Cigarette Smoke Induces Gefitinib Resistance in NSCLC Cells via ROS/Sirt3/SOD2 Pathway / 中国肺癌杂志

BACKGROUND@#Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC.@*METHODS@#PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8.@*RESULTS@#CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05).@*CONCLUSIONS@#The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.

BMXΔN mediates gefitinib resistance of lung cancer cells through ERK/MAPK signaling pathway / 国际肿瘤学杂志

Objective:To explore the mechanism of a novel BMX splicing variant induced epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) gefitinib resistance in lung cancer.Methods:Stable transgenic cell line PC9-BMXΔN and HCC827-BMXΔN were constructed by lentivirus infection of PC9 and HCC827 cells carrying EGFR mutation. The cells were divided into PC9-Vec group (PC9 cells transfected with empty vector), PC9-BMX group (PC9 cells stably expressing BMX), PC9-BMXΔN group (PC9 cells stably expressing BMXΔN) and HCC827-Vec group (HCC827 cells transfected with empty vector), HCC827-BMXΔN group (HCC827 cells stably expressing BMXΔN). Quantitative real-time PCR was used to detect the expression levels of mRNA. The protein expression levels in each group were detected by Western blotting. The cells in the PC9-Vec group and PC9-BMXΔN group were treated with 0, 0.01, 2.00, 50.00, 100.00, 200.00 nmol/L and 2.00, 4.00 μmol/L gefitinib. The cells in the HCC827-Vec group and HCC827-BMXΔN group were treated with 0, 0.01, 1.00, 10.00, 100.00 nmol/L and 1.00 μmol/L gefitinib. MTT method was used to detect cell viabilities.Results:The PC9-BMXΔN cells were scattered and showed a fibroblast-like morphology. Compared with the PC9-Vec cells, the relative expression levels of fibronectin, N-cadherin, vimentin, Snail, Slug and TWIST 2 mRNA in PC9-BMXΔN cells were up-regulated. Compared with the PC9-Vec cells and PC9-BMX cells, the expression levels of fibronectin and vimentin protein in PC9-BMXΔN cells were up-regulated; while the expression level of E-cadherin protein in PC9-BMXΔN cells was significantly down-regulated. Compared with the PC9-Vec cells, the cell viabilities of PC9-BMXΔN cells treated with 0.01 nmol/L [(99.11±2.16)% vs. (91.29±1.91)%, t=-4.701, P=0.011], 2.00 nmol/L [(80.41±1.48)% vs. (63.36±2.14)%, t=-11.324, P<0.001], 50.00 nmol/L [(80.83±5.38)% vs. (60.22±3.61)%, t=-5.507, P=0.005], 100.00 nmol/L [(75.54±3.46)% vs. (59.93±1.91)%, t=-6.836, P=0.002], 200.00 nmol/L [(77.57±6.53)% vs. (56.70±2.88)%, t=-5.064, P=0.007], 2.00 μmol/L [(70.22±3.45)% vs. (53.14±0.89)%, t=-8.309, P=0.001], 4.00 μmol/L [(68.66±4.67)% vs. (52.30±2.59)%, t=-4.882, P=0.008] gefitinib were significantly increased, with statistically significant differences. Similarly, compared with the HCC827-Vec cells, the cell viabilities of HCC827-BMXΔN cells treated with 1.00 nmol/L [(64.36±2.49 )% vs. (47.13±4.21)%, t=-7.067, P=0.019], 10.00 nmol/L [(63.25±5.87)% vs. (43.28±2.95)%, t=-5.267, P=0.006], 100.00 nmol/L [(49.47±5.74)% vs. (37.12±4.92)%, t=-2.830, P=0.047], 1.00 μmol/L [(49.05±3.34)% vs. (32.06±4.73)%, t=-5.073, P=0.007] gefitinib were significantly increased, with statistically significant differences. Gefitinib treatment could significantly inhibit the expression levels of p-EGFR and p-ERK1/2 both in PC9-Vec cells, PC9-BMX cells and PC9-BMXΔN cells. Compared with the PC9-Vec cells and PC9-BMX cells, the expression level of p-EGFR in PC9-BMXΔN cells was significantly increased after gefitinib treatment for 8 h (0.91±0.04 vs. 0.81±0.04 vs. 0.80±0.05, all P<0.05); the expression levels of p-ERK1/2 in PC9-BMXΔN cells were significantly increased after gefitinib treatment for 2 h (0.64±0.06 vs. 0.38±0.12 vs. 0.37±0.14), 4 h (1.28±0.06 vs. 1.08±0.06 vs. 1.11±0.07), and 8 h (0.75±0.04 vs. 0.55±0.05 vs. 0.60±0.07), with statistically significant differences (all P<0.05). Conclusion:BMXΔN is involved in EGFR-TKI gefitinib resistance in lung cancer, which may be achieved by inducing cells to undergo epithelial-mesenchymal transition and activating the ERK/MAPK signaling pathway.

Targeting PAK2 Reverses Gefitinib Resistance in Non-small Cell Lung Cancer Cells / 中国生物化学与分子生物学报

EGFR tyrosine kinase inhibitor (EGFR-TKI)-targeted therapy has been playing an important role in the treatment of non-small cell lung cancer (NSCLC). However, unavoidable therapeutic resistance significantly limits the clinical efficacy of TKI. As an important member of the PAK family of serine/threonine kinases, p21-activated protein kinase 2 (PAK2) plays a critical role in tumorigenesis and tumor development. The aim of this work is to investigate the effect and molecular mechanism of inhibition of PAK2 on the reversal of gefitinib resistance in NSCLC. Firstly, Western blotting was used to detect the expression and phosphorylation level of PAK2 in gefitinib-resistant HCC827/GR cells. The results showed that the phosphorylation level of PAK2 was significantly increased in HCC827/GR cells compared with HCC827 cells (P < 0. 01), while the total protein level of PAK2 did not change. Then, HCC827/GR cells were treated with PAK2 inhibitors FRAX597 or G5555. The MTS assay and clone formation assay results showed that FRAX597 or G5555 significantly increased the sensitivity of HCC827/GR cells to gefitinib (P < 0. 01). Flow cytometry analysis showed that treatment of FRAX597 could induce cell cycle arrest in G

FOXC1 Knockdown Reverses Gefitinib Resistance in Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#Lung cancer is the malignant tumor with the highest incidence and mortality in China, among which non-small cell lung cancer (NSCLC) accounts for about 80%. Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeted therapy has been playing an important role in treatment of NSCLC. However, unavoidable therapeutic resistance significantly limits the clinical efficacy of EGFR-TKI. As a key member of the forkhead box protein family, FOXC1 is aberrantly expressed in NSCLC and involved in NSCLC progression. The aim of this work is to investigate the effect and potential mechanism of FOXC1 on gefitinib resistance in NSCLC.@*METHODS@#Western blot was performed to assess the expression of FOXC1 protein in HCC827/GR cells. Immunohistochemistry (IHC) assays were performed in human NSCLC tissues with gefitinib resistance. HCC827/GR cells were transfected with shRNA specifically targeting FOXC1 mRNA and stable cell lines were established. The effects of FOXC1 on cell viability and apoptosis were analyzed using a new methyl thiazolyl tetrazolium assay (MTS assay) and flow cytometry. Self-renewal ability was determined by mammosphere-formation analysis. Quantitative real-time PCR (qRT-PCR) and Western blot were employed to detect the expression of SOX2, Nanog, OCT4 and CD133. Flow cytometry analysis were further used to detect the level of CD133. IHC assays were used to detect the levels of SOX2 and CD133 in NSCLC tissues with genfitiinb resistance. Correlations of the expressions of FOXC1, CD133 and SOX2 with each other in lung adenocarcinoma samples were analyzed based on The Cancer Genome Atlas (TCGA) database.@*RESULTS@#The expression of FOXC1 is significantly increased in HCC827/GR cells compared with HCC827 cells (P<0.05). IHC results showed FOXC1 was highly expressed in NSCLC tissues with gefitinib resisitance. Knockdown of FOXC1 significantly increased the sensitivity of HCC827/GR cells to gefitinib. The cell viability was decreased and the apoptosis was promoted (P<0.05). Moreover, FOXC1 knockdown apparently inhibited the expression of SOX2 and CD133, and decreased the mammosphere-formation capacity in HCC827/GR cells. In NSCLC tissues with gefitinib resistance, the expressions of SOX2 and CD133 were significantly higher compared with gefitinib-sensitive tissues (P<0.01). Meanwhile, the expressions of FOXC1, CD133 and SOX2 with each other were positively correlated (P<0.05).@*CONCLUSIONS@#FOXC1 could increase gefitinib resitance in NSCLC, by which mechanism is related to the regulation of cancer stem cell properties.

Effect and significance of sunitinib on gefitinib resistance of lung cancer cells / 中国药学杂志

OBJECTIVE: To explore effection and significance of sunitinib on gefitinib resistance of lung cancer cells. METHODS: Cell proliferation inhibition of 28 gefitinib resistance lung cancer patients tumor tissues extracted with sunitinib treatment were measured using MTT assay. After sunitinib treatment, sensitivity of gefitinib resistance cells were measured by CellTiter-Glo method;The accumulation of Rh-123 in gefitinib resistant cells with sunitinib treatment were measured by FCM method. Accumulation of multi-drug resistance protein (MDR), transcription factors κB(NF-κB)and vascular endothelial growth factor(VEGF) were examined using Western blotting after sunitinib treatment on gefitinib resistance cells. RESULTS: Cell proliferation were inhibited by dose dependent after sunitinib treatment, IC50 is (6.73±1.57) μmol·L-1. Sensitivity of gefitinib resistance cells were significantly increased, and decreased the IC50 of gefitinib for inhibiting resistance cells after 1 and 2 μmol·L-1 sunitinib combine with gefitinib treatment, IC50 of gefitinib is (38.64±1.29) and (20.37±1.75) μmol·L-1 respectively. The accumulation of Rh-123 in gefitinib resistant cells with 1 μmol·L-1 sunitinib treatment were significantly increased(P<0.05), fluorescent density increased by 2.44 times following treatment with 1 μmol·L-1 of sunitinib, accumulation of Rh-123 in gefitinib resistant cells with 2 μmol·L-1 sunitinib treatment is larger significantly increased(P<0.01), fluorescent density increased by 5.64 times following treatment with 2 μmol·L-1 of sunitinib; expression level of MDR, NF-κB and VEGF proteins were significantly reduced after 1 and 2 μmol·L-1 sunitinib treatment. CONCLUSION: Sunitinib is able to reverse the gefitinib resistance, the theoretical foundation is provided for clinical treatment of patients with gefitinib resistance.

miR-124 modulates gefitinib resistance through SNAI2 and STAT3 in non-small cell lung cancer / 华中科技大学学报（医学）（英德文版）

Gefitinib is used as a first-line treatment for advanced non-small cell lung cancer (NSCLC). Unfortunately, most NSCLC patients inevitably develop gefitinib resistance during treatment. In addition to EGFR mutation status, the mechanisms involved are largely unknown. In this study, we showed that miR-124, a tumor suppressor, was significantly down-regulated in gefitinib-resistant NSCLC patients and cell lines compared with gefitinib-sensitive patients and cell lines. In addition, the miR-124 depletion induced gefitinib resistance, and miR-124 overexpression sensitized gefitinib-resistant cells to gefitinib. Mechanistic analysis revealed that miR-124 decreased SNAI2 and STAT3 expression by directly targeting their 3'UTRs and that knocking down SNAI2 or STAT3 partly reversed the gefitinib resistance induced by miR-124 depletion. Our data demonstrate that the miR-124 plays a new critical role in acquired resistance to gefitinib and that the manipulation of miR-124 might provide a therapeutic strategy for reversing acquired gefitinib resistance.