Cigarette Smoke Condensate Research Articles

Copper oxide nanoparticles exacerbate chronic obstructive pulmonary disease by activating the TXNIP-NLRP3 signaling pathway.

Although copper oxide nanoparticles (CuONPs) offer certain benefits to humans, they can be toxic to organs and exacerbate underlying diseases upon exposure. Chronic obstructive pulmonary disease (COPD), induced by smoking, can worsen with exposure to various harmful particles. However, the specific impact of CuONPs on COPD and the underlying mechanisms remain unknown. In this study, we investigated the toxic effects of CuONPs on the respiratory tract, the pathophysiology of CuONPs exposure-induced COPD, and the mechanism of CuONPs toxicity, focusing on thioredoxin-interacting protein (TXNIP) signaling using a cigarette smoke condensate (CSC)-induced COPD model. In the toxicity study, CuONPs exposure induced an inflammatory response in the respiratory tract, including inflammatory cell infiltration, cytokine production, and mucus secretion, which were accompanied by increased TXNIP, NOD-like receptor protein 3 (NLRP3), caspase-1, and interleukin (IL)-1β. In the COPD model, CuONPs exposure induced the elevation of various indexes related to COPD, as well as increased TXNIP expression. Additionally, TNXIP-knockout (KO) mice showed a significantly decreased expression of NLRP3, caspase-1, and IL-1β and inflammatory responses in CuONPs-exposed COPD mice. These results were consistent with the results of an in vitro experiment using H292 cells. By contrast, TNXIP-overexpressed mice had a markedly increased expression of NLRP3, caspase-1, and IL-1β and inflammatory responses in CuONPs-exposed COPD mice. We elucidated the exacerbating effect of CuONPs exposure on the respiratory tract with underlying COPD, as well as related signaling transduction via TXNIP regulation. CuONPs exposure significantly increased inflammatory responses in the respiratory tract, which was correlated with elevated TXNIP-NLRP3 signaling.

MitoQ relieves mitochondrial dysfunction in UVA and cigarette smoke-induced Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD), a degenerative corneal condition, is characterized by the droplet-like accumulation of the extracellular matrix, known as guttae and progressive loss of corneal endothelial cells ultimately leading to visual distortion and glare. FECD can be influenced by environmental stressors and genetic conditions. However, the role of mitochondrial dysfunction for advancing FECD pathogenesis is not yet fully studied. Therefore, in the present study we sought to determine whether a combination of environmental stressors (ultraviolet-A (UVA) light and cigarette smoke condensate (CSC)) can induce mitochondrial dysfunction leading to FECD. We also investigated if MitoQ, a water-soluble antioxidant, can target mitochondrial dysfunction induced by UVA and CSC in human corneal endothelial cells mitigating FECD pathogenesis. We modeled the FECD by increasing exogenous oxidative stress with CSC (0.2%), UVA (25J/cm2) and a combination of UVA + CSC and performed a temporal analysis of their cellular and mitochondrial effects on HCEnC-21T immortalized cells in vitro before and after MitoQ (0.05 μM) treatment. Interestingly, we observed that a combination of UVA + CSC exposure increased mitochondrial ROS and fragmentation leading to a lower mitochondrial membrane potential and increased levels of cytochrome c release leading to apoptosis and cell death. MitoQ intervention successfully mitigated these effects and restored cell viability. The UVA + CSC model could be used to study stress induced mitochondrial dysfunction. Additionally, MitoQ can serve as a viable antioxidant in attenuating mitochondrial dysfunction, underscoring its potential as a molecular-focused treatment approach to combat FECD pathogenesis.

Mitochondrial free radicals contribute to cigarette smoke condensate-induced impairment of oxidative phosphorylation in the skeletal muscle in situ

Oxidative stress plays a critical role in cellular dysfunction associated with cigarette smoke exposure and aging. Some chemicals from tobacco smoke have the potential to amplify mitochondrial ROS (mROS) production, which, in turn, may impair mitochondrial respiratory function. Accordingly, the present study tested the hypothesis that a mitochondria-targeted antioxidant (MitoTEMPO, MT) would attenuate the inhibitory effects of cigarette smoke on skeletal muscle respiratory capacity of middle-aged mice. Specifically, mitochondrial oxidative phosphorylation was assessed using high-resolution respirometry in permeabilized fibers from the fast-twitch gastrocnemius muscle of middle-aged C57Bl/6J mice. Before the assessment of respiration, tissues were incubated for 1hr with a control buffer (CON), cigarette smoke condensate (2 % dilution, SMOKE), or MitoTEMPO (10 μM) combined with cigarette smoke condensate (MT + SMOKE). Cigarette smoke condensate (CSC) decreased maximal-ADP stimulated respiration (CON: 60 ± 15 pmolO2.s−1.mg−1 and SMOKE: 33 ± 8 pmolO2.s−1.mg−1; p = 0.0001), and this effect was attenuated by MT (MT + SMOKE: 41 ± 7 pmolO2.s−1.mg−1; p = 0.02 with SMOKE). Complex-I specific respiration was inhibited by CSC, with no significant effect of MT (p = 0.35). Unlike CON, the addition of glutamate (ΔGlutamate) had an additive effect on respiration in fibers exposed to CSC (CON: 0.9 ± 1.1 pmolO2.s−1.mg−1 and SMOKE: 5.4 ± 3.7 pmolO2.s−1.mg−1; p = 0.008) and MT (MT + SMOKE: 8.2 ± 3.8 pmolO2.s−1.mg−1; p ≤ 0.01). Complex-II specific respiration was inhibited by CSC but was partially restored by MT (p = 0.04 with SMOKE). Maximal uncoupled respiration induced by FCCP was inhibited by CSC, with no significant effect of MT. These findings underscore that mROS contributes to cigarette smoke condensate-induced inhibition of mitochondrial respiration in fast-twitch gastrocnemius muscle fibers of middle-aged mice thus providing a potential target for therapeutic treatment of smoke-related diseases. In addition, this study revealed that CSC largely impaired muscle respiratory capacity by decreasing metabolic flux through mitochondrial pyruvate transporter (MPC) and/or the enzymes upstream of α-ketoglutarate in the Krebs cycle.

Protein inhibitor of activated signal transducer and activator of transcription 2 is an oncoprotein in oral squamous cell carcinoma and related to cigarette smoking - An in vitro study

Background/purposeOral cancer is one of the most prevalent malignant tumors in Taiwan. Due to the heterogeneity of oral cancer cells, the five-year survival rate of patients is only 50%. Post-translational modifications contribute to protein diversity and directly influence cell functions. The protein inhibitor of activated signal transducer and activator of transcription 2 (PIAS2) is known to undergo post-translational modifications, yet its impact on oral cancer remains unclear. Materials and methodsPIAS2 expression was modulated by transfecting cells with a PIAS2 expression vector or by knocking down PIAS2 using siRNA with low and high PIAS2 expression, respectively. These cells were subjected to invasion, migration, and proliferation assays to evaluate the effects of PIAS2. Changes in genotype, such as epithelial-mesenchymal transition (EMT) markers, were also examined. Additionally, the effect of cigarette smoke condensate (CSC) on PIAS2 expression in oral squamous cell carcinoma (OSCC) cells was investigated. ResultsOverexpression of PIAS2 significantly increased the malignant behaviors of oral cancer cells. In YD38 and SAS cells with low PIAS2 expression, overexpression of PIAS2 enhanced proliferation, invasion, and migration. PIAS2 overexpression also affected EMT gene expression, suppressing E-cadherin and increasing fibronectin expression. Conversely, PIAS2 knockdown in OECM1 and SCC25 cells suppressed malignant behaviors and reversed EMT markers, increasing E-cadherin and decreasing fibronectin expression. Furthermore, a dose-dependent increase in PIAS2 expression was observed when OSCC cells were treated with CSC. ConclusionPIAS2 functions as an oncogene in oral cancer, and cigarette smoking induces PIAS2 expression. Increased PIAS2 levels lead to enhanced malignancy in oral cancer.

Cigarette smoking-related OLC1 overexpression associated with poor prognosis in bladder urothelial carcinoma

AimsTo explore the clinical significance of OLC1 and cigarette smoking in bladder urothelial carcinoma (UBC). Materials and methodsOLC1 mRNA expression was detected in 106 UBC samples by mRNA array or reverse real-time PCR. OLC1 protein expression in 114 UBC samples was detected by immunohistochemical staining. Wild-type C57BL/6J mice were injected with cigarette smoke condensate (n = 12) or exposed to cigarette smoke (n = 6) to investigate the correlations between cigarette smoking and OLC1 expression using mRNA array. Key findingsThe mRNA and protein expression of OLC1 were higher in tumor samples (p < 0.01) and significantly correlated with tumor stage (p < 0.05). OLC1 protein expression and smoking history were correlated with disease-free survival (p < 0.05). OLC1 expression was significantly elevated in smoking patients with higher smoking intensity on both mRNA and protein levels (p < 0.05). Cigarette smoke exposure experiments revealed that OLC1 mRNA overexpressed in bladder uroepithelium of mice. SignificanceOLC1 could serve as a potential prognosis biomarker of UBC, especially for smoking patients.

The Cytochrome P450 2C8*3 Variant (rs11572080) Is Associated with Improved Asthma Symptom Control in Children and Altered Lipid Mediator Production and Inflammatory Response in Human Bronchial Epithelial Cells.

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence, and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiologic processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 (n = 207) for patients with one or more copies of the CYP2C8*3 allele versus 4.42 (n = 965) for CYP2C8*1/*1 (P = 0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid. Interestingly, 9(10)- and 12(13)- dihydroxyoctadecenoic acid, the hydrolyzed metabolites of 9(10)- and 12(13)- epoxyoctadecenoic acid, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. SIGNIFICANCE STATEMENT: These findings suggest a role for CYP2C8 in regulating the epoxyoctadecenoic acid:dihydroxyoctadecenoic acid ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.

Tobacco Smoke Condensate Induces Morphologic Changes in Human Papillomavirus-Positive Cervical Epithelial Cells Consistent with Epithelial to Mesenchymal Transition (EMT) with Activation of Receptor Tyrosine Kinases and Regulation of TGFB.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10-6-100 μg/mL). We found CSC (10-3 or 10 μg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2, PDGFRB, and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

MiRNAs mediate the impact of smoking on dental pulp stem cells via the p53 pathway.

Cigarette smoke changes the genomic and epigenomic imprint of cells. In this study, we investigated the biological consequences of extended cigarette smoke exposure on dental pulp stem cells (DPSCs) and the potential roles of miRNAs. DPSCs were treated with various doses of cigarette smoke condensate (CSC) for up to 6 weeks. Cell proliferation, survival, migration, and differentiation were evaluated. Cytokine and miRNA expression were profiled. The results showed that extended exposure to CSC significantly impaired the regenerative capacity of the DPSCs. Bioinformatic analysis showed that the cell cycle pathway, cancer pathways (small cell lung cancer, pancreatic, colorectal, and prostate cancer), and pathways for TNF, TGF-β, p53, PI3K-Akt, mTOR, and ErbB signal transduction, were associated with altered miRNA profiles. In particular, 3 miRNAs has-miR-26a-5p, has-miR-26b-5p, and has-miR-29b-3p fine-tune the p53 and cell cycle signaling pathways to regulate DPSC cellular activities. The work indicated that miRNAs are promising targets to modulate stem cell regeneration and understanding miRNA-targeted genes and their associated pathways in smoking individuals have significant implications for disease control and prevention.

Abstract 5445: Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach

Abstract Introduction: The cancer stem cell hypothesis explains the heterogeneity, drug resistance, and metastatic potential of Small Cell Lung Cancer (SCLC). To address limited surgical tissue availability, we developed an induced pluripotent stem cell (iPSC) model for investigating SCLC stemness mechanisms and potential novel therapies. Cigarette smoke condensates (CSC) have previously been linked to stem cell activation in lung epithelial cells. We aimed to transform normal lung iPSCs (Lu-iPSC) through prolonged CSC exposure to identify candidate genes in the transition to cancer stem cells. Methods: Four Lu-iPSC clones derived from normal human small airway epithelial cells (SAEC) of two healthy donors were exposed to CSC at different concentrations for short- (7 days) and long-term (6 months) periods. RNA-seq was performed to identify differentially expressed genes (DEGs) relative to respective SAEC. Gene expression data of 23 SCLC cell lines and 38 NSCLC from the public databases was extracted and analyzed relative to SAEC. In vitro tri-lineage differentiation studies, drug sensitivity assays, and phenotypic characterization by flow cytometry was performed in this study. Results: Gene expression profiles of Lu-iPSCs closely resembled those of SCLC rather than NSCLC. Short-term CSC exposure reduced Lu-iPSC proliferation while increasing the expression of key stemness markers such as SALL1 in a dose-dependent manner. Concurrently, CSC exposure downregulated the expression of nodal and its inhibitors, LEFTY1 and LEFTY2, which can be considered as the earliest signs of differentiation events in Lu-iPSC. Long term CSC exposure induced expression of epithelial mesenchymal transition markers including ZEB1 and ZEB2. In-vitro trilineage differentiation studies revealed that CSC particularly decreased expression of endodermal lineage specific markers (GATA4, SOX17, and FOXA2) without affecting mesodermal and ectodermal markers. Importantly, CSC induced the phenotypic expression of several lung cancer stem cell markers (CD44, CD133, and CD338) along with resistance to cisplatin and topotecan. RNA seq analysis of long-term CSC exposed Lu-iPSCs identified 92 common DEGs (53 up and 39 down) relative to their controls. Gene set enrichment analysis on DEGs indicated upregulation of Notch signaling, IGFBPs, TGF beta receptor signaling, and NCAM1 interactions while GABA receptor activation and GPCR signaling were downregulated. Comprehensive RNA seq analysis of CSC exposed and unexposed Lu-iPSCs relative to SCLC and NSCLC, identified potential candidate genes associated with SCLC stemness. Conclusion: Our findings suggest that this iPSC model shows promise for investigating mechanisms mediating stemness in SCLC and identifying candidate genes and their associated pathways as potential targets for preclinical development of novel treatment regimens for these highly lethal neoplasms. Citation Format: Sudheer Kumar Gara, Haitao Wang, Anand Singh, Tuana Tolunay, Vivek Shukla, Ruihong Wang, Mary Zhang, Stephanie Shiffka, David S. Schrump. Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5445.

Abstract 1463: Characterizing inflammation-induced epigenetic and transcriptomic alterations driving NSCLC using lung organoid models

Abstract Chronic inflammation is a major driver of lung cancer and contributes to its pathogenesis by inducing genetic and epigenetic alterations. These, in turn, modulate the microenvironment to facilitate emergence of tumor promoting immune evasive mechanisms. The evolution of epigenetic events and their role, both alone, and in association with lung cancer specific genetic drivers in initiation of lung cancer is not well delineated. Specific pathological subtypes of non-small cell lung cancer (NSCLC) have distinct cells of origin, suggestive of cell type-specific changes in driving their development. We employed lung organoid models with chronic exposure to cigarette smoke condensate (CSC) to elucidate sequential, cell type-specific epigenetic and transcriptomic alterations and their role in co-operating with genetic events to drive development of NSCLC. We examined epigenomic and transcriptomic alterations over a 6-month exposure period by DNA methylation arrays, ATAC-seq, and RNA-seq. We observe that CSC exposure causes distinct morphological changes in organoid structure and composition, accompanied by an increase in proliferative potential. Using flow cytometry and immunofluorescent staining approaches, we demonstrate a chronic inflammation-driven shift in stem cell populations accompanied by reduction in differentiated cell types. Bulk and single-cell RNA sequencing approaches identified distinct temporal changes in transcriptional signatures in CSC exposed organoids suggestive of evolution of an immune evasive and pro-tumorigenic phenotype. Gene set enrichment analysis revealed a downregulation of interferon signaling and upregulation of Myc targets and oxidative phosphorylation pathways in CSC treated organoids. Notably, co-culturing organoids with key immune cell populations, demonstrate the ability of the CSC exposed organoids to modulate shifts in immune cells from a pro- to anti- inflammatory state. In accordance with this shift in immune cell phenotype, CSC treated organoids secreted decreased levels of pro-inflammatory cytokines as measured by multiplex ELISA assays. Genome wide DNA methylation analysis reveal that CSC methylated genes were associated with differentiation and cell fate commitment as well as transcription factor activity. Finally, we demonstrate that introduction of key NSCLC-specific driver events at the 6-month time point sensitize only CSC treated organoids to tumor formation in vivo, leading to the development of distinct genetic driver-specific subtypes of NSCLC. Altogether, our results demonstrate the ability of chronic inflammation to drive key cell type-specific transcriptomic and epigenetic changes leading to the development of distinct subtypes of NSCLC. Our findings will help identify molecular correlates for early detection and aid development of novel therapeutic strategies for disease interception. Citation Format: Na Wang, Kara Lombardo, Raksha Padaki, Ray-Whay Chiu Yen, Malcolm V. Brock, Leslie Cope, Hariharan Easwaran, Stephen B. Baylin, Michelle Vaz. Characterizing inflammation-induced epigenetic and transcriptomic alterations driving NSCLC using lung organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1463.

Abstract 4830: Predicting drug treatment response in African American lung cancer patients who smoke

Abstract Background: Lung cancer is the third most commonly diagnosed cancer in the US and the leading cause of cancer-related death. African Americans (AAs) have higher lung cancer incidence and mortality rates, and lower survival rates, when compared to their European American (EA) counterparts. It is widely known that cigarette smoking is the strongest risk factor for lung cancer development, but it also plays a significant role in clinical outcome. Cigarette smoking after lung cancer diagnosis (up to 30% patients) comes with several risks, including increased secondary tumors and mortality rates (by 20%), as well as poor treatment outcomes (higher radiation and chemotherapy failure and higher drug clearance). There are limited transcriptomic studies that describe molecular characterizations of AA smokers with lung cancer to predict treatment outcomes. Hypothesis: Cigarette smoking leads to greater drug resistance-related gene expression in AA lung cancer patients. Methods: mRNA expression changes in an AA (NCI-H2172) and EA (NCI-H1944) lung cancer cell line pair exposed to liquid cigarette smoke condensate (CSC) were profiled. The cell lines were matched based on age (62 years), sex (female), histology (lung adenocarcinoma, LUAD, the most common subtype), and culture properties (adherent). The CSC was derived from Murty Pharmaceuticals and prepared by smoking University of Kentucky's 3R4F Standard Research Cigarette on an FTC Smoke Machine. A total of 500,000 cells per well were treated for one hour under standard incubation conditions (37°C, pH 7.0, 5% CO2). Cells were pelleted, RNAs were isolated, treated with DNAse-I to remove contaminating genomic DNA, quantified, and sent for bulk mRNA-sequencing (Novogene Co). Three biological replicates and technical triplicates were performed. Partek Flow and Partek Genomics Suite workflows were used to identify population-specific differentially expressed gene (DEG) signatures and pathway enrichment after CSC exposure. DEG were profiled in LUAD tissues from current smokers in an AA and EA TCGA cohort (n = 18 AAs, 86 EAs). Results: Population-specific gene expression changes were discovered (n = 52 AA DEGs, n = 31 EA DEGs; P &amp;lt;0.001). All of the DEGs were mutually exclusive with no overlap. Some pathways were shared between both populations and others were unique. PI3K/Akt/mTOR signaling pathway enrichment was unique in AA LUAD cells and ECM receptor interactions were distinct in EA LUAD cells. AKT2 (which activates mTOR) had significantly higher expression in LUAD tumor tissues from current AA smokers compared to current EA smokers. Conclusion: Population-specific PI3K/Akt/mTOR pathway alterations may help explain drug resistance in LUAD tumors from AA patients. PI3K signaling is considered a significant cause of resistance to chemotherapy, targeted therapy, and immunotherapy. Future Directions: Expose AA and EA LUAD cell lines to CSC and PI3K/Akt/mTOR pathway inhibitors. Citation Format: Mia Ray, Isaiah Osei-Gyening, Kwabena Acheampong, Marisa Riebesell, Khadijah A. Mitchell. Predicting drug treatment response in African American lung cancer patients who smoke [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4830.

Abstract 7082: Epidemiology meets epitranscriptomics: Exploring the cancer-related role of a novel TERT-antisense transcript and its regulation by RNA methylation

Abstract Human telomerase reverse transcriptase (TERT) maintains telomeres at chromosome ends and its dysregulation is implicated in various human health conditions. Multiple GWAS signals are detected within the TERT genomic region at chr 5p15.33. As a potential proxy for these signals, we investigated a highly polymorphic variable number tandem repeat (VNTR)2-1 within intron 2 of TERT, with 57-143 copies of a 42 bp repeat unit per allele. By functional annotation of VNTR2-1, we predicted that it generates an intronic transcript antisense to TERT with a variable-size open reading frame (ORF) of 2502-5211 bp (834-2038 aa). Specific to the TERT-antisense transcript, we predicted N6-methyladenosine (m6A) consensus motifs within each 42 bp unit of VNTR2-1, with longer VNTR2-1 regions carrying more m6A motifs. In a stranded RNA-seq dataset of African pediatric Burkitt lymphoma (BL) tumors, which have high TERT expression, we detected the VNTR2-1-containing TERT-antisense transcript in a subset of tumors (n=35/113, 30.9%), confirming the expression of this novel transcript. VNTR2-1 expression was also experimentally validated by 5’-RACE/Nanopore-seq in Raji (BL cell line) and UMUC3 (bladder cancer cell line). Additionally, the VNTR2-1 transcript was visualized in UMUC3 cells using RNA-FISH. The consensus 42 bp VNTR2-1 repeat sequence and its m6A-deficient variants were cloned into a m6A-deficient dual-luciferase reporter vector (m-psiCHECK-2) and expressed in UMUC3 cells, showing an m6A-motif dose-dependent response driving luciferase protein expression. Transient treatment with STM2457, an inhibitor of the m6A writer METTL3, reduced the expression of the luciferase reporter, confirming that m6A motifs within VNTR2-1 are methylated by METTL3. Additionally, Long-term cigarette smoke condensate (CSC) treatment of UMUC3 reduced luciferase expression, suggesting alterations of VNTR2-1 m6A due to smoke exposure. We explored potential peptides produced from VNTR2-1 transcript by analysis of public mass spectrometry datasets using PepQuery and identified several unique VNTR2-1 peptide fragments in human tumors. Next, we cloned a synthetic FLAG-tagged gene containing three VNTR2-1 consensus repeats with native Kozak sequence and translation start/stop codons. Western blotting confirmed the translation of the VNTR2-1 peptide that was ablated by start codon mutation, suggesting that the native Kozak sequence is sufficient for VNTR2-1 translation. These results support VNTR2-1 as a peptide-producing transcript. In conclusion, we identified a novel intronic transcript produced by VNTR2-1 antisense to TERT. This m6A-regulated transcript could function through several mechanisms, such as a lncRNA or translated peptide, potentially regulating TERT expression and telomerase function in health and disease conditions, including cancer. Citation Format: Brenen W. Papenberg, Oscar Florez-Vargas, Michelle Ho, Kaitlin Forsythe, Chi Zhang, Chia-Han Lee, Sam Mbulaiteye, Pedro J. Batista, Ludmila Prokunina-Olsson. Epidemiology meets epitranscriptomics: Exploring the cancer-related role of a novel TERT-antisense transcript and its regulation by RNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7082.

Repeated intratracheal instillation of whole-cigarette smoke condensate to assess lung damage in a rat model.

Cigarette smoke induces an inflammatory response in the lungs by recruiting inflammatory cells, leading to lung diseases such as lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis. Existing inhalation exposure methods for assessing the adverse effects of cigarette smoke require expensive equipment and are labor-intensive. Therefore, we attempted to develop a novel method to assess these adverse effects using intratracheal instillation (ITI) of whole cigarette smoke condensate (WCSC). The WCSC (0, 5, 10, or 20 mg/mL) was administered by ITI once daily for 6 or 12 days using an automatic video instillator. Repeated WCSC ITI increased the lung weight, and monocyte chemoattractant protein-1 (MCP-1), neutrophil, and lymphocyte levels within bronchoalveolar lavage fluid compared to the control. In the histopathological analysis of the lung tissue, a mild inflammatory response was observed in the 6 and 12 days 20 mg/mL WCSC exposure groups. The genome-wide RNA-seq expression patterns revealed that inflammatory and immune response-related genes, such as the chemokine signaling pathway, Th1/Th2 cell differentiation, and cytokine-cytokine receptor interaction, were employed following WCSC exposure. In addition, MCP-1 was time-dependent and increased in the 10 mg/mL exposure group compared to the control group. These results suggested that the WCSC might induce the potential pulmonary inflammatory response. Furthermore, we proposed that ITI may be a rapid and effective method of evaluating the adverse effects of WCSC within a short exposure period (less than 2 weeks), and it can be used to evaluate cigarette inhalation toxicity studies as an alternative method.

Korean Red Ginseng suppresses emphysematous lesions induced by cigarette smoke condensate through inhibition of macrophage-driven apoptosis pathways

BackgroundCigarette smoke is generally accepted as a major contributor to chronic obstructive pulmonary disease (COPD), which is characterized by emphysematous lesions. In this study, we investigated the protective effects of Korean Red Ginseng (KRG) against cigarette smoke condensate (CSC)-induced emphysema. MethodsMice were instilled with 50 mg/kg of CSC intranasally once a week for 4 weeks, KRG was administered to the mice once daily for 4 weeks at doses of 100 or 300 mg/kg, and dexamethasone (DEX, positive control) was administered to the mice once daily for 2 weeks at 3 mg/kg. ResultsKRG markedly decreased the macrophage population in bronchoalveolar lavage fluid and reduced emphysematous lesions in the lung tissues. KRG suppressed CSC-induced apoptosis as revealed by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining and Caspase 3 immunohistochemistry. Additionally, KRG effectively inhibited CSC-mediated activation of Bcl-2-associated X protein/Caspase 3 signaling, followed by the induction of cell survival signaling, including vascular endothelial growth factor/phosphoinositide 3-kinase/protein kinase B in vivo and in vitro. The DEX group also showed similar improved results in vivo and in vitro. ConclusionTaken together, KRG effectively inhibits macrophage-mediated emphysema induced by CSC exposure, possibly via the suppression of pro-apoptotic signaling, which results in cell survival pathway activation. These findings suggest that KRG has therapeutic potential for the prevention of emphysema in COPD patients.

Investigating the Impact of Smoking Traditional and E-Cigarettes on Acute Myeloid Leukemia Progression

Acute myeloid leukemia (AML) is a highly heterogeneous disease with a variable prognosis. The exact mediators of AML are not fully understood but there are several factors that may contribute to the development of the disease. These can be split into genetic and non-genetic causes. cytogenetic abnormalities have been identified such as FLT 3 gene which frequently mutated in AML and associated with a poor prognosis. As for non-genetic causes: prior exposure to chemotherapy or radiation therapy have been implicated. Lately cigarette smoking in AML has been shown to have poor survival outcome for AML patients relative to non-smokers. Cigarette smoke contains many compounds, some of which have been associated with increased risk for AML. Benzene, radioactive components, and possibly other carcinogens are present in tobacco and tobacco smoke which lead to production of chromosomal defects. Cigarette smoke exposure globally alters DNA methylation in blood cells and these changes can persist for decades. All commercial tobacco products, including smokeless tobacco, cigarette smoke and E cigarettes contain tobacco-specific nitrosamines such asN′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK and NNN promotes tumor growth by enhancing and deregulating cell proliferation, survival, migration, and invasion, thereby creating a microenvironment for tumor growth. This calls for a need for to understand the molecular changes in AML especially when exposed to agents that can impact survival. Therefore, we hypothesize when FLT-3 AML model is exposed to smoking metabolites such as cigarettes smoking condensate (CSC), NNN, NNK and E cigarettes can lead to increased leukemia burden. To test this in vitro CSC treatment of AML cells was used to evaluate how chemicals from cigarette smoke can impact AML cells without the high heat combustion from cigarettes. NNN and NNK treatment were separately used for treatment of AML cells. Two FLT-3 AML cell lines used (MOLM-13 and MV-4-11) and non-FLT-3 AML cell line (OCI-AML3) as control.Cells were treated in vitro with 10 ug/ml cigarette smoke condensate (CSC) or 1uM NNN or 1 uM NNK or vehicle (DMSO). Viability and cell proliferation were measured with each passage every 48-72 hours. There was no difference in cell proliferation and viability with CSC, NNN or NNK exposure. This suggests possibly that microenvironment is needed to evaluate the impact of smoking on AML progression. To test this in vivo, luciferase tagged MOLM13 cells were treated in vitro with 10ug/ml cigarette smoke condensate (CSC) or vehicle DMSO for two weeks then injected into mice. Mice injected with MOLM 13 cells treated with CSC had significantly more leukemia burden than control. Similar findings were replicated in MV-4-11 mouse model. Next, we wanted to evaluate if this increased leukemia burden leads to DNA methylation changes. We Found 8 genes to be significantly hypermethylated from the CSC two-week in vitro treatment. This was also found in the vivo experiment where mice were exposed to Cigarette smoking in vivo. (Table 1) This study shows that CSC treated AML cell lines can lead to increase leukemia burden in vivo but not in vitro. There are difference in DNA methylation between AML cells lines exposed to CSC compared to control. Our future directions will include evaluate leukemia burden in FLT-3 AML mouse model exposed to NNN, NNK and E- cigarettes Study ROS changes, protein expression and DNA methylation impacted by smoking.

P14-23: Effects of repeated heat-not-burn-whole cigarette smoke condensate treatment in rat liver in vivo

P14-23: Effects of repeated heat-not-burn-whole cigarette smoke condensate treatment in rat liver in vivo

Cigarette smoking exposure disrupts the regenerative potential of dental pulp stem cells.

Smoking is known to alter the regenerative and immunomodulatory properties of many types of mesenchymal stem cells (MSCs). This study investigates the impact of cigarette smoke exposure on the regenerative potential of dental pulp stem cells (DPSCs). DPSCs were treated with various doses of cigarette smoke condensate (CSC) or nicotine. Cell proliferation and survival were evaluated by a water-soluble tetrazolium salt (WST-1) and a survival assay. DPSC migration, cytokine expression, mutagenesis, and the signaling pathway were also measured during CSC and nicotine treatment. Low concentrations of CSC and nicotine did not impair cell proliferation, but higher concentrations reduced cell proliferation. CSC and nicotine could impede DPSC survival and migration in a dose-dependent manner. In addition, the cytokine secretion expression profile was altered with CSC or nicotine treatments. In particular, secretion of IL-6, TNF-α, and IL-10 significantly increased, while TGF-β1 levels showed different patterns after exposure to CSC or nicotine, as shown by ELISA and quantitative PCR. Nicotine treatment increased AKT (also known as protein kinase B) and extracellular signal-regulated kinase (ERK) phosphorylation. Finally, CSC induced higher levels of mutagenicity than nicotine, as shown by the Ames test. These findings suggest that cigarette smoke exposure alters the regenerative abilities of DPSCs in various ways. Future studies are warranted to further characterize the underlying molecular mechanisms of smoking-mediated damage to DPSCs, which will guide the personalized stem cell treatment plan for smoking patients.

Cryptotanshinone alleviates lipopolysaccharide and cigarette smoke-induced chronic obstructive pulmonary disease in mice via the Keap1/Nrf2 axis

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity worldwide. Cigarette smoking, which leads to abnormalities in the airways or alveoli and persistent obstruction of the airway's flow, is a significant risk factor of COPD. Cryptotanshinone (CTS) is the active ingredient in Salvia miltiorrhiza (Danshen) and has many pharmacological properties including anti-inflammatory, antitumor, and antioxidant properties, but its impact on COPD is uncertain. In the present study, the potential effect of CTS on COPD was investigated in a modified COPD mice model induced with cigarette smoke (CS) and lipopolysaccharide (LPS) exposure. CTS significantly reversed the decline in lung function, emphysema, inflammatory cell infiltration, small airway remodeling, pulmonary pathological damage, and airway epithelial cell proliferation in CS- and LPS-exposed mice. Additionally, CTS decreased inflammatory cytokines such as tumor necrosis factor α (TNF α), interleukins IL-6 and IL-1β, and keratinocyte chemoattractant (KC), increased the activities of superoxide dismutase (SOD), Catalase (CAT) and L-Glutathione (GSH), and repressed the expression of protein hydrolases matrix metalloprotein (MMP)− 9 and − 12 in pulmonary tissue and bronchoalveolar lavage fluid (BALF). The protective effects of CTS were also observed in human bronchial epithelial cell line BEAS-2B simulated with cigarette smoke condensate (CSC) and LPS. Mechanistically, CTS can repress the protein level of Keap1, resulting to activation of erythroid 2-related factor (Nrf2), finally alleviating COPD. In summary, the present findings demonstrated that CTS dramatically ameliorates COPD induced by CS and LPS via activating Keap1/Nrf2 pathway.

Development and assessment of a novel standardized method for preparation of whole cigarette smoke condensate (WCSC) for toxicity testing of cigarette smoke

Cigarette smoke condensates provide toxic effects data for cigarette products, but the lack of a standardized preparation method yields inconsistent results. This study developed a standardized whole cigarette smoke condensate (WCSC) preparation method, encompassing both particulate and gaseous phases. The goal of this study was to obtain a WCSC of consistent quality by maximizing cigarette smoke concentration. The effectiveness of the method was verified by evaluating the concentrations of total particulate matter (TPM) and organic compounds (OCs: nicotine, triacetin, glycerin, acetaldehyde, and acetone) in the samples at each step of preparing the WCSC from cigarette smoke: (1) generating cigarette smoke using the HCI method, (2) collecting particulate and gaseous phases in cigarette smoke using the Cambridge filter (using modified ISO method) and Dulbecco’s phosphate buffered saline (DPBS; the maximum allowable TPM mass per the one Cambridge filter is 537 mg), (3) extracting TPM collected on the Cambridge filter using methanol (the maximum TPM mass that can be effectively extracted using 5 mL of methanol is 1085 mg), (4) removing methanol from the TPM extract using the N2 evaporation method (the relative recovery of OCs by N2 evaporation is 97.3 ± 0.96 %), and (5) mixing the TPM residue and DPBS-absorbing gaseous phase of cigarette smoke (relative recovery of OCs in the WCSC compared to the TPM residue and the DPBS is 94.7 ± 0.52 % with fairly good reproducibility and RSD values below 5 %).

WITHDRAWN: Evaluation of the effects of e-cigarette aerosol extracts and tobacco cigarette smoke extracts on human gingival epithelial cells

Smoking increases the risk of a number of diseases, including cardiovascular, oral and lung diseases. E-cigarettes are gaining popularity among young people as an alternative to cigarettes, but there is debate over whether they are less harmful to the mouth than e-cigarettes. In this study, human gingival epithelial cells (HGECs) were treated with four commercially available e-cigarette aerosol condensates (ECAC) or commercially available generic cigarette smoke condensates (CSC) with different nicotine concentrations. Cell viability was determined by MTT assay. Cell apoptosis was observed by acridine orange (AO) and Hoechst33258 staining. The levels of type I collagen, matrix metalloproteinase (MMP-1, MMP-3), cyclooxygenase 2 and inflammatory factors were detected by ELISA and RT-PCR. Finally, ROS levels were analyzed by ROS staining. The different effects of CSC and ECAC on HGECs were compared. The results showed that higher nicotine concentration of CS significantly reduced the activity of HGECs. By contrast, all ECAC had no significant effect. The levels of matrix metalloproteinase, COX-2, and inflammatory factors were higher in HGECs treated with CSC than those treated with ECAC. In contrast, the level of type I collagen was higher in HGECs treated with ECAC than those treated with CSC. In conclusion, all four flavors of e-cigarettes were less toxic to HGE cells than tobacco, but further clinical studies are needed to determine whether e-cigarettes are less harmful to oral health than conventional cigarettes.