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Objective To explore antitumor effect of 131I-Trastuzumab on human epidermal growth factor receptor(HER) 2 overexpressing breast cancer cells and investigate its possible mechanism.Methods The expression levels of HER2 of three different breast cancer cell lines (BT474,MCF-7,HCC1937) were detected with immunofluorescence.Trastuzumab was labeled with 131I using the Iodogen method and 131I-Trastuzumab was isolated with ultrafiltration membrane,then the labeling efficiency,radiochemical purity and immunoreactivity were measured.The effects of 131I,Trastuzumab and 131I-Trastuzumab on viability of BT474 cells were evaluated with cell counting kit-8 (CCK-8) assay.The levels of total Akt and phosphorylated Akt (p-Akt) were detected with Western blot analysis.One-way analysis of variance (ANOVA),ANOVA for factorial design,Bonferroni correction and Pearson correlation analysis were used for data analysis.Results The expression level of HER2 in BT474 cells was much higher than those in HCC1937 and MCF-7 cells.The labeling efficiency,radiochemical purity and immunoreactivity of 131I-Trastuzumab were (89.71± 2.93)％,(91.80±1.43)％ and (58.84±3.35)％ respectively.131I (4.625 GBq/L),Trastuzumab(125.0 rmg/L) and 131I-Trastuzumab(4.625 GBq/L) exhibited a dose-dependent cytotoxicity against BT474 cells (r =-0.964,-0.912,-0.618;all P＜0.05).The cell viability of 131I-Trastuzumab treated gourp (34.73％ ±5.03％) was significantly lower than those of 131I and Trastuzumab treated groups (64.36％± 1.51％ and 58.09％±4.14％;t=10.373 and 8.180,both P＜0.05),and the cell viability of control group was (100.00±4.54)％.131I-Trastuzumab shown a positive multiplicative interaction between 131I and Trastuzumab (F=9.226,P＜0.05;CDI =0.929).Western blot results showed that there was no significant difference of total Akt expression among the control group,131I group,Trastuzumab group and 131I-Trastuzmab group (F=0.208,P＞0.05).P-Akt expression in both Trastuzumab group and 131I-Trastuzumab group were much lower than those of control group and 131I group (t=12.524,15.984,7.347,10.807;all P＜0.05),while there was no significant difference of p-Akt expression between Trastuzumab group and 131I-Trastuzumab group(t =3.460,P＞0.05).Conclusions 131I-Trastuzumab may kill HER2 overexpressing breast cancer cells more effectively than Trastuzumab alone.The underlying mechanism may be attributed to that 131I-Trastuzumab may enhance the radiosensitivity by the inhibitory effect on PI3K/Akt pathway and thus exert synergistic effects with 131I.
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Objective To prepare 99Tcm-human epidermal growth factor receptor 2 (HER2) affibody (ABH2) and explore its feasibility as an imaging agent in HER2-positive breast cancer.Methods Sodium glucoheptonate and SnCl2 · 2H2O were used to label ABH2 with 99Tcm.The labeling yield and radiochemical purity of 99Tcm-ABH2 were determined.The stability of 99Tcm-ABH2 was tested in PBS and serum.The equilibrium disassociation constant (Kd) of 99Tcm-ABH2 was measured with MBA-MD-361 breast cancer cells.SPECT/CT imaging was carried out at 1.0 h and 4.5 h after injection of 37 MBq 99Tcm-ABH2 in nude mice (n=4) xenografted with MBA-MD-361 breast cancer.The T/NT (liver,brain,lung,heart,bone and muscle) ratios were deduced from SPECT/CT acquired data.For the blocking experiment,200 μg ABH2 was injected intravenously before 99Tcm-ABH2 injection.SPECT/CT imaging was performed in the same way.The T/NT ratios were compared between non-blocked and blocked groups by one-way analysis of variance.Results Fhe labeling yield of 99Tcm-ABH2 was above 99％.99Tcm-ABH2 was substantially stable in PBS and serum;the radiochemical purity was (95.0± 1.0)％ after incubation with serum at 37 ℃ for 6.0 h.The Kd of 99Tcm-ABH2 was 1.7 nmol/L.The radioactive uptake in cancer was visualized at 1.0 h and 4.5 h after injection of 99Tcm-ABH2 in HER2 positive MDA-MB-361 breast cancer.99Tcm-ABH2 was cleared out mainly through urinary system.The ratios of tumor to liver,lung,brain,heart,muscle and bone were 1.81± 0.60,8.95±1.13,20.08±6.12,7.61±0.56,10.62±1.78,11.42±2.07,respectively at 4.5 h after 99Tcm-ABH2 injection.After ABH2 blocking,the corresponding T/NT ratios were 0.60±0.23,3.05± 1.38,5.24±2.17,2.42±1.02,8.16±2.66,2.76±0.48 (F=29.38,P＜0.05) respectively.Conclusion 99Tcm-ABH2 could be synthesized with high purity,and this new agent could be used to image HER2-positive breast cancer specifically.
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Objective To prepare the 99Tcm-labeled human epidermal growth factor receptor type 2 (HER2) affibody molecule ZHER2:342 and evaluate its receptor binding specificity in vitro.Methods The molecular ZHERa:342 was labeled with 99Tcm using the ligand exchange method.The labeling efficiency and radiochemical purity were measured by HPLC.The major factors,such as the mass of SnC12 and NaOH and reaction time were analyzed,and the optimal method was summarized.Cell binding kinetics and cellular retention of the probe were investigated in HER2-expressing SKOV-3 cells and MDA-MB-231 cells with low HER2 expression respectively.HER2 binding specificity of 99Tcm-ZHER2:342 was analyzed by a pre-injection of excess unlabeled ZHER2:342 to saturate HER2 receptors.One-way analysis of variance and two-sample t test were used.Results The optimal labeling procedure was as follows:5 μg (1 g/L) of ZHER2:342 was mixed with 5 μg of NaOH (1 g/L),then 8.8 μg SnC12(1 g/L,solution) was added,followed by 150 μl (37 MBq) 99TcmO4-solution,and finally the mixture was slightly vortexed and incubated for 1 h at room temperature.99TcmZHER2:342 was stable in vitro with a high labeling efficiency of (98.10± 1.73)％.The radiochemical purity was ＞ 98％,and was more than 85％ after the incubation for 24 h in saline and fresh human serum.The cell binding of 99Tcm-ZHER2:342 with HER2-expressing SKOV-3 cells gradually increased over time with a peak of (9.95± 1.02)％ at 6 h.The binding of 99Tcm-ZHER2:342 in SKOV-3 cells was significantly higher than that in MDA-MB-231 cells at every time point (5.68-9.88 vs 0.56-2.11 ; t:from-34.50 to-13.14,all P＜0.01).The labeled molecular probe retained the capacity to bind specifically to HER2-expressing SKOV-3 cells since the cell binding decreased from (9.95 ± 1.02) ％ to (2.11 ±0.27) ％ after receptor saturation (t =-13.14,P＜0.01).Conclusions 99Tcm-ZHER2:342 has a high labeling efficiency,good stability and optimal binding specificity.These characteristics enable it to be a promising molecular probe for HER2-targeting imaging.
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Objective To study the biodistribution of anti-HER-2/neu monoclonal antibody Herceptin labeled by 131I(131I-Herceptin) in healthy KM mice and nude mice bearing human ovarian cancer xenografts and radioimmunoimaging (RII) of the nude xenografts-bearing mice.Methods 131I-Herceptin was prepared using Iodogen method.The labeling efficiency, radiochemical purity, stability and immunocompetence were measured.The percentage activity of injection dose per gram of tissue (%ID/g) and the radioactivity ratio of tumor to non-tumor tissue (T/NT) were calculated for each time point.The optimal time for imaging was investigated by comparing the 131I-Herceptin SPECT for the nude mouse models bearing ovarian cancer xenografts at different time points.Results The labeling efficiency and radiochemical purity of 131I-Herceptin were 89.8% and 98.4%, respectively.The labeling was stable and had good immunocompetence.131 I-Herceptin was cleared rapidly mainly through liver, spleen and kidneys, consistent with first order two-compartment model.The uptake of 131I-Herceptin in the tumors bearing human SKOV-3 xenografts was much higher than that in nontumor tissue.The% ID/g was 18.08 in the tumor at 24 h post injection.The T/NT ratio increased with time and was 27.27 at 72 h post injection.The tumors in nude mice bearing SKOV-3 xenografts could be visualized on 131I-Herceptin SPECT imaging 2 h post injection; definitely identiffed 48 h post injection and the radioactivity ratio of tumor to contralateral tissue was 11.44 at 120 h post injection.However, the tumor in nude mice bearing HO-8910 xenografts did not show abnormal uptake of 131 I-Herceptin at each time point.Conclusions 131 I-Herceptin is a good radiopharmaceutical targeting SK-OV-3 xeuografts and it may be useful in imaging carcinoma of ovary and target therapy of its metastases with high HER-2/neu expression.
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Objective To study the expression of cyclin E and epidermal growth factor receptor (EGFR) in breast carcinoma and their correlation with metastasis,relapse and survival time. [WT5”HZ]Methods Cyclin E and EGFR expressions at protein level were determined by immunohistochemistry technique in 110 patients with breast carcinoma. Results Of the 110 patients,cyclin E and EGFR positive expression were both in 60 (54.55%),and there was a positive relationship between cyclin E and EGFR expression (r s=0.823,P =0.001);Cyclin E and EGFR expression level were correlated with clinical stage (? 2=12.86,P =0.005;? 2=14.21,P =0.004),tumor histological grading (? 2=8.86,P =0.005;? 2=4.90,P =0.04),lymph node metastasis (? 2=10.22,P =0.001;? 2=9.62,P =0.002),ER expression (? 2=29.8,P =0.001;? 2=32.08,P =0.001) and PR expression (? 2=19.56,P =0.001;? 2=26.92,P =0.001). The rate of local relapse and distant metastasis in cases with positive cyclin E and EGFR expression were significantly higher than that in cases with negative expression (? 2=7.33,P =0.01;? 2=7.88,P =0.005);The mean survival time and 5-year survival rate in cases with positive cyclin E and EGFR was significantly shorter than that in cases with negative expression. [WT5”HZ]ConclusionCyclin E and EGFR expression can predict the relapse and metastasis of breast carcinoma. They can be used as markers of prognosis of breast carcinoma in clinical practice.
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AIM: To investigate the effect of antisense RNA on osteopontin (OPN) expression in renal tubular epithelial cells. METHODS: Cell clone expressing stably OPN antisense RNA was formed by transfering retroviral vector expressing OPN antisense RNA into renal tubular epithelial cells, NRK52E cells, using liposome, with cell clones transfected by empty vector and vector expressing OPN sense RNA as controls. Ribonuclease protection assay(RPA), Western Blot, ELISA and assay of OPN activity were performed to detect expression of OPN mRNA and protein in above clones cultured with or without epidermal growth factor(EGF). RESULTS: The antisense RNA was only expressed by antisense clone. Antisense clone, sense clone and empty clone all expressed OPN mRNA. EGF enhanced expression of OPN mRNA, but not OPN antisense RNA or OPN sense RNA in above clones. OPN protein was not expressed in antisense clone cultured with or without EGF and empty clone cultured without EGF, but was expressed in sense clone cultured with or without EGF and empty clone cultured with EGF. CONCLUSION: Antisense RNA can inhibit OPN protein expression by means of preventing OPN mRNA translation, but not inhibit OPN mRNA transcription in renal tubular epithelial cells.
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Objective: To study the expressien and heterogeneity of epidenmoid growth factor receptor(EGFR) in human mucoepidermoid carcinoma MEC 1 cells. Methods: EGFR1 McAb (MaH) immunofluorescent stain and flowcytometer (FCM) were used to detect the positive percentage and heterogeneity of EGFR expression in MEC 1 cells. Results: Positive expression of EGFR was found in 100% of MEC 1 cells. Average peak channel of relative fluorescent intensity was 8.006?4.410. Double peaks of MEC 1 cells from single parameter histogram was observed with FCM. Conclusion: All MEC 1 cells express EGFR and there are heterogeneous cell populations of EGFR expression in MEC 1 cell line.
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Objective:To evaluate the expression of EGFR and gene nm23H1 and their relation to the tumor oncogenesis and progress of cholangiocarcinoma.Methods:SABC immunohistochemistry was used to detect the expression of EGFR and nm23H1 in cholangiocarcinoma and cholangitis tissue.Results:The positive rate of nm23H1 in cholangiocarcinoma was lower than cholangitis(P
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AIM: To determine EGF contents in human milk, frech cow's milk and cow's milk-based infant formulas and the relationship between EGF content of human milk and neonatal maturity.METHODS: EGF contents in 57 human colostrum from mothers delivering prematurely and at term, 4 different fresh cow's milk and 8 different cow's-milk-based infant formulas with hydrolyzed and non-hydrolyzed proteins were determined by radioimmunoassay (RIA). RESULTS: Human milk from mothers of premature infants had higher EGF content compared to that from mothers of term infants[(28.2?10.3) nmol/L vs(17.3?9.6) nmol/L]. There was a negative correlation between EGF content of human milk and gestational age, birth weight of neonates. The values in fresh cow's milk [(16.6?3.8) nmol/L]were similar to that in human term milk. The contents in non-hydrolyzed protein formulas[(7.5?1.9) nmol/L]were much lower than that in human milk and fresh cow's milk. No immunoreactive EGF was detected in all hydrolyzed protein formulas. CONCLUSION: The occurrence of high EGF concentration in premature milk may represent a maternal compensatory mechanism to accelerate the growth and maturation in immature infants. Lack of EGF in formulas suggests that they may not suitable for those newborns with immature or damaged gastrointestinal tract.
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Objective To investigate the expressions of vascular endothelial growth factor (VEGF) , hypoxia inducible factor 1 alpha (HIF 1?) and epidermal growth factor (EGF) in hepatocellular carcinoma (HCC) and their clinical significance. Methods The expressions of VEGF, HIF 1? and EGF in 36 cases of HCC and corresponding paraneoplastic tissues and normal liver tissues (6 cases) were studied by immunohistochemistry assay. ResultsThe expression rate of VEGF, HIF 1? and EGF in HCC tissue was 89%, 67% and 75% respectively, higher than those in paraneoplastic tissues and normal liver tissues ( P
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Objective To investigate the role of heparin binding epidermal growth factor-like growth factor(HB-EGF)in active psoriasis vulgaris.Methods HB-EGF mRNA and protein were detected by hy-bridization in situ and immunohistochemistry in normal skin tissues,lesional and non-lesional psoriatic skin of progressive stage.Results In normal skin tissues,the stain of HB-EGF mRNA and protein was located in the basal layer of epidermis(100.00%)and there was only a little expression in the suprabasal layers(16.67%).Focal overexpression was found in the suprabasal layers of non-lesional and peri-lesional psoriat-ic skin(88.00%,80.00%respectively);however,there was no HB-EGF mRNA protein expression in the superabasal layers of the central part of psoriatic lesions(0),and nearly no expression in the basal layer(4.00%).Conclusion HB-EGF may play an important role in the pathogenesis of early psoriasis.
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Objective To investigate the relationship between keratinocyte growth factor (KGF), nerve growth factor(NGF)and psoriasis;and to study the interaction between keratinocytes and interstitial cells in psoriasis. Methods The expression of KGF, KGF receptor (KGFR), NGF, NGF receptor (NGFR), epidermal growth factor receptor (EGFR) and proliferating cell neuclear antigen (PCNA) was studied with immunohistochemical technique (SP) in lesional skin, non lesional skin and normal controls. Results Significant overexpression of KGF and KGFR was present in psoriatic basal cell layers and suprabasal cell layers compared to that in non lesional and normal controls (P0.05). Expression of KGF and KGFR was absent in dermis. Expression of NGF and NGFR was observed mainly in granular cell layers and upper spinous layers, and was significantly different between normal controls and non lesional, between non lesional and lesional skin (P
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Objective To investigate expression of p63?PCNA and epidermal growth factor receptor(EGFr)in skin squamous cell carcinoma(SCC) . Method Tissue specimens from 68 cases of SCC were studied using immunohistochemistry staining method with monoclonal antibodies to p63?PCNA and EGFr. Results The expression of p63 protein and PCNA was principally restricted to basal cells with high proliferative potentiality and was absent in cells of stratum corneum that were undergoing terminal differentiation in normal or carcinoma epidermis. In highly differentiated tumors, the staining of p63 formed a ring surrounding carcinoma nests. In low differentiated tumors, p63 positive cells were increased and disorderly arranged. Diffuse expression of PCNA and EGFr was found through out all carcinoma nests. Expression of p63?PCNA and EGFr was significantly stronger in SCC and intraepithelial neoplasm(SIN)level Ⅲthan those in normal or proliferative epidermis near the carcinoma. A significant correlation was found between expression of p63,PCNA,EGFr and degree of differentiation of SCC,and between p63 and EGFr,p63 and PCNA,PCNA and EGFr, respectively. Conclusion Overexpression of p63?PCNA?EGFr may be contributed to acquisition of enhanced capability of proliferation, aggression and anaplasia in SCC.
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AIM: To construct a recombinant hEGF-hbFGF(78-154aa)fusion protein, which not only has the heparin-binding ability, but also promotes the growth of the cells, and to express the fusion protein in E. coli expression system with high expression level.METHODS: hEGF gene was joined with 231 bp fragment coding hbFGF(78-154aa) and expressed in E. coli. The fusion protein was purified using affinity chromatography of heparin-Hyper D and analyzed with western blot. The pI value and the biological activity were both assayed.RESULTS: The fusion protein was expressed in a high expression level of about 30% of the total cell protein, as estimated by SDS-PAGE. Western analysis results showed that the antigenicity of fusion protein was similar to hEGF. Fusion protein could not only bind heparin but also promote the growth of 3T3 cell. The pI value of fusion protein was 5.2.CONCLUSION: The recombinant hEGF-hbFGF(78-154aa) fusion protein possessed the characteristics of both hEGF and hbFGF. This new-designed protein would become a good object for the research on the relationship between the structure and the function of the growth factor.
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AIM: To examine the effect of protamine sulfate, lipopolysaccharide(LPS),tumor necrosis factor(TNF), epidermal growth factor(EGF) and " Bushen Huoxue Xiezhuo" (BHX) decoction on the proliferation of extracorporeal cultured rat glomerular epithelial cells (GEC). METHODS: Their action on the proliferation of rat GEC were investigated using the -TdR incorporation. Meanwhile, the serum of rats treated with BHX decoction was extracted pharmacologically and its effects on the growth of GEC were also studied. RESULTS: LPS, protamine sulfate, TNF-? and EGF could significantly inhibit the -TdR incorporation of GEC in a dose- and time-dependent manner. However, this inhibition could be efficiently reversed by the serum containing BHX decoction. CONCLUSION: GEC is one of the main target cells on which BHX decoction act, and the protection on GEC might be one of the mechanisms underlying the role of BHX decoction in preventing the progression of nephrosis.
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AIM:To observe the expression of epidermal growth factor receptors(EGFR) of gingival tissue in periodontitis. METHODS: The expression of EGFR was determined by using immunohistochemical techniques in gingival tissue of 15 healthy individual, 32 cases with adult periodontitis (AP) and 12 cases with juvenile periodontitis (JP). RESULTS: Expression of EGFR was mainly located on basal cell membranes in healthy gingiva, and the staining intensity was faint. In AP cases, expression of high level EGFR was mostly observed on the membranes of epithelial cell in the periodontal endopocket or junctional epithelium, intensity of staining appeared to decrease gradually with the differentiation of keratinocytes, and the horny cell layer was not stained by the antibody. In JP cases, strong positive staining was present on membrane of epithelial cells in the germinative layer of gingival tissue. There was an apparent difference between healthy gingiva and AP or JP (P＜ 0.01 ), or AP and JP (P＜ 0.01). CONCLUSION: The distribution and expression of EGFR in gingival epithelium of periodontitis showed obvious differ- euce. The data indicated that EGFR may affect apical migration of junctional epithelium, and may play a role in development of AP and JP.