Nicotine inhibits CD24 expression in Lewis lung carcinoma cells by upregulation of RAS expression
- Authors:
- Published online on: May 22, 2018 https://doi.org/10.3892/ijo.2018.4415
- Pages: 815-822
Abstract
Introduction
Lung cancer is a major cause of cancer-related mortality in China (1,2). The overall 5-year survival rate for patients with non-small cell lung cancer (NSCLC) is ~17.1% (2). In the past years, extensive research has been performed to obtain a better understanding of the underlying molecular biology of lung cancer (3–5). Recent studies have shed light on the role of cancer stem cells (CSCs) in lung cancer (6–8).
CSCs are a subpopulation of cells within a tumor that possess self-renewal and tumor-initiating capacities (9). Cluster of ddifferentiation 24 (CD24) is a small membrane glycoprotein that has emerged as a major determinant of stemness in various cancer types (10,11). Although CD24 has been used extensively in combination with other putative markers to isolate CSCs (12–14), the lack of their universal expression limits their usage to lung cancer (15,16). However, CD24 has been suggested as a biomarker for carcinoma progression in lung cancer (17,18).
The aim of the present study was to assess the tumor promotion roles of CD24 in the subtypes of lung cancer. Firstly, the significance of CD24 mRNA in human lung cancer was evaluated using the Oncomine database. Secondly, CD24high and CD24low cells were isolated from Lewis lung carcinoma (LLC) cells and the tumorigenic ability of these cells in vitro and in vivo was identified. Furthermore, a focus was placed on the roles of nicotine in CD24 expression, and the associated molecular signaling pathways were investigated. The findings of this study may be useful in improving the clinical effectiveness for a better prognosis in patients with lung adenocarcinoma.
Materials and methods
Oncomine database analysis
CD24 mRNA levels in NSCLC tissues were compared with their matched normal tissues using the Oncomine database (http://www.oncomine.org). The threshold used to obtain the most significant probes of the queried gene for each microarray data included a 2-fold difference in expression between cancer and normal tissues, with a P-value of <1×10−4.
Kaplan-Meier plotter analysis
The prognostic value of CD24 mRNA in lung cancer was analyzed using Kaplan-Meier (KM)-Plotter (http://kmplot.com/analysis/). Overall survival of the patients with high and low levels of CD24 mRNA was shown by the log-rank test.
Cell culture
LLC cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were maintained in a humidified cell incubator with 5% CO2 at 37°C.
Fluorescence-activated cell sorting (FACS)
Sorting of the side population (SP) cells from LLC cells was performed as described previously (19). SP cells were washed twice with PBS and suspended in 100 µl assay buffer [PBS, 0.5% bovine serum albumin (BSA), 2 mM EDTA (pH 7.2)] with 10 µl phycoerythrin (PE)-conjugated anti-CD24 antibody (1:50; catalog no. 555428; BD Biosciences, Franklin Lakes, NJ, USA) and 20 µl fluorescein isothiocyanate-conjugated anti-CD133/2 (clone 293C3) antibody [1:20; catalog no. 130-104-322; Miltenyi Biotec Technology and Trading (Shanghai) Co., Ltd. Shanghai, China]. The cells were then incubated in the dark at 4°C for 30 min, washed twice with 1 ml assay buffer and centrifuged at 300 × g for 10 min. The cell pellet was subjected to FACS using a BD Aria II sorter (BD Biosciences).
Reverse transcription-polymerase chain reaction (PCR)
Total RNA was isolated from cells using an RNeasy Mini kit (Beijing Bomed Gene Technology Co., Ltd., Beijing, China). cDNA was reverse transcribed from 1 µg total RNA using a Takara Reverse Transcription kit (Takara Biotechnology, Co., Ltd., Dalian, China), and then amplified using the following primers: CD24 sense, 5′-ACTCAGGCCAGGAAACGTCTCT-3′ and antisense, 5′-AACAGCCAATTCGAGGTG GAC-3′; ATP-binding cassette subfamily G member 2 (Junior blood group) (ABCG2) sense, 5′-AGCTGCAAGGAAAGATC CAA-3′ and antisense 5′-TCCAGACACACCACGGATAA-3′; and GAPDH sense, 5′-AGAAGGCTGGGGCTCATTTG-3′ and antisense, 5′-AGGGGCCATCCACAGTCTTC-3′. The PCR products were electrophoresed on a 1.5% agarose gel, and visualized by ethidium bromide staining under an ultraviolet imaging system (UVP, LLC, Phoenix, AZ, USA). The RT-PCR conditions were as follows: 10 min at 95°C for dena-turation, followed by 35 cycles of 20 sec at 95°C, 40 sec at 56°C and 30 sec at 72°C, and a final extension step of 5 min at 72°C.
Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 30 min, followed by washing twice in PBS at room temperature (RT) for 5 min. Non-specific binding sites were blocked with 3% BSA in PBS for 1 h at RT. PE-conjugated anti-CD24 antibody (as aforementioned), diluted in 3% BSA/PBS, was applied overnight at 4°C. For every coverslip, the cells were observed and images were captured in 5 random fields using an Olympus CX71 fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Colony-formation assay
Cells were plated at a density of 2×105 cells/well in 24-well plates under serum-free, colony-specific conditions. Fresh aliquots of epidermal growth factor and basic fibroblast growth factor were added every day. Subsequent to culturing the cells for 3 weeks, colonies were visible under a light microscope (Olympus CX31; Olympus).
Transwell assay
Cell suspension (200 µl; 1×105 cells/ml, RPMI-1640 medium with 1% FBS) was placed into the upper chamber of a Transwell (8-µM pore size polycarbonate membrane; Cell Biolabs, San Diego, CA, USA). In the lower chamber, RPMI-1640 medium with 10% FBS was added. Subsequent to culture for 24 h, the cells that had migrated through and adhered to the lower surface of the membrane were fixed with paraformaldehyde for 15 min and stained with 0.1% crystal violet for 10 min at RT. Next, five fields of view were randomly selected for the counting of cells under a light microscope (Olympus CX31; Olympus).
Nicotine treatment and Ras inhibitor (salirasib)
According to the method used in the study by Chu et al (20), CD24low and CD24high cells were split every 3 days with medium supplemented with 0.5 µM nicotine to keep the drug at a constant concentration. For the controls, CD24low and CD24high cells treated with nicotine were incubated with salirasib (100 µM; 162520-00-5; Tocris Bioscience, Bristol, UK) for 24 h at 37°C.
In vivo assays
Liaoning Medical University Ethics Committee (Jinzhou, Liaoning, China) approved the research protocols performed in this study. NOD SCID mice (25–40 g; 4 to 6-weeks-old; male; NOD.CB17-Prkdcscid/NcrCrl; Charles River Laboratories, Inc., Wilmington, MA, USA) received standard laboratory food and water ad libitum and were maintained in micro-isolator cages with filtered air and handled under sterile conditions under a laminar flow hood. A subcutaneous injection of cells [1×107 cells in 200 µl PBS, including main population (MP), SP, CD24high, CD24low, CD133highCD24high or CD133highCD24low; 30 mice in each treatment group] was administered into the flank of each mouse. Tumors were measured using calipers, and tumor volumes were calculated (tumor volume = length × width2 × 0.52) (21). Once the tumor diameters had reached 3–5 mm, the mice were used in the following studies. According to the methods used in the study by Cavarra et al (22), male mice injected with CD24high or CD24low cells were exposed to the smoke of four cigarettes/day at 10 a.m. and 4 p.m. for 3 months (1.2 mg of nicotine; Honghe filter cigarettes; Honghe Cigarette Factory, Yunnan, China), 1 month prior to cell inoculation and 2 months after cell inoculation), in specially designed cages. The mice were examined at 0, 10, 20, 30, 40, 50 and 60 days, and tumor growth was evaluated by measuring the length and width of the tumor mass. Subsequently, the animals were euthanized with pentobarbital sodium via the tail vein (100 mg/kg). The survival state of the immunodeficient mice was observed day and night, and euthanasia was available to use at the first sign of any mental or dietary problems.
Immunohistochemical staining
Endogenous peroxidase activity was blocked in 4-µm tumor sections with 3% hydrogen peroxide for 30 min at RT. Antigen retrieval was performed in citrate buffer (10 mM; pH 6.0) for 30 min at 95°C in a pressure cooker. Primary antibodies were incubated with sections at 1:500 overnight at 4°C (Table I). Sections were then incubated with horseradish peroxidase-labeled polymer-conjugated goat anti-mouse/goat anti-rabbit secondary antibody (1:100; catalog no. A0208/A0216; Beyotime Institute of Biotechnology, Beijing, China) for 60 min at RT, followed by incubation with a streptavidin horseradish peroxidase complex (Beyotime Institute of Biotechnology) for 60 min at RT. Bound antibody was visualized with 3,3′-diaminobenzidine tetrahydrochloride (Beyotime Institute of Biotechnology). Sections were also counterstained with hematoxylin for 30 sec at RT (Beyotime Institute of Biotechnology). The results were visible under a light microscope (Olympus CX31; Olympus).
Western blot analysis
Protein was extracted in lysis buffer (P0013B; Beyotime Institute of Biotechnology) for 30 min on ice. The extract was centrifuged at 4,000 × g for 5 min at 4°C to remove debris. Total protein concentration was determined using the bicinchoninic acid protein assay kit (P0010; Beyotime Institute of Biotechnology). Extracted proteins (30 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked in 5% BSA/Tris-buffered saline plus Tween-20 at RT for 1 h and then incubated with primary antibodies at 4°C overnight (Table I). The reaction was followed by probing with peroxidase-coupled secondary antibodies at 1:1,000 dilution (catalog no. A0216; goat anti-mouse; and catalog no. A0208; goat anti-rabbit; Beyotime Institute of Biotechnology), and binding results were visualized by enhanced chemiluminescence kit (Amersham; GE Healthcare, Chicago, IL, USA).
Ingenuity Pathway Analysis
The Ingenuity Pathway Analysis software (Ingenuity® Systems; www.ingenuity.com) was used to build networks and identify pathways of CD24 based on data mining.
Statistical analysis
Each experiment was performed in triplicate. Data were analyzed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Statistical analysis was performed using one-way analysis of variance and Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
Associations between CD24 status and clinical parameters of lung cancer patients
Oncomine analysis of cancer versus normal tissue showed that the CD24 mRNA level was higher in lung adenocarcinoma, large cell lung carcinoma, squamous cell lung carcinoma and small cell lung carcinoma (Fig. 1). No difference in CD24 mRNA was found between male and female lung cancer patients (data not shown). Notably, the patients aged 40 to 49 years old exhibited a higher CD24 mRNA level compared with that of patients of other age groups (data not shown). Another notable result was that smoking decreased CD24 expression in lung cancer patients (Fig. 2).
In addition, the prognostic values of CD24 mRNA were analyzed in subtypes of lung cancer by KM-plotter. CD24 mRNA can be used as a prognostic marker for patients with lung adenocarcinoma (P=0.0029) (Fig. 3). No influence of CD24 mRNA on squamous cell lung carcinoma was found (P=0.65) (Fig. 3). A high level of CD24 mRNA was shown to improve the survival rate of male lung cancer patients (P=0.0066) (Fig. 3). A high level of CD24 mRNA could also improve the prognosis of never smokers (P=0.00036), while no influence of CD24 mRNA on former smokers and current smokers was found (P=0.06) (Fig. 3).
CD133highCD24low and CD133highCD24high fractions in LLC cells
In our previous study, SP cells were isolated from LLC cells using Hoechst 33342 efflux analysis (19). ABCG2 protein is a surface marker of SP cells (19). In the present study, SP cells were further isolated by use of two CSC-specific markers, CD133 and CD24 (Fig. 4A). In addition, it was found that CD24 mRNA and protein expression was significantly higher in the CD133highCD24high cells compared with that in the CD133highCD24low cells (Fig. 4B and C). ABCG2 mRNA and protein was higher in CD133highCD24high cells and CD133highCD24low cells than that in MP cells. Immunofluorescence results showed that CD24 was localized in the membrane of CD133highCD24high and CD133highCD24low cells (Fig. 4D).
Tumorigenic ability of CD133highCD24low and CD133highCD24high LLC cells in vitro and in vivo
Colony formation assays were performed to detect the proliferation of MP, SP, CD133highCD24high and CD133highCD24low cells. Sphere clusters were clearly observed in SP, CD133highCD24high and CD133highCD24low cells (P<0.05), with no difference among these three cell types (Fig. 5A). Migration of MP, SP, CD133highCD24high and CD133highCD24low cells was detected using Transwell assay. It was found that more SP, CD133highCD24high and CD133highCD24low cells migrated to the lower membrane compared with MP cells (P<0.05) (Fig. 5B). No difference in mobility was found among SP, CD133highCD24high and CD133highCD24low cells (Fig. 5B). Furthermore, it was found that SP, CD133highCD24high and CD133highCD24low cells had similar abilities to transfer the tumors into immunocompromised mice (Fig. 6A and B). The diameters of the largest single subcutaneous tumors observed in the SP, CD133highCD24high and CD133highCD24low groups were 4.4±0.3, 4.6±0.3 and 4.7±0.2 mm. These results indicated that CD24 expression did not increase the tumor-forming ability of the LLC cells.
Potential mechanism of nicotine-inhibited CD24 expression
Based on the results of Ingenuity Pathways Analysis software (version 6.3; Ingenuity Systems, Redwood, CA, USA), the differentially expressed proteins in CD24-expressing cells were determined (data not shown). In addition, immunohistochemistry (IHC) results showed that the level of RAS was markedly higher in the cancer tissues of CD133highCD24low-injected mice compared with that in the MP group, while almost no RAS expression was found in the CD133highCD24high group (Fig. 6C). The results also revealed that CD24 expression was associated with HSP90 expression (Fig. 6C). Low E-cadherin expression was found in all MP, SP, CD133highCD24high and CD133highCD24low groups (Fig. 6C).
Furthermore, it was found that nicotine treatment inhibited CD24 expression in vitro (Fig. 7A). The level of RAS was moderately increased in CD24low and CD24high LLC cells after nicotine treatment (Fig. 7A). To test the hypothesis that CD24 expression partly depended on downregulation of RAS in LLC cells, salirasib was used as a RAS blocker. Downregulation of RAS using salirasib could induce CD24 expression in CD24low LLC cells (Fig. 7A). Altogether, these results suggest that nicotine could inhibit CD24 expression in LLC cells via activating RAS.
CD24high and CD24low cell-injected mice were subjected to cigarette smoke, as aforementioned. The tumor volumes of these two groups exhibited no significant changes from the beginning to the end of the experiment (Fig. 6B). IHC results also showed that RAS and CD24 expression were not changed in the mice subjected to smoke (Fig. 7B).
Discussion
In the present study, the prognostic value of CD24 mRNA in NSCLC was analyzed using the Oncomine database. A number of previous studies have discussed the roles of CD24 in lung cancer (17,23). In the study by Karimi-Busheri et al (17), upregulation of CD24 was observed in >75% of NSCLC patients. Kristiansen et al (23) reported a higher incidence of CD24 expression in NSCLC tissues. Consistent with these previous results, the present study also found higher CD24 mRNA expression in lung cancer tissues compared with that in matched normal tissues. Kristiansen et al (23) found that CD24 expression is an independent predictor of a shortened survival time in NSCLC patients. Lee et al (24) demonstrated a significant association between CD24 expression and shorter NSCLC patient survival times. However, in the present study, it was found that CD24 mRNA was associated with a longer survival time in the patients with lung adenocarcinoma. The main reason for this difference may be that dynamic changes of CD24 protein throughout the development of cancer (25). CD24 is a heavily glycosylated protein that also demonstrates increased additional structural flexibility in its mature form (25).
Previous studies showed that CD24 could not be used as a CSC marker for human lung adenocarcinoma (16,26). Roudi et al (16) found that CD24 could not be considered a potential marker for isolating CSCs in the human lung adenocarcinoma A549 cell line. Xu et al (15) also found that CD24− A549 cells possess partial CSC properties, but actually are not CSCs. In the present study, another lung adenocarcinoma cell line, LLC, was used to analyze the roles of CD24 in the stemness of lung adenocarcinoma. The in vitro and in vivo experiments demonstrated that CD24high LLC cells showed no significant differences in terms of metastasis and tumorgenicity compared with CD24low cells. These results indicated that CD24 could not be used to isolate CSCs from lung adenocarcinoma cells.
The most important result of the present study is that CD24 expression is critically dependent on the smoking status of lung cancer patients. It was found that nicotine could inhibit CD24 expression in LLC cells by upregulation of RAS. Nicotine is believed to promote the tumorigenesis of lung cancer cells (15). An increase in Ras activity/expression is frequently found in numerous cancer types (27,28). More and more evidence indicates that nicotine is able to activate Ras upon its interaction with nicotine acetylcholine receptors (15). In agreement with previous studies, the present study also confirmed that nicotine could induce RAS expression in LLC cells. In previous studies, the activation of RAS was able to downregulate CD24 expression at the mRNA and protein levels (29,30). The present study also demonstrated that the expression of oncogenic Ras directly downregulated the expression of CD24 at the protein level. Furthermore, inhibition of RAS could partially restore CD24 expression in LLC cells. These results provided an integrated insight regarding the mechanism of nicotine-inhibited CD24 expression in LLC cells. Nicotine is well known to be an addictive component of cigarettes. Notably, no effects of smoking on CD24 expression were found in vivo. The main reason for this is that the level of nicotine may have been too low to influence established xenograft tumor models.
In summary, the principal findings of the present study were that: i) CD24 could be used as a prognostic marker in lung adenocarcinoma; ii) in vitro and in vivo experiments did not find a significant influence of CD24 on tumorgenicity of LLC cells; and iii) nicotine inhibited CD24 expression in LLC cells by upregulation of RAS. However, the downstream proteins of RAS should be analyzed in further studies.
Acknowledgments
Not applicable.
Funding
This study was supported by the National Natural Scientific Foundation of China (grant no. 81502558), the President Fund of Liaoning Medical University (grant no. XZJJ20140102) and the Biological Anthropology Innovation Team Project of Jinzhou Medical University (grant no. JYLJ201702).
Availability of data and materials
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.
Authors' contributions
PX and FR were responsible for the study design, original article drafting and editing, data acquisition and data analysis. DHL, MA and BLB performed the experiments. DHL and PX were responsible for data analysis. DHL, MA and BLB were responsible for data acquisition. DHL, MA, BLB and PX were responsible for data interpretation and methodology. PX and FR were responsible for supervision of the whole study and funding acquisition. PX revised the manuscript. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The Ethical Committee of Jinzhou Medical University approved this investigation.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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