Background
In the United States, human, non-melanoma skin cancers are most frequently diagnosed in Caucasians, accounting for over 3.5 million cases each year [
1]. American Cancer Society estimates indicated 11,980 deaths from skin cancer in 2011 [
2]. The major causative factor for skin cancer is UV radiation from sunlight [
3,
4]. Both experimental and epidemiological evidences suggest UVB is an important component of solar radiation that acts as a complete carcinogen by initiating and promoting skin cancer [
5,
6]. Estimates show that one among five Americans will develop skin cancer [
7]. UV radiation, besides resulting in characteristic DNA damage, also causes tumor promotion by inducing various signal transduction pathways which can lead to distinct cellular responses including cell proliferation, transformation, and cell death [
8,
9]. Mechanisms that suppress tumorigenesis involve the modulation of signal transduction pathways leading to arrest in the cell cycle progression or induction of apoptosis. It has been proposed that sunscreens alone are not sufficient in preventing skin cancer, thus there is a need for more effective ways to prevent this malignancy [
10,
11]. For this reason, chemoprevention of skin cancer by natural compounds has gained importance in recent years [
12,
13]. More than 1000 phytochemicals have shown chemopreventive effects against cancer [
14‐
16], and one such phytochemical is magnolol, whose effects are investigated for the prevention of skin cancer in this study.
Magnolol and honokiol are phenolic compounds obtained from the bark and seed cones of
Magnolia officinalis which has been used in traditional Chinese medicine. Recently, we have reported the chemopreventive effects of honokiol on UVB-induced skin cancer development in mice [
17]. Honokiol and magnolol are isomers and share a number of biological properties. Studies have demonstrated that magnolol has multiple pharmacological properties such as antioxidant [
18], anti-inflammatory [
19], and central nervous system depressant effects [
20]. It has been reported that magnolol delayed the formation of papillomas in mouse skin initiated by 7,12-dimethylbenz (α) anthracene (DMBA) and promoted by 12-
O-tetradecanoyl phorbol-13-acetate (TPA) [
21].
For the first time, in this study, we reported the effects of magnolol on UVB-induced skin cancer development in SKH-1 mice. Since UVB induces squamous cell carcinoma in mice, the effects of magnolol on human epidermoid squamous cell carcinoma A431 cells were investigated to elucidate the possible mechanisms of action. The effects of magnolol were investigated on UVB-induced skin carcinogenesis in SKH-1 mice, a model relevant to human cancer where UVB acts as complete carcinogen. Loss of apoptosis and rapid cell proliferation are major factors responsible for tumorigenesis [
22], therefore, the present study focuses on the effects of magnolol on apoptosis, cell survival pathways and cell cycle arrest.
Signal transduction and activators of transcription 3 (STAT 3) and mitogen-activated protein kinase (MAPK) signaling play a major role in apoptosis, proliferation, and tumor promotion [
23,
24]. Overactivity of the MAPK pathway has been shown to be involved in cancer promotion and development [
25‐
27]. Therefore, we investigated the effects of magnolol on the modulation of STAT3 and MAPK signaling pathways.
Methods
Reagents
Magnolol was purchased from Nacalai tesque (Kyoto, Japan). Thiazolyl blue tetrazolium bromide (MTT) and other chemicals of analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO). Cell proliferation ELISA kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Vibrant Apoptosis Kit 2 and APO-BrdU TUNEL assay kit were purchased from Molecular Probes (Eugene, OR). The primary antibodies such as cleaved caspase-3, cleaved caspase-8, pSTAT3-Tyr705, pSTAT3-Ser445, pMEK1/2, B-Raf and cleaved PARP were purchased from Cell Signaling Technology (Beverly, MA). Primary Antibodies such as Cdc25A, Cdc25B, Cdc25C, p-Cdc25C, CDK-2, CDK-4, Cyclin B1, Cyclin A, Cdc2p34, p-ERK1/2, p-AKT, PCNA, anti-mouse IgG horseradish peroxidase-linked and anti-rabbit IgG horseradish peroxidase-linked secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Kip1/p27 antibody was purchased from BD-Pharmingen (San Diego, CA) and anti-Cip1/p21 antibody from Upstate Biotechnology (Lake Placid, NY).
Cell culture
Human epidermoid carcinoma A431 cells were purchased from American Type Culture Collection (Manassas, VA). A431 cells were cultured in DMEM supplemented with 10% heat inactivated fetal bovine serum, 100 unit/ml of penicillin and 100 ug/ml of streptomycin in a humidified atmosphere containing 95% air and 5% CO2 at 37°C. For treatments of cell cultures, magnolol was dissolved in DMSO to make a 50 mM stock solution, this stock solution was diluted in DMEM at different concentrations and was immediately used. In all assays the final concentration of DMSO in DMEM was 0.4%.
Animals
Five to six week old female SKH-1 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Institutional Animal Care and Use Committee (IACUC) approvals were obtained for all experimental protocols. Mice were housed under climate-controlled environment with a 12 hours light/dark cycle and were provided with free access to food and water during the experiment.
UVB Light Source
Four FS-40-T-12 UVB lamps were used as UVB light source. UVB exposure dose was controlled by integrating dosimeters manufactured by Daavlin Corporation (Bryan, OH, USA).
UVB-induced skin tumor development protocol
Five to six weeks old female SKH-1 mice were randomly divided into four groups of 20 each. Carcinogenesis was initiated and promoted by UVB, dose (30 mJ/cm
2) for 5 days a week (monday-friday). This is a dose close to the UVB human exposure causing cancer development [
28]. Group 1 served as control and received 200 μ l of acetone, group 2, group 3 and group 4 received 30 μ g, 45 μ g and 60 μ g of magnolol in 200 μ l of acetone respectively. Treatments were administered topically one hour before UVB exposure. The experiment was carried out for 25 weeks. Tumor counts and body weights were recorded on weekly basis for 25 weeks. Results were analyzed for tumor incidence, multiplicity and area.
Histopathological analysis of mice tumors
Mice were euthanized by cervical dislocation at the end of the above mentioned protocol. Skin collected from five animals per group was fixed by immersion in 10% neutral buffered formalin for three days at room temperature. Fixed tissues were processed into paraffin-wax blocks, sectioned and stained with hematoxylin-eosin (HE) and then evaluated under light microscope.
Effects of magnolol on tumor area in SKH-1 mice
Tumor areas were quantified as described earlier [
17,
29] by using images from tumor bearing mice which were taken at the end of 25 weeks. By using Photoshop CS3 (Adobe systems, San Jose, CA, USA) tumor boundaries were determined and areas were measured by using Image-Pro Plus 5.1 (Media Cybernetics, Inc, Bethesda, MD, USA).
MTT assay for cell viability
A431 cells (9000 cells/well) were plated in 96 well plates. After 24 h, cells were treated with different concentrations of magnolol (75, 100, 125 μM) for 12 h, 24 h and 48 h, using cells treated with growth medium 0.4% DMSO as control. Cell viability was determined at the end of each treatment by using MTT assay as previously reported [
30].
BrdU assay for cell proliferation
A431 cells (9000 cells/well) were plated in 96 well plates. After 24 h, cells were treated with different concentrations of magnolol (75, 100, 125 μM) or treated with growth medium 0.4% DMSO as control, for 48 h. At the end of the treatment, Bromodeoxyuridine (BrdU) incorporation assay was carried out using ELISA kit (Roche Diagnostics, GmbH, Manheim, Germany) using manufacturer protocol as previously reported from our laboratory [
30]. The experiment was repeated three times.
Quantification of apoptosis by Annexin V/PI staining
Apoptosis was quantified by using Vibrant Apoptosis Kit 2 (Molecular Probes) as previously reported from our laboratory [
30]. A431 cells (2 × 10
5)/well were plated in six well plates and after 24 h were treated with magnolol (100, 150 μM) for 48 h. Then cells were collected, washed and stained with annexin-V labeled with a fluorophore that binds to phosphatidylserine exposed on apoptotic cells. Also cells were treated with propidium iodide (PI) a DNA intercalator dye that stains dead cells. Samples were analyzed after staining with both dyes in BD FACScan flow cytometry and the percentages of apoptotic cells were evaluated using CellQuest software (BD Biosciences, San Jose, CA).
Quantitation of DNA fragmentation by TUNEL assay
Apo-BrdU TUNEL assay kit (Molecular Probes) was used to quantify the amount of DNA fragmentation in magnolol treated A431 cells by using manufacturer's protocol as previously reported [
30]. Positive and negative control cells were run with each assay.
Cell Cycle analysis
Subconfluent A431 cells plated in six well plates were treated with different concentrations of magnolol (75, 100, 125 μM) or control media for 12, 24 and 48 h. After each treatment, cells were harvested, washed and fixed in 70% ethanol in DPBS. Fixed cells were treated with 100 μ l of RNase A (1mg/ml) for 30 min at 37°C. After incubation, 900 μ l of staining buffer and 20 μ l of PI (Propidium iodide 1 mg/ml) were added to each sample and incubated for 30 min in the dark. The samples were analyzed with BD FACScan flow cytometry (BD Biosciences, San Jose, CA) using Cell Quest Software (BD Biosciences, San Jose, CA) as previously reported [
31,
32].
Preparation of tissues and cell lysates for immunoblotting
Tissue samples: Mice were sacrificed by cervical dislocation, then treated skin was collected, fat from skin was removed by scalpel, and then skin was homogenized in 0.1 mM Tris-HCl/0.15 M NaCl (pH 7.4). The homogenate was filtered and centrifuged at 10000g for 45 min in a Beckman J2-21 centrifuge (Brea, CA.), the obtained pellet was combined with 5% SDS, 0.5% leupeptin and pepstatin and 1% PMSF, then was passed through a 25G needle and centrifuged at 13000g for 20 min, the obtained supernatant was heated for 5 min at 100°C. Finally, protein concentrations were determined by BCA protein assay (Pierce, Rockford, IL), then separated by SDS-PAGE and analyzed by Western blot.
For A431 cell lysates, 1.5 × 106 cells were plated in 100 mm culture dish. Subconfluent A431 cells were treated with varying concentrations of magnolol (75, 100, 125 μM) and DMEM 0.4% DMSO as control, for 24 and 48h. At the end of each treatment cells were lysed.
Protein concentrations for tissues and cells' proteins were determined by BCA protein assay kit (Pierce, Rockford, IL) with albumin as standard.
The tissues or cells' proteins (50 μ g) were resolved by SDS-PAGE and were transferred onto nitrocellulose membranes. The membranes were probed with appropriate antibodies followed by secondary antibody and detection by ECL plus detection system (Amersham Biosciences, Piscataway, NJ). Equal protein loading is ensured by reprobing each membrane with β-actin antibody. Western blotting was repeated for 3-5 samples and representative bands from all replicated experiments are reported. Western blots were detected and quantified by using a UVB Biochem Gel Documentation system (UVP, Inc., Upland, CA) and this data were analyzed statistically.
Statistical Analysis
INSTAT software (Graph Pad, San Diego, CA) was used to analyze data. Chi square analysis was used for the data on tumor incidence. Analysis of variance followed by Tukey's test was used for tumor multiplicity and area, as well as for Western blots and for various in vitro assays. Significance in all experiments was considered at p < 0.05. All values were expressed as mean ± standard error.
Discussion
Magnolol, a hydroxylated biphenolic compound isolated from
Magnolia officinalis, most commonly used in traditional Chinese medicine has been investigated for its effects on skin carcinogenesis. In this study, we determined the effect of magnolol in UVB-induced skin cancer in SKH-1 mice and on a human epidermoid skin cancer cell line
in vitro. Neolignans from
Magnolia officinalis delayed papilloma formation in skin tumor promotion by TPA [
21,
37]. We investigated the effects of magnolol in a UVB-induced skin carcinogenesis model with a UVB dose of 30 mJ/cm
2/day which is more translational and relevant to human skin cancer as compared to previous studies that used higher doses of UV radiation [
5,
15]. Magnolol 30 μg and 60 μg in 200 μl of acetone showed a protective effect in a dose dependent manner when applied topically. In this study interestingly, 45 μg magnolol did not have any effect on tumor incidence and lower effects than the 30 μg application in tumor multiplicity. Magnolol may have biphasic effects on various target proteins not investigated in this study, thus the middle dose is less effective than the lower dose. Further studies with an increased range of magnolol doses are needed to fully understand this biphasic effect. We used very low doses (in micrograms) of magnolol compared to other chemopreventive agents which use milligrams per applications [
5,
13] thus indicating the higher potency of magnolol over other agents. The results demonstrated that magnolol delayed the onset of tumorigenesis when compared to the control. Tumor multiplicity was reduced by 27-55% for 30 μg and 60 μg of magnolol respectively compared to the control.
Mechanistic studies showed that magnolol induced apoptosis through extrinsic pathway and affected tumor development by causing cell cycle arrest at G2/M phase in our animal models.
To gain insight and have understanding of signaling mechanisms involved in the magnolol anticarcinogenic effect, we used human epidermoid A431 cells as an
in vitro model. Magnolol inhibits cell viability and proliferation which together contributed to overall inhibition of cell growth in A431 cells at concentrations 75-125 μM for 12-48 h. Cancer development involves deregulation in cell cycle progression. Control of the cell cycle plays an important role in controlling tumor growth [
22,
38]. As such, effects of magnolol on the cell cycle and its related proteins were investigated in A431 cells. The results obtained demonstrate that magnolol induced G2/M cell cycle arrest, is one mechanism of inhibition of cell viability and proliferation. As cyclins/cyclin dependent kinases tightly regulate the cell cycle progression [
39,
40], the effects of magnolol on cell cycle proteins were investigated. Our findings revealed that treatment of cells with magnolol resulted in a significant decrease in cyclin A, cyclin B1, CDK2, CDK4, Cdc2 and increase in Cip1/p21 expression at all concentrations compared to control. Our studies on honokiol [
17], an isomer of magnolol have indicated similar anticarcinogenic effects as magnolol. However, honokiol caused cell cycle arrest at G0/G1 phase in A431 cells [
41] unlike magnolol which caused cell cycle arrest at G2/M phase.
Anticarcinogenic effects are modulated by two major events: inhibition of cell proliferation and induction of apoptosis [
25,
42]. Accordingly, the effects of magnolol on induction of apoptosis in A431 cells were investigated. During apoptosis, cells undergo changes such as loss of phospholipids asymmetry of the plasma membrane, cell shrinkage, proteases activation and finally DNA fragmentation [
43]. Our flow cytometry data demonstrated that magnolol significantly induced apoptosis in A431 cells as assessed by annexin-V/PI staining which detects apoptotic cells by their loss of phospholipids plasma membrane asymmetry. Then, later we examined DNA fragmentation in apoptotic cell by using TUNEL assay. Magnolol 48h treatment induced DNA fragmentation in A431 cells at higher concentrations (150 μM).
There are two reported pathways for the induction of apoptosis. In the extrinsic or death receptor pathway of apoptosis, activation of death receptors by ligands leads to activation of caspase-8. This activated caspase-8 can activate caspase-3, an executioner caspase. Activated caspase-3 can cleave PARP and thereby results in apoptosis [
44,
45]. Consistent with the above reports, magnolol treatment to A431 cells activated caspase-8 and caspase-3 in a concentration dependent manner that led to PARP cleavage. These observations suggest that magnolol induced apoptosis through extrinsic pathway and are consistent with the results obtained from animal experiments.
The STAT pathway regulates the transcription of a wide variety of genes involved in proliferation, development, and tumorigenesis [
46,
47]. Among different STAT family members, STAT3 is implicated in tumorigenesis [
47] and it plays an important role in skin cancer development [
48]. STATs are activated either by serine or tyrosine phosphorylation by JAK kinases, then they undergo dimerization followed by nuclear translocation and regulation of the expression of target genes [
49]. Our results showed that treatment of A431 cells with magnolol inhibited the phosphorylation of STAT3 at tyrosine residues. Downstream targets of p-STAT3 include cyclin D1, our results showed that magnolol decreased cyclin D1 expression, and this may lead to cell cycle arrest [
50]. In the present study, our
in vitro data has shown that magnolol treatment increased the phosphorylation of ERK protein in A431 cells, suggesting activation of ERK and upregulation of p21 by magnolol as a mechanism for cell cycle arrest [
51]. However, further studies are needed to study the effects of magnolol on the phosphorylation of these proteins at very early stages instead of at 24h and 48h.
Magnolol inhibited cell proliferation through regulation of Cip1/p21 in human glioblastoma cells [
52], induced apoptosis via inhibition of EGFR, PI3K/AKT signaling pathways in human prostate cancer cells [
36] and inhibited MMP-9 expression through the transcription factor NF-kB in TNF-α- induced human urinary bladder cancer cells [
53]. Magnolol induces apoptosis via activation of both mitochondrial and death receptor pathways in A375-S2 malignant melanoma cells [
54]. Recent studies by Tanaka et al. [
55], have shown the preventive effects of magnolol on UV-induced photoaging by inhibiting the expression of NF-kB. A recent study by Kuo et al. [
35] showed that magnolol down-regulated TPA induced iNOS and COX-2 gene expression in mouse skin suggesting that magnolol could be novel agent preventing inflammation associated tumorigenesis.
Our studies for the first time provided the evidence that magnolol pretreatment at very low doses (micrograms per applications compared to most other agents which are used in milligrams per application) prevents UVB-induced skin cancer development in SKH-1 mice by both inducing apoptosis and decreasing cell proliferation through modulation of various signaling pathways. The sunscreen effects of magnolol have not been investigated in this study which may contribute to anticarcinogenic effects. Future studies involving various inhibitors, antisense oligonucleotides and dominant negative mutants or siRNA are needed to map the pathways to conclude the signaling involved in the anticancer effects of magnolol. Magnolol has a great potential to be a safe and potent chemopreventive agent against skin cancer development in human.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CC has performed UVB induced skin cancer development in SKH-1mice, in vitro assays and Western blots for various proteins for A431cells and wrote the manuscript; RG performed Western blots for cell cycle and apoptotic proteins from tissue samples. XZ helped with Western blots. RSK and AY helped with flow cytometric analysis; DZ performed the histopathological examination of samples; MH helped with image pro analysis; HF identified the magnolol for the studies; and CD overall conceived and executed the idea, and prepared the final draft of manuscript. All authors read and approved the final manuscript.