Richard Tobin

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition

BackgroundIt has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination.MethodsThe Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay.ResultsIn our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells.ConclusionMcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors.

Intrauterine growth restriction and prematurity influence regulatory T cell development in newborns

PURPOSE The aim of this study was to determine the relationship of birth weight and gestational age with regulatory T cells (Tregs) in cord blood of human newborns. METHODS Cord blood mononuclear cells (CBMCs) of 210 newborns were analyzed using flow cytometry to identify Tregs (CD3(+), CD4(+), CD25(high), FoxP3(high)) and measure FoxP3 mean fluorescence intensity (MFI). Suppressive index (SI) was calculated as FoxP3 MFI per Treg. RESULTS Mode of delivery had no significant effect on Tregs at birth. Term babies with growth restriction had fewer Tregs than their appropriate weight counterparts but equivalent SI. Preterm babies had higher percentages of Tregs, but lower SI than term controls. SI steadily increased through gestation. CONCLUSIONS Intrauterine growth restriction is correlated with fewer circulating Tregs and prematurity with decreased functionality of Tregs compared to term appropriate weight infants. This may have implications in diseases such as necrotizing enterocolitis that disproportionately affect premature and lower birth weight infants.

Inhibition of Fas-Fas ligand interaction attenuates microvascular hyperpermeability following hemorrhagic shock.

ABSTRACT Hemorrhagic shock (HS)–induced microvascular hyperpermeability poses a serious challenge in the management of trauma patients. Microvascular hyperpermeability occurs mainly because of the disruption of endothelial cell adherens junctions, where the “intrinsic” apoptotic signaling plays a regulatory role. The purpose of this study was to understand the role of the “extrinsic” apoptotic signaling molecules, particularly Fas–Fas ligand interaction in microvascular endothelial barrier integrity. Rat lung microvascular endothelial cells (RLMECs) were exposed to HS serum in the presence or absence of the Fas ligand inhibitor, FasFc. The effect of HS serum on Fas receptor and Fas ligand expression on RLMECs was determined by flow cytometry. Endothelial cell permeability was determined by monolayer permeability assay and the barrier integrity by &bgr;-catenin immunofluorescence. Mitochondrial reactive oxygen species formation was determined using dihydrorhodamine 123 probe by fluorescent microscopy. Mitochondrial transmembrane potential was studied by fluorescent microscopy as well as flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Rat lung microvascular endothelial cells exposed to HS serum showed increase in Fas receptor and Fas ligand expression levels. FasFc treatment showed protection against HS serum-induced disruption of the adherens junctions and monolayer hyperpermeability (P < 0.05) in the endothelial cells. Pretreatment with FasFc also decreased HS serum-induced increase in mitochondrial reactive oxygen species formation, restored HS serum-induced drop in mitochondrial transmembrane potential, and reduced HS serum-induced caspase 3 activity in RLMECs. These findings open new avenues for drug development to manage HS-induced microvascular hyperpermeability by targeting the Fas–Fas ligand–mediated pathway.

Microvascular endothelial cell hyperpermeability induced by endogenous caspase 3 activator staurosporine.

BACKGROUND Microvascular hyperpermeability following conditions such as hemorrhagic shock occurs mainly owing to disruption of the adherens junctional protein complex in endothelial cells. The objective of this study was to examine the action of staurosporine, a potent activator of endogenous caspase 3 on the adherens junction and the cellular pathway through which it causes possible endothelial cell barrier dysfunction. METHODS Rat lung microvascular endothelial cell (RLMEC) permeability was measured by fluorescein isothiocyanate-albumin flux across the monolayer in a Transwell plate. Integrity of the endothelial cell adherens junctions was studied using immunofluorescence of &bgr;-catenin and vascular endothelial-cadherin. Mitochondrial reactive oxygen species formation was determined by using dihydrorhodamine 123 and mitochondrial transmembrane potential by JC-1 fluorescent probe and flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Cell death assay in RLMECs was performed using propidium iodide staining and analyzed by flow cytometry. RESULTS Staurosporine (1 µM)–treated RLMEC monolayers showed significant increase in permeability, which was decreased by pretreatment with caspase 3 specific inhibitor, Z-DEVD-FMK (p < 0.05). Immunofluorescence studies showed staurosporine induced disruption of the adherens junction, which was reversed by Z-DEVD-FMK. Staurosporine treatment led to an increase in mitochondrial reactive oxygen species formation and a decrease in mitochondrial transmembrane potential. Furthermore, staurosporine induced a significant increase in caspase 3 activity (p < 0.05) but not cell death in RLMECs (p < 0.05). CONCLUSION Staurosporine-induced disruption of the adherens junction and microvascular endothelial cell hyperpermeability is associated with the activation of mitochondrial “intrinsic” apoptotic signaling cascade but without causing endothelial cell death. Our results suggest that prevention of mitochondrial-mediated activation of caspase 3 has therapeutic potential against microvascular hyperpermeability.

Vitamin D3 Suppresses Class II Invariant Chain Peptide Expression on Activated B-Lymphocytes: A Plausible Mechanism for Downregulation of Acute Inflammatory Conditions

Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP+ B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (P value = 0.01)]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings.