Intuitive repositioning of an anti-depressant drug in combination with tivozanib: precision medicine for breast cancer therapy.

Despite the existing therapies and lack of receptors such as HER-2, estrogen receptor and progesterone receptor, triple-negative breast cancer is one of the most aggressive subtypes of breast cancer. TNBCs are known for their highly aggressive metastatic behavior and typically migrate to brain and bone for secondary site propagation. Many diseases share similar molecular pathology exposing new avenues in molecular signaling for engendering innovative therapies. Generation of newer therapies and novel drugs are time consuming associated with very high resources. In order to provide personalized or precision medicine, drug repositioning will contribute in a cost-effective manner. In our study, we have repurposed and used a neoteric combination of two drug molecules namely, fluvoxamine and tivozanib, to target triple-negative breast cancer growth and progression. Our combination regime significantly targets two diverse but significant pathways in TNBCs. Subsequent analysis on migratory, invasive, and angiogenic properties showed the significance of our repurposed drug combination. Molecular array data resulted in identifying the specific and key players participating in cancer progression when the drug combination was used. The innovative combination of fluvoxamine and tivozanib reiterates the use of drug repositioning for precision medicine and subsequent companion diagnostic development.

Current updates on precision therapy for breast cancer associated brain metastasis: Emphasis on combination therapy.

Cancer therapies have undergone a tremendous progress over the past decade. Precision medicine provides a more tailored approach, making the combination of existing therapies more precise. Different types of cancers are characterized by unique biomarkers that are targeted using various genomic approaches by clinicians and companies worldwide to achieve efficient treatment with minimal side effects. Precision medicine has two broad approaches namely stratified and personalized medicine. The driver mutations could vary within a subtype while the same driver mutations could be found across different subtypes. Precision medicine has recently gained a lot of importance for breast cancer therapy. Various kinds of mutations like hotspot mutations, gene alterations, gene amplification mutations are targeted to design a more specific therapy. Apart from these known gene mutations there are various unknown mutations. Thus, tumor heterogeneity can pose a challenge to precision medicine. For breast cancer, one of the most successful models developed in case of precision medicine is the anti-HER2 therapies as HER2 was considered to have the worst prognosis being highly malignant. But now due to the advent of HER2 receptor targeted therapies, it has a good prognosis. Moreover, precision medicine helps in identifying if the drug molecules being used for the treatment of one kind of cancer can be beneficial in the treatment of another kind of cancer as well, considering the signaling pathways and machinery is similar in most of the cancers. This reduces the time for new drug development and is economically more feasible. Precision medicine will prove to be very advantageous in case of brain metastasis.

Perspectives on the role of brain cellular players in cancer-associated brain metastasis: translational approach to understand molecular mechanism of tumor progression.

Brain metastasis is one of the leading causes of death among cancer patients. Cancer cells migrate to various sites and harbor different niche in the body which help cancer cells in their survival. The brain is one of the safest place where cancer cells are protected from immune cells. Breast, lung, and melanoma cancer cells have high propensity to migrate towards the brain. To enter the brain, cancer cells have to cross the blood brain barrier. Survival and finding new niche in the brain are directed by several mechanisms in which different cellular players take part such as astrocytes, microglia, Schwann cells, satellite cells, oligodendrocytes, and ependymal cells. Usually, cancer cells highjack the machinery of brain cellular players to survive in the brain environment. It has been shown that co-culture of M2 macrophage with cancer cells leads to increased proliferation and survival of cancer cells. One of the challenges of understanding brain metastasis is appropriate model system to understand dynamic interaction of cancer cells and brain cellular players. To meet this challenge, microfluidic-based devices are employed which can mimic the dynamic conditions as well as can be used for culturing human cells for personalized therapy. In this review, we have systematically reviewed the current status of the role of cellular players in brain metastasis along with explaining how translational approach of microfluidics can be employed for finding new drug target as well as biomarker for brain metastasis. Finally, we have also commented on the mechanism of action of drugs against brain metastasis.

Integrative natural medicine inspired graphene nanovehicle-benzoxazine derivatives as potent therapy for cancer.

Natural products from medicinal plants have always attracted a lot of attention due to their diverse and interesting therapeutic properties. We have employed the principles of green chemistry involving isomerization, coupling and condensation reaction to synthesize a class of compounds derived from eugenol, a naturally occurring bioactive phytophenol. The compounds were characterized structurally by 1H-, 13C-NMR, FT-IR spectroscopy and mass spectrometry analysis. The purity of compounds was detected by HPLC. The synthesized compounds exhibited anti-cancer activity. A 10-12-fold enhancement in efficiency of drug molecules (~ 1 µM) was observed when delivered with graphene oxide (GO) as a nanovehicle. Our data suggest cell death via apoptosis in a dose-dependent manner due to increase in calcium levels in specific cancer cell lines. Interestingly, the benzoxazine derivatives of eugenol with GO nanoparticle exhibited enhanced therapeutic potential in cancer cells. In addition to anti-cancer effect, we also observed significant role of these derivatives on parasite suggesting its multi-pharmacological capability.

cAMP regulated EPAC1 supports microvascular density, angiogenic and metastatic properties in a model of triple negative breast cancer.

Breast cancer is a leading cause of cancer-related mortality in women. Triple-negative breast cancer (TNBC; HER2-, ER-/PR-) is an aggressive subtype prone to drug resistance and metastasis, which is characterized by high intratumor microvascular density (iMVD) resulting from angiogenesis. However, the mechanisms contributing to the aggressive phenotypes of TNBC remain elusive. We recently reported that down-regulation of exchange factor directly activated by cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in metastatic properties including proliferation and cell migration in TNBC cell lines. Here, we report that EPAC1 supports TNBC-induced angiogenesis, tumor cell migration and invasiveness as well as pro-metastatic phenotypes in endothelial cells induced through the tumor secretome. Using an approach that integrates proteomics with bioinformatics and gene ontologies, we elucidate that EPAC1 supports a tumor-secreted network of angiogenic, cell adhesion and cell migratory pathways. Using confocal microscopy, we show that signaling molecules involved in focal adhesion, including Paxillin and MENA, are down-regulated in the absence of EPAC1, and electric cell substrate impedance sensing technique confirmed a role for EPAC1 on TNBC-induced endothelial cell permeability. Finally, to provide a translational bridge, we studied iMVD and therapy response using a primary human tumor explant assay, CANscriptTM, which suggests a link between therapy-modulated neovascularization and drug sensitivity. These data provide mechanistic insight into the role of EPAC1 in regulating the tumor microenvironment, iMVD and cancer cell-induced angiogenesis, a dynamic mechanism under drug pressure that may associate to treatment failure.

Perspective on nanochannels as cellular mediators in different disease conditions.

Tunnelling nanotubes (TNTs), also known as membrane nanochannels, are actin-based structures that facilitate cytoplasmic connections for rapid intercellular transfer of signals, organelles and membrane components. These dynamic TNTs can form de novo in animal cells and establish complex intercellular networks between distant cells up to 150 µm apart. Within the last decade, TNTs have been discovered in different cell types including tumor cells, macrophages, monocytes, endothelial cells and T cells. It has also been further elucidated that these nanotubes play a vital role in diseased conditions such as cancer, where TNT formation occurs at a higher pace and is used for rapid intercellular modulation of chemo-resistance. Viruses such as HIV, HSV and prions also hijack the existing TNT connections between host cells for rapid transmission and evasion of the host immune responses. The following review aims to describe the heterogeneity of TNTs, their role in different tissues and disease conditions in order to enhance our understanding on how these nanotubes can be used as a target for therapies.

Insights into exchange factor directly activated by cAMP (EPAC) as potential target for cancer treatment.

Cancer remains a global health problem and approximately 1.7 million new cancer cases are diagnosed every year worldwide. Although diverse molecules are currently being explored as targets for cancer therapy the tumor treatment and therapy is highly tricky. Secondary messengers are important for hormone-mediated signaling pathway. Cyclic AMP (cAMP), a secondary messenger responsible for various physiological processes regulates cell metabolism by activating Protein kinase A (PKA) and by targeting exchange protein directly activated by cAMP (EPAC). EPAC is present in two isoforms EPAC1 and EPAC2, which exhibit different tissue distribution and is involved in GDP/GTP exchange along with activating Rap1- and Rap2-mediated signaling pathways. EPAC is also known for its dual role in cancer as pro- and anti-proliferative in addition to metastasis. Results after perturbing EPAC activity suggests its involvement in cancer cell migration, proliferation, and cytoskeleton remodeling which makes it a potential therapeutic target for cancer treatments.

Benzoxazine derivatives of phytophenols show anti-plasmodial activity via sodium homeostasis disruption.

Development of new class of anti-malarial drugs is an essential requirement for the elimination of malaria. Bioactive components present in medicinal plants and their chemically modified derivatives could be a way forward towards the discovery of effective anti-malarial drugs. Herein, we describe a new class of compounds, 1,3-benzoxazine derivatives of pharmacologically active phytophenols eugenol (compound 3) and isoeugenol (compound 4) synthesised on the principles of green chemistry, as anti-malarials. Compound 4, showed highest anti-malarial activity with no cytotoxicity towards mammalian cells. Compound 4 induced alterations in the intracellular Na+ levels and mitochondrial depolarisation in intraerythrocytic Plasmodium falciparum leading to cell death. Knowing P-type cation ATPase PfATP4 is a regulator for sodium homeostasis, binding of compound 3, compound 4 and eugenol to PfATP4 was analysed by molecular docking studies. Compounds showed binding to the catalytic pocket of PfATP4, however compound 4 showed stronger binding due to the presence of propylene functionality, which corroborates its higher anti-malarial activity. Furthermore, anti-malarial half maximal effective concentration of compound 4 was reduced to 490 nM from 17.54 µM with nanomaterial graphene oxide. Altogether, this study presents anti-plasmodial potential of benzoxazine derivatives of phytophenols and establishes disruption of parasite sodium homeostasis as their mechanism of action.

Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis.

Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP-EPAC1-AKAP9 direction to the development of additional biotherapeutics for breast cancer.

A novel spiroindoline targets cell cycle and migration via modulation of microtubule cytoskeleton.

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.

Identification of noreremophilane-based inhibitors of angiogenesis using zebrafish assays.

Noreremophilanes are a rare class of cis-hydrindanes produced by genus Ligularia herbaceous plants which are known to exhibit interesting biological activities. We synthesized cis-hydrindanes based on a naturally occurring noreremophilane scaffold using a Diels-Alder/aldol sequence and screened them for multiple biological activities using high-content zebrafish embryonic development assays. We discovered a noreremophilane that has strong anti-angiogenic effects on the developing zebrafish embryos as well as on tumor-induced angiogenesis in a zebrafish xenograft model. We synthesized several derivatives of this class of noreremophilanes and performed structure-activity relationship studies in zebrafish to identify more potent and less toxic analogs of the original structure.

R-spondin3 prevents mesenteric ischemia/reperfusion-induced tissue damage by tightening endothelium and preventing vascular leakage.

Inflammation and vascular injury triggered by ischemia/reperfusion (I/R) represent a leading cause of morbidity and mortality in a number of clinical settings. Wnt and its homolog partners R-spondins, in addition to regulating embryonic development have recently been demonstrated to serve as wound-healing agents in inflammation-associated conditions. Here we ask whether R-spondins could prevent inflammation-associated tissue damage in ischemic disorders and thus investigate the role of R-spondin3 (R-spo3) in a mouse model of mesenteric I/R. We demonstrate that R-spo3 ameliorates mesenteric I/R-induced local intestinal as well as remote lung damage by suppressing local and systemic cytokine response and deposition of IgM and complement in intestinal tissues. We also show that decreased inflammatory response is accompanied by tightening of endothelial cell junctions and reduction in vascular leakage. We conclude that R-spo3 stabilizes endothelial junctions and inhibits vascular leakage during I/R and thereby mitigates the inflammatory events and associated tissue damage. Our findings uniquely demonstrate a protective effect of R-spo3 in I/R-related tissue injury and suggest a mechanism by which it may have these effects.

AKAP9 regulation of microtubule dynamics promotes Epac1-induced endothelial barrier properties.

Adhesive forces at endothelial cell-cell borders maintain vascular integrity. cAMP enhances barrier properties and controls cellular processes through protein kinase A bound to A-kinase anchoring proteins (AKAPs). It also activates exchange protein directly activated by cAMP (Epac1), an exchange factor for Ras-related protein 1 (Rap1) GTPases that promotes cadherin- and integrin-mediated adhesion through effects on the actin cytoskeleton. We demonstrate that AKAP9 facilitates the microtubule polymerization rate in endothelial cells, interacts with Epac1, and is required for Epac1-stimulated microtubule growth. AKAP9 is not required for maintaining barrier properties under steady-state conditions. Rather, it is essential when the cell is challenged to make new adhesive contacts, as is the case when Epac activation enhances barrier function through a mechanism that, surprisingly, requires integrin adhesion at cell-cell contacts. In the present study, defects in Epac-induced responses in AKAP9-silenced cells were evident despite an intact Epac-induced increase in Rap activation, cortical actin, and vascular endothelial-cadherin adhesion. We describe a pathway that integrates Epac-mediated signals with AKAP9-dependent microtubule dynamics to coordinate integrins at lateral borders.

p120-Catenin regulates leukocyte transmigration through an effect on VE-cadherin phosphorylation.

Vascular endothelial-cadherin (VE-cad) is localized to adherens junctions at endothelial cell borders and forms a complex with alpha-, beta-, gamma-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form short-lived "gaps" during leukocyte transendothelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endothelial cells transduced with an adenovirus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leukocyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions.

The omission of comprehensive care: an analysis of the Nursing Home Reform Act of 1987.

With the large increase in the older adult population, the number of people in need of nursing home services is expected to grow rapidly. The challenge is to attain or maintain the highest possible physical, mental and psychological well-being of residents through provision of medically-related social services as required by the Nursing Home Reform Act (NHRA) of 1987. However, the basis of psycho-social service provision in NHRA is the size of the nursing home, which is questionable and is therefore making legislation less equitable. This policy analysis of NHRA will include suggestions of feasible policy alternatives to make the current legislation more equitable and effective. The article recognizes and endorses the contribution of social workers in psycho-social service provision and advocates for an increase in the ratio of mental health professionals to residents, therefore meeting the psycho-social service needs of the residents.

Amalgamation of PI3K and EZH2 blockade synergistically regulates invasion and angiogenesis: combination therapy for glioblastoma multiforme.

Glioblastoma multiforme is known as the primary malignant and most devastating form of tumor in central nervous system of adult population. Amongst all CNS cancers, Glioblastoma multiforme GBM is a rare grade IV astrocytoma and it has the worst prognosis initiated by metastasis to supra-tentorial region of the brain. Current options for the treatment include surgery, radiation therapy and chemotherapy. Substantial information of its pathology and molecular signaling exposed new avenues for generating innovative therapies. In our study, we have undertaken a novel combination approach for GBM treatment. PI3K signaling participates in cancer progression and plays a significant role in metastasis. Here, we are targeting PI3K signaling pathways in glioblastoma along with EZH2, a known transcriptional regulator. We found that targeting transcriptional regulator EZH2 and PI3K affect cellular migration and morphological changes. These changes in signatory activities of cancerous cells led to inhibit its progression in vitro. With further analysis we confirmed the angiogenic inhibition and reduction in stem-ness potential of GBM. Later, cytokine proteome array analysis revealed several participants of metastasis and tumor induced angiogenesis using combination regime. This study provides a significant reduction in GBM progression investigated using Glioblastoma Multiforme U-87 cells with effective combination of pharmacological inhibitors PI-103 and EPZ-6438. This strategy will be further used to combat GBM more innovatively along with the existing therapies.

Social Work Role in Pain Management with Hospice Caregivers: A National Survey.

This article reports on an exploratory study of hospice social workers' assessment and collaborative practices related to pain management; especially caregiver concerns about patient pain. A non-randomized national survey indicated that social workers assess the components of pain but are not able to devote as much attention to it as they feel is needed. While most reported assessing patient and family needs, many do not use standardized assessment instruments. These data suggested that while social workers may understand their role in pain management they struggled for the time and tools needed to help address caregiver concerns related to pain management. This study suggests that the development of standardized assessment instruments for hospice social workers would be helpful and points to the value of team training and discussion about ways the social worker can best assist caregivers with pain management issues. Research is needed on social work interventions with caregivers related to pain to establish an evidence base for hospice social work, cement a role identity for social work in pain management, and facilitate increased interdisciplinary collaboration to improve the team response to all aspects of pain.

Stable Dispersions of Covalently Tethered Polymer Improved Graphene Oxide Nanoconjugates as an Effective Vector for siRNA Delivery.

Conjugates of poly(amidoamine) (PAMAM) with modified graphene oxide (GO) are attractive nonviral vectors for gene-based cancer therapeutics. GO protects siRNA from enzymatic cleavage and showed reasonable transfection efficiency along with simultaneous benefits of low cost and large scale production. PAMAM is highly effective in siRNA delivery but suffers from high toxicity with poor in vivo efficacy. Co-reaction of GO and PAMAM led to aggregation and more importantly, have detrimental effect on stability of dispersion at physiological pH preventing their exploration at clinical level. In the current work, we have designed, synthesized, characterized and explored a new type of hybrid vector (GPD), using GO synthesized via improved method which was covalently tethered with poly(ethylene glycol) (PEG) and PAMAM. The existence of covalent linkage, relative structural changes and properties of GPD is well supported by Fourier transform infrared (FTIR), UV-visible (UV-vis), Raman, X-ray photoelectron (XPS), elemental analysis, powder X-ray diffraction (XRD), thermogravimetry analysis (TGA), dynamic light scattering (DLS), and zeta potential. Scanning electron microscopy (SEM), and transmission electron microscopy (TEM) of GPD showed longitudinally aligned columnar self-assembled ∼10 nm thick polymeric nanoarchitectures onto the GO surface accounting to an average size reduction to ∼20 nm. GPD revealed an outstanding stability in both phosphate buffer saline (PBS) and serum containing cell medium. The binding efficiency of EPAC1 siRNA to GPD was supported by gel retardation assay, DLS, zeta potential and photoluminescence (PL) studies. A lower cytotoxicity with enhanced cellular uptake and homogeneous intracellular distribution of GPD/siRNA complex is confirmed by imaging studies. GPD exhibited a higher transfection efficiency with remarkable inhibition of cell migration and lower invasion than PAMAM and Lipofectamine 2000 suggesting its role in prevention of breast cancer progression and metastasis. A significant reduction in the expression of the specific protein against which siRNA was delivered is revealed by Western blot assay. Furthermore, a pH-triggered release of siRNA from the GPD/siRNA complex was studied to provide a mechanistic insight toward unloading of siRNA from the vector. Current strategy is a way forward for designing effective therapeutic vectors for gene-based antitumor therapy.

Visualization and quantification of Plasmodium falciparum intraerythrocytic merozoites.

Malaria, a leading parasitic killer, is caused by Plasmodium spp. The pathology of the disease starts when Plasmodium merozoites infect erythrocytes to form rings, that matures through a large trophozoite form and develop into schizonts containing multiple merozoites. The number of intra-erythrocytic merozoites is a key-determining factor for multiplication rate of the parasite. Counting of intraerythrocytic merozoites by classical 2-D microscopy method is error prone due to insufficient representation of merozoite in one optical plane of a schizont. Here, we report an alternative 3-D microscopy based automated method for counting of intraerythrocytic merozoites in entire volume of schizont. This method offers a considerable amount of advantages in terms of both, ease and accuracy.

Predicting stable functional peptides from the intergenic space of E. coli.

Expression of synthetic proteins from intergenic regions of E. coli and their functional association was recently demonstrated (Dhar et al. in J Biol Eng 3:2, 2009. doi:10.1186/1754-1611-3-2). This gave birth to the question: if one can make 'user-defined' genes from non-coding genome-how big is the artificially translatable genome? (Dinger et al. in PLoS Comput Biol 4, 2008; Frith et al. in RNA Biol 3(1):40-48, 2006a; Frith et al. in PLoS Genet 2(4):e52, 2006b). To answer this question, we performed a bioinformatics study of all reported E. coli intergenic sequences, in search of novel peptides and proteins, unexpressed by nature. Overall, 2500 E. coli intergenic sequences were computationally translated into 'protein sequence equivalents' and matched against all known proteins. Sequences that did not show any resemblance were used for building a comprehensive profile in terms of their structure, function, localization, interactions, stability so on. A total of 362 protein sequences showed evidence of stable tertiary conformations encoded by the intergenic sequences of E. coli genome. Experimental studies are underway to confirm some of the key predictions. This study points to a vast untapped repository of functional molecules lying undiscovered in the non-expressed genome of various organisms.