To evaluate the therapeutic potential of honokiol, three human lung cancer cell lines H460, A549, and H358 cells were cultured with an increasing concentration of honokiol for 72 h, and then cell viability was determined by MTT assay. Honokiol inhibited the growth of H460, A549 and H358 cells in a dose-dependent manner (Figure 1B), with 50% inhibition concentration (IC₅₀) at 72 h of 30.42 ± 8.47, 50.58 ± 4.93, and 59.38 ± 6.75 μM, respectively, but it showed low toxicity to two normal lung cells (CCD19-Lu and BEAS-2B) (Figure 1A). Subsequently, we examined the effect of honokiol on cell colony formation, in accordance with the cell cytotoxicity, honokiol significantly inhibited the colony formation capacity in a dose-dependent fashion in KRAS mutated cell lines (Figure 1B).

To investigate whether the induction of apoptosis also contributed to honokiol-mediated growth inhibition of KRAS mutated cells, we used Annexin V-FITC/PI flow cytometry to analyze the population of apoptotic cells. Results showed that honokiol-induced apoptosis in the three KRAS mutant cell lines in a concentration dependent manner (Figure 2A). To further demonstrate the mechanism by which honokiol induced apoptosis in these KRAS mutant cell lines, western blotting assay was performed to evaluate the expression of several well-characterized apoptotic proteins. As shown in Figure 2B, honokiol increased the expression of pro-apoptotic protein Bax, while decreased the expression of anti-apoptotic protein Bcl-2 in these KRAS mutation cell lines. In addition, PARP cleavage in honokiol treated cells, further confirmed that honokiol induced apoptosis in KRAS mutated cells.

Because growth factor-mediated activation of KRAS is known to activate the RAF/MEK/ERK and RAF/PI3K/AKT pathway (Castellano et al., 2013; Samatar and Poulikakos, 2014), we next examined the effect of honokiol on RAS mediated signaling transduction in KRAS mutated cells, such as the phosphorylation status of c-RAF, AKT, and ERK. The results showed that treatment with honokiol led to a reduction in c-RAF, ERK, and AKT phosphorylation in three KRAS mutated lung cancer cell lines (Figure 3). Our data indicated that honokiol inhibited KRAS mutated cell line growth and survival via regulating KRAS downstream signaling pathways.

We next evaluated the cell cycle profile of KRAS mutant cells treated with honokiol. Cells (H460, A549, and H358) were treated with or without 60 μM honokiol for 24 h and cell cycle analysis was performed using flow cytometry. Results showed that treatment with honokiol led to cell cycle arrest at the G0/G1 phase in KRAS mutated cell lines (Figure 4A). Furthermore, we determined the effect of honokiol on cycle-related protein levels of G0/G1 phase by immunoblotting to gain insights into the mechanism of honokiol-induced cell cycle arrest in KRAS mutated cell lines. As shown in Figure 4B, cyclin-dependent kinase inhibitors p21 and p27 exhibited a promotion in the honokiol-treated three KRAS mutated cell lines with a marked reduction of cyclin D1. These results suggested that G1 arrest induced by honokiol might be attributed to the effect on p21, p27 as well as cyclin D1 and resulted in the inhibition of cell proliferation.

Stiudies reported that honokiol can trigger autophagy (Cheng et al., 2016; Yeh et al., 2016). In order to determine whether honokiol could induce autophagy in KRAS mutated cell lines, we assessed one of the key hallmarks of autophagy: the conversion of soluble LC3-I to lipid bound LC3-II (Tanida et al., 2004). As illustrated in Figure 5A, treatment with honokiol distinctly increased the conversion of LC3-I to LC3-II of KRAS mutated cells in a concentration-dependent manner, and this accumulation of LC3-II induced by honokiol was relieved in the presence of autophagy inhibitor 3-methyladenine (3-MA), a class III PI3K inhibitor (Figure 5B). AMPK is a sensor of cellular energy status and it is activated under high intracellular AMP conditions, thereby induces autophagy via the AMPK-mTOR dependent pathway (Mihaylova and Shaw, 2011). To investigate whether the effects of honokiol on KRAS mutated cells relied on this signaling pathway, we examined the activity of honokiol on the representative signaling cascades. The results showed that treatment of cells with honokiol suppressed mTOR phosphorylation, leading to inhibition of P70S6K kinase activity, which signals cell survival and growth in transduction pathway (Asnaghi et al., 2004), with concomitant up-regulation of phospho-AMPK (Figure 5C). Taken together, our results suggested that honokiol induced autophagy in KRAS mutated cells was mediated through the AMPK-mTOR signaling pathway.

Although many anti-cancer agents can activate autophagy in different types of cancers, it remains controversial whether autophagy promotes cell death or acts as a pro-survival mechanism (Hippert et al., 2006). To test whether autophagy is required for honokiol-mediated growth inhibition in KRAS mutated cells, we evaluated the impact of autophagy on honokiol-mediated cytotoxicity by inhibiting autophagy with 3-MA. As shown in Figure 6, compared with use of honokiol alone, co-treatment with autophagy inhibitor 3-MA led to the decreased apoptosis in KRAS mutant cells, suggesting that the honokiol-induced autophagy contributed to its antitumor activity.

Previous results have indicated that Sirt3 acts as a tumor suppressor against lung adenocarcinoma tumor tumorigenesis by maintaining mitochondrial integrity and efficient oxidative metabolism (Xiao et al., 2013; Chen Y. et al., 2014). Our data further confirmed Sirt3 mRNA levels were significantly downregulated in KRAS mutated cell lines compared with normal lung epithelial cells (Figure 7A). Similarly, a marked downregulation of Sirt3 protein levels was observed in KRAS mutated cell lines (Figure 7C). Recently, researchers discovered that honokiol might be a pharmacological activator of Sirt3 (Pillai et al., 2015), so we wondered whether honokiol can regulate Sirt3 levels in KRAS mutated cell lines. Sirt3 could mediate metabolic reprogramming in human breast cancer cells by destabilizing Hif-1α (Bell et al., 2011), The results showed that treatment of cells with honokiol increased both mRNA and protein levels of Sirt3 (Figure 7B), accompanied by a reduction of Hif-1a expression (Figure 7D), which can be destabilized by Sirt3 mediated metabolic reprogramming in human breast cancer cells (Bell et al., 2011). Collectively, these observations indicated that the anti-cancer effect of honokiol is mediated by activating Sirt3 expression.

H460, A549, H358, H2122, BEAS-2B, NIH3T3, CCD19-Lu cells were obtained from the American Type Culture Collection and cultured in an environment of 5% CO₂ at 37°C in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), CCD19-Lu cells were grown in MEM medium supplemented with 10% FBS, BEAS-2B cells were maintained in BEBM containing 0.01 mg/ml fibronectin, 0.03 mg/ml bovine collagen type I and 0.01 mg/ml bovine serum albumin. All cells were added 100 units/ml penicillin, and 100 μg/ml streptomycin. Honokiol was purchased from Selleck Chemicals. Antibodies to GAPDH, C-RAF, p-C-RAF, p-AKT (Ser473), p-ERK (Thr202/Thy204), P21, P27, Cyclin D1, Sirt3, Hif-1α, and ERK were purchased from cell signaling technology. Anti-AKT, KRAS antibody were acquired from Santa Cruz Biotechnology.

Cell viability was assayed using 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT). Briefly, KRAS mutant cells (H460, A549, and H358) were seeded in a 96-well plate at a density of 3 × 10³ cells/well for 24 h, respectively, and were cultured overnight for cell adhesion, then treated with different concentrations honokiol (0–80 μM) for another 72 h. At the end of treatment, 10 μl MTT (5 mg/ml) solution was added to each well and further incubated at 37°C for 4 h, then 100 μl of the resolved solution (10% SDS and 0.1 mm HCL) was added to each well to dissolve the MTT formazan crystals. Absorbance was measured at a wavelength of 570nm with BioRad microplate reader (FluoDia T70, Photon Technology International, Lawrenceville, NJ, USA), the cell viability is calculated as the percentages change of the absorbance of treated cells divided by the absorbance of untreated cells. The IC₅₀ value for the compound was determined by GraphPad Prim5.0 software.

Briefly, KRAS mutant cells (H460, A549, and H358) were seeded in 6-well plates (500 cells/well), after attachment overnight, cells were exposed to various concentration of honokiol with medium changes every 3 days until visible colonies formed. The colonies were washed with cold PBS, then fixed in 4% paraformaldehyde (PFA) for 15 min, and stained with 0.5% crystal violet (1% PFA, 0.5% crystal violet, and 20% methanol in ddH2O) for 20 min. The colonies were photographed.

KRAS mutant cells (H460, A549, and H358) were treated with various concentrations of honokiol (0, 10, 20, 40, and 60 μM) for 48 h. Cells were washed with PBS and resuspended in 1× binding buffer (100 μl). One microliter of annexin V (AV)-fluorescein isothiocyanate solution (2.5 μg/ml) and 1 μl of dissolved propidium iodide (PI) (50 μg/ml) were added to the cell suspensions vortexed and incubated at room temperature in the dark for 15 min. 400 μl of chilled 1× binding buffer was added and mixed gently prior to the examination of the cell preparations by flow cytometry (FACSCalibur, BD Biosciences), a minimum of 10,000 events were collected and analyzed, and data were analyzed with the Flow J software.

Briefly, KRAS mutant cells (H460, A549, and H358) were seeded on a 6-well plate at 1 × 10⁵ cells/well for 24 h, respectively, after treatment with 60 μM honokiol for another 24 h. The cells collected and washed with PBS, followed by fixation with 70% (v/v) ethanol at –20°C overnight. Thereafter, cells were washed and resuspended in 500 μl PBS containing 50 μg/ml PI and 1 mg/ml RNaseA for 30 min at room temperature in the dark. In total, 10,000 events were analyzed immediately in each sample subjected to flow cytometer (FACSCalibur, BD Biosciences), and the percentages of cells in G1, S and G2/M phases were calculated.

Whole-cell lysates were rinsed with ice-cold PBS and lysed in RIPA Lysis buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1 mM EDTA, 0.25% sodium deoxycholate, 1% NP-40,) containing protease (Roche) and phosphatase (Roche) inhibitors. Lysates were centrifuged at 14,100 × g for 10 min at 4°C. Thirty microgram of total protein from each sample was separated on a 10-12% polyacrylamide gel, and then transferred to a nitrocellulose membrane (Millipore). Immunoblots were blocked with 5% skim milk in TBS/Tween 20 (0.05%, v/v) for 1 h at RT, followed by overnight incubation at 4°C with primary anti-bodies. After washing three times with TBST, the membranes were incubated with secondary rabbit or mouse fluorescent antibodies for 1 h in the dark, then the signal intensity of the membranes was scanned on a LI-COR Odessy imaging system (Belfast). All primary antibodies were diluted in 1:1000, while their recommended secondary antibodies were diluted in 1:10000.

Total RNA was extracted from the cultured cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA (1 μg) was converted into cDNA using the PrimeScript^TM RT reagent kit with gDNA Eraser (Takara) according to the manufacturer’s instructions. RT-PCR was performed using SYBR^® Premix Ex Taq^TM II (Takara) in the ABI PRISM^® 7300 real-time PCR system (Applied Biosystems). The 2^-ΔΔCt method was used to calculate relative expression level.

GraphPad Prism 5.0 software was used to analysis data statistic. The results were presented as the mean ± standard deviation (SD). Statistical analysis was carried out using Student’s t-test or one-way analysis of variance. P-values < 0.05 were regarded statistically significant.