1.Gefitinib inhibits glycolysis and induces programmed cell death in non-small cell lung cancer cells.
Qiao ZHOU ; Jiahui LI ; Jinlong PANG ; Fangtian FAN ; Shanshan LI ; Hao LIU
Journal of Southern Medical University 2020;40(6):884-892
OBJECTIVE:
To observe the cell death pattern induced by gefitinib in non-small cell lung cancer A549 and H1975 cells and explore the possible mechanism in light of glycolysis.
METHODS:
The inhibitory effects of gefitinib at 20, 30, or 40 μmol/L in A549 cells and at 20, 40, or 80 μmol/L in H1975 cells were examined using MTT assay. The changes of lactic acid level in the cells were determined with a lactic acid kit, and the expression levels of glycolysis-related proteins (PKM2 and HK2) and the proteins in PI3K-Akt-mTOR signaling pathway were detected using Western blotting. 2-NBDG was used for detecting glucose uptake capacity of the cells, and ATP kit was used to detect the intracellular ATP level. The mitochondrial membrane potential of the cells was examined with the JC-1 kit, and cell apoptosis was analyzed with Annexin V-FITC/PI double staining. The relative expression levels of the apoptotic proteins Bax and Bcl-2 and the autophagy marker protein LC3B were detected with Western blotting.
RESULTS:
MTT assay showed that gefitinib inhibited the proliferation of A549 and H1975 cells in a time- and dose-dependent manner ( < 0.05). The IC of gefitinib at 24, 48 and 72 h was 48.6, 28.6 and 19.7 μmol/L in A549 cells and was 321.6, 49.1 and 14.6 μmol/L in H1975 cells, respectively. Gefitinib significantly lowered intracellular lactic acid level of the cells ( < 0.05) and down-regulated the expressions of PKM2 and HK2 proteins ( < 0.05) and PI3K-Akt-mTOR signaling pathway-associated proteins ( < 0.05). Gefitinib obviously inhibited glucose uptake and ATP levels in both A549 and H1975 cells ( < 0.05). Treatment with gefitinib induced obviously enhanced apoptosis in the cells, resulting in apoptosis rates of (10.77± 1.0)%, (14.5±0.4)%, (17.4±0.2)% and (32.1±0.6)% at 0, 20, 30 and 40 μmol/L in A549 cells ( < 0.05) and of (10.5±0.6)%, (13.2± 0.92)%, (18.9±0.98)% and (35.1±1.4)% at 0, 20, 40 and 80 μmol/L in H1975 cells, respectively ( < 0.05). The protein expression of Bax increased and that of Bcl-2 decreased following gefitinib treatment in the cells ( < 0.05). Gefitinib significantly increased autophagy in A549 and H1975 cells as shown by increased LC3B expressions following the treatment ( < 0.05).
CONCLUSIONS
Gefitinib can inhibit the proliferation, induce apoptosis and increase autophagy in A549 and H1975 cells. Gefitinib induces apoptosis of the cells possibly by affecting glycolysis and PI3K-Akt-mTOR signaling pathway.
Apoptosis
;
Carcinoma, Non-Small-Cell Lung
;
Cell Line, Tumor
;
Cell Proliferation
;
Gefitinib
;
Glycolysis
;
Humans
;
Lung Neoplasms
;
Phosphatidylinositol 3-Kinases
2.Effect of down-regulation of growth arrest and DNA damage inducible protein 45β on PC9 lung adenocarcinoma cells.
Hao HU ; Kailin QUE ; Hao PENG ; Jia LIU ; Cheng HAN ; Na ZHANG ; Tao HOU ; Chunhong HU ; Jin'an MA
Journal of Central South University(Medical Sciences) 2018;43(11):1209-1215
To explore the effect of down-regulation of growth arrest and DNA damage inducible protein 45β (GADD45β) on the PC9 lung adenocarcinoma cells.
Methods: GADD45β gene siRNA sequence was designed and synthesized, which was transfected into PC9 lung adenocarcinoma cells through lentivirus transfection. Quantitative real-time PCR (qRT-PCR) and Western blot are used to examine the mRNA and protein levels of GADD45β in PC9 cells before and after the transfection. Annexin V-allophycocyanin (APC) double-staining flow cytometry was used to detect the apoptosis level after the transfection. The intracellular DNA content after transfection was detected by flow cytometry. The percentage of the cells at each period of cell cycle was calculated, and the effect of RNA interference on the cell growth were analyzed. The effects of RNA interference on the tumor-formation ability of cells were tested by counting the number of clones. MTT assay was used to test the half maximal inhibitory concentration (IC50) of PC9 cells for gefitinib.
Results: The 5'-AAATCCACTTCACGCTCAT-3' sequence was identified as the effective sequence for GADD45β gene RNA interference. The mRNA and protein expression levels of GADD45β were markedly decreased (both P<0.05) at 48 h after transfection of GADD45β-siRNA, which resulted in the increased apoptosis rate (P<0.05), decreased tumor clone number (P<0.05) and increased percentage of PC9 cell at the S stage and G2/M stage (P<0.05). The IC50 for gefitinib was decreased obviously (P<0.05).
Conclusion: Down-regulation of GADD45β can reduce the colony-forming ability of PC9 cells, promote the cell apoptosis, and enhance the sensitivity of PC9 cells to gefitinib.
Adenocarcinoma of Lung
;
Antigens, Differentiation
;
genetics
;
metabolism
;
Antineoplastic Agents
;
pharmacology
;
Apoptosis
;
drug effects
;
Cell Line, Tumor
;
Cell Proliferation
;
Down-Regulation
;
Gefitinib
;
pharmacology
;
Humans
;
RNA, Small Interfering
4.Expression of Acetaldehyde Dehydrogenase in Gefitinib-resistant Human Lung Adenocarcinoma HCC-827/GR Cells.
Tingting YANG ; Jingjing GU ; Ting LIU ; Haibin MA ; Xiaona MA ; Jin TAO ; Yiran JIN ; Xueyun LIANG
Chinese Journal of Lung Cancer 2018;21(6):431-436
BACKGROUND:
Tumor recurrence and drug resistance are the main causes of death in tumor patients. The family of acetaldehyde dehydrogenase (ALDH) is closely related to the proliferation, migration, invasion and resistance of tumor cells, and different ALDH subtypes are expressed in different tumor cells. The aim of this study is to elucidate the ALDH subtype in human lung adenocarcinoma HCC-827/GR cells, which resistant to the gefitinib.
METHODS:
The human lung adenocarcinoma HCC-827 cells were used to generate the gefitinib-resistant HCC-827/GR cells; the expression of ALDH subtype in either HCC-827 or HCC-827/GR was detected by flow cytometry; The proliferative capacity and sensitivity to gefitinib of hcc-827/GR cells were analyzed by MTT assay before and after treatment with 100 μmol/L diethyllaminaldehyde (DEAB); Real-time quantitative PCR was used to detect the expression of ALDH subtypes at mRNA levels in hcc-827 cells and hcc-827/GR cells.
RESULTS:
Compared with HCC-827 cells, the positive rate of ALDH in HCC-827/GR cells increased. The proliferation ability of HCC-827/GR cells decreased after treatment with 100 μmol/L DEAB. Compared with HCC-827 cells, the expression of ALDH1A1 and ALDH1L1 mRNA was increased in hcc-827/GR cells, but the ALDH3B2 expression was decreased.
CONCLUSIONS
ALDH might be used as a molecular biomarker to test the gefitinib-resistant to lung adenocarcinoma cancer cells, and the ALDH1A1 may play a role in gefitinib resistance in lung cancer.
Adenocarcinoma
;
pathology
;
Adenocarcinoma of Lung
;
Aldehyde Oxidoreductases
;
antagonists & inhibitors
;
genetics
;
Cell Line, Tumor
;
Drug Resistance, Neoplasm
;
genetics
;
Enzyme Inhibitors
;
pharmacology
;
Gefitinib
;
Gene Expression Regulation, Neoplastic
;
drug effects
;
Humans
;
Lung Neoplasms
;
pathology
;
Quinazolines
;
pharmacology
5.Role of EZH2 Inhibitor Combined with Gefitinib in EGFR-TKIs Resistant Lung Cancer Cells.
Hao GONG ; Yin YUAN ; Yongwen LI ; Hongbing ZHANG ; Ying LI ; Weiting LI ; Pan WANG ; Ruifeng SHI ; Chao LIU ; Liyuan CUI ; Hongyu LIU ; Jun CHEN
Chinese Journal of Lung Cancer 2019;22(5):255-263
BACKGROUND:
Lung cancer is one of the common malignant tumors that impair human health. With the development of epigenetics, the researchers found that enhancer of Zeste homolog 2 (EZH2) is highly expressed in lung cancer tissue and its expression is closely related to the prognosis. EZH2 inhibitor can also enhance the sensitivity of tumor cells to a variety of anti-tumor drugs. The purpose of this study is to investigate the effect of combination of EZH2 inhibitor and gefitinib on the proliferation, apoptosis and migration of Gefitinib-resistant lung cancer cells.
METHODS:
PC9 and PC9/AB2 cells were used for this study. CCK-8 and EdU experiment were used to detect combined treatment on cell viability and proliferation activity; Wound healing assay and Transwell chamber experiment were used to determine the effects of combination therapy on cell migration ability; Flow cytometry was used to detect the effect of combination therapy on EZH2 and apoptosis; Western blot was used to observe the effect of combination therapy on epidermal growth factor receptor (EGFR) signaling pathway-related proteins expression.
RESULTS:
In gefitinib-resistant cell line PC9/AB2, gefitinib combined with EZH2 inhibitor GSK343 can significantly inhibit cell viability, reduce cell migration and increase cell apoptosis. At the same time, combination therapy can significantly inhibit the expression of EZH2 and phosphorylation EGFR proteins.
CONCLUSIONS
The combination of EZH2 inhibitor GSK343 and gefitinib sensitize PC9/AB2 cell to gefitinib response. This study also suggests that synergistic therapy plays a role in the reversal of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) resistance in lung cancer.
Antineoplastic Agents
;
pharmacology
;
Cell Line, Tumor
;
Cell Movement
;
drug effects
;
Cell Proliferation
;
drug effects
;
Cell Survival
;
drug effects
;
Drug Resistance, Neoplasm
;
drug effects
;
Drug Synergism
;
Enhancer of Zeste Homolog 2 Protein
;
antagonists & inhibitors
;
ErbB Receptors
;
antagonists & inhibitors
;
Gefitinib
;
pharmacology
;
Humans
;
Lung Neoplasms
;
pathology
;
Protein Kinase Inhibitors
;
pharmacology
6.Clinical Observation of Gefitinib with Pericardial Perfusion for Advanced Non-small Cell Lung Cancer.
Xiaomeng WANG ; Jin CHEN ; Jiaqi YAO ; Renhua GUO
Chinese Journal of Lung Cancer 2018;21(1):37-42
BACKGROUND:
Epidermal growth factor receptor (EGFR) mutation non-small cell lung cancer (NSCLC) is an important subtype of lung cancer. The incidence of malignant pericardial effusion (MPCE) in EGFR-mutant NSCLC patients is high. However, there are few researches on the treatmentof this type of patients.
METHODS:
We collected data on clinical characteristics and treatment of advanced NSCLC patients who harboring EGFR mutants and MPCE between January 2010 and December 2016. The treatments were divided into three groups: oral gefitinib combined with pericardial perfusion of hydroxycamptotheci (HCPT) group (gefitinib/HCPT); intravenous chemotherapy combined with pericardial perfusion of HCPT group (chemotherapy/HCPT) and gefitinib monotherapy group. And we retrospectively analyzed patients' outcomes in three groups.
RESULTS:
In 273 advanced NSCLC patients with EGFR mutations, 29 cases had pericardial effusion, among which 6 patients with small amount of pericardial effusion were excluded, and 23 patients were analyzed. Median pericardium progression free survival (PFS) was 247 days. PFS for gefitinib/HCPT group (460 days) was superior to PFS for chemotherapy/HCPT group (94 days, P=0.008) and gefitinib monotherapy group (131 days, P=0.032). As for the efficacy of primary pulmonary lesions, the efficacy in gefitinib/ HCPT group was superior to chemotherapy/HCPT group [objective response rate (ORR): 33.3% vs 12.5%; disease control rate (DCR): 86.7% vs 62.5%]. There is no difference of ORR and DCR between gefitinib/HCPT group and gefitinib monotherapy group. No obvious adverse reaction was observed in all three groups.
CONCLUSIONS
First-line gefitinib therapy combined with pericardial perfusion of HCPT can improve pericardium PFS for advanced NSCLC patients who harboring EGFR mutants andmalignantpericardial effusion. This finding should be confirmed further through multicenter, prospective clinical trials with large sample size.
Adult
;
Aged
;
Aged, 80 and over
;
Carcinoma, Non-Small-Cell Lung
;
complications
;
drug therapy
;
metabolism
;
pathology
;
Disease-Free Survival
;
ErbB Receptors
;
metabolism
;
Female
;
Gefitinib
;
Humans
;
Lung Neoplasms
;
complications
;
drug therapy
;
metabolism
;
pathology
;
Male
;
Middle Aged
;
Perfusion
;
Pericardial Effusion
;
complications
;
Pericardium
;
Quinazolines
;
administration & dosage
;
therapeutic use
;
Retrospective Studies
;
Treatment Outcome
7.STE029 Overcomes EGFR-TKI Resistance in Human Lung Adenocarcinoma.
Lin HUANG ; Mei HOU ; Jiewei LIU ; Yang LI ; Wang SHEN ; Qinghua ZHOU
Chinese Journal of Lung Cancer 2022;25(11):771-781
BACKGROUND:
Acquired and primary resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is still the bottleneck of clinical treatment of advanced non-small cell lung cancer (NSCLC). STE029 is a novel anticancer drug which consists of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) inhibitor and novel cancer cell membrane targeting molecular. This study aimed to investigate the reversal mechanism of EGFR-TKI resistance by STE029 in lung adenocarcinoma.
METHODS:
CCK8 test was used to test the cell viability and survival rate of EGFR mutated PC9 cell (Gefitinib sensitive), PC9/BB4 cell (acquired Gefitinib resistant), and EGFR wild type A549 cell after treatment of STE029, Gefitinib or combination of both. EdU test was applied to detect changes in cell cycle and Hoechst 33258 was applied to detect apoptosis rate in overcoming the EGFR-TKI resistance. The activity of EGFR/PI3K/Akt, cell cycle and apoptosis signal pathways were examined. In vivo, nude mice were exposed to STE029, Gefitinib and STE029+Gefitinib for 5 wk. And the the tumor volume was measured and tumor weight was obtained on the last day.
RESULTS:
(1) PC9 cells was highly sensitive to Gefitinib, while PC9/BB4 and A549 cell showed significant resistance to Gefitinib treatment; (2) STE029+Gefitinib treatment could significantly decrease the 50% inhibitory concentrarion (IC₅₀) of Gefitinib in PC9, PC9/BB4 and A549 cells (P<0.05, respectively); (3) In PC9 and PC9/BB4 cells, STE029+Gefitinib can block cell cycle and inhibit cell proliferation (P<0.001), while there was no significant difference in apoptosis rate among three drug intervention groups (P>0.05); However, apoptosis rate was increased in STE029+Gefitinib group in A549 cell (P<0.01), while no significance detected in cell proliferation (P>0.05). (4) In PC9 and PC9/BB4 cells, the combination of STE029 and Gefitinib could downregulate p-EGFR, p-Akt, p-Cyclin D1 and Cyclin D1 (P<0.001), and upregulate the expression of GSK-3β (P<0.001), and the expression of cleaved caspase-8, caspase-8 cleaved caspase-9, caspase-9 showed no difference among groups (P>0.05). In A549 cells, the combination of STE029 and Gefitinib could downregulate p-Akt (P<0.001) and upregulate cleaved caspase-8 and cleaved caspase-9 (P<0.001); (5)In vivo, the combination of STE029 and Gefitinib effectively inhibited tumor development and progression compared to STE029 alone or Gefitinib alone, with significant difference (P<0.05) in PC9 and PC9/BB4 xenografted tumor.
CONCLUSIONS
STE029 could sensitize Gefitinib by inhibiting EGFR/PI3K/Akt pathway, blocking the tumor cell cycle and proliferation and inducing apoptosis through caspase-8 and caspase-9 dependent pathway. STE029 deserves further investigations in overcoming EGFR-TKI resistance in lung cancer.
Animals
;
Mice
;
Humans
;
Gefitinib/pharmacology*
;
Caspase 9
;
Caspase 8
;
Cyclin D1
;
Carcinoma, Non-Small-Cell Lung
;
Glycogen Synthase Kinase 3 beta
;
Mice, Nude
;
Phosphatidylinositol 3-Kinases
;
Proto-Oncogene Proteins c-akt
;
Lung Neoplasms/genetics*
;
Adenocarcinoma of Lung/drug therapy*
;
Protein Kinase Inhibitors/pharmacology*
;
ErbB Receptors/genetics*
8.Cigarette Smoke Induces Gefitinib Resistance in NSCLC Cells via ROS/Sirt3/SOD2 Pathway.
Yawan ZI ; Ke LIAO ; Hong CHEN
Chinese Journal of Lung Cancer 2023;26(4):245-256
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.
Humans
;
Gefitinib/therapeutic use*
;
Carcinoma, Non-Small-Cell Lung/metabolism*
;
Sirtuin 3/therapeutic use*
;
Lung Neoplasms/metabolism*
;
Reactive Oxygen Species/therapeutic use*
;
Antineoplastic Agents/therapeutic use*
;
Cigarette Smoking
;
Sincalide/therapeutic use*
;
ErbB Receptors/metabolism*
;
Drug Resistance, Neoplasm/genetics*
;
Cell Line, Tumor
9.Effect of SLC7A11 gene downregulation on the gefitinib resistance of lung adenocarcinoma PC9/GR cells and its mechanism.
Yun Long JIA ; Yan ZHAO ; Shu Man ZHEN ; Zi Shuo CHENG ; Bo Yang ZHENG ; Yue Ping LIU ; Li Hua LIU
Chinese Journal of Oncology 2023;45(9):779-786
Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.
Humans
;
Gefitinib/therapeutic use*
;
Antineoplastic Agents/therapeutic use*
;
Lung Neoplasms/pathology*
;
Down-Regulation
;
Quinazolines/therapeutic use*
;
Drug Resistance, Neoplasm/genetics*
;
ErbB Receptors/metabolism*
;
Adenocarcinoma of Lung
;
Protein Kinase Inhibitors/therapeutic use*
;
RNA, Small Interfering/genetics*
;
Cell Line, Tumor
;
Amino Acid Transport System y+/metabolism*
10.Effect of SLC7A11 gene downregulation on the gefitinib resistance of lung adenocarcinoma PC9/GR cells and its mechanism.
Yun Long JIA ; Yan ZHAO ; Shu Man ZHEN ; Zi Shuo CHENG ; Bo Yang ZHENG ; Yue Ping LIU ; Li Hua LIU
Chinese Journal of Oncology 2023;45(9):779-786
Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.
Humans
;
Gefitinib/therapeutic use*
;
Antineoplastic Agents/therapeutic use*
;
Lung Neoplasms/pathology*
;
Down-Regulation
;
Quinazolines/therapeutic use*
;
Drug Resistance, Neoplasm/genetics*
;
ErbB Receptors/metabolism*
;
Adenocarcinoma of Lung
;
Protein Kinase Inhibitors/therapeutic use*
;
RNA, Small Interfering/genetics*
;
Cell Line, Tumor
;
Amino Acid Transport System y+/metabolism*