Cisplatin‐resistance induces lung squamous carcinoma cell growth by nicotine‐mediated α7nAchR/HDAC1/Cyclin D1/pRb cell cycle activation

The majority of adenocarcinoma lung cancer is found in nonsmokers. A history of tobacco use is more common in squamous cell carcinoma of the lung. The aim of this study is to identify the cisplatin (CDDP)‐resistance that promotes lung squamous carcinoma cell growth through nicotine‐mediated HDAC1/7nAchR/E2F/pRb cell cycle activation. Squamous cell carcinoma (NCI‐H520 and NCI‐H157) cells were examined after cisplatin and nicotine treatment by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide assay, cell migration assay, immunofluorescence staining, western blot analysis, and immunoprecipitation analysis. Consequently, CDDP is released from DNA and Rb phosphorylated pRb as a result of nicotine‐induced cancer cell proliferation through 7nAchR, which then triggers the opening of the HDAC1 cell cycle. The cell cycle is stopped when CDDP adducts are present. Nicotine exerts cancer cytoprotective effects by allowing HDAC1 repair mechanisms to re‐establish E2F promoting DNA stimulation cell cycle integrity in the cytosol and preventing potential CDDP and HDAC1 suppressed in the nuclear. Concentration expression of nicotine causes squamous carcinoma cell carcinogens to emerge from inflammation. COX2, NF‐KB, and NOS2 increase as a result of nicotine‐induced squamous carcinoma cell inflammation. Nicotine enhanced the cell growth‐related proteins such as α7nAchR, EGFR, HDAC1, Cyclin D, Cyclin E, E2F, Rb, and pRb by western blot analysis. It also induced cancer cell inflammation and growth. As a result, we suggest that nicotine will increase the therapeutic resistance effects of CDDP. This has the potential to interact with nicotine through α7nAchR receptors and HDAC1/Cyclin D/E2F/pRb potentially resulting in CDDP therapy resistance, as well as cell cycle‐induced cancer cell growth.

Lung cancer is a nicotine-dose response disease that develops when environmental exposures, such as cigarette smoke cause lung cancer cell growth. 1,2Lung cancer, with distinct subtypes like adenocarcinoma and squamous cell carcinoma, poses unique challenges in comprehending its molecular intricacies.This study delves into the intricate relationship between nicotine exposure, cisplatin resistance, and cell growth in lung squamous carcinoma, with a specific focus on nicotine-mediated histone deacetylase 1 (HDAC1) activation.However, nicotine exhibits cocarcinogenic properties and promotes the growth of tumors and malignant transformation was doses lower than 1 µM of nicotine. 2Adenocarcinoma is commonly found in nonsmokers, while squamous cell carcinoma is frequently associated with tobacco use.The majority of adenocarcinoma lung cancer is found in nonsmokers occur.Lung cancer, characterized by diverse subtypes and various etiological factors, poses a significant health challenge.Nicotine associated with cancer in humans is according to squamous carcinoma cells. 3Nicotine stimulates cancer cell growth by binding to α7 nicotinic-acetylcholine receptors (α7nAchR) or endothelial production of endothelial growth factor receptors (EGFR), resulting in the activation of HDAC1, an enzyme that leads to histone chemical modification after translation is still unclear.CDDP is a form of platinum therapy used to treat NSCLC. 4,5CDDP is frequently used after NSCLC surgery, however, it has the disadvantage of causing renal toxicity. 6,7CDDP through these mechanisms leads to the development of chemoresistance resulting in therapeutic failure to mediate nicotine. 8Nicotine induces a cell cycle to provide associated HDAC1 because of lung malignancies that are CDDP-resistant. 9nce, CDDP has several anticancer mechanisms.In human cancer cells, HDACs may have additional independent roles. 10,11Nicotine also stimulates angiogenesis and tumor growth, which is also mediated by α7nAChR, possibly involving EGFR.Stimulating the receptors in the cancer cells leads to downstream activation of several signaling cascades that promote cancer cell survival, migration, and metastasis in a tumor-specific manner.The aim is to identify the CDDP resistance that induces lung squamous carcinoma cell growth by nicotine-mediated α7nAchR or EGFR to regular HDAC1induced E2F/Rb cell cycle activation.

| Cell culture
Cell culture with nicotine treatment NCI-H520 and NCI-H157 cells by transfection HDAC1 +/+ , transfection small interfering RNA (siRNA) HDAC1 plasmid, valproic acid (VPA), and CDDP.HDAC1 plasmid (HDAC1 +/+ , 3 µg/mL) transfected to cells by Lipofectamine™ 2000 (Sigma) was used to treat human lung squamous carcinoma cells (NCI-H520 and NCI-H157).Cells were treated with 1 mM VPA, an inhibitor of HDACs to inhibit the expression of HDAC1 in a 10 cm dish with 10 mL media.Cisplatin, a chemotherapeutic drug (CDDP, 50 μM) was used to treat lung cancer cell lines with nicotine for 24 h to establish cisplatin-resistance cell lines.After transfection, siRNA lentivirus plasmid-A was found.Lung cancer cells were transiently transfected with an HDAC1 siRNA plasmid (1 µg/mL).HDAC1 small interfering RNAs (HDAC1 siRNAs): Lipofectamine 2000 reagent (Invitrogen) was used to transfect 5′-CCCAUAACUUGCUGUUAAA-3′ which was then extracted for further analysis after 24 h.NCI-H520 and NCI-H157 lung squamous carcinoma cells were exposed to 1.0 µM nicotine for 24 h with or without treatment as determined by western blot and immunoprecipitation analyses.

| Statistical analysis
All statistical data analyses were performed in Excel and GraphPad Prism 6.All quantitative results were presented as the mean ± SEM.
The statistical significance of the difference between means was determined using a two-way analysis of variance (ANOVA) followed by post hoc analysis using Student t-tests as comparison tests.*p < .05,**p < .01 were statistically significant difference compared to control.# p < .05,## p < .01 were considered statistically significant when compared with HDAC1 plasmid (HDAC1 +/+ ) transfected.

| The effects of nicotine dose on NCI-H520 carcinoma cell growth or cytotoxicity
In this study, we examined nicotine concentrations ranging from 0.1 to 1.0 µM, which is similar to nicotine concentrations that would be achieved in cell culture studies after nicotine exposure.Figure 1 shows that nicotine concentration (<1.0 µM) promotes cell growth and proliferation, while a high concentration (>1.0 µM) with consistent cytotoxic effects appears to induce cancer cell death.Epidemiological clinical data implicate that the patients with Kaplan-Meier Survival Analysis descriptive procedure for continued smoking and never smoking causes progression of time-to-event variables (Supporting Information S1: Figure 1S1).According to Figure 1A, nicotine has the highest cancer growth at 24 h induced by cell viability (%) at 0.5 μM (p < .01)and 1.0 μM (p < .05)by MTT assay.
Toxic effects were discovered at nicotine concentrations greater than 1.0 μM after 24 h, especially prolonged high-dose exposure to nicotine.We discovered nicotine-induced lung cancer growth at 1.0 M.However, the reduction was statistically different above 1.0 M nicotine (p = .01). Figure 1B shows that the cytotoxicity effects of nicotine occurring at final concentrations of 1.5, 2.5, 5.0, and 10.0 μM disrupts the essential balance of cancer cell growth.In this study, we want to know the effects of nicotine resistance on CDDPinduced apoptosis in human NSCLC carcinoma cells (Supporting Information S1: Figure 1S2).Additionally, we used immunofluorescence staining to identify HDAC1 protein expression levels involved in the nucleus when the H520 cell was exposed to nicotine concentrations of 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 µM, respectively.Findings from immunofluorescence staining showed that HDAC1 at 1.0 μM nicotine has higher protein expression than other dosages (Figure 1C).There was nicotine-related HDAC1 expression in the nucleus.Cytochrome c P450 2A6 (CYP2A6) and cytochrome c P450 2A13 (CYP2A13) protein expression levels increased by nicotine concentrations at 2.5 µM (p < .05)and 3.0 µM (p < .01),while cytochrome c P450 1B1 (CYP1B1) decreased by H520 NSCLC cell toxicity by western blot analysis at 1.0, 1.5, 2.0, 2.5 and 3.0 µM final nicotine concentration, respectively (Figure 1D).While lower concentrations of nicotine at 1.0 µM are reported to enhance tumor growth, higher concentrations of nicotine influx at 1.0 µM act as potent cytotoxicity.Several enzymes such as CYP2A6, CYP2A13, and CYP1B1 can metabolize nicotine to cause cytotoxicity in the lung.Furthermore, the results showed that nicotine concentration induced α7nAChR or EGFR receptor signaling in NSCLC, which accelerated the growth of lung cancer growth and the associated nicotine underlying mechanisms.According to the result, we showed that α7nAChR was activated as the nicotine dose increased (p < .05)(Figure 2A).All of the dose-dependent effects of nicotine treatments were postulated to be mediated by α7nAChR.However, EGFR protein levels increased significantly differently with the addition of

| Nicotine may be recommended as a chemotherapy drug, CDDP, resistance in NCI-H520 squamous cancer cells
In this study, the NCI-H520 group of squamous carcinoma cells may have developed E2F-targeting CDDP as a result of detecting HDAC1 activity.Results presented in Figure 3 showed that CDDP and VPA treatment without nicotine resulted in higher levels of HDAC1 +/+ protein expression in the transfected plasmid than in the control (Figure 3A).The carcinogens possess the ability to block apoptosis, an important mechanism in the development of tumors and resistance to chemotherapy (Supporting Information S1: Figure 3S1).According to some reports, cell cycle-related proteins may be used to regulate cancer cell growth.Cell cycle-related proteins, E2F, Rb, and pRb, were detected using western blot analysis.E2F, Rb, and pRb, and protein expression levels increased by HDAC1 +/+ transfection.However, Figure 3B showed that E2F, Rb, and pRb were suppressed by VPA, CDDP, and VPA + CDDP, while Figure 3C showed that HDAC1 was located in the nucleus and not in the cytosol.On the other hand, cyclin-dependent kinase inhibitors (p53, p21, and p27) have been associated with HDAC1 regulation.Figure 3D,E,H showed that in NCI-H520, compared with HDAC1 +/+ transfection, protein expression levels of p21, p27, and p53 have increased by VPA, CDDP, and VPA + CDDP (*p < .05)(Figure 3D,E,H).The ATM protein may be suppressed by HDAC1 as a result of cell growth inhibition.ATM protein was observed to increase by VPA, but not CDDP, or combining VPA and CDDP (Figure 3F).BAX protein expression level was increased by VPA, CDDP, and VPA + CDDP. Figure 3G showed that VPA, CDDP, and the combination of VPA and CDDP increased the protein expression level of BAX (p < .05).However, significant differences were observed when compared with HDAC1 +/+ transfection, protein expression levels of p21, p27, p53, ATM, and BAX (p < .05).The expression of HDAC1 is significantly higher in NSCLC than in lung cancer cells.Increases in HDAC1 and NF-κB were observed after HDAC1 +/+ transfection using immunofluorescence staining (Figure 3I,J).Furthermore, we want to identify the nicotine and cell cycle protein interaction expression in the cytosol and nucleus (Figure 4).Western blot analysis increased cell cycle staining and predicted an improved squamous cancer cell group.Nicotine may have tumor-promoting or cocarcinogenic activities.Following the effects of the nicotine dose treatment, we examined the squamous cancer cells.Our findings suggested that nicotine dosage effects from cigarette smoke extract may cause cancer cell growth and cytotoxicity in NCI-H157 cells.When HDAC1 moved from the nucleus to the cytosol, CDDP-binding DNA was freed.Notably, nicotine may have influenced chemotherapeutic drug resistance processes to accelerate lung cancer growth resulting in CDDP's loss of lung cancer cell-killing abilities.
3.3 | Nicotine exposure induced cancer cell growth activity to resistance on CDDP in NCI-H520 and NCI-H157 squamous cell carcinomas Using western blot analysis, NCI-H520 cancer cells will be newly formed by the following indicated cell-cycle arrest, apoptosis induction, and activation of cancer-suppressor genes.VPA is an inhibitor of HDACs.HDAC1 inhibited E2F transcription resulting in GDDP arrest G2/M cell cycle.After transfecting NCI-H520 cells with the HDAC1 +/+ plasmid, VPA, CDDP, and CDDP combining VPA at a 24 h nicotine treatment, we conducted western blot analysis to identify the cells.Results showed that transfected HDAC1 +/+ plasmid has significantly increased cyclin D/Rb/cyclin E/E2F cell cycle protein expression levels at 24 h nicotine treatment (Figure 4).We discovered that nicotine stimulated NCI-H520 cell growth and CDDP-resistance from G2 to M phase and that VPA, CDDP, and VPA + CDDP increased Cyclin D/Rb/Cyclin E/E2F protein expression levels.It should be mentioned that nicotine-induced CDDPresistance at 24 h treatment in NCI-H520 cell, Cyclin D/Rb/Cyclin E/E2F has a significantly different increase by VPA, CDDP, and VPA + CDDP.To examine the effects of the HDACs-related on nicotine by HDAC1 +/+ plasmid, VPA, CDDP, VPA + CDDP, and HDAC1 +/+ + VPA + CDDP in human lung NCI-H520 cell.The result showed that nicotine-induced not only HDAC1/HDAC2 protein expression levels increase by CDDP, but also increased HDAC3 (Figure 4E).In contrast, CDDP induced cell resistance in the G2/M phase, thus, we observed that Cyclin A/Cyclin B was increased by VPA, CDDP, and VPA + CDDP (Figure 4F,G).The protein expression levels of NF-KB increased by CDDP, VPA + CDDP, and HDAC1 +/+ + VPA + CDDP (Figure 4H).Besides, we observed that developing treatment strategies for the interplay of p27 and p21 with nicotine F I G U R E 3 Cisplatin (CDDP) induces a cell cycle arrest which is the molecular mechanism of histone deacetylase 1 (HDAC1) in the NCI-H520 cells squamous lung cancer cells cytosol.(A) Protein expression levels of HDAC1 relative to HDAC2 using western blot analysisby HDAC1 +/+ transfection, VPA, CDDP, HDAC1 +/+ + CDDP, VPA + CDDP, and HDAC1 +/+ + VPA + CDDP.(B) Protein expression levels of E2F, Rb, and pRb using western blot analysisby HDAC1 +/+ transfection, VPA, CDDP, HDAC1 +/+ + CDDP, VPA + CDDP, and HDAC1 +/+ + VPA + CDDP.(C) HDAC1 +/+ transfection location is in the nucleus but not in the cytosol.(D) Western blot analysisof p21 protein expression level by HDAC1 +/+ transfection, VPA, CDDP, and VPA + CDDP.Statistical analysis of protein expression levels of p21 in H520 cells.Data were expressed as mean ± scanning electron microscope (SEM), *p < .05,was significantly different compared with the control.# p < .05,was significantly different compared with HDAC1 +/+ transfection.(E) Western blot analysisof p27 protein expression level by HDAC1 +/+ transfection, VPA, CDDP, and VPA + CDDP.Statistical analysis of protein expression levels of p27 in H520 cells.All data were expressed as mean ± SEM. *p < .05 was significantly different compared with control.# p < .05 was significantly different compared with HDAC1 +/+ transfection.(F) Western blot analysisof ATM protein expression level by HDAC1 +/+ transfection, VPA, CDDP, and VPA + CDDP.Statistical analysis of protein expression levels of ATM in NCI-H520 cells.All data were expressed as mean ± SEM. *p < .05 was significantly different compared with control.# p < .05 was significantly different compared with HDAC1 +/+ transfection.(G) Western blot analysisof BAX protein expression level by HDAC1 +/+ transfection, VPA, CDDP, and VPA + CDDP.Statistical analysis of protein expression levels of BAX in NCI-H520 cells.Data were expressed as mean ± SEM. *p < .05 was significantly different compared with control.# p < .05 was significantly different compared with HDAC1 +/+ transfection.(H) Western blot analysisof p53 protein expression level by HDAC1 +/+ transfection, VPA, CDDP, and VPA + CDDP.Statistical analysis of protein expression levels, p53, in H520 cells.Data were expressed as mean ± SEM. *p < .05 was significantly different compared with control.# p < .05 was significantly different compared with HDAC1 +/+ transfection.(I) Immunofluorescent staining of HDAC1 and NF-κB in NCI-H157 squamous lung cancer cells by HDAC1 +/+ transfection and CDDP in the cytosol and nucleus.HDAC1 +/+ transfection, CDDP, Cisplatin; VPA, valproic acid.We detected cell cycle and related proteins by western blot analysiswhich mediates HDAC1 plasmid (HDAC1 +/+ ) transfection, VPA, CDDP, and VPA + CDDP into NCI-H520 cells.potentially resulted in therapy resistance.We found that p27 and p21 are still suppressed by VPA, CDDP, VPA + CDDP, and HDAC1 +/+ + VPA + CDDP (Figure 4I).The research further elucidates the underlying mechanisms for nicotine resistance CDDP in squamous cell carcinoma.A high nicotine concentration (>1.0 μM) appeared to have consistent cytotoxic effects in inducing cell death.Low concentrations of nicotine that promote cells correspond to cancer cell growth in NCI-H157 squamous cell carcinomas (Figure 5A, Supporting Information S1: Figures 5S1 and 5S2).The maximum cancer growth enhanced by MTT assay was seen at 1.0 M nicotine doses after 72 h.We observed that CDDP, VPA, and CDDP + VPA have a CDDP effect that suppresses cancer cell growth, whereas nicotine effects can allow cancer cell growth (Figure 5B).In NCI-H157 squamous carcinoma cells, we observed a CDDP-resistance effect in NCI-H157 squamous carcinoma cells at 24 and 48 h; nicotine did not significantly increase cell viability.Figure 5C showed that the nicotine contribution at 24 and 48 h did not increase the cancer cell survival rate limited in those with NCI-H157 squamous cell lung cancer.Additionally, we detected NCI-HDAC1 protein expression levels with nicotine and CDDP using immunofluorescent staining.Results showed that HDAC1 fluorescent positive cells increased in NCI-H157 cells by nicotine, but not CDDP (Figure 5C).
At the same time, we discovered that NCI-H157 cell migration at 72 h has higher distance than control group (Figure 5D).For the carcinogenic interaction of nicotine and HDAC1 in a cell cycle regulation, the fundamental mechanisms responsible for both nicotine, as well as possible, cell growth in NCI-H157 NSCLC, have been elucidated.The results showed that HDAC1 may possibly be regulated by α7nAChR receptors, but not from EGFR with nicotine.by western blot analysis (Figure 5E).By transfecting HDAC1 siRNA, CDDP, and HDAC1 siRNA + CDDP into NCI-H157 cells, it is possible to significantly reduce α7nAChR, EGFR, and HDAC1 in the absence of nicotine.On the other hand, we used western blot analysis to identify the levels of protein expression that control the cell cycle in relation to HDAC1.Results showed that in comparison to controls, HDAC1 siRNA, CDDP, and combining HDAC1 siRNA and CDDP raised the protein expression levels of pRb, E2F, Cyclin D, and Cyclin E with nicotine by western blot analysis.At the same time, we observed that Cyclin A, Cyclin B, and p27 decreased after exposure of nicotine to HDAC1 siRNA, CDDP, and HDAC1 siRNA + CDDP (Figure 5F).As a result of nicotine treatment, CDDP was freed from DNA, and HDAC1 and pRb were able to restore E2F DNA transcription.Hence, we can find CDDP ineffectiveness on NCI-H157 cancer cells and HDAC1-induced cell cycle activation.Cell cycle suppressor proteins, p53, p27, and p21 accumulate in the cytoplasm to trigger apoptosis.Consequently, nicotine increases CDDP resistance by inducing p53, p27, and p21 (Figure 5F).HDAC1 siRNA, CDDP, and HDAC1 siRNA + CDDP suppressed Rb, E2F, Cyclin D, Cyclin E, Cyclin A, and Cyclin B in the absence of nicotine administration, while HDAC1 siRNA, CDDP, and HDAC1 siRNA + CDDP, on the other hand, increased the levels of the suppression genes p21, p53, and p27.Hence, we suggest that nicotine resistance CDDP was inhibited and cancer cell growth function was arrested by the cell cycle.However, EGFR, HDAC1, Rb, E2F, Cyclin D, and Cyclin E protein expression levels with nicotine increased by HDAC1 siRNA, CDDP, and combining HDAC1 siRNA and CDDP, while Cyclin A, Cyclin B, p53, p21, and p27 protein expression levels with nicotine decreased by HDAC1 siRNA, CDDP, and combining HDAC1 siRNA and CDDP in squamous cancer H157 cells.

| Nicotine interaction with CDDP-induced NCI-H157 lung cancer cell growth in nucleus localization and membrane localization
Nicotine can stimulate tumor growth which is also mediated through EGFR, possibly actively involving HDAC1.As a result, we identified the expression of the CDDP and HDAC1 protein interaction in the nucleus by nicotine resistance.In the NCI-H520 squamous carcinoma cells, we observed and confirmed the presence of nicotine resistance chemotherapy and the ability of CDDP to trigger cell-cycle arrest and apoptosis in lung cancer cells with nicotine resistance.After nicotine exposure in the NCI-H157 cells, HDAC1 leaves the nucleus, while CDDP is released from the nucleus.Therefore, we discovered that HDAC1 protein expression level increased in the cytosol and was suppressed by CDDP in the nucleus.E2F protein expression level increased by HDAC1 +/+ transfection and decreased by CDDP and HDAC1 +/+ + CDDP in the nucleus.Thus, Figure 6A showed pRb has decreased by CDDP protein expression in the cytosol and resistance CDDP adducts in the nucleus.On the other hand, CDDP-induced NF-ΚκB expression level is increased by nicotine (Figure 6B).When compared to the control, γ-H2AX increased by CDDP expression in the nucleus and cytosol (Figure 6B).However, nicotine binds through cell membrane receptors, α7nAchR or EGFR, resulting in HDAC1 activation.Results showed that nicotine-induced α7nAchR in the nucleus decreased expression level by CDDP resistance, but EGFR activation in cells cytosol by HDAC1 +/+ (Figure 6C).While HDAC1 +/+ transfection by nicotine could identify elevated EGFR protein expression in the cytosol and nucleus, nicotine could stimulate HDAC1 expression.After nicotine treatment, we found that the super-shift mobility HDAC1 expression by HDAC1 +/+ using immunoprecipitation analysis (Figure 6D).We might conclude that Rb coupled with HDAC1 each other led to E2F-induced cell growth by nicotine.At the same time, we found nicotine-boosted protein expression levels in α7nAchR and EGFR, which are situated at the cell membrane (Figure 6E).

| DISCUSSION
The data given in this study go beyond this paradigm to show that exposure to nicotine-induced squamous cell carcinoma develops CDDP resistance and the relationship between cigarette smoke and HDAC1 that interacted with nicotine.Adenocarcinoma is often prevalent in nonsmokers, while squamous cell carcinoma is primarily associated with tobacco use.We identified the interaction between the carcinogens in cigarettes that may have contributed to the treatment of CDDP resistance, particularly in squamous cell carcinoma.This research delves into the nuanced connections between nicotine exposure, cisplatin resistance, and cell growth in lung squamous carcinoma.The unraveling of these intricate molecular interactions may pave the way for the development of personalized and targeted treatment modalities for squamous cell carcinoma.This study discovered nicotine-induced chemotherapeutic resistance in squamous cancer NCI-H520 cells and NCI-H157.4] There are several mechanisms that interact with both nicotine and CDDP when exposed to cancer cells.CDDP's ability to crosslink with the purine bases on DNA has been connected to cell cycle arrest in the G2/M phase-induced cancer cell apoptosis.7][18][19] Nicotine stimulates the migration, growth of NSCLC cancer cell lines. 20A low concentration (1.0 µM) of nicotine has been reported to cause CDDP resistance (Figures 1 and 2).HDAC1 had to be recruited by HDAC2 to the HDAC2 gene under repression. 21AC2 was not necessary for HDAC1 to connect to sites that were activated by HDAC2 (Figure 3).A low concentration (1.0 µM) of nicotine has been reported to cause CDDP resistance.Some papers reported that HDAC2 has required for the recruitment of HDAC1 to the repressed HDAC2.22 Figure 3 showed that HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets.Subsequently, nicotine binds to E2F in the nucleus to the cytosol through the inducing cell receptor protein, α7nAChR.Thus, CDDP release results from nicotine-mediated activation of pRb hyperphosphorylation.The findings shed light on potential mechanisms influencing chemoresistance and tumor progression, offering crucial insights for the development of targeted therapeutic strategies in the management of squamous cell carcinoma.Intriguingly, we discovered that nicotine promotes lung growth of cancer by increasing the chemosensitivity of CDDP-resistant cells through the cell cycle signaling mediated by HDAC1 activation.Thus, nicotine-mediated squamous cancer cell growth provides one of the linkages to α7nAChR effects on increased cell cycle and increased cancer cell growth (Figure 4).This study demonstrates that nicotine stimulates tumor growth and is mediated by the α7nAChR, but possibly involves nicotine-resistance CDDP-induced cell growth effects mediated need (Figure 5).This study unveils the active role of nicotine in inducing cisplatin resistance in lung cancer, emphasizing the importance of understanding the molecular dynamics of HDAC1 activation.The CDDP causes HDAC1 to activate E2F cancer-suppressor genes, inhibiting their transcription.23,[24][25] These results suggest that nicotine may be necessary for HDAC1 carcinogenic contentmediated advanced development or chemotherapy treatment failure in lung cancer.26 The intricate relationship between nicotine, HDAC1, and cisplatin resistance carries significant therapeutic implications.
The study not only illuminates the complex mechanisms underlying nicotine-induced effects but also provides potential avenues for targeted interventions in the treatment of squamous cell carcinoma.
Nicotine binds to α7nAChR on the surface of tumor cells to induce HDAC1 activity of tumor cells promoting tumor growth function (Figure 6).Nicotine effects on cancer growth allowing HDAC1 repair Nicotine is active in highly cisplatin (CDDP)-resistant cancer cells.The mechanisms of the nicotine treatment of CDDPresistant lung cancer for histone deacetylase 1 (HDAC1) activity.HDAC1 transfected cells were directly binding between cytosol and nucleus distribution.Here, in cell viability and migration methods, we first found nicotineregulated cisplatin-resistant cell growth by targeting HDAC1.Expression of cisplatin was negatively correlated with HDAC1.HDAC1 inhibitor, VPA, in the cancer cells, was predicted.Besides, CDDP inhibited cell growth and reduced the expression of E2F proteins.However, nicotine increases the detoxification of CDDP and induces CDDP resistance to induce lung cancer cell growth through α7nAchR and EGFR receptors.On the other hand, nicotine regulates HDAC1 by α7nAchR receptors in nuclear, but EGFR receptors in the cell cytosol.then processed, permeabilized, and incubated with the HDAC1 (1:200, sc-81598FITC; Santa Cruz Lab) and NF-κB (1:200, sc-3061; Santa Cruz Lab) primary antibodies at 4°C overnight by fluorescein isothiocyanateconjugated secondary antibody (1:500; Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (1:200, sc-3598; Santa Cruz Lab).A merged image is the photographic region captured in the first and second images.After 24 h of treatment with nicotine and CDDP, NCI-H157 cells were extensively incubated with HDAC1.NCI-H157 were incubated with HDAC1 primary antibody incubated with FITC-/Texas Red-conjugated secondary antibody at room temperature (2 h).Phosphate-buffered saline washing was followed by Fluoromount G and then examined under a fluorescence microscope (Zeiss Axioskop fluorescent microscope).A merged image is made up of the photographic region captured in the first and second images.The photographed region was acquired in the image by fluorescence microscope (Zeiss Axioskop fluorescent microscope).

0. 1
μM nicotine (p < .01).Low concentrations of nicotine are reported to induce inflammation leading to tumor growth, while high nicotine influx acts as a potent tumor repressor, leading to cytotoxicity and apoptosis.Nicotine, like anything that enters the body, can be metabolized in the lung and kidney by several enzymes, including CYP2A6, CYP2A13, and CYP1B1 boosted cell proliferation to the point where a low concentration was required.The concentration of nicotine stimulated cell growth to correspond to low concentration F I G U R E 1 Nicotine dose-response NCI-520 squamous lung cancer cells growth and cytotoxicity.(A) Cell viability (%) in different nicotine concentrations at 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 µM for 24 h in NCI-H520 cell using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide.Data were expressed as mean ± scanning electron microscope (SEM), *p < .05,**p < .01,were significantly different compared with control.(B) The concentration of nicotine-induced tumor growth and cytotoxicity.Nicotine-stimulated effects at 0, 0.1, 0.5, 1.0, 1.5, 2.5, 5.0, 10.0 µM by NCI-H520 non-small-cell lung cancer (NSCLC) cell growth and cytotoxicity.(C) Histone deacetylase 1 (HDAC1) protein at 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 µM nicotine concentration by immunofluorescence staining.4′,6-diamidino-2-phenylindole is represented by a blue color.HDAC1 is represented by a green color.(D) Cell cytotoxicity proteins, CYP2A6, CYP2A13, and CYP1B1 at 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2. 5, 3.0 µM nicotine concentration in a 10 cm dish 10 mL medium in NCI-H520 NSCLC cell by western blot analysis.Quantification of protein expression levels by western blot proteins.All data were expressed as mean ± SEM, *p < .05,**p < .01,were significantly different compared to the control.when needed.Nicotine also stimulates tumor growth mediated through α7nAChR and EGFR, potentially through COX2 and NF-KB inflammatory production (Figure 2B).Our findings indicated that the cell cycle (Cyclin D/Cyclin E) and growth inhibition (p27/p21) play a role in how nicotine exposure affects cell growth.Figure 2D showed that Cyclin D/Cyclin E and p27/p21 were dose-response curve increases and decreases at 1.0 μM concentration of nicotine.Adenocarcinoma and squamous lung cancer cells, however, were among the NSCLC cells.Figure 2C shows that with the activation of c-jun and p38α and the reduction of Bcl2/p-Akt, HDACs inactivation F I G U R E 2 The nicotine-induced inflammation in NCI-520 cell by dose concentration.(A) α7nAchR and endothelial growth factor receptors (EGFR) protein expression levels relative to nicotine treatment dose by western blot analysis.Quantification of α7nAchR and EGFR proteins levels.All data were expressed as mean ± SEM, *p < .05,**p < .01 were significantly different compared to the control without nicotine.(B) Concentration of nicotine-induced inflammation factor, NOS2, and nuclear factor-κB (NF-κB), proteins at 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 µM as the final nicotine concentration in NCI-H520 cell by western blot analysis.Quantification of NOS2 and NF-κB protein expression levels.All data were expressed as mean ± SEM, * p < .05,**p < .01 were significantly different compared to the control without nicotine.(C) HDAC2, HDAC4, and HDAC5 induce by nicotine in NCI-H520 cancer cells by western blot analysis.Quantification of HDAC2, HDAC4, and HDAC5 protein expression levels.All data were expressed as mean ± SEM, *p < .05,**p < .01 were significantly different compared to the control without nicotine.(D) Nicotine-induced cell cycle relative proteins, Cyclin D1 and Cyclin E, and their suppressed proteins, p27, and p21, c-jun, and p38α protein expression using western blot analysisin NCI-H520 NSCLC cancer cells.Quantification of protein expression levels by western blot analysis.All data were expressed as mean ± SEM, *p < .05,**p < .01 were significant difference compared to the control without nicotine.led to a decrease in the development of tumor cells and the induction of cellular death.

F I G U R E 6
The relationship between the molecular mechanisms of histone deacetylase 1 (HDAC1) with nicotine resistance cisplatin (CDDP) in NCI-H157 cells localization expression in the cytosol and nucleus.(A) HDAC1, E2F, and Rb/pRb by HDAC1 +/+ transfection, CDDP, and HDAC1 +/+ + CDDP in the cytosol and nucleus by western blot analysis.(B) Protein expression levels of NF-KκB, p-γH2AX, and γH2AX relative to HDAC1 with nicotine in the NCI-H157 cytosol and nucleus by HDAC1 +/+ transfection, CDDP, and HDAC1 +/+ + CDDP by western blot analysis.(C) Cell membrane receptors, α7nAchR and endothelial growth factor receptors (EGFR), protein expression levels in the NCI-H157 cytosol and nucleus by HDAC1 +/+ transfection, CDDP, and HDAC1 +/+ + CDDP by western blot analysis.(D) Interaction of HDAC1 and Rb in the NCI-H157 nucleus by HDAC1 +/+ transfection, CDDP, and HDAC1 +/+ + CDDP by immunoprecipitation analysis.(E) α7nAchR and EGFR protein expression with nicotine located at the cell membrane.HDAC1 +/+ transfection.mechanisms to re-establish E2F/pRb promoting cell growth resulting in released potentially CDDP released and DNA open cell cycle.The NCI-H520 squamous carcinoma cell line is examined for CDDP resistance induced by nicotine.Results demonstrate increased HDAC1 expression and CDDP resistance, potentially mediated through α7nAChR and EGFR receptors.Inhibition of HDAC1 with VPA suppresses cell cycle-related proteins, indicating a promising avenue for therapeutic intervention.

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CONCLUSIONNicotine has an important role in regulating the activities of chemotherapeutic medications.27There are several mechanisms by which chemotherapeutic drugs interact with nicotine.We suggest that nicotine may increase the therapeutic resistance effects of CDDP-based treatments.Consequently, we discovered nicotineinduced HDAC1 protein interaction with CDDP expression in the nucleus.Specifically, the study focuses on the intricate role of nicotine-induced HDAC1 activation, aiming to uncover potential molecular mechanisms contributing to chemoresistance and tumor progression.Nicotine has been shown to have tumor-promoting or co-carcinogenic properties through α7nAChR and EGFR receptors (Figure7).This comprehensive understanding may pave the way for more effective and targeted therapeutic approaches in the management of squamous cell carcinoma.Nicotine treatment increased pRb hyperphosphorylation and rapidly increasing HDAC1 expression, and subsequently cell cycle activation.F I G U R E 7 Lung squamous carcinoma cell growth by nicotinemediated HDAC1/E2F/Rb cell cycle.Cisplatin (CDDP) adducts induce a cell cycle arrest in the G2/M phases of the cell cycle.The mechanisms of nicotine's influence on the effectiveness of antilung cancer cells.The main nicotine effect is mediated by membranous α7-nicotinic acetylcholine receptors (α7nAChR) whose stimulation leads to sustained activation of intracellular pathways.Nicotine induces α7nAChR into the nucleus-led CDDP released and then pRb hyperphophorylation into the cytosol binding HDAC1 and activation of Cyclin D/CyclinE/E2F/pRb activity enhanced cell cycle promotes stimulation of cancer cell growth.VPA, valproic acid.