Cannabinoid receptor expression in non-small cell lung cancer. Effectiveness of tetrahydrocannabinol and cannabidiol inhibiting cell proliferation and epithelial-mesenchymal transition in vitro.
- 作者列表："Milian L","Mata M","Alcacer J","Oliver M","Sancho-Tello M","Martín de Llano JJ","Camps C","Galbis J","Carretero J","Carda C
BACKGROUND/OBJECTIVE:Patients with non-small cell lung cancer (NSCLC) develop resistance to antitumor agents by mechanisms that involve the epithelial-to-mesenchymal transition (EMT). This necessitates the development of new complementary drugs, e.g., cannabinoid receptors (CB1 and CB2) agonists including tetrahydrocannabinol (THC) and cannabidiol (CBD). The combined use of THC and CBD confers greater benefits, as CBD enhances the effects of THC and reduces its psychotropic activity. We assessed the relationship between the expression levels of CB1 and CB2 to the clinical features of a cohort of patients with NSCLC, and the effect of THC and CBD (individually and in combination) on proliferation, EMT and migration in vitro in A549, H460 and H1792 lung cancer cell lines. METHODS:Expression levels of CB1, CB2, EGFR, CDH1, CDH2 and VIM were evaluated by quantitative reverse transcription-polymerase chain reaction. THC and CBD (10-100 μM), individually or in combination (1:1 ratio), were used for in vitro assays. Cell proliferation was determined by BrdU incorporation assay. Morphological changes in the cells were visualized by phase-contrast and fluorescence microscopy. Migration was studied by scratch recolonization induced by 20 ng/ml epidermal growth factor (EGF). RESULTS:The tumor samples were classified according to the level of expression of CB1, CB2, or both. Patients with high expression levels of CB1, CB2, and CB1/CB2 showed increased survival reaching significance for CB1 and CB1/CB2 (p = 0.035 and 0.025, respectively). Both cannabinoid agonists inhibited the proliferation and expression of EGFR in lung cancer cells, and CBD potentiated the effect of THC. THC and CBD alone or in combination restored the epithelial phenotype, as evidenced by increased expression of CDH1 and reduced expression of CDH2 and VIM, as well as by fluorescence analysis of cellular cytoskeleton. Finally, both cannabinoids reduced the in vitro migration of the three lung cancer cells lines used. CONCLUSIONS:The expression levels of CB1 and CB2 have a potential use as markers of survival in patients with NSCLC. THC and CBD inhibited the proliferation and expression of EGFR in the lung cancer cells studied. Finally, the THC/CBD combination restored the epithelial phenotype in vitro.
背景/目的: 非小细胞肺癌 (NSCLC) 患者通过涉及上皮-间质转化 (EMT) 的机制对抗肿瘤药物产生耐药性。这需要开发新的补充药物，例如大麻素受体 (CB1 和CB2) 激动剂，包括四氢大麻酚 (THC) 和大麻二酚 (CBD)。THC和CBD的组合使用赋予更大的益处，因为CBD增强了THC的作用并减少了其精神活性。我们评估了CB1 和CB2 的表达水平与NSCLC患者的临床特征之间的关系，以及THC和CBD (单独和组合) 对增殖的影响，a549 、H460 和H1792 肺癌细胞系的EMT和体外迁移。 方法: 通过定量逆转录-聚合酶链反应评估CB1 、CB2 、EGFR、CDH1 、CDH2 和VIM的表达水平。单独或组合 (1:1 比例) 的THC和CBD (10-100 μ m) 用于体外测定。通过BrdU掺入测定法测定细胞增殖。通过相差和荧光显微镜观察细胞的形态学变化。通过 20 ng/ml表皮生长因子 (EGF) 诱导的划痕再着色来研究迁移。 结果: 根据CB1 、CB2 或两者的表达水平对肿瘤样品进行分类。CB1 、CB2 和CB1/CB2 高表达水平的患者显示CB1 和CB1/CB2 达到显著性的生存增加 (分别为p = 0.035 和 0.025)。两种大麻素激动剂都抑制肺癌细胞中EGFR的增殖和表达，并且CBD增强了THC的作用。THC和CBD单独或组合恢复上皮表型，如通过CDH1 的表达增加和CDH2 和VIM的表达减少以及通过细胞骨架的荧光分析所证明的。最后，两种大麻素降低了所使用的三种肺癌细胞系的体外迁移。 结论: CB1 和CB2 的表达水平作为NSCLC患者的生存标志物具有潜在的应用价值。THC和CBD抑制研究的肺癌细胞的增殖和EGFR的表达。最后，THC/CBD组合在体外恢复上皮表型。
METHODS::Pulmonary artery sling is a rare congenital anomaly of the origin and course of the left pulmonary artery. Patients with this condition typically present with respiratory failure in young infancy, and asymptomatic cases are uncommon. We describe the case of an adult patient with a lung adenocarcinoma of the right upper lobe, extending into the hilum and superior mediastinum, and with a previously unknown pulmonary artery sling anomaly. The local invasiveness of the tumor and the peculiar vascular anatomy contributed to a unique surgical scenario, wherein multiple reconstructive procedures were required.
METHODS::Patients with idiopathic pulmonary fibrosis (IPF) have higher risk of developing lung cancer, for example, squamous cell carcinoma (SCC), and show poor prognosis, while the molecular basis has not been fully investigated. Here we conducted DNA methylome analysis of lung SCC using 20 SCC samples with/without IPF, and noncancerous lung tissue samples from smokers/nonsmokers, using Infinium HumanMethylation 450K array. SCC was clustered into low- and high-methylation epigenotypes by hierarchical clustering analysis. Genes hypermethylated in SCC significantly included genes targeted by polycomb repressive complex in embryonic stem cells, and genes associated with Gene Ontology terms, for example, "transcription" and "cell adhesion," while genes hypermethylated specifically in high-methylation subgroup significantly included genes associated with "negative regulation of growth." Low-methylation subgroup significantly correlated with IPF (78%, vs. 17% in high-methylation subgroup, p = 0.04), and the correlation was validated by additional Infinium analysis of SCC samples (n = 44 in total), and data from The Cancer Genome Atlas (n = 390). The correlation between low-methylation subgroup and IPF was further validated by quantitative methylation analysis of marker genes commonly hypermethylated in SCC (HOXA2, HOXA9 and PCDHGB6), and markers specifically hypermethylated in high-methylation subgroup (DLEC1, CFTR, MT1M, CRIP3 and ALDH7A1) in 77 SCC cases using pyrosequencing (p = 0.003). Furthermore, low-methylation epigenotype significantly correlated with poorer prognosis among all SCC patients, or among patients without IPF. Multivariate analysis showed that low-methylation epigenotype is an independent predictor of poor prognosis. These may suggest that lung SCC could be stratified into molecular subtypes with distinct prognosis, and low-methylation lung SCC that significantly correlates with IPF shows unfavorable outcome.
METHODS::The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-13 C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.