Y.M. Choy

Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells

Green tea catechins (GTCs) including (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epicatechin (EC) were shown to suppress cell growth and induce apoptosis in various cell systems in addition to their chemo-preventive effect. In this study, except EC which was inactive, green tea extract (TE) and other 3 GTCs were found to suppress the growth and induce apoptosis in human prostate cancer DU145 cells largely through an increase in reactive oxygen species formation and mitochondrial depolarization. The conclusion was supported by the fact that the profiles for different GTCs in growth suppression, apoptosis induction, ROS formation and mitochondrial depolarization are in a similar order, i.e. ECG > EGCG > EGC > EC. Although the molecular mechanisms are still not clear, apoptosis induced by GTCs is not related to the members of BCL-2 family as EGCG did not alter the expression of BCL-2, BCL-X(L) and BAD in DU145 cells.

The isolation and characterization of an immunomodulatory and anti-tumor polysaccharide preparation from Flammulina velutipes.

Alkaline-soluble antitumor polysaccharide was prepared from the cell wall of the mushroom Flammulina velutipes. The backbones) of the polysaccharide is mainly composed of beta-(1-->3)-D-linked glucose and its molecular weight was estimated to be about 200 kD. The polysaccharide was found to be non-toxic by brine shrimp assay. When injected into mice intraperitoneally, the polysaccharide triggered proliferation of splenic lymphocytes and also vascular dilation and hemorrhage (VDH) response. The polysaccharide exhibited potent anti-tumor activity against sarcoma SC-180 in vivo but not in vitro.

Immunomodulatory and Anti-Tumour Polysaccharides from Medicinal Plants

Many Chinese medicinal plants have immunomodulatory and anti-tumour activities. Most of the anti-tumour activities of these Chinese herbs are probably due to their immunostimulating polysaccharide components. A general scheme for the isolation and purification of the bioactive polysaccharides from naturally occurring medicinal plants is described. Hot-water extraction followed by various chromatographic methods are usually used to purify the bioactive polysaccharides. The different fractions separated from Chinese medicinal plants show a range of immunomodulatory and anti-tumour activities. The analytical methods used for monosaccharide sequence determination and structural elucidation of the bioactive polysaccharides are described, as are the tests used to evaluate their immunopharmacological activities, both in vitro and in vivo. The purification, characterization and structural elucidation of immunomodulatory polysaccharides from medicinal plants may have important implications in the immunotherapy of cancer and in the treatment of various other diseases.

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c.

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by α-D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.

The immunostimulating activities of anti-tumor Polysaccharides from Pseudostellaria heterophylla

We have previously shown that a mitogenic fraction (PH-I) separated from Pseudostellaria heterophylla exhibits both immunomodulatory and anti-tumor activities. In the present study, PH-I was further purified by gel filtration chromatography and the resulting three fractions (PH-I A, PH-I B and PH-I C) were assessed for their anti-tumor activity in vivo. It was found that fraction PH-I C from P. heterophylla could markedly suppress the growth of EAT cells in vivo. Mechanistic studies have shown that i.p. injection of PH-I C into mice could enhance the phagocytic activity of thioglycollate-elicited peritoneal macrophages. Moreover, PH-I C showed a potent activating effect on the cytotoxic activity of natural killer (NK) cells and alloreactive cytotoxic T cells (Tc) as well as increased the MurIL-2-induced lymphokine activated killer cell (LAK) activity in vitro. In addition, PH-I C could increase the number of tumor infiltrating lymphocytes (TILs) in the tumor site of WEHI-164-bearing mice. Finally, i.v. injection of PH-I C significantly elevated the levels of IFN-gamma and IL-4 in sera of EAT-bearing mice.

Slow Increase in Intranuclear and Cytosolic Free Calcium Concentrations in L929 Cells Is Important in Tumour Necrosis Factor-Alpha-Mediated Cell Death

The potential involvement of cellular calcium in the signalling pathway of tumour necrosis factor-alpha (TNF) was assessed in L929 cells using 45Ca2+ and confocal laser scanning microscopy with fluorescence calcium indicators. Our data indicate that the effect of TNF on intracellular Ca2+ mobilization is a slow process with no discernible increase in the cytosolic free Ca2+ concentration ([Ca2+]c) and intranuclear Ca2+ level ([Ca2+]n) within the 1st min of TNF (25 ng/ml) administration. However, prolonged exposure (2 h) of L929 cells to TNF brought about pronounced increase in cytosolic and intranuclear [Ca2+] even in the absence of external Ca2+. The increase in intracellular [Ca2+] was more apparent when cells were treated with thapsigargin, an inhibitor of microsomal Ca2+-ATPase. Interestingly, most of the Ca2+ released was around and confined to the nucleus. Following the pretreatment of cells with thapsigargin, a synergistic killing effect was obtained when cells were cultured with TNF. The use of 45Ca2+ also revealed that TNF enhanced the 45Ca2+ uptake in a time-dependent manner. Calcium channel blockers, verapamil and diltiazem, could alleviate both the TNF-mediated 45Ca2+-uptake and killing activity. Our results therefore suggest that an increase in cellular Ca2+ is a crucial factor in the TNF cytotoxicity.

Mitogenic and tumor necrosis factor producing activities of Pseudostellaria heterophylla

Separation of mitogenic fraction (PH-I) from Pseudostellaria heterophylla and characterization of its biological activities were investigated. PH-I was isolated as an alcohol-insoluble fraction from the hot water extract obtained by heating the roots of P. heterophylla in water at 80 degrees C for 4 h. It is a water-soluble substance consisting of mainly carbohydrates (56.8%) and a small amount of proteins (7.6%). The incorporation of tritiated thymidine into the DNA of murine spleen lymphocytes could be stimulated by PH-I in a dose-dependent manner. PH-I could act as a priming agent for the release of tumor necrosis factor (TNF) in mice. Moreover, PH-I exhibited potent anti-tumor activities against Ehrlich ascites tumor (EAT) cells in vivo but not in vitro.

Changes in surface carbohydrates of erythrocytes during in vivo aging

Abstract Erythrocyte aging is accompanied by an overall reduction in surface carbohydrate content. A decrease in sialic acid and an increase in D-galactose and N-acetylgalactosamine in the terminal position of the glycoprotein polysaccharide chains are also observed in aged erythrocytes. In the light of these and other observations, it is proposed that the newly exposed galactose/galactosamine residues in the desialylated glycoproteins may serve as recognition signals triggering the elimination of senescent erythrocytes from circulation.

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997.

Suppression of narcotic withdrawals and plasma ACTH by auricular electroacupuncture

Abstract Auricular electroacupuncture (AES) has been found to be successful in the suppression of withdrawal symptoms of morphine-addicted mice. In abrupt withdrawals precipited by naloxone, the plasma adrenocorticotropin (ACTH) rises to a high level which can also be effectively suppressed by AES. This elevation of plasma ACTH is not due to naloxone, as naloxone has no effect on the ACTH level in non-addicted mice. The possible physiological effect produced by AES is discussed.