Elsevier

Food and Chemical Toxicology

Volume 126, April 2019, Pages 223-232
Food and Chemical Toxicology

Auriculasin sensitizes primary prostate cancer cells to TRAIL-mediated apoptosis through up-regulation of the DR5-dependent pathway

https://doi.org/10.1016/j.fct.2019.02.030Get rights and content

Highlights

  • Auriculasin effectively sensitized TRAIL-induced apoptotic cell death in RC-58T/h/SA#4 primary prostate cancer cells.

  • However, treatment of auriculasin, and TRAIL did not affect proliferation in RWPE-1 prostate epithelial cells.

  • Combined treatment of auriculasin, and TRAIL induced caspase-dependent and -independent apoptosis in RC-58T/h/SA#4 cells.

  • Auriculasin sensitized TRAIL-induced apoptosis through up-regulation of DR5 signaling pathway.

  • Combined treatment of auriculasin, and TRAIL suppressed phosphorylation of PI3K/Akt/mTOR pathway.

Abstract

Primary prostate cancer cells frequently develop resistance toward chemotherapy as well as most chemotherapeutics have been reported to induce undesirable cytotoxicity in normal cells. In this study, we performed sensitizing activity analysis of auriculasin (AC) to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in RC-58T/h/SA#4 primary prostate cancer cells without significant cytotoxicity in RWPE-1 prostate epithelial cells. Combined treatment with AC and TRAIL at optimal concentrations resulted in tumor-specific apoptotic cell death in RC-58T/h/SA#4 cells, characterized by DNA fragmentation, accumulation of apoptotic cell population, and nuclear condensation. Compared to single treatment with AC or TRAIL, co-treatment with AC and TRAIL significantly increased expression of Bax, cleaved PARP, AIF, endo G, and cytochrome c but decreased expression of phosphorylation of AKT and mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K), Bcl-2 and caspases-9, -8, -3, and -10. The sensitizing effect of AC to TRAIL was well correlated with inhibition of death receptor 5 (DR5) CHOP, and p53 expression. Moreover, pre-treatment with a chimeric blocking antibody for DR5 effectively reduced AC-TRAIL-induced cell death and apoptosis-related protein expression. These results suggest that non-toxic concentrations of AC sensitize TRAIL-resistant primary prostate cancer cells to TRAIL-mediated apoptosis via up-regulation of DR5 and downstream signaling pathways.

Introduction

Numerous cancer species are characterized by extremely aggressive growth in early stages of development to nearby and distant organs (Massagué and Obenauf, 2016). Thus, failure to treat primary cancer at an early stage can increase morbidity and mortality of prostate cancer patients (Berger et al., 2011). In recent years, effective cancer strategies applying chemotherapeutics to cancer stem cells or primary cancer cells have attracted attention (Zhang et al., 2018; Zhang et al., 2018). However, drug resistance and normal cell toxicity are known as major hurdles in early stages of primary cancer therapy. Since primary and metastases cancer cells show molecular differences (Ramaswamy et al., 2003), it is also urgent to articulate novel therapeutic strategies for primary prostate cancer with minimal cytotoxicity in normal cells.

Prostate cancer is the most common cancer species in the world as well as a significant cause of cancer-induced death in men. Although various chemotherapy drugs have shown transient efficacy for prostate cancer, significant side effects are induced by continuous utilization of cancer therapeutics (Karavelioglu et al., 2016; Frederiks et al., 2015). To avoid the side effects of conventional chemotherapies for cancer treatment, development of ideal therapeutics that induce cancer-selective apoptosis has been carried out by numerous researchers. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand superfamily of cytokines broadly expressed by neutrophils, natural killer cells, and lymphocytes (Guimarães et al., 2018). TRAIL can bind to death domain-containing receptors, such as death receptor 4 (DR4) and death receptor 5 (DR5), and non-death domain-containing receptors, such as designated decoy receptor 1 (DcR1), DcR2, and DcR3 (Johnstone et al., 2008). Upon binding of TRAIL to extracellular DR4 and DR5, a cascade of downstream proteins is activated after development of death-inducing signal complex (DISC) and FAS-associated protein death domain (FADD) (Bertsch et al., 2014). Consequently, TRAIL triggers caspase-dependent and/or -independent apoptosis directly activating caspase-8 as well as truncates BH3-interacting domain death agonist (BID) (Woo et al., 2017).

However, certain types of cancer cells have demonstrated TRAIL resistance caused by mutational inactivation of pro-apoptotic genes (Bax, and Bak), overexpression of anti-apoptotic genes (Bcl-2, and Bcl-XL), and dysfunction of death receptors (DR4, and DR5) (Burris HA 3rd, 2013). This diverse range of resistance mechanisms presents new challenges for long-term tumor control. Furthermore, Kim et al. (2011) reported that primary prostate cancer cells are less sensitive to TRAIL-induced apoptosis than metastatic prostate cancer cells, and Lemke et al. (2014) reported that TRAIL can trigger non-apoptotic signaling pathways, which can induce malignancy in some TRAIL-resistant primary cancer cells. To overcome TRAIL resistance in various cancer cells, numerous strategies that exert promising cancer suppressing activity have been developed and are at the pre-clinical stage. Especially, a number of researchers have shown that cancer cells with TRAIL resistance can be sensitized by bioactive compounds from natural products, including curcumin (Jung et al., 2006), methylseleninic acid (Yamagucci et al., 2005), and ursolic acid (Shin and Park, 2013). Therefore, anti-cancer reports using combined treatments with non-toxic concentrations of natural compounds and TRAIL in TRAIL-resistant primary prostate cancer cells have drawn increasing interest.

Auriculasin (AC) is a prenylated isoflavone in various food ingredients, such as roots of Flemingia philippinensis (Wang et al., 2013), stem bark of Erythrina senegalensis (Oh et al., 1998), and osage orange fruits (Peter and Krammer, 1998). It is known to activate the caspase-independent signaling pathway and inhibit proliferation of prostate cancer cells, confirming its strong anti-tumor activity (Wang et al., 2013; Cho et al., 2018a, Cho et al., 2018b). In our previous study, AC treatment (5 μM) significantly induced caspase-independent apoptosis in LNCaP metastatic prostate cancer cells without significant RWPE-1 prostate epithelial cell toxicity (Cho et al., 2018a, Cho et al., 2018b). Although AC is an interesting candidate to suppress metastatic prostate cancer due to its lack of normal cell cytotoxicity, the molecular mechanisms underlying the anti-cancer activity of AC in TRAIL-resistant primary prostate cancer cells have not been fully elucidated.

The present study investigated the anti-cancer activity of single or combined treatment with AC and TRAIL against TRAIL-resistant primary prostate cancer cells as well as identified potential mechanisms. Interestingly, non-cytotoxic concentrations of AC efficiently induced DR5-mediated apoptosis by TRAIL in RC-58T/h/SA#4 primary prostate cancer cells. Collectively, our results elucidate the mechanisms behind the synergistic anti-cancer activities of AC and TRAIL at non-toxic concentrations in primary prostate cancer cells and could help facilitate the development of promising cancer strategies without significant side effects.

Section snippets

Isolation of AC

AC was isolated as described previously (Wang et al., 2013). Briefly, the air-dried root bark of F. philippinensis (0.3 kg) was chopped, and extracted with MeOH (3 L × 3) at room temperature for 7 days. The combined filtrate was concentrated in vacuum to yield a dark red gum (51 g, 17.1%). The MeOH extract (25 g) was subjected to column chromatography (CC) on silica gel using a hexane to ethyl acetate gradient (50:1-1:1) to give 8 fractions (A-H, 500 mL/each). Fraction D (3.2 g) was

AC sensitizes TRAIL-resistant RC-58T/h/SA#4 cells to TRAIL-induced cell death

In order to determine the inhibitory effect of combined treatment with AC and TRAIL on proliferation of prostate cancer cells, both RC-58T/h/SA#4 and RWPE-1 cells were treated with various doses of AC with or without TRAIL (Fig. 1). AC and TRAIL-induced suppression of cell viability was confirmed by SRB assay. RC-58T/h/SA#4 cells were moderately resistant to 2.5–10 μM AC and 50–200 ng/mL of TRAIL (Fig. 1A). However, co-treatment with AC (5–10 μM) and TRAIL (50–200 ng/mL) significantly reduced

Discussion

In the present study, AC isolated from F. philippinensis was screened by testing its synergistic apoptosis activity in combination with TRAIL in primary prostate cancer cells. AC was found to improve TRAIL-mediated apoptosis in TRAIL-resistant RC-58T/h/SA#4 primary prostate cancer cells. In addition, involvement of the DR5 apoptosis pathway in co-treatment with AC and TRAIL was investigated for the first time. Hence, the results of the current study, in which AC sensitized primary prostate

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgement

This study was financially supported by the Dong-A University Research Fund.

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