The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy / 中西医结合学报

OBJECTIVE@#Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.@*METHODS@#A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA 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 rAAV production were determined.@*RESULTS@#The presence of 122T 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 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.@*CONCLUSION@#HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Genetically-engineered "all-in-one" vaccine platform for cancer immunotherapy

An essential step for cancer vaccination is to break the immunosuppression and elicit a tumor-specific immunity. A major hurdle against cancer therapeutic vaccination is the insufficient immune stimulation of the cancer vaccines and lack of a safe and efficient adjuvant for human use. We discovered a novel cancer immunostimulant, trichosanthin (TCS), that is a clinically used protein drug in China, and developed a well-adaptable protein-engineering method for making recombinant protein vaccines by fusion of an antigenic peptide, TCS, and a cell-penetrating peptide (CPP), termed an "all-in-one" vaccine, for transcutaneous cancer immunization. The TCS adjuvant effect on antigen presentation was investigated and the antitumor immunity of the vaccines was investigated using the different tumor models. The vaccines were prepared

Analysis of quality markers based on efficacy and material base of different medicinal parts of Trichosanthes kirilowii / 中草药

Different parts of Trichosanthes kirilowii can all be used as medicines, including the fruits (Trichosanthis Fructus), pericarps (Trichosanthis Pericarpium), seeds (Trichosanthis Semen) and roots (Trichosanthis Radix). Modern research has confirmed that the main active ingredients of Trichosanthis Pericarpium are flavonoids and amino acids; Trichosanthis Semen mainly contains terpenoids and sterols; Trichosanthis Radix mainly contains protein, steroids and polysaccharides. And the pharmacological effects of various medicinal parts are also different. This paper summarizes the traditional efficacy, chemical composition and modern pharmacological effects of different medicinal parts of T. kirilowii, analyzes the relationship between them, so as to analyze and predict the quality marker of T. kirilowii.

The anti-cancerous activity of recombinant trichosanthin on prostate cancer cell PC3

CONTEXT: Trichosanthin produced in the root tube of Trichosanthes kirilowii shows anti-tumor activity on a series of cancer cells including Hela, MCF-7, HL-60. But there is little information about its effect on the carcinogenesis of prostate cancer. OBJECTIVE: This work was designed to study the role of trichosanthin on prostate cancer cells PC3. MATERIALS AND METHODS: Trichosanthin was expressed in BL21 strain and purified by affinity chromatography. MTT assay was designed to determine the effect of trichosanthin on growth of PC3 cells at doses of 10, 20, 40, 60, 80, and 120 µg/ml.Then the effect of 50 µg/ml rTCS alone or combined with 2 µM IL-2 on PC3 cell proliferation was analyzed. And the mechanism of rTCS was studied by western blot. After that the in vivo effect of rTCS combined with IL-2 was explored in mice bearing PC3 xenograft tumor. RESULTS: Trichosanthin was successfully expressed in BL21 and purified by 100 mM imidazole. It was shown to inhibit proliferation of PC3 cells in a dose-dependent manner with IC50 50.6 µg/ml. When combined with cytokine IL-2, a significant synergic effect was obtained. The inhibition rate on PC3 was around 50 % in combination group while only 35.5 % in single rTCS group at 50 µg/ml. Further, the expression of full length caspase-8 and Bcl-2 decreased significantly while cleaved caspase-8 and Bax were up-regulated, which suggest that caspase-8-mediated apoptosis pathway may be activated by rTCS in PC3 cells. Moreover, our data demonstrated that tumor volume and tumor weight were significantly reduced in rTCS-treated or rTCS/IL-2-treated nude mice bearing PC3 xenograft tumor compared with control. And significant difference was also found between rTCS and rTCS/IL-2 group. CONCLUSIONS: This study demonstrates that rTCS is a potential agent with high in vitro and in vivo anti-tumor activity on PC3 cells. And rTCS combined with IL-2 is a promising strategy in treating patients with prostate cancer in future.

Study on Anti-tumor Activities of Trichosanthin Mediated by Low Molecular Weight Protamine / 广州中医药大学学报

Objective To investigate the anti-tumor activities of cell-penetrating peptide ( CPP) - mediated trichosanthin ( TCS) , which is a recombinant protein obtained from Radix Trichosanthis. Methods Cysteine residue was introduced to the C-terminus of TCS by protein recombinant technique, and then with the newly-formed terminal as the modification site, TCS was coupled with CPP. As a target protein, CPP-mediated TCS was isolated and purified by affinity chromatography. The expression of the target protein and its responsiveness to reducing substances were detected by using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cellular uptake rate of CPP-mediated TCS was determined by using cell uptake test, and its anti-tumor activity was measured by using methyl thiazolyl tetrazolium (MTT) assay. Results The TCS-CPP compound had been successfully developed in this study, and showed certain reducing responsiveness. After modified with CPP, TCS had higher cellular uptake rate and stronger anti-tumor effect on HeLa and MCF-7 cells. Conclusion TCS modified by CPP can enhance the anti-tumor activities of TCS.

Effects of trichosanthin on apoptosis and cytoskeleton microtubule structure reconfiguration in lung cancer A549 cells / 中国肿瘤临床

Objective:This study aimed to explore the effects of trichosanthin on the proliferative inhibition and apoptosis induc-tion in human lung carcinoma A549 cells. Methods:A549 cells were treated with various concentrations of TCS. The inhibitory effects in proliferation were detected by the MTT method. The microfilament changes were observed by transmission electron microscopy. Apoptosis rate and cell cycle were determined by flow cytometry. Results:A549 cells treated with TCS presented apoptotic changes and decreased cell activity. When the concentration increased and time was prolonged, the inhibition rate increased correspondingly. Conclusion:Pharmacological doses of TCS inhibited the proliferation and differentiation in lung carcinoma A549 cells and affected the function in A549 cells by changes in the cytoskeleton.

Cloning of trichosanthin gene and its induction effects on the apoptpsis of colorectal carcinoma LoVo cell / 复旦学报（医学版）

Objective To clone cDNA of trichosanthin (TCS) and purify TCS, and to study its influence on apoptosis and growth inhibition of colorectal carcinoma LoVo cells in vitro. Methods MTT assay was adopted to measure the growth inhibition ratio of LoVo cells treated with TCS, and apoptosis was assayed by agarose gel eletrophoresis. Results The results showed that the higher concentration of TCS and the longer testing time, the stronger growth inhibition of LoVo cells. DNA agarose gel eletrophoresis showed a gradient, which confirmed that TCS could induce the apoptosis of LoVo cells. Conclusions TCS can inhibit the growth of LoVo cells in vitro and induce its apoptosis. Our study provides evidence for the application of TCS in clinical treatment of human colorectal carcinoma.

Purification of EGF-TCS recombinant fusion protein and its targeting action on human tumor cells in vitro / 第三军医大学学报

Objective To express and purify EGF-TCS fusion protein and observe the targeted and selective killing effect on cancer cells of the fusion protein.Methods The recombinant expression plasmid PQE30/EGF-TCS was transformed to E.coli.M15 and the fusion protein(EGF-TCS) was expressed.Ni-NTA Agrose affinity chromatography was used to purify the protein,flow cytometry to detect EGFR expression rate in cancer cells(BEL-7402,MCF-7,BGC-823) and normal liver LO2 cells,and the killing test to verify selective killing ability of EGF-TCS;The cell apoptosis detection by flow cytometry and microscopic observation were used to confirm the selective killing ability of EGF-TCS.Results Recombinant expression plasmid PQE30/EGF-TCS was expressed in E.coli.M15 stably and effectively.The purity of EGF-TCS was over 95% by chromatography.EGFR expression rate was highest in hepatoma cells BEL-7402(72.33%) and lowest in normal liver LO2 cells(5.51%).The killing ability of recombinant protein was more effective to cancer cells(IC50 of BEL-7402,MCF-7 and BGC-823 was 11.4,22.47 and 12.53 ?g/ml respectively) and was weak to normal cells(IC50 53.19 ?g/ml).Conclusion The recombinant protein EGF-TCS that induces apoptosis of cancer cells was successfully constructed by gene engineering technology.

Trichosanthin-induced apoptosis in melanoma B-16 cell line / 中国病理生理杂志

AIM: To investigate the antitumor effect and mechanism of trichosanthin (TCS) on melanoma B-16 cells. METHODS: (1) The injury of B-16 cells by trichosanthin was observed with SCGE and hoechst33258 staining method. (2) LCSM and specificity fluorescent probe Fluo-3/AM, H2DCF-DA, DAF-FM diacetate were applied to analyze the dynamic changes of Ca2+, ROS and NO in single cell cultured with TCS. Simultaneously, the relationship between ROS, NO and increase of Ca2+ was also revealed. RESULTS: (1)When treated with TCS (50 mg/L) for 3 h and 6 h, neither cytotoxicity assay nor SCGE showed the differences compared with control group. After 12 h incubation, specificity phenomena of DNA injury-comet tail appeared in SCGE and chromatin condensation even apoptotic body formation were seen by Hoechst33258 staining. (2) TCS (50 mg/L) evoked rapid enhancement of the production of Ca2+, ROS and NO in the cell and the differences between TCS and control group had statistical significance (P

Clinical Observation of the Effect Intracervical and Intramuscular Injection of Trichosanthin on Tubal Pregnancy / 上海第二医科大学学报

ObjectiveTo evaluate Trichosanthin(TCS) in treating ectopic pregnancy by com-paring to clinical efficacy and side effects between the intramuscular and intracervical adiministrationgroup. MethodsTCS was given to the patients by either the intracervical or the intramuscular route.Compare the clinical results. ResultsThere was a 90% cure rate in the intramuscular injection groupand a 94% cure rate in the intracervical injection group. Side effects were slighter in the intracervical in-jection group than intramuscular injection, especially in fever and its sustained time (P<0.05)。ConclusionsThe clinical efficacy was consistent in two groups but the side effects of the intra - cervicalgroup were slighter.

Comparison of the relative content of trichosanthin between root and callus derived from Trichosanthes kirilowii / 中国药学杂志

OBJECTIVE：The relative content of trichosanthin (TCS) of the calli induced from the leaves of Trichosanthes kirilowii Maxim. was measured and a comparison between the calli and the root was made. METHODS：TCS was obtained by the fractional precipitate with acetone from the homogenate of the root or the calli. To examine and measure TCS, several methods, such as immuno-precipitation reaction, SDS-PAGE and electrophoregram scanning, were usde. RESULTS：The results of immuno-precipitation reaction and SDS-PAGE showde that TCS existed in the calli and in the root of T.kirilowii Maxim.. It was found that TCS was the richest component in the acetone precipitated crude extract of the calli with a relative content of 44.22% TCS in the extract, though the absolute content of TCS in the calli was less than that in the root. CONCLUSION:Extracting TCS from the calli derived from leaves has not been reported previously. The absolute content of TCS in the root is 2.66 times more than that in the calli.

The effect of Trichosanthin on cytokines production and ion channel of human PB-MC / 中国免疫学杂志

Abstract Objective: To study the effect of Trichosanthin on cytokines production and ion channel of human PBMC, to demonstrate its immune regulatory mechanism.Methods: The levels of IL-2 and IL-6 produced by PBMC under stimulation of PHA-M were measured by radio-immunoassay after cultured with Trichosanthin. The current intensity of voltage-dependent potassium channel [K(V) channel] were recorded by patch-clamp technique after added Trichosanthin.Results: Both IL-2 and IL-6 production were enhanced by Trichosanthin, and the current in-tensity of K(v) channel were markedly increased from (210.2 ?53.6)PA to (2 223.0 ?310.7)PA.Conclusion: Trichosanthin can enhance the production of both IL-2 and IL-6 of human PBMC and increase the current intensity of K( v) channel on T lymphocyte membrane, which im-prove cell-mediated immunity and humoral immunity.

Apoptosis in gastric cancer cells induced by trichosanthin is related with down-expression of bcl-2 / 中华消化杂志

Objective To investigate the apoptosis in gastric cancer induced by trichosanthin, and the relationship between this apoptosis and expression of bcl2. Methods In in vitro experiments, morphologic test and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of gastric adenocarcinoma cell line SGC7901 before and after the trichosanthin treatment. Immunohistochemical staining method and Northern Blot hybridization were used for detecting expression status of apoptosisrelated genes bcl2, before and after trichosanthin treatment. Results When SGC7901 cells were treated with trichosanthin (0.1 ?g/ml, 36 h), they presented some typical apoptotic morphologic changes observed by fluorescent staining. These morphologic changes include nuclear condensation, nucleosomal fragments forming a lunate body under nuclear membrane, etc. When SGC7901 cells were treated with trichosanthin at the concentration 0.1 ?g/ml for 36 h,42 h and 48 h, respectively, TUNEL staining showed a significant increase of apoptotic index (AI), from 3.78%?1.11%, 3.98%?1.12%,3.85%?1.08%, respectively, to 11.30% ? 2.33%, 10.22% ?2.00%,11.18%?1.85%(P

In vitro introduction of recombinant trichosanthin on demethylation of p27 in HeLa cells / 中草药

Objective To investigate the effects of recombinant trichosanthin(rTCS) on methylation status and expression level of p27 gene in HeLa cells.Methods HeLa cells was treated by different concentration(20 ?g/mL,40 ?g/mL,and 80 ?g/mL) of rTCS for 48 h and then methylation-specific polymerase chain reaction(MSP) was used to detect the promoter methylation status of the p27 gene,real-time PCR was used to detect levels of p27 and DNMT1 mRNA,and Western blotting assay was used to detect expression level of p27 protein before and after treatment with rTCS.Results Low expression level and promoter methylation status of the p27 gene were detected in HeLa cells.Treatment with 40 ?g/mL rTCS totally demethylated p27 promoter.Treatment with 20 ?g/mL,40 ?g/mL or 80 ?g/mL rTCS resulted in a 2.22-,4.00-or 6.03-folds increase in p27 mRNA level,respectively,and also a great increase in p27 protein level.A high DNMT1 expression level was observed in HeLa cells and treatment with 40 ?g/mL rTCS resulted in a 78% decrease at the DNMT1 mRNA expression.Conclusion rTCS could reverse promoter hypermethylation and re-activate the expression of p27 gene by inhibiting DNMT1 expression in HeLa cells,which indicates its potential use in cancer therapy.

Molecular mechanism study of Trichosanthin induced apoptosis in human cervical carcinoma HeLa cells / 中国药理学通报

Aim To explore effects of TCS on the gene expression profiles of human cervical carcinoma HeLa cells with microarray technique and further investigate its molecular mechanism. Methods RNA from both control and treated HeLa cells were isolated and reversely transcribed to cDNA with the incorporation of cy3 and cy5-labeled dUTP, probes were hybridized with BiostarH-Ⅰ cDNA microarray, chips were scanned and then analyzed by GenePix Pro 3.0 software. Results 78 significantly differently expressed genes were screened out of which up-and down-regulated genes were 62 and 16 respectively. the up-regulated genes were closely related with apoptosis (such as NOP56、TNFSF10、CASP9、DFFB,etc) and the down-regulated genes were associated with the adhesion and interaction between cells (such as COL9A3、LGALS3BP、 MGST3,etc). Conclusion TCS could result in differential expression of multiple genes in HeLa cells, and DNA microarray technique can provide valuable insight into the molecular mechanism of TCS-induced apoptosis.

Study on the relevancy between OPCML and DAPK gene methylation and CasKi cell apoptosis / 中国药理学通报

Aim To study tumor suppressor gene OPCML,DAPK methylation changes during the process of CasKi cell apoptosis induced by trichosanthin, and to explore the correlation and the role between cervical cancer cell apoptosis and tumor suppressor gene methylation so as to find new demethylation drugs.Methods ① MTT was applied to assay the inhibition of TCS to CasKi cell proliferation and flow cytometry was used to analyze cervical CasKi cell apoptosis induced by trichosanthin; ② Methylation-specific PCR(MSP) technology was applied to detect cervical cancer and during cell apoptosis process OPCML and DAPK gene promoter methylation status of CpG islands.Results In CasKi cervical cancer cells,OPCML and DAPK gene promoter region showed a high degree of CpG island methylation status, by trichosanthin treatment,the growth of CasKi markedly was inhibited, and flow cytometry analysed the characteristic sub-G1 peak,OPCML and DAPK gene promoter region showed no CpG island methylation of performance.Conclusions During the process of CasKi cell apoptosis induced by trichosanthin,OPCML and DAPK gene demethylated significantly,accordingly, trichosanthin might be a new methylation inhibitor,and there might be some correlation between cell apoptosis and tumor suppressor gene methylation.And OPCML and DAPK gene methylation tests might become new clinical indicators for the early detection of cervical cancer.

Study on the antigenic determinants of trichosanthin fragments / 中国免疫学杂志

The antigenic determinants of Trichosanthin (TCS) were studied using cyanogen bromide cleavage of TCS and immunoblotting technique. 21 McAbs prepared against TCS could be divided into 5 groups according to the patterns of their reactivities with CNBr-fragments of TCS (CBTf1-4). (1) 7 antibodies bind to CBTf 1;(2) 2 antibodies bind to CBTf2;(3)2 antibodies bind to CBTf 4;(4)7 antibodies are cross-reactive with these fragments; (5) 3 antidodies bind to none of 4 fragments. Using two fragments CBTf1 and CBTf2 as competitors in a ELISA competition test, quantitative determination of antigenicity of TCS fragments by 4 McAbs to native TCS has shown that the maximal amount of bound antibody for CBTf1 with 2 McAbs reached 50% and for CBTf2 with another McAb nearly 50%. From these data, we concluded that TCS fragments have kept antigenic determinants as native TCS and it is very essential for us to study the structure-function relationship of TCS.

In vitro inhibition of trichosanthin-monoclonal antibody conjugate on human melanoma cells / 中国免疫学杂志

We have constructed an immunotoxin(Ng76-TCS),which was composed of a monoclonalantibody directed against human melanoma and trichosanthin(TCS)——a single chain ribosomeinactivating protein.The cultured human melanoma cells(M21)were inhibited effectively byNg 76-TCS.The cytotoxicity of Ng76-TCS to M21 cells was 2,000-fold higher than that of free TCS and Ng76 mixture.A conjugate,which was prepared with normal mice immunoglobulinand TCS(NIgG-TCS),was 160-fold less cytotoxic to M21 cells.Meanwhile Ng76-TCS was125-fold less cytotoxic to nontarget cells Hela.These results showed that the immunotoxinNg76-TCS was a potent and specific anti-human melanoma agent.

Healthy subjects distinguishable as CD 8-meddiators and non-mediators for capabilty of diminishing trichosanthin induced immune suppression after depleting or blocking CD 8~+ cells / 中国免疫学杂志

Trichosanthin (Tk), a crystal plant protein isolated and purified from root tubes of a Chinese medicinal herb Trichosanthes kiritowii Maxim (Cucurbitaceae), was demonstrated to be capable of inducing immune suppression in man, and the suppression could be eased up by depleting the culture of CD 8~+ cells with monoclonal anti bodies as reported in our previous work (1). This paper desc ribes that the diminition of suppression,or the restoration of low- responsiveness, however, was restricted in certain subjects we studied. For the others, the suppression kept unchanged or even got more vigorous with the same treatment. For this reason, healthy subjects were categoried as CD 8 -mediators (M~+) and non-mediators (M ~- ). In addition, there were some interesting points revealed as follows. 1 ) M~+ and M~- were determined as a phenotipically stable and reproduceable traits; 2) The difference between two categories can only be observed in the MLR- Tk testing system but not in the non-specific PHA- Tk system in which PHA initiated lymphoproliferation was depressed by T K; 3 ) Instead of stimu lator, only responding PBMC in one-way MLC was affectad by depleting CD8 cells, which resulted in the distinguishable phenotypes of M~+ and M~-; 4) The supernatants collected from Tk sensitized. cultured PBMC could show suppressive activity if the cell donors were M~+, but not M~-.