Autophagy and the functional roles of Atg5 and beclin-1 in the anti-tumor effects of 3β androstene 17α diol neuro-steroid on malignant glioma cells

Abstract

In this study, we demonstrate that the anti-tumor activity of the neuro-steroid, 3β androstene 17α diol (17α-AED) on malignant glioma cells is mediated by the induction of autophagy. 17α-AED can inhibit the proliferation an induce cell death of multiple, unrelated gliomas with an IC₅₀ between 8 and 25 μM. 17α-AED treatment induced the formation of autophagosomes and acidic vesicular organelles in human malignant gliomas which was blocked by bafilomycin A1 or 3-methyladenine. Cleavage of microtubule-associated protein-light chain 3 (LC3), an essential step in autophagosome formation, was detected in human malignant glioma cells exposed to 17α-AED. In 17α-AED treated T98G glioma cells there was an increase in the autophagy related proteins Atg5 and beclin-1. Silencing of ATG5 or beclin-1 with small interfering RNA significantly reduced the incidence of autophagy in 17α-AED treated malignant gliomas and attenuated the cytotoxic effects of the neuro-steroid indicating that the induction of autophagy mediates the anti-glioma activity of 17α-AED rather than serving as a cyto-protective response. These results demonstrate that 17α-AED possesses significant anti-glioma activity when used at pharmacologically relevant concentrations in vitro and the cytotoxic effects are resultant from the induction of autophagy.

Introduction

Glioblastomas are grade IV astrocytic neoplasms derived from the glial lineage; are the most frequent of primary central nervous system tumors, accounting for 69% of the glial tumors; and are the most malignant [1]. Traditional treatment for this disease is surgical resection accompanied by radiation and is generally considered palliative with the 5-year survival rate of 2% [2]. A recent advance in the treatment of this brain cancer is the use of the alkylating agent, temozolomide, in combination with radiation therapy which has shown to extend the median survival of malignant glioma patients an additional 2.5 months [3], [4].

Androstene steroids are produced in neuro-ectodermal tissue by the metabolism of dehydroepiandrosterone [5]. The androstene steroids exist in α- and β-epimeric forms and, although chemically identically, the biological actions of the two epimers are distinctly different. 3β androstene 17β diol (17β-AED) is an enhancer of immune regulation [6], [7], [8], [9], [10]. In contrast, 3β androstene 17α diol (17α-AED) possesses potent anti-tumor activity but no known immune function. 17α-AED can inhibit the proliferation and induce cell death of human MDA-MB231 and ZR75-1 breast adenocarcinomas; U937 and HL-60 lymphomas; and T98G and U251MG malignant gliomas [11], [12], [13]. However, the mechanism of the anti-tumor effects of 17α-AED is different in malignant gliomas as compared to that of the other tumors. In this regard, 17α-AED induces apoptotic type I programmed cell death in the breast tumors and the lymphomas but not in malignant gliomas [11], [12], [13]. Human malignant glioma cells exposed to 17α-AED do not display hallmark features of apoptosis such as DNA fragmentation and caspase activation, suggesting that an alternate cell death pathway is utilized in these cells [13].

Autophagy is generally associated with a cell survival response to starvation or a toxic environment and is a ubiquitous process in which cells degrade cytosolic materials such as proteins and organelles [14], [15], [16], [17]. In this process, cytoplasmic constituents are sequestered within the developing membrane of a nascent autophagosomal vesicle which then fuses with a lysosome to form an autolysosome. Here, the contents of the vesicle are degraded and recycled. Autophagosome formation involves two functional groups of Atg proteins. The first group is a lipase signaling complex. In mammalian cells, the class III phosphatidylinositol-3 kinase (PI3K) and p150 form a complex with beclin-1 (orthologue of yeast Atg6) to initiate the nucleation of the pre-autophagosome [18], [19]. The second group consists of an ubiquitin-like conjugation system which is required for pre-autophagosome expansion and completion. It is composed of the Atg12-Atg5 conjugation system and microtubule-associated protein 1 light chain 3 (LC3) which is the mammalian orthologue of yeast Atg8. LC3 is shuttled to the autophagosomal membrane by the Atg12-Atg5 complex. LC3-I (18 kDa) is converted to LC3-II (16 kDa) which is integrated into the autophagosome membrane [16], [18]. Both conjugation systems are interdependent and autophagy does not proceed if there is a defect in one of the systems.

Although autophagy is generally viewed as a cell survival mechanism, in some cases it can be a form of type II programmed cell death [17], [20], [21]. The role of autophagy in cancers such as malignant glioma is controversial. In this regard, the induction of autophagy may be a cyto-protective mechanism in response to cytotoxic agents or the nutrient-poor tumor micro-environment. On the other hand, autophagy may act as a tumor suppressor mechanism and induce cell death which has been such as observed when apoptosis is blocked [14], [20], [21], [22].

Here, we show that 17α-AED can inhibit the proliferation and induce cell death of multiple, unrelated glioma cell lines. Based upon our recent findings, we hypothesized that, in human malignant gliomas, the in vitro anti-tumor effects of 17α-AED may be mediated by autophagic type II programmed cell death. To test this, we analyzed 17α-AED treated malignant glioma cells for the presence of autophagosomes; elevated levels of autophagy associated proteins and the conversion of LC3-I to LC3-II. Our findings revealed that 17α-AED induced autophagic cell death in malignant glioma cells. Furthermore, the disruption of the autophagic process in malignant glioma cells with small interfering RNA (siRNA) targeting ATG5 or beclin-1 attenuated the biological effects of 17α-AED.