Phenylbutyric acid induces the cellular senescence through an Akt/p21WAF1 signaling pathway

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Abstract

It has been well known that three sentinel proteins – PERK, ATF6 and IRE1 – initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21WAF1 induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21WAF1 pathway by PERK inhibition.

Highlights

► Phenylbutyric acid induces cellular senescence. ► Phenylbutyric acid activates Akt kinase. ► The knockdown of PERK also can induce cellular senescence. ► Akt/p21WAF1 pathway activates in PERK knockdown induced cellular senescence.

Introduction

The endoplasmic reticulum (ER) is a major site not only for protein and lipid synthesis but also for cellular homeostasis regulation. After translocation of secreted or sorted proteins to the ER, folding process is monitored by quality control system in the lumen [1]. The accumulation of unfolded or misfolded proteins in the ER induces ER stress which activates the unfolded protein response (UPR) to halt protein translocation into the ER and increases the production of molecular chaperones. Prolonged ER stress resulted from protein aggregation leads UPR to induce apoptosis [2]. It was recently reported that various tumors are under high levels of ER stress and UPR is distinct from normal cells. Because of the rapid growth rate and glucose metabolism, signals generated by misfolded or unfolded proteins occur often in cancer cells [3]. Thus, it has been suggested that cancer cells utilize the UPR for survival during tumor growth.

ER stress responses can be initiated by activation of three different ER membrane-anchored proteins, namely PKR-like ER kinase (PERK) [4], inositol-requiring enzyme 1 (IRE1) [5], [6] and activating transcription factor 6 (ATF6) [7]. Therefore, these proteins serve as sentinels for ER stress, which can threaten cell survival. The luminal domains of these three proteins are known to interact with Grp78/Bip, and accumulation of unfolded or misfolded proteins in the ER lumen is responsible for initiating UPR signaling, which decreases the interactions between the sentinel proteins and Grp78/Bip [8]. Activated PERK phosphorylates translational initiation factor eIF2α, resulting in reduction of global protein biosynthesis. Also, phosphorylated eIF2α induces specific mRNA translation, such as ATF4, which upregulates ER stress target genes [9]. ER-embedded ATF6 is separated from Grp78/Bip and translocates into the Golgi apparatus, where it is cleaved to form the nuclear ATF6. The nuclear form of ATF6 is involved in the induction of the UPR responsive gene by binding to the ER stress response element (ERSE) in the promoter [10]. Activated IRE1 functions as an endonuclease to cleave the mRNA of the X-box binding protein 1 (XBP1) into a form that encodes a functional bZIP transcription factor [11]. Subsequently, the spliced XBP1 protein upregulates several genes, including ER chaperones and enzymes, which provide relief during ER stress [12].

Generally, it has been thought that cellular senescence can occur through various upstream triggers, such as oncogenes and genotoxic reagents (Reactive oxygen species (ROS), UV, γ-irradiation, doxorubicine etc.) [13]. Although it is widely accepted that both p53/p21WAF1 and Rb/p16INK4a pathways are major signaling pathways in cellular senescence [14], the precise mechanisms in ER stress induced senescence have not yet been determined [15]. However, recent evidence has suggested the possibility that ER stress is likely linked to the cellular senescence pathway. In addition, protein folding and the generation of ROS, as a protein oxidation product, occur in the endoplasmic reticulum [16], [17]. HRASG12V-driven senescence in melanocyte is mediated by the ER-associated UPR [18], [19]. Also, upregulation of ER stress has been shown to be responsible for oxidative stress induced chondrocyte senescence [20].

In this study, we identified a chemical chaperone, called PBA, that induces cellular senescence in some cancer cells, and demonstrated that the molecular mechanisms of this compound is related with the inactivation of PERK, which permits Akt/p21WAF1 activation.

Section snippets

Long exposure to PBA leads to cellular senescence in both MCF-7 and HT1080 cells

Because of the high proliferation rate, cancer cells require increased translation ability in comparison with normal cells. Moreover, cancer cells are more often exposed to severe conditions, such as hypoxia and nutrient starvation, during their lifetime, which results in the accumulation of unfolded or misfolded proteins in the ER. Thus, to adapt to this condition, cancer cells seems to upregulate the UPR. Therefore, we hypothesized that a reduction of ER stress in cancer cells may facilitate

Cell culture

Cells were cultured in RPMI-1640 (Human breast cancer MCF-7) or DMEM (Human fibrosarcoma HT1080), supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA), penicillin (100 units/mL) and streptomycin (100 μg/ml) (Invitrogen, USA). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2.

Antibodies and reagents

Antibodies against p21WAF1, p38MAPK, p53, CHOP, Bip, Akt and PARP were obtained from Santa Cruz Biotechnology (USA) and anti-α-tubulin was from Calbiochem (USA). Antibodies against pp38, pAkt, cleaved

Acknowledgment

This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (2009-0086319, 20110026379).

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    These authors contributed equally to this work.

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