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Objective To explore the mechanism by which the anti-human P185erbB2 scFv-Fc-IL-2(HFI)modulates tumor surface molecules and activates immune effector cells in vitro. MethodsMTT assay was used to test the proliferation and the LAK-like cytotoxicity. Flow cytometry assay was used to test the expression of ICAM-1, Fas and erbB2 receptors in tumor cells and the expression levels of CD molecules FasL and LFA-1 in human PBMC. Antibody-dependent cell-mediated cytotoxicity(ADCC)mediated by HFI against SKOV3, MCF-7 and SGC-7901 tumor cells was explored hv LDH release assay. Results The expression levels of ICAM-1 and Fas on SKOV3 cell treated with HFI were upregulated, from 24.85％ and 0.53％ to 85.36％ and 59.19％ respectively, while the expression levels of erbB2 on SKOV3, MCF-7 and SGC-7901 tumor cells treated with HFI were downregulated, from 98.48％, 42.60％ and 36.66％ to 94.01％,30.95％ and 12.36％ respectively. HFI could significantly enhance the proliferation activity of human PBMC, and CD3+ CD8+ T cells and CD3- CD16+ CD56+ NK cells were elevated, from 24.37％ and 6.90％ to 38.80％ and 13.45％ respectively. The expression levels of CD25, LFA-1 and FasL were significantly enhanced from 3.99％, 86.52％ and 5.02％ to 12.96％, 99.06％ and 16.19％. The LAK-like cytotoxicity of human PBMC treated with HFI against SKOV3, MCF-7,SGC-7901 tumor cells was significantly improved:HFI was effective in mediating ADCC against SKOV3,MCF-7 and SGC-7901 tumor cells which expressed high,medium and low levels of erbB2,respectively,and HFI-induced ADCC was correlated with the degrees of erbB2 expression on the tumor cells. Conclusion The expression levels of ICAM-1 and Fas on SKOV3 cell treated with HFI are significantly upregulated. The expression levels of erbB2 on SKOV3, MCF-7 and SGC-7901 tumor cells treated with HFI are downregulated. HFI can significantly enhance the proliferation activity of human PBMC. The LAK-like eytotoxicity of human PBMC treated with HFI against tumor cells is significantly enhanced. HFI iS effective in mediating ADCC and the activity of HFI-induced ADCC is correlated with the degrees of erbB2 expression on the tumor cells.
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Objective:To study the effects of ICA on HepG2.2.15 cell proliferation, their sensitivity to the lysis by CD3AK effector cell, to investigate the reversal action of ICA on hepatocarcinoma cells from immune escape through Fas/FasL pathway.To provide the theoretical and experimental bases for ICA development.Methods:MTT assay was used to detect cell proliferation and CD3AK cells cytotoxicity activity;flow cytometry assay was used to examine expression of surface molecules and apoptosis rate of HepG2.2.15 cells.Results:When HepG2.2.15 cells line was treated with 50 ?g/ml ICA,a significant reduction of the rate of cell proliferation was observed. Inhibition rate at 48h was 22.04%,and 29.68% at 72h.Kinetic study showed that inhibition of cell proliferation was time dependent (P0.05).ICA could inhibit apoptosis of Jurkat cells induced by HepG2.2.15 cells. In the co-culture system of HepG2.2.15 cell and Jurkat T cell, apoptosis ratio of Jurkat cell was reduced from 46.66% to 18.20% by ICA (P
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Human CD3AK cells were prepared from peripheral blood mononuclear cells by culturing with recombinant IL-2 and antiCD3AK McAb. The mechanism and regulation of CD3AK cytotoxic activity with cytokines (rhIFN-?, rhIFN-?, TNF) and chernotherapeutic agents (CDDP or ADM) were observed by LDH-release assay, ABC-CELISA and the flow cytometric assay. The results showed: (1) Adhesion molecules ICAM-l/LFA-1 participated in CD3AK-mediated killing of tumor cells, hrlFN-? and TNF enhanced cytotoxicity of CD3AK through this pathway. (2) CD3AK could indirectly kill tumor cells by releasing soluable cytotoxic factors. (3) The membrane-associated TNF may be involved in CD3AK-mediated cytotoxicity. (4) CD3AK cells could induce the apoptosis of tumor cells. (5) Pretreatment of tumor cells with CDDP or ADM resulted in the increased vulnerability of tumor cells to CD3AK-mediated killing, the enhancement of CD3AK-mediated cytotoxicity by CDDP was relative to the increased expression of ICAM-1, HLA-ABC on tumor cell membrane.
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Objective: To investigate the influence of Icarrin(ICA) on transforming growth factor ?2(TGF?2) expression by PG cells and the mechanisms of reversion of TGF?2 mediated immunosuppression by ICA.Methods: Cell proliferation and cytotoxici- ty were assayed by MTT assay. mRNA level of TGF?2 and perforin was detected by RT-PCR assay. assay.Expression of TGF?2 and IL- 2R? was analysed by folw cytometry assay. Results: ICA could inhibit the protein and mRNA expression of TGF?2 in PG cells. It could also reverse the supressed cytotoxicity of LAK, CD3AK cells by TGF?2. The IL-2R? expression on LAK and perforin gene transcription and proliferation of CD3AK inhibited by TGF?2 were also partly recovered. Conclusion: The anti-anti-tumor activity of ICA is at least partly due to its capability of inhibiting TGF?2 expression in tumor cells and restoring the cytotoxicity of immunocompetent cells inhibited by TGF?2.
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AIM Mechanism of icariin (ICA) and pseudomonas jinanensis agent (PJA) on anti-metasta-sis of a highly metastatic human lung tumor cell line (PG) and the effect type of the combination of ICA,PJA were studied. METHODS PG cells were treated with ICA、PJA, or the combination of both in vitro. Adhesion assay, invasion assay, migration assay, flow cytometry (FCM) and immunohistochemical straining were used. The effects of ICA and PJA combination were evaluated with Q method of Jin Zheng-Jun. RESULTS ICA、PJA could decrease PG cells adhesive ratio to laminin substrate in a time-dependent manner ( P
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Objective:To study inhibited tecomerase activity of HL-60 cells in human cancer cell by Icarrin(ICA) and its mechanism.Methods:MTT assay,NBT assay, TRAP-PCR,RT-PCR assay,Flow Cytometry.Results:ICA inhibited telomerase activity in HL-60 cells significantly.It was negative correlation between telomerase activity and the expression ratio of CD11b antigen in HL-60 cells.It can induced HL-60 cells to differentiate into mature granulocytes.It can changed cell cycle distribution of HL-60 cells,which was reflected by the accumulation of the vast majority of cells in the G0/G1phase and the loss of cells in the S phase. It can downregulated cell proliferation related gene c-myc;upregulated p21 gene protein and mRNA expression.Conclusion:This study proved mechanism of ICA on inhibited telomerase activity from gene-protein-effect of cell level in HL-60 cells.