The oncogenic role of CB2 in the progression of non-small-cell lung cancer

The oncogenic role of CB2 in the progression of non-small-cell lung cancer Shaohua Xua, Hanchen Maa, Yuhong Bob, Mingju Shaoc,a

Department of Respiratory, The Second Hospital of Shandong University, Jinan 250033, Shandong Province, ChinabWeihai Municipal Hospital, Weihai 264200, Shandong Province, ChinacDepartment of Emergency Medicine, The Second Hospital of Shandong University, No. 247 Beiyuan Street, Jinan 250033, Shandong Province, China

ARTICLE INFO Keywords:NSCLCCB2Tumor progressionAkt/mTOR/P70S6K pathwayABSTRACTBackground:Several studies have verified the important role of cannabinoid and cannabinoid receptor agonistsin tumor progression. However, little is known about the precise role of CB2 expression level in the progressionof non-small-cell lung cancer (NSCLC).Methods:The expression of CB2 in NSCLC tissues and corresponding paracancerous tissues was examined usingimmunohistochemical staining assay. The expression of CB2 was silenced by siRNA interference and loss-of-function assays were performed to investigate the biological function of CB2 in the proliferation, migration,invasion, and apoptosis of NSCLC cells. The expression of related proteins was detected using western blotanalysis.Results:In this study, we observed that CB2 was up-regulated in NSCLC tissues and the up-regulation wascorrelated with tumor size and advanced NSCLC pathological grading. Moreover, compared with the controlgroup, silencing of CB2 decreased the proliferation, migration and invasion abilities of A549 and H1299 cells,and induced apoptosis by regulation of Bcl-2/Bax axis and active Caspase3. Furthermore, CB2 knockdown in-activated the Akt/mTOR/P70S6K pathway by decreasing the level of p-Akt, p-mTOR and expression of P70S6Kin A549 and H1299 cells.Conclusion:Our data suggested that targeting CB2 may inhibit the growth and survival of NSCLC cells, which theAkt/mTOR/P70S6K pathway may be involved in. These results confer the pro-oncogenic role of CB2 in theprogression of NSCLC, thus improving our understanding of CB2 in tumor progression.1. IntroductionNon-small-cell lung cancer (NSCLC) has the highest cancer mor-tality worldwide [1]. Lung adenocarcinoma accounts for 40%70%cases of NSCLC [2]. Despite the progress in diagnosis and treatmentstrategies, the patients with NSCLC still have an unfavorable prognosiswith the 5 and 10 year survival rates of < 15% and < 7% [3]. There-fore, it is necessary to further study the molecular mechanisms relatedto the carcinogenesis and progression of NSCLC, and identify effectivetargets for developing novel therapeutic strategies.CB2, also known as Cannabinoid CB2 receptor gene (CNR2), is aspecific G-protein coupled receptor (GPCR), which is expressed inperipheral and inflammatory tissues and involved in the anti-in-flammatory effects of cannabinoids [4]. Since increasing studies havesuggested that cannabinoids can be used in the treatment of cancer, therole of CB2 in tumor progression has also been noticed. CB2 agonists,such as JWH-133, have been proved to play an antitumor effect inseveral cancers [5, 6]. Recently, CB2 is reported to be up-regulated invarious tumor tissues, including melanoma [7], colon cancer [8], breastcancer [9], bladder cancer [10], and hepatocellular carcinoma [11]. Inaddition, Martínez-Martínez et al.find that up-regulation of CB2 isassociated with poor prognosis of patients with colon cancer, and tu-mors with higher levels of the CB2 have a higher proliferation level [8],indicating an inconsistent function of CB2 in colon cancer progressionwith the in vitro experiments.No study has clarified the precise role of CB2 expression level in theprogression of NSCLC. Based on these reports, we aimed to investigatethe relationship between CB2 expression level and progression ofNSCLC, and assess the role of CB2 involved in progression of NSCLC.For thefirst time, our data revealed that CB2 was up-regulated inNSCLC and suggested the pro-oncogenic role of CB2 in the progressionof NSCLC.https://doi.org/10.1016/j.biopha.2019.109080Received 10 May 2019; Received in revised form 31 May 2019; Accepted 2 June 2019Corresponding author.E-mail address:sddxsmj@yeah.net(M. Shao).Biomedicine & Pharmacotherapy 117 (2019) 1090800753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).T

2. Materials and methods2.1. Patients and clinical samplesNSCLC tissues and corresponding paracancerous tissues were col-lected from 68 cases of patients who underwent NSCLC resection in theSecond Hospital of Shandong University from Jun 2014 to April 2017.The clinicopathological information of patients with NSCLC, includingage, gender, tumor size, and pathological grading, was summarized inTable 1. This study was approved by the Research Ethics Committee ofThe Second Hospital of Shandong University (China). Written informedconsents were obtained from all patients. All specimens were handledand made anonymous according to the ethical and legal standards.2.2. Immunohistochemical stainingTissues was stained by EliVisionplus kit (Maixin, China) accordingto the manufacturers instruction. Anti-CB2 was obtained fromProteintech (USA). The CB2 protein expression level was assessed basedon the staining intensity and percentage of positive stained cells. Thescoring criteria were as follows. The staining intensity was graded asfollows: no staining = 0; weak staining = 1; moderate staining = 2;and strong staining = 3; the percentage of positive stained cells wasgraded as follows: grade 1 = 1%10%; grade 2 = 11%50%; grade3 = 51%80%; and grade 4 = more than 80%. Samples were scored bymultiplying the percentage of CB2-positive cells and staining intensity,and more than 6 score as CB2 up-regulated group.2.3. Cell culture and transfectionThe human NSCLC cell lines A549 and H1299 were obtained fromthe Cell Bank of Chinese Academy of Sciences in Shanghai and main-tained in RPMI-1640 (HyClone, USA) containing 10% FBS (Gibco, USA)and antibiotics (Sigma-Aldrich, Germany). Cells were transfected with50 nM siRNA-CB2 (Oligobio, China) or scrambled siRNA (negativecontrol, NC) using Lipofectamine 2000 (Invitrogen, USA) following themanufacturers protocol.2.4. Western blotting analysisTransfected cells were lysed using RIPA Lysis Buffer (CWBIO, China)for protein extraction. 20μg protein of each sample was electro-phoresed on 10% SDS-PAGE gel, and transferred to a PVDF membrane(Millipore, USA) afterwards. The membrane was blocked with 5% non-fat milk for 1 h prior to incubation with primary antibodies at 4 °Covernight. Following incubation with secondary antibody for 1 h, thesignal was developed using enhanced chemiluminescence detection kit(CWBIO). Primary antibodies against CB2, Bcl-2, Bax, active Caspase3,P70S6K, andβ-tubulin were purchased from Proteintech Group (USA).Akt, p-Akt, mTOR and p-mTOR antibodies were obtained from CellSignaling Technology (USA). All horseradish peroxidase-conjugatedsecondary antibodies were obtained from Proteintech Group.2.5. CCK8 assayCell viability were assessed by CCK8 assay. Briefly, about 1 × 103cells were seeded in each well of 96-well plate and transfected withsiRNA for 24 h. Following the transfection, cell were cultured withCCK8 reagent (CWBIO) at 37 °C for 90 min, the absorbance was mea-sured every 24 h at 450 nm.2.6. Colony formation assayAfter being transfected for 24 h, cells were seeded into culturedishes containing 5 ml medium at a density of 200 cells per dish, andcultured at 37 °C with 5% CO2until cells had formed sufficiently largeclones. The medium was removed and 0.1% crystal violet was per-formed applied for staining the colony. The colonies were imaged andcounted under a light microscope.2.7. Wound-healing assayWound-healing assay was used for assessment of cell migrationability. Transfected cells were seeded in 6-well plates at a density of5×105cells/well for 12 h. After that, wounds were generated usingpipette tips and cells were cultured in serum-free medium for 24 h. Thewound closure was imaged and analyzed using ImageJ software(National Institutes of Health, USA).2.8. Cell invasion assayTranswell chambers (Millipore, USA) were performed to assessmigration ability of A549 and H1299 cells. Following the transfectionwith siRNA for 24 h, resuspended cells in serum-free culture mediumwere transferred to the upper chamber, and complete medium wasadded to the lower chamber. After incubation for 24 h, migratory cellswerefixed with 4% paraformaldehyde, and stained with 0.1% crystalviolet for 20 min. Photograph and count the number of migratory cellsunder the microscope.2.9. Cell apoptosis assayFor assessment of cell apoptosis, transfected cells were cultured inserum-free medium for 24 h for starvation and stained with Annexin V-FITC and PI using the In Situ Cell Death Detection Kit (Roche, USA)according to the instructions. The apoptosis rate was analyzed byflowcytometer (BD FACSC anto II, BD Biosciences, USA), and calculated byBD FACSDiva software.2.10. Statistical analysisAll experiments were repeated in triplicate and the values werepresented as Mean ± SD. The SPSS 18.0 statistical software was per-formed to carry out statistical analysis. The comparison between theexpression level of CB2 and clinicopathological parameters of patientswith lung adenocarcinoma were analyzed using Chi-square test.Studentsttest or one-way ANOVA was performed for statistical ana-lysis between groups, andP< 0.05 was considered statistically sig-nificant.Table 1Correlation between expression level of CB2 and clinicopathologic parametersin the patients with NSCLC.VariablesTotal cases(n = 68)CB2 expressionP-valueUp-regulated(n = 52)Intact(n = 16)Gender0.118Male45378Female23158Age (years)0.264<60302196038317Tumor diameter(cm)0.045*< 5432914525232Pathologicalgrading0.044*III483315IIIIV20191* P < 0.05.S. Xu, et al.Biomedicine & Pharmacotherapy 117 (2019) 1090802

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