Frankis G. Almaguel

Extracellular, cell-permeable survivin inhibits apoptosis while promoting proliferative and metastatic potential

The tumour microenvironment is believed to be involved in development, growth, metastasis, and therapy resistance of many cancers. Here we show survivin, a member of the inhibitor of apoptosis protein (IAP) family, implicated in apoptosis inhibition and the regulation of mitosis in cancer cells, exists in a novel extracellular pool in tumour cells. Furthermore, we have constructed stable cell lines that provide the extracellular pool with either wild-type survivin (Surv-WT) or the previously described dominant-negative mutant survivin (Surv-T34A), which has proven pro-apoptotic effects in cancer cells but not in normal proliferating cells. Cancer cells grown in conditioned medium (CM) taken from Surv-WT cells absorbed survivin and experienced enhanced protection against genotoxic stresses. These cells also exhibited an increased replicative and metastatic potential, suggesting that survivin in the tumour microenvironment may be directly associated with malignant progression, further supporting survivins function in tumourigenesis. Alternatively, cancer cells grown in CM taken from the Surv-T34A cells began to apoptose through a caspase-2- and caspase-9-dependent pathway that was further enhanced by the addition of other chemo- and radiotherapeutic modalities. Together our findings suggest a novel microenvironmental function for survivin in the control of cancer aggressiveness and spread, and should result in the genesis of additional cancer treatment modalities.

Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

BackgroundHormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality.ResultsWe investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity.ConclusionThese results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC.

Activation and reversal of lipotoxicity in PC12 and rat cortical cells following exposure to palmitic acid

Lipotoxicity involves a series of pathological cellular responses after exposure to elevated levels of fatty acids. This process may be detrimental to normal cellular homeostasis and cell viability. The present study shows that nerve growth factor‐differentiated PC12 cells (NGFDPC12) and rat cortical cells (RCC) exposed to high levels of palmitic acid (PA) exhibit significant lipotoxicity and death linked to an “augmented state of cellular oxidative stress” (ASCOS). The ASCOS response includes generation of reactive oxygen species (ROS), alterations in the mitochondrial transmembrane potential, and increase in the mRNA levels of key cell death/survival regulatory genes. The observed cell death was apoptotic based on nuclear morphology, caspase‐3 activation, and cleavage of lamin B and PARP. Quantitative real‐time PCR measurements showed that cells undergoing lipotoxicity exhibited an increase in the expression of the mRNAs encoding the cell death‐associated proteins BNIP3 and FAS receptor. Cotreatment of NGFDPC12 and RCC cells undergoing lipotoxicity with docosahexaenoic acid (DHA) and bovine serum albumin (BSA) significantly reduced cell death within the first 2 hr following the initial exposure to PA. The data suggest that lipotoxicity in NGFDPC12 and cortical neurons triggers a strong cell death apoptotic response. Results with NGFDPC12 cells suggest a linkage between induction of ASCOS and the apoptotic process and exhibit a temporal window that is sensitive to DHA and BSA interventions.

Expression of E-FABP in PC12 cells increases neurite extension during differentiation: involvement of n-3 and n-6 fatty acids.

Epidermal fatty acid‐binding protein (E‐FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E‐FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E‐FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n‐3 docosahexaenoic acid (DHA), C20:5n‐3 eicosapentaenoic acid (EPA), and C20:4n‐6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E‐FABP expression that is blocked by mitogen‐activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor‐differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12‐AS) which exhibit low E‐FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12‐S). Replenishing NGFDPC12‐AS cells with biotinylated recombinant E‐FABP (biotin‐E‐FABP) protein restores normal neurite outgrowth. Cellular localization of biotin‐E‐FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E‐FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12‐S than in NGFDPC12‐AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E‐FABP.

Metabolomics uncovers dietary omega-3 fatty acid-derived metabolites implicated in anti-nociceptive responses after experimental spinal cord injury.

Chronic neuropathic pain is a frequent comorbidity following spinal cord injury (SCI) and often fails to respond to conventional pain management strategies. Preventive administration of docosahexaenoic acid (DHA) or the consumption of a diet rich in omega-3 polyunsaturated fatty acids (O3PUFAs) confers potent prophylaxis against SCI and improves functional recovery. The present study examines whether this novel dietary strategy provides significant antinociceptive benefits in rats experiencing SCI-induced pain. Rats were fed control chow or chow enriched with O3PUFAs for 8weeks before being subjected to sham or cord contusion surgeries, continuing the same diets after surgery for another 8 more weeks. The paw sensitivity to noxious heat was quantified for at least 8weeks post-SCI using the Hargreaves test. We found that SCI rats consuming the preventive O3PUFA-enriched diet exhibited a significant reduction in thermal hyperalgesia compared to those consuming the normal diet. Functional neurometabolomic profiling revealed a distinctive deregulation in the metabolism of endocannabinoids (eCB) and related N-acyl ethanolamines (NAEs) at 8weeks post-SCI. We found that O3PUFAs consumption led to a robust accumulation of novel NAE precursors, including the glycerophospho-containing docosahexaenoyl ethanolamine (DHEA), docosapentaenoyl ethanolamine (DPEA), and eicosapentaenoyl ethanolamine (EPEA). The tissue levels of these metabolites were significantly correlated with the antihyperalgesic phenotype. In addition, rats consuming the O3PUFA-rich diet showed reduced sprouting of nociceptive fibers containing CGRP and dorsal horn neuron p38 mitogen-activated protein kinase (MAPK) expression, well-established biomarkers of pain. The spinal cord levels of inositols were positively correlated with thermal hyperalgesia, supporting their role as biomarkers of chronic neuropathic pain. Notably, the O3PUFA-rich dietary intervention reduced the levels of these metabolites. Collectively, these results demonstrate the prophylactic value of dietary O3PUFA against SCI-mediated chronic pain.

Differential effect of proIGF-II and IGF-II on resveratrol induced cell death by regulating survivin cellular localization and mitochondrial depolarization in breast cancer cells

Insulin-like growth factor II (IGF-II) plays a pivotal role in fetal and cancer development by signaling through the IGF-I and insulin receptors and activating the estrogen signaling cascade. We previously showed that precursor IGF-II (proIGF-II, the predominant form expressed in cancer) and not mature IGF-II (mIGF-II) blocks resveratrol (RSV) (a phytoalexin/anticancer agent)-induced cell death in MCF-7 cells. We hypothesize that proIGF-II regulates antiapoptotic proteins and/or the mitochondria to inhibit RSV actions and promote cell survival. This study examines the effect of mIGF-II and proIGF-II on survivin expression and mitochondrial polarization in response to RSV. RSV inhibits survivin expression and stimulates mitochondrial depolarization, caspase 7 activation and cell death. These effects were completely blocked by the addition of proIGF-II. RSV treatment had no effect on transfected MCF-7 cells constitutively expressing proIGF-II, while IGF-II siRNA transfection decreased survivin levels. Our results provide new insights for the potential use of proIGF-II as target for new anticancer therapies.

Lipotoxicity-mediated cell dysfunction and death involve lysosomal membrane permeabilization and cathepsin L activity.

Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity.

Precursor IGF-II (proIGF-II) and mature IGF-II (mIGF-II) induce Bcl-2 and Bcl-XL expression through different signaling pathways in breast cancer cells

IGF-II plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor. We have shown that inhibition of IGF-II by resveratrol (RSV) induced apoptosis and that proIGF-II (highly expressed in cancer) was more potent than mIGF-II in inhibiting this effect. Thus, we hypothesized that IGF-II differentially regulates the signaling cascade of the IGF-I receptor to stimulate the anti-apoptotic proteins Bcl-2 and Bcl-XL to prevent apoptosis. RSV treatment to breast cancer cells inhibited Bcl-2 and Bcl-XL expression and induced mitochondrial membrane depolarization. ProIGF-II was more potent than mIGF-II in: (1) activating the PI3/Akt pathway, (2) regulating Bcl-2 and Bcl-XL expression, and (3) inducing phosphorylation/nuclear translocation of Cyclic AMP-responsive element binding protein. Furthermore, IGF-II differentially regulated the intracellular translocation of Bcl-2 and Bcl-XL, a critical process in breast cancer progression to hormone-independence. Our study provides a novel mechanism of how proIGF-II promotes progression and chemoresistance in breast cancer development.

230 PALMITIC ACID TRIGGERS CELL DEATH IN PC12 CELLS THROUGH THE ACTIVATION OF MITOCHONDRIAL PROAPOPTOTIC GENES.

The present study uses PC12 cells to investigate the mechanism by which pathological concentrations of saturated free fatty acids (FFAs) trigger cell death. Fatty acid-induced apoptotic cell death has been proposed to play a role in the neuronal loss observed following traumatic injury in the CNS and PNS. To further study this observation, we performed a series of c-DNA array experiments using mRNA of PC12 cells exposed to palmitic acid complexed with bovine serum albumin (BSA) (2:1 ration). Our data show that palmitic acid changes the levels of expression of several mRNAs of proteins that have been associated with apoptosis such as BNIP3 and Bax. Quantitative real-time RT-PCR (QRTPCR) experiments revealed an up-regulation of the proapoptotic mRNA BNIP3 that reached a maximum of 4-fold induction and Bax that reached a maximum of 3.5 induction during the course of the exposure of PC12 cells to palmitic acid. Western blots results also show an induction in BNIP3 and Bax protein. Additional activation of BNIP3/Bax can induce cell death by heterodimerization with antiapoptotic Bcl-2/Bcl-xl or by opening of the permeability transition pore resulting in mitochondrial dysfunction. Our results suggest that saturated FFAs induced cell death through the activation of proapoptotic mitochondrial genes like BNIP3 and Bax.

181 PALMITIC ACID-INDUCED CELL DEATH IN NERVE GROWTH FACTOR DIFFERENTIATED PC12 CELLS INVOLVES THE UP-REGULATION OF HYPOXIA-INDUCING FACTOR 1 AND βNIP-3.

Hypoxic-ischemic injury in the nervous system is associated with the degradation of membrane phospholipids with subsequent release of free fatty acids (FFAs). It has been proposed that this increase in the release of FFA can cause fatty acid-induced apoptotic neuronal cell loss and would be at least in part responsible for the observed secondary injury seen following hypoxia/ischemia and nerve injury insult. Purpose The present study uses NGF differentiated PC12 (NGFDPC12) cells to study neuronal apoptosis and to investigate the mechanism by which pathological concentrations of saturated FFA trigger neuronal cell death. We previously reported that NGFDPC12 cells exposed to palmitic acid (PA) show a dramatic loss of viability as early as 12 hours after treatment and exhibit an increase in the expression levels of several mRNAs of apoptosis-associated proteins such as BAK and βNIP-3 and hypoxic-inducing factor 1 alpha (HIF-1). Methods To further study this observation, we performed a series of Western blots experiments using cell extract of NGFDPC12 cells exposed to PA bound to bovine serum albumin (BSA) (2:1 ratio) to determine the levels of βNIP-3 and its activator HIF-1. Cells were treated with the PA for 6, 9, 12, and 24 hours. Cells were then harvested and lysed in an extraction buffer with protease inhibitors. Next, 20 μg of total cellular protein were electrophoresed on 4-12% gradient SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane and blocked with 7.5% nonfat milk in TTBS for 1 hour. Blots were then probed with antibodies for HIF-1 or βNIP-3 followed by a secondary antibody conjugated to horseradish peroxidase. Antigen-antibody complexes were visualized with enhanced chemiluminescence. Results Our Western blot results show an increase in both proteins HIF-1 and βNIP-3. HIF-1 and βNIP-3 have a maximum expression at 6 hours after PA treatment and gradually decreased thereafter. Conclusion These findings suggest that these two apoptosis-associated proteins may play a role in the apoptotic cell damage mediated by saturated FFA in NGFDPC12 cells. This work is supported in part by NIGMS award 2R25GM060507-05 to M.D.L.