Fig 1.
Effects of RBDS and gramisterol on cell viability and apoptosis.
(A), (B) MTT assay of WEHI-3 cells treated with different concentrations of RBDS for 24, 48 and 72 h, and gramisterol for 24 h. Percent cell viability was calculated relative to the untreated control. The experiments were performed in triplicate and data are presented as mean±SEM. Significant difference was considered at p<0.05 compared with the control. (C) Phase contrast photographs and DAPI-DNA staining of WEHI-3 cells after treatment with RBDS 0, 50, 100 and 200 μg/mL for 48 h. The apoptotic bodies are indicated by white arrowheads. (D) Gel electrophoresis of DNA extracted from RBDS treated-WEHI-3 for 48 h. DNA ladder or fragmentation indicated apoptosis event in the cells. (E) Flow cytometry analysis of WEHI-3 cells after 48 h RBDS-treatment and annexin-V staining. Percent cell count of apoptosis detection was demonstrated from the average of two experiments.
Fig 2.
Effects of RBDS and gramisterol on the cell cycle.
(A) Representative flow cytometry analysis of DNA contents (PI staining) in WEHI-3 cells after 24 h treatment with RBDS (0, 50 and 100 μg/mL) and gramisterol (0, 1 and 2 μM). (B) Bar graphs and histograms show DNA distribution in each phase of cell cycle. The experiments were performed in duplicate and data are presented as average.
Fig 3.
Effects of RBDS and gramisterol on cell cycle and apoptosis proteins.
(A) Representative immunoblotting assay of WEHI-3 cells treated with RBDS (0, 50, 100 and 200 μg/mL) and gramisterol (0, 1, 2 and 3 μM) for 24 h. The experiments were performed in triplicate. (B) The protein bands density were measured semi-quantitatively and analyzed using ImageJ software. Data are expressed as mean±SEM (n = 3).
Fig 4.
Effects of RBDS treatment on the body weight and percent survival.
(A) Body weight of leukemic mice treated with RBDS (WEHI-3/RBDS 3 mg/kg and WEHI-3/RBDS 9 mg/kg) compared with normal mice and untreated-leukemic mice (WEHI-3). (B) Percent survival of WEHI-3/RBDS 3 mg/kg and WEHI-3/RBDS 9 mg/kg mice were significantly higher than the untreated-WEHI-3. Data are presented as mean±SEM (n = 10). **p< 0.01, ***p< 0.001 were considered significant differences compared to WEHI-3.
Fig 5.
Pathological changes in spleen and liver.
(A) Representative photograph of mice spleen and liver in situ. (B) Organ index was calculated from organ weight (g)/body weight (g) × 100. Data are expressed as mean±SEM. (n = 10). (C) Histopathological features of mice spleen and liver stained with Hematoxylin & Eosin (original magnification 20X; insets 40X). In spleen section: black arrows indicate invasion of leukemic cells into white pulps forming tumor necrotic foci. Black and white arrowheads indicate leukemic cells and lymphocytes, respectively. Abbreviations: Wp = white pulp, Rp = red pulp, C = capsule. In liver section: black and white arrowheads indicate leukemic cells and hepatocytes, respectively.
Fig 6.
Effect of RBDS on normal immune cells population.
The relative amount of normal immune cells in the peripheral blood assessed from the levels of surface markers: CD3+ (for T cells), CD19+ (for B cells), CD11b+ (for monocytes and neutrophils) and Mac3+ (for macrophages) in RBDS-treated and untreated leukemic mice. Data are expressed as mean±SEM (n = 10).
Fig 7.
The serum cytokines released from the immune function-related cells of leukemic mice.
The induced-leukemic mice were treated with or without RBDS supplementation. Serum cytokines were measured individually from each mouse (four mice per group) by ELISA. Data are expressed as the mean±SEM.
Fig 8.
Effect of RBDS containing gramisterol on immune cell population and function.
(A) The percentage of peritoneal immune cells assessed from the surface markers level (CD11b+) in RBDS-treated and untreated leukemic mice. The experiment was performed in triplicate. Data are expressed as mean±SEM (n = 10). (B) Flow cytometry analysis of peritoneal macrophages phagocytic activity as measured in pHrodo Red positive cells. Data are expressed as mean±SEM (n = 10). (C) Immunofluorescence intensity illustrated by pHrodo Red positive cells represented the phagocytotic activity of the cells.
Fig 9.
Pathway analysis of gramisterol effect on immune cells.
Western blot analysis of pSTAT1 level in spleen cells (lanes 1–4) and peritoneal cells (lanes 5–8) after treatments with i) gramisterol, ii) culture supernatant containing cytokines released from RBDS-treated leukemic mice spleen cells plus a minor supplementation of cytokine IFN-γ, iii) the combination of both gramisterol and the cytokines. β-actin was used as an internal control. The experiment was performed in triplicate. The relative expression of pSTAT1 is shown as mean±SEM.
Fig 10.
Gramisterol and IFN-γ inhibited STAT3 phosphorylation in tumor cells.
Western blot analysis of pSTAT3 signaling activation in WEHI-3 cells after treatment with i) gramisterol, ii) culture supernatant containing cytokines released from RBDS-treated leukemic mice spleen cells plus a minor supplementation of cytokine IFN-γ, iii) the combination of both gramisterol and the cytokines. β-actin was used as an internal control. The experiment was performed in triplicate. The relative expression of pSTAT3 was shown as mean±SEM.