| BackgroundLung cancer is the most prevalent malignancy worldwide, with an annual incidence of 1.5 million and roughly 85% being non-small cell lung cancer (NSCLC). Recent studies have revealed that the epigenetic alterations in tumor suppressor genes and the consequent cellular transformations are closely related to lung cancer progression and metastasis. Given their dynamic and reversible characteristics, epigenetic variations are potential targets in lung cancer chemoprevention and treatment.Among epigenetic mechanisms, histone acetylation and DNA methylation are probably the most extensively studied in lung cancer. Histone acetylation, which is mediated by histone deacetylases (HDACs) and histone acetyltransferases, plays an important role in the initiation and development of NSCLC. HDAC enzymes in the human body could be divided into four classes:class I (HDACs 1-3 and 8), class II (HDACs 4-7,9, and 10), class III (Sirtl-Sirt7), and class IV (HDAC 11). Class I HDACs, which are almost overexpressed in various tumor tissues, principally promote tumor cell proliferation and invasion, which ultimately lead to poor prognosis and drug resistance. Therefore, extensive studies on lung cancer have identified some HDAC inhibitors (HDACi), especially those targeting class I HDACs. These inhibitors serve as effective antitumor agents for lung cancer treatment by regulating cell apoptosis, cell cycle progression, angiogenesis, metastasis, and invasion. HDACi enhance the sensitivity of tumor cell apoptosis by activating the TRAIL pathway, which increases the surface expression of one or both of the agonistic TRAIL receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5), and initiates the caspase cascade.To date, the U.S. Food and Drug Administration has approved several synthetic HDACi, including valproic acid, trichostatin A (TSA), suberoylanilide hydroxamic acid, MS-275, and trapoxin A, for cancer treatment. However, the side effects and chronic toxicities of these drugs limit their wide application. Accumulating evidence has implied that dietary and nutritional compounds possessing low toxicities and strong bioactivities substantially contribute to lung cancer inhibition by influencing the epigenetic landscape. Therefore, exploring novel anticancer agents derived from natural plants or food, which could also mediate epigenetic interventions, is necessary for successful NSCLC treatment and prevention.Magnolol, a bioactive compound extracted from Magnolia officinalis, possesses multiple pharmacological effects, such as antitumor, antidepressant, antioxidant, and anti-inflammatory properties. Overwhelming evidence has revealed that magnolol can serve as a therapeutic agent in several cancer types, including lung, prostate, colon, and breast cancers. Furthermore, most molecular mechanisms underlying the antitumor effects of magnolol focus on the caspase-independent and HIF-1α/VEGF signaling pathways. Polyphenol mixture (PM) composed of various active constituents, such as honokiol, magnolol, and obovatol, can also be derived from M. officinalis. PM can significantly suppress tumor progression in xenograft models. However, PM has many constituents which contain magnolol and honokiol, it is not understand which active ingredient has antitumor effect or PM has better antitumor effect than one of the main component.It has been reported that honokiol, an isomer of magnolol, can hinder the epigenetic deacetylating activities of class I HDACs in NSCLC. However, despite this development of honokiol, the mechanisms underlying the antitumor effects of magnolol and PM, specifically on their capacities of epigenetic modifications, remain to be clarified.ObjectivesThe present study aimed to investigate the effects of magnolol and PM on the expression levels of class I HDACs in NSCLC cell lines (A549 and H1299). The potential of magnolol and PM to serve as class I HDACi and whether or not the underlying mechanism of this process involves tumor cell apoptosis induction were also evaluated in vitro and in vivo. The discovery and development of new and effective nontoxic herb-derived components that target epigenetic regulation are crucial for lung cancer treatment and chemoprevention.MethodsMTT assay, real-time PCR analysis, cell cycle analysis, apoptosis analysis, ChIP, nude mice tumor xenograft study, immunohistochemical detection of TUNEL-positive cells and flow cytometry assays were employed to investigate the antitumor and mechanisms effects of magnolol and PM on NSCLC in vitro and in vivo.1. MTT cell proliferation assayA549, H1299 and H226 cells,4000 cells per well were cultivated in 96-well plates and grown 24h, followed by treated with various magnolol (0,10,20,40,60, 80,100,120,150μM) and PM (0,4,8,16,21,26,32,40 μg/mL) concentrations or 48 h. Thereafter, cells were harvested and incubated with 100 μL of MTT (0.5 mg/mL) at 37℃ for an additional 4 h, the resulting formazan crystals were dissolved in 150 μL of DMSO. The absorbance was recorded at 570 nm. The effect of magnolol and PM on cell viability was determined relative to the viability of control-treated cells that were assigned a value of 100%.2. Flow cytometry was used to analyze cell cycleAfter magnolol (0,15,30 and 60 μM) or PM (0,4,8 and 16 μg/mL) treatment for 48 h, the cells were then harvested, washed with cold PBS, and processed for ice-cold 75% ethanol in PBS at 4℃ overnight. The cells were collected and washed with 1 mL cold PBS, then the cells were incubated with 1.5 mL containing 500 μL PI which has RNase A at 37℃ for 30 min. The cell cycle phase distribution was determined using a FACSCalibur instrument equipped with FlowJo v7.6 software.3. Flow cytometry was used to analyze cell apoptosisAfter magnolol (0,15,30 and 60 μM) or PM (0,4,8 and 16μg/mL) treatment for 48 h, the cells were then harvested and washed with PBS, then the cells were re-suspended with 100 μl lx Binding buffer and incubated with 5 μL Annexin V and 5 μl PI double-labeling at 37℃ for 15 min in the dark. After incubation, the cells were added 400 μL 1x Binding buffer. The apoptosis cell analysis was performed using a FACSCalibur instrument with FlowJo v7.6 software.4. Real-time PCR analysis was used to analyze the gene levels of class I HDACs, DR4, DR5 and TRALAfter treatments of (0,15,30 and 60 μM) and PM (0,4,8 and 16 μg/mL) for 48h, cellular RNA was isolated using the TRIzol extraction method. cDNA was synthesized from total RNA using a reverse transcription kit. SYBR Green real-time PCR amplification and detection were then performed using an ABI 7500 system.5. Analysis of the protein expression levels to use Western blotA549 and H1299 cells were treated with magnolol (0,15,30 and 60 μM), PM (0, 4,8 and 16 μg/mL) or TSA (500 nM) for the desired period of time, and cell lysates were prepared as detailed previously. Proteins were subjected to SDS-PAGE (5% stacking gel and 12% separating gel, respectively) and transferred onto the polyvinylidene fluoride membrane. Western blot analysis was performed to detect the expression levels of target proteins (class I HDACs, Ac-H3, Ac-H4, AC p53, AC p65 and apoptosis checkpoint proteins) as detailed previously. Loading of equal protein on the gels was verified by reprobing the membrane with antibodies against β-actin or histone H3. Other detailed procedures are described in previous literature. ECL chemiluminescence detection agent was applied to obtain the blot signals, following the manufacturer’s instructions. The relative intensity of the protein bands was scanned and quantified using Quantity One Program.6. Chromatin immunoprecipitation assay (ChIP)After treatments of magnolol and PM, A549 and H1299 cells were fixed using 1% formaldehyde, lysed and sonicated and precleared using a PierceTM Agarose ChIP Kit (Thermo), following the manufacturer’s instructions. The lysate was precipitated with the primary antibodies H3K27ac (4μg) overnight at 4℃. Purified DNA was measured using gene specific primers and PCR amplification. The primers specific to promoter of DR5 (-1bp～-2500bp,2.5kb) were used as follows:forward senesce 5’-GGCGGCAGAGAAGACTTAAT-3’and reverse senesce 5’-CCTGAGGATGGAGACCTTATTTC-3’. For each group,10% of the chromatin was assayed for equal loading (input). Input was conducted for each group tested.7. A549 tumor xenograft model was used to analyze the antitumor effects of magnolol and PMExponentially growing A549 cells in 200 μL of PBS containing 2 × 106 cells were injected s.c. in the right flank of each mouse. When the tumor volume reached an average of 50-100 mm3, mice were randomly divided into four groups with seven mice in each group. Experimental animals were treated by oral gavage with 20 and 20 mg/kg of magnolol and PM each day in 200 μL of corn oil for five times a week. The 5 mg/kg of afatinib was suspended in 0.5%(w/v) methylcellulose containing 0.4% Tween 80 also by oral gavage for five times a week. The control mice group received an equal volume of corn oil by gavage. The mice were orally administered five times a week for four weeks. At the termination of the experiment, mice were sacrificed to harvest the tumor from each mouse, and the wet weight of the tumor was recorded. Tumor volume (TV) was measured every 3 days using micrometer calipers. TV was calculated using the following formula:TV (mm3)=1/2 × (L×W 2), where L is the long diameter, and W is the short diameter. The tumor tissues were used to investigated immunohistochemical and protein expression levels.8. Immunohistochemical detection of TUNEL-positive cellsQuantitative assessment of apoptotic cells in tumor sections was assessed by the TUNEL method In Situ Cell Death Detection Kit, POD, following the manufacturer’s instructions and as detailed previously. TUNEL-positive cells were detected and counted using a fluorescence microscope.9. Chemical induced model was used to analyze prevention of magnolol and PMC57/B6 mice were used and these were housed at the Institute of Laboratory Animals. The animals were divided into three groups with twenty mice per group. The group 1:the control group. The group 2 and group 3:magnolol (lOmg/kg) and PM (10mg/kg). The urethane (600 mg/kg) were injected into mice via the abdominal once a week for 15 successive weeks to induce lung cancer by 30th week The next day, the mice were given were given drug (orally). Mice were sacrificed after 30 weeks and take the lung tissue.10. Statistical analysisThe statistical significance of the data was determined by one-way ANOVA using SPSS software. All data are shown as mean ± SD. In each case, p< 0.05 was considered statistically significant.Results1. Magnolol and PM suppressed tumor cell viabilityMTT and flow cytometry assays were employed to investigate the effect of magnolol and PM on NSCLC cells. Treatment with magnolol or PM for 48 h significantly suppressed the cell viabilities of A549, H1299 and H226 cells in a dose-dependent manner.2. Magnolol and PM induced G0/G1 phase cell cycle arrest and apoptosisNext, we determined the effect of cell cycle and apoptosis. Magnolol and PM dose-dependently induced the G0/G1 arrest of A549 and H1299 cells, induced the S phase of H226 cells. Furthermore, the cell apoptosis was markedly induced by the increasing concentrations of magnolol and PM in A549 and H1299 cells.3. Magnolol and PM decreased class I HDACs protein levels and functionsTo explore further the mechanism underlying tumor cell apoptosis induced by magnolol and PM, the protein and gene expression of class I HDACs in the nucleus was determined. Comparing with the control group, magnolol and PM dose-dependently decreased the nuclear protein expression levels of HDAC1, HDAC2, HDAC3, and HDAC8 in A549 and H1299 cells. Compared with the control treatment, the magnolol treatment markedly reduced the protein expression levels of HDAC2, HDAC3, and HDAC8 in the nucleus of A549 cells. But magnolol and PM didn’t affect the gene levels of HDACs.Considering the most vital function of class I HDACs is to deacetylate the gene of histones or non-histones, subseguently, the effects of magnolol and PM on the acetylation of histones (H3 and H4) and non-histones (p53 and p65) were determined. As expected, both magnolol and PM significantly increased the protein levels of the acetylated histone H3 and H4 in A549 and H1299 cells relative to the control. Interestingly, magnolol significantly increased the protein expression level of AC histone H3 to a greater extent than that of AC histone H4. Compared with the control treatment, magnolol and PM treatments increased the expression levels acetylation of non-histones such as AC p53 and AC p65 in A549 cells. Meanwhile, we only analyzed p65 protein expression level in the H1299 cells where p53 was deleted. Magnolol and PM significantly increased the protein expression of AC p65 in a dose-dependent manner.4. Magnolol and PM partially induced apoptosis through the TRAIL pathwayThe basal levels of key proteins involved in TRAIL-mediated apoptosis were studied to ascertain the effects of class I HDACs reduction induced by magnolol and PM. Magnolol and PM significantly increased the gene expression levels of DR5. Compared with the control treatment, magnolol or PM treatment significantly increased the expression level of the pro-apoptotic protein Bax while suppressed that of the anti-apoptotic protein Bcl-2. Furthermore, magnolol and PM treatments increased the protein levels of DR5, caspase 3, cleaved caspase 3, and cleaved PARP significantly in A549 and H1299 cells when compared with the control treatment. However, the expression levels of caspase 8 were not markedly affected by magnolol and PM treatments.5. Inhibition of class Ⅰ HDACs partially prevented tumor cell from apoptosis induced by magnolol and PMTo determine whether magnolol- and PM-triggered apoptosis is caused by the inhibition of class Ⅰ HDACs, the flow cytometry and Western blot assays were used. The significant apoptosis ratio with 60μM magnolol in A549 and H1299 cells for 48 h were 64.40±0.73% and 13.67± 0.62%, respectively. While A549 and H1299 cells were pretreated with HDAC inhibitor TSA, followed by treated with magnolol and PM for additional 48 h. The class Ⅰ HDACs proteins were significantly decreased in cells treated with TSA alone, but did not induce cell death in H1299, indicating TSA at concentration of 500 nM could inhibit HDACs activities. Notably, when cells were pretreated with TSA, the proportion of apoptosis induced by magnolol were significantly decreased to 39.05±1.48% and 42.50±0.71% in A549 and H1299 cells. Similarly, after treatment of TSA, the percentage of A549 and H1299 apoptosis cells were significantly decreased to 7.61±0.86% and 8.79±0.95% induced by 16 μg/mL PM, respectively. These results suggested TSA partially prevented tumor cell from apoptosis induced by magnolol and PM, and inhibition of HDACs might contributed to the apoptotic effect of magnolol and PM, but not exclusive mechanism. After treating A549 and H1299 cells with magnolol and PM, respectively, the H3K27 site was hyperacetylated within DR5 gene. The increased band density observed in ChIP assays suggested that magnolol and PM treatments could directly enhanced histone acetylation in the promoter of DR5 gene via significantly suppressing HDACs catalytic activities.6. Magnolol and PM suppressed tumor growth by inhibiting class I HDACsXenograft animal models were established to determine the effects of magnolol and PM on the growth of A549 cells in vivo. Compared with the control treatment, the other treatments induced no body weight loss, whereas afatinib, magnolol, and PM treatments significantly decreased the tumor volumes. Magnolol and PM treatments markedly inhibited tumor weight by 44.40% and 35.45%, respectively, relative to the control. Compared with the control treatment, magnolol and PM treatments significantly decreased the protein levels of class Ⅰ HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) in the tumor tissues in vivo. In addition, magnolol and PM treatments significantly increased the protein levels of AC histone H3 and H4, as well as those of AC p53 and AC p65. Immunohistochemical analysis was also conducted to explore the apoptosis ratio in tumor tissues after treatment. The percentages of TUNEL-positive cells were significantly higher after the magnolol and PM treatments than after the control treatment, especially in the magnolol-treated tumor tissues. Compared with the control treatment, magnolol and PM treatments significantly increased the protein levels of DR5, Bax, caspase 3, cleaved caspase 3, cleaved PARP, and caspase 8 in the tumor tissues but decreased the expression level of Bcl-2.7. Magnolol and PM prevented the occurrence and development of lung cancerCompared with control group, magnolol and PM can effectively reduce the pulmonary nodules, and no obvious toxicity showed that magnolol and PM can be used as prevention drugs for prevented the occurrence and development of lung cancer.ConclusionsOur findings first proposed that magnolol and PM could act as class I HDACi through the TRAIL pathway and arrest cell in the G0/G1 phase to inhibit NSCLC development.1. Magnolol and PM have stronger inhibition on A549, H1299 and H226, which the strongest inhibition of magnolol and PM were A549 and H1299, and can induce apoptosis of A549 and H1299 cells.2. The protein levels of class I HDACs (HDAC1, HDAC2, HDAC3 and HDAC8) were suppressed by magnolol and PM. Meanwhile, the expression levels of acetylation of histones (H3 and H4) and non-histones (AC p53 and AC p65) were induced by magnolol and PM.3. Magnolol and PM significantly decreased the G0/G1 cycle key protein cyclin D1 to block the cell cycle in G0/G1 cycle.4. Magnolol and PM could act as class I HDACi through the TRAIL pathway to induce cells apoptosis, but not exclusive mechanism. Magnolol and PM treatments could directly enhanced histone acetylation in the promoter of DR5 gene via significantly suppressing HDACs catalytic activities5. Magnolol and PM suppressed class I HDACs to inhibit A549 tumor xenograft growth and can induce cell apoptosis through TRAIL pathway in vivo. Magnolol and PM also prevented the occurrence and development of lung cancer. |
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