Protein-bound polysaccharide K (PSK) was kindly provided by Kureha Chemical Ind. Co. (Tokyo, Japan). It is prepared by extracting cultured mycelia of Coriolus versicolor with hot water. The precipitate is separated from the clear supernatant with saturated ammonium sulfate, then desalted and dried 23. Protein-bound polysaccharide K was dissolved in RPMI medium or water and heated at 50°C for 20–30 min until a clear solution appeared. The PSK preparation was filter-sterilized and diluted in culture medium or water to the desired concentration. Protein-bound polysaccharide K was previously titrated in NKL cells 16 and the working dilution was 100 μg/mL. PSK extract digested with neuraminidase was also tested, digesting 100 μg of PSK with 4 μl (Sigma) and incubating for 3 h at 37°C. Our group previously showed that PSK is composed of two bands of very high molecular weight 22. After digestion with neuraminidase, these bands are reduced to a single band of about 12 kd. These results indicate that PSK is probably composed of a single 12-kd protein, and that this protein is highly glycosylated 22. Two different extracts of PSK were also used: one rich in sugars and other rich in proteins.

The following tumour cell lines were studied: B16 murine melanoma, B9 murine MCA-induced fibrosarcoma, Ando-2 human melanoma, AGS human gastric cancer, A-549 human lung cancer, Hela human cervical adenocarcinoma and Jurkat T lymphoma leukemia. The NKL studied was established from PBLs of a patient with LGL leukemia 19. All cell lines were obtained from the American Type Culture Collection (Manassas, USA) except for the B9 cell line, which was generated at our laboratory, and the Ando-2 and NKL cell lines, kindly provided by P. Coulie (Unite de Genetique Cellulaire, Louvain University, Brussels, Belgium), F. X. Real (Instituto Municipal de Investigaciones Medicas, Barcelona, Spain) and Dr. M. Lopet-Botet (Universidad Pompeu-Fabra, Barcelona, Spain), respectively.

Cell lines derived from solid tumours were grown at 37°C in a humidified atmosphere of 5% CO₂ in DMEM culture medium (Gibco, Paisley UK) supplemented with 10% heat-inactivated foetal bovine serum (Life Technologics, Milan Italy), antibiotics and glutamine. Jurkat T cell leukemia was cultured in RPMI 1640 with 10% heat-inactivated fetal bovine serum. The NKL cell line was cultured in RPMI 1640 with 10% heat-inactivated human AB serum (Sigma Chemical, St Louis, MO; USA) and human recombinant IL-2 (100 U/ml; purity > 97%, specific activity, 2 × 10⁶ U/mg) (Roche, Nutley, NJ; USA).

The effect of PSK on tumour cell proliferation was assessed by measuring BrdU incorporation with the BrdU colorimetric ELISA Cell Proliferation Kit (Roche Diagnostic). Cells were plated in 96-well microculture plates (5 × 10³ cells/well). Every 48 h, the culture medium was replaced and PSK was added. After 48–96 h, BrdU labelling reagent was added and cultured for a further 1–3 h. Assays were also performed by counting viable cells using Trypan Blue. Briefly, cancer cell lines were seeded into culture tissue-flask (1.5–2 × 10⁵/culture tissue-flask) and incubated for 24 h at 37°C in a humidified atmosphere of 5% CO₂. Cells were then treated with 100 μg/ml of PSK in the culture medium, which was replaced every 48 h. After 4–6 days, cells were collected by centrifugation and a small sample of cell suspension was diluted in 0.4% Trypan Blue, counting cells in a haemocytometer chamber. Each cell sample was counted in this way at least three times and each assay was repeated at least three times.

Human lymphocytes were isolated from venous blood by the Ficoll-Hystopaque separation method. Proliferation of PBLs was analyzed in vitro using 5-bromo-2'-deoxyuridine (BrdU) labelling of DNA-synthesizing cells with the above-mentioned kit. PBLs were seeded in 96-well microculture plates at a cell density of 5 × 10⁴ per well. Two different concentrations of PSK were used, 100 μg/ml and 50 μg/ml. Concanavalin A (5 μg/ml, Sigma) and IL-2 were used as positive controls. PSK was also used in combination with IL-2 or Concanavalin A. After 48 h of culture in presence or absence of PSK, BrdU labelling reagent (final concentration 10 μM) was added and cells were cultured for 24 h. Cells were then fixed for 30 min and incubated with anti-BrdU for 1 h at 37°C. 100 μl of tetramethyl-benzidine (TMB) was used as substrate. Optical densities were determined at 370 nm by means of an ELISA microplate reader (Biotek, Power-Wave XS). Controls were the culture medium, cells cultured only in medium and cells incubated with anti-BrdU in absence of BrdU. All experiments were repeated at least three times.

Briefly, cells were plated in six-well plates (5 × 10⁵ per well) or in culture tissue-flask (15 × 10⁵) and continuously exposed for 4 days to 100 μg/ml of PSK. The DNA synthesis rate was examined by BrdU incorporation method using FITC BrdU Flow Kit (BD Pharmingen) according to manufacturer's instructions. BrdU was then detected by DNase cell treatment using FITC-conjugated anti-BrdU antibody. Cells were washed with 1 ml 1 × BD Perm/Wash Buffer, and 20 μl 7-amino-actinomycin D was added. Analysis was performed with 50000 cells using Cell Quest Software and FACScan flow cytometer (Becton-Dickinson).

After treatment of cancer cells with PSK for four days, cells were detached from the culture tissue-flask with PBS containing 3 mM EDTA. These cells were then collected together with floating cells, washed twice with cold PBS and resuspended in binding buffer at a concentration of 1 × 10⁶ cells per ml; 100 μl of solution was incubated for 30 min at 4°C with 5 μl of Annexin V-PE antibody (BD Biosciences), and 5 μl of 7-amino-actynomycin D was then added. Cells were incubated for 15 min in darkness, and 400 μl of staining buffer was added before flow cytometry analysis. Apoptosis was analyzed by quadrant statistics as follows: Annexin V- and 7-AAD-negative cells are alive; Annexin V-positive and 7-AAD-negative cells are in early stages of apoptosis; Annexin V-negative and 7-AAD-positice cells are dead but not by apoptosis; and Annexin V-positive and 7-AAD-positive cells are in mid- or end-stage apoptosis.

FITC conjugated monoclonal anti-active-caspase-3 antibody (BD Biosciences) was used to determine whether the protease caspase-3 is involved in PSK-induced apoptosis. After 4-day treatment with PSK, cancer cells were washed twice with cold PBS and fixed and permeabilized in Cytofix/Cytoperm buffer. Then, cells were incubated with FITC-conjugated monoclonal rabbit anti-active human-caspase-3 antibody for 30 min. Cells were washed twice and 500 μl of 1 × Perm Wash Buffer was added before analysis by flow cytometry.

Values are expressed as means ± SD. Student's t-test was used for statistical comparisons, considering a significance value of P < 0.05.

Tumour cell lines were cultured in 96-well plate for 48–72 h (2.5–5 × 10³ cells) in medium alone (control) or with PSK (100 μg/ml or 50 μg/ml) for 4–6 days. Cell proliferation was then measured by BrdU incorporation (absorbance), which was significantly lower in PSK-treated versus untreated tumour cells (Fig. 1). AGS and A549 cell lines showed a strong decrease in absorbance after treatment with 100 μg/ml PSK that was less marked after treatment with 50 μg/ml of PSK. Inhibition of proliferation was around 65% in melanoma cell lines B16 and Ando-2, lower in Hela and Jurkat cell lines and lowest (20%) in B9 murine fibrosarcoma (Fig. 1). PSK-treated tumour cells showed morphological changes (rounded and granulated morphology, increased vacuolisation, cell shrinkage) and a large number of the cells detached from culture flasks.

These assays were repeated in cell culture flasks (1.5–2.5 × 10⁵cells), and viable cells were counted in Haemacytometer chamber using Trypan Blue. As shown in Table 1, there was a significant decrease in the final number of viable cells, with a proliferation inhibition of 22%–84% versus control cells. There was an excellent correlation between the results obtained with the two assays (absorbance and cell count). In Hela tumour cells, proliferation inhibition was higher after treatment with 50 μg/ml versus 100 μg/ml of PSK. In all other tumour cell lines, proliferation inhibition was similar or higher at 100 μg/ml PSK.

A dose-response analysis was performed to determine the in vitro effect of PSK on human PBLs. PBLs (5 × 104) were plated in 96-well tissue plate for 48–72 h with eight serially diluted extractions ranging from 500 μg/ml (concentration n°8) to 3.9 μg/ml (concentration n°1). Concentration n°0 represents cells cultured in medium alone. BrdU incorporation during DNA synthesis was then measured by ELISA. Optical densities were very similar between treated and untreated PBLs (data not shown). However, simultaneous treatment of PBLs with IL-2 (100 U/ml) + PSK (100 μg/ml) produced a higher proliferation rate (4.5-fold) versus PBLs treated with IL-2 alone (3-fold) (Fig. 2). Untreated and Concanavalin A-treated PBLs served as controls (Fig. 2).

Tumour cell proliferation inhibition was compared among different PSK variants. Neuraminidase treatment digests glicosylated proteins. A549 tumour cell line was cultured in medium alone (control) or with PSK (100 μg/ml) or neuraminidase-treated PSK (100 μg/ml) for 4–6 days and then counted using trypan blue. No significant differences in proliferation inhibition were found between PSK and neuraminidase-treated PSK (Fig. 3a). The same results were found for sugar-rich and protein-rich PSK variants as for PSK (data not shown).

It was previously reported that PSK induces proliferation and activation of NKL cells 16. Treatment of NKL cells with 100 μg/ml PSK or neuraminidase-treated PSK for 96 h induced a similar increase in their proliferation (Fig. 3b), which was slightly higher than that obtained after culture of NKL with IL-2 alone (Fig. 3b). Induction of NKL proliferation was slightly lower in sugar-rich and protein-rich PSK variants; this difference was not significant (data not shown)

Mechanisms of PSK cytotoxic activity were analysed by flow cytometry in order to study the effect on cell cycle phase distribution. Culture of AGS tumour cell line with 100 μg/ml of PSK produced total cell cycle arrest with cell accumulation in G₀/G₁ phase and no cells in S phase (Fig. 4). Cell cycle phase distributions were: 32.2% G₀/G₁, 31.1% S and 16.2% G₂/M in control AGS cells and 60.8% G₀/G₁, 0% S and 14.1% G₂/M in PSK-treated AGS cells. Similar results were found in Ando-2, A549 and B16 tumour cell lines (Table 2). Results in B9 fibrosarcoma showed a slowing rather than an arrest of the cell cycle (Fig.4), with a partial accumulation in G₀/G₁ phase (49.15% untreated cells and 63.17% PSK-treated cells) at the expense of a decrease in S phase (20.54% vs. 15.14%) and G₂/M phase (12.6% vs. 6.96%). Similar results were found in Hela and Jurkat tumour cells (Table 2). These results indicate that PSK produces arrest or slowing of the cell cycle according to the tumour cell histology.

Cancer cell lines were treated with 100 μg/ml PSK for 4 days to examine the capacity of PSK to induce apoptosis. Untreated or PSK-treated cancer cells were incubated with Annexin V-PE in a buffer containing 7-amino-actinomycin (7-AAD) and analyzed by flow cytometry. Figure 5 depicts representative results for AGS and B9 tumour cells. PSK increased apoptosis from 4.32% (untreated cells) to 37.52% in AGS cells but not in B9 tumour cells (untreated cells, 11.37% vs. PSK-treated cells, 12.11%). Table 3 depicts the results for other tumour cell lines, showing that PSK induces apoptosis in A549, B16 and Ando-2 tumour cells.

Caspases are the main enzymes involved in the apoptotic pathway and the participation of active caspase-3 in PSK-induced apoptosis was evaluated. Tumour cells were treated with PSK (100 μg/ml) for 4 days, then permeabilized, fixed and stained for active human caspase-3 and analyzed by flow cytometry. In the AGS cell line, untreated cells were negative for presence of active-caspase-3, whereas around 36% of PSK-treated cells showed detectable active caspase-3 (Fig. 6). However, in tumour cell lines in which PSK did not produce apoptosis, e.g., B9 tumour cells, no caspase-3 expression was detected after PSK treatment (Fig. 6). Table 4 depicts the results obtained with the other tumour cell lines analysed.