Recently, transcriptome profiling is a method of large-scale investigation of gene expression levels and has been widely used to identify cellular states, to find genes with similar expression patterns or to investigate molecular mechanisms of action of anticancer agents. The differentially expressed genes screened by RNA-sequencing can offer a new insight into the complex mechanisms of action of anticancer drugs (Wacker et al., 2012; Yang et al., 2013). RNA-sequencing is a technique for transcriptome profiling that uses next generation sequencing (NGS) technologies and measures transcript levels. The gene ontology (GO) term enrichment was applied to find the biological functions of differentially expressed genes, and the kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was utilized to determine the related signal pathways. NGS is now generally accepted as a method to analyze total mRNA expression and elucidate cellular processes at the molecular level. Therefore, NGS allowed the identification of transcript levels that are involved in the induction of cell death in cancer cells to find the biological functions of differentially expressed genes.

In recent years, there has been renewed interest in natural products for the discovery and development of new anticancer drugs. Mushrooms have caught researchers’ attention due to their anticancer activity demonstrated in a variety of human cancer cells including breast, prostate and colorectal cancer cells in vitro (Hu et al., 2002; Stanley et al., 2005; Hsu et al., 2008). The anti-proliferative and apoptotic properties of mushrooms have been proposed relying on their tumor cell growth inhibitory activity (Sarangi et al., 2006; Israilides et al., 2008). White genius mushroom is a white strain of H. marmoreus (also known as bunashimeji and hon-shimeji) and is very popular edible mushroom in Taiwan. Originally, H. marmoreus was cultivated with a polypropylene plastic bottle in environmental control system in Japan. White genius mushroom was cultivated with a modified polypropylene plastic bags and environmental control system. H. marmoreus cultivated with the new methods was named “white genius mushroom” in Taiwan. The white genius mushroom was harvested when the stalk length is more than 15 cm and the yield of fruiting bodies of a cultivation bag is about 270–350 g. The mycelium incubation time of white genius mushroom is about 120 days, it needed about 26–30 days from primordial mushroom fruiting bodies formation to harvest. It has been reported that the extracts of H. marmoreus had an anticancer activity (Bao and You, 2011). However, the exact mechanism underlying H. marmoreus-produced anticancer effect remains to be elucidated. To identify the critical genes and pathways that are involved in the WGM extracts-induced cell death of Hep3B cells and to better understand the underlying mechanisms, the transcriptomes were analyzed in control and WGM extracts-treated cells using the next-generation sequencing technology.

Reactive oxygen species (ROS) are highly reactive species that are generated from oxygen metabolism. Excessive production of ROS may lead to oxidative stress, loss of cell function and ultimately cell death. ROS have been proved to be signaling molecules involved in the regulation of cell survival and cell death (Szatrowski and Nathan, 1991; Trachootham et al., 2009; You et al., 2014). It has also been reported that ROS play an important role in various signal transduction pathways of autophagy and apoptosis (Zhang et al., 2016; Dhanasekaran and Reddy, 2017). Particularly, producing ROS participated in the drug-induced cancer cell apoptosis and autophagy has been receiving increasing attention (Chen et al., 2008; Azad et al., 2009). Although autophagy has been considered as a cell survival mechanism, there is accumulating evidence for cross-talk in the regulation of apoptosis and induction of autophagy (Gump and Thorburn, 2011; Mukhopadhyay et al., 2014; Wei et al., 2014). Furthermore, the occurrence of autophagy can promote apoptosis and, thus, accelerate cell death (Rubinstein et al., 2011; Mukhopadhyay et al., 2014).

According to the report provided by the Ministry of Health and Welfare in Taiwan, liver cancer was the second leading cause of death in 2020. Since liver cancer presents a serious public health problem and economic burdens on the society, many investigators have exerted efforts to prevent the development of the cancer. Although H. marmoreus was found to have anticancer activities, there is no substantial evidence relating H. marmoreus to treatment of cancer. In this study, WGM extracts was examined for its anticancer and chemopreventive activities in hepatocarcinoma cells.

White genius mushroom is a white strain of H. marmoreus (also known as bunashimeji and hon-shimeji) and is very popular edible mushroom in Taiwan. In this study, we explored the mechanisms of WGM extracts-induced cell death in Hep3B cancer cells. Recently, transcriptome profiling has been employed extensively to identify cellular states or to find genes with similar expression patterns (Wu et al., 2019; Shi et al., 2020). In general, GO and KEGG pathway enrichment analysis were subsequently conducted to elucidate cellular processes at the molecular level and further to investigate the key determinants for drugs-induced gene expression profiles. GO analysis is a commonly used method for the functional annotation of large amounts of genomic data (Gene Ontology Consortium, 2006). The KEGG pathways database is a comprehensive and well recognized database with a wide range of biochemical pathways (Kanehisa and Goto, 2000). The enrichment of dysfunctional signaling pathways was screened by the KEGG pathway analysis. In this study, next generation sequencing (NGS) technologies was used to examine differentially expressed genes between WGM extracts-treated and control cells. According to the result of NGS analysis, 8711 differentially expressed genes were identified including 5125 upregulated genes and 3586 downregulated genes screened in 150 μg/ml WGM extracts-treated samples compared with control samples. GO analysis showed that the DEGs were found to be involved in biological process, cellular component and molecular function. KEGG pathway enrichment analysis was performed in the present study to systematically determine the enrichment of signaling pathways of the differentially expressed genes in WGM extracts-treated cells. A total of 18 KEGG pathways with enriched DEGs were obtained. According to the KEGG pathway enrichment analysis, apoptosis, autophagy and mitophagy pathways were identified as significant in WGM extracts-treated cells compared to control cells. Among the 3675 DEGs induced by WGM extracts, 105 DEGs are related to apoptosis, 170 DEGs are related to mitophagy and 226 DEGs are related to autophagy. It suggested that WGM extracts might be able to induce autophagy in Hep3B cells in this study.

To further demonstrate the possible role of autophagy of WGM extracts-induced cell death, the present study examined the protein expression of the endogenous autophagy markers p62 and LC3B. P62 can shuttle ubiquitinated cargo for autophagic degradation that is used as a reporter of autophagy activity (Komatsu and Ichimura, 2010; Katsuragi et al., 2015). Phosphorylation of p62 on serine 403 (p62p(S403)) is thought to increase its binding affinity for ubiquitin chains and is a key component in the autophagic degradation of poly-ubiquitinated proteins (Katsuragi et al., 2015). LC3B has been widely used to be an autophagosomal marker for monitoring autophagy. LC3BII, the lipid modified form of LC3B, is believed to be involved in autophagosome membrane expansion and fusion events and be a standard marker for autophagosomes (Kabeya et al., 2000). In this study, WGM extracts induced a concentration-dependent accumulation of LC3BII, p62 and p62p(S403) proteins which are consistent with those of other studies reporting that an increase in LC3BII or p62 expression is required for autophagy induction (Puissant et al., 2010; Katsuragi et al., 2015; Martino et al., 2019). We then examined the protein levels of beclin 1, a key regulator of autophagosome formation (Kang et al., 2011). The protein levels of beclin 1 was gradually reduced as the WGM extracts concentration increased, indicating that WGM extracts-induced autophagy in Hep3B cell is a beclin 1-independent process. Although beclin 1 was often perceived to be a key player in autophagy execution and its knock down blocks autophagic cell death (Yue et al., 2003), accumulating evidences showed that beclin 1-independent autophagy is invariably associated with cell death (Scarlatti et al., 2008; Shrivastava et al., 2011).

Excessive production of ROS may lead to oxidative stress, loss of cell function and ultimately cell death. In fact, many anti-cancer drugs have been shown to have an anti-tumor effects via ROS-dependent autophagic and apoptotic cell death in various cancer cells (Chen et al., 2008; Azad et al., 2009). It has also been reported that reactive oxygen species play an important role in various signal transduction pathways, such as JNK-signaling and NF-κB pathway, of autophagy and apoptosis (Zhang et al., 2016; Dhanasekaran and Reddy, 2017). Autophagy and particularly mitophagy have been shown to play an essential role in reducing the mitochondrial ROS production, thereby allowing mitochondria to avoid oxidative damage (Kurihara et al., 2012; Bin-Umer et al., 2014). In this study, we demonstrated that autophagy, mitophagy and apoptosis pathways were involved in WGM extracts-induced Hep3B cell death according to the KEGG pathway enrichment analysis. Although beclin 1 expression was required for ROS-induced autophagy (Azad et al., 2009), it has also been reported that anticancer agents can induce ROS-mediated beclin 1-independent autophagy in various cancer cell types (Al Dhaheri et al., 2014; Martino et al., 2019). The reduced levels of beclin 1 are believed to inhibit autophagy and prevent the turnover of damaged mitochondria, leading to further production of ROS and oxidative stress (Kurihara et al., 2012; Bin-Umer et al., 2014). Since mitophagy and a dose-dependent reduction in beclin 1 protein expression were observed after 24 h treatment with WGM extracts, the ROS production was determined in this study. The present study demonstrated that there was a dose- and time-dependent increase in the production of ROS after treatment with WGM extracts in Hep3B cells. It seemed to suggest that mitophagy cannot effectively eliminate the mitochondrial damage during WGM extracts-induced cell death, which leads to further production of ROS and oxidative stress, resulting in a dramatic loss in cell viability.

To further investigate whether the effect of WGM extracts on the cell death of Hep3B cells could be linked to the production of ROS, reduced glutathione, a key antioxidant, and N-acetylcysteine, a precursor of glutathione, were used in this study. The present study demonstrated that glutathione or N-acetylcysteine significantly reversed the WGM extracts-mediated Hep3B cell death. It suggested that ROS production be involved in WGM extracts-induced Hep3B cancer cell death. The phenotypic characteristics of WGM extracts-treated Hep3B cells were also examined by microscopic inspection of overall morphology. It is noteworthy that treatment with WGM extracts results in a significant dose-dependent increase in the formation of membrane-enclosed vacuoles within cells. We have observed that WGM extracts at 150 μg/ml causes the accumulation of big cytoplasmic vacuoles and cell death in Hep3B cells, while pretreatment with glutathione or N-acetylcysteine reduced vacuole formation and cell death in the cells. Furthermore, a reduction in WGM extracts-mediated ROS production was observed in the experiments of glutathione or N-acetylcysteine pretreatment. Based on the above data, we guess that vacuole formation and cell death are related to the production of ROS during the WGM extracts-induced cell death.

We further examined whether WGM extracts-induced the formation of membrane-enclosed vacuoles is associated with the induction of autophagy by WGM extracts in Hep3B cells. In this study, inhibitor of autophagy, 3-methyladenine (3-MA), was used to confirm whether membrane-enclosed vacuoles induced by WGM extracts were related to autophagy. 3-MA has been widely used as autophagic inhibitor due to its inhibitory effect on autophagosome formation. In this study, 3-MA cannot relieve the membrane-closed vacuoles induced by WGM extracts. It seems to indicate that there is no observable connection between the membrane-enclosed vacuoles induction and autophagy. Our results also showed that 3-MA had no effect on the WGM extracts-induced cell death, indicating autophagy is probably not the primary signal involved in WGM extracts-induced cell death. Furthermore, 3-MA alone slightly induced Hep3B cell death. Although autophagy has been considered as a cell survival mechanism, there is accumulating evidence for cross-talk in the regulation of apoptosis and induction of autophagy (Gump and Thorburn, 2011; Mukhopadhyay et al., 2014; Wei et al., 2014). Furthermore, the occurrence of autophagy can promote apoptosis and, thus, accelerate cell death (Rubinstein et al., 2011; Mukhopadhyay et al., 2014). Although autophagy has been considered as cell survival mechanism, our study had shown that autophagy involved in WGM extracts-induced Hep3B cell death. Since 3-MA cannot alleviate or inhibit cell death caused by WGM extracts, we guessed that autophagy induced by WGM extracts could be an indication that the cells attempted to survive through induction autophagy in this study.

LysoPC is a monoglycerophospholipid in which a phosphorylcholine moiety is attached to the sn-3 position of glycerol. LysoPC can have different long-chain fatty acids, which is the 16, 18 and 20 carbon atoms, bonded to the sn-1 position of glycerol. (18:2(9Z,12Z)) lysoPC is a linoleic acid at the sn-1 position. LysoPC is normally generated from phosphatidylcholine (PC) through the catalysis of phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins and atherosclerotic tissues. Previous studies had demonstrated that the downregulation of PC and upregulation of lysoPC could induce cell apoptosis (Anthony et al., 1999; Cui and Houweling, 2002; Takahashi et al., 2002). In the research of the antitumor effects of PP242, an inhibitor of mechanistic target of rapamycin (mTOR), in a colon cancer xenograft mouse model, Rashid et al. (2020) indicated that lysoPC (18:2) in the PP242-treated mice was significantly increased after mTOR inhibition. It has also been suggested that lysoPC (16:0) induces apoptosis in human endothelial cells through a p38-MAPK-dependent pathway (Takahashi et al., 2002). (2E,6E)-3,7,11,15,19,23,27,31,35-nonamethylhexatriaconta-2,6,34-triene-1,11,15,19,23,27,31-heptol is a polyhydroxy isoprenoid and has been described to be a novel structural class of inhibitors of the human neurokinin-1 (NK-1) and neurokinin-2 (NK-2) receptors (Hegde et al., 1997). The neurokinin receptors, also known as tachykinin receptors, have played an important role in the regulation of a variety of fundamental biological processes such as neuronal activity, cell proliferation, inflammation, etc (Campos and Calixto, 2000; Pennefather et al., 2004; Muñoz et al., 2019). Recently, it has been reported that the NK-1 receptor (NK1R) played a pivotal in the development of cancer, including pancreatic cancer, thyroid cancer, gastrointestinal cancer and breast carcinoma (Muñoz and Coveñas, 2014; Muñoz and Coveñas, 2016; Ebrahimi et al., 2020; Isorna et al., 2020). Furthermore, new evidence suggested that NK1R is overexpressed in human hepatoma and can be antagonized to exhibit a significant inhibitory effect on the proliferation of cancer cells in vivo and in vitro (Garnier et al., 2016). Therefore, NK-1R is currently considered to be an important target for the treatment of hepatoblastoma. Based on the above data and reasons, in this study, we have tested the hypothesis that these two anti-cancer components of WGM extracts may be the reason of cell death caused by WGM extracts.

White genius mushroom is very popular edible mushroom in Taiwan. We hope that WGM may be developed into a dietary supplement or dietary chemopreventive agent for the cancer treatment. For defining WGM as a promising chemopreventive agent against human liver cancer, we explored the mechanisms of WGM extracts-induced cell death in human hepatoma Hep3B cancer cells. Next generation sequencing was used for identifying the target pathways associated with WGM extracts in Hep3B cells. According to the KEGG pathway enrichment analysis, autophagy, mitophagy and apoptosis pathways were identified as significant in WGM extracts-treated cells compared to control cells. In this study, protein expression of the endogenous autophagy markers LC3BII, p62 and p62p(S403) was elevated subsequent to WGM extracts treatment, suggesting that WGM extracts indeed induce autophagy in Hep3B cells. The present study also demonstrated that WGM extracts-induced cell death and ROS production is partially inhibited by GSH or NAC pretreatment in Hep3B cells, but membrane-enclosed vacuoles formation is completely prevented. Therefore, there is observable connection between the production of ROS and membrane-enclosed vacuoles formation or cell death. In the current study, we provided evidence that WGM extracts induced beclin 1-independent autophagic cell death through ROS generation.