Oral cancer cells secrete stress neurotransmitter and proliferate in response to tobacco carcinogen NNK through a cellular adrenergic pathway.

Oral cancer cells secrete stress neurotransmitter and proliferate in response to tobacco carcinogen NNK through a cellular adrenergic pathway. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oral cancer cells secrete stress neurotransmitter and proliferate in response to tobacco carcinogen NNK through a cellular adrenergic pathway. Flávia Alves Verza, Ana Lívia Santos-Sousa, Sandra Helena Penha Oliveira, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3934938/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Although there is a growing body of evidence showing the effects of stress-related catecholamines on oral cancer progression, to date there are no studies that have investigated whether oral squamous cells carcinoma (OSCC)-cells are capable of producing these hormones and whether this phenomenon is modulated by tobacco-related nitrosamines. In this study we investigated whether keratinocytes (HaCaT) and OSCC-derived cell lines (SSC9 and SCC25) are able to secrete the neurotransmitter norepinephrine (NE) as also the effects of the tobacco carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the NE secretion and OSCC proliferation. Supernatant from the HaCaT, SCC9, and SCC25 cells showed higher NE levels (6-, 14.9- and 15.1-fold more, respectively) compared to culture media without cells. When the cells were stimulated with NNK, a tobacco-specific carcinogen, there were increases in the levels of NE secretion by HaCaT and SCC25 cells, but not by SCC9 cells. NNK (10 µM) induced cell proliferation in the HaCaT, SCC9, and SCC25 cell lines and these effects were totally inhibited by blocking β-adrenergic receptors with propranolol. The NNK-induced OSCC cell proliferation was further dependent on nicotinic acetylcholine receptors α4 (nAChR-α4) activation (totally in SCC9 cells and partially in SCC25 cells), but not dependent on nAChR-α7 activation. Inhibition of the β-adrenergic receptors, nAChR-α4 and nAChR-α7 did not block NNK-induced HaCaT proliferation. Our findings suggest that oral cancer cells secrete the neurotransmitter norepinephrine and the tobacco nitrosamine NNK promotes increased cell proliferation through a stress-related cellular adrenergic pathway. Oral squamous cell carcinoma Norepinephrine Beta-Adrenergic Receptor Nicotinic Receptors Stress Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Tobacco consumption is a worldwide epidemic with 155 million tobacco smokers in the world aged 15–24 years[ 1 , 2 ]. Smoking is one of the main cancer inducing factors, being related to 29% of all cancers deaths[ 3 ]. Cigarette smoking increases the risk of developing tumors by almost all organs, being a strong factor for development of oral squamous cell carcinoma (OSCC)[ 4 ]. According to International Agency for Research on Cancer data, is estimated 658.392 new cases of oral cancer worldly in 2023 and 460.00 deaths resulting from this malignancy[ 5 ]. Tobacco contain multiple types of chemicals and carcinogens associated to smoking-induced cancer. Tobacco-specific nitrosamines (TSNAs), including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), have been associated with carcinogenic activity, likewise tumorigenic and mutagenic abilities[ 6 ]. Nicotine is one of the main components of tobacco and is considered a genotoxic and tumor-promoting substance[ 7 ]. The nicotinic acetylcholine receptors (nAChR) are found in neuron synaptic transmission, keratinocytes and bronchial epithelium, suggesting that these receptors have features that go beyond neurotransmission[ 8 ]. Nicotine and NNK presents in tobacco are nAChR agonists and plays a relevant role in mediating malignant transformation of oral epithelial cells[ 9 ]. The activation of nAChRs modulate the expression of genes encoding proteins involved in signal transduction, cell cycle regulation, apoptosis, and cell adhesion[ 10 , 11 ]. NNK is a pro-carcinogen that requires metabolic activation to exert its carcinogenic functions. Multiple cytochrome P450 enzymes catalyze metabolic activation of NNK and others nitrosamine derivatives[ 12 ]. This activation induce metabolites that can promote a series of damage of nucleobases in DNA and form DNA adducts[ 13 ]. In addition to DNA adduction, NNK can also promote increased cell proliferation, survival and migration of mutated cells and cancer cells invasion[ 14 ]. Neurotransmitters derived from chronic stress affects many biological functions, including cancer progression[ 15 ]. In addition to activation of the hypothalamic pituitary adrenal (HPA) axis, the stress response is mediated by the sympathetic nervous system (SNS) resulting in increased release of the neurotransmitters norepinephrine (NE) and epinephrine (E) in different tissues and organs[ 16 , 17 ]. The biological effects of stress-related neurotransmitters are mediated by beta-adrenergic receptors (β-ARs), detected directly on tumor cells in numerous malignancies[ 17 ]. The stress neurotransmitters incites the tumor production of pro-inflammatory cytokines and growth factors, affecting cancer progression [ 18 , 19 ]. NE upregulate expression of vascular endothelial growth factor (VEGF)[ 20 ], interleukin-6 (IL-6)[ 21 , 22 ] and are responsible for the modulation of the matrix metalloproteinases (MMPs) in tumor microenvironment[ 23 ]. Patients with oral cancer display SNS hyperactivation with high systemic levels of NE and E[ 24 ]. Increased catecholamine levels are able to enhance proliferation, migration and invasion of OSCC cells in preclinical models[ 21 , 25 ]. Moreover, recent findings from our group identified that NE was able to induce chemoresistance in OSCC cells through β-ARs[ 26 ]. The structure of tobacco nitrosamine NNK is similar to NE and E, being able to act as an agonist for β-ARs and signaling pathways that activate transcription, cell proliferation and survival[ 27 , 28 ]. Previous studies have shown that different cell lines ( i.e. colon cancer[ 29 ], pancreatic cancer[ 30 ], small airways epithelial cells[ 31 ] and breast cancer[ 32 – 34 ]) are able to produce and release stress-related neurotransmitter, an event that can be potentiated by tobacco nitrosamines such as NNK. Although there is a growing body of evidence showing the effects of stress-related catecholamines on OSCC progression, to date there are no studies that have investigated whether OSCC cells are capable of producing these hormones and whether this phenomenon is modulated by tobacco-related nitrosamines. In this sense, the present study investigated the autocrine production of NE in human cell lines derived from OSCC and normal keratinocyte, as well as the NNK effects on the neurotransmitter secretion and cell proliferation mediated by the β-ARs and nAChRs. MATERIALS AND METHODS Cell Culture The cell lines derived from keratinocytes (HaCaT) and oral squamous cells carcinoma (OSCC)-derived cell lines (SSC9 and SCC25) were obtained from American Type Cell Collection (ATCC). The culture media used in this experiment was Dulbecco's modified Eagle - DMEM (Invitrogen - Life Technologies, Carlsbad, CA, USA) supplemented with 0.5% or 10% FBS (GIBCO - Life Technologies, Carlsbad, CA, USA), 100/mL penicillin, 100ug/mL streptomycin and 0.1% gentamicin to HaCaT cells; and equal parts DMEM and F12 culture medium (Invitrogen, Carlsbad, CA, USA) (DMEM/F12) supplemented with 0.5% or 10% FBS, 100/mL penicillin, 100 ug/mL streptomycin, 0.1% gentamycin and 400 pg/mL hydrocortisone for SCC9 and SCC25 cell lines. To assess whether HaCaT, SCC9 and SCC25 cells are capable of secreting the neurotransmitter NE, the cell lines were cultured with 10% FBS, seeded in 6-well plates and incubated at 37°C in a moist atmosphere 5% CO 2 . After reaching approximately 80% confluency, the respective culture media were changed and the cells were cultured for 48-hours at normal (10%) or reduced FBS concentrations (0.5%). As a control, 6-well plates containing the same medium used for cell lines but without cells, were maintained in the same conditions and periods used for cell cultivation. The supernatants from the cultures and control plates were collected and stored at -80°C for NE measurements. To evaluate if the concentrations of NE in the medium are dependent on the amount of Fetal Bovine Serum (FBS), NE levels were measured in the culture media without cells supplemented with normal FBS concentration (10%) or reduced concentration (0.5%). Measurement of NE concentration To evaluate the NE levels in the culture media (supernatants) with or without cells in two different concentrations of FBS, hormone concentrations were quantified using a specific kit (EIAb Science Co. Ltd., China) through the enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's recommendations. The assay was performed in triplicate. The minimum detectable concentration of the assay (assay sensitivity) was less than 7.5 pg/mL, and intra-assay and inter-assay coefficient of variation (CV) were < 4.3% and 7.5%, respectively. The optical density (OD) was read in a spectrophotometer set to the wavelength of 450 nm. Treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) To evaluate the effects of carcinogen NNK on the NE secretion and cell proliferation rates in HaCaT, SCC9 and SCC25 cells, two different experimental sets were carried out. First, the cells were expanded in 24-well plates at a density of 2x10 5 in specific culture medium (10% FBS) in accordance with the respective cell line. After the cells reached 80% confluency, FBS concentration was reduced to 0.5% and the cells were incubated for another 24 hours. Then, the cells were stimulated with NNK (Sigma, St. Louis, MO) at concentrations of 100 nM, 1 µM, or 10 uM for 6 hours. After this period, cell supernatant from each line was collected and NE levels were measured by enzyme immunoassay as described above. Cell Proliferation assays To evaluate the effects of NNK on the proliferation rates of HaCaT, SCC9 and SCC25 cells, they were plated in 96-well plates (1x10 4 cells/well) in specific culture medium (10% FBS) in accordance with the respective cell lines. After the cells reached 60% confluency the FBS concentration was reduced to 0.5%. After 24 hours the cells were incubated in the absence or presence of NNK (Sigma St. Louis, MO) at concentrations of 1 nM, 10 nM, 100 nM, 1 µM and 10 µM for 48 hours. After 36 hours of this period, cells were incubated with BrdU for 2 hours, and after the end time, the BrdU incorporation levels were measured by ELISA using a specific kit (Millipore, Billerica, MA USA) according to the manufacturer's recommendations. The cell proliferation rates were represented by the BrdU incorporation levels, which were detected according to a resulting color. The optical density values (OD) were analyzed by spectrophotometry. Role of β-adrenergic and nicotinic receptors nAChR-α7 and nAChR-α4 on NNK-induced cell proliferation To investigate whether the effects of NNK on HaCaT and oral cancer cells proliferation were dependent on β-adrenergic receptors and nicotinic nAChR-α7 and nAChR-α4, the cells were expanded in 98 well plates until they reach 60% confluency. Then, the FBS concentration was reduced to 0.5%. Thirty minutes before NNK treatment, the cells were treated or not with the antagonists for β-adrenergic receptors (propranolol at 10 µM) and for nAChR-α4 and nAChR-α7receptors (dihydro-β-erythroidine at 200 nM and α-bungarotoxin at 100 nM, respectively). Then the cells were stimulated with 10 uM NNK. This NNK concentration was chosen for having determined the best stimulatory effect on cell proliferation in previous tests. Cell proliferation after the treatment with NNK in association or not with antagonist drugs were assessed by BrdU incorporation rates as previously described. Statistical Analysis Data were checked for normality, and t-student test or one-way analysis of variance (ANOVA) followed by the Bonferroni’s multiple-comparison test were used to determine the statistical significance of differences between groups. P values < 0.05 were considered significant. RESULTS Culture medium for normal keratinocytes and oral cancer cell lines contains measurable norepinephrine levels that are dependent on FBS concentrations. To evaluate whether culture medium used for cultivation of HaCaT (DMEM), SCC9 and SCC25 (DMEM/F12 for both) cells have basal levels (culture medium without cells) of NE, samples of the two types of mediums in normal (10%) and reduced (0.5%) FBS concentrations were tested for hormone measurement. Our results indicated that both DMEM and DMEM/F12 medium supplemented with FBS had measurable NE levels even before being placed in contact with the cell lines ( Fig. 1 ) . Furthermore, NE levels were correlated to FBS concentrations in the medium, since for both studied culture medium, those with the highest concentration of FBS (10%) had higher hormone levels than those with reduced FBS concentrations (0.5%) ( p < 0.05) ( Fig. 1 ) . DMEM culture medium showed higher NE levels than DMEM/F12 when they were compared with the same FBS concentrations, but these differences did not reach statistical significance ( p > 0.05) ( Fig. 1 ) . Oral cancer cells and normal keratinocytes secrete norepinephrine. To assess whether the HaCaT, SCC9 and SCC25 cell lines ( Fig. 1 A, 1 B and 1 C ) secrete NE, the cells were cultured in normal culture medium (10% FBS) or in reduced FBS concentration (0.5%). The NE levels in the cell supernatant for both FBS conditions were measured and compared with the hormonal levels found in the culture media without cells. In general, the results indicated that the supernatants from all cell lines showed higher NE levels compared to culture medium without cells ( Fig. 1 D, 1 E and 1 F ) . The supernatant from the HaCaT cells showed higher NE concentration compared to culture medium without cells, both for the medium with lower FBS concentration (0.5%) (31.11 ± 5.2 pg/mL vs 4.70 ± 2.0 pg/mL, respectively; p < 0.01), and for medium with 10% FBS (51.72 ± 08.03 vs. 16.73 ± 3.6 pg/ml, respectively; p < 0.01) ( Fig. 1 D ) . Following the same profile which was observed with the cell-free culture media, the supernatant from the HaCaT cells cultured with 10% FBS showed higher NE level when compared to supernatant from cells cultured with 0.5% FBS ( p < 0.05) ( Fig. 1 D ) . Similar results were found for SCC9 ( Fig. 1 E ) and SCC25 ( Fig. 1 F) cells. Regarding SCC9 cells, the cell supernatant at 0.5% FBS exhibited a NE level about 15 times higher (17.3 ± 2.6 pg/mL) than the medium without SCC9 cells (1.41 ± 0.3 pg/mL) ( p < 0.01) ( Fig. 1 E ) . When tested in culture medium DMEM/F12 with 10% FBS, the NE levels in the SCC9 cells supernatant was 4.3 times higher (45.23 ± 3.4pg/mL) than the basal culture medium without cells (10.34 pg/mL ± 0.6) ( p < 0.001) ( Fig. 1 E ) . Supernatant from the SCC9 cells cultured with 10% FBS showed increased NE levels after 48 hours compared to supernatant from the same cells cultured with 0.5% FBS ( p < 0.001) ( Fig. 1 E ) . The results from the SCC25 cells were very similar to those found with SCC9 cells. The supernatant from the SCC25 cells showed NE level 15 times higher than medium without cells ( p < 0.01) when SCC25 cells were cultured 0.5% FBS ( Fig. 1 F. ) ; and about 6 times than culture medium in the absence of cells using 10% FBS condition ( p < 0.001) ( Fig. 1 F ) . Similar to what was observed for SCC9 cells, NE levels in the supernatant from the SCC25 cells cultured with 10% FBS were higher than the hormone levels found when FBS 0.5% was used ( p < 0.001) ( Fig. 1 F ) . In order to assess which cell line showed higher potential to secrete NE, we have estimated the percentage of increase of NE levels for each line when the cell supernatant was compared to its respective culture media in basal conditions (without cells). The data showed that depending on the FBS concentration, there were changes in cellular response and consequent NE levels in the supernatant. For example, when HaCaT cells were cultured with 0.5% FBS they increased in about 6 times the NE levels after 48 hours of cultivation. The OSCC cells secreted higher NE levels in the supernatant compared to HaCaT cells (14.9 times for SCC9 cells and 15.1 times for SCC25 cells) ( Fig. 1 G ) . As for the cells cultured with 10% FBS the percentage of increasing of NE levels in the supernatant was lower than when the cells were cultured in 0.5% FBS, although maintaining the greatest potential for NE secretion by the cancer cells (4.3 times for OSCC9 cells and 6.1 times for the OSCC25 cells) compared to keratinocytes (3.0 times) ( Fig. 1 H ) . These data suggest that cancer cells may secrete greater amount of NE than non-neoplastic cells HaCaT. NNK induces NE secretion in OSCC cells and normal keratinocytes. After we have demonstrated that normal keratinocytes and OSCC cells are able to secrete NE, we investigated whether the NNK carcinogen could stimulate the hormone secretion. For this analysis, the cells were treated with NNK in different concentrations for 6 hours. After this time, the supernatant from each cell line was collected and tested for NE levels measurement. Our results indicated that NNK at 100 nM and 10 uM stimulated increased NE secretion by the HaCaT cells when compared to unstimulated cells ( p < 0.05), with 10 uM concentration eliciting the most robust NE increase ( Fig. 2 A ) . NNK at 100 nM and 10 uM induced significant NE secretion by the SCC9 and SCC25 cells, respectively ( p < 0.05) ( Fig. 2 C ) . NNK at 100nM induced increased secretion of NE in OSCC9 cells ( p 0.05) ( Fig. 2 B ) . NNK increases keratinocytes and OSCC cells proliferation. In order to evaluate the effects of NNK on keratinocytes and OSCC cells proliferation, the cells were treated with different concentrations of NNK. The results showed that NNK in highest concentration tested (10 µM) induced a significant increase of cellular proliferation in all cell lines tested ( Fig. 3 A, 3 B and 3 C ) . All NNK concentrations increased the proliferation rates of HaCaT cells in a dose-dependent manner, but only the 10 µM NNK induced a significant increase in cell proliferation relative to untreated cells with the carcinogen ( p < 0.05) ( Fig. 3 A ) . The results for the SCC9 cells demonstrated that 1 µM and 10 µM NNK increased in 69% and 142% cell proliferation rates compared to unstimulated cells, respectively ( p < 0.05) ( Fig. 3 B ) . The same results profile was found in the SCC25 cells ( Fig. 3 C ) . NNK at different concentrations induced increased proliferation levels of SCC25 cells, buy only 10 µM NNK reached statistical significance increasing 26% the cell proliferation rate ( p 0.05) ( Fig. 3 C ) . NNK-induced OSCC cells proliferation are dependent on β-adrenergic receptors. To evaluate whether NNK effects on the HaCaT, SCC9 and SCC25 cells are dependent on the β1- and β2-adrenergic receptors and α4- and α7-AChRs, we carried out cell proliferation assays with NNK-treated cells in the absence or presence of antagonists for these receptors. For this purpose, the cells were stimulated with 10 µM NNK with or without the following inhibitors: α-bungarotoxin (inhibitor of nAChR-α7 receptor), dihydro-β-Erythroidine (inhibitor of nAChR-α4 receptor) and propranolol (inhibitor of β1- and β2-adrenergic receptors. The concentration of 10 µM NNK was selected as it has promoted the most intense stimulatory effects on the proliferation rates in the tested cells. The results showed that none of the three antagonists tested had significant effect on the HaCaT NNK-induced cell proliferation (p > 0.05) (data not shown). Nevertheless, propranolol (a nonspecific β1- and β2-adrenergic receptors inhibitor) completely blocked the effects caused by NNK on the SCC9 and SCC25 cells proliferation ( Fig. 4 A and 4 B ) . Dihydro-β-erythroidine completely inhibited the NNK effect on the SCC9 cell proliferation ( p 0.05) ( Figs. 4 A and 4 B ) . Interestingly, α-bungarotoxin had no effect on the NNK-induced proliferation in any of the OSCC cell lines ( p > 0.05) ( Figs. 4 A and 4 B ) . These results suggest that NNK effect on the OSCC cells proliferation are dependent on β-adrenergic and nAChR-α4 receptors activation. DISCUSSION In this study, our results reveled the ability of autocrine secretion of the neurotransmitter NE by oral cancer cells, which can play an autocrine catecholamine loop. Cancer cells showed increased secretion of NE compared to HaCaT cells, and the supernatant of HaCaT and OSCCs cell lines had significantly higher levels of NE in relation to the culture medium without cells. The higher NE levels secreted by OSCC cell lines compared to non-malignant cells may represent an adrenergic hyperactivation on cancer cells. Non-tumorigenic and tumorigenic human breast cell[ 32 ], pancreatic cancer and normal pancreatic ductal epithelial cells[ 30 ], and immortalized human small airway epithelial cell line[ 31 ] are capable to produce norepinephrine with any stimulation. Nonetheless, this is the first time that this ability is described in OSCC cells. The NNK is a highly specific tobacco carcinogen nitrosamine, but the mechanisms involved in oral carcinogenesis and the proliferation of oral cancer cells induced by NNK are poorly known. Our results revealed that NNK in highest concentration induced a significant increase of cellular proliferation in keratinocytes (HaCaT) and OSCC cells (OSCC9 and OSCC25). NNK has the ability to induce proliferation in others cell lines, including colon cancer cell lines[ 35 ], oral cancer (SCC-15, HSC-13, OSC-19, and UM1)[ 36 ], endometrial adenocarcinoma[ 37 ] and neuroendocrine cells[ 38 ]. Non-neoplastic alveolar epithelial cells subjected to prolonged exposure to NNK cells led to acquire increasingly malignant properties such as increased proliferation and cell motility[ 39 ]. Likewise, the treatment of Ishikawa cells with NNK induces cell cycle progression and proliferation by up-regulating cyclin E and cyclin D expression, as well as by down-regulating p21 and p27[ 37 ]. NNK has a high affinity for β-adrenergic receptors because of its structural resemblance to classical beta-adrenergic agonists[ 40 ]. When blocking β1- and β2-AR in esophageal cancer cells, there was a reverse in the blocking on oncogenic effects of NNK[ 41 ]. The activation of β-adrenergic receptors in response to NNK has been demonstrated in the growth of pancreatic carcinoma[ 42 ], colon cancer[ 35 ], and esophageal squamous cell carcinoma[ 43 ] cell lines. In our study, we conduct cell proliferation assays in NNK-treated cells in the presence of inhibitor of β-adrenergic receptors types 1 and 2. Our results showed that propranolol blocked the effects caused by NNK on the proliferation of the SCC9 and SCC25 cells, but not in HaCaT cells. These results suggest that tobacco-related nitrosamines may influence OSCC progression via beta-adrenergic activation, a recognized stress-associated pathway. The β-adrenergic receptors are involved in the proliferation of various types of cancer as pancreas[ 44 ], colorectal[ 35 ], stomach[ 45 ] and lung[ 27 ]. The effects of cell proliferation in these studies was blocked by propranolol. In our findings, NNK induces SCC-25 and HaCaT cells secreting NE. The same was not observed in SCC-9 cells indicating that this cell line may already have reached a maximum limit of neurotransmitter secretion under basal conditions without NNK stimulation. NE secretion from human small airway epithelial cells is regulated by nAChRs, and there is a greater increase in receptor responsiveness when stimulated with NNK[ 46 ]. Pancreatic cancer cells and normal pancreatic duct epithelia display an autocrine loop catecholamine that stimulates their proliferation by β-ARs and nAChRs-α3, -α5, and -α7[ 30 ]. Al-Wadei and colleagues[ 47 ] reported that the neurotransmitter γ-aminobutyric inhibitory neurotransmitter (GABA) and its synthesizing-enzyme glutamate decarboxylase 65 were suppressed in NNK-induced small airway-derived pulmonary adenocarcinoma cells and in pancreatic ductal adenocarcinoma. Despite the suppression of GABA, protein expression of the nAChR-α4 and –α7 was upregulated[ 47 ]. This study showed that the tested antagonists of nAChR-α4 and nAChR-α7 had no significant effects on the NNK-induced proliferation in HaCaT cells. However, the dihydro-β-Erythroidine inhibited NNK effect on SCC9 cells proliferation and, in part, its effect of NNK OSCC25 on the proliferation of cells. The α-bungarotoxin had no effect on cell proliferation induced by NNK in any cell line cancer. These results indicate that the NNK effect on the proliferation of OSCC cells can be dependent on nAChR-α4. Schuller and colleagues (2007) discuss about the human small cell lung cancer (SCLC) exposition to NNK increased expression of the nAChR-α7 and caused influx of Ca2+. This cascade induces the activation of PKC, Raf-1, ERK1/2, and c-myc, increasing of cell proliferation[ 48 ]. The nitrosamines in tobacco as NNK can promote the activation of the serine/threonine kinase Akt and contribute to carcinogenesis of the normal human airway through nAChRs-α3, -α4 and -α7, respectively[ 31 ]. In the present study, we demonstrated for the first time that OSCC cells secrete the stress-related NE and this event can be super regulated by tobacco carcinogen NNK. NNK also increases the proliferation of HaCaT and OSCC cells, mainly through β-adrenergic receptors, the same receptors activated by NE. These finding together suggest that OSCC development may be related to an imbalance of the NE secretion and activation of its receptors directly inducing changes related to the malignant phenotype. In conclusion, this study reveals that OSCC cells secrete the stress-related neurotransmitter norepinephrine and the tobacco carcinogen NNK induces increased cell proliferation through a stress-related cellular adrenergic pathway. Declarations Conflicts of interest: Not applicable. Ethics approval: Not applicable Informed consent: Not applicable. Funding: This study was supported by the São Paulo State Research Foundation (FAPESP). FAPESP grant 2016/25255-0 designated to Daniel G Bernabé supported Psychosomatic Research Center's studies. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contribution Flávia Alves Verza: original draft, material preparation, data collection and analysis. Ana Lívia Santos-Sousa: assisted in material analysis. Sandra Helena Penha de Oliveira: edition and review the manuscript. Daniel Galera Bernabé: conceptualization, resources, formal analysis, supervision, funding acquisition, project administration, writing-review and editing. The paper was critically reviewed and approved by all authors. Acknowledgements: not applicable References Reitsma MB, Flor LS, Mullany EC, Gupta V, Hay SI, Gakidou E (2021) Spatial, temporal, and demographic patterns in prevalence of smoking tobacco use and initiation among young people in 204 countries and territories, 1990–2019. Lancet Public Health [Internet]. Published by Elsevier Ltd. ;6:e472–81. http://dx.doi.org/10.1016/S2468-2667(21)00102-X World Health Organization. Tobacco [Internet] (2022) Available from: https://www.who.int/en/news-room/fact-sheets/detail/tobacco . Acessed 21 jan 2024 Danaei G, Vander Hoorn S, Lopez AD, Murray CJL, Ezzati M (2005) Causes of cancer in the world: Comparative risk assessment of nine behavioural and environmental risk factors. Lancet 366:1784–1793 Jethwa AR, Khariwala SS (2017) Tobacco-related carcinogenesis in head and neck cancer. Cancer Metastasis Rev [Internet] 36:411–423 Ferlay J, Laversanne M, Ervik M, Lam F, Colombet M, Mery L et al (2022) Global Cancer Observatory: Cancer Tomorrow [Internet]. Int. Agency Res. Cancer. Lyon, France; Available from: https://gco.iarc.fr/tomorrow/en . Acessed 21 jan 2024 Hecht SS (2003) Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer [Internet] 3:733–744 Grando SA (2014) Connections of nicotine to cancer. Nat Rev Cancer Nat Publishing Group 14:419–429 Zoli M, Pucci S, Vilella A, Gotti C (2018) Neuronal and Extraneuronal Nicotinic Acetylcholine Receptors. Curr Neuropharmacol 16:338–349 Kalantari-Dehaghi M, Bernard H-U, Grando SA (2012) Reciprocal effects of NNK and SLURP-1 on oncogene expression in target epithelial cells. Life Sci [Internet] 91:1122–1125 Lin C, Lin L, Chen T, Ye Y, Chiang B (2023) The expression of nicotinic acetylcholine receptor subunits and their associations with local immune cells and prognosis in oral squamous cell carcinoma. Cancer Med [Internet] 12:18918–18930 Hildegard M, Schuller (2013) Regulatory Role of the α7nAChR in Cancer. Curr Drug Targets 13:680–687 Yamazaki H, Inui Y, Yun CH, Guengerich FP, Shimada T (1992) Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis 13:1789–1794 Phillips DH (2002) Smoking-related DNA and protein adducts in human tissues. Carcinogenesis 23:1979–2004 Xue J, Yang S, Seng S (2014) Mechanisms of cancer induction by tobacco-specific NNK and NNN. Cancers (Basel) 6:1138–1156 Antoni MH, Lutgendorf SK, Cole SW, Dhabhar FS, Sephton SE, McDonald PG et al (2006) The influence of bio-behavioural factors on tumour biology: pathways and mechanisms. Nat Rev Cancer [Internet] 6:240–248 Itoi K, Sugimoto N (2010) The brainstem noradrenergic systems in stress, anxiety and depression. J Neuroendocrinol 22:355–361 Armaiz-Pena GN, Cole SW, Lutgendorf SK, Sood AK (2013) Neuroendocrine influences on cancer progression. Brain Behav Immun [Internet] 30:S19–25 Lutgendorf SK, Andersen BL (2015) Biobehavioral approaches to cancer progression and survival: Mechanisms and interventions. Am Psychol [Internet]. Nature Publishing Group; ;70:186–97. http://dx.doi.org/10.1016/j.bbi.2012.06.005 Bernabé DG (2021) Catecholamines Mediate Psychologic Stress–Induced Cancer Progression. Cancer Res 81:5144–5146 Thaker PH, Han LY, Kamat AA, Arevalo JM, Takahashi R, Lu C et al (2006) Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat Med 12:939–944 Bernabé DG, Tamae AC, Biasoli ÉR, Oliveira SHP (2011) Stress hormones increase cell proliferation and regulates interleukin-6 secretion in human oral squamous cell carcinoma cells. Brain Behav Immun 25:574–583 Yang EV, Kim Sjae, Donovan EL, Chen M, Gross AC, Webster Marketon JI et al (2009) Norepinephrine upregulates VEGF, IL-8, and IL-6 expression in human melanoma tumor cell lines: Implications for stress-related enhancement of tumor progression. Brain Behav Immun [Internet]. Elsevier Inc.; ;23:267–75. http://dx.doi.org/10.1016/j.bbi.2008.10.005 Moreno-Smith M, Lutgendorf SK, Sood AK (2010) Impact of stress on cancer metastasis. Futur Oncol [Internet]. ;6:1863–81. Available from: https://www.futuremedicine.com/doi/ 10.2217/fon.10.142 Bastos DB, Sarafim-Silva BAM, Sundefeld MLMM, Ribeiro AA, Brandão JDP, Biasoli ÉR et al (2018) Circulating catecholamines are associated with biobehavioral factors and anxiety symptoms in head and neck cancer patients. Multhoff G, editor. PLoS One [Internet]. ;13:e0202515. https://dx.plos.org/10.1371/journal.pone.0202515 Xie H, Li C, He Y, Griffin R, Ye Q, Li L (2015) Chronic stress promotes oral cancer growth and angiogenesis with increased circulating catecholamine and glucocorticoid levels in a mouse model, vol 51. Oral Oncol [Internet]. Elsevier Ltd, pp 991–997 Tjioe KC, Cardoso DM, Oliveira SHP, Bernabé DG (2022) Stress hormone norepinephrine incites resistance of oral cancer cells to chemotherapy. Endocr Relat Cancer [Internet] 29:201–212 Schuller HM, Tithof PK, Williams M, Plummer H (1999) The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone is a β-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via β-adrenergic receptor-mediated release of arachidonic acid. Cancer Res 59:4510–4515 Al-Wadei HAN, Plummer HK, Ullah MF, Unger B, Brody JR, Schuller HM (2012) Social Stress Promotes and γ-Aminobutyric Acid Inhibits Tumor Growth in Mouse Models of Non–Small Cell Lung Cancer. Cancer Prev Res [Internet] 5:189–196 Wong HPS, Yu L, Lam EKY, Tai EKK, Wu WKK, Cho CH (2007) Nicotine promotes cell proliferation via α7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells. Toxicol Appl Pharmacol 221:261–267 Al-Wadei MH, Al-Wadei HAN, Schuller HM (2012) Pancreatic Cancer Cells and Normal Pancreatic Duct Epithelial Cells Express an Autocrine Catecholamine Loop that Is Activated by Nicotinic Acetylcholine Receptors α3, α5, and α7. Mol Cancer Res [Internet] 10:239–249 Al-Wadei HAN, Al-Wadei MH, Masi T, Schuller HM (2010) Chronic exposure to estrogen and the tobacco carcinogen NNK cooperatively modulates nicotinic receptors in small airway epithelial cells. Lung Cancer [Internet] Elsevier Irel Ltd 69:33–39 Amaro F, Silva D, Reguengo H, Oliveira JC, Quintas C, Vale N et al (2020) Β-Adrenoceptor Activation in Breast Mcf-10a Cells Induces a Pattern of Catecholamine Production Similar To That of Tumorigenic Mcf-7 Cells. Int J Mol Sci 21:1–16 Chiba T, Maeda T, Fujita Y, Takeda R, Kikuchi A, Kudo K et al (2019) The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells. Endocrinology 160:1–16 Shi M, Liu D, Duan H, Qian L, Wang L, Niu L et al (2011) The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells. Breast Cancer Res Treat 125:351–362 Wu WKK, Wong HPS, Luo SW, Chan K, Huang FY, Hui MKC et al (2005) 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone from cigarette smoke stimulates colon cancer growth via β-adrenoceptors. Cancer Res 65:5272–5277 Guo J, Zhou S, Huang P, Xu S, Zhang G, He H et al (2020) NNK-mediated upregulation of DEPDC1 stimulates the progression of oral squamous cell carcinoma by inhibiting CYP27B1 expression. Am J Cancer Res [Internet] 10:1745–1760 Kim SM, Hwang KA, Choi DW, Choi KC (2018) The cigarette smoke components induced the cell proliferation and epithelial to mesenchymal transition via production of reactive oxygen species in endometrial adenocarcinoma cells. Food Chem Toxicol [Internet]. Elsevier; ;121:657–65. https://doi.org/10.1016/j.fct.2018.09.023 Schuller HM (1989) Cell type specific, receptor-mediated modulation of growth kinetics in human lung cancer cell lines by nicotine and tobacco-related nitrosamines. Biochem Pharmacol 38:3439–3442 Mennecier G, Torres LN, Cogliati B, Sanches DS, Mori CM, Latorre AO et al (2014) Chronic exposure of lung alveolar epithelial type II cells to tobacco-specific carcinogen NNK results in malignant transformation: A new in vitro lung carcinogenesis model. Mol Carcinog 53:392–402 Schuller HM (2007) Neurotransmitter receptor-mediated signaling pathways as modulators of carcinogenesis. Prog Exp Tumor Res 39:45–63 GUO K, MA Q, WANG L, HU H, LI J, ZHANG D et al (2009) Norepinephrine-induced invasion by pancreatic cancer cells is inhibited by propranolol. Oncol Rep [Internet] 22:1265–1270 Weddle DL, Tithoff P, Williams M, Schuller HM (2001) β-Adrenergic growth regulation of human cancer cell lines derived from pancreatic ductal carcinomas. Carcinogenesis 22:473–479 Zhang N, Sun X, Sun M, Zhu S, Wang L, Ma D et al (2015) 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Promotes Esophageal Squamous Cell Carcinoma Growth Via Beta-Adrenoceptors in Vitro and in Vivo. PLoS ONE 10:1–16 Shin VY, Jin HC, Ng EKO, Yu J, Leung WK, Cho CH et al (2008) Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and β-adrenergic receptor signaling pathways. Toxicol Appl Pharmacol [Internet] Elsevier Inc 233:254–261 Shin VY, Wu WKK, Chu KM, Koo MWL, Wong HPS, Lam EKY et al (2007) Functional role of β-adrenergic receptors in the mitogenic action of nicotine on gastric cancer cells. Toxicol Sci 96:21–29 Egleton RD, Brown KC, Dasgupta P (2009) Angiogenic activity of nicotinic acetylcholine receptors: Implications in tobacco-related vascular diseases. Pharmacol Ther [Internet]. Elsevier Inc.; ;121:205–23. http://dx.doi.org/10.1016/j.pharmthera.2008.10.007 Al-Wadei HA, Schuller HM (2009) Nicotinic receptor-associated modulation of stimulatory and inhibitory neurotransmitters in NNK-induced adenocarcinoma of the lungs and pancreas. J Pathol [Internet]. ;218:437–45. Available from: https://onlinelibrary.wiley.com/doi/ 10.1002/path.2542 Schuller HM (2007) Nitrosamines as nicotinic receptor ligands. Life Sci [Internet]. Elsevier Inc.; ;80:2274–80. http://dx.doi.org/10.1016/j.lfs.2007.03.006 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3934938","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271460763,"identity":"9977a2f3-8f96-4712-8cc0-7ea6af6bca70","order_by":0,"name":"Flávia Alves Verza","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Flávia","middleName":"Alves","lastName":"Verza","suffix":""},{"id":271460764,"identity":"276d5db2-a1cc-4797-b6f5-51fdac898d2e","order_by":1,"name":"Ana Lívia Santos-Sousa","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Lívia","lastName":"Santos-Sousa","suffix":""},{"id":271460765,"identity":"430b9657-18bc-47b2-8636-92c0f6379699","order_by":2,"name":"Sandra Helena Penha Oliveira","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"Helena Penha","lastName":"Oliveira","suffix":""},{"id":271460766,"identity":"f7824453-0139-468d-a4c3-8b610b40f53d","order_by":3,"name":"Daniel Galera Bernabé","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYDCCAyBUwJAAYjMzMNhARBkbCGkxgGtJI04LA5KWw4S18B0/e/DABwOGPH7pswc/F1ScT9zO3v7sAeOOezi1SJ7JSzg4w4ChWLIvL1l6xpnbiTt7zpgbMJ4pxqnF4ECOwWEeA4bEDWd4DKR5224nbriRwybB2JaAW8v5NwaH/wC17D/DY/yb99+5xA33nz/Dr+UG0BYGkC08PGbSvA0HgLYwmOHVInnjjcHBHgOJYokzPGbWM44lG284k2NukHgGtxa+8znGH35U2OTx9/AY3y6osZPdcPz4swcfd+DWAgUSKDw2BoIa0AEbqRpGwSgYBaNgeAMAqXtbc3EiiZgAAAAASUVORK5CYII=","orcid":"","institution":"São Paulo State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"Galera","lastName":"Bernabé","suffix":""}],"badges":[],"createdAt":"2024-02-06 20:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3934938/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3934938/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50860078,"identity":"32244e6d-e5de-46a8-9083-b4595771ed07","added_by":"auto","created_at":"2024-02-08 13:25:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":942716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOralcancer cells and keratinocytes secrete the stress-related neurotransmitter norepinephrine. \u003c/strong\u003eMicroscopy aspects of the cell lines used in the study: HaCaT \u003cstrong\u003e(A)\u003c/strong\u003e, SCC9 \u003cstrong\u003e(B)\u003c/strong\u003e and SCC25 \u003cstrong\u003e(C)\u003c/strong\u003e. NE secretion by the HaCaT \u003cstrong\u003e(D)\u003c/strong\u003e, SCC9 \u003cstrong\u003e(E)\u003c/strong\u003e and SCC25 cells \u003cstrong\u003e(F)\u003c/strong\u003e. NE levels were measured in the HaCaT and OSCC cells supernatants, in normal concentrations of FBS (10%) or in reduced concentration (0.5%). Cell supernatants from the three cell lines tested showed higher NE concentration compared to culture medium without cells (adjusted for normal or reduced FBS concentration). **P \u0026lt; 0.01, ***P \u0026lt; 0.001. \u003cstrong\u003eG \u003c/strong\u003eand \u003cstrong\u003eD\u003c/strong\u003e. The data represent the percentage of increase of NE levels in the cell supernatants from each cell line compared to their respective culture media without cells with FBS at 0.5% (G) or 10% (H). The NE level value found in the culture medium without cells (control) was fixed in 100%, and used to show how many times the cell line increased the hormone levels.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3934938/v1/b183453581a70064d108fb33.png"},{"id":50859803,"identity":"cc206d36-baed-4405-b9bc-aa2cd8868334","added_by":"auto","created_at":"2024-02-08 13:17:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNNK promotes NE secretion in HaCaT and SCC25 cells.\u003c/strong\u003e HaCaT (A), SCC9 (B) and SCC25 (C) cells were cultivated for 24 hours in reduced FBS concentration (0.5%), and then they were stimulated by different concentrations of NNK (100 nM, 1 µM and 10 µM) at 6 hours. \u003cstrong\u003eA\u003c/strong\u003e. NNK (100 nM and 10 µM) induced increased NE secretion by the HaCaT cells compared to the control (nonstimulated cells). \u003cstrong\u003eB. \u003c/strong\u003eNNK at 100 nM induced significantly NE secretion the by the SCC9 cells. \u003cstrong\u003eC. \u003c/strong\u003eNNK (10 µM) induced higher NE secretion by the SCC25 cells compared to the control. *indicates statistically significant differences between cells stimulated with NNK and non-stimulated cells (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3934938/v1/ed5b338d30aa8b3a0b18e020.png"},{"id":50860077,"identity":"96ed420b-a527-4a7e-8c0c-f8d27a70b004","added_by":"auto","created_at":"2024-02-08 13:25:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNNK effects on the keratinocytes and OSCC cells proliferation.\u003c/strong\u003e The HaCaT, SCC9 and SCC25 cells were cultivated for 24 hours in reduced FBS concentration (0.5%) and then stimulated with 1 nM, 10 nM, 100 nM, 1 µM and 10 µM NNK for 48 hours. NNK at 10 µM increased proliferation of the HaCaT (A), SCC9 (B) and SCC25 cells (C) compared to control (non-stimulated cells). NNK at 1µM also induced SCC9 cells proliferation (B). *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3934938/v1/11836ded09406c929edfbd4a.png"},{"id":50859804,"identity":"13b6e266-731d-4b8c-8607-3815e8db954a","added_by":"auto","created_at":"2024-02-08 13:17:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of the β-adrenergic receptors and nicotinic acetylcholine receptors (nAChR) α4 and α7 on the NNK-induced OSCC cells proliferation.\u003c/strong\u003e To evaluate whether NNK-induced SCC9 and SCC25 cells proliferation are dependent on the β-adrenergic receptors and nAChRs-α4 and -α7, the cells were treated with 10 µM NNK in presence or absence of the respective antagonists: Propranolol (Prop) (10 µM), Diidro-β-eritroidine (DhβE) (200 nM) and α-Bungarotoxine (BTX) (100 nM). Inhibition of the ß-adrenergic receptors, nAChRs-α4 and -α7 did not block NNK-induced HaCaT proliferation (p\u0026gt;0.05) (data not shown). \u003cstrong\u003eA. \u003c/strong\u003eIn the SCC9 cells, NNK-induced cell proliferation was significantly inhibited by DhβE and Propranolol. \u003cstrong\u003eB.\u003c/strong\u003e Propranolol (a non-selective β1- and β2-adrenergic receptors) also blocked the NNK-induced proliferation by the SCC25 cells. The α4-nAChR antagonist DhβE reduced the NNK-induced SCC25 cells proliferation, but this effect did not reach statistical significance (p\u0026gt;0.05). *p\u0026lt;0.05; **\u0026lt;0.01; ***p0.001.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3934938/v1/8a2839b76787d5b83b1ce827.png"},{"id":51922232,"identity":"43c22a88-4d64-4f78-b164-83cd16e14508","added_by":"auto","created_at":"2024-03-03 22:44:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1559357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3934938/v1/09f12646-138e-47f7-b624-36f596c184bd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oral cancer cells secrete stress neurotransmitter and proliferate in response to tobacco carcinogen NNK through a cellular adrenergic pathway.","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTobacco consumption is a worldwide epidemic with 155\u0026nbsp;million tobacco smokers in the world aged 15\u0026ndash;24 years[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Smoking is one of the main cancer inducing factors, being related to 29% of all cancers deaths[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Cigarette smoking increases the risk of developing tumors by almost all organs, being a strong factor for development of oral squamous cell carcinoma (OSCC)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. According to International Agency for Research on Cancer data, is estimated 658.392 new cases of oral cancer worldly in 2023 and 460.00 deaths resulting from this malignancy[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Tobacco contain multiple types of chemicals and carcinogens associated to smoking-induced cancer. Tobacco-specific nitrosamines (TSNAs), including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), have been associated with carcinogenic activity, likewise tumorigenic and mutagenic abilities[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nicotine is one of the main components of tobacco and is considered a genotoxic and tumor-promoting substance[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The nicotinic acetylcholine receptors (nAChR) are found in neuron synaptic transmission, keratinocytes and bronchial epithelium, suggesting that these receptors have features that go beyond neurotransmission[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNicotine and NNK presents in tobacco are nAChR agonists and plays a relevant role in mediating malignant transformation of oral epithelial cells[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The activation of nAChRs modulate the expression of genes encoding proteins involved in signal transduction, cell cycle regulation, apoptosis, and cell adhesion[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. NNK is a pro-carcinogen that requires metabolic activation to exert its carcinogenic functions. Multiple cytochrome P450 enzymes catalyze metabolic activation of NNK and others nitrosamine derivatives[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This activation induce metabolites that can promote a series of damage of nucleobases in DNA and form DNA adducts[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition to DNA adduction, NNK can also promote increased cell proliferation, survival and migration of mutated cells and cancer cells invasion[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeurotransmitters derived from chronic stress affects many biological functions, including cancer progression[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition to activation of the hypothalamic pituitary adrenal (HPA) axis, the stress response is mediated by the sympathetic nervous system (SNS) resulting in increased release of the neurotransmitters norepinephrine (NE) and epinephrine (E) in different tissues and organs[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The biological effects of stress-related neurotransmitters are mediated by beta-adrenergic receptors (β-ARs), detected directly on tumor cells in numerous malignancies[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The stress neurotransmitters incites the tumor production of pro-inflammatory cytokines and growth factors, affecting cancer progression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. NE upregulate expression of vascular endothelial growth factor (VEGF)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], interleukin-6 (IL-6)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and are responsible for the modulation of the matrix metalloproteinases (MMPs) in tumor microenvironment[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePatients with oral cancer display SNS hyperactivation with high systemic levels of NE and E[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Increased catecholamine levels are able to enhance proliferation, migration and invasion of OSCC cells in preclinical models[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, recent findings from our group identified that NE was able to induce chemoresistance in OSCC cells through β-ARs[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The structure of tobacco nitrosamine NNK is similar to NE and E, being able to act as an agonist for β-ARs and signaling pathways that activate transcription, cell proliferation and survival[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have shown that different cell lines (\u003cem\u003ei.e.\u003c/em\u003e colon cancer[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], pancreatic cancer[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], small airways epithelial cells[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and breast cancer[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]) are able to produce and release stress-related neurotransmitter, an event that can be potentiated by tobacco nitrosamines such as NNK. Although there is a growing body of evidence showing the effects of stress-related catecholamines on OSCC progression, to date there are no studies that have investigated whether OSCC cells are capable of producing these hormones and whether this phenomenon is modulated by tobacco-related nitrosamines. In this sense, the present study investigated the autocrine production of NE in human cell lines derived from OSCC and normal keratinocyte, as well as the NNK effects on the neurotransmitter secretion and cell proliferation mediated by the β-ARs and nAChRs.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eThe cell lines derived from keratinocytes (HaCaT) and oral squamous cells carcinoma (OSCC)-derived cell lines (SSC9 and SCC25) were obtained from American Type Cell Collection (ATCC). The culture media used in this experiment was Dulbecco's modified Eagle - DMEM (Invitrogen - Life Technologies, Carlsbad, CA, USA) supplemented with 0.5% or 10% FBS (GIBCO - Life Technologies, Carlsbad, CA, USA), 100/mL penicillin, 100ug/mL streptomycin and 0.1% gentamicin to HaCaT cells; and equal parts DMEM and F12 culture medium (Invitrogen, Carlsbad, CA, USA) (DMEM/F12) supplemented with 0.5% or 10% FBS, 100/mL penicillin, 100 ug/mL streptomycin, 0.1% gentamycin and 400 pg/mL hydrocortisone for SCC9 and SCC25 cell lines. To assess whether HaCaT, SCC9 and SCC25 cells are capable of secreting the neurotransmitter NE, the cell lines were cultured with 10% FBS, seeded in 6-well plates and incubated at 37\u0026deg;C in a moist atmosphere 5% CO\u003csub\u003e2\u003c/sub\u003e. After reaching approximately 80% confluency, the respective culture media were changed and the cells were cultured for 48-hours at normal (10%) or reduced FBS concentrations (0.5%). As a control, 6-well plates containing the same medium used for cell lines but without cells, were maintained in the same conditions and periods used for cell cultivation. The supernatants from the cultures and control plates were collected and stored at -80\u0026deg;C for NE measurements. To evaluate if the concentrations of NE in the medium are dependent on the amount of Fetal Bovine Serum (FBS), NE levels were measured in the culture media without cells supplemented with normal FBS concentration (10%) or reduced concentration (0.5%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of NE concentration\u003c/h2\u003e \u003cp\u003eTo evaluate the NE levels in the culture media (supernatants) with or without cells in two different concentrations of FBS, hormone concentrations were quantified using a specific kit (EIAb Science Co. Ltd., China) through the enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's recommendations. The assay was performed in triplicate. The minimum detectable concentration of the assay (assay sensitivity) was less than 7.5 pg/mL, and intra-assay and inter-assay coefficient of variation (CV) were \u0026lt;\u0026thinsp;4.3% and 7.5%, respectively. The optical density (OD) was read in a spectrophotometer set to the wavelength of 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTreatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of carcinogen NNK on the NE secretion and cell proliferation rates in HaCaT, SCC9 and SCC25 cells, two different experimental sets were carried out. First, the cells were expanded in 24-well plates at a density of 2x10\u003csup\u003e5\u003c/sup\u003e in specific culture medium (10% FBS) in accordance with the respective cell line. After the cells reached 80% confluency, FBS concentration was reduced to 0.5% and the cells were incubated for another 24 hours. Then, the cells were stimulated with NNK (Sigma, St. Louis, MO) at concentrations of 100 nM, 1 \u0026micro;M, or 10 uM for 6 hours. After this period, cell supernatant from each line was collected and NE levels were measured by enzyme immunoassay as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation assays\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of NNK on the proliferation rates of HaCaT, SCC9 and SCC25 cells, they were plated in 96-well plates (1x10\u003csup\u003e4\u003c/sup\u003e cells/well) in specific culture medium (10% FBS) in accordance with the respective cell lines. After the cells reached 60% confluency the FBS concentration was reduced to 0.5%. After 24 hours the cells were incubated in the absence or presence of NNK (Sigma St. Louis, MO) at concentrations of 1 nM, 10 nM, 100 nM, 1 \u0026micro;M and 10 \u0026micro;M for 48 hours. After 36 hours of this period, cells were incubated with BrdU for 2 hours, and after the end time, the BrdU incorporation levels were measured by ELISA using a specific kit (Millipore, Billerica, MA USA) according to the manufacturer's recommendations. The cell proliferation rates were represented by the BrdU incorporation levels, which were detected according to a resulting color. The optical density values (OD) were analyzed by spectrophotometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRole of β-adrenergic and nicotinic receptors nAChR-α7 and nAChR-α4 on NNK-induced cell proliferation\u003c/h2\u003e \u003cp\u003eTo investigate whether the effects of NNK on HaCaT and oral cancer cells proliferation were dependent on β-adrenergic receptors and nicotinic nAChR-α7 and nAChR-α4, the cells were expanded in 98 well plates until they reach 60% confluency. Then, the FBS concentration was reduced to 0.5%. Thirty minutes before NNK treatment, the cells were treated or not with the antagonists for β-adrenergic receptors (propranolol at 10 \u0026micro;M) and for nAChR-α4 and nAChR-α7receptors (dihydro-β-erythroidine at 200 nM and α-bungarotoxin at 100 nM, respectively). Then the cells were stimulated with 10 uM NNK. This NNK concentration was chosen for having determined the best stimulatory effect on cell proliferation in previous tests. Cell proliferation after the treatment with NNK in association or not with antagonist drugs were assessed by BrdU incorporation rates as previously described.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were checked for normality, and t-student test or one-way analysis of variance (ANOVA) followed by the Bonferroni\u0026rsquo;s multiple-comparison test were used to determine the statistical significance of differences between groups. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eCulture medium for normal keratinocytes and oral cancer cell lines contains measurable norepinephrine levels that are dependent on FBS concentrations.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate whether culture medium used for cultivation of HaCaT (DMEM), SCC9 and SCC25 (DMEM/F12 for both) cells have basal levels (culture medium without cells) of NE, samples of the two types of mediums in normal (10%) and reduced (0.5%) FBS concentrations were tested for hormone measurement. Our results indicated that both DMEM and DMEM/F12 medium supplemented with FBS had measurable NE levels even before being placed in contact with the cell lines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Furthermore, NE levels were correlated to FBS concentrations in the medium, since for both studied culture medium, those with the highest concentration of FBS (10%) had higher hormone levels than those with reduced FBS concentrations (0.5%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. DMEM culture medium showed higher NE levels than DMEM/F12 when they were compared with the same FBS concentrations, but these differences did not reach statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOral cancer cells and normal keratinocytes secrete norepinephrine.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess whether the HaCaT, SCC9 and SCC25 cell lines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e secrete NE, the cells were cultured in normal culture medium (10% FBS) or in reduced FBS concentration (0.5%). The NE levels in the cell supernatant for both FBS conditions were measured and compared with the hormonal levels found in the culture media without cells. In general, the results indicated that the supernatants from all cell lines showed higher NE levels compared to culture medium without cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. The supernatant from the HaCaT cells showed higher NE concentration compared to culture medium without cells, both for the medium with lower FBS concentration (0.5%) (31.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 pg/mL vs 4.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 pg/mL, respectively; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and for medium with 10% FBS (51.72\u0026thinsp;\u0026plusmn;\u0026thinsp;08.03 vs. 16.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 pg/ml, respectively; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Following the same profile which was observed with the cell-free culture media, the supernatant from the HaCaT cells cultured with 10% FBS showed higher NE level when compared to supernatant from cells cultured with 0.5% FBS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Similar results were found for SCC9 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e and SCC25 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) cells. Regarding SCC9 cells, the cell supernatant at 0.5% FBS exhibited a NE level about 15 times higher (17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 pg/mL) than the medium without SCC9 cells (1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 pg/mL) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. When tested in culture medium DMEM/F12 with 10% FBS, the NE levels in the SCC9 cells supernatant was 4.3 times higher (45.23\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4pg/mL) than the basal culture medium without cells (10.34 pg/mL\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Supernatant from the SCC9 cells cultured with 10% FBS showed increased NE levels after 48 hours compared to supernatant from the same cells cultured with 0.5% FBS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. The results from the SCC25 cells were very similar to those found with SCC9 cells. The supernatant from the SCC25 cells showed NE level 15 times higher than medium without cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) when SCC25 cells were cultured 0.5% FBS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF.\u003cb\u003e)\u003c/b\u003e; and about 6 times than culture medium in the absence of cells using 10% FBS condition (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Similar to what was observed for SCC9 cells, NE levels in the supernatant from the SCC25 cells cultured with 10% FBS were higher than the hormone levels found when FBS 0.5% was used (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. In order to assess which cell line showed higher potential to secrete NE, we have estimated the percentage of increase of NE levels for each line when the cell supernatant was compared to its respective culture media in basal conditions (without cells). The data showed that depending on the FBS concentration, there were changes in cellular response and consequent NE levels in the supernatant. For example, when HaCaT cells were cultured with 0.5% FBS they increased in about 6 times the NE levels after 48 hours of cultivation. The OSCC cells secreted higher NE levels in the supernatant compared to HaCaT cells (14.9 times for SCC9 cells and 15.1 times for SCC25 cells) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. As for the cells cultured with 10% FBS the percentage of increasing of NE levels in the supernatant was lower than when the cells were cultured in 0.5% FBS, although maintaining the greatest potential for NE secretion by the cancer cells (4.3 times for OSCC9 cells and 6.1 times for the OSCC25 cells) compared to keratinocytes (3.0 times) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. These data suggest that cancer cells may secrete greater amount of NE than non-neoplastic cells HaCaT.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNNK induces NE secretion in OSCC cells and normal keratinocytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter we have demonstrated that normal keratinocytes and OSCC cells are able to secrete NE, we investigated whether the NNK carcinogen could stimulate the hormone secretion. For this analysis, the cells were treated with NNK in different concentrations for 6 hours. After this time, the supernatant from each cell line was collected and tested for NE levels measurement. Our results indicated that NNK at 100 nM and 10 uM stimulated increased NE secretion by the HaCaT cells when compared to unstimulated cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with 10 uM concentration eliciting the most robust NE increase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. NNK at 100 nM and 10 uM induced significant NE secretion by the SCC9 and SCC25 cells, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. NNK at 100nM induced increased secretion of NE in OSCC9 cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Stimulation with 10uM NNK also increased NE secretion of in SCC9 cells, but this result did not reach statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNNK increases keratinocytes and OSCC cells proliferation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to evaluate the effects of NNK on keratinocytes and OSCC cells proliferation, the cells were treated with different concentrations of NNK. The results showed that NNK in highest concentration tested (10 \u0026micro;M) induced a significant increase of cellular proliferation in all cell lines tested \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. All NNK concentrations increased the proliferation rates of HaCaT cells in a dose-dependent manner, but only the 10 \u0026micro;M NNK induced a significant increase in cell proliferation relative to untreated cells with the carcinogen (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The results for the SCC9 cells demonstrated that 1 \u0026micro;M and 10 \u0026micro;M NNK increased in 69% and 142% cell proliferation rates compared to unstimulated cells, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The same results profile was found in the SCC25 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. NNK at different concentrations induced increased proliferation levels of SCC25 cells, buy only 10 \u0026micro;M NNK reached statistical significance increasing 26% the cell proliferation rate (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. NNK at 10 nM reduced SCC25 cell proliferation, but this effect was not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNNK-induced OSCC cells proliferation are dependent on β-adrenergic receptors.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate whether NNK effects on the HaCaT, SCC9 and SCC25 cells are dependent on the β1- and β2-adrenergic receptors and α4- and α7-AChRs, we carried out cell proliferation assays with NNK-treated cells in the absence or presence of antagonists for these receptors. For this purpose, the cells were stimulated with 10 \u0026micro;M NNK with or without the following inhibitors: α-bungarotoxin (inhibitor of nAChR-α7 receptor), dihydro-β-Erythroidine (inhibitor of nAChR-α4 receptor) and propranolol (inhibitor of β1- and β2-adrenergic receptors. The concentration of 10 \u0026micro;M NNK was selected as it has promoted the most intense stimulatory effects on the proliferation rates in the tested cells. The results showed that none of the three antagonists tested had significant effect on the HaCaT NNK-induced cell proliferation (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (data not shown). Nevertheless, propranolol (a nonspecific β1- and β2-adrenergic receptors inhibitor) completely blocked the effects caused by NNK on the SCC9 and SCC25 cells proliferation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Dihydro-β-erythroidine completely inhibited the NNK effect on the SCC9 cell proliferation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and partly the NNK-induced SCC25 cells proliferation, but in this cell line, the result did not reach statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Interestingly, α-bungarotoxin had no effect on the NNK-induced proliferation in any of the OSCC cell lines (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. These results suggest that NNK effect on the OSCC cells proliferation are dependent on β-adrenergic and nAChR-α4 receptors activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, our results reveled the ability of autocrine secretion of the neurotransmitter NE by oral cancer cells, which can play an autocrine catecholamine loop. Cancer cells showed increased secretion of NE compared to HaCaT cells, and the supernatant of HaCaT and OSCCs cell lines had significantly higher levels of NE in relation to the culture medium without cells. The higher NE levels secreted by OSCC cell lines compared to non-malignant cells may represent an adrenergic hyperactivation on cancer cells. Non-tumorigenic and tumorigenic human breast cell[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], pancreatic cancer and normal pancreatic ductal epithelial cells[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and immortalized human small airway epithelial cell line[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] are capable to produce norepinephrine with any stimulation. Nonetheless, this is the first time that this ability is described in OSCC cells.\u003c/p\u003e \u003cp\u003eThe NNK is a highly specific tobacco carcinogen nitrosamine, but the mechanisms involved in oral carcinogenesis and the proliferation of oral cancer cells induced by NNK are poorly known. Our results revealed that NNK in highest concentration induced a significant increase of cellular proliferation in keratinocytes (HaCaT) and OSCC cells (OSCC9 and OSCC25). NNK has the ability to induce proliferation in others cell lines, including colon cancer cell lines[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], oral cancer (SCC-15, HSC-13, OSC-19, and UM1)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], endometrial adenocarcinoma[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and neuroendocrine cells[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Non-neoplastic alveolar epithelial cells subjected to prolonged exposure to NNK cells led to acquire increasingly malignant properties such as increased proliferation and cell motility[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Likewise, the treatment of Ishikawa cells with NNK induces cell cycle progression and proliferation by up-regulating cyclin E and cyclin D expression, as well as by down-regulating p21 and p27[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNNK has a high affinity for β-adrenergic receptors because of its structural resemblance to classical beta-adrenergic agonists[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. When blocking β1- and β2-AR in esophageal cancer cells, there was a reverse in the blocking on oncogenic effects of NNK[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The activation of β-adrenergic receptors in response to NNK has been demonstrated in the growth of pancreatic carcinoma[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], colon cancer[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and esophageal squamous cell carcinoma[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] cell lines. In our study, we conduct cell proliferation assays in NNK-treated cells in the presence of inhibitor of β-adrenergic receptors types 1 and 2. Our results showed that propranolol blocked the effects caused by NNK on the proliferation of the SCC9 and SCC25 cells, but not in HaCaT cells. These results suggest that tobacco-related nitrosamines may influence OSCC progression via beta-adrenergic activation, a recognized stress-associated pathway. The β-adrenergic receptors are involved in the proliferation of various types of cancer as pancreas[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], colorectal[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], stomach[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and lung[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The effects of cell proliferation in these studies was blocked by propranolol.\u003c/p\u003e \u003cp\u003eIn our findings, NNK induces SCC-25 and HaCaT cells secreting NE. The same was not observed in SCC-9 cells indicating that this cell line may already have reached a maximum limit of neurotransmitter secretion under basal conditions without NNK stimulation. NE secretion from human small airway epithelial cells is regulated by nAChRs, and there is a greater increase in receptor responsiveness when stimulated with NNK[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Pancreatic cancer cells and normal pancreatic duct epithelia display an autocrine loop catecholamine that stimulates their proliferation by β-ARs and nAChRs-α3, -α5, and -α7[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Al-Wadei and colleagues[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] reported that the neurotransmitter γ-aminobutyric inhibitory neurotransmitter (GABA) and its synthesizing-enzyme glutamate decarboxylase 65 were suppressed in NNK-induced small airway-derived pulmonary adenocarcinoma cells and in pancreatic ductal adenocarcinoma. Despite the suppression of GABA, protein expression of the nAChR-α4 and \u0026ndash;α7 was upregulated[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study showed that the tested antagonists of nAChR-α4 and nAChR-α7 had no significant effects on the NNK-induced proliferation in HaCaT cells. However, the dihydro-β-Erythroidine inhibited NNK effect on SCC9 cells proliferation and, in part, its effect of NNK OSCC25 on the proliferation of cells. The α-bungarotoxin had no effect on cell proliferation induced by NNK in any cell line cancer. These results indicate that the NNK effect on the proliferation of OSCC cells can be dependent on nAChR-α4. Schuller and colleagues (2007) discuss about the human small cell lung cancer (SCLC) exposition to NNK increased expression of the nAChR-α7 and caused influx of Ca2+. This cascade induces the activation of PKC, Raf-1, ERK1/2, and c-myc, increasing of cell proliferation[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The nitrosamines in tobacco as NNK can promote the activation of the serine/threonine kinase Akt and contribute to carcinogenesis of the normal human airway through nAChRs-α3, -α4 and -α7, respectively[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, we demonstrated for the first time that OSCC cells secrete the stress-related NE and this event can be super regulated by tobacco carcinogen NNK. NNK also increases the proliferation of HaCaT and OSCC cells, mainly through β-adrenergic receptors, the same receptors activated by NE. These finding together suggest that OSCC development may be related to an imbalance of the NE secretion and activation of its receptors directly inducing changes related to the malignant phenotype. In conclusion, this study reveals that OSCC cells secrete the stress-related neurotransmitter norepinephrine and the tobacco carcinogen NNK induces increased cell proliferation through a stress-related cellular adrenergic pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest:\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval:\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent:\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported by the S\u0026atilde;o Paulo State Research Foundation (FAPESP). FAPESP grant 2016/25255-0 designated to Daniel G Bernab\u0026eacute; supported Psychosomatic Research Center's studies. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFl\u0026aacute;via Alves Verza: original draft, material preparation, data collection and analysis. Ana L\u0026iacute;via Santos-Sousa: assisted in material analysis. Sandra Helena Penha de Oliveira: edition and review the manuscript. Daniel Galera Bernab\u0026eacute;: conceptualization, resources, formal analysis, supervision, funding acquisition, project administration, writing-review and editing. The paper was critically reviewed and approved by all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003enot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eReitsma MB, Flor LS, Mullany EC, Gupta V, Hay SI, Gakidou E (2021) Spatial, temporal, and demographic patterns in prevalence of smoking tobacco use and initiation among young people in 204 countries and territories, 1990\u0026ndash;2019. Lancet Public Health [Internet]. Published by Elsevier Ltd. ;6:e472\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/S2468-2667(21)00102-X\u003c/span\u003e\u003cspan address=\"10.1016/S2468-2667(21)00102-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. Tobacco [Internet] (2022) Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/en/news-room/fact-sheets/detail/tobacco\u003c/span\u003e\u003cspan address=\"https://www.who.int/en/news-room/fact-sheets/detail/tobacco\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Acessed 21 jan 2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanaei G, Vander Hoorn S, Lopez AD, Murray CJL, Ezzati M (2005) Causes of cancer in the world: Comparative risk assessment of nine behavioural and environmental risk factors. Lancet 366:1784\u0026ndash;1793 \u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJethwa AR, Khariwala SS (2017) Tobacco-related carcinogenesis in head and neck cancer. Cancer Metastasis Rev [Internet] 36:411\u0026ndash;423\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerlay J, Laversanne M, Ervik M, Lam F, Colombet M, Mery L et al (2022) Global Cancer Observatory: Cancer Tomorrow [Internet]. Int. Agency Res. Cancer. Lyon, France; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gco.iarc.fr/tomorrow/en\u003c/span\u003e\u003cspan address=\"https://gco.iarc.fr/tomorrow/en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Acessed 21 jan 2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHecht SS (2003) Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer [Internet] 3:733\u0026ndash;744\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrando SA (2014) Connections of nicotine to cancer. Nat Rev Cancer Nat Publishing Group 14:419\u0026ndash;429\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZoli M, Pucci S, Vilella A, Gotti C (2018) Neuronal and Extraneuronal Nicotinic Acetylcholine Receptors. Curr Neuropharmacol 16:338\u0026ndash;349\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalantari-Dehaghi M, Bernard H-U, Grando SA (2012) Reciprocal effects of NNK and SLURP-1 on oncogene expression in target epithelial cells. Life Sci [Internet] 91:1122\u0026ndash;1125\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin C, Lin L, Chen T, Ye Y, Chiang B (2023) The expression of nicotinic acetylcholine receptor subunits and their associations with local immune cells and prognosis in oral squamous cell carcinoma. Cancer Med [Internet] 12:18918\u0026ndash;18930\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHildegard M, Schuller (2013) Regulatory Role of the α7nAChR in Cancer. Curr Drug Targets 13:680\u0026ndash;687\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamazaki H, Inui Y, Yun CH, Guengerich FP, Shimada T (1992) Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis 13:1789\u0026ndash;1794\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillips DH (2002) Smoking-related DNA and protein adducts in human tissues. Carcinogenesis 23:1979\u0026ndash;2004\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue J, Yang S, Seng S (2014) Mechanisms of cancer induction by tobacco-specific NNK and NNN. Cancers (Basel) 6:1138\u0026ndash;1156\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntoni MH, Lutgendorf SK, Cole SW, Dhabhar FS, Sephton SE, McDonald PG et al (2006) The influence of bio-behavioural factors on tumour biology: pathways and mechanisms. Nat Rev Cancer [Internet] 6:240\u0026ndash;248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eItoi K, Sugimoto N (2010) The brainstem noradrenergic systems in stress, anxiety and depression. J Neuroendocrinol 22:355\u0026ndash;361\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmaiz-Pena GN, Cole SW, Lutgendorf SK, Sood AK (2013) Neuroendocrine influences on cancer progression. Brain Behav Immun [Internet] 30:S19\u0026ndash;25\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLutgendorf SK, Andersen BL (2015) Biobehavioral approaches to cancer progression and survival: Mechanisms and interventions. Am Psychol [Internet]. Nature Publishing Group; ;70:186\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.bbi.2012.06.005\u003c/span\u003e\u003cspan address=\"10.1016/j.bbi.2012.06.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernab\u0026eacute; DG (2021) Catecholamines Mediate Psychologic Stress\u0026ndash;Induced Cancer Progression. Cancer Res 81:5144\u0026ndash;5146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThaker PH, Han LY, Kamat AA, Arevalo JM, Takahashi R, Lu C et al (2006) Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat Med 12:939\u0026ndash;944\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernab\u0026eacute; DG, Tamae AC, Biasoli \u0026Eacute;R, Oliveira SHP (2011) Stress hormones increase cell proliferation and regulates interleukin-6 secretion in human oral squamous cell carcinoma cells. Brain Behav Immun 25:574\u0026ndash;583\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang EV, Kim Sjae, Donovan EL, Chen M, Gross AC, Webster Marketon JI et al (2009) Norepinephrine upregulates VEGF, IL-8, and IL-6 expression in human melanoma tumor cell lines: Implications for stress-related enhancement of tumor progression. Brain Behav Immun [Internet]. Elsevier Inc.; ;23:267\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.bbi.2008.10.005\u003c/span\u003e\u003cspan address=\"10.1016/j.bbi.2008.10.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreno-Smith M, Lutgendorf SK, Sood AK (2010) Impact of stress on cancer metastasis. Futur Oncol [Internet]. ;6:1863\u0026ndash;81. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.futuremedicine.com/doi/\u003c/span\u003e\u003cspan address=\"https://www.futuremedicine.com/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2217/fon.10.142\u003c/span\u003e\u003cspan address=\"10.2217/fon.10.142\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBastos DB, Sarafim-Silva BAM, Sundefeld MLMM, Ribeiro AA, Brand\u0026atilde;o JDP, Biasoli \u0026Eacute;R et al (2018) Circulating catecholamines are associated with biobehavioral factors and anxiety symptoms in head and neck cancer patients. Multhoff G, editor. PLoS One [Internet]. ;13:e0202515. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dx.plos.org/10.1371/journal.pone.0202515\u003c/span\u003e\u003cspan address=\"https://dx.plos.10.1371/journal.pone.0202515\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie H, Li C, He Y, Griffin R, Ye Q, Li L (2015) Chronic stress promotes oral cancer growth and angiogenesis with increased circulating catecholamine and glucocorticoid levels in a mouse model, vol 51. Oral Oncol [Internet]. Elsevier Ltd, pp 991\u0026ndash;997\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTjioe KC, Cardoso DM, Oliveira SHP, Bernab\u0026eacute; DG (2022) Stress hormone norepinephrine incites resistance of oral cancer cells to chemotherapy. Endocr Relat Cancer [Internet] 29:201\u0026ndash;212\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuller HM, Tithof PK, Williams M, Plummer H (1999) The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone is a β-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via β-adrenergic receptor-mediated release of arachidonic acid. Cancer Res 59:4510\u0026ndash;4515\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Wadei HAN, Plummer HK, Ullah MF, Unger B, Brody JR, Schuller HM (2012) Social Stress Promotes and γ-Aminobutyric Acid Inhibits Tumor Growth in Mouse Models of Non\u0026ndash;Small Cell Lung Cancer. Cancer Prev Res [Internet] 5:189\u0026ndash;196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong HPS, Yu L, Lam EKY, Tai EKK, Wu WKK, Cho CH (2007) Nicotine promotes cell proliferation via α7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells. Toxicol Appl Pharmacol 221:261\u0026ndash;267\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Wadei MH, Al-Wadei HAN, Schuller HM (2012) Pancreatic Cancer Cells and Normal Pancreatic Duct Epithelial Cells Express an Autocrine Catecholamine Loop that Is Activated by Nicotinic Acetylcholine Receptors α3, α5, and α7. Mol Cancer Res [Internet] 10:239\u0026ndash;249\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Wadei HAN, Al-Wadei MH, Masi T, Schuller HM (2010) Chronic exposure to estrogen and the tobacco carcinogen NNK cooperatively modulates nicotinic receptors in small airway epithelial cells. Lung Cancer [Internet] Elsevier Irel Ltd 69:33\u0026ndash;39\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmaro F, Silva D, Reguengo H, Oliveira JC, Quintas C, Vale N et al (2020) Β-Adrenoceptor Activation in Breast Mcf-10a Cells Induces a Pattern of Catecholamine Production Similar To That of Tumorigenic Mcf-7 Cells. Int J Mol Sci 21:1\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiba T, Maeda T, Fujita Y, Takeda R, Kikuchi A, Kudo K et al (2019) The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells. Endocrinology 160:1\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi M, Liu D, Duan H, Qian L, Wang L, Niu L et al (2011) The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells. Breast Cancer Res Treat 125:351\u0026ndash;362\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu WKK, Wong HPS, Luo SW, Chan K, Huang FY, Hui MKC et al (2005) 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone from cigarette smoke stimulates colon cancer growth via β-adrenoceptors. Cancer Res 65:5272\u0026ndash;5277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo J, Zhou S, Huang P, Xu S, Zhang G, He H et al (2020) NNK-mediated upregulation of DEPDC1 stimulates the progression of oral squamous cell carcinoma by inhibiting CYP27B1 expression. Am J Cancer Res [Internet] 10:1745\u0026ndash;1760\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SM, Hwang KA, Choi DW, Choi KC (2018) The cigarette smoke components induced the cell proliferation and epithelial to mesenchymal transition via production of reactive oxygen species in endometrial adenocarcinoma cells. Food Chem Toxicol [Internet]. Elsevier; ;121:657\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fct.2018.09.023\u003c/span\u003e\u003cspan address=\"10.1016/j.fct.2018.09.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuller HM (1989) Cell type specific, receptor-mediated modulation of growth kinetics in human lung cancer cell lines by nicotine and tobacco-related nitrosamines. Biochem Pharmacol 38:3439\u0026ndash;3442\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMennecier G, Torres LN, Cogliati B, Sanches DS, Mori CM, Latorre AO et al (2014) Chronic exposure of lung alveolar epithelial type II cells to tobacco-specific carcinogen NNK results in malignant transformation: A new in vitro lung carcinogenesis model. Mol Carcinog 53:392\u0026ndash;402\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuller HM (2007) Neurotransmitter receptor-mediated signaling pathways as modulators of carcinogenesis. Prog Exp Tumor Res 39:45\u0026ndash;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGUO K, MA Q, WANG L, HU H, LI J, ZHANG D et al (2009) Norepinephrine-induced invasion by pancreatic cancer cells is inhibited by propranolol. Oncol Rep [Internet] 22:1265\u0026ndash;1270\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeddle DL, Tithoff P, Williams M, Schuller HM (2001) β-Adrenergic growth regulation of human cancer cell lines derived from pancreatic ductal carcinomas. Carcinogenesis 22:473\u0026ndash;479\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang N, Sun X, Sun M, Zhu S, Wang L, Ma D et al (2015) 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Promotes Esophageal Squamous Cell Carcinoma Growth Via Beta-Adrenoceptors in Vitro and in Vivo. PLoS ONE 10:1\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin VY, Jin HC, Ng EKO, Yu J, Leung WK, Cho CH et al (2008) Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and β-adrenergic receptor signaling pathways. Toxicol Appl Pharmacol [Internet] Elsevier Inc 233:254\u0026ndash;261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin VY, Wu WKK, Chu KM, Koo MWL, Wong HPS, Lam EKY et al (2007) Functional role of β-adrenergic receptors in the mitogenic action of nicotine on gastric cancer cells. Toxicol Sci 96:21\u0026ndash;29\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEgleton RD, Brown KC, Dasgupta P (2009) Angiogenic activity of nicotinic acetylcholine receptors: Implications in tobacco-related vascular diseases. Pharmacol Ther [Internet]. Elsevier Inc.; ;121:205\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.pharmthera.2008.10.007\u003c/span\u003e\u003cspan address=\"10.1016/j.pharmthera.2008.10.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Wadei HA, Schuller HM (2009) Nicotinic receptor-associated modulation of stimulatory and inhibitory neurotransmitters in NNK-induced adenocarcinoma of the lungs and pancreas. J Pathol [Internet]. ;218:437\u0026ndash;45. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://onlinelibrary.wiley.com/doi/\u003c/span\u003e\u003cspan address=\"https://onlinelibrary.wiley.com/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/path.2542\u003c/span\u003e\u003cspan address=\"10.1002/path.2542\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuller HM (2007) Nitrosamines as nicotinic receptor ligands. Life Sci [Internet]. Elsevier Inc.; ;80:2274\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.lfs.2007.03.006\u003c/span\u003e\u003cspan address=\"10.1016/j.lfs.2007.03.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oral squamous cell carcinoma, Norepinephrine, Beta-Adrenergic Receptor, Nicotinic Receptors, Stress","lastPublishedDoi":"10.21203/rs.3.rs-3934938/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3934938/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough there is a growing body of evidence showing the effects of stress-related catecholamines on oral cancer progression, to date there are no studies that have investigated whether oral squamous cells carcinoma (OSCC)-cells are capable of producing these hormones and whether this phenomenon is modulated by tobacco-related nitrosamines. In this study we investigated whether keratinocytes (HaCaT) and OSCC-derived cell lines (SSC9 and SCC25) are able to secrete the neurotransmitter norepinephrine (NE) as also the effects of the tobacco carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the NE secretion and OSCC proliferation. Supernatant from the HaCaT, SCC9, and SCC25 cells showed higher NE levels (6-, 14.9- and 15.1-fold more, respectively) compared to culture media without cells. When the cells were stimulated with NNK, a tobacco-specific carcinogen, there were increases in the levels of NE secretion by HaCaT and SCC25 cells, but not by SCC9 cells. NNK (10 \u0026micro;M) induced cell proliferation in the HaCaT, SCC9, and SCC25 cell lines and these effects were totally inhibited by blocking β-adrenergic receptors with propranolol. The NNK-induced OSCC cell proliferation was further dependent on nicotinic acetylcholine receptors α4 (nAChR-α4) activation (totally in SCC9 cells and partially in SCC25 cells), but not dependent on nAChR-α7 activation. Inhibition of the β-adrenergic receptors, nAChR-α4 and nAChR-α7 did not block NNK-induced HaCaT proliferation. Our findings suggest that oral cancer cells secrete the neurotransmitter norepinephrine and the tobacco nitrosamine NNK promotes increased cell proliferation through a stress-related cellular adrenergic pathway.\u003c/p\u003e","manuscriptTitle":"Oral cancer cells secrete stress neurotransmitter and proliferate in response to tobacco carcinogen NNK through a cellular adrenergic pathway.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-08 13:17:51","doi":"10.21203/rs.3.rs-3934938/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8aeb051f-2c00-4185-8914-02530591eff9","owner":[],"postedDate":"February 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-03T22:36:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-08 13:17:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3934938","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3934938","identity":"rs-3934938","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}