Figure 5

Honokiol increases LKB1 expression, LKB1:STRAD interaction, cytosolic translocation, and depletion of LKB1 abrogates honokiol-mediated modulation of AMPK, inhibition of migration, and invasion of breast cancer cells. (a) MCF7 and MDA-MB-231 cells were treated with 2.5 μM honokiol for indicated time intervals. U, untreated cell. Total protein was isolated, and equal amounts of proteins were resolved with SDS-PAGE and subjected to immunoblot analysis by using specific antibodies for LKB1. The membranes were reblotted by using actin antibody as control. The blots are representative of multiple independent experiments. The histogram is the mean of densitometric analysis showing relative density units (RDUs) of the Western blot signals for LKB1 normalized to actin in three independent experiments. *P < 0.005, compared with untreated controls. (b) MCF7 cells were treated with 2.5 μM honokiol or untreated and subjected to immunoprecipitation assay by using IgG or LKB1 antibodies, as indicated. Immunoprecipitates were analyzed by using anti-STRAD antibodies. The histogram is the mean of densitometric analysis showing relative density units (RDUs) of the Western blot signals for STRAD in three independent experiments. *P < 0.005, compared with untreated controls. (c) MCF7 and MDA-MB-231 cells were treated with honokiol (HNK), and LKB1 protein was analyzed with immunofluorescence by using LKB1 antibody; 4'6-diamidino-2-phenylindole staining was used to determine the nuclear localization. These results are representative of multiple independent experiments. (d) LKB1 was depleted in MCF7 and MDA-MB-231 cells by using two different lentiviral LKB1 short-hairpin RNA (shRNA1 and shRNA2) constructs and a negative control construct that was created in the same vector system (pLKO.1). Stable pools of LKB1-depleted (LKB1^shRNA) and vector control (pLKO.1) cells were used for total protein isolation, and equal amounts of proteins were subjected to immunoblot analysis by using specific antibodies for LKB1. Actin was used as control. (e) MDA-MB-231-LKB1^shRNA (LKB1-sh1 and LKB1-sh2) and MDA-MB-231-pLKO.1 (pLKO.1) cells were treated with honokiol (HNK, 2.5 μM), and phosphorylation of AMPK was analyzed with Western blot analysis. Total AMPK antibody was used as control. (f) MDA-MB-231-LKB1^shRNA (LKB1-sh1 and LKB1-sh2) and MDA-MB-231-pLKO.1 (pLKO.1) cells were grown to confluence, scratched with a pipette tip, and photographed immediately after scratching (0 hours). Culture media were replaced with media containing honokiol (HNK, 2.5 μM) or untreated media (U). The plates were photographed at the identical location of the initial image (0 hours) at 24 hours. The results shown are representative of three independent experiments performed in triplicate. (g) MDA-MB-231-LKB1^shRNA (LKB1-sh1 and LKB1-sh2) and MDA-MB-231-pLKO.1 (pLKO.1) cells were cultured in Matrigel invasion chambers followed by treatment with honokiol (HNK, 2.5 μM) for 24 hours. The number of cells that invaded through the Matrigel was counted in five different regions. The slides were blinded to remove counting bias. The result shows the mean of three independent experiments performed in triplicate. *P < 0.005, compared with untreated controls.