A polysaccharide from Lentinus edodes inhibits human colon cancer cell proliferation and suppresses tumor growth in athymic nude mice

Abstract

The antitumor effect of Lentinan is thought rely on the activation of immune responses; however, little is known about whether Lentinan also directly attacks cancer cells. We therefore investigated the direct antitumor activity of SLNT (a water-extracted polysaccharide from Lentinus edodes) and its probable mechanism. We showed that SLNT significantly inhibited proliferation of HT-29 colon cancer cells and suppressed tumor growth in nude mice. Annxein V-FITC/PI, DAPI, AO/EB and H&E staining assays all showed that SLNT induced cell apoptosis both in vitro and in vivo. SLNT induced apoptosis by activating Caspase-3 via both intrinsic and extrinsic pathways, which presented as the activation of Caspases-9 and -8, upregulation of cytochrome c and the Bax/Bcl-2 ratio, downregulation of NF-κB, and overproduction of ROS and TNF-α in vitro and in vivo. Pretreatment with the caspase-3 inhibitor Ac-DEVD-CHO or antioxidant NAC blocked SLNT-induced apoptosis. These findings suggest that SLNT exerts direct antitumor effects by inducing cell apoptosis via ROS-mediated intrinsic and TNF-α-mediated extrinsic pathways. SLNT may thus represent a useful candidate for colon cancer prevention and treatment.

Keywords: ROS; apoptosis; colon cancer; lentinan; nude mice.

Figure 1. SLNT inhibited HT-29 cell proliferation and induced apoptosis

The cell viability was evaluated by MTT assay (A). Annexin V-FITC/PI staining was used to analyze apoptosis; representative results (B) and summative data (C) from three independent experiments were shown. Cells treated with 400, 800 and 1600 μg/mL of SLNT or without for 48 h were stained with AO/EB (green/orange) or DAPI (blue) (D). Each bar represents means ± SD (n = 3). ^*p < 0.05, ^***p < 0.001 versus negative control (NC) group.

Figure 2. Inhibition and apoptotic induction effect of SLNT on HT-29 xenografts

Tumor volume from day 5 to day 21 (A) and tumor weights at the end of treatments were measured (B) Columns, mean ± SD (n = 6); ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus NC group. Photographs of nude mice and tumors of each group were taken at end of experiment (C) Representative histopathological changes of apoptosis in tumors were analyzed by H&E staining (400×, D).

Figure 3. SLNT induced HT-29 cell apoptosis by activation of caspase-3 in cells and in tumors

Activity of caspase-3 in cells (A) and in tumor xenografts (B) was measured by western blot. Immunohistochemistry analysis of caspase-3 in tumors of SLNT (5.0 mg/kg) and NC were shown (C, 400×). HT-29 cells were incubated with SLNT (800 μg/mL), a caspase-3 inhibitor Ac-DEVD-CHO (25 μM) or combination for 48 h, representative results (D) and summative data (E) were shown. Each bar represents means ± SD (n = 3). ^**p < 0.01, ^***p < 0.001 versus NC; ^###p < 0.001 versus combination group.

Figure 4. Effect of SLNT on intrinsic apoptotic-related proteins in cells and in tumors

Expression of Bax, Bcl-2, cytosolic Cytochrome c and Cleaved caspase-9 in cells (A, B) and in tumor xenografts (C, D) were analyzed by western blot in three independent experiments. Each bar represents means ± SD (n = 3), ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus NC group.

Figure 5. SLNT induced the loss of mitochondrial membrane potential (MMP) in cells

The change of MMP was analyzed by flow cytometry (A) and fluorescence microscope (B, 400×). Representative images of NC and SLNT (800 μg/mL) taken by fluorescence microscope were shown.

Figure 6. SLNT increased the generation of ROS in cells and in nude mice

Cellular ROS was measured by DCFH-DA using flow cytometry: a mixture image of NC (black), 400 μg/mL (red), 800 μg/mL (blue) and 1600 μg/mL (purple) of SLNT was shown (A), and fluorescence intensity were summarized (B); representative images (C, 400×) were taken by fluorescence microscope. The level of ROS in serum of nude mice was detected by ELISA assay (D). Each bar represents means ± SD (n = 3–6), ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus NC group.

Figure 7. ROS was responsible for the SLNT-induced intrinsic apoptosis

HT-29 cells were incubated with SLNT (800 μg/mL), an antioxidant NAC (5 μM), or combination for 48 h. Annexin V-FITC/PI staining was used to analyze cell apoptosis (A, B), ^***p < 0.001 versus combination group, ^###p < 0.001 versus NAC group. The expression of Bax, Bcl-2 and cytosol Cytochrome c were measured by western blot (C) and summarized (D). Each bar represents means ± SD (n = 3), ^*p < 0.05, ^**p < 0.01 versus combination group.

Figure 8. SLNT activated caspase-8, decreased nuclear NF-κB and increased TNF-α in vitro and in vivo

Expression of cleaved caspase-8 and nuclear NF-κB in cells (A) and in tumors (B) were analyzed by western blot. Activity of caspase-8 in cells was further measured by Caspase-8 activity kit (C). Immunohistochemistry analysis of cleaved caspase-8 in NC and in SLNT (5.0 mg/kg) tumors was shown (D). The level of TNF-α in serum of nude mice (E) and in cell culture medium (F) was measured by ELISA assay. Each bar represents means ± SD, n = 3–6, ^*p < 0.05, ^***p < 0.001 versus NC group.

Figure 9. SLNT inhibited NF-κB p65 nuclear translocation both in vitro and in vivo

Figure 10. Flow diagram of the probable mechanism of direct antitumor effect of SLNT on HT-29 cells