Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization

In eukaryotic cells, microtubule is the main component of the cellular cytoskeleton, plays important roles in cell shape maintenance, intracellular transport, cell motility, meiosis, and mitosis [1, 2]. Specifically, microtubule is required for the process of mitosis in orchestrating the separation and segregation of chromosomes, which suggests that microtubule is a rational target for the development of anticancer drugs [3]. Microtubule-targeted drugs disrupt the dynamics of microtubule and induce mitosis arrest of tumor cells. These drugs are commonly classified into two major groups. The first group drugs inhibit microtubule polymerization, such as colchicine and vinorelbine, while the second group drugs stimulate microtubule depolymerization, such as paclitaxel and docetaxel [4].

Lung cancer is the most common malignant tumor and the leading cause of cancer-related death in the world [5, 6]. More than 1.5 million new cases of lung cancer are diagnosed every year, approximately 80% of which are non-small cell lung cancer (NSCLC) [7-9]. The morbidity is rapidly increasing mainly due to environmental pollution and unhealthy lifestyles (smoking, occupational exposure, diet, et al). Therefore, there is an urgent need to identify biomarkers and drug targets for early diagnosis and treatment for NSCLC [10]. Currently, chemotherapy plays a crucial role in comprehensive NSCLC therapy. Cisplatin-based drugs are the most widely used chemotherapy medicine for NSCLC [11, 12]. Additionally, some chemo-drugs targeting at microtubule including docetaxel, cabazitaxel, epothilones and vinorelbine have been used for NSCLC treatment [13]. These agents have demonstrated overall survival benefit clinically [3]. Nevertheless, drug resistance often appears after prolonged treatment [14-16]. Therefore, development of novel generation of anti-microtubule candidates is a promising and urgent task for NSCLC treatment.

Magnolia officinalis belongs to Magnoliaceae family and distributes over China, Japan, and South Korea. Magnolia officinalis is used as a folk remedy for gastrointestinal disorders, cough, acute pain, anxiety, and allergic diseases. Magnolol (5,5′-diallyl-2,2′-dihydroxybiphenyl, Fig. 1A, MW: 308.3495), a hydroxylated biphenyl compound isolated from the root and stem bark of Magnolia officinalis, was shown to have muscle relaxant, anti-oxidative, anti-atherosclerosis, anti-inflammatory, and anti-microbial effects [17-22]. Recent studies have shown that Magnolol exhibits anti-cancer properties by inhibiting proliferation, inducing differentiation and apoptosis, suppressing angiogenesis, countering metastasis, and reversing multidrug resistance [23-28]. However, the function and mechanism underlying the antitumor activity of Magnolol against lung cancer remain unknown. In this study, we investigated the efficacy of Magnolol against human NSCLC cells both in vitro and in vivo. Our results indicated that Magnolol induced G2/M phase cell cycle arrest through inhibiting microtubule polymerization. Further study demonstrated that Magnolol treatment led to inhibition of proliferation, apoptosis activation and autophagy induction of NSCLC cells. Importantly, Magnolol suppressed xenograft of A549 tumor growth in vivo. All these results suggest that Magnolol is a promising candidate for NSCLC treatment.

Human NSCLC cell lines PGCL3, SK-MES-1, NCI-H460, A549, NCI-1299 and immortalized human normal bronchial epithelium cell line 16HBE were purchased from ATCC [29]. A549 cells were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific). PGCL3, SK-MES-1, NCI-H460, NCI-1299 and 16HBE cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific). Both mediums were supplemented with 10% fetal bovine serum (Wisent). All cells were incubated at 37°C with 5% humidified CO₂. Mice were obtained from National Rodent Laboratory Animal Resources, Shanghai Branch of China. Cdc 2, p-Cdc 2, Cdc 25c, Cyclin B1 and actin antibodies were purchased from Cell Signaling Technology, and phospho-Ser/Thr-Pro MPM-2 antibody were obtained from Millipore. Magnolol with a purity of up to 98% was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Magnolol was dissolved in DMSO (Sigma) at a stock solution of 100 mM and stored at –20°C.

Cell cycle analysis was performed according to previous report [30]. Cells were treated with four different doses (0, 5, 10, 20 μM) of Magnolol for 24 hrs. After washed with PBS and digested with trypsin, adherent and floating cells were collected, washed once with PBS, and then fixed in cold 70% ethanol overnight in 4°C. After ethanol fixation, cells were washed in PBS once and suspended in PBS with 200 μg/mL RNAase and 50μg/mL propidium iodide (PI) in the dark for 30 minutes. Then cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences).

Immunofluorescence staining assay was performed as previously described [31] with minor modification. Briefly, Then cells treated were incubated with 4% paraformaldehyde for 20 min, washed with PBS, and treated with 0.5% Triton-X 100, washed with PBS. After blocking in 0.5% BSA, cells were incubated with primary antibody overnight at 4oC before further incubation with secondary antibody at 37°C in the dark. Images were taken by confocal microscopy (Leica).

Cells were seeded on gelatin-coated coverslips. After fixed with 4% paraformaldehyde for 20 min at room temperature, cells were permeabilized with 0.2% Triton-X 100 in PBS for 5 min. The nuclei were stained with DAPI. After washed with PBS, cells were visualized and photographed with Leica microscopy. Five random fields were counted and analyzed. Mitotic index was calculated by dividing the total number of examined cells by the number of cells in mitosis.

Cell proliferation was assessed by Sulforhodamine B (SRB) assay. Briefly, 4000 cells per well were seeded in 96-well plates. After 24 hrs, cells were exposed to five different dose (0, 1, 5, 10, 20 μM) of Magnolol for 72 hrs. Cells were fixed with 10% trichloroacetic acid for 1 hr at 4 oC, washed five times with flowing water, and then air-dried. Cells were stained with 50 µL 0.4% (w/v) SRB for 20 min at room temperature, washed five times with 1% acetic acid, and then air-dried. 100 µL 10 mM Tris was added per well, and absorbance was measured at 515 nm.

Cells were seeded in 6-well dishes. Then cells were allowed to attach overnight and then exposed to different doses of Magnolol for a week. After fixed with 4% paraformaldehyde for 20 min room temperature, cells were stained with 0.2% crystal violet. The number of cell colonies was calculated and analyzed as the ratio of the number of treated samples to untreated samples. Triplicate wells were set up for each dose.

Cells were exposed to drug treatment for indicated time and lysed in RIPA buffer as detailed in previous report [32]. Lysate was mixed with sample loading buffer and heated at 100oC for 10 min. After separated by SDS-polyacrylamide gel electrophoresis (PAGE), extracted protein were transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk in PBS and 0.1% Tween-20 and then incubated with specific primary antibodies overnight at 4oC. Then membranes were exposed to secondary antibodies for 2 hrs at room temperature.

Apoptosis analysis was carried out by flow cytometry (FACSCalibur; BD Biosciences). Cells were treated with different doses of Magnolol for 48 hrs or with 10 µM Magnolol for different times. The procedure was performed as previous report [8]. Cells were washed with PBS, then harvested by 0.25% trypsin and washed with PBS again. After that, 5µL Annexin V fluorescein isothiocyanate and 5 µL propidium iodide were added and the mixture was kept in the dark for 15 min at room temperature, then 400 µL of binding buffer was added and analyzed immediately with flow cytometry (FACSCalibur; BD Biosciences).

Viability assay was performed using the live/dead viability/cytotoxicity kit (Molecular Probes). This kit contains calcein-AM to stain the living cells (green) and ethidium homodimer-1(EthD-1) to stain the dead cells (red). Briefly, cells were exposed to Magnolol treatment for 48 hrs, then 2 µM calcein-AM and 4 µM EthD-1 working solution was added. After incubated at room temperature for 30 min, the green living cells and the red dead cells were visualized by fluorescence microscopy and photographed. Cells from three random areas per sample were counted for statistical analysis.

In vitro microtubule polymerization assay was performed according to previous reports with modification [33, 34]. A549 and NCI-1299 cells were seeded on 6-well plates. Then cells were exposed to treatment of Magnolol, 50 nM colchicine and 50 nM paclitaxel for 24 hrs, respectively. The cells were washed with PBS for three times before adding lysis buffer containing 20 mM Tris-HCL (PH6.8), 1 mM MgCl₂, 2 mM EGTA and Protease inhibitor and phosphatase inhibitor and 0.5% Nonidet. Supernatants were collected after centrifugation at 14000 rpm for 10 min. Supernatant and the pellet were dissolved in SDS-PAGE sampling loading buffer at 95oC for 10 min. Supernatant containing soluble tubulin and the pellet (the polymerized tubulin lysates) were subjected to 10% SDS-PAGE before Western blotting.

A549 cells (2×10⁶) were implanted subcutaneously on the right side of the dorsal area of 4-week-old male nude mice. 10 days later, mice were divided into two groups (n=8) randomly as reported [35]. Magnolol (25 mg/kg) was injected intraperitoneally every other day. Control group was treated with DMSO. Twenty days later, mice were sacrificed, tumors were removed and images were taken. The growth rate of the tumor xenograft was evaluated by determining the tumor volume using digital caliper everyday. Tumor growth rate was measured as the following equation, volume=length×width2×0.52. Mice were continually observed and were treated consecutively for 20 days with the measurement of body weight until they were sacrificed.

All values are given as means±standard error of the mean (s.e.m.) or standard deviation (s.d.) as indicated. Comparisons between two groups were made either by Student’s t-test or Mann–Whitney U-Test; for more than two groups’ one way analysis of variance (ANOVA) with Tukey’s multiple comparison test or Kruskal–Wallis test with Dunn’s multiple comparison test was performed. Tumor growth was analyzed with two-way analysis of variance and Bonferroni multiple comparison testing. For the Kaplan–Meier analysis a log-rank test was performed. P<0.05 was accepted as statistically significant. For all graphs: *P<0.05; **P<0.01; ***P<0.001.

Firstly we performed SRB assay to examine the anti-proliferative effect of Magnolol against NSCLC cells (PGCL3, SK-MES-1, NCI-H460, A549, NCI-1299 cells) and human normal bronchial epithelium cells (16HBE cells). As shown in Fig. 1B, Magnolol inhibited the proliferation of PGCL3, SK-MES-1, NCI-H460, A549 and NCI-1299 cells, while it showed slightly inhibitory effect on the 16HBE cells. Notably, the half maximal inhibitory concentrations (IC₅₀) of Magnolol at 72hrs was ∼5 µM on both NCI-1299 and A549 NSCLC cells, but the toxicity on 16HBE cells was negligible. Next, we sought to investigate the anti-proliferative effect of Magnolol on lung cancer cells by using colony formation assay. In accordance with the result of SRB assay, as shown in Fig. 1C, Magnolol treatment with low concentration (5 μm) led to significant inhibition on A549 and H1299 cells colony formation compared to the controls. Taken together, these results demonstrate that Magnolol not only has strong efficacy on the proliferation of NSCLC cells but also possesses a great selectivity against NSCLC cells over the normal bronchial cells.

It is commonly known that cell proliferation defect is mainly attributed to cell cycle alteration. Subsequently, the effect of Magnolol on cell cycle progression of NSCLC cells was assessed. After 24 hrs’ treatment, the flow cytometry analysis revealed that Magnolol treatment caused dramatic accumulation of cell populations at the G2/M phase on both A549 and NCI-H1299 cells in a dose-dependent manner. Accordingly, cell populations in S and G1 phases decreased under Magnolol treatment (Fig. 2A). To elucidate the molecular mechanisms through with Magnolol induced the G2/M phase arrests, the levels of the G2/M phase regulatory proteins were evaluated by western blotting. As expected, the data showed that treatment of A549 and NCI-1299 cells with Magnolol for 24 hrs resulted in a dose-dependent downregulation of phosphorylated Cdc2, total Cdc25c, and an accumulation of Cyclin B1 (Fig. 2B). Nevertheless, the protein level of total Cdc2 was not affected by the treatment of Magnolol. In addition, we examined the other key cell cycle regulators under the treatment. As shown in Fig. 2C, the reduction of Cyclin A2, Cyclin D1, Cdk1 and Cdk4 was observed when the cells treated with Magnolol, but Cdk2, p21 and p27 increased as compared with untreated control. Taken together, these findings indicate that the effect of Magnolol on the G2/M phase cell cycle arrest is associated with accumulation of Cyclin B1/ Cdk2/p21/p27 and suppression of phosphorylated Cdc2/Cdc25c/Cyclin A2/Cyclin D1/ Cdk1/Cdk4 in the NSCLC cells.

Next, the mitosis index assay [36] that stains the cells with DAPI was performed to investigate whether Magnolol treatment could affect cell mitosis. As shown in Fig. 3A and B, with treatment of an increasing doses of Magnolol, more A549 cells were accumulated at mitotic (M) phase as indicated by condensed nuclei DAPI staining. In addition, NCI-H1299 cells showed the similar M phase cells accumulation responding to Magnolol treatment (data not shown). Furthermore, we found a marked increase in mitosis-specific MPM-2 expression by western blotting in Magnolol-treated cells (Fig. 3C), further suggesting Magnolol treatment could arrest cells at M phase.

Microtubules are composed of α-tubulin and β-tubulin heterodimers and play critical roles in regulating cell cycle and cell proliferation [4]. Conventionally, α-tubulin and β-tubulin heterodimers exist in two forms, soluble monomer and polymerized tubulin heterodimers. Our above results showed that the inhibitory effect of Magnolol on NSCLC cell growth was associated with cell mitotic arrest, which might be related to microtubular dynamics. Hence, the in vitro microtubule polymerization assay was performed to investigate the effect of Magnolol on microtubule polymerization in A549 and NCI-H1299 cells. Herein, 50 nM treatment of Colchicine or Paclitaxel was used as positive control or negative control, respectively. As shown in Fig. 4A, Magnolol treatment resulted in polymerized tubulin decrease in a dose-dependent manner. Immunofluorescent staining of the cellular microtubule at interphase and mitotic phase was performed to further examine how Magnolol affected microtubule polymerization. As shown in Fig. 4B, in the absence of Magnolol treatment, A549 cells that were at interphase exhibited the normal microtubule organization and arrangement. In contrast, the Magnolol-treated cells presented disorganized microtubule distribution and more diffused staining pattern throughout the cytoplasm, which was similar to those Colchicines-treated cells. Mechanistically, Colchicines inhibits microtubule polymerization by binding to tubulin directly. In Fig. 4C, mitotic A549 cells without Magnolol treatment showed intact spindle assembly, while the Magnolol-treated cells showed disrupted spindle structure and irregular organization. These observations suggest that Magnolol depolymerizes microtubules in NSCLC cells.

It is well documented that there is a crosstalk between anti-microtubule activity with apoptotic induction [37, 38]. Hence we sought to explore the effect of Magnolol on NSCLC cell apoptosis. As shown in Fig. 5A and B, the Annexin V-FITC/PI staining and flow cytometric assay revealed that Magnolol treatment induced apoptosis of A549 cells in both concentration-dependent and time-dependent manners. Western blotting analysis further indicated that Magnolol treatment up-regulated the protein levels of several apoptotic markers such as Cleaved-caspase 9, cleaved-PARP and Bax. Instead, anti-apoptotic Bcl-2 protein level was downregulated in the Magnolol-treated cells compared to the control (Fig. 5C). The similar result was obtained when we treated NCI-H1299 cells with Magnolol (data not shown). P53 gene is null in NCI-H1299 cells, suggesting that the apoptosis induction in NSCLC cells upon Magnolol treatment was not dependent on p53 status. Interestingly, we also found treatment of A549 cells with Magnolol led to activation of ATG5, ATG12 and LC3B-II/-I ratio, suggesting cellular autophagy induction. The mTOR kinase serves as a signal hub for autophagy induction and the activation of mTOR by Akt suppresses autophagy. As shown in Fig. 5C, both p-Akt (S473) and mTOR levels decreased in Magnolol-treated cells compared to the control, indicating Magnolol induced autophagy via downregulation of the Akt/mTOR pathway. Since both apoptosis activation and autophagy induction could provoke cell death, we hypothesized Magnolol treatment would lead to cell death of NSCLC cells. Following Magnolol treatment, the A549 and NCI-H1299 NSCLC cells were co-stained with Calcine AM and propidium iodide (PI) to differentiate live (green) and dead (red) cells, respectively. As revealed in Fig. 6A and B, in the case of control cells, no acute cell death or loss of cell viability was observed. In contrast, the percentage of dead cells increased under Magnolol treatment for 48 hrs in A549 and NCI-H1299 NSCLC cells. These data indicate that Magnolol provokes apoptosis and autophagy which could lead to cell death in NSCLC cells.

To further evaluate the tumor-suppressing effect of Magnolol in vivo, a model for tumorigenicity of A549 NSCLC cells in nude mice was established. A549 cells (2×10⁶) were implanted subcutaneously into 4-week-old male nude mice. The effect of Magnolol (25mg/kg) on tumor xenograft was examined as indicated in Fig. 7A. As anticipated, Magnolol treatment significantly reduced the tomor sizes compared to the control. Specifically, the average tumor weight of control group was 312.54 ± 64.13mg, whereas the tumor weight in Magnolol-treated group was 176.32 ± 10.25 mg. For the average tumor size, the control group showed 2510.62 ± 800.91 mm3, whereas Magnolol-treated group was 1190.85 ± 296.91 mm³ (Fig. 7B). Notably, Magnolol treatment at the given concentration had little effect on the body weight of the mice (Fig. 7C). Therefore, in animal model, Magnolol also exhibited strong tumor suppression efficacy and low toxicity as it did in in vitro cell culture system.

Natural sources provide beneficial pharmacotherapy for people worldwide. In this study, we reported for the first time that the effect of Magnolol, a hydroxylated biphenyl compound isolated from the root and stem bark of M. officinalis, against NSCLC cells both in vitro and in vivo. Several previous studies have documented the anti-cancer activity of Magnolol [23-28]. For example, Magnolol-induced apoptosis of HCT-116 colon cancer cells was associated with the AMP-activated protein kinase signaling pathway. In PC-3 prostate carcinoma cells, Magnolol reduced MMP-2/-9 and cyclooxygenase-2 at the mRNA level. In this current work, we demonstrated that Magnolol significantly inhibited NSCLC cell proliferation, growth, and colony formation, as well as suppressed tumor growth in a xenograft model of human lung cancer, suggesting its potential in lung cancer control. Our work further proved that Magnolol might exert its anti-tumor efficacy through the disruption of cellular polymerization of microtubule.

Lung cancer especially NSCLC causes more deaths than any other types of cancer in both men and women worldwide. In the past few years, great strides have been made in the discovery of effective and new therapies for NSCLC [39, 40]. However, most NSCLC cases ultimately relapsed and progressed to intractable late-stage metastatic disease. The successful use of docetaxel and cabazitaxel in NSCLC chemotherapy suggested that microtubule-targeting agents might be a feasible strategy. Drugs that disrupt microtubule dynamics have been used in the clinic as anti-cancer drugs for over twenty years. These drugs bind to tubulin and thus cause an increase or decrease in the interphase microtubule mass at high concentrations [3]. However, drug resistance and concomitant side effects hinder the clinical use for effective cancer therapy [41]. Therefore, it is necessary to develop new agents that overcome these hurdles.

In our study, we showed that cellular polymerized tubulin decreased significantly under the Magnolol exposure in microtubule polymerization assay (Fig. 4A). It was similar when we treated the cells with Colchicine, a known chemical inhibitor for microtubule polymerization, suggesting Magnolol is a potential microtubule-targeting agent. Further Immunofluorescence staining for tubulin in cells treated with Magnolol revealed disorganized microtubule distribution at interphase and disrupted spindle at mitotic phase (Fig. 4B and C). In accordance with the fact that microtubule-targeting agents arrest the cell cycle at G2/M phase, our results showed that Magnolol induced a G2/M blockade, as indicated by flow cytometry analysis (Fig. 2A). We further investigated the in-depth molecular mechanism of cell cycle arrest induced by Magnolol treatment. Previous reports have shown that Cyclin B and Cdc2 kinase regulate the start of M phase. The activation of Cdc2 kinase is dependent on accumulation of Cyclin B1 and dephosphorylation of Cdc2 [42]. We observed an obvious dose-dependent decrease of phosphorylated Cdc2 and total Cdc25c, while an accumulation of Cyclin B1 protein under Magnolol treatment, suggesting cell cycle was blocked at mitophase. Another evidence is that we found MPM-2, a protein marker for mitotic phase [43], increased upon Magnolol treatment in a dose-dependent manner.

Cell cycle arrest at mitotic phase might be an upstream event leading to apoptosis. Under Magnolol treatment, we indeed found activation of cellular apoptosis by using Annexin V-FITC/PI staining (Fig. 5A and B). Western blotting analysis confirmed the change of apoptotic protein markers (Cleaved-caspase 9, Cleaved-PARP, Bax, Bcl-2). Meanwhile, we also detected the strong induction of autophagy in cells treated with Magnolol. The Akt-mTOR signaling pathway is critical for controlling autophagy induction. We found that Magnolol treatment resulted in down-regulation of both p-Akt and mTOR levels but activation of autophagy marker LC3B-II. Calcein AM is a nonfluorescent dye that could be hydrolyzed by intracellular esterases to a green fluorescent calcein dye in live cells. Propidium iodide (PI), a dead cell staining, is generally excluded from viable cells. It binds to the DNA of dead cells, generating red fluorescence. Our live-dead staining assay suggested that cell death increased upon Magnolol treatment (Fig. 6). Since apoptosis and autophagy are two important pathways led to cell death, we speculate that induction of cell cycle arrest and apoptosis, along with promoted activation of autophagy might mechanistically underlie the efficacy of Magnolol on NSCLC cells. Furthermore, we used mouse xenograft model to confirm the Magnolol’s efficacy on NSCLC cells in vivo.

In addition to Magnolol’s remarkable anticancer efficacy shown both in vitro and in vivo, another advantage of Magnolol is its acceptable toxicity. Current commercial microtubule targeting agents are widely used in the clinic but side effects are significant due to drug toxicity. Although Magnolol also induces cell cycle arrest in 16HBE cells, an immortalized human normal bronchial epithelium cell line, the effect was negligible compared to the effect on the cancer cells. Besides, in subcutaneous xenograft tumor model, in vivo treatment of Magnolol at the effective concentration had little effect on the body weight of the mice (Fig. 7C).

Although Magnolol had the inhibitory effect on microtubule polymerization, we still did not know the specific molecular mechanism. It is not clear how Magnolol exerted its inhibitive function on microtubule polymerization. Does Magnolol bind to microtubule directly or through binding to other indirect molecules? In addition, although there was a report to test some symbolic parameters of pharmacokinetics and pharmacodynamics (PDPK) on Magnolol in vivo [44], there is still an urgent need to determine more specific PDPK characteristics before its clinical utilization in NSCLC treatment.

In conclusion, our data demonstrate Magnolol is efficacious in suppressing the growth of NSCLC cells both in vitro and in vivo via perturbing the microtubule polymerization. These findings suggest Magnolol may foster novel therapeutic application for NSCLC treatment.

This work was supported by Hubei Provincial Natural Science Foundation of China (No. 2016CFB210) and the Startup Scientific Research Foundation for Doctors of Hubei University of Science and Technology (No. BK1506).

The authors declare no conflict of interest.

J. Shen, H. Ma and T. Zhang contributed equally to this work.