Novel effect and the mechanistic insights of fruiting body extract of medicinal fungus Antrodia cinnamomea against T47D breast cancer

doi:10.1016/j.phymed.2016.11.006

Phytomedicine

Volume 24, 15 January 2017, Pages 39-48

https://doi.org/10.1016/j.phymed.2016.11.006 Get rights and content

Abstract

Introduction

Tamoxifen, an anti-oestrogenic drug for estrogen receptor positive (ER+) breast cancer, was observed to stimulate tumor growth or drug resistance in patients. Antrodia cinnamomea (AC), a precious medicinal fungus has been traditionally used as a folk remedy for cancers in Asian countries. The objective of this study was to investigate the bioefficacy and the underlying molecular mechanisms of the AC fruiting bodies extracts (AC-3E) against human ER+ T47D breast cancer cells, and compare the effect with that of tamoxifen.

Methods

Cell proliferation, migration, TUNEL assay, western blotting, time-lapse confocal microscopy analyses, chorioallantoic membrane assay, and a xenograft BALB/c nude mouse system were used in this study. Chemical fingerprinting of AC-3E was established using LC-MS.

Results

AC-3E attenuated T47D breast cancer cell activity by deregulating the PI3K/Akt/mTOR signaling pathway and key cell-cycle mediators, and inducing apoptosis. AC-3E also effectively inhibited tube-like structures of endothelial cells, blood vessel branching and microvessel formation ex vivo and in vivo. Significant preventive and therapeutic effects against T47D mammary tumor growth of AC-3E was observed comparable or superior to tamoxifen treatment in xenograft BALB/c nude mice. Dehydroeburicoic acid (2) was characterized as the main chemical constituent in AC-3E against breast cancer.

Conclusion

This study suggests that AC-3E extracts can be employed as a double-barreled approach to treat human ER+ breast cancer by attacking both cancer cells and tumor-associated blood vessel cells.

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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¹: Both authors contributed equally to this article.

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Original ArticleNovel effect and the mechanistic insights of fruiting body extract of medicinal fungus Antrodia cinnamomea against T47D breast cancer

Abstract

Introduction

Methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

Cell lines and culture conditions

AC-3 extract inhibits T47D cell proliferation

Discussion

Author contributions

Conflict of interest

Acknowledgments

Biochem. Pharmacol.

J. Ethnopharmacol.

Phytomedicine

Am. J. Pathol.

Food Chem. Toxicol.

Food Chem. Toxicol.

Food Chem. Toxicol.

Cell

Free Radic. Biol. Med.

Eur. J. Pharmacol.

J. Ethnopharmacol.

Cancer Lett.

Food. Chem. Toxicol.

Mol. Cell

Regulation of ERalpha-mediated transcription of Bcl-2 by PI3K-AKT crosstalk: implications for breast cancer cell survival

Int. J. Oncol.

New targets for therapy in breast cancer: mammalian target of rapamycin (mTOR) antagonists

Breast Cancer Res.

Original Article
Novel effect and the mechanistic insights of fruiting body extract of medicinal fungus Antrodia cinnamomea against T47D breast cancer