Human RCC 786-0 cells were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium containing penicillin (50 U/ml), streptomycin (50 U/ml) and 10% FBS according to the ATCC procedures. Media came from ATCC. Supplements and FBS were obtained from Gibco (Grand Island, NY, USA). Honokiol 98% (HonoPure^®) was provided by EcoNugenics, Inc. (Santa Rosa, CA, USA) and dissolved in DMSO at a concentration of 80 mM then stored at −20°C. Rho-kinase inhibitor Y-27632 was purchased from Calbiochem (Darmstadt, Germany). Rhodamine phalloidin was purchased from Molecular Probes (Grand Island, NY, USA). Methanol-free formaldehyde solution 16% was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). DMSO and other reagents were purchased from Sigma (St. Louis, MO, USA). Anti-RhoA, anti-phospho-RhoA and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-MLC2 and anti-phospho-MLC2 antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Human RCC A-498 and 786-0 cells were treated with indicated concentrations of honokiol for 24 h and cell proliferation was determined as described (21). Cell viability was determined after incubation with honokiol for 24 h by staining with trypan blue as described (22). Data are the mean ± SD from three independent experiments.

Cell migration of 786-0 cells treated with honokiol (0–40 μM) or honokiol (20 μM) + Y-27632 (10 μM) or Y-27632 (10 μM) was performed in Transwell chambers according to established method (23). Briefly, 786-0 cells (0.2×10⁶) suspended in serum-free medium were added to the upper chamber of an insert, and the insert was placed in a 24-well plate containing medium with 10% FBS. Migration assays were carried out for 3 h. Data points represent the mean ± SD of three individual filters within one representative experiment repeated at least twice.

The Rho Activation Assay Kit (EMD Millipore, Billerica, MA, USA) was used to determine whether honokiol could modulate RhoA activity in 786-0 cells according to the manufacturer’s instructions. In brief, cells were exposed to vehicle or honokiol (20 μM) for 30 min, rinsed in ice-cold TBS and lysed in the lysis buffer provided. For Rho pull-down assay, cell lysates were incubated with glutathione-agarose beads bounding to a GST-tagged Rho binding domain of Rhotekin. The precipitated GTP-bound forms of proteins were analyzed by western blot analysis with antibody specific for RhoA. Activated RhoA was normalized to the total RhoA. 786-0 cell extract loaded with GTPγS was used as a positive control.

786-0 cells were treated with vehicle or honokiol (20 μM) for 30, 60 or 120 min, respectively. Protein extracts isolated from cells were prepared and western blot analysis with anti-phospho-RhoA, anti-RhoA, anti-phospho-MLC2, anti-MLC2 or anti-β-actin antibodies was performed as previously described (21). Western blots were quantified with HP-Scanjet 550c and analyzed by UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT, USA).

786-0 cells (6.0×10⁴) were plated on the Millicell EZ Slide 4-well glass slide (EMD Millipore, Darmstadt, Germany) and incubated for 24 h. After exposure to honokiol (20 μM) or honokiol (20 μM) + Y-27632 (10 μM) or Y-27632 (10 μM) for 1 h, cells were washed in PBS and fixed with 4% formaldehyde for 15 min. The cells were then permeabilized with PBS containing 0.1% Triton X-100 for 5 min and stained with rhodamine phalloidin for 20 min. Nucleus was stained with DAPI for 2 min and further washed with PBS. Detection of actin stress fibers was achieved using an inverted microscope (Leica DMR type 020-525-024 fluorescence microscope; Leica Microsystems GmbH, Wetzlar, Germany) and a confocal microscope (Bio-Rad Radiance 2100 laser scanning system; Bio-Rad, Hercules, CA, USA).

All statistical analysis was performed using SigmaPlot 11.2.0 (Systat Software, Inc., San Jose, CA, USA). Data are presented as the mean ± SD. Statistical comparisons were carried out using ANOVA with the significance level adjusted using the repeated t-tests with Bonferroni correction. P<0.05 was considered to be significant.

As honokiol exhibits anticancer effects in different cancer types (6,24–28), its effect on the growth of human RCC was evaluated in this study. A-498 and 786-0 cells were treated with honokiol (0–80 μM) for 24 h and proliferation was determined as described in ‘Materials and methods’. We found that honokiol suppressed the proliferation of A-498 (IC₅₀, 37.17 μM) and 786-0 cells (IC₅₀, 51.28 μM) dose-dependently (Fig. 1). Moreover, honokiol does not affect the viability of A-498 and 786-0 cells after treatment of 24 h (Fig. 1), suggesting cytostatic effect of honokiol on human RCC.

Comparing with A-498, the 786-0 cells exhibit higher expression of LIM and SH3 protein 1 (LASP-1), which correlated with aggressive phenotype and poor prognosis in RCC (20). Thus, the more aggressive cell line 786-0 was selected to investigate whether honokiol inhibits cell migration under the incubation time (3 h) and concentrations (0–40 μM) that do not affect viability of 786-0 cells. As shown in Fig. 2, honokiol significantly inhibits cell migration in a dose-dependent manner. Taken together, our data indicate that honokiol not only inhibits proliferation of human RCC but also suppresses migration, an initial important step in cancer metastasis (29).

To investigate the effect of honokiol and its possible mechanisms of action with regard to human RCC, we focused on the Rho GTPases, which play key roles in coordinating the cellular responses required for cell migration (30). Rho pull-down assay showed that exposure of 786-0 cells to honokiol (20 μM) resulted in a strong activation of RhoA (Fig. 3A). GTPγS, a hydrolysis-resistant GTP analog, was used as positive control (Fig. 3A). Because activity of RhoA can also be negatively regulated by its phosphorylation at Ser188 (14), the phosphorylation status of RhoA with honokiol treatment was determined in our study. Honokiol (20 μM) suppressed the phosphorylation level of RhoA after 60 min without changing the level of total RhoA (Fig. 3B). Moreover, GTPase activation coincided with phosphorylation of MLC2, detected by western blot analysis with antibodies specific for phosphorylated Thr18 and Ser19 (Fig. 4). Thus, we considered that RhoA/ROCK/MLC signaling was activated by honokiol in 786-0 cells.

Stress fibers, which look like bundles of actin filaments, are an actin-myosin-based contractile system regulated by the RhoA/ROCK/MLC signaling (31). Excessive formation of actin stress fibers (rhodamine phalloidin-positive staining of actin fibers) was identified in 786-0 cells treated with honokiol (20 μM) compared with vehicle control (Fig. 5A, B, E and F). Interestingly, this phenomenon disappeared when cells were treated with the ROCK inhibitor Y-27632 (10 μM) and honokiol (Fig. 5C and G). This inhibition can also be identified in 786-0 cells treated with Y-27632 only (Fig. 5D and H).

To determine whether the inhibition of cell migration by honokiol is mediated by the activation of RhoA/ROCK/MLC signaling in 786-0 cells, we pre-treated 786-0 cells with Y-27632 for 60 min and then determined cell migration with honokiol as described in ‘Materials and methods’. In accordance with the change in actin stress fibers, the effect of honokiol on migration of 786-0 cells was significantly abrogated by Y-27632 (Fig. 6A). These results further proved our hypothesis that Honokiol-induced RhoA/ROCK/MLC activation plays an integral role in honokiol-mediated inhibition of migration potential in RCC (Fig. 6B).