Anti-tumor metastatic activity of β-glucan purified from mutated Saccharomyces cerevisiae

Abstract

The β-glucans isolated from Saccharomyces cerevisiae (S. cerevisiae) enhance the innate immune system, but there is little evidence for its antitumor activity. To examine the antitumor and immunostimulating activities of β-glucan (IS-2) purified from mutated S. cerevisiae, we made an experiment on innate immune response against metastasis of cancer cells by comparing with the β-glucan from wild-type S. cerevisiae. In experimental lung metastasis of colon 26-M3.1 carcinoma or B16-BL6 melanoma cells, prophylactic administration of β-glucan purified from mutated S. cerevisiae significantly inhibited lung metastasis in a dose-dependent manner. Furthermore, therapeutic administration of IS-2 also significantly inhibited the colon 26-M3.1 cell growth in mice. In an assay of liver and spleen metastasis produced by i.v. inoculation of L5178Y-ML25 lymphoma cells, IS-2 also significantly inhibited metastasis in CDF1 mice. Furthermore, pretreatment with IS-2 two days before tumor inoculation significantly prolonged the survival time of tumor-bearing mice. In an in vitro cytotoxicity analysis, IS-2 (up to 100 μg/ml) did not affect the growth of colon 26-M3.1 cells. In contrast, IS-2 enhanced splenocyte proliferating activity in a dose-dependent manner. Peritoneal macrophages stimulated with IS-2 produced various cytokines, such as IL-1β, IFN-γ, and IL-12. In addition, treatment with IS-2 (20 μg/mouse) induced tumoricidal activity of peritoneal macrophages against colon 26-M3.1 cells. In an assay for natural killer (NK) cell activity, IS-2 (20 μg/mouse, i.v.) significantly augmented NK cytotoxicity against Yac-1 tumor cells at 2 days after IS-2 treatment. The depletion of NK cells by injection of rabbit anti-asialo GM1 serum abolished the inhibitory effect of IS-2 on lung metastasis of colon 26-M3.1 cells. These data suggest that IS-2 inhibits tumor metastasis via activation of macrophages and NK cells.

Introduction

Many efforts have been made to develop and improve immunotherapy strategies for the treatment of malignancies. The use of biological response modifiers (BRMs) for enhancing host defense responses against tumors is one of the most attractive alternatives to cytotoxic drugs [1]. Metastasis is one of the characteristics that distinguish malignant tumors from benign neoplasm. Progressive metastasis of tumor cells in the host results in biological hetõerogeneity for immunogenicity, such as growth rate, cell markers, and sensitivity to chemotherapeutic drugs [2]. Therefore, preventing metastasis is one of the most crucial problems in cancer treatment. Activation of the innate immune system protects against foreign antigens, including tumors [3]. Stimulation of the innate immune system has been attempted with a number of strategies, including cytokines [4] constituents isolated from microorganisms [5] and herbal plants [6], synthetic adjuvants [7], and oligonucleotides [8], and some have reached the clinical trials. The primary mechanism of immunostimulation by BRMs is activation of macrophages or NK cells [3], [4], [5], [6], [7], which can then lyse or inhibit the growth of tumor cells. Indeed, many experimental studies and clinical trials showed that natural immunity played an important role in blocking metastasis from primary tumors [9]. NK cells and macrophages are responsible for the natural immunity against tumors [10], [11]. In addition, various cytokines, such as IL-12, TNF-α, or IL-1β from macrophages, augment NK cell responses, and these proinflammatory cytokines can induce activation of adaptive immunity in part through stimulation of IFN-γ production from NK cells [12], [13], [14].

β-glucan derived from the yeast cell wall of species such as Saccharomyces cerevisiae can modulate immune responses in vitro and in vivo[15]. Administration of β-glucan isolated from S. cerevisiae increases host resistance to various foreign antigens, such as viral, bacterial, and parasitic infections, and protects against tumors via immunostimulation [16], [17], [18].

Here we compared the inhibitory potential on tumor metastasis of a mutated and wild-type β-glucan from S. cerevisiae using colon 26-M3.1 carcinoma, B16-BL6 melanoma, and L5178Y-ML25 lymphoma cells in syngeneic mice. We also analyzed the involvement of NK cell and macrophage activation to clarify the immunostimulation effects.

Section snippets

Preparation of glucans from mutated S. cerevisiae

Treatment for the mutation and growth conditions of S. cerevisiae, and the β-glucan preparation process have been described [19], [20]. Briefly, cultured S. cerevisiae in YDP medium were centrifuged at 7000 rpm for 15 min, diluted to 1–2 × 10⁸ cells/ml, and washed 3 times with distilled water. The cells were then treated with NTG (N-metyl-N′-nitro-N-nitrosoguanidine, Sigma) to induce a 0.1% survival rate. Surviving yeast were washed with distilled water and cultured in YDP medium containing

Inhibitory effect of IS-2 on experimental lung metastasis

We first compared the effects of mutated and wild-type S. cerevisiae β-glucans in a colon 26-M3.1 lung metastasis model. Glucan treatment (0.8–100 μg, i.v.) from mutated yeast dramatically inhibited lung metastasis of colon26-M3.1 cells in a dose-dependent manner. Administration of 20 and 100 μg of IS-2 inhibited lung metastasis over 90%, and 100 μg of wild-type β-glucan inhibited lung metastasis over 83% compared to the tumor control group. Treatment with 20 μg of wild-type showed partial

Discussion

Immunological approaches to treat cancer are broadly classified into innate and adaptive, in which cancer cell-specific and non-specific immunological mechanisms are involved. Biological response modifiers (BRM) such as natural products, oriental medicines, cytokines, and synthetic adjuvants, can suppress experimental tumor growth and metastasis [16], [22]. In fact, BRMs that enhance host defense systems have been applied to cancer immunotherapy [1], [5], [14], [15], [22].

β-glucans isolated

Acknowledgements

This work was supported by the Regional Innovation Center Program of the Ministry of Commerce, Industry, and Energy through the Bio-Food & Drug Research Center at Konkuk University, Korea.