Sigma-2 receptor agonist derivatives of 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) induce cell death via mitochondrial superoxide production and caspase activation in pancreatic cancer

Abstract Background Despite considerable efforts by scientific research, pancreatic cancer is the fourth leading cause of cancer related mortalities. Sigma-2 receptors, which are overexpressed in several tumors, represent promising targets for triggering selective pancreatic cancer cells death. Methods We selected five differently structured high-affinity sigma-2 ligands (PB28, PB183, PB221, F281 and PB282) to study how they affect the viability of diverse pancreatic cancer cells (human cell lines BxPC3, AsPC1, Mia PaCa-2, and Panc1 and mouse Panc-02, KCKO and KP-02) and how this is reflected in vivo in a tumor model. Results Important cytotoxicity was shown by the compounds in the aggressive Panc02 cells, where cytotoxic activity was caspase-3 independent for four of the five compounds. However, both cytotoxicity and caspase-3 activation involved generation of Reactive Oxygen Species (ROS), which could be partially reverted by the lipid antioxidant \u3b1-tocopherol, but not by the hydrophilic N-acetylcysteine (NAC) indicating crucial differences in the intracellular sites exposed to oxidative stress induced by sigma-2 receptor ligands. Importantly, all the compounds strongly increased the production of mitochondrial superoxide radicals except for PB282. Despite a poor match between in vitro and the in vivo efficacy, daily treatment of C57BL/6 mice bearing Panc02 tumors resulted in promising effects with PB28 and PB282 which were similar compared to the current standard-of-care chemotherapeutic gemcitabine without showing signs of systemic toxicities. Conclusions Overall, this study identified differential sensitivities of pancreatic cancer cells to structurally diverse sigma-2 receptor ligands. Of note, we identified the mitochondrial superoxide pathway as a previously unrecognized sigma-2 receptor-activated process, which encourages further studies on sigma-2 ligand-mediated cancer cell death for the targeted treatment of pancreatic tumors

New roles for the major human 3'-5' exonuclease TREX1 in human disease

Aicardi-Goutières syndrome (AGS), Systemic Lupus Erythematosus (SLE), Familial Chilblain Lupus (FCL) and Retinal Vasculopathy and Cerebral Leukodystrophy (RVCL) {a new term encompassing three independently described conditions with a common etiology—Cerebroretinal Vasculopathy (CRV), Hereditary Vascular Retinopathy (HVR) and Hereditary Endotheliopathy, Retinopathy and Nephropathy (HERNS)}—have previously been regarded as distinct entities. However, recent genetic analysis has demonstrated that each of these diseases maps to chromosome 3p21 and can be caused by mutations in TREX1, the major human 3'–5' exonuclease. In this review, we discuss the putative functions of TREX1 in relationship to the clinical, genetic and functional characteristics of each of these conditions

Modification of titanium implants surface by microarc oxidation in presence of polymeric solutions. The study of immune response

In the present study, we performed the in vitro evaluation of titanium samples with four types of coatings obtained by microarc oxidation (MAO): pure calcium phosphate (CaP), CaP- chitosan, CaP- hyaluronic acid, CaP - polyvinylpyrrolidone coatings. AlamarBlue test and TNFα secretion analysis (ELISA) were performed to investigate the immune response. It was shown that formation of MAO hybrid calciumphosphate coatings increases the biocompatibility of samples. Adding of polyvinylpyrrolidone and hyaluronic acid to the electrolyte solution presumably makes it possible to obtain hybrid coatings with anti-inflammatory properties

Lysosomal membrane permeabilization is an early event in sigma-2 receptor ligand mediated cell death in pancreatic cancer

BACKGROUND: Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. RESULTS: Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. CONCLUSIONS: Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and α-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways

Mesothelin\u27s minimal MUC16 binding moiety converts TR3 into a potent cancer therapeutic via hierarchical binding events at the plasma membrane

TRAIL has been extensively explored as a cancer drug based on its tumor-selective activity profile but it is incapable per se of discriminating between death receptors expressed by normal host cells and transformed cancer cells. Furthermore, it is well documented that surface tethering substantially increases its biologic activity. We have previously reported on Meso-TR3, a constitutive TRAIL trimer targeted to the biomarker MUC16 (CA125), in which the entire ectodomain of human mesothelin was genetically fused to the TR3 platform, facilitating attachment to the cancer cells via the MUC16 receptor. Here, we designed a truncation variant, in which the minimal 64 amino acid MUC16 binding domain of mesothelin was incorporated into TR3. It turned out that the dual-domain biologic Meso64-TR3 retained its high MUC16 affinity and bound to the cancer cells quickly, independent of the TR3/death receptor interaction. Furthermore, it was substantially more potent than Meso-TR3 and TR3 in vitro and in a preclinical xenograft model of MUC16-dependent ovarian cancer. Phenotypically, Meso64-TR3 is more closely related to non-targeted TR3, evident by indistinguishable activity profiles on MUC16-deficient cancers and similar thermal stability characteristics. Overall, Meso64-TR3 represents a fully human, MUC16-targetd TRAIL-based biologic, ideally suited for exploring preclinical and clinical evaluation studies in MUC16-dependent malignancies

The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine

<p>Abstract</p> <p>Background</p> <p>Sigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy.</p> <p>Results</p> <p>The binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM <it>in vitro</it>. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our <it>in vivo </it>model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities.</p> <p>Conclusions</p> <p>This study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.</p

The Targeted SMAC Mimetic SW IV-134 is a strong enhancer of standard chemotherapy in pancreatic cancer

Abstract Background Pancreatic cancer is a lethal malignancy that frequently acquires resistance to conventional chemotherapies often associated with overexpression of inhibitors of apoptosis proteins (IAPs). We have recently described a novel means to deliver second mitochondria-derived activator of caspases (SMAC) mimetics selectively to cancer cells employing the sigma-2 ligand/receptor interaction. The intrinsic death pathway agonist SMAC offers an excellent opportunity to counteract the anti-apoptotic activity of IAPs. SMAC mimetics have been used to sensitize several cancer types to chemotherapeutic agents but cancer-selective delivery and appropriate cellular localization have not yet been considered. In our current study, we tested the ability of the sigma-2/SMAC drug conjugate SW IV-134 to sensitize pancreatic cancer cells to gemcitabine. Methods Using the targeted SMAC mimetic SW IV-134, inhibition of the X-linked inhibitor of apoptosis proteins (XIAP) was induced pharmacologically and its impact on cell viability was studied alone and in combination with gemcitabine. Pathway analyses were performed by assessing caspase activation, PARP cleavage and membrane blebbing (Annexin-V), key components of apoptotic cell death. Single-agent treatment regimens were compared with combination therapy in a preclinical mouse model of pancreatic cancer. Results The sensitizing effect of XIAP interference toward gemcitabine was confirmed via pharmacological intervention using our recently designed, targeted SMAC mimetic SW IV-134 across a wide range of commonly used pancreatic cancer cell lines at concentrations where the individual drugs showed only minimal activity. On a mechanistic level, we identified involvement of key components of the apoptosis machinery during cell death execution. Furthermore, combination therapy proved superior in decreasing the tumor burden and extending the lives of the animals in a preclinical mouse model of pancreatic cancer. Conclusion We believe that the strong sensitizing capacity of SW IV-134 in combination with clinically relevant doses of gemcitabine represents a promising treatment option that warrants clinical evaluation

Novel treatment option for MUC16-positive malignancies with the targeted TRAIL-based fusion protein Meso-TR3

BACKGROUND: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. METHODS: Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. RESULTS: Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. CONCLUSIONS: Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies