Control of FLIPL expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells

Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP_L) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP_L levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP_L upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP_L expression. FLIP_L upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP_L upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP_L levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated.     

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-x_L counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-x_L observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA.     

The involvement of mitochondria and the caspase-9 activation pathway in rituximab-induced apoptosis in FL cells

Despite the wide use of anti-CD20 antibody rituximab in the cancer treatment of B cell malignancies, the signalling pathways of CD20-induced apoptosis are still not understood. By using dominant negative (DN)-caspase-9 overexpressing follicular lymphoma cells we demonstrated that the activation of caspase-9 was essential for rituximab-mediated apoptosis. The death receptor pathway mediated by caspase-8 activation was not involved in rituximab-mediated apoptosis since overexpression of FLIP_short or FLIP_long proteins, inhibitors of caspase-8 activation, could not inhibit rituximab-induced apoptosis. However, the death receptor pathway activation by anti-Fas antibodies showed an additive effect on rituximab-induced apoptosis. The stabilisation of the mitochondrial outer membrane by Bcl-x_L overexpression inhibited cell death, showing the important role of mitochondria in rituximab-induced apoptosis. Interestingly, the rituximab-induced release of cytochrome c and collapse of mitochondrial membrane potential were regulated by caspase-9. We suggest that caspase-9 and downstream caspases may feed back to mitochondria to amplify mitochondrial disruption during intrinsic apoptosis.     

A role for c-FLIPL in the regulation of apoptosis,autophagy, and necroptosis in T lymphocytes

Caspase 8 plays a dual role in the survival of T lymphocytes. Although active caspase 8 mediates apoptosis upon death receptor signaling, the loss of caspase 8 activity leads to receptor-interacting protein (RIP)-1/RIP-3-dependent necrotic cell death (necroptosis) upon TCR activation. The anti-apoptotic protein c-FLIP (cellular caspase 8 (FLICE)-like inhibitory protein) suppresses death receptor-induced caspase 8 activation. Moreover, recent findings suggest that c-FLIP is also involved in inhibiting necroptosis and autophagy. It remains unclear whether c-FLIP protects primary T lymphocytes from necroptosis or regulates the threshold at which autophagy occurs. Here, we used a c-FLIP isoform-specific conditional deletion model to show that c-FLIP_L-deficient T cells underwent RIP-1-dependent necroptosis upon TCR stimulation. Interestingly, although previous studies have only described necroptosis in the absence of caspase 8 activity, we found that pro-apoptotic caspase 8 activity and apoptosis were also enhanced in c-FLIP_L-deficient T lymphocytes. Furthermore, c-FLIP_L-deficient T cells exhibited enhanced autophagy, which served a cytoprotective function. Together, these findings indicate that c-FLIP_L plays an important antinecroptotic role and is a key regulator of apoptosis, autophagy, and necroptosis in T lymphocytes.     

Cellular FLIPL plays a survival role and regulates morphogenesis in breast epithelial cells

Strong evidences support the inhibitory activity of cellular FLICE-inhibitory protein (FLIP) in the apoptotic signalling by death receptors in tumor cells. However, little is known about the role of FLIP in the regulation of apoptosis in non-transformed cells. In this report, we demonstrate that FLIP_L plays an important role as a survival protein in non-transformed breast epithelial cells. Silencing of FLIP_L by siRNA methodology enhances TRAIL-R2 expression and activates a caspase-dependent cell death process in breast epithelial cells. This cell death requires the expression of TRAIL, TRAIL-R2, FADD and procaspase-8 proteins. A mitochondria-operated apoptotic pathway is partially required for FLIP_L siRNA-induced apoptosis. Interestingly, FLIP_L silencing markedly abrogates formation of acinus-like structures in a three-dimensional basement membrane culture model (3D) of the human mammary MCF-10A cell line through a caspase-8 dependent process. Furthermore, over-expression of FLIP_L in MCF-10A cells delayed lumen formation in 3D cultures. Our results highlight the central role of FLIP in maintaining breast epithelial cell viability and suggest that the mechanisms regulating FLIP levels should be finely controlled to prevent unwanted cell demise.     

Abamectin induces apoptosis and autophagy by inhibiting reactive oxygen species‐mediated PI3K/AKT signaling in MGC803 cells

Abamectin (ABA) is one of the most widely used compounds in agriculture and veterinary medicine. However, the cytotoxicity of ABA in human gastric cells is utterly unknown. In this study, ABA suppressed the proliferation of MGC803 cells by arresting the cell cycle at the G0/G1‐phase. Moreover, ABA induced mitochondrial‐mediated apoptosis by inducing the loss of mitochondrial membrane potential, upregulation of Bax/Bcl‐2, and activation of caspase‐3. ABA significantly improved the LC3‐II/LC3‐I ratio and reduced P62 protein expression in a dose‐dependent manner. Through detection of the reactive oxygen species (ROS) levels, we found ABA induced the accumulation of intracellular ROS and then reduced PI3K/AKT signaling activation related to MGC803 cell apoptosis and autophagy. Our results indicate that ABA exerts cytotoxic effects on human MGC803 cells through apoptosis and autophagy by inhibiting ROS‐mediated PI3K/AKT signaling. Furthermore, ABA may be a potential risk to human gastric health.     

Proliferative versus apoptotic functions of caspase-8 : Hetero or homo: The caspase-8 dimer controls cell fate

Caspase-8, the initiator of extrinsically-triggered apoptosis, also has important functions in cellular activation and differentiation downstream of a variety of cell surface receptors. It has become increasingly clear that the heterodimer of caspase-8 with the long isoform of cellular FLIP (FLIP_L) fulfills these pro-survival functions of caspase-8. FLIP_L, a catalytically defective caspase-8 paralog, can interact with caspase-8 to activate its catalytic function. The caspase-8/FLIP_L heterodimer has a restricted substrate repertoire and does not induce apoptosis. In essence, caspase-8 heterodimerized with FLIP_L prevents the receptor interacting kinases RIPK1 and -3 from executing the form of cell death known as necroptosis. This review discusses the latest insights in caspase-8 homo- versus heterodimerization and the implication this has for cellular death or survival. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

BIM-mediated AKT phosphorylation is a key modulator of arsenic trioxide-induced apoptosis in cisplatin-sensitive and -resistant ovarian cancer cells

Background

Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to the effective treatment of human ovarian cancer. Previous reports indicated that arsenic trioxide (ATO) induces cell apoptosis in both drug-sensitive and -resistant ovarian cancer cells.

Principal Findings

In this study, we determined the molecular mechanism of ATO-induced apoptosis in ovarian cancer cells. Our data demonstrated that ATO induced cell apoptosis by decreasing levels of phosphorylated AKT (p-AKT) and activating caspase-3 and caspase-9. Importantly, BIM played a critical role in ATO-induced apoptosis. The inhibition of BIM expression prevented AKT dephosphorylation and inhibited caspase-3 activation during cell apoptosis. However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation. Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation. Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression.

Conclusions

We demonstrated the roles of BIM in ATO-induced apoptosis and the molecular mechanisms of BIM expression regulated by ATO during ovarian cancer cell apoptosis. Our findings suggest that BIM plays an important role in regulating p-AKT by activating caspase-3 and that BIM mediates the level of AKT phosphorylation to determine the threshold for overcoming cisplatin resistance in ovarian cancer cells.     

ASK1 is activated by arsenic trioxide in leukemic cells through accumulation of reactive oxygen species and may play a negative role in induction of apoptosis

Arsenic trioxide (ATO) is remarkably effective for treating acute promyelocytic leukemia. Here, we find that ATO treatment of NB4 and K562 leukemic cells induces activation of ASK1. ASK1 activation was induced most significantly at low concentrations of ATO, where G2/M arrest but not apoptosis was induced. On the other hand, ATO barely activated ASK1 at high concentrations, where apoptosis as well as activation of JNK and p38 was induced significantly. ATO-induced accumulation of reactive oxygen species (ROS), while the ASK1 activation was suppressed by cotreatment with an antioxidant, N-acetyl-l-cysteine. Murine embryonic fibroblasts (MEFs) from ASK1-deficient mice were more susceptible to ATO-induced apoptosis than control MEFs. Furthermore, ATO at the low concentration induced significant apoptosis in K562 cells when ASK1 was knocked down by siRNA. These results indicate that ASK1 is activated by ATO through ROS accumulation and may negatively regulate apoptosis in leukemic cells without activating p38 and JNK.     

Silencing FLIPL modifies TNF-induced apoptotic protein expression

Death-receptor induced apoptosis is regulated by FLIP [FLICE (Fas-associated protein with death domain-like IL-1β-converting enzyme)-inhibitory protein] via modification of caspase-8 activation. As an important modulator of apoptosis, the long isoform, FLIP_L, regulates life and death in many various types of normal and tumor cells and tissues to render resistance to death receptor-mediated apoptosis. In addition, FLIP_L has been shown to be involved in regulation of intrinsic (mitochondrial) pathways of apoptosis as well as regulating other proteins involved in cytoprotection and cell cycle progression. Therefore, understanding the role of FLIP_L in complex regulatory networks of cell survival/death mechanisms is vital for future developments to control diseases such as cancer. Here, we shown that silencing FLIP_L in HEK 293 cells changed the expression levels of proteins that are involved in both extrinsic and intrinsic apoptosis, as well as regulating tumor necrosis factor-α (TNF)-mediated apoptotic patterns. We also show that FLIP_L-silenced cells have a lower rate of proliferation and cell cycle progression when compared to control cells. Moreover, treatment with TNF restored proliferation rates in FLIP_L-silenced cells back to more normal levels when compared to control cells. These results suggest that cells have evolved complex compensatory mechanisms to overcome the absence of a key apoptotic regulatory proteins.     

Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 μM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1–2 μM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. B. Ozpolat and U. Akar contributed equally.     

Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells

We have investigated the involvement of intracellular pH (pH_i) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na⁺/H⁺ exchanger1 (NHE1) inhibitor cariporide and the “high K⁺” buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH_i than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH_i and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH_i recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH_i and P-gp in K562/DOX cells.     

Erk is involved in the differentiation induced by diallyl disulfide in the human gastric cancer cell line MGC803

Diallyl disulfide (DADS) is a major constituent of garlic. Previously, we found that DADS both inhibited proliferation in human gastric cancer cells in vitro and in vivo, and induced G2/M arrest. In this study, we investigated whether this differentiation effect was induced by DADS in human gastric cancer MGC803 cells, and whether it was related to an alteration in ERK activity. The results showed that the growth of MGC803 cells was inhibited by DADS. Cells treated with DADS displayed a lower nucleocytoplasmic ratio and tended to form gland and intercellular conjunction structures. The ConA-mediated cell agglutination ratio and cells’ ALP specific activity decreased. In MGC803 cells, dye transfer was limited to a few cells neighbouring the dye-injected cell and to a depth of 1–2 layers beneath the scrape site. However, after treatment with DADS, the LY (Lucifer Yellow) was transferred to several cells immediately neighbouring the microinjected cell and to a depth of 2–4 cell layers from the scrape site. This indicated that DADS induced differentiation in MGC803 cells. Western blot analysis revealed that although DADS did not influence the quantity of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls. At 30 mg·L⁻¹, DADS inhibited the activation of ERK1/2 in 15–30 min. These results suggested that the DADS-induced differentiation of MGC803 cells involved an alteration of the ERK1/2 signaling pathway.     

CFLAR/c-FLIPL

Necroptosis, a caspase-independent, receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1)/RIPK3-dependent necrotic cell death, occurs in cells when apoptosis is blocked. A high level of macroautophagy (herein referred to as autophagy) is usually detected in necroptotic cells, although it is still controversial as to whether excessive autophagy leads to cell death or is cytoprotective. In a recently published paper, we show that the anti-apoptotic protein CFLAR (CASP8 and FADD-like apoptosis regulator) long isoform (CFLAR_L) plays a critical role in all three fundamental intracellular processes: autophagy, necroptosis, and apoptosis in T lymphocytes. CFLAR_L-deficient T cells suffer from severe cell death upon T cell receptor stimulation, in which both apoptosis and necroptosis are involved. Autophagy is enhanced in both naïve and activated CFLAR_L-deficient T cells and plays a cytoprotective function. Here, we summarize our findings and discuss the future direction in the study of the interplay of autophagy, apoptosis and necroptosis in T lymphocytes.     

Antioxidant Ascorbate Is Stabilized by NADH-Coenzyme Q10 Reductase in the Plasma Membrane

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ₁₀ and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ₁₀ reductase activity and its internal sequence being identical to cytochrome b ₅ reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ₁₀ content in isolated plasma membranes. We show here the role of CoQ₁₀ and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b ₅-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ₁₀ and its NADH-dependent reductase.