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Cudraticusxanthone A isolated from the roots of Cudrania tricuspidata inhibits metastasis and induces apoptosis in breast cancer cells
Journal of Ethnopharmacology 194 (2016) 57–62
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Cudraticusxanthone A isolated from the roots of Cudrania tricuspidata inhibits metastasis and induces apoptosis in breast cancer cells Soo-Myeong Jeon a,1, Dong-Sung Lee b,1, Gil-Saeong Jeong a,n a b
College of Pharmacy, Keimyung University, 1095 Dalgubeol-daero, Daegu 42601, Republic of Korea College of Pharmacy, Chosun University, Dong-gu, Gwangju 61452, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 8 March 2016 Received in revised form 8 August 2016 Accepted 22 August 2016 Available online 29 August 2016
Ethnopharmacological relevance: The roots of Cudrania tricuspidata is a deciduous tree found in Korea, China, and Japan. C. tricuspidata contains an abundance of various minerals, B vitamins, and flavonoids to help prevent diverse cancers. Cudratricusxanthone A (CTXA), a compound isolated from the roots of C. tricuspidata, has potent anti-proliferative, antioxidative, and monoamine oxidase inhibitory effects. Aim of the study: In the present study, cudratricusxanthone A (CTXA) is a xanthone isolated from the bioassay-guided fractionation of the EtOH extract of C. tricuspidata with strong anti-cancer activity in breast cancer cells. The effect of CTXA on cell migration and apoptosis were evaluated in the MCF-7 and MDA-MB-231 human breast carcinoma cell lines. Materials and methods: Effects of CTXA on phorbol 12-myristate 13-acetate (PMA)-induced MCF-7 and MDA-MB-231 cells. Flow cytometric measurements of CTXA-induced apoptosis in breast cancer cells. Results: The results show that CTXA gradually reduced viability of the two breast cancer cell lines and induced apoptosis in a concentration-dependent manner. Moreover, CTXA effectively blocked breast cancer cell migration and invasion. CTXA decreased the expression of matrix metalloproteinase-9, extracellular regulated kinases 1 and 2 and phosphorylation of the inhibitor IκBα in the MCF-7 and MDAMB-231 cell lines. Conclusions: Collectively, these results indicate that CTXA possesses anti-cancer activities and provide a basis for developing effective therapeutic agents to inhibit growth and metastasis of breast cancer. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cudrania tricuspidata Cudratricusxanthone A Metastasis Apoptosis MCF-7 cells MDA-MB-231 cells
1. Introduction Breast cancer induces significant morbidity among women worldwide, and metastasis commonly occurs in these patients (Abdullah et al., 2014). The limited number of efficient therapeutic strategies to treat breast cancers has lead to a high rate of cancerrelated deaths in women. The 5-year survival rate for breast cancer in women has increased to 80% over the last decade (Kim et al., 2014). One-third of patients with breast cancer die from tumors that metastasize to other organs because of the high invasion rate (DeSantis et al., 2011). Moreover, breast cancer is highly malignant with substantial probability for metastasis, and metastatic breast cancer is a primary cause of female mortality (Lee et al., 2008). No current treatment effectively controls recurrence and metastasis of breast cancer; therefore, new therapies are needed. n
Corresponding author. E-mail addresses: [email protected] (S.-M. Jeon), [email protected] (D.-S. Lee), [email protected] (G.-S. Jeong). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2016.08.042 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
Xanthone compounds are secondary metabolites of plants and microorganisms with antioxidant, anti-microbial, anti-allergic, and anti-cancer activities, based on their diverse structures. The biological efficacy of xanthones has been reported by many scientists who have isolated these compounds to develop potential new drug agents (Lee et al., 2005). Cudrania tricuspidata Bureau (Cudrang; mandarin melon berry) is a deciduous tree found in Korea, China, and Japan. The cortex and roots of this species have been used to treat hepatitis and inflammation and protect against functional β-cell damage (Lee et al., 1996; Lee and Jeong, 2014). C. tricuspidata was the principle medicine for treating women sickness in ancient times due to its non-toxic nature and sweet taste. Moreover, the plant contains an abundance of various minerals, B vitamins, and flavonoids to help prevent diverse cancers (Cho et al., 2003). Cudratricusxanthone A (CTXA), a compound isolated from the roots of C. tricuspidata, has potent anti-proliferative, antioxidative, and monoamine oxidase inhibitory effects (Hwang et al., 2007). In addition, the anticoagulant, antiplatelet, and profibrinolytic properties of CTXA have been studied in tumor necrosis factor-α activated human umbilical vein endothelial cells
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S.-M. Jeon et al. / Journal of Ethnopharmacology 194 (2016) 57–62
(Yoo et al., 2013). However, the effects of CTXA on breast cancers, tumor metastasis, and apoptosis and its associated molecular mechanism have not been described. In this study, we discovered that CTXA induced apoptosis and suppressed migration and invasion by breast cancer cells. Furthermore, CTXA controlled breast tumor growth and decreases metastases in vitro by inhibiting metastasis and inducing apoptosis. Our data show that CTXA may be a potential agent for treating breast cancer.
atmosphere (37 °C and 5% CO2). A 10 ml aliquot of MTT (3-[4,5dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide) was added to , 100 ml of cell suspension and incubated for 4 h. The medium was removed, 100 ml of dimethyl sulfoxide (DMSO) was added, and absorbance was determined at 595 nm using a microplate reader (Tecan Trading AG, Basel, Switzerland). Cell viability was described as the relative percentage of the control. 2.4. Invasion assay
2. Materials and methods 2.1. Plant material and isolation of CTXA The roots of C. tricuspidata were purchased from the Yangnyeong herbal medicine Market, Daegu, Korea, in June 2014, and air-dried. A voucher specimen (KMU-2014–06-08) of the plant was deposited at the College of Pharmacy in Keimyung University. The dried roots (1.5 kg) of C. tricuspidata were extracted with EtOH (10 L 3) at room temperature for 5 days. The dried EtOH extract (50 g) was suspended in H2O, and the resulting H2O layer was partitioned with hexane, CH2Cl2, and EtOAc. The CH2Cl2-soluble fraction (15.7 g) was chromatographed on a silica gel column with CHCl3/MeOH/H2O (9:1:0.1– 4:1:0.1–6:4:1) to obtain 6 fractions (Fr. A–F). Fr. C (1.02 g) was subjected to silica gel column chromatography (eluent: CHCl3/MeOH, 40:1–10:1) to afford 5 fractions (Fr. C1–C5). Fr. C3 (127 mg) was purified by Sephadex LH-20 column chromatography with CHCl3/MeOH (15:1) to give CTXA (12.8 mg). The structure of CTXA (Fig. 1A) was identified by comparison with spectral data in the literature (An et al., 2006). CTXA was determined to be 497.3% pure by HPLC analysis.
The invasion assay was performed using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. The Matrigel coating was re-hydrated in 0.5 ml medium for 2 h in a humidified incubator at 37 °C and 5% CO2. Approximately 2 104 cells/0.5 ml were added to the upper chamber, and different CTXA concentrations in media were added to the bottom wells. The chambers were incubated for 22 h. Cells on the upper side of the chamber were fixed and stained with Toluidine blue solution. Invaded cells were counted in four random areas of the membrane using an inverted microscope (Leica Microsystems CMS GmbH, Zena, Germany). 2.5. Wound healing assay Cells were seeded in a 6-well plate and grown overnight to confluence. The cells were scratched with a 200 ml pipette tip to create a wound, the cells were washed in medium and the medium was replaced with-serum free medium containing different concentrations of CTXA. After 24 h, movement of the cells was confirmed under a microscope. Results are expressed as the mean number of migrating cells/field.
2.2. Cell culture and treatment 2.6. Western blot analysis The MCF-7 and MDA-MB-231 human breast cancer cell lines were obtained from the Korean Cell Line Bank (Seoul, South Korea) and cultured in high Dulbecco's modified Eagle's medium and RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum and antibiotics (penicillin/streptomycin) and incubated at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. Cell viability assay Cells (1 104 cells/ml) were cultured in 96-well plates with various concentrations of CTXA for 18 h in a humidified
Cells lysates were prepared by suspending the cells in lysis buffer. Equal amounts of protein were prepared and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk and incubated with primary and horseradish peroxidase-conjugated secondary antibodies. The membranes were washed three times for 15 min each with TBS-T buffer and then incubated suing the reagents in the Western Bright ECL Western blotting detection kit (Advansta Inc., Menlo Park, CA, USA) according to manufacturer's instructions and
Fig. 1. Effects of EtOH extract and fractions from C. tricuspidata on cell viability in MCF-7 cells. (A) MCF-7 cells were incubated for 24 h with various concentrations of EtOH extract of C. tricuspidata. (B) MCF-7 cells were incubated for 24 h with fractions from EtOH extract of C. tricuspidata. Data are mean values of three experiments 7 standard deviations. *Po 0.05 vs. control.
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Fig. 2. Effects of cudraticusxanthone A (CTXA) on viability of MCF-7 and MDA-MB-231 cells. (A) Chemical structure of CTXA. (B, C) MCF-7 and MDA-MB-231 cells were incubated for 24 h with various concentrations of CTXA (1–50 μΜ) and cell viability was determined. Data are mean values of three experiments 7 standard deviations. * Po 0.05 vs. control.
Fig. 3. Cudraticusxanthone A (CTXA) inhibits phorbol 12-myristate 13-acetate (PMA)-induced cell invasion and migration. (A) CTXA inhibited Matrigel invasion by PMA differentiated MCF-7 and MDA-MB-231 cells. After 24 h incubation, cells on the underside of the filter were fixed and stained. (B) MCF-7 and MDA-MB-231 cells were seeded in a 6 well plate and cultured overnight. The cells were treated with PMA and CTXA 5 μM for 24 h, and cell migration was observed under a microscope. The number of cells that migrated in to a cell-free gap was calculated. *Po 0.05 vs. PMA treated groups.
Fig. 4. Cudraticusxanthone A (CTXA) inhibits phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase (MMP) 9 and phosphor-IκBα expression in MCF-7 and MDA-MB-231 cells. Cells were treated with the indicated CTXA concentrations in the presence of PMA for 24 h. After 24 h of starvation in serum-free media, MCF-7 and MDA-MB-231 cells were incubated with CTXA for 2 h. (A) Cell lysates were analyzed by Western blot with an anti-MMP-9 antibody. (B) The cells were collected, and the level of phosphor-IκBα was determined by Western blotting with phosphor-specific antibodies.
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Fig. 5. Cudraticusxanthone A (CTXA) specifically inhibits activation of p38/JNK in breast cancer cells. After 24 h of starvation in serum-free media, MCF-7 and MDA-MB-231 cells were incubated with CTXA for 2 h and were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 15 min (A, B) Cells were collected, and the levels of phosphor-JNK, p38, and ERK1/2 were determined by Western blotting with phosphor-specific antibodies.
visualized with Imagequant LAS4000 system (Fujifilm Life Science, Tokyo, Japan).
3.2. Effects of CTXA on cell viability, invasion and migration in MCF-7 and MDA-MB-231 cells
2.7. Annexin V and propidium iodide (PI) staining
The structure of CTXA (Fig. 2A) was identified by comparison with spectral data in the literature (An et al., 2006), and it has high purity at 4 97.3% by HPLC analysis. Viability of the two cancer cell lines relative to controls is expressed as percentage of live cells (Fig. 2B and C). All experiments were performed in triplicate. CTXA reduced cell viability both in MCF-7 and MDAMB-231 cells. Therefore, CTXA was selected for further studies elucidate the underlying mechanism of cell death induction in MCF-7 and MDA-MB-231 cells. Based on the cell viability results, CTXA was used in further experiments. CTXA (10 mM) tended to decrease phorbol 12-myristate 13-acetate (PMA)-induced MCF-7 and MDA-MB-231 cell invasion. These results indicate that CTXA exerts anti-tumor effects by suppressing PMAinduced cell invasion (Fig. 3).
Annexin V staining was performed using the fluorescein isothiocyanate (FITC)-Annexin V staining kit (BD Biosciences) following the manufacturer's instructions. Briefly, CTXA-treated cells were washed with PBS and resuspended in 1 binding buffer containing Annexin V and PI. Fluorescence intensity was measured by flow cytometry (BD Biosciences). 2.8. Statistical analysis Data are expressed as mean 7standard deviation of at least three independent experiments, and differences were detected using the Student's t-test. A p-value o0.05 was considered statistically significant.
3. Results 3.1. Isolation of CTXA from the bioassay-guided fractionation of the EtOH extract of C. tricuspidata. Initially, we checked the MCF-7 cell death by EtOH extract of C. tricuspidata. EtOH extract significantly decreased the MCF-7 cell viability (Fig. 1A). To find what components are involved in the anti-cancer effects of EtOH extract of C. tricuspidata, EtOH extract was suspended in H2O, and the resulting H2O layer was partitioned with hexane, CH2Cl2, and EtOAc. Among the fractions, CH2Cl2 fraction increased MCF-7 cell death more than other fractions (Fig. 1B). From this primary result, CH2Cl2 fraction was selected for further studies to isolate the active compound. The CH2Cl2-soluble fraction was chromatographed on a silica gel column, and CTXA was obtained, respectively.
3.3. Effects of CTXA on PMA-induced matrix metalloproteinase (MMP) 9 secretion, phosphor-IκBα activation and the mitogen-activated protein kinase (MAPK) pathway in MCF-7 and MDA-MB-231 cells The effects of CTXA on the PMA-induced MMP-9 secretion and activation of phosphor-Iκ Bα were investigated by Western blot assay. MMP-9 expression increased significantly in response to 100 nM PMA, whereas CTXA decreased MMP-9 expression in a dose-dependent manner in MCF-7 and MDA-MB231 cells (Fig. 4). We also determined whether CTXA inhibited the MAPK and IκBα signaling pathways. CTXA effectively inhibited phosphorylation of ERK1/2, p38, and JNK in PMA-induced MCF-7 and MDA-MB-231 cells (Fig. 5). Moreover, CTXA strongly inhibited phosphorylation of IκBα in PMA-stimulated breast cancer cells (Fig. 4). These results clearly show that CTXA inhibits MAPK and NF-κB signaling.
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Fig. 6. Flow cytometric measurements of cudraticusxanthone A (CTXA)-induced apoptosis in breast cancer cells. Human breast cancer cells were treated with 1, 5, 10, and 20 μM CTXA for 24 h and then stained with Annexin V and propidium iodide (PI). (A) Apoptotic cells were detected by flow cytometry. Effects of CTXA on the levels of apoptosis-related proteins in MCF-7 and MDA-MB-231 cells. (B) Cell lysates were analyzed by Western blot with anti-apoptosis antibody. A Western blot analysis was done to detect the levels of poly ADP ribose polymerase (PARP) and cleaved PARP. *P o0.05 vs. control.
3.4. Effects of CTXA on apoptosis signaling in MCF-7 and MDA-MB231 cells A fluorescence-assisted cell sorting analysis was done to elucidate whether CTXA-mediated cytotoxicity induced apoptosis. MCF-7 and MDA-MB-231 cells treated with CTXA (1, 5, 10, and 20 mM) for 24 h were analyzed by flow cytometry using double staining with Annexin V and propidium iodide (PI) to quantify the cells undergoing apoptosis (Fig. 6A). CTXA induced activation of caspase-3, cleaved caspase-3, poly ADP ribose polymerase (PARP), and cleavage of PARP in MCF-7 and MDA-MB-231 cells (Fig. 6B), indicating the involvement of mitochondria in CTXA-induced apoptosis.
4. Discussion Cancer is the leading cause of death worldwide (Jemal et al., 2011). Among cancers, breast cancer is highly malignant and has high metastatic potential and cancer-related mortality. Despite recent developments in breast cancer therapy, no targeted
treatments are accessible for some patients. Accordingly, identifying novel drug candidates to prevent cancer metastasis is needed. Highly abundant natural compounds derived from plants are being investigated to protect against the side effects of chemotherapeutic agents. The current study evaluated the effects of CTXA, which was derived from the roots of C. tricuspidata, against human breast cancer cells and elucidate the mechanism of action. We used MCF-7 and MDA-MB-231 human breast cancer cell line to analyze the effects of CTXA on breast cancer cell invasion and apoptosis. The results show that CTXA significantly suppressed migration and induced apoptosis in MCF-7 and MDA-MB-231 cells. The induction of apoptosis is related to repression of antiapoptotic proteins and upregulation of pro-apoptotic proteins (Singh et al., 2014). The Bcl-2 protein helps regulate the mitochondrial-associated apoptotic pathway. Downregulation of Bcl2 results in damage to the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol to activate caspase-9 (Marsden et al., 2004). Cleaved-caspase-9 activates caspase-3, which is a critical enzyme in the apoptotic pathway, leading to subsequent apoptotic events. Decreased Bcl-2 and increased cleaved-caspase-3 expression levels in breast cancer
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cells treated with CTXA were involved in inducing apoptosis through the mitochondrial-associated apoptotic signaling pathway. CTXA effectively suppressed breast cancer cell migration and invasion. Proteolytic enzymes, which degrade the surrounding extracellular matrix, help the metastatic spread and migration of cancer cells through the basement membrane. MMPs are involved in matrix remodeling (Hurst and Welch, 2011). Our results demonstrate that CTXA downregulated MMP-9 expression and activity. Consequently, CTXA suppressed MMP-9 activity to impair the migration and invasion abilities of breast cancer cells. Activation of NFκB is vital for migration and invasion of cancer cells (Im et al., 2014). CTXA inhibited NFκB activity and suppressed breast cancer invasion. The phosphorylation of IκBα releases NFκB, which is transferred from the cytoplasm to the nucleus, to control target genes (Wrighton et al., 1996). In this study, we observed that the transfer of phosphorylated IκBα from the cytoplasm to the nucleus was inhibited by CTXA, demonstrating that CTXA suppressed NFκB activity and downregulated MMP-9 expression. The MAPK signaling cascade, including ERK1/2, p38, and JNK, has been implicated in the invasion and migration of tumors. In this study, we hypothesized that CTXA would antagonize the MAPK pathways to suppress invasion and migration of breast cancer cells. The results indicate that CTXA inhibited activation of JNK and p38 signaling in a dose-dependent manner, but had little effect on ERK1/2 signaling. In conclusion, we investigated that CTXA is a xanthone isolated from the bioassay-guided fractionation of the EtOH extract of C. tricuspidata with strong anti-cancer activity in breast cancer cells. CTXA exerted anti-tumor effects on breast cancer cells by suppressing cell migration and invasion and inducing apoptosis in vitro. The abilities of CTXA to induce apoptosis by activating the mitochondrial-associated apoptotic signaling pathway and inhibit cell invasion and migration by downregulating MMP-9 suggest that it may be a novel anti-tumor drug for breast cancer therapy.
Authors’ contributions E. Choi and D.-S. Lee. performed the experiments related to biological evaluation and wrote the manuscript; G.-S.J. organized this work and contributed to writing the manuscript.
Conflicts of interest The authors declare no conflict of interest.
Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. NRF-2015M3A9A5031091 and NRF-2015R1C1A1A02036465).
References Abdullah, A.-S., Mohammed, H., Abdullah, A.S., Mirghani, R., Al-Qubaisi, M, M.E.S., 2014. Cytotoxic effects of Mangifera indica L. kernel extract on human breast cancer (MCF-7 and MDA-MB-231 cell lines) and bioactive constituents in the crude extract. BMC Complement. Altern. Med. 14, 199. An, R.B., Sohn, D.H., Kim, Y.C., 2006. Hepatoprotective compounds of the roots of Cudrania tricuspidata on tacrine-induced cytotoxicity in Hep G2 Cells. Biol. Pharm. Bull. 29, 838–840. Cho, E.J., Yokozawa, T., Rhyu, D.Y., Kim, S.C., Shibahara, N., Park, J.C., 2003. Study on the inhibitory effects of Korean medicinal plants and their main compounds on the 1,1-diphenyl-2-picrylhydrazyl radical. Phytomedicine 10, 544–551. DeSantis, C., Siegel, R., Bandi, P., Jemal, A., 2011. Breast Cancer Statistics 61. A Cancer Journal for Clinicians, CA, pp. 409–418. Hurst, D.R., Welch, D.R., 2011. Metastasis suppressor genes at the interface between the environment and tumor cell growth. Int. Rev. Cell Mol. Biol. 286, 107–180. Hwang, J.H., Hong, S.S., Han, X.H., Hwang, J.S., Lee, D., Lee, H., Yun, Y.P., Kim, Y., Ro, J. S., Hwang, B.Y., 2007. Prenylated xanthones from the root bark of Cudrania tricuspidata. J. Nat. Prod. 70, 1202–1209. Im, N.K., Jang, W.J., Jeong, C.H., Jeong, G.S., 2014. Delphinidin suppresses PMA-induced MMP-9 expression by blocking the NF-ĸB activation through MAPK signaling pathways in MCF-7 human breast carcinoma cells. J. Med. Food 17, 855–861. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., 2011. Global cancer statistics. CA: A Cancer J. Clin. 61, 69–90. Kim, J.A., Kim, D.H., Hossain, M.A., Kim, M.Y., Sung, B., Yoon, J.H., Suh, H., Jeong, T.C., Chung, H.Y., Kim, N.D., 2014. HS-1793, a resveratrol analogue, induces cell cycle arrest and apoptotic cell death in human breast cancer cells. Int. J. Oncol. 44, 473–480. Lee, B.W., Lee, J.H., Lee, S.T., Lee, H.S., Lee, W.S., Jeong, T.S., Park, K.H., 2005. Antioxidant and cytotoxic activities of xanthones from Cudrania tricuspidata. Bioorg. Med. Chem. Lett. 15, 5548–5552. Lee, D.S., Jeong, G.S., 2014. Arylbenzofuran isolated from Dalbergia odorifera suppresses lipopolysaccharide-induced mouse BV2 microglial cell activation, which protects mouse hippocampal HT22 cells death from neuroinflammationmediated toxicity. Eur. J. Pharmacol. 728, 1–8. Lee, H.S., Seo, E.Y., Kang, N.E., Kim, W.K., 2008. [6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells. J. Nutr. Biochem. 19, 313–319. Lee, I., Kim, C., Song, K., Kim, H., Koshino, H., Uramoto, M., Yoo, I., 1996. Cytotoxic benzyl dihydroflavonols from Cudrania tricuspidata. Phytochemistry 41, 213–216. Marsden, V.S., Ekert, P.G., Van, D.M., Vaux, D.L., Adams, J.M., De, S.H., 2004. Bcl-2regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9. J. Cell Biol. 165, 775–780. Singh, L., Pushker, N., Saini, N., Sen, S., Sharma, A., Bakhshi, S., Chawla, B., Kashyap, S., 2014. Expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins in human retinoblastoma. Clin. Exp. Ophthalmol. 43, 259–267. Wrighton, C.J., Hofer, W.R., Eytner, R., Moll, T., Bach, F.H., Martin, R., 1996. Inhibition of endothelial cell activation by adenovirus-mediated expression of IκBα, an inhibitor of the transcription factor NF-κB. J. Exp. Med. 183, 1013–1022. Yoo, H., Ku, S.K., Lee, W., Kwak, S., Baek, Y.D., Min, B.W., Jeong, G.S., Bae, J.S., 2013. Antiplatelet, anticoagulant, and profibrinolytic activities of cudratricusxanthone A. Arch. Pharmacal Res. 37, 1069–1078.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Cudraticusxanthone A isolated from the roots of Cudrania tricuspidata inhibits metastasis and induces apoptosis in breast cancer cells Soo-Myeong Jeon a,1, Dong-Sung Lee b,1, Gil-Saeong Jeong a,n a b
College of Pharmacy, Keimyung University, 1095 Dalgubeol-daero, Daegu 42601, Republic of Korea College of Pharmacy, Chosun University, Dong-gu, Gwangju 61452, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 8 March 2016 Received in revised form 8 August 2016 Accepted 22 August 2016 Available online 29 August 2016
Ethnopharmacological relevance: The roots of Cudrania tricuspidata is a deciduous tree found in Korea, China, and Japan. C. tricuspidata contains an abundance of various minerals, B vitamins, and flavonoids to help prevent diverse cancers. Cudratricusxanthone A (CTXA), a compound isolated from the roots of C. tricuspidata, has potent anti-proliferative, antioxidative, and monoamine oxidase inhibitory effects. Aim of the study: In the present study, cudratricusxanthone A (CTXA) is a xanthone isolated from the bioassay-guided fractionation of the EtOH extract of C. tricuspidata with strong anti-cancer activity in breast cancer cells. The effect of CTXA on cell migration and apoptosis were evaluated in the MCF-7 and MDA-MB-231 human breast carcinoma cell lines. Materials and methods: Effects of CTXA on phorbol 12-myristate 13-acetate (PMA)-induced MCF-7 and MDA-MB-231 cells. Flow cytometric measurements of CTXA-induced apoptosis in breast cancer cells. Results: The results show that CTXA gradually reduced viability of the two breast cancer cell lines and induced apoptosis in a concentration-dependent manner. Moreover, CTXA effectively blocked breast cancer cell migration and invasion. CTXA decreased the expression of matrix metalloproteinase-9, extracellular regulated kinases 1 and 2 and phosphorylation of the inhibitor IκBα in the MCF-7 and MDAMB-231 cell lines. Conclusions: Collectively, these results indicate that CTXA possesses anti-cancer activities and provide a basis for developing effective therapeutic agents to inhibit growth and metastasis of breast cancer. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cudrania tricuspidata Cudratricusxanthone A Metastasis Apoptosis MCF-7 cells MDA-MB-231 cells
1. Introduction Breast cancer induces significant morbidity among women worldwide, and metastasis commonly occurs in these patients (Abdullah et al., 2014). The limited number of efficient therapeutic strategies to treat breast cancers has lead to a high rate of cancerrelated deaths in women. The 5-year survival rate for breast cancer in women has increased to 80% over the last decade (Kim et al., 2014). One-third of patients with breast cancer die from tumors that metastasize to other organs because of the high invasion rate (DeSantis et al., 2011). Moreover, breast cancer is highly malignant with substantial probability for metastasis, and metastatic breast cancer is a primary cause of female mortality (Lee et al., 2008). No current treatment effectively controls recurrence and metastasis of breast cancer; therefore, new therapies are needed. n
Corresponding author. E-mail addresses: [email protected] (S.-M. Jeon), [email protected] (D.-S. Lee), [email protected] (G.-S. Jeong). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2016.08.042 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
Xanthone compounds are secondary metabolites of plants and microorganisms with antioxidant, anti-microbial, anti-allergic, and anti-cancer activities, based on their diverse structures. The biological efficacy of xanthones has been reported by many scientists who have isolated these compounds to develop potential new drug agents (Lee et al., 2005). Cudrania tricuspidata Bureau (Cudrang; mandarin melon berry) is a deciduous tree found in Korea, China, and Japan. The cortex and roots of this species have been used to treat hepatitis and inflammation and protect against functional β-cell damage (Lee et al., 1996; Lee and Jeong, 2014). C. tricuspidata was the principle medicine for treating women sickness in ancient times due to its non-toxic nature and sweet taste. Moreover, the plant contains an abundance of various minerals, B vitamins, and flavonoids to help prevent diverse cancers (Cho et al., 2003). Cudratricusxanthone A (CTXA), a compound isolated from the roots of C. tricuspidata, has potent anti-proliferative, antioxidative, and monoamine oxidase inhibitory effects (Hwang et al., 2007). In addition, the anticoagulant, antiplatelet, and profibrinolytic properties of CTXA have been studied in tumor necrosis factor-α activated human umbilical vein endothelial cells
58
S.-M. Jeon et al. / Journal of Ethnopharmacology 194 (2016) 57–62
(Yoo et al., 2013). However, the effects of CTXA on breast cancers, tumor metastasis, and apoptosis and its associated molecular mechanism have not been described. In this study, we discovered that CTXA induced apoptosis and suppressed migration and invasion by breast cancer cells. Furthermore, CTXA controlled breast tumor growth and decreases metastases in vitro by inhibiting metastasis and inducing apoptosis. Our data show that CTXA may be a potential agent for treating breast cancer.
atmosphere (37 °C and 5% CO2). A 10 ml aliquot of MTT (3-[4,5dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide) was added to , 100 ml of cell suspension and incubated for 4 h. The medium was removed, 100 ml of dimethyl sulfoxide (DMSO) was added, and absorbance was determined at 595 nm using a microplate reader (Tecan Trading AG, Basel, Switzerland). Cell viability was described as the relative percentage of the control. 2.4. Invasion assay
2. Materials and methods 2.1. Plant material and isolation of CTXA The roots of C. tricuspidata were purchased from the Yangnyeong herbal medicine Market, Daegu, Korea, in June 2014, and air-dried. A voucher specimen (KMU-2014–06-08) of the plant was deposited at the College of Pharmacy in Keimyung University. The dried roots (1.5 kg) of C. tricuspidata were extracted with EtOH (10 L 3) at room temperature for 5 days. The dried EtOH extract (50 g) was suspended in H2O, and the resulting H2O layer was partitioned with hexane, CH2Cl2, and EtOAc. The CH2Cl2-soluble fraction (15.7 g) was chromatographed on a silica gel column with CHCl3/MeOH/H2O (9:1:0.1– 4:1:0.1–6:4:1) to obtain 6 fractions (Fr. A–F). Fr. C (1.02 g) was subjected to silica gel column chromatography (eluent: CHCl3/MeOH, 40:1–10:1) to afford 5 fractions (Fr. C1–C5). Fr. C3 (127 mg) was purified by Sephadex LH-20 column chromatography with CHCl3/MeOH (15:1) to give CTXA (12.8 mg). The structure of CTXA (Fig. 1A) was identified by comparison with spectral data in the literature (An et al., 2006). CTXA was determined to be 497.3% pure by HPLC analysis.
The invasion assay was performed using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. The Matrigel coating was re-hydrated in 0.5 ml medium for 2 h in a humidified incubator at 37 °C and 5% CO2. Approximately 2 104 cells/0.5 ml were added to the upper chamber, and different CTXA concentrations in media were added to the bottom wells. The chambers were incubated for 22 h. Cells on the upper side of the chamber were fixed and stained with Toluidine blue solution. Invaded cells were counted in four random areas of the membrane using an inverted microscope (Leica Microsystems CMS GmbH, Zena, Germany). 2.5. Wound healing assay Cells were seeded in a 6-well plate and grown overnight to confluence. The cells were scratched with a 200 ml pipette tip to create a wound, the cells were washed in medium and the medium was replaced with-serum free medium containing different concentrations of CTXA. After 24 h, movement of the cells was confirmed under a microscope. Results are expressed as the mean number of migrating cells/field.
2.2. Cell culture and treatment 2.6. Western blot analysis The MCF-7 and MDA-MB-231 human breast cancer cell lines were obtained from the Korean Cell Line Bank (Seoul, South Korea) and cultured in high Dulbecco's modified Eagle's medium and RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum and antibiotics (penicillin/streptomycin) and incubated at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. Cell viability assay Cells (1 104 cells/ml) were cultured in 96-well plates with various concentrations of CTXA for 18 h in a humidified
Cells lysates were prepared by suspending the cells in lysis buffer. Equal amounts of protein were prepared and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk and incubated with primary and horseradish peroxidase-conjugated secondary antibodies. The membranes were washed three times for 15 min each with TBS-T buffer and then incubated suing the reagents in the Western Bright ECL Western blotting detection kit (Advansta Inc., Menlo Park, CA, USA) according to manufacturer's instructions and
Fig. 1. Effects of EtOH extract and fractions from C. tricuspidata on cell viability in MCF-7 cells. (A) MCF-7 cells were incubated for 24 h with various concentrations of EtOH extract of C. tricuspidata. (B) MCF-7 cells were incubated for 24 h with fractions from EtOH extract of C. tricuspidata. Data are mean values of three experiments 7 standard deviations. *Po 0.05 vs. control.
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Fig. 2. Effects of cudraticusxanthone A (CTXA) on viability of MCF-7 and MDA-MB-231 cells. (A) Chemical structure of CTXA. (B, C) MCF-7 and MDA-MB-231 cells were incubated for 24 h with various concentrations of CTXA (1–50 μΜ) and cell viability was determined. Data are mean values of three experiments 7 standard deviations. * Po 0.05 vs. control.
Fig. 3. Cudraticusxanthone A (CTXA) inhibits phorbol 12-myristate 13-acetate (PMA)-induced cell invasion and migration. (A) CTXA inhibited Matrigel invasion by PMA differentiated MCF-7 and MDA-MB-231 cells. After 24 h incubation, cells on the underside of the filter were fixed and stained. (B) MCF-7 and MDA-MB-231 cells were seeded in a 6 well plate and cultured overnight. The cells were treated with PMA and CTXA 5 μM for 24 h, and cell migration was observed under a microscope. The number of cells that migrated in to a cell-free gap was calculated. *Po 0.05 vs. PMA treated groups.
Fig. 4. Cudraticusxanthone A (CTXA) inhibits phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase (MMP) 9 and phosphor-IκBα expression in MCF-7 and MDA-MB-231 cells. Cells were treated with the indicated CTXA concentrations in the presence of PMA for 24 h. After 24 h of starvation in serum-free media, MCF-7 and MDA-MB-231 cells were incubated with CTXA for 2 h. (A) Cell lysates were analyzed by Western blot with an anti-MMP-9 antibody. (B) The cells were collected, and the level of phosphor-IκBα was determined by Western blotting with phosphor-specific antibodies.
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Fig. 5. Cudraticusxanthone A (CTXA) specifically inhibits activation of p38/JNK in breast cancer cells. After 24 h of starvation in serum-free media, MCF-7 and MDA-MB-231 cells were incubated with CTXA for 2 h and were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 15 min (A, B) Cells were collected, and the levels of phosphor-JNK, p38, and ERK1/2 were determined by Western blotting with phosphor-specific antibodies.
visualized with Imagequant LAS4000 system (Fujifilm Life Science, Tokyo, Japan).
3.2. Effects of CTXA on cell viability, invasion and migration in MCF-7 and MDA-MB-231 cells
2.7. Annexin V and propidium iodide (PI) staining
The structure of CTXA (Fig. 2A) was identified by comparison with spectral data in the literature (An et al., 2006), and it has high purity at 4 97.3% by HPLC analysis. Viability of the two cancer cell lines relative to controls is expressed as percentage of live cells (Fig. 2B and C). All experiments were performed in triplicate. CTXA reduced cell viability both in MCF-7 and MDAMB-231 cells. Therefore, CTXA was selected for further studies elucidate the underlying mechanism of cell death induction in MCF-7 and MDA-MB-231 cells. Based on the cell viability results, CTXA was used in further experiments. CTXA (10 mM) tended to decrease phorbol 12-myristate 13-acetate (PMA)-induced MCF-7 and MDA-MB-231 cell invasion. These results indicate that CTXA exerts anti-tumor effects by suppressing PMAinduced cell invasion (Fig. 3).
Annexin V staining was performed using the fluorescein isothiocyanate (FITC)-Annexin V staining kit (BD Biosciences) following the manufacturer's instructions. Briefly, CTXA-treated cells were washed with PBS and resuspended in 1 binding buffer containing Annexin V and PI. Fluorescence intensity was measured by flow cytometry (BD Biosciences). 2.8. Statistical analysis Data are expressed as mean 7standard deviation of at least three independent experiments, and differences were detected using the Student's t-test. A p-value o0.05 was considered statistically significant.
3. Results 3.1. Isolation of CTXA from the bioassay-guided fractionation of the EtOH extract of C. tricuspidata. Initially, we checked the MCF-7 cell death by EtOH extract of C. tricuspidata. EtOH extract significantly decreased the MCF-7 cell viability (Fig. 1A). To find what components are involved in the anti-cancer effects of EtOH extract of C. tricuspidata, EtOH extract was suspended in H2O, and the resulting H2O layer was partitioned with hexane, CH2Cl2, and EtOAc. Among the fractions, CH2Cl2 fraction increased MCF-7 cell death more than other fractions (Fig. 1B). From this primary result, CH2Cl2 fraction was selected for further studies to isolate the active compound. The CH2Cl2-soluble fraction was chromatographed on a silica gel column, and CTXA was obtained, respectively.
3.3. Effects of CTXA on PMA-induced matrix metalloproteinase (MMP) 9 secretion, phosphor-IκBα activation and the mitogen-activated protein kinase (MAPK) pathway in MCF-7 and MDA-MB-231 cells The effects of CTXA on the PMA-induced MMP-9 secretion and activation of phosphor-Iκ Bα were investigated by Western blot assay. MMP-9 expression increased significantly in response to 100 nM PMA, whereas CTXA decreased MMP-9 expression in a dose-dependent manner in MCF-7 and MDA-MB231 cells (Fig. 4). We also determined whether CTXA inhibited the MAPK and IκBα signaling pathways. CTXA effectively inhibited phosphorylation of ERK1/2, p38, and JNK in PMA-induced MCF-7 and MDA-MB-231 cells (Fig. 5). Moreover, CTXA strongly inhibited phosphorylation of IκBα in PMA-stimulated breast cancer cells (Fig. 4). These results clearly show that CTXA inhibits MAPK and NF-κB signaling.
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Fig. 6. Flow cytometric measurements of cudraticusxanthone A (CTXA)-induced apoptosis in breast cancer cells. Human breast cancer cells were treated with 1, 5, 10, and 20 μM CTXA for 24 h and then stained with Annexin V and propidium iodide (PI). (A) Apoptotic cells were detected by flow cytometry. Effects of CTXA on the levels of apoptosis-related proteins in MCF-7 and MDA-MB-231 cells. (B) Cell lysates were analyzed by Western blot with anti-apoptosis antibody. A Western blot analysis was done to detect the levels of poly ADP ribose polymerase (PARP) and cleaved PARP. *P o0.05 vs. control.
3.4. Effects of CTXA on apoptosis signaling in MCF-7 and MDA-MB231 cells A fluorescence-assisted cell sorting analysis was done to elucidate whether CTXA-mediated cytotoxicity induced apoptosis. MCF-7 and MDA-MB-231 cells treated with CTXA (1, 5, 10, and 20 mM) for 24 h were analyzed by flow cytometry using double staining with Annexin V and propidium iodide (PI) to quantify the cells undergoing apoptosis (Fig. 6A). CTXA induced activation of caspase-3, cleaved caspase-3, poly ADP ribose polymerase (PARP), and cleavage of PARP in MCF-7 and MDA-MB-231 cells (Fig. 6B), indicating the involvement of mitochondria in CTXA-induced apoptosis.
4. Discussion Cancer is the leading cause of death worldwide (Jemal et al., 2011). Among cancers, breast cancer is highly malignant and has high metastatic potential and cancer-related mortality. Despite recent developments in breast cancer therapy, no targeted
treatments are accessible for some patients. Accordingly, identifying novel drug candidates to prevent cancer metastasis is needed. Highly abundant natural compounds derived from plants are being investigated to protect against the side effects of chemotherapeutic agents. The current study evaluated the effects of CTXA, which was derived from the roots of C. tricuspidata, against human breast cancer cells and elucidate the mechanism of action. We used MCF-7 and MDA-MB-231 human breast cancer cell line to analyze the effects of CTXA on breast cancer cell invasion and apoptosis. The results show that CTXA significantly suppressed migration and induced apoptosis in MCF-7 and MDA-MB-231 cells. The induction of apoptosis is related to repression of antiapoptotic proteins and upregulation of pro-apoptotic proteins (Singh et al., 2014). The Bcl-2 protein helps regulate the mitochondrial-associated apoptotic pathway. Downregulation of Bcl2 results in damage to the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol to activate caspase-9 (Marsden et al., 2004). Cleaved-caspase-9 activates caspase-3, which is a critical enzyme in the apoptotic pathway, leading to subsequent apoptotic events. Decreased Bcl-2 and increased cleaved-caspase-3 expression levels in breast cancer
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cells treated with CTXA were involved in inducing apoptosis through the mitochondrial-associated apoptotic signaling pathway. CTXA effectively suppressed breast cancer cell migration and invasion. Proteolytic enzymes, which degrade the surrounding extracellular matrix, help the metastatic spread and migration of cancer cells through the basement membrane. MMPs are involved in matrix remodeling (Hurst and Welch, 2011). Our results demonstrate that CTXA downregulated MMP-9 expression and activity. Consequently, CTXA suppressed MMP-9 activity to impair the migration and invasion abilities of breast cancer cells. Activation of NFκB is vital for migration and invasion of cancer cells (Im et al., 2014). CTXA inhibited NFκB activity and suppressed breast cancer invasion. The phosphorylation of IκBα releases NFκB, which is transferred from the cytoplasm to the nucleus, to control target genes (Wrighton et al., 1996). In this study, we observed that the transfer of phosphorylated IκBα from the cytoplasm to the nucleus was inhibited by CTXA, demonstrating that CTXA suppressed NFκB activity and downregulated MMP-9 expression. The MAPK signaling cascade, including ERK1/2, p38, and JNK, has been implicated in the invasion and migration of tumors. In this study, we hypothesized that CTXA would antagonize the MAPK pathways to suppress invasion and migration of breast cancer cells. The results indicate that CTXA inhibited activation of JNK and p38 signaling in a dose-dependent manner, but had little effect on ERK1/2 signaling. In conclusion, we investigated that CTXA is a xanthone isolated from the bioassay-guided fractionation of the EtOH extract of C. tricuspidata with strong anti-cancer activity in breast cancer cells. CTXA exerted anti-tumor effects on breast cancer cells by suppressing cell migration and invasion and inducing apoptosis in vitro. The abilities of CTXA to induce apoptosis by activating the mitochondrial-associated apoptotic signaling pathway and inhibit cell invasion and migration by downregulating MMP-9 suggest that it may be a novel anti-tumor drug for breast cancer therapy.
Authors’ contributions E. Choi and D.-S. Lee. performed the experiments related to biological evaluation and wrote the manuscript; G.-S.J. organized this work and contributed to writing the manuscript.
Conflicts of interest The authors declare no conflict of interest.
Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. NRF-2015M3A9A5031091 and NRF-2015R1C1A1A02036465).
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