Role of PI3K/Akt/mTOR and MEK/ERK pathway in Concanavalin A induced autophagy in HeLa cells

Highlights

• Con A induces robust autophagy in HeLa dose and time dependently.

• Con A induces fraction of HeLa cell death through autophagy induced apoptosis.

• Con A suppresses PI3K/Akt/mTOR pathway and activates Raf/MEK/ERK pathway.

Abstract

Concanavalin A (Con A), a mannose or glucose specific legume lectin, is well known for its anti-proliferative and cytotoxic effect on different types of cancer cells, through its binding to the membrane receptors leading to a major stimulus for the induction of distinct metabolic responses. Recently it has been also been proved that, Con A induces autophagy in hepatoma cells through internalization and mitochondria mediated pathway involving a mitochondrial interacting protein named Bcl2/E1B-19 kDa protein-interacting protein 3 (BNIP3). Through this current endeavor, we propose a membrane associated pathway involved in Con A induced autophagy, taking Human cervical cancer (HeLa) cell as a cancer model. Here, we deciphered the role of membrane mediated phosphatidylinositol 3 kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin) and MEK/Extracellular signal-regulated kinases (ERK) pathway in Con A induced autophagy in HeLa cells. Subsequently, we found that Con A treatment suppresses the PI3K/Akt/mTOR and up regulates the MEK/ERK pathway leading to the activation of autophagy. This study will further help us to understand the mechanism behind the autophagic pathway induced by Con A and simultaneously it will strengthen its effective use as a prospective cancer chemo-therapeutic.

Introduction

Autophagy is a conserved eukaryotic cellular degradative process that helps in maintaining the cellular metabolism and homeostasis [1]. Apart from maintaining the cellular homeostasis, it plays a vital role in different cellular functions. Autophagy ameliorates cellular damage during the stress conditions by removing the damaged organelles and protein aggregates and recycling them to produce essential nutrients and energy for the cells [2]. Given the importance of autophagy in cellular context, failure in any part of autophagic machinery leads to various diseases such as cancer [3], [4], [5], neurodegenerative diseases [6], [7], heart diseases [8], [9] and autoimmunity diseases [10], [11]. Thereby, autophagy modulating compounds provide an attractive drug target for the treatment of those diseases.

Plant lectins hold a special place as autophagy inducers for their specific capability to bind various cellular sugar moieties and activate number of cellular pathways [12]. Concanavalin A, a well known member of legume lectin family with a mannose/glucose binding specificity has shown distinct anti-proliferative and anti-tumor activities in human melanoma A375 cells and human hepatocellular liver carcinoma HepG2 cells [13]. Recently, it has been established that Con A induces autophagic cell death in a murine in situ hepatoma model through internalization and mitochondria-mediated pathway [14]. This pathway involves a mitochondrial interacting protein named BNIP3, which is known for its pro-death functions [15]. On the contrary, previous studies have reported that the binding of Con A to cell surface receptors provide the major stimulus for the induction of distinct metabolic responses [16], [17]. It has been clearly established that the binding is the only reason for the cytotoxicity in murine hepatocytes and even blocking its internalization produce the same extent of cytotoxicity [18]. Combining these two propositions, it can be inferred that there exists two separate pathways of Con A induced cytotoxicity. One pathway advocates the involvement of mitochondria and mitochondria-interacting proteins and another, suggests the involvement of membrane receptors without elaborating the detailed underlying mechanisms. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including autophagy. It has been shown that growth factor increases the interaction of ERK cascade components with autophagy (ATG) proteins in both cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes and ERK phosphorylation is upregulated by lipidation of autophagic protein LC3 [19]. On the contrary, Akt signaling is known to suppress autophagy [20]. Akt negatively regulates autophagy in response to mitogens via activation of target of rapamycin (mTOR), which inhibits multiple autophagy-promoting proteins via phosphorylation [21]. Inhibition of the Akt signaling pathway and enhanced activity of ERK1/2 have previously been implicated in controlling induction of macroautophagy in mammalian cancer cells [22]. Based on this previous finding, we wanted to study whether the intertwining PI3K/Akt and MEK/ERK pathways have any role to play in Con A induced autophagy. Through the current study, we propose a membrane associated pathway involved in Con A induced autophagy, taking human cervical cancer (HeLa) cell as a model. We observed that in presence of autophagy inhibitor 3MA, Con A induced apoptosis was inhibited which indicated that Con A induced autophagy contributes to apoptosis in human cancer cells. Moreover, it was demonstrated that Con A induced autophagy through upregulation of MEK/ERK and downregulation of PI3K/Akt signaling pathway concurrently. Our study helps to reveal a different mechanism behind the autophagic pathway induced by Con A and strengthens the prospective of projecting it as a future anti-neoplasitc drug.