Hericium erinaceus enhances doxorubicin-induced apoptosis in human hepatocellular carcinoma cells
Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the fourth leading cause of cancer-related mortality worldwide, with the highest incidence in Asia [1]. Despite surgical management and application of non-surgical therapeutic modalities, the incidence of HCC continues to rise [2]. Doxorubicin (Dox) is an effective chemotherapeutic drug for the treatment of patients with HCCs [3]. However, Dox has been shown to cause high systemic toxicity to normal tissues, as well as immunosuppression, secondary cardiomyopathy, and development of drug resistance during cancer therapy, thereby limiting successful outcomes of cancer chemotherapy [4], [5]. Accordingly, several new therapeutic strategies are being developed to treat cancer. Recently, combination (rather than single-agent) chemotherapy has been found to be a superior treatment strategy that offers the potential for lowering the dose of chemotherapeutic drugs to reduce side effects. A related strategy is to evaluate the mechanisms of natural medicines [6], [7]. Many studies have demonstrated that polysaccharides from basidiomycetes mushrooms had highly beneficial therapeutic effects including: (1) preventing oncogenesis after administration of peroral medications developed from these mushrooms or their extracts, (2) direct anti-tumor activity against various tumors, (3) synergistic anti-tumor activity in combination with chemotherapy, and (4) preventive effects on tumor metastasis [8], [9], [10], [11]. In addition, unlike existing cancer chemotherapeutic drugs, mushroom-derived polysaccharides are known to have no toxic side effects. Most of the related clinical evidence was determined for the commercial polysaccharides lentinan (Lentinus edodes), krestin (Coriolus versicolor), and schizophyllan (Schizophyllum commune) [12]. Data on polysaccharides isolated from Hericium erinaceus (HE) is highly impressive, and these polysaccharides show promise for use as cancer therapeutics. Studies have shown that HE extracts strongly suppress the growth of various tumors in vitro and in vivo[13], [14]. However, the molecular signaling involved in HE-mediated anti-tumor activity has never been investigated in HepG2 human hepatocellular carcinoma cells.
Apoptosis, or programmed cell death, is an important physiological process of cell death and occurs during tissue remodeling, immune regulation, and tumor regression. Most of the chemotherapeutic agents kill cancer cells by inducing apoptotic death pathways [15]. Cells undergoing apoptosis show a sequence of cardinal morphological features, including membrane blebbing, cellular shrinkage, and condensation of chromatin [16]. The two primary apoptotic pathways are the extrinsic death receptor-mediated pathway and the intrinsic mitochondria-mediated pathway. These two pathways, when stimulated, lead to the release of cytochrome c from the mitochondria and to the activation of the death signal [17]. Apoptotic signaling and execution through these two pathways depends on caspases, or aspartate-specific cysteine proteases, which are the key effector molecules in the apoptotic process [18]. It has been suggested that Dox, an anticancer agent, elicits a caspase cascade, leading to apoptosis [19], [20]. A member of the mitogen-activated protein kinase (MAPK) family, c-Jun NH2-terminal kinase (JNK), is rapidly phosphorylated and subsequently activated by a diverse spectrum of different cell stimuli. The JNK pathway has been shown to be closely linked to apoptosis by activation of the mitochondria-mediated apoptosis pathway and to regulate the degradation of cellular Fas-associated death domain (FADD) interleukin-1 β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP), which is an important caspase cascade inhibitor [21], [22], [23].
To improve chemotherapeutic treatment, much effort has been made to identify chemosensitisers; that is, agents that are able to overcome multi-drug resistance (MDR) [24]. Interestingly, apart from MDR, nuclear factor κB (NF-κB) has been shown to be involved in chemoresistance in different cell types, suggesting a possible role of this transcription factor in the chemosensitising effects of agents [25].
Although there are many therapeutic strategies to treat cancer, including chemotherapy, high systemic cytotoxic toxicity and drug resistance have limited the successful outcomes in most cases. Recently, TNF-related apoptosis-inducing ligand (TRAIL) and non-steroidal anti-inflammatory drugs (NSAIDs) have been found to sensitize Dox-induced apoptosis in various types of tumors [26], [27], [28]. In addition, the combination treatment of Dox and natural products dramatically augments the therapeutic effects against breast cancer, prostate cancer, and hepatocellular carcinoma [29], [30].
In this study, we investigated the ability of HE to synergize the inhibitory action of Dox in human hepatocellular carcinoma HepG2 cell lines. We also evaluated the molecular mechanisms underlying this synergistic effect and sought evidence of synergy between Dox and HE.
Section snippets
Materials
The human hepatocellular carcinoma cell line, HepG2 (ATCC HB-8065), was obtained from Korean Cell Line Bank (Seoul, Korea). Fetal bovine serum, penicillin G, streptomycin, and RPMI 1640 were obtained from GIBCO (Grand Island, NY, USA). Dox, acridine orange, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cell-permeable inhibitor peptide (NF-κB SN50), Bay 11-7082, and SP600125 were obtained from Calbiochem (La
HE and doxorubicin, at a low dose, synergise to induce apoptosis in human hepatocellular carcinoma cells
To investigate the anti-tumor effect of HE on hepatocellular carcinoma, we tested the effect of HE or Dox treatment on human hepatocellular carcinoma cells. HepG2 cells were either left untreated, or treated with different doses of HE or Dox for 72 h. After treatment with HE alone, a moderately dose-dependent growth-inhibitory effect on HepG2 cells was observed. A similar phenomenon was observed following Dox treatment (Fig. 1A). To evaluate whether or not the growth-inhibitory effect of HE or
Discussion
In this study, we showed that low doses of HE and Dox synergize to induce apoptosis in human hepatocellular carcinoma HepG2 cells. Caspase 3 was activated in the apoptotic process induced by the combination treatment of HE and Dox. The suppression of caspase activity by a general caspase inhibitor z-VAD-fmk confirmed that the promotion of apoptosis by combination treatment involved a caspase-dependent pathway. Moreover, reducing the expression level of c-FLIP through upregulation of JNK
Conflicts of interest
None declared.
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