RESUMEN
BACKGROUND & OBJECTIVE: Epidermal growth factor receptor (EGFR) is expressed in most human epithelial cancers and is involved in the development of cancer cell resistance to irradiation. We used gefitinib, a selective EGFR tyrosine kinase inhibitor (EGFR-TKI), to investigate its effects and mechanisms in enhancing the radiosensitivity of human gastric cancer cell lines in vitro. METHODS: The expression of EGFR protein in 7 human gastric cell lines (MKN45, SGC7901, SNU-1, N87, AGS, SNU-16, and KATO-III) was determined by Western blot, in which 2 cell lines with high expression of EGFR were selected for additional test. The inhibitory effect of gefitinib on cell proliferation was measured by MTT assay. Cell survival was determined by clonogenic assay, and then the radiosensitivity parameters were calculated. The effects of gefitinib in combination with radiation on cell apoptosis and cell cycle distribution were analyzed by flow cytometry. RESULTS: Of the 7 gastric cancer cell lines, the expression of EGFR in MKN45 and SGC7901 cells were the highest. The 50% inhibition concentrations (IC(50)) of gefitinib were 0.4 mmol/L for MKN45 cells and 0.8 mmol/L for SGC7901 cells. Cell survival was significantly decreased with the elevation of gefitinib concentration or radiation dose (P<0.05). When treated with 0.1x and 0.2 x IC(50) of gefitinib, the radiosensitization enhancement ratios (SER) of MKN45 cells were 1.102 and 1.154, and those of SGC7901 cells were 1.092 and 1.176, respectively. Either gefitinib or radiation induced cell apoptosis, reduced the percentage of cells at S phase and increased the percentage of cells at G(2)/M phase (P<0.01). CONCLUSIONS: Gefitinib followed by radiation could increase the radiosensitivity of MKN45 and SGC7901 cells with high expression of EGFR and inhibit cell proliferation, induce apoptosis, and alter cell phase distribution. Gefitinib could be a radiation sensitizer for gastric tumors with high expression of EGFR.
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Receptores ErbB/metabolismo , Quinazolinas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Neoplasias Gástricas/patología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Receptores ErbB/antagonistas & inhibidores , Gefitinib , Humanos , Aceleradores de Partículas , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
BACKGROUND: Bcl-2, the anti-apoptotic protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of Bcl-2, RNA interference (RNAi) was used to inhibit Bcl-2 expression in the human gastric cancer cells in vitro and in vivo. METHODS: Bcl-2 small interfering RNA (siRNA) was transfected into human gastric cancer cells SGC-7901, and Bcl-2 expression was monitored by real-time polymerase chain reaction (PCR) and Western blot. Cell proliferation, apoptosis, and telomerase activity were examined by MTT, flow cytometry, and TRAP assay, respectively. Gastric cancer cells treated with 100 nmol/L Bcl-2 siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed. RESULTS: Bcl-2 siRNA significantly inhibited the expression of Bcl-2 in human gastric cancer cells at both mRNA and protein levels in a time- and dose-dependent manner. Bcl-2 siRNA also decreased telomerase activity (by 78.76%) and increased the rate of apoptosis (by 37.47%). SGC-7901 cell growth was also significantly suppressed in vivo and in vitro. CONCLUSIONS: Bcl-2 expression knockdown suppressed the growth of gastric cancer cells. Thus, Bcl-2 may play a very important role in carcinogenesis of gastric cancer and its knockdown may offer a new potential gene therapy approach for human gastric cancer in future.
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Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Neoplasias Gástricas/terapia , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , Neoplasias Gástricas/patología , TransfecciónRESUMEN
OBJECTIVE: To evaluate the feasibility and efficacy of intraperitoneal chemotherapy for malignant ascites caused by different types of abdominal cancers guided by chemo-sensitivity methyl tetrojolium coloremetric (MTT) assay in vitro. METHODS: Cancer cells in the malignant ascites were collected for MTT assay to determine the chemo-sensitivity. The drug producing the highest or the second highest inhibition rate was selected for intraperitoneal chemotherapy. The correlation between the results of MTT assay and the response of malignant ascites, the clinical features, Karnofsky performance score (KPS) and prognosis were analyzed. RESULTS: MTT assay indicated that Taxotere (TXT) and Hydroxycamptothecin (HCPT) were the most effective to cancer cells in malignant ascites, and HCPT was mostly frequently used for intraperitoneal chemotherapy (56.9%). Twenty-four patients showed response by intraperitoneal chemotherapy (complete response: 7; partial response: 17) with a slightly significant correlation between the results of MTT assay and response of malignant ascites (P = 0. 014). The KPS of the responders was improved significantly (P < 0.001), and the response of malignant ascites to intraperitoneal chemotherapy was demostrated as an independent prognostic factor by multi-variate analysis in this series. CONCLUSION: In vitro chemo-sensitivity MTT assay guided intraperitoneal chemotherapy for malignant ascites is simple, effective and safe, which can improve the KPS and prognosis of the responders.
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Adenocarcinoma/tratamiento farmacológico , Ascitis/tratamiento farmacológico , Camptotecina/análogos & derivados , Neoplasias Gástricas/tratamiento farmacológico , Taxoides/uso terapéutico , Adenocarcinoma/patología , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Ascitis/patología , Camptotecina/administración & dosificación , Camptotecina/farmacología , Camptotecina/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Docetaxel , Femenino , Humanos , Inyecciones Intraperitoneales , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Gástricas/patología , Taxoides/administración & dosificación , Taxoides/farmacología , Células Tumorales CultivadasRESUMEN
In this preliminary study, we evaluated the impact of hyperthermia (HT) and hyperthermic chemotherapy (HTCT) on six human gastric cancer cell lines and explored the mechanisms of cell-killing effect under HTCT. Treatment conditions were categorized into 4 modes: i) normothermic control (NT), ii) HT, iii) normothermic chemotherapy (NTCT) and iv) HTCT. According to the data of MTT and LM observations, isolated HT only temporarily inhibited cell proliferation and had no cell-killing effect on gastric cancer cell lines employed in our study except for SNU-1. Combining with HT enhanced the cytotoxicity of CDDP in all gastric cell lines and the concentration inhibiting cell proliferation and inducing cell death of HTCT was much lower than that of NTCT. There was a synergistic effect of HT and chemotherapy on inhibiting proliferation in each cell line in a certain range of CDDP concentration. The data of TEM and FCM proved that HTCT induced cell death with two modes - apoptosis and necrosis, and apoptosis was the major type. Microarray illustrated that, under HTCT, a total of 58 gene expressions were regulated according to the filtering criteria, including 10 extra genes with an expression change below the threshold or even unchanged when treated with either HT or CDDP alone. Five of these 10 genes were verified by QRT-PCR. These genes may include the target genes for the enhancing effect of HT on chemotherapy and their effects should be further validated by functional analysis.
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Antineoplásicos/farmacología , Cisplatino/farmacología , Hipertermia Inducida , Neoplasias Gástricas/terapia , Apoptosis , Proliferación Celular , Terapia Combinada , Humanos , Necrosis , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To compare the expression and activities of urokinase type plasminogen activator (uPA) among different gastric cancer cell lines and investigate their relations with peritoneal metastatic potency. METHODS: The uPA expression in 4 gastric cancer cell lines (AGS, SGC7901, MKN45, MKMN28) was detected using ELISA and Western blot methods. uPA activity was detected simultaneously using uPA activity kit. The gastric cancer cells were cultured with confluent mesothelial cells in 24-well plates or Boyden chambers for different times. The adhesive cells were counted directly under a microscope. The motility and invasion of gastric cancer cells were determined by MTT assay. RESULTS: Among four gastric cancer lines,the highest expression of uPA was found in SGC7901 and the highest uPA activity in MKN45, while the lowest expression and activity of uPA in AGS. Compared with the other three lines, MKN45 had stronger adhesion than MKMN28 (P< 0.05), SGC7901 (P< 0.05), and AGS (P< 0.01), but there were no significant differences in motility and invasion among MKN45, MKN28 and SGC7901. The adhesion,motility and invasion of AGS were weaker compared with those of the other three cell lines. CONCLUSION: The uPA expression and activity are significantly different among 4 gastric cancer cell lines, and positively correlated with their peritoneal metastatic potency.
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Neoplasias Peritoneales/metabolismo , Neoplasias Gástricas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Western Blotting , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis de la Neoplasia , Neoplasias Peritoneales/secundario , Neoplasias Gástricas/clasificación , Neoplasias Gástricas/patologíaRESUMEN
BACKGROUND & OBJECTIVE: Drug-resistance is a major factor of influencing treatment efficacy of advanced gastric cancer. This study was to evaluate in vitro antitumor effects of different chemotherapeutic drugs on fresh human gastric cancer cells, and explore their relationships with expressions of P-glycoprotein (P-gp), and glutathione S transferase -pi (GST-pi) in human gastric cancer tissue. METHODS: A total of 39 specimens of highly purified gastric cancer cells were separately exposed to 5-fluorouracil (5-FU), cisplatin (DDP), mitomycin C (MMC), adriamycin (ADM), and hydroxycamptothecin (HCPT). Inhibitory rate of cells was detected by MTT assay. Metabolic activity of cells was detected by trypan blue exclusive assay. Cell apoptosis was detected by in situ terminal deoxynucleotidyl transferase assay (TdT assay). Expressions of GST-pi and P-gp in gastric cancer tissues were detected by immunohistochemistry. RESULTS: After exposure to antitumor drugs, morphologic changes, decrease of metabolic activity, and apoptosis were appeared in gastric cancer cells. Inhibitory rates of cancer cells exposed to MMC, DDP, 5-FU were significantly higher than those of cells exposed to ADM, and HCPT [(38.6+/-7.7)%, (38.1+/-8.8)%, and (37.8+/-10.3)% vs. (31.9+/-10.4)%, and (29.7+/-10.2)%, P < 0.01]. Apoptosis rate of cancer cells exposed to CDDP, 5-FU, and MMC were significantly higher than those of cells exposed to HCPT, and ADM [(32.1+/-7.7)%, (31.1+/-8.8)%, and (29.8+/-6.3)% vs. (21.9+/-7.4)%, and (19.9+/- 7.4)%, P < 0.05]. Positive rate of GST-pi was 66.7%(26/39), that of P-gp was 59.0%(23/39). GST-pi positive cells showed resistance to DDP and MMC, P-gp positive cells showed resistance to ADM and HCPT. CONCLUSIONS: Overexpressions of P-gp and GST-pi might contribute to drug-resistance of tumor. Detection of GST-pi and P-gp, together with MTT chemosensitivity test, might be useful for selecting more effective chemotherapeutic drugs.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Gutatión-S-Transferasa pi/metabolismo , Neoplasias Gástricas/patología , Camptotecina/análogos & derivados , Camptotecina/farmacología , Núcleo Celular/metabolismo , Cisplatino/farmacología , Citoplasma/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Femenino , Fluorouracilo/farmacología , Humanos , Masculino , Persona de Mediana Edad , Mitomicina/farmacología , Neoplasias Gástricas/metabolismo , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To study the correlation between expression of urokinase-type plasminogen activator (uPA) and capability of tumor cell seeding to the peritoneal membrane by different gastric cancer lines. METHODS: Expression of uPA in 4 human gastric cancer cell lines was examined by semi-quantitative RT-PCR, ELISA and Western blot. uPA activity was determined by an assay kit. After ip inoculation of cancer cells to nude mice, tumors on peritoneal membrane was grossly examined for tumor cell seedings. RESULTS: SGC7901 was the highest in uPA expression among human gastric cancer cell lines AGS, SGC7901, MKN45, and MKN28. MKN45 had the strongest uPA activity, while AGS was lowest in both uPA expression and activity. Peritoneal seeding tumors of various sizes were observed in mice inoculated with SGC7901 and MKN45 cells. In addition to peritoneal seedings, bloody ascites was present in mice inoculated with MKN28. The MKN45-inoculated mice took the least time to develop tumors and had the shortest surviving period. No peritoneal seeding was seen in mice inoculated with AGS cells. CONCLUSION: Three of 4 human gastric cancer cell lines studied express uPA mRNA and activity, which correlate with their peritoneal seeding potentials.
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Adenocarcinoma/enzimología , Siembra Neoplásica , Neoplasias Peritoneales/secundario , Neoplasias Gástricas/enzimología , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Adenocarcinoma/secundario , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Neoplasias Gástricas/patología , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
AIM: To study the expression of vascular endothelial growth factor C (VEGF-C) and chemokine receptor CCR7 in gastric carcinoma and to investigate their associations with lymph node metastasis of gastric carcinoma and their values in predicting lymph node metastasis. METHODS: The expression of VEGF-C and CCR7 in gastric carcinoma tissues obtained from 118 patients who underwent curative gastrectomy was examined by immunohistochemistry. Among these patients, 39 patients underwent multi-slice spiral CT (MSCT) examination. RESULTS: VEGF-C and CCR7 were positively expressed in 52.5 and 53.4% of patients. VEGF-C expression was more frequently found in tumors with lymph node metastasis than those without it (P<0.001). VEGF-C expression was also closely related to lymphatic invasion (P<0.001), vascular invasion (P<0.01), and TNM stage (P<0.001). However, there was no significant correlation between VEGF-C expression and age at surgery, gender, tumor size, tumor location, Lauren classification, and depth of invasion. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P<0.001) and was also associated with tumor size (P<0.01), depth of invasion (P<0.001), lymphatic invasion (P<0.001), and TNM stage (P<0.001). However, the presence of CCR7 had no correlation to age at surgery, gender, tumor location, Lauren classification, and vascular invasion. Among the 39 patients who underwent MSCT examination, only CCR7 expression was related to lymph node metastasis determined by MSCT (P<0.05). In the current retrospective study, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of VEGF-C and CCR7 expression in the diagnosis of lymph node metastasis for patients with gastric carcinoma were 73.8%, 70.2%, 72.6%, 71.4% and 72.0%, and 82.0%, 77.2%, 79.4%, 80.0% and 79.7%, respectively. After subdivision according to the combination of VEGF-C and CCR7 expression, receiver operating characteristic (ROC) analysis showed that the accuracy of the combined examination of VEGF-C and CCR7 expression in predicting lymph node metastasis was relatively high (area under ROC curve [Az]=0.83). CONCLUSION: The expression of VEGF-C and CCR7 is related to lymph node metastasis of gastric carcinoma and both of them may become new targets for the treatment of gastric carcinoma. Furthermore, the combined examination of VEGF-C and CCR7 expression in endoscopic biopsy specimens may be useful in predicting lymph node metastasis of gastric carcinoma and deciding the extent of surgical lymph node resection.
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Carcinoma/metabolismo , Receptores de Quimiocina/metabolismo , Neoplasias Gástricas/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Humanos , Metástasis Linfática , Pronóstico , Receptores CCR7RESUMEN
AIM: To determine the expression levels of three metabolic enzymes of fluoropyrimidines: thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines, and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd). METHODS: TS, TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR, TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50), representing the sensitivity to drugs, were determined by MTT assay. RESULTS: IC50 values ranged from 1.28 to 12.26 microM for 5-FU, and from 5.02 to 24.21 microM for FdUrd, respectively. Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P<0.05), but neither TS nor TP correlated with 5-FU IC50 (P>0.05). Only TS mRNA level was sharply related with FdUrd sensitivity (P<0.05), but TP and DPD were not (P>0.05). A correlation was found between mRNA and protein levels of DPD (P<0.05), but not TP (P<0.05). CONCLUSION: DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd, respectively.
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Antimetabolitos Antineoplásicos/farmacología , Neoplasias Colorrectales , Enzimas/metabolismo , Fluorouracilo/farmacología , Neoplasias Gástricas , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Dihidrouracilo Deshidrogenasa (NADP)/genética , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Resistencia a Antineoplásicos , Enzimas/genética , Floxuridina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Timidina Fosforilasa/genética , Timidina Fosforilasa/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
AIM: To evaluate the effect of reactive oxygen species such as hydrogen peroxide on the progression of human colon cancer. METHODS: Human colon carcinoma cell lines, LS174T and HCT8, were treated respectively with 10(-5), 10(-7) or 10(-9) mol x L(-1) hydrogen peroxide for 24h,and co-cultured with human endothelial cell line ECV-304. The migration of ECV-304 induced by cancer cells was calculated and the expression level of vascular endothelial growth factor in cancer cells was determined by RT-PCR analysis and ELISA. Dactinomycin of 1.5mg x L(-1) which could block transcription of cancer cells was applied to observing the effects of H(2)O(2) on transcriptional activity and the relative half-life of VEGF mRNA. Finally,to evaluate the effect of H2O2 on NF-kappaB activity in colon cancer cells, NF-kappaB in cytoplasm and nucleus of the cells were detected with FITC-tagged antibody and its presence in the nucleus(Fn) vs cytoplasm(Fc) was monitored by measuring the green fluorescence integrated over the nucleus by laser scanning cytometry(LSC). RESULTS: Exogenouse hydrogen peroxide of low concentration increased the migration of endothelial cell induced by colon cancer cells. When cancer cells were treated with 10(-5) mol x L(-1) H2O2, the migration number of endothelial cells induced by LS174T cells was 203+/-70 and the number induced by HCT8 cells was 145+/-65. The two values were significantly higher than those treated with other concentrations of H2O2 (P<0.01). The expression of vascular endothelial growth factor in cancer cells, which could be blocked by dactinomycin, were increased to a certain degree, while the relative half-life of VEGF mRNA was not prolonged after treatment with hydrogen peroxide. The activity of NF-kappaB in colon cells rose after the cells were exposed to hydrogen peroxide for 24h. The Fn values in HCT8 cells were 91+/-13 (0 mol x L(-1) H2O2) and 149+/-40(10(-5) mol x L(-1) H2O2)(P<0.05), in LS174T cells were 127+/-35(0 mol x L(-1) H2O2) and 192+/-11(10(-5)mol x L(-1) H2O2) (P<0.05). It is similar to the case of VEGF expression in cancer cells. CONCLUSION: Hydrogen peroxide increases vascular endothelial growth factor expression in colon cancer cells, and it is likely that reactive oxygen species such as hydrogen peroxide facilitates the development of colon cancer.