Biomedical subjects

Ling Tian

Publications and source records attributed to Ling Tian.

At least 55 records · Page 3Linked to original sources

PubMed2004

[Constructing the tumor cell vaccine based on homologous matrix metalloproteinase-2 and exploring its effects].

OBJECTIVE: To construct the tumor cell vaccine based on homologous matrix metalloproteinase-2 (MMP-2), and explore its anti-tumor effects. METHODS: The tumor cell vaccine was constructed by transfecting chicken MMP-2 to tumor cells. MTT colorimetric assay was used to evaluate the activation of the transfectants. Then we observed the formative tumor response in vivo and evaluated the effects of the tumour cell vaccine in immunized mice rechallenged with parental cells. RESULTS: Using RT-PCR, we picked out the cell strain with the highest expression level from the transfectants. The MTT colorimetric assay revealed that the transfectants increased the expression of the MMP-2 of tumor cells while they did not change the trend of tumor cells' growth. And in animal experiments the vaccine was preliminarily found to be effective for inhibiting the tumour growth and prolonging the survival time of animals. CONCLUSION: We have constructed the tumor cell vaccine based on homologous matrix metalloproteinase-2. The experiments demonstrated that breaking the immune tolerance of MMP-2 by means of active immunity should be a useful approach to cancer therapy.

Adenocarcinoma↗

PubMed2004

[Serological screening of xenogeneic antigens from rat testis].

OBJECTIVE: It is the intent of this study to analyzed the rat testis library by employing a modified SEREX (serological analysis of recombinant cDNA expression library) approach to find the xenogeneic homologous tumor antigens which could be useful in developing cancer vaccines. METHODS: The screening serum was obtained from the immunized rabbits with human ovarian cancer cells, and 10 positive clones were isolated from the rat testis cDNA library using SEREX technology. RESULTS: We found these 10 clones encoded seven different proteins by means of bioinformatic analysis. Among them, OV-2 and OV-4 encode proteins related to carcinoma in human, yet OV-6 and OV-7 are novel and their encoded proteins remain unknown. CONCLUSION: This study indicates that the utilization of xenogeneic immunized serum in the serological screening of xenogeneic homologous tumor antigens may expand the application of traditional SEREX technology.

Animals↗

PubMed2004

[Construction of pcDNA-BLC and expression of BLC in tumor cells].

OBJECTIVE: To establish a stable transformants of B-lymphocyte chemoattractant (BLC) with the eukaryotic expression vector for investigation of its anti-tumor function further. METHODS: The BLC full-length cDNA was amplified by RT-PCR from the total RNA isolated from the spleen of mouse. After sequence determination, the BLC full-length cDNA was cloned into the eukaryote expression vector pcDNA3.1 (+), the resulted recombinant was designated as pcDNA-BLC. Then pcDNA-BLC was transfected into murine tumor cell line colon 26 by LIPOFECTIN. The stable transformants were selected by G418 and identified by immunoblot assay. The bioactivity of BLC was confirmed by chemotaxis assay. RESULTS: The recombinant pcDNA-BLC was successfully constructed. The stable transformants of BLC can express the bioactive BLC in tumor cell lines. CONCLUSION: The stable transformants can stably express BLC in tumor cell lines. These stable transformants will be useful for further study of the BLC biological effect on tumor and for the development of cancer vaccine.

Adenocarcinoma↗

PubMed2004

[Construction of eukaryotic expression plasmid pSecTag2B-msBlyS expressing mouse soluble B lymphocyte stimulator].

OBJECTIVE: The purpose of this study was to clone the soluble form of the mouse BlyS (msBlyS) and insert it into a eukaryotic expression vector pSecTag2B in order to further elucidat the antitumor activity induced by msBlyS expressed by the recombined plasmid pSecTag2B-msBlyS. METHODS: Full length cDNA of mouse soluble BlyS (msBlyS) was amplified by reverse transcription-PCR from total RNA of mouse spleen. The PCR product was ligated directly with linearized vector pCR2.1 supplied in the TA cloning kit. The recombined plasmid pCR2.1-msBlyS which was selected and identified using blue-white screening method and restriction map analysis and the purified original plasmid pSecTag2B were both cut by HindIII and EcoR I. The digested fragments were extracted and purified from low-melting temperature agarose and ligated by T4DNA ligase. The recombined plasmid pSecTag2B-msBlyS were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing. RESULTS: The sequencing data indicated that inserted msBlyS gene had correct DNA sequence and orientation. CONCLUSION: Eukaryotic expression vector pSecTag2B. Expressing mouse BlyS have successfully been cloned. This will provide us an opportunity to do further research work on BlyS.

Animals↗

PubMed2004

[Construction of recombinant adenovirus for the angiogenic inhibitor, vasostatin, and its expression in vitro].

OBJECTIVE: To construct a recombinant replication-deficient adenovirus for the angiogenic inhibitor, vasostatin and assay its expression in vitro. METHODS: The cDNA for vasostatin was got by PCR amplification, then it was cloned into T vector and confirmed by enzymatic analysis and direct sequencing. Subsequently the cDNA was subcloned into the shuttle plasmid, and co-transformed into 293 cells with backbone plasmid. The resulting recombinant andenovirus was confirmed by PCR and western blot. The activity was assayed by inhibition of HUVEC growth. RESULTS: The PCR product was about 590 bp in length, and sequencing result was identical to that reported. The construction of recombinant replication-deficient adenovirus was confirmed by PCR. Western blot analysis showed the expression of vasostatin in vitro. The supernatant from transduced HeLa cells inhibited the growth of HUVEC specifically. CONCLUSION: The recombinant adenovirus for vasostatin efficiently mediated the expression of the protein in vitro.

Adenoviridae↗

PubMed2004

[The mechanism of anti-tumor immune response against mouse melanoma to xenogeneic vaccination].

OBJECTIVE: To investigate the immunological mechanism for inhibiting melanoma growth in mouse by vaccination with xenogeneic melanocytes. METHODS: Xenogeneic vaccine was prepared from pig eye melanocytes. By means of indirect ELISA the antibodies against pig melanocytes and B16 melanoma cells in immunized mice sera were detected and the immunoglobulin subclass were analyzed. Then after purification, the immunoglobulins were used for the inhibition of cell proliferation in vitro. Analyses of cross-reactive antigen in both pig melanocytes and B16 melanoma cells were performed by Western blot. Xenogeneic vaccine was used before B16 melanoma challenge in C57 BL/c mice and then the growth of tumor was monitored. Meanwhile, other mice immunized with xenogeneic vaccine were depleted of NK cells or CD4+ or CD8+ T lymphocytes. RESULTS: The antibodies against pig melanocytes and B16 melanoma cells in mice sera were not detected by indirect ELISA until 2 weeks after first xenogeneic vaccination, and after the first finding, the antibody titers increased with the time of immunization. The anti-tumor activity and production of autoantibodies, conspicuously those of the elevated IgG, could be abrogated by the depletion of CD4+ T lymphocytes. The cross-reactive antigen with 180 kda protein in both pig melanocytes and B16 melanoma cells was confirmed. Xenogeneic vaccination resulted in inhibition of tumor growth in 90% of the immunized mice. The protective immune response elicited in this fashion was dispelled in the mice depleted of CD4+ T lymphocytes. However this response was found in 70% of the mice depleted of CD8+ T lymphocytes, and the depletion NK cells did not influence the anti-tumor effect of the vaccine. CONCLUSION: The anti-tumor immune response is capable of inhibiting melanoma growth; both humoral immunity and cellular immunity could be induced by xenogeneic melanocytes vaccination. This immune response is mainly mediated by CD4+ T lymphocytes.

Animals↗

PubMed2004

[Screening and identification of stable transfectants of mouse soluble B lymphocyte stimulator].

Mouse colon cancer cells CT26 were transfected with constructed plasmid expressing mouse soluble B lymphocyte stimulator (msBlyS). Single cell clones were selected with 100 microg/ml Zeosin and subcloned by serial limiting dilution. Eight resistant transfectants were isolated and expanded, and five of them displayed the desirable msBlyS cDNA band amplified by semi-quantitative RT-PCR assay. Western blot analysis showed that only msBlyS molecules of the expected size were detected in the cell lysates from transfectants. The supernatant of transfectants could costimulate B cell proliferation in standard costimulation assay. Thus we have successfully screened the stable transfectants expressing high levels of msBlyS in CT26 cells, which could be used as cancer vaccines for further anti-tumor immunotherapy.

Animals↗

PubMed2004

Cancer immunotherapy of targeting angiogenesis.

Tumor growth and metastasis are angiogenesis-dependent. Anti-angiogenic therapy may be a useful approach to cancer therapy. This review discussed tumor angiogenesis and immunotherapy of targeting tumor angiogenesis from two main aspects: (1) active vaccination to induce effective anti-angiogenesis immunity; (2) passive immunotherapy with anti-pro-angiogenic molecules relevant antibody. Evidence from the recent years suggested that anti-angiogenic therapy should be one of the most promising approaches to cancer therapy.

Angiogenesis Inhibitors↗

PubMed2003

Jun activation domain-binding protein 1 expression in breast cancer inversely correlates with the cell cycle inhibitor p27(Kip1).

The Jun activation domain-binding protein 1 (JAB1), aside from being an activator protein 1 coactivator, is involved in degradation of the cyclin-dependent kinase inhibitor p27. We examined JAB1 and p27 protein expression in invasive breast carcinoma specimens and the association of this expression with clinical outcome. JAB1 was detected immunohistochemically in 43 of 53 (81%) tumors; 32 (60%) breast carcinomas showed high JAB1 expression (>50% of cells positive) and reduced or absent p27 levels (P = 0.02, Mann-Whitney U test). Tumors with high p27 expression were rarely positive for JAB1. All eight patients with JAB1-negative tumors had no evidence of relapse or disease progression at a median follow-up of 70 months. Immunoblotting showed strong JAB1 expression in breast carcinoma samples but not in paired normal breast epithelial samples. Targeted overexpression of JAB1 by regulated adenovirus in breast cancer cell lines also reduced p27 levels by accelerating degradation of p27. Thus, the JAB1:p27 ratio may be a novel indicator of aggressive, high-grade tumor behavior, and control of JAB1 could be a novel target for experimental therapies.

Adenoviridae↗

PubMed2003

Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2.

The breaking of immune tolerance of "self-antigens" associated with angiogenesis is an attractive approach to cancer therapy by active immunity. We used vascular endothelial growth factor receptor-2 (VEGFR-2) as a model antigen to explore the feasibility of the immunotherapy with a vaccine based on a xenogeneic homologous protein. To test this concept, we prepared a quail homologous VEGFR-2 protein vaccine (qVEGFR) based on quail VEGFR-2. At the same time, a protein vaccine based on the corresponding ligand-binding domain of mouse self-VEGFR-2 (mVEGFR) was also prepared and used as a control. We found that immunotherapy with qVEGFR was effective at protective and therapeutic antitumor immunity in several solid and hematopoietic tumor models in mice. Autoantibodies against mouse VEGFR-2 (Flk-1) were identified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Anti-VEGFR antibody-producing B cells were detectable by ELISPOT. Endothelial deposition of immunoglobulins developed within tumor. VEGF-mediated endothelial cell proliferation was inhibited in vitro by immunoglobulins from qVEGFR-immunized mice. Antitumor activity was caused by the adoptive transfer of the purified immunoglobulins. Antitumor activity and production of autoantibodies against Flk-1 could be abrogated by the depletion of CD4+ T lymphocytes. Angiogenesis was apparently inhibited within the tumors, and the vascularization of alginate beads was also reduced. No marked toxicity was found in the immunized mice. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factor receptor associated with angiogenesis in a cross-reaction with single xenogeneic homologous protein.

Animals↗

PubMed2003

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition.

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.

Antibodies, Monoclonal↗

PubMed2003

Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice.

Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction.

Alginates↗

PubMed2003

Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor.

The breaking of immune tolerance against self epidermal growth factor receptor (EGFr) should be a useful approach for the treatment of receptor-positive tumors with active immunization. To test this concept, we constructed a plasmid DNA encoding extracellular domain of xenogeneic (human) EGFr (hEe-p) or corresponding control mouse EGFr (mEe-p) and empty vector (c-p). Mice immunized with hEe-p showed both protective and therapeutic antitumor activity against EGFr-positive tumor. Sera isolated from the hEe-p-immunized mice exhibited positive staining for EGFr-positive tumor cells in flow cytometric analysis and recognized a single 170-kDa band in Western blot analysis. Ig subclasses responded to rEGFr proteins were elevated in IgG1, Ig2a, and Ig2b. There was the deposition of IgG on the tumor cells. Adoptive transfer of the purified Igs showed the antitumor activity. The increased killing activity of CTL against EGFr-positive tumor cells could be blocked by anti-CD8 or anti-MHC class I mAb. In vivo depletion of CD4(+) T lymphocytes could completely abrogate the antitumor activity, whereas the depletion of CD8(+) cells showed partial abrogation. The adoptive transfer of CD4-depleted (CD8(+)) or CD8-depleted (CD4(+)) T lymphocytes isolated from mice immunized with hEe-p vaccine showed the antitumor activity. In addition, the increase in level of both IFN-gamma and IL-4 was found. Taken together, these findings may provide a new vaccine strategy for the treatment of EGFr-positive tumors through the induction of the autoimmune response against EGFr in a cross-reaction between the xenogeneic homologous and self EGFr.

Adoptive Transfer↗

PubMed2003

Sustained activation of JNK/p38 MAPK pathways in response to cisplatin leads to Fas ligand induction and cell death in ovarian carcinoma cells.

The efficacy of cisplatin in cancer chemotherapy is limited by the development of resistance. Although the molecular mechanisms involved in chemoresistance are poorly understood, cellular response to cisplatin is known to involve activation of MAPK and other signal transduction pathways. An understanding of early signal transduction events in the response to cisplatin could be valuable for improving the efficacy of cancer therapy. We compared cisplatin-induced activation of three MAPKs, JNK, p38, and ERK, in a cisplatin-sensitive human ovarian carcinoma cell line (2008) and its resistant subclone (2008C13). The JNK and p38 pathways were activated differentially in response to cisplatin, with the cisplatin-sensitive cells showing prolonged activation (8-12 h) and the cisplatin-resistant cells showing only transient activation (1-3 h) of JNK and p38. In the sensitive cells, inhibition of cisplatin-induced JNK and p38 activation blocked cisplatin-induced apoptosis; persistent activation of JNK resulted in hyperphosphorylation of the c-Jun transcription factor, which in turn stimulated the transcription of an immediate downstream target, the death inducer Fas ligand (FasL). Sequestration of FasL by incubation with a neutralizing anti-FasL antibody inhibited cisplatin-induced apoptosis. In contrast, chemoresistance in 2008C13 cells was associated with failure to up-regulate FasL. Moreover, in these cells, selective stimulation of the JNK/p38 MAPK pathways by adenovirus-mediated delivery of recombinant MKK7 or MKK3 led to sensitization to apoptosis through reactivating FasL expression. Thus, the JNK > c-Jun > FasL > Fas pathway plays an important role in mediating cisplatin-induced apoptosis in ovarian cancer cells, and the duration of JNK activation is critical in determining whether cells survive or undergo apoptosis.

Apoptosis↗

PubMed2003

Active immunogene therapy of cancer with vaccine on the basis of chicken homologous matrix metalloproteinase-2.

Matrix metalloproteinase (MMP) family, in particular MMP-2, may play a key role in angiogenesis and tumor growth. It is conceivable that the breaking of immune tolerance of MMP-2 should be a useful approach to cancer therapy by active immunity. To test this concept, we constructed a plasmid DNA encoding chicken homologous MMP-2 (c-MMP-2) and control vectors. We found that the vaccine based on chicken homologous MMP-2 as a model antigen could induce both protective and therapeutic antitumor immunity. Autoantibodies against MMP-2 in sera of mice immunized with c-MMP-2 could be found by Western blotting analysis and ELISA assay. There was the deposition of autoantibodies within the tumor. IgG1 and IgG2b were substantially increased in response to c-MMP-2 immunization. The elevation of MMP-2 in the sera of tumor-bearing mice was abrogated with the vaccination of c-MMP-2. Transmigration of human endothelial cells and tumor cells through gelatin-coated filters was inhibited with immunoglobulins isolated from mice immunized with c-MMP-2. The gelatinase activity of MMP-2, including both latent MMP-2 (M(r) 72,000) and active MMP-2 (M(r) 66,000) derived from tumor tissues, was apparently inhibited by the vaccination with c-MMP-2. The antitumor activity and the inhibition of angiogenesis were acquired by the adoptive transfer of the purified immunoglobulins. The antitumor activity and production of autoantibodies against MMP-2 could be abrogated by the depletion of CD4(+) T lymphocytes. Angiogenesis was apparently inhibited within tumor, and chick CAMs angiogenesis was also inhibited. Thus, our findings may provide a vaccine strategy for cancer therapy through the induction of an autoimmune response against MMP-2 in a cross-reaction by the immunization with the single xenogeneic homologous MMP-2 gene and may be of importance in the additional exploration of the application of other xenogeneic homologous genes identified in human and other animal genome projects in cancer therapy.

Animals↗

PubMed2003

Cisplatin resistance in an ovarian carcinoma is associated with a defect in programmed cell death control through XIAP regulation.

Chemoresistance is a major impediment to the successful treatment of cancer. It involves various mechanisms, including defects in the apoptosis program that is induced by anticancer drugs. To further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin (CDDP) treatment, we compared the effect of CDDP on expression of X-linked inhibitor of apoptosis protein (XIAP), a direct inhibitor of caspase-3, -7, and -9, Fas, Fas-ligand (Fas-L), and pro- and antiapoptotic proteins in a CDDP-sensitive human ovarian carcinoma cell line (2008) and its CDDP-resistant subclone (2008C13). In this article, we show that cisplatin treatment led to a differential expression of distinct apoptotic targets in the CDDP-sensitive cell line (2008) and its CDDP-resistant subclone (2008C13). The acquisition of cisplatin resistance was associated with the ability of the treated cells to enhanced expression of XIAP, whereas the death inducer Fas-L was abrogated in 2008C13 following treatment with CDDP. However, the CDDP-sensitive cells failed to activate XIAP but increased Fas-L expression, indicating that distinct regulatory mechanisms are operative. These findings suggest that the expression of XIAP and downregulation of Fas-L are linked to chemoresistance in ovarian carcinoma cells and may represent one of the potential antiapoptotic mechanisms involved during this process.

Antineoplastic Agents↗

PubMed2003

[Expression, purification and refolding of extracellular domain of mouse epidermal growth factor receptor].

A DNA fragment encoding extracellular domain of mouse epidermal growth factor receptor (EGFR) was obtained by PCR from a previous recombinant plasmid. The DNA fragment was then ligated into prokaryotic expression vector, and expressed in Escherichia Coli. The recombinant protein was purified under denature conditions by affinity chromatography, and refolded with gradient dialysis. The recombinant protein could produce antibodies to recognize extracellular domain and full-length of mouse EGFR, and form homodimer in the presence of EGF detected by western blot analysis. These findings provide evidence that the renatured recombinant extracellular domain of mouse epidermal growth factor receptor is immunogenetic and may be important for further application of this protein in functional and immunological research.

Animals↗

PubMed2003

[Synthesis of N-[2-(cholesteryloxycarbonylamino) ethyl] carbamoylmethylated mannan].

OBJECTIVE: To study the synthesis of chol-AECM-mannan. METHODS: Using mannan as the main material, we synthesized a compound with polysaccharides partially modified by cholesterol (namely chol-AECM-mannan) through carboxymethylating, carbamoylating and condensation reaction. RESULTS: The product was confirmed by IR spectrum and 1H nuclear magnetic resonance (1HNMR). The proton integral ratio of mannan and cholesterol is 100: 5, that is to say, the numbers of the substituted cholesterol per 100 mannose units are 1. 1. The production rate is 24%. CONCLUSION: The method is a effective pathway in term of synthesizing chol-AECM-mannan.

Cholesterol↗