Recurrent Multivalvular Staphylococcus Lugdunensis Endocarditis Causing Complete Heart Block after TAVR.

Prosthetic valve endocarditis after transcatheter aortic valve replacement (PVE after TAVR) is a feared complication most often observed during the early postprocedural period. We report a case of severe, multivalvular PVE after TAVR with complete heart block caused by an uncommon organism. A 78-year-old female with prior Streptococcus agalactiae mitral valve endocarditis treated with antibiotics presented one year later with severe, symptomatic aortic insufficiency. She subsequently underwent TAVR given high surgical risk. Six weeks post-TAVR, she presented with syncope, fever, and complete heart block. Transthoracic echocardiogram was not demonstrative of vegetation. Blood cultures were positive for Staphylococcus lugdunensis. Transesophageal echocardiogram (TEE) demonstrated vegetations of the aortic, mitral, and tricuspid valves and aorto-mitral continuity. While awaiting surgery, the patient developed cardiac arrest; she was resuscitated and taken to surgery emergently. The patient underwent TAVR explantation, bovine pericardial tissue aortic and porcine bioprosthetic mitral valve replacements, and tricuspid valve repair. Additionally, left main coronary artery endarterectomy was performed due to presence of infectious vegetative material. Staphylococcus lugdunensis is an unusual but virulent organism that may damage both native and prosthetic valves. Early surgery is recommended for PVE after TAVR, especially in cases with perivalvular disease causing conduction abnormalities. Learning Objectives. TAVR has revolutionized the management of severe aortic stenosis and has even been successfully utilized in select cases of aortic regurgitation. Unfortunately, there are a number of associated complications that can be difficult to diagnose, such as prosthetic valve endocarditis (PVE). We emphasize maintaining a high clinical suspicion for PVE after TAVR in patients presenting with conduction abnormalities and highlight the importance of early surgical management in cases complicated by heart block, abscesses, or destructive penetrating lesions.

Metastasis of breast tumor cells to brain is suppressed by targeting RLIP alone and in combination with 2'-Hydroxyflavanone.

Brain metastasis is an important cause of morbidity and mortality in cancer-patients. Breast tumor cells frequently metastasize to brain and initiate severe therapeutic complications. In the present study, we evaluated the anti-metastatic effects of 2'-hydroxyflavanone (2HF) alone and in combination with RLIP targeted therapy in a novel murine model of breast tumor metastasis. The MDA-MB231Br (brain-seeking) breast cancer (BC) cells stably-transfected with luciferase were injected into the left-ventricle of NSG mouse heart and the migration of cells to brain was monitored using a non-invasive bioluminescent imaging system. To evaluate the tumor growth suppressive effects, mice were given 2HF (50 mg/kg, b.w., alternate days orally), RLIP-antibody (Rab; 5 mg/kg, b.w., weekly i.p.) or combination of 2HF+Rab starting day1 after intra-cardiac injection. Our results reveal that 2HF and Rab significantly prevented the metastasis of BC cells to brain. Further, mice treated with combination of 2HF+Rab exhibited no metastasis as compared to either or the single agent-treated mice. This study for the first time demonstrates the anti-metastatic effects of 2HF and RLIP-inhibition in-vivo in a novel breast tumor metastasis model and provides the rationale for further clinical investigation.

Targeting the mercapturic acid pathway and vicenin-2 for prevention of prostate cancer.

Prostate cancer (CaP) is often androgen-sensitive malignancy and regresses upon inhibition of androgen signaling. However, CaP, nearly always develops androgen resistance and progresses to aggressive and lethal androgen-independent CaP, which lacks satisfactory therapy. For metastatic CaP, patients are often treated with Taxotere (docetaxel), a cytoskeleton-targeted chemotherapy drug, that provides transient palliative benefit but to which patients rapidly develop drug-resistance. Combination chemotherapy may be used instead, but is more toxic and adds little clinically relevant benefit over docetaxel. Therefore, novel strategies to enhance docetaxel efficacy are needed to effectively treat patients with metastatic CaP. The mercapturic acid pathway, which metabolizes genotoxic and pro-apoptotic toxins, is over-expressed in CaP and plays an important role in carcinogenesis, metastasis and therapy-resistance of CaP. Vicenin-2, a flavonoid derived from Tulsi (holy basil) as an active compound, inhibits the growth of CaP and increases the anti-tumor activity of docetaxel in-vitro and in-vivo. Taken together, the combination of vicenin-2 and docetaxel could be highly effective in the treatment of advanced and metastatic CaP due to their multi-targeting anti-tumor potential.

RLIP76 Inhibition: A Promising Developmental Therapy for Neuroblastoma.

Refractory and relapsed neuroblastoma (NB) present with significant challenges in clinical management. Though primary NBs largely with wild-type p53 respond well to interventions, dysfunctional signaling in the p53 pathways in a MYCN oncogene driven background is found in a number of children with NB. The p53-mutant NB is largely unresponsive to available therapies and p53-independent targeted therapeutics represents a vital need in pediatric oncology. We analyzed the findings on mercapturic acid pathway (MAP) transporter RLIP76, which has broad and critical effects on multiple pathways as essential for carcinogenesis, oxidative stress and drug-resistance, is over-expressed in NB. RLIP76 inhibition by antibodies or depletion by antisense causes apoptosis and sensitization to chemo-radiotherapy in many cancers. In addition, recent studies indicate that the interactions between p53, MYCN, and WNT regulate apoptosis resistance and protein ubiquitination. RLIP76 and p53 interact with each other and colocalize in NB cells. Targeted depletion/inhibition of RLIP76 causes apoptosis and tumor regression in NB irrespective of p53 status. In the present review, we discuss the mechanisms and the role of RLIP76 in oxidative stress, drug-resistance and clathrin-dependent endocytosis (CDE), and analyze the molecular basis for the role of RLIP76 targeted approaches in the context principal drivers of NB pathogenesis, progression and drug-resistance. The evidence from RLIP76 studies in other cancers, when taken in the context of our recent RLIP76 focused mechanistic studies in NB, provides strong basis for further characterization and development of RLIP76 targeted therapies for NB.

Didymin: an orally active citrus flavonoid for targeting neuroblastoma.

Neuroblastoma, a rapidly growing yet treatment responsive cancer, is the third most common cancer of children and the most common solid tumor in infants. Unfortunately, neuroblastoma that has lost p53 function often has a highly treatment-resistant phenotype leading to tragic outcomes. In the context of neuroblastoma, the functions of p53 and MYCN (which is amplified in ~25% of neuroblastomas) are integrally linked because they are mutually transcriptionally regulated, and because they together regulate the catalytic activity of RNA polymerases. Didymin is a citrus-derived natural compound that kills p53 wild-type as well as drug-resistant p53-mutant neuroblastoma cells in culture. In addition, orally administered didymin causes regression of neuroblastoma xenografts in mouse models, without toxicity to non-malignant cells, neural tissues, or neural stem cells. RKIP is a Raf-inhibitory protein that regulates MYCN activation, is transcriptionally upregulated by didymin, and appears to play a key role in the anti-neuroblastoma actions of didymin. In this review, we discuss how didymin overcomes drug-resistance in p53-mutant neuroblastoma through RKIP-mediated inhibition of MYCN and its effects on GRK2, PKCs, Let-7 micro-RNA, and clathrin-dependent endocytosis by Raf-dependent and -independent mechanisms. In addition, we will discuss studies supporting potential clinical impact and translation of didymin as a low cost, safe, and effective oral agent that could change the current treatment paradigm for refractory neuroblastoma.

Comparison of Modular PEG Incorporation Strategies for Stabilization of Peptide-siRNA Nanocomplexes.

Nanoparticulate systems have shown great promise in overcoming the considerable trafficking barriers associated with systemic nucleic acid delivery, which must be addressed to unlock the full potential of technologies such as RNAi and gene editing in vivo. In addition to mediating the cytoplasmic delivery of nucleic cargo and shielding it from nuclease degradation and immunostimulation, nucleic-acid-containing nanomaterials delivered intravenously must also be stable in the bloodstream after administration to avoid toxicity and off-target delivery. To this end, the hydrophilic molecule polyethylene glycol (PEG) has been deployed in many different nanoparticle systems to prevent aggregation and recognition by the reticuloendothelial system. However, the optimal strategy for incorporating PEG into self-assembled nucleic acid delivery systems to obtain nanoparticle stability while retaining important functions such as receptor targeting and cargo activity remains unclear. In this work, we develop substantially improved formulations of tumor-penetrating nanocomplexes (TPNs), targeted self-assembled nanoparticles formulated with peptide carriers and siRNA that have been shown to mitigate tumor burden in an orthotopic model of ovarian cancer. We specifically sought to tailor TPNs for intravenous delivery by systematically comparing formulations with three different classes of modular PEG incorporation (namely PEG graft polymers, PEG lipids, and PEGylated peptide), each synthesized using straightforward bioconjugation techniques. We found that the addition of PEG lipids or PEGylated peptide carriers led to the formation of small and stable nanoparticles, but only nanoparticles formulated with PEGylated peptide carriers retained substantial activity in a gene silencing assay. In vivo, this formulation significantly decreased accumulation in off-target organs and improved initial availability in circulation compared to results from the original non-PEGylated particles. Thus, from among a set of candidate strategies, we identified TPNs with admixed PEGylated peptide carriers as the optimal formulation for systemic administration of siRNA on the basis of their performance in a battery of physicochemical and biological assays. Moreover, this optimized formulation confers pharmacologic advantages that may enable further translational development of tumor-penetrating nanocomplexes, highlighting the preclinical value of comparing formulation strategies and the relevance of this systematic approach for the development of other self-assembled nanomaterials.

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling.

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.

Associated Anisotropy Decays of Ethidium Bromide Interacting with DNA.

Ethidium Bromide (EB) is a commonly used dye in a deoxyribonucleic acid (DNA) study. Upon an intercalation, this dye significantly increases its brightness and fluorescence lifetime. In this report we have studied the time-resolved fluorescence properties of EB existing simultaneously in free and DNA-bound forms in the solution. Fluorescence intensity decays were fitted globally to a double exponential model with lifetimes corresponding to free (1.6ns) and bound (22ns) forms, and molar fractions were determined for all used solutions. Anisotropy decays displayed characteristic time dependence with an initial rapid decline followed by recovery and slow decay. The short-lived fraction associated with free EB molecules decreases faster than long-lived fraction associated with EB bound to DNA. Consequently, contribution from fast rotation leads to initial rapid decay in anisotropy. On the other hand bound fraction, due to slow rotation helps recover anisotropy in time. This effect of associated anisotropy decays in systems such as EB free/EB-DNA is clearly visible in a wide range of concentrations, and should be taken into account in polarization assays and biomolecule dynamics studies.

Associated anisotropy decays of ethidium bromide interacting with DNA.

Ethidium Bromide (EB) is a commonly used dye in a deoxyribonucleic acid (DNA) study. Upon an intercalation, this dye significantly increases its brightness and fluorescence lifetime. In this report we have studied the time resolved fluorescence properties of EB existing simultaneously in free and DNA-bound forms in the solution. Fluorescence intensity decays were fitted globally to a double exponential model with lifetimes corresponding to free (1.6 ns) and bound (22 ns) forms, and molar fractions were determined for all used solutions. Anisotropy decays displayed characteristic time dependence with an initial rapid decline followed by recovery and slow decay. The short-lived fraction associated with free EB molecules decreases faster than long-lived fraction associated with EB bound to DNA. Consequently, contribution from fast rotation leads to initial rapid decay in anisotropy. On the other hand bound fraction, due to slow rotation helps recover anisotropy in time. This effect of associated anisotropy decays in systems such as EB free/EB-DNA is clearly visible in a wide range of concentrations, and should be taken into account in polarization assays and biomolecule dynamics studies.

COH-SR4 reduces body weight, improves glycemic control and prevents hepatic steatosis in high fat diet-induced obese mice.

Obesity is a chronic metabolic disorder caused by imbalance between energy intake and expenditure, and is one of the principal causative factors in the development of metabolic syndrome, diabetes and cancer. COH-SR4 ("SR4") is a novel investigational compound that has anti-cancer and anti-adipogenic properties. In this study, the effects of SR4 on metabolic alterations in high fat diet (HFD)-induced obese C57BL/J6 mice were investigated. Oral feeding of SR4 (5 mg/kg body weight.) in HFD mice for 6 weeks significantly reduced body weight, prevented hyperlipidemia and improved glycemic control without affecting food intake. These changes were associated with marked decreases in epididymal fat mass, adipocyte hypertrophy, increased plasma adiponectin and reduced leptin levels. SR4 treatment also decreased liver triglycerides, prevented hepatic steatosis, and normalized liver enzymes. Western blots demonstrated increased AMPK activation in liver and adipose tissues of SR4-treated HFD obese mice, while gene analyses by real time PCR showed COH-SR4 significantly suppressed the mRNA expression of lipogenic genes such as sterol regulatory element binding protein-1c (Srebf1), acetyl-Coenzyme A carboxylase (Acaca), peroxisome proliferator-activated receptor gamma (Pparg), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr), as well as gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) in the liver of obese mice. In vitro, SR4 activates AMPK independent of upstream kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). Together, these data suggest that SR4, a novel AMPK activator, may be a promising therapeutic compound for treatment of obesity, fatty liver disease, and related metabolic disorders.

Rlip76 transports sunitinib and sorafenib and mediates drug resistance in kidney cancer.

RLIP76 is a stress-responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It represents the predominant glutathione-conjugate (GS-E) transporter in cells. We have shown that RLIP76 plays a crucial role in defending cancer cells from radiation and chemotherapeutic toxin-mediated apoptosis, and that its inhibition by antibodies or depletion by siRNA or antisense causes apoptosis in a number of cancer cell types. We demonstrated for the first time that the striking anti-neoplastic effects with no evident toxicity in terms of either weight loss or metabolic effects are also demonstrable for the antibody, antisense and siRNA in a renal cell xenografts model of Caki-2 cells (Singhal et al., Cancer Res., 2009, 69: 4244). Present studies were performed to determine if RLIP76 targeting is more broadly applicable in other kidney cancer cell lines, to compare the signaling effects of RLIP76 antisense with kinase inhibitors used in treatment of renal cell carcinoma, and to determine whether kinase inhibitors were substrates for transport by RLIP76. Results of these studies show that sorafenib as well as sunitinib are substrates for transport by RLIP76 thus are competitive inhibitors of GS-E transport. Furthermore, kinase inhibition in the ERK as well as PI3K pathways by RLIP76 depletion is more profound and consistent and is more widely apparent in a number of renal carcinoma cell lines. These studies offer strong support for our overall hypothesis that RLIP76 is an overarching anti-apoptosis mechanism that, if inhibited, can be more broadly effective in the treatment of renal cell carcinoma.

Role of RLIP76 in doxorubicin resistance in lung cancer.

Lung cancer is still a major cause of cancer deaths in spite of considerable efforts in its systemic therapy. Chemotherapy, along with local irradiation is frequently employed but as a palliative therapy. Inherent and acquired resistance in NSCLC and SCLC towards chemotherapeutic agents further makes chemotherapy an incommodious problem. The resistance mechanisms responsible for inherent DOX-resistance of NSCLC and acquired DOX-resistance in SCLC have been the subject of numerous investigations. This review will focus on the recent studies done for understanding the mechanism(s) of inherent and acquired resistance in NSCLC and SCLC and how these can be exploited for the future development of more effective novel biologic agents for the treatment of lung cancer.