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1.
Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell-expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.  相似文献   

2.
Gonadotrophin-releasing hormone (GnRH) is the prime decapeptide hormone in the regulation of mammalian reproduction. Active immunization against GnRH has been a good treatment option to fight against hormone-dependent disease such as breast cancer. We designed and purified a novel protein vaccine Hsp65–GnRH6 containing heat shock protein 65 (Hsp65) and six copies of GnRH in linear alignment. Immunization with Hsp65–GnRH6 evoked strong humoral response in female mice. The generation of specific anti-GnRH antibodies was detected by ELISA and verified by western blot. In addition, anti-GnRH antibodies effectively neutralized endogenous GnRH activity in vivo, as demonstrated by the degeneration of the ovaries and uteri in the vaccinated mice. Moreover, the growth of EMT-6 mammary tumor allografts was inhibited by anti-GnRH antibodies. Histological examinations have shown that there was increased focal necrosis in tumors. Taken together, our results showed that immunization with Hsp65–GnRH6 elicited high titer of specific anti-GnRH antibodies and further led to atrophy of reproductive organs. The specific antibodies could inhibit the growth of EMT-6 murine mammary tumor probably via an indirect mechanism that includes the depletion of estrogen. In view of these results, the protein vaccine Hsp65–GnRH6 appears to be a promising candidate vaccine for hormone-dependent cancer therapy.  相似文献   

3.
High luminescence quantum yield water‐soluble CdTe/ZnS core/shell quantum dots (QDs) stabilized with thioglycolic acid were synthesized. QDs were chemically coupled to fully humanized antivascular endothelial growth factor165 monoclonal antibodies to produce fluorescent probes. These probes can be used to assay the biological affinity of the antibody. The properties of QDs conjugated to an antibody were characterized by ultraviolet and visible spectrophotometry, fluorescent spectrophotometry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transmission electron microscopy and fluorescence microscopy. Cell‐targeted imaging was performed in human breast cancer cell lines. The cytotoxicity of bare QDs and fluorescent probes was evaluated in the MCF‐7 cells with an MTT viability assay. The results proved that CdTe/ZnS QD–monoclonal antibody nanoprobes had been successfully prepared with excellent spectral properties in target detections. Surface modification by ZnS shell could mitigate the cytotoxicity of cadmium‐based QDs. The therapeutic effects of antivascular endothelial growth factor antibodies towards cultured human cancer cells were confirmed by MTT assay. Copyright © 2014 John Wiley & Sons, Ltd.  相似文献   

4.
DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.  相似文献   

5.
The expression of GnRH (GnRH-I, LHRH) and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the ovary. The proliferation of human ovarian cancer cell lines is time- and dose-dependently reduced by GnRH and its superagonistic analogs. The classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of GnRH analogs in these cancer cells. The GnRH receptor rather interacts with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity via activation of a phosphotyrosine phosphatase resulting in downregulation of cancer cell proliferation. In addition GnRH activates nucleus factor κB (NFκB) and protects the cancer cells from apoptosis. Furthermore GnRH induces activation of the c-Jun N-terminal kinase/activator protein-1 (JNK/AP-1) pathway independent of the known AP-1 activators, protein kinase (PKC) or mitogen activated protein kinase (MAPK/ERK).  相似文献   

6.
BACKGROUND: Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. AIM: Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. METHOD: First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. RESULTS: a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. CONCLUSIONS: GnRH is synthesised and released by trophoblastic cells.  相似文献   

7.
8.
So WK  Cheng JC  Poon SL  Leung PC 《The FEBS journal》2008,275(22):5496-5511
The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) is well known for its role in the control of pituitary gonadotropin secretion, but the hormone and receptor are also expressed in extrapituitary tissues and tumor cells, including epithelial ovarian cancers. It is hypothesized that they may function as a local autocrine regulatory system in nonpituitary contexts. Numerous studies have demonstrated a direct antiproliferative effect on ovarian cancer cell lines of GnRH and its synthetic analogs. This effect appears to be attributable to multiple steps in the GnRH signaling cascade, such as cell cycle arrest at G(0)/G(1). In contrast to GnRH signaling in pituitary gonadotropes, the involvement of G(alpha q), protein kinase C and mitogen-activated protein kinases is less apparent in neoplastic cells. Instead, in ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin-sensitive protein G(alpha i), leading to the activation of protein phosphatase, which in turn interferes with growth factor-induced mitogenic signals. Apoptotic involvement is still controversial, although GnRH analogs have been shown to protect cancer cells from doxorubicin-induced apoptosis. Recently, data supporting a regulatory role of GnRH analogs in ovarian cancer cell migration/invasion have started to emerge. In this minireview, we summarize the current understanding of the antiproliferative actions of GnRH analogs, as well as the recent observations of GnRH effects on ovarian cancer cell apoptosis and motogenesis. The molecular mechanisms that mediate GnRH actions and the clinical applications of GnRH analogs in ovarian cancer patients are also discussed.  相似文献   

9.
RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and the corresponding rat anti-id Mabs were generated and characterized. Following immunizations in mice, antisera raised against anti-id antibodies were analyzed by typical immunoassays. It was observed that mouse anti-anti-id sera (Ab3) revealed binding affinity and specificity to CA215 that are comparable to those of RP215. Both RP215 and Ab3 were shown to induce apoptosis of cultured cancer cells in vitro by TUNEL and MTT assays. These experimental observations were consistent with that of in vivo tumor growth inhibition by RP215 in previous nude mouse experiments. Therefore, heterologous or homologous anti-id antibodies of RP215 that contain the internal image of its specific epitope in CA215 may serve as effective anti-cancer vaccines for therapeutic treatments of various cancers in humans. The relative stability of RP215-specific carbohydrate-associated epitope was compared to that of human IgG at extreme pH’s (≤2 or ≥12) or following NaIO4 treatments. The major molecular forms of CA215 were further documented with various enzyme immunoassays and found to have similar secondary structures to those of normal human immunoglobulin G.  相似文献   

10.
11.
Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys6, desGly10, Pro9‐NH2]‐GnRH, [d ‐Cys6, desGly10, Pro9‐NHEt]‐GnRH, and [d ‐Cys6, α‐aza‐Gly10‐NH2]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys6, desGly10, Pro9‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys6, desGly10, Pro9‐NH2]‐GnRH‐Dox) and conjugate III ([d ‐Cys6, α‐aza‐Gly10‐NH2]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys6‐des‐Gly10‐Pro9‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys6‐des‐Gly10‐Pro9‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.  相似文献   

12.
Diagnosis of many infectious, autoimmune diseases and cancers depends on the detection of specific antibodies against peptide epitope by enzyme-linked immunosorbent assay (ELISA). However, small peptides are difficult to be coated on the plate surfaces. In this study, we selected GnRH as a model hapten to evaluate whether VEGF121 would be suitable as an irrelevant hapten-carrier to develop a universal platform for specific antibodies detection. Firstly, GnRH was fused to the C terminus of VEGF121 and the resultant fusion protein VEGF–GnRH expressed effectively as inclusion bodies in Escherichia coli. Thereafter, VEGF–GnRH was easily purified to near homogeneity with a yield of about 235 mg from 2.1 L induced culture. At last, VEGF–GnRH was used to perform ELISA and western blot, and our results suggested that VEGF–GnRH was capable of detecting anti-GnRH antibodies in sera both qualitatively and quantitatively. Indeed, previous studies of our laboratory had demonstrated that other fusion proteins such as VEGF–Aβ10, VEGF–GRP, VEGF–CETPC, and VEGF–βhCGCTP37 were able to detect their corresponding antibodies specifically. Therefore, VEGF121 may be a suitable irrelevant fusion partner of important diagnostic peptide markers. Our works would shed some light on the development of a universal platform for detection of specific antibodies.  相似文献   

13.
The α2-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-α2-macroglobulin receptor monoclonal antibodies bind to 90–100% of the blood monocyte population and not to other blood cells. This defines the α2-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18–33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.  相似文献   

14.
Lung cancer is one of the most common malignancies worldwide. In this Letter, novel MOM-ether analogs of isosteviol were designed and synthesized to be tested for anticancer activities against H1299 lung cancer cell lines. The effects of these derivatives were studied in H1299 human large cell lung carcinoma cells that are null for p53 and compared to normal counterparts NL-20 normal lung epithelial cells. The initial screening of twelve MOM-ether analogs of isosteviol derivatives on H1299 lung cancer cells by MTT assay revealed that two derivatives (an ester and a carbamate) were the most potent in reducing cell viability. The IC50 values for these derivatives were determined to be 14 and 21 μM respectively. We compared the cytotoxicity of the best derivatives in H1299 lung cancer cells and NL-20 normal lung epithelial cells. Both derivatives showed lower cytotoxic effects on NL-20 normal lung epithelial cells. Moreover, both derivatives induced apoptosis in H1299 lung cancer cells more than NL-20 normal lung epithelial cells.  相似文献   

15.
South American camelids are induced ovulators and require a stimulus to trigger the LH surge responsible for the ovulation. Seminal plasma (SP) of fertile alpacas (Lama pacos) was tested using a bioassay of pituitary cells to study the effect of seminal plasma on LH release. Plates containing rat pituitary cells (2 x 10(5) cells/90-95% viability) were cultured adding: (A) whole SP (WSP) treated with charcoal-dextran, or 1:2 or 1:4 proportions diluted in culture medium (DMEM/HEPES + antibiotics), or (B) 1:2 SP + anti-GnRH rabbit serum (inhibitory potency 10(-5) M), or (C) 1:2 SP + anti-GnRH + 100 nM synthetic GnRH (buserelin acetate) or (D) 100 nM, 50 nM, 10 nM, and 1 nM synthetic GnRH. Concentration (ng/ml) of LH secreted (Sec) and contained (Con) was analyzed using RIA 125I and the percentage of Sec and Con in each experiment was determined. The results of LH Sec for the cells treated with 50, 10, and 1 nM GnRH were 39, 13, and 1.5%, respectively (r2 = 98.41%, r = 0.9920) but cells treated with 100 nM GnRH secreted 10% of LH. With WSP, 1:2, or 1:4 SP the LH Sec was of 44.5% (3.25 ng/ml), 27% (1.9 ng/ml), and 18% (1.2 ng/ml), respectively. The exposure of cells to 1:2 SP + anti-GnRH, or to 1:2 SP + anti-GnRH/100 nM GnRH produced 31% (2.20 ng/ml) and 30% (1.8 ng/ml) of LH Sec, respectively. These results suggest that the SP of alpacas could have some factor(s) different from GnRH that would contribute to the mechanisms of LH secretion and to the induced ovulation in the female alpaca.  相似文献   

16.
Summary A 3-dimensional tumor growth inhibition assay [18] has been adapted to test the cytotoxic activity of a panel of monoclonal antibodies directed to various antigenic determinants on the surface of mouse mammary tumor cells. Target cells can be prepared from either cultured cells or from pieces of fresh tumor. Antibody and complement are added when cells are growing actively and cell growth can be measured, non destructively, over a 7–10-day period. Effective diffusion of antibody through collagen gel and binding to target cells embedded in the gel is demonstrated by indirect immunofluorescent staining. The specificity of monoclonal antibody AMT 101 cytotoxicity for mouse mammary tumor cells is the same in trypan blue exclusion assays of single-cell suspensions as in collagen gel assays, with complete killing seen in the collagen gel assay only. The collagen gel assay allows the testing of repeated treatments in vitro, as well as combined treatment with multiple antibodies. It also allows cell-cell interaction and preserves all cell components in the tumor. The collagen gel assay has potential as a method of predicting the outcome of monoclonal antibody treatment of solid tumors.  相似文献   

17.
软骨多糖诱导MCF-7乳腺癌细胞凋亡的实验研究   总被引:1,自引:0,他引:1  
研究软骨多糖诱导MCF-7乳腺癌细胞凋亡及其作用机理。方法:选用MCF-7人类乳腺癌细胞系体外培养,应用MTT法检测细胞生长抑制率,TUNEL法检测细胞凋亡率,HE染色法观察细胞形态学改变,流式细胞仪检测细胞周期的变化,免疫荧光方法检测BCL-2BAD及波形蛋白Vimentin的表达率。结果:软骨多糖对MCF-7细胞体外生长具有明显的抑制作用,且呈时间和浓度依赖性;软骨多糖可诱导MCF-7细胞发生凋亡并伴随有凋亡小体出现等形态学变化;软骨多糖促进BCL-2蛋白的表达水平下降,BAD表达水平上升,及Vimentin的降解。结论:软骨多糖能够在体外诱导MCF-7细胞凋亡,是一种新型的抗乳腺癌活性物质。  相似文献   

18.
Two gonadotropin-releasing hormone (GnRH) peptides with a cystein substitution of the first (C1-GnRH) or tenth (C10-GnRH) amino acid were conjugated to ovalbumin and equine serum albumin, respectively, via the sulfhydryl group of the introduced cysteine. Animals were immunized three times at 3-wk intervals with both conjugates in either saline (n = 5), Freund's complete adjuvant (FCA; n = 5), Havlogen (n = 6), Ribi adjuvant system (RAS; n = 5), dimethyl dioctadecyl ammonium bromide (DDA; n = 4), Alhydrogel (n = 5) or Regressin (n = 5). Animals immunized with conjugates in saline or RAS did not produce anti-GnRH titers. The highest anti-GnRH titers were produced by animals treated with FCA. The Alhydrogel and DDA treatments stimulated the production of GnRH antibodies in all animals treated, but titers were lower than in animals immunized with FCA. When vaccines were formulated with Havlogen or Regressin, anti-GnRH titers were low or absent. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were depressed in FCA and in Alhydrogel treated animals. The antisera raised were predominantly directed against either the carboxy- or the amino-terminal end of the GnRH peptide, or directed equally against both, depending on the individual animal. Results suggest that no epitope of GnRH dominates the immune response in cattle and show that the best alternative to FCA is Alhydrogel.  相似文献   

19.
GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His(5), Trp(7), Tyr(8)) were introduced singly or in pairs into GnRH I. Tyr(5) replacement by His(5) produced the highest increase in the antiproliferative potency of GnRH I. Tyr(8) substitution of Arg(8) produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys(6) and D-Arg(6)) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg(8) of GnRH I makes contact with Asp(302), whereas Tyr(8) of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.  相似文献   

20.
Background  The aim of this study was to screen and identify novel B cell epitopes within the human heparanase protein and to investigate the impact of self-developed anti-heparanase polypeptide antibodies on growth and invasion of HCCLM6 human hepatocellular carcinoma cells in vitro. Methods  The flexible regions of secondary structure and the B cell epitopes of the human heparanase amino acid sequence were predicted by DNAStar and Bcepred software.The multiple antigenic peptides (MAP) of the epitopes were synthesized in eight-branched form. Rabbits were immunized with the eight-branched MAPs mixed with the universal T-helper epitope human IL-1β peptide (VQGEESNDK, amino acid 163–171). The immunogenicity of the synthesized peptides was evaluated by ELISA, western blot and immunohistochemistry. The impact of the self-developed rabbit anti-heparanase polyclonal antibodies on growth and invasion ability of HCCMLM6 cells were analyzed in a cell culture model. The cells were first treated with one of the three antibodies, respectively, and then measured by using MTT, flow cytometry, plate clone formation, invasion assay and heparan sulfate degrading enzyme assay. Results  The three amino acid sequences 1–15 (MAP1), 279–293 (MAP2), and 175–189 (MAP3) in the large subunit of the human heparanase protein were predicted as its most potential epitopes. ELISA, western blot and immunohistochemistry analysis showed that all three MAPs were capable to induce high titer of serum antibodies. Antibodies induced by MAP1 and MAP2 were high specific. Furthermore, anti-MAP2 antibodies showed the strongest avidity towards liver cancer tissues. Under the treatment with the three anti-heparanase antibodies, respectively, the growth, cell cycle and clone formation of the cells remained unchanged when compared with a treatment with normal rabbit IgG. However, an inhibition of cell invasiveness and heparanase activity could be detected under the treatment with anti-MAP1- or anti-MAP2-antibody (with a terminal concentration of 100 μg/ml). The cell invasiveness was decreased by 54 and 38%, respectively, the heparanase activity by 43 and 39%, respectively. Conclusion  The multiple antigenic peptides MAP1 (AC 1–15) and MAP2 (AC 279–293) may be the dominant B cell epitopes in the human heparanase protein. The induced polypeptide antibodies can effectively inhibit the heparanase activity of HCCLM6 liver cancer cells and therefore influence their invasion ability, which provides a theoretic basis for the development of anti-heparanase antibodies and their clinical use as vaccine.  相似文献   

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