Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model

Abstract

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-β/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)⁺ liver cell foci and the numbers of Ki-67⁺ proliferating cells within GST-P⁺ foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P⁺ foci (area) to 48.9–49.4% and Ki-67⁺ cells to 55.5–61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN^– foci, phospho-Akt substrate⁺ foci, phospho-Smad3^– foci and Smad4^– foci in GST-P⁺ foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN^– foci in GST-P⁺ foci to 59.8–72.2% and Smad4^– foci to 62.4–71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate⁺ foci in GST-P⁺ foci to 75.2–75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-β/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.

Introduction

Piperonyl butoxide (PBO) is a pesticide synergist widely used with pyrethroids for grain protection and a non-genotoxic hepatocarcinogen in F344 rats (Takahashi et al., 1994). PBO has been known to induce cytochrome P450 (CYP) 1A1 and also CYP2B (Tasaki et al., 2010), both of which generate reactive oxygen species (ROS) as a byproduct of microsomal oxidation (Puntarulo and Cederbaum, 1998, Nishikawa et al., 2002, Waxman and Azaroff, 1992, Kinoshita et al., 2003), and it is suspected that ROS production caused by PBO administration is considered to be responsible for hepatocarcinogenic or liver tumor-promoting potential in rats (Muguruma et al., 2007). Moreover, we have previously reported that non-genotoxic hepatocarcinogens including PBO can activate phosphatase and tensin homolog (PTEN)/Akt signaling and disrupt Smad-dependent signaling in preneoplastic liver cell foci expressing glutathione S-transferase placental form (GST-P) produced by tumor promotion in an initiation-promotion model in rats (Taniai et al., 2009, Ichimura et al., 2010).

Phytochemicals, such as flavonoids and carotenoids, are natural chemical substances existing in plants that are known to exert various beneficial health effects through alteration of intracellular signaling regulation on cell proliferation, inflammation and apoptosis (Surh, 2003, Aggarwal and Shishodia, 2006). Among the phytochemicals, anthocyanin, which is a group of secondary metabolite from plants and the most important subclass of flavonoids (He and Giusti, 2010), is inherent to many foods, such as berries, grape, red cabbage, purple sweet potato and bilberry. In particular, bilberry (Vaccinium myrtillus L.) is known as one of the richest natural sources of anthocyanin that potentiate beneficial effects against various diseases, such as cancer and diabetes (Lala et al., 2006, Lankinen et al., 2011). Examples of beneficial effects of anthocyanin-rich fruit extracts include anti-hepatocarcinogenic effects in rats promoted with phenobarbital (Bishayee et al., 2011) and anti-skin tumor-promoting effects in CD-1 mouse by inhibition of NF-κB (Afaq et al., 2005) in two-stage carcinogenesis models.

Enzymatically modified isoquercitrin (EMIQ) is a quercetin-glycoside mixture consisting of isoquercitrin and its α-glucosylated derivatives, with 1–10 additional linear glucose moieties (Akiyama et al., 2000). EMIQ is produced from rutin by enzymatic glycosylation (Formica and Regelson, 1995). It is effective as an antioxidant in vivo, and we have previously shown that it has chemopreventive potential against the development of GST-P⁺ liver cell foci induced in rats by promotion with oxfendazole, phenobarbital, or β-naphthoflavone, all of which cause oxidative cellular responses as a mechanism of tumor promotion (Morita et al., 2011, Nishimura et al., 2010, Shimada et al., 2010). We have also reported that EMIQ exerts tumor suppressive effect by facilitation of apoptotic liver cells and suppression of proliferating liver cells within GST-P⁺ foci produced by promotion with thioacetamide, a representative hepatocarcinogen (Fujii et al., 2013, Kimura et al., 2013).

The present study was performed to assess whether bilberry extracts (BBE) and EMIQ exert tumor suppressive effects on the hepatocarcinogenic process of PBO through suppressing oxidative stress responses using a two-stage liver carcinogenesis model in N-diethylnitrosamine (DEN)-initiated and PBO-promoted rats. For this purpose, we focused on the antioxidant effects of BBE and EMIQ, and in addition, we investigated the change of expression of cell signaling, such as PTEN/Akt and Smad signaling, during the early stages of hepatocellular tumor promotion with or without co-treatment with BBE or EMIQ.