- Email: [email protected]
Curcumin inhibits the migration and invasion of human A549 lung cancer cells through the inhibition of matrix metalloproteinase-2 and -9 and Vascular Endothelial Growth Factor (VEGF)
Cancer Letters 285 (2009) 127–133
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Curcumin inhibits the migration and invasion of human A549 lung cancer cells through the inhibition of matrix metalloproteinase-2 and -9 and Vascular Endothelial Growth Factor (VEGF) Song-Shei Lin a, Kuang-Chi Lai b,c, Shu-Chun Hsu d, Jai-Sing Yang e, Chao-Lin Kuo f, Jing-Pin Lin g, Yi-Shih Ma h, Chih-Chung Wu i, Jing-Gung Chung j,* a
Department of Medical Imaging and Radiological Science, Central Taiwan University of Science and Technology, Taichung 406, Taiwan School of Medicine, China Medical University, Taichung 404, Taiwan c Department of Surgery, China Medical University Beigang Hospital, Yunlin 651, Taiwan d School of Nutrition, China Medical University, Taichung 404, Taiwan e Department of Pharmacology, China Medical University, Taichung 404, Taiwan f School of Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan g School of Chinese Medicine, China Medical University, Taichung 404, Taiwan h Graduate Institute of Chinese Medical Science, China Medical University, Taichung 404, Taiwan i Department of Health Sciences, Chang Jung Christian University, Tainan 711, Taiwan j Department of Biological Science and Technology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan b
a r t i c l e
i n f o
Article history: Received 6 February 2009 Received in revised form 25 April 2009 Accepted 29 April 2009
Keywords: Curcumin Migration Invasion Matrix metalloproteinases A549 cancer cells
a b s t r a c t It is well known that matrix metalloproteinases (MMPs) act an important role in the invasion, metastasis and angiogenesis of cancer cells. Agents suppressed the MMPs could inhibited the cancer cells migration and invasion. Numerous evidences had shown that curcumin (the active constituent of the dietary spice turmeric) has potential for the prevention and therapy of cancer. Curcumin can inhibit the formation of tumors in animal models of carcinogenesis and act on a variety of molecular targets involved in cancer development. There is however, no available information to address the effects of curcumin on migration and invasion of human lung cancer cells. The anti-tumor invasion and migration effects of lung cancer cells induced by curcumin were examined. Here, we report that curcumin suppressed the migration and invasion of human non-small cell lung cancer cells (A549) in vitro. Our findings suggest that curcumin has anti-metastatic potential by decreasing invasiveness of cancer cells. Moreover, this action was involved in the MEKK3, p-ERK signaling pathways resulting in inhibition of MMP-2 and -9 in human lung cancer A549 cells. Overall, the above data shows that the anticancer effect of curcumin is also exist for the inhibition of migration and invasion in lung cancer cells. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In Taiwan, lung cancer is the major causes of cancer-related deaths in men and women and about 32.8 persons per 100 thousand die annually from lung, based on reports * Corresponding author. Tel.: +886 4 22053366x2500; fax: +886 4 22053764. E-mail address: [email protected] (J.-G. Chung). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.04.037
from the People’s Health Bureau of Taiwan. Although the treatment of lung cancer have been improved but the mortality in lung cancer patients remains high. In order to decrease cancer mortality we should focused on health initiatives including prevention, early detection, and effective treatments. A growing body of research suggests that numerous naturally occurring compounds act as antioxidants, cancer preventative and therapeutic agents [1–3]. Based on the epidemiological studies demonstrate that
128
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
consumption of a phytochemical-rich diet (fruits and vegetables) can lead to decrease the risk of certain types of human cancers [4,5]. In clinical treatment of cancer patients, many prescription drugs are derived from natural plant species [6,7]. Curcumin (diferuloylmethane), an active component of the spice turmeric (Curcuma longa), possess biological activities include chemopreventive and therapeutic properties against many tumors in both in vitro and in vivo models and including clinical trials [8,9]. Curcumin is able to suppress cell proliferation and inflammation, induce apoptosis, and sensitize tumor cells to cancer therapies [10–12] and it also suppress invasion, angiogenesis, and metastasis of cancer cells [13]. Many mechanisms are involved in the biological activities of curcumin including the nuclear factor-jB, IjBa kinase, Akt, activator protein1, mitogen-activated protein kinases (MAPK), cyclooxygenase-2 lipoxygenase, inducible nitric-oxide synthase, urinary plasminogen activator, tumor necrosis factor, chemokines, and cell cycle machinery have been suggested as the targets of curcumin [11,14–17]. Recently, it was reported that curcumin inhibits Akt/mTOR signaling in various tumor cells [18–21]. There is no dose-limiting toxicity after curcumin is administered at doses up to 10 g/day and when the serum concentration of curcumin was 1.77 ± 1.87 lmol/L at the dose of 8 g/day [22]. Based on these reports it may suggest that curcumin could be a potential in cancer therapy. However, to our knowledge the cancer inhibitory potential of curcumin has not been evaluated against migration and invasion of lung cancer 549 cells in vitro systems. In the present study, we investigated the molecular mechanisms by which curcumin suppress migration and invasion in A549 lung cancer cells in vitro. Our results show that curcumin concentration- and timedependently inhibits the migration and invasion in human A549 lung cancer cells. 2. Materials and methods 2.1. Materials and chemicals Curcumin, dimethyl sulfoxide (DMSO), trypan blue and triton X-100, pyruvate, penicillin G and streptomycin were obtained from Sigma Chemical. (St. Louis, MO, USA). AntiMMP-2, anti-PI3 K, anti-PKC, anti-VEGF, anti-c-jun-p, antiRas, anti-GRB2, anti-MEKK3, anti-FAK, anti-MKK7, antiJNK, anti-ERK, anti-MMP-9, anti-MMP-2 and anti-Rho A were purchased from Santa Cruz Biotechnology. Materials and chemicals for electrophoresis were obtained from BioRad.
2.3. Migration determination The A549 cells (1 104 per well) were plated in 6-well plates for 24 h then cells in the individual wells were wounded by scratching with a sterile pipette tip (to remove the cells but both side still have original cells) and incubated with F12K medium containing no FBS and then cells were treated with or without curcumin (10 or 20 lM) for 0, 24 and 48 h. Cells were photographed under phase-contrast microscopy (100) as previously described [23]. 2.4. Migration and invasion determinations Cell migration and invasion were determined with or without matrigel-coated transwell cell culture chambers (8 lm pore size) (Millipore, Billerica, MA, USA) as described previously [23,24]. A549 cells were kept for 24 h in serumfree-medium then were trypsinized and resuspended in serum-free F12K medium and placed in the upper chamber of the transwell insert (5 104 cells/well) and incubated with 0.5% DMSO or curcumin (0, 10 or 20 lM) and F12K medium containing 10% FBS was added to the lower chamber. The plates were incubated in a humidified atmosphere with 95% air and 5% CO2 at 37 °C for 24 or 48 h, non-invasive cells in the upper chamber were removed by wiping with a cotton swab, and invasive cells were fixed with 4% formaldehyde in PBS and were stained with 2% crystal violet in 2% ethanol. Cells in the lower surface of the filter which penetrated through the matrigel were counted under a light microscope at 200 [23]. 2.5. Western blotting A549 cells (1 106/wells) in F12K medium were plated onto 6-well plates then were treated with 30 lM curcumin for 0, 6, 12, 24 and 48 h. Following each time point, cells from each well were harvested and isolated by centrifugation in 1500 rpm for 5 min. The isolated cells were lysed individually and the total protein levels were quantified. Then to determine the proteins levels associated with invasion and migration (PI3K, PKC, VEGF, c-jun-p, Ras, GRB2, MEKK3, FAK, MKK7, JNK, ERK, MMP-9, MMP-2 and Rho A). All samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), as described previously [23]. 2.6. Statistical analysis The differences between the curcumin-treated and control group were analyzed by Student’s t-test.
2.2. Cell culture 3. Results
Human lung carcinoma cell line (A549) was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were placed onto 75 cm2 tissue culture flasks in F12K medium supplemented with 10% FBS, 1% penicillin–streptomycin (100 units/ml penicillin and 100 lg/ml streptomycin) and 1% glutamine and grown at 37 °C under a humidified 5% CO2 and 95% air at one atmosphere.
3.1. Curcumin-inhibited the migration of A549 cells in vitro The inhibitions of A549 cell migration by curcumin were examined by using transwell and wound-healing assays and results are shown in Fig. 1A and B. Higher dose (20 lM) curcumin and longer (48 h) incubation time period led to greater inhibition of cell migration in A549 cells. The percentage of inhibition ratio is 83–95% and these effects were doseand time-dependent manners.
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
129
Fig. 1. Curcumin-inhibited the migration of A549 cells in vitro. A549 cells (1 104 cells/well; 12-well plates) were treated with 0, 10 and 20 lM curcumin for 24 and 48 h. Cells were wounded by scratching with a pipette tip or using transwell cell culture chambers, and the cells were incubated with F12K medium containing no FBS and incubation with or without curcumin for 48 h. Cells were photographed under phase-contrast microscopy (200) (Panel A) and quantitation (B). P < 0.01, significant difference between curcumin-treated groups and the control.
3.2. Curcumin-inhibited the invasion of A549 cells in vitro The inhibitions of A549 cells invasion by curcumin were examined and results are shown in Fig. 2A and B. The Fig. 2A shows that A549 cells invased from the upper to the lower chamber in majority of cells from the control group (absence of curcumin) but the penetration of the EHScoated filter by A549 cells was inhibited in the presence of curcumin. This inhibitory effect was higher at a curcumin concentration of 20 lM than 10 lM. The quantification of cells in the lower chamber from Fig. 2B indicated that curcumin significantly inhibited A549 cell invasion, the percentage of inhibition ratio is 95–98% and this effect was dose-dependent. 3.3. Curcumin-inhibited levels of PI3K, PKC, VEGF, c-jun-p, Ras, GRB2, MEKK3, FAK, MKK7, JNK, ERK, MMP-9, MMP-2 and RhoA in A549 cells Levels of invasion- and migration-associated proteins in A549 cells after treatment with curcumin were determined and quantitated by Western blotting. Results were shown in Fig. 3A–C indicate that the levels of VEGF, c-jun-p, and GRB2 (Fig. 3A), MEKK3, FAK and MKK7 (Fig. 3B), MMP2 and MMP-9 and Rho A (Fig. 3C) were lower than the corresponding control group. However, JNK and PERK (Fig. 3B) was higher than that of control. MMP-2 and -9 play an important role of cell invasion and migration. These effects may have led to the inhibition of invasion and migration from A549 cells after exposure to curcumin at 30 lM.
4. Discussion It is well known that curcumin perform anticancer activity in many cancer cells in vitro and in vivo. Our previous studies also showed that curcumin decreased the percentage of viable A549 cells through G2/M phase arrest and apoptosis and those effects are dose- and time-dependent manners. Although it was reported that curcumin can inhibit human lung adenocarcinoma (CL1-5) cell invasion and metastasis through activation of the tumor suppressor DnaJ-like heat shock protein 40 (HLJ1) through activation of the JNK/JunD pathway [20]. However, actions of curcumin on migration and invasion of human A549 lung cancer cells and the possible signaling pathways have not been addressed. In this study, we found that curcumin suppressed the migration and invasion of A549 cells through the MEEK3, FAK, Rho A and MKK7 pathway then led to the inhibition of MMP-2. Cell migration is involving several growth factors, for binding to receptors on cell surface, stimulating down-
130
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
Fig. 2. Curcumin-inhibited the invasion of A549 cells in vitro. A549 cells (104 cells/0.4 ml F12K medium) were placed and cells that penetrated through the matrigel to the lower surface of the filter were stained with crystal violet and were photographed under a light microscope at 200 (Panel A). Quantification of cells in the lower chamber, was performed by counting cells at 200 (Panel B). P < 0.001, significant difference between curcumin-treated groups and the control.
stream signaling pathways and resulting in cytoskeletal reorganization and stimulation of motility machinery of the cell [25]. Therefore, this cellular process provides a multiple targets for the development of therapeutic agents to inhibit cancer invasion and metastasis [26,27]. It was reported that cancer cell–matrix interaction is play an initial and critical step for promoting cell migration, proliferation and degradation [28,29]. Matrix metalloproteinases (MMPs) are a multigene family of zinc-dependent endopeptidases that to play the degrading essentially all extracellular matrix (ECM) components, allowing tumor cells to invade surrounding tissues and enter the blood stream to travel to distant sites [30]. MMPs play an important role in ECM degradation for tumor growth, invasion and tumor-induced angiogenesis [31]. It is well known that: (1) the derangement of ECM by MMPs plays an initial key role in cell movement which via the alteration of cell-ECM interactions; (2) MMPs breakdown the basement membrane for cancer cells to
migration and invasion. Among MMPs, MMP-2 and MMP-9 play the most important for basement membrane type IV collagen degradation [28,29,32]. In particular, MMP-2 expression is associated with tumor invasion, angiogenesis, metastasis and recurrence [33,34]. It was reposted that MMP-2 and MMP-9 are correlated with an aggressive, invasive or metastatic tumor phenotype [35– 37]. It is well known that MMP inhibitors block endothelial cell activities which are essential for new vessel development leading to proliferation and invasion [38,39]. Therefore, MMP-2 and MMP-9 are thought to be therapeutic targets of anticancer drugs based on the degrading action of both enzymes on gelatins which are major components of the basement membrane. Thus, it seems quite likely that curcumin could inhibit or delay cancer invasion and migration in A549 cells via modulation of MMPs. Our results showed that curcumin suppressed the levels of ERK, MMP-2 and -9 protein levels in A549 cells. It has
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
131
Fig. 3. Representative Western blot showing changes in the levels of associated proteins in migration and invasion of A549 cells after exposure to curcumin. The A549 cells (1 106 cells/well) were treated with curcumin at 30 lM for 0, 6, 12, 24 and 48 h then the total protein was prepared and determined, as described in Section 2. The levels of PI3K, PKC, VEGF, C-jun-p, Ras and GRB2 (A), MEKK3, FAK, MKK7, JNK1/2, ERK1/2 (B) and MMP-2, MMP-9 and Rho A (C) expressions were estimated by Western blotting, as described in Section 2.
been reported that the proteins of MMP-9 and uPA require NF-jB [40,41] and AP-1. It is well known that the activation of ERK will stimulates two cis-acting regulatory elements including the binding sites of AP-1 and NF-jB which play an important role in controlling MMP-9 gene expression [42]. Anti-metastatic compounds inhibited MMPs through the different mechanisms [43] those mechanisms include: (1) direct inhibition of the enzymatic activity of MMPs; (2) interference in the activation of pro-MMPs; and (3) reduced expression of MMP genes and protein levels. Other factors such as the pharmacokinetics of curcumin suggest that a poor bioavailability in
humans due to its rapid metabolism in the liver and intestinal wall, when taken orally [44–47]. Therefore, the exact molecular signaling pathway for curcumin-inhibited the migration and invasion in A549 cells still need further investigation. Taken together, our data for the first time showed that curcumin could inhibit migration and invasion of human A549 lung cancer cells through MEKK and ERK signaling pathways resulting in the inhibition of MMP-2 and -9 as summarized in Fig. 4. Curcumin could be recognized to be a therapeutic agent for inhibiting the metastasis and invasion of lung.
132
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
Fig. 4. Molecular signaling pathways for curcumin-inhibited invasion and migration in human lung cancer A549 cells.
Conflict of interest None declared. Acknowledgment This work was supported by Grant NSC95-2745-B-039002-URD from National Science Council, Taiwan. References [1] D.M. Parkin, P. Pisani, J. Ferlay, Estimates of the worldwide incidence of 25 major cancers in 1990, Int. J. Cancer 80 (1999) 827–841. [2] M. Hashibe, P. Brennan, S. Benhamou, X. Castellsague, C. Chen, M.P. Curado, L. Dal Maso, A.W. Daudt, E. Fabianova, L. Fernandez, V. Wunsch-Filho, S. Franceschi, R.B. Hayes, R. Herrero, S. Koifman, C. La Vecchia, P. Lazarus, F. Levi, D. Mates, E. Matos, A. Menezes, J. Muscat, J. Eluf-Neto, A.F. Olshan, P. Rudnai, S.M. Schwartz, E. Smith, E.M. Sturgis, N. Szeszenia-Dabrowska, R. Talamini, Q. Wei, D.M. Winn, D. Zaridze, W. Zatonski, Z.F. Zhang, J. Berthiller, P. Boffetta, Alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium, J. Natl. Cancer Inst. 99 (2007) 777–789. [3] M. Rahman, J. Sakamoto, T. Fukui, Calculation of population attributable risk for bidi smoking and oral cancer in south Asia, Prev. Med. 40 (2005) 510–514. [4] F.L. Meyskens Jr., E. Szabo, Diet and cancer: the disconnect between epidemiology and randomized clinical trials, Cancer Epidemiol. Biomark. Prev. 14 (2005) 1366–1369. [5] K.A. Steinmetz, J.D. Potter, Vegetables, fruit, and cancer. I. Epidemiology, Cancer Causes Control 2 (1991) 325–357. [6] R. Room, T. Babor, J. Rehm, Alcohol and public health, Lancet 365 (2005) 519–530. [7] P. Balaram, H. Sridhar, T. Rajkumar, S. Vaccarella, R. Herrero, A. Nandakumar, K. Ravichandran, K. Ramdas, R. Sankaranarayanan, V. Gajalakshmi, N. Munoz, S. Franceschi, Oral cancer in southern India: the influence of smoking, drinking, paan-chewing and oral hygiene, Int. J. Cancer 98 (2002) 440–445. [8] T.O. Khor, Y.S. Keum, W. Lin, J.H. Kim, R. Hu, G. Shen, C. Xu, A. Gopalakrishnan, B. Reddy, X. Zheng, A.H. Conney, A.N. Kong, Combined inhibitory effects of curcumin and phenethyl isothiocyanate on the growth of human PC-3 prostate xenografts in immunodeficient mice, Cancer Res. 66 (2006) 613–621.
[9] R.A. Sharma, A.J. Gescher, W.P. Steward, Curcumin: the story so far, Eur. J. Cancer 41 (2005) 1955–1968. [10] D. Karunagaran, R. Rashmi, T.R. Kumar, Induction of apoptosis by curcumin and its implications for cancer therapy, Curr. Cancer Drug Targets 5 (2005) 117–129. [11] R.L. Thangapazham, A. Sharma, R.K. Maheshwari, Multiple molecular targets in cancer chemoprevention by curcumin, AAPS J. 8 (2006) E443–E449. [12] R.K. Srivastava, Q. Chen, I. Siddiqui, K. Sarva, S. Shankar, Linkage of curcumin-induced cell cycle arrest and apoptosis by cyclindependent kinase inhibitor p21 (/WAF1/CIP1), Cell Cycle 6 (2007) 2953–2961. [13] L.G. Menon, R. Kuttan, G. Kuttan, Anti-metastatic activity of curcumin and catechin, Cancer Lett. 141 (1999) 159–165. [14] H.W. Chen, H.C. Huang, Effect of curcumin on cell cycle progression and apoptosis in vascular smooth muscle cells, Br. J. Pharmacol. 124 (1998) 1029–1040. [15] S. Aggarwal, H. Ichikawa, Y. Takada, S.K. Sandur, S. Shishodia, B.B. Aggarwal, Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation, Mol. Pharmacol. 69 (2006) 195–206. [16] S.Y. Kim, S.H. Jung, H.S. Kim, Curcumin is a potent broad spectrum inhibitor of matrix metalloproteinase gene expression in human astroglioma cells, Biochem. Biophys. Res. Commun. 337 (2005) 510– 516. [17] B.B. Aggarwal, A. Kumar, A.C. Bharti, Anticancer potential of curcumin: preclinical and clinical studies, Anticancer Res. 23 (2003) 363–398. [18] C.S. Beevers, F. Li, L. Liu, S. Huang, Curcumin inhibits the mammalian target of rapamycin-mediated signaling pathways in cancer cells, Int. J. Cancer 119 (2006) 757–764. [19] H. Aoki, Y. Takada, S. Kondo, R. Sawaya, B.B. Aggarwal, Y. Kondo, Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways, Mol. Pharmacol. 72 (2007) 29–39. [20] H.W. Chen, J.Y. Lee, J.Y. Huang, C.C. Wang, W.J. Chen, S.F. Su, C.W. Huang, C.C. Ho, J.J. Chen, M.F. Tsai, S.L. Yu, P.C. Yang, Curcumin inhibits lung cancer cell invasion and metastasis through the tumor suppressor HLJ1, Cancer Res. 68 (2008) 7428–7438. [21] J.H. Kim, C. Xu, Y.S. Keum, B. Reddy, A. Conney, A.N. Kong, Inhibition of EGFR signaling in human prostate cancer PC-3 cells by combination treatment with beta-phenylethyl isothiocyanate and curcumin, Carcinogenesis 27 (2006) 475–482. [22] A.L. Cheng, C.H. Hsu, J.K. Lin, M.M. Hsu, Y.F. Ho, T.S. Shen, J.Y. Ko, J.T. Lin, B.R. Lin, W. Ming-Shiang, H.S. Yu, S.H. Jee, G.S. Chen, T.M. Chen, C.A. Chen, M.K. Lai, Y.S. Pu, M.H. Pan, Y.J. Wang, C.C. Tsai, C.Y. Hsieh, Phase I clinical trial of curcumin, a chemopreventive agent, in
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
[23]
[24]
[25] [26]
[27] [28] [29] [30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
patients with high-risk or pre-malignant lesions, Anticancer Res. 21 (2001) 2895–2900. S.C. Hsu, C.L. Kuo, J.P. Lin, J.H. Lee, C.C. Lin, C.C. Su, M.D. Yang, J.G. Chung, Crude extracts of Euchresta formosana radix inhibit invasion and migration of human hepatocellular carcinoma cells, Anticancer Res. 27 (2007) 2377–2384. Y.T. Huang, J.J. Hwang, L.T. Lee, C. Liebow, P.P. Lee, F.C. Ke, T.B. Lo, A.V. Schally, M.T. Lee, Inhibitory effects of a luteinizing hormonereleasing hormone agonist on basal and epidermal growth factorinduced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells, Int. J. Cancer 99 (2002) 505–513. B. Anand-Apte, B. Zetter, Signaling mechanisms in growth factorstimulated cell motility, Stem Cells 15 (1997) 259–267. J.T. Price, E.W. Thompson, Mechanisms of tumour invasion and metastasis: emerging targets for therapy, Expert Opin. Ther. Targets 6 (2002) 217–233. G. Fenteany, S. Zhu, Small-molecule inhibitors of actin dynamics and cell motility, Curr. Top Med. Chem. 3 (2003) 593–616. F.G. Giancotti, E. Ruoslahti, Integrin signaling, Science 285 (1999) 1028–1032. W.G. Stetler-Stevenson, Type IV collagenases in tumor invasion and metastasis, Cancer Metast. Rev. 9 (1990) 289–303. L.A. Liotta, P.S. Steeg, W.G. Stetler-Stevenson, Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation, Cell 64 (1991) 327–336. J. Westermarck, V.M. Kahari, Regulation of matrix metalloproteinase expression in tumor invasion, FASEB J. 13 (1999) 781–792. Z.S. Zeng, A.M. Cohen, J.G. Guillem, Loss of basement membrane type IV collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis, Carcinogenesis 20 (1999) 749–755. K. Komatsu, Y. Nakanishi, N. Nemoto, T. Hori, T. Sawada, M. Kobayashi, Expression and quantitative analysis of matrix metalloproteinase-2 and -9 in human gliomas, Brain Tumor Pathol. 21 (2004) 105–112. M. Wang, T. Wang, S. Liu, D. Yoshida, A. Teramoto, The expression of matrix metalloproteinase-2 and -9 in human gliomas of different pathological grades, Brain Tumor Pathol. 20 (2003) 65–72. A.S. Papathoma, V. Zoumpourlis, A. Balmain, A. Pintzas, Role of matrix metalloproteinase-9 in progression of mouse skin carcinogenesis, Mol. Carcinogen. 31 (2001) 74–82. M.I. Cockett, G. Murphy, M.L. Birch, J.P. O’Connell, T. Crabbe, A.T. Millican, I.R. Hart, A.J. Docherty, Matrix metalloproteinases and metastatic cancer, Biochem. Soc. Symp. 63 (1998) 295–313. F.J. Bianco Jr., D.C. Gervasi, R. Tiguert, D.J. Grignon, J.E. Pontes, J.D. Crissman, R. Fridman, D.P. Wood Jr., Matrix metalloproteinase-9
[38]
[39]
[40]
[41]
[42]
[43] [44]
[45]
[46]
[47]
133
expression in bladder washes from bladder cancer patients predicts pathological stage and grade, Clin. Cancer Res. 4 (1998) 3011–3016. R. Benelli, R. Adatia, B. Ensoli, W.G. Stetler-Stevenson, L. Santi, A. Albini, Inhibition of AIDS-Kaposi’s sarcoma cell induced endothelial cell invasion by TIMP-2 and a synthetic peptide from the metalloproteinase propeptide: implications for an anti-angiogenic therapy, Oncol. Res. 6 (1994) 251–257. A.N. Murphy, E.J. Unsworth, W.G. Stetler-Stevenson, Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation, J. Cell Physiol. 157 (1993) 351–358. A.R. Farina, A. Tacconelli, A. Vacca, M. Maroder, A. Gulino, A.R. Mackay, Transcriptional up-regulation of matrix metalloproteinase9 expression during spontaneous epithelial to neuroblast phenotype conversion by SK-N-SH neuroblastoma cells, involved in enhanced invasivity, depends upon GT-box and nuclear factor kappaB elements, Cell Growth Differ. 10 (1999) 353–367. M. Bond, R.P. Fabunmi, A.H. Baker, A.C. Newby, Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B, FEBS Lett. 435 (1998) 29–34. J.W. Rhee, K.W. Lee, W.J. Sohn, Y. Lee, O.H. Jeon, H.J. Kwon, D.S. Kim, Regulation of matrix metalloproteinase-9 gene expression and cell migration by NF-kappa B in response to CpG-oligodeoxynucleotides in RAW 264.7 cells, Mol. Immunol. 44 (2007) 1393–1400. M. Hidalgo, S.G. Eckhardt, Development of matrix metalloproteinase inhibitors in cancer therapy, J. Natl. Cancer Inst. 93 (2001) 178–193. G. Garcea, D.P. Berry, D.J. Jones, R. Singh, A.R. Dennison, P.B. Farmer, R.A. Sharma, W.P. Steward, A.J. Gescher, Consumption of the putative chemopreventive agent curcumin by cancer patients: assessment of curcumin levels in the colorectum and their pharmacodynamic consequences, Cancer Epidemiol. Biomark. Prev. 14 (2005) 120–125. R.A. Sharma, S.A. Euden, S.L. Platton, D.N. Cooke, A. Shafayat, H.R. Hewitt, T.H. Marczylo, B. Morgan, D. Hemingway, S.M. Plummer, M. Pirmohamed, A.J. Gescher, W.P. Steward, Phase I clinical trial of oral curcumin: biomarkers of systemic activity and compliance, Clin. Cancer Res. 10 (2004) 6847–6854. G. Garcea, D.J. Jones, R. Singh, A.R. Dennison, P.B. Farmer, R.A. Sharma, W.P. Steward, A.J. Gescher, D.P. Berry, Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration, Br. J. Cancer 90 (2004) 1011– 1015. R.A. Sharma, H.R. McLelland, K.A. Hill, C.R. Ireson, S.A. Euden, M.M. Manson, M. Pirmohamed, L.J. Marnett, A.J. Gescher, W.P. Steward, Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer, Clin. Cancer Res. 7 (2001) 1894–1900.
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Curcumin inhibits the migration and invasion of human A549 lung cancer cells through the inhibition of matrix metalloproteinase-2 and -9 and Vascular Endothelial Growth Factor (VEGF) Song-Shei Lin a, Kuang-Chi Lai b,c, Shu-Chun Hsu d, Jai-Sing Yang e, Chao-Lin Kuo f, Jing-Pin Lin g, Yi-Shih Ma h, Chih-Chung Wu i, Jing-Gung Chung j,* a
Department of Medical Imaging and Radiological Science, Central Taiwan University of Science and Technology, Taichung 406, Taiwan School of Medicine, China Medical University, Taichung 404, Taiwan c Department of Surgery, China Medical University Beigang Hospital, Yunlin 651, Taiwan d School of Nutrition, China Medical University, Taichung 404, Taiwan e Department of Pharmacology, China Medical University, Taichung 404, Taiwan f School of Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan g School of Chinese Medicine, China Medical University, Taichung 404, Taiwan h Graduate Institute of Chinese Medical Science, China Medical University, Taichung 404, Taiwan i Department of Health Sciences, Chang Jung Christian University, Tainan 711, Taiwan j Department of Biological Science and Technology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan b
a r t i c l e
i n f o
Article history: Received 6 February 2009 Received in revised form 25 April 2009 Accepted 29 April 2009
Keywords: Curcumin Migration Invasion Matrix metalloproteinases A549 cancer cells
a b s t r a c t It is well known that matrix metalloproteinases (MMPs) act an important role in the invasion, metastasis and angiogenesis of cancer cells. Agents suppressed the MMPs could inhibited the cancer cells migration and invasion. Numerous evidences had shown that curcumin (the active constituent of the dietary spice turmeric) has potential for the prevention and therapy of cancer. Curcumin can inhibit the formation of tumors in animal models of carcinogenesis and act on a variety of molecular targets involved in cancer development. There is however, no available information to address the effects of curcumin on migration and invasion of human lung cancer cells. The anti-tumor invasion and migration effects of lung cancer cells induced by curcumin were examined. Here, we report that curcumin suppressed the migration and invasion of human non-small cell lung cancer cells (A549) in vitro. Our findings suggest that curcumin has anti-metastatic potential by decreasing invasiveness of cancer cells. Moreover, this action was involved in the MEKK3, p-ERK signaling pathways resulting in inhibition of MMP-2 and -9 in human lung cancer A549 cells. Overall, the above data shows that the anticancer effect of curcumin is also exist for the inhibition of migration and invasion in lung cancer cells. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In Taiwan, lung cancer is the major causes of cancer-related deaths in men and women and about 32.8 persons per 100 thousand die annually from lung, based on reports * Corresponding author. Tel.: +886 4 22053366x2500; fax: +886 4 22053764. E-mail address: [email protected] (J.-G. Chung). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.04.037
from the People’s Health Bureau of Taiwan. Although the treatment of lung cancer have been improved but the mortality in lung cancer patients remains high. In order to decrease cancer mortality we should focused on health initiatives including prevention, early detection, and effective treatments. A growing body of research suggests that numerous naturally occurring compounds act as antioxidants, cancer preventative and therapeutic agents [1–3]. Based on the epidemiological studies demonstrate that
128
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
consumption of a phytochemical-rich diet (fruits and vegetables) can lead to decrease the risk of certain types of human cancers [4,5]. In clinical treatment of cancer patients, many prescription drugs are derived from natural plant species [6,7]. Curcumin (diferuloylmethane), an active component of the spice turmeric (Curcuma longa), possess biological activities include chemopreventive and therapeutic properties against many tumors in both in vitro and in vivo models and including clinical trials [8,9]. Curcumin is able to suppress cell proliferation and inflammation, induce apoptosis, and sensitize tumor cells to cancer therapies [10–12] and it also suppress invasion, angiogenesis, and metastasis of cancer cells [13]. Many mechanisms are involved in the biological activities of curcumin including the nuclear factor-jB, IjBa kinase, Akt, activator protein1, mitogen-activated protein kinases (MAPK), cyclooxygenase-2 lipoxygenase, inducible nitric-oxide synthase, urinary plasminogen activator, tumor necrosis factor, chemokines, and cell cycle machinery have been suggested as the targets of curcumin [11,14–17]. Recently, it was reported that curcumin inhibits Akt/mTOR signaling in various tumor cells [18–21]. There is no dose-limiting toxicity after curcumin is administered at doses up to 10 g/day and when the serum concentration of curcumin was 1.77 ± 1.87 lmol/L at the dose of 8 g/day [22]. Based on these reports it may suggest that curcumin could be a potential in cancer therapy. However, to our knowledge the cancer inhibitory potential of curcumin has not been evaluated against migration and invasion of lung cancer 549 cells in vitro systems. In the present study, we investigated the molecular mechanisms by which curcumin suppress migration and invasion in A549 lung cancer cells in vitro. Our results show that curcumin concentration- and timedependently inhibits the migration and invasion in human A549 lung cancer cells. 2. Materials and methods 2.1. Materials and chemicals Curcumin, dimethyl sulfoxide (DMSO), trypan blue and triton X-100, pyruvate, penicillin G and streptomycin were obtained from Sigma Chemical. (St. Louis, MO, USA). AntiMMP-2, anti-PI3 K, anti-PKC, anti-VEGF, anti-c-jun-p, antiRas, anti-GRB2, anti-MEKK3, anti-FAK, anti-MKK7, antiJNK, anti-ERK, anti-MMP-9, anti-MMP-2 and anti-Rho A were purchased from Santa Cruz Biotechnology. Materials and chemicals for electrophoresis were obtained from BioRad.
2.3. Migration determination The A549 cells (1 104 per well) were plated in 6-well plates for 24 h then cells in the individual wells were wounded by scratching with a sterile pipette tip (to remove the cells but both side still have original cells) and incubated with F12K medium containing no FBS and then cells were treated with or without curcumin (10 or 20 lM) for 0, 24 and 48 h. Cells were photographed under phase-contrast microscopy (100) as previously described [23]. 2.4. Migration and invasion determinations Cell migration and invasion were determined with or without matrigel-coated transwell cell culture chambers (8 lm pore size) (Millipore, Billerica, MA, USA) as described previously [23,24]. A549 cells were kept for 24 h in serumfree-medium then were trypsinized and resuspended in serum-free F12K medium and placed in the upper chamber of the transwell insert (5 104 cells/well) and incubated with 0.5% DMSO or curcumin (0, 10 or 20 lM) and F12K medium containing 10% FBS was added to the lower chamber. The plates were incubated in a humidified atmosphere with 95% air and 5% CO2 at 37 °C for 24 or 48 h, non-invasive cells in the upper chamber were removed by wiping with a cotton swab, and invasive cells were fixed with 4% formaldehyde in PBS and were stained with 2% crystal violet in 2% ethanol. Cells in the lower surface of the filter which penetrated through the matrigel were counted under a light microscope at 200 [23]. 2.5. Western blotting A549 cells (1 106/wells) in F12K medium were plated onto 6-well plates then were treated with 30 lM curcumin for 0, 6, 12, 24 and 48 h. Following each time point, cells from each well were harvested and isolated by centrifugation in 1500 rpm for 5 min. The isolated cells were lysed individually and the total protein levels were quantified. Then to determine the proteins levels associated with invasion and migration (PI3K, PKC, VEGF, c-jun-p, Ras, GRB2, MEKK3, FAK, MKK7, JNK, ERK, MMP-9, MMP-2 and Rho A). All samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), as described previously [23]. 2.6. Statistical analysis The differences between the curcumin-treated and control group were analyzed by Student’s t-test.
2.2. Cell culture 3. Results
Human lung carcinoma cell line (A549) was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were placed onto 75 cm2 tissue culture flasks in F12K medium supplemented with 10% FBS, 1% penicillin–streptomycin (100 units/ml penicillin and 100 lg/ml streptomycin) and 1% glutamine and grown at 37 °C under a humidified 5% CO2 and 95% air at one atmosphere.
3.1. Curcumin-inhibited the migration of A549 cells in vitro The inhibitions of A549 cell migration by curcumin were examined by using transwell and wound-healing assays and results are shown in Fig. 1A and B. Higher dose (20 lM) curcumin and longer (48 h) incubation time period led to greater inhibition of cell migration in A549 cells. The percentage of inhibition ratio is 83–95% and these effects were doseand time-dependent manners.
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
129
Fig. 1. Curcumin-inhibited the migration of A549 cells in vitro. A549 cells (1 104 cells/well; 12-well plates) were treated with 0, 10 and 20 lM curcumin for 24 and 48 h. Cells were wounded by scratching with a pipette tip or using transwell cell culture chambers, and the cells were incubated with F12K medium containing no FBS and incubation with or without curcumin for 48 h. Cells were photographed under phase-contrast microscopy (200) (Panel A) and quantitation (B). P < 0.01, significant difference between curcumin-treated groups and the control.
3.2. Curcumin-inhibited the invasion of A549 cells in vitro The inhibitions of A549 cells invasion by curcumin were examined and results are shown in Fig. 2A and B. The Fig. 2A shows that A549 cells invased from the upper to the lower chamber in majority of cells from the control group (absence of curcumin) but the penetration of the EHScoated filter by A549 cells was inhibited in the presence of curcumin. This inhibitory effect was higher at a curcumin concentration of 20 lM than 10 lM. The quantification of cells in the lower chamber from Fig. 2B indicated that curcumin significantly inhibited A549 cell invasion, the percentage of inhibition ratio is 95–98% and this effect was dose-dependent. 3.3. Curcumin-inhibited levels of PI3K, PKC, VEGF, c-jun-p, Ras, GRB2, MEKK3, FAK, MKK7, JNK, ERK, MMP-9, MMP-2 and RhoA in A549 cells Levels of invasion- and migration-associated proteins in A549 cells after treatment with curcumin were determined and quantitated by Western blotting. Results were shown in Fig. 3A–C indicate that the levels of VEGF, c-jun-p, and GRB2 (Fig. 3A), MEKK3, FAK and MKK7 (Fig. 3B), MMP2 and MMP-9 and Rho A (Fig. 3C) were lower than the corresponding control group. However, JNK and PERK (Fig. 3B) was higher than that of control. MMP-2 and -9 play an important role of cell invasion and migration. These effects may have led to the inhibition of invasion and migration from A549 cells after exposure to curcumin at 30 lM.
4. Discussion It is well known that curcumin perform anticancer activity in many cancer cells in vitro and in vivo. Our previous studies also showed that curcumin decreased the percentage of viable A549 cells through G2/M phase arrest and apoptosis and those effects are dose- and time-dependent manners. Although it was reported that curcumin can inhibit human lung adenocarcinoma (CL1-5) cell invasion and metastasis through activation of the tumor suppressor DnaJ-like heat shock protein 40 (HLJ1) through activation of the JNK/JunD pathway [20]. However, actions of curcumin on migration and invasion of human A549 lung cancer cells and the possible signaling pathways have not been addressed. In this study, we found that curcumin suppressed the migration and invasion of A549 cells through the MEEK3, FAK, Rho A and MKK7 pathway then led to the inhibition of MMP-2. Cell migration is involving several growth factors, for binding to receptors on cell surface, stimulating down-
130
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
Fig. 2. Curcumin-inhibited the invasion of A549 cells in vitro. A549 cells (104 cells/0.4 ml F12K medium) were placed and cells that penetrated through the matrigel to the lower surface of the filter were stained with crystal violet and were photographed under a light microscope at 200 (Panel A). Quantification of cells in the lower chamber, was performed by counting cells at 200 (Panel B). P < 0.001, significant difference between curcumin-treated groups and the control.
stream signaling pathways and resulting in cytoskeletal reorganization and stimulation of motility machinery of the cell [25]. Therefore, this cellular process provides a multiple targets for the development of therapeutic agents to inhibit cancer invasion and metastasis [26,27]. It was reported that cancer cell–matrix interaction is play an initial and critical step for promoting cell migration, proliferation and degradation [28,29]. Matrix metalloproteinases (MMPs) are a multigene family of zinc-dependent endopeptidases that to play the degrading essentially all extracellular matrix (ECM) components, allowing tumor cells to invade surrounding tissues and enter the blood stream to travel to distant sites [30]. MMPs play an important role in ECM degradation for tumor growth, invasion and tumor-induced angiogenesis [31]. It is well known that: (1) the derangement of ECM by MMPs plays an initial key role in cell movement which via the alteration of cell-ECM interactions; (2) MMPs breakdown the basement membrane for cancer cells to
migration and invasion. Among MMPs, MMP-2 and MMP-9 play the most important for basement membrane type IV collagen degradation [28,29,32]. In particular, MMP-2 expression is associated with tumor invasion, angiogenesis, metastasis and recurrence [33,34]. It was reposted that MMP-2 and MMP-9 are correlated with an aggressive, invasive or metastatic tumor phenotype [35– 37]. It is well known that MMP inhibitors block endothelial cell activities which are essential for new vessel development leading to proliferation and invasion [38,39]. Therefore, MMP-2 and MMP-9 are thought to be therapeutic targets of anticancer drugs based on the degrading action of both enzymes on gelatins which are major components of the basement membrane. Thus, it seems quite likely that curcumin could inhibit or delay cancer invasion and migration in A549 cells via modulation of MMPs. Our results showed that curcumin suppressed the levels of ERK, MMP-2 and -9 protein levels in A549 cells. It has
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
131
Fig. 3. Representative Western blot showing changes in the levels of associated proteins in migration and invasion of A549 cells after exposure to curcumin. The A549 cells (1 106 cells/well) were treated with curcumin at 30 lM for 0, 6, 12, 24 and 48 h then the total protein was prepared and determined, as described in Section 2. The levels of PI3K, PKC, VEGF, C-jun-p, Ras and GRB2 (A), MEKK3, FAK, MKK7, JNK1/2, ERK1/2 (B) and MMP-2, MMP-9 and Rho A (C) expressions were estimated by Western blotting, as described in Section 2.
been reported that the proteins of MMP-9 and uPA require NF-jB [40,41] and AP-1. It is well known that the activation of ERK will stimulates two cis-acting regulatory elements including the binding sites of AP-1 and NF-jB which play an important role in controlling MMP-9 gene expression [42]. Anti-metastatic compounds inhibited MMPs through the different mechanisms [43] those mechanisms include: (1) direct inhibition of the enzymatic activity of MMPs; (2) interference in the activation of pro-MMPs; and (3) reduced expression of MMP genes and protein levels. Other factors such as the pharmacokinetics of curcumin suggest that a poor bioavailability in
humans due to its rapid metabolism in the liver and intestinal wall, when taken orally [44–47]. Therefore, the exact molecular signaling pathway for curcumin-inhibited the migration and invasion in A549 cells still need further investigation. Taken together, our data for the first time showed that curcumin could inhibit migration and invasion of human A549 lung cancer cells through MEKK and ERK signaling pathways resulting in the inhibition of MMP-2 and -9 as summarized in Fig. 4. Curcumin could be recognized to be a therapeutic agent for inhibiting the metastasis and invasion of lung.
132
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
Fig. 4. Molecular signaling pathways for curcumin-inhibited invasion and migration in human lung cancer A549 cells.
Conflict of interest None declared. Acknowledgment This work was supported by Grant NSC95-2745-B-039002-URD from National Science Council, Taiwan. References [1] D.M. Parkin, P. Pisani, J. Ferlay, Estimates of the worldwide incidence of 25 major cancers in 1990, Int. J. Cancer 80 (1999) 827–841. [2] M. Hashibe, P. Brennan, S. Benhamou, X. Castellsague, C. Chen, M.P. Curado, L. Dal Maso, A.W. Daudt, E. Fabianova, L. Fernandez, V. Wunsch-Filho, S. Franceschi, R.B. Hayes, R. Herrero, S. Koifman, C. La Vecchia, P. Lazarus, F. Levi, D. Mates, E. Matos, A. Menezes, J. Muscat, J. Eluf-Neto, A.F. Olshan, P. Rudnai, S.M. Schwartz, E. Smith, E.M. Sturgis, N. Szeszenia-Dabrowska, R. Talamini, Q. Wei, D.M. Winn, D. Zaridze, W. Zatonski, Z.F. Zhang, J. Berthiller, P. Boffetta, Alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium, J. Natl. Cancer Inst. 99 (2007) 777–789. [3] M. Rahman, J. Sakamoto, T. Fukui, Calculation of population attributable risk for bidi smoking and oral cancer in south Asia, Prev. Med. 40 (2005) 510–514. [4] F.L. Meyskens Jr., E. Szabo, Diet and cancer: the disconnect between epidemiology and randomized clinical trials, Cancer Epidemiol. Biomark. Prev. 14 (2005) 1366–1369. [5] K.A. Steinmetz, J.D. Potter, Vegetables, fruit, and cancer. I. Epidemiology, Cancer Causes Control 2 (1991) 325–357. [6] R. Room, T. Babor, J. Rehm, Alcohol and public health, Lancet 365 (2005) 519–530. [7] P. Balaram, H. Sridhar, T. Rajkumar, S. Vaccarella, R. Herrero, A. Nandakumar, K. Ravichandran, K. Ramdas, R. Sankaranarayanan, V. Gajalakshmi, N. Munoz, S. Franceschi, Oral cancer in southern India: the influence of smoking, drinking, paan-chewing and oral hygiene, Int. J. Cancer 98 (2002) 440–445. [8] T.O. Khor, Y.S. Keum, W. Lin, J.H. Kim, R. Hu, G. Shen, C. Xu, A. Gopalakrishnan, B. Reddy, X. Zheng, A.H. Conney, A.N. Kong, Combined inhibitory effects of curcumin and phenethyl isothiocyanate on the growth of human PC-3 prostate xenografts in immunodeficient mice, Cancer Res. 66 (2006) 613–621.
[9] R.A. Sharma, A.J. Gescher, W.P. Steward, Curcumin: the story so far, Eur. J. Cancer 41 (2005) 1955–1968. [10] D. Karunagaran, R. Rashmi, T.R. Kumar, Induction of apoptosis by curcumin and its implications for cancer therapy, Curr. Cancer Drug Targets 5 (2005) 117–129. [11] R.L. Thangapazham, A. Sharma, R.K. Maheshwari, Multiple molecular targets in cancer chemoprevention by curcumin, AAPS J. 8 (2006) E443–E449. [12] R.K. Srivastava, Q. Chen, I. Siddiqui, K. Sarva, S. Shankar, Linkage of curcumin-induced cell cycle arrest and apoptosis by cyclindependent kinase inhibitor p21 (/WAF1/CIP1), Cell Cycle 6 (2007) 2953–2961. [13] L.G. Menon, R. Kuttan, G. Kuttan, Anti-metastatic activity of curcumin and catechin, Cancer Lett. 141 (1999) 159–165. [14] H.W. Chen, H.C. Huang, Effect of curcumin on cell cycle progression and apoptosis in vascular smooth muscle cells, Br. J. Pharmacol. 124 (1998) 1029–1040. [15] S. Aggarwal, H. Ichikawa, Y. Takada, S.K. Sandur, S. Shishodia, B.B. Aggarwal, Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation, Mol. Pharmacol. 69 (2006) 195–206. [16] S.Y. Kim, S.H. Jung, H.S. Kim, Curcumin is a potent broad spectrum inhibitor of matrix metalloproteinase gene expression in human astroglioma cells, Biochem. Biophys. Res. Commun. 337 (2005) 510– 516. [17] B.B. Aggarwal, A. Kumar, A.C. Bharti, Anticancer potential of curcumin: preclinical and clinical studies, Anticancer Res. 23 (2003) 363–398. [18] C.S. Beevers, F. Li, L. Liu, S. Huang, Curcumin inhibits the mammalian target of rapamycin-mediated signaling pathways in cancer cells, Int. J. Cancer 119 (2006) 757–764. [19] H. Aoki, Y. Takada, S. Kondo, R. Sawaya, B.B. Aggarwal, Y. Kondo, Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways, Mol. Pharmacol. 72 (2007) 29–39. [20] H.W. Chen, J.Y. Lee, J.Y. Huang, C.C. Wang, W.J. Chen, S.F. Su, C.W. Huang, C.C. Ho, J.J. Chen, M.F. Tsai, S.L. Yu, P.C. Yang, Curcumin inhibits lung cancer cell invasion and metastasis through the tumor suppressor HLJ1, Cancer Res. 68 (2008) 7428–7438. [21] J.H. Kim, C. Xu, Y.S. Keum, B. Reddy, A. Conney, A.N. Kong, Inhibition of EGFR signaling in human prostate cancer PC-3 cells by combination treatment with beta-phenylethyl isothiocyanate and curcumin, Carcinogenesis 27 (2006) 475–482. [22] A.L. Cheng, C.H. Hsu, J.K. Lin, M.M. Hsu, Y.F. Ho, T.S. Shen, J.Y. Ko, J.T. Lin, B.R. Lin, W. Ming-Shiang, H.S. Yu, S.H. Jee, G.S. Chen, T.M. Chen, C.A. Chen, M.K. Lai, Y.S. Pu, M.H. Pan, Y.J. Wang, C.C. Tsai, C.Y. Hsieh, Phase I clinical trial of curcumin, a chemopreventive agent, in
S.-S. Lin et al. / Cancer Letters 285 (2009) 127–133
[23]
[24]
[25] [26]
[27] [28] [29] [30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
patients with high-risk or pre-malignant lesions, Anticancer Res. 21 (2001) 2895–2900. S.C. Hsu, C.L. Kuo, J.P. Lin, J.H. Lee, C.C. Lin, C.C. Su, M.D. Yang, J.G. Chung, Crude extracts of Euchresta formosana radix inhibit invasion and migration of human hepatocellular carcinoma cells, Anticancer Res. 27 (2007) 2377–2384. Y.T. Huang, J.J. Hwang, L.T. Lee, C. Liebow, P.P. Lee, F.C. Ke, T.B. Lo, A.V. Schally, M.T. Lee, Inhibitory effects of a luteinizing hormonereleasing hormone agonist on basal and epidermal growth factorinduced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells, Int. J. Cancer 99 (2002) 505–513. B. Anand-Apte, B. Zetter, Signaling mechanisms in growth factorstimulated cell motility, Stem Cells 15 (1997) 259–267. J.T. Price, E.W. Thompson, Mechanisms of tumour invasion and metastasis: emerging targets for therapy, Expert Opin. Ther. Targets 6 (2002) 217–233. G. Fenteany, S. Zhu, Small-molecule inhibitors of actin dynamics and cell motility, Curr. Top Med. Chem. 3 (2003) 593–616. F.G. Giancotti, E. Ruoslahti, Integrin signaling, Science 285 (1999) 1028–1032. W.G. Stetler-Stevenson, Type IV collagenases in tumor invasion and metastasis, Cancer Metast. Rev. 9 (1990) 289–303. L.A. Liotta, P.S. Steeg, W.G. Stetler-Stevenson, Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation, Cell 64 (1991) 327–336. J. Westermarck, V.M. Kahari, Regulation of matrix metalloproteinase expression in tumor invasion, FASEB J. 13 (1999) 781–792. Z.S. Zeng, A.M. Cohen, J.G. Guillem, Loss of basement membrane type IV collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis, Carcinogenesis 20 (1999) 749–755. K. Komatsu, Y. Nakanishi, N. Nemoto, T. Hori, T. Sawada, M. Kobayashi, Expression and quantitative analysis of matrix metalloproteinase-2 and -9 in human gliomas, Brain Tumor Pathol. 21 (2004) 105–112. M. Wang, T. Wang, S. Liu, D. Yoshida, A. Teramoto, The expression of matrix metalloproteinase-2 and -9 in human gliomas of different pathological grades, Brain Tumor Pathol. 20 (2003) 65–72. A.S. Papathoma, V. Zoumpourlis, A. Balmain, A. Pintzas, Role of matrix metalloproteinase-9 in progression of mouse skin carcinogenesis, Mol. Carcinogen. 31 (2001) 74–82. M.I. Cockett, G. Murphy, M.L. Birch, J.P. O’Connell, T. Crabbe, A.T. Millican, I.R. Hart, A.J. Docherty, Matrix metalloproteinases and metastatic cancer, Biochem. Soc. Symp. 63 (1998) 295–313. F.J. Bianco Jr., D.C. Gervasi, R. Tiguert, D.J. Grignon, J.E. Pontes, J.D. Crissman, R. Fridman, D.P. Wood Jr., Matrix metalloproteinase-9
[38]
[39]
[40]
[41]
[42]
[43] [44]
[45]
[46]
[47]
133
expression in bladder washes from bladder cancer patients predicts pathological stage and grade, Clin. Cancer Res. 4 (1998) 3011–3016. R. Benelli, R. Adatia, B. Ensoli, W.G. Stetler-Stevenson, L. Santi, A. Albini, Inhibition of AIDS-Kaposi’s sarcoma cell induced endothelial cell invasion by TIMP-2 and a synthetic peptide from the metalloproteinase propeptide: implications for an anti-angiogenic therapy, Oncol. Res. 6 (1994) 251–257. A.N. Murphy, E.J. Unsworth, W.G. Stetler-Stevenson, Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation, J. Cell Physiol. 157 (1993) 351–358. A.R. Farina, A. Tacconelli, A. Vacca, M. Maroder, A. Gulino, A.R. Mackay, Transcriptional up-regulation of matrix metalloproteinase9 expression during spontaneous epithelial to neuroblast phenotype conversion by SK-N-SH neuroblastoma cells, involved in enhanced invasivity, depends upon GT-box and nuclear factor kappaB elements, Cell Growth Differ. 10 (1999) 353–367. M. Bond, R.P. Fabunmi, A.H. Baker, A.C. Newby, Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B, FEBS Lett. 435 (1998) 29–34. J.W. Rhee, K.W. Lee, W.J. Sohn, Y. Lee, O.H. Jeon, H.J. Kwon, D.S. Kim, Regulation of matrix metalloproteinase-9 gene expression and cell migration by NF-kappa B in response to CpG-oligodeoxynucleotides in RAW 264.7 cells, Mol. Immunol. 44 (2007) 1393–1400. M. Hidalgo, S.G. Eckhardt, Development of matrix metalloproteinase inhibitors in cancer therapy, J. Natl. Cancer Inst. 93 (2001) 178–193. G. Garcea, D.P. Berry, D.J. Jones, R. Singh, A.R. Dennison, P.B. Farmer, R.A. Sharma, W.P. Steward, A.J. Gescher, Consumption of the putative chemopreventive agent curcumin by cancer patients: assessment of curcumin levels in the colorectum and their pharmacodynamic consequences, Cancer Epidemiol. Biomark. Prev. 14 (2005) 120–125. R.A. Sharma, S.A. Euden, S.L. Platton, D.N. Cooke, A. Shafayat, H.R. Hewitt, T.H. Marczylo, B. Morgan, D. Hemingway, S.M. Plummer, M. Pirmohamed, A.J. Gescher, W.P. Steward, Phase I clinical trial of oral curcumin: biomarkers of systemic activity and compliance, Clin. Cancer Res. 10 (2004) 6847–6854. G. Garcea, D.J. Jones, R. Singh, A.R. Dennison, P.B. Farmer, R.A. Sharma, W.P. Steward, A.J. Gescher, D.P. Berry, Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration, Br. J. Cancer 90 (2004) 1011– 1015. R.A. Sharma, H.R. McLelland, K.A. Hill, C.R. Ireson, S.A. Euden, M.M. Manson, M. Pirmohamed, L.J. Marnett, A.J. Gescher, W.P. Steward, Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer, Clin. Cancer Res. 7 (2001) 1894–1900.