| Objective:Lung cancers have been the leading cause of cancer-related death all over the world. Accounting for 80% of all lung cancers are non-small cell lung cancer(NSCLC). Most NSCLC patients have been diagnosed at advanced stage, the five year overall suvival rate was less than 15%. Platinum-based doubled chemotherapy strategy is a conventional therapy for NSCLC patients with advanced stage, but chemotherapies have low efficacy with side effects. Recent advances in molecular targeted therapies have provided alternative therapeutic options for locally advanced or metastatic NSCLC. The epidermal growth factor receptor(EGFR) belongs to the receptor tyrosine kinase HER family which consist of four members of HER-1/EGFR, HER-2, HER-3 and HER-4. After binding with its ligand, EGFRs dimerize by homo-dimerization or hetero-dimerization, lead to EGFR intracellular area automatically phosphorylation and subsequently activation of a series of downstream signaling pathways, such as Ras-Raf-MAPK, PI3K-Akt pathways, which promote cancer proliferation, differentiation, migration, invasion and so on. Recent studies have shown that epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs), such as gefitinib and erlotinib, show notable effects against non-small cell lung cancers(NSCLCs) harboring EGFR-activating mutations in exons 19(deletion mutation), exon 21(L858R substitution mutation). Thus EGFR-TKIs have been widely used in the clinical treatment of advanced NSCLC. However, the majority of NSCLC patients who initially respond eventually acquire resistance to EGFR-TKIs. These reports suggested that there may be other molecules affect EGFR signaling pathways, which play an important role in NSCLC. Caveolin-1(Cav-1), a 21-24 KDa scaffold protein, is a structural component of caveolae. It is composed of 178 amino acid residues, where both C-terminal and N-terminal are exposed to the cytoplasm, hydrophobic amino acid residues(102-134), which located in the middle of the peptide chain, form a hairpin structure, anchor Cav-1 on the cell membrane. N-terminal is the main functional region of Cav-1, binding to EGFR, participated in proliferation, adhesion, migration, and signal pathways transduction. Therefore, we hypothesize that the expression of Cav-1 may affect the sensitivity to EGFR-TKIs in NSCLC. In order to verify the hypothesis whether the amounts of Cav-1 expression influence the sensitivity to EGFR-TKIs, the following experiments were performed in NSCLC cell line PC-9 cells:(1)Stably transfected cell line with overexpression of Cav-1(PC-9/Cav-1) and control cell line(PC-9/pc DNA3.1) were established and verified by Western blot.(2) Effects of erlotinib on the biological behavior of stable cell lines, including proliferation, migration and invasion were examined by MTS, wound healing assay and transwell cell invasion assay.(3)Western blot was used to detect the effects of erlotinib on the inhibition of EGFR phosphorylation and EGF-induced downstream EGFR signaling.Methods:1 Proving the stably transfected cell lines with overexpression of Cav-1 by Western blot The expression efficiency of Cav-1 was measured by Western blot. 2 Proliferation assay MTS assay was used to examine the proliferation abilities of stably transfected cell lines affected by erlotinib. PC-9/Cav-1 and PC-9/pc DNA3.1 cells were seeded in 96-well plates, respectively. Cells were treated with different concentrations of erlotinib(0, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32μmol/L) and continue to culture for 48 h, MTS assay was used to measure the OD values and to calculate the growth inhibition rate and the half maximal inhibitory concentration(IC50) of erlotinib. 3 Migration assay Wound healing assay was used to examine the migration abilities of stably transfected cell lines affected by erlotinib. PC-9/Cav-1 and PC-9/pc DNA3.1 cells were seeded in 24-well plates, respectively. After 24 hours cells were scratched with 10μl pipette tips and washed with PBS, then treated with or without erlotinib. Images of cells at the same field were taken every 6 hours until the closure of the scratch. The migration rate of cells were calculated by the following formula: Cell migration rate =(initial width of the scratch- current width) /2/ initial width of the scratch×100%. 4 Invasion assay The invasion assays were carried out using Transwell chamber with 10 mm diameter and 8μm pore size polycarbonate membrane coated with matrigel. After starved with serum-free 1640 medium for 4 hours, PC-9/Cav-1 and PC-9/pc DNA3.1 cells were seeded into upper chamber with serum-free 1640 medium with or without erlotinib, respectively. Lower chamber were added into 10% serum 1640 medium. After incubated for 48 hours, the invaded cells were fixed with 95% ice-cold ethanol and then stained with hematoxylin and eosin(HE). 5 Western blotting PC-9/Cav-1 cells and PC-9/pc DNA3.1 cells were seeded into 35 mm dishes. After the treatment of erlotinib(0.04 μmol/L) for different time(0 hour and 4 hour), total proteins were extracted from cells. Western blot was used to examine the levels of Cav-1, p-EGFR, EGFR, p-ERK, and ERK, p-Akt, and Akt. β-actin was used as a loading control.Results:1 Cav-1 protein levels of stably transfected cell lines The relative levels of Cav-1 protein in PC-9/Cav-1 cells(1.74±0.06) were significantly higher than in PC-9/pc DNA3.1 group cells(0.61±0.99)(P<0.05). 2 The sensitivity to erlotinib in stably transfected cell lines 2.1 The IC50 value of erlotinib in stably transfected cell lines The cell growth inhibition rates of PC-9/Cav-1 cells at different concentrations(0, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32μmol/L) of erlotinib were markedly lower than those of PC-9/pc DNA3.1 cells, there was significant difference in the cell growth inhibition between the two cell lines(P<0.05, respectively). The IC50 value of PC-9/Cav-1 cells(0.06±0.01) were significantly higher than that of PC-9/pc DNA3.1 cells(0.03±0.01)(P<0.05). These results indicated that the sensitivity of erlotinib in PC-9/Cav-1 cells was lower than PC-9/pc DNA3.1 cells. 2.2 Migration abilities of stably transfected cell lines treated with or without erlotinib Migration rates of PC-9/Cav-1 cells without erlotinib for 6h, 12 h and 18h(14.81±0.84%,27.98±0.73%,39.34±0.54%) were significantly higher than that of PC-9/pc DNA3.1(12.89±0.81%,19.93±1.99%,26.07±0.68%)(P<0.05, respectively). Migration rates of PC-9/Cav-1 cells with erlotinib(0.04μmol/L) exposure for 6h, 12 h and 18h(10.66±1.19%,14.61±2.23%,19.77±0.83%) were significantly higher than that in PC-9/pc DNA3.1 cells(4.80±1.04%,9.34±1.77%,14.14±2.61%)(P<0.05, respectively). 2.3 Invasion abilities of stably transfected cell lines treated with or without erlotinib The average invasion cell numbers of PC-9/Cav-1 cells without erlotinib(79.00±4.60) were significantly more than that in PC-9/pc DNA3.1 cells(56.33±6.50)(P<0.05). The average invasion cell numbers of PC-9/Cav-1 group with erlotinib(0.04μmol/L) exposure(49.00±3.60) were significantly more than that in PC-9/pc DNA3.1 group(34.33±4.90)(P<0.05). 3 Levels of EGFR phosphorylation and the downstream signal molecules in stably transfected cell lines treated with erlotinib The levels of different proteins in stably transfected cells were detected by Western blot. After treating with erlotinib for 0μmol/L and 0.04μmol/L, the levels of p Y-EGFR in PC-9/Cav-1 group( 2.25±0.29, 2.01±0.13) were significantly higher than PC-9/pc DNA3.1 group(1.54±0.09, 1.02±0.05)(P<0.05, respectively). The levels of p-ERK in PC-9/Cav-1 group(2.24±0.05,0.80±0.15) were significantly higher than PC-9/pc DNA3.1 group(1.36±0.02,0.28±0.02)(P<0.05, respectively). The levels of p-Akt in PC-9/Cav-1 group(0.52±0.03,0.30±0.03) were significantly higher than PC-9/pc DNA3.1 group(0.83±0.02,0.64±0.01)(P<0.05, respectively).Conclusion:1 Cav-1reduce inhibitory effects to erlotinib in PC-9 cells on the proliferation, migration and invasion abilities. 2 Cav-1 reduce the sensibility to erlotinib in PC-9 cells and the inhibitory effects of erlotinib in PC-9 cells via decreasing the inhibition of erlotinib on the EGFR phosphorylation and its downstream Ras/Raf/MEK/ERK and PI3K/Akt signal pathways. |
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