In Vitro Suppression of Growth of Murine WEHI-3 Leukemia Cells and in Vivo Promotion of Phagocytosis in a Leukemia Mice Model by Indole-3-carbinol

Abstract

Indole-3-carbinol (I3C), a potential anticancer substance, can be found in cruciferous (cabbage family) vegetables, mainly cauliflower and Chinese cabbage. However, the bioactivity of I3C on the apoptotic effects of murine leukemia WEHI-3 cells and promotion of immune responses in leukemia mice model are unclear. In this study, we investigated the effect of I3C on cell-cycle arrest and apoptosis in vitro and immunomodulation in vivo. I3C decreased the viable WEHI-3 cells and caused morphological changes in a concentration- and time-dependent manner. I3C also led to G0/G1 phase arrest, decreased the levels of cyclin A, cyclin D, and CDK2, and increased the level of p21(WAF1/CIP1). Flow cytometric analyses further proved that I3C promoted ROS and intracellular Ca(2+) production and decreased the levels of ΔΨ(m) in WEHI-3 cells. Cells after exposure to I3C for 24 h showed DNA fragmentation and chromatin condensation. Comet assay also indicated that I3C induced DNA damage in examined cells. I3C increased the levels of cytochrome c, FADD, GADD153, GRP78, and caspase-12 as well as induced activities of caspase-3, -8, and -9. Moreover, I3C attenuated NF-κB DNA binding activity in I3C-treated WEHI-3 cells as shown by EMSA and Western blotting analyses. In the in vivo study, we examined the effects of I3C on WEHI-3 leukemia mice. Results showed that I3C increased the level of T cells and decreased the level of macrophages. I3C also reduced the weights of liver and spleen, and it promoted phagocytosis by macrophages as compared to the nontreated leukemia mice group. On the basis of our results, I3C affects murine leukemia WEHI-3 cells both in vitro and in vivo.

Laminarin Promotes Immune Responses and Reduces Lactate Dehydrogenase but Increases Glutamic Pyruvic Transaminase in Normal Mice In Vivo

“…Animals were kept with 12 h light/dark cycles at 25˚C in the animal center of China Medical University (Taichung, Taiwan). Before performing experiments, the animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of China Medical University (approval ID: 104-11-B) and were cared for following the institutional animal ethical guidelines of the China Medical University as described previously (10). Thirty-six mice were randomly separated into four groups of nine mice.…”

“…Laminarin was administered orally every 2 days for 14 days (total of seven times) via a steel gavage tube and the body weight was recorded. After completion of treatment, mice from each group were weighed and sacrificed as described previously (10).…”

“…After treatment, all mice were individually weighed and blood samples, liver and spleen were individually collected. Spleens were used for isolating splenocytes and for measuring NK cell activity as described previously (10). Blood from each mouse (1 ml) was lysed to destroy the red blood cells with 1x Pharm Lyse™ lysing buffer (BD Biosciences Pharmingen Inc., San Diego, CA, USA) as per guidelines from BD Biosciences.…”

“…Leukocytes from each mouse were collected and stained with phycoerythrin (PE)-labeled anti-mouse CD3 and CD19, fluorescein isothiocyanate (FITC)-labeled anti-mouse CD11b and Mac-3 antibodies (BD Biosciences Pharmingen Inc.) for 30 min. All samples were washed with phosphate-buffered saline (PBS) and were analyzed by flow cytometry to measure the percentage of cell marker (population) as previously described (10).…”

“…Based on the PHAGOTEST® kit manufacturer's instructions (Orpegen Pharma, Heidelberg, Germany), all isolated macrophages were placed in plates containing 10 μl of target Escherichia coli-FITC and were individually mixed and then were analyzed for phagocytosis using flow cytometery. Quantification of phagocytosis was performed by CellQuest software as described previously (10).…”