Estrogen/estrogen receptor activation protects against DEN-induced liver fibrosis in female rats via modulating TLR-4/NF-kß signaling.

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- ß, and IL-10 were evaluated. Expression of NF-kß, CD68 (a marker for macrophage infiltration), ER-ß 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-kß 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-ß 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-kß, in the alleviation of LF pathogenesis.

Design, Synthesis of (±)-Millpuline†A, and Biological Evaluation for the Lung Cell Protective Effects through SRC.

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.

NNK from tobacco smoking enhances pancreatic cancer cell stemness and chemoresistance by creating a ß2AR-Akt feedback loop that activates autophagy.

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 ß2-adrenergic receptor (ß2AR)-Akt axis. Finally, we proved that NNK intervention could not only activate ß2AR, but also increase its expression, making ß2AR and Akt form a feedback loop. Overall, these findings show that the NNK-induced ß2AR-Akt feedback loop promotes stemness and gemcitabine resistance in pancreatic cancer cells.

Identification and validation of a cigarette smoke-related five-gene signature as a prognostic biomarker in kidney renal clear cell carcinoma.

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.

Involvement of m6A regulatory factor IGF2BP1 in malignant transformation of human bronchial epithelial Beas-2B cells induced by tobacco carcinogen NNK.

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.

Chemopreventive Potential of Myrtenal against Nitrosamine-Initiated, Radiation-Promoted Rat Bladder Carcinogenesis.

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.

Orchestrating Nitric Oxide and Carbon Monoxide Signaling Molecules for Synergistic Treatment of MRSA Infections.

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.

Lung Tumor Growth Promotion by Tobacco-Specific Nitrosamines Involves the ß2-Adrenergic Receptors-Dependent Stimulation of Mitochondrial REDOX Signaling.

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 (ß2-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.

Iron oxide nanoparticles exert inhibitory effects on N-Bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung tumorigenesis in rats.

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.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Histone Acetylation via α7nAChR-Mediated PI3K/Akt Activation and Its Impact on γ-H2AX Generation.

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.

Stress hormones promote DNA damage in human oral keratinocytes.

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.

Synthesis, anti-microbial, toxicity and molecular docking studies of N-nitroso-N-phenylhydroxylamine (cupferron) and its derivatives.

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.

Exposure to a mixture of cigarette smoke carcinogens disturbs gut microbiota and influences metabolic homeostasis in A/J mice.

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.

Mass Spectrometric Quantitation of Apurinic/Apyrimidinic Sites in Tissue DNA of Rats Exposed to Tobacco-Specific Nitrosamines and in Lung and Leukocyte DNA of Cigarette Smokers and Nonsmokers.

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 (N7POBdG) 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 108 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 N7POBG 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.

Differential modulation of the contextual conditioned emotional response by CB1 and TRPV1 receptors in the ventromedial prefrontal cortex: Possible involvement of NMDA/nitric oxide-related mechanisms.

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.

Synergistic Effect between Human Papillomavirus 18 and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone on Malignant Transformation of Immortalized SHEE Cells.

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.

A lysosome specific theranostic NO donor inhibits cancer cells by stimuli responsive molecular self-decomposition with an on-demand fluorescence pattern.

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.

Investigation of Precise Molecular Mechanistic Action of Tobacco-Associated Carcinogen `NNK´ Induced Carcinogenesis: A System Biology Approach.

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 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.

Dose-Related Modulatory Effects of Polymeric Black Tea Polyphenols (PBPs) on Initiation and Promotion Events in B(a)P and NNK-Induced Lung Carcinogenesis.

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.

Determination of genomic N3-methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser-induced fluorescence.

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.