p27 specifically decreases in squamous carcinoma, and mediates NNK-induced transformation of human bronchial epithelial cells.

Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.

Circular RNA circNIPBL regulates TP53-H179R mutations in NNK-induced bronchial epithelial carcinogenesis.

Exposure to environmental carcinogens is a significant contributor to cancer development, with genetic and epigenetic alterations playing pivotal roles in the carcinogenic process. However, the interplay between epigenetic regulation and genetic changes in carcinogenesis has yet to receive comprehensive attention. This study investigates the impact of continuous exposure to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on bronchial epithelial cells, leading to malignant transformation. Our findings reveal the down-regulation of the tumor suppressor-like circular RNA circNIPBL during oncogenic processes concomitant with the accumulation of the TP53-H179R, a single nucleotide variant. Diminished circNIPBL expression enhances the proliferative, distant metastatic, and tumor-forming capabilities of NNK-induced cancerous cells and lung cancer cell lines (A549, H1299), while also promoting the accumulation of TP53-H179R during NNK-induced carcinogenesis. Mechanistic investigations demonstrate that circNIPBL interacts with HSP90α to regulate the translocation of AHR into the nucleus, which may be a potential regulatory mechanism for NNK-induced carcinogenesis and TP53-H179R accumulation. This study introduces a novel perspective on the interplay between genetic alterations and epigenetic regulation in chemical carcinogenesis, which provides novel insight into the etiology of cancer.

LncRNA XIST modulates miR-328-3p ectopic expression in lung injury induced by tobacco-specific lung carcinogen NNK both in vitro and in vivo.

BACKGROUND AND PURPOSE: It is well acknowledged that tobacco-derived lung carcinogens can induce lung injury and even lung cancer through a complex mechanism. MicroRNAs (MiRNAs) are differentially expressed in tobacco-derived carcinogen nicotine-derived nitrosamine ketone (NNK)-treated A/J mice. EXPERIMENTAL APPROACH: RNA sequencing was used to detect the level of long non-coding RNAs (lncRNAs). Murine and human lung normal and cancer cells were used to evaluate the function of lncRNA XIST and miR-328-3p in vitro, and NNK-treated A/J mice were used to test their function in vivo. In vivo levels of miR-328-3p and lncRNA XIST were analysed, using in situ hybridization. miR-328-3p agomir and lncRNA XIST-specific siRNA were used to manipulate in vivo levels of miR-328-3p and lncRNA XIST in A/J mice. KEY RESULTS: LncRNA XIST was up-regulated in NNK-induced lung injury and dominated the NNK-induced ectopic miRNA expression in NNK-induced lung injury both in vitro and in vivo. Either lncRNA XIST silencing or miR-328-3p overexpression exerted opposing effects in lung normal and cancer cells regarding cell migration. LncRNA XIST down-regulated miR-328-3p levels as a miRNA sponge, and miR-328-3p targeted the 3'-UTR of FZD7 mRNA, which is ectopically overexpressed in lung cancer patients. Both in vivo lncRNA XIST silencing and miR-328 overexpression could rescue NNK-induced lung injury and aberrant overexpression of the lung cancer biomarker CK19 in NNK-treated A/J mice. CONCLUSIONS AND IMPLICATIONS: Our results highlight the promotive effect of lncRNA XIST in NNK-induced lung injury and elucidate its post-transcriptional mechanisms, indicating that targeting lncRNA XIST/miR-328-3p could be a potential therapeutic strategy to prevent tobacco carcinogen-induced lung injury in vivo.

Nicotine-derived NNK promotes CRC progression through activating TMUB1/AKT pathway in METTL14/YTHDF2-mediated m6A manner.

Cigarette smoking substantially promotes tumorigenesis and progression of colorectal cancer; however, the underlying molecular mechanism remains unclear. Among 662 colorectal cancer patients, our investigation revealed a significant correlation between cigarette smoking and factors, such as large tumor size, poor differentiation, and high degree of invasion. Among the nicotine-derived nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) emerged as the most critical carcinogen, which significantly promoted the malignant progression of colorectal cancer both in vivo and in vitro. The results of methylated RNA immunoprecipitation and transcriptome sequencing indicated that NNK upregulated transmembrane and ubiquitin-like domain-containing protein 1 (TMUB1) via N6-adenosine methylation, which was regulated by methyltransferase-like 14 (METTL14) and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Elevated TMUB1 levels were associated with a higher risk of cancer invasion and metastasis, leading to a high mortality risk in patients with colorectal cancer. Additionally, TMUB1 promoted lysine63-linked ubiquitination of AKT by interacting with AMFR, which led to the induction of malignant proliferation and metastasis in colorectal cancer cells exposed to NNK. In summary, this study provides a new insight, indicating that targeting TMUB1 expression via METTL14/YTHDF2 mediated N6-adenosine methylation may be a potential therapeutic and prognostic target for patients with colorectal cancer who smoke.

From cultivation to cancer: formation of N-nitrosamines and other carcinogens in smokeless tobacco and their mutagenic implications.

Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific N-nitrosamines (TSNAs), such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.

Diallyl Disulfide Blocks Cigarette Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Lung Tumorigenesis via Activation of the Nrf2 Antioxidant System and Suppression of NF-κB Inflammatory Response.

Chemoprevention is a potential strategy to reduce lung cancer incidence and death. Recently, we reported that garlic oil significantly inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis. Diallyl disulfide (DADS) is a bioactive ingredient in garlic. Our goal was to examine the chemopreventive effectiveness and mechanism of DADS on NNK-triggered lung cancer in vivo and in vitro in the current investigation. The results indicated that DADS significantly reduced the number of lung nodules in the NNK-induced A/J mice. Consistent with the in vivo results, DADS markedly inhibited NNK-induced decrease of MRC-5 cells' viability. Mechanistically, DADS could promote Nrf2 dissociated from the Keap1-Nrf2 complex and accelerate Nrf2 nuclear translocation, which in turn upregulates its downstream target genes. Besides, DADS further inhibited the NF-κB signaling cascade, thus reducing the accumulation of inflammatory factors. Collectively, these discoveries supported the potential of DADS as a novel candidate for the chemoprevention of tobacco-carcinogen-induced lung cancer.

Hypermethylation of the ADIRF promoter regulates its expression level and is involved in NNK-induced malignant transformation of lung bronchial epithelial cells.

The carcinogenic mechanism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a well-known tobacco carcinogen, has not been fully elucidated in epigenetic studies. 5-Methylcytosine (5mC) modification plays a major role in epigenetic regulation. In this study, the 5mC level increased in both BEAS-2B human bronchial epithelium cells treated with 100 mg/L NNK for 24 h and NNK-induced malignant-transformed BEAS-2B cells (2B-NNK cells), suggesting that 5mC modification is associated with the malignant transformation mechanism of NNK. Using a combination of Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq), RNA sequencing (RNA-seq), and bioinformatics analysis of data from the Genomic Data Commons database, we found that the Adipogenesis regulatory factor (ADIRF) promoter region was abnormally hypermethylated, yielding low ADIRF mRNA expression, and that ADIRF overexpression could inhibit the proliferation, migration, and invasion of 2B-NNK cells. This finding suggests that ADIRF plays a tumor suppressor role in the NNK-induced malignant transformation of cells. Subsequently, using 5-Aza-2'-deoxycytidine (5-Aza-2'-dC) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Catalytically Dead Cas9 (dCas9 system), we verified that the demethylation of the ADIRF promoter region in 2B-NNK cells inhibited the proliferation, migration, and invasion ability of the cells and increased their apoptosis ability. These results suggest that abnormal 5mC modification of the ADIRF promoter plays a positive regulatory role in the pathogenesis of NNK-induced lung cancer. This study offers a new experimental basis for the epigenetic mechanism of NNK-induced lung cancer.

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.

A novel agonist of 4-1BB costimulatory receptor shows therapeutic efficacy against a tobacco carcinogen-induced lung cancer.

Immunotherapy utilizing checkpoint inhibitors has shown remarkable success in the treatment of cancers. In addition to immune checkpoint inhibitors, immune co-stimulation has the potential to enhance immune activation and destabilize the immunosuppressive tumor microenvironment. CD137, also known as 4-1BB, is one of the potent immune costimulatory receptors that could be targeted for effective immune co-stimulation. The interaction of the 4-1BB receptor with its natural ligand (4-1BBL) generates a strong costimulatory signal for T cell proliferation and survival. 4-1BBL lacks costimulatory activity in soluble form. To obtain co-stimulatory activity in soluble form, a recombinant 4-1BBL protein was generated by fusing the extracellular domains of murine 4-1BBL to a modified version of streptavidin (SA-4-1BBL). Treatment with SA-4-1BBL inhibited the development of lung tumors in A/J mice induced by weekly injections of the tobacco carcinogen NNK for eight weeks. The inhibition was dependent on the presence of T cells and NK cells; depletion of these cells diminished the SA-4-1BBL antitumor protective effect. The number of lung tumor nodules was significantly reduced by the administration of SA-4-1BBL to mice during ongoing exposure to NNK. The data presented in this paper suggest that utilizing an immune checkpoint stimulator as a single agent generate a protective immune response against lung cancer in the presence of a carcinogen. More broadly, this study suggests that immune checkpoint stimulation can be extended to a number of other cancer types, including breast and prostate cancers, for which improved diagnostics can detect disease at the preneoplastic stage.

Mitochondria-targeted metformin (mitomet) inhibits lung cancer in cellular models and in mice by enhancing the generation of reactive oxygen species.

Lung cancer is the leading cause of cancer-related mortality in the United States. Although some epidemiological studies have shown an inverse relationship between the use of metformin, a widely used antidiabetic drug, and the incidence of lung cancer, the real benefits of the drug are unclear as the efficacy is low and the outcomes are quite heterogeneous. To develop a more potent form of metformin, we synthesized mitochondria-targeted metformin (mitomet) and tested its efficacy in in vitro and in vivo models of lung cancer. Mitomet was cytotoxic to transformed bronchial cells and several non-small cell lung cancer (NSCLC) cell lines but relatively safe to normal bronchial cells, and these effects were mediated mainly via induction of mitochondrial reactive oxygen species. Studies using isogenic A549 cells showed that mitomet was selectively toxic to those cells deficient in the tumor suppressor gene LKB1, which is widely mutated in NSCLC. Mitomet also significantly reduced the multiplicity and size of lung tumors induced by a tobacco smoke carcinogen in mice. Overall, our findings showed that mitomet, which was about 1000 and 100 times more potent than metformin, in killing NSCLC cells and reducing the multiplicity and size of lung tumors in mice, respectively, is a promising candidate for the chemoprevention and treatment of lung cancer, in particular against LKB1-deficient lung cancers which are known to be highly aggressive.

Brain distribution and metabolic profiling of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats investigated by UHPLC-HRMS/MS following peripheral administration.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known to be a tobacco-specific N-nitrosamine and has peripheral carcinogenic properties. It can also induce oxidative stress, glial cell activation, and neuronal damage in the brain. However, the distribution and metabolic characteristics of NNK in the central nervous system are still unclear. Here, a sensitive and effective UHPLC-HRMS/MS method was established to identify and investigate the metabolites of NNK and their distribution in the rat brain. In addition, the pharmacokinetic profiles were simultaneously investigated via blood-brain synchronous microdialysis. NNK and its seven metabolites were well quantified in the hippocampus, cortex, striatum, olfactory bulb, brain stem, cerebellum, and other regions of rat brain after peripheral exposure (5 mg/kg, i.p.). The average content of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in all brain regions was at least threefold higher than that of NNK, indicating a rapid carbonyl reduction of NNK in the brain. Lower concentrations of pyridine N-oxidation products in the cortex, olfactory bulb, hippocampus, and striatum might be related to the poor detoxification ability in these regions. Compared to α-methyl hydroxylation, NNK and NNAL were more inclined to the α-methylene hydroxylation pathway. Synchronous pharmacokinetic results indicated that the metabolic activity of NNK in the brain was different from that in the blood. The mean α-hydroxylation ratio in the brain and blood was 0.037 and 0.161, respectively, which indicated poor metabolic activity of NNK in the central nervous system.

Molecular docking and dynamic simulation studies of α4ß2 and α7 nicotinic acetylcholine receptors with tobacco smoke constituents nicotine, NNK and NNN.

Smoking constitutes a major global health problem. As it triggers various health hazards including cancers, cardiac and pulmonary illness, it is imperative to understand the mechanism of action of various smoke constituents on our cellular processes. Various in vitro studies have compiled the affinity of cigarette smoke constituents on various nicotinic acetylcholine receptors (nAChRs). But the nature of the intermolecular interactions contributing to this affinity and the key amino acids in the receptor active sites involved in this are not investigated so far. Here, we are examining the interaction of α7nAChR and α4ß2nAChR on nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosornicotine (NNN), the physiologically significant constituents in smoke, through molecular docking and dynamics simulations study. The docking of α4ß2nAChR structure with the ligands nicotine, NNK and NNN yielded docking scores of -41.45 kcal/mol, -59.28 kcal/mol and -54.60 kcal/mol, respectively, and that of α7nAChR receptor molecule with the ligands yielded docking scores of -59.54 kcal/mol, -71.06 kcal/mol and -70.86 kcal/mol, respectively. The study showed that NNK exhibited the highest affinity with the ligands which was confirmed by dynamics simulation. But higher stability of interactions as surmised from Molecular dynamics simulations was found for nicotine with α4ß2nAChR and NNN with α7nAChR. The findings validate the in vitro studies comparing the affinities of these compounds. The study will be useful in formulating effective nAChR agonists to treat neurological disorders and antagonists for smoke deaddiction and improve health standards.Communicated by Ramaswamy H. Sarma.

Proteome derangement in malignant epithelial cells and its stroma following exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Discovering novel changes in the proteome of malignant lung epithelial cells and/or the tumor-microenvironment is paramount for diagnostic, prognostic, and/or therapy development. A time-dependent 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumor model was used to screen the proteome of lung tumors. NNK-transformed human lung epithelial BEAS-2B cells were then established to evaluate the epithelial cell-specific protein changes. A duration-dependent increase of tumor burden was observed in NNK-treated mice, 2/12 (17%), 8/12 (67%), 9/12 (75%), and 10/10 (100%) at weeks 8, 12, 16, and 20 after the NNK exposure, respectively. A total of 25 differentially expressed proteins (≥ twofold change), predominantly structural, signaling, and metabolic proteins, were detected by two-dimensional difference gel electrophoresis and identified by mass spectrometry. Calregulin, ezrin, histamine releasing factor (HRF), and inorganic pyrophosphatase 1 (PPA1) exhibited changes and were further confirmed via immunoblotting. In addition, immunohistochemistry (IHC) analysis indicated upregulated E-cadherin and decreased vimentin expression in epithelial cells of tumor tissues. Acquisition of a neoplastic phenotype in NNK-transformed BEAS-2B cells was demonstrated by enhanced wound closure and increased anchorage independent colony formation. In transformed BEAS-2B cells, protein expression of E-cadherin, ezrin, and PPA1 (but not calregulin and HRF) was upregulated, as was observed in tumor tissues IHC staining using mouse lung tumor tissues further revealed that HRF upregulation was not lung epithelial cell specific. Altogether, tumorigenesis after NNK exposure may be initiated by protein dysregulation in lung epithelial cells together with proteome derangement derived from other cell types existing in the tumor-microenvironment.

Recovering the angiogenic/angiostatic balance in NNK-induced lung carcinoma via 12 weeks of submaximal swimming and Nigella sativa nanocapsule.

Background: Carcinogen nitrosamine 4-(methyl-trosamino)-1-(3-pyridyl)-1-butanone (NNK) remarkably affects the actions of growth factors: EGFR, VEGFR-2, as well as the natural tumor suppressors: TGFß-1 and TIMP-1. We propose that utilizing non-chemical interventions such as swimming and Nigella sativa nanocapsule play role in controlling cancer progression through direct effects on tumor-inherent factors. Material and methods: Male rats were randomly placed into seven groups: Control (C), Solvent (S), (NNK), NNK+N.sativa (NNK+NS), NNK+Exercise (NNK+E), N.sativa+Exercise (NS+E), NNK+N.sativa+Exercise (NNK+NS+E). The exercise program consisted of 12 weeks of submaximal swimming. NNK and NS groups received weekly doses of 12/5 mg/kg and 125 µg/kg of NNK and N.sativa, respectively. By the end of the protocol, the levels of VEGFR-2, and TIMP-1 were determined using immunohistochemistry method and EGFR, and TGFß-1 levels were measured by RT-PCR assay. Results: In comparison with control group, there was a significant increase in the levels of VEGFR-2 in NNK, NNK+E, NNK+NS, NS+E, and NNK+NS+E groups (P ≤ 0.001), also TGFß-1 levels of NNK+E and NS+E groups significantly increased (P ≤ 0.001). While EGFR levels did not change remarkably (P˃0.05), except in NNK group (P ≤ 0.001), TIMP-1 in NNK, NNK+E, NS+E, NNK+NS+E groups significantly decreased (P ≤ 0.001). Conclusion: We recommend 12 weeks of submaximal swimming and 125 µg/kg N.sativa nanocapsule are safe interventions to recover the balance of selected angiogenic/ angiostatic markers and to control tumor initiation, growth, and metastasis in lung carcinoma induced by 12/5 mg/kg of NNK injection.

Managing MMP-2, MMP-9, VEGFR-2, TGFß-1, and TIMP-1 in NNK-induced lung carcinoma by nonchemical interventions in female rats.

Background: Smart and flexible methods are attracting remarkable interest in cancer-related biological and chemical therapies. To achieve a safer, affordable, and more effective cancer treatment, we evaluated the application of submaximal swimming and Nigella sativa (NS) nano-drug on lung tissues of female rats induced by NNK. Material and methods: A 12-weeks protocol of submaximal swimming was performed in pathologic and non-pathologic groups. NNK and NS groups, respectively received weekly doses of 12/5 mg/kg and 125 µg/kg of body weight. By the end of the protocol, the ratios of MMP-2, MMP-9, and TIMP-1 determined by using immunohistochemistry essay, and RT-PCR analysis for VEGFR-2 and TGFß-1. Results: As a result, treatment with exercise and NNK resulted in VEGFR-2 overexpression (P ≤ 0.001 and P ≤ 0.05, respectively). In NNK, NNK+E, NNK+NS, and NNK+NS+E groups, protein expression of MMP-2 and MMP-9 significantly increased, despite the reduction of TIMP-1 levels in the same groups compared to control (P ≤ 0.001). TGFß-1 ratio significantly increased following preformed interventions in non-pathologic groups: E (P ≤ 0.001) and NS+E (P ≤ 0.01). Conclusion: IHC and gene assays indicate a favorable and acceptable effect of the designed training protocol besides the treatment with N.sativa nano-drug, by which cancer development could be restricted through recovering the natural balance of angiogenic and angiostatic markers.

Effects of cigarette smoke extract derived from heated tobacco products on the proliferation of lung cancer stem cells.

Epidemiological studies have suggested that cigarette smoking can increase a person's risk of developing several types of cancer, including lung cancer. Lung cancer originates from cancer stem cells (CSCs), which constitute a minor cell population in tumors, and contribute to drug resistance and recurrence. Heated tobacco products (HTPs) produce aerosols that contain nicotine and toxic chemicals. Current evidence, however, is insufficient to accurately determine if HTPs are less harmful than burned cigarettes. This study has investigated the effects of cigarette smoke extract (CSE) from HTPs on lung CSCs in lung cancer cell lines. We found that CSEs induced the proliferation of lung CSCs and increased the expression levels of stem cell markers. In addition, CSE induced epithelial-mesenchymal transition (EMT) expression and cytokine production. These results suggest that HTPs can induce lung CSCs in vitro.

Regression of Lung Cancer in Mice by Intranasal Administration of SARS-CoV-2 Spike S1.

This study underlines the importance of SARS-CoV-2 spike S1 in prompting death in cultured non-small cell lung cancer (NSCLC) cells and in vivo in lung tumors in mice. Interestingly, we found that recombinant spike S1 treatment at very low doses led to death of human A549 NSCLC cells. On the other hand, boiled recombinant SARS-CoV-2 spike S1 remained unable to induce death, suggesting that the induction of cell death in A549 cells was due to native SARS-CoV-2 spike S1 protein. SARS-CoV-2 spike S1-induced A549 cell death was also inhibited by neutralizing antibodies against spike S1 and ACE2. Moreover, our newly designed wild type ACE2-interacting domain of SARS-CoV-2 (wtAIDS), but not mAIDS, peptide also attenuated SARS-CoV-2 spike S1-induced cell death, suggesting that SARS-CoV-2 spike S1-induced death in A549 NSCLC cells depends on its interaction with ACE2 receptor. Similarly, recombinant spike S1 treatment also led to death of human H1299 and H358 NSCLC cells. Finally, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) intoxication led to the formation tumors in lungs of A/J mice and alternate day intranasal treatment with low dose of recombinant SARS-CoV-2 spike S1 from 22-weeks of NNK insult (late stage) induced apoptosis and tumor regression in the lungs. These studies indicate that SARS-CoV-2 spike S1 may have implications for lung cancer treatment.

Acute cytotoxicity test of PM2.5, NNK and BPDE in human normal bronchial epithelial cells: A comparison of a co-culture model containing macrophages and a mono-culture model.

BACKGROUND: Based on extensive research on cytotoxicity of exogenous compounds in vitro, it is essential to develop a cell model that better mimics environment in vivo to explore cytotoxic mechanisms of exogenous compounds. METHODS: A co-culture system was established using a transwell system with Beas-2B and U937 cells. Cells were treated with fine particulate matter (PM2.5; 25, 50 and 100 µg/mL), nicotine-derived nitrosamine ketone (NNK; 50, 100 and 200 µg/mL) and benzo(a)pyrene diol epoxide (BPDE; 0.5, 2 and 8 µM) for 24 h. Cell proliferation, apoptosis and cell cycle, DNA damage were detected by CCK-8 and EdU, flow cytometry, and comet assay, respectively. Differentially expressed transcript and cytokine concentrations were determined by transcriptome sequencing and Cytokine Array, respectively. RESULTS: Compared with mono-culture, cell proliferation increased, apoptosis decreased, and DNA damage decreased in a dose-response relationship in co-culture. Gene expression profile was significantly different in co-culture, with significantly increased expression levels of 48 cytokines in co-culture. CONCLUSION: Cytotoxic damage to Beas-2B cells induced by exogenous carcinogens, including PM2.5, NNK and BPDE, was significantly reduced in a co-culture system compared with a mono-culture system. The mechanism may be related to changes in expression of cytokines, such as LIF, and activation of related pathways, such as TNF signaling pathway. Cytotoxic damage to Beas-2B induced by PM2.5, NNK and BPDE, was significantly reduced in co-culture. The mechanism may be related to changes in expression of cytokines and activation of related pathways. These findings provide new insights into cytotoxicity and experimental basis for safety evaluations of exogenous carcinogens.

Nicotine Inhibits the Cytotoxicity and Genotoxicity of NNK Mediated by CYP2A13 in BEAS-2B Cells.

Both tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nicotine can be metabolized by cytochrome P450 2A13 (CYP2A13). Previous studies have shown that nicotine has a potential inhibitory effect on the toxicity of NNK. However, due to the lack of CYP2A13 activity in conventional lung cell lines, there had been no systematic in vitro investigation for the key target organ, the lung. Here, BEAS-2B cells stably expressing CYP2A13 (B-2A13 cells) were constructed to investigate the effects of nicotine on the cytotoxicity and genotoxicity of NNK. The results showed more sensitivity for NNK-induced cytotoxicity in B-2A13 cells than in BEAS-2B and B-vector cells. NNK significantly induced DNA damage, cell cycle arrest, and chromosomal damage in B-2A13 cells, but had no significant effect on BEAS-2B cells and the vector control cells. The combination of different concentration gradient of nicotine without cytotoxic effects and a single concentration of NNK reduced or even counteracted the cytotoxicity and multi-dimensional genotoxicity in a dose-dependent manner. In conclusion, CYP2A13 caused the cytotoxicity and genotoxicity of NNK in BEAS-2B cells, and the addition of nicotine could inhibit the toxicity of NNK.

The Effect of Tobacco Smoke N-Nitrosamines, NNK and NDEA, and Nicotine, on DNA Mismatch Repair Mechanism and miRNA Markers, in Hypopharyngeal Squamous Cell Carcinoma: An In Vivo Model and Clinical Evidence.

Deregulation of the DNA mismatch repair (MMR) mechanism has been linked to poor prognosis of upper aerodigestive tract cancers. Our recent in vitro data have provided evidence of crosstalk between deregulated miRNAs and MMR genes, caused by tobacco smoke (TS) N-Nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in hypopharyngeal cells. Here, we explored whether chronic exposure to TS components can affect MMR mechanism and miRNA profiles in hypopharyngeal mucosa. Using a mouse model (C57Bl/6J wild type) of in vivo 14-week exposure to NNK (0.2 mmol/L) and N-Nitrosodiethylamine (NDEA; 0.004 mmol/L), with or without nicotine (0.02 µmol/L), we provide direct evidence that TS components can promote dysplasia, significant downregulation of Msh2 and Mlh1 genes and deregulation of miR-21, miR-155, miR-34a, and miR-451a. By analyzing eight human specimens from tobacco smokers and eight controls, we provide clinical evidence of a significant reduction in hMSH2 and hMLH1 mRNAs in hypopharyngeal squamous cell carcinoma (HSCC). In summary, deregulation of the MMR mechanism and miRNAs is caused by chronic exposure to TS-related N-Nitrosamines, with or without nicotine, in the early stages of upper aerodigestive tract carcinogenesis, and can also be detected in human HSCC. Thus, we encourage future studies to further elucidate a possible in vivo dose-dependent effect of individual or combined N-Nitrosamines, NNK and/or NDEA, and nicotine, on the MMR mechanism and their clinical testing to elaborate prognosis and risk assessment.