Investigation of the autophagy and apoptosis relation through BAG-1, BcL-2 and Beclin 1

BAG (Bcl-2 associated athanogene) family belongs to a group of multifunctional proteins conserved in yeasts, plants and animals and has significant roles in crucial metabolic pathways such as apoptosis, tumourigenesis, signaling, cell proliferation, and autophagy. BAG-1 is a member of BAG family proteins and known as an anti-apoptotic protein. BAG-1 has three major isoforms including BAG-1L, BAG-1M ve BAG-1S that are translated owing to the use of alternative translation start sites. BAG-1 localizes in different parts of cells and interacts with several molecular targets. Thereby, BAG-1 modulates important regulatory pathways including transcription, apoptosis, cell proliferation, hormone action, cell migration with its interaction partners. Apoptosis, which is an important mechanism for maintaining homeostasis, induces morphological changes and programmed cell death due to external stimuli. Apoptosis takes place through two major mechanisms, which are extrinsic and intrinsic pathways. Extrinsic pathway is activated by binding of Fas ligand to Fas receptor on the cell membrane, followed by activation of caspase-8 and release of pro-apoptotic factors from the mitochondria. Intrinsic pathway is activated by release of cytochrome-c from mitochondria due to membrane permeability, followed by apoptosome formation. Cytochrome-c release occurs due to the imbalance between pro-apoptotic and anti-apoptotic proteins on the mitochondria. There are many factors influencing this imbalance. These proteins include pro-apoptotic Bcl-2 members Bax, BAK, BAD, and anti-apoptotic Bcl-2 and Bcl-xL. BAG-1 is accepted as a factor in apoptotic pathways due to its interaction with Bcl-2. One of the major roles of BAG-1 is to prevent the release of pro-apoptotic factors such as cytochrome-c from the mitochondria. Furthermore, by forming a complex consisting of Akt, c-Raf, Hsp70 and several proteins leads to Bad phosphorylation and induces cell survival. In addition, autophagy is one of the mechanisms for adaptating to stress conditions and maintaining homeostasis. Autophagy means "self-eating", and has roles in cell differentiation, aging, regulation of metabolism, and cell death. Moreover, it has been shown to involved in development of cancer and neurodegenerative diseases. Atg family proteins and Beclin 1 are important proteins involved in autophagy mechanism. Beclin 1, which is discovered as Bcl-2 partner, has roles in many mechanisms, especially in tumourigenesis. Beclin 1 has BH3 domain, which is specific to Bcl-2 protein family, and interacts also with Bcl-xL. Cells make a decision between apoptosis and autophagy due to disrupted interaction between Beclin 1 and Bcl-2 or Bcl-xL. The aim of this study is to investigate the interaction of BAG-1, with Beclin 1 and Bcl-2, and its role on balance between apoptosis and autophagy. To this end, in BAG-1 overexpressed and silenced breast cancer cells, BAG-1 isoforms were investigated. Then, Beclin 1 expression and phosphorylation levels were checked upon altered BAG-1 expression. Beclin 1 phosphorylation levels were decreased in Bag-1 silenced MCF-7 and MDA-MB-231 breast cancer cells. Besides, to understand whether BAG-1 and Bcl-2 interact, immunoprecipitation assays were carried out by using IgG beads. BAG-1 isoforms and Beclin 1 were observed together. Besides, probable interaction of BAG-1 and Beclin 1 was predicted via PRISM server. The BH3 domain of Beclin 1 and BAG domain of BAG-1 interacted. To figure out the possible role of BAG-1 in autophagic regulation, Atg proteins expression levels were examined by immunoblotting. Also, breast cancer cells treated with autophagy inhibitor 3-MA and c-Raf inihibitor GW-5074 then, Akt and Bad expression and phosphorylation levels were analyzed. C-Raf inhibitor did not result in a change in the phosphorylation level of Bad. However, phosphorylation level of Bad was decreased under 3-MA treatment although BAG-1 overexpression. As a result, Akt might be the kinase in the complex, where BAG-1 is involved and phosphorylate Bad to induce cell survival. As a conclusion, BAG-1 might be interacting with Beclin 1. By this study, it is highlighted that BAG-1 might have a role in autophagic regulation mechanism which contents Akt pathway in cell survival. BAG-1 is a remarkable protein - as a potential biomarker especially in molecular mechanisms underlying breast cancer and further - studies are required to understand the role of BAG-1 in breast cancer.