AstragalosideII inhibits autophagic flux and enhance chemosensitivity of cisplatin in human cancer cells

Abstract

Inhibition of autophagy has been daily served as a promising anti-cancer treatment strategies. AstragalosideII (ASII), a main compound isolated from traditional Chinese medicine Radix Astragali, has been demonstrated to inhibit autophagy and reverse multidrug resistance in human hepatic cancer cells Bel-7402/5-FU. In this study, we inspected the function and mechanisms of ASII and cisplatin on autophagy in human cancer cells, and assessed the effect of ASII on cisplatin-induced apoptosis. We found ASII increased LC3II protein level, p62 protein level and GFP-LC3 puncta accumulation in human cancer cells. Furthermore, we found that ASII downregulated the expression of lysosomal cathepsinB/L (CTSB/L) in EBSS medium and affected the lysosomal acidification. Finally, we demonstrated that cisplatin induced protective autophagy which was involved of PI3K/Akt/mTOR pathway. Moreover, ASII in conjunction with cisplatin significant reduced cell viability, arrested in S phase and increased apoptosis. In conclusion, these findings suggested that ASII served as autophagy inhibitor which restored chemosensitivity of anticancer agent cisplatin and enhanced tumor cell death.

Introduction

Macroautophagy (hereafter referred to as autophagy) is a conserved catabolic process characterized by sequestration of cytoplasmic components including damaged long-lived proteins and organelles in double membrane vesicles called autophagosomes, which are transported to and degraded in lysosome [1]. Autophagy is also a dynamic process which consists of four successive stages including isolation membrane, vesicle elongation, autophagosome and autolysosome [2]. Recently, more and more evidence has illustrated that autophagy is not only associated with growth, differentiation and apoptosis of cell, but also related with the development of variously significant human diseases, such as metabolic diseases, neurodegenerative diseases, infectious diseases and cancers [3]. However, the function of autophagy in cancer is controversial and intricate. Autophagy is considered as a tumor suppressor [4], on the other hand, autophagy will offer a survival superiority to counteract cell death under the condition of chemotherapy, radiotherapy, starvation or hypoxia stress [5], [6], [7], [8]. Autophagy is aslo a complex regulatory process, which is related to a variety of upstream regulating signaling pathways [9], [10]. It is worth noting that the classic PI3K/AKT/mTOR signaling pathway is a archetypal survival pathway that is constitutively activated in various cancer types [11], while PI3K/Akt/mTOR pathway inhibitors are currently being considered in clinical trials for treatment of a great number of cancers [12]. Moreover, increasing evidence shows that the activation of autophagy is associated with PI3K/AKT/mTOR pathway [13]. Currently, chemotherapy is one of the major means that oncologists used to treat and cure cancer patients, especially when a metastatic disease is diagnosed. Among many anti-cancer chemotherapeutic agents, cisplatin is one of the most significant anticancer drugs which is used to treat many solid tumors including gastric cancer, breast cancer, non-small cell lung cancer, etc. [14], [15], [16]. The antitumor mechanism of cisplatin is to induce cell apoptosis by damaging DNA [17], while chemotherapy resistance reduces clinical efficacy. Recent studies have shown that cisplatin can induce autophagy in cancer cells, and the function of which is a protective mechanism [18], [19]. However, molecular mechanisms of cisplatin-induced autophagy were unknown in human gastric cancer SGC-7901 cells.

At present, compounds from natural plants are very important resources for drugs. Astragaloside is one of the main active ingredients in Radix Astragali, a traditional Chinese medicine which has wide pharmacological effects including immunomodulatory, antiviral, antioxidants, anticancer and so on [20], [21], [22], [23]. ASII(3b,6a,16b,20R,24S)-3-((2-O-acetyl-beta-d-xylopy-xylopyranosyl)oxy)-20,24-epoxy-16,25-dihydroxy-9,19-cyclolanostan-6-ylbetad-glucopyranoside) (Fig. 1A), a member of the triterpenoid glycosides is treated as a marker monomeric compound in Radix Astragali. We have demonstrated that ASII inhibited autophagy in human hepatocarcinoma Bel-7402/5-FU and reversed P-glycoprotein-mediated multidrug resistance of human hepatoma cells Bel-7402/5-FU by regulating MAPK signaling pathway [24], [25].

In this study, we demonstrated that cisplatin can induce protective autophagy which is involved PI3K/Akt/mTOR-mediated signaling pathways and ASII is capable of suppressing the late stage of autophagy by affecting lysosomal functions, through which it increases human cancer cells to the sensitivity of cisplatin-induced cell death. At present, autophagy inhibitors in combination with chemotherapy drugs has been increasingly known as a promising strategy for the treatment of cancer. Our results will provide a new vision for anticancer drug development via targeting autophagy.