Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.     

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

Metformin (Met), an AMP-activated protein kinase (AMPK) inducer, is primarily transported by organic cation transporters expressed at the surface of renal proximal tubular epithelial cells. However, the implication of Met in renal function remains poorly understood. Interestingly, AICAR, another AMPK inducer, has been shown to inhibit the Unfolded Protein Response (UPR) generated by tunicamycin in cardiomyocytes in an AMPK-kinase dependent fashion suggesting metformin may also block the UPR. In this work, we have examined the effect of metformin on the expression of UPR-related markers (GRP94 and CHOP) induced by glucosamine (GlcN), 2-deoxyglucose (2-DOG) and tunicamycin (TUNI) in renal proximal tubular epithelial cells and in murine mesangial cells. Met attenuated GRP94 and CHOP expression induced by GlcN and 2-DOG, but not TUNI only in renal epithelial cells, even though the AMPK activation was observed in both renal epithelial and mesangial cells. Met did not require the contribution of its AMPK kinase inducing activity to block UPR markers expression. This report has identified a novel inhibitory function of metformin on UPR, which may have a beneficial impact on kidney homeostatic function.     

Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.     

Modulation of endoplasmic reticulum (ER) stress-induced autophagy by C/EBP homologous protein (CHOP) and inositol-requiring enzyme 1α (IRE1α) in human colon cancer cells

To explore the relationship between UPR and autophagy in intestinal epithelial cells, we investigated whether autophagy was induced by endoplasmic reticulum (ER) stress in colon cancer cell lines. We demonstrated that autophagy was induced by ER stress in HT29, SW480, and Caco-2 cells. In these cells, inositol-requiring enzyme1α (IRE1α) and C/EBP homologous protein (CHOP) were involved in the ER stress–autophagy pathway, and CHOP was a regulator of IRE1α protein expression. Our findings suggest that CHOP promotes IRE1α and autophagy especially in ER stress conditions. This study will provide important insights into the disclosure of the ER stress–autophagy pathway.     

Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.     

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.     

Inhibition of stearoyl-CoA desaturase 1 expression induces CHOP-dependent cell death in human cancer cells

Background

Cancer cells present a sustained de novo fatty acid synthesis with an increase of saturated and monounsaturated fatty acid (MUFA) production. This change in fatty acid metabolism is associated with overexpression of stearoyl-CoA desaturase 1 (Scd1), which catalyses the transformation of saturated fatty acids into monounsaturated fatty acids (e.g., oleic acid). Several reports demonstrated that inhibition of Scd1 led to the blocking of proliferation and induction of apoptosis in cancer cells. Nevertheless, mechanisms of cell death activation remain to be better understood.

Principal Findings

In this study, we demonstrated that Scd1 extinction by siRNA triggered abolition of de novo MUFA synthesis in cancer and non-cancer cells. Scd1 inhibition-activated cell death was only observed in cancer cells with induction of caspase 3 activity and PARP-cleavage. Exogenous supplementation with oleic acid did not reverse the Scd1 ablation-mediated cell death. In addition, Scd1 depletion induced unfolded protein response (UPR) hallmarks such as Xbp1 mRNA splicing, phosphorylation of eIF2α and increase of CHOP expression. However, the chaperone GRP78 expression, another UPR hallmark, was not affected by Scd1 knockdown in these cancer cells indicating a peculiar UPR activation. Finally, we showed that CHOP induction participated to cell death activation by Scd1 extinction. Indeed, overexpression of dominant negative CHOP construct and extinction of CHOP partially restored viability in Scd1-depleted cancer cells.

Conclusion

These results suggest that inhibition of de novo MUFA synthesis by Scd1 extinction could be a promising anti-cancer target by inducing cell death through UPR and CHOP activation.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.     

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.     

The Unfolded Protein Response Plays a Predominant Homeostatic Role in Response to Mitochondrial Stress in Pancreatic Stellate Cells

Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5–2.5 μM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 μM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGFα were unaffected, and the immune modulator IL-4 was markedly upregulated. These data imply that metabolic stress-induced PaSC reprogramming differentially modulates neighboring cells in the tumor microenvironment.     

C/EBP homologous protein drives pro-catabolic responses in chondrocytes

Introduction

Excess C/EBP homologous protein (CHOP) expression is one feature of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. Here, we focused on CHOP expression and function in chondrocytes.

Methods

We studied human knee osteoarthritis (OA) cartilage, bovine chondrocytes cultured in alginate and subjected to sub-lethal biomechanical injury, and knee chondrocytes of human autopsy donors. We performed siRNA knockdown and transfection.

Results

UPR activation was increased in human knee OA cartilage in situ, and in biomechanically injured cultured chondrocytes in vitro. In normal human chondrocytes, CHOP “gain of function” sensitized chondrocytes to IL-1β induced nitric oxide (NO) and matrix metalloproteinase (MMP)-3 release without inducing these responses by itself. Excess CHOP expression, by itself, induced superoxide production and apoptosis. Conversely, siRNA knockdown of CHOP and the UPR-specific mediator X-box binding protein (XBP1) inhibited NO release by >80% (P <0.0005) in response to IL-1β, and blunted MMP-3 release, whereas there were only minimal effects of the UPR mediator GRP78 on these responses. The anti-inflammatory metabolic “super-regulator” AMP kinase (AMPK) is known to limit UPR activation in vascular muscle cells. Here, CHOP supported the capacity of IL-1β to suppress AMPK activity in chondrocytes. We also observed that inhibition of AMPK activity promoted an increase in chondrocyte CHOP expression. Conversely, pharmacologic activation of AMPK by 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) blunted chondrocyte CHOP expression in response to biomechanical injury.

Conclusions

Biomechanical injury and IL-1 signaling stimulate UPR activation in chondrocytes. CHOP mediates chondrocyte catabolic and apoptotic responses to IL-1β, and does so partly by inhibiting AMPK activity. Conversely, development of excess CHOP activity is limited by AMPK activity in chondrocytes. Our findings suggest a mechanism for potential chondroprotection by AICAR and other AMPK activators. The work is of translational relevance for OA, since several drugs that activate AMPK are already in the clinic for arthritis (for example, allosteric AMPK activators sodium salicylate and high dose aspirin, and methotrexate, which activates AMPK by generating AICAR).     

Modulation of the Akt Pathway Reveals a Novel Link with PERK/eIF2α, which Is Relevant during Hypoxia

The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.     

The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis.     

Metformin promotes survivin degradation through AMPK/PKA/GSK-3β-axis in non–small cell lung cancer

Metformin, a first-line antidiabetic drug, has been reported with anticancer activities in many types of cancer. However, its molecular mechanisms remain largely unknown. As a member of inhibitor of apoptosis proteins, survivin plays an important role in the regulation of cell death. In the present study, we investigated the role of survivin in metformin-induced anticancer activity in non–small cell lung cancer in vitro. Metformin mainly induced apoptotic cell death in A549 and H460 cell lines. It remarkably suppressed the expression of survivin, decreased the stability of this protein, then promoted its proteasomal degradation. Moreover, metformin greatly suppressed protein kinase A (PKA) activity and induced its downstream glycogen synthase kinase 3β (GSK-3β) activation. PKA activators, both 8-Br-cAMP and forskolin, significantly increased the expression of survivin. Consistently both GSK-3β inhibitor LiCl and siRNA restored the expression of survivin in lung cancer cells. Furthermore, metformin induced adenosine 5′-monophosphate-activated protein kinase (AMPK) activation. Suppression of the activity of AMPK with Compound C reversed the degradation of survivin induced by metformin, and meanwhile, restored the activity of PKA and GSK-3β. These results suggest that metformin kills lung cancer cells through AMPK/PKA/GSK-3β-axis–mediated survivin degradation, providing novel insights into the anticancer effects of metformin.     

Divergent Effects of PERK and IRE1 Signaling on Cell Viability

Protein misfolding in the endoplasmic reticulum (ER) activates a set of intracellular signaling pathways, collectively termed the Unfolded Protein Response (UPR). UPR signaling promotes cell survival by reducing misfolded protein levels. If homeostasis cannot be restored, UPR signaling promotes cell death. The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood. The ER-resident proteins, PERK and IRE1, control two key UPR signaling pathways. Protein misfolding concomitantly activates PERK and IRE1 and has clouded insight into their contributions toward life or death cell fates. Here, we employed chemical-genetic strategies to activate individually PERK or IRE1 uncoupled from protein misfolding. We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis. By contrast, equivalent durations of IRE1 signaling enhanced cell proliferation without promoting cell death. These results demonstrate that extended PERK and IRE1 signaling have opposite effects on cell viability. Differential activation of PERK and IRE1 may determine life or death decisions after ER protein misfolding.     

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.