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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-human results using 177Lu-FAP-2286 for peptide-targeted radionuclide therapy (PTRT). Methods: PTRT using 177Lu-FAP-2286 was performed in 11 patients with advanced adenocarcinomas of pancreas, breast, rectum and ovary after prior confirmation of uptake on 68Ga-FAP-2286/-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 doses were 0.07 ± 0.02 Gy/GBq (range 0.04 – 0.1). The mean absorbed doses for kidneys and red marrow were 1.0 ± 0.6 Gy/GBq (range 0.4 – 2.0) and 0.05 ± 0.02 Gy/GBq (range 0.03 – 0.09), respectively. Significant uptake and long tumor retention of 177Lu-FAP-2286 resulted in high absorbed tumor doses, e.g., 3.0 ± 2.7 Gy/GBq (range 0.5 – 10.6) in bone metastases. No grade (G) 4 adverse events were observed. G3 events occurred in 3 patients – 1 pancytopenia, 1 leukocytopenia and 1 pain flare-up; 3 patients reported 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.

Radiopharmaceutical therapy utilizing

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

(89)Zr immuno-PET continues to be assessed in numerous clinical trials. This report evaluates the use of (89)Zr-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: (89)Zr-DFO-trastuzumab and (89)Zr-DFO-cetuximab were prepared using (89)ZrCl(4). 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(−1) n-acetyl-l-cysteine (NAC), or 0.25 M NaOAc containing 5 mg·mL(−1) l-methionine (L-MET). To assess antigen reactivity, (89)Zr-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 (89)ZrCl(4), (89)Zr-DFO-trastuzumab and (89)Zr-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, (89)Zr-DFO-trastuzumab remained reactive toward antigen after being prepared with (89)ZrCl(4). Conclusion: (89)ZrCl(4) facilitated the radiosynthesis of (89)Zr immuno-PET agents with increased specific activity. L-MET enhanced long-term solution stability better than all other formulations examined, and (89)Zr-DFO-trastuzumab remained reactive toward antigen. Although further evaluation is necessary, these initial results suggest that (89)ZrCl(4) may be useful in immuno-PET radiochemistry as radiolabeled monoclonal antibodies are increasingly integrated into precision medicine strategies.

// Anirudh Sattiraju 1, 2, * , Kiran Kumar Solingapuram Sai 1, 2, * , Ang Xuan 4, * , Darpan N. Pandya 2 , Frankis G. Almaguel 1 , Thaddeus J. Wadas 1, 3 , Denise M. Herpai 2, 3 , Waldemar Debinski 2, 3 and Akiva Mintz 1, 2 1 Department of Radiology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2 Brain Tumor Center of Excellence, Wake Forest University Comprehensive Cancer Center, Winston-Salem, NC 27157, USA 3 Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 4 Department of Nuclear Medicine and Radiology, The People’s Hospital of Zhengzhou University, Zhengzhou, Henan 450003, China * These authors contributed equally to this work Correspondence to: Akiva Mintz, email: amintz@wakehealth.edu Keywords: glioblastoma, IL13RA2, alpha particle therapy, Actinium-225, Copper-64 Received: January 10, 2017 Accepted: April 15, 2017 Published: May 11, 2017 ABSTRACT 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 [ 64 Cu]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 [ 225 Ac]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 [ 225 Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [ 225 Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

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.

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.

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

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.

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.

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-X(L) to prevent apoptosis. RSV treatment to breast cancer cells inhibited Bcl-2 and Bcl-X(L) 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-X(L) 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-X(L), 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.

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.

Glioblastoma (GBM) is the most aggressive and common primary malignant astrocytoma which is characterized by tumor heterogeneity, infiltrating margins. Radiotherapy, chemotherapy and experimental targeted therapies have been ineffective at meaningfully increasing patient survival. One significant shortcoming of systemically delivered therapies is their inability to cross the blood brain barrier (BBB) and access infiltrating tumor cells. Therefore, in this work, we tested the potential of locoregionally targeting GBM via IL13Rα2, which we discovered to be expressed on greater than 75% of GBMs. To accomplish this, we intracranially infused copper-64 (64Cu) radiolabeled IL13Rα2 specific peptide Pep-1L, previously developed by Pandya et al., into mice bearing IL13Rα2 expressing orthotopic GBMs. Small animal micro PET/CT imaging showed ~2-fold greater tumor specific localization and lower volume of distribution of 64Cu-Pep-1L within brains of mice. Post-PET biodistribution study showed greater retention of 64Cu-Pep-1L 4 hours (%ID/g = 20.85± 0.65) and 24 hours (%ID/g = 14.65± 0.30) post infusion when compared to similarly infused 64Cu radiolabeled scrambled control peptide 4 hours (%ID/g = 10.73± 2.02) and 24 hours (%ID/g = 5.97± 1.47) post infusion. These results demonstrate that Pep-1L efficiently targets IL13Rα2 expressing GBMs in vivo upon loco-regional delivery and can effectively deliver potential therapeutic agents to GBM tumors while sparing normal brain. This work was supported by the American Cancer Society Mentored Research Scholar grant # 124443-MRSG-13-121-01-CDD (Mintz), 1R01CA179072-01A1 (Mintz), P30 CA012197 (Pasche), R01CA07414519 (Debinski) and the Translational Imaging Program (TIP) of the Wake Forest CTSA (UL1TR001420). Citation Format: Anirudh Sattiraju, Ang Xuan, Frankis Almaguel, Denise Herpai, Waldemar Debinski, Akiva Mintz, Kiran Kumar Solingapuram Sai. PET/CT imaging of interleukin-13 receptor alpha-2-targeted peptide to glioblastoma after locoregional delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1831. doi:10.1158/1538-7445.AM2017-1831

CPI-613 is a lipoate analogue that has been shown to inhibit the pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes selectively in tumor cells (reviewed in Exp.Rev.Clin.Pharma. 7, 837). These two enzymes control the vast majority of carbon flow into the tricarboxylic acid (TCA) cycle and play a central role in mitochondrial metabolism. PDH converts pyruvate into acetyl-CoA, which in turn can enter the TCA cycle for cellular respiration. Since pyruvate is the final product of glycolysis, PDH serves to link glycolysis to the TCA cycle. Early clinical trials with CPI-613 have demonstrated very promising clinical responses in pancreatic cancer, leukemia, and lymphoma when used in combination with standard chemotherapy. Moreover, apparently homeostatic responses of tumor cells to CPI-613 inhibition of mitochondrial metabolism include compensatory upregulation of glucose uptake. Thus, we hypothesized that we can use 18F-Fludeoxyglucose (FDG)-PET/CT, a translational imaging approach that measures cellular glucose uptake, as a biomarker for CPI-613 cellular delivery and its targeting of mitochondrial metabolism. Therefore, in this work we evaluated the in vitro and in vivo glucose uptake in cancer cells and tumor xenografts after treatment with CPI-613. To measure glucose uptake in vitro we pulsed BxPc3 pancreatic cancer cells with 3H 2-deoxyglucose for 15 or 60 minutes following 2 hours of treatment with CPI-613. We observed a significant upregulation (~100% increase) of cellular 2-deoxyglucose uptake, consistent with a compensatory increase in glucose uptake as a result of successful targeting of mitochondrial metabolism. We therefore examined whether this upregulation occurs in vivo using FDG-PET/CT. Mice bearing BxPc3 flank tumors were treated with 50mg/kg of CPI-613 and underwent FDG-PET/CT scans 4 hours and 24 hours post-CPI-613 treatment. Similar to the in vitro response, tumors treated with CPI-613 exhibited a 75% increase in 18F-FDG uptake compared to untreated controls at 4 hours post therapy. In the 24 hour post-therapy scans, tumor 18F-FDG was significantly decreased, indicating tumor cell killing, which corresponds to previously published data that demonstrated efficacy of CPI-613 against BxPc3 tumor xenografts. These results indicate the potential for using the initial FDG flare seen on PET imaging as a biomarker to detect mitochondrial targeting by CPI-613 immediately after CPI-613 treatment, a strategy that may be used, after further clinical validation, to stratify responders to this novel mitochondrial inhibitor. Citation Format: Kiran Solingapuram Sai, Zuzana Zachar, Frankis Almaguel, Shawn D. Stuart, Michael S. Dahan, Moises Guardado, Stephanie Rideout, Minghui Wang, Anirudh Sattiraju, Paul M. Bingham, Boris Pasche, Akiva Mintz. FDG-PET imaging as a potential biomarker of mitochondrial targeting by CPI-613, a novel inhibitor of mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2867. doi:10.1158/1538-7445.AM2017-2867

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.

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.

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 survivin's 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.

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.

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.

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.