OBJECTIVE: To determine the sensitivity of cetuximab induced apoptosis in laryngeal squamous carcinoma cells Hep-2, and to evaluate the synergistic killing effects and regulation mechanism of cetuximab alone or cetuximab in combination with chemotherapeutic agents (cisplatin) or radiation means on Hep-2 cells. METHOD: To investigate the cytotoxicities of cetuximab, cisplatin and radiation, cell counting kit-8 (CCK-8) assay was used for the detection of cell growth inhibition ratio, and fluorescence activated cell sorter FACS for the apoptotic rate and cell cycle distribution. RESULT: Cetuximab had inhibitive effect on Hep-2 cells within a certain range of concentration in a time- and dose-dependence manner. The inhibition concentration 50% (IC50) of cetuximab on Hep-2 cells for 24 h was 1 036.84 microg/ml. For application of cisplatin and radiation, the apoptotic rate of Hep-2 cell was higher by combining with cetuximab than their single or combined administration. Moreover, the cell cycle arrested at G0/G1 phase. CONCLUSION Laryngeal cancer Hep-2 cells was sensitive to the cetuximab induced apoptosis. Cetuximab combined with cisplatin and/or radiation can increase the antiproliferative effects on Hep-2 cells. These findings suggest the synergistic combination of cetuximab and cytotoxic agents was sequence depended.
Antibodies, Monoclonal, Humanized ; pharmacology ; Apoptosis ; drug effects ; radiation effects ; Cell Line, Tumor ; Cetuximab ; Cisplatin ; pharmacology ; Combined Modality Therapy ; Humans

OBJECTIVETo investigate the role of epidermal growth factor receptor (EGFR) expression level in cetuximab cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) effect against A549 lung cancer cell line.

METHODSA549 cell line and NKTm cells were used as the target cell and the effector cell, respectively. pEGFR-EGFP plasmids were transfected into A549 cells by nucleofector method. EGFR expression levels were measured by immunohistochemistry. The ADCC activity induced by cetuximab was assessed by cell counting kit-8 assay.

RESULTSA549 cells transfected with pEGFR-EGFP plasmids expressed higher level of EGFR protein on membrane and were more sensitive to ADCC activity mediated by cetuximab (P<0.05). The inhibition rate of A549 cells showed no significant difference between transfection group and wild-type group when treated with cetuximab alone (P> 0.05).

CONCLUSIONEGFR expression level influences the sensitivity of A549 lung cancer cell line to ADCC activity mediated by cetuximab but not to cetuximab alone.

Adenocarcinoma ; pathology ; Antibodies, Monoclonal, Humanized ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cetuximab ; Humans ; Immunohistochemistry ; Lung Neoplasms ; pathology ; Receptor, Epidermal Growth Factor ; metabolism ; Tumor Cells, Cultured

OBJECTIVE: This research aims to investaigate the effect of cetuximab and dendritic cells (DCs) to kill the head and neck squamous cell (HNSCC), in order to provide a new way for the patients of HNSCC. METHOD: DCs were induced from peripheral blood monocytes by rhIL-4, rhGM-CSF and TNF-alpha in vitro, 7days later, detecting the surface marks of DCs for example CD83, CD86, and then using MTT and flow cytometry detecting the effect T lymphocytes induced by DCs combining cetuximab to kill HNSCC; EGFR and pEGFR in each group were anlysised by Western blot. RESULT: It is successful to induce DCs in vitro. Mature DCs (mDCs) expressed the suface mark such as CD83, CD86 higher compared with immature DCs (imDCs). Compared with other groups, cetuximab combined with DCs significantly enhanced the cytotoxicty and apoptosis to HNSCC (P < 0.05). pEGFR were gradually reduced as the concenetration of cetuximab increasing (P < 0.05). However, comparing with the group of cetuximab, the group of cetuximab combined with DC has no significant difference at the same concentration of cetuximab. In each group EGFR also has no significant diference (P > 0.05). CONCLUSION Cetuximab and DCs have synergistic effects, which can significantly enhance the killing effect of HNSCC.
Antibodies, Monoclonal, Humanized ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; pathology ; Cetuximab ; Dendritic Cells ; immunology ; Head and Neck Neoplasms ; pathology ; Humans ; Squamous Cell Carcinoma of Head and Neck ; Tumor Cells, Cultured

OBJECTIVETo establish a cetuximab-resistant human nasopharyngeal carcinoma 5-8F/Erbitux cell line and preliminarily study the relationship between the insulin-like growth factor 1 receptor (IGF-1R) signaling pathway and the resistance of nasopharyngeal carcinoma to cetuximab.

METHODSA nasopharyngeal cancer cell line, 5-8F, with high epidermal growth factor receptor (EGFR) expression and cetuximab sensitivity, was selected as study object. The cetuximab-resistant 5-8F/Erbitux cell line was induced by stepwise selection after exposure to increasing doses of cetuximab. The IC(50) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the resistance index (RI) was calculated. The growth curves of 5-8F and 5-8F/Erbitux cells were plotted and the doubling times were calculated by cell counting assay. The cell cycle was detected by flow cytometry. Cross-resistance profiles of 5-8F/Erbitux cells to 5-Fu, Taxol and DDP were tested by MTT assay. Expression levels of P-gP, IGF-1R and P-IGF-1R of 5-8F and 5-8F/Erbitux cells were determined by Western blot analysis and MDR1 gene by real-time fluorescent quantitative PCR.

RESULTSA cetuximab-resistant human nasopharyngeal carcinoma cell line 5-8F/Erbitux was successfully established and their resistance index (RI) were 1.2 and 1.1, respectively, at 3 d and 5 d of the cetuximab treatment. The doubling times of 5-8F and 5-8F/Erbitux cells were 26.63 h and 142.30 h, respectively. Flow cytometry demonstrated that 5-8F/Erbitux cells showed an increased population at G(0)/G(1) phase (P < 0.001) and reduced population at S phase (P < 0.001), compared with 5-8F cells. The 5-8F/Erbitux cells showed cross-resistance to 5-Fu (RI = 3.95, P < 0.01) and some resistance to Taxol as well as enhanced sensitivity to DDP (P > 0.05 for all). The 5-8F/Erbitux cells also had increased levels of P-gP, IGF-1R and P-IGF-1R compared with 5-8F cells (P < 0.001 for all). Expression of MDR1 gene was not detected in 5-8F cells and only very weak expression in 5-8F/Erbitux cells.

CONCLUSIONCetuximab-resistant 5-8F/Erbitux cells have no common multidrug resistance like that induced by traditional chemotherapeutic drugs. The excessive activation of the IGF-1R signaling pathway is probably one of the mechanisms that caused resistance of 5-8F/Erbitux cells to cetuximab.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cetuximab ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Humans ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Paclitaxel ; pharmacology ; Phosphorylation ; Receptor, IGF Type 1 ; metabolism ; Signal Transduction

OBJECTIVETo explore the correlation of ras gene to the drug resistance of nasopharyngeal carcinoma to cetuximab.

METHODSCultured 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab. MTT assay was used to determine the IC50 (half inhibitory concentration) of cetuximab and the drug resistance index (RI). Western blotting was employed to detect the protein levels of H-ras and K-ras. Real-time PCR was used to detect the expression of H-ras and K-ras. Gene sequencing was performed to identify potential mutations in H-ras and K-ras genes.

RESULTSWe successfully induced cetuximab-resistant 5-8F/Erbitux hNPC cells by stepwise exposure to increasing doses of cetuximab. After treatment with cetuximab for 3 and 5 days, the RI of 5-8F/Erbitux cells was 1.2 and 1.1, respectively. The 5-8F/Erbitux cells had increased levels of H-ras and K-ras protein expressions (P<0.001) and enhanced gene expressions of H-ras (P=0.016) and ras-p21 (P=0.113) with decreased K-ras gene expression (P=0.000). Sequence analysis identified no mutations in the H-ras and K-ras genes in codons 12, 13, 59, and 61.

CONCLUSIONGene amplification and overexpression of H-ras is the major mechanism that causes the drug resistance of 5-8F/Erbitux cells to cetuximab.

Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Cell Line, Tumor ; Cetuximab ; Drug Resistance, Neoplasm ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genes, ras ; genetics ; Humans ; Nasopharyngeal Neoplasms ; genetics ; pathology

OBJECTIVEThis experiment aims to study the anti-angiogenic ability of vinorelbine combined with cetuximab in vitro and in vivo.

METHODSHuman lung adenocarcinoma A549 cells were used as control group. Proliferation of human umbilical vein endothelial cells (HUVEC) was assessed by MTT assay. Furthermore, we used Transwell chambers, capillary tube formation and flow cytometry to observe the effects of vinorelbine combined with cetuximab on HUVEC migration, tube formation and cell apoptosis, respectively. In addition, the anti-angiogenic ability of the drugs was checked using chicken chorioallantoic membrane (CAM) model.

RESULTSThe inhibitory rate of HUVEC growth was 25.8%, 39.2%, 54.0% for vinorelbine at the concentration of 0.1 ng/ml, 0.4 ng/ml, and 0.8 ng/ml, respectively; that of 0.25 microg/ml cetuximab was 19.7%, and that of 0.1 ng/ml vinorelbine + 0.25 microg/ml cetuximab, 0.4 ng/ml vinorelbine + 0.25 microg/ml cetuximab and 0.8 ng/ml vinorelbine + 0.25 microg/ml cetuximab was 29.5%, 46.4%, 64.6%, respectively. The inhibitory rates of the drugs at the above mentioned combinations of migration and tube formation of HUVEC were 51.9%, 68.2%, 95.0%, respectively. The inhibitory rate of 0.1 ng/ml + 0.25 microg/ml cetuximab and 0.4 ng/ml vinorelbine + 0.25 microg/ml cetuximab on tube formation of HUVEC was 38.8% and 57.7%, respectively, showing a sub-additive effect, and that of combination of 0.8 ng/ml vinorelbine + 0.25 microg/ml cetuximab was 78.9%, showing a synergistic effect. In addition, the apoptotic rate of HUVEC induced by 0.8 ng/ml vinorelbine + 0.25 microg/ml cetuximab was 59.9%, showing a synergistic effect. The in vivo experiment also showed that the combination of the two drugs had a synergistic anti-angiogenic effect.

CONCLUSIONBoth low dose vinorelbine and cetuximab have an anti-angiogenic effect in vitro and in vivo, and the combination of the two drugs has sub-additive or synergistic inhibitory effect on angiogenesis.

Adenocarcinoma ; blood supply ; pathology ; Angiogenesis Inhibitors ; pharmacology ; Animals ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cetuximab ; Chick Embryo ; Drug Synergism ; Endothelial Cells ; cytology ; Humans ; Lung Neoplasms ; blood supply ; pathology ; Neovascularization, Pathologic ; prevention & control ; Umbilical Veins ; cytology ; Vinblastine ; analogs & derivatives ; pharmacology

OBJECTIVE: To determine an approach enriching cancer stem cells from laryngeal cancer cell line. To investigate whether laryngeal cancer stem cells in chemoradiotherapy have the characteristic of resistance. METHOD: CD133+ cells and CD133- cells was detected and isolated from Hep-2 cell line by fluorescence activated cell sorting technology. The cytotoxicities of cisplatin and radiation were investigated by cell counting kit-8(CCK-8) assay. The apoptosis and cell cycle was analyzed with flow cytometry. RESULT: CD133+ cells accounted for a fraction of (2.43 +/- 0.77)% in Hep-2 cell line. CD133+ cells have a more obvious characteristics of cancer stem cells. Different cisplatin and radiation concentrations of for two cell have inhibition, in a certain concentration range and the dosage dependence. Cisplatin and radiation had synergistic inhibitory effects with CD133- cells on the growth of two cell. Moreover, cell cycle arrest at G0/G1 phase and more apoptosis was induced by synergistic combination. Different concentrations of cetuximab for Hep-2 cells have inhibition, in a certain concentration range and time and the dosage dependence. The half maxial inhibitory concentration (IC50) of cetuximab to Hep-2 cells on 24 h was 1 036.84 microg/L. Cisplatin and radiation had synergistic inhibitory effects with cetuximab on the growth of Hep-2 cell line. Moreover, cell cycle arrest at G0/G1 phase and more apoptosis was induced by synergistic combination. CONCLUSION Compared with CD133- cells, CD133+ cells subpopulation exhibited extraordinary cancer stem.
AC133 Antigen ; Antibodies, Monoclonal, Humanized ; pharmacology ; Antigens, CD ; analysis ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cetuximab ; Chemoradiotherapy ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Flow Cytometry ; Glycoproteins ; analysis ; Humans ; Laryngeal Neoplasms ; therapy ; Neoplastic Stem Cells ; drug effects ; radiation effects ; Peptides ; analysis ; Radiation Tolerance

OBJECTIVEEGFR-mediated tumor proliferation plays an important role in the development of cancer, and is a key candidate for targeted therapy. The aim of this study is to evaluate the impact of EGFR monoclonal antibody Cetuximab (C225) on the growth, proliferation and apoptsis of gastric cancer xenograft in nude mice, and its possible mechanisms.

METHODSA gastric cancer cell line SGC-7901 with high EGFR expression level was screened from 7 gastric cancer cell lines. Gastric cancer xenografts in nude mice were established, and randomly divided into C225 treatment group and PBS control group. Tumor growth curves were calculated, the impact of C225 on the tumor growth, proliferation and angiogenesis was evaluated by immunohistochemical (IHC) staining Ki67 and CD34, respectively. The effect of C225 on apoptosis in the gastric cancer cells was evaluated by TUNEL assay. The expression levels of EGFR and its transcription factor Sp1 were detected by IHC staining and Western blot.

RESULTSAfter C225 treatment, the proliferation and growth of gastric cancer xenograft in nude mice were significantly decreased. In the contrast, the apopotic indexes in C225 treatment group and PBS control group were (16.4% +/- 0.3%) and (3.1% +/- 0.9%), respectively, with a significant difference (P < 0.001). There was no significant difference of the densities of CD34-positive microvessels between C225 treatment group and control group. Elevated expression of EGFR and Sp1 after C225 treatment was observed by IHC staining and Western blot assay.

CONCLUSIONEGFR monoclonal antibody cetuximab (C225) can effectively inhibit the growth of gastric cancer xenografts in nude mice, and trigger its apoptosis. Yet, C225 treatment may upregulate the expression of EGFR and its transcription factor Sp1. A "block-transcription activation-compensation" mechanism may exist to explain the molecular mechanism of acquired resistance of a single target blockade treatment.

Animals ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cetuximab ; Humans ; Ki-67 Antigen ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; pathology ; Neovascularization, Pathologic ; prevention & control ; Random Allocation ; Receptor, Epidermal Growth Factor ; immunology ; metabolism ; Sp1 Transcription Factor ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays

OBJECTIVETo explore the changes of the sensitivity of cetuximab-resistant human nasopharyngeal carcinoma (hNPC) cells 5-8F/Erbitux to cetuximab by silencing H-ras gene with RNA interference (RNAi).

METHODSThe 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect the protein levels of H-ras and K-ras. Real-time PCR was employed to detect the expression of H-ras and K-ras. H-ras-shRNA plasmids (shRNA vector carrying the H-ras gene) were constructed and transferred into 5-8F/Erbitux cells. The gene and protein expression levels of H-ras and the changes of the sensitivity of 5-8F/Erbitux cells to cetuximab after transfection were measured, respectively.

RESULTSAfter treatment with cetuximab for 3 and 5 days, the resistance index (RI) of the 5-8F/Erbitux cells was 1.2 and 1.1, and the protein levels of H-ras and K-ras in 5-8F/Erbitux cells were 0.798 +/- 0.019 and 0.190 +/- 0.011, respectively, significantly higher than that in the 5-8F cells (P<0.001). The gene expressions of H-ras and K-ras in 5-8F/Erbitux cells were 1.260 +/- 0.114 and 0.850 +/- 0.006, respectively. Compared with 5-8F cells, the former was higher (P = 0.016) and the latter was lower (P = 0.000). After transfection with H-ras-shRNA plasmid, the 5-8F/Erbitux cells showed reduced levels of H-ras gene and protein, and the cell apoptosis and inhibition rates increased significantly (P<0.001).

CONCLUSIONSH-ras siRNA can reverse cetuximab-resistance of 5-8F/Erbitux cells through down-regulation of H-ras gene expression, indicating that the generation of cetuximab-resistance in 5-8F/Erbitux cells is associated with amplification and overexpression of the H-ras gene.

Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma ; Cell Line, Tumor ; Cell Proliferation ; Cetuximab ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Genes, ras ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVE: To explore the association between expression of ADAM17 and cetuximad resistance in human colorectal cancer SW480 cells. METHODS: The expression of ADAM17 was detected using Western blotting in different human colorectal cancer cell lines, and the cells highly expressing ADAM17 were selected as the target cells. SW480 cells were transfected with ADAM17-siRNA 1 and ADAM17-siRNA 2 and the changes in the expression of ADAM17 protein were detected using Western blotting. SW480 cells were exposed to cetuximad for 24 h and the cell apoptosis was analyzed using flow cytometry. Transwell assay was used to examine the migration ability of SW480 cells with different expression levels of ADAM17; Western blotting was used to analyze the changes in the expressions of AKT signaling pathway-related proteins in the treated cells. RESULTS: The baseline expressions of ADAM17 were significantly higher in SW480 cells than in the other human colorectal cancer cell lines tested ( < 0.05). Both ADAM17-siRNA 1 and 2 effectively reduced the expression of ADAM17 protein in SW480 cells. Knockdown of ADAM17 with siRNA 1 significantly increased the sensitivity of SW480 cells to tocetuximad ( < 0.05), obviously inhibited the cell proliferation, migration and invasion, and significantly reduced the expressions of p-EGFR and p-AKT in the cells ( < 0.001). CONCLUSIONS ADAM17 knockdown obviously inhibits EGFR-AKT signaling pathway and increases the sensitivity of SW480 cells to tocetuximad.
ADAM17 Protein ; genetics ; metabolism ; Antineoplastic Agents, Immunological ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cetuximab ; pharmacology ; Colorectal Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Drug Resistance, Neoplasm ; genetics ; ErbB Receptors ; metabolism ; Gene Knockdown Techniques ; Humans ; Neoplasm Invasiveness ; Oncogene Protein v-akt ; metabolism ; RNA, Small Interfering ; Signal Transduction ; Transfection ; methods