Anti-tumor activity and immunological modification of ribosome-inactivating protein （RIP） from Momordica charantia by covalent attachment of polyethylene glycol

Ribosome-inactivating proteins （RIPs） are a family of enzymes that depurinate rRNA and inhibit protein biosynthesis. Here we report the purification, apoptosisinducing activity, and polyethylene glycol （PEG） modification of RIP from the bitter melon seeds. The protein has a homogenous N-terminal sequence of N- Asp-Val-Ser-Phe-Arg. Moreover, the RIP displayed strong apoptosis-inducing activity and suppressed cancer cell growth. This might be attributed to the acti- vation of caspases-3. To make it available for in vivo application, the immunogenicity of RIP was reduced by chemical modification with 20 kDa （mPEG）2-Lys-NHS. The inhibition activity of both PEGylated and non- PEGylated RIP against cancer cells was much stronger than against normal cells, and the antigenicity of PEGylated RIP was reduced significantly. Our results suggested that the PEGylated RIP might be potentially developed as anti-cancer drug.     

Antitumor effects of the molecule-downsized immunoconjugate composed of lidamycin and Fab′fragment of monoclonal antibody directed against type IV collagenase

Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab'-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab' fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab'-LDM conjugate was 65 kD with a 1 : 1 molecular ratio of Fab' and LDM. The Fab'-LDM conjugate maintained most part of the immunoreactivity of Fab' fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab'-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab'-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment     

Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to ?stensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the fur- ther study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Ana- baena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.     

Enhanced antitumor effects of tumor antigen-pulsed dendritic cells by their transfection with GM-CSF gene

To investigate the biological characterization and antitumor activitites of GM-CSF gene-transfected dendritic cells, the splenic dendritic cells were infected with GM-CSF recombinant replication-deficient adenoviruses in vitro . Their enhanced expression of B7 was demonstrated by FACS analysis, and more potent stimulatory activity was confirmed by allogeneic MLR. Immunization of dendritic cells pulsed with irradiated B16 melanoma cells induced sig-nificant CTL and enabled host to resist the challenge of wild-type B16 cells. When they were transfected with GM-CSF gene subsequently, the induced CTL activity was higher, and the produced protection against B16 cell challenge and therapeutic effect on the mice with preestablished pulmonary melastases more effective. These data suggest that the dendritic cells pulsed with tumor antigen then transfected with GM-CSF gene can be used as an effective vaccine in tumor immunotherapy.     

黑色素抑制流感病毒诱导宿主细胞凋亡

The apoptosis induced by influenza virus in cultured MDCK cells was reported and the selective inhibitory effect of melamin on the apoptosis induced by influenza virus was investigated. The results showed that the DNA ladder could be first detected at 6 h post-infection (p.i.), accompanied by nuclear condensation and nuclear fragmentation could be easily detected at 12 h p.i. In addition, the apoptosis-induced activity of influenza virus A1/Jingfang 86-1 strain was more potent than that of B/Hufang 93-1 strain (P＜0.05). In the range of 20-125 μg/mL, melanin was found to significantly (P＜0.001) inhibit apoptosis induced by 64 hemagglutination unit influenza virus infection with a inhibitory rate comparable to that obtained by virazole and showed no cytotoxicity. The inital results suggested that the mechanism of melanin against the apoptosis induced by influenza virus was related to the blockage of viruses' adsorbtion to the host cells.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulat-ing factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GM-CSF-dependent cell line TF1 and was chemotactic for monocytes. The in vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combina-tion of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. The in vivo anti-tumor effect indicated that     

Selective proliferation of human γδ T cells in vitro

The effect of monoethylphosphate (MEP,commercial available or synthesized) together with IL-2 on the selective proliferation of human γδT cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated.The γδT cells were stimulated by MEP to proliferate in a dose-dependent manner.The effect of synthesized MEP was 10 times greater than that of commercial MEP.When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP,the total cell number increased about 1000-3000 fold;and the ratio of γδT cells reached to 70-80%.The selective expansion of γδT cells depended on the synergic action of MEP and IL-2.The bulk cultured γδT cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5,K562 and Daudi) and autologous tumor cells.The culture system described here not only offers a simple method for obtaining a large number of γδT cells which may become a new effector in the adoptive immunotherapy,but also provides a useful model for the further studies of the structure and function of γδT cells in vitro.     

Human immune suppression is inducible by trichosanthin via CD8 cell—mediated pathway

Trichosanthin(Tk),a polypeptide with 249 amino acid residues isolated and purified from a Chinese medicinal herb,showed the capability of inducing abortion and was able to inhibit tumor growth and HIV replication.Owing to sequence homology of the peptide with a ribosomeinactivating protein,the downward activity of Tk was suggested to be related to its cytotoxic property.We report here,however,that Tk could exert potent inhibitory effects on human lymphoproliferative responses in vitro to allogeneic,mitogenic and soluble antigens with 50% inhibition doses ranged between 0.05 and 0.5μg／ml.The lowresponsivenesss caused by Tk was not due to toxic cytolysis.Rather,evidences suggested that,in the dose range adopted,the Tk-induced inhibition was attributable,at least in part,to immune suppression,in view of (1) Tk was more effective in the early stage of alloreactivity;(2)Suppression also occurred if responder cells were pulsetreated with Tk rather than cocultured;(3)Irradiated Tk-pulsed cells were capable of inducing suppression in a Tk-free culture;(4)Suppression could also be transferred by the supernatants of Tk-pulsed cultured cells;(5)Tk-induced immune suppression was diminished by depletion of CD8^ cells from the culture,and,finally;(6)Adding CD8^ cells back to the culture could restore the suppres sion.Thus the possibility that Tk might function as a down-regulator by immunological mechanisms in human immune responses is discussed.     

Over-expression of fibroblast activation protein alpha increases tumor growth in xenografts of ovarian cancer cells

Fibroblast activation protein alpha （FAPα） is a 95-kDa serine protease of post-prolyl peptidase family on cell surface. FAPoL is widely expressed in tumor microenviron- ment. The wide spread association of FAPα expression with cancer suggests that it has important functions in the disease. However, the nature of FAPα＇s roles in cancer cell activity is not well-determined. It has been showed that FAPα silencing in SKOV3 cells induces ovarian tumors but significantly reduces tumor growth in a xenograft mouse model. To further determine the role of FAPoL in epithelial ovarian cancer cells, SKOV3-FAPα and HO8910-FAPα cell lines, which over-expressed FAPα stably, were con- structed and then their biological behaviors were investi- gated. It was found that FAPoL promoted ovarian cancer cell proliferation, drug resistance, invasiveness, and migra- tion in vitro. Immunochemistry assay showed that FAPα significantly facilitated tumor growth in xenograft tumor tissues. These results suggested that FAPα might directly promote tumor growth and invasiveness in ovarian cancer cells.     

Study on docetaxel-loaded nanoparticles with high antitumor efficacy against malignant melanoma

Docetaxel （Doc） has extraordinary activities against a variety of solid tumors. However, the clinical efficacy of Doc is limited due to its poor solubility, low selective distribution, fast elimination in vivo, etc. In the present study, Doc was incorporated into the core-shell structure of nanoparticles prepared based on our previous work. The obtained docetaxel-loaded nanoparticles （DOCNP） were characterized with various biophysical methodologies, and its antitumor efficacy against malignant melanoma was evaluated both in vitro and in vivo. Our results indicated that Doc could be incorporated into the nanoparticles with high encapsulation efficiency （〉90%）. The incorporated Doc can be released from DOCNP in a sustained manner. In vitro cytotoxicity studies indicated that DOCNP could effectively kill B16 cells and show a dose- and time-dependent efficacy. Furthermore, intratumoral administration revealed that DOCNP has significantly higher antitumor effect and lower toxicity to normal cells and tissues than free Doe. These results suggest that DOCNP may be a promising drug delivery system in therapy for malignant melanoma.     

Expression of a soluble form of CTLA4 on macrophage and its biological activity.

Interaction between cytotoxic T lymphocyte-associated antigen-4(CTLA4,CD152) and B7 molecules (B7-1 and B7-2) is of importance in the cellular events of lymphocyte,including antigen-specific T-cell activation and induction of autoreactive T-cell.We describe haere the first introduction of a murine soluble CTLA4 gene,CTLA4Ig,to Mm1 cells,a macrophagic cell line.CTLA4Ig was successfully expressed on Mm1 cells and the expressed CTLA4Ig was found to be functionally active in their binding to B7 molecules by flow cytometry and immunofluorescence studies.The biological activity of CTLA4Ig from the transfected Mm1 cells was studied and showed inhibitory activity on mixed lymphocyte culture.A high CTLA4Ig producing macrophagic cell line was obtained.As Mm1 cells were regarded as difficult for gene transfection and there has so far been no report on expression of CTLA4Ig gene on Mm1 cells,these results suggested that the CELA4Ig expressing Mm1 cells could be useful for analysis of CTLA4 and B8 molecule interaction in both macrophage and T-cell.     

Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.     

Effects of a chemically derived homo zwitterionic polysaccharide on immune activation in mice

In this study, a chemically modified homo zwitterionic polysaccharide （ZPS）, sulfated chitosan, was used to examine its effects on murine immune response. The results showed that homoZPS could markedly promote the proliferation of both splenic T/B cells and adhesive cells. In particular, flow cytometry assay demonstrated that the sulfated chitosan could non-specifically activate CD3＋ and CD8＋ T cells proliferation in vitro. The effectiveness of sulfated chitosan as adjuvant was tested using bovine serum albumin （BSA） and diphtheria toxin （DT） as antigens and compared with that of aluminum hydroxide. The levels of specific antibodies to BSA and DT significantly increased after homoZPS vaccination. Both homoZPS and aluminum hydroxide adjuvants could protect mice against the attack of DT from edemas of spleen and tail. The present findings demonstrated the chemically derived homoZPS could be a potential candidate in the development of T-lymphocyte dependent vaccine adjuvants.     

Construction of a novel fusion protein harboring mouse inter- feron γ and epidermal growth factor receptor binding domain and enhancement of its antitumor activity

A novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain was constructed with the method of genetic and protein engineering. The fusion protein kept complete antiviral activity with the titer of 108 IU per liter of culture. The EGF-RBD of the fusion protein exhibited competitive binding activity against 125I-mEGF for mEGF receptors on A431 cells. The fusion protein was shown to be more potent in in-hibiting the growth of cultured mouse breast carcinoma cells than interferon γ. Experimental data on mouse B16 malig-nant melanoma model indicated that the tumor weight of fusion protein-treated group was statistically significantly smaller than that of interferon γ-treated group. The work here provides a necessarily reliable clue for the upcoming clinical employment of a novel class of targeting interferons.     

Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi,a Sapindaceae Deciduous Tree

A vegetative storage protein （VSP） with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions： nitrogen storage and defense.     

Activity identification of ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in cell-free system and in hepatic stellate cells

Transforming growth factorβ1 (TGFβ1) is known to be intimately involved in many cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimeric hammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulate TGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions of both ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activity in vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozyme inhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. So it indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the research on the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidate for TGFβ1-related diseases.     

Comparison of two in vitro angiogenesis assays for evaluating the effects of netrin-1 on tube formation

Netrin-1 is a neural guidance cue that also regulates vascu- lar development. Controversial results, however, have been obtained concerning the roles of netrin-1 in vascular devel- opment both in vivo and in vitro. In the present study, two in vitro angiogenesis assays were compared to evaluate the effects of netrin-1 secreted by retrovirally transduced mel- anoma cells （Mel2a-netrinl） on tube formation. The results showed that there was no obvious difference in tube forma- tion induced by conditioned media （CM） from the control, Mel2a-netrinl and Mel2a cells in a matrigel assay. The results of another in vitro assay, in which endothelial cells were co-cultured with human fibroblasts, however, showed that Mel2a-netrinl CM inhibited the tube formation, sup- posedly through blocking the elongation and coalescence of human umbilical vein endothelial cells （HUVECs）. These results confirmed that the matrigel assay is not able to demonstrate the anti-angiogenic roles of netrin-1.     

The kinetics of IL—4 and IFN—γ gene expression in Mice after Trichosansin immunization

Trichosanthin (TCS) is a potent allergen to mice.According to our previous experiments,it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization,while OVA alone could not induce IgE to it.In this work,the kinetics of interleukin 4(IL-4) and interferon γ(IFN-γ) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method.It indicated that TCS induced significant IL-4 gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response.In contrast,the IFN-γ gene expression was suppressed.Furthermor,the IL-4 gene expression in the secondary response was lower than that in the primary response.Thus the presence of IgE memory B cells were studied.Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation,while in the MLN of the mice primed 30 days before and without boost,it was almost as the same amount of the unimmunized control.These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.