Ganoderma Tsugae Induced ROS-Independent Apoptosis and Cytoprotective Autophagy in Human Chronic Myeloid Leukemia Cells T
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Food and Chemical Toxicology 124 (2019) 30–44 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Ganoderma tsugae induced ROS-independent apoptosis and cytoprotective autophagy in human chronic myeloid leukemia cells T You-Cheng Hseua,b,c,d, Yi-Chun Shene, Ming-Ching Kaof, Dony Chacko Mathewa, ∗ Palaniyandi Karuppaiyae, Mei-Ling Lie, Hsin-Ling Yange, a Department of Cosmeceutics, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung, 40402, Taiwan b Department of Health and Nutrition Biotechnology, Asia University, Taichung, 41354, Taiwan c Chinese Medicine Research Center, China Medical University, Taichung, 40402, Taiwan d Research Center of Chinese Herbal Medicine, China Medical University, Taichung, 40402, Taiwan e Institute of Nutrition, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung, 40402, Taiwan f Department of Biological Science and Technology, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung, 40402, Taiwan ARTICLE INFO ABSTRACT Keywords: The medicinal fungus Ganoderma, known in Chinese as Lingzhi or Reishi, traditionally has various medicinal Ganoderma tsugae uses and has been employed in cancer treatment in Asia for centuries. This study used ethanol-extracted Human chronic myeloid leukemia Ganoderma tsugae (GT) and examined its antitumor activities on human chronic myeloid leukemia cells as well as G2/M arrest its molecular mechanism of action. Treatment with GT (200–400 μg/mL) significantly reduced cell viability and Apoptosis caused G2/M arrest in K562 cells. In addition, GT induced mitochondrial and death receptor mediated apoptosis, Autophagy correlated with DNA fragmentation, followed by cytochrome c release, caspase-3/8/9 activation, PARP clea- vage, Fas activation, Bid cleavage, and Bax/Bcl-2 dysregulation. Cytoprotective autophagy was found to be induced by GT, as was revealed by increased LC3-II accumulation, Beclin-1/Bcl-2 dysregulation, acidic vesicular organelle formation, and p62/SQSTM1 activation. Notably, pretreatment of cells with the autophagy inhibitors 3-MA and CQ enhanced GT-induced apoptosis. Interestingly, reactive oxygen species production in cells was not triggered by GT administration; equally, the antioxidant N-acetylcysteine was found to be incapable of pre- venting apoptosis and autophagy induced by GT treatment. Finally, this study discovered that cytoprotective autophagy induced by GT was associated with EGFR and PI3K/AKT/mTOR signaling cascade suppression. In summary, GT demonstrated antitumor activity against human chronic myeloid leukemia. 1. Introduction Accumulating evidence has been obtained that a variety of phyto- chemicals such as alkaloids, saponins, flavonoids, terpenoids, lignans, Chronic myeloid leukemia (CML) is one of the deadliest malignant saponins, peptides, polyketides, and plant extracts inhibit CML pro- hematological stem-cell disorders in adults and is characterized by liferation and induce apoptosis (Khajapeer and Baskaran, 2016). myeloid proliferation and Philadelphia chromosomal aberration Therefore, the development of novel drugs or agents from natural re- (Nowell, 2007). For most patients with CML, allogeneic hematopoietic sources is critical to generating therapeutic regimens for patients with stem-cell transplantation and tyrosine kinase inhibitor (TKI) therapy CML. are the accepted treatment. However, a few such patients fail to re- Clinical trials have evaluated the anticarcinogenic property of spond to TKIs because of their genetic mutations. Patients who undergo medicinal mushroom extracts (Lu et al., 2016). Polysaccharides derived TKI therapy often report cutaneous side effects (Webb et al., 2017). from medicinal mushrooms are best known for their anticancer and Phytocomponents are receiving increasing attention in the field of immunomodulatory properties. In addition, scholars have demon- cancer biology due to their minor or nontoxicity and cost effectiveness. strated that these phytocompounds and extracts from medicinal Scholars are thus currently investigating whether natural products mushrooms induce apoptosis, autophagy, and cell-cycle arrest in var- could be invaluable for the treatment of cancer and infection. ious types of cancerous cells and can be used as a chemotherapeutic ∗ Corresponding author. Institute of Nutrition, College of Biopharmaceutical and Food Sciences, China Medical University, 91 Hsueh Shih Road, Taichung, 40402, Taiwan. E-mail address: [email protected] (H.-L. Yang). https://doi.org/10.1016/j.fct.2018.11.043 Received 27 July 2018; Received in revised form 18 October 2018; Accepted 18 November 2018 Available online 19 November 2018 0278-6915/ © 2018 Elsevier Ltd. All rights reserved. Y.-C. Hseu et al. Food and Chemical Toxicology 124 (2019) 30–44 agent (Hsu et al., 2008; Patel and Goyal, 2012; Wang et al., 2017; Wu USA). From Santa Cruz Biotechnology, Inc. (Heidelberg, Germany), we et al., 2012; Yu et al., 2012). Eukaryotic cell-cycle progression is a obtained mouse monoclonal antibodies against Bax and β-actin and fundamental mechanism determining cell proliferation; the subsequent rabbit polyclonal antibodies against cytochrome c, Bcl-2, Fas, and β- activation of cyclin-dependent kinases and cyclin is known to mediate tubulin. Mouse monoclonal antibodies against Bid were procured from this mechanism (Pan et al., 2002). Several chemotherapeutic agents Cell Signaling Technology, Inc. (MA, USA), as were rabbit monoclonal have been derived from non-toxic natural products, and these agents antibodies against caspase-3, caspase-8, caspase-9, Bax, and PARP. inhibit cell growth and proliferation in cancerous cells through Antibodies against Beclin-1, p62/SQSTM1, p-mTOR, p-PI3K, PI3K, prompting cell-death signaling cascades (Ricci and Zong, 2006). LC3I/II, AKT, and p-AKT came from Cell Signaling Technology, Inc. Apoptosis and autophagy play crucial physiological and patholo- Antibody against p-EGFR was from Life Technologies (Gaithersburg, gical roles in numerous diseases including cancer and are part of a se- MD, USA). Antibody against GAPDH was procured from Cusabio quence of biochemical events that cause changes to cell structure and Biotechnology, Inc. (Wuhan, China). All secondary antibodies were eventually resulting in cell death. During apoptosis, chromatin is con- bought from Santa Cruz Biotechnology (CA, USA). The chemicals 3- densed, caspases are activated, and internucleosomal DNA is cleaved methyladenine (3-MA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- (Taylor et al., 2008). Autophagy involves the degradation of long- trazolium bromide (MTT), N-acetylcysteine (NAC), propidium iodide standing cytosolic proteins and organelles as well as material recycling (PI), acridine orange (AO), chloroquine (CQ), and 2‘,7‘-dihydro- for maintaining cellular component quality (Maiuri et al., 2007). fluorescein-diacetate (DCFH2-DA) were from Sigma-Aldrich (MO, USA). However, exaggerated autophagy leads to programmed cell death of We obtained Z-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) from type II; this is because mitochondria and other survival molecules are Calbiochem (CA, USA). The remaining chemicals used in this experi- intensely degraded (Morselli et al., 2009). Drugs and treatments against ment were reagent or HPLC grade and were obtained from Sigma- cancer cause cellular death in cancer cells by triggering autophagy ra- Aldrich or Merck & Co., Inc. (Darmstadt, Germany). ther than apoptosis (Gozuacik and Kimchi, 2004; Kondo et al., 2005). How apoptosis and autophagy proceed simultaneously is complex and 2.2. Ganoderma tsugae extract preparation dependent on the cellular context. Accumulating evidence indicates that excessive reactive oxygen species (ROS) relentlessly damage DNA Ganoderma tsugae was generously gifted by Luo-Gui Ying of the and proteins and reduce the potential of the mitochondrial membrane Fungi Agriculture Farm in Taoyuan, Taiwan. GT extracts were derived potential (ΔΨm), leading to apoptosis and autophagy (Ly et al., 2003; using a method reported earlier (Hsu et al., 2008). The extracts were Park et al., 2012). Thus, the main objectives of chemoprevention are standardized as mentioned by Tseng et al. (2018) (Tseng et al., 2018). blocking cancer-cell initiation and promotion and inducing either or In brief, a powder of the fruiting body of GT (30 g) was immersed in both autophagy and apoptosis (Sharma, 2012). 300 mL of 99.9% ethanol, with the solution then mixed and shaken Used in traditional Chinese medicine for more than 4000 years in using a rotating shaker for 24 h. After being centrifuged, the filtrate Asian countries, Ganoderma, a popular chemopreventive medicinal underwent filtering using filter paper (Whatman, cat. No. 1001–110) mushroom genus, has recently strongly garnered the attention of and the residues were extracted with ethanol two times. The collected Taiwan's cancer research (and more broadly, health care) communities filtrates were concentrated in a rotary evaporator under reduced pres- (Hsu et al., 2008; Yu et al., 2012). Of the numerous bioactive com- sure. The yield of the GT extracts was approximately 13.8%. GT stock pounds that are present in mushrooms of this genus, polysaccharides solution was prepared with ethanol (200 mg/mL) and, until further use, and triterpenoids are the most promising for therapy against many stored at −80 °C. diseases, cancer being one (Zhou et al., 2007). Ganoderma tsugae (GT), a medicinal