Modulation of Cellular Hsp72 Levels in Undifferentiated and Neuron-Like SH-SY5Y Cells Determines Resistance to Staurosporine-Induced Apoptosis

Lesley Cheng,Danielle J Smith,Phillip Nagley,Robin L Anderson,Ferdinando Di Cunto

doi:10.1371/journal.pone.0024473

Lesley Cheng, Danielle J Smith + Show 3 more

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https://doi.org/10.1371/journal.pone.0024473

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Abstract

Increased expression of Hsp72 accompanies differentiation of human neuroblastoma SH-SY5Y cells to neuron-like cells. By modulating cellular levels of Hsp72, we demonstrate here its anti-apoptotic activity both in undifferentiated and neuron-like cells. Thermal preconditioning (43°C for 30 min) induced Hsp72, leading to cellular protection against apoptosis induced by a subsequent treatment with staurosporine. Preconditioned staurosporine-treated cells displayed decreased Bax recruitment to mitochondria and subsequent activation, as well as reduced cytochrome c redistribution from mitochondria. The data are consistent with Hsp72 blocking apoptosis upstream of Bax recruitment to mitochondria. Neuron-like cells (with elevated Hsp72) were more resistant to staurosporine by all measured indices of apoptotic signaling. Use of stable transfectants ectopically expressing moderately elevated levels of Hsp72 revealed that such cells in the undifferentiated state showed enhanced resistance to staurosporine-induced apoptosis, which was even more robust after differentiation to neuron-like cells. Overall, the protective effects of differentiation, thermal preconditioning and ectopic Hsp72 expression were additive. The strong inverse correlation between cellular Hsp72 levels and susceptibility to apoptosis support the notion that Hsp72 acts as a significant neuroprotective factor, enabling post-mitotic neurons to withstand potentially lethal stress that induces apoptosis.

Highlights

Apoptosis in neurons contributes to pathological conditions, such as the acute brain injury that occurs in stroke, or the chronic injury in neurodegenerative disorders [1]
Applying a Cell Titer-Blue cell viability assay, undifferentiated SH-SY5Y cells subjected to thermal preconditioning showed modest protection against death induced by 50 nM STS for 12 h, whereas neuron-like SH-SY5Y cells treated with 50 nM STS show significantly greater cell survival when thermally preconditioned (p,0.01) (Figure 1A)
Induction of apoptosis in SH-SY5Y cells by STS was indicated by characteristic changes in nuclear morphology (Figure 1B)

Summary

Introduction

Apoptosis in neurons contributes to pathological conditions, such as the acute brain injury that occurs in stroke, or the chronic injury in neurodegenerative disorders [1]. The mitochondrial pathway of apoptosis can be elicited by cellular stresses, including DNA damage or loss of survival-inducing intracellular or extracellular signaling pathways [2,3]. In response to cellular stresses, Bax is recruited to the mitochondria where it is activated, leading to redistribution of intermembrane space proteins such as cytochrome c (cyt c) from the mitochondria to the cytosol [3]. Hsp is often induced during cellular stress to repair damage, maintain cellular homeostasis and facilitate the recovery of cells from otherwise lethal stimuli [4,5]. Hsp is upregulated in injured and damaged areas of the brain during a variety of external stresses such as hyperthermia, stroke, ischemia and acute brain injury [6]. Hsp is upregulated in neuronal cells under thermal preconditioning, a non-lethal thermal stress that protects cells from a subsequent, otherwise lethal, cellular insult [7,8,9,10,11]

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