To investigate the role of Janus kinase (JAK) inhibitor AG490 in the anti-proliferation and cell cycle in human retinoblastoma HXO-RB44 cell lines in vitro, and to explore its effect on the expression of JAK2/signal transducer and activator of transcription 3 (STAT3).
 Methods: Cells were divided into an experiment group and a control group, and the experiment group was further divided into 6 sub-groups according to different AG490 concentrations (6.25, 12.50, 25.00, 50.00 or 100.00 μmol/L). Cell proliferation in the different groups was analyzed by cell vitality determination. Cell cycle distribution and apoptosis rate were examined by flow cytometry. The protein levels of STAT3, p-STAT3 and vascular endothelial growth factor (VEGF) were detected by Western blot.
 Results: After 48 h treatment with AG490, the viability of HXO-RB44 cells was reduced in a concentration-dependent manner. Compared with the control group, there was no significant difference in the experiment groups except the 6.25 μmol/L group (all P>0.05). The apoptosis rates in the experiment groups were significantly increased with increase in concentration of AG490 compared with that in the control group (all P<0.05). The cell ratio in the G1 phase in 50 or 100 μmol/L group was increased, whereas the cell ratio in the S phase was decreased. Western blot results showed that the expressions of STAT3 and p-STAT3 in the experiment groups were dramatically reduced with the increase in concentration of AG490 compared with that in the control group (all P<0.05). VEGF expression didn't obviously change in the experiment groups with AG490 concentration less than 12.5 μmol/L compared with that in the control group (both P>0.05), but there were significant differences in the other experiment groups (all P<0.05). 
 Conclusion: JAK inhibitor AG490 can inhibit proliferation and promote apoptosis of the retinoblastoma HXO-RB44 cells through down-regulation of JAK2/STAT3 signaling pathway.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Janus Kinase 2 ; genetics ; metabolism ; Retinoblastoma ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; drug effects ; Tyrphostins ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism

To investigate the effect of diosgenin (Dgn) on chondrocytes and its relation to JAK2/STAT3 signaling pathway in mice with osteoarthritis (OA).Fifteen male C57BL/6 mice were randomly divided into three groups:control group, OA group and OA+Dgn group. After 4 weeks of treatment, the histopathological changes of cartilage tissue were observed by toluidine blue staining under light microscopy and the ultrastructure of chondrocytes was observed under electron microscopy. The primarily cultured chondrocytes of OA mice were randomly divided into 4 groups:(1) OA group, (2) Dgn group, (3) Dgn+AG490 group, (4) AG490 group. The expression of p-JAK2, p-STAT3, Bax, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) were detected by Western blotting, and superoxide dismutase (SOD) was detected using colorimetric method.The morphological observation showed that the chondrocytes of OA group presented considerable pathological changes, while the chondrocytes in OA+Dgn group maintained intact membrane. Electron microscopy observation found obvious injury in cartilage tissues of OA group, while that in OA+Dgn group remained smooth. Compared with OA group, the expressions of p-JAK2 and p-STAT3 in chondrocytes of Dgn group were increased (all<0.05), and the expressions of Bax protein, SDH, COX and SOD were decreased (all<0.05). While compared with Dgn group, the expressions of p-JAK2, p-STAT3, SDH, COX and SOD in chondrocytes of Dgn+AG490 group were decreased (all<0.05), and the expression of Bax protein was increased (<0.05).Diosgenin can inhibit apoptosis and increase mitochondrial oxidative stress capacity of chondrocytes in mice with osteoarthritis, which is closely related to the activation of JAK2/STAT3 signaling pathway.
Animals ; Apoptosis ; drug effects ; Cartilage ; drug effects ; pathology ; Chondrocytes ; chemistry ; drug effects ; pathology ; Diosgenin ; pharmacology ; Electron Transport Complex IV ; metabolism ; Janus Kinase 2 ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondria ; drug effects ; genetics ; Osteoarthritis ; genetics ; physiopathology ; Oxidative Stress ; drug effects ; STAT3 Transcription Factor ; drug effects ; Signal Transduction ; Succinate Dehydrogenase ; metabolism ; Superoxide Dismutase ; metabolism ; Tyrphostins ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate the effects of combined inhibition of signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor-1α (HIF-1α) in the enhancement of chemosensitivity of the model of human laryngeal squamous cacinoma in nude mice. METHOD: Model nude mice were divided into six groups randomly: control group(A) , cisplatin group(B) , cisplatin and AG490 group(C) , cisplatin and HIF-1α⁻/⁻ group (D), cisplatin combined AG490 and HIF-1α⁻/⁻ group (E), HIF-1α⁻/⁻ group (F) (only in calculating tumor inhibition rate). 3mg/kg cisplatin was administered by peritoneal injection for 3 days. Then cisplatin and 10 mg/kg AG490 were administered every other day for 12 days. The expression of Ki67 and HIF-1α was detected by immunocytochemical method. Western blot was used to detect the expression of p-STAT3. RESULT: The expression of HIF-1α in group C and group D were lower than that in group B, and there were significant difference respectively (t₁ = 2.782, t₂ = 3.873, P < 0.05); The expression of HIF-1α in group E was lower than that in group C and group D respectively, and there were significant difference respectively (t₁ = 6.140, t₂ = 3.667, P < 0.01). The expression level of p-STAT3 in group C was markedly lower compared with that in group B, and there were significant difference between them (t = 17.840, P < 0.01); There were no difference between the expression level of p-STAT3 in group D and that in group B (t = 0.038, P > 0.05); The expression level of p-STAT3 in group E was significantly lower compared with that in group C and group D respectively (P < 0.01). Tumor inhibition rate of group E was higher than that in group B, group C , as well as group D respectively and there were significant difference respectively (t₁ = 5.509, P < 0.01; t₂ = 3.422, P < 0.05; t₃ = 2.718, P < 0.05 ). Ki67 index of group E was lower than that in group B, group C as well as group D respectively and there were significant difference respectively(t₁ = 8.307, P < 0.01; t₂ = 3.736, P < 0.05; t₃ = 4.524, P < 0.01). CONCLUSION Combined inhibition of STAT3 and HIF-1α could enhance chemo-sensitivity in the model of human laryngeal squamous cacinoma in nude mice.
Animals ; Antineoplastic Agents ; pharmacology ; Carcinoma, Squamous Cell ; drug therapy ; metabolism ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Ki-67 Antigen ; metabolism ; Laryngeal Neoplasms ; drug therapy ; metabolism ; Mice ; Mice, Nude ; Neoplasms, Experimental ; drug therapy ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Tyrphostins ; pharmacology

The type 1 insulin-like growth factor receptor (IGF-1R) and its downstream signaling components have been increasingly recognized to drive the development of malignancies, including non-small cell lung cancer (NSCLC). This study aimed to investigate the effects of IGF-1R and its inhibitor, AG1024, on the progression of lung cancer. Tissue microarray and immunohistochemistry were employed to detect the expressions of IGF-1 and IGF-1R in NSCLC tissues (n=198). Western blotting was used to determine the expressions of IGF-1 and phosphorylated IGF-1R (p-IGF-1R) in A549 human lung carcinoma cells, and MTT assay to measure cell proliferation. Additionally, the expressions of IGF-1, p-IGF-1R and IGF-1R in a mouse model of lung cancer were detected by Western blotting and real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), respectively. The results showed that IGF-1 and IGF-1R were overexpressed in NSCLC tissues. The expression levels of IGF-1 and p-IGF-1R were significantly increased in A549 cells treated with IGF-1 as compared to those treated with IGF-1+AG1024 or untreated cells. In the presence of IGF-1, the proliferation of A549 cells was significantly increased. The progression of lung cancer in mice treated with IGF-1 was significantly increased as compared to the group treated with IGF-1+AG1024 or the control group, with the same trend mirrored in IGF-1/p-IGF-1R/IGF-1R at the protein and/or mRNA levels. It was concluded that IGF-1 and IGF inhibitor AG1024 promotes lung cancer progression.
Adult ; Aged ; Animals ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Proliferation ; Disease Models, Animal ; Disease Progression ; Female ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Male ; Mice ; Middle Aged ; Receptor, IGF Type 1 ; antagonists & inhibitors ; physiology ; Tyrphostins ; pharmacology

OBJECTIVETo investigate the role of TGF-beta1 and STAT3 signaling in liver fibrosis using a rat model system and to determine the therapeutic mechanism of AG490 in relation to this signaling pathway.

METHODSRats were randomly divided into a control group and DENA-induced liver fibrosis model group, and then subdivided into AG490 treatment groups. During fibrosis development, liver tissue samples were collected at different time points (0, 4 and 8 weeks) and evaluated according to the Scheuer scoring system. Expression of STAT3, TGFbeta1, alpha-SMA, E-cadherin, MMP2 and TIMP1 was measured by PCR (mRNA) and immunohistochemistry and western blotting (protein).

RESULTSIncreasing degrees of inflammation and fibrosis were observed in liver tissues of DENA-treated rats throughout model establishment. The mRNA expression of TGFbeta1 and STAT3 was significantly increased in DENA-induced rats with advanced fibrosis (AF) compared to those with early fibrosis (EF) (P = 0.034 and P = 0.012 respectively). The protein expression of TGF-beta1, phospho-Smad2, alpha-SMA, E-cadherin, STAT3 and phospho-STAT3 was significantly increased in DENA-induced rats with AF compared to the unmodeled control group (P = 0.048, P = 0.003, P = 0.002, P = 0.028, P = 0.009 and P = 0.039). The protein expression of E-cadherin was lower in the DENA-induced rats with AF than in those with EF (P = 0.026). STAT3 and TGF-beta1 co-expression was detected in AF tissues. DENA-induced AG490-treated rats with AF showed substantially lower protein expression of STAT3, TGF-beta1, MMP2 and TIMP1 compared to DENA-induced untreated rats with AF (P = 0.006, P = 0.018, P = 0.010 and P = 0.005); in addition, the degrees of fibrosis and inflammation were also greatly reduced in the DENA-induced AG490-treated rats with AF compared to DENA-induced untreated rats with AF (P = 0.042 and P = 0.021). Conclusions STAT3 signal transduction may regulate the TGF-beta1 pathway and affect liver fibrosis, especially in the advanced phase. AG490 can inhibit TGFbeta1-STAT3 signaling, resulting in reversal of liver fibrosis.

Animals ; Disease Models, Animal ; Liver Cirrhosis ; chemically induced ; metabolism ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Transforming Growth Factor beta1 ; metabolism ; Tyrphostins ; pharmacology

OBJECTIVETo investigate the antitumour molecular mechanisms of WP1066 (STAT-3 inhibitor ) to human tongue squamous cell carcinoma in vitro and in vivo.

METHODSWP1066 was used to inhibit the p-STAT-3 expression in Tscca human tongue squamous cell carcinoma cell line .Real-time PCR was used to detect the microRNA-21 expression after treatment with DMSO and WP1066. Methyl thiazolyl tatrozolium (MTT) assay was employed to determine cell survival. Flow cytometry (FCM) was used to measure apoptosis. The expression level of STAT-3/p-STAT-3, programmed cell death protein 4 (PDCD-4), antigen 67 (Ki-67), B cell lymphoma 2 (Bcl-2) and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) was examined by Western blotting. Luciferase reporter gene assay was conducted to verify the regulation of STAT-3 to microRNA-21. Tscca xenograft tumor model was established in BALB/c nude mice and the tumors were divided into control, DMSO and WP1066 treated groups. The tumor tissues were measured by immunohistochemistry stain and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay.

RESULTSSTAT-3/p-STAT-3 protein was suppressed after treatment with WP1066 (STAT-3:F = 15.336, P = 0.004, p-STAT-3: F = 52.837, P = 0.000). MicroRNA-21 relative expression level was down-regulated (F = 8.197, P = 0.019). Cell survival rate was significantly reduced after treatment with WP1066 than control groups (F = 94.388, P = 0.000). Early apoptosis rate increased after treatment with WP1066 (F = 217.080, P = 0.000) . PDCD-4 and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) protein expression was increased after treatment with WP1066 (PDCD-4:F = 8.771, P = 0.017; CCASP-3: F = 26.611, P = 0.001) .Ki-67 and Bcl-2 protein was down regulated (Ki-67:F = 5.854, P = 0.039; Bcl-2:F = 125.502, P = 0.000). Luciferase reporter gene assay proved that STAT-3 combined with specific promoter region of microRNA-21.In vivo, the tumor volume after treatment with WP1066 was significantly smaller than control groups by the end of observation (F = 15.390, P = 0.000) .Immunological histological chemistry indicated that PDCD-4 and CCASP-3 protein expression was up-regulated simultaneously while Ki-67 and Bcl-2 protein of tumor tissue was down-regulated after treatment with WP1066 than control groups. TUNEL assay suggested that apoptosis index rose after treatment with WP1066 than control groups (F = 133.368, P = 0.000) .

CONCLUSIONSWP1066 affected Tscca cancer cell proliferation and apoptosis via inhibiting STAT-3/microRNA-21.WP1066 provided new direction and possibility to human tongue squamous cell carcinoma therapy.

Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; biosynthesis ; Carcinoma, Squamous Cell ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Heterografts ; Humans ; In Vitro Techniques ; Mice ; Mice, Nude ; Pyridines ; pharmacology ; Real-Time Polymerase Chain Reaction ; STAT3 Transcription Factor ; biosynthesis ; Signal Transduction ; Tongue Neoplasms ; pathology ; Tyrphostins ; pharmacology

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.
Animals ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Survival ; drug effects ; ErbB Receptors ; antagonists & inhibitors ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; MCF-7 Cells ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Minor Histocompatibility Antigens ; Mitogen-Activated Protein Kinases ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Transplantation, Heterologous ; Tyrphostins ; pharmacology

OBJECTIVETo detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).

METHODSStat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed.

RESULTSSuppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012).

CONCLUSIONSIn a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Survival ; drug effects ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Neoplasm Grading ; Neoplasm Staging ; Phosphorylation ; RNA, Small Interfering ; genetics ; STAT3 Transcription Factor ; antagonists & inhibitors ; genetics ; metabolism ; Tyrphostins ; pharmacology

OBJECTIVETo explore the effects of EGFR-TKI AG1478 on the expression of FoxMl and FOXO3a genes in non-small cell cancer (NSCLC) cell lines, and explore the effect on cell proliferation and drug sensitivity to AG1478 after down-regulation of FOXMl and FOXO3a expression by RNAi technique.

METHODSHuman lung cancer cells were treated with AG1478 at different concentrations. RT-PCR and Western blot were used to examine the expression of P-EGFR, FOXM1, FOXO3a mRNA and protein. After transient transfection of FOXM1 and FOXO3a siRNA, RT-PCR and Western blot were employed to determine the transfection efficiency and expression of the related proteins. CCK-8 assay, colony formation assay and flow cytometry were performed to evaluate the cell proliferation, colony formation ability and the changes in cell cycle distribution.

RESULTSThe expressions of FOXM1 mRNA and protein were inhibited by AG1478 in a dose-dependent manner (both P < 0.05). After transfection with FOXM1 siRNA, the expressions of FOXM1 mRNA and protein, and proteins of cyclin B1, c-Myc, and Bcl-2 were significantly down-regulated, and the expressions of p21 and cleaved-PARP proteins were significantly up-regulated (all P < 0.05). The colony number of FOXM1siRNA transfection group was 37.3 ± 8.6, significantly lower than that of the blank control (135.3 ± 7.0) and negative control group (125.3 ± 7.5, P < 0.05). The colony formation inhibition rate was (7.40 ± 0.94)% in the negative control group and (72.4 ± 6.09)% in the FOXM1 siRNA transfection group. FOXM1siRNA transfection induced cell cycle arrest at G2/M phase with a percentage of (55.6 ± 4.83)%, significantly higher than that of the blank control [(24.30 ± 1.95)%] and negative control group [(21.3 ± 2.06)%, P < 0.05]. Additionally, the FOXM1siRNA transfection significantly increased the chemosensitivity of A549 cells to AG1478 (P < 0.05). Besides, AG1478 induced expression and nuclear relocation of FOXO3a. After the FOXO3a siRNA transfection, the expression of FOXM1 protein was significantly up-regulated, and resulted in a reduction of AG1478-induced inhibition of FOXM1.

CONCLUSIONSThe expression of FOXM1 is down-regulated by AG1478 via FOXO3a in the NSCLC cell lines, and then increases the chemosensitivity of A549 cells to AG1478. It suggests that FOXM1 could be a potential target for the therapy and drug exploitation for NSCLC.

Adenocarcinoma ; metabolism ; pathology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Dose-Response Relationship, Drug ; Down-Regulation ; Forkhead Box Protein M1 ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Quinazolines ; administration & dosage ; pharmacology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; Transfection ; Tyrphostins ; administration & dosage ; pharmacology

OBJECTIVETo investigate the relationship between EGFR activation and down-regulation of miRNA-145 in lung cancer.

METHODSNormal human lung epithelia cell line (BEAS-2B), human lung adenocarcinoma cell lines with wild-type EGFR (A549 and H292) and human lung adenocarcinoma cell lines with EGFR mutation (H1975 and H1650) were chosen in this study. The levels of miRNA-145 and p-EGFR were determined by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively, and the relationship between p-EGFR and miRNA-145 levels was analyzed. The miRNA-145 levels were determined by qRT-PCR after activating EGFR with EGF or blocking EGFR signal pathway with AG1478. In addition, ERK1/2 inhibitor U0126 was used to inhibit ERK1/2 activation and then the expression of miRNA-145 was detected.

RESULTSThe miRNA-145 levels were closely negatively related with p-EGFR in lung cancer cells (r = -0.926, P = 0.024). EGF down-regulated miRNA-145 expression, particularly in BEAS-2B cells (53.0%; t = 30.993, P = 0.001) and A549 cells (42.6%; t = 14.326, P = 0.005).The miRNA-145 was up-regulated after inhibiting p-EGFR with AG1478, and significantly enhanced by 67.5% in H1975 cells when treated with AG1478 (t = 8.269, P = 0.014). The ERK1/2 signal pathway was activated by p-EGFR. U0126 restored the miRNA-145 down-regulation induced by EGFR-activation in lung cancer cells.

CONCLUSIONThe activation of EGFR down-regulates miRNA-145 expression through ERK1/2 in lung cancer cells.

Butadienes ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Down-Regulation ; Enzyme Activation ; drug effects ; Enzyme Inhibitors ; pharmacology ; Epidermal Growth Factor ; pharmacology ; Epithelial Cells ; metabolism ; Humans ; Lung ; cytology ; Lung Neoplasms ; metabolism ; pathology ; MAP Kinase Signaling System ; MicroRNAs ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Nitriles ; pharmacology ; Phosphorylation ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; metabolism ; Tyrphostins ; pharmacology