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.
Animals ; Dependovirus/genetics* ; Genetic Therapy ; Genetic Vectors/genetics* ; HEK293 Cells ; Humans ; Mice ; MicroRNAs/genetics* ; Trichosanthin

OBJECTIVELittle effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine (TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus (rAAV) vectors has not been attempted.

METHODSWe synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The protein expression was confirmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into rAAV vectors, under the control of a liver cancer-specific promoter. The liver tumor growth was then monitored following intratumor administration of the rAAV vectors.

RESULTSThe expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin (TCS) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of rAAV vectors containing the TCS gene significantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.

CONCLUSIONOur studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; physiology ; Cell Line, Tumor ; DNA, Complementary ; Dependovirus ; Genetic Vectors ; Humans ; Liver Neoplasms ; physiopathology ; Medicine, Chinese Traditional ; methods ; Mice ; Neoplasms ; physiopathology ; Trichosanthin ; genetics ; pharmacology

OBJECTIVETo observe the effects of high expression of recombinant trichosanthin (rTCS) on the cell proliferation and cell cycle of human cervical cancer Caski cells.

METHODEukaryotic expression plasmid pcDNA3.1(-)/6His-TCS was constracted and stably transfected into Caski cells. RT-PCR,Western-blot were used to select the clones with rTCS high-expressing. Using pcDNA3.1(-)-transfected cells as the control, MTT assay and flowcytometry were used to elucidate the effects of rTCS high expression on cell growth and cycle regulation in Caski cells.

RESULTThe Caski cells with stable high expression of rTCS was successfully established, which could inhibit the cell growth (P<0.01) and arrest Caski cells in G1 and G2 phases (P<0.05) obviously.

CONCLUSIONHigh expression of rTCS can inhibit the growth of Caski cervical cancer cells, which might provide a new pathway for the therapy of cervical cancer.

Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Humans ; Recombinant Proteins ; pharmacology ; Transfection ; methods ; Trichosanthin ; pharmacology ; Uterine Cervical Neoplasms ; pathology

The clinical or experimental study proves that Chinese medicine such as Tripteryglum wilfordii, Lignum Sappan, Caulis Sinomenii, Radix Trichosanthis and Herba Artemisiae Annuae have good immunosuppressive activity. Further researches on the immunosuppressive active components from Chinese medicine have been the main direction in recent years. The recent researches on immunosuppressive effect and possible mechanisms for the monomers such as triperine, triptolide, bazilein, potosappanin A, sinomenine, trichosanthin and artemisinin extracted from those Chinese medicine are introduced in this review.
Animals ; Artemisinins ; pharmacology ; therapeutic use ; Diterpenes ; pharmacology ; therapeutic use ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Epoxy Compounds ; pharmacology ; therapeutic use ; Humans ; Immunosuppressive Agents ; pharmacology ; therapeutic use ; Morphinans ; pharmacology ; therapeutic use ; Phenanthrenes ; pharmacology ; therapeutic use ; Plants, Medicinal ; chemistry ; Trichosanthin ; pharmacology ; therapeutic use

OBJECTIVETo prepare the anticolon carcinoma vaccine with rich chaperone peptide and to examine its anticancer immunological efficacy.

METHODSCT26 colon carcinoma cells were cultured in 1 mg/L Trichosanthin 1640 medium at different temperatures to induce the chaperone expression and promote the synthesis of antigen peptides. Groups of these cells treated under the different condition were lysed by the sonic disintegration, and the lysates were centrifuged. The rawpurified proteins were obtained from the supernatants by precipitating with saturated ammonium sulfate and removing the molecules below 50,000 and above 300,000 in molecular weight via dialysis. Furthermore, the proteins with the molecular weights in 70,000, 90,000, 95,000, 110,000 and 170,000 were collected through gel filtration and SDS-PAGE. The purified proteins were analysed by Western blotting, and inspected on the anticancer immunological effects including lymphocyte proliferation and the activities of NK and CTL.

RESULTSMajor of the chaperone peptides of anticancer effects in CT26 cells, including antigen peptides joining with HSP70, HSP90, gp96, HSP110 and HSP170, was satisfactorily extracted and condensed, and rich chaperone peptide composites were successfully obtained. The composites prepared under various condition could all enhance lymphocyte proliferation and the activities of CTL and NK(P<0.01).

CONCLUSIONSThe rich chaperone peptide composites are successfully prepared via dialysis, salt fractionation and gel filtration combined with SDS-PAGE. Both the heat stress and Trichosanthin can increase the composites, which treated by 42 centi-degree heat stress and Trichosanthin are found to possess the strongest anticancer efficacy.

Animals ; Cancer Vaccines ; immunology ; Cells, Cultured ; Heat Stress Disorders ; metabolism ; Heat-Shock Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Chaperones ; immunology ; Trichosanthin ; pharmacology

OBJECTIVETo evaluate the anti-tumor effect of recombinant toxin EGF-TCS against transplanted human hepatocellular carcinoma in nude mice.

METHODSHuman hepatocellular carcinoma BEL-7,402 cells were inoculated subcutaneously in the right axillary region of nude mice, and 6 days later, EGF-TCS was injected intravenously at 100, 50, and 25 microg/kg. The mice were executed on the next day of drug withdrawal and the tumors were weighed and the tumor inhibition rate calculated. Immunohistochemistry was also performed on the tumor tissues to provide clue for the possible pathways of tumor inhibition.

RESULTSEGF-TCS markedly inhibited the tumor growth in nude mice, with a tumor inhibition rate of 71.3%, 60.87% and 45.22% corresponding to EGF-TCS dosage of 100, 50, and 25 microg/kg, respectively. Variance analysis suggested that EGF-Linker-TCS could significantly inhibit the tumor growth in the mice (F=8.712, P=0.006), and immunohistochemistry showed significantly inhibited angiogenesis in the tumors by EGF-TCS. No blood vessels were found in the tumor tissues in high dosage group, and there were also reduced blood vessels in the other two smaller dose groups in comparison with the untreated model group, indicating that EGF-TCS inhibited tumor growth and migration by inhibiting tumor angiogenesis.

CONCLUSIONEGF-TCS can inhibit the growth of solid tumors in nude mice, suggesting the potential value of this preparation in cancer therapy.

Animals ; Antineoplastic Agents ; metabolism ; therapeutic use ; Carcinoma, Hepatocellular ; drug therapy ; Cell Line, Tumor ; Disease Models, Animal ; Epidermal Growth Factor ; Female ; Humans ; Immunotoxins ; genetics ; metabolism ; therapeutic use ; Liver Neoplasms ; drug therapy ; Male ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; metabolism ; therapeutic use ; Trichosanthin ; genetics ; metabolism ; therapeutic use

The study was aimed to investigate the mechanism of cytotoxic effect of trichosanthin (TCS) on leukemia or lymphoma cell lines. Trypan blue exclusion was used to measure the effect of TCS on cell growth and flow cytometry was used to detect the effects of TCS on cell apoptosis and cell cycle. The results indicated that the TCS could inhibit proliferation of various leukemia/lymphoma cell lines at 12.5 microg/ml concentration, but the effect of TCS on T-lymphocyte and macrophage cell lines showed inducing cell apoptosis and the effect of TCS on B lymphocyte cell line showed inhibiting cell growth. Detection of the cell cycle revealed that TCS could arrest B lymphocytes at S phase, but not had this effect on T lymphocytes at the same condition. It is concluded that TCS can kill leukemia/lymphoma cells through different mechanisms such as inducing cell apoptosis or arresting cell cycle according to cell types.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Humans ; Leukemia ; pathology ; Lymphoma ; pathology ; Trichosanthin ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo construct a recombinant expression vector of the fusion protein epidermal growth factor (EGF)-Linker-trichosanthin (TCS) and achieve its expression in E. coli to obtain purified EGF-linker-TCS fusion protein.

METHODSThe gene fragments of EGF-linker were amplified by PCR and inserted into the expression plasmid PQE30-TCS, followed by transformation of the recombinant plasmid into E. coli M15 for expression of the fusion protein. Ni-FF column chromatography was utilized for purification of the expressed product.

RESULTSThe recombinant plasmid PQE30-EGF-linker-TCS was stably and highly expressed in E. coli M15. The expressed product existed in the form of soluble protein accounting for about 40% of total cellular protein and reached a purity of above 95% after purification with Ni-FF column chromatography.

CONCLUSIONThe recombinant plasmid PQE30/EGF-linker-TCS has been successfully constructed, which provides a basis for further structural and functional study of EGF and TCS and their potential clinical application for cancer therapy.

Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Epidermal Growth Factor ; genetics ; metabolism ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Trichosanthin ; genetics ; metabolism

OBJECTIVETrichosanthin (TCS), a ribosome-inactivating protein extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii maximowicz, has various pharmacological properties including abortifacient, anti-tumor and anti-virus. This study aimed to evaluate the effects of TCS on infectious brain injury induced by Herpes simplex virus-1 (HSV-1) in mice.

METHODSNinety mice were randomly assigned into three groups: Normal control group (n=30), Model group (n=30) and TCS-treated group (n=30). Viral encephalitis was induced by intracranial inoculation of HSV-1 in the latter two groups. The TCS-treated group was injected with TCS 30 minutes before HSV-1 inoculation. The water content of brain tissue was measured at 1, 12, 24 and 48 hrs, and at 4 and 7 days after HSV-1 inoculation. The viral titer of brain tissue and brain histopathological changes were detected at 7 days after HSV-1 inoculation. The neurological deficient scores were determined daily.

RESULTSThe water content of brain tissue in the TCS-treated group between 48 hrs and 7 days after HSV-1 inoculation was significantly lower than that in the Model group (P < 0.05), although it was significantly higher than that in the Normal control group (P < 0.05). The viral titer of brain tissue in the TCS-treated group was markedly lower than that in the Model group (1.16 +/- 0.45 vs 2.89 +/- 0.44; P < 0.05) 7 days after HSV-1 inoculation. The neurological deficient scores of the TCS-treated group after 24 hrs of HSV-1 inoculation were significantly lower than that in the Model group but were higher than those of the Normal control group. TCS treatment resulted in alleviated pathological changes of brain tissue compared with the Model group 7 days after HSV-1 inoculation.

CONCLUSIONSTCS has protective effects against infectious brain injury induced by HSV-1 in mice.

Animals ; Body Water ; metabolism ; Brain ; metabolism ; pathology ; virology ; Encephalitis, Viral ; drug therapy ; Female ; Herpes Simplex ; drug therapy ; Herpesvirus 1, Human ; Male ; Mice ; Neuroprotective Agents ; therapeutic use ; Trichosanthin ; therapeutic use

OBJECTIVETo explore a new molecular method to authenticate Radix Trichosanthis.

METHODThree 20 mer primers based on the ITS sequence was designed. The PCR reaction system was optimized and applied to nineteen different sources of Radix Trichosanthis and nine adulterants and substitutes.

RESULTPolymorphic map of Radix Trichosanthis and its adulterants was obtained from primer TKS1-64. 560 bp and 960 bp bands were authentic markers for Radix Trichosanthis.

CONCLUSIONPrimer TKS1-64F possesses the advantages of good stability and reproducibility. This new method is named as anchored primer amplification polymorphism DNA(APAPD). It was a potential method to used in molecular identification of other meteria medica.

DNA Primers ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; Drug Contamination ; prevention & control ; Polymorphism, Genetic ; Random Amplified Polymorphic DNA Technique ; methods ; Reproducibility of Results ; Trichosanthes ; chemistry ; classification ; genetics ; Trichosanthin ; genetics ; isolation & purification