CLDN1 Increases Drug Resistance of Non-Small Cell Lung Cancer by Activating Autophagy via Up-Regulation of ULK1 Phosphorylation - PubMed (original) (raw)
CLDN1 Increases Drug Resistance of Non-Small Cell Lung Cancer by Activating Autophagy via Up-Regulation of ULK1 Phosphorylation
Zhenhuan Zhao et al. Med Sci Monit. 2017.
Abstract
BACKGROUND The aim of this study was to investigate the expression of CLDN1 in non-small cell lung cancer (NSCLC) and its mechanism of action in cisplatin resistance. MATERIAL AND METHODS A total of 55 patients with NSCLC admitted to our hospital between October 2013 and October 2015 were included. NSCLC tissues and tumor-adjacent tissues (≥5 cm from tumor edge) were collected. Among the 55 patients, 37 had adenocarcinoma and 18 had squamous cell carcinoma. Quantitative real-time polymerase chain reaction was used to determine mRNA expression, and protein expression was examined using Western blotting. CCK-8 assay was used to determine cell proliferation and Transwell assay was used to detect migration and invasion of the cells. Confocal microscopy was used to observe autophagosomes. RESULTS Increased CLDN1 expression promoted the development and metastasis of NSCLC. CLDN1 expression in A549/CDDP cells was up-regulated at both transcriptional and translational levels. Reduced CLDN1 expression decreased the drug resistance, proliferation, migration, and invasion abilities of A549/CDDP cells. Decreased CLDN1 expression promoted the apoptosis of A549/CDDP cells. CLDN1 enhanced CDDP drug resistance of A549 cells by activating autophagy. CLDN1 promoted the autophagy of A549 cells by up-regulating the phosphorylation level of ULK1. CONCLUSIONS The present study demonstrates that expression of CLDN1 in NSCLC is up-regulated and it is correlated with clinicopathological features. CLDN1 activates autophagy through up-regulation of ULK1 phosphorylation and promotes drug resistance of NSCLC cells to CDDP.
Conflict of interest statement
Conflict of interest
None.
Figures
Figure 1
CLDN1 mRNA expression in non-small cell lung cancer (NSCLC) and its clinical significance. (A) Relative expression of CLDN1 in NSCLC tissues and tumor-adjacent normal tissues. * P<0.05 compared with normal tissues. (B) Relative expression of CLDN1 in patients with lymphatic metastasis (N1) or without lymphatic metastasis (N0). * P<0.05 compared with patients without lymph node metastasis. (C) Relative expression of CLDN1 in patients at TNM stages I/II and III/IV. * P<0.05 compared with stage I/II.
Figure 2
Relative expression of CLDN1 in A549 cells and A549/CDDP cells. (A) Relative expression of CLDN1 mRNA. * P<0.05 compared with A549 cells. (B) Relative expression of CLDN1 protein. * P<0.05 compared with A549 cells. Intermittent impact method was used to construct A549/CDDP cells. After incubation with medium concentration of CDDP (1 μg/ml) for 24 h, dead cells were washed away and fresh RPMI-1640 complete medium was added. When the cells were stable, the same concentration of CDDP was added for impact, which was repeated until tumor cells were able to stably proliferate in RPMI-1640 complete medium containing 0.5 μg/ml CDDP.
Figure 3
Relative expression of CLDN1 protein in A549/CDDP cells transfected with siR-NC or siR-CLDN1. Western blotting was performed to measure protein expression. * P<0.05 compared with siR-NC group.
Figure 4
Effect of silencing of the expression of CLDN1 on the proliferation of A549/CDDP cells. Cells were seeded into 96-well plates at a density of 1×104 cells per well in triplicate. For every 24 h, the cells were incubated with CCK-8 reagent (Beyotime, Shanghai, China) for 30 min. Absorbance at 490 nm was read on a microplate reader (168-1000; Model 680, Bio-Rad, Hercules, CA, USA) at 24 h, 48 h, and 72 h, and proliferation curves were plotted using absorbance values at each time point. * P<0.05 compared with siR-NC group.
Figure 5
Effect of silencing of the expression of CLDN1 on the migration and invasion of A549/CDDP cells. Transwell assay was used to determine the migration and invasion ability of cells. * P<0.05 compared with siR-NC group.
Figure 6
Effect of silencing of the expression of CLDN1 on the apoptosis of A549/CDDP cells. Flow cytometry was used to detect the apoptosis rate of cells. * P<0.05 compared with siR-NC group.
Figure 7
Effect of autophagy on CDDP resistance of A549 cells. (A) Expression of LC3B I and II and their ratio in A549 cells, A549/CDDP cells, and A549/CDDP cells transfected with siR-NC or siR-CLDN1. Western blotting was used to determine the expression of LC3B I and II. (B) Morphology and number of autophagosomes in A549 cells, A549/CDDP cells, and A549/CDDP cells transfected with siR-NC or siR-CLDN1.Confocal microscopy was used to observe autophagosomes. * P<0.05 compared with A549 cells; # P<0.05 compared with siR-NC group.
Figure 8
Effect of CLDN1 expression on the phosphorylation level of ULK1. Western blotting was performed to measure the expression of ULK1 and phosphorylated ULK1 in A549/CDDP cells in siR-NC, siR-CLDN1, or CLDN1 overexpression groups. * P<0.05 compared with siR-NC group.
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