Polyphenolics from mango (Mangifera indica L.) suppress breast cancer ductal carcinoma in situ proliferation through activation of AMPK pathway and suppression of mTOR in athymic nude mice

Abstract

The objective of this study was to assess the underlying mechanisms of mango polyphenol decreased cell proliferation and tumor volume in ductal carcinoma in situ breast cancer. We hypothesized that mango polyphenols suppress signaling along the AKT/mTOR axis while up-regulating AMPK. To test this hypothesis, mango polyphenols (0.8 mg gallic acid equivalents per day) and pyrogallol (0.2 mg/day) were administered for 4 weeks to mice xenografted with MCF10DCIS.com cells subcutaneously (n=10 per group). Tumor volumes were significantly decreased, both mango and pyrogallol groups displayed greater than 50% decreased volume compared to control. There was a significant reduction of phosphorylated protein levels of IR, IRS1, IGF-1R, and mTOR by mango; while pyrogallol significantly reduced the phosphorylation levels of IR, IRS1, IGF-1R, p70S6K, and ERK. The protein levels of Sestrin2, which is involved in AMPK-signaling, were significantly elevated in both groups. Also, mango significantly elevated AMPK phosphorylation and pyrogallol significantly elevated LKB1 protein levels. In an in vitro model, mango and pyrogallol increased reactive oxygen species (ROS) generation and arrested cells in S phase. In silico modeling indicates that pyrogallol has the potential to bind directly to the allosteric binding site of AMPK, inducing activation. When AMPK expression was down-regulated using siRNA in vitro, pyrogallol reversed the reduced expression of AMPK. This indicates that pyrogallol not only activates AMPK, but also increases constitutive protein expression. These results suggest that mango polyphenols and their major microbial metabolite, pyrogallol, inhibit proliferation of breast cancer cells through ROS-dependent up-regulation of AMPK and down-regulation of the AKT/mTOR pathway.

Introduction

It is estimated that breast cancer constitutes approximately 30% of newly diagnosed cancer—with 232,670 cases diagnosed in 2014 [1], contributing to approximately 40,000 deaths [2]. Ductal carcinoma in situ (DCIS) is a non-invasive, or non-infiltrating carcinoma considered as a precursor to invasive breast cancer [3], [4]. It was reported that 20–50% of DCIS progressed to invasive disease [3], and the mortality rate from DCIS was 3.3% after 20 years from diagnosis [5]. Given the high incidence and mortality associated with breast cancer and DCIS, an ongoing effort in drug development along with the refinement of current treatment options is necessary to provide the most effective therapeutic strategies. Currently, available treatment options commonly cause adverse side effects contributing to both a decreased quality of life and decreased adherence to treatment amongst patients [6]. Due to demonstrated anti-tumor efficacy and a lack of deleterious side effects, dietary polyphenols are strong, potential candidates for alternative treatments to conventional, small, synthetic molecules[7], [8].

Mango (Mangifera indica L.) polyphenols exhibit anti-inflammatory and cancer-cytotoxic properties in multiple cancer types, including malignancies of the colon and breast [9], [10], [11], [12]. Mango polyphenolics are rich in gallic acid, gallotannins, galloyl glycosides, and flavonoids [13]. However, pyrogallol is the major microbial metabolite of gallotannins detected in human urine following mango intake in previous human clinical studies [14]. Gallotannins are initially hydrolyzed to release free gallic acid in intestinal pyrogallol generation [15], which can be decarboxylated to pyrogallol by intestinal microflora [16]. Pyrogallol has been shown to exhibit health promoting properties in different disease models [17], [18], and is therefore considered a major bioactive metabolite in gallotannins.

The AKT/mTOR and 5′ AMP-activated protein kinase (AMPK) signaling are dysregulated in breast cancer and has been a target for breast cancer therapy [19], [20]. Previously, our lab reported that the anti-inflammatory and anti-cancer effects of mango polyphenols was due to modulation of the AKT/mTOR pathway [9], [21]. This study further investigates the link between the modulation of the AKT/mTOR pathway and AMPK signaling by mango polyphenols. AMPK signaling is responsible for maintaining energy homeostasis within the cells, and its activation culminates in the inhibition of the mTOR pathway, leading to autophagy [22]. It is well established that the AMPK signaling can be activated by reactive oxygen species (ROS), and the ROS-activated AMPK pathway may be modulated by pro-oxidants [23], [24], [25]. Pyrogallol was previously shown to induce cell cycle arrest and apoptosis due to its pro-oxidant properties [26]. Therefore, pyrogallol would be expected to induce ROS-dependent AMPK activation and subsequent suppression of the AKT/mTOR pathway.

This experiment seeks to determine if mango polyphenols and their major intestinal metabolite, pyrogallol, inhibit cell proliferation of breast cancer cells in a xenograft tumor model. Our hypothesis was that mango polyphenols and pyrogallol may have anti-cancer activity on ductal carcinoma in situ through inhibition of AKT/mTOR axis and activation of AMPK. This was the first time that the inhibition of ductal carcinoma in situ breast cancer by mango polyphenols and pyrogallol had been investigated in vivo.