The influence dose of nicotine exposure on H520 smoking-related Non-Small-Cell Lung carcinoma cell growth and toxicity

Background and Objectives: Nicotine exposure may affect NSCLC is associated with lung cancer in humans. Whether nicotine exhibits carcinogenesis promoted activities in tumor growth still unknown. Nicotine is known to have dichotomous effects on cancer biology, acting like a proor antiPreprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 June 2020 doi:10.20944/preprints202006.0033.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. carcinogenesis agent. There are different functions between adenocarcinoma and squamous NSCLC cancer cells. Excess generation of nicotine may inhibit mitochondrial metabolism, protein modification, and DNA cleavage. Materials and Methods: We used the H520 NSCLC line obtained from human lung epithelial cells to detected nicotine growth and toxicity using MTT assay and western blotting. The concentration of nicotine stimulated cell growth to correspond to low concentration, while high concentration was cytotoxic. Results: According to MTT assay results, at 1.0 μM nicotine has significantly enhanced the H520 cell viability (%). Nicotine induced lung cancer carcinogenesis through mechanisms of α7nAchR, EGFR, HDAC2/4/5, Cyclin D/Cyclin E, Bcl-2, p-Akt, and inflammatory proteins of NF-KappaB and COX2 increases at 1.0 μM. Apoptosis proteins were decreases at 1.0 μM nicotine by p21, p27, c-jun, and p38α using western blotting. Nicotine stimulates tumor growth is mediated through α7nicotinic-acetylcholine receptors (α7nAChR), possibly involving inflammation. On the other hand, at high nicotine concentrations (> 1.0 μM) with consistent cytotoxic effects and appeared to be due to direct cell kill. Nicotine can prevent apoptosis induced by NSCLC. Conclusion: Therefore, the effects on chemotherapeutics by NSCLC malignant cell lines, nicotine in concentrations as low as 1.0 μM decreased. These mechanisms are responsible for the genotoxic effects caused by nicotine. This leads to downstream effects on decreased apoptosis, increased cell proliferation and transformation. The malignant NSCLC cells respond to the treatment with nicotine in lung cancer, the nicotine-mediated induction of growth may provide one of its links to α7nAchR or EGFR.


Introduction
Cigarette smoking has been considered one of risk factors for lung cancer cells.
Nicotine constitutes the dry weight of cigarette. Nicotine exposure is an important component in cigarette but is a non-carcinogen [1]. Nicotine exerts its biologic function mainly through nicotinic acetylcholine receptors (nAChR) and/or EGF receptors (EGFR) in the lung cancer cells. Among various subtypes of nicotinic receptors, α7nAChR, can bind nicotine with highest affinity and mediate multiple effects of nicotine in lung cancer. α7nAChR and EGFR expressed on lung carcinoma form a part of a proliferative network facilitating the growth of NSCLC cells [2]. Nicotine could induce the proliferation of a variety of lung carcinoma cell clines, but there is no evidence that nicotine itself provokes cancer. Nicotine alone is generally accepted as a tumor promoter, but not a tumor initiator in carcinogenesis [3]. Effects of nicotine on cancer growth have been demonstrated on NSCLC cells. Nicotine promoted NSCLC lung cancer in all patients, the role of nicotine underlying mechanisms through α7nAChR nicotine receptor signaling in lung cancer [4]. Nicotine increases migration and invasion of lung cancer cells through activation of the α7nAChR.
Nicotine binds to α7nicotinic-acetylcholine receptors (α7nAchR) and EGF receptor, leading to activation of the HDAC2/4/5 and cell cycle signaling pathway [5]. Nicotine directly regulated COX2 expression in a α7nAchR dependent manner. Its activation resulted in regression of tumor cell growth and inactivation of cellular apoptosis via cell cycle arrest phase in lung cancer cells [6,7]. Cellular cytotoxicity was associated with inhibition of DNA synthesis, not stimulation of DNA synthesis [8]. The role of the NF-kappa B activation in protecting the cells is involved in cell survival, proliferation, and apoptosis by activating anti-apoptotic mechanisms [9]. Nitric oxide (NO) from L-arginine by NO synthase (NOS2) contributed in inflammatory response [10]. The activities of c-jun and p38 MAP kinases show to be involved in apoptosis [11]. The specific development of cancer growth roles of individual HDACs is closely linked to regulation of cell proliferation, apoptosis, and cell cycle. Therefore, nicotine alone is a generally accepted as a tumor promoter, but not a tumor initiator in carcinogenesis.

Materials and Methods
Cell culture and nicotine treatment H520 NSCLC lung cancer lines were obtained from human lung epithelial cells.
Lung cancer cells grown in RPMI-1640 medium with 10% fetal bovine serum containing 100 units/mL penicillin, and 100 μg/mL streptomycin, in a 5% CO2 atmosphere at 37 °C. Modified to contain Earle's Balanced Salt Solution, nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate. This reduced level of sodium bicarbonate (NaHCO3, 1.5 g/L) is intended for use in 5% CO2 in air. Additional sodium bicarbonate may be required for use in incubators containing higher percentages of CO2.  Permeabilised with 0.1% Triton X-100 for 20 min, and blocked with 10% BSA in PBS for 30 min. Followed processing lung cancer cells incubated with HDAC2 primary antibody at 4°C overnight and FITC-conjugated secondary antibody (1:500, Invitrogen), and nuclei were labeled with DAPI. Extensive washing with PBS will be performed between each step and examined by confocal microscope (OLYMPUS FV500/BX Laser Scanning Confocal Microscope).

Statistical analysis
All data are presented as the mean ± SEM. Three independent repeats were conducted in MTT assay. Error bars represent these repeats. Statistical analyses were performed using the Student t test. P < 0.05, P < 0.01 was accepted as statistically significant. All statistical analyses were performed with the GraphPad Prism 6 software package.  Figure 3B showed HDAC2 at 1.0 μM nicotine has higher than other dose. This express was HDAC2 signaling related nicotine in the cytosol and nuclear. Result showed α7 nicotinic-acetylcholine receptor (α7nAChR) was with nicotine increasing doses.

During
Although nicotine has been implicated as a potential factor in the pathogenesis of human NSCLC lung cancer, because nicotine contribute to carcinogenesis by activating α7nAChR or EGFR on lung tumors and epithelial cells, its mechanism of action in the development of lung cancer remains largely unknown, we detected whether differential expression of α7nAChR or EGFR expression in H520 NSCLC cells after nicotine treatment could explain the different association with smoking. We detected EGFR and HDAC2/4/5 expression in H520 NSCLC cells, EGFR was pivotal in inducing tumor and 4B), but suppression genes, p21 and p27, have significant difference decreases at 1.0 μM nicotine concentration (p<0.01 and p<0.05, respectively) ( Figure 4C). As dose increased above 1.0 μM concentration, p21 and p27 protein were increases by nicotine induced cell apoptosis. The same results also observed in pro-inflammation of c-jun and p38α decreased at 1.0 μM nicotine concentration in H520 squamous cancer cell ( Figure 4D). Higher than 1.0 ~ 3.0 μM concentration, p21, p27, c-jun and p38α were induced H520 NSCLC cancer cell apoptosis (p<0.01). Higher nicotine concentration will induce cell cytotoxicity. CYP 2A6 and CYP 2A13 have higher expression at above 1.0 μM nicotine concentration in H520 squamous cancer cell ( Figure 5B).
The production of NO was assessed by NOS2. As low as the concentration of 0.1 μM nicotine, it increased the pro-inflammatory cytokines, NO production, NOS2 (p<0.01) ( Figure 5A) and CYP1B1 (p<0.01) ( Figure 5B). In contrast, above 0.1 μM nicotine concentration, NOS2 and CYP1B1 have significant difference decreases (p<0.01). That may suggest nicotine induced cytotoxicity increases lead to cell apoptosis, not cancer therapy effects.

Discussion
Develop cigarette smoking strategies for the interplay lung cancer with nicotine as well as potentially cell growth in NSCLC. The underlying mechanisms responsible of the nicotine dose-experimental works has described for the carcinogenic potential of HDACs content in apoptosis and cell cycle regulation of H520 NSCLC cells [12]. Low concentrations of nicotine are reported to promote inflammation led to tumor growth, whereas high nicotine influx acts as a potent tumor repressor, leading to cytotoxicity and apoptosis [13,14]. Like anything that enters the body, nicotine can be metabolized in lung and kidney via several enzymes such as cytochrome P450 2A6 (CYP2A6), cytochrome P450 2A13 (CYP2A13), cytochrome P450 1B1 (CYP1B1) [15]. The nicotine concentration in blood plasma of smokers varies. The average human plasma concentration of nicotine in heavy smokers is 0.6 μM. The half-life of nicotine in human plasma is around 2 h [16,17]. The concentration of nicotine stimulated cell growth to correspond to low concentration was needed [18,19]. Exposure

Conclutions
Standardizing cigarette smoking levels is also likely to be important. Nicotine increases binding the receptors in a dose-and duration-dependent manner.
Nicotine is unusual in comparison to most drugs, as its concentration profile changes from induced tumor growth from inflammation to cytotoxic with doses.
There is increasing interest in developing nicotine releasing materials as potent tumoricidal agents in which high and localized concentrations of nicotine may be directly released in a sustained manner to the tumor site. Therefore, a suggested activity that increases your metabolic rate can help speed up the clearance of nicotine.       CYP2A6, CYP2A13 and CYP1B1 analysis. All data was expressed mean ± SEM, * p<0.05, ** p<0.01 significant difference compared with control without treatment nicotine.