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Introduction
Protein-bound polysaccharide-K (PSK) is a protein-bound polysaccharide K (Krestin), extracted from cultured mycelia of Coriolus versicolor (CM101) and the average molecular weight is approximately 100,000 (1). PSK is one of the biological response modifiers (BRM), and is used clinically in combination therapy for gastrointestinal cancer and small cell lung carcinoma (1–4).
The most important and widely reported property is the immunomodulatory effect, as direct antitumor activity is weak. A few previous reports have indicated that PSK induces cytostatic effects on growth and invasion by modulating the expression of major histocompatibility complex (MHC) class I and inhibition of nuclear factor kappaB (NF-κB) (5).
Our previous study demonstrated that PSK inhibits cellular proliferation in a cell type-specific manner and that the inhibition is most profound for promyelomonocytic leukemia HL-60 cells (6). We have shown that the underlying mechanism of the inhibition in cellular proliferation is due, in part, to caspase-3-mediated apoptosis.
Apoptosis is one of the main mechanisms for inhibition of cancer growth and proliferation. The execution of apoptosis, or programmed cell death, is associated with characteristic morphological and biochemical changes mediated by a series of gene regulations and cell-signaling pathways (7). Introduction of apoptosis included activation of caspase, change of mitochondrial signaling pathway and regulation of Bcl-2 family members. Recently, perturbation of mitochondrial function has been shown to be a key event in the apoptotic cascade. Anticancer drugs may damage the mitochondria by increasing the permeability of the outer mitochondrial membrane, which is associated with the collapse of the mitochondrial membrane potential (ΔΨm) (8).
Therefore, the present study investigated the mechanism of the apoptosis induced by PSK and focused on the mitochondrial and p38 mitogen-activated protein kinase (MAPK)-dependent pathway in HL-60 cells.
Materials and methods
Cells
The promyelomonocytic leukemia cell line HL-60 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in RPMI-1640 (Gibco-BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Life Technologies, Milan, Italy).
Reagents
PSK was manufactured at Kureha Corp. (Tokyo, Japan), and was dissolved in Dulbecco’s phosphate-buffered saline (DPBS) at a concentration of 100 mg/ml (Gibco-BRL). In each experiment, the PSK solution was freshly prepared and further diluted with medium. The p38 MAPK inhibitor SB203580 was purchased from LC Laboratories (Woburn, MA, USA), and dissolved...