Antcin-A Modulates Epithelial-to-Mesenchymal Transition and Inhibits Migratory and Invasive Potentials of Human Breast Cancer Cells via p53-Mediated miR-200c Activation

Abstract

Antcin-A (ATA) is a steroid-like phytochemical isolated from the fruiting bodies of a precious edible mushroom Antrodia cinnamomea. We previously showed that ATA has strong anti-inflammatory and anti-tumor effects; however, other possible bioactivities of this unique compound remain unexplored. In the present study, we aimed to investigate the modulation of epithelial-to-mesenchymal transition (EMT), anti-migration, and anti-invasive potential of ATA against human breast cancer cells in vitro. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were incubated with ATA for 24 h. Wound healing, trans-well invasion, western blot, q-PCR, F-actin staining, and immunofluorescence assays were performed. We found that treatment with ATA significantly blocked EMT processes, as evidenced by upregulation of epithelial markers (E-cadherin and occludin) and downregulation of mesenchymal markers (N-cadherin and vimentin) via suppression of their transcriptional repressor ZEB1. Next, we found that ATA could induce miR-200c, which is a known player of ZEB1 repression. Further investigations revealed that ATA-mediated induction of miR-200c is associated with transcriptional activation of p53, as confirmed by the fact that ATA failed to induce miR-200c or suppress ZEB1 activity in p53 inhibited cells. Further in vitro wound healing and trans-well invasion assays support that ATA could inhibit migratory and invasive potentials of breast cancer cells, and the effect was likely associated with induced phenotypic modulation. Taken together, the present study suggests that antcin-A could be a lead phyto-agent for the development of anti-metastatic drug for breast cancer treatment.

Key words

antcin-A - Antrodia cinnamomea - Fomitopsidaceae - breast cancer - epithelial-to-mesenchymal transition - miR-200c - ZEB1 - p53

• References

• 1 International Agency for Research on Cancer. Cancer Tomorrow 2018: estimated number of incident cases from 2018 to 2030, breast, females, all ages. Available at: http://gco.iarc.fr/tomorrow Accessed September 12, 2018
• 2 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019; 69: 7-34
• 3 DeSantis CE, Bray F, Ferlay J, Lortet-Tieulent J, Anderson BO, Jemal A. International variation in female breast cancer incidence and mortality rates. Cancer Epidemiol Biomarkers Prev 2015; 24: 1495-1506
• 4 Weigelt B, Peterse JL, van ʼt Veer LJ. Breast cancer metastasis: markers and models. Nat Rev Cancer 2005; 5: 591-602
• 5 Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 2014; 15: 178-196
• 6 Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 2009; 10: 704-714
• 7 Ma L. MicroRNA and metastasis. Adv Cancer Res 2016; 132: 165-207
• 8 Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 2008; 22: 894-907
• 9 Weidle UH, Dickopf S, Hintermair C, Kollmorgen G, Birzele F, Brinkmann U. The role of micro RNAs in breast cancer metastasis: preclinical validation and potential therapeutic targets. Cancer Genom Proteom 2018; 15: 17-39
• 10 Abbasi B, Iqbal J, Mahmood T, Khalil A, Ali B, Kanwal S, Shah S, Ahmad R. Role of dietary phytochemicals in modulation of miRNA expression: natural swords combating breast cancer. Asian Pac J Trop Med 2018; 11: 501-509
• 11 Ao ZH, Xu ZH, Lu ZM, Xu HY, Zhang XM, Dou WF. Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases. J Ethnopharmacol 2009; 121: 194-212
• 12 Geethangili M, Tzeng YM. Review of pharmacological effects of Antrodia camphorata and its bioactive compounds. Evid Based Complement Alternat Med 2011; 2011: 17
• 13 Lu MC, El-Shazly M, Wu TY, Du YC, Chang TT, Chen CF, Hsu YM, Lai KH, Chiu CP, Chang FR, Wu YC. Recent research and development of Antrodia cinnamomea . Pharmacol Ther 2013; 139: 124-156
• 14 Shanmugavadivu M, Velmurugan BK. Pharmalogical activities of antroquinonol – mini review. Chem Biol Interact 2018; 297: 8-15
• 15 Zhang BB, Hu PF, Huang J, Hu YD, Chen L, Xu GR. Current advances on the structure, bioactivity, synthesis, and metabolic regulation of novel ubiquinone derivatives in the edible and medicinal mushroom Antrodia cinnamomea . J Agric Food Chem 2017; 65: 10395-10405
• 16 Chen YC, Liu YL, Li FY, Chang CI, Wang SY, Lee KY, Li SL, Chen YP, Jinn TR, Tzen JT. Antcin A, a steroid-like compound from Antrodia camphorata, exerts anti-inflammatory effect via mimicking glucocorticoids. Acta Pharmacol Sin 2011; 32: 904-911
• 17 Kumar KJ, Vani MG, Chueh PJ, Mau JL, Wang SY. Antrodin C inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via suppression of Smad2/3 and beta-catenin signaling pathways. PLoS One 2015; 10: e0117111
• 18 Manni A. Tamoxifen therapy of metastatic breast cancer. J Lab Clin Med 1987; 109: 290-299
• 19 Chang CJ, Chao CH, Xia W, Yang JY, Xiong Y, Li CW, Yu WH, Rehman SK, Hsu JL, Lee HH, Liu M, Chen CT, Yu D, Hung MC. p53 regulates epithelial-mesenchymal transition and stem cell properties through modulating miRNAs. Nat Cell Biol 2011; 13: 317-323
• 20 Hseu YC, Chang GR, Pan JY, Rajendran P, Mathew DC, Li ML, Liao JW, Chen WT, Yang HL. Antrodia camphorata inhibits epithelial-to-mesenchymal transition by targeting multiple pathways in triple-negative breast cancers. J Cell Physiol 2019; 234: 4125-4139
• 21 Hseu YC, Chao YH, Lin KY, Way TD, Lin HY, Thiyagarajan V, Yang HL. Antrodia camphorata inhibits metastasis and epithelial-to-mesenchymal transition via the modulation of claudin-1 and Wnt/beta-catenin signaling pathways in human colon cancer cells. J Ethnopharmacol 2017; 208: 72-83
• 22 Liu YM, Liu YK, Huang PI, Tsai TH, Chen YJ. Antrodia cinnamomea mycelial fermentation broth inhibits the epithelial-mesenchymal transition of human esophageal adenocarcinoma cancer cells. Food Chem Toxicol 2018; 119: 380-386
• 23 Liu YW, Lu KH, Ho CT, Sheen LY. Protective effects of Antrodia cinnamomea against liver injury. J Tradit Complement Med 2012; 2: 284-294
• 24 Yue PY, Wong YY, Chan TY, Law CK, Tsoi YK, Leung KS. Review of biological and pharmacological activities of the endemic Taiwanese bitter medicinal mushroom, Antrodia camphorata (M. Zang et C. H. Su) Sh. H. Wu et al. (higher Basidiomycetes). Int J Med Mushrooms 2012; 14: 241-256
• 25 Lu ZM, Xu ZH. Antcin A contributs to anti-inflammatory effect of Niuchangchih (Antrodia camphorata). Acta Pharmacol Sin 2011; 32: 981-982
• 26 Gokila Vani M, Kumar KJ, Liao JW, Chien SC, Mau JL, Chiang SS, Lin CC, Kuo YH, Wang SY. Antcin C from Antrodia cinnamomea protects liver cells against free radical-induced oxidative stress and apoptosis in vitro and in vivo through Nrf2-dependent mechanism. Evid Based Complement Alternat Med 2013; 2013: 296082
• 27 Huo Y, Win S, Than TA, Yin S, Ye M, Hu H, Kaplowitz N. Antcin H protects against acute liver injury through disruption of the interaction of c-Jun-N-terminal kinase with mitochondria. Antioxid Redox Signal 2017; 26: 207-220
• 28 Tien AJ, Chien CY, Chen YH, Lin LC, Chien CT. Fruiting bodies of Antrodia cinnamomea and its active triterpenoid, antcin K, ameliorates N-nitrosodiethylamine-induced hepatic inflammation, fibrosis and carcinogenesis in Rats. Am J Chin Med 2017; 45: 173-198
• 29 Senthil KK, Gokila VM, Mau JL, Lin CC, Chu FH, Wei CC, Liao VH, Wang SY. A steroid like phytochemical antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1. Oncotarget 2016; 7: 62836-62861
• 30 Chen YF, Chang CH, Huang ZN, Su YC, Chang SJ, Jan JS. The JAK inhibitor antcin H exhibits direct anticancer activity while enhancing chemotherapy against LMP1-expressed lymphoma. Leuk Lymphoma 2019; 60: 1193-1203
• 31 Chiu KY, Chen TH, Wen CL, Lai JM, Cheng CC, Liu HC, Hsu SL, Tzeng YM. Antcin-H isolated from Antrodia cinnamomea inhibits renal cancer cell invasion partly through inactivation of FAK-ERK-C/EBP-beta/c-Fos-MMP-7 pathways. Evid Based Complement Alternat Med 2017; 2017: 5052870
• 32 Hsieh YC, Rao YK, Whang-Peng J, Huang CY, Shyue SK, Hsu SL, Tzeng YM. Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress coincident with activation of intrinsic and extrinsic apoptotic pathway. J Agric Food Chem 2011; 59: 10943-10954
• 33 Lai CI, Chu YL, Ho CT, Su YC, Kuo YH, Sheen LY. Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. J Tradit Complement Med 2016; 6: 48-56
• 34 Huang YL, Chu YL, Ho CT, Chung JG, Lai CI, Su YC, Kuo YH, Sheen LY. Antcin K, an Active triterpenoid from the fruiting bodies of basswood-cultivated Antrodia cinnamomea, inhibits metastasis via suppression of integrin-mediated adhesion, migration, and invasion in human hepatoma cells. J Agric Food Chem 2015; 63: 4561-4569
• 35 Senthil Kumar KJ, Gokila Vani M, Hsieh HW, Lin CC, Liao JW, Chueh PJ, Wang SY. MicroRNA-708 activation by glucocorticoid receptor agonists regulate breast cancer tumorigenesis and metastasis via downregulation of NF-kappaB signaling. Carcinogenesis 2019; 40: 335-348
• 36 Lv ZD, Kong B, Li JG, Qu HL, Wang XG, Cao WH, Liu XY, Wang Y, Yang ZC, Xu HM, Wang HB. Transforming growth factor-beta 1 enhances the invasiveness of breast cancer cells by inducing a Smad2-dependent epithelial-to-mesenchymal transition. Oncol Rep 2013; 29: 219-225
• 37 Srivastava SK, Arora S, Averett C, Singh S, Singh AP. Modulation of microRNAs by phytochemicals in cancer: underlying mechanisms and translational significance. BioMed Res Inter 2015; 2015: 9
• 38 Wang Q, Cheng Y, Wang Y, Fan Y, Li C, Zhang Y, Wang Y, Dong Q, Ma Y, Teng YE, Qu X, Liu Y. Tamoxifen reverses epithelial-mesenchymal transition by demethylating miR-200c in triple-negative breast cancer cells. BMC Cancer 2017; 17: 492
• 39 Liu PL, Liu WL, Chang JM, Chen YH, Liu YP, Kuo HF, Hsieh CC, Ding YS, Chen WW, Chong IW. MicroRNA-200c inhibits epithelial-mesenchymal transition, invasion, and migration of lung cancer by targeting HMGB1. PLoS One 2017; 12: e0180844
• 40 Schubert J, Brabletz T. p53 Spreads out further: suppression of EMT and stemness by activating miR-200c expression. Cell Res 2011; 21: 705-707
• 41 Knezevic J, Pfefferle AD, Petrovic I, Greene SB, Perou CM, Rosen JM. Expression of miR-200c in claudin-low breast cancer alters stem cell functionality, enhances chemosensitivity and reduces metastatic potential. Oncogene 2015; 34: 5997-6006
• 42 Zhu H, Evans B, OʼNeill P, Ren X, Xu Z, Hait WN, Yang JM. A role for p53 in the regulation of extracellular matrix metalloproteinase inducer in human cancer cells. Cancer Biol Ther 2009; 8: 1722-1728
• 43 Gu W, Xu W, Sun X, Zeng B, Wang S, Dong N, Zhang X, Chen C, Yang L, Chen G, Xin A, Ni Z, Wang J, Yang J. Anordrin eliminates tamoxifen side effects without changing its antitumor activity. Sci Rep 2017; 7: 43940
• 44 Wu Y, Tian WJ, Gao S, Liao ZJ, Wang GH, Lo JM, Lin PH, Zeng DQ, Qiu DR, Liu XZ, Zhou M, Lin T, Chen HF. Secondary metabolites of petri-dish cultured Antrodia camphorata and their hepatoprotective activities against alcohol-induced liver injury in mice. Chin J Nat Med 2019; 17: 33-42
• 45 Li ZW, Kuang Y, Tang SN, Li K, Huang Y, Qiao X, Yu SW, Tzeng YM, Lo JY, Ye M. Hepatoprotective activities of Antrodia camphorata and its triterpenoid compounds against CCl4-induced liver injury in mice. J Ethnopharmacol 2017; 206: 31-39
• 46 Zhang Y, Verbeek FJ. Comparison and integration of target prediction algorithms for microRNA studies. J Integr Bioinform 2010; 7: 127