Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy

Purpose

PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo.

Experimental Design

We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitonealy administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated.

Results

We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed.

Conclusion

Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.     

Identification and characterization of cells with high angiogenic potential and transitional phenotype in calcific aortic valve

Recent data suggest that angiogenesis plays an important role in the pathogenesis of valvular disease. However, the cellular mechanisms underlying this process remain unknown. This study aimed at identifying and characterizing the cellular components responsible for pathological neovascularization in calcific aortic valves (CAV). Immunohistochemical analysis of uncultured CAV tissues revealed that smooth muscle alpha-actin (alpha-SMA)-positive cells, which coexpressed Tie-2 and vascular endothelial growth factor receptor-2 (VEGFR-2), can be identified prior to the initiation of capillary-like tube formation. In a second step, leaflets of CAV and non-calcific aortic valves (NCAV) were cultured and the cells involved in capillary-like tube formation were isolated. The majority of these cells displayed the same phenotype as non-cultured cells identified in CAV tissues, i.e., expression of alpha-SMA, Tie-2, and VEGFR-2. In comparison to cells isolated from cultures of NCAV leaflets, these cells showed enhanced angiogenic activity as demonstrated by migration and tube assays. The coexpression of VEGFR-2 and Tie-2 together with alpha-SMA suggests both endothelial and mesenchymal properties of the angiogenically activated cells involved in valvular neovascularization. Hence, our findings might provide new insights into the process of pathological angiogenesis in cardiac valves.     

Novel strategies for inhibition of bacterial biofilm in chronic rhinosinusitis

An important role has been recently reported for bacterial biofilm in the pathophysiology of chronic diseases, such as chronic rhinosinusitis (CRS). CRS, affecting sinonasal mucosa, is a persistent inflammatory condition with a high prevalence around the world. Although the exact pathological mechanism of this disease has not been elicited yet, biofilm formation is known to lead to a more significant symptom burden and major objective clinical indicators. The high prevalence of multidrug-resistant bacteria has severely restricted the application of antibiotics in recent years. Furthermore, systemic antibiotic therapy, on top of its insufficient concentration to eradicate bacteria in the sinonasal biofilm, often causes toxicity, antibiotic resistance, and an effect on the natural microbiota, in patients. Thus, coming up with alternative therapeutic options instead of systemic antibiotic therapy is emphasized in the treatment of bacterial biofilm in CRS patients. The use of topical antibiotic therapy and antibiotic eluting sinus stents that induce higher antibiotic concentration, and decrease side effects could be helpful. Besides, recent research recognized that various natural products, nitric oxide, and bacteriophage therapy, in addition to the hindered biofilm formation, could degrade the established bacterial biofilm. However, despite these improvements, new antibacterial agents and CRS biofilm interactions are complicated and need extensive research. Finally, most studies were performed in vitro, and more preclinical animal models and human studies are required to confirm the collected data. The present review is specifically discussing potential therapeutic strategies for the treatment of bacterial biofilm in CRS patients.     

Extracellular micro/nanovesicles rescue kidney from ischemia-reperfusion injury

Acute renal failure (ARF) is a clinical challenge that is highly resistant to treatment, and its high rate of mortality is alarming. Ischemia–reperfusion injury (IRI) is the most common cause of ARF. Especially IRI is implicated in kidney transplantation and can determine graft survival. Although the exact pathophysiology of renal IRI is unknown, the role of inflammatory responses has been elucidated. Because mesenchymal stromal cells (MSCs) have strong immunomodulatory properties, they are under extensive investigation as a therapeutic modality for renal IRI. Extracellular vesicles (EVs) play an integral role in cell-to-cell communication. Because the regenerative potential of the MSCs can be recapitulated by their EVs, the therapeutic appeal of MSC-derived EVs has dramatically increased in the past decade. Higher safety profile and ease of preservation without losing function are other advantages of EVs compared with their producing cells. In the current review, the preliminary results and potential of MSC-derived EVs to alleviate kidney IRI are summarized. We might be heading toward a cell-free approach to treat renal IRI.     

Effect of single nucleotide polymorphisms in SEPS1 and SEPP1 on expression in the protein level in metabolic syndrome in subjects with cardiovascular disease

Molecular Biology Reports - Metabolic syndrome (MetS) results from the interaction between environmental and genetic factors. Several previous studies considered the role of selenium in developing...     

Role of thrombin in the pathogenesis of atherosclerosis

Atherosclerosis is an arterial disease associated with inflammation. Thrombin is a procoagulant and proinflammatory serine protease that contributes to the pathology of atherosclerosis by enhancing the expression of cell adhesion molecules, inducing the secretion of proinflammatory cytokines, activating inflammatory responses in atherosclerotic plaques, stimulating proliferation of aortic smooth muscle cells, and exacerbating vascular lesions at sites of injury. Hence, thrombin appears to be an important target for treatment of atherosclerosis and thrombin pharmacological inhibitors have significant therapeutic potency for suppressing inflammatory responses in cardiovascular diseases. This review summarizes the proinflammatory signaling functions of thrombin as well as the therapeutic potency of thrombin inhibitors in the pathogenesis of atherosclerosis and hence their potential therapeutic value in this condition.     

Relative contribution of hemopoietic and pulmonary parenchymal cells to lung inducible nitric oxide synthase (inos) activity in murine endotoxemia

Acute lung injury is an important feature of sepsis and increased iNOS expression and NO production contribute to the pathogenesis of this syndrome. We generated bone marrow-transplanted chimeric mice with iNOS expression limited to either inflammatory or pulmonary parenchymal cells, and assessed pulmonary iNOS activity and systemic levels of NO metabolites in an endotoxemic model of sepsis. We found that while both pulmonary parenchymal cells and inflammatory cells contribute to the increased lung iNOS activity in endotoxemia, pulmonary parenchymal cells contribute to a significantly greater degree. Using measurement of plasma NO(-)(x), whole body NO production was assessed in this model. We found that the main source of NO(-)(x) was again, parenchymal cells and not inflammatory cells. This is the first study to demonstrate that most of the increased NO production in this model of endotoxemic sepsis derives from parenchymal cells rather than inflammatory cells.     

Differential expression of human basic fibroblast growth factor in Escherichia coli: potential role of promoter

Four different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (hbFGF), using Escherichia coli as host organism. The hbfgf structural gene was cloned into four expression vectors, pET-3a, pTrc99A, pPR37 and pKK223-3 differing only in their promoters, which were T7, trc, PR and tac respectively. Expression of the gene was induced by adding 0.5 mM (final concentration) of isopropyl--D-thio-galactopyranoside (IPTG) for the vectors carrying T7, trc and tac promoters or by a temperature shift from 30 to 42 °C for the vector carrying PR. The highest level of expression was observed in pET-1005 (a pET-3a derivative)/BL21 (DE3) system with 18.5 mg/l rhbFGF and the second high level expression was in pR37-1007 (pPR37 derivative) BL21 (DE3) system with 5 mg of rhbFGF/l. Since in the latter system a temperature shift was used for induction, 29% of the hbFGF was recovered as inclusion bodies in the insoluble cell fraction. The level of expression for the two other systems (pTrc-1006 and pKK-1008) was very low.     

Partitioning glucose distribution/transport, disposal, and endogenous production during IVGTT

We have separated the effect of insulin on glucose distribution/transport, glucose disposal, and endogenous production (EGP) during an intravenous glucose tolerance test (IVGTT) by use of a dual-tracer dilution methodology. Six healthy lean male subjects (age 33 +/- 3 yr, body mass index 22.7 +/- 0.6 kg/m(2)) underwent a 4-h IVGTT (0.3 g/kg glucose enriched with 3-6% D-[U-(13)C]glucose and 5-10% 3-O-methyl-D-glucose) preceded by a 2-h investigation under basal conditions (5 mg/kg of D-[U-(13)C]glucose and 8 mg/kg of 3-O-methyl-D-glucose). A new model described the kinetics of the two glucose tracers and native glucose with the use of a two-compartment structure for glucose and a one-compartment structure for insulin effects. Insulin sensitivities of distribution/transport, disposal, and EGP were similar (11.5 +/- 3.8 vs. 10.4 +/- 3.9 vs. 11.1 +/- 2.7 x 10(-2) ml small middle dot kg(-1) small middle dot min(-1) per mU/l; P = nonsignificant, ANOVA). When expressed in terms of ability to lower glucose concentration, stimulation of disposal and stimulation of distribution/transport accounted each independently for 25 and 30%, respectively, of the overall effect. Suppression of EGP was more effective (P < 0.01, ANOVA) and accounted for 50% of the overall effect. EGP was suppressed by 70% (52-82%) (95% confidence interval relative to basal) within 60 min of the IVGTT; glucose distribution/transport was least responsive to insulin and was maximally activated by 62% (34-96%) above basal at 80 min compared with maximum 279% (116-565%) activation of glucose disposal at 20 min. The deactivation of glucose distribution/transport was slower than that of glucose disposal and EGP (P < 0.02) with half-times of 207 (84-510), 12 (7-22), and 29 (16-54) min, respectively. The minimal-model insulin sensitivity was tightly correlated with and linearly related to sensitivity of EGP (r = 0.96, P < 0.005) and correlated positively but nonsignificantly with distribution/transport sensitivity (r = 0.73, P = 0.10) and disposal sensitivity (r = 0.55, P = 0.26). We conclude that, in healthy subjects during an IVGTT, the two peripheral insulin effects account jointly for approximately one-half of the overall insulin-stimulated glucose lowering, each effect contributing equally. Suppression of EGP matches the effect in the periphery.