randomly divided into 5 groups of 20 animals each, and given a basal diet and drinking water containing 0 or 0.1% of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for 2 weeks. Two weeks later, the rats were intratracheally instilled magnetite 7 times at an interval of 4 weeks, at the doses of 0, 1.0 or 5.0 mg/kg body weight, and sacrificed at the end of the experimental period of 30 weeks. The multiplicities of macroscopic lung nodules and histopathologically diagnosed bronchiolo-alveolar hyperplasia, induced by DHPN, were both significantly decreased by the high dose of magnetite. The expression of minichromosome maintenance (MCM) protein 7 in non-tumoral alveolar epithelial cells, and the number of CD163-positive macrophages in tumor nodules were both significantly reduced by magnetite. It is suggested that magnetite exerts inhibitory effects against DHPN-induced lung tumorigenesis, by the reduction of alveolar epithelial proliferation and the M2 polarization of tumor-associated macrophages., (Copyright © 2021. Published by Elsevier Inc.)

The local delivery of gaseous signaling molecules (GSMs) has shown promising therapeutic potential. However, although GSMs have a subtle interplay in physiological and pathological conditions, the co-delivery of different GSMs for therapeutic purposes remains unexplored. Herein, we covalently graft a nitric oxide (NO)-releasing N-nitrosamine moiety onto the carbon monoxide (CO)-releasing 3-hydroxyflavone (3-HF) antenna, resulting in the first NO/CO-releasing donor. Under visible light irradiation, photo-mediated co-release of NO and CO reveals a superior antimicrobial effect toward Gram-positive bacteria with a combination index of 0.053. The synergy of NO and CO hyperpolarizes and permeabilizes bacterial membranes, which, however, shows negligible hemolysis and no evident toxicity toward normal mammalian cells. Moreover, the co-release of NO and CO can efficiently treat MRSA infection in a murine skin wound model, showing a better therapeutic capacity than vancomycin., (© 2021 Wiley-VCH GmbH.)

The present study was undertaken to evaluate the chemopreventive activity of myrtenal, a natural monoterpene, against bladder carcinoma in rats induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and promoted with γ-ionizing radiation (γ-IRR) as well as to assess the involvement of inflammation, apoptosis and oxidative damage in tumor development. Histopathological examination of rat bladder revealed the presence of noninvasive papillary transitional cell carcinoma (Grade 2) in sections from BBN group indicating the credibility of the applied carcinogenesis model. Myrtenal treatment caused improvement in urinary bladder mucosa with cells more likely in Grade 1. Administration of myrtenal to BBN-treated rats exhibited downregulation in the expressions of COX-2, NF-kB and STAT-3 associated with suppression of inflammatory cytokines levels of TNF-α and IL-6 as well as biomarkers of oxidative damage (MDA & NO). In addition, myrtenal treatment caused a significant increase in caspase-3 activity and Bax/Bcl-2 ratio. Data obtained suggested that the anti-inflammatory effect and the induction of apoptosis contributed largely to the beneficial antitumor effects of myrtenal in rats with BBN/γ-IRR-induced bladder carcinoma. Present findings, in addition to benefits described in other pathologies, indicated myrtenal as a potential adjuvant natural compound for the prevention of tumor progression of bladder cancer.

A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Bacterial resistance to antimicrobial agents is increasing at an alarming rate globally and requires new lead compounds for antibiotics. In this study, N-phenyl-N-nitroso hydroxylamine (cupferron) and its derivatives have been synthesised using readily available starting materials. The compounds were obtained in high yield and purity. They show activity towards a range of Gram-positive and Gram-negative pathogenic bacteria, with minimum inhibitory concentration (MIC) values as low as 2 μg.mL -1 against the tested organisms, especially for Gram-positive species. Toxicity studies on the lead compound 3b indicate insignificant effects on healthy cell lines. Molecular docking studies on the lead compound identify possible binding modes of the compound, and the results obtained correlate with those of in vitro and MIC studies. The lead compound shows excellent drug-likeness properties., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chronic stress increases the systemic levels of stress hormones norepinephrine and cortisol. As well as tobacco-specific carcinogen NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone), they can induce expressive DNA damage contributing to the cancer development. However, it is unknown whether stress hormones have genotoxic effects in oral keratinocytes. This study investigated the effects of stress hormones on DNA damage in a human oral keratinocyte cell line (NOK-SI). NOK-SI cells stimulated with norepinephrine or cortisol showed higher DNA damage compared to untreated cells. Norepinephrine-induced DNA damage was reversed by pre-treatment with beta-adrenergic blocker propranolol. Cells treated with NNK combined to norepinephrine displayed reduced levels of caspases 3 and 7. Cortisol also reduced the activity of pro-apoptotic enzymes. NNK or norepinephrine promoted single-strand breaks and alkali-label side breaks in the DNA of NOK-SI cells. Pre-treatment of cells with propranolol abolished these effects. Carcinogen NNK in the presence or absence of cortisol also induced DNA damage of these cells. The genotoxic effects of cortisol alone and hormone combined with NNK were blocked partially and totally, respectively, by the glucocorticoid receptor antagonist RU486. DNA damage promoted by NNK or cortisol and carcinogen combined to the hormone led to intracellular γH2AX accumulation. The effects caused by NNK and cortisol were reversed by propranolol and glucocorticoid receptor antagonist RU486, respectively. Propranolol inhibited the oxidation of basis induced by NNK in the presence of DNA-formamidopyrimidine glycosylase. DNA breaks induced by norepinephrine in the presence or absence of NNK resulted in higher 8OHdG cellular levels. This effect was also induced through beta-adrenergic receptors. Together, these findings indicate that stress hormones induce DNA damage of oral keratinocytes and could contribute to oral carcinogenesis., (© 2021. The Author(s).)

An increased risk of developing lung cancer has been associated with exposure to cigarette smoke carcinogens and alteration in the gut microbiota. However, there is limited understanding about the impact of exposure to NNK and BaP, the two important components of cigarette smoke carcinogens, on gut microbiota in lung cancer. The present study characterized the influence of exposure to a mixture of NNK plus BaP on lung cancer, feces metabolite composition, and gut microbiota in the A/J mice. The A/J mice were administered NNK plus BaP, and the changes in gut microbiota and feces metabolic profiles were characterized using 16S rRNA gene sequencing and metabolomics, respectively. Results presented here illustrated that a mixture of NNK plus BaP exposure triggered lung carcinogenesis as shown by light microscopy and histopathological evaluation. 16S rRNA sequencing of gut microbiota indicated that exposure to NNK plus BaP could modified fecal bacterial composition. Elevated levels of Actinobacteria, Bifidobacterium, and Intestinimonas and reduced levels of Alistipes, Odoribacter, and Acetatifactor are associated with NNK plus BaP triggered lung cancer. In addition, metabolomics profile revealed the regulation of metabolism including purine metabolism, phenylalanine metabolism, primary bile acid biosynthesis, steroid hormone biosynthesis, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and others. In conclusion, the results provide some guidance for using gut microbes as biomarkers to assess the progression of lung cancer, and lead to interventional targets to control the development of the disease in the future., (Copyright © 2021. Published by Elsevier B.V.)

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N '-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N 7 POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O -(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 10 8 nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and nonsmokers. Rats treated with 5 or 21 mg/kg bw NNK for 4 days by s.c. injection had 2-6 and 2-17 times more AP sites than controls in liver and lung DNA ( p < 0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks ( p < 0.05) but not for 50 and 70 weeks to 5 ppm of NNK in their drinking water. Levels of N 7 POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm ( S )-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA ( p < 0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to nonsmokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals and to evaluate AP sites in DNA of smokers and nonsmokers.

Background: Blockade of cannabinoid CB1 or vanilloid TRPV1 receptors in the ventromedial prefrontal cortex of rats respectively increases or decreases the conditioned emotional response during re-exposure to a context previously paired with footshocks. Although these mechanisms are unknown, they may involve local modulation of glutamatergic and nitrergic signaling., Aim: We investigated whether these mechanisms are involved in the reported effects of CB1 and TRPV1 modulation in the ventromedial prefrontal cortex., Methods: Freezing behavior and autonomic parameters were recorded during the conditioned response expression., Results: The CB1 receptors antagonist NIDA, or the TRPV1 agonist capsaicin (CPS) in the ventromedial prefrontal cortex increased the conditioned emotional response expression, and these effects were prevented by TRPV1 and CB1 antagonism, respectively. The increased conditioned emotional response evoked by NIDA and CPS were prevented by an NMDA antagonist or a neuronal nitric oxide synthase inhibitor. A nitric oxide scavenger or a soluble guanylate cyclase inhibitor prevented only the NIDA effects and the CPS effect was prevented by a non-selective antioxidant drug, as nitric oxide can also induce reactive oxygen species production., Conclusion: Our results suggest that CB1 and TRPV1 receptors in the ventromedial prefrontal cortex differently modulate the expression of conditioned emotional response through glutamatergic and nitrergic mechanisms, although different pathways may be involved.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific nitrosamine (TSNA) that induces malignant tumors in rodents. High-risk human papillomavirus (hr-HPV) infection is an important cause of several human cancers. Epidemiological evidence has shown that HPV cooperatively induces carcinogenesis with tobacco smoke. In the present study, the synergistic carcinogenesis of NNK and HPV18 was investigated. Immortalized human esophageal epithelial SHEE cells containing the HPV18 E6E7 gene were constructed by lentiviral transfection. SHEE-E6E7 cells were exposed to NNK along with SHEE-V cells without HPV18 E6E7 as a negative control. The cooperation of NNK and HPV was examined by wound-healing, transwell, and colony-forming assays. The results showed that NNK exposure promoted the migration, invasion, and proliferation abilities of both SHEE-E6E7 and SHEE-V cells; however, the changes in these phenotypic features were remarkably stronger in SHEE-E6E7 cells than those in SHEE-V cells. Our findings indicate that NNK promotes malignant transformation of human esophageal epithelial cells and suggest a synergistic carcinogenesis with the HPV18 E6E7 oncogene. As reported previously, the formation of pyridyloxybutylated DNA adducts is a crucial step in NNK-mediated carcinogenesis. In order to clarify the influence of HPV on the formation of NNK-induced DNA adducts, the amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts were determined using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. We observed that the levels of HPB-releasing adducts in SHEE-E6E7 cells were significantly higher ( p < 0.01) than those of SHEE-V cells, which was in line with results of the phenotypic assays. In conclusion, this study provides direct evidence that NNK and HPV18 exhibit a synergistic effect on formation of DNA adducts, resulting in malignant transformation of esophageal epithelial cells. Such knowledge on the interaction between infection and smoking habits in the development of cancers informs cancer-prevention strategies. Further studies to delineate the molecular mechanism and to identify specific intervention targets are worthwhile.

The anticancer mechanism of NO is difficult to study owing to its short lifetime and high reactivity. Thus, a theranostic anticancer NO donor assembled with NO on-demand release abilities, accurate lysosome location capabilities and signal feedback behavior was developed. Profiting from the theranostic properties, the specific mechanism was comprehensively studied. Spectral and cell imaging studies revealed that the as prepared NO donors could release NO in solution or within cancer cells. Fluorescence co-dyeing experiments demonstrated that Mo-Nap-NO entered lysosomes specifically and disrupted them after being triggered by light. Upon irradiation with 460 nm visible light, both the donors demonstrated considerable in vitro anticancer effects. A further mechanistic study showed that after entering the lysosome and being triggered by 460 nm irradiation, NO ruptured the lysosome, resulting in the release of cathepsin D into the cytosol, which activated the caspase3 mediated apoptosis pathway.

Cancer is the second deadliest disease listed by the WHO. One of the major causes of cancer disease is tobacco and consumption possibly due to its main component, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). A plethora of studies have been conducted in the past aiming to decipher the association of NNK with other diseases. However, it is strongly linked with cancer development. Despite these studies, a clear molecular mechanism and the impact of NNK on various system-level networks is not known. In the present study, system biology tools were employed to understand the key regulatory mechanisms and the perturbations that will happen in the cellular processes due to NNK. To investigate the system level influence of the carcinogen, NNK rewired protein-protein interaction network (PPIN) was generated from 544&nbsp;reported proteins drawn out from 1317 articles retrieved from PubMed. The noise was removed from PPIN by the method of modulation. Gene ontology (GO) enrichment was performed on the seed proteins extracted from various modules to find the most affected pathways by the genes/proteins. For the modulation, Molecular COmplex DEtection (MCODE) was used to generate 19 modules containing 115 seed proteins. Further, scrutiny of the targeted biomolecules was done by the graph theory and molecular docking. GO enrichment analysis revealed that mostly cell cycle regulatory proteins were affected by NNK., Competing Interests: The authors declare no conflict of interest.

Methylating substances alter DNA by forming N3-methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), we synthesized the N3mT-3'-phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N'-nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5-hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5-methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE-LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Quantitative measurement of DNA adducts in carcinogen-exposed cells provides the information about the frequency of formation and the rate of removal of DNA lesions in vivo, which yields insights into the initial events of mutagenesis. Metabolic activation of tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), leads to pyridyloxobutylation and pyridylhydroxybutylation of DNA. In this study, we employed a highly robust nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the isotope-dilution method for simultaneous quantification of O 6 -[4-(3-pyridyl)-4-hydroxylbut-1-yl]-2'-deoxyguanosine ( O 6 -PHBdG) and O 2 - and O 4 -[4-(3-pyridyl)-4-hydroxylbut-1-yl]-thymidine ( O 2 -PHBdT and O 4 -PHBdT). Cultured mammalian cells were exposed to a model pyridylhydroxybutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALOAc), followed by DNA extraction, enzymatic digestion, and sample enrichment prior to nLC-nESI-MS/MS quantification. Our results demonstrate, for the first time, that O 4 -PHBdT is quantifiable in cellular DNA and naked DNA upon NNALOAc exposure. We also show that nucleotide excision repair (NER) machinery may counteract the formation of O 2 -PHBdT and O 4 -PHBdT, and O 6 -alkylguanine DNA alkyltransferase (AGT) may be responsible for the repair of O 6 -PHBdG and O 4 -PHBdT in mammalian cells. Together, our study provides new knowledge about the occurrence and repair of NNAL-induced DNA lesions in mammalian cells.

Combination of photosensitizers (PS) for photodynamic therapy with NO photodonors (NOPD) is opening intriguing horizons towards new and still underexplored multimodal anticancer and antibacterial treatments not based on "conventional" drugs and entirely controlled by light stimuli. In this contribution, we report an intriguing molecular hybrid based on a BODIPY light-harvesting antenna that acts simultaneously as PS and NOPD upon single photon excitation with the highly biocompatible green light. The presented hybrid offers a combination of superior advantages with respect to the other rare cases reported to date, meeting most of the key criteria for both PSs and NOPDs in the same molecular entity such as: (i) capability to generate 1 O 2 and NO with single photon excitation of biocompatible visible light, (ii) excellent 1 O 2 quantum yield and NO quantum efficiency, (iii) photogeneration of NO independent from the presence of oxygen, (iv) large light harvesting properties in the green region. Furthermore, this compound together with its stable photoproduct, is well tolerated by both normal and cancer cells in the dark and exhibits bimodal photomortality of cancer cells under green light excitation due to the combined action of the cytotoxic 1 O 2 and NO., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Many of the toxicants in tobacco smoke undergo biotransformation in the lungs of smokers, both to reactive and to detoxified derivatives. Human air-liquid-interface (ALI) airway tissue models have emerged as an advanced in vitro model for evaluating the toxicity of inhaled substances; however, the metabolic potential of these cultures has not been evaluated extensively. In this study, we compared the metabolic activities of an ALI tissue model to the undifferentiated normal human primary bronchial epithelial (NHBE) cells from which it was derived. Measurement of the basal levels of gene expression for 84 phase I drug metabolism enzymes indicated that most genes were upregulated in ALI cultures compared to NHBE cells. Furthermore, the enzymatic activities of three cytochrome P450s involved in the bioactivation of tobacco-specific nitrosamines were higher in the ALI cultures, and the bioactivation of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), as measured by the formation of two of its major metabolites, i.e., keto acid and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was significantly greater in the ALI cultures. Finally, NNK was a direct-acting genotoxicant in the ALI cultures, while the genotoxicity of NNK was detected in NHBE cells only in the presence of an exogenous liver S9 activation system. Taken together, our findings demonstrate the greater metabolic potential of well-differentiated ALI cultures than primary NHBE cells, supporting the potential use of ALI airway cultures as an alternative in vitro model for evaluating inhaled toxicants that require metabolic transformation., (Published by Elsevier Ltd.)

The role of niacin's metabolite, nicotinamide adenine dinucleotide (NAD), in DNA repair via base-excision repair pathway is well documented. We evaluated if niacin deficiency results in genetic instability in normal human fetal lung fibroblasts (MRC-5), and further, does it leads to enhanced accumulation of cigarette smoke-induced genetic damage? MRC-5 cells were grown discretely in niacin-proficient/deficient media, and exposed to nicotine-derived nitrosamine ketone (NNK, a cigarette smoke carcinogen). Niacin deficiency abated the NAD polymerization, augmented the spontaneous induction of micronuclei (MN) and chromosomal aberrations (CA) and raised the expression of 10 genes and suppressed 12 genes involved in different biological functions. NNK exposure resulted in genetic damage as measured by the induction of MN and CA in cells grown in niacin-proficient medium, but the damage became practically marked when niacin-deficient cells were exposed to NNK. NNK exposure raised the expression of 16 genes and suppressed the expression of 56 genes in cells grown in niacin-proficient medium. NNK exposure to niacin-deficient cells raised the expression of eight genes including genes crucial in promoting cancer such as FGFR3 and DUSP1 and suppressed the expression of 33 genes, including genes crucial in preventing the onset and progression of cancer like RASSF2, JUP, and IL24, in comparison with the cells grown in niacin-proficient medium. Overall, niacin deficiency interferes with the DNA damage repair process induced by chemical carcinogens like NNK, and niacin-deficient population are at the higher risk of genetic instability caused by cigarette smoke carcinogen NNK., (© 2018 Wiley Periodicals, Inc.)

Our understanding of dose-related effects of polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, is limited. In the present study, the effect of various doses of black tea (0.75, 1.5, and 3%)-derived PBP-rich extract on biochemical parameters and lung carcinogenicity in A/J mice was investigated. Pretreatment with PBPs showed the dose-related decrease in B(a)P-induced expression and activity of CYP1A1 in the liver while CYP1A2 expression and activity in the lung. Dose-dependent significant increase in PBP-mediated over-expression and activity of GSTs (alpha in the liver while pi in the lung) were observed in polyphenol-treated groups. Significant dose-related decrease in number and intensity of BPDE-DNA adducts were observed in liver and lung. Black tea (1.5%, 3%)-derived PBPs showed dose-mediated decrease in lung tumor incidence and multiplicity which was further correlated with different molecular markers like cell proliferation and apoptosis in B(a)P and NNK model. In conclusion, dose-dependent chemopreventive effects of PBPs, both anti-initiating (induction of phase II and inhibition of carcinogen-induced phase-I enzymes leading to decrease in BPDE-DNA adducts) and anti-promoting (decreased cell proliferation and increased apoptosis lowering incidence and/or multiplicity of lung lesions), were observed in A/J mice without significant toxicity.

Background: The present study describes glycerosomes (vesicles composed of phospholipids, glycerol and water) as a novel drug delivery system for topical application of celecoxib (CLX) and cupferron (CUP) compound., Aim: The goal of this research was to design topical soft innovative vesicles loaded with CLX or CUP for enhancing the efficacy and avoiding systemic toxicity of CLX and CUP., Methods: CLX and CUP loaded glycerosomes were prepared by hydrating phospholipid-cholesterol films with glycerol aqueous solutions (20-40%, v/v). Box-Behnken design, using Design-Expert® software, was the optimum choice to statistically optimize formulation variables. Three independent variables were evaluated: phospholipid concentration (X 1 ), glycerol percent (X 2 ) and tween 80 concentration (X 3 ). The glycerosomes particle size (Y 1 ), encapsulation efficiency percent (Y 2 : EE %) and drug release (Y 3 ) were selected as dependent variables. The anti-inflammatory effect of CLX and CUP glycerosomal gel was evaluated by carrageenan-induced rat paw edema method followed by histopathological studies., Results: The optimized formulations (CLX2* and CUP1*) showed spherical morphology under transmission electron microscopy, optimum particle size of 195.4 ± 3.67 nm, 301.2 ± 1.75 nm, high EE of 89.66 ± 1.73%, 93.56 ± 2.87%, high drug release of 47.08 ± 3.37%, 37.60 ± 1.89% and high cumulative amount of drug permeated in 8 h of 900.18 ± 50.24, 527.99 ± 34.90 µg.cm -2 through hairless rat skin, respectively. They also achieved significant remarkable paw edema inhibition in comparison with the control group (p < .05)., Conclusion: Finally, the administration of CLX2* and CUP1* loaded glycerosomal gel onto the skin resulted in marked reduction of edema, congestive capillary and inflammatory cells and this approach may be of value in the treatment of different inflammatory disorders.

Objectives: The aim of this study was to comparatively analyze the effects of different concentrations of cigarette smoke condensate (CSC, a standardized tobacco extract) and ethanol on intracellular enzyme activation, cell necrosis, alteration of cytosolic calcium concentration ([Ca]c), and amylase secretion in pancreatic acinar cells., Methods: The effects of CSC (1 μg/mL to 0.4 mg/mL) and ethanol (10-100 mM) on intracellular enzyme activity, cell necrosis, and [Ca]c were measured by fluorescence assays in isolated pancreatic acinar cells. Amylase secretion was evaluated by spectrophotometry. Supramaximal concentrations of cholecystokinin (10-100 nM) were used as positive control., Results: Neither CSC nor ethanol induced trypsin or elastase activation. Both CSC (0.1-0.4 mg/mL) and ethanol (10-75 mM) significantly increased [Ca]c. Amylase secretion was increased only in CSC-treated cells (0.3 and 0.4 mg/mL). After 60 minutes, CSC (0.3 and 0.4 mg/mL) significantly increased acinar cell necrosis at a similar percentage to that induced by cholecystokinin. Ethanol did not induce any significant cell necrosis., Conclusions: Cigarette smoke condensate induces acinar cell injury and increases [Ca]c and amylase secretion, independently of intracellular enzyme activation, suggesting that tobacco could induce several main early events of pancreatitis in pancreatic acinar cells. However, ethanol only induces increases [Ca]c, having no effect on cell injury, amylase secretion, or intracellular enzyme activation.

Long-term nicotine-derived nitrosamine ketone (NNK) and arecoline exposure promotes carcinogenesis and head and neck squamous cell carcinoma (HNSCC) progression, although most associated data on the two were analyzed individually. The molecular mechanisms underlying tumor progression associated with the synergistic effects of NNK and arecoline remain unclear. We treated SCC-25 and FaDu cells with NNK and arecoline (separately or in combination) for 3 months. Comparative analysis was performed to investigate the mechanism underlying the acquisition of properties related to tumor promotion, including stemness, anti-apoptosis, and resistance to HNSCC therapeutics. Long-term exposure to NNK and arecoline resulted in an increase in cancer stem cell properties, anti-apoptosis, and the resistance to cisplatin in HNSCC. We detected abundant epidermal growth factor receptor (EGFR) expression in HNSCC cells after combined treatment with NNK and arecoline. EGFR was pivotal in inducing tumor promotion and anti-apoptosis in cancer cells by inducing pAKT and NFκB. Combined treatment with NNK and arecoline synergistically facilitated tumor aggressiveness via EGFR-AKT signaling. Targeting EGFR-AKT signaling may be a feasible strategy for treating HNSCC., Competing Interests: The authors have declared that no competing interests exist.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are carcinogenic in animal models and are believed to play an important role in human lung carcinogenesis for cigarette smokers. Cytochrome P450-mediated metabolism of these tobacco-specific nitrosamines produces reactive species that alkylate DNA in the form of pyridyloxobutyl (POB)- or pyridylhydroxybutyl (PHB)-DNA adducts. Understanding the formation mechanism and overall levels of these adducts can potentially enhance cancer prevention methods through the identification of particularly susceptible smokers. Previous studies have identified and measured a panel of POB- and PHB-DNA base adducts of dGuo, dCyd, and Thd; however, dAdo adducts have yet to be determined. In this study, we complete this DNA adduct panel by identifying and quantifying levels of NNK- and NNAL-derived dAdo adducts in vitro and in vivo. To accomplish this, we synthesized standards for expected dAdo-derived DNA adducts and used isotope-dilution LC-ESI + -MS/MS to identify POB adducts formed in vitro from the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) with calf thymus DNA. Adduct levels were then quantified in lung and liver DNA of rats chronically treated with NNK or NNAL for 50 weeks using similar LC-MS detection methods. The in vitro studies identified N 6 -POB-dAdo and N 1 -POB-dIno as products of the reaction of NNKOAc with DNA, which supports our proposed mechanism of formation. Though both N 6 -dAdo and N 1 -dIno adducts were found in vitro, only N 6 -dAdo adducts were found in vivo, implying possible intervention by DNA repair mechanisms. Analogous to previous studies, levels of N 6 -POB-dAdo and N 6 -PHB-dAdo varied both with tissue and treatment type. Despite the adduct levels being relatively modest compared to most other POB- and PHB-DNA adducts, they may play a biological role and could be used in future studies as NNK- and NNAL-specific DNA damage biomarkers.

Background/aim: Our aim was to investigate whether tissue with fatty infiltration within the lobes of the pancreas (scattered FI) is sensitive to carcinogen-induced pancreatic ductal proliferation., Materials and Methods: Seven-week-old female C57BL/6J, C57BL/6J-A y , KK-A y , and ICR mice were subcutaneously treated with N-nitrosobis(2-oxopropyl) amine at a dose of 80 mg/kg body weight, and the differences in damage-induced cell proliferation and their biochemical data were compared 2 days after., Results: Scattered FI in the pancreas was obvious only in KK-A y mice, which have high serum lipid, leptin and insulin levels, and cell proliferation both in pancreatic and common bile ducts was enhanced only in KK-A y mice by the carcinogen treatment., Conclusion: Scattered FI in the pancreas per se can be an important factor for carcinogenesis. The genetic background causing scattered FI of the pancreas should be further investigated., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Nitric oxide (NO) potentially plays a regulatory role in mitochondrial fusion and fission, which are vital to cell survival and implicated in health, disease, and aging. Molecular tools facilitating the study of the relationship between NO and mitochondrial dynamics are in need. We have recently developed a novel NO donor (NOD550). Upon photoactivation, NOD550 decomposes to release two NO molecules and a fluorophore. The NO release could be spatially mapped with subdiffraction resolution and with a temporal resolution of 10 s. Due to the preferential localization of NOD550 at mitochondria, morphology and dynamics of mitochondria could be monitored upon NO release from NOD550.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL, or (R)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290-4510, 872-1120, and 763-1430 fmol/mg DNA, accounting for 15-38%, 8%, and 5-9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents.

Background: The high levels of blood alcohol achieved with chronic plus binge alcohol exposures are somewhat reduced by co-administration of tobacco-specific Nicotine-Derived Nitrosamine Ketone (NNK) suggesting that NNK may alter alcohol metabolism., Objective: We examined ethanol and acetaldehyde-metabolizing enzyme activities and malondialdehyde adduct formation in rats exposed to ethanol (chronic + binge), NNK, or both., Methods: 4-week old Long Evans rats were fed liquid diets containing 0% or 26% caloric ethanol for 8 weeks. Ethanol-fed rats were binge-administered ethanol (2 g/kg; on Mondays, Wednesdays, and Fridays) by intraperitoneal (i.p.) injection, while control group administered saline in weeks 7 and 8 (n=12/group). Six rats from each group were administered i.p. injections of NNK (2 mg/kg) or saline on Tuesdays, Thursdays, and Saturdays of weeks 3 through 8. Alcohol dehydrogenase, catalase, and aldehyde dehydrogenase activities were measured using commercial assays. Cytochrome P450 mRNA levels (17 isoforms) were measured by quantitative reverse transcription-polymerase chain reaction. Malondialdehyde immunoreactivity was measured by enzyme-linked immunosorbent assay., Results: Dual exposures to ethanol and NNK significantly increased frontal lobe ADH activity relative to control (P=0.01) and ethanol only (P=0.04) treatments, and ALDH relative to control (P=0.02). In contrast, malondialdehyde-protein expression was not significantly altered by ethanol+NNK. Ethanol decreased CYP1A1 mRNA expression relative to control (P=0.02), and combined ethanol+NNK exposures decreased the expression of CYP1A1 (P=0.01) and CYP2C6 (P=0.03)., Conclusion: Dual exposures to ethanol and NNK increase brain ethanol metabolism and inhibit the expression of CYP450s that regulate xenobiotic metabolism., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Molsidomine is a well-known vasodilatating, antianginal drug. Despite earlier studies with its metabolites (3-morpholino-syndnonimine (SIN-1) and N-nitroso-N-morpholino-amino-acetonitrile (SIN-1A)), which indicated a potential favorable cardioprotective activity, a lot of controversy remains. The aim of our research was to compare molsidomine, SIN-1, SIN-1A, and lidocaine influence on arrhythmias and hemodynamic parameters in 2 experimental models in rats. In the Langendorff heart study, SIN-1A markedly elevated left ventricular systolic pressure, maximum rise and fall of the first pressure derivative, coronary flow, and myocardial oxygen consumption. In addition, SIN-1A more so than SIN-1 significantly lowered creatine kinase release. The antiarrhythmic action of SIN-1 was observed, while lidocaine significantly diminished ventricular arrhythmias duration in comparison with the control. In the ischemia-reperfusion-induced arrhythmias model, hypotensive action of molsidomine was observed as well as the reduction in pressure rate product. Molsidomine also prolonged ventricular tachycardia duration. On the other hand, no significant effects on hemodynamic parameters as well as on ventricular arrhythmias were found in any of the SIN-1 and SIN-1A groups. In conclusion, our research suggests a possible direct, cardioprotective action of SIN-1A. It seems worthwhile to further investigate molsidomine derivatives, especially SIN-1A, because of its potential use in invasive cardiology procedures such as percutaneous transluminal coronary angioplasty.

N'-Nitrosonornicotine (NNN) is considered to be one of the most carcinogenic compounds of the four conventionally measured tobacco-specific N-nitrosamines (TSNAs). In order to evaluate the significance of metabolic activation for the carcinogenic potential of NNN, its catalysis by different phase I enzymes and its interaction with nicotine and nicotine-derived TSNAs need to be investigated. Using an in vitro model system, NNN was found to interact with various cytochrome P450 enzymes, predominantly CYP2A13. Mass-spectrometric analysis confirmed the presence of various predicted NNN metabolites, including 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid) but little amount of 4-oxo-4-(3-pyridyl) butanal (OPB), which was somewhat different from in vitro NNK metabolism. Addition of nicotine, N'-Nitrosoanatabine (NAT), N'-Nitrosoanabasine (NAB) resulted in a competitive inhibition for NNN metabolism. The inhibition constant Ki value was calculated as 0.98 μM (nicotine), 1.37 μM (NAT), 0.71 μM (NAB) for HPB formation, 1.35 μM (nicotine), 1.35 μM (NAT), 1.01 μM (NAB) for hydroxy acid formation and 8.40 μM (nicotine), 3.40 μM (NAT), 3.04 μM (NAB) for OPB formation, respectively. These results implied that CYP2A13 is the most efficient enzyme to metabolize NNN in vitro and structurally similar tobacco constitutes including nicotine, NAT and NAB influence the metabolic activation of NNN, which may further interfere in its carcinogenicity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log 10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log 10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log 10 fold, 18 strains showed enhanced growth of 0.3-0.97 log 10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log 10 fold, 8 strains showed enhanced growth of 0.3-1.0 log 10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 μg/ml; while the growth of P. micros was enhanced 0.31-0.54 log 10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log 10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria., (Published by Elsevier Ltd.)

Hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) has been found in various types of human cancers, including head and neck cancer (HNC). Although smoking is critical in the development and progression of HNC, how tobacco components activate STAT3 is unclear. We demonstrated that exposure of HNC cell lines to a tobacco extract induced a rapid Y705 phosphorylation of STAT3 and a rapid increase in the SUMO protease SENP3 that depended on a simultaneous increase in reactive oxygen species. We identified that SUMOylation at the lysine 451 site facilitated STAT3 binding to the phosphatase TC45 through an SUMO-interacting motif of TC45. SENP3 could thus enhance STAT3 phosphorylation by de-conjugating the SUMO2/3 modification of STAT3. Knocking-down of SENP3 greatly impaired basal and induced STAT3 phosphorylation by tobacco extract or interleukin 6. A correlation between SENP3 protein levels and STAT3 Y705 phosphorylation levels in human laryngeal carcinoma specimens was found, which was more significant in the specimens derived from the smoker patients and with poor clinicopathological parameters. Our data identified SUMOylation as a previously undescribed post-translational modification of STAT3 and SENP3 as a critical positive modulator of tobacco- or cytokine-induced STAT3 activation. These findings provide novel insights into the hyperphosphorylation of STAT3 in development of HNC.

Nitric oxide (NO), hydrogen sulfide and polysulfides have been proposed to contribute to redox signaling by activating the Keap-1/Nrf2 stress response system. Nitrosopersulfide (SSNO - ) recently emerged as a bioactive product of the chemical interaction of NO or nitrosothiols with sulfide; upon decomposition it generates polysulfides and free NO, triggering the activation of soluble guanylate cyclase, inducing blood vessel relaxation in vitro and lowering blood pressure in vivo. Whether SSNO - itself interacts with the Keap-1/Nrf2 system is unknown. We therefore sought to investigate the ability of SSNO - to activate Nrf2-dependent processes in human vascular endothelial cells, and to compare the pharmacological effects of SSNO - with those of its precursors NO and sulfide at multiple levels of target engagement. We here demonstrate that SSNO - strongly increases nuclear levels, binding activity and transactivation activity of Nrf2, thereby increasing mRNA expression of Hmox-1, the gene encoding for heme oxygenase 1, without adversely affecting cell viability. Under all conditions, SSNO - appeared to be more potent than its parent compounds, NO and sulfide. SSNO - -induced Nrf2 transactivation activity was abrogated by either NO scavenging with cPTIO or inhibition of thiol sulfuration by high concentrations of cysteine, implying a role for both persulfides/polysulfides and NO in SSNO - mediated Nrf2 activation. Taken together, our studies demonstrate that the Keap-1/Nrf2 redox system is a biological target of SSNO - , enriching the portfolio of bioactivity of this vasoactive molecule to also engage in the regulation of redox signaling processes. The latter suggests a possible role as messenger and/or mediator in cellular sensing and adaptations processes., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Activation of receptor tyrosine kinases (RTKs) is associated with carcinogenesis, but its contribution to smoking-associated lung carcinogenesis is poorly understood. Here we show that a tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced insulin-like growth factor 1 receptor (IGF-1R) activation via β-adrenergic receptor (β-AR) is crucial for smoking-associated lung carcinogenesis. Treatment with NNK stimulated the IGF-1R signaling pathway in a time- and dose-dependent manner, which was suppressed by pharmacological or genomic blockade of β-AR and the downstream signaling including a Gβγ subunit of β-AR and phospholipase C (PLC). Consistently, β-AR agonists led to increased IGF-1R phosphorylation. The increase in IGF2 transcription via β-AR, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-kappa B (NF-κB) was associated with NNK-induced IGF-1R activation. Finally, treatment with β-AR antagonists suppressed the acquisition of transformed phenotypes in lung epithelial cells and lung tumor formation in mice. These results suggest that blocking β-AR-mediated IGF-1R activation can be an effective strategy for lung cancer prevention in smokers.

Introduction: Cigarette smoke (CS) is associated with a broad range of diseases including lung cancer. Many researchers have suggested that cigarette smoke condensate (CSC) may be more toxic compared to cigarette smoke extract (CSE) because CSC contains the lipid-soluble faction of smoke while CSE contains the hydrophilic or gas phase. The aim of this research is to investigate the effects of CSC on the disruption of endoplasmic reticulum (ER)-Golgi homeostasis in normal lung epithelial cells., Methods: CS was generated according to the ISO 3308 method. To ascertain the mechanistic effects of CSC on lung toxicity, normal lung epithelial cells of the cell line 16HBE14o- were treated with CSC (0.1mg/mL) for 48 hours. The toxic effects of CSC on ER-Golgi homeostasis and GOLPH3 expression were observed through diverse molecular tools including transmission electron microscope analysis., Results: Our results demonstrated that CSC treatment increased reactive oxygen species generation in lung cells and led to the alteration of ER-Golgi homeostasis in conjunction with increased autophagy. In particular, GOLPH3, known as an oncogene and a marker protein for the trans-Golgi network, was upregulated in CSC-treated cells. GOLPH3 protein overexpression was also confirmed in the lungs of human lung cancer patients as well as NNK-treated mice., Conclusion: Our study revealed that CSC caused lung damage through the disruption of ER-Golgi homeostasis and autophagy induction. The expression level of the trans-Golgi marker protein GOLPH3 could serve as a reliable bio-indicator for CS-related lung cancer., Implications: CS is a harmful factor in the development of many diseases including cancer. In this research, we demonstrated that CSC treatment led to malfunction of the ER-Golgi network, with the disrupted ER and Golgi causing GOLPH3 overexpression and abnormal autophagy accumulation. In addition, although the value of GOLPH3 as a predictor remains to be fully elucidated, our data suggest that GOLPH3 levels may be a novel prognostic biomarker of tobacco related lung disease., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tobacco-sourced carcinogen including benzopyrene (B[a]P) in lung cancer metastasis has not been fully reported. In this study, lung carcinoma A549 cell line was used to investigate the potential roles of tobacco-sourced B[a]P on cell metastasis and invasion and to assess its underlying mechanism. Effects of tobacco-sourced carcinogen on A549 cell proliferation, metastasis, and invasion were analyzed using MTT assay, Transwell assay, and scratch method, respectively. The effects of tobacco-sourced carcinogen on cytokines and chemokines secretion were detected using enzyme-linked immunosorbent assay. Moreover, correlation between inflammatory factor expression and cancer cell migration and invasion was assessed using siRNA-mediated gene silencing. Data showed that both B[a]P and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone either at high or low dose performed no significant difference on A549 cell proliferation with time increasing. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone performed no significant difference on A549 cell migration and invasion while B[a]P significantly increased A549 cell migration and invasion compared to the control group (P < 0.05). Consequently, except for IL-6, IL-8, CCL-2, and CCL-3, secretions were significantly increased by B[a]P treatment compared to the control (P < 0.05). Furthermore, when CCL-2 and CCL-3 were silenced, the migrated and invasive A549 cells were significantly decreased compared to the control, respectively (P < 0.05), while silenced IL-8 drastically decreased the migrated and invasive cells compared to the control (P < 0.01). Taken together, this study illustrated that there may be significant correlation between smoking and lung cancer metastasis. B[a]P maybe an excellent contributor for lung cancer metastasis through up-regulating IL-8, CCL-2, and CCL-3 expression., (© 2016 by the Society for Experimental Biology and Medicine.)

A tobacco-specific component, 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK), is a major risk factor for many cancers. Recent reports have demonstrated that NNK exposure may be associated with tumor progression and chemoresistance in certain cancers. However, the underlying NNK-induced mechanism contributing to the aggressiveness of colorectal cancer (CRC) has not been thoroughly studied. In this study, we used HT29 cells treated with NNK to simulate the long-term exposure of cigarette smoke. A comparative analysis was performed to evaluate cell proliferation, migration, and invasion as well as epithelial-mesenchymal transition (EMT) markers and drug-resistance genes expression, cancer stem cell (CSC) properties, and anti-apoptotic activity. Signaling pathways related to chemoresistance were also investigated. As a result, NNK exposure dose-dependently stimulates cell proliferation, enhance abilities of migration and invasion, induce EMT phenomenon, and attenuate apoptosis. Furthermore, NNK exposure also promotes the capabilities of sphere formation, upregulation of Snail, and overexpression of CD133, Nanog, OCT4, and the drug-resistant genes. Knockdown of Snail results in upregulation of Raf kinase inhibitor protein (RKIP), increased apoptosis, reversal of EMT phenomenon, and reducation of expression of CSC markers, all of which contribute to a decrease of chemoresistance. Our study demonstrates a number of related mechanisms that mediate the effect of NNK exposure on increasing CRC therapeutic resistance via the Snail signaling pathway. Targeting Snail may provide a feasible strategy for the treatment of CRC., Competing Interests: The authors declared no conflicts of interest.

Cigarette smoking is a risk factor for pancreatic cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, we show that smoking, HIF-1α expression and β2-adrenogenic receptor (β2-AR) expression are negatively correlated with the overall survival of pancreatic cancer patients. Moreover, HIF-1α expression and β2-AR expression are positively correlated with smoking status, different histological differentiation and among the tumor node metastasis (TNM) stages in pancreatic cancer patients. NNK increases HIF-1α expression in pancreatic cancer in vitro and in vivo. Furthermore, knockdown of HIF-1α and ICI118, 551 (a β2-AR selective antagonist) abrogates NNK-induced pancreatic cancer proliferation and invasion in vitro and inhibits NNK-induced pancreatic cancer growth in vivo. However, using CoCl2 (a HIF-1α stabilizing agent which decreases HIF-1α degradation under normoxia conditions) reverses ICI118, 551 induced effects under NNK exposure. Thus, our data indicate that β2-AR signaling regulates NNK-induced pancreatic cancer progression via upregulation of HIF-1α. Taken together, β2-AR signaling and HIF-1α may represent promising therapeutic targets for preventing smoking induced pancreatic cancer progression.

(S)-N'-Nitrosonornicotine [(S)-NNN] and racemic NNN are powerful oral and esophageal carcinogens in the F344 rat, whereas (R)-NNN has only weak activity. Tumor formation in these tissues of rats treated with racemic NNN was far greater than the sum of the activities of the individual enantiomers. We hypothesized that metabolites of (R)-NNN enhanced levels of DNA adducts produced by (S)-NNN. A test of that hypothesis necessitated the development of a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for the analysis of O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo), a highly mutagenic DNA adduct not previously quantified in rats treated with NNN. The new method, with a limit of detection of 6.5 amol for diluted standard and 100 amol for DNA samples, was applied in this study. Groups of nine F344 rats were treated with doses as follows: 7 ppm (R)-NNN, 7 ppm (S)-NNN, and 14 ppm racemic NNN; 14 ppm (R)-NNN, 14 ppm (S)-NNN, and 28 ppm racemic NNN; or 28 ppm (R)-NNN, 28 ppm (S)-NNN, and 56 ppm racemic NNN for 5 weeks, and tissues were analyzed for DNA adducts. We found statistically significant, but modest, synergistic enhancement of levels of O(6)-POB-dGuo in the esophagus but not the oral cavity of rats treated with racemic NNN (low and median doses only) compared to the sum of the amounts formed in these tissues of rats treated with (S)-NNN or (R)-NNN. There was no synergy in the formation of other POB-DNA adducts of NNN in oral cavity and esophagus, nor was there any evidence for synergy in nasal respiratory and olfactory epithelium, lung, or liver. Our results provide the first quantitation of O(6)-POB-dGuo in DNA from tissues of rats treated with NNN and evidence for synergy in DNA adduct formation as one possible mechanism by which (R)-NNN enhances the carcinogenicity of (S)-NNN in rats.

Background: Recent studies suggest that long-term exposure of the carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) found in tobacco smoke is involved in the progression of head and neck squamous cell carcinoma (HNSCC). The underlying nicotine-mediated mechanism remains unclear., Methods: An analysis of SCC-25 and Fadu cells with or without NNK exposure focusing on the evaluation of migration and invasion abilities, the expression of epithelial-mesenchymal transition, drug-resistance-related genes, properties of cancer stem cells (CSCs), and anti-apoptosis was performed., Results: Long-term NNK exposure enhances migration and invasion with morphological alterations in a dose-dependently manner. Furthermore, NNK exposure also upregulates Snail, promotes sphere-forming ability, and overexpresses aldehyde dehydrogenase 1 (ALDH1), Nanog, OCT4, ABCG2, and MDR1., Conclusion: The current study confirmed that long-term NNK exposure plays a role in HNSCC by increasing anti-apoptosis and therapeutic resistance via the Snail-RKIP signaling pathway. Our data also suggest that α7 nicotinic acetylcholine receptor (α7-nAChR) inhibition or targeting Snail may provide a feasible rationale for preventing the progression of HNSCC., (© 2015 Wiley Periodicals, Inc.)

Aims: Since epidemiologic studies suggest that tobacco smoke toxins, e.g. the nicotine-derived nitrosamine ketone (NNK) tobacco-specific nitrosamine, can be a co-factor in alcohol-related brain disease (ARBD), we examined the independent and additive effects of alcohol and NNK exposures on spatial learning/memory, and brain insulin/IGF signaling, neuronal function and oxidative stress., Methods: Adolescent Long Evans rats were fed liquid diets containing 0 or 26% caloric ethanol for 8 weeks. During weeks 3-8, rats were treated with i.p. NNK (2 mg/kg, 3×/week) or saline. In weeks 7-8, ethanol groups were binge-administered ethanol (2 g/kg; 3×/week). In week 8, at 12 weeks of age, rats were subjected to Morris Water Maze tests. Temporal lobes were used to assess molecular indices of insulin/IGF resistance, oxidative stress and neuronal function., Results: Ethanol and NNK impaired spatial learning, and NNK ± ethanol impaired memory. Linear trend analysis demonstrated worsening performance from control to ethanol, to NNK, and then ethanol + NNK. Ethanol ± NNK, caused brain atrophy, inhibited insulin signaling through the insulin receptor and Akt, activated GSK-3β, increased protein carbonyl and 3-nitrotyrosine, and reduced acetylcholinesterase. NNK increased NTyr. Ethanol + NNK had synergistic stimulatory effects on 8-iso-PGF-2α, inhibitory effects on p-p70S6K, tau and p-tau and trend effects on insulin-like growth factor type 1 (IGF-1) receptor expression and phosphorylation., Conclusions: Ethanol, NNK and combined ethanol + NNK exposures that begin in adolescence impair spatial learning and memory in young adults. The ethanol and/or NNK exposures differentially impair insulin/IGF signaling through neuronal growth, survival and plasticity pathways, increase cellular injury and oxidative stress and reduce expression of critical proteins needed for neuronal function., (© The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

Unlabelled: Smoking carcinogen N-nitrosamines such as 4-methylnitrosamino-l-3-pyridyl-butanone (NNK) require metabolic activation to exert their genotoxicity. The first activation step is mainly catalyzed by cytochrome P450 (CYP) family. Estrogen receptor α (ERα) plays a role in lung pathology. The association between them is unknown. In this study, we explored the relationship and function of CYP1B1 and ERα in NNK-induced lung tumorigenesis. CYP1B1 and ERα expression was analyzed in human lung cancer tissues and NNK-induced lung tumor of A/J mice. Cell lines NCI-H23 and NCI-H460 were employed to further study the responsible mechanisms using various cellular and molecular approaches. Our in vivo experiments demonstrated that CYP1B1 and ERα were over-expressed at the early stage of NNK-induced lung tumorigenesis. Microarray analysis found that ERα was involved in the extracellular-signal-regulated kinase (ERK)/MAPK pathway. NNK activated RAS/ERK/AP1 as it remarkably increased the levels of p-ERK, c-Fos, and c-Jun but inhibited multiple negative regulators of Ras/ERK/AP1, Pdcd4, Spry1, Spry2, and Btg2 through up-regulating miR-21. Both CYP1B1 siRNA and ERK-specific inhibitor U0126 suppressed NNK-mediated ERα up-regulation, suggesting that ERα was downstream of CYP1B1 and ERK. ERK inactivation led to the accumulation of CYP1B1, indicating that CYP1B1 was upstream of ERK activation. Inhibition of ERK or ERα decreased NNK-induced cell proliferation. Blockage of CYP1B1 or ERα induced apoptosis of lung cancer cells. Collectively, NNK-mediated ERα induction is via CYP1B1 and ERK and contributes to the lung carcinogenesis. The inhibition of CYP1B1, ERK, or ERα may arrest the lung cancer cell growth, implicating a pivotal strategy for the treatment of lung cancer., Key Messages: Smoking carcinogen NNK requires metabolic activation to exert their genotoxicity. CYP1B1 is the enzyme to catalyze NNK. NNK activates CYP1B1 and ERK to induce ERα. Inhibition of CYP1B1, ERK, or ERα arrests the lung cancer cell growth.

Recent research has demonstrated that the nicotinergic signaling network of mammary epithelium can both mediate the physiological control of normal breast epithelial cells (BECs) and exhibit tumor-promoting effects on malignant BECs. Therefore, mammary nicotinic acetylcholine (ACh) receptors (nAChRs) may become a specific target for novel anti-breast cancer therapies. Toward this goal, we investigated the difference in the ACh receptor repertoires between normal and malignant BECs, determined effects of nicotinic ligands on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-dependent activation of ERK1/2 and tumorigenic transformation of MCF10A cells, and characterized reciprocal effects of NNK and SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor related protein-1)-1 on the expression of nAChR subunits and several oncogenes and tumor-suppressing genes in BECs. Both the non-malignant MCF10A and malignant MCF7 breast cells expressed α3, α5, α7, α9, α10, β1, β2, γ, δ and ε nAChR subunits and M(1), M(3), M(4) and M(5) muscarinic receptor subtypes. The malignancy was associated with expression of α1, α4 and β4 nAChR subunits and M(2) subtype. Malignant transformation of BECs was also associated with overexpression of α7-, and α9-made nAChRs. NNK upregulated ERK1/2 phosphorylation, stimulated expression of the gene encoding the tumor-promoter HGF, downregulated expression of the tumor suppressor gene CDKN2A, and induced tumorigenic transformation of MCF10A cells. Compared to the canonical nAChR antagonists, SLURP-1 showed the highest ability to abolish the nAChR-mediated effects of NNK in both cell-signaling and cell-transformation assays and reversed many effects of NNK on gene expression. SLURP-1 also markedly upregulated the tumor suppressor genes CDKN2B, RUNX3 and TP73. Altogether, the obtained results provided new insight into the molecular mechanisms of nAChR-mediated oncogenic effects of NNK on BECs and demonstrated the ability to abolish or reverse these effects by SLURP-1., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Before the 1930s, squamous cell carcinoma (SCC) of the oesophagus was almost unknown among black South Africans. From the 1930s the annual frequency rose. A dietary cause was sought, the staple diet of black people having changed from sorghum to maize (corn), with traditional beer being brewed from maize. Carcinogenic N-nitrosamines in traditional beer were suggested as a cause of SCC of the oesophagus, with Fusarium moniliforme, a corn saprophyte, thought to play a role., Objectives: To confirm the presence of N-nitrosamines in traditional beer and demonstrate a mechanism for the oncogenesis of oesophageal carcinoma., Methods: Analysis by high-performance liquid chromatography was conducted for the identification of nitrosamines in traditional beer samples, and molecular docking studies were employed to predict the affinity between N-nitrosamines and the S100A2 protein., Results: Carcinogenic N-nitrosamines were identified in all six samples of traditional beer examined (N=18 analyses), and docking studies confirmed a high affinity of the nitrosamine N-nitrosopyrrolidone with the S100A2 protein. This may result in the altered expression of the S100A2 protein, leading to tumour progression and prognosis., Conclusion: It is suggested that carcinogenic N-nitrosamines in traditional beer are a major factor in the causation of SCC of the oesophagus in black South Africans. N-nitrosamines have been shown to produce cancer experimentally, but there has not been conclusive epidemiological evidence that N-nitrosamines are carcinogenic to humans. This study is the first to demonstrate the potential link between N-nitrosamines and a human tumour.

The tobacco-specific nitrosamine NNK is a potent carcinogen found in tobacco smoke and implicated in the development of lung cancer. The major route of NNK metabolism is carbonyl reduction by AKR1C1, AKR1C2, CBR1, and 11β-HSD1 to form NNAL. This study investigated the potential role of variants in this pathway on lung cancer risk by examining 53 tag-SNPs representing the common variations in AKR1C1, AKR1C2, CBR1, and HSD11B1 in 456 lung cancer cases and 807 controls. One SNP in CBR1 (rs2835267) was significantly associated with overall risk of lung cancer, but did not pass multiple testing adjustment (OR: 0.76 95% CI: 0.58-0.99, P = 0.048, FDR P = 0.20). After stratification and multiple testing correction, three SNPs showed significance. One SNP (rs2835267) in CBR1 showed a significant decreased risk for smokers with a high pack-years (OR: 0.3595% CI: 0.17-0.69, P = 0.018) and in SCC (OR: 0.4895% CI: 0.29-0.76, P = 0.018). Another SNP located in CBR1 (rs3787728) also showed a significant decreased risk in SCC (OR: 0.4695% CI: 0.26-0.80, P = 0.024) and small cell carcinoma (only in current smokers) (OR: 0.06895% CI: 0.01-0.42, P = 0.028). The HSD11B1 SNP (rs4844880) showed a significant increased risk for adenocarcinoma within former smokers (OR: 3.9495% CI: 1.68-9.22, P = 0.011). Haplotype analysis found significance with six haplotypes and lung cancer risk. These findings indicate that select variants in genes in the carbonyl reduction pathway of NNK may alter the risk of lung cancer., (© 2014 Wiley Periodicals, Inc.)

Our previous study showed that chromosome region maintenance 1 (CRM1), a nuclear export receptor for various cancer-associated "cargo" proteins, was important in regulating lung carcinogenesis in response to a tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The objectives of this study are to comprehensively evaluate the significance of CRM1 in lung cancer development and investigate the therapeutic potential of targeting CRM1 for lung cancer treatment using both in vitro and in vivo models. We showed that CRM1 was overexpressed not only in lung tumor tissues from both lung cancer patients and mice treated with NNK but also in NNK-transformed BEAS-2B human bronchial epithelial cells. Furthermore, stable overexpression of CRM1 in BEAS-2B cells by plasmid vector transfection led to malignant cellular transformation. Moreover, a decreased CRM1 expression level in A549 cells by short hairpin siRNA transfection led to a decreased tumorigenic activity both in vitro and in nude mice, suggesting the potential to target CRM1 for lung cancer treatment. Indeed, we showed that the cytotoxic effects of cisplatin on A549 cells with CRM1 down-regulated by short hairpin siRNA were significantly increased, compared with A549 cells, and the cytotoxic effects of cisplatin became further enhanced when the drug was used in combination with leptomycin B, a CRM1 inhibitor, in both in vitro and in vivo models. Cancer target genes were significantly involved in these processes. These data suggest that CRM1 plays an important role in lung carcinogenesis and provides a novel target for lung cancer adjuvant therapy.