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trichosanthin enhances anti-tumor immune response in a murine Lewis lung cancer model by boosting the interaction between TSLC1 and CRTAM

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Cellular & Molecular Immunology 2011年第4期8卷 359-367页

作者： Yuchan Cai Shudao Xiong Yijie Zheng Feifei Luo Pei Jiang Yiwei Chu Department of Immunology Shanghai Medical CollegeKey Laboratory of Molecular Medicine of Ministry of EducationFudan UniversityShanghaiChina

trichosanthin(TCS),extracted from the Chinese medicinal herb Trichosanthes kirilowi,has shown promise for the inhibition of tumor growth.However,its immunomodulatory effect on tumor–host interaction remains unknown.In this study,we focused on the effect of TCS on murine anti-tumor immune response in the 3LL Lewis lung carcinoma tumor model and explored the possible molecular pathways involved.In addition to inhibiting cell proliferation and inducing apoptosis in the 3LL tumor,TCS retarded tumor growth and prolonged mouse survival more significantly in C57BL/6 immunocompetent mice than in nude mice.This reflected the fact that the host immune system was involved in tumor eradication.Using FACS analysis,we found that TCS increased the percentage of effector T cells,particularly Interferon-gamma(IFN-c)producing CD41 and CD81 T cells from tumor-bearing mice.TCS also promoted the vigorous proliferation of antigen-specific effector T cells,markedly increased Th1 cytokine secretion and elicited more memory T cells in tumor-bearing mice,consequently enhancing the anti-tumor response and inducing immune protection.Furthermore,we found that TCS upregulated the expression of tumor suppressor in lung cancer 1(TSLC1)in 3LL tumor cells and the expression of its ligand,class I-restricted T cell-associated molecule(CRTAM),in effector T cells.Blocking TSLC1 expression with small interfering RNA(siRNA)significantly eliminated the effects of TCS on the proliferation and cytokine secretion of effector T cells,suggesting that TCS enhances anti-tumor immune response at least partially by boosting the interaction between TSLC1 and CRTAM.Collectively,our data demonstrate that TCS not only affects tumor cells directly,but also enhances anti-tumor immunity via the interaction between TSLC1 and CRTAM.These findings may lead to the development of a novel approach for tumor regression.

关键词： anti-tumor immunity CRTAM immune protection trichosanthin TSLC1

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trichosanthin Induced Th2 Polarization Status

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Cellular & Molecular Immunology 2006年第4期3卷 297-301页

作者： Shuying Zhao Yanyan Wang Haiming Wei School of Life Sciences University of Science and Technology of China Hefei 230027 Anhui China

trichosanthin is extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii maximowicz （Tian Hua Fen）. TCS has abortifacient, anti-tumor, anti-HIV and immunoregulatory functions. It has been proved that it could inhibit immune response and arouse a T helper 2 response in the draining lymph node. In the current study the effect of TCS on mouse splenocytes was investigated. We stimulated C57BL/6 mice with TCS both in vivo and in vitro and analyzed the change of type 1 and type 2 cytokines in mouse splenocytes. The results showed that TCS could induce the expression of IL-4, one of the major T helper 2 （Th2） cytokines, and inhibit the expression of IFN-γ, an important Thl cytokine in spleen lymphocytes both in vivo and in vitro. It is also shown the kinetics of Thl-to-Th2 transition after TCS stimulation in vivo in C57BL/6 mice. We found that type 2 cytokines, such as IL-10, TGF-β and IL-4 were increased regularly but IFN-γ, was decreased at day 3 and then increased. However the mechanism for cytokine change is not clear.

关键词： trichosanthin IL-4 IFN-γ Th

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trichosanthin inhibits T cell activation by interfering with the recruitment of ZAP-70 to CD3 ζchain

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Cell Research 1998年第1期8卷 33-39页

作者： HONG JIAN SAI LI FU ZHONG YI SHEN PEI HUA LU KUANG YEN CHOU (Shanghai Institute of Immunology, Shanghai Second Medical University, Shanghai China) Shanghai Institute of Immunology Shanghai Second Medical University Shanghai China

Plant protein trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb. By examination of tyrosine phosphorylation of cell lysate, we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3initiated signal transduction in addition to blocking the phosphorylation of PKC. As shown in our experiment,the expression intensity of ZAP-70, a kind of protein tyrosine kinase, was not changed but its phosphorylation could be inhibited. When physical link between CD3(chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk, the anti-CD3 McAb-induced recruitment of ZAP70 to CD3 ζ chain was observed to be blocked in some extent. This may account for, at least in part, how trichosanthin was able to inhibit the TCR-triggered T cell proliferation.

关键词： trichosanthin TCR signaling PTK tyrosine phosphorylation

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Refined Structure of trichosanthin at 1. 73 A Resolution

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Science China Chemistry 1994年第1期37卷 59-73页

作者：高奔马星奇王耀萍陈世芝吴伸董贻诚 Institute of Biophysics Academia Sinica Beijing 100101 PRC

An X-ray reflection data set of the orthorhombic crystal of trichosanthin(TCS) at 1. 73 A resolution had been collected using the area detector. We determined TCS crystal structure by the molecular replacement method using the data of the known α-momorcharin model and TCS intensities , and then TGS structure refinement at 1. 73 A resolution was performed with the restrained least-squares refinement. A final R-factor of 0.186 was obtained with a model obeying standard geometry within 0. 013A in bond lengths and 2. 48° in bond angles. The final model contains 133 solvent molecules in the asymmetric unit. This paper gives a detailed description of TCS molecular structure, temperature factors, hydrogen bonds, bound water, the distribution of conserved residues and the interactions between the TCS molecules. The hydrogen bond between the hydroxyl group of conserved residue 14Tyr and O157 plays an important role in maintaining the active site conformation. Conserved residue 160Glu and its conformation at

关键词： trichosanthin molecular replacement method least-squares refinement.

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The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

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Journal of Integrative Medicine 2021年第6期19卷 515-525页

作者： Gai Ran Xi-lin Feng Yi-lin Xie Qing-yun Zheng Peng-peng Guo Ming Yang Ying-lu Feng Chen Ling Li-qing Zhu Chen Zhong State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology(Ministry of Education) School of Life SciencesZhongshan HospitalFudan UniversityShanghai 200438China Division of Cellular and Molecular Therapy Department of PediatricsUniversity of FloridaGainesvilleFL 32611USA Department of Traditional Chinese Medicine Chinese People’s Liberation Army 971 HospitalQingdao 266071Shandong ProvinceChina Department of Clinical Laboratory The First Affiliated Hospital of Wenzhou Medical UniversityWenzhou 325000Zhejiang ProvinceChina

Objective:Plant-derived cytotoxic transgene expression,such as trichosanthin(tcs),regulated by recombinant adeno-associated virus(r AAV)vector is a promising cancer gene therapy.However,the cytotoxic transgene can hamper the vector production in the r AAV producer cell line,human embryonic kidney(HEK293)cells.Here,we explored micro RNA-122(miR122)and its target sequence to limit the expression of the cytotoxic gene in the r AAV producer cells.Methods:A miR122 target(122 T)sequence was incorporated into the 30 untranslated region of the tcs c DNA sequence.The firefly luciferase(fluc)transgene was used as an appropriate control.Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells.The effects of miR122 overexpression on cell growth,transgene expression,and r AAV production were determined.Results:The presence of 122 T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line(in vitro),fresh human hepatocytes(ex vivo),and mouse liver(in vivo).Also,the normal liver physiology was unaffected by delivery of 122 T sequence by r AAV vectors.Compared with the parental cells,the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122 T,as well as the ability to produce liver-targeting r AAV vectors.Fascinatingly,the yield of r AAV vectors carrying the tcs-122 T gene was increased by 77.7-fold in HEK293-mir122 cells.Moreover,the tcs-122 T-containing r AAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.Conclusion:HEK293-mir122 cells along with the 122 T sequence provide a potential tool to attenuate the cytotoxic transgene expression,such as tcs,during r AAV vector production.

关键词： trichosanthin miR122 target sequence Recombinant adeno-associated virus vector Gene therapy Liver cancer

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trichosanthin can spontaneously penetrate phospholipid monolayer under acid condition

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Chinese Science Bulletin 1999年第20期44卷 1892-1895页

作者： Ricky N.S.Wong Biological Department Hong Kong Baptist UniversityHong KongChina

Tianhuafen is a kind of traditional Chinese medicine for abortion, which has long been used in China. The basic protein trichosanthin (TCS) is responsible for such activity. As a ribosome inactivating protein (RIP), trichosanthin removes A4304 in the 28s rRNA via the N-glycosidase activity and inactivates the ribosome. However, it remains unclear how TCS

关键词： trichosanthin (TCS) lipid monolayer critical insertion pressure.

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Using Toad Oocytes as an Ultrasensitive System for Estimating the Biological Activity of trichosanthin

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Science China Chemistry 1993年第4期36卷 402-410页

作者：熊用周朱光左嘉客王亚辉 Shanghai Institute of Cell Biology Academia Sinica Shanghai 200031 PRC

A sensitive bioassay for estimating the activity of trichosanthin was developed by using intraoocyte microinjection as the technique and germinal vescicle breakdown as a marker. The GVBD inhibitory activity assay was million-fold more sensitive than the routinely used abortifacient activity assay. The detectable amount of trichosanthin ranged from 0.5 to 100 pg. Since the least detectable dose was 0.5 pg, it made the assay suitable to analyse the microquantity of pure trichosanthin used for studying the structure and function of the drug.

关键词： trichosanthin toad oocyte maturation promoting factor germinal vesicle breakdown.

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Cloning and Sequencing of trichosanthin Gene and Its Expression in Escherichia coli and Tobacco Plant

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Science China Chemistry 1993年第6期36卷 669-676页

作者：鲍一明储瑞银韩晋华张会潘乃穟顾孝诚陈章良 The National Laboratory of Protein Engineering and Plant Genetic Engineering Department of Biology Peking University Beijing 100871 PRC

A trichosanthin gene was cloned from Trichosanthes kirilowii genomic DNA by polymerase chain reaction (PCR). Nucleotide sequence data indicated that we obtained the coding region of the mature trichosanthin peptide as well as its signal peptide at the N-terminus. Comparisons of our sequence with the previously reported nucleotide sequences of this gene showed 99.25% homology, yet there were notable differences between the previously reported amino acid sequence and our deduced result. This gene was subcloned into a highlevel expression plasmid (pJLA502) of E. coli under the control of a P_RP_L promoter, and we observed the gene product after temperature induction. The gene was further cloned into plant intermediate vector pE3 under the control of a CaMV 35S promoter, and transferred into a tobacco genome using the agrobacterium-mediated gene transfer system. Western blotting analysis of the protein extracted from Escherichia coli and transgenic tobacco plants proved that the trichosanthin gene has

关键词： trichosanthin polymerase chain reaction nucleotide sequence analysis gene expression transgenic plant

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Chemical synthesis of a structural gene coding for trichosanthin

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Chinese Journal of Chemistry 1995年第4期13卷 349-357页

作者： CHEN, HB XIA, Y JING, JP JIANG, K BAO, JS CHINESE ACAD SCI SHANGHAI INST ORGAN CHEMCOMP CHEM LABSTATE KEY LAB BIOORGAN & NAT PROD CHEMSHANGHAI 200032PEOPLES R CHINA

A structural gene (750 bp), which codes for a type I ribosome inactivating protein, trichosanthin, has been designed according to the codon usage of highly expressed gene in E. coli and chemically synthesized. In the synthesized gene, twenty-seven unique restriction sites were evenly dispersed with an average distance between two adjacent sites less than 50 bp to facilitate a systematic investigation on structure-functional relationship of this protein by site-directed mutagenesis. To synthesize it, the whole gene was divided into three large fragments (EP, PN and NH) which were assembled from several chemical synthetic oligonucleotides by enzymatic method. The assembly of both the fragment EP from six oligonucleotides (A-F) and the fragment PN from four oligomers (G-J) was catalyzed by T-4 DNA ligase in using the single stranded DNA method [Chen, H.-B. et al., Nucl. Acids Res., 18, 871(1990)]. And fragment NH was formed from three duplexes K, L and M by the classical double stranded DNA method. Finally, each fragment was cloned into vector pUC18 in succession to form the plasmid, pC0TCS, to complete the whole gene synthesis, The sequencing data for the synthetic gene coincides with the designed one.

关键词： RIBOSOME INACTIVATING PROTEIN trichosanthin GENE DESIGN GENE SYNTHESIS SYNTHETIC GENE

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Crystallization and Preliminary Crystallographic Analysis of β-trichosanthin,an Isoform of trichosanthin from the Root Tuber of Trichosanthe kirilowii

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Chinese Journal of Structural Chemistry 2007年第11期26卷 1303-1307页

作者：侯晓敏陈明晃彭奇陈荔清 Meehan Edward J 黄明东 State Key Laboratory of Structural Chemistry Fujian Institute of Research on the Structure of MatterThe Chinese Academy of Sciences Laboratory for Structural Biology Department of ChemistryGraduate Programs of BiotechnologyChemistry and Materials ScienceUniversity of Alabama in Huntsville

β-trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms using the hanging-drop vapor-diffusion method. The form A and form B crystals belong to the orthorhombic space group P212121 and monoclinic space group P21, respectively. X-ray data have been collected to 1.6 and 1.2 A resolution for form A and form B crystals, respectively, using a synchrotron source.

关键词： ribosome-inactivating protein isoform trichosanthin crystallization

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