Available online at www.sciencedirect.com
ScienceDirect Journal of Nutritional Biochemistry 25 (2014) 177 – 185
Curcumin inhibits lung cancer cell migration and invasion through Rac1-dependent signaling pathway Qing-yong Chen a,⁎, 1 , Ying Zheng b, c, 1 , De-min Jiao a, 1 , Fang-yuan Chen d , Hui-zhen Hu a , Yu-quan Wu a , Jia Song a , Jie Yan a , Li-jun Wu a , Gui-yuan Lv b,⁎ a
Department of Respiratory Disease, The 117th Hospital of PLA, Hangzhou, Zhejiang 310013, P.R. China b Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, P.R. China c Department of Pharmacy, The 117th Hospital of PLA, Hangzhou, Zhejiang, 310013, P.R. China d The Second Affiliated Hospital of Shaanxi Chinese Medicine University, Xianyang, Shaanxi, 712000, P.R. China
Received 27 April 2013; received in revised form 16 September 2013; accepted 4 October 2013
Abstract Curcumin, a natural and crystalline compound isolated from the plant Curcuma longa with low toxicity in normal cells, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about antimetastasis effects and mechanism of curcumin in lung cancer. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis. In this study, we examined the influence of curcumin on in vitro invasiveness of human lung cancer cells and the expressions of Rac1. The results indicate that curcumin at 10 μM slightly reduced the proliferation of 801D lung cancer cells but showed an obvious inhibitory effect on epidermal growth factor or transforming growth factor β1-induced lung cancer cell migration and invasion. Meanwhile, we demonstrated that the suppression of invasiveness correlated with inhibition of Rac1/PAK1 signaling pathways and matrix metalloproteinase (MMP) 2 and 9 protein expression by combining curcumin treatment with the methods of Rac1 gene silence and overexpression in lung cancer cells. Laser confocal microscope also showed that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. At last, through xenograft experiments, we confirmed the connection between Rac1 and the growth and metastasis inhibitory effect of curcumin in vivo. In summary, these data demonstrated that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through inhibition of Rac1/PAK1 signaling pathway and MMP-2 and MMP-9 expression, which provided a novel insight into the molecular mechanism of curcumin against lung cancer. © 2014 Elsevier Inc. All rights reserved. Keywords: Curcumin; Migration; Invasion; Rac1; Actin cytoskeleton; MMP-2/9
1. Introduction Lung cancer, the leading cause of cancer deaths, has the most rapidly increasing incidence rate in the developed country and in China. Clinical data showed that most lung cancer patients eventually suffered from relapse and/or metastasis after complete excision of the cancer, even if they were at stage IA [1]. Despite great progresses have been made in the last decades, the detailed mechanism of lung cancer relapse and metastasis is not fully understood. Rac1, an important small Rho GTPases family protein, has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis [2]. Overexpression of Rac1 is ⁎ Corresponding authors. Qing-yong Chen is to contacted at Department of Respiratory Disease, The 117th hospital of PLA, Hangzhou, Zhejiang 310013, P.R. China. Tel./fax: +86 57187340861. Gui-yuan Lv, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China. Tel./fax: +86 57186613601. E-mail addresses: [email protected] (Q. Chen), [email protected] (G. Lv). 1 These authors contributed equally to this work. 0955-2863/$ - see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2013.10.004
considered as an independent predictor of adverse outcome of some carcinomas [3]. Inhibition of Rac1 expression or disruption of its function significantly reduces cancer cells metastasis in many tumor models [4,5]. Our recent studies also showed that overexpression of Rac1 was widespread in primary lung cancer patients. Silence of Rac1 expression by shRNA suppressed lung cancer cells migration, invasion and induced rearrangements of the actin cytoskeleton in lung cancer cells [6]. Rac1 regulates cellular functions such as cytoskeletal dynamics, cell adhesion and transcription via activating PAK1 (one of the best characterized member of Rac1 effectors) and other downstream signaling molecules. Therefore, agents that inhibiting Rac1 or its downstream targets might have anticancer metastatic effect. Curcumin (diferuloylmethane), an active component of the spice turmeric (Curcuma longa), has chemopreventive and therapeutic properties against many tumors both in vitro and in vivo [7,8]. Several studies have shown that curcumin induces apoptosis more potently in cancer cells than in normal cells and attributed its inhibitory effect to the inhibition of angiogenesis nitric oxide synthase, receptor tyrosine kinase and protein kinase C activities and regulation of certain gene transcriptional factors, such as c-Jun/AP-1, JNK, Nuclear factor κB
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(NF-κB) and P53 [8–11]. Previous studies from our lab showed that curcumin inhibited cell proliferation and induced cell apoptosis in lung cancer through the modulation of lysosomal pathway and reactive oxygen species-dependent mitochondrial signaling pathway [7,12]. Recently, some studies on the anticancer effect of curcumin have focused on anti-invasion and antimetastasis aspect [13,14]. However, the effects of curcumin on lung cancer metastasis and underlying molecular mechanism remains remain largely unknown. The Rac1 signaling pathway is closely associated with tumor invasion and metastasis, and curcumin showed a potent inhibitive role on metastasis in various cancer cells, which implies an inner relationship between inhibitory effects of curcumin and Rac1 signaling. In present study, we determined the effects of curcumin on the migration and invasion of lung cancer cells both in vitro and in vivo systems and further demonstrated that the inhibitory effects of curcumin were relate to the inhibition of Rac1/PAK1 signaling pathways, matrix metalloproteinase (MMP) 2/9 expression and actin cytoskeleton rearrangements. These results provide a novel insight into the molecular mechanisms of curcumin in inhibition of lung cancer cell migration and invasion, and also show potential therapeutic value of curcumin in preventing lung cancer metastasis. 2. Materials and methods 2.1. Reagents and cell culture Curcumin was purchased from Sigma Chemical Co. Rac1 antibody was purchased from Upstate Biotechnology Inc (New York, NY, USA). PAK1, p-PAK1, MMP-2 and MMP-9 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Human epidermal growth factor (EGF) and transforming growth factor β1 (TGF-β1) were obtained from (PreproTech GmbH, Hamburg, Germany). A Rac1L61 plasmid, encoding active form of Rac1, was provided by Dr. Liang Fan (Nanfang Hospital affiliated to Southern Medical University, China). Plasmid expressing green fluorescent protein (GFP)-tagged negative shRNA or Rac1 shRNA was generated as described previously [6]. Human large cell lung carcinoma 801D cell line was purchased from the Cell Bank at the Chinese Academy of Sciences, which was first developed by Dr. Lezhen Chen [15], The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin and streptomycin at 37°C in a humidified atmosphere of 5% CO2.
2.5. Cell migration assay Cell migration was assayed by wound healing assay as described previously [9]. Initially, cells were allowed to grow to 80%–90% confluence in 12-well plates precoated with 0.1% gelatin (Sigma) and then starved with serum-free RPMI 1640 medium overnight. After that, cells were scraped by pipette tips to create a denuded zone (gap) of constant width. Subsequently, cellular debris was washed with PBS, and the cells were exposed to indicated concentrations of EGF or TGF-β1. The wound closure was monitored and photographed at 0 and 24 h under a Leica inverted microscope. To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation. Artificial lines fitting the cutting edges were drawn on pictures of the original wounds and overlaid on the pictures of cultures after incubation. Migrated cells across the white lines were counted in six random fields from each triplicate treatment, and data are presented as mean±S.D. 2.6. Cell invasion assay Invasion assay was carried out using modified matrigel Boyden chambers consisting of 24-well Millicell (Millipore Corporation, Shanghai, China) membrane filter (8-μm pore size) as described previously [13]. Dilute Matrigel (50 μl; BectonDickinson; 1:3) in serum-free cold cell culture media and applied to the top side of filter. Briefly, cells were trypsinized and resuspended in serum-free medium. Two hundred microliters of the cell suspension (105 cells) with 10 μM curcumin was added to the upper chamber of each well and 0.1% of dimethyl sulfoxide (DMSO) as the solvent control. The bottom chambers were filled with 500 μl RPMI 1640 medium supplemented with 10% fetal bovine serum and growth factor (EGF or TGF-β1). The chamber was incubated for 20 h at 37°C. At the end of incubation, the cells in the upper surface of the membrane were carefully removed with a cotton swab. Cells invading across the matrigel to the lower surface of the membrane were fixed with methanol and stained with 0.5% crystal violet. The invading cells on the lower surface of the membrane filter were counted with a light microscope. The data presented are the average number of cells attached to the bottom surface from five random fields. Each experiment was carried out in triplicate. 2.7. Laser confocal microscope To determine the effect of curcumin on cell morphology and cytoskeleton, cells were plated in six-well plates containing 12-mm glass coverslips and grown for 16 h and then treated with curcumin and indicated concentrations of growth factor (EGF or TGF-β1). After the exposure period, the medium was removed, and the cells were washed with PBS and then fixed with 4% formaldehyde dissolved in PBS for 10 min at room temperature and permeabilized for 10 min with 0.2% TritonX-100. Cells were incubated with 500 ng/ml tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma) at room temperature in PBS containing 0.1% Triton X-100 for 45 min. The cells were examined and photographed by confocal microscopy. 2.8. Reverse transcription polymerase chain reaction assay
2.2. Real-time cell analysis cytotoxicity testing Real-time cell analysis (RTCA) system (Roche Applied Science, Indianapolis, IN, USA) was employed for dynamic assessment of curcumin toxicity. The principle and use of the RTCA system have been described previously [16]. Briefly, under the control of RTCA software, the sensor analyzer automatically selected wells to be measured and continuously monitored changes in electrode impedance of cultivated cells in the electronic sensor plate. Cell index (CI) is used to represent cell status based on electrical impedance, which is proportional to the culture area covered by attached cells so that toxicity-induced cell shrinkage and detachment of cells from the plate due to cell death result in a lower CI value. In the study, the effect of the curcumin was monitored dynamically for every 15 min. The CI against the time was plotted.
2.3. Assessment of cell proliferation by MTT assay The antiproliferative effect of curcumin on 801D lung cancer cells was also determined by the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye uptake method. Briefly, the cells were seeded in quintuplicate at a density of 5×103 cells per well in a 96-well plate in a final volume of 0.2 ml medium and incubated for 24 h at 37°C. Curcumin was added to each well and incubated for another 24 h. The cells were washed with phosphate-buffered saline (PBS), and 20 μl MTT solution (5 mg/ ml) was added to each well. After a 4-h incubation at 37°C, 150 μl isopropanol was added. The absorbance of MTT formazan was measured at 490 nm.
2.4. Cell transfection Cells plated in 6-well plates were transfected with indicated plasmids using Lipofectamin 2000 (Invitrogen) according to the manufacturer's instructions. After 24 h of transfection, GFP positive cells were sorted using fluorescence activated cell sortor (FACS) and used for different experiments.
Total RNA was isolated from each group of cells using Trizol reagent (Invitrogen) according to the manufacturer's instruction. Obtained complementary DNAs were amplified using specific primers. The polymerase chain reaction (PCR) from each sample was performed by the following conditions: 5 min at 94°C, 30 cycles of 30 s at 94°C, 1 min at 55°C, and final extension for 1 min at 72°C. Primers for Rac1 are as follows: 5′-ATGCAGGCCATCAAGTGTGTGGTG-3′ (sense) and 5′-TTACAACAGCAGGCATTTTCTCTTCC-3′ (antisense). 2.9. Western blot analysis The whole-cell extracts were prepared in RIPA buffer [20 mM Tris, 2.5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 40 mM NaF, 10 mM Na4P2O7 and 1 mM phenylmethanesulfonylfluoride (PMSF)]. Thirty micrograms of cellular protein of each sample was applied to immunoblot following 10% SDSpolyacrylamide gel electrophoresis and probed with specific antibodies as indicated, followed by a horseradish peroxidase-conjugated goat antimouse or antirabbit antibody (Millipore). Immunoreactive bands were visualized by enhanced chemiluminescence (Millipore) according to the manufacturer's instructions. Quantification of reactive protein bands was performed by densitometric analysis, and the fold change was calculated by normalizing with control β-actin levels. 2.10. In vivo animal studies The cell viability and cell number of 801D cells were calculated by trypan blue. Then, 1×107 live cells in RPMI-1640 medium were injected subcutaneously into the flanks of mice (nude mice, average 25 g, purchased from Shanghai Laboratory Animal Center, Shanghai, China). All experimental procedures used in this study had been approved by the ethics committee in the 117th Hospital of PLA, and all animal experiments had been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The authors who performed experiments had given their informed consent
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prior to the study and had followed “principles of laboratory animal care” (National Institutes of Health publication no. 86-23, revised 1985). Tumor-bearing mice were then divided randomly into treatment groups (three mice per group) and treatment initiated when the xenografted solid tumors reached a volume of about 50 mm3. Each mouse was injected intraperitoneally every 3 days with either 30 μl of control vehicle (DMSO), curcumin (30 and 45 mg/kg) or cisplatin (DDP, 8 mg/kg). Mice exhibiting tumors were monitored, and tumor size was measured once every 3 days using calipers. The tumor volume in each animal was estimated according to the formula: tumor volume (mm3)=L×W2/2 (where L is the length and W is the width), with the final measurement taken 4 weeks after curcumin treatment, and at the same time, the body weight of each animal was measured once every day. At the end of the experiment (4 weeks after curcumin treatment), the mice were anesthetized by CO2 and sacrificed. Tumors from each animal were removed, measured and weighed individually. Part of the mice tumor tissue was fixed in 4% paraformaldehyde in PBS and then embedded in paraffin. Serial sections were cut at 6-μm thickness and stained with
Fig. 1. Cytotoxic effect of curcumin on 801D cell line. (A) CI was measured every 15 min by the RTCA system; dose- and time-dependent cytotoxicity was caused by curcumin in 801D cells. (B) Cell viability based on MTT assay after 24-h curcumin exposure. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control). (C) Cell viability based on microscopic observation after 24-h curcumin exposure; photographs were taken at a ×100 magnification.
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hematoxylin and eosin (H&E) and immunohistochemistry. Immunohistochemical staining and evaluation were described in previous studies analysis [6].
2.11. Statistical analysis All experiments were performed in triplicate and analyzed by T test or ANOVA (SPSS, version 16.0) for significant differences. P values less than .05 were considered statistically significant. Where appropriate, the data are presented as the mean±S.D.
Fig. 2. Curcumin inhibited EGF-induced cell migration and invasion. (A) 801D confluent monolayer cells were scratched with a pipette tip and then treated with 10 μM curcumin in the presence of 100 ng/ml EGF or not. Representative images showing the inhibitory effect of curcumin on EGF-induced cell migration at 24 h. (B) The invasion ability of 801D was determined by invasion assay. Cells in low surface of the Boyden chamber were stained and photographed under a light microscope at ×100 magnification, and invaded cells were quantified by counting the number of stained cells. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
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3. Results
3.2. Curcumin inhibits EGF- or TGF-β1-induced migration and invasion of lung cancer cells
3.1. Curcumin inhibits the viability of lung cancer cells First, we used the RTCA to examine the antiproliferative effects of curcumin on lung cancer cells. Fig. 1A shows typical RTCA curve of 801D cells responding to curcumin treatment (CI vs. exposure time). The index curve culminated to the maximum gradually without curcumin treatment, indicating that the untreated cells continue to grow and proliferate with time. However, the CI curves were dramatically different in response to curcumin treatment (10, 20, 40, and 60 μM). The results showed that curcumin treatment resulted in a time- and dose-dependent loss of cell viability, which was similar to the results of MTT assay (Fig. 1B) and microscopic observation (Fig. 1C). Interestingly, we also found that antiproliferative effect of curcumin on lung cancer cells is more obvious than on normal lung bronchial epithelial cells (data not shown). Curcumin (10 μM) was shown to have a weak effect on the growth of 801D lung cancer cells as shown in our results. Therefore, the concentration was used in the subsequent experiments.
Epidermal growth factor receptor (EGFR) and TGF-β-dependent signaling pathways are pivotal in promoting lung cancer cell growth and metastasis [17,18]. EGF and TGF-β receptors were also reported two important targets of curcumin [19,20]. In the study, we used EGF and TGF-β1 to enhance the motor abilities of 801D lung cancer cells and evaluate the effects of curcumin on cell migration and invasion. As shown in Fig. 2 and Supplementary Material Fig. S1, 801D cells exposed to either EGF or TGF-β1 enhanced the 801D cell migration into a wounded area and across the transwell membrane. However, in the presence of 10 μM curcumin, cell migration and invasion stimulated by EGF and TGF-β1 were significantly reduced. Apparently, curcumin can effectively inhibit EGF- or TGF-β1-induced migration and invasion of lung cancer cells. 3.3. Reduced expression of Rac1 protein in association with curcumin treatment Rac1 is an important GTPase that has been implicated in many cellular processes such as cytoskeleton rearrangement, cell adhesion and transcriptional activation. Besides, it also has been suggested to be involved in cancer cell migration, invasion and metastasis. In this study, we performed reverse transcriptase PCR and Western blot assays to determine whether Rac1 is regulated by curcumin, and the results showed that the expression of Rac1 in 801D cells can be inhibited by curcumin both at the messenger RNA (mRNA) and protein levels (Fig. 3A). Importantly, similar inhibition effects were also found in other lung cancer cells (data not shown). In addition, either EGF or TGF-β1 stimulation can elevate the expression of Rac1 protein in 801D cells, and pretreatment of 801D cells with 10 μM curcumin partly abolished EGF or TGF-β1-induced Rac1 overexpression (Fig. 3B and Supplementary Material Fig. S2), suggesting that Rac1 is likely to be the downstream effector of curcumin and may be involved in the migration and invasion suppression by curcumin. 3.4. Altered Rac1 protein expression affects lung cancer cell migration and invasion In our previous and present studies, we have been found that either silence of Rac1 by shRNA or the use of specific inhibitor of Rac1 (NSC23766) suppressed 801D lung cancer cells migration and invasion (Fig. 4A, B). To further confirm the involvement of Rac1 in the inhibitory effect of curcumin on cell migration and invasion, we measured cell migration and invasion after transfection with constitutive active Rac1L61 plasmid. As we expected, Rac1L61 plasmid significantly elevated the cell migration and invasion ability compared with the negative control (Fig. 4C, D) and can be also attenuated by curcumin treatment. Thus again, we found that curcumin inhibited cell migration and invasion by interfering with the Rac1-dependent signaling pathway. 3.5. Cell actin cytoskeleton arrangement induced by curcumin treatment
Fig. 3. Curcumin inhibits endogenous and EGF-induced Rac1 expression. (A) Curcumin treatment for 24 h significantly inhibited the expression of Rac1 both at the mRNA and protein levels. (B) Pretreatment of 801D cells with 10 μM curcumin resulted in inhibition of EGF-induced Rac1 expression. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
It is well established that actin polymerization and depolymerization (actin dynamics) play a crucial function in cell motility. Rac1 has been widely implicated in cytoskeleton rearrangements. EGF and TGF-β1-induced phenotype change is associated with actin remodeling and has been considered as a characteristic of invasive and metastatic cells [17,21]. Therefore, we then examined the effect of curcumin on cytoskeleton changes stimulated by EGF or TGF-β1. As shown in Fig. 5 and Supplementary Material Fig. S3, both EGF (100 ng/ ml) and TGF-β1 (6 ng/ml) increased polymerization of actin within
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Fig. 4. Rac1 regulated cell migration and invasion in vitro. (A) Compared with controls, transfection with Rac1shRNA or treatment with Rac1 inhibitor (NSC23766, 100 μM) significantly inhibited the migration of 801D cells in wound healing assay. (B) For invasion assay, the invasive cell number of 801D transfected with Rac1shRNA was significantly less than that of the negative control, Rac1 inhibitor (NSC23766) has the same effect as Rac1shRNA. C-) Transfection with Rac1L61 plasmid obviously enhanced the cell migration and invasion ability compared with the negative control, and curcumin clearly inhibited the migration and invasion of Rac1L61-transfected cells. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
indicated time as visualized by TRITC-phalloidin staining, whereas Rac1 shRNA treated cells exhibited a decrease of polymerization of actin. Treatment with curcumin also inhibited TGF-β1-induced actin polymerization, suggesting that Rac1 is involved in this actin polymerization and curcumin can inhibit EGF- or TGF-β1-induced migration through actin remodeling in 801D cells.
but no changes were detected at total PAK1 level. Similar results were also found in Rac1-silenced cells. Conversely, cells transfected with constitutive active Rac1L61 enhanced the phosphorylation of PAK1 and were significantly inhibited by curcumin, suggesting that the antimetastatic function of curcumin in the lung cancer cells is likely, at least in part, through the Rac1-regulated phosphorylation of PAK1.
3.6. Curcumin inhibits PAK1 phosphorylation, a Rac1 downstream protein related to cell invasion
3.7. Curcumin inhibited MMP-2 and MMP-9 expression
Here, we also explored whether curcumin affects the expression of PAK1, a Rac1 downstream functional protein, which has been shown to involve in cell invasion and migration. As shown in Fig. 6B, PAK1 phosphorylation was inhibited significantly in curcumin-treated cells,
The Rho families of small GTPases and MMPs are the two protein families that play key roles in cell movement. Rac1 was shown to mediate the expression of MMPs in different cell types [22]. In our study, we also found that the expression of MMP-2 and MMP-9 significantly increased in cells transfected with constitutive active
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Fig. 5. Curcumin and Rac1 silencing inhibited EGF-induced cytoskeleton changes. Thirty minutes of EGF stimulation induced a significant increase of filopodia and lamellipodia formation. However, this effect was inhibited by curcumin treatment or Rac1 shRNA transfection.
Rac1L61 plasmid, but abolished by Rac1 knockdown or curcumin treatment. Furthermore, curcumin inhibited the expression of MMP-2 and MMP-9 in the Rac1L61-transfected cells (Fig. 7). Therefore, we believe that curcumin inhibits cell-associated expression of MMP-2 and MMP-9 through the regulation of Rac1 activity. 3.8. Curcumin inhibits lung cancer growth and invasion in vivo Overgrowth and metastasis are two major characteristics of malignant tumors. We therefore investigated whether curcumin could suppress lung cancer tumor growth and invasion in vivo. Cisplatin (DDP), a commonly used medicine for clinical chemotherapy, was also used as a positive control in our study. As shown in Fig. 8A and B, the volume and average weight of tumors in control group increased more obviously than those in curcumin-treated group. In addition, H&E staining showed that the tumors and lung tissues in curcumin-treated groups had clear boundaries with less invasiveness. In contrast, tumors and lung tissues arising from control groups displayed characteristics of invasion, indicating that curcumin inhibited invasive lung cancer in vivo (Fig. 8C).
Fig. 6. Effects of curcumin inhibited the levels of phospho-PAK1. Cells were treated with curcumin (10–20 μM) or transfected with indicated plasmids and then treated with or without curcumin; the expressions of Rac1 (A) and phosphorylated and total PAK1 (B) were examined by Western blot. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
To determine the relationship among Rac1 expression, lung tumor growth, metastasis and curcumin treatment in vivo, we examined the expression of Rac1 and Ki-67 (a tumor marker related to malignant degree) in the nude mice xenograft model. We found that formed tumors showed strongly positive Rac1 and Ki-67 staining in the controls, but the tumors exhibited only weak immunoreactions in curcumin-treated cells (Fig. 8C). These data suggest that Rac1 is involved in lung cancer growth and invasion, and Rac1 expression can also be inhibited by curcumin in vivo.
4. Discussion Recently, some studies on the anticancer effect of curcumin have focused on anti-invasion and antimetastasis aspect [13,14,23]. However, the effects of curcumin on lung cancer metastasis
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Fig. 7. Curcumin inhibited MMP-2 and MMP-9 expressions in 801D cells. The protein expressions of MMP-2 and MMP-9 were significantly inhibited by curcumin in Rac1L61 transfection and nontransfection cells. Rac1 shRNA showed same inhibitory effects as curcumin on MMP-2 and MMP-9 expression. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
and underlying molecular mechanism remains remain largely unknown. In the present study, we found that low-toxic levels of curcumin (10 μM) could efficiently inhibit EGF- or TGF-β1induced migration and invasion of lung cancer cells in vitro, and this effect is related to inhibition of the Rac1/PAK1 signaling pathway. Furthermore, we examined the effects of curcumin in xenograft mouse lung tumor model and found that Rac1 is also involved in lung cancer growth and metastasis in vivo. To our knowledge, this is the first study reporting that Rac1 and PAK1 were found to be involved in the antimetastasis action of curcumin in lung cancer cells. Initially, we measured the effect of curcumin on cell proliferation. As shown in Fig. 1, curcumin dramatically inhibited cell growth of 801D lung cancer cells in a dose- and time-dependent manner. Meanwhile, cell viability inhibition of curcumin in 801D lung cancer cells was not significant when curcumin was used as low as 10 μM, suggesting that cell death or apoptosis is not the main factor for inhibiting lung cancer cell migration by lower concentrations of curcumin, which was used in our study. Rac-1 belongs to the Rho family of GTPases, and it functions as a signal regulator to the actin cytoskeleton. Several lines of evidence have shown that Rac1 can be activated by various growth factor receptors (EGFR [24], α6β4 integrin receptor [25], TGF-β receptor [26] and so on through guanine nucleotide exchange factors such as Sos proteins, Tiam1 and Vav2. Interestingly, all these growth factor receptors are also reported as the targets of curcumin [19,20,27,28]. The exact role of these receptors in anti-invasion effects of curcumin in lung cancer needs more researches. In our study, we genetically modulated the expression levels of the Rac1 gene in lung cancer 801D cells by either up- or down-regulation (Rac1L61, Rac1 inhibitor and Rac1 shRNA). Then, the results from curcumin treatment supported the evidence that the effect of inhibiting lung cancer cell migration and invasion by curcumin is at least partly due to the inhibition of Rac1 expression.
Fig. 8. Curcumin inhibited the invasion and metastasis of lung cancer in vivo. (A) All mice of the untreated groups developed tumors approximately 30 days after injection. The tumors formed in the curcumin-treated group grew more slowly. (B) The tumor weight of the untreated groups was heavier than that of the curcumin-treated groups. (*Pb.05 and **Pb.01 vs. untreated groups; n=5). (C) H&E staining revealed the cellular heterogeneity in pathological sections of the excised tumor tissue. Untreated groups displayed characteristics of invasion. In contrast, curcumin-treated groups showed clear boundaries with less invasiveness (original magnification, ×200). Immunohistochemistry showed that the tumors formed by the untreated groups were positive or strongly positive with Ki-67 and Rac1 stain, whereas the tumors formed by the curcumin-treated groups were weakly positive with Ki-67 and Rac1 stain (original magnification, ×200).
The organized polymerization of actin filaments is believed to be an important mechanism required for cell migration [29]. Rac1 is reported to be a key regulator of actin dynamics that lead to organized actin-based structures associated with cell migration [30]. In this study, we investigated the effect of curcumin on actin cytoskeleton by phalloidin stain in 801D cells. The results showed that curcumin
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dramatically inhibited the formation of stress fiber, filopodia, and membrane ruffles in EGF- or TGF-β1-treated 801D cells. These effects mimicked those of Rac1 shRNA transfection and NSC23766 treatment, suggesting that the blocking of Rac1 expression and the disruption of microfilament skeleton rearrangement are both involved in curcumin-induced cell migration inhibition. It have been reported that the inhibitory effect of Rac1 on myosin contractility can be mediated by the Rac1 effector PAKs [31]. PAK1 is well known as a downstream effector of Rac1. It binds to Rac1 in a GTP-dependent manner. Activated PAK1 (phospho-PAK1) regulates cellular functions such as cytoskeletal dynamics, cell adhesion and transcription. The formation of actin meshwork lamellipodia stimulated by phospho-PAK1 in the leading edge is crucial for cell migration [6]. Downstream targets for PAKs include myosin light-chain kinase (MLCK). MLCK phosphorylates the myosin light chain, which is important in regulating actin cytoskeletonl dynamics [32]. In addition, PAK phosphorylates and activates LIM kinase, which, in turn, phosphorylates and inactivates the actin-severing protein cofilin, thus promoting filament assembly [33]. In the present study, we found that curcumin significantly attenuated PAK1 phosphorylation. This inhibition might lead to PAK1 inactivation and, in turn, affect the downstream cytoskeletal targets of PAK1. These effects can also been detected in Rac1 shRNA-transfected cells. Moreover, Western blotting results also showed that cells transfected with Rac1L61 enhanced the phosphorylation of PAK1 compared with the negative control. Curcumin inhibited the phosphorylation of PAK1 in Rac1L61-transfected cells, suggesting that curcumin exerted its antimetastatic activity via regulating the activity of Rac1 downstream functional proteins. MMP-2 and MMP-9 play the most important role for basal membrane type IV collagen degradation, a critical step for cancer invasion and metastasis. In our study, we also used Western blotting to test the expression of these two invasion associated proteins. The results showed that the expression of MMP-2 and MMP-9 was decreased significantly when curcumin was used. These data are in agreement with previous studies where curcumin inhibited the migration and invasion of other tumor cells via down-regulation of MMP-9 or MMP-2 expression [23,34,35]. Rac1 was shown to mediate the activation of MMPs in different cell types [36,37]. To further investigate whether down-regulation of MMP-9 and MMP-2 expression by curcumin was through Rac1 signaling in lung cancer cells, 801D cells were transfected with Rac1 shRNA or Rac1L61 plasmid, and the expression levels of MMP-2 and MMP-9 were examined by Western blotting. The results showed that MMP-2 and MMP-9 expression increased significantly by Rac1L61 plasmid. Conversely, it was diminished by Rac1 shRNA transfection, suggesting that MMP-2- and MMP-9-mediated cell invasion may be regulated by Rac1. However, how Rac1 affected the MMPs is still not clear. Previous study has shown the linkage of Rac1 and MMP activity through NF-κB-dependent signaling [36]. Recently, a study reported that Rac1 may regulate MMP-9 expression via H2O2 production primary supplied by mitochondria [37]. The experiment regarding the relationship of MMP-2 and MMP-9 expression with NF-κB signaling and/or H2O2 production is undergoing in our laboratory. In conclusion, we identified a novel mechanism for the inhibitory effect of curcumin on the invasion and migration of lung cancer cells. The reduction in the metastasis ability observed in curcumin-treated cells is at least partly due to the inhibition of Rac1/PAK1 pathway signaling and the decreased MMP-2 and MMP-9 expression. The above findings and concepts disclosed here provide an important basis for a further exploration toward understanding the action mechanisms of curcumin and possibly its beneficial effect in the prevention of tumor metastasis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jnutbio.2013.10.004.
Acknowledgments This work was supported by the Zhejiang Traditional Chinese Medicine Administration Bureau (Project no. 2008CA077) and the Nanjing military area commands “334” high-level scientific and technological personnel training plan. We would like to thank Drs. Yanyi Wang and Yingke Xu for their technical assistance and discussion.
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[31] Bokoch GM. Biology of the p21-activated kinases. Annu Rev Biochem 2003;72:743–81. [32] Sanders LC, Matsumura F, Bokoch GM, de Lanerolle P. Inhibition of myosin light chain kinase by p21-activated kinase. Science 1999;283:2083–5. [33] Edwards DC, Sanders LC, Bokoch GM, Gill GN. Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1999;1:253–9. [34] Liao S, Xia J, Chen Z, Zhang S, Ahmad A, Miele L, et al. Inhibitory effect of curcumin on oral carcinoma CAL-27 cells via suppression of Notch-1 and NF-kappaB signaling pathways. J Cell Biochem 2011;112:1055–65. [35] Yodkeeree S, Chaiwangyen W, Garbisa S, Limtrakul P. Curcumin, demethoxycurcumin and bisdemethoxycurcumin differentially inhibit cancer cell invasion through the down-regulation of MMPs and uPA. J Nutr Biochem 2009;20:87–95. [36] Kheradmand F, Werner E, Tremble P, Symons M, Werb Z. Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science 1998;280:898–902. [37] Murthy S, Ryan A, He C, Mallampalli RK, Carter AB. Rac1-mediated mitochondrial H2O2 generation regulates MMP-9 gene expression in macrophages via inhibition of SP-1 and AP-1. J Biol Chem 2010;285:25062–73.
ScienceDirect Journal of Nutritional Biochemistry 25 (2014) 177 – 185
Curcumin inhibits lung cancer cell migration and invasion through Rac1-dependent signaling pathway Qing-yong Chen a,⁎, 1 , Ying Zheng b, c, 1 , De-min Jiao a, 1 , Fang-yuan Chen d , Hui-zhen Hu a , Yu-quan Wu a , Jia Song a , Jie Yan a , Li-jun Wu a , Gui-yuan Lv b,⁎ a
Department of Respiratory Disease, The 117th Hospital of PLA, Hangzhou, Zhejiang 310013, P.R. China b Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, P.R. China c Department of Pharmacy, The 117th Hospital of PLA, Hangzhou, Zhejiang, 310013, P.R. China d The Second Affiliated Hospital of Shaanxi Chinese Medicine University, Xianyang, Shaanxi, 712000, P.R. China
Received 27 April 2013; received in revised form 16 September 2013; accepted 4 October 2013
Abstract Curcumin, a natural and crystalline compound isolated from the plant Curcuma longa with low toxicity in normal cells, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about antimetastasis effects and mechanism of curcumin in lung cancer. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis. In this study, we examined the influence of curcumin on in vitro invasiveness of human lung cancer cells and the expressions of Rac1. The results indicate that curcumin at 10 μM slightly reduced the proliferation of 801D lung cancer cells but showed an obvious inhibitory effect on epidermal growth factor or transforming growth factor β1-induced lung cancer cell migration and invasion. Meanwhile, we demonstrated that the suppression of invasiveness correlated with inhibition of Rac1/PAK1 signaling pathways and matrix metalloproteinase (MMP) 2 and 9 protein expression by combining curcumin treatment with the methods of Rac1 gene silence and overexpression in lung cancer cells. Laser confocal microscope also showed that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. At last, through xenograft experiments, we confirmed the connection between Rac1 and the growth and metastasis inhibitory effect of curcumin in vivo. In summary, these data demonstrated that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through inhibition of Rac1/PAK1 signaling pathway and MMP-2 and MMP-9 expression, which provided a novel insight into the molecular mechanism of curcumin against lung cancer. © 2014 Elsevier Inc. All rights reserved. Keywords: Curcumin; Migration; Invasion; Rac1; Actin cytoskeleton; MMP-2/9
1. Introduction Lung cancer, the leading cause of cancer deaths, has the most rapidly increasing incidence rate in the developed country and in China. Clinical data showed that most lung cancer patients eventually suffered from relapse and/or metastasis after complete excision of the cancer, even if they were at stage IA [1]. Despite great progresses have been made in the last decades, the detailed mechanism of lung cancer relapse and metastasis is not fully understood. Rac1, an important small Rho GTPases family protein, has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis [2]. Overexpression of Rac1 is ⁎ Corresponding authors. Qing-yong Chen is to contacted at Department of Respiratory Disease, The 117th hospital of PLA, Hangzhou, Zhejiang 310013, P.R. China. Tel./fax: +86 57187340861. Gui-yuan Lv, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China. Tel./fax: +86 57186613601. E-mail addresses: [email protected] (Q. Chen), [email protected] (G. Lv). 1 These authors contributed equally to this work. 0955-2863/$ - see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2013.10.004
considered as an independent predictor of adverse outcome of some carcinomas [3]. Inhibition of Rac1 expression or disruption of its function significantly reduces cancer cells metastasis in many tumor models [4,5]. Our recent studies also showed that overexpression of Rac1 was widespread in primary lung cancer patients. Silence of Rac1 expression by shRNA suppressed lung cancer cells migration, invasion and induced rearrangements of the actin cytoskeleton in lung cancer cells [6]. Rac1 regulates cellular functions such as cytoskeletal dynamics, cell adhesion and transcription via activating PAK1 (one of the best characterized member of Rac1 effectors) and other downstream signaling molecules. Therefore, agents that inhibiting Rac1 or its downstream targets might have anticancer metastatic effect. Curcumin (diferuloylmethane), an active component of the spice turmeric (Curcuma longa), has chemopreventive and therapeutic properties against many tumors both in vitro and in vivo [7,8]. Several studies have shown that curcumin induces apoptosis more potently in cancer cells than in normal cells and attributed its inhibitory effect to the inhibition of angiogenesis nitric oxide synthase, receptor tyrosine kinase and protein kinase C activities and regulation of certain gene transcriptional factors, such as c-Jun/AP-1, JNK, Nuclear factor κB
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(NF-κB) and P53 [8–11]. Previous studies from our lab showed that curcumin inhibited cell proliferation and induced cell apoptosis in lung cancer through the modulation of lysosomal pathway and reactive oxygen species-dependent mitochondrial signaling pathway [7,12]. Recently, some studies on the anticancer effect of curcumin have focused on anti-invasion and antimetastasis aspect [13,14]. However, the effects of curcumin on lung cancer metastasis and underlying molecular mechanism remains remain largely unknown. The Rac1 signaling pathway is closely associated with tumor invasion and metastasis, and curcumin showed a potent inhibitive role on metastasis in various cancer cells, which implies an inner relationship between inhibitory effects of curcumin and Rac1 signaling. In present study, we determined the effects of curcumin on the migration and invasion of lung cancer cells both in vitro and in vivo systems and further demonstrated that the inhibitory effects of curcumin were relate to the inhibition of Rac1/PAK1 signaling pathways, matrix metalloproteinase (MMP) 2/9 expression and actin cytoskeleton rearrangements. These results provide a novel insight into the molecular mechanisms of curcumin in inhibition of lung cancer cell migration and invasion, and also show potential therapeutic value of curcumin in preventing lung cancer metastasis. 2. Materials and methods 2.1. Reagents and cell culture Curcumin was purchased from Sigma Chemical Co. Rac1 antibody was purchased from Upstate Biotechnology Inc (New York, NY, USA). PAK1, p-PAK1, MMP-2 and MMP-9 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Human epidermal growth factor (EGF) and transforming growth factor β1 (TGF-β1) were obtained from (PreproTech GmbH, Hamburg, Germany). A Rac1L61 plasmid, encoding active form of Rac1, was provided by Dr. Liang Fan (Nanfang Hospital affiliated to Southern Medical University, China). Plasmid expressing green fluorescent protein (GFP)-tagged negative shRNA or Rac1 shRNA was generated as described previously [6]. Human large cell lung carcinoma 801D cell line was purchased from the Cell Bank at the Chinese Academy of Sciences, which was first developed by Dr. Lezhen Chen [15], The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin and streptomycin at 37°C in a humidified atmosphere of 5% CO2.
2.5. Cell migration assay Cell migration was assayed by wound healing assay as described previously [9]. Initially, cells were allowed to grow to 80%–90% confluence in 12-well plates precoated with 0.1% gelatin (Sigma) and then starved with serum-free RPMI 1640 medium overnight. After that, cells were scraped by pipette tips to create a denuded zone (gap) of constant width. Subsequently, cellular debris was washed with PBS, and the cells were exposed to indicated concentrations of EGF or TGF-β1. The wound closure was monitored and photographed at 0 and 24 h under a Leica inverted microscope. To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation. Artificial lines fitting the cutting edges were drawn on pictures of the original wounds and overlaid on the pictures of cultures after incubation. Migrated cells across the white lines were counted in six random fields from each triplicate treatment, and data are presented as mean±S.D. 2.6. Cell invasion assay Invasion assay was carried out using modified matrigel Boyden chambers consisting of 24-well Millicell (Millipore Corporation, Shanghai, China) membrane filter (8-μm pore size) as described previously [13]. Dilute Matrigel (50 μl; BectonDickinson; 1:3) in serum-free cold cell culture media and applied to the top side of filter. Briefly, cells were trypsinized and resuspended in serum-free medium. Two hundred microliters of the cell suspension (105 cells) with 10 μM curcumin was added to the upper chamber of each well and 0.1% of dimethyl sulfoxide (DMSO) as the solvent control. The bottom chambers were filled with 500 μl RPMI 1640 medium supplemented with 10% fetal bovine serum and growth factor (EGF or TGF-β1). The chamber was incubated for 20 h at 37°C. At the end of incubation, the cells in the upper surface of the membrane were carefully removed with a cotton swab. Cells invading across the matrigel to the lower surface of the membrane were fixed with methanol and stained with 0.5% crystal violet. The invading cells on the lower surface of the membrane filter were counted with a light microscope. The data presented are the average number of cells attached to the bottom surface from five random fields. Each experiment was carried out in triplicate. 2.7. Laser confocal microscope To determine the effect of curcumin on cell morphology and cytoskeleton, cells were plated in six-well plates containing 12-mm glass coverslips and grown for 16 h and then treated with curcumin and indicated concentrations of growth factor (EGF or TGF-β1). After the exposure period, the medium was removed, and the cells were washed with PBS and then fixed with 4% formaldehyde dissolved in PBS for 10 min at room temperature and permeabilized for 10 min with 0.2% TritonX-100. Cells were incubated with 500 ng/ml tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma) at room temperature in PBS containing 0.1% Triton X-100 for 45 min. The cells were examined and photographed by confocal microscopy. 2.8. Reverse transcription polymerase chain reaction assay
2.2. Real-time cell analysis cytotoxicity testing Real-time cell analysis (RTCA) system (Roche Applied Science, Indianapolis, IN, USA) was employed for dynamic assessment of curcumin toxicity. The principle and use of the RTCA system have been described previously [16]. Briefly, under the control of RTCA software, the sensor analyzer automatically selected wells to be measured and continuously monitored changes in electrode impedance of cultivated cells in the electronic sensor plate. Cell index (CI) is used to represent cell status based on electrical impedance, which is proportional to the culture area covered by attached cells so that toxicity-induced cell shrinkage and detachment of cells from the plate due to cell death result in a lower CI value. In the study, the effect of the curcumin was monitored dynamically for every 15 min. The CI against the time was plotted.
2.3. Assessment of cell proliferation by MTT assay The antiproliferative effect of curcumin on 801D lung cancer cells was also determined by the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye uptake method. Briefly, the cells were seeded in quintuplicate at a density of 5×103 cells per well in a 96-well plate in a final volume of 0.2 ml medium and incubated for 24 h at 37°C. Curcumin was added to each well and incubated for another 24 h. The cells were washed with phosphate-buffered saline (PBS), and 20 μl MTT solution (5 mg/ ml) was added to each well. After a 4-h incubation at 37°C, 150 μl isopropanol was added. The absorbance of MTT formazan was measured at 490 nm.
2.4. Cell transfection Cells plated in 6-well plates were transfected with indicated plasmids using Lipofectamin 2000 (Invitrogen) according to the manufacturer's instructions. After 24 h of transfection, GFP positive cells were sorted using fluorescence activated cell sortor (FACS) and used for different experiments.
Total RNA was isolated from each group of cells using Trizol reagent (Invitrogen) according to the manufacturer's instruction. Obtained complementary DNAs were amplified using specific primers. The polymerase chain reaction (PCR) from each sample was performed by the following conditions: 5 min at 94°C, 30 cycles of 30 s at 94°C, 1 min at 55°C, and final extension for 1 min at 72°C. Primers for Rac1 are as follows: 5′-ATGCAGGCCATCAAGTGTGTGGTG-3′ (sense) and 5′-TTACAACAGCAGGCATTTTCTCTTCC-3′ (antisense). 2.9. Western blot analysis The whole-cell extracts were prepared in RIPA buffer [20 mM Tris, 2.5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 40 mM NaF, 10 mM Na4P2O7 and 1 mM phenylmethanesulfonylfluoride (PMSF)]. Thirty micrograms of cellular protein of each sample was applied to immunoblot following 10% SDSpolyacrylamide gel electrophoresis and probed with specific antibodies as indicated, followed by a horseradish peroxidase-conjugated goat antimouse or antirabbit antibody (Millipore). Immunoreactive bands were visualized by enhanced chemiluminescence (Millipore) according to the manufacturer's instructions. Quantification of reactive protein bands was performed by densitometric analysis, and the fold change was calculated by normalizing with control β-actin levels. 2.10. In vivo animal studies The cell viability and cell number of 801D cells were calculated by trypan blue. Then, 1×107 live cells in RPMI-1640 medium were injected subcutaneously into the flanks of mice (nude mice, average 25 g, purchased from Shanghai Laboratory Animal Center, Shanghai, China). All experimental procedures used in this study had been approved by the ethics committee in the 117th Hospital of PLA, and all animal experiments had been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The authors who performed experiments had given their informed consent
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prior to the study and had followed “principles of laboratory animal care” (National Institutes of Health publication no. 86-23, revised 1985). Tumor-bearing mice were then divided randomly into treatment groups (three mice per group) and treatment initiated when the xenografted solid tumors reached a volume of about 50 mm3. Each mouse was injected intraperitoneally every 3 days with either 30 μl of control vehicle (DMSO), curcumin (30 and 45 mg/kg) or cisplatin (DDP, 8 mg/kg). Mice exhibiting tumors were monitored, and tumor size was measured once every 3 days using calipers. The tumor volume in each animal was estimated according to the formula: tumor volume (mm3)=L×W2/2 (where L is the length and W is the width), with the final measurement taken 4 weeks after curcumin treatment, and at the same time, the body weight of each animal was measured once every day. At the end of the experiment (4 weeks after curcumin treatment), the mice were anesthetized by CO2 and sacrificed. Tumors from each animal were removed, measured and weighed individually. Part of the mice tumor tissue was fixed in 4% paraformaldehyde in PBS and then embedded in paraffin. Serial sections were cut at 6-μm thickness and stained with
Fig. 1. Cytotoxic effect of curcumin on 801D cell line. (A) CI was measured every 15 min by the RTCA system; dose- and time-dependent cytotoxicity was caused by curcumin in 801D cells. (B) Cell viability based on MTT assay after 24-h curcumin exposure. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control). (C) Cell viability based on microscopic observation after 24-h curcumin exposure; photographs were taken at a ×100 magnification.
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hematoxylin and eosin (H&E) and immunohistochemistry. Immunohistochemical staining and evaluation were described in previous studies analysis [6].
2.11. Statistical analysis All experiments were performed in triplicate and analyzed by T test or ANOVA (SPSS, version 16.0) for significant differences. P values less than .05 were considered statistically significant. Where appropriate, the data are presented as the mean±S.D.
Fig. 2. Curcumin inhibited EGF-induced cell migration and invasion. (A) 801D confluent monolayer cells were scratched with a pipette tip and then treated with 10 μM curcumin in the presence of 100 ng/ml EGF or not. Representative images showing the inhibitory effect of curcumin on EGF-induced cell migration at 24 h. (B) The invasion ability of 801D was determined by invasion assay. Cells in low surface of the Boyden chamber were stained and photographed under a light microscope at ×100 magnification, and invaded cells were quantified by counting the number of stained cells. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
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3. Results
3.2. Curcumin inhibits EGF- or TGF-β1-induced migration and invasion of lung cancer cells
3.1. Curcumin inhibits the viability of lung cancer cells First, we used the RTCA to examine the antiproliferative effects of curcumin on lung cancer cells. Fig. 1A shows typical RTCA curve of 801D cells responding to curcumin treatment (CI vs. exposure time). The index curve culminated to the maximum gradually without curcumin treatment, indicating that the untreated cells continue to grow and proliferate with time. However, the CI curves were dramatically different in response to curcumin treatment (10, 20, 40, and 60 μM). The results showed that curcumin treatment resulted in a time- and dose-dependent loss of cell viability, which was similar to the results of MTT assay (Fig. 1B) and microscopic observation (Fig. 1C). Interestingly, we also found that antiproliferative effect of curcumin on lung cancer cells is more obvious than on normal lung bronchial epithelial cells (data not shown). Curcumin (10 μM) was shown to have a weak effect on the growth of 801D lung cancer cells as shown in our results. Therefore, the concentration was used in the subsequent experiments.
Epidermal growth factor receptor (EGFR) and TGF-β-dependent signaling pathways are pivotal in promoting lung cancer cell growth and metastasis [17,18]. EGF and TGF-β receptors were also reported two important targets of curcumin [19,20]. In the study, we used EGF and TGF-β1 to enhance the motor abilities of 801D lung cancer cells and evaluate the effects of curcumin on cell migration and invasion. As shown in Fig. 2 and Supplementary Material Fig. S1, 801D cells exposed to either EGF or TGF-β1 enhanced the 801D cell migration into a wounded area and across the transwell membrane. However, in the presence of 10 μM curcumin, cell migration and invasion stimulated by EGF and TGF-β1 were significantly reduced. Apparently, curcumin can effectively inhibit EGF- or TGF-β1-induced migration and invasion of lung cancer cells. 3.3. Reduced expression of Rac1 protein in association with curcumin treatment Rac1 is an important GTPase that has been implicated in many cellular processes such as cytoskeleton rearrangement, cell adhesion and transcriptional activation. Besides, it also has been suggested to be involved in cancer cell migration, invasion and metastasis. In this study, we performed reverse transcriptase PCR and Western blot assays to determine whether Rac1 is regulated by curcumin, and the results showed that the expression of Rac1 in 801D cells can be inhibited by curcumin both at the messenger RNA (mRNA) and protein levels (Fig. 3A). Importantly, similar inhibition effects were also found in other lung cancer cells (data not shown). In addition, either EGF or TGF-β1 stimulation can elevate the expression of Rac1 protein in 801D cells, and pretreatment of 801D cells with 10 μM curcumin partly abolished EGF or TGF-β1-induced Rac1 overexpression (Fig. 3B and Supplementary Material Fig. S2), suggesting that Rac1 is likely to be the downstream effector of curcumin and may be involved in the migration and invasion suppression by curcumin. 3.4. Altered Rac1 protein expression affects lung cancer cell migration and invasion In our previous and present studies, we have been found that either silence of Rac1 by shRNA or the use of specific inhibitor of Rac1 (NSC23766) suppressed 801D lung cancer cells migration and invasion (Fig. 4A, B). To further confirm the involvement of Rac1 in the inhibitory effect of curcumin on cell migration and invasion, we measured cell migration and invasion after transfection with constitutive active Rac1L61 plasmid. As we expected, Rac1L61 plasmid significantly elevated the cell migration and invasion ability compared with the negative control (Fig. 4C, D) and can be also attenuated by curcumin treatment. Thus again, we found that curcumin inhibited cell migration and invasion by interfering with the Rac1-dependent signaling pathway. 3.5. Cell actin cytoskeleton arrangement induced by curcumin treatment
Fig. 3. Curcumin inhibits endogenous and EGF-induced Rac1 expression. (A) Curcumin treatment for 24 h significantly inhibited the expression of Rac1 both at the mRNA and protein levels. (B) Pretreatment of 801D cells with 10 μM curcumin resulted in inhibition of EGF-induced Rac1 expression. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
It is well established that actin polymerization and depolymerization (actin dynamics) play a crucial function in cell motility. Rac1 has been widely implicated in cytoskeleton rearrangements. EGF and TGF-β1-induced phenotype change is associated with actin remodeling and has been considered as a characteristic of invasive and metastatic cells [17,21]. Therefore, we then examined the effect of curcumin on cytoskeleton changes stimulated by EGF or TGF-β1. As shown in Fig. 5 and Supplementary Material Fig. S3, both EGF (100 ng/ ml) and TGF-β1 (6 ng/ml) increased polymerization of actin within
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Fig. 4. Rac1 regulated cell migration and invasion in vitro. (A) Compared with controls, transfection with Rac1shRNA or treatment with Rac1 inhibitor (NSC23766, 100 μM) significantly inhibited the migration of 801D cells in wound healing assay. (B) For invasion assay, the invasive cell number of 801D transfected with Rac1shRNA was significantly less than that of the negative control, Rac1 inhibitor (NSC23766) has the same effect as Rac1shRNA. C-) Transfection with Rac1L61 plasmid obviously enhanced the cell migration and invasion ability compared with the negative control, and curcumin clearly inhibited the migration and invasion of Rac1L61-transfected cells. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
indicated time as visualized by TRITC-phalloidin staining, whereas Rac1 shRNA treated cells exhibited a decrease of polymerization of actin. Treatment with curcumin also inhibited TGF-β1-induced actin polymerization, suggesting that Rac1 is involved in this actin polymerization and curcumin can inhibit EGF- or TGF-β1-induced migration through actin remodeling in 801D cells.
but no changes were detected at total PAK1 level. Similar results were also found in Rac1-silenced cells. Conversely, cells transfected with constitutive active Rac1L61 enhanced the phosphorylation of PAK1 and were significantly inhibited by curcumin, suggesting that the antimetastatic function of curcumin in the lung cancer cells is likely, at least in part, through the Rac1-regulated phosphorylation of PAK1.
3.6. Curcumin inhibits PAK1 phosphorylation, a Rac1 downstream protein related to cell invasion
3.7. Curcumin inhibited MMP-2 and MMP-9 expression
Here, we also explored whether curcumin affects the expression of PAK1, a Rac1 downstream functional protein, which has been shown to involve in cell invasion and migration. As shown in Fig. 6B, PAK1 phosphorylation was inhibited significantly in curcumin-treated cells,
The Rho families of small GTPases and MMPs are the two protein families that play key roles in cell movement. Rac1 was shown to mediate the expression of MMPs in different cell types [22]. In our study, we also found that the expression of MMP-2 and MMP-9 significantly increased in cells transfected with constitutive active
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Fig. 5. Curcumin and Rac1 silencing inhibited EGF-induced cytoskeleton changes. Thirty minutes of EGF stimulation induced a significant increase of filopodia and lamellipodia formation. However, this effect was inhibited by curcumin treatment or Rac1 shRNA transfection.
Rac1L61 plasmid, but abolished by Rac1 knockdown or curcumin treatment. Furthermore, curcumin inhibited the expression of MMP-2 and MMP-9 in the Rac1L61-transfected cells (Fig. 7). Therefore, we believe that curcumin inhibits cell-associated expression of MMP-2 and MMP-9 through the regulation of Rac1 activity. 3.8. Curcumin inhibits lung cancer growth and invasion in vivo Overgrowth and metastasis are two major characteristics of malignant tumors. We therefore investigated whether curcumin could suppress lung cancer tumor growth and invasion in vivo. Cisplatin (DDP), a commonly used medicine for clinical chemotherapy, was also used as a positive control in our study. As shown in Fig. 8A and B, the volume and average weight of tumors in control group increased more obviously than those in curcumin-treated group. In addition, H&E staining showed that the tumors and lung tissues in curcumin-treated groups had clear boundaries with less invasiveness. In contrast, tumors and lung tissues arising from control groups displayed characteristics of invasion, indicating that curcumin inhibited invasive lung cancer in vivo (Fig. 8C).
Fig. 6. Effects of curcumin inhibited the levels of phospho-PAK1. Cells were treated with curcumin (10–20 μM) or transfected with indicated plasmids and then treated with or without curcumin; the expressions of Rac1 (A) and phosphorylated and total PAK1 (B) were examined by Western blot. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
To determine the relationship among Rac1 expression, lung tumor growth, metastasis and curcumin treatment in vivo, we examined the expression of Rac1 and Ki-67 (a tumor marker related to malignant degree) in the nude mice xenograft model. We found that formed tumors showed strongly positive Rac1 and Ki-67 staining in the controls, but the tumors exhibited only weak immunoreactions in curcumin-treated cells (Fig. 8C). These data suggest that Rac1 is involved in lung cancer growth and invasion, and Rac1 expression can also be inhibited by curcumin in vivo.
4. Discussion Recently, some studies on the anticancer effect of curcumin have focused on anti-invasion and antimetastasis aspect [13,14,23]. However, the effects of curcumin on lung cancer metastasis
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Fig. 7. Curcumin inhibited MMP-2 and MMP-9 expressions in 801D cells. The protein expressions of MMP-2 and MMP-9 were significantly inhibited by curcumin in Rac1L61 transfection and nontransfection cells. Rac1 shRNA showed same inhibitory effects as curcumin on MMP-2 and MMP-9 expression. Columns, mean from three different experiments with three duplicates; bars, S.E. (*Pb.05 and **Pb.01 vs. respective control).
and underlying molecular mechanism remains remain largely unknown. In the present study, we found that low-toxic levels of curcumin (10 μM) could efficiently inhibit EGF- or TGF-β1induced migration and invasion of lung cancer cells in vitro, and this effect is related to inhibition of the Rac1/PAK1 signaling pathway. Furthermore, we examined the effects of curcumin in xenograft mouse lung tumor model and found that Rac1 is also involved in lung cancer growth and metastasis in vivo. To our knowledge, this is the first study reporting that Rac1 and PAK1 were found to be involved in the antimetastasis action of curcumin in lung cancer cells. Initially, we measured the effect of curcumin on cell proliferation. As shown in Fig. 1, curcumin dramatically inhibited cell growth of 801D lung cancer cells in a dose- and time-dependent manner. Meanwhile, cell viability inhibition of curcumin in 801D lung cancer cells was not significant when curcumin was used as low as 10 μM, suggesting that cell death or apoptosis is not the main factor for inhibiting lung cancer cell migration by lower concentrations of curcumin, which was used in our study. Rac-1 belongs to the Rho family of GTPases, and it functions as a signal regulator to the actin cytoskeleton. Several lines of evidence have shown that Rac1 can be activated by various growth factor receptors (EGFR [24], α6β4 integrin receptor [25], TGF-β receptor [26] and so on through guanine nucleotide exchange factors such as Sos proteins, Tiam1 and Vav2. Interestingly, all these growth factor receptors are also reported as the targets of curcumin [19,20,27,28]. The exact role of these receptors in anti-invasion effects of curcumin in lung cancer needs more researches. In our study, we genetically modulated the expression levels of the Rac1 gene in lung cancer 801D cells by either up- or down-regulation (Rac1L61, Rac1 inhibitor and Rac1 shRNA). Then, the results from curcumin treatment supported the evidence that the effect of inhibiting lung cancer cell migration and invasion by curcumin is at least partly due to the inhibition of Rac1 expression.
Fig. 8. Curcumin inhibited the invasion and metastasis of lung cancer in vivo. (A) All mice of the untreated groups developed tumors approximately 30 days after injection. The tumors formed in the curcumin-treated group grew more slowly. (B) The tumor weight of the untreated groups was heavier than that of the curcumin-treated groups. (*Pb.05 and **Pb.01 vs. untreated groups; n=5). (C) H&E staining revealed the cellular heterogeneity in pathological sections of the excised tumor tissue. Untreated groups displayed characteristics of invasion. In contrast, curcumin-treated groups showed clear boundaries with less invasiveness (original magnification, ×200). Immunohistochemistry showed that the tumors formed by the untreated groups were positive or strongly positive with Ki-67 and Rac1 stain, whereas the tumors formed by the curcumin-treated groups were weakly positive with Ki-67 and Rac1 stain (original magnification, ×200).
The organized polymerization of actin filaments is believed to be an important mechanism required for cell migration [29]. Rac1 is reported to be a key regulator of actin dynamics that lead to organized actin-based structures associated with cell migration [30]. In this study, we investigated the effect of curcumin on actin cytoskeleton by phalloidin stain in 801D cells. The results showed that curcumin
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dramatically inhibited the formation of stress fiber, filopodia, and membrane ruffles in EGF- or TGF-β1-treated 801D cells. These effects mimicked those of Rac1 shRNA transfection and NSC23766 treatment, suggesting that the blocking of Rac1 expression and the disruption of microfilament skeleton rearrangement are both involved in curcumin-induced cell migration inhibition. It have been reported that the inhibitory effect of Rac1 on myosin contractility can be mediated by the Rac1 effector PAKs [31]. PAK1 is well known as a downstream effector of Rac1. It binds to Rac1 in a GTP-dependent manner. Activated PAK1 (phospho-PAK1) regulates cellular functions such as cytoskeletal dynamics, cell adhesion and transcription. The formation of actin meshwork lamellipodia stimulated by phospho-PAK1 in the leading edge is crucial for cell migration [6]. Downstream targets for PAKs include myosin light-chain kinase (MLCK). MLCK phosphorylates the myosin light chain, which is important in regulating actin cytoskeletonl dynamics [32]. In addition, PAK phosphorylates and activates LIM kinase, which, in turn, phosphorylates and inactivates the actin-severing protein cofilin, thus promoting filament assembly [33]. In the present study, we found that curcumin significantly attenuated PAK1 phosphorylation. This inhibition might lead to PAK1 inactivation and, in turn, affect the downstream cytoskeletal targets of PAK1. These effects can also been detected in Rac1 shRNA-transfected cells. Moreover, Western blotting results also showed that cells transfected with Rac1L61 enhanced the phosphorylation of PAK1 compared with the negative control. Curcumin inhibited the phosphorylation of PAK1 in Rac1L61-transfected cells, suggesting that curcumin exerted its antimetastatic activity via regulating the activity of Rac1 downstream functional proteins. MMP-2 and MMP-9 play the most important role for basal membrane type IV collagen degradation, a critical step for cancer invasion and metastasis. In our study, we also used Western blotting to test the expression of these two invasion associated proteins. The results showed that the expression of MMP-2 and MMP-9 was decreased significantly when curcumin was used. These data are in agreement with previous studies where curcumin inhibited the migration and invasion of other tumor cells via down-regulation of MMP-9 or MMP-2 expression [23,34,35]. Rac1 was shown to mediate the activation of MMPs in different cell types [36,37]. To further investigate whether down-regulation of MMP-9 and MMP-2 expression by curcumin was through Rac1 signaling in lung cancer cells, 801D cells were transfected with Rac1 shRNA or Rac1L61 plasmid, and the expression levels of MMP-2 and MMP-9 were examined by Western blotting. The results showed that MMP-2 and MMP-9 expression increased significantly by Rac1L61 plasmid. Conversely, it was diminished by Rac1 shRNA transfection, suggesting that MMP-2- and MMP-9-mediated cell invasion may be regulated by Rac1. However, how Rac1 affected the MMPs is still not clear. Previous study has shown the linkage of Rac1 and MMP activity through NF-κB-dependent signaling [36]. Recently, a study reported that Rac1 may regulate MMP-9 expression via H2O2 production primary supplied by mitochondria [37]. The experiment regarding the relationship of MMP-2 and MMP-9 expression with NF-κB signaling and/or H2O2 production is undergoing in our laboratory. In conclusion, we identified a novel mechanism for the inhibitory effect of curcumin on the invasion and migration of lung cancer cells. The reduction in the metastasis ability observed in curcumin-treated cells is at least partly due to the inhibition of Rac1/PAK1 pathway signaling and the decreased MMP-2 and MMP-9 expression. The above findings and concepts disclosed here provide an important basis for a further exploration toward understanding the action mechanisms of curcumin and possibly its beneficial effect in the prevention of tumor metastasis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jnutbio.2013.10.004.
Acknowledgments This work was supported by the Zhejiang Traditional Chinese Medicine Administration Bureau (Project no. 2008CA077) and the Nanjing military area commands “334” high-level scientific and technological personnel training plan. We would like to thank Drs. Yanyi Wang and Yingke Xu for their technical assistance and discussion.
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