Polysaccharide Krestin Enhances Manganese Superoxide Dismutase Activity and mRNA Expression in Mouse Peritoneal Macrophages

American Journal of Chinese Medicine, Summer-Fall, 2000 by Zhan-Jun Pang, Yuan Chen, Mei Zhou

Abstract: Manganese superoxide dismutase (MnSOD), an inductive antioxidant enzyme, can protect cells from oxidative injury to the mitochondria. The elevation of MnSOD activity in cells can effectively prevent many diseases associated with oxidative stress. Polysaccharide Krestin (PSK), a kind of protein-bound polysaccharide extracted from Coriolus versicolor, is used as an immune response modifier in anti-tumor therapy. We have previously found that PSK could alleviate the oxidative injury that oxidized low density lipoprotein (Ox-LDL) brought to monocytes/macrophages, and therefore had some preventive or therapeutic effect on atherosclerosis. In order to find out if the effects of PSK were associated with the alteration of antioxidant enzymes, we investigated its effect on MnSOD activity and gene expression in mouse peritoneal macrophages. The results showed that PSK could enhance SOD activity and increase the contents of MnSOD mRNA in mouse peritoneal macrophages. Furthermore, the induction of MnSOD by PSK could be blocked by cycloheximide and actinomycin D.

Aerobic life-style offers both benefits and risks to living cells. The major risk comes from the formation of reactive oxygen intermediates during normal oxygen metabolism. Reactive oxygen species (ROS, i.e. superoxide radical, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]; hydrogen peroxide, [H.sub.2][O.sub.2]; and hydroxyl radical, *OH), can react with DNA, proteins and unsaturated fatty acids resulting in DNA strand breaks and oxidative damage, protein-protein or protein-DNA crosslinks. They are thought to be involved in cancer, aging and various inflammatory disorders including bacterial or viral infections, amyotrophic lateral sclerosis, ischemic heart disease, Alzheimer's disease, Down's syndrome, Parkinson's disease and atherosclerosis (Valentine et al., 1998; Saran et al., 1998; Multhaup et al., 1997). Fortunately, cells in the body possess a wide range of interlinked antioxidant defense mechanisms to protect themselves from damage by ROS. Among these mechanisms, several antioxidant enzymes including catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPx, EC 1.11.1.9) and glutathione S-transferase (GST, EC 2.5.1.18), play an important role in scavenging ROS produced in cells.

Three forms of SOD exist in cells and tissues: copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and extracellular superoxide dismutase (EC-SOD) (Fridovich et al., 1989). Evidence suggests that both CuZnSOD and MnSOD are important in cell defense against oxygen toxicity. But only MnSOD was reported to be induced in response to stimuli, such as tumor necrosis factor (TNF). MnSOD is a superoxide anion scavenger located in mitochondria. Increased expression of MnSOD can diminish oxygen radical-mediated injuries and the cytotoxic effects of TNF [Alpha] (Liochev et al., 1997), ionizing radiation, and certain chemotherapeutic agents. Reports in recent years also proved MnSOD to be a new type of tumor suppressor gene (Zhong et al., 1997; Liu et al., 1997; Kiningham et al., 1997).

Polysaccharide Krestin (PSK) is protein-bound polysaccharide extracted from a mushroom Coriolus versicolor, and was used as an immune response modifier in anti-tumor therapy by many researchers (Ng TB., 1998). It was found that PSK could suppress tumor growth and metastasis. In our previous work, we found that PSK could alleviate the oxidative injury that oxidized low density lipoprotein (Ox-LDL) brought to monocytes/ macrophages, and therefore have some prophylactic or therapeutic effects on atherosclerosis, which is now considered as a kind of multicellular inflammatory response, a result from an imbalance between antioxidant and oxidant-generating systems (Chen et al., 1996). Monocyte/macrophages are always viewed to play an important role in regulating immunological response in the body, especially in defending inflammatory diseases and tumors. Therefore, in order to uncover the mechanisms that PSK exerted on diseases, we investigated the effect of PSK on MnSOD gene expression in mouse peritoneal macrophages.

Materials and Methods

Reagents

DMEM (Dulbecco's Modified Eagle's Medium), pyrogallol, actinomycin D, cycloheximide, acetovanilone, PMSF, aprotinin, diethyl pyrocarbonate (DEPC), guanidinium thiocyanate, sarcosyl were obtained from Sigma Co. (St. Louis Mo, USA). DIG DNA Labeling and Detection kit, DIG Easy Hyb, positively charged nylon filter, digoxigenin-labeled 1.5kb [Beta]-actin RNA probe were purchased from Boehringer Mannheim Biochemicals (Mannheim, Germany). PSK, (the average molecular weight is 1,200 kD and the content of saccharide is about 78%), was extracted from the mushroom Coriolus versicolor in our laboratory according to the method of Xi et al. (Xi et al., 1993), and was prepared as 1.5% (W/V) solution in sterile saline. Endotoxin was not detectable at the concentration used in the Limulus amoebocyte lysate assay (Association of Cape Cod, Woods Hole, MA, USA), which had a sensitivity level of 50 pg/ml. All other chemicals were of analytical grade. The plasmid containing a 0.737 kb MnSOD cDNA insert was a gift from Dr. Huang (U.S.A)