PSMA-Targeted Radiopharmaceuticals for Prostate Cancer Diagnosis and Therapy.

ABSTRACT: Prostate cancer (PCa) is the most common noncutaneous malignancy in men. Until recent years, accurate imaging of men with newly diagnosed PCa, or recurrent or low-volume metastatic disease, was limited. Further, therapeutic options for men with advanced, metastatic, castration-resistant disease were increasingly limited as a result of increasing numbers of systemic therapies being combined in the upfront metastatic setting. The advent of urea-based, small-molecule inhibitors of prostate-specific membrane antigen (PSMA) has partially addressed those shortcomings in diagnosis and therapy of PCa. On the diagnostic side, there are multiple pivotal phase III trials with several different agents having demonstrated utility in the initial staging setting, with generally modest sensitivity but very high specificity for determining otherwise-occult pelvic nodal involvement. That latter statistic drives the utility of the scan by allowing imaging interpreters to read with very high sensitivity while maintaining a robust specificity. Other pivotal phase III trials have demonstrated high detection efficiency in patients with biochemical failure, with high positive predictive value at the lesion level, opening up possible new avenues of therapy such as metastasis-directed therapy. Beyond the diagnostic aspects of PSMA-targeted radiotracers, the same urea-based chemical scaffolds can be altered to deliver therapeutic isotopes to PCa cells that express PSMA. To date, one such agent, when combined with best standard-of-care therapy, has demonstrated an ability to improve overall survival, progression-free survival, and freedom from skeletal events relative to best standard-of-care therapy alone in men with metastatic, castration-resistant PCa who are post chemotherapy. Within the current milieu, there are a number of important future directions including the use of artificial intelligence to better leverage diagnostic findings, further medicinal chemistry refinements to the urea-based structure that may allow improved tumor targeting and decreased toxicities, and the incorporation of new radionuclides that may better balance efficacy with toxicities than those nuclides that are available.

177Lu-PSMA Therapy.

Radiopharmaceutical therapy using 177Lu-prostate-specific membrane antigen (PSMA) is an effective prostate cancer treatment that was recently approved by the U.S. Food and Drug Administration. This method leverages the success of PSMA-targeted PET imaging, enabling delivery of targeted radiopharmaceutical therapy; has demonstrated a clear benefit in large prospective clinical trials; and promises to become part of the standard armamentarium of treatment for patients with prostate cancer. This review highlights the evidence supporting the use of this agent, along with important areas under investigation. Practical information on technology aspects, dose administration, nursing, and the role of the treating physician is highlighted. Overall, 177Lu-PSMA treatment requires close collaboration among referring physicians, nuclear medicine technologists, radiopharmacists, and nurses to streamline patient care.

The Role of Microbiome in Brain Development and Neurodegenerative Diseases.

Hundreds of billions of commensal microorganisms live in and on our bodies, most of which colonize the gut shortly after birth and stay there for the rest of our lives. In animal models, bidirectional communications between the central nervous system and gut microbiota (Gut-Brain Axis) have been extensively studied, and it is clear that changes in microbiota composition play a vital role in the pathogenesis of various neurodevelopmental and neurodegenerative disorders, such as Autism Spectrum Disorder, Alzheimer's disease, Parkinson's disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, anxiety, stress, and so on. The makeup of the microbiome is impacted by a variety of factors, such as genetics, health status, method of delivery, environment, nutrition, and exercise, and the present understanding of the role of gut microbiota and its metabolites in the preservation of brain functioning and the development of the aforementioned neurological illnesses is summarized in this review article. Furthermore, we discuss current breakthroughs in the use of probiotics, prebiotics, and synbiotics to address neurological illnesses. Moreover, we also discussed the role of boron-based diet in memory, boron and microbiome relation, boron as anti-inflammatory agents, and boron in neurodegenerative diseases. In addition, in the coming years, boron reagents will play a significant role to improve dysbiosis and will open new areas for researchers.

Boron Chemicals in Drug Discovery and Development: Synthesis and Medicinal Perspective.

A standard goal of medicinal chemists has been to discover efficient and potent drug candidates with specific enzyme-inhibitor abilities. In this regard, boron-based bioactive compounds have provided amphiphilic properties to facilitate interaction with protein targets. Indeed, the spectrum of boron-based entities as drug candidates against many diseases has grown tremendously since the first clinically tested boron-based drug, Velcade. In this review, we collectively represent the current boron-containing drug candidates, boron-containing retinoids, benzoxaboroles, aminoboronic acid, carboranes, and BODIPY, for the treatment of different human diseases.In addition, we also describe the synthesis, key structure-activity relationship, and associated biological activities, such as antimicrobial, antituberculosis, antitumor, antiparasitic, antiprotozoal, anti-inflammatory, antifolate, antidepressant, antiallergic, anesthetic, and anti-Alzheimer's agents, as well as proteasome and lipogenic inhibitors. This compilation could be very useful in the exploration of novel boron-derived compounds against different diseases, with promising efficacy and lesser side effects.

Feasibility, Biodistribution, and Preliminary Dosimetry in Peptide-Targeted Radionuclide Therapy of Diverse Adenocarcinomas Using 177Lu-FAP-2286: First-in-Humans Results.

Fibroblast activation protein (FAP) is a promising target for diagnosis and therapy of numerous malignant tumors. FAP-2286 is the conjugate of a FAP-binding peptide, which can be labeled with radionuclides for theranostic applications. We present the first-in-humans results using 177Lu-FAP-2286 for peptide-targeted radionuclide therapy (PTRT). Methods: PTRT using 177Lu-FAP-2286 was performed on 11 patients with advanced adenocarcinomas of the pancreas, breast, rectum, or ovary after prior confirmation of uptake on 68Ga-FAP-2286 or 68Ga-FAPI-04 PET/CT. Results: Administration of 177Lu-FAP-2286 (5.8 ± 2.0 GBq; range, 2.4-9.9 GBq) was well tolerated, with no adverse symptoms or clinically detectable pharmacologic effects being noticed or reported in any of the patients. The whole-body effective dose was 0.07 ± 0.02 Gy/GBq (range, 0.04-0.1 Gy/GBq). The mean absorbed doses for kidneys and red marrow were 1.0 ± 0.6 Gy/GBq (range, 0.4-2.0 Gy/GBq) and 0.05 ± 0.02 Gy/GBq (range, 0.03-0.09 Gy/GBq), respectively. Significant uptake and long tumor retention of 177Lu-FAP-2286 resulted in high absorbed tumor doses, such as 3.0 ± 2.7 Gy/GBq (range, 0.5-10.6 Gy/GBq) in bone metastases. No grade 4 adverse events were observed. Grade 3 events occurred in 3 patients-1 with pancytopenia, 1 with leukocytopenia, and 1 with pain flare-up; 3 patients reported a pain response. Conclusion:177Lu-FAP-2286 PTRT, applied in a broad spectrum of cancers, was relatively well tolerated, with acceptable side effects, and demonstrated long retention of the radiopeptide. Prospective clinical studies are warranted.

Alpha-Enolase: Emerging Tumor-Associated Antigen, Cancer Biomarker, and Oncotherapeutic Target.

Alpha-enolase, also known as enolase-1 (ENO1), is a glycolytic enzyme that "moonlights" as a plasminogen receptor in the cell surface, particularly in tumors, contributing to cancer cell proliferation, migration, invasion, and metastasis. ENO1 also promotes other oncogenic events, including protein-protein interactions that regulate glycolysis, activation of signaling pathways, and resistance to chemotherapy. ENO1 overexpression has been established in a broad range of human cancers and is often associated with poor prognosis. This increased expression is usually accompanied by the generation of anti-ENO1 autoantibodies in some cancer patients, making this protein a tumor associated antigen. These autoantibodies are common in patients with cancer associated retinopathy, where they exert pathogenic effects, and may be triggered by immunodominant peptides within the ENO1 sequence or by posttranslational modifications. ENO1 overexpression in multiple cancer types, localization in the tumor cell surface, and demonstrated targetability make this protein a promising cancer biomarker and therapeutic target. This mini-review summarizes our current knowledge of ENO1 functions in cancer and its growing potential as a cancer biomarker and guide for the development of novel anti-tumor treatments.

Psychosocial Stress, Glucocorticoid Signaling, and Prostate Cancer Health Disparities in African American Men.

Recent advances in our understanding of racial disparities in prostate cancer (PCa) incidence and mortality that disproportionately affect African American (AA) men have provided important insights into the psychosocial, socioeconomic, environmental, and molecular contributors. There is, however, limited mechanistic knowledge of how the interplay between these determinants influences prostate tumor aggressiveness in AA men and other men of African ancestry. Growing evidence indicates that chronic psychosocial stress in AA populations leads to sustained glucocorticoid signaling through the glucocorticoid receptor (GR), with negative physiological and pathological consequences. Compelling evidence indicates that treatment of castration-resistant prostate cancer (CRPC) with anti-androgen therapy activates GR signaling. This enhanced GR signaling bypasses androgen receptor (AR) signaling and transcriptionally activates both AR-target genes and GR-target genes, resulting in increased prostate tumor resistance to anti-androgen therapy, chemotherapy, and radiotherapy. Given its enhanced signaling in AA men, GR-together with specific genetic drivers-may promote CRPC progression and exacerbate tumor aggressiveness in this population, potentially contributing to PCa mortality disparities. Ongoing and future CRPC clinical trials that combine standard of care therapies with GR modulators should assess racial differences in therapy response and clinical outcomes in order to improve PCa health disparities that continue to exist for AA men.

89Zr-Chloride Can Be Used for Immuno-PET Radiochemistry Without Loss of Antigen Reactivity In Vivo.

89Zr immuno-PET continues to be assessed in numerous clinical trials. This report evaluates the use of 89Zr-chloride in the radiolabeling of monoclonal antibodies conjugated with desferrioxamine B (DFO), describes its effects on radiopharmaceutical reactivity toward antigen, and offers guidance on how to ensure long-term stability and purity. Methods:89Zr-DFO-trastuzumab and 89Zr-DFO-cetuximab were prepared using 89ZrCl4 The stability of each was evaluated for 7 d in 20 mM histidine/240 mM sucrose buffer, 0.25 M sodium acetate (NaOAc) buffer containing 5 mg·mL-1n-acetyl-l-cysteine (NAC), or 0.25 M NaOAc containing 5 mg·mL-1 l-methionine (L-MET). To assess antigen reactivity, 89Zr-DFO-trastuzumab was evaluated using the Lindmo method and tested in PET/CT imaging of mouse models of human epidermal growth factor receptor 2-positive or -negative lung cancer. Results: Using 89ZrCl4, 89Zr-DFO-trastuzumab and 89Zr-DFO-cetuximab were prepared with increased specific activity and retained purities of 95% after 3 d when formulated in NaOAc buffer containing L-MET. Based on Lindmo analysis and small-animal PET/CT imaging, 89Zr-DFO-trastuzumab remained reactive toward antigen after being prepared with 89ZrCl4Conclusion:89ZrCl4 facilitated the radiosynthesis of 89Zr immuno-PET agents with increased specific activity. L-MET enhanced long-term solution stability better than all other formulations examined, and 89Zr-DFO-trastuzumab remained reactive toward antigen. Although further evaluation is necessary, these initial results suggest that 89ZrCl4 may be useful in immuno-PET radiochemistry as radiolabeled monoclonal antibodies are increasingly integrated into precision medicine strategies.

Peptide-based PET imaging of the tumor restricted IL13RA2 biomarker.

Peptides that target cancer cell surface receptors are promising platforms to deliver diagnostic and therapeutic payloads specifically to cancer but not normal tissue. IL13RA2 is a tumor-restricted receptor found to be present in several aggressive malignancies, including in the vast majority of high-grade gliomas and malignant melanoma. This receptor has been successfully targeted for diagnostic and therapeutic purposes using modified IL-13 ligand and more recently using a specific peptide, Pep-1L. In the current work, we establish the in vitro and in vivo tumor binding properties of radiolabeled Pep-1L, designed for tumor imaging. We radiolabeled Pep-1L with Copper-64 and demonstrated specific cell uptake in the IL13RA2-over expressing G48 glioblastoma cell line having abundant IL13RA2 expression. [64Cu]Pep-1L binding was blocked by unlabeled ligand, demonstrating specificity. To demonstrate in vivo tumor uptake, we intravenously injected into tumor-bearing mice and demonstrated that [64Cu]Pep-1L specifically bound tumors at 24 hours, which was significantly blocked (3-fold) by pre-injecting unlabeled peptide. To further demonstrate specificity of Pep-1L towards IL13RA2 in vivo, we exploited an IL13RA2-inducible melanoma tumor model that does not express receptor at baseline but expresses abundant receptor after treatment with doxycycline. We injected [64Cu]Pep-1L into mice bearing IL13RA2-inducible melanoma tumors and performed in vivo PET/CT and post-necropsy biodistribution studies and found that tumors that were induced to express IL13RA2 receptor by doxycycline pretreatment bound radiolabeled Pep-1L 3-4 fold greater than uninduced tumors, demonstrating receptor specificity. This work demonstrates that [64Cu]Pep-1L selectively binds hIL13RA2-expressing tumors and validates Pep-1L as an effective platform to deliver diagnostics and therapeutics to IL13RA2-expressing cancers.

Automated synthesis of 1-[11C]acetoacetate on a TRASIS AIO module.

We automated radiochemical synthesis of 1-[11C]acetoacetate in a commercially available radiochemistry module, TRASIS AllInOne by [11C]carboxylation of the corresponding enolate anion generated in situ from isopropenylacetate and MeLi, and purified by ion-exchange column resins.1-[11C]acetoacetate was synthesized with high radiochemical purity (95%) and specific activity (~ 66.6GBq/µmol, n = 30) with 35% radiochemical yield, decay corrected to end of synthesis. The total synthesis required ~ 16min. PET imaging studies were conducted with 1-[11C]acetoacetate in vervet monkeys to validate the radiochemical synthesis. Tissue uptake distribution was similar to that reported in humans.

IL13RA2 targeted alpha particle therapy against glioblastomas.

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [64Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [225Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups (p < 0.05). GBM tissue extracted from mice treated with [225Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [225Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

Radiolabeling and initial biological evaluation of [18F]KBM-1 for imaging RAR-α receptors in neuroblastoma.

Retinoic acid receptor alpha (RAR-α) plays a significant role in a number of diseases, including neuroblastoma. Children diagnosed with high-risk neuroblastoma are treated13-cis-retinoic acid, which reduces risk of cancer recurrence. Neuroblastoma cell death is mediated via RAR-α, and expression of RAR-α is upregulated after treatment. A molecular imaging probe that binds RAR-α will help clinicians to diagnose and stratify risk for patients with neuroblastoma, who could benefit from retinoid-based therapy. In this study, we report the radiolabeling, and initial in vivo evaluation of [18F]KBM-1, a novel RAR-α agonist. The radiochemical synthesis of [18F]KBM-1 was carried out through KHF2 assisted substitution of [18F]- from aryl-substituted pinacolatoesters-based retinoid precursor. In vitro cell uptake assay in human neuroblastoma cell line showed that the uptake of [18F]KBM-1 was significantly inhibited by all three blocking agents (KBM-1, ATRA, BD4) at all the selected incubation times. Standard biodistribution in mice bearing neuroblastoma tumors demonstrated increased tumor uptake from 5min to 60min post radiotracer injection and the uptake ratios for target to non-target (tumor: muscle) increased 2.2-fold to 3.7-fold from 30min to 60min post injection. Tumor uptake in subset of 30min blocking group was 1.7-fold lower than unblocked. These results demonstrate the potential utility of [18F]KBM-1 as a RAR-α imaging agent.

Cerebral Misery Perfusion on Susceptibility Weighted Imaging in Acute Carotid Dissection.

The cerebral vasculature incorporates several fail-safes that must be breached before an irreversible ischemic event takes place. In particular, when autoregulatory vasodilatation fails secondary to falling cerebral perfusion pressure (CPP; stage I hemodynamic failure), increases in the oxygen extraction fraction work to maintain the cerebral metabolic rate of oxygen. Previously, failure of this mechanism, stage II hemodynamic failure, or misery perfusion, has been imaged via positron emission tomography/computed tomography (PET/CT). Current susceptibility-weighted sequences (SWI) allow for more efficient imaging of this physiology. In this case, we identify an incident of reversible ischemia caused by spontaneous carotid artery dissection using a combination of diffusion weighted imaging (DWI) and SWI. The level of hemodynamic failure identified by the imaging sequences elevated the urgency of neurointervention, expediting the patient's arrival to the neurointerventional table and thus avoiding impending irreversible ischemia.

Docosahexanoic acid antagonizes TNF-α-induced necroptosis by attenuating oxidative stress, ceramide production, lysosomal dysfunction, and autophagic features.

OBJECTIVE: It was previously reported that docosahexanoic acid (DHA) reduces TNF-α-induced necrosis in L929 cells. However, the mechanisms underlying this reduction have not been investigated. The present study was designed to investigate cellular and biochemical mechanisms underlying the attenuation of TNF-α-induced necroptosis by DHA in L929 cells. METHODS: L929 cells were pre-treated with DHA prior to exposure to TNF-α, zVAD, or Necrostatin-1 (Nec-1). Cell death and survival were assessed by MTT and caspase activity assays, and microscopic visualization. Reactive oxygen species (ROS) were measured by flow cytometry. C16- and C18-ceramides were measured by mass spectrometry. Lysosomal membrane permeabilization (LMP) was evaluated by fluorescence microscopy and flow cytometry using Acridine Orange. Cathepsin L activation was evaluated by immunoblotting and fluorescence microscopy. Autophagy was assessed by immunoblotting of LC3-II and Beclin. RESULTS: Exposure of L929 cells to TNF-α alone for 24 h induced necroptosis, as evidenced by the inhibition of cell death by Nec-1, absence of caspase-3 activity and Lamin B cleavage, and morphological analysis. DHA attenuated multiple biochemical events associated with TNF-α-induced necroptosis, including ROS generation, ceramide production, lysosomal dysfunction, cathepsin L activation, and autophagic features. DHA also attenuated zVAD-induced necroptosis but did not attenuate the enhanced apoptosis and necrosis induced by the combination of TNF-α with Actinomycin D or zVAD, respectively, suggesting that its protective effects might be limited by the strength of the cell death insult induced by TNF-α. CONCLUSIONS: DHA effectively attenuates TNF-α-induced necroptosis and autophagy, most likely via its ability to inhibit TNF-α-induced sphingolipid metabolism and oxidative stress. These results highlight the role of this Omega-3 fatty acid in antagonizing inflammatory cell death.

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.

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

BACKGROUND: Hormone-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. RESULTS: We 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. CONCLUSION: These 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.

Characterization of methyl-beta-cyclodextrin toxicity in NGF-differentiated PC12 cell death.

Cyclodextrins (CDs) are used to deliver hydrophobic molecules in aqueous environments. Methyl-beta-cyclodextrin (MbetaCD), a member of this family of molecules, has been proposed to be a good carrier to deliver fatty acids to cells in culture. This report focuses on studying the in vitro effects of MbetaCD on nerve growth factor-differentiated PC12 (NGFDPC12) cells, a tissue culture model to study neuronal survival and differentiation. The main findings are: (1) NGFDPC12 cells have normal viability when exposed to 0.12% MbetaCD but showed a significant loss in cell viability at higher concentrations; (2) NGFDPC12 cells exposed to 0.25% MbetaCD exhibit nuclear condensation, blebbing and apoptotic bodies, and whole cell lysates exhibited an increase in caspase-3-like activity and high levels of Bax and Bcl-X(L) protein expression compared to control. Cultures treated with 0.25% MbetaCD also showed cleavage of normal 21-kDa Bax protein into a 18-kDa fragment. (3) Experiments using 0.12% MbetaCD to deliver oleic acid did not affect cell viability, in contrast NGFDPC12 cultures in which 0.25% MbetaCD concentration is used exhibited similar loss of cell viability as observed with 0.25% MbetaCD alone. Treating these cultures with caspase-3 inhibitor z-VAD-fmk did not protect the cells from MbetaCD toxic effects. (4) Immortalized Schwann cells (iSC) exposed to MbetaCD 0.12% did not show loss of cell viability while 0.25% MbetaCD triggered a significant toxicity but with a different dose and time course dynamic than NGFDPC12 cells. Thus, NGFDPC12 or iSC cell cultures exposed to 0.12% MbetaCD exhibits normal viability while higher concentrations increase in cell death and apoptosis.