Polysaccharide-K augments docetaxel-induced tumor suppression and antitumor immune response in an immunocompetent murine model of human prostate cancer

Abstract

Advanced castration-resistant prostate cancer has high mortality rates and limited treatment options. Novel therapies are needed to better contend with this disease. Polysaccharide-K® (PSK), an extract of the mushroom Trametes versicolor, has immunomodulatory and tumor suppressive activities. PSK is used in Asia as a cancer immunotherapy. However, its benefit in combination with taxanes for prostate cancer is unknown. We examined whether PSK would enhance docetaxel-induced apoptosis and augment anti-tumor immune responses in orthotopic tumors using transgenic adenocarcinoma of the mouse prostate (TRAMP)-C2-bearing mice. Combining PSK with docetaxel induced significantly higher tumor suppression than either treatment alone (p<0.05), including a reduction in tumor proliferation and enhanced apoptosis. Combined PSK and docetaxel treatment led to a lower decrease in number of white blood cells than docetaxel alone, an effect accompanied by increased numbers of tumor-infiltrating CD4+ and CD8+ T cells. PSK with or without docetaxel significantly enhanced mRNA expression of IFN-γ compared to control, but did not significantly alter T-regulatory FoxP3 mRNA expression in tumors. PSK also augmented docetaxel-induced splenic natural killer cell cytolytic activity against YAC-1 target cells (p=0.045). This study is the first to show that PSK enhances docetaxel-induced prostate cancer tumor suppression, apoptosis and antitumor responses.

Figures

Figure 1

Oral administration of PSK plus docetaxel induces tumor regression. C57BL/6 mice with established tumors (50 mg) were treated with oral saline control daily, oral PSK (300 mg/kg) daily, i.p. injection of docetaxel (2x weekly) or a combination of PSK and docetaxel for a period of 10–12 days. Mean tumor weight ± standard error measurement (mean ± SEM) are shown for each treatment group of mice (n=7/group). PSK combined with docetaxel significantly suppressed tumor growth as compared to saline control, PSK or docetaxel treatments alone (P

Figure 2

Ki67 and TUNEL expression in tumors treated with docetaxel and PSK. Mice with established prostate tumors were treated with oral saline daily, oral PSK (300 mg/kg) daily, i.p. docetaxel injection (2x weekly) or a combination of PSK and docetaxel for a period of 10–12 days. Tumors were stained for the Ki67 proliferation marker and TUNEL expression. Shown are representative staining results and summary graphs showing the number of tumor cells positive for each marker (mean ± SEM) viewed at magnification ×40 for a total of 10 views in each of 6–8 tumors (hpf, per high powered field; H&E, hematoxylin and eosin staining). Combining PSK with docetaxel induced a reduction in proliferating Ki67+ cells compared to docetaxel alone (p<0.05), and significantly enhanced the apoptotic, TUNEL+ cells compared to PSK alone (p<0.01) or docetaxel alone (p<0.05).

Figure 3

Change in murine weights with combination therapy. Mice used in the same experiment as in Fig. 2 were weighed every other day for 12 days. Data are shown as mouse weight (mean ± sem) of each group (n=7 per group). Saline control or docetaxel treatments alone led to significant loss in murine weight by day 12, while PSK alone or PSK combined with docetaxel treatment maintained murine weight over this time period.

Figure 4

WBC in tumor bearing mice treated with PSK and docetaxel. Groups of mice (n=7) bearing established TRAMP-C2 tumors (12 days) were treated with either oral saline, subtherapeutic dose of docetaxel (5 mg/kg twice weekly), oral gavage of PSK (300 mg/kg), or a combination of docetaxel and PSK. WBC were assessed at the end of the experiment (mean ± SEM). Docetaxel alone induced a 25% reduction in WBC. Addition of PSK decreased the docetaxel-suppressive effect by ~50%, a difference that was statistically significant compared to docetaxel alone (p=0.03).

Figure 5

PSK combined with docetaxel enhances tumor infiltrating T cells. Mice bearing orthotopic TRAMP-C2 tumors were treated with either: 1) saline; 2) PSK (300 mg/kg daily; 3) docetaxel (5 mg/kg); or 4) PSK and docetaxel. Mice were sacrificed on day 12 after treatment and tumors were harvested. (A) Fluorescent immunohistochemistry was performed for CD4+ and CD8+ T cells (examples of positive cells indicated by arrows). Populations of both CD4+ (B) and CD8+ (C) T cell subsets were significantly higher in mice treated with docetaxel and PSK compared to PSK or docetaxel alone (p<0.05).

Figure 6

Effects of PSK and docetaxel treatments on mRNA expression of markers of antitumor immune response. Groups of mice (n=8 control; n=5 docetaxel; n= 6 PSK; n=4 PSK + docetaxel) bearing established TRAMP-C2 tumors (12 days) were treated with either oral saline, subtherapeutic dose of docetaxel (5 mg/kg twice weekly), oral gavage of PSK (300 mg/kg), or a combination of docetaxel and PSK. RNA was extracted from isolated tumors and real-time RT-PCR performed using primers and probes for several different cytokines and antitumor immune response markers. Shown are the effects of the different treatment groups on mRNA expression of IFN-γ (A), IL-2 (B), and TNF-α (C). The values shown are relative cytokine mRNA per 1,000 copies of β-actin. Mice treated with PSK, either alone (p=0.04) or with docetaxel (p=0.01) significantly induced IFN-γ mRNA expression in TRAMP-C2 tumors compared to saline control.

Figure 7

PSK in combination with docetaxel enhances NK cell activity of splenocytes from TRAMP-C2 tumor bearing mice. Splenocytes from mice treated with saline control or PSK and docetaxel alone and in combination were isolated and cultured in vitro with YAC-1 murine tumor target cells and assessed for NK cell killing of YAC-1 cells. Splenic cells from animals treated with PSK and docetaxel combination therapy had the highest level of NK cell activity at all effector:target ratios. NK cell activity induced by DTX/PSK was significantly enhanced at the 50:1 E:T ratio (p=0.045 using a linear mixed model to analyze data from 17 mice).