Selective killing of choriocarcinoma cells in vitro by trichosanthin, a plant protein purified from root tubers of the Chinese medicinal herb Trichosanthes kirilowii

Trichosanthin is an abortifacient plant protein purified from the Chinese medicinal herb Tian-hua-fen, obtained from root tubers of Trichosanthes kirilowii (Cucurbitaceae). Recently, trichosanthin has also been used in the treatment of trophoblastic tumours, including hydatidiform mole, invasive mole and choriocarcinoma. We have examined the effects of trichosanthin on cultured cells and observed selective cytotoxic effects on choriocarcinoma cells. Mouse melanoma cells were also sensitive to its cytotoxic action. Cytotoxic effects on cultured cells were usually not prominent after 24 hr treatment. Continued incubation for 48 hr in control culture medium after treatment with trichosanthin enhances the cytotoxicity on cells. Our findings suggest that trichosanthin exerts specific cytotoxic activity towards trophoblastic cells.     

Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants.

1. The biochemical characteristics and biological activities of eight Cucurbitaceae plant proteins designated trichosanthin (isolated from tubers of Trichosanthes kirilowii), beta-trichosanthin (isolated from tubers of Trichosanthes cucumeroides), alpha- and beta-momorcharins (isolated from seeds of Momordica charantia), momorchochin (isolated from tubers of Momordica cochinchinensis), luffaculin (isolated from seeds of Luffa acutangula) and luffin-a and luffin-b (isolated from seeds of Luffa cylindrica), were reviewed. 2. The isolation procedures for all eight proteins are based on aqueous extraction, acetone fractionation and ion exchange chromatography. Ammonium sulfate precipitation and gel filtration are steps which may be included to improve purification. 3. The proteins are basic in nature and possess a molecular weight of approx. 30,000. All except trichosanthin are glycoproteins. The content of Asx and Glx residues is high. The N-terminal amino acid residue is Asp. Their amino acid compositions and N-terminal amino acid sequences are similar. 4. Circular dichroism spectroscopic studies revealed that trichosanthin, alpha- and beta-momorcharins possess similar secondary but different tertiary structures. 5. Most of the proteins are immunologically distinct. 6. The proteins exhibit abortifacient, antitumor, ribosome inactivating and immunomodulatory activities. Trichosanthin manifests anti-human immunodeficiency virus activity.     

Isolation and characterization of an abortifacient protein,momorcochin, from root tubers of Momordica cochinchinensis (Family Cucurbitaceae)

A glycoprotein with a molecular weight of 32 000 as estimated by SDS-polyacrylamide gel electrophoresis, and characterized by an abundance of Asp and Glu residues and an absence of Cys residues in its amino acid analysis, was isolated from fresh root tubers of Momordica cochinchinensis using a procedure that involved acetone precipitation, ammonium sulfate precipitation, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-75. The protein was capable of inducing mid-term abortion in mice. The characteristics of this protein were compared and contrasted with those of the abortifacient proteins isolated from other plants of the Cucurbitaceae family.     

Isolation of a ribosome-inactivating and abortifacient protein from seeds of Luff a acutangula

A glycoprotein with a molecular weight of 28 000 as estimated by SDS-polyacrylamide gel electrophoresis was isolated from seeds of Luffa acutangula using a procedure that involved acetone precipitation, ion exchange chromatography on CM Sepharose CL-6B and gel filtration on Sephadex G-50. In immunodiffusion studies it was found to be immunologically distinct from abortifacient proteins isolated from other members of the Cucurbitaceae family including Momordica charantia, Momordica cochinchinensis, Trichosanthes kirilowii and Trichosanthes cucumeroides. There were some differences in amino acid composition among the proteins although there was a gross similarity. The protein from L. acutangula was capable of inducing mid-term abortion in mice and inhibiting protein synthesis in a cell-free system.     

Purification of three isolectins from root tubers of Trichosanthes kirilowii (Tianhuafen)

Proteins precipitated by acetone from an aqueous extract of Trichosanthes kirilowii root tubers contained abortifacient and erythroagglutinating activities. The abortifacient protein was unadsorbed while the erythroagglutinins were adsorbed on DEAE-Sepharose equilibrated nd eluted with 0.02 M NH₄HCO₃ containing 0.1 M NaCl. The erythroagglutinins eluted from the ion exchanger were further fractionated on Lacto-gel and subsequently on Concanavalin A-Sepharose to yield three lectins.     

Trichosanthin, α-momorcharin and β-momorcharin: identity of abortifacient and ribosome-inactivating proteins

The abortifacient proteins trichosanthin, α-momorcharin and β-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.     

Chemical and biological characterization of the galactose binding lectins from Trichosanthes kirilowii root tubers

Three galactose binding isolectins have been isolated from Trichosanthes kirilowii root tubers. Two of the isolectins, TK-I and TK-II, are similar in many aspects including molecular weight, amino acid composition, NH₂-terminal amino acid residue, blood group and carbohydrate specificities, immunodiffusion and immunoelectrophoretic behavior, hemagglutinating and insulinomimetic activities, and in possessing subunits with different molecular weights. Compared to TK-I and TK-II, lectin TK-III has a larger molecular weight, subunits with the same molecular weight, a single and distinctive NH₂-terminal amino acid residue, a different isoelectric point and lower hemagglutinating activity. The three lectins share common antigenic determinants in their structures. β-Linked terminal oligosaccharides containing D-galactose inhibit hemagglutination induced by the lectins with a higher potency than α-linked oligosaccharides. The lectins are non-mitogenic and did not inhibit the concanavalin-A induced mitogenic response of lymphocytes.     

中药天花粉及其类似品的鉴别研究

本文对天花粉的使用概况作了介绍。据本草考证,自古以来中药天花粉的原植物主要来源即为栝楼Trichosanthes kirilowii Maxim.,但约自南宋之后天花粉的来源有所发展,因此天花粉商品又是多来源的。据作者在21省、区的100余市、县调查,国内约有30种植物根部曾作天花粉入药(包括地区习惯用药及混淆品)。本文对较多见的17种真伪天花粉列表简介,并对其中10种主要商品或混淆品的鲜根外形、药材或干燥品性状及显微特征(包括横切面及解离组织)进行了描述比较,同时找出各种外形及组织上的鉴别特征;列表比较其间的异同点。此外我们已经对其他6种天花粉进行了鉴别研究,待拉丁学名确定后再行报道。     

Studies on antitumor and immunopotentiating activities of polysaccharides from Trichosanthes rhizome

The polysaccharide fraction from the rhizome ofTrichosanthes kirilowii (Cucurbitaceae) showed marked antitumor and cytotoxic activity with immunopotentiating activity. It was evidenced by the increase in the number of circulating leucocytes and peritoneal exudate cells and the recovery of lowered antibody forming activity in mice. The polysaccharide was mainly composed of glucose, galactose, fructose, mannose and xylose and a samll amount of protein.     

Trichosanthin induces atresia of ovarian follicles and inhibits steroidogenesis in gonadotropin-primed immature mice.

1. Immature mice were induced to ovulate by injections of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin spaced 48 hr apart. 2. Trichosanthin (TCS), a protein purified from tubers of Trichosanthes kirilowii (Family Cucurbitaceae), was administered respectively 24 hr before, on the same day as, 24 hr after and 48 hr after the PMSG injection. 3. The results of TCS injections at different times were generally similar. The number of maturing follicles, corpora lutea and ovulated oocytes, and the ovarian weight, remained unaltered. 4. However, there was an increased incidence of follicular atresia and degeneration of ovulated oocytes, and a lowering of serum estradiol-17 beta and progesterone levels.     

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.     

Recent advances in trichosanthin, a ribosome-inactivating protein with multiple pharmacological properties.

Trichosanthin (TCS), a ribosome-inactivating protein extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii Maximowicz, has multiple pharmacological properties including abortifacient, anti-tumor and anti-HIV. It is traditionally used to induce abortion but its antigenicity and short plasma half-life have limited the repeated clinical administration. In this review, work to locating antigenic sites and prolonging plasma half-life are discussed. Studies on structure-function relationship and mechanism of cell entry are also covered. Recently, TCS has been found to induce apoptosis, enhance the action of chemokines and inhibit HIV-1 integrase. These findings give new insights on the pharmacological properties of TCS and other members of ribosome-inactivating proteins.     

A novel extraction of trichosanthin from Trichosanthes kirilowii roots using three-phase partitioning and its in vitro anticancer activity

Context: Three-phase partitioning (TPP), a unique technique which has been explored for protein separation, was used for extraction of trichosanthin (TCS).

Objective: TPP was used to optimize the TCS extraction and to determine its anticancer activity.

Materials and methods: The process consists of the simultaneous addition of t-butanol and ammonium sulfate to the aqueous slurry of Trichosanthes kirilowii Maxim (Cucurbitaceae) root powder. The extraction of TCS was optimized with respect to the concentration of ammonium sulfate loading, the ratio of t-butanol to slurry, extraction time and pH. The anticancer activity was performed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay in vitro.

Results: The extraction time with this technique is lower in comparison to conventional solvent extraction. The optimized protocol resulted in maximum recovery of 98.68% (w/w) protein within 1?h. The in vitro cytotoxic activity of the TCS was evaluated against HepG2 and WRL 68 cancer cell line and results showed that TCS possesses quite highly significant anticancer activity having IC₅₀ values of 10.38 and 15.45?μmol/l, respectively, comparable to standard drugs.

Conclusion: This framework is utilized as a basis for optimization for protein separation using TPP technique which is economical and eco-friendly.     

The mechanisms of action of Tianhua™ on antitumor activity in lung cancer cells

Context: Tianhua (TH-R) is extracted from Trichosanthes kirilowii Maxim (Cucurbitaceae) containing trichosanthin, a traditional Chinese medicine, which has been locally reported to have good anticancer effects in vivo in both animal and human models. However, there have been several reports that trichosanthin has an anticancer effect involving apoptosis.

Objective: To investigate other anticancer effects of TH-R, various tumorigenesis parameters were verified.

Materials and methods: Telomerase activity, anti-apoptosis, anti-migration and immunomodulatory activity were estimated by telomeric repeat amplification protocol assay (TRAP), flow cytometry, Boyden chamber assay and ELISA assay, respectively.

Results: In our studies, we are the first to find that TH-R had a cytotoxic effect on lung cancer cells in MTS assays; it could change the cell cycle distribution of human lung cancer cells (A549 cell line) and induce apoptosis. Further anti-telomerase effects in human lung adenocarcinoma A549 cells using the TRAP assay were noted. TH-R also had an aggregation effect on peripheral blood lymphocytes, but no effect on stimulating peripheral lymphocytes to produce human interferon-γ(IFN-γ). TH-R could inhibit the migration, or metastatic ability, of A549 cells by Boyden chamber assay. In the oral feeding therapy of an in vivo mouse model, there was an initial inhibition of A549 cancer cell growth, but no statistical difference after one month of therapy.

Discussion and conclusion: It has been proven that medicinal herbs such as Tianhua have positive effects against cancer through preventing or inhibiting the process of lung tumorigenesis.     

Conformation similarities of ricin A-chain and trichosanthin

The conformation of ricin A-chain from castor bean was studied by circular dichroism at pH 4.7, 7 and 9 and compared with that of trichosanthin from the Chinese herb Tianhuafen. The CD spectra of ricin A-chain and trichosanthin were nearly identical at each of the three pHs. Analysis of the data indicated that, like trichosanthin, ricin A-chain had about 29%α-helix and 42%β-sheet but no β-turn. However, there was a subtle difference in the CD spectra in 20 mm sodium dodecyl sulfate, the addition of which at pH 7 slightly increased the helicity and decreased the content of β-sheet of ricin A-chain in contrast to a larger increase in helicity at the expense of β-sheet for trichosanthin, thus indicating a different stability against the surfactant. Native ricin A-chain and trichosanthin had about the same amount of secondary structure, which supports the belief that a high degree of sequence homology of the two proteins [Zhang & Wang (1986) Nature 321, 477–478] may lead to a conformational similarity between them, even though the two proteins are not taxonomically related.     

Studies on the constituents of "Trichosanthes root". IV. Constituents of roots of Trichosanthes multiloba Miq., Trichosanthes miyagii Hay. and Chinese crude drug "karo-kon"

A mixture of alpha-spinasterol, stigmast-7-en-3-beta-ol (1) and its 3-O-beta-D-glucopyranoside (2), bryonolic acid (3), 23,24-dihydrocucurbitacin B (4), cucurbitacin B (5), cucurbitacin D (6) were obtained from the fresh roots of Trichosanthes multiloba Miq. (Cucurbitaceae). 1, 2, 4, 5 and 6 were also obtained from the fresh roots of Trichosanthes miyagii Hay. From the chinese crude drug "Karo-Kon", which was considered to be prepared from the roots of Trichosanthes kirilowii Maxim., 1, 2 and 3 were provided.     

天花粉治疗糖尿病物质基础及作用机制研究进展

天花粉为葫芦科植物栝楼或双边栝楼的干燥根,具有清热泻火、生津止渴等功效,临床多用于热病烦渴、肺热燥咳和内热消渴等症,是治疗糖尿病之要药。然而,目前中药治疗糖尿病仍以传统方剂或中成药为主,天花粉在糖尿病的新药开发进展缓慢。本文通过对天花粉及其复方制剂治疗糖尿病的物质基础和作用机制进行归纳总结,为下一步天花粉治疗糖尿病的新药开发提供参考。     

REVERSIBLE MODIFICATION OF LYSINE IN β-LACTOGLOBULIN USING CITRACONIC ANHYDRIDE

Acylation of lysine in β-lactoglobulin-B with citraconic anhydride resulted in a loss of free sulfhydryl groups. These were not regenerated under the conditions used to remove the modifying groups from lysine. Gel filtration and polyacryl- amide gel electrophoresis of the citraconylated and decitraconylated β-lactoglobulin showed the presence of high molecular weight components. Modification of sulfhydryl groups with N-ethylmaleimide prior to citraconylation prevented the formation of these high molecular weight components. The heterogeneity of the decitraconylated protein was attributed to a combination of inter-molecular disulfide bonding of subunits caused by structural changes occurring during lysine modification and to alkylation of free sulfhydryl groups via the citraconyl double bond.     

The effect of planting depth,disbudding, and root pruning on root weight and aconite alkaloid content of the tuberous root of aconite

The tuberous root of aconite, Aconiti tuber, contains tonic but poisonous aconite alkaloids, such as aconitine, hipaconitine and mesaconitine. The standardization of the cultivation protocols must be established, since the quality of the crude drug, especially the content of aconite alkaloids, is influenced by cultivation conditions. Here, we report the effects of planting depth, disbudding and root pruning on the content of the aconite alkaloids as well as on the weight of the root tuber of aconite. The daughter root tubers of shallow-planted plants showed a significantly higher content of aconitine, hipaconitine and sum of aconite alkaloids per unit weight than those of deeply planted root tubers (control group), while the average weight per root tuber was lower than that of the control group. The total weight of the daughter root tubers per plant of the disbudding group was significantly higher than that of other groups, including the root-pruning group whose average weight per tuber was the highest. In conclusion, disbudding is effective in achieving both higher content of aconite alkaloids and heavier total weight of the root tuber.     

The triterpenoid fraction from Trichosanthes dioica root suppresses experimentally induced inflammatory ascites in rats

Abstract

Context. Trichosanthes dioica Roxb. (Cucurbitaceae), called pointed gourd in English, is a dioecious climber found wild throughout the plains of the Indian subcontinent and traditionally used in India for several medicinal purposes.

Objective: The present study evaluated the protective effect of the triterpenoid enriched fraction from T. dioica root (CETD) against experimentally induced acute inflammatory ascites in Wistar albino rats.

Materials and methods: The CETD was administered orally at the different doses (25, 50 and 100?mg/kg body weight) to overnight fasted rats, and then ascites was induced by intraperitoneal administration of formalin solution. After 7?h, the rats were sacrificed and the volume of ascitic fluid was measured.

Results: The CETD demonstrated significant (p?<?0.01) reduction of ascitic fluid formation in a dose-dependent manner as compared with control.

Conclusion: The CETD produced significant and dose-dependent inhibition of experimentally induced inflammatory ascites in Wistar albino rats.