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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Biomed Pharmacother. 2021 Oct 19;144:112312. doi: 10.1016/j.biopha.2021.112312

Nuclear factor kappa-B contributes to cigarette smoke tolerance in pancreatic ductal adenocarcinoma through cysteine metabolism

Venugopal Gunda 1, Yashpal S Chhonker 2, Nagabhishek Sirpu Natesh 1, Pratima Raut 1, Sakthivel Muniyan 1, Todd A Wyatt 3,4,5, Daryl J Murry 2,6, Surinder K Batra 1,6, Satyanarayana Rachagani 1
PMCID: PMC8599650  NIHMSID: NIHMS1751771  PMID: 34678726

Abstract

Background:

Retrospective studies revealed that cigarette smoking enhances risk of incidence and worsens prognosis in pancreatic cancer (PC) patients. Poor prognosis in smoker cohort of PC patients indicates prevalence of cigarette smoke stimulated survival mechanisms yet to be explored in PC. In this study, cigarette smoke induced metabolic pathways were explored and targeted in PC.

Methods:

Human pancreatic ductal adenocarcinoma cell (PDAC) lines, genetically engineered mice models (GEMMs), mass spectrometry based heavy isotope-based metabolite analysis, cytotoxicity assays and Nuclear factor kappa-B (NF-kB) targeting were utilized in this study. Cigarette smoke extract (CSE) was prepared fresh each day by bubbling cell culture media with the smoke emitted from 85 mm, filtered, Code 1R6F reference cigarettes and used for in vitro procedures. High dose cigarette smoke exposure of GEMMs was achieved by daily exposure of animals to similar cigarettes, 6 h/day for a total period of 180 days.

Findings:

We observed that PDAC cells upregulate glutathione anabolism through cysteine uptake and glutamate cysteine ligase (GCLM), supporting survival of PDAC cells. In vivo, cigarette smoke exposure leads to concomitant upregulation of GCLM and activated NF-kB in the PDAC consistent with in vitro, in CSE-exposed PDAC. Finally, either inhibition of NF-kB or depletion of cysteine impaired PDAC cell survival in cigarette smoke exposed conditions through suppression of glutathione and ROS enhancement, reverted by glutathione supplementation.

Interpretation:

Our findings demonstrate scope for targeting smoke induced, NF-kB mediated, cysteine and glutathione metabolism for improving the survival of smoke addicted PDAC.

Funding:

The authors obtained funding from the National Institutes of Health and the National Cancer Institute (R01 CA247763, R21 CA238953, P01 CA217798, R01 CA 228524, and R01 CA206444).

Keywords: Pancreatic cancer, cigarette smoke, NF-κB, cysteine, glutathione

Graphical Abstract

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1. Introduction

Cancers of pancreas are listed among the top ten leading cancer types affecting patient survival in the United States (1). According to recent statistical projections, there will be high percentage of mortality (8%) in pancreatic cancer (PC) patients compared to the percent affected with this cancer which would be about 3% (1). Identifying the contributions of individual risk factors facilitating poor prognosis in pancreatic ductal adenocarcinoma (PDAC, which is the most common among PC cases) would be useful for improving early screening and awareness on risks associated with etiological factors contributing to poor outcomes in PDAC patients (2). Somatic mutation of KRAS is the most common in PDAC which supports initial transformation of pancreatic acinar and ductal cells (3). Subsequent progression of PDAC into aggressive tumors depends on contribution of collateral genetic mutations (4–6), as well as etiological factors which play key role in mediating KRAS-driven PDAC through diverse mechanisms (7, 8).

Cigarette smoking leads to poor prognosis in PC patients indicating the critical role of smoking in etiology of PC and its association with increased risk of PC (9). Comparison of survival trends among the PC patients with different magnitudes of cigarette smoking such as, longer duration of cigarette smoking, and higher number of cigarettes smoked per day during smoking period revealed significant negative impact of the high dose cigarette smoking on survival of PC patients (10). These negative impacts of chronic and high dose cigarette smoking on the survival of PC patients have been echoed in retrospective studies (10, 11) indicating that either chronic or high dosage of cigarette smoking evident among PC patients (12), is deleterious in PC patients. Despite of such absolute negative impact of high dose of cigarette smoke or chronic smoke exposure in PC patients, mechanisms through which PC cells respond to the high dose of cigarette smoke were least studied. Especially, availability of PC cell lines and genetically modified mice models of PC harboring mutant, KRASG12D as well as wild type KRAS genes could be utilized for fortifying the effects of high dosage of cigarette smoke on PC.

Components of cigarette smoke, such as cotinine, stimulate oncogenic signaling mechanisms that facilitate survival of transformed cells, which in turn, promote oncogene-driven tumor initiation and progression in cancer (13). Chronic and daily higher consumption of cigarettes leads to accumulation of the cigarette smoke derived carcinogens in the tissues of cigarette smokers (14). Such high cigarette smoke derived carcinogen levels were achieved for research studies in vivo in mice models by exposing mice to higher doses of cigarette smoke (250 mg/m3 of total particulate material) (15) and by culturing cancer cell lines harboring oncogenes to concentrated smoke extract (CSE) for in vitro evaluations (16). Human PC cell lines and the genetically engineered mice models (GEMMs) of PC harbor genetic mutations prevalent in PC (14, 16). Among the oncogenes promoting PDAC, KRASG12D facilitates transformation of non-malignant pancreatic intraepithelial neoplasms (PanINs) to intraductal papillary mucinous neoplasms (IPMNs) with additional mutations (TP53, SMAD4 and CDKN2A loss) resulting in malignant pancreatic ductal adenocarcinoma (PDAC). Furthermore, KRASG12D also supports tumor promoting metabolism as established in in vivo models of PC (17). Smoke exposure also leads to metabolic alterations in cancer (18). However, the effects of smoke on metabolism in KRASG12D as well as KRAS wild type models of PDAC are not known. This is significant in lieu of fact that both KRASG12D and smoke have independent and negative impact on PC survival (12, 19).

Mutant KRAS supported tumor progression involves regulation of cytostatic stress mechanisms which were well reported in PDAC (20, 21). Thus, existence of the stress regulatory mechanisms in PDAC implies that high dose cigarette smoke-induced stress could be well tolerated in PDAC. However, direct evidence showing suppression of high dose cigarette smoke-induced stress mechanisms in PDAC were not yet reported in the literature and form the basis of the present study. Cellular mechanisms that are common in cigarette smoke-induced pathology and mutant KRAS-driven cancer models would provide clues regarding the significance of cigarette smoke-induced pathology in PDAC. For instance, signaling and survival pathways mediated through Nuclear factor-κ-beta (NF-κB) regulate cellular response and survival in smoke-exposed models (22). In the context of PDAC, NF-κB was identified to play a key role downstream of KRASG12D (23). Thus, both cigarette smoke-induced stress and mutant-KRAS elicited mechanisms involve NF-κB mediated pathways which lead us to hypothesize that cigarette smoke could either potentiate mechanisms fueled by KRAS mutation or activate metabolism identical to metabolism driven by KRAS mutation.

We evaluated our hypothesis of exploring survival mechanisms in high dose cigarette smoke exposed PDAC using in vitro and in vivo models of PDAC. Our study elaborated the metabolism, survival and stress mechanisms elicited by cigarette smoke in PDAC models. Our in vitro findings demonstrate that pancreatic tumor cells exposed to cigarette smoke upregulate their glutathione anabolism. The upregulation of glutathione anabolism in turn depends on cysteine uptake in cigarette smoke exposed PC cells. The de novo glutathione biosynthesis is essential for the survival of PC cells in cigarette smoke exposure which could be abrogated by inhibiting NF-κB as discussed in detail. Thus, we also explored the upregulation of NF-κB, regulation of glutathione metabolism and ROS by NF-κB and the effects of their inhibition on the survival of cigarette smoke exposed, PDAC cells.

2. Materials and Methods

2.1. Cell culture

Human pancreatic cancer cell lines Colo357, Capan1, CD18/HPAFII/HPAF2, T3M4 and CFPAC1 were obtained from American Type Culture Collection (ATCC), validated by short tandem repeat (STR) DNA profiling at the University of Nebraska Medical Center (UNMC) before performing experiments, and cell lines were tested for mycoplasma contamination. Cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM), except for CFPAC1 and Capan1, which were cultured in Isovec Modified Dulbecco Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin) at 37°C with 5% CO2 in a humidified atmosphere. Cells were maintained in common media (DMEM with defined amino acid concentrations) for metabolomics and survival assays.

2.2. Cigarette smoke extract preparation for in vitro studies

Cigarette smoke extract (CSE) was prepared fresh each day for use in all in vitro experimental procedures. The reference cigarettes (85 mm, filtered, Code 1R6F) used in this study were obtained from the Center for Tobacco Reference Products (CTRP), a division of the Kentucky Tobacco Research & Development Center (KTRDC) at the University of Kentucky (Lexington, KY). One cigarette was connected to a peristaltic pump (Model ATS-P, Bentley Laboratories, Santa Anna, CA), lighted, and bubbled through 50 mL of sterile DMEM (pH 7.4) equilibrated at a burn rate of 6 minutes, or 160 cm3/min in 50 mL tube closed with parafilm. At approximately 60–75 mm consumed cigarette, the pump is turned off and the 50 mL tube is capped as 100% CSE (24). Undilute CSE media was then sterile filtered (0.22 μm) and diluted into sterile culture media to a final concentration of 10, 25, 50 and 100% by volume. All CSE was used within 12 h of preparation. Cells were initially seeded in normal DMEM and maintained in incubator for 24 h to facilitate cell adhesion. Cell culture media were mixed with fresh and filtered 100% CSE to achieve desired concentration (10, 25, 50 and 100% by volume) of CSE in DMEM. Cells attached to culture dishes were rinsed with sterile phosphate buffered saline (PBS) and fresh media containing different concentrations of CSE were added and the cells were maintained in incubator till the end of the experiments. Cell viability was >95% for 48 h treatment with up to 10% CSE dilution.

2.3. Steady state metabolomics

Cells (2.5 × 106) were cultured for 24 h in normal DMEM, and culture medium was exchanged with either normal media or media containing 25% CSE. Cells were maintained in different incubators to avoid diffusion of smoke into cells with normal media. Metabolites were analyzed using polar metabolomics methodology as described recently (25). Cell pellets obtained during metabolite extraction were processed for DNA extraction (26) and used for the analysis of normalized metabolite data using Metaboanalyst (27).

2.4. Cell survival assays

Cells were maintained in DMEM with 10% serum for 24 h before initiating cell survival assays. Around 2 × 103 cells per well were seeded in 96-well plates and allowed to attach overnight, followed by exchanging with different media. Normal media contained DMEM (composition based on SH3002201, MilliporeSigma, St. Louis, MO) and cysteine-deprived media were prepared by reconstituting DMEM deprived of methionine and cysteine (D0422, MilliporeSigma) with methionine. Cells seeded in 96-well plates were allowed to grow in defined media [normal, Cys-deprived, CSE mixed media, Erastin (E7781, MilliporeSigma) treated, DL-Butylthionine sulfoximine (2640, MilliporeSigma) treated, BMS345541 (B596080, Toronto Research Chemicals) treated, N-acetylcysteine (A0737, MilliporeSigma) supplemented and reduced glutathione ethylester (GSH-EE, G1404, MilliporeSigma) supplemented media] for 48 h and 96 well plates were analyzed with MTT assay. Additional assays were performed in 12 well plates for 72 h in defined media for microscopic imaging at 10X magnification using EVOS FL Auto Imaging system and acquisition software (ThermoFisher).

2.5. Metabolite labeling analysis

Cells were seeded in 6 cm culture dishes in normal DMEM and allowed to attach for 8 h. Media reconstituted with 13C3,15N-Cys, 10% dialyzed FBS either containing vehicle, Erastin or BMS345541 were added for 4 h and cells were extracted for metabolites using 80% methanol. Metabolite analysis was conducted using a Shimadzu Nexera ultra-high-performance liquid chromatography (UPLC) system (Columbia, MD). Peak resolution and separation for all samples were achieved by using a Discovery HS F5–3 (2.1 mm × 150, 3um, Supelco, Bellefonte, PA) equipped with a C18 guard column, gradient of 0.1% formic acid (mobile phase A) and 0.1% Formic acid in Acetonitrile (mobile phase B) at a total flow rate of 0.25 mL/min. Mass spectrometric detection using multiple reaction monitoring (MRM) for Cysteine, M+4 Cysteine, GSH and M+4 GSH was performed on an LC-MS/MS 8060 system (Shimadzu Scientific).

2.6. In vivo smoke exposure and immunohistochemical analysis

Experiments with KC animals (K-rasG12D; Pdx-1cre) were reviewed and approved by the University of Nebraska Medical Center, Institutional Animal Care and Use Committee (IACUC). Animals at 10 weeks of age were divided into two groups comprising Sham and cigarette smoke exposure groups. Animals in smoke group were exposed to cigarette (University of Kentucky, Reference Cigarette, CTRP 1R6F) smoke for 180 days, 6 h each day (250 mg total suspended particles/m3) which is high dose smoke in each burn cycle modified as per the method described in previous publication (28). Body weights of mice, pancreas weights and pancreatic histology were analyzed at the end of the smoke exposure. Pancreatic tissues were formalin fixed and paraffin embedded for subsequent analysis. Immunohistochemical staining was carried out with anti-GCLM rabbit polyclonal antibody (Abcam-ab194806) and anti-NF-κB rabbit monoclonal antibody (Abcam-ab81283) as described previously (28).

2.7. Evaluation of reactive oxygen species (ROS)

Cells (2 × 104) were seeded in clear bottom, black wall 96 well plates and incubated overnight in normal DMEM for attachment. Media were aspirated and fresh media added for control, CSE, Erastin, BMS345541 and GSH-EE treatments for 1 h. Following incubation, cells were rinsed with PBS and PBS containing 10 μM H2DCFDA (2’−7’-dichlorodihyrofluorescin diacetate, D399, ThermoFisher) was added to the treated cells. Cells were incubated for additional 30 min in DCFDA and rinsed twice with PBS before either imaging or quantifying fluorescence using EVOS FL Auto Imager or SYNERGY neo2 multi-mode reader (BIOTEK, Winooski, VT).

2.8. Western blotting

Cells were treated with normal, CSE and BMS345541 for either 24 h or 1 h and washed twice with cold PBS before lysis in radioimmunoprecipitation assay lysis buffer at 4°C for 30 min. Cell pellets were separated by centrifugation (15,000×g, 10 min) and supernates were estimated for protein concentration. Protein lysates were separated by electrophoresis using SDS-PAGE followed by Western blotting for proteins of interest using primary antibodies against CBS (MA5–17273, ThermoFisher), GCLM (JM93–61, ThermoFisher), NF-κB (8424T, Cell Signaling Technologies, CST, Danvers, MA) and β-ACTIN (A1978, MilliporeSigma). Nuclear and cytosolic protein fractions were isolated using Cell fractionation kit (ab109719, Abcam) and utilized for Western blotting using antibodies for GAPDH (5174S, CST) and Lamin A/C (2032S, CST), respectively.

2.9. Statistical Analysis

All in vitro assays for cell survival and ROS experiments were performed with three biological replicates and statistical analysis was performed using Prism 7.0b (GraphPad Software Inc., San Diego, CA). Normalized data were expressed as mean ± standard error of the mean (SEM) and multiple comparisons using one-way analysis of variance (ANOVA) were applied for comparisons. For metabolites data and other two-group comparisons a two-tailed, unpaired Student’s t-test was applied, and statistical significance values were derived within Excel (Microsoft).

3. Results

3.1. Cigarette smoke exposure alters common metabolic pathways in pancreatic cancer cells

Cellular response to concentrated cigarette smoke extract (CSE) manifests as alterations in cellular mechanisms including metabolism (29) which was observed in PC cells subjected to CSE (Fig.1a). Pancreatic cancer cells cultured with 25% CSE for 24 h, demonstrated altered metabolic profiles as per the discriminant analysis and heatmaps presented in Fig.1a and b, respectively. The metabolic profiles of vehicle vs. CSE-treated Colo357 and CD18/HPAFII cells segregated with similar magnitudes as evident from component-1 values of discrimination in partial least square discriminant analysis (PLS-DA) (Fig.1a). Summarizing the relative abundance profiles of individual metabolites across the vehicle and CSE-treated Colo357 and CD18/HPAFII revealed that metabolite abundances fluctuated in both cell lines upon exposure to CSE (Fig.1b). Furthermore, fluctuations in relative abundance of individual metabolites, in turn, impacted metabolic pathways in CSE-exposed cells as depicted by pathway impact analysis (Fig.1c). Of note, metabolic pathways comprising glutathione, cysteine and methionine were commonly and significantly altered in both Colo357 and CD18/HPAFII, PC cells treated with 25% CSE. In addition to the common pathway alterations in our cell line models, unique pathways such as glycine, serine, and purine metabolism were exclusively and significantly altered in Colo357 and CD18/HPAFII cells, respectively (Fig.1c). Based on these significant alterations, we analyzed the relative abundances of individual metabolites corresponding to cysteine, methionine, and glutathione metabolic pathways along with NADPH, as glutathione metabolism impacts NADPH in redox metabolism. Comparison of individual metabolite levels revealed that cysteine was significantly upregulated in CSE-treated cell lysates from both the cell lines, whereas, methionine, S-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH) and cystathionine, oxidized glutathione (GSSG), NADP, NDAPH, and the ratio of NADP/NADPH were significantly altered only in the CSE-exposed, Colo357 cells (Fig.1d & e). Overall, exposure of PC cell lines CD18/HPAFII and Colo-357 to CSE resulted in upregulation of cysteine and glutathione metabolic pathways.

Fig.1. Cigarette smoke extract (CSE) alters cysteine metabolism in pancreatic cancer.

Fig.1.

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(a) Partial least discriminant analysis showing metabolic shift in PDAC cell lines exposed to CSE. Each colored circle indicates a biological replicate used for metabolite analysis and three similar colored replicates indicate biological replicates of same condition, (b) Heatmaps deciphering metabolic differences between 24 h vehicle and CSE-treated PDAC. Each row represents a metabolite and each heatmap column represents the profile from a replicate. The heatmap scale on the right side indicates relative change in the metabolite level with red indicating an increase and green indicating decrease in metabolite levels, respectively, (c) Pathway impact analyses revealing significantly altered metabolic pathways in CSE-exposed PDAC cells. The color in circles indicates combined impact of the respective pathway with red showing high impact and yellow showing lower impact. The size of each circle indicates number of metabolites detected in corresponding pathways and (d & e) Bar plots showing relative changes in metabolites (n=3, *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively).

3.2. Glutathione anabolism elevates in CSE-exposed pancreatic cancer cells

Glutathione synthesis is a critical route of cellular utilization of cysteine entering into cells through the cystine transporter, SLC7A11 (Fig.2a). Increased cellular cysteine levels in both PC cell lines exposed to cigarette smoke (Fig.1d) indicate possibilities for either utilization of cysteine for GSH biosynthesis or other routes of cysteine utilization. Therefore, we probed for upregulation of glutathione biosynthesis in CSE-exposed PC cells through Western blot analyses and observed an increase in Glutamate-cysteine ligase (GCLM) protein involved in glutathione biosynthesis (Fig.2b), but not the Cystathionine beta-synthase (CBS) protein which is upstream of GCLM in the glutathione biosynthesis pathway. To further investigate the impact of CSE on GSH biosynthesis, we compared the uptake and incorporation of heavy isotope labeled cysteine (13C3,15N-Cysteine) into GSH in vehicle and CSE-exposed PC cells. Relative comparison of intracellular 13C3,15N-Cys in vehicle and CSE-exposed PC cells revealed higher abundance of labeled cysteine in CSE-exposed, Colo357 and CD18/HPAFII cells (Fig.2c). However, glutathione labeling derived from labeled 13C3,15N-Cys was only elevated in the CSE-treated Colo357 cells within 4 h of exposure to cigarette smoke extract (Fig.2d). Because GSH is rapidly utilized in cells, we also compared the ratio of heavy isotope labeled-GSH to unlabeled-GSH in vehicle and CSE-exposed cells and observed that the ratio of 13C3,15N-GSH/12C,14N-GSH was significantly higher in both the Colo357 and CD18/HPAFII cells exposed to CSE for 4 h (Fig2.e). Furthermore, pretreatment of cells with a cellular Cys uptake inhibitor, Erastin, reduced the intracellular, labeled-Cys in CSE exposed Colo357 and CD18/HPAFII cells (Fig.2f). Treatment with Erastin also reduced the labeled-GSH and ratio of labeled-GSH to unlabeled-GSH in CSE-exposed cells as shown in Fig.2g and Fig.2h, respectively. Based on these results, we conclude that PC cells have higher cysteine uptake for facilitating de novo GSH synthesis in smoke exposed conditions.

Fig.2. Cigarette smoke extract (CSE) enhances glutathione anabolism from cysteine in pancreatic cancer.

Fig.2.

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(a) Metabolic route of GSH anabolism from Cysteine, (b) Western blot showing upregulation of GCLM in CSE-exposed PDAC cells, β-ACTIN was used as loading control, (c-d) Bar charts showing relative changes in: Intracellular labeled Cysteine, labeled GSH and intracellular labeled GSH/unlabeled GSH ratios in PDAC cells treated with specified conditions (n=3, *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively).

3.3. Cysteine modulates PC cell survival through glutathione during cigarette smoke exposure

Cellular adaptability to CSE is contingent on the quantity and duration of cell exposure to CSE (30). We explored the tolerance of PC cells to different dosages of CSE in the abundance and depletion of Cys through survival analyses. We noticed an inverse trend in survival of PC cells with increase in percent of CSE from 10–100% (Fig.3a). The survival of Colo357 and CD18/HPAFII cells was least affected by 10% CSE at 48 h in cell survival assays (Fig.3a) which was negatively affected by the depletion of Cys from culture media. Depletion of Cys reduced cell survival in vehicle treated PC cells followed by further reduction in 10% CSE exposed cells at 48 h as determined by survival analysis. However, Cys depletion was more effective in reducing survival of only Colo357 cells with 48 h exposure to 50 and 100% CSE, which was not evident in CD18/HPAFII cells within same interval of exposure. Therefore, we extended the survival analyses, post 72 h incubation with 25% CSE and Cys-depletion, which revealed moderate decrease in survival of Colo357 and CD18/HPAFII cells incubated with either CSE or Cys-depletion, but there were lack of cellular growth and cell detachment in CSE+Cys depleted condition (Fig.3b). The negative impact of Cys-depletion on the survival of PDAC cell lines subjected to 25% CSE was further confirmed through decrease in the survival of cells (Capan1, CFPAC and T3M4) after incubation with 25% CSE and Cys depletion for 72 h (SF1.a). Based on the dependence of PDAC cells on Cys for survival and increase in labeled-GSH in CSE exposed conditions, we further investigated the role of GSH in promoting survival of PDAC cells in similar conditions. We noticed ablation of inhibitory effects of Cys-depletion and CSE on cell survival when media were supplemented with N-acetylcysteine (NAC, the precursor for cellular GSH) and cell permeable, Glutathione ethyl ester (GSH-EE) in survival analyses (Fig.3c). We have also recapitulated the impact of Cys depletion on cellular GSH, using the GSH depleting agent, Butylthiosulfoxime (BSO), resulting in reduced cell survival of Colo357 and CD18/HPAFII cells exposed to CSE for 72 h in survival assays (SF1.b). We confirmed the impact of BSO on cell survival is through depletion of GSH by observing the reversal of survival in PDAC cells with GSH-EE supplementation in BSO treated conditions (SF1.c). We further observed that the GSH-EE treatment in combination with BSO reversed the death of PDAC cells in Cys-depleted and CSE treated conditions (Fig.3c). In summary, these results demonstrate that PDAC cells require cysteine uptake and cysteine-derived, GSH for their survival in cigarette smoke exposed conditions.

Fig.3. Cysteine derived glutathione regulates pancreatic cancer cell survival in response to cigarette smoke extract (CSE).

Fig.3.

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(a) Bar charts showing relative changes in survival of PDAC cells in CSE and cysteine-depleted media, (b) Micrographs showing changes in survival of PDAC cells in CSE and cysteine-depleted media with scale bar indicating 400 μM and (c) Bar charts showing relative changes in survival of PDAC cells in CSE, cysteine-depleted and metabolite supplemented media. For bar plots: n=3, *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively.

3.4. Glutathione upregulation mechanisms in cigarette smoke exposed PDAC mice

Spontaneous mice models recapitulate the initiation and progression of human PDAC providing an in vivo platform for unraveling the role of oncogenes and etiological factors in PDAC (31). In our in vivo approach, we exposed 10 weeks old KrasG12D,Pdx-Cre (KC) mice to high dose of cigarette smoke 250 TSP/m3 or sham (air-exposed) for 180 days (Fig.4a). We observed that mice exposed to cigarette smoke had significant lowered body weights (Fig.4b) along with increase in weights of pancreas (Fig.4c). Smoke exposure enhanced tumor incidence in pancreas of mice (n=14) exposed to cigarette smoke with 14 mice developing pancreatic tumors out of 14 mice exposed to smoke in contrast to 4 mice developing pancreatic tumors out of 14 mice exposed to sham (air exposed) (Fig.4d). Exposure to cigarette smoke also significantly enhanced the frequency and penetrance of pancreatic lesions within the pancreas (Fig.4e), which were mostly confined to the ductal lesions of pancreas (Fig.4f). In par to the increase in GCLM that we observed in our in vitro smoke-exposed PC cells, we also observed increased expression of GCLM in PanINs and pancreatic tumors from smoke exposed mice. Especially, the pancreatic ductal lesions (PanIN) in smoke-exposed mice group showed higher GCLM expression compared to the PanIN lesions from mice maintained in sham-exposed conditions (Fig.4g). The increased incidence of PanINs was also an indicator of inflammation which could be regulated through multiple factors. As per the reported study, Nuclear factor-κ-B (NF-κB) is a transcription factor upregulated in response to inflammation and, in turn, regulates the expression of GCLM (32). Therefore, we validated the expression of NF-κB in PanIN lesions from sham and cigarette smoke-exposed mice and observed an increase in staining for NF-κB in the nuclei of pancreatic ductal cells from mice exposed to cigarette smoke (Fig.4g). In contrast to smoke exposed mice, staining for nuclear NF-κB was very low in the epithelial lining of PanIN lesions of sham-exposed mice. Therefore, we concluded that cigarette smoke exposure enhances pancreatic lesions in GEMMs of PDAC. Furthermore, smoke exposure upregulates NF-κB-GCLM axis in the PanIN lesions of PDAC which supports the notion that these cancer cells survive in CSE exposed conditions.

Fig.4. Cigarette smoke aggravates pancreatic lesions in pancreatic cancer model in vivo.

Fig.4.

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(a) Scheme of smoke exposure in KC mice models, (b, c & e) Box and whisker plots showing differences in body mass, pancreatic mass and PanIN frequency in Sham (n=14) and smoke-exposed (n=10) mice (** & *** indicate p<0.01 and 0.001, respectively). (d) Stacking bars showing difference in tumor frequency of Sham (n =14) and Smoke-exposed (n=10) mice, (f) and (g) show PanINs from pancreas (20x magnification, scale bar = 100 μm), IHC analysis for GCLM in ductal lesions of PanINs (indicated by black arrows) and nuclear staining for NF-κB indicated by black arrows in ductal lesions of PanINs from mice pancreas at 40x magnification (scale bar = 50 μm), respectively.

3.5. Smoke-induced glutathione synthesis requires NF-κB mediated GCLM induction

Upregulation of GCLM and increase in nuclear localization of NF-κB in PanIN lesions from cigarette smoke-exposed KC mice led us to validate the role of NF-κB in regulating GSH biosynthesis in PDAC models. We identified that treatment of PDAC cells with NF-κB inhibitor, BMS345541, for 60 min reduces the nuclear localization of NF-κB in either vehicle or CSE exposed conditions (Fig.5a). Utilizing this inhibition approach, we pre-treated the Colo357 and CD18/HPAFII cells with BMS345541 for 60 min followed with exposure of either vehicle or 25% CSE. We noticed that exposure to CSE upregulates the GCLM protein in both Colo357 and CD18/HPAFII cells which was inhibited by pre-exposure to BMS345541 in vehicle as well as CSE-treated PDAC cells (Fig.5b). Based on the inhibition of GCLM expression through NF-κB inhibition, we explored the effects of BMS345541, on Cys and GSH levels in CSE-treated PDAC cell lines. Intriguingly, intracellular labeled-Cys was not significantly affected in either vehicle or CSE exposed Colo357 cells (Fig.5c). In contrast to Cys in Colo357 cells, labeled-Cys was significantly higher in BMS345541 treated CD18/HPAFII cells which was not altered in BMS345541 and CSE treated conditions (Fig.5d). Finally, analysis of heavy-isotope labeled-GSH from Colo357 and CD18/HPAFII cells revealed a significant decrease in intracellular labeled-GSH levels in BMS345541-treated conditions compared to the vehicle and CSE-treated cells (Fig.4e & f), indicating that upsurge in GSH biosynthesis in CSE exposed PDAC cells is dependent on NF-κB mediated GCLM expression.

Fig.5. NF-κB regulates cigarette smoke extract (CSE)-mediated glutathione de novo synthesis from cysteine.

Fig.5.

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(a & a) Western blots showing changes in NF-κB and GCLM in vehicle, CSE and BMS345541 treated PDAC cells. GAPDH, LaminA/C and β-ACTIN were used as loading controls and (c-f) Bar charts showing relative changes in: Intracellular labeled Cysteine and labeled GSH in PDAC cells treated with specified conditions (n=3, *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively).

3.6. NF-κB inhibition abrogates survival of PC cells through ROS regulation

Cellular GSH facilitates redox homeostasis through suppression of reactive oxygen species (ROS). Therefore, we evaluated the changes in cellular ROS levels in our in vitro models and noticed that PC cells show increased staining for intracellular fluorescent DCFDA (an indicator of ROS) in CSE treated conditions. However, treating cells with either Erastin or BMS345541 (NF-κB inhibitor) alone enhanced the DCFDA signal significantly, only in CD18/HPAFII cells (Fig.6a–d). Furthermore, treating Colo357 and CD18/HPAFII cells with CSE in combination with either Erastin or BMS345541 significantly enhanced the ROS signals in both cell lines in comparison to the vehicle and control cells. Such increase was reversed by supplementing cells with GSH-EE which reduced the ROS upregulation observed in CSE, Erastin and BMS345541 alone or in combination treated conditions (Fig.6a–d). Finally, treatment with BMS345541 reduced the survival in Colo357 and CD18/HPAFII cells in CSE exposed cells, which was reversed with supplementation of GSH-EE in CSE- and BMS3455541-treated conditions (Fig.6e), indicating that NF-κB-mediated regulation of GSH synthesis supports ROS suppression and survival in PDAC (Fig.6f). Thus, we concluded that NF-κB-mediated regulation of GSH synthesis is essential for the survival of PC cells in cigarette smoke exposed conditions.

Fig.6. NF-κB mediated glutathione anabolism regulates survival in pancreatic cancer cells in response to cigarette smoke extract (CSE).

Fig.6.

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(a & c) Representative images of DCFDA staining in CSE, Erastin, BMS3455541 and GSH-EE treated PDAC cell lines at 10X magnifications, (b & d) Bar charts showing relative changes in intracellular ROS levels of PDAC cells treated with specified conditions (n=3 and *** indicates p<0.001), (e) Micrographs showing changes in survival of PDAC cells in CSE, BMS345541 and GSH-EE treated conditions, scale bars indicate 400 μM and (f) Summary of NF-κB mediated glutathione regulates survival in smoke-exposed PDAC.

4. Discussion

Mutations driving PC are among the intrinsic, stress inducing factors that lead to proliferative and oxidative stress in pancreatic cancer cells (33). Such mutation-induced oxidative stress gets compensated through upregulation of glutathione metabolism facilitated by mutant KRas (17). In addition to the oncogenes, components of cigarette smoke also induce oxidative stress which affects metabolism in smoke affected cells, tissues and systemic metabolism (34). Our results demonstrate that CSE alters PC cell metabolism, and most significantly, cysteine and glutathione metabolic pathways were perturbed in CSE-exposed, KRAS wild type (Colo357) as well as KRASG12D mutation harboring, CD18/HPAFII PC cell lines. Increase in GSH and its precursor Cys are indicative of stress-induced metabolic response (35), as previously reported in mutation driven PC (17, 36) and cigarette smoke exposed cells (37, 38). Our results demonstrate similar metabolite changes, along with decrease in NADPH in CSE exposed pancreatic cancer cells (Fig.1d & e), reflecting metabolic deviation as a compensation for oxidative stress in CSE exposed PDAC.

Metabolic fluctuations enable cancer cells to survive in stressed conditions (39). In our study, upregulation of Cys and GSH metabolism in PDAC cells upon exposure to CSE (Fig.3a&b and SF.1) indicates metabolic modulation as a survival adaptation in cigarette smoke exposed PDAC. N-acetylcysteine (NAC) serves as a substitute for Cys in generating GSH (40) and GSH-EE is a cell-permeable source of GSH which replenishes GSH in cancer cells (41) and we justify the role of Cys metabolism in supporting survival of PDAC in smoke-exposed conditions through survival recovery of PDAC cells exposed to cigarette smoke and Cys-deprived media in the presence of NAC and GSH-EE supplementation. Thus, our findings are in agreement with a recent report indicating reliance of PDAC on cysteine for survival under stress conditions (42).

Upregulation of glutathione metabolism in cigarette smoke-exposed PDAC was further substantiated from in vivo evaluations in cigarette smoke-exposed KC mice (Fig.4). Firstly, increase in incidence of PanINs and pancreatic tumor burden in cigarette smoke-exposed KC mice revealed that smoke exposure enhances survival as well as progression of transformed cells into pancreatic lesions as reported previously (28). In addition to accelerating the incidence and progression of PanIN lesions, smoke exposure also increased GCLM expression within the ductal cells of PanIN lesions in KC mice (Fig.4). As these intraductal mucinous neoplasms are indicative of inflammatory and progressively developing pancreatic lesions which progress to pancreatic tumors in mice models of pancreatic cancer (43), we concluded that cigarette smoke exposure aggravates PanIN lesions in PDAC models. Furthermore, increased GCLM could be indicative of stress-induced survival response based on both increased GCLM levels as well as high PanIN frequency in smoke exposed KC models of PDAC.

Cigarette smoke exposure not only supports carcinogenesis, but high smoke concentration could also increase ROS levels which, in turn, leads to oxidative damage and death in cells exposed to excess smoke concentrations (44). In our study cell death observed in high concentrations of CSE reflects that PDAC cells could only adapt to limited concentration of acute smoke exposure, wherein cellular defense against smoke induced oxidative stress facilitates their survival in these conditions. Glutathione synthesis through GCLM regulates smoke-induced carcinogenesis as reported in in vivo mice models (45). Glutathione synthesis replenishes tissue GSH which could be utilized for detoxification of cytotoxic ROS, smoke adducts and oxidized thiols generated in cigarette smoke exposure (46–48) and we conclude that PDAC cells possess ample GSH for suppressing the cytotoxic effects of smoke components. Furthermore, pancreatic cancer cells effectively acquire exogenous Cys required for maintaining redox homeostasis (42, 49). Thus, upregulation of the glutathione synthesis and Cys uptake in smoke-exposed PDAC demonstrates that PDAC upregulates Cys-GSH metabolism under stress conditions.

Based on the concomitant increase of glutathione synthesis, GCLM expression and survival of PDAC cells in CSE-treated conditions, we hypothesized that stress-inducible transcription factors with a prominent role in regulating GCLM and cell survival would regulate cigarette smoke-induced stress in PDAC. Nuclear factor-κB is a redox-sensitive transcription factor upregulated for supporting survival mechanisms in smoke-exposed as well as KRASG12D models (22, 50). Stress-induced translocation of NF-κB to the nucleus enhances GCLM expression and increases glutathione synthesis in cancer cells (32). Our in vivo results showed increased nuclear localization of NFκB in the PanIN lesions and tumoral zones of smoke-exposed KC mice models (Fig.4f). Mice models of PDAC recapitulate the stage-wise transformation of pancreatic tumors from initial pancreatic lesions termed as PanINs to dismal PDACs wherein PanINs reflect the initial stages of ductal transformation (51). Thus, increase in PanIN frequency along with the expression of GCLM and NF-κB within PanINs reflects a possible role for NF-κB in cigarette smoke-induced GCLM expression. We further confirmed the role of NF-κB in regulating cigarette smoke-induced GCLM through our in vitro findings as inhibiting nuclear translocation of NF-κB using BMS345541 both reduced GCLM in smoke-exposed PDAC (Fig.5a&b) and decreased de novo GSH synthesis under similar conditions (Fig.5f). Thus, our results demonstrate NF-κB mediated GCLM regulation is stimulated by smoke exposure in KRAS wild type as well as KRASG12D cell line models. However, NF-κB inhibition did not alter the Cys uptake in either vehicle or cigarette smoke-treated PDAC cell lines, and CD18/HPAFII cells showed increased labeled-Cys with BMS345541 treatment (Fig.5e). Furthermore, GCLM, but not CBS, involved in GSH synthesis was upregulated in CSE-exposed PDAC cells (Fig.2b). These results demonstrate modulation of GSH synthesis in smoke-exposed PDAC is through GCLM, which in turn depends on activation of NF-κB.

Our study also demonstrates that Cys depletion augments ROS in cigarette smoke-exposed, PDAC which was enabled through inhibition of SLC7A11 similar to the NF-κB inhibition (Fig.6a–d). Thus, inhibition of Cys uptake as well as NF-κB inhibition have similar effects on ROS upregulation in smoke-exposed PDAC models which was established in our study through reversal of ROS induction in Erastin and BMS345541-treated PDAC cells by GSH-EE (Fig.6). Increase in cellular ROS levels is an indicator of stress induction that, in turn, supports carcinogenesis in cigarette smoke-exposed models (52). Thus, we conclude that GSH synthesis enables pancreatic cancer cells to cope with smoke-induced ROS through NF-κB-mediated GCLM upregulation.

5. Conclusions

Smoking tobacco is an established and independent risk factor that leads to poor prognosis in pancreatic cancer. However, mechanisms supporting PDAC in cigarette smoke exposure were previously not widely explored (16, 53). Identification of cigarette smoke-induced mechanisms would facilitate developing translational strategies for controlling PDAC progression in cigarette smokers. We elaborated on the role played by glutathione anabolism and the significance of cysteine in regulating smoke-induced ROS in pancreatic cancer. Depletion of glutathione was previously reported to enhance inflammation in smoke exposure models (54). Based on our findings, upregulation of cellular ROS in cigarette smoke-exposed PDAC cells induces cytotoxicity and cell death either through NF-κB inhibition or Cys depletion indicating that cysteine restriction and NF-κB inhibitors appear as promising possibilities to restrict cigarette smoke-induced PDAC progression which need further exploration using pre-clinical models.

Supplementary Material

1

SF1. Cysteine-derived glutathione regulates pancreatic cancer cell survival in response to cigarette smoke extract (CSE). (a) Micrographs showing changes in survival of PDAC cells in CSE and cysteine-depleted media with scale bar indicating 400 μM, (b) Bar charts showing relative changes in survival of PDAC cells in CSE and BSO treated conditions (n=3) and (c) Micrographs showing changes in survival of PDAC cells in CSE, BSO and GSH-EE treated conditions, scale bars indicate 400 μM.

Highlights:

  • Pancreatic ductal adenocarcinoma (PDAC) tolerates high dose cigarette smoke

  • Cysteine uptake enhances in PDAC exposed to high dose cigarette smoke

  • De novo glutathione synthesis protects cigarette smoke exposed PDAC

  • NF-κB regulates glutathione synthesis in cigarette smoke exposed PDAC

  • Targeting NF-κB impairs ROS balance in cigarette smoke exposed PDAC

  • PDAC succumbs to cigarette smoke upon cysteine depletion

Acknowledgements

The authors are thankful to Geoffrey A. Talmon for guidance in pathology analysis, Deanna D. Mosley, for helping with CSE preparation and Kavita Mallya, for helping in Biochemical reagent arrangements.

Footnotes

Conflicts of Interest

S.K.B. is a co-founder of Sanguine Diagnostics and Therapeutics, Inc., located in Omaha, NE. The other authors declare that they have no competing interests.

Declaration of Interest

S.K.B. is a co-founder of Sanguine Diagnostics and Therapeutics, Inc., located in Omaha, NE. The other authors declare that they have no competing interests.

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References

  • 1.Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer Statistics, 2021. CA Cancer J Clin. 2021;71(1):7–33. [DOI] [PubMed] [Google Scholar]
  • 2.Yang J, Xu R, Wang C, Qiu J, Ren B, You L. Early screening and diagnosis strategies of pancreatic cancer: a comprehensive review. Cancer Commun (Lond). 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Storz P, Crawford HC. Carcinogenesis of Pancreatic Ductal Adenocarcinoma. Gastroenterology. 2020;158(8):2072–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Izeradjene K, Combs C, Best M, Gopinathan A, Wagner A, Grady WM, et al. Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas. Cancer Cell. 2007;11(3):229–43. [DOI] [PubMed] [Google Scholar]
  • 5.Collins MA, Bednar F, Zhang Y, Brisset JC, Galban S, Galban CJ, et al. Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice. J Clin Invest. 2012;122(2):639–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Schofield HK, Zeller J, Espinoza C, Halbrook CJ, Del Vecchio A, Magnuson B, et al. Mutant p53R270H drives altered metabolism and increased invasion in pancreatic ductal adenocarcinoma. JCI Insight. 2018;3(2). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kolodecik T, Shugrue C, Ashat M, Thrower EC. Risk factors for pancreatic cancer: underlying mechanisms and potential targets. Front Physiol. 2013;4:415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Slebos RJ, Hoppin JA, Tolbert PE, Holly EA, Brock JW, Zhang RH, et al. K-ras and p53 in pancreatic cancer: association with medical history, histopathology, and environmental exposures in a population-based study. Cancer Epidemiol Biomarkers Prev. 2000;9(11):1223–32. [PubMed] [Google Scholar]
  • 9.Pandol SJ, Apte MV, Wilson JS, Gukovskaya AS, Edderkaoui M. The burning question: why is smoking a risk factor for pancreatic cancer? Pancreatology. 2012;12(4):344–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Yuan C, Morales-Oyarvide V, Babic A, Clish CB, Kraft P, Bao Y, et al. Cigarette Smoking and Pancreatic Cancer Survival. J Clin Oncol. 2017;35(16):1822–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Molina-Montes E, Van Hoogstraten L, Gomez-Rubio P, Lohr M, Sharp L, Molero X, et al. Pancreatic Cancer Risk in Relation to Lifetime Smoking Patterns, Tobacco Type, and Dose-Response Relationships. Cancer Epidemiol Biomarkers Prev. 2020;29(5):1009–18. [DOI] [PubMed] [Google Scholar]
  • 12.Weissman S, Takakura K, Eibl G, Pandol SJ, Saruta M. The Diverse Involvement of Cigarette Smoking in Pancreatic Cancer Development and Prognosis. Pancreas. 2020;49(5):612–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Momi N, Kaur S, Ponnusamy MP, Kumar S, Wittel UA, Batra SK. Interplay between smoking-induced genotoxicity and altered signaling in pancreatic carcinogenesis. Carcinogenesis. 2012;33(9):1617–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Peretti-Watel P, Constance J, Guilbert P, Gautier A, Beck F, Moatti JP. Smoking too few cigarettes to be at risk? Smokers’ perceptions of risk and risk denial, a French survey. Tob Control. 2007;16(5):351–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Rubin RL, Hermanson TM, Bedrick EJ, McDonald JD, Burchiel SW, Reed MD, et al. Effect of cigarette smoke on autoimmunity in murine and human systemic lupus erythematosus. Toxicol Sci. 2005;87(1):86–96. [DOI] [PubMed] [Google Scholar]
  • 16.Nimmakayala RK, Seshacharyulu P, Lakshmanan I, Rachagani S, Chugh S, Karmakar S, et al. Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1. Gastroenterology. 2018;155(3):892–908 e6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ying H, Kimmelman AC, Lyssiotis CA, Hua S, Chu GC, Fletcher-Sananikone E, et al. Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism. Cell. 2012;149(3):656–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Salem AF, Al-Zoubi MS, Whitaker-Menezes D, Martinez-Outschoorn UE, Lamb R, Hulit J, et al. Cigarette smoke metabolically promotes cancer, via autophagy and premature aging in the host stromal microenvironment. Cell Cycle. 2013;12(5):818–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kim ST, Lim DH, Jang KT, Lim T, Lee J, Choi YL, et al. Impact of KRAS mutations on clinical outcomes in pancreatic cancer patients treated with first-line gemcitabine-based chemotherapy. Mol Cancer Ther. 2011;10(10):1993–9. [DOI] [PubMed] [Google Scholar]
  • 20.Kim-Fuchs C, Le CP, Pimentel MA, Shackleford D, Ferrari D, Angst E, et al. Chronic stress accelerates pancreatic cancer growth and invasion: a critical role for beta-adrenergic signaling in the pancreatic microenvironment. Brain Behav Immun. 2014;40:40–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Grabocka E, Bar-Sagi D. Mutant KRAS Enhances Tumor Cell Fitness by Upregulating Stress Granules. Cell. 2016;167(7):1803–13 e12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hasnis E, Bar-Shai M, Burbea Z, Reznick AZ. Mechanisms underlying cigarette smoke-induced NF-kappaB activation in human lymphocytes: the role of reactive nitrogen species. J Physiol Pharmacol. 2007;58 Suppl 5(Pt 1):275–87. [PubMed] [Google Scholar]
  • 23.Prabhu L, Mundade R, Korc M, Loehrer PJ, Lu T. Critical role of NF-kappaB in pancreatic cancer. Oncotarget. 2014;5(22):10969–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kashyap R, Floreani AA, Heires AJ, Sanderson SD, Wyatt TA. Protein kinase C-alpha mediates cigarette smoke extract- and complement factor 5a-stimulated interleukin-8 release in human bronchial epithelial cells. J Investig Med. 2002;50(1):46–53. [DOI] [PubMed] [Google Scholar]
  • 25.Gunda V, Pathania AS, Chava S, Prathipati P, Chaturvedi NK, Coulter DW, et al. Amino Acids Regulate Cisplatin Insensitivity in Neuroblastoma. Cancers (Basel). 2020;12(9). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sidorova JV, Biderman BV, Nikulina EE, Sudarikov AB. A simple and efficient method for DNA extraction from skin and paraffin-embedded tissues applicable to T-cell clonality assays. Exp Dermatol. 2012;21(1):57–60. [DOI] [PubMed] [Google Scholar]
  • 27.Chong J, Xia J. Using MetaboAnalyst 4.0 for Metabolomics Data Analysis, Interpretation, and Integration with Other Omics Data. Methods Mol Biol. 2020;2104:337–60. [DOI] [PubMed] [Google Scholar]
  • 28.Kumar S, Torres MP, Kaur S, Rachagani S, Joshi S, Johansson SL, et al. Smoking accelerates pancreatic cancer progression by promoting differentiation of MDSCs and inducing HB-EGF expression in macrophages. Oncogene. 2015;34(16):2052–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Agarwal AR, Yin F, Cadenas E. Short-term cigarette smoke exposure leads to metabolic alterations in lung alveolar cells. Am J Respir Cell Mol Biol. 2014;51(2):284–93. [DOI] [PubMed] [Google Scholar]
  • 30.Bourgeois JS, Jacob J, Garewal A, Ndahayo R, Paxson J. The Bioavailability of Soluble Cigarette Smoke Extract Is Reduced through Interactions with Cells and Affects the Cellular Response to CSE Exposure. PLoS One. 2016;11(9):e0163182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Rachagani S, Torres MP, Kumar S, Haridas D, Baine M, Macha MA, et al. Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy. J Hematol Oncol. 2012;5:68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Meng Q, Peng Z, Chen L, Si J, Dong Z, Xia Y. Nuclear Factor-kappaB modulates cellular glutathione and prevents oxidative stress in cancer cells. Cancer Lett. 2010;299(1):45–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Storz P. KRas, ROS and the initiation of pancreatic cancer. Small GTPases. 2017;8(1):38–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Liu G, Lee DP, Schmidt E, Prasad GL. Pathway Analysis of Global Metabolomic Profiles Identified Enrichment of Caffeine, Energy, and Arginine Metabolism in Smokers but Not Moist Snuff Consumers. Bioinform Biol Insights. 2019;13:1177932219882961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Bansal A, Simon MC. Glutathione metabolism in cancer progression and treatment resistance. J Cell Biol. 2018;217(7):2291–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Lo M, Ling V, Wang YZ, Gout PW. The xc- cystine/glutamate antiporter: a mediator of pancreatic cancer growth with a role in drug resistance. Br J Cancer. 2008;99(3):464–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Ji X, Qian J, Rahman SMJ, Siska PJ, Zou Y, Harris BK, et al. xCT (SLC7A11)-mediated metabolic reprogramming promotes non-small cell lung cancer progression. Oncogene. 2018;37(36):5007–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Altraja S, Mahlapuu R, Soomets U, Altraja A. Cigarette smoke-induced differential regulation of glutathione metabolism in bronchial epithelial cells is balanced by an antioxidant tetrapeptide UPF1. Exp Toxicol Pathol. 2013;65(6):711–7. [DOI] [PubMed] [Google Scholar]
  • 39.Daher B, Vucetic M, Pouyssegur J. Cysteine Depletion, a Key Action to Challenge Cancer Cells to Ferroptotic Cell Death. Front Oncol. 2020;10:723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Atkuri KR, Mantovani JJ, Herzenberg LA, Herzenberg LA. N-Acetylcysteine--a safe antidote for cysteine/glutathione deficiency. Curr Opin Pharmacol. 2007;7(4):355–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Levy EJ, Anderson ME, Meister A. Transport of glutathione diethyl ester into human cells. Proc Natl Acad Sci U S A. 1993;90(19):9171–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Badgley MA, Kremer DM, Maurer HC, DelGiorno KE, Lee HJ, Purohit V, et al. Cysteine depletion induces pancreatic tumor ferroptosis in mice. Science. 2020;368(6486):85–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Hruban RH, Adsay NV, Albores-Saavedra J, Compton C, Garrett ES, Goodman SN, et al. Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions. Am J Surg Pathol. 2001;25(5):579–86. [DOI] [PubMed] [Google Scholar]
  • 44.Morsch A, Wisniewski E, Luciano TF, Comin VH, Silveira GB, Marques SO, et al. Cigarette smoke exposure induces ROS-mediated autophagy by regulating sestrin, AMPK, and mTOR level in mice. Redox Rep. 2019;24(1):27–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Yamamoto ML, Chapman AM, Schiestl RH. Effects of side-stream tobacco smoke and smoke extract on glutathione- and oxidative DNA damage repair-deficient mice and blood cells. Mutat Res. 2013;749(1–2):58–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Gould NS, Min E, Gauthier S, Martin RJ, Day BJ. Lung glutathione adaptive responses to cigarette smoke exposure. Respir Res. 2011;12:133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Kode A, Rajendrasozhan S, Caito S, Yang SR, Megson IL, Rahman I. Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2008;294(3):L478–88. [DOI] [PubMed] [Google Scholar]
  • 48.Higashi T, Elmeligy E, Mai Y, Noya Y, Terada K, Mazaki Y, et al. Glutathione and cysteines suppress cytotoxicity of gas phase of cigarette smoke by direct reacting with unsaturated carbonyl compounds in the gas phase. Biochem Biophys Res Commun. 2019;509(4):988–93. [DOI] [PubMed] [Google Scholar]
  • 49.Sharbeen G, McCarroll JA, Akerman A, Kopecky C, Youkhana J, Kokkinos J, et al. Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition. Cancer Res. 2021. [DOI] [PubMed] [Google Scholar]
  • 50.Doppler H, Panayiotou R, Reid EM, Maimo W, Bastea L, Storz P. The PRKD1 promoter is a target of the KRas-NF-kappaB pathway in pancreatic cancer. Sci Rep. 2016;6:33758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Singh K, Pruski M, Bland R, Younes M, Guha S, Thosani N, et al. Kras mutation rate precisely orchestrates ductal derived pancreatic intraepithelial neoplasia and pancreatic cancer. Lab Invest. 2021;101(2):177–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Valavanidis A, Vlachogianni T, Fiotakis K. Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles. Int J Environ Res Public Health. 2009;6(2):445–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Zhang J, Bai R, Li M, Ye H, Wu C, Wang C, et al. Excessive miR-25–3p maturation via N(6)-methyladenosine stimulated by cigarette smoke promotes pancreatic cancer progression. Nat Commun. 2019;10(1):1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Gould NS, Min E, Huang J, Chu HW, Good J, Martin RJ, et al. Glutathione Depletion Accelerates Cigarette Smoke-Induced Inflammation and Airspace Enlargement. Toxicol Sci. 2015;147(2):466–74. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

1

SF1. Cysteine-derived glutathione regulates pancreatic cancer cell survival in response to cigarette smoke extract (CSE). (a) Micrographs showing changes in survival of PDAC cells in CSE and cysteine-depleted media with scale bar indicating 400 μM, (b) Bar charts showing relative changes in survival of PDAC cells in CSE and BSO treated conditions (n=3) and (c) Micrographs showing changes in survival of PDAC cells in CSE, BSO and GSH-EE treated conditions, scale bars indicate 400 μM.

NIHMS1751771-supplement-1.jpg (1.2MB, jpg)

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