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PubMed1993

Neurological reactions in HIV-infected patients treated with trichosanthin.

Trichosanthin is a ribosome-inactivating protein that is being studied as a possible treatment for patients infected with human immunodeficiency virus (HIV). We report the clinical and pathological features in two patients who experienced neurological reactions to trichosanthin. Both patients were neurologically asymptomatic prior to treatment but developed coma and multifocal neurological deficits after treatment. Neuropathological examination revealed regions of severe, multifocal necrosis with histiocytic infiltrates. These reactions to trichosanthin may be mediated by soluble factors released by HIV-infected macrophages.

Acquired Immunodeficiency Syndrome↗
PubMed1998

[Study on Trichosanthin induced apoptosis of leukemia K562 cells].

Trichosanthin (TCS), an eukaryotic ribosome-inactivating protein isolated from the root tuber of Trichosanthes plant, has various biological activities including abortion induction, antitumor, and anti-HIV. In this study, cultured human leukemia K562 cells treated with trichosanthin were examined. Analysis of the cells by single laser flow cytometry showed the sub-G1 peak. DNA extracted from these cells formed a characteristic "ladder" on agarose gel electrophoresis. Under electromicroscope, typical morphological changes of apoptosis were also observed. From all of these findings, we concluded that trichosanthin was able to induce apoptosis in K562 cells.

Antineoplastic Agents, Phytogenic↗
PubMed1993

[Studies on injury-mechanism of trichosanthin on trophoblast cells and choriocarcinoma cells in culture].

For analysing the injurious mechanism of Trichosanthin (TCS) on trophoblast cells, cytotrophoblast cells were separated from placental villi in human early pregnancy and cultured on millipore filters coated with collagen in dual-environmental culture chambers. After 7-10 days culture the cells grew into confluent monolayer. A lot of multinucleated giant cells (synthetial like) could be found under light microscopy. Cytotrophoblast cells and the giant one were characterized as epithelial type by indirect immuno-fluorescent staining with anti-keratin. Because the procedure of separation of trophoblast cells is laborious and the choriocarcinoma cells (JAR) were as sensitive as trophoblast cells to trichosanthin, so the choriocarcinoma cells were used instead of the trophoblast cells in the later experiments. The internalization and distribution of TCS conjugated to 15 nm (in diameter) gold particles were examined. Electron microscopy showed that the TCS-gold particles were bound to the cell membrane and entered via coated pit and then internalized into coated vesicles (endosomes) within 30-60 minutes after treatment. Nevertheless, a number of free TCS-gold particles entered into the cell membrane nonspecifically. In the series of 60-120 minutes treatment, the TCS-gold particles presented in the multivesicular bodies. It is worthy to emphasize that the TCS-gold particles entered the cytosol from the endosomes and distributed nearby the rough endoplasmic reticulum (RER) and ribosomes within 120-180 minutes. In the meanwhile, the cells showed pathological changes markedly. With the same method, human liver carcinoma cells (BE 7402), kidney cells of African green monkey (CV-1) and rat embryonic liver epithelial cells (LW 13) were treated by TCS-gold particles as control. However, no particles had been found on the cell surface or in the cytosol within 60-120 minutes (see Table 1). In addition, trophoblast cells were treated by BSA-gold particles and transferrin-gold particles separately there still no particles could be found. (See Table 2). It is more interesting that TCS-Hepama-1-gold particles internalized into the choriocarcinoma cells with the same manner as TCS-gold entered, and it could not be affected by pretreatment with Hepama-1 an hour. But, TCS-Hepama-1-gold particles bound to the microvilli of human liver carcinoma cells, and it can be inhibited competitively by pretreatment with Hepama-1 for an hour (see Table 3). These results indicated consistently that trichosanthin possesses a high affinity to the membrane of trophoblast cells and choriocarcinoma cells, and the internalization of this plant toxic protein into its target cells characterized by receptor-mediated endocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Trichosanthin, a ribosome inactivating and anti-HIV protein, does not alter the concentration of beta-endorphin in the mouse brain.

Trichosanthin was administered intramuscularly into mature male ICR mice at a dosage of 0.2mg/25g, daily for three days. The mice were sacrificed four hours after the last injection. Their brains were dissected out and divided into three regions: A(thalamus and hypothalamus), B(cerebral cortex) and C(cerebellum and brainstem). Their pituitaries were also removed. The pituitaries and the various brain regions were extracted with hot 1M acetic acid. The extracts were lyophilized and reconstituted in radioimmunoassay (RIA) buffer before assay for beta-endorphin by RIA. It was found that there was a significant elevation of beta-endorphin concentration after trichosanthin treatment only in brain region A. The data suggest that trichosanthin does not adversely affect the brain.

Animals↗
PubMed1993

[The physical properties, pharmacology and antifertility action of crystal trichosanthin protein liposome].

The crystal trichosanthin protein liposome (TPL) was prepared by an emulsion-forming method. The rate of entrapment was 33.0-44.8% and the rate of recovery was 95.15-99.58%. The determination of trichosanthin protein was accomplished by spectrophotometry at 650 nm, thus eliminating completely the interference of lipids. The distribution of particle size of TPL was mainly in the range of 1-2 microns (96.9%). TPL was sterilized by 60Co radiation at 4 degrees C for 90 min and was stable when stored at 4 degrees C, 25 degrees C and 37 degrees C. The seepage rate of trichosanthin from the liposome was 15.22% after storage for 38 days at room temperature. Experiments showed that TPL had a slight allergic reaction, but was safe by hemolysis and coagulation tests. When the mice were given TPL ip 8 micrograms per animal 6 days after pregnancy, the rate of termination of early pregnancy was 83.3%. It thus appears that TPL can terminate early pregnancy in mice distinctly. This indicates that TPL may be expected to be developed into a new type of anti-early pregnancy preparation.

Abortion, Induced↗
PubMed1993

The crystal and molecular structure of trichosanthin at 2.6 A resolution.

The crystallographic refinement of trichosanthin has been performed at 2.6 A resolution. The crystal and molecular structure of trichosanthin is described in detail in this paper. On summarizing the regularity of the amino acid sequences of eight kinds of ribosome inactivating proteins and combining with the crystal and molecular structure of trichosanthin, fifteen most conservative amino acid residues are analyzed. It is found that four most conservative polar amino acid residues Gln156, Glu160, Arg163 and Glu189 gather on the molecular surface on the boundary of the large and small domains, thus forming the active center of the protein molecule.

Amino Acid Sequence↗
PubMed1993

Using toad oocytes as an ultrasensitive system for estimating the biological activity of trichosanthin.

A sensitive bioassay for estimating the activity of Trichosanthin was developed by using intra-oocyte microinjection as the technique and germinal vesicle 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 micro-quantity of pure Trichosanthin used for studying the structure and function of the drug.

Abortion, Induced↗
PubMedDate not supplied

Effect of trichosanthin an anti-leukemia protein on normal mouse spleen cells.

Trichosanthin, from Trichosanthes kirilowii is a member of the Cucurbitaceae family. It has been reported to have anti-cancer activity. The effects of Trichosanthin peptides (15 amino acids in length) on ConA stimulated incorporation of 3[H] thymidine into cultured, primary mouse spleen cells and L1210 cells were determined. Both spleen cells and L1210 cells were plated at 7.5 x 10(5) cells/ml in a microtiter plate. Different concentrations of the peptide (0.5, 5, 25, and 50 micrograms/ml) and ConA (1 microgram/ml) were added with serum free media, and half of the samples were labeled with 1 microCi/well of 3[H] thymidine. The additional half of the cell samples were used to determine cell number and % viability. After 24 hours incubation, the N-terminal peptides (amino acid residues #1-15 and 16-30) caused increases in ConA stimulated incorporation of 3[H] thymidine into normal spleen cells at low concentrations of the peptides (5 micrograms/ml). The viability of spleen cells and L1210 cells were not affected by these peptides at 5 micrograms/ml. These N-terminal peptides (#1-15 and 16-30) were tested for in vivo anti-tumor activity. There was a delay of tumor formation in the treated vs control group. The results suggest that N-terminal sequences of trichosanthin have anti-tumor activity.

Amino Acid Sequence↗
PubMed1985

[Application of trichosanthin in 179 difficult cases of artificial abortion].

This article introduces the application of compound crystal trichosanthin to 179 difficult cases of artificial abortion including cases with extreme anteversion and anteflexion of uterus with a fine and long cervix, cases with uterine deformations, cases within 1 year after cesarotomy, and cases of recently induced abortion. Past experiences have indicated that there have been considerable difficulties in inducing abortion in such cases as the above, and that accidents have been liable to occur. The authors have recently employed compound crystal trichosanthin, composed of crystal trichosanthin, testosterone propionate and reserpine, and succeeded in bringing about drug expansion of uterus and abortion, and safe abortion at that. The success roles were as follows: 97.96% for the group with extreme anteversion and anteflexion of uterus with a fine and long cervix, 100% for the one with uterine deformations, 95.08% for the one with past cesarotomy and 96.72% for the one with recently induced abortion.

Abortifacient Agents↗
PubMed1998

Application of free-flow electrophoresis to the purification of trichosanthin from a crude product of acetone fractional precipitation.

The application of free-flow electrophoresis (FFE) to the purification of trichosanthin (TCS) from a crude product of acetone fractional precipitation was investigated. An electrophoresis technique, combining field step electrophoresis (FSE) and zone electrophoresis (ZE) to a one-step procedure, was optimized until a satisfactory purification factor (1.35), high resolution, and purity (>99%) were achieved. Testing several separation buffer systems revealed that a throughput of 14.2 mg/h can be obtained when the very basic TCS (pI 10.1) was dissolved and electrophoresed in a phosphate buffer system of pH 4. The purity of electrophoresed trichosanthin was proved by a variety of analytical methods, such as sodium dodecyl sulfate (SDS)-gel electrophoresis, capillary isoelectric focusing (CIEF), and sequencing of N- and C-termini. The high purity and large throughput achieved at low cost by using FFE indicates that this method can be employed for TCS purification.

Acetone↗
PubMed1994

Structure of trichosanthin at 1.88 A resolution.

Trichosanthin (TCS) is one of the single chain ribosome-inactivating proteins (RIPs). The crystals of the orthorhombic form of trichosanthin have been obtained from a citrate buffer (pH 5.4) with KCl as the precipitant. The crystal belongs to the space group P2(1)2(1)2(1) with a = 38.31, b = 76.22, c = 79.21 A. The structure was solved by molecular replacement method and refined using the programs XPLOR and PROLSQ to an R-factor of 0.191 for the reflections within the 6-1.88 A resolution range. The bond length and bond angle in the protein molecule have root-mean-square deviations from ideal value of 0.013 A and 3.3 degrees, respectively. The refined model includes 247 residues and 197 water molecules. The TCS molecule consists of two structural domains. The large domain contains six alpha-helices, a six-stranded sheet, and an antiparallel beta-sheet. The small domain has a largest alpha-helix, which shows a distinct bend. The possible active site of the molecule located on the cleft between two domains was proposed. In the active site Arg-163 and Glu-160, Glu-189 and Arg-122 form two ion pairs, Glu-189 and Gln-156 are hydrogen bonded to each other. Three water molecules are bonded to the residues in the active site region. The structures of TCS molecule and ricin A-chain (RTA) superimpose quite well, showing that the structures of the two protein molecules are homologous. Comparison of the structures of the TCS molecule in this orthorhombic crystal with that in the monoclinic crystal indicates that there are no essential differences of the structures between the two protein crystals.

Amino Acid Sequence↗
PubMed2001

Identification of epitopes of trichosanthin by phage peptide library.

The phage displayed random peptide library has recently emerged as a powerful technique for analyzing Ab-Ag interactions. In this study, the method was employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were selected and a phage-peptide library with nine amino acids (9 aa) was used to screen the positive phage clones that have high affinity to the mAbs. Two groups of phage clones that carried peptide-specific binding to mAbs were identified by the screen. The identified phage clones carried peptide-specific binding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the serum from phage-immunized mice. They all showed positive results. The conserved interaction motifs were deduced from the peptide sequences of each group of selected phage clones. When compared the motif sequence with the sequence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further confirmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable to bind 4B5 mAb. The results suggested that the phage display peptide library is useful to identify Ag epitopes and to raise Ab in disease diagnosis and treatment.

Amino Acid Sequence↗
PubMed1997

Acute renal failure and proximal tubule lesions after trichosanthin injection in rats.

The structural basis of the recently recognized renal impairment after infusion of trichosanthin (TCS), a type I ribosome inactivating protein, is uncertain, but functionally it appears to be related to a lesion in the renal tubules. In this study, renal dysfunction in experimental rats was induced by a single dose of TCS. Creatinine clearance and tubular proteinuria were used to assess renal function. Light microscopy and ultrastructure of the kidneys were examined and apoptosis in proximal tubules was evaluated by the in situ TdT-mediated nick end labeling technique. TCS-treated rats demonstrated a significant dose-dependent decrease in creatinine clearance together with a mild degree of low-molecular-weight proteinuria. The proximal convoluted tubule was the site of lesions showing individual tubular cell death, which was more abundant in rats receiving high doses of TCS. Apoptotic cell death, together with heterophagosomes and large residual bodies, was observed. DNA fragmentation was confirmed by the in situ technique. There was also a dose-dependent density of apoptotic cells. Other portions of the nephron were spared, and it was not accompanied by any inflammatory infiltrate. In conclusion, these findings are consistent with TCS-induced proximal tubular toxicity resulting in reduction of glomerular filtration rate and tubular proteinuria. The extent of injury is dosage dependent. Both necrotic cell death and apoptosis participated in the loss of cells from the proximal tubules. Such toxicity may be mediated through intracellular events induced by trichosanthin.

Acute Kidney Injury↗
PubMed1995

Identification of a stable complex of trichosanthin with nicotinamide adenine dinucleotide phosphate.

Trichosanthin, a type I ribosome-inactivating protein with RNA N-glycosidase activity, forms a stable complex with nicotinamide adenine dinucleotide phosphate, a substrate analog. Difference UV spectroscopy, fluorescence spectroscopy, and 31P NMR are used to identify the formation of the complex, followed by a crystal structure analysis carried out to elucidate the active-site structure of trichosanthin. The determination of germinal vesicle breakdown indicates that the complex does not, at least for abortion-inducing activity, result in competitive inhibition to the protein.

Abortifacient Agents, Nonsteroidal↗
PubMed1990

Toxicities of trichosanthin and alpha-momorcharin, abortifacient proteins from Chinese medicinal plants, on cultured tumor cell lines.

Trichosanthin and alpha-momorcharin are abortifacient proteins extracted from Chinese medicinal herbs. Study of their in vitro cytotoxicities showed that the two proteins selectively injured choriocarcinoma and melanoma cells. Hepatoma cells represented the most resistant cell line among the various cell lines investigated. Cytotoxicity profiles of trichosanthin and alpha-momorcharin differed from those of anti-cancer drugs which interfere with DNA metabolism such as cisplatin, methotrexate and 5-fluorouracil. Radioactive precursor incorporation studies suggested that the two abortifacient proteins inhibited cellular protein synthesis. The marked decrease in secretion of human chorionic gonadotrophin and progesterone by choriocarcinoma cells after treatment with the proteins could be attributed mainly to loss of cells.

Abortifacient Agents, Nonsteroidal↗
PubMed1981

Prostaglandin E and F2 alpha levels in plasma and amniotic fluid during mid-trimester abortion induced by trichosanthin.

This paper gives a brief account concerning the estimation of PGE and PGF2 alpha concentrations in both plasma and amniotic fluid by radioimmunoassay in mid-trimester abortion induced by Trichosanthin. The significant change in amniotic fluids as compared with that in plasma is brought out for discussion as an aid to the study of mechanism of abortion by Trichosanthin.

Abortifacient Agents, Nonsteroidal↗
PubMed1993

The biological activities of trichosanthin after coupling to dextran.

1. The major biological and pharmacological activities of trichosanthin (TCS) were retained after its covalent coupling to dextran T40. The potency and toxicity was generally reduced. 2. A dose of TCS as low as 0.02 mg/25 g induced 100% mid-term abortion whereas 20 times as much dextran-trichosanthin (DX-TCS) is required to produce the same effect. 3. A third of the mice died at a TCS dose of 0.1 mg/25 g but 4 times as much DX-TCS did not cause any death. 4. DX-TCS suppressed Con A or PHA induced lymphocyte transformation in a dose dependent manner. The potency was decreased as compared to TCS. 5. Both TCS and DX-TCS exhibited a cytostatic effect on cultured tumour cells (PU5 and hepatoma), the former being more potent.

Abortifacient Agents↗
PubMed2000

Capillary electrophoretic determination of apoptosis of HeLa cells induced by trichosanthin.

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. The observation of apoptosis is very important in the research of cancer and cancer therapy. The traditional observation method of apoptosis was agarose gel electrophoresis, which is depending on the determination of ladder-liking DNA fragments extracted from apoptotic cells. It is time-consuming and low-sensitive. Recently, the sieving capillary electrophoresis has been used to detect apoptosis too. However, the problem of DNA fragments contamination is still existing. Here, we have developed a capillary electrophoresis method that could detect apoptosis of whole cell directly and do not need to extract DNA fragments from cells. Apoptosis of adherent cell HeLa cell of carcinoma induced by cyclophosphamide was used as the model to establish the method. The effluence of medicine concentration on apoptosis of cells was studied in detail. It was also found that the method could detect the change of cells in the early period of apoptosis. The induction of apoptosis of HeLa cell by trichosanthin was determined with the method, and the result of flow cytometry was also proved that trichosanthin could result in apoptosis of HeLa cells.

Apoptosis↗