Polyphenols Targeting MAP Kinase Signaling Pathway in Neurological Diseases: Understanding Molecular Mechanisms and Therapeutic Targets.

Polyphenols are a class of secondary metabolic products found in plants that have been extensively studied for how well they regulate biological processes, such as the proliferation of cells, autophagy, and apoptosis. The mitogen-activated protein kinase (MAPK)-mediated signaling cascade is currently identified as a crucial pro-inflammatory pathway that plays a significant role in the development of neuroinflammation. This process has been shown to contribute to the pathogenesis of several neurological conditions, such as Alzheimer's disease (AD), Parkinson's disease (PD), CNS damage, and cerebral ischemia. Getting enough polyphenols through eating habits has resulted in mitigating the effects of oxidative stress (OS) and lowering the susceptibility to associated neurodegenerative disorders, including but not limited to multiple sclerosis (MS), AD, stroke, and PD. Polyphenols possess significant promise in dealing with the root cause of neurological conditions by modulating multiple therapeutic targets simultaneously, thereby attenuating their complicated physiology. Several polyphenolic substances have demonstrated beneficial results in various studies and are presently undergoing clinical investigation to treat neurological diseases (NDs). The objective of this review is to provide a comprehensive summary of the different aspects of the MAPK pathway involved in neurological conditions, along with an appraisal of the progress made in using polyphenols to regulate the MAPK signaling system to facilitate the management of NDs.

The Humanised NPY-mRFP RBL Reporter Cell Line Is a Fast and Inexpensive Tool for Detection of Allergen-Specific IgE in Human Sera.

Rat basophilic leukaemia (RBL) cells have been used for decades as a model of high-affinity Immunoglobulin E (IgE) receptor (FcεRI) signalling. Here, we describe the generation and use of huNPY-mRFP, a new humanised fluorescent IgE reporter cell line. Fusion of Neuropeptide Y (NPY) with monomeric red fluorescent protein (mRFP) results in targeting of fluorescence to the granules and its fast release into the supernatant upon IgE-dependent stimulation. Following overnight sensitisation with serum, optimal release of fluorescence upon dose-dependent stimulation with allergen-containing extracts could be measured after 45 min, without cell lysis or addition of any reagents. Five substitutions (D194A, K212A, K216A, K226A, and K230A) were introduced into the FcεRIα cDNA used for transfection, which resulted in the removal of known endoplasmic reticulum retention signals and high surface expression of human FcεRIα* in huNPY-mRFP cells (where * denotes the penta-substituted variant), comparable to the ~500,000 FcεRIα molecules per cell in the RS-ATL8 humanised luciferase reporter, which is a human FcεRIα/FcεRIγ double transfectant. The huNPY-mRFP reporter was used to demonstrate engagement of specific IgE in sera of Echinococcus granulosus-infected individuals by E. granulosus elongation factor EgEF-1ß and, to a lesser extent, by EgEF-1δ, which had been previously described as IgE-immunoreactive EgEF-1ß/δ.

Unusual manifestations of extra-pulmonary tuberculosis: a pictorial essay.

Purpose: Tuberculosis is quite common in India. About 40% of all Indians are infected with mycobacterium tuberculosis, with 2.5 million active tuberculosis cases. Tuberculosis primarily affects the lungs, but it can affect any organ. Extra-pulmonary involvement occurs 15-20% of cases in immune-competent patients and in more than 50% in HIV patients. The most common extra-pulmonary sites are lymph nodes, pleura, abdomen, genitourinary tract, skin, joints and bones, or meninges. Case reports: In this pictorial review article we present 8 atypical cases of tuberculosis and describe their imaging features and histopathology. Conclusions: Extra-pulmonary tuberculosis can affect virtually any organ and can mimic various inflammatory and neoplastic disorders apart from other infective conditions. A strong index of suspicion is required particularly in those countries endemic to tuberculosis.

Statins decrease the expression of c-Myc protein in cancer cell lines.

Statins are potent inhibitors of the mevalonate/cholesterol biosynthetic pathway and are widely prescribed for the prevention of cardiovascular diseases. Here, we carried out a comprehensive analysis of the effects of three statins, simvastatin, atorvastatin, and lovastatin, on six different cancer cell lines that include a P-glycoprotein-expressing, multidrug resistant variant of an ovarian cancer cell line. Incubation of all cancer cell lines with statins resulted in suppression of cell proliferation without inducing apoptotic cell death. The cell proliferation arrest could be reversed upon transfer of cells to statin-free growth media as well as by the supplementation of the growth media with mevalonate. Further analysis suggested that statins induced cell cycle arrest at G0/G1 phase in four cancer cell lines and the loss of c-Myc protein in three cancer cell lines. The c-Myc expression and the progression of cell division cycle were restored upon the addition of mevalonate to the culture media containing statins. Finally, cells incubated with statins contained an increased level of phosphorylated histone H2AX, an observation previously correlated to cellular senescence. Together, these data demonstrate that statins inhibit the mevalonate pathway which is tightly coupled to oxidative branch of the pentose phosphate pathway, c-Myc expression, cell division cycle progression, and cellular senescence. Implications of these observations in the application of statins as cancer therapeutics are discussed.

Functional characterization of the cytochrome P450 monooxygenase CYP71AU87 indicates a role in marrubiin biosynthesis in the medicinal plant Marrubium vulgare.

BACKGROUND: Horehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds. RESULTS: Mining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare. CONCLUSIONS: In a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.

A 6-week laboratory research rotation in pharmacogenomics: a model for preparing pharmacy students to practice precision medicine.

Comparison of human genome sequences from different individuals has unraveled that genes involved in the drug efficacy and metabolism are polymorphic, harboring mutations, splicing variations and other alterations. These data provide a reasonable explanation for the inter-individual variations observed in drug therapy. Thus, a detailed molecular analysis and an in-depth knowledge of these genes is a prerequisite to practice pharmacogenomics-based medicine. We have introduced a 6-week laboratory research rotation to train students in the expression analysis of different pharmacogenes combined with bioinformatics tools. Students were first introduced to the bioinformatics tools to identify appropriate DNA primers to amplify specific pharmacogenes from the laboratory cancer cell lines. The amplified DNA fragments were sequenced. Finally, students were trained in bioinformatics tools to establish the identity of these DNA sequences. The possible implications of this laboratory training in developing problem-solving skills needed in the implementation of pharmacogenomics knowledge in the clinic, are discussed.

An NGO-Implemented Community-Clinic Health Worker Approach to Providing Long-Term Care for Hypertension in a Remote Region of Southern India.

Poor blood pressure control results in tremendous morbidity and mortality in India where the leading cause of death among adults is from coronary heart disease. Despite having little formal education, community health workers (CHWs) are integral to successful public health interventions in India and other low- and middle-income countries that have a shortage of trained health professionals. Training CHWs to screen for and manage chronic hypertension, with support from trained clinicians, offers an excellent opportunity for effecting systemwide change in hypertension-related burden of disease. In this article, we describe the development of a program that trained CHWs between 2014 and 2015 in the tribal region of the Sittilingi Valley in southern India, to identify hypertensive patients in the community, refer them for diagnosis and initial management in a physician-staffed clinic, and provide them with sustained lifestyle interventions and medications over multiple visits. We found that after 2 years, the CHWs had screened 7,176 people over age 18 for hypertension, 1,184 (16.5%) of whom were screened as hypertensive. Of the 1,184 patients screened as hypertensive, 898 (75.8%) had achieved blood pressure control, defined as a systolic blood pressure less than 140 and a diastolic blood pressure less than 90 sustained over 3 consecutive visits. While all of the 24 trained CHWs reported confidence in checking blood pressure with a manual blood pressure cuff, 4 of the 24 CHWs reported occasional difficulty documenting blood pressure values because they were unable to write numbers properly. They compensated by asking other CHWs or members of their community to help with documentation. Our experience and findings suggest that a CHW blood pressure screening system linked to a central clinic can be a promising avenue for improving hypertension control rates in low- and middle-income countries.

Surface-bound galectin-4 regulates gene transcription and secretion of chemokines in human colorectal cancer cell lines.

One long-term complication of chronic intestinal inflammation is the development of colorectal cancer. However, the mechanisms linking inflammation to the colorectal tumorigenesis are poorly defined. Previously, we have demonstrated that galectin-4 is predominantly expressed in the luminal epithelia of the gastrointestinal tract, and its loss of expression plays a key role in the colorectal tumorigenesis. However, the mechanism by which galectin-4 regulates inflammation-induced tumorigenesis is unclear. Here, we show that galectin-4 secreted by the colorectal cancer cell lines was bound to the cell surface. Neutralization of surface-bound galectin-4 with anti-galectin-4 antibody resulted in increased cell proliferation with concomitant secretion of several chemokines into the extracellular medium. Neutralization of the surface-bound galectin-4 also resulted in the up-regulation of transcription of 29 genes, several of which are components of multiple inflammation signaling pathways. In an alternate experiment, binding of recombinant galectin-4 protein to cell surface of the galectin-4-negative colorectal cancer cells resulted in increased p27, and decreased cyclin D1 and c-Myc levels, leading to cell cycle arrest and apoptosis. Together, these data demonstrated that surface-bound galectin-4 is a dual function protein-down-regulating cell proliferation and chemokine secretion in galectin-4-expressing colorectal cancer cells on one hand and inducing apoptosis in galectin-4-negative colorectal cancer cells on the other hand.

Heterogeneity in the expression of receptors in the human breast cancer metastasized to the brain.

Assessment of the human epidermal growth factor receptor-2 (Her2/ErbB2) and estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer has been an accepted standard to predict clinical outcome. Expression of these receptors in primary breast cancer has also been an important predictor of visceral organ metastasis. Many studies of breast cancer have reported risk factors for brain metastasis that include Her2/ErbB2 positivity, ER negativity, and negativity for all the above three receptors. However, it is not clear whether expression of these receptors would persist subsequent to brain metastasis. To address this possibility, we analyzed different breast cancer brain metastases (BCBM) for the expression of Her2/ErbB2, ER, and PR by immunohistochemistry procedure. The results showed that BCBM are heterogeneous in the receptor expression: Five BCBMs were Her2/ErbB2-positive and one negative; four BCBMs were ER-positive, and two were negative; five BCBMs were PR-positive and one negative. However, expression of these receptors in their combination is also heterogeneous: Four BCBMs were positive for all of the Her2/ErbB2, ER, and PR; one BCBM was positive for Her2/ErbB2 and PR but negative for ER; one BCBM was positive for PR but negative for Her2/ErbB2/ER. Similar heterogeneity in the expression of these receptors was also observed in primary tumors. Importantly, BCBM tumors that were assigned as ER- and PR-positive contained tumor cells that lacked expression of these receptors in other regions of the biopsies. Taken together, our findings indicate that the BCBM exhibit heterogeneity in the expression amounts of Her2/ErbB2, ER, and PR, which could be a result of the influence of tumor microenvironment in the brain or different tumor cells populating the metastatic region.

The influence of metastatic site on the expression of CEA and cellular localization of ß-catenin in colorectal cancer.

BACKGROUND AND AIM: The usefulness of carcinoembryonic antigen (CEA) in the diagnosis and prognosis of colorectal cancer (CRC) is unclear. The aim was to analyze changes in the expression of CEA during CRC progression and metastasis, so as to determine the influence of tumor metastatic organ on the CEA expression by CRC cells. METHODS: The human biopsies of adenocarcinomas in colon and CRC liver and lung metastases were analyzed by immunohistochemistry for the expression of CEA. Expression of E-cadherin and ß-catenin was also analyzed to localize the CRC neoplastic glands in metastatic tissues. RESULTS: The CRC neoplastic glands in colon and liver expressed significantly higher amount of CEA compared with crypts in normal colon. In contrast, CRC neoplastic glands formed in lung expressed low CEA level. However, CEA expression was high in areas of tumor necrosis in lung. E-cadherin and ß-catenin were cell membrane-bound in normal crypts and CRC neoplastic glands in colon and liver. Although these two proteins were also cell membrane-bound in a majority of CRC neoplastic glands in lungs, a significant proportion of these expressed ß-catenin in the nucleus, which lacked either E-cadherin or ß-catenin at the cell membrane. CONCLUSION: Our findings indicate that lung microenvironment is unique in that it suppresses the expression of CEA by CRC cells forming neoplastic glands. In addition, lung microenvironment promotes nuclear localization of ß-catenin, suggesting that the Wnt signaling pathway is relatively active highly in CRC metastasized to lung, when compared with liver or colon.

Luteolin induces apoptosis in multidrug resistant cancer cells without affecting the drug transporter function: involvement of cell line-specific apoptotic mechanisms.

Bioflavonoids are of considerable interest to human health as these serve as antioxidant and anticancer agents. Although epidemiological and experimental studies suggest that luteolin, a natural bioflavonoid, exhibits chemopreventive properties, its effectiveness as an antiproliferative agent against multidrug resistant (MDR) cancers is unclear. Thus, we assessed the antiproliferative effects of luteolin and associated molecular mechanisms using two MDR cancer cell lines that express high levels of P-glycoprotein and ABCG2. In this article, we demonstrate that luteolin induces apoptosis in P-glycoprotein- and ABCG2-expressing MDR cancer cells without affecting the transport functions of these drug transporters. Analysis of various proliferative signaling pathways indicated that luteolin-induced apoptosis involves reactive oxygen species generation, DNA damage, activation of ATR → Chk2 → p53 signaling pathway, inhibition of NF-kB signaling pathway, activation of p38 pathway and depletion of antiapoptotic proteins. Importantly, use of luteolin in these analyses also identified specific molecular characteristics of NCI-ADR/RES and MCF-7/Mito(R) cells that highlight their different tissue origins. These results suggest that luteolin possesses therapeutic potential to control the proliferation of MDR cancers without affecting the physiological function of drug transporters in the body tissues.

Galectin-4 functions as a tumor suppressor of human colorectal cancer.

Development of colorectal cancer (CRC) involves a series of genetic alterations with altered expression of proteins and cell signaling pathways. Here, we identified that galectin-4 (gal-4), a marker of differentiation, was down-regulated in CRC. The goal of this work was to determine the function of gal-4 in CRC. Toward this goal, the human colon biopsies and tissue microarrays containing a gradient of pathology were analyzed for gal-4 expression by immunohistochemistry. Cell proliferation, migration, motility, forced expression, knockdown, cell cycle and apoptosis assays were used to characterize gal-4 function. Immunohistochemistry identified that gal-4 expression was significantly down-regulated in adenomas and was essentially absent in invasive carcinomas. Forced expression of gal-4 in gal-4 -ve cells induced cell cycle arrest and retarded cell migration and motility. Further, gal-4 sensitized the cells to camptothecin-induced apoptosis. Gal-4 knockdown resulted in increased cell proliferation, migration and motility. Gal-4 was found to be associated with Wnt signaling proteins. Finally, gal-4 expression led to down-regulation of Wnt signaling target genes. This study demonstrates that loss of gal-4 is a common and specific event in CRC. This study also shows that gal-4 exhibits tumor suppressive effects in CRC cells in vitro. Through its ability to interact with and down-regulate the functions of Wnt signaling pathway, gal-4 reveals a new dimension in the control of the Wnt signaling pathway. Thus, gal-4 may prove to be an important molecule in understanding the biology of CRC.

Synthesis, spectral characterization, in vitro microbial and cytotoxic studies of lanthanum(III) and thorium(IV) complexes with 1,2,4-triazole Schiff bases.

A series of metal complexes of La(III) and Th(IV) have been synthesized with newly derived biologically active ligands. These ligands were synthesized by the condensation of 3-substituted-4-amino-5-hydrazino-1,2,4-triazole with 8-formyl-7-hydroxy- 4-methylcoumarin. The structure of the complexes has been proposed by elemental analyses, spectroscopic data i.e. i.r., (1)H nmr, Uv-Vis, FAB-mass and thermal studies. The elemental analyses of the complexes conform to the stoichiometry of the type [La(L).3H2O].2H2O and [Th(L)(NO3).2H2O].2H2O where (L = L(I)-L(IV)). All the complexes are soluble in DMF and DMSO and are non-electrolytes in DMF and DMSO. All these ligands and their complexes have also been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Staphylococcus pyogenes and Pseudomonas aeruginosa) and antifungal activities (Aspergillus niger, Aspergillus flavus and cladosporium) by the MIC method. The brine shrimp bioassay was also carried out to study their invitro cytotoxic properties.

Type 1 TNF receptor forms a complex with and uses Jak2 and c-Src to selectively engage signaling pathways that regulate transcription factor activity.

The type 1 TNFR (TNFR1) contains a death domain through which it interacts with other death-domain proteins to promote cellular responses. However, signaling through death-domain proteins does not explain how TNFR1 induces the tyrosine phosphorylation of intracellular proteins, which are important to cellular responses induced by TNFR1. In this study, we show that TNFR1 associates with Jak2, c-Src, and PI3K in various cell types. Jak2 and c-Src constitutively associate with and are constitutively active in the TNFR1 complex. Stimulation with TNF induces a time-dependent change in the level of Jak2, c-Src, and PI3K associated with TNFR1. The tyrosine kinase activity of the complex varies with the level of tyrosine kinase associated with TNFR1. TNFR1/c-Src plays a role in activating Akt, but not JNK or p38 MAPK, whereas TNFR1/Jak2 plays a role in activating p38 MAPK, JNK, and Akt. TNFR1/c-Src, but not TNFR1/Jak2, plays an obligate role in the activation of NF-kappaB by TNF, whereas TNFR1/Jak2, but not TNFR1/c-Src, plays an obligate role in the activation of STAT3. Activation of TNFR1 increased the expression of vascular endothelial growth factor, p21(WAF1/CIP1), and manganese superoxide dismutase in MCF7 breast cancer cells, and increased the expression of CCl2/MCP-1 and IL-1beta in THP-1 macrophages. Inhibitors of Jak2 and c-Src impaired the induction of each of these target proteins. These observations show that TNFR1 associates with and uses nonreceptor tyrosine kinases to engage signaling pathways, activate transcription factors, and modulate gene expression in cells.

Varied expression and localization of multiple galectins in different cancer cell lines.

Galectins play a key role in oncogenic processes. Although several galectins are known, their relative expression at the mRNA and protein levels, the subcellular localization, and their relationship to the oncogenic manifestation remains unclear. Herein we report a comprehensive characterization of the expression of major galectins in human breast cancer (drug-sensitive MCF-7 and drug-resistant MCF-7/Adr(R)), colon cancer (HCT-116 and HT-29), and glioma (T98G) cell lines, as these cells are common model systems for studying oncogenic processes. The expected approximately 14.5 kDa galectin-1, predominantly cytosolic, was detected in the cancer and normal cell lines. Notably, two different molecular forms of galectin-1 with molecular masses of approximately 13.5 and 15 kDa were detected in T98G cells, the latter being in the extracellular medium, perhaps a result of post-translational processing. Immunocytochemistry indicated that the extracellular galectin-1 bound to the cell surface was punctated in appearance, suggesting that it was bound to specific receptors. Immunohistological studies indicated that metastasizing carcinomas express high levels of galectin-1. On the other hand, galectin-3 was readily detectable in all cancer cell lines but undetectable in normal cell lines, indicating that galectin-3 is a cancer-specific biomarker protein. Galectin-3 was a cytosolic protein but was not detected in the extracellular medium, indicating that cancer cells do not secrete this galectin. Finally, despite the RT-PCR analysis suggesting the presence of two transcripts of galectin-8 in all cancer cell lines, the corresponding approximately 36 kDa protein was only detectable in the nuclear and cytosolic fractions upon cell fractionation. Notably, a different molecular form of galectin-8 of approximately 18 kDa was immunoprecipitated from the extracellular media, suggesting that the secreted galectin-8 undergoes post-translational processing. These results highlight the expression of galectins in different molecular forms in cancers, warranting caution in interpreting the results of functional studies of individual galectins, particularly because these proteins function redundantly in cancer pathways.

Bap29varP, a variant of Bap29, influences the cell surface expression of the human P-glycoprotein.

P-glycoprotein (Pgp), a plasma membrane (PM) glycoprotein, is responsible for the development of multidrug resistance. The mechanism by which Pgp is targeted to the PM is not defined. To identify proteins that influence Pgp trafficking, we utilized the yeast two-hybrid analysis procedure, which identified a new isoform of endoplasmic reticulum (ER)-bound Bap29, termed Bap29varP, as an interacting protein with the N-terminus of Pgp. The drug-resistant human breast cancer MCF-7 (MCF-7/Adr(R)) cells express both Bap29varP and approximately 170 kDa Pgp, which are however absent in the drug-sensitive MCF-7 cells. When Bap29varP was overexpressed in MCF-7/Adr(R) cells, Pgp was predominantly localized in the ER and intracellular vesicles, suggesting Bap29varP influences Pgp trafficking. When Pgp was expressed in MCF-7 cells, it was exclusively found in the ER with a molecular mass of approximately 160 kDa slightly smaller than that of the molecular mass of Pgp expressed in MCF-7/Adr(R) cells. On the other hand, when Pgp was expressed in Bap29varP-containing human colon adenocarcinoma HT-29 cells, it was localized at the PM. These findings together suggest that Bap29varP acts as an essential chaperone, influencing the processing and trafficking of Pgp to the cell surface.

RNF2 interacts with the linker region of the human P-glycoprotein.

The human P-glycoprotein (Pgp) is a drug-efflux pump responsible for innate or acquired multidrug resistance in many cancers. Pgp contains a unique approximately 75 amino acid long linker region in its middle, which is critically important for its drug transport and ATPase functions. To identify cellular proteins that bind to this linker region and modulate Pgp function, a yeast two-hybrid analysis was carried out. This procedure identified RNF2 (RING finger protein 2), an E3 ubiquitin ligase, as a prominent Pgp-interacting protein. Co-expression of RNF2 with Pgp in Sf9 insect cells resulted in decreased ATPase activity and proteolytic protection of the transporter protein. Immunoprecipitation experiments confirmed the physical interaction between these two proteins. Confocal microscopy showed the presence of RNF2 in the cytoplasm of the Pgp-negative, drug-sensitive MCF-7 breast cancer cells. However, it was undetectable in the Pgp-positive and drug-resistant MCF-7 cells. We suggest that RNF2 regulates the cellular abundance of Pgp, and plays a key role in the development of cancer drug resistance through its own down-regulation.

Tissue transglutaminase interacts with protein kinase A anchor protein 13 in prostate cancer.

We have previously described that tissue transglutaminase (tTG) is a high level phenotypic biomarker in prostate cancer, which is down regulated in prostate cancer and surrounding premalignant field compared to benign prostate glands. To understand the function of tTG in prostate cancer, we sought to identify proteins that interact with the transglutaminase moiety of tTG using a human prostate cancer complementary deoxyribonucleic acid library in a Yeast 2-Hybrid system. The Yeast 2-Hybrid experiments identified a strong and novel interaction between the transglutaminase moiety and protein kinase A anchor protein 13 (AKAP13), which was quantified by beta-galactosidase assay, confirmed in vitro by immunoprecipitation experiments using PC3 prostate cancer cell lysates, and in vivo colocalization was confirmed by immunofluorescence studies in PC3 cells. Because AKAP plays a major role in protein kinase A and Rho protein mediated signaling, functional studies are underway to elucidate the significance of tTG-AKAP13 interaction in prostate cancer.

Characterization of a new antibody raised against the NH2 terminus of P-glycoprotein.

PURPOSE: Cancers exposed to chemotherapy develop multidrug resistance, a major cause for chemotherapy failure. One mechanism of multidrug resistance development is due to overexpression of P-glycoprotein (Pgp) in these cancer cells. Thus, a prechemotherapy evaluation of Pgp in cancer cells aids in the design of alternative regimens that can circumvent such failure. As few Pgp-specific antibodies are available in detecting low levels of Pgp, there is a need for preparing an antibody that allows the detection of Pgp by various immunologic methods. EXPERIMENTAL DESIGN: We selected the amino acid stretch 11 to 34 in the cytoplasmically located NH2 terminus of Pgp as antigen, which was chemically synthesized and used to raise an antibody in a rabbit, termed NH2 11 antibody. We compared the properties of NH2 11 antibody with that of the well-characterized Pgp-specific antibody, C219, by Western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. RESULTS: Immunoblotting analysis suggested that NH2 11 antibody efficiently interacts with both recombinant and constitutively expressed Pgp in cancerous and noncancerous human cells. Immunoprecipitation reactions indicated that the NH2 11 antibody selectively immunoprecipitates Pgp. Immunocytochemical analyses indicated that the NH2 11 antibody detects Pgp in drug-resistant breast cancer cells as well as in human prostate and breast adenocarcinoma tissue sections. CONCLUSION: As the NH2 11 antibody detects Pgp present in cells and tissues, we conclude that the amino acid sequence to which this antibody was raised is highly antigenic and the antibody is useful in the detection of Pgp by a variety of immunologic methods.

E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer.

The cadherin family of transmembrane glycoproteins plays a critical role in cell-to-cell adhesion and cadherin dysregulation is strongly associated with cancer metastasis and progression. In this study, we report a novel interaction between protein kinase D1 [PKD1; formerly known as protein kinase C mu (PKCmu)] and E-cadherin. PKD1 is a serine/threonine-specific kinase known to play a role in multiple cellular processes including apoptosis, cytoskeleton remodeling, and invasion. Our study shows that PKD1 colocalizes with E-cadherin at cell junctions in LNCaP prostate cancer cells and coimmunoprecipitates with E-cadherin from lysates of LNCaP cells. In vitro kinase assays have shown that PKD1 phosphorylates E-cadherin. Inhibition of PKD1 activity by the selective inhibitor Gö6976 in LNCaP cells resulted in decreased cellular aggregation and overexpression of PKD1 in C4-2 prostate cancer cells increased cellular aggregation and decreased cellular motility. We also validated the PKD1 and E-cadherin colocalization in human prostate cancer tissue by confocal laser scanning microscopy. Our study has identified E-cadherin as a novel substrate of PKD1, and phosphorylation of E-cadherin by PKD1 is associated with increased cellular aggregation and decreased cellular motility in prostate cancer. Because both E-cadherin and PKD1 are known to be dysregulated in prostate cancer, our study identified an important protein-protein interaction influencing the signal transduction system associated with cell adhesion in prostate cancer.