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PURPOSE: c-Met and its ligand, hepatocyte growth factor (HGF), play a critical role in oncogenesis and metastatic progression. The aim of this study was to identify inhibited enzymogram and to test the antitumor activity of SIM-89 (a c-Met receptor tyrosine kinase inhibitor) in non-small cell lung cancer. MATERIALS AND METHODS: Z′-LYTE kinase assay was employed to screen the kinase enzymogram, and mechanism of action (MOA) analysis was used to identify the inhibited kinases. Cell proliferation was then analyzed by CCK8 assay, and cell migration was determined by transwell assay. The gene expression and the phosphorylation of c-Met were examined by realtime-PCR and western blotting, respectively. Finally, the secretion of HGF was detected by ELISA assay. RESULTS: c-Met, activated protein kinase (AMPK), and tyrosine kinase A (TRKA) were inhibited by SIM-89 with the IC₅₀ values of 297 nmol/L, 1.31 µmol/L, and 150.2 nmol/L, respectively. SIM-89 exerted adenosine triphosphate (ATP) competitive inhibition on c-Met. Moreover, the expressions of STAT1, JAK1, and c-Met in H460 cells were decreased by SIM-89 treatment, and c-Met phosphorylation was suppressed in A549, H441, H1299, and B16F10 cells by the treatment. In addition, SIM-89 treatment significantly decreased the level of HGF, which accounted for the activation of c-Met receptor tyrosine kinase. Finally, we showed cell proliferation inhibition and cell migration suppression in H460 and H1299 cells after SIM-89 treatment. CONCLUSION: In conclusion, SIM-89 inhibits tumor cell proliferation, migration and HGF autocrine, suggesting it's potential antitumor activity.
Subject(s)
Adenosine Triphosphate , Blotting, Western , Carcinogenesis , Carcinoma, Non-Small-Cell Lung , Cell Movement , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Gene Expression , Hepatocyte Growth Factor , Lung Neoplasms , Phosphorylation , Phosphotransferases , Protein Kinases , Protein-Tyrosine KinasesABSTRACT
Objective: To observe the effect of a novel targeted agent enzastaurin alone or in combination with gefitinib on ge-fitinib-resistant human non-small cell lung cancer cells to explore the rational drug combination. Methods: CCK-8 assay was used to measure the cell proliferation. Combination index ( CI) was calculated by Chou-Talalay method to assess the efficacy of the combination therapy. The flow cytometry (FCM) was used to analyze the change in the cell cycle. Results:In 72h, the IC50 value of gefitinib and enzastaurin for the lung cancer NCI-H460 cells was 6. 99μmol·L-1 (95%CI:3. 55-13. 79μmol·L-1 ) and 7. 25μmol·L-1 (95%CI:4. 77-1. 02 μmol·L-1), respectively. The inhibition effect on the cell proliferation was stronger in the combination treatment than that in the monotherapy (P<0. 05), and the simultaneous treatment showed the most significant inhibition effect (P<0. 01). The IC50 value of gefitinib for H460 cells in the simultaneous administration group, the sequential administration group with gefitinib used first and the sequential administration group with enzastaurin used first was 0.006 μmol·L-1(95%CI:0.002-0.020μmol·L-1), 0.02μmol·L-1(95%CI:0.011-0.037 μmol·L-1) and 0.085 μmol·L-1(95% CI:0.042-0.170μmol·L-1, respectively. The CI of the simultaneous administration group was lower than one when the gefitinib concentration was above 0. 05μmol·L-1 . The cell cycle distribution result indicated that the simultaneous administration group had significantly increased G0/G1 proportion (P<0. 05) and induced cell cycle arrest at G1 phase. Conclusion:Protein kinase C inhibitor enzastaurin combined with EGFR inhibitor gefitinib shows a synergistic effect, suggesting that the combination treatment of the two drugs might be a new strategy for the follow-up therapy of gefitinib-resistant non-small cell lung cancer.
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Objective To explore the changes of proteomic spectra from plasma of patients with lung cancer or benign lung diseases and health controls in order to establish a primary diagnosis model of lung cancer. Methods The proteomic spectra from plasma of 108 patients with lung cancer, 40 patients with benign lung diseases and 22 healthy individuals were analysed by surface-enhanced laser desorption/ionization time of flight mass spectrometry ( SELDI-TOF-MS). The best decision tree model was established by cluster analysis and principal component analysis. Then the model was blindly validated by the protein of 21 patients with lung benign diseases and 47 patients with stage I lung cancer. Results Twenty-three significantly differentially expressed protein peaks were successfully detected (P <0.001). Blinded validation suggested that the accuracy for diagnosing lung cancer was 72. 06%, the sensitivity and specificity were 72. 34% and 71.43%, respectively, and the positive predictive value and negative predictive value were 85. 0% and 78. 95%, respectively. Conclusion SELDI-TOF-MS protein chip technology provides a new tool for the early diagnosis of lung cancer.
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To explore the dual regulatory effects of Shuanghuang Shengbai Granule, a compound traditional Chinese herbal medicine, on cell cycle in Lewis-bearing mice with chemotherapy-induced myelosuppression.
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Objective To explore the effects of inducible co-stimulator (ICOS) gene on the cytotoxic activity of cytokine-induced killer (CIK) cells against cholangiocarcinoma cells. Methods CIK-ICOS cells were obtained by stable transfecting ICOS genes into CIK cells through the adenovirus vector whereas untransfected and EGFP-transfected CIK cells were treated as controls. The proliferation and apoptosis of different CIK cells, as well as their cytotoxicity against cholangiocarcinoma cells in the three groups were detected. The expressions of IFN-T, IL-2 and TNF-α in the supernatant of different CIK cells were measured by ELISA. SCID mice with cholangiocarcinoma were randomly divided into CIK group, CIK-EGFP group, CIK-ICOS group and normal saline group. The cytotoxic activity of CIK-ICOS cells against cholangiocarcinoma cells in vivo was observed. Results CIK-ICOS cells displayed better proliferation than CIK cells and CIK-EGFP cells. At day 20 and 23 of culture, the apoptosis rate of CIK-ICOS cells was 0.69% and 0.89%, respectively, while that of the CIK cells was 2.90% and 4.92%. The cytotoxic effect of CIK-ICOS cells at different E: T ratio against cholangiocarcinoma cells was significantly stronger than that of CIK cells and CIK-EGFP cells (F=13.37, 6.46, 25.51, P<0.05). The concentration of IFN-γ in CIK-ICOS cultured supernatant was (49.50±4.73)μg/L, which was significantly higher than that in the cultured supernatant of CIK cells [(30.53±3.73)μg/L] and CIK-EGFP cells [(30.12±2.64)μg/L](F=38.89, P<0.05). The growth of cholangiocarcinoma was significantly slower in CIK-ICOS group than that in CIK group and CIK-EGFP group, whereas the necrosis area of tumor was larger and the CIK cells in CIK-ICOS group was more than those in the other two groups. Conclusions CIK cells had the function of killing cholangiocarcinoma cells in vitro and in vivo. After ICOS genes were transfected into CIK cells, the survival time of CIK cells in vitro was prolonged and the proliferation of CIK cells was enhanced, as well as the secretion of IFN-γ was increased so that the cytotoxicity of CIK cells against cholangiocarcinoma cells in vitro and in vivo was enhanced.
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<p><b>BACKGROUND</b>Protein kinase C(PKC) is a potentially important target for can-cer therapeutics due to its potential role in carcinogenesis.Abnormal expression and increasing activity of PKC-α are present in non-small cell lung cancer(NSCLC).PKC inhibitor can show anti-tumor effects through inducing tumor cell apoptosis,enhancing cytotoxic effects and down-regulating expressions of multidrug resistance gene.By observing the effects of PKC inhibitor chelerythrine chloride(CH) on drug-sensitivity to cisplatin of four NSCLC cell lines its mechanism of effect initially is explored.</p><p><b>METHODS</b>NSCLC cell lines(H1299,H460,A549 and cisplatin-resistant A549) were dealed with PKC inhibitor CH respectively.The expressions of PKC-α mRNA and protein in NSCLC cell lines were examined by reverse transcription polymerase chain reaction(RT-PCR) and Western blot.The apoptosis rates of NSCLC cells lines were detected by flow cytometry.The drug-sensitivity to cisplatin of NSCLC cells lines was measured by methabenzthiazuron(MTT) assay.</p><p><b>RESULTS</b>The expression levels of PKC-α mRNA and protein in cisplatin-resistant A549 cell lines were significantly higher than H1299,H460 and parent A549 cell lines(P < 0.05).The expression levels of PKC-α mRNA and protein in four NSCLC cell lines decreased at different extent.The apoptosis rates of cisplatin-resistant A549 cell lines increased obviously after treating with CH for 4 and 24 hours,but it was not seen in H1299,H460 and parent A549 cell lines.The IC50 value of cisplatin of NSCLC cell lines decreased at different degree after treating with CH and it was more obvious in cisplatin-resistant A549 cell lines(P < 0.05).</p><p><b>CONCLUSIONS</b>High expressions of PKC-α mRNA and protein exist in all four NSCLC cell lines.PKC inhibitor CH can enhance the drug-sensitivity of NSCLC cell lines to cisplatin by inhibiting their expression of PKC-α mRNA and protein.When compared with parent A549 cell lines,cisplatin-resistant A549 cell line's drug-sensitivity to cisplatin is increasing more efficiently by PKC inhibitor CH through inhibition of PKC-α protein's expression and elevation of tumor cell apoptosis rates.</p>
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BACKGROUND: Vascular endothelial growth factor (VEGF) is able to effectively treat the ischemic heart disease, but in vivo VEGF cannot be maintained effective concentration. OBJECTIVE: To detect the expression of VEGF mRNA and protein in human bone marrow mensenchymal stem cells transferred by VEGF-165 gene. DESIGN, TIME AND SETTING: The empirical study was conducted from March 2006 to April 2007 at Shanghai Chest Hospital. MATERIALS: Human bone marrow mesenchymal stem cell line and VEGF were offered by Basic Laboratory, Thoracic Tumor Institute, Shanghai Chest Hospital. METHODS: hVEGF165 gene was reconstructed in pcPGK-vector and transferred into human bone marrow mensenchymal stem cells (BMSCs) by liposome-mediated method, clone screening by G418. MAIN OUTCOME MEASURES: The mRNA and protein of VEGF gene in transferred cells was detected by reverse transcription-polymerase chain reaction (RT-PCR), Real time PCR, Western Blot, enzyme linked immunosorbent assay (ELISA) in 3 stem cells of pcPGK-VEGF165-IRES-GFP and pcPGK- IRES-GFP, respectively. RESULTS: pcPGK-hVEGF165 vector was reconstructed and transferred into hMSCs successfully. The expression of hVEGF165 in the transfected hMSCs was demonstrated with RT-PCR and Real time PCR. Western Blot and ELISA demonstrated that the expression of hVEGF165 in the transfected hMSCs and VEGF protein in supernatant were significantly more than untransfected hMSCs. CONCLUSION: hVEGF165 can be successfully transfected into BMSCs by using liposome mediated gene transfer. Stably expressed VEGF165 cell line can be obtained..
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<p><b>BACKGROUND</b>Dendritic cell (DC)-based immunotherapy is a new approach and effective for some malignant tumors. The aim of this study is to observe the efficacy and toxicity of immunotherapy with carcinoembryonic antigen (CEA) peptide-pulsed DCs in patients with refractory advanced lung cancer.</p><p><b>METHODS</b>Lung cancer patients with high CEA expression were enrolled into this project. Autologous DCs were generated from patients' plastic-adherent peripheral blood mononuclear cells and loaded with CEA 5 days later. Cytokine-induced killer cells (CIK) were cultured from non-adherent peripheral blood mononuclear cells. DCs and CIK were transfused to patients. Responses and toxicities were observed.</p><p><b>RESULTS</b>A total of 22 patients with lung cancer received DCs immunotherapy. DCs doses were 2.5×10⁶-9.6×10⁷ (5.03×10⁶). CIK doses were 3.4×10⁸-46×10⁸. CD3, CD8, NK and IFN-γ levels obviously increased after treatment (P < 0.05). The 1-year survival rate was 68.2% (15/22). Main toxicities were fever and rash.</p><p><b>CONCLUSIONS</b>DCs-based immunotherapy is feasible and safe to patients with lung cancer.</p>
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OBJECTIVE: To study the function of Qiyeling Decoction in inducing apoptosis of transplanted human lung adenocarcinoma cells A549 in nude mice. METHODS: Nude mice with transplanted A549 tumor were randomly divided into the untreated control group (group A), chemotherapy treated group (group B), chemotherapy plus Qiyeling Decoction treated group (group C), Qiyeling Decoction treated group (group D) and managed correspondingly. The tumor volume was measured and calculated into tumor weight. The apoptosis of tumor cells were examined using in situ cell apoptosis detection kit. RESULTS: The tumor weight was lower obviously in groups B, C and D than that in group A (P<0.05). The apoptosis of tumor cells was lower obviously in groups B, and C than that in group D (P<0.05). Cells in group A appeared perfect differentiation during the early stage and apoptosis later. CONCLUSION: Qiyeling Decoction can induce A549 cell apoptosis in nude mice.
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<p><b>OBJECTIVE</b>To evaluate the effect of vascular endothelia1 growth factor (VEGF) on the hematogenous metastasis of non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The identification of lung cancer cells in the peripheral blood were carried out by cytological, immunohistocytologica1 and immunofluorecent stains respectively, following isolation of cytokeratin-expressing cells with magnetic activated cell sorting. The quantification of cancer cells in the blood was performed according to the established flow cytometric assay. The plasma VEGF was measured by commercially available ELISA kit.</p><p><b>RESULTS</b>The lung cancer cells in the blood, showing a remarkable nuclear polymorphism, expressed the epithelial marker cytokeratin and telomerase reverse transcriptase (hTERT). These cells were stained positive by an NSCLC-specific monoclonal antibody S5Al0-2, but negative by antibodies against CD34 and CD45 antigens. Using the flow cytometric assay, 44 cases (28.6%) of l54 NSCLC patients were found to have cancer cells in their blood, with the incidence of positive cases correlated with the stage of disease. The plasma VEGF level was significantly increased in NSCLC patients in comparison with healthy individuals and patients with benign pulmonary diseases. This level was correlated with the stages of disease in patients with adenocarcinoma. In patiens with cancer cells in their blood, a higher level of plasma VEGF was related with an increased number of cancer cells.</p><p><b>CONCLUSION</b>The plasma VEGF level is increased in NSCLC patients with approximate1y one fourth to have cancer cells in the peripheral blood. In these patients, increased VEGF level promotes hematogenous tumor metastasis, as indicated by a much higher number of cancer cells in the blood.</p>