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Oct 7, 2014 - (7) O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that removes the most cytotoxic lesions generated by TMZ, has been widely ...
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Pine (Pinus morrisonicola Hayata) Needle Extracts Sensitize GBM8901 Human Glioblastoma Cells to Temozolomide by Downregulating Autophagy and O6‑Methylguanine-DNA Methyltransferase Expression Chia-Leng Liao,† Chien-Min Chen,‡ Yan-Zin Chang,§ Guang-Yaw Liu,∥ Hui-Chih Hung,⊥ Tung-Ying Hsieh,§ and Chih-Li Lin*,§,# †

Department of Neurology, Jen-Ai Hospital, Taichung 412, Taiwan Department of Neurosurgery, Changhua Christian Hospital, Changhua 500, Taiwan § Institute of Medicine, and ∥Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung 402, Taiwan ⊥ Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung 402, Taiwan # Department of Medical Research, Chung Shan Medical University Hospital, Taichung 402, Taiwan ‡

ABSTRACT: Pine needle extracts of Pinus morrisonicola (Hayata) are commonly used as a functional health beverage. However, it remains unclear what the mechanism is underlying the antitumor activity of pine needle extract. The aims of present study were to investigate the anti-glioblastoma effects of pine needle extracts as well as its bioactive compounds. From three different solvent extracts of pine needles, the water extract displayed the strongest cytotoxicity effects on GBM8901 glioblastoma cells. The isolated compounds were identified as pinocembrin, chrysin, and tiliroside. Chrysin was the most active ingredient of pine needle extract for the induction of apoptosis and suppression of migration and invasion. It also markedly inhibited temozolomide (TMZ)-induced autophagy and O6-methylguanine-DNA methyltransferase (MGMT) expression. Because both autophagy and MGMT overexpression have been implicated to TMZ-induced drug resistance in glioblastoma, our results showed that pine needle extract and chrysin may serve as a potential anticancer agent against glioblastoma, especially with regard to sensitizing glioblastoma cells resistant to TMZ. KEYWORDS: pine, glioblastoma, chrysin, temozolomide (TMZ), autophagy

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INTRODUCTION Polyphenol compounds are abundantly present as micronutrients in our diet, and evidence is available on the role of these compounds in promoting health benefits and preventing various diseases. Several polyphenols, which are responsible for the unique properties of herbs, have been identified in dietary sources. Pinus morrisonicola Hayata, also called as Taiwan white pine or Taiwan short-leaf pine, is endemic to Taiwan and has diverse healing properties. For decades, pine needles have been used as an important functional health food that provides potentially positive health benefits beyond basic nutrition.1 Many studies have shown that pine needles have the most biological activity;2 therefore, they are widely used for the production of wine and soft drinks having health-promoting effects. Studies have also reported its physiological activities and therapeutic effects, including antioxidant, anti-inflammatory, antibacterial, and anticancer properties.3 Pine needle extracts contain high levels of phytochemicals, especially flavonoids. Although interest in this area is growing, limited data are available on the effectiveness of pine needle extracts for the prevention and treatment of cancer. Therefore, were performed this study to elucidate the underlying mechanisms and to identify individual compounds having anticancer benefits. Glioblastoma multiforme (GBM) is the most deadly form of human brain cancer. At present, significant tumor migration, © 2014 American Chemical Society

invasion, and chemoresistance are the major barriers to the effective treatment of glioblastoma.4 Patients with such highly invasive tumors show poor prognosis, with a median survival time of only approximately 9−12 months, until the initiation of chemotherapy with temozolomide (TMZ), a standard treatment for glioblastoma.5 TMZ is a DNA-alkylating agent that mispairs nucleotide bases during DNA replication, thus preventing tumor cell division and inducing apoptotic pathways.6 This improvement in median survival by TMZ treatment is extremely significant but modest. The limited success of TMZ in glioblastoma appears to be related to the occurrence of chemoresistance and the inability of TMZ in inducing tumor cell death. Consequently, the tumor recurs within 6−8 months. At cellular and molecular levels, several mechanisms are responsible for the recurrence of glioblastoma.7 O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that removes the most cytotoxic lesions generated by TMZ, has been widely implicated in inducing TMZ resistance in glioblastoma cells.8 In addition, resistance of glioblastoma cells to TMZ may be related to the induction of autophagy.9,10 However, the Received: Revised: Accepted: Published: 10458

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cally at 550 nm. The cell viability was determined as the percentage of control cells treated with the vehicle alone. High-Performance Liquid Chromatography (HPLC) and Tandem Mass Spectrometry (MS/MS) Analyses. Sample separation was performed using a HPLC system (JASCO model PU-2080, Tokyo, Japan). Isocratic chromatography was performed using separate reversed-phase (RP)-HPLC by employing the following conditions: fractionation in a LUNA C18 analytical column packed with 5 μm particles (250 × 4.6 mm, Phenomenex, Torrance, CA) and elution with a mobile phase (deionized water/methanol/formic acid = 79.1:20:0.1) at a flow rate of 1.0 mL/min that was monitored at a wavelength of 280 nm [SPD-10 AV ultraviolet−visible (UV−vis) detector, Shimadzu, Kyoto, Japan]. MS was performed using an API 3000 triple quadruple mass spectrometer equipped with a Turbo IonSpray interface operated in negative electrospray ionization (ESI, AB SCIEX, Framingham, MA). All quantitative and qualitative analyses were performed in a multiple reaction monitoring (MRM) scan. Western Blot Analysis. GBM8901 cells were harvested and homogenized in lysis buffer [50 mM Tris−HCl at pH 8.0, 5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol] for 30 min at 4 °C. Equal amounts of total cellular proteins (50 μg) were resolved by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE), transferred onto polyvinylidene difluoride membranes (Millipore), and probed using primary antibodies, followed by horseradish-peroxidase-conjugated secondary antibodies. The immunocomplexes were visualized using enhanced chemiluminescence kits (Millipore). Nucleus Morphology. Changes in cell nuclei morphology that were characteristic of apoptosis were examined using a fluorescence microscope (AXioskop 2, Zeiss) on cells grown on coverslips. For this, cells were fixed in 4% paraformaldehyde after 24 h of treatment with the indicated compound, permeabilized in ice-cold methanol, and incubated with 1 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) staining under the fluorescence microscope for 15 min at room temperature. Apoptosis by Flow Cytometry. Cells were cultured as described above. The percentage of sub-G1 apoptotic cells was examined by the propidium iodide (PI) technique using flow cytometry. Briefly, GBM8901 cells (1 × 106/mL) were fixed in 70% methanol, resuspended in phosphate-buffered saline (PBS) incubated with 50 μg/mL PI for 0.5 h, and analyzed by FACScan (Becton Dickinson, San Jose, CA). Cell Migration and Invasion Assay. The cell migration and invasion assays were performed in a 24-well Boyden chamber with an 8 μm pore size polycarbonate membrane (Corning, NY). For the migration assay, 1 × 105 cells in 200 μL serum-free medium were added to the upper compartment of the chamber; the lower compartment was filled with 600 μL of RPMI 1640 supplemented with 10% fetal bovine serum (FBS). After incubation at 37 °C for 24 h, GBM8901 cells remaining in the upper chamber were removed using swabs. The cells on the lower surface of the membrane were fixed with methanol, stained with 0.1% crystal violet, photographed, and counted using a light microscope. The invasion assay was performed using the same procedure, except that the membrane was coated with Matrigel (BD Biosciences) to form a matrix barrier and 3 × 104 GBM8901 cells were added to the upper compartment of the chamber. Acridine Orange Staining. Acridine orange is a dye that specifically labels intracellular acidic vesicles. GBM8901 cells were treated with the indicated compounds for 24 h. The cells were then stained with 1 μg/ mL acridine orange for 15 min, washed with RPMI 1640 medium, and examined under the fluorescence microscope. Protein Expression Knockdown by siRNA Technology. The beclin1 and Atg7 siRNA gene silencer (human) dsRNA was obtained from Santa Cruz Biotechnology (sc-44286 and sc-41448). Neuroblastoma cells were transfected with dsRNAs using siRNA transfection reagent (Santa Cruz) and incubated for 6 h. Afterward, the cells were analyzed by immunoblot for protein expression. Statistical Analysis. All data were obtained from at least three separate experiments and were expressed as the mean ± standard error of the mean (SEM). Statistical comparison of the differences among multiple groups was performed using one-way analysis of variation

precise role of autophagy in the death of tumor cells, especially TMZ-treated glioblastoma cells, is not yet entirely clear. Because previous studies have shown the potential anticancer effects of pine needle extracts, we performed this study to investigate the anti-glioblastoma effects as well as the bioactive compounds present in pine needle extracts. In particular, chrysin (5,7-dihydroxyflavone), a major phenolic compound in pine needle extract, may be responsible for the anticancer properties of pine needle extracts.11 Chrysin exhibits antioxidant and antiviral activities, reduces blood lipids, and prevents mutagenesis. In one study, chrysin showed antitumor activities by suppressing tumor cell proliferation and inducing apoptosis.12 However, studies on the effects of chrysin on human glioblastoma are rare. In this study, we showed that chrysin was the most active ingredient in pine needle extract that induced apoptosis and suppressed the migration and invasion of glioblastoma cell line GBM8901. Furthermore, chrysin markedly inhibited autophagy and reduced MGMT expression, which may contribute to TMZ chemoresistance. Taken together, our results suggested that pine needle extracts containing chrysin might provide potential beneficial effects against glioblastoma and attenuate resistance to TMZ in a synergistic behavior. This may help in developing anti-proliferative and anti-recurrence strategies by sensitizing glioblastoma cells resistant to TMZ.

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MATERIALS AND METHODS

Materials. Chrysin, TMZ, 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), 3-methyladenine (3-MA), chloroquine (CQ), and rapamycin were purchased from Sigma-Aldrich (München, Germany). Caspase inhibitor zVAD-fmk was obtained from R&D Systems (Minneapolis, MN). Antibodies to β-actin, anti-pmTOR, antimTOR, beclin1, p62, and MGMT were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cleaved PARP, cleaved caspase 3, anti-pAkt, and anti-Akt antibodies were obtained from Millipore (Bedford, MA). Anti-LC3, Atg5-Atg12, and Atg7 antibodies were purchased from Novus Biologicals (Littleton, CO). Acridine orange was obtained from Life Technologies (Rockville, MD). Sample Preparation. Fresh leaves (pine needles) of P. morrisonicola Hayata were collected from Taiwanese mountains in Nantou county at elevations of 300−2300 m. The preparation of pine needle extracts for this study was modified from a previous report.13 Briefly, pine needles were air-dried, homogenized, and ground. The obtained powder (200 g) was extracted with water, methanol, or ethanol at room temperature for 48 h. Next, the respective solutions were filtered and dried by evaporation of the filtrates in vacuum at 40 °C. The pine needle extracts of water, methanol, and ethanol were named pine water extract (PWE), pine methanol extract (PME), and pine ethanol extract (PEE), respectively. The dried samples were stored at −20 °C until further use. Cell Culture and Viability Assay. GBM8901 human glioblastoma cells, EOC 13.31 glia cells, and Detroit 551 and NIH3T3 fibroblast cells were obtained from Bioresources Collection and Research Center (Hsinchu, Taiwan). GBM8901 cells were maintained in RPMI-1640 medium; Detroit 551 cells were maintained in minimum essential medium (MEM); and EOC 13.31 and NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco). Primary mixed glial cell cultures were established from neonatal rat brain tissue according to a previous protocol.14 All cultures were supplemented with 10% fetal calf serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine and incubated at 37 °C in a humidified atmosphere of 5% CO2. In the viability assay, the cells were seeded in 96well plates at a density of 1 × 104 cells/well and were incubated overnight. Next, the cells were treated with indicated conditions. After 24 h, the tetrazolium salt MTT was added to the medium according to the instructions of the manufacturer. Only viable cells metabolized MTT to a purple product formazan, which was quantified spectrophotometri10459

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Figure 1. P. morrisonicola Hayata needle extracts have cytotoxic effects on GBM8901 cells. (A) Cytotoxic potential of the three solvent extracts. GBM8901 cells were incubated with 0−200 μg/mL pine extracts for 24 h. Cytotoxicity was measured using the MTT method, and the values are represented as the mean ± SEM of triplicates. PME, pine methanol extract; PEE, pine ethanol extract; PWE, pine water extract. (B) Major ingredients of PWE were identified by HPLC. Three compounds, pinocembrin [retention time (RT) = 5.2 min], chrysin (RT = 5.5 min), and tiliroside (RT = 11.8 min), were identified in the extracts. (C) MS/MS product ion profiles of pinocembrin, chrysin, and tiliroside. (∗) p < 0.05 and (∗∗) p < 0.01 when compared to non-treated cells using the one-way ANOVA test.

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(ANOVA) tests, and differences between pairs of groups were compared using Student’s t test. Statistical significance was assumed when the p value was ≤0.05. All of the statistical analyses were performed using the SPSS statistical software (SPSS, Inc., Chicago, IL).

RESULTS Cytotoxic Effects of Various Solvent Extracts of P.

morrisonicola (Hayata) on the GBM8901 Cell Line. 10460

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Figure 2. Chrysin-induced apoptosis in GBM8901 cells. (A) Effects of PWE, pinocembrin, chrysin, and tiliroside against GBM8901 cells after 24 h of treatment. Among these compounds, chrysin showed the most powerful anti-glioblastoma activity by reducing cell viability. (B) Chrysin exerted cytotoxicity in GBM8901 cells in a dose-dependent manner but only limited the effect in Detroit 551 human normal skin fibroblasts at a concentration of 100 μM. Measuring apoptosis by (C) fragmented nuclei stained and (D) flow cytometry analysis. (E) Caspase 3 and PARP activation were determined by immunoblotting and evaluated in chrysin-treated GBM8901 cells. Results are expressed as the mean ± SEM of three independent experiments performed in triplicates. (∗) p < 0.05 and (∗∗) p < 0.01 when compared to non-treated cells using the one-way ANOVA test. Proteins from three independent western blot experiments were quantified using densitometry and normalized to actin band intensity. The scale bar represents 50 μm.

Phenolic compounds are commonly present in pine and are reported to have multiple biological effects, such as growth suppression and death induction. To determine whether pine needle extracts have antiproliferative effects on glioblastoma cells, the ability of crude pine needle extracts to inhibit the

viability of GBM8901 cells was examined. Figure 1A shows the cytotoxic effect of various solvent extracts of pine needles, including PME, PEE, and PWE. The IC50 values of PWE, PME, and PEE were 121.0, 208.5, and 243.7 μg/mL, respectively. Of these different solvent extracts, the inhibitory effect of PME was 10461

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Figure 3. Chrysin inhibits the migration and invasion of GBM8901 cells. Transwell migration and Matrigel invasion assays showed that chrysin treatment resulted in a significant inhibition of (A) 74% in migration and (B) 68% in invasion. Experiments were performed in triplicates, and values are presented as the mean ± SEM. (#) p < 0.05 for one-way ANOVA or (∗) p < 0.05 and (∗∗) p < 0.01 for Student’s t test.

lines, including Detroit 551, NIH3T3, EOC 13.31, and rat primary mixed glial cells, to 100 μM chrysin did not induce significant growth inhibition. These results confirmed that chrysin might inhibit the growth of glioblastoma cancer cells without significantly affecting normal cells. To determine further the relationship between growth inhibition and apoptosis, cells were stained with the nuclear staining cytochemistry dye DAPI to analyze the morphology of the nuclei. As shown in Figure 2C, observation of stained fragmented nuclei confirmed that chrysin induced apoptosis in GBM8901 cells. Apoptotic cells were simultaneously quantified by flow cytometry, and there was a significant increase in the percentage of cells in the sub-G1 phase after treatments of chrysin (Figure 2D). Apoptosis induction by chrysin was further examined by western blotting. Figure 2E shows a dose-dependent cleavage of caspase 3 and poly(ADPribose)-polymerase (PARP) in GBM8901 cells treated with chrysin. Thus, a higher concentration of chrysin (100 μM) induced apoptosis after 24 h of treatment. Chrysin Inhibits Migration and Invasion by GBM8901 Cells. The above results showed that chrysin induced apoptosis in glioblastoma cells through caspase 3 and PARP pathways. However, a low concentration of chrysin (25 μM) did not show this strong apoptosis-inducing effect. Most studies have reported that glioblastoma is the most migratory and invasive brain tumor and is invariably fatal for affected patients.15 Therefore, GBM8901 cells were treated with chrysin to determine their

similar to that of PEE at a higher concentration. However, the IC50 value of PWE on GBM8901 cell viability was the lowest. To determine the active ingredients on pine needle extracts that act against GBM8901 cells, the PWE was further analyzed using HPLC (Figure 1B). Using C18 column material and an acidic mobile phase, all of the major constituents of PWE were wellresolved within 20 min. HPLC signals were assigned by standard comparison of UV spectra and were then used directly for MS/ MS studies to identify the specific compounds. Using these methods, three compounds were identified from the extract: pinocembrin, chrysin, and tiliroside. These results indicate that the three phenolic compounds were predominantly present in PWE and represented approximately 29% of the total flavonoid content. Chrysin Reduces the Proliferation and Induces Apoptosis in GBM8901 Cells. To determine the most active component of PWE, the cytotoxicity of these pure individual compounds was examined. As shown in Figure 2A, all of the compounds inhibited the proliferation of GBM8901 cells significantly. However, chrysin showed the highest inhibitory activity against GBM8901 cells, indicating that chrysin has a greater anti-glioblastoma activity in PWE. Figure 2B shows that chrysin concentrations ranging from 25 to 100 μM inhibited the growth of GBM8901 cells in a time-dependent manner. The growth inhibition was approximately 50% at a concentration of 100 μM after 24 h. However, exposure of several normal cell 10462

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Figure 4. continued

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Figure 4. Chrysin sensitizes GBM8901 cells to TMZ-induced cytotoxicity. (A) Cells were treated for 24 h with the indicated concentrations of TMZ and chrysin (20 μM) or PWE (50 μg/mL). (∗) p < 0.05 and (∗∗) p < 0.01 when compared to TMZ-treated cells for the one-way ANOVA test. (B) Fluorescence microscopy analysis of GBM8901 cells treated with TMZ and chrysin (20 μM), PWE (50 μg/mL), 3-MA (1 mM), or CQ (50 μM) for 24 h, followed by acridine orange staining. The formation of acridine-orange-accumulating acidic vacuoles (orange−red fluorescence) in TMZ-treated cells was significantly suppressed by chrysin, PWE, and autophagic inhibitors 3-MA or CQ treatments. (C) Western blots showing autophagy induction by monitoring LC3, Atg12−Atg5 conjugate, Atg7, beclin1, and p62 levels in GBM8901 cells treated or not treated with TMZ (100 μM) alone or in combination with PWE (50 μg/mL), chrysin (20 μM), or 3-MA (1 mM) for 24 h. (D) Fluorescent microscopy analysis of cells LC3 treated with TMZ (100 μM) alone or combined with chrysin (20 μM) or rapamycin (200 nM) co-treatments for 24 h. (E) Beclin1 and Atg7 siRNA (20 nmol/L pool) knockdown was confirmed via western blot, and autophagy blockade was validated using LC3-II western blot. Enhanced apoptosis in response to TMZ (100 μM) was determined via PARP cleavage. (F) Western blot analysis showed p-Akt/Akt, p-mTOR/mTOR, and MGMT in GBM8901 cells alone or treated with increasing concentrations of chrysin in combination with TMZ (100 μM). (G) Both rapamycin (200 nM) and zVAD-fmk (10 μM) treatments for 24 h blocked chrysin-induced cell death significantly. However, 3-MA or CQ treatments enhance cell death similar to that in chrysintreated groups. (∗) p < 0.05 and (∗∗) p < 0.01 when compared to cells treated with TMZ + chrysin for Student’s t test. The mean ± SEM of three independent experiments performed in triplicates is shown. Proteins from three independent western blot experiments were quantified using densitometry and normalized to actin band intensity. The scale bar represents 50 μm.

combination of PWE (50 μg/mL) or chrysin (20 μM) with TMZ had synergistic cytotoxic effects on GBM8901 cells. This suggested that PWE or chrysin might be effective TMZsensitizing agents. Studies have been reported that therapeutic resistance of glioblastoma is attributed to the activation of druginduced autophagy. Therefore, we first used acridine orange staining to examine whether acidic vacuoles were induced in GBM8901 cells after treatment with TMZ. After exposure to TMZ (100 μM) for 24 h, cells showed an increase in orange fluorescence, indicating the accumulation of acridine orange in the acidic compartments of TMZ-treated cells. However, cotreatment with PWE or chrysin significantly decreased the orange fluorescence (Figure 4B). Similar to PWE or chrysin, treated cells with the autophagy inhibitors 3-methyladenine (3MA) and chloroquine (CQ) can both significantly reduce the number of detected orange acidic vesicles. To verify the obtained results, western immunoblotting was performed to probe the autophagy hallmark LC3. TMZ-induced autophagy was determined by the shift in the molecular weight of LC3 from LC3-I to LC3-II. On the other hand, PWE or chrysin markedly inhibited TMZ-induced LC3-II formation. Similar reduction was observed in Atg7, Atg12−Atg5 conjugate, beclin1, and p62. Besides, autophagy inhibition was accomplished using 3-MA, which caused similar effects compared to chrysin groups (Figure

migratory and invasive capacities, which are the two most important features of malignant cells. As shown in Figure 3A, chrysin treatment remarkably suppressed the migration of GBM8901 cells by approximately 40−70% in a dose-dependent manner compared to that in control groups. Because cell migration is believed to be a fundamental step in tumor invasion, we next examined the effect of chrysin on the invasive capacity of GBM8901 cells. Treatment of Matrigel-coated wells with chrysin reduced cell invasion significantly in a dose-dependent manner. Because we used a lower dose of chrysin, the above antimetastatic effects of chrysin were not mainly attributed to its cytotoxicity, as shown in Figure 2B. Our findings suggest that low levels of chrysin effectively inhibited cell migration and invasion by GBM8901 cells. Chrysin Sensitizes GBM8901 Cells to TMZ by Inhibiting Autophagy and Attenuating MGMT Expression. Because TMZ therapy is the current standard of care for patients with malignant glioblastoma, we next examined whether TMZ can decrease cell growth of the GBM8901 cell line. As shown in Figure 4A, TMZ was unable to decrease the growth of GBM8901 cells even at concentrations of up to 200 μM. These results are consistent with the results of previous studies, which showed that GBM8901 cells are grade-IV GBM cells with an inherent tendency of drug resistance and recurrence.16 In contrast, a 10464

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The therapeutic benefit of TMZ also depends upon its ability to alkylate/methylate the DNA in tumor cells. MGMT expression in glioblastoma cells is the most critical determinant of TMZ resistance.20 MGMT counteracts the effects of TMZ by removing DNA methyl adducts, suggesting that downregulation of MGMT is effective for the management of TMZ-resistant glioblastoma.21 To date, many clinical trials have aimed to modulate MGMT expression for increasing TMZ efficiency. In this regard, our present study showed that chrysin administration suppressed MGMT expression. Previous studies have suggested that Akt activation contributes to MGMT overexpression in glioblastoma.22 These findings indicated that chrysin-inhibited Akt signaling may be a strong and selective approach against TMZ-resistant glioblastoma.23 Logically, activation of AktmTOR signaling inhibits autophagy. However, our data indicated that mTOR phosphorylation was unaltered but phosphorylation of Akt at Ser473 was significantly inactivated by chrysin treatment. This indicates chrysin-inhibited autophagy through an Akt-mTOR signaling-independent pathway. Interestingly, accumulating studies indicated that autophagy is mediated in many circumstances by negative modulation of apoptosis.24 Moreover the autophagy-associated protein, LC3, and Akt were maintained during the treatment with TMZ, suggesting that glioblastoma cells escape from TMZ-induced cell death because of Akt signaling pathways.25 Our results demonstrated that chrysin induces apoptosis and downregulates Akt of GBM8901 cells significantly, suggesting that chrysininduced apoptotic signaling serves to inhibit autophagy. This result is consistent with those of previous studies, which have shown that pharmacologic inhibition of stress-induced autophagy was reported to promote chemotherapeutic response.26 However, the mechanisms mediating the counter-regulation of apoptosis and autophagy are not yet fully understood. In addition, recent studies have reported that chrysin inhibits nuclear factor E2-related factor 2 (Nrf2) by downregulating the PI3K/Akt/Nrf2 pathway.27 The Nrf2 signaling is known to activate autophagy-related genes independent of mTOR.28 Thus, our data indicated that chrysin decreased the expression of Akt, which may have contribute to the inhibition of autophagy by a similar pathway in GBM8901 cells. However, these ideas remain to be examined further. Chrysin, a natural, biologically active dietary flavonoid extracted from propolis and other plants, has various beneficial health effects. Chrysin exerts an anticancer effect through various mechanisms, including inhibition of the PI3K/Akt pathway and activation of mitogen/stress-activated kinase 1 (MSK1) signaling.29,30 Chrysin also selectively sensitizes cancer cells to apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by inhibiting STAT3 and Mcl-1.31 Moreover, chrysin decreases the expression of vimentin, suggesting that it attenuates epithelial−mesenchymal transition and suppresses metastases.29 This results indicate the potential of chrysin in treating cancer and overcoming therapeutic resistance. In this study, we reported that chrysin is an active compound in pine needle extracts and potentially inhibits autophagy and MGMT expression, thus sensitizing glioblastoma cells to TMZ. Similar results were obtained by Markiewicz-Ż ukowska et al., who showed antiproliferative properties of chrysin-rich ethanolic extract of propolis in glioblastoma and its synergistic action with TMZ.32 On the basis of these results, we proposed that chrysin may be a useful monotherapy to enhance brain tumor apoptosis and inhibit tumor migration/invasion or a component of combination therapy to overcome TMZ resistance and prolong

4C). Furthermore, an increase of LC3 punctate dots was observed in TMZ-only groups but abolished by co-treating of chrysin (Figure 4D). In contrast, chrysin-induced autophagy blockade returned to levels similar to those of control for the rapamycin-treated cells. Thus, these independent approaches suggested that TMZ-induced autophagy in GBM8901 cells was inhibited by the co-administration of PWE or chrysin. To further confirm the observations, knockdown of the autophagy constituent, beclin1 and Atg7, was conducted using targetspecific siRNAs and cells were treated with or without TMZ, as shown in Figure 4E. Both beclin1 and Atg7 knockdown resulted in pronounced GBM8901 cell apoptosis in response to TMZ treatment. One study reported that TMZ-induced autophagy in glioblastoma cells is dependent upon MGMT.17 Because MGMT overexpression is a principle mechanisms contributing to TMZ resistance in glioblastoma cells, we examined whether chrysin regulated MGMT expression to increase sensitivity to TMZ. Immunoblotting results shown in Figure 4F indicated that GBM8901 cells expressed higher levels of MGMT, irrespective of TMZ treatment. However, application of chrysin markedly suppressed MGMT expression by inhibiting AKT signaling in a dose-dependent manner. Besides, the expression of phosphomTOR (Ser2448) was unaltered by chrysin, indicating chrysininhibited autophagy through a mTOR signaling-independent pathway. To further elucidate the impact of autophagy on chrysin-mediated cell death, GBM8901 cells were treated with autophagy inducers or inhibitors to determine cell viability. As shown in Figure 4G, 3-MA or CQ enhanced glioblastoma cell death similar to that in chrysin-treated groups. However, both rapamycin and zVAD-fmk treatments blocked chrysin-induced cell death, indicating that autophagy supported the viability of GBM8901 cells against chrysin-induced apoptosis. Taken together, these data showed that chrysin might sensitize GBM8901 cells to TMZ by attenuating MGMT expression and TMZ-induced autophagy simultaneously.

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DISCUSSION Glioblastoma is the most malignant human brain tumor, characterized by increased growth and invasion. The standard therapy for glioblastoma is maximal resection, followed by radiotherapy and concurrent chemotherapy with drugs, such as TMZ. However, glioblastoma is still associated with poor prognosis because of high recurrence after TMZ chemotherapy. To enhance the benefit of TMZ in the treatment of aggressive glioblastoma, effective combination strategies that sensitize glioblastoma cells to TMZ are important to prevent the recurrence of these tumors. In this regard, natural products, such as flavonoids, have received considerable attention because of their lower side effects.18 In this study, we evaluated the inhibitory effects of pine needle extracts on a malignant glioblastoma cell line GBM8901. Our results showed that chrysin, a major biologically active component in pine needle extracts, inhibited the growth and invasiveness of the glioblastoma cell line. We further observed that chrysin also suppressed TMZ-induced autophagy, which protects glioblastoma cells against chronic TMZ treatment. Signaling through both the Akt pathway and autophagy is frequently activated in glioblastoma cells.19 Because autophagy is a prosurvival response that makes glioblastoma cells resistant to chemotherapy, our results suggested that use of the nutraceutical chrysin for inhibiting Akt activation and stress-induced autophagy may be a potential therapeutic approach for treating glioblastoma, especially in cases of TMZ-resistant tumor recurrence. 10465

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the survival of patients with malignant glioblastoma. Pine needle extracts and chrysin are reported to have various effects on the central nervous system (CNS),13,33 suggesting that chrysin may have the ability to cross the blood−brain barrier (BBB). In fact, most flavonoids are amphipathic molecules that appears to be capable of passing the BBB and reaching the CNS.34 However, further studies, especially in in vivo models, are needed to elucidate the efficacy of the anti-glioblastoma effects of chrysin. In conclusion, we identified an efficient method to extract chrysin from pine needles and evaluated the anti-glioblastoma effects of chrysin, which could be potentially used as an efficacious component in functional food products. Our study also showed a novel role of chrysin in sensitizing glioblastoma cells to TMZ. These findings are consistent with those obtained from a model that simultaneously suppresses TMZ-induced autophagy and MGMT expression in TMZ-resistant glioblastoma, suggesting that modulation of the autophagic pathway may be a novel way to modify resistance to TMZ. Therefore, chrysin in combination with TMZ chemotherapeutic agent warrants further examination.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +886-4-2473-0022, ext. 12405. Fax: +886-4-24723229. E-mail: [email protected]. Funding

This work was supported by grants from the Chung Shan Medical University−Jen-Ai Hospital (CSMU-JAH-101-002), the Changhua Christian Hospital (101-CCH-IRP-23), and the Ministry of Science and Technology (98-2320-B-040-015-MY3 and 101-2320-B-040-015-MY3). The fluorescence microscope and imaging analyzer were performed in the Instrument Center of Chung Shan Medical University, which is supported by the Ministry of Science and Technology, Ministry of Education, and Chung Shan Medical University. Notes

The authors declare no competing financial interest.

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