J Breast Cancer.  2017 Sep;20(3):234-239. 10.4048/jbc.2017.20.3.234.

Crotonis Fructus Extract Inhibits 12-O-Tetradecanoylphorbol-13-Acetate-Induced Expression of Matrix Metalloproteinase-9 via the Activator Protein-1 Pathway in MCF-7 Cells

• Song HK
• Lee GS
• Park SH
• Noh EM
• Kim JM
• Ryu DG
• Jung SH
• Youn HJ
• Lee YR
• Kwon KB

• KMID: 2438989
• DOI: http://doi.org/10.4048/jbc.2017.20.3.234

Abstract

PURPOSE
Metastatic cancers spread from the primary site of origin to other parts of the body. Matrix metalloproteinase-9 (MMP-9) is essential in metastatic cancers owing to its major role in cancer cell invasion. Crotonis fructus (CF), the mature fruits of Croton tiglium L., have been used for the treatment of gastrointestinal disturbance in Asia. In this study, the effect of the ethanol extract of CF (CFE) on MMP-9 activity and the invasion of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells was examined.
METHODS
The cell viability was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The expression of MMP-9 was examined by Western blotting, zymography, and real-time polymerase chain reaction. An electrophoretic mobility gel shift assay was performed to detect activator protein-1 (AP-1) DNA binding activity and cell invasiveness was measured by an in vitro Matrigel invasion assay.
RESULTS
CFE significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, CFE attenuated the TPA-induced activation of AP-1.
CONCLUSION
The results indicated that the inhibitory effects of CFE against TPA-induced MMP-9 expression and MCF-7 cell invasion were dependent on the protein kinase C δ/p38/c-Jun N-terminal kinase/AP-1 pathway. Therefore, CFE could restrict breast cancer invasiveness owing to its ability to inhibit MMP-9 activity.

Keyword

Crotonis fructus; Matrix metalloproteinase 9; MCF-7 cells; Neoplasm invasiveness; Transcription factor AP-1

MeSH Terms

Asia
Blotting, Western
Breast Neoplasms
Cell Survival
Croton
DNA
Ethanol
Fruit
In Vitro Techniques
Matrix Metalloproteinase 9*
MCF-7 Cells*
Neoplasm Invasiveness
Protein Kinase C
Real-Time Polymerase Chain Reaction
Transcription Factor AP-1*
DNA
Ethanol
Matrix Metalloproteinase 9
Protein Kinase C
Transcription Factor AP-1

Figure

• 

  Figure 1 CFE inhibits TPA-induced MMP-9 expression in MCF-7 cells. (A) To assess the cytotoxicity of CFE, cells were treated with various concentrations of CFE for 24 hours. An EZ-cytox enhanced cell viability assay kit was used to detect the. (B) CFE inhibits TPA-induced MMP-9 expression in MCF-7 cells. MCF-7 cells grown in monolayer culture were treated with the indicated CFE concentrations in the presence of TPA for 24 hours. Cell lysates were analyzed by Western blot with anti-MMP-9 antibody and β-actin as a loading control. Conditioned medium was prepared and used for gelatin zymography (Zymo) to assess the effect of CFE on MMP-9 activity in MCF-7 cells. Cells were pretreated with CF for 1 hour and then stimulated with TPA for 24 hours. (C) MMP-9 mRNA levels were analyzed by RT-qPCR, and GAPDH was used as an internal control. Values are shown as mean±SEM of three independent experiments. CFE=Crotonis fructus extract; TPA=12-O-tetradecanoylphorbol-13-acetate; MMP-9=matrix metalloproteinase-9; Zymo=zymography; RT-qPCR=real-time quantitative polymerase chain reaction; GAPDH=glyceraldehyde 3-phosphate dehydrogenase; SEM=standard error of the mean. *p<0.01 vs. TPA.

• 

  Figure 2 CFE inhibits TPA-induced activation of PKCδ. MCF-7 cells were pretreated with CFE for 1 hour and then with TPA for 1 hour. Western blot analysis was performed to detect the levels of PKCα, PKCβ, PKCδ, and Na-K ATPase as a loading control in the membrane fractions. CFE=Crotonis fructus extract; TPA=12-O-tetradecanoylphorbol-13-acetate; PKC=protein kinase C.

• 

  Figure 3 CFE inhibits TPA-induced AP-1 activation in MCF-7 cells. (A) MCF-7 cells were pretreated with CFE for 1 hour and then stimulated with TPA for 15 minutes. The expressions of p-p38, p38, p-JNK, JNK, p-ERK and ERK were analyzed by using Western blotting. (B) Cells were pretreated with CFE for 1 hour before TPA treatment. After stimulation with TPA for 3 hours, the cell nuclear extracts were obtained and subjected to Western blotting to determine the nuclear levels of NF-κB (p50 and p65) and AP-1 (p-c-Jun) subunits. (C) AP-1 DNA binding activity was determined by electrophoretic mobility gel shift assay. CFE=Crotonis fructus extract; TPA=12-O-tetradecanoylphorbol-13-acetate; AP-1=activator protein-1; p=phosphorylated; JNK=c-Jun N-terminal kinase; ERK=extracellular signal-regulated kinase; NF-κB=nuclear factor-κB; PCNA=proliferating cell nuclear antigen.

• 

  Figure 4 CFE inhibits TPA-induced Matrigel invasion in MCF-7 cells. Cells were seeded in the upper chamber of chamber dishes and were either treated with CFE or left untreated. After 24 hours of incubation, (A) cells on the bottom of the chamber membrane were fixed, stained. (B) The number of counted cells was graphed on the basis of the TPA treatment group. Values represent mean±SEM of three independent experiments. CFE=Crotonis fructus extract; TPA=12-O-tetradecanoylphorbol-13-acetate; SEM=standard error of the mean. *p<0.01 vs. TPA.

Reference

1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015; 136:E359–E386.

2. Yücel B, Bahar S, Kaçan T, Şeker M, Celasun MG, Bahçeci A, et al. Importance of metastasis site in survival of patients with breast cancer. Austin J Med Oncol. 2014; 1:7.

3. Eckhardt BL, Francis PA, Parker BS, Anderson RL. Strategies for the discovery and development of therapies for metastatic breast cancer. Nat Rev Drug Discov. 2012; 11:479–497.

4. Woessner JF Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991; 5:2145–2154.

5. Park HI, Ni J, Gerkema FE, Liu D, Belozerov VE, Sang QX. Identification and characterization of human endometase (matrix metalloproteinase-26) from endometrial tumor. J Biol Chem. 2000; 275:20540–20544.

6. Mylona E, Nomikos A, Magkou C, Kamberou M, Papassideri I, Keramopoulos A, et al. The clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-9 according to their localization in invasive breast carcinoma. Histopathology. 2007; 50:338–347.

7. Tsai JC, Tsai S, Chang WC. Effect of ethanol extracts of three Chinese medicinal plants with anti-diarrheal properties on ion transport of the rat intestinal epithelia. J Pharmacol Sci. 2004; 94:60–66.

8. Wang X, Lan M, Wu HP, Shi YQ, Lu J, Ding J, et al. Direct effect of croton oil on intestinal epithelial cells and colonic smooth muscle cells. World J Gastroenterol. 2002; 8:103–107.

9. Kim JH, Lee SJ, Han YB, Moon JJ, Kim JB. Isolation of isoguanosine from Croton tiglium and its antitumor activity. Arch Pharm Res. 1994; 17:115–118.

10. Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. Amplification of IL-1 beta-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-kappa B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunol. 2000; 165:5788–5797.

11. Lungu G, Covaleda L, Mendes O, Martini-Stoica H, Stoica G. FGF-1-induced matrix metalloproteinase-9 expression in breast cancer cells is mediated by increased activities of NF-kappaB and activating protein-1. Mol Carcinog. 2008; 47:424–435.

12. Nabeshima K, Inoue T, Shimao Y, Sameshima T. Matrix metalloproteinases in tumor invasion: role for cell migration. Pathol Int. 2002; 52:255–264.

13. Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst. 1997; 89:1260–1270.

14. Hetter GP. An examination of the phenol-croton oil peel: part I. dissecting the formula. Plast Reconstr Surg. 2000; 105:227–239.

15. Jeon CM, Min GW, Park JH, Yoon CH, Jeong JC, Kang JJ, et al. Effect of the methanol extract of Dansameum on the ammonia-induced peptic ulcer in rats. Korean J Orient Int Med. 2000; 21:597–604.

16. Kim MS, Kim HR, So HS, Lee YR, Moon HC, Ryu DG, et al. Crotonis fructus and its constituent, croton oil, stimulate lipolysis in OP9 adipocytes. Evid Based Complement Alternat Med. 2014; 2014:780385.

17. Kim KC, Lee CH. MAP kinase activation is required for the MMP-9 induction by TNF-stimulation. Arch Pharm Res. 2005; 28:1257–1262.

18. Hong S, Park KK, Magae J, Ando K, Lee TS, Kwon TK, et al. Ascochlorin inhibits matrix metalloproteinase-9 expression by suppressing activator protein-1-mediated gene expression through the ERK1/2 signaling pathway: inhibitory effects of ascochlorin on the invasion of renal carcinoma cells. J Biol Chem. 2005; 280:25202–25209.

19. Karin M, Liu Zg, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol. 1997; 9:240–246.

20. Crawford HC, Matrisian LM. Mechanisms controlling the transcription of matrix metalloproteinase genes in normal and neoplastic cells. Enzyme Protein. 1996; 49:20–37.

21. Kim JM, Noh EM, Kwon KB, Kim JS, You YO, Hwang JK, et al. Curcumin suppresses the TPA-induced invasion through inhibition of PKC-alpha-dependent MMP-expression in MCF-7 human breast cancer cells. Phytomedicine. 2012; 19:1085–1092.