Original ArticleNovel effect and the mechanistic insights of fruiting body extract of medicinal fungus Antrodia cinnamomea against T47D breast cancer
Graphical abstract
Introduction
Breast cancer is the most frequently diagnosed life-threatening cancer in women and the leading cause of cancer death among women worldwide. Current cancer therapies, including endocrine therapy, chemotherapy, and targeted therapy, play an important role in the treatment of breast cancer; however, drug resistance, severe side effects, high recurrence rate, and metastases remain threats to breast cancer patients. Systemic tamoxifen (endocrine) treatment, for instance, was found to cause tamoxifen-stimulated tumor growth or drug resistance (Schafer et al., 2000). Due to the shortcomings of the current therapies, there is a pressing need for development of new therapeutic or preventive agents for breast cancer. Phytomedicines or phytoagents with novel molecular mechanisms provide one potential area for development.
Antrodia cinnamomea (AC) (known as Niu-Chang-Chih in Chinese medicine) is a precious medicinal fungus endemic to Taiwan that has been popularly used as a folk medicine for various disorders. Recently, the therapeutic effect of AC fruiting body against chronic alcohol consumption-induced liver damage in rats was reported (Huang et al., 2010) and other hepatoprotective activities of fruiting body extracts or mycelium from solid or submerged cultivation have been reviewed (Ao et al., 2009, Geethangili and Tzeng, 2011). Also, AC extract has been shown to protect normal mouse spleen immune cells from radiation-mediated side effects, but intriguingly, the extract selectively enhanced radiation-induced inflammation and cytotoxicity in human colorectal cancer cells (Cheng et al., 2014).
Several reports on the anti-cancer activities of fruiting body extracts of A. cinnamomea have shown that total AC extracts prepared by organic solvents such as methanol, chloroform, ethanol or ethylacetate exhibit various anti-cancer activities against HepG2, PLC/PRF/5 liver cancer cells (Hsu et al., 2007), colon, Jurkat or prostate cancer cell lines (Rao et al., 2007), or leukemia HL60 cells (Hseu et al., 2004), through regulation of Bcl-2 family proteins, and activation of caspases or NF-κB protein. In one recent study, programmed cell death via the autophagic pathway was observed in AC extract-treated head and neck cancer cells (Chang et al., 2013). A report of A. cinnamomea anti-human breast cancer cell activities focused on study of the fermented culture broth of AC against triple negative MDA-MB-231 cell activity (Yang et al., 2012b). The mode of action of these activities has been found to be through inhibition of the expression of COX-2 and induction of cell cycle arrest or apoptosis in MDA-MB-231 cells in vitro or in a xenograft mouse model (Hseu et al., 2008, Hseu et al., 2007), or through inhibition of the MAPK signaling pathway (Yang et al., 2011). So far, only the fermented culture broth of AC has been reported to induce apoptosis in estrogen receptor positive (ER+) breast cancer MCF-7 cells (Yang et al., 2006). Little or no information concerning the bioactivity and mode of action of the fruiting body extracts of AC against ER+ breast cancer cells, such as T47D cells has been reported.
Because approximately 70% of breast cancer patients are diagnosed with ER+ cancer, and the current ER antagonistic drug (tamoxifen) shows drug resistance or side effects (Ring and Dowsett, 2004), this study aimed to investigate the pharmacological activity and the underlying modes of action of the fruiting body extract of A. cinnamomea against human ER+ T47D breast cancer cells. Further, to address the quality control of this folk medicine, the chemical fingerprint and active constituent(s) in the A. cinnamomea extract were also characterized.
Section snippets
Cell lines and culture conditions
T47D, a human mammary ductal carcinoma cell line obtained from American Type Culture Collection, ATCC, Manassas, VA, USA, was grown in Roswell Park Memorial Institute 1640 medium (RPMI-1640; Life Technologies, Grand Island, NY, USA); H184B5F5/M10 (ATCC), a human mammary epithelial cell line, was cultured in minimum essential medium (MEM; Life Technologies, USA). All cell lines were cultured in specific media supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin in a humidified
AC-3 extract inhibits T47D cell proliferation
The anti-cancer cell proliferation activity on T47D (ER+) cells of the fruiting body extracts of A. cinnamomea grown for 3 months (AC-3E), 6 months (AC-6E), or 9 months (AC-9E) was compared using MTT assay. Tamoxifen (Tam), an estrogen antagonistic drug, was used as a reference control in this study. The result in Fig. 1 shows that, at 48 h treatment, the magnitude of cell cytotoxicity against T47D cells is AC-3E > AC-6E > AC-9E, with an IC50 of 52.7 µg/ml, 122.6 µg/ml, 141.9 µg/ml,
Discussion
In this study, A. cinnamomea fruiting body extracts were shown to attenuate proliferation, migration, and tumor growth of estrogen receptor positive T47D human breast cancer cells in vitro and in vivo by deregulating the PI3K/Akt signaling pathway and cell-cycle mediators, inducing apoptosis, and inhibiting tumor angiogenesis. This is the first report to demonstrate that AC extracts exert similarly potent activity to that of the standard estrogen antagonism drug tamoxifen against estrogen
Author contributions
K. M. S., T. H. S. and L. F. S. designed the research study.
K. M. S., T. H. S., W. L. L., C. Y. C., B. Y. H. and S. Y. W. performed the studies and analyzed the data.
W. W. H. cultivated and collected the AC fruiting body.
K. M. S., T. H. S., W. L. L., and L. F. S. wrote the manuscript.
L. F. S. supervised the research.
Conflict of interest
The authors declare that they have no conflict of interests. The authors declare no competing financial interests.
Acknowledgments
This study was supported by institutional grant funding from Academia Sinica, Taiwan. The authors thank Dr. Jung-Yaw Lin for his support on this project and Mr. Chung-Chih Yang for his technical assistance on the tube formation experiments.
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Both authors contributed equally to this article.