Role of PI3K/Akt/mTOR and MEK/ERK pathway in Concanavalin A induced autophagy in HeLa cells
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.
Section snippets
Reagents
Concanavalin A was purchased from Himedia (Mumbai, India). Acridine orange, LY294002 and PD98059 and 3-Methyladenine were purchased from Sigma (St. Louis, MO, USA). Anti-β-actin, anti phospho-Akt at Ser473, anti-total Akt (11E7), anti-phospho-p70S6K at Ser235/236, anti-phospho-MEK1/2 at Ser217/221 and anti-total MEK1/2 antibodies were purchased from Cell Signaling Technology. Secondary goat anti-rabbit IgG-HRP was purchased from Santa Cruz Biotechnology.
Cell culture
The human cervical cancer (HeLa) cell
Con A induces cell death through apoptosis in HeLa cells
The inhibitory effect of Concanavalin A on HeLa cells was studied by MTT assay and a remarkable inhibition was found only at 36 h in a dose-dependent manner. The IC50 value was determined to be 54 μg/ml at 36 h (Fig. 1A). Con A has been reported to induce both apoptotic and autophagic death in cancer cell lines [14]. Further study was directed to establish a relation between Con A induced autophagy and apoptosis in HeLa cells. Cell cycle analysis by flow cytometry showed an increase in apoptosis
Discussion
It has been proven that Con A induces differential consequences like mitogenic, toxic, immune activation and autophagy in different type of cell line. Interestingly in some cell line dose dependently it shows both mitogenic and also toxic activity [14]. Similarly Con A at above 20 μg/ml concentration induces autophagy and at above 50 μg/ml induces cytotoxicity in HeLa cells. The nature of cell death depends on duration of the treatment like Con A at long time (after 24 h time) induces both
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported in part by grant from Council of Scientific & Industrial Research, India and Department of Biotechnology, Govt. of India.
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