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Anti-metastatic effect of deoxyelephantopin from Elephantopus scaber in A549 lung cancer cells in vitro a
b
b
A.K. Farha , S.R. Dhanya , S. Nair Mangalam & P. Remani
a
a
Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India b
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CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India Published online: 17 Feb 2015.
To cite this article: A.K. Farha, S.R. Dhanya, S. Nair Mangalam & P. Remani (2015): Anti-metastatic effect of deoxyelephantopin from Elephantopus scaber in A549 lung cancer cells in vitro, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1012165 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1012165
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1012165
SHORT COMMUNICATION Anti-metastatic effect of deoxyelephantopin from Elephantopus scaber in A549 lung cancer cells in vitro A.K. Farhaa, S.R. Dhanyab, S. Nair Mangalamb and P. Remania* a
Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India; CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India b
Downloaded by [Lakehead University] at 06:56 13 March 2015
(Received 4 November 2014; final version received 20 January 2015)
In this study, we focused on the in vitro anti-metastatic effects of deoxyelephantopin (DOE), a sesquiterpene lactone from Elephantopus scaber on lung cancer A549 cells. DOE significantly decreased the metastatic potential of A549 cells as demonstrated by transwell invasion and migration assay. DOE inhibited the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor at transcript level. Tissue inhibitors of metalloproteinase-2 (TIMP-2) mRNA levels was up-regulated in A549 tumour cells without any change in TIMP-1 expression after DOE treatment. DOE inhibited the protein levels of p-ERK1/2 and p-Akt in A549 cells but it activated p-JNK, p-p38 protein expression. NF-kB and IkBa expressions were down-regulated in DOE-treated cells. All these results demonstrated that DOE has shown anti-metastatic activity against A549 tumour cells. Keywords: deoxyelephantopin; anti-migratory; anti-invasive; MMP; uPA; Elephantopus scaber; TIMP
1. Introduction Lung cancer is the leading cause of cancer mortality worldwide (Jemal et al. 2010). Non-small cell lung cancer (NSCLC) accounts for nearly 80% of all lung cancers. The progression of cancer from benign and delimited growth to invasive and metastatic growth is the major cause of poor clinical outcome in lung cancer patients (Liu et al. 2010). Conventional treatments of NSCLC are fairly ineffective. Consequently, a great demand for alternate therapy in the treatment of lung cancer is felt. Metastasis is a multistep process in which tumour cells from primary tumour detach from its origin, invading the surrounding tissue matrix, enters into the circulatory systems of blood and
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Downloaded by [Lakehead University] at 06:56 13 March 2015
2
A.K. Farha et al.
lymph and locate in distant parts of the body and form secondary tumour. The crucial steps in metastasis are degradation of extracellular matrix (ECM) and basement membrane (BM). Matrix metalloproteinases (MMPs) abundantly expressed in many cancer cells contribute to metastasis by degrading ECM. Tissue inhibitors of metalloproteinase (TIMPs) regulate the activities of MMPs (Figueira et al. 2009). Urokinase-type plasminogen activator (uPA), an essential protein that promotes invasion and metastasis, is involved in the hydrolysis of BM and ECM. Thus, metastasis may be prevented by targeting MMP-2, MMP-9 and uPA system. The mitogenactivated protein kinases (MAPKs) have been implicated in cell invasion, migration and angiogenesis, events that are essential for the successful completion of metastasis (Reddy et al. 2003). In addition, PI3K/Akt signal transduction pathway is involved in the development, progression and metastasis of tumours (Chen et al. 2014). NF-kB is constitutively expressed in tumour cells and plays a key role in tumour progression and invasion (Karin et al. 2002). Natural products of plant origin were intensively described to prevent cancer metastasis (Ogasawara et al. 2001). Elephantopus scaber is a well-known herb widely used in South-East Asia for the treatment of neoplasm and other infectious diseases (Kabeer & Prathapan 2014). The main constituents of this herb are sesquiterpene lactones of which deoxyelephantopin (DOE) is the major component. Recent studies suggest that DOE inhibited the growth of breast cancer cells and nasopharyngeal carcinoma cells by apoptosis induction and G2/M cell cycle arrest (Huang et al. 2010; Su et al. 2011). We have reported that DOE has shown anti-tumour effect on A549 lung cancer cells and SiHa cervical cancer cells (Kabeer et al. 2013; Farha et al. 2014). Regarding the anti-metastatic effects of DOE on lung cancer cells, no literature reports are available yet. In this study, anti-metastatic potential of DOE on A549 cells and molecular mechanisms were investigated. 2. Results and discussion 2.1. Effect of DOE on the migration and invasion property of A549 cells Despite advances in the therapeutic options, high mortality of lung cancer is mainly attributed to metastasis. Thus, the search for agents capable of reducing the metastatic potential of tumour cell is a better option for lung cancer treatment. In this study, we investigated the anti-metastatic potential of DOE (Figure 1) on A549 cells. In vitro wound closure assay and migration assay were performed to analyse the effect of DOE on the migrating property of A549 cells. Untreated A549 cells migrated to close the wound while the migration was inhibited by DOE treatment. After 48 h, the area of wound created in the cell monolayer was decreased in control cells, whereas in treated cells the area was increased (Figure S1A). A significant decrease in the number of migrating cells through the filters was observed after 24 h of treatment with DOE ( p , 0.001) (Figure S1A). In vitro invasion assay was used to investigate whether DOE inhibits the invasiveness of A549 cells. The invasion rate of cells treated with DOE was lowered as compared with control, indicating that DOE suppressed tumour cell invasion (Figure S1B).
Figure 1. Structure of DOE isolated from E. scaber.
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Migration and invasion assays have demonstrated that DOE showed the anti-metastatic potentials by inhibiting the steps involved in metastasis. 2.2. Effects of DOE on MMP-2, MMP-9, uPA, uPAR, TIMP-1 and TIMP-2 mRNA expression in A549 cells MMPs, associated with cancer cell migration and invasion, are inhibited by endogenous TIMPs such as TIMP-1 and TIMP-2 (Deryugina & Quigley 2006). Disturbance in the balance of MMPs and TIMPs is found in cancer (Lambert et al. 2004). In this study, DOE significantly downregulated the mRNA expression level of MMP-2 and MMP-9 and up-regulated TIMP-2 expressions without any change in TIMP-1 mRNA level (Figure S2A). The plasmin generated as a consequence of binding of uPA on its receptor urokinase-type plasminogen activator receptor (uPAR) is known to activate MMPs thereby triggering ECM degradation leading to metastasis (Schweinitz et al. 2004). DOE reduced uPA and uPAR mRNA expression and inhibited the metastatic ability of A549 cells (Figure S2A). This study indicates that DOE could inhibit the migration and invasion property of tumour cells, probably through a decrease in MMPs and uPA system expressions, and simultaneous increase of the TIMP expression. 2.3. Effect of DOE on MAPKs and Akt signalling pathways in A549 cells MAPK signalling pathways known to mediate metastasis includes extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinases (JNK) and p38 (Chang & Karin 2001). The PI3K-Akt signalling pathway plays another crucial role in MMPs for uPA gene regulation, cell survival and invasion (Kim et al. 2001). To clarify the involvement of MAPKs and Akt signalling pathways in DOE-mediated metastatic inhibition of A549 cells, we have evaluated the protein levels of MAPKs (JNK, p38 and ERK) and Akt by western blotting. Exposure of cells to DOE reduced the protein levels of both p-ERK 1/2 and p-Akt. The protein level of p-p38 and pJNK was up-regulated (Figure S2B). The total protein level of ERK1/2, JNK, p38 and Akt was not affected by DOE treatment. DOE suppressed the phosphorylation of Akt and ERK1/2 effectively. 2.4. Effects of DOE on NF-kB mRNA expression NF-kB, present in the cytoplasm as an inactive heterotrimer, consists of p50, p65 and IkBa subunits. On activation, IkBa protein undergoes degradation and NF-kB heterodimer translocates into the nucleus where it regulates the transcriptional activation of genes involved in cell proliferation, metastasis and angiogenesis (Bjorklund & Koivunen 2005). NF-kB is constitutively activated in NSCLC and is associated with metastasis and poor prognosis (Chen et al. 2011). Treatment of A549 cells with DOE resulted in a strong inhibition of NF-kB activation with a decrease in gene expression of both NF-kB and IkBa at transcript level (Figure S2A). As a transcription factor, NF-kB is known to regulate the expression of various genes involved in invasion such as MMP-2 and MMP-9, uPA and uPAR (Al Dhaheri et al. 2013). Consistent with these results, DOE treatment also inhibited the downstream targets of genes such as MMP-2 and MMP-9, uPA and uPAR resulting in metastasis inhibition. 3. Conclusion These results indicate that anti-metastatic effect of DOE in A549 cells is associated with the down-regulation of NF-kB, IkBa MMP-2, MMP-9, uPA and uPAR and up-regulation of TIMP2 at the transcript level. DOE suppressed the activation of ERK1/2 and Akt, but promoted the
4
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activation of JNK and p38 and resulted in the inhibition of metastasis. Thus, DOE might be a strong candidate for the treatment of lung cancer. Supplementary material Experimental details, figures and table relating to this article are available online.
Funding The first author thanks the University Grant Commission (UGC) for financial support. This study was supported by research grant from the Indian Council of Medical Research (ICMR), New Delhi, India [grant number 59/47/2011/BMS/TRM].
Downloaded by [Lakehead University] at 06:56 13 March 2015
Conflict of interest There is no conflict of interest to declare.
References Al Dhaheri Y, Attoub S, Arafat K, AbuQamar S, Viallet J. 2013. Anti-metastatic and anti-tumor growth effects of Origanum majorana on highly metastatic human breast cancer cells: inhibition of NFkB signaling and reduction of Nitric Oxide production. PLoS ONE. 8:e68808. doi:10.1371/journal.pone.0068808. Bjorklund M, Koivunen E. 2005. Gelatinase-mediated migration and invasion of cancer cells. Biochim Biophys Acta. 1755:37–69. Chang L, Karin M. 2001. Mammalian MAP kinase signaling cascades. Nature. 410:37–40. doi:10.1038/35065000. Chen SC, Chien YC, Pan CH, Sheu JH, Chen CY, Wu CH. 2014. Inhibitory effect of dihydroaustrasulfone alcohol on the migration of human non-small cell lung carcinoma A549 cells and the antitumor effect on a Lewis lung carcinoma-bearing tumor model in C57BL/6J mice. Mar Drugs. 12:196–213. doi:10.3390/md12010196. Chen W, Li Z, Bai L, Lin Y. 2011. NF-kB in lung cancer, a carcinogenesis mediator and a prevention and therapy target. Front Biosci. 16:1172–1185. doi:10.2741/3782. Deryugina EI, Quigley JP. 2006. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev. 25:9–34. doi:10.1007/s10555-006-7886-9. Farha AK, Dhanya SR, Mangalam SN, Geetha BS, Latha PG, Remani P. 2014. Deoxyelephantopin impairs growth of cervical carcinoma SiHa cells and induces apoptosis by targeting multiple molecular signaling pathways. Cell Biol Toxicol. 30:331–343. doi:10.1007/s10565-014-9288-z. Figueira RC, Gomes LR, Neto JS, Silva FC, Silva ID, Sogayar MC. 2009. Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential. BMC Cancer. 9:20. doi:10.1186/1471-2407-9-20. Huang CC, Lo CP, Chiu CY, Shyur LF. 2010. Deoxyelephantopin, a novel multifunctional agent, suppresses mammary tumour growth and lung metastasis and doubles survival time in mice. Br J Pharmacol. 159:856– 871. doi:10. 1111/j.1476-5381.2009.00581.x. Jemal A, Siegel R, Xu J, Ward E. 2010. Cancer statistics, 2010. Cancer J Clin. 60:277–300. doi:10.3322/caac.20073. Kabeer FA, Prathapan R. 2014. Phytopharmacological profile of Elephantopus scaber. Pharmacologia. 5:272–285. Kabeer FA, Sreedevi GB, Nair MS, Rajalekshmi DS, Gopalakrishnan LP, Kunjuraman S, Prathapan R. 2013. Antineoplastic effects of deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, on lung adenocarcinoma (A549) cells. J Integr Med. 11:269– 277. doi:10.3736/jintegrmed2013040. Karin M, Cao Y, Greten FR, Li ZW. 2002. NF-kB in cancer: from innocent bystander to major culprit. Nat Rev Cancer. 2:301–310. doi:10.1038/nrc780. Kim D, Kim S, Koh H, Yoon SO, Chung AS. 2001. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J. 15:1953–1962. doi:10.1096/fj.01-0198com. Lambert E, Dasse´ E, Haye B, Petitfre`re E. 2004. TIMPs as multifacial proteins. Crit Rev Oncol Hematol. 49:187–198. doi:10.1016/j.critrevonc.2003.09.008. Liu P-L, Tsai J-R, Hwang J-J, Chou S-H, Cheng Y-J, Lin F-Y, Chen Y-L, Hung C-Y, Chen W-C, Chen Y-H, Chong I-W. 2010. High-Mobility Group Box 1-mediated matrix metalloproteinase-9 expression in non-small cell lung cancer contributes to tumor cell invasiveness. Am J Respir Cell Mol Biol. 43:530–538. doi:10.1165/rcmb.20090269OC. Ogasawara M, Matsubara T, Suzuki H. 2001. Screening of natural compounds for inhibitory activity on colon cancer cell migration. Biol Pharm Bull. 24:720–723. doi:10.1248/bpb.24.720.
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Reddy KB, Nabha SM, Atanaskova N, Gomes LR, Terra LF, Wailemann RA, Labriola L, Sogayar MC. 2003. Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev. 22:395 – 403. doi:10.1023/ A:1023781114568. Schweinitz A, Steinmetzer T, Banke IJ, Arlt MJ, Sturzebecher A, Schuster O, Geissler A, Giersiefen H, Zeslawska E, Jacob U. 2004. Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as anti-metastatic agents. J Biol Chem. 279:33613–33622. doi:10.1074/jbc. M314151200. Su M, Chung HY, Li Y. 2011. Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells. Biochem Biophys Res Commun. 411:342– 347. doi:10.1016/j.bbrc. 2011.06.144.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Anti-metastatic effect of deoxyelephantopin from Elephantopus scaber in A549 lung cancer cells in vitro a
b
b
A.K. Farha , S.R. Dhanya , S. Nair Mangalam & P. Remani
a
a
Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India b
Click for updates
CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India Published online: 17 Feb 2015.
To cite this article: A.K. Farha, S.R. Dhanya, S. Nair Mangalam & P. Remani (2015): Anti-metastatic effect of deoxyelephantopin from Elephantopus scaber in A549 lung cancer cells in vitro, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1012165 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1012165
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Lakehead University] at 06:56 13 March 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1012165
SHORT COMMUNICATION Anti-metastatic effect of deoxyelephantopin from Elephantopus scaber in A549 lung cancer cells in vitro A.K. Farhaa, S.R. Dhanyab, S. Nair Mangalamb and P. Remania* a
Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India; CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India b
Downloaded by [Lakehead University] at 06:56 13 March 2015
(Received 4 November 2014; final version received 20 January 2015)
In this study, we focused on the in vitro anti-metastatic effects of deoxyelephantopin (DOE), a sesquiterpene lactone from Elephantopus scaber on lung cancer A549 cells. DOE significantly decreased the metastatic potential of A549 cells as demonstrated by transwell invasion and migration assay. DOE inhibited the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor at transcript level. Tissue inhibitors of metalloproteinase-2 (TIMP-2) mRNA levels was up-regulated in A549 tumour cells without any change in TIMP-1 expression after DOE treatment. DOE inhibited the protein levels of p-ERK1/2 and p-Akt in A549 cells but it activated p-JNK, p-p38 protein expression. NF-kB and IkBa expressions were down-regulated in DOE-treated cells. All these results demonstrated that DOE has shown anti-metastatic activity against A549 tumour cells. Keywords: deoxyelephantopin; anti-migratory; anti-invasive; MMP; uPA; Elephantopus scaber; TIMP
1. Introduction Lung cancer is the leading cause of cancer mortality worldwide (Jemal et al. 2010). Non-small cell lung cancer (NSCLC) accounts for nearly 80% of all lung cancers. The progression of cancer from benign and delimited growth to invasive and metastatic growth is the major cause of poor clinical outcome in lung cancer patients (Liu et al. 2010). Conventional treatments of NSCLC are fairly ineffective. Consequently, a great demand for alternate therapy in the treatment of lung cancer is felt. Metastasis is a multistep process in which tumour cells from primary tumour detach from its origin, invading the surrounding tissue matrix, enters into the circulatory systems of blood and
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Downloaded by [Lakehead University] at 06:56 13 March 2015
2
A.K. Farha et al.
lymph and locate in distant parts of the body and form secondary tumour. The crucial steps in metastasis are degradation of extracellular matrix (ECM) and basement membrane (BM). Matrix metalloproteinases (MMPs) abundantly expressed in many cancer cells contribute to metastasis by degrading ECM. Tissue inhibitors of metalloproteinase (TIMPs) regulate the activities of MMPs (Figueira et al. 2009). Urokinase-type plasminogen activator (uPA), an essential protein that promotes invasion and metastasis, is involved in the hydrolysis of BM and ECM. Thus, metastasis may be prevented by targeting MMP-2, MMP-9 and uPA system. The mitogenactivated protein kinases (MAPKs) have been implicated in cell invasion, migration and angiogenesis, events that are essential for the successful completion of metastasis (Reddy et al. 2003). In addition, PI3K/Akt signal transduction pathway is involved in the development, progression and metastasis of tumours (Chen et al. 2014). NF-kB is constitutively expressed in tumour cells and plays a key role in tumour progression and invasion (Karin et al. 2002). Natural products of plant origin were intensively described to prevent cancer metastasis (Ogasawara et al. 2001). Elephantopus scaber is a well-known herb widely used in South-East Asia for the treatment of neoplasm and other infectious diseases (Kabeer & Prathapan 2014). The main constituents of this herb are sesquiterpene lactones of which deoxyelephantopin (DOE) is the major component. Recent studies suggest that DOE inhibited the growth of breast cancer cells and nasopharyngeal carcinoma cells by apoptosis induction and G2/M cell cycle arrest (Huang et al. 2010; Su et al. 2011). We have reported that DOE has shown anti-tumour effect on A549 lung cancer cells and SiHa cervical cancer cells (Kabeer et al. 2013; Farha et al. 2014). Regarding the anti-metastatic effects of DOE on lung cancer cells, no literature reports are available yet. In this study, anti-metastatic potential of DOE on A549 cells and molecular mechanisms were investigated. 2. Results and discussion 2.1. Effect of DOE on the migration and invasion property of A549 cells Despite advances in the therapeutic options, high mortality of lung cancer is mainly attributed to metastasis. Thus, the search for agents capable of reducing the metastatic potential of tumour cell is a better option for lung cancer treatment. In this study, we investigated the anti-metastatic potential of DOE (Figure 1) on A549 cells. In vitro wound closure assay and migration assay were performed to analyse the effect of DOE on the migrating property of A549 cells. Untreated A549 cells migrated to close the wound while the migration was inhibited by DOE treatment. After 48 h, the area of wound created in the cell monolayer was decreased in control cells, whereas in treated cells the area was increased (Figure S1A). A significant decrease in the number of migrating cells through the filters was observed after 24 h of treatment with DOE ( p , 0.001) (Figure S1A). In vitro invasion assay was used to investigate whether DOE inhibits the invasiveness of A549 cells. The invasion rate of cells treated with DOE was lowered as compared with control, indicating that DOE suppressed tumour cell invasion (Figure S1B).
Figure 1. Structure of DOE isolated from E. scaber.
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Downloaded by [Lakehead University] at 06:56 13 March 2015
Migration and invasion assays have demonstrated that DOE showed the anti-metastatic potentials by inhibiting the steps involved in metastasis. 2.2. Effects of DOE on MMP-2, MMP-9, uPA, uPAR, TIMP-1 and TIMP-2 mRNA expression in A549 cells MMPs, associated with cancer cell migration and invasion, are inhibited by endogenous TIMPs such as TIMP-1 and TIMP-2 (Deryugina & Quigley 2006). Disturbance in the balance of MMPs and TIMPs is found in cancer (Lambert et al. 2004). In this study, DOE significantly downregulated the mRNA expression level of MMP-2 and MMP-9 and up-regulated TIMP-2 expressions without any change in TIMP-1 mRNA level (Figure S2A). The plasmin generated as a consequence of binding of uPA on its receptor urokinase-type plasminogen activator receptor (uPAR) is known to activate MMPs thereby triggering ECM degradation leading to metastasis (Schweinitz et al. 2004). DOE reduced uPA and uPAR mRNA expression and inhibited the metastatic ability of A549 cells (Figure S2A). This study indicates that DOE could inhibit the migration and invasion property of tumour cells, probably through a decrease in MMPs and uPA system expressions, and simultaneous increase of the TIMP expression. 2.3. Effect of DOE on MAPKs and Akt signalling pathways in A549 cells MAPK signalling pathways known to mediate metastasis includes extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinases (JNK) and p38 (Chang & Karin 2001). The PI3K-Akt signalling pathway plays another crucial role in MMPs for uPA gene regulation, cell survival and invasion (Kim et al. 2001). To clarify the involvement of MAPKs and Akt signalling pathways in DOE-mediated metastatic inhibition of A549 cells, we have evaluated the protein levels of MAPKs (JNK, p38 and ERK) and Akt by western blotting. Exposure of cells to DOE reduced the protein levels of both p-ERK 1/2 and p-Akt. The protein level of p-p38 and pJNK was up-regulated (Figure S2B). The total protein level of ERK1/2, JNK, p38 and Akt was not affected by DOE treatment. DOE suppressed the phosphorylation of Akt and ERK1/2 effectively. 2.4. Effects of DOE on NF-kB mRNA expression NF-kB, present in the cytoplasm as an inactive heterotrimer, consists of p50, p65 and IkBa subunits. On activation, IkBa protein undergoes degradation and NF-kB heterodimer translocates into the nucleus where it regulates the transcriptional activation of genes involved in cell proliferation, metastasis and angiogenesis (Bjorklund & Koivunen 2005). NF-kB is constitutively activated in NSCLC and is associated with metastasis and poor prognosis (Chen et al. 2011). Treatment of A549 cells with DOE resulted in a strong inhibition of NF-kB activation with a decrease in gene expression of both NF-kB and IkBa at transcript level (Figure S2A). As a transcription factor, NF-kB is known to regulate the expression of various genes involved in invasion such as MMP-2 and MMP-9, uPA and uPAR (Al Dhaheri et al. 2013). Consistent with these results, DOE treatment also inhibited the downstream targets of genes such as MMP-2 and MMP-9, uPA and uPAR resulting in metastasis inhibition. 3. Conclusion These results indicate that anti-metastatic effect of DOE in A549 cells is associated with the down-regulation of NF-kB, IkBa MMP-2, MMP-9, uPA and uPAR and up-regulation of TIMP2 at the transcript level. DOE suppressed the activation of ERK1/2 and Akt, but promoted the
4
A.K. Farha et al.
activation of JNK and p38 and resulted in the inhibition of metastasis. Thus, DOE might be a strong candidate for the treatment of lung cancer. Supplementary material Experimental details, figures and table relating to this article are available online.
Funding The first author thanks the University Grant Commission (UGC) for financial support. This study was supported by research grant from the Indian Council of Medical Research (ICMR), New Delhi, India [grant number 59/47/2011/BMS/TRM].
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Conflict of interest There is no conflict of interest to declare.
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