PPARγ‐inactive Δ2‐troglitazone independently triggers ER stress and apoptosis in breast cancer cells

Abstract

Our aim was to better understand peroxisome proliferator-activated receptor gamma (PPARγ)-independent pathways involved in anti-cancer effects of thiazolidinediones (TZDs). We focused on Δ2-troglitazone (Δ2-TGZ), a PPARγ inactive TZD that affects breast cancer cell viability. Appearance of TUNEL positive cells, changes in mitochondrial membrane potential, cleavage of poly(ADP-ribose) polymerase (PARP)-1 and caspase-7 revealed that apoptosis occurred in both hormone-dependent MCF7 and hormone-independent MDA-MB-231 breast cancer cells after 24 and 48 h of treatment. A microarray study identified endoplasmic reticulum (ER) stress as an essential cellular function since many genes involved in ER stress were upregulated in MCF7 cells following Δ2-TGZ treatment. Δ2-TGZ-induced ER stress was further confirmed in MCF7 cells by phosphorylation of pancreatic endoplasmic reticulum kinase-like endoplasmic reticulum kinase (PERK) and its target eIF2α after 1.5 h, rapid increase in activating transcription factor (ATF) 3 mRNA levels, splicing of X-box binding protein 1 (XBP1) after 3 h, accumulation of binding immunogloblulin protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) after 6 h. Immunofluorescence microscopy indicated that CHOP was relocalized to the nucleus of treated cells. Similarly, in MDA-MB-231 cells, overexpression of ATF3, splicing of XBP1, and accumulation of BiP and CHOP were observed following Δ2-TGZ treatment. In MCF7 cells, knock-down of CHOP or the inhibition of c-Jun N-terminal kinase (JNK) did not impair cleavage of PARP-1 and caspase-7. Altogether, our results show that ER stress is an early response of major types of breast cancer cells to Δ2-TGZ, prior to, but not causative of apoptosis.

Δ2-Troglitazone promotes cytostatic rather than pro-apoptotic effects in breast cancer cells cultured in high serum conditions

“…In our previous studies performed in low serum conditions, we showed that D2-TGZ treatment was followed early by the activation of PERK/eIF2a and IRE1/XBP-1 axes of the UPR in both MDA-MB-231 and MCF-7 cells. 17 We also observed in both cell lines a later increase in the expression of the chaperone BiP as well as the induction of CHOP, the latter being translocated in the nucleus. In this context, we showed that ER stress was not causative of apoptosis.…”

“…17 In order to investigate if D2-TGZ still induced ER stress in 10% FCS-conditions, cells were treated with D2-TGZ during a time course in 10% FCS-containing medium and several players of the unfolded protein response (UPR) pathway that is activated in case of ER stress were studied. We focused on the pancreatic endoplasmic reticulum kinase-like endoplasmic reticulum kinase (PERK) and the eukaryotic initiation factor 2a (eIF2a) that are responsible for a slowing down of protein translation and the inositol requiring enzyme 1 (IRE1)-dependent X-box-binding protein-1 (XBP-1) mRNA cleavage that is required for the synthesis of a highly active transcription factor responsible for the expression of the immunoglobulin heavy chain binding protein (BiP), a chaperone that binds to unfolded proteins.…”

“…17 In order to determine if D2-TGZ also induced apoptosis in 10% FCS-conditions, both cell lines were treated for 72 h with D2-TGZ (75 mM for MDA-MB-231 cells and 60 mM for MCF-7 cells) in 10% FCS-containing medium. Control cells received DMSO.…”

“…Nevertheless, apoptosis did not seem to be a consequence of ER stress in MCF-7 cells. 17 Serum starvation is an experimental condition applied to limit undesirable effects on cell response due to the complex and non-standardized composition of serum. 18 It is also a routine procedure carried out to synchronize proliferating cells and to reduce basal cellular activity.…”

“…This compound was tested previously either in high serum conditions (5% or 10% fetal calf serum (FCS)-containing medium) 14,22 or in a low serum environment (1% or 0% FCS-containing medium). 10,12,13,[15][16][17] Most data from our laboratory were obtained from breast cancer cell lines (MDA-MB-231 and MCF-7) cultured in 1% FCS-containing medium. In order to determine the impact of serum deprivation, we studied the effects of D2-TGZ on cells maintained in 10% FCS-containing medium.…”