Article pubs.acs.org/jnp
Gigantol, a Bibenzyl from Dendrobium draconis, Inhibits the Migratory Behavior of Non-Small Cell Lung Cancer Cells Sopanya Charoenrungruang,† Pithi Chanvorachote,†,‡ Boonchoo Sritularak,§ and Varisa Pongrakhananon*,†,‡ †
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand ‡ Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand § Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand ABSTRACT: Lung cancer is one of the most common causes of cancer death due to its high metastasis potential. The process of cancer migration is an early step that is required for successful metastasis. The discovery and development of natural compounds for cancer therapy have garnered increasing attention in recent years. Gigantol (1) is a bibenzyl compound derived from the Thai orchid, Dendrobium draconis. It exhibits significant cytotoxic activity against several cancer cell lines; however, until recently, the role of 1 on tumor metastasis has not been characterized. This study demonstrates that 1 suppresses the migratory behavior of non-small cell lung cancer H460 cells. Western blot analysis reveals that 1 downregulates caveolin-1 (Cav-1), activates ATP-dependent tyrosine kinase (phosphorylated Akt at Ser 473), and cell division cycle 42 (Cdc42), thereby suppressing filopodia formation. The inhibitory effect of 1 on cell movement is also exhibited in another lung cancer cell line, H292, but not in normal human keratinocytes (HaCat). The inhibitory activity of 1 on lung cancer migration suggests that this compound may be suitable for further development for the treatment of cancer metastasis.
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Akt activation and Cdc42 in cell motility.13 Phosphorylated Akt contributes to up-regulation or activation of Cdc42 through actin reorganization and filopodia formation at the edge of moving cells.14 Recent evidence suggests that Cav-1 plays an essential role in cancer aggressiveness and metastasis, and its overexpression is closely associated with increased cancer migration.15−17 High levels of Cav-1 are found in many cancer types, including lung cancer,16,18−20 and overexpression of Cav1 results in increased motility of H460 human lung cancer cells, while knockdown of the protein causes the opposite effect.16 Taken together, these molecular pathways are potential targets for the inhibition of cell motility and, thus, the prevention of cancer metastasis. Several naturally derived compounds, including 1, exhibit promising anticancer activity.21−23 1 is a bibenzyl component isolated from the Thai orchid, Dendrobium draconis,24 and it was reported to have several pharmacological activities, such as inhibition of platelet aggregation, anti-inflammatory, and induction of cancer cell death and apoptosis.21,25−27 Currently, the antimetastasis activity of 1 on lung cancer remains unknown. This study aims to investigate the negative regulatory
orldwide, lung cancer is one of the most common causes of cancer-related death, and metastasis accounts for more than 90% of deaths from this type of cancer.1 Metastasis, which is a dissemination of primary tumors to secondary sites, consists of several steps, including cell migration, invasion, intravasation, extravasation, and establishment of secondary tumors. Cancer migration is recognized as an important prerequisite process that is necessary for successful metastasis, and a growing body of in vivo evidence has demonstrated that the inhibition of cancer motility can result in metastasis suppression.2,3 Several signaling proteins, such as a protein tyrosine kinase called focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), cell division cycle 42 (Cdc42), and caveolin-1 (Cav-1), are known to be responsible for cell migration.4−7 Increased FAK activation is tightly associated with enhanced migratory behavior and cancer metastasis,8,9 and FAK signaling regulates the formation and turnover of focal adhesions in cells in a moving state.10 The phosphorylation of FAK at Y397 triggers the recruitment of Src, which initiates signaling cascades that regulate cell migration.11 Likewise, increased Akt phosphorylation is associated with metastasis behavior in some cancer cells and has been shown, in certain cases, to be the downstream effector of FAK.12 Emerging evidence also implicates a linkage between © XXXX American Chemical Society and American Society of Pharmacognosy
Received: January 30, 2014
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in Hoechst-positive apoptosis cells, whereas PI-positive necrotic cells were not detected when compared with nontreated control cells (Figure 1B). Consistently, 1 at concentrations of less than 50 μM had only minimal effects on the number of apoptotic cells. Because the movement of the cells toward unoccupied areas may be due to their proliferative ability, the proliferative effect of 1 was further clarified. Figure 1C shows that no significant change in cell proliferation was observed in the cells treated with nontoxic doses of 1 over the treatment time. Nontoxic concentrations of 1 were further tested for their effects on cancer migration. Gigantol (1) Suppresses Lung Cancer H460 Cell Migration and Invasion. Cancer cell migration, which occurs after cell detachment from their primary site and prior to the establishment of secondary tumors,29 plays an important role during metastasis.28 Attenuation of migration is, thus, a potential therapeutic approach against cancer metastasis. To investigate the effect of 1 on H460 cell migration, cells were treated with various nontoxic doses of 1 (0−20 μM) for 24, 48, or 72 h, and the migratory behavior was analyzed using woundhealing and Boyden chamber assays. Wound-healing assays demonstrated that 1 was able to suppress cell migration into the wound space in a dose-dependent manner compared with nontreated controls (Figure 2A and C). Approximately 30, 50, and 70% reductions in motile activity were found in the cells treated with 5, 10, or 20 μM for 24 h, respectively (Figure 2A). Inhibition of H460 cell migration by 1 was also observed in a time-dependent manner compared with nontreated cells at each time point (Figure 2B and C). Furthermore, Boyden chamber assays revealed a similar trend of 1 limiting the migration of cells through the membrane. Compound 1 at 5,
role of 1 on lung cancer H460 cell migration and invasion using wound healing, Boyden chamber, and invasion assays. Western blot analysis of migratory-related proteins and immunostaining of filopodia formation were performed to identify the underlying mechanisms.
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RESULTS AND DISCUSSION Cytotoxicity of Gigantol (1) on Lung Cancer H460 Cells. Previous work suggests that 1 has the ability to induce lung cancer apoptosis through a mitochondria-dependent pathway.21 However, scientific evidence regarding the antimetastasis activity of 1 is required to support the development of the compound for further clinical study. To determine the effect of 1 on cancer migration, its cytotoxicity was investigated. H460 cells were incubated with various concentrations of 1 (0−500 μM) for 24 h, and cell viability was examined using the MTT assay. Figure 1A shows that 1 at concentrations of more than 50 μM causes a significant decrease in viability, with approximately 83, 80, and 20% of cells remaining viable in response to 50, 100 and, 500 μM 1, respectively, while concentrations of less than 20 μM showed no significant effect (Figure 1A). Data analysis indicated that the IC50 of 1 was approximately 247.55 ± 4.94 μM. Analysis of the mode of cell death further showed that 50 and 100 μM 1 caused an increase
Figure 1. Cytotoxic effect of gigantol (1) on human lung cancer H460 cells. (A) H460 cells were treated with various concentrations of 1 (0−500 μM) for 24 h. Cell viability was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The viability of untreated cells was represented as 100%. (B) H460 cells were treated with 1 (0−100 μM) for 24 h. Apoptotic and necrotic cell death were evaluated using Hoechst 33342/PI staining and calculated as a percentage compared with nontreated control cells. (C) H460 cells were treated with 1 (0−20 μM) for 12, 24, 48, and 72 h. Cell viability was determined by an MTT assay. The viability of nontreated cells was represented as 100%. Data represent mean ± SD (n = 4). *p < 0.05 versus nontreated control cells. B
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Figure 2. Effect of gigantol (1) on human lung cancer H460 cell migration. A confluent monolayer of H460 cells was wounded using a 1 mm width tip and (A) incubated with nontoxic doses of 1 (0−20 μM) for 24 h or (B) cultured in the presence or absence of 20 μM 1 for 24, 48, and 72 h. Wound space was analyzed and represented as migration level relative to the change of those in nontreated cells. Data represent the mean ± SD (n = 4). *p < 0.05 versus nontreated control cells. (C) After the indicated treatment, migrating cells in the denuded zone were photographed. (D) H460 cell migration was examined by Boyden chamber assay. Cells were treated with 1 (0−20 μM) for 24 h. Migratory cells at the basolateral side of the membrane were stained with Hoechst 33342 for 30 min and visualized by fluorescence microscopy. Data were plotted as an average number of cells in each field and represented the mean ± SD (n = 4). *p < 0.05 versus nontreated control cells.
with 1 (0−20 μM) for 24 h, and, filopodia protrusions were examined using a phalloidin staining assay. Figure 4A shows that H460 cells exhibited a substantial number of filopodia at the edge of their cell membranes that were significantly decreased in the presence of 1 (10−20 μM). These data suggest that 1 suppresses filopodia formation, and this effect, at least in part, inhibits cancer motility. Emerging evidence suggests that the accumulation of filopodia at the border of moving cells is regulated by Cdc42.31 To test whether the decrease in the number of filopodia protrusions caused by 1 involved Cdc42, cells were treated with 1 (0−20 μM) for 24 h and, subjected to protein expression analysis. Western blot analysis revealed that Cdc42 was substantially down-regulated in response to 1, compared with nontreated cells (Figure 4B). Decreased Cdc42 expression has been reported to impede cell migration,33 which supports the data indicating that 1 reduces the migratory behavior of lung cancer cells via Cdc42 attenuation and filopodia suppression.
10, or 20 μM resulted in approximately 0.7-, 0.4-, and 0.3-fold reductions in cell migration, respectively (Figure 2D). Because cancer migration and invasion are integral steps required for cancer metastasis,30 the possible anti-invasive effect of 1 was further tested using an extracellular matrix-coated transwell assay. Cells were added onto the coated Transwell insert and incubated with various concentrations of 1 (0−20 μM) for 24−48 h. Figure 3 shows that treatment with 1 gradually reduced the number of invaded H460 cells in a doseand time-dependent manner, compared with nontreated control cells. These results indicate the potential role of 1 in the suppression of cancer migration and invasion. Gigantol (1) Down-regulates Cdc42 and Attenuates Filopodia Protrusion. It has been reported that membrane protrusions, called filopodia, play an important role in cell movement and is tightly associated with cancer metastasis.31,32 To investigate whether the migration-inhibiting effect of 1 was involved with the regulation of filopodia, cells were incubated C
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Figure 3. Effect of gigantol (1) on human lung cancer H460 cell invasion. (A) H460 cells were treated with various nontoxic doses of 1 (0−20 μM) for 24 h. (B) H460 cells were treated with 1 (0−20 μM) or left untreated as control for various times (24 and 48 h). Cell invasion was evaluated using Transwell-coated inserts with matrigel as described in the Experimental Section. Data were plotted as an average number of cells in each field and represent the mean ± SD (n = 4). *p < 0.05 versus nontreated control cells. (C) Invading cells across the membrane were stained with Hoechst 33342 for 30 min and visualized by fluorescence microscopy.
Effect of Gigantol (1) on Migratory-Related Proteins. Previous research indicates that phosphorylation of Akt at Ser 473 is critical for cancer migration.34−36 Indeed, phosphorylated Akt regulates actin remodeling by phosphorylating several substrates, including Cdc42, which leads to cell motility, in part, by enhancing the formation of actin-rich structures known as filopodia at the focal adhesion site at the edge of the cell membrane.37,38 In addition, Cav-1 overexpression was found in advanced stage cancer and associated with metastasis.19 To study the specific proteins involved in the ability of 1 to inhibit cell motility, H460 cells were incubated with various concentrations of 1 (0−20 μM) for 24 h and migratoryrelating proteins were examined by Western blot analysis. Figures 4C and D show that treatment with 1 caused a gradual decrease of Cav-1 and phosphorylated Akt, whereas unphosphorylated Akt was not altered when compared with the nontreated control. In contrast, neither FAK phosphorylated at Tyr397 nor unphosphorylated FAK was affected by treatment with 1. Others have shown that signaling initiated through Src and FAK Tyr397 plays a central role in cancer cell motility.11 However, results from the present study indicate that 1 can abrogate the migratory activity of H292 cells in a manner associated with Akt down-regulation of Ser473 phosphorylation and Cav-1 expression but independent of the down-regulation of FAK Tyr397 (Figure 4C). To confirm the findings indicating that 1 inhibits migratory behavior through Cav-1 down-regulation, H460 cells were stably transfected with Cav-1 overexpressing, Cav-1 shRNA, or control plasmid. The cells were then treated with various doses of 1, and cell motility was examined using a wound healing assay. The data presented in Figure 5 illustrate that Cav-1-
overexpressing (H460/Cav-1) cells exhibited a greater migrating rate compared with control H460 (H460/ctrl) cells. In contrast, Cav-1 knockdown (H460/shCav-1) cells with the lowest level of Cav-1 exhibited minimal migratory activity (Figure 5B). Interestingly, 1 administration significantly attenuated cell movement to near the wound space in all Cav-1modulating cells (Figure 5B). Previous studies have reported that Cav-1 plays an essential role in cancer metastasis through Akt activation,16,20,39 and these data demonstrate a similar trend of activated Akt and Cav-1 expression in response to treatment with 1. To clarify whether 1 down-regulates Cav-1 expression and consequently suppresses Akt activation, phosphorylated Akt in Cav-1 modulating cells was examined. The data in Figure 5A demonstrate that phosphorylation of Akt was substantially decreased in Cav-1 knockdown cells. In contrast, phosphorylation of Akt was significantly increased in Cav-1-overexpressing cells compared with control cells, whereas total Akt was unchanged (Figure 5A). In addition, Akt phosphorylation was tightly correlated with motility (Figure 5B). These findings illustrate a novel molecular activity of 1 in the regulation of cancer migration via Cav-1 and Akt. The Antimigratory Activity of Gigantol (1) on Lung Cancer H292 Cells and Human Keratinocyte HaCat Cells. To confirm the inhibitory effect of 1 on lung cancer motility, an additional human lung cancer H292 cell line was investigated. H292 cells were treated with 1 (0−20 μM) for 24 h. Woundhealing assay data indicate that 1 could attenuate H292 cell motility (Figure 6A), which is consistent with the results using H460 cells and strengthens the evidence of the effect of 1 on lung cancer migration. In addition, the effect of 1 was studied in D
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Figure 4. Effect of gigantol (1) on filopodia formation and migratory-related proteins. (A) After treatment with a nontoxic dose of 1 for 24 h, cells were stained with either phalloidin or Hoechst 33342 and visualized by fluorescence microscopy. Filopodia protrusions are indicated by arrows and represented as an average number of filopodia per cell relative to nontreated cells. Values are mean of samples ± SD *p < 0.05 versus nontreated control cells. (B) After indicated treatment, H460 cells were collected and analyzed for cell division cycle 42 (Cdc42) or (C) focal adhesion kinase (FAK), phosphorylated FAK (Tyr 397; p-FAK), ATP dependent tyrosine kinase (Akt), phosphorylated Akt (Ser 473; p-Akt) and Caveolin-1 (Cav1) expression by Western blot analysis. The blots were reprobed with β-actin to confirm equal loading. The immunoblot signals were quantified by densitometry and mean data from four independent experiments were presented. Values are mean of samples ± SD *p < 0.05 versus nontreated control cells. Cells Culture. NCI-H460 and NCI-H292 human lung carcinoma cells and human keratinocyte HaCat cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). H460 and H292 cells were cultured in RPMI-1640 medium, and HaCat cells were cultured in DMEM medium at 37 °C in a 5% CO2 humidified incubator. Both mediums were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/mL streptomycin (Gibco, MD, USA). Cell Viability and Cell Proliferation Assay. Cell viability was analyzed using a colorimetric MTT assay. Ten thousand cells in 100 μL of RPMI medium were seeded onto each well of a 96-well plate and incubated overnight for cell attachment. Subsequently, the cells were treated with different concentrations of 1 (0−500 μM) for 24 h for the cell viability assays and for 12, 24, 48, and 72 h for the cell proliferation assays. After the indicated times, the medium was removed and replaced with MTT solution for 4 h at 37 °C. Formazan product was solubilized with 100 μL DMSO, and the intensity was measured at 570 nM using a microplate reader (Anthros, Durham, NC, USA). Viable cells were represented as the percentage cell viability relative to control cells as per the equation below. The IC50 was analyzed from four independent experiments using GraphPad Prism 5.0 (La Jolla, CA).
normal human keratinocyte HaCat cells. Treatment with 1 caused no inhibitory effect on HaCat cell movement (Figure 6B), suggesting that 1 selectivity inhibits cancer cell migratory behavior. In conclusion, this study demonstrates that 1 can suppress lung cancer cell migration through a Cav-1-dependent pathway. The down-regulation of Cav-1 expression in response to 1 leads to the attenuation of Akt phosphorylation and Cdc42 expression and, thereby, inhibits filopodia formation. The data presented here on the antimigratory activity of 1 could provide the basis for the future development of this compound as a novel therapy for overcoming cancer metastasis.
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EXPERIMENTAL SECTION
Chemicals and Antibodies. 1 was isolated from Dendrobium draconis as previously described,24 and its purity was determined using HPLC and NMR spectroscopy. 1 with more than 95% purity was used in this study. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, propidium iodide (PI), phalloidin tetramethylrhodamine B isothiocyanate, bovine serum albumin (BSA), and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical, Inc. (St. Louis, MO, USA). Antibodies for phosphorylated Akt (Ser473), Akt, phosphorylated FAK (Tyr397), FAK, Cdc42, Cav-1 and β-actin, and peroxidase-conjugated secondary antibodies were obtained from Cell Signaling (Denvers, MA, USA).
%Cell viability =
OD570 of treated cells x100 OD570 of untreated control cells
Apoptosis Assay. Apoptosis and necrosis were identified using a fluorescent DNA staining assay with Hoechst 33342 and propidium E
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Transwell filter (8 μm pore) in RPMI medium containing 0.1% FBS and incubated with various concentrations of 1. RPMI medium containing 10% FBS was added to the lower chamber. Following incubation, the nonmigratory cells on the upper side of the membrane were removed by wiping with a cotton swab, and the cells that had migrated to the underside of the membrane were stained with 10 μg/ mL Hoechst 33342 for 30 min, visualized, and scored using fluorescence microscopy (Olympus IX51 with DP70). Invasion Assay. Cells were seeded in 24-well plates at a density of 1.5 × 105 cells/well onto the upper filter of Matrigel-coated Transwell (8 μm pore) filters in RPMI medium containing 0.1% FBS and then incubated with various concentrations of 1. RPMI medium containing 10% FBS was added to the lower chamber. Following the incubation, the cells on the upper side of the membrane were removed, and those that invaded the underside of the membrane were stained with 10 μg/ mL Hoechst 33342 for 30 min, visualized, and scored using fluorescence microscopy. Cell Morphology Characterization. Cells were seeded at a density of 5 × 104 cells/well onto a six-well plate and incubated overnight for attachment. The cells were treated with various concentrations of 1 for 24 h, washed with PBS, fixed in 4% paraformaldehyde in PBS, permeabilized by 0.1% Triton-X100 in PBS, and blocked with 0.2% BSA for 30 min. The cells were then incubated with rhodamine-phalloidin diluted 1:100 in PBS for 15 min and rinsed three times with PBS. Cells were cytospun onto a glass slide, mounted, and imaged using fluorescence microscopy. Plasmids and Transfection. The Cav-1 expression and control plasmids were obtained from the American Type Culture Collection (Manassas, VA, USA), and the Cav-1 knockdown plasmid (shCav-1) was obtained from Santa Cruz Biotechnology. Stable transfection was established by culturing the cells in a six-well plate until they reached 60−70% confluency. Fifteen microliters of Lipofectamine 2000 reagent and 2 μg of Cav-1, shCav-1, or control plasmid were used to transfect the cells in the absence of serum. After 12 h, the medium was replaced with culture medium containing 10% fetal bovine serum. Approximately 2 days after the beginning of transfection, the cells were released by 0.025% trypsin, plated onto 75 cm2 culture flasks, and cultured for 30 days with antibiotic selection. The stable transfectants were pooled, and the expression of the Cav-1 protein in the transfectants was confirmed by Western blotting. The cells were cultured in G418-free RPMI 1640 medium for at least two passages before use in each experiment. Western Blot Analysis. Cells were seeded at a density of 5 × 105 cells/well onto six-well plates overnight. After specific treatments, the cells were washed twice with cold PBS and incubated with lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM sodium chloride (NaCl), 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail for 40 min on ice. Cell lysates were collected, and the protein content was determined using the BCA protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein from each sample (60 μg) were denatured by heating at 95 °C for 5 min in Laemmli loading buffer and subsequently loaded onto 10% SDS-polyacrylamide gels. After separation, the proteins were transferred onto 0.45 μm nitrocellulose membranes, and the transferred membranes were blocked in 5% nonfat dry milk in TBST (25 mM Tris-HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20) for 1 h and subsequently incubated with a specific primary antibody overnight at 4 °C. The membranes were washed three times with TBST for 10 min and incubated with horseradish peroxidase (HRP)conjugated anti-rabbit or anti-mouse IgG for 2 h at room temperature. After three washes with TBST, the immune complexes were detected using chemiluminescence (Supersignal West Pico; Pierce) and quantified using the analyst/PC densitometry software (Bio-Rad). Statistical Analysis. The data were obtained from at least four independent experiments and are presented as the mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA and a post hoc test (Tukey’s test) at a significance level of p < 0.05. SPSS 17.0 was used for all statistical analyses.
Figure 5. Effects on cell migration of Cav-1 overexpression and knockdown. (A) H460 cells were stably transfected with Cav-1, shCav1 or control plasmid as described in the Experimental Section. Expression of Cav-1, phosphorylated Akt (Ser473; p-Akt) and Akt in the transfected cells was determined by Western blotting. The blots were reprobed with β-actin to confirm equal loading. The immunoblot signals were quantified by densitometry and mean data from four independent experiments are presented. Data represent the mean ± SD (n = 4). *p < 0.05 versus control transfected (H460/Ctrl) cells. (B) Confluent monolayer of Cav-1 overexpressing (H460/Cav-1) Cav-1 knockdown (H460/shCav-1) and control (H460/Ctrl) cells were wounded using a 1 mm width tip and incubated with nontoxic dose of 1 (20 μM) for 24 h. Migrating cells in the denuded zone were photographed and wound space was analyzed and represented as migration level relative to the change of those in nontreated cells. Data represent the mean ± SD (n = 4). *p < 0.05 versus nontreated cells. iodide (PI). Ten thousand cells in 100 μL of RPMI medium were seeded onto each well of a 96-well plate and incubated overnight for cell attachment. The cells were then treated with various concentrations of 1 (0−500 μM). At the indicated times, the cells were incubated with 10 μg/mL Hoechst 33342 and 5 μg/mL PI for 30 min. The cells were then visualized using fluorescence microscopy (Olympus IX51 with DP70) as previously described.40 Migration Determination. Migration was determined using wound healing and Boyden chamber assays. For the wound healing assay, the cells were seeded at a density 2.5 × 105 cells/well in a 24well plate. After the cell monolayer was formed, a micropipette tip was used to scratch the attached cells to generate a wound space. The cells were then washed with PBS and replaced with serum-free RPMI medium containing various concentrations of 1. The progress of cell migration into the wound was photographed using an inverted microscope at the indicated times. The average wound size represented the relative cell migration and was calculated by dividing the change of the wound space of the sample by that of the control cells in each experiment. For the Boyden chamber assay, cells were seeded at 5 × 104 cells/well onto an upper 24-well plate of the F
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Figure 6. Effect of gigantol (1) on human nonsmall cell lung cancer H292 and human keratinocyte HaCat cell migration. Confluent monolayer of (A) H292 cells and (B) HaCat cells were wounded using a 1 mm width tip and incubated with nontoxic doses of 1 (0−20 μM) for 24 h. Migrating cells in the denuded zone were photographed and wound space was analyzed and represented as migration level relative to the change of those in nontreated cells. Data represent the mean ± SD (n = 4). *p < 0.05 versus nontreated cells.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +662-218-8341. Fax: +662-218-8340. E-mail: varisa.p@ pharm.chula.ac.th. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to the 90th Anniversary of Chulalongkorn University, Ratchadaphisaksomphot Endownment Funds, and Faculty of Pharmaceutical Sciences, Chulalongkorn University (Phar2556-RG07; V.P.).
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