Knockdown of the Inhibitor of Apoptosis BRUCE Sensitizes Resistant Breast Cancer Cells to Chemotherapeutic Agents.

BACKGROUND AND OBJECTIVES: Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles. RESULTS AND METHODS: IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone. CONCLUSIONS: Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

ARTS and Siah collaborate in a pathway for XIAP degradation.

ARTS (apoptosis-related protein in the TGF-ß signaling pathway) is a mitochondrial protein that binds XIAP (X-linked inhibitor of apoptosis protein) upon entering the cytosol, thus promoting cell death. Expression of ARTS is lost in some malignancies. Here, we show that ARTS binds to XIAP at BIR1, a domain distinct from the caspase-binding sites. Furthermore, ARTS interacts with the E3 ligase Siah-1 (seven in absentia homolog 1) to induce ubiquitination and degradation of XIAP. Cells lacking either Siah or ARTS contain higher steady-state levels of XIAP. Thus, ARTS serves as an adaptor to bridge Siah-1 to XIAP, targeting it for destruction.

XIAP mediates NOD signaling via interaction with RIP2.

NOD1 and NOD2 are members of the NOD-like receptor (NLR) protein family that are involved in sensing the presence of pathogens and are a component of the innate immune system. Upon activation by specific bacterial peptides derived from peptidoglycans, NODs interact via a CARD-CARD interaction with the receptor-interacting protein kinase RIP2, an inducer of NF-kappaB activation. In this report, we show that NOD signaling is dependent on XIAP, a member of the inhibitor of apoptosis protein (IAP) family. Cells deficient in XIAP exhibit a marked reduction in NF-kappaB activation induced by microbial NOD ligands and by over-expression of NOD1 or NOD2. Moreover, we show that XIAP interacts with RIP2 via its BIR2 domain, which could be disrupted by XIAP antagonists SMAC and SMAC-mimicking compounds. Both NOD1 and NOD2 associated with XIAP in a RIP2-dependent manner, providing evidence that XIAP associates with the NOD signalosome. Taken together, our data suggest a role for XIAP in regulating innate immune responses by interacting with NOD1 and NOD2 through interaction with RIP2.

Novel quinazoline-based compounds impair prostate tumorigenesis by targeting tumor vascularity.

Previous evidence showed the ability of the quinazoline-based alpha(1)-adrenoreceptor antagonist doxazosin to suppress prostate tumor growth via apoptosis. In this study, we carried out structural optimization of the chemical nucleus of doxazosin and a subsequent structure-function analysis toward the development of a novel class of apoptosis-inducing and angiogenesis-targeting agents. Our lead compound, DZ-50, was effective at reducing endothelial cell viability via a nonapoptotic mechanism. Treatment with DZ-50 effectively prevented in vitro tube formation and in vivo chorioallantoic membrane vessel development. Confocal microscopy revealed a significantly reduced ability of tumor cells to attach to extracellular matrix and migrate through endothelial cells in the presence of DZ-50. In vivo tumorigenicty studies using two androgen-independent human prostate cancer xenografts, PC-3 and DU-145, showed that DZ-50 treatment leads to significant suppression of tumorigenic growth. Exposure to the drug at the time of tumor cell inoculation led to prevention of prostate cancer initiation. Furthermore, DZ-50 resulted in a reduced formation of prostate-tumor derived metastatic lesions to the lungs in an in vivo spontaneous metastasis assay. Thus, our drug discovery approach led to the development of a class of lead (quinazoline-based) compounds with higher potency than doxazosin in suppressing prostate growth by targeting tissue vascularity. This new class of quinazoline-based compounds provides considerable promise as antitumor drugs for the treatment of advanced prostate cancer.

Identification of ABR-215050 as lead second generation quinoline-3-carboxamide anti-angiogenic agent for the treatment of prostate cancer.

BACKGROUND: Linomide, Figure 1, produces robust and consistent in vivo growth inhibition of prostate cancer models via its anti-angiogenic activity and inhibition of autoimmune encephalomyelitis models of multiple sclerosis (MS). MS clinical trials were discontinued because of unacceptable toxicity, due to dose-dependent induction of proinflammation. METHODS: Therefore, linomide analogs were initially screened to determine their in vivo potency to inhibit growth of the Dunning R-3327 AT-1 rat prostate cancer model in rats and their potency to inhibit angiogenesis in a Matrigel assay in mice. RESULTS: Based upon its superior potency (i.e., 30- to 60-fold more potent than linomide) in these assays and its lack of a proinflammation in the Beagle-dog, ABR-215050 (tasquinimod), Figure 1, was characterized for dose-response ability to inhibit the growth of a series of four additional human and rodent prostate cancer models in mice. Pharmacokinetic analysis following oral dosing documented that blood and tumor tissue levels of ABR-215050 as low as 0.5-1 microM are therapeutically effective. This efficacy is correlated with inhibition of angiogenesis in a variety of assays (endothelial capillary tube formation, aortic ring assay, chorioallantoic membrane assay, real-time tumor blood flow and PO(2) measurements, tumor blood vessel density, and tumor hypoxic and apoptotic fractions). CONCLUSIONS: Based upon its robust and consistent anti-angiogenic activity and thus tumor growth, ABR-215050 has entered clinical trials for the treatment of prostate cancer.

Doxazosin induces apoptosis of benign and malignant prostate cells via a death receptor-mediated pathway.

Quinazoline-based alpha1-adrenoceptor antagonists such as doxazosin and terazosin have been previously shown to induce apoptosis in prostate cancer cells via an alpha1-adrenoceptor-independent pathway, involving activation of transforming growth factor-beta1 (TGF-beta1) signaling. In this study, the molecular events initiating this apoptotic effect were further investigated in vitro using the human androgen-independent prostate cancer cells PC-3 and the human benign prostate epithelial cells BPH-1. Quantitative microarray assays were done in PC-3 and BPH-1 cells after treatment with doxazosin (25 micromol/L, 6 and 24 hours) to identify the early gene changes. Transient changes in the expression of several apoptosis regulators were identified, including up-regulation of Bax and Fas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3. Moreover, there were significant changes in the expression pattern of signaling components of the extracellular matrix such as integrins alpha2, alphaV, beta1, and beta8. Western blot analysis revealed activation of caspase-8 and caspase-3 within the first 6 to 12 hours of treatment with doxazosin in both PC-3 and BPH-1 cells. Doxazosin-induced apoptosis was blocked by specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of Fas-associated death domain (FADD) and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of the effect of doxazosin in prostate cells. These results show that doxazosin exerts its apoptotic effects against benign and malignant prostate cells via a death receptor-mediated mechanism with a potential integrin contribution towards cell survival outcomes.

Three-dimensional myofiber architecture of the embryonic left ventricle during normal development and altered mechanical loads.

Mechanical load influences embryonic ventricular growth, morphogenesis, and function. To date, little is known regarding how the embryonic left ventricular (LV) myocardium acquires a three-dimensional (3D) fiber architecture distribution or how altered mechanical load influences local myofiber architecture. We tested the hypothesis that altered mechanical load changes the maturation process of local 3D fiber architecture of the developing embryonic LV compact myocardium. We measured transmural myofiber angle distribution in the LV compact myocardium in Hamburger-Hamilton stages 21, 27, 31, and 36 chick embryos during normal development or following either left atrial ligation (LAL; LV hypoplasia model) or conotruncal banding (CTB; LV hyperplasia model). The embryonic LV was stained with f-actin and then z-serial optical sectioning was performed using a laser confocal scanning microscope. We reconstructed local 3D myofiber images and computed local transmural myofiber angle distribution. Transmural myofiber angles in compact myocardium (in LV sagittal sections) were oriented in a circumferential direction until stage 27 (-10 to 10 degrees). Myofibers in the outer side of compact myocardium shifted to a more longitudinal direction by stage 36 (10 to 40 degrees), producing a transmural gradient in myofiber orientation. Developmental changes in transmural myofiber angle distribution were significantly delayed following LAL, while the changes in angle distribution were accelerated following CTB. Results suggest that mechanical load modulates the maturation process of myofiber architecture distribution in the developing LV compact myocardium.

Doxazosin inhibits human vascular endothelial cell adhesion, migration, and invasion.

The quinazoline-derived alpha1-adrenoceptor antagonists, doxazosin and terazosin have been recently shown to induce an anoikis effect in human prostate cancer cells and to suppress prostate tumor vascularity in clinical specimens [Keledjian and Kyprianou, 2003]. This study sought to examine the ability of doxazosin to affect the growth of human vascular endothelial cells and to modulate vascular endothelial growth factor (VEGF)-mediated angiogenesis. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro model to determine the effect of doxazosin on cell growth, apoptosis, adhesion, migration, and angiogenic response of endothelial cells. The effect of doxazosin on cell viability and apoptosis induction of human endothelial cells, was evaluated on the basis of trypan blue and Hoechst 33342 staining, respectively. Doxazosin antagonized the VEGF-mediated angiogenic response of HUVEC cells, by abrogating cell adhesion to fibronectin and collagen-coated surfaces and inhibiting cell migration, via a potential downregulation of VEGF expression. Furthermore there was a significant suppression of in vitro angiogenesis by doxazosin on the basis of VEGF-mediated endothelial tube formation (P < 0.01). Fibroblast growth factor-2 (FGF-2) significantly enhanced HUVEC cell tube formation (P < 0.01) and this effect was suppressed by doxazosin. These findings provide new insight into the ability of doxazosin to suppress the growth and angiogenic response of human endothelial cells by interfering with VEGF and FGF-2 action. This evidence may have potential therapeutic significance in using this quinazoline-based compound as an antiangiogenic agent for the treatment of advanced prostate cancer.

Pharmacological exploitation of the alpha1-adrenoreceptor antagonist doxazosin to develop a novel class of antitumor agents that block intracellular protein kinase B/Akt activation.

The alpha1-adrenoreceptor antagonist doxazosin induces apoptosis in malignant cells with moderate potency via an alpha1-adrenoreceptor-independent mechanism. Here, we demonstrate that the ability of doxazosin to induce apoptosis in PC-3 prostate cancer cells was, in part, attributable to the inhibition of protein kinase B (PKB)/Akt activation. The separation of the effect of doxazosin on apoptosis from its original pharmacological activity provides molecular underpinnings to develop novel antitumor agents. Replacement of the (2,3-dihydro-benzo[1,4]dioxane)-carbonyl moiety of doxazosin with aryl-sulfonyl functions dramatically improves the potency in facilitating Akt deactivation and inducing apoptosis. The optimal compounds, 33 and 44, were effective in apoptosis induction at low micromolar concentrations irrespective of androgen dependency and p53 functional status. Both agents were active in suppressing the growth of a panel of 60 cancer-cell lines with IC50 values of 2.2 and 1.5 microM, respectively. Together, these in vitro efficacy data suggest the translational potential of these agents in prostate cancer treatment.

Novel targeting of apoptosis pathways for prostate cancer therapy.

Selection of treatment options for clinically localized prostate cancer is based on a host of factors including the patient's age, overall health status, potential complications, clinical tumor stage and Gleason score. It is widely acknowledged that androgen independent disease remains the main obstacle to improving the survival and quality of life in patients with advanced prostate cancer. Apoptosis as a genetically regulated process has a critical endpoint that coincides with the therapeutic goal of successful treatment of androgen-dependent and androgen-independent prostate cancer. Opportunities to alter the apoptotic threshold of prostate cancer cells using antisense technology and gene therapy certainly exist, but the scope and extent of their applicability and action depends upon research delineating the many subtleties within the apoptotic pathway. Most epithelial and endothelial cells undergo apoptosis when they loose contact with the extracellular matrix (ECM), via the phenomenon of anoikis. Signaling interaction between growth factor apoptosis-signaling pathways and cellular effectors of anoikis potential and tumor vascularity provides a new molecular basis for optimizing combination approaches for the effective treatment of advanced prostate cancer. Agents that induce epithelial or endothelial cell apoptosis by antagonizing integrin binding are considered for cancer therapy via their ability to inhibit tumor vascularization. This review summarizes the current knowledge of the therapeutic benefit of apoptosis induction within the context of tumor neovascularization inhibition, and provides an insight into the consequences of anoikis induction (by different agents) in targeting angiogenesis in prostate cancer cells.

Regional passive ventricular stress-strain relations during development of altered loads in chick embryo.

Mechanical load influences embryonic ventricular growth, morphogenesis, and function. However, little is known about changes in regional passive ventricular properties during the development of altered mechanical loading conditions in the embryo. We tested the hypothesis that regional mechanical loads are a critical determinant of embryonic ventricular passive properties. We measured biaxial passive right and left ventricular (RV and LV, respectively) stress-strain relations in chick embryos at Hamburger-Hamilton stages 21 and 27 after conotruncal banding (CTB) to increase biventricular pressure load or left atrial ligation (LAL) to reduce LV volume load and increase RV volume load. In the RV, wall strains at end-diastolic (ED) pressure normalized whereas ED stresses increased after either CTB or LAL during development. In the left ventricle, both ED strain and stress normalized after CTB, whereas both remained reduced with significantly increased myocardial stiffness after LAL. These results suggest that the embryonic ventricle adapts to chronically altered mechanical loading conditions by changing specific RV and LV passive properties. Thus regional mechanical load has a critical role during cardiogenesis.

Advancements in immersive VR as a tool for preoperative planning for laparoscopic surgery.

The utility of three-dimensional (3D) models for planning laparoscopic surgery and surgical training has been demonstrated. (1) Computed tomography (CT) scans with oral and intravenous contrast medium are frequently used for preoperative evaluation of patients undergoing complex laparoscopic surgery. Immersive 3D VR overcomes many of the conceptual limitations encountered when conveying or teaching 3D relationships via 2D images traditionally produced by these scans. Over the past year we have made advancements in several areas. First, we have improved the quality of our datasets by utilizing higher resolution multi-detector scans and altering the protocols used. Second, we now register multiple isosurface views with standard axial views and volume textured views to provide additional information and perspective. Third, we now routinely use auto-segmentation techniques to visualize individual structures.