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Objective@#To detect the expressions of epidermal growth factor-like domain 7 (EGFL7) and P185 protein in breast cancer tissues and serum, and to analyze the correlation between the expression levels of EGFL7 and P185 in tissues and clinicopathological parameters of breast cancer patients.@*Methods@#Sixty patients with breast cancer in Hunan Cancer Hospital from March 2016 to March 2018 were collected as observation group, and 60 patients with breast benign lesions in the hospital during the same period were selec-ted as control group. The expressions of EGFL7 and P185 protein in tissues of patients in the two groups were detected by immunohistochemical two-step method, and the levels of EGFL7 and P185 protein in serum of patients in the two groups were detected by enzyme-linked immunosorbent assay (ELISA). The relationships between the expressions of EGFL7 and P185 protein and clinicopathological parameters of breast cancer patients were analyzed.@*Results@#The positive rates of EGFL7 in tissues in the observation group and the control group were 65.00% (39/60) and 28.33% (17/60), and there was a significant difference between the two groups (χ2=16.205, P<0.001). The positive rates of P185 in tissues in the two groups were 43.33% (26/60) and 15.00% (9/60), and there was a significant difference between the two groups (χ2=11.657, P=0.001). The serum levels of EGFL7 protein in the observation group and the control group were (3.39±0.38) μg/ml and (2.75±0.31) μg/ml respectively, with a significant difference (t=10.109, P<0.001). The serum levels of P185 protein in the two groups were (7.12±0.75) μg/ml and (6.08±0.62) μg/ml respectively, with a significant difference (t=8.279, P<0.001). The positive expression of EGFL7 protein was closely related to tumor size (χ2=6.128, P=0.013), TNM stage (χ2=7.781, P=0.005) and metastasis (χ2=5.444, P=0.020). The positive expression of P185 protein was closely related to tumor size (χ2=8.910, P=0.003) and TNM stage (χ2=8.024, P=0.005).@*Conclusion@#The levels of EGFL7 and P185 protein are high in breast cancer tissues and serum, and their positive expressions are related to tumor size and TNM stage. EGFL7 and P185 proteins play important roles in the progression of breast cancer.
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Objective To detect the expressions of epidermal growth factor-like domain 7 (EGFL7) and P185 protein in breast cancer tissues and serum,and to analyze the correlation between the expression levels of EGFL7 and P185 in tissues and clinicopathological parameters of breast cancer patients. Methods Sixty patients with breast cancer in Hunan Cancer Hospital from March 2016 to March 2018 were collected as observation group,and 60 patients with breast benign lesions in the hospital during the same period were selec-ted as control group. The expressions of EGFL7 and P185 protein in tissues of patients in the two groups were detected by immunohistochemical two-step method,and the levels of EGFL7 and P185 protein in serum of patients in the two groups were detected by enzyme-linked immunosorbent assay (ELISA). The relationships between the expressions of EGFL7 and P185 protein and clinicopathological parameters of breast cancer patients were analyzed. Results The positive rates of EGFL7 in tissues in the observation group and the control group were 65. 00％ (39 / 60)and 28. 33％ (17 / 60),and there was a significant difference between the two groups (χ2 = 16. 205,P < 0. 001). The positive rates of P185 in tissues in the two groups were 43. 33％ (26 / 60)and 15. 00％ (9 / 60),and there was a significant difference between the two groups (χ2 = 11. 657,P = 0. 001). The serum levels of EGFL7 protein in the observation group and the control group were (3. 39 ± 0. 38)μg/ ml and (2. 75 ± 0. 31)μg/ ml respectively,with a significant difference (t = 10. 109,P < 0. 001). The serum levels of P185 protein in the two groups were (7. 12 ± 0. 75)μg/ ml and (6. 08 ± 0. 62)μg/ ml respectively, with a significant difference (t = 8. 279,P < 0. 001). The positive expression of EGFL7 protein was closely related to tumor size (χ2 = 6. 128,P = 0. 013),TNM stage (χ2 = 7. 781,P = 0. 005)and metastasis (χ2 =5. 444,P = 0. 020). The positive expression of P185 protein was closely related to tumor size (χ2 = 8. 910, P = 0. 003)and TNM stage (χ2 = 8. 024,P = 0. 005). Conclusion The levels of EGFL7 and P185 protein are high in breast cancer tissues and serum,and their positive expressions are related to tumor size and TNM stage. EGFL7 and P185 proteins play important roles in the progression of breast cancer.
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Objective: Overexpression of the HER2/neu oncogene is a frequent molecular event in multiple human cancers. Being a cancer antigen, p185 erbB2 is an ideal target for immunotherapy. In order to decrease the immunogenicity of mouse anti-p185 erbB2 monoclonal antibody in human cancer therapy, we constructed the eukaryotic expression vector of anti-p185 erbB2 chimeric monoclonal antibody and verified expression of the chimeric antibody in CHO-dhfr - cell. Methods: The variable regions of light chain and heavy chain were amplified with RT-PCR and inserted into the chimeric antibody vector pWSD2. After CHO-dhfr - cells were transfected with recombination plasmid by lipofectAMINE, the chimeric antibody expressing level was identified with RT-PCR, indirect-ELISA, and Western blot. The specificity of the anti-p185 erbB2 chimeric antibody was testified with ELISA assay and immunoprecipitation. Moreover, the effects of chimeric antibody on the proliferation of breast cancer cell line SKBR3, which is overexpressing p185 erbB2 , were measured with MTT assay in vitro. Results: The anti-p185 erbB2 chimeric antibody eukaryotic expression vector was constructed successfully and the expression of the chimeric antibody in CHO-dhfr - was verified by RT-PCR, indirect-ELISA, and Western blot. ELISA assay showed that chimeric antibody reacted with cells overexpressing p185 erbB2 specifically, but did not react with that non-overexpressing p185 erbB2 . Immunoprecipitation test confirmed that the chimeric antibody could bind to p185 erbB2 specifically. The MTT assay demonstrated that the chimeric antibody could inhibit the growth of SKBR3 cells overexpressing p185 erbB2 . Conclusion: The anti-p185 erbB2 mouse/human chimeric antibody that was expressed in CHO-dhfr - cells can bind to p185 erbB2 specifically and inhibit proliferation of SKBR3 cells overexpressing p185 erbB2 . It has a potential application in biotherapy of cancer.
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PURPOSE: The HER2 gene encodes a 185-kd transmembrane glycoprotein receptor (p185(HER2)) that has partial homology with the epidermal growth factor receptor (EGFR) and shares intrinsic tyrosine kinase activity. The HER2 gene has been found to be amplified in various human cancers and to be associated with poor prognosis. The authors investigated the correlation between clinicopathologic factors and the overexpression of the p185(HER2) in Korean gastric adenocarcinoma patients, and determined whether the antiproliferative effects of anti- p185(HER2) antibody can also be observed on gastric cancer cell lines that overexpress this growth factor receptor. MATERIALS AND METHODS: We evaluated the relationship between p185(HER2) overexpression and clinicopathological features in 94 (M: F=52: 42) gastric adenocarcinoma patients (median age 59 years). Protein expression was analysed by immunohistochemical staining in paraffin embedded tissues with monoclonal antibody for p185(HER2). To explore the role of humanized anti-p185(HER2) monoclonal antibody trastuzumab (Herceptin ) in vitro, the growth curve of Korean gastric cancer cells that overexpress the p185(HER2) protein was studied and a cell cycle analysis was performed. RESULTS: p185(HER2) overexpression correlates positively with lymph node metastasis (p=0.002), distant metastasis (p=0.01), AJCC classification (p=0.01), higher relapse rate p=0.001), and a tendential association with the pT stage (p=0.054). p185(HER2) overexpression was found to be more frequent in advanced gastric cancer than early gastric cancer (54.1% vs 24.2%, p=0.008). Patients with overexpression of p185(HER2) were found to have significantly lower relapse-free (p=0.003) and overall survival (p= 0.0004) than patients without overexpression. Among several Korean gastric cancer cell lines, SNU-1, SNU-5, and SNU-620 overexpress p185(HER2). Trastuzumab inhibited the proliferation of p185(HER2) overexpressed Korean gastric cancer cell line by 21% with down-regulation of p185(HER2) protein expression. DNA fluorescence flow cytometry of propidium iodide-stained nuclei showed a reduction in the fraction of the S phase following treatment with trastuzumab. CONCLUSIONS: Taken together, our observations suggest the potential prognostic significance of p185(HER2) overexpression in Korean gastric adenocarcinoma patients and point to the need for further research on this mechanism. This suggests the possible use of p185(HER2) as a therapeutic target in gastric cancer.
Subject(s)
Humans , Adenocarcinoma , Cell Cycle , Cell Line , Classification , DNA , Down-Regulation , Flow Cytometry , Fluorescence , Genes, erbB-2 , Glycoproteins , Lymph Nodes , Neoplasm Metastasis , Paraffin , Prognosis , Propidium , Protein-Tyrosine Kinases , ErbB Receptors , Recurrence , S Phase , Stomach Neoplasms , TrastuzumabABSTRACT
Objective The purpose of this study was to investigate the relationship of p185 and p53 protein overexpression with pathological types and recurrence.Antibodies against p185 and p53 proteins were used to measure expression of these proteins in the nuclei or cell membrane of cells from sections of the giant cell tumor of bone GCT.It showed that 11 out of 52 tumors overexpressed p185 protein and 14 out of 52 tumors had abnormally high levels of p53 protein,4 cases had abnormally high levels of both p185 and p53 proteins,positive expression rates of p185 and p53 in cases with recurrence and cases without reccarrence were 46 2%,20 5% and 38 5%,15 4% respectively.However,there was no association between p185 and p53-positive cases and pathological degree.There was significant correlation between the expression of p185 and p53 protein in the giant cell tumor of bone and recurrence.(? 2=6 125,P=0 047).However,there was no statitically significance between the expression of p185 and p53 protein in the giant cell tumor of bone and pathological types.So that,we consider that the clinical significance for p185,p53 overexpression in GCT to be researched further.
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PURPOSE: Anti-p185HER2/neu monoclonal antibody (mAb) that can induce phenotypic reversion and monoclonal antibodies specific for the p185HER2/neu growth factor receptor that are able to diminish its kinase-signaling properties represent a specific advance in the therapy for p185HER2/neu-expressing human cancers. With mAb treatment, down-regulation of p185HER2/neu surface receptors and attenuation of the kinase-signaling properties have been observed and regarded as a basic phenomenon; however, the mechanisms for mAb-induced phenotypic reversion are not clear. METHODS: We used human tumor-cell lines of SK-BR-3, T6-17, and U373MG. With immunoprecipitation and Western blotting, we investigated the changes in p185HER2/neu receptor phosphorylation and the expression of signal-regulatory proteins (SIRPs) after mAb treatment. To identify the proteins interacting with Tat-binding protein-1 (TBP1), we used the Clonotech Gal4 matchmaker two-hybrid system. RESULTS: Minimal to moderate reduction in phosphotyrosine (pTyr) content was observed in SK-BR-3 and T6-17 cells with short-term (10-30 minutes) incubation after mAb treatment, but that did not alter total p185HER2/neu receptor density. SIRPs phosphorylation after peptide treatment was increased. With mAb treatment, three proteins were shown to interact with TBP1, and all of the interacting proteins are subunits of proteasome 26S. Collectively, anti- p185HER2/neu mAb or peptide down-regulates the surface receptors and attenuates the kinase signaling, which then both induces higher proteasome activity through increased TBP1 and increases SIRPs expres sion. CONCLUSION: Increased proteasomal activity may degrade abnormal proteins and increased SIRPs may regulate signal transduction toward the norm. Therefore, activation of a protein-degradation pathway and induction of signal-regulatory proteins may be possible mechanisms for the ultimate anti-tumor effects of the anti-p185HER2/neu mAb or peptide.
Subject(s)
Humans , Antibodies, Monoclonal , Blotting, Western , Down-Regulation , Immunoprecipitation , Phosphorylation , Phosphotransferases , Phosphotyrosine , Proteasome Endopeptidase Complex , Signal TransductionABSTRACT
Objective:To clone Fab genes of anti-p185 monoclonal antibody 5E12 and express it in E.coli.Methods:Fd and ? genes were cloned by RT-PCR, inserted into Fab expression vector and expressed in E.coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis. The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry.Results:Fd and ? genes were cloned and expressed in E.coli. But the bacterially expressed Fab fragments showed no antigen binding activity. After the N-terminal sequences of V regions was corrected to original sequences, the Fab expressed in bacterial was able to target HER2/neu-expressing cells(NIH3T3/erbB-2 cells). Correction of Fd N-terminal sequences could partially resume the antigen binding activity. But correction of ? chain N terminal sequences was shown no expected result.Conclusion:Successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the V region N terminal changes introduced with PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.
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To study the anti-cancer activity of the three anti-oncoprotein p185 mAbs established by surface epitope masking method so as to provide some basic information for clinical tumor targeting therapy.Methods: Immnune flurorence staining, MTT assay, soft-ar-gar culture and long-term cell proliferation assay detected the suppressive activities of the mAbs against the breast cancer cells overexpressing p185 and p185-transfected fibroblast cells, and the inducing effects of the mAbs on tumors sensitivity to chemotherapy.Results:The 3 mAbs specifically suppressed tumor cell proliferation and enhanced the sensitivity of tumors to chemotherapeutics. Conclusion:These mAbs have the potentials to develop into the clinical tumor targeting therapeutic reagents of the tumors overexpressing p185.
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Objective:To explore the enhanced sensitization to chemotherapeutic agent paclitaxel-induced apoptotic effect on p185-overexpressing human malignant breast cancer cell lines SKBr3 by anti-p185c-erbB-2/neu engineered antibody treatment and to study its emerging mechanism.Methods:The proliferative inhibitory effect was assessed by MTS assay; Cells stained with AnnexinV-FITC and PI were used to qualify the apoptotic cell number by FACS(Fluorescence-activated cell sorting) analysis; Phosphorylation of Ser473 AKt and p65 NF-?B subunit were determined by Western blot; NF-?B-DNA binding activity was demonstrated by EMSA(Electrophoretic mobility shift assay).Results:Anti-p185c-erbB-2/neu engineered antibody rendered SKBr3 cells more susceptible to paclitaxel-induced proliferative inhibition, which further attributed to apoptotic induction; Furthermore, combination of the engineered antibody and paclitaxel also showed effective suppression of AKt/NF-?B pathway in SKBr3 cells.Conclusion:The aggressiveness of AKt/NF-?B pathway was partly attributed to its resistance to paclitaxel-induced apoptosis. Anti-p185c-erbB-2/neu engineered antibody plus paclitaxel combination rendered p185-overexpressing human malignant breast cancer cells SKBr3 more susceptible to paclitaxel-induced apoptosis by efficient suppression of AKt/NF-?B pathway.