Curcumin Encapsulated into Biocompatible Co-Polymer PLGA Nanoparticle Enhanced Anti-Gastric Cancer and Anti-Helicobacter Pylori Effect.

BACKGROUND: The current disadvantages (high cost, toxicity, resistance) of chemotherapy for gastric cancer opted people for alternative therapy from natural source. Curcumin (natural product) possess multiple biological activities but low bio-availability limits their uses as therapeutic. The Nano-formulation of curcumin increased the bioavailability and productivity of anti-cancer and anti-bacterial properties. The present study was initiated to determine the anti-cancer and anti-bacterial effect of Nano curcumin against gastric cancer and H. pylori. METHODS: Curcumin loaded PLGA nanoparticles (CUR-NPs) was prepared by single emulsion solvent evaporation method. The MIC were determined using agar dilution method to find the anti-H. Pylori activity of Nano curcumin. The cytotoxicity of Nano curcumin was evaluated by MTT assay and the apoptotic effect (cell cycle arrest and morphology change) was shown by PI staining and microscopy. RESULTS: The MIC of nanocurcumin and curcumin for all four H. pylori strains were 8 µg/ml and 16 µg/ml respectively. The inhibition rate of gastric cancer cells after treatment with curcumin was increased from 6% to 67% for 24h, from 8% to 75% for 48h, from 10% to 83% for 72h. In case of nanocurcumin, the inhibition rate increased from 7% to 69% for 24h, 11% to 87% for 48h and 16% to 97% for 72h. The IC50 of curcumin and Nano-curcumin were 24.20 µM and 18.78 µM respectively for 72 h. The population of cells in sub-G0 population increased from 4.1% in the control group to 24.5% and 57.8% when treated with curcumin and nanocurcumin respectively. After 72h of treatment with nanocurcumin, the apoptotic cells population increased as compared to native curcumin treated cells. CONCLUSION: The Nano curcumin might be used as a potential therapeutics against gastric cancer and H. Pylori. There is need of further in vivo study in order to validate CUR-NPs activity.

Novel virulence factor dupA of Helicobacter pylori as an important risk determinant for disease manifestation: An overview.

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative, human gastric pathogen found usually in the mucous lining of stomach. It infects more than 50% of the world's population and leads to gastroduodenal diseases. The outcome of disease depends on mainly three factors: Host genetics, environment and bacterial factors. Among these, bacterial virulence factors such as cagA, vacA are well known for their role in disease outcomes. However, based on the global epidemiological results, none of the bacterial virulence (gene) factors was found to be associated with particular diseases like duodenal ulcer (DU) in all populations. Hence, substantial importance has been provided for research in strain-specific genes outside the cag pathogenicity island, especially genes located within the plasticity regions. dupA found within the plasticity regions was first demonstrated in 2005 and was proposed for duodenal ulcer development and reduced risk of gastric cancer in certain geographical regions. Due to the discrepancies in report from different parts of the world in DU development related to H. pylori virulence factor, dupA became an interesting area of research in elucidating the role of this gene in the disease progression. In this review, we shed light on the detailed information available on the polymorphisms in dupA and their clinical relevance. We have critically appraised several pertinent studies on dupA and discussed their merits and shortcomings. This review also highlights dupA gene as an important biomarker for DU in certain populations.

Low prevalence of clarithromycin-resistant Helicobacter pylori isolates with A2143G point mutation in the 23S rRNA gene in North India.

Resistance of Helicobacter pylori to clarithromycin is associated with a single base substitution in the 23S rRNA gene. In this study, clarithromycin-resistant H. pylori isolates were analysed for the presence of 23S rRNA gene mutations. H. pylori were isolated from 68 patients suffering from various gastroduodenal diseases in North India. Minimum inhibitory concentrations (MICs) were determined by the agar dilution method, and point mutations in clarithromycin-resistant strains were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. Clarithromycin resistance was observed in 11.8% (8/68) of the H. pylori isolates in North India. The A2143G point mutation in the 23S rRNA gene was found in 87.5% (7/8) of the clarithromycin-resistant strains, and the A2142G mutation in association with the T2182C mutation was found in 12.5% (1/8). In conclusion, the continued high prevalence of clarithromycin-sensitive H. pylori strains (88.2%) observed in this study allows the use of the triple-therapy regimen for the treatment of H. pylori infection in this region. Surveillance studies need to be conducted at regular intervals for clarithromycin resistance in the population. To our knowledge, this is the first study in India to report that point mutations at position A2143G and at A2142G in association with T2182C are associated with clarithromycin resistance, confirming reports from other parts of the world.

Helicobacter pylori plasticity region genes are associated with the gastroduodenal diseases manifestation in India.

BACKGROUND: Almost all Helicobacter pylori infected person develop gastritis and severe gastritis is supposed to be the denominator of peptic ulcer diseases, which may lead to gastric cancer. However, it is still an enigma why few strains are associated with ulcer formation, while others are not related with any disease outcome. Although a number of putative virulence factors have been reported for H. pylori, there are contradictory results regarding their connotation with diseases. Recently, there has been a significant attention in strain-specific genes outside the cag pathogenicity island, especially genes within plasticity regions. Studies demonstrated that certain genes in this region may play important roles in the pathogenesis of H. pylori-associated diseases. The aim of this study was to assess the role of selected genes (jhp0940, jhp0945, jhp0947 and jhp0949) in the plasticity region in relation to risk of H. pylori-related diseases in Indian population. METHODS: A total of 113 H. pylori strains isolated from duodenal ulcer (DU) (n = 61) and non-ulcer dyspepsia (NUD) subjects (n = 52) were screened by PCR and Dot-Blot to determine the presence of these genes. The comparative study of IL-8 production and apoptosis were also done by co-culturing the AGS cells with H. pylori strains of different genotype. RESULTS: PCR and Dot-Blot results indicated that the prevalence rates of jhp0940, jhp0945, jhp0947 and jhp0949 in the H. pylori strains were 9.8, 47.5, 50.8, 40.9 % and 17.3, 28.8, 26.9, 19.2 % isolated from DU and NUD, respectively. IL-8 production and apoptotic cell death were significantly higher in H. pylori strains containing jhp0945, jhp0947 and jhp0949 than the strains lacking those genes. Results indicated that the prevalence of jhp0945, jhp0947 and jhp0949 are associated with increased risk of severe diseases in India. CONCLUSION: Our study showed that presence of jhp0945, jhp0947 and jhp0949 were significantly associated with symptomatic expressions along with the increased virulence during in vitro study whereas jhp0940 seems to be negatively associated with the disease. These results suggest that jhp0945, jhp0947 and jhp0949 could be useful prognostic markers for the development of duodenal ulcer in India.

'Modulation of the enzymatic activities of replicative helicase (DnaB) by interaction with Hp0897: a possible mechanism for helicase loading in Helicobacter pylori'.

DNA replication in Helicobacter pylori is initiated from a unique site (oriC) on its chromosome where several proteins assemble to form a functional replisome. The assembly of H. pylori replication machinery is similar to that of the model gram negative bacterium Escherichia coli except for the absence of DnaC needed to recruit the hexameric DnaB helicase at the replisome assembly site. In the absence of an obvious DnaC homologue inH. pylori, the question arises as to whether HpDnaB helicase is loaded at theHp-replication origin by itself or is assisted by other unidentified protein(s). A high-throughput yeast two-hybrid study has revealed two proteins of unknown functions (Hp0897 and Hp0340) that interact with HpDnaB. Here we demonstrate that Hp0897 interacts with HpDnaB helicase in vitro as well as in vivo Furthermore, the interaction stimulates the DNA binding activity of HpDnaB and modulates its adenosine triphosphate hydrolysis and helicase activities significantly. Prior complex formation of Hp0897 and HpDnaB enhances the binding/loading of DnaB onto DNA. Hp0897, along with HpDnaB, colocalizes with replication complex at initiation but does not move with the replisome during elongation. Together, these results suggest a possible role of Hp0897 in loading of HpDnaB at oriC.

Helicobacter pylori strains harboring babA2 from Indian sub population are associated with increased virulence in ex vivo study.

BACKGROUND: The babA2 gene along with the cagA and vacA of Helicobacter pylori has been considered as a risk factor for the disease outcome in certain populations. This study was aimed to understand the role of babA2 of H. pylori with the background of cagA and vacA in disease manifestations in Indian sub population. METHODS: A total of 114 H. pylori strains isolated from duodenal ulcer (DU) (n = 53) and non-ulcer dyspepsia (NUD) patients (n = 61) were screened for the prevalence of these virulence markers by PCR. The comparative study of IL-8 production and apoptosis were done by co-culturing the AGS cell line with H. pylori strains with different genotypes. Adherence assay was performed with babA2 positive and negative strains. Two isogenic mutants of babA2 were constructed and the aforesaid comparative studies were carried out. RESULTS: PCR results indicated that 90.6 % (48/53), 82 % (50/61) and 73.6 % (39/53) strains from DU patients were positive for cagA, vacA, and babA2, respectively. Whereas the prevalence of these genes in NUD subjects were 70.5 % (43/61); 69.8 % (37/53), and 65.6 % (39/61), respectively. Although adherence to AGS cells was comparable among strains with babA2 positive and negative genotypes, but the triple positive strains could induce highest degree of IL-8 production and apoptosis, followed by the cagA (-)/vacA (-)/babA2 (+) strains and triple negative strains, respectively. The wild type strains showed significantly higher IL-8 induction as well as apoptosis in ex vivo than its isogenic mutant of babA2. CONCLUSION: PCR study demonstrated that there was no significant association between the distribution of babA2 genotype or of triple positive strains and disease outcome in this sub population. The adherence assay showed that there was no significant difference in the extent of adherence to AGS cells among babA2 positive and negative strains. But the ex vivo study indicated that the triple positive or even the babA2 only positive strains are involved in increased virulence. The wild type strains also exhibited increased virulence compared to the babA2 mutant strains. This inconsistency demonstrated that bacterial genotype along with host genetic polymorphisms or other factors play important role in determining the clinical manifestation of H. pylori infections.

Association of Intact dupA (dupA1) rather than dupA1 cluster with duodenal ulcer in Indian population.

BACKGROUND: The duodenal ulcer promoting gene (dupA) and dupA cluster in Helicobacter pylori have been described as a risk factor for duodenal ulcer development in some populations. Polymorphic gene dupA can be divided into two groups, intact dupA1 (long or short type based on the presence or absence of 615-bp extra sequences at the 5' region) having complete reading frame and other truncated dupA2 having frame-shift mutation. This study was aimed to elucidate the role of dupA of H. pylori and their clusters in the disease manifestation of Indian population. METHODS: A total of 170 H. pylori strains were screened for the presence of dupA, dupA alleles and dupA cluster by PCR and sequencing. Pro-inflammatory cytokine (IL-8) with different dupA variant H. pylori stimulated gastric epithelial cells (AGS cells) was measured by ELISA. RESULTS: A total of 50 strains (29.4%) were positive for dupA among the tested 170 strains. The prevalence of dupA1 in duodenal ulcer (DU) and non-ulcer dyspepsia (NUD) populations was found to be 25.5% (25/98) and 11.1% (8/72), respectively and 16.4% (28/170) of the tested strains had dupA1, cagA and vacAs1m1 positive. The distribution of long and short type dupA1 has not been significantly associated with the disease outcome. The dupA cluster analysis showed that 10.2% (10/98) and 8.3% (6/72) strains were positive among DU and NUD, respectively. IL-8 production was significantly higher in dupA1(+) , cagA (+), vacA (+) (902.5 ± 79.01 pg/mL) than dupA2 (+) , cagA (+) , vacA (+) (536.0 ± 100.4 pg/mL, P = 0.008) and dupA (-), cagA (+), vacA (+) (549.7 ± 104.1 pg/mL, P = 0.009). Phylogenetic analysis of dupA indicated that the Indian H. pylori strains clustered with East Asian strains but distinct from Western strains. This is the first known genetic element of Indian H. pylori that is genetically closer to the East Asian strains but differed from the Western strains. CONCLUSIONS: The intact dupA1 was significantly associated with DU than NUD (P = 0.029) but the dupA1 cluster has no role in the disease manifestation at India (P = 0.79). Thus, dupA1 can be considered as a biomarker for DU patients in India.

Role of nitric oxide and CCAAT/enhancer-binding protein transcription factor in statin-dependent induction of heme oxygenase-1 in mouse macrophages.

The effect of statins on heme oxygenase-1 (HO-1) was compared in 2 murine cell lines, RAW 264.7 and J774A.1 cell lines, and in primary peritoneal macrophages of BALB/c or C57BL/6 mice. The role of endogenous nitric oxide and the type of transcription factors involved were explored. Simvastatin and fluvastatin induced HO-1. Pretreatment of cells with l-NMMA or 1400 W, two different nitric oxide synthase inhibitors, partially blocked statin-dependent induction of HO-1 in RAW 264.7 and J774A.1 but not in primary peritoneal macrophages. Induction of HO-1 by statins was dependent on p-38 MAP kinase activation in all types of macrophages. In RAW 264.7 cells, both statins increased the activity of reporter genes linked to the proximal 1.3 kbp promoter of HO-1 (EC50 of 1.4±0.3 µM for simvastatin and 0.6±0.03 µM for fluvastatin). This effect was significantly blocked by 1400 W (80±5.2% inhibition, p<0.02) and mevalonate, the direct metabolite of HMGCoA reductase. Gel retardation experiments implicated C/EBPß, AP-1 but not USF, for both RAW 264.7 and primary peritoneal macrophages of C57BL/6 mice. Collectively we showed a differential role of endogenous nitric oxide between macrophage cell lines and primary macrophages and an effect of statins in the protection against inflammation by increasing HO-1 expression.

Reduction of Blood Pressure by AT1 Receptor Decoy Peptides.

BACKGROUND: We previously identified the binding of the chaperone protein gamma-aminobutyric acid receptor-associated protein (GABARAP) to a sequence on the carboxy-terminus of the angiotensin II AT1 receptor (AT1R) and showed that this binding enhances AT1R trafficking to the cell surface as well as angiotensin signaling. METHODS: In this study, we treated sodium-depleted mice with decoy peptides consisting either of a fusion of the cell-penetrating peptide penetratin and the GABARAP/AT1R binding sequence or penetratin fused to a mutated AT1R sequence. We used telemetry to measure blood pressure. RESULTS: Systolic and diastolic pressure fell during the 24 hours following decoy peptide injection but not after control peptide injection. Active cell-penetrating decoy peptide decreased 24-hour average systolic blood pressure from 129.8 ± 4.7 mmHg to 125.0 ± 6.0 mmHg (mean ± standard deviation). Diastolic blood pressure fell from 99.0 ± 7.1 mmHg to 95.0 ± 9.2 mmHg (n=5). Administration of the control peptide raised systolic blood pressure from 128.7 ± 1.3 mmHg to 131.7 ± 2.9 mmHg and diastolic pressure from 93.9 ± 4.5 mmHg to 95.9 ± 4.2 mmHg (n=5). The decreases in both systolic and diastolic blood pressure after active peptide administration were statistically significant compared to control peptide administration (P<0.05, two-tailed Wilcoxon rank-sum test). CONCLUSION: These results indicate the physiological and potentially therapeutic relevance of inhibitors of GABARAP/AT1R binding.

Consolidated academic and research exposition: a pilot study of an innovative education method to increase residents' research involvement.

BACKGROUND: Internal medicine residents at the Ochsner Clinic Foundation stay engaged with clinical work and have difficulty initiating and completing research and publishing their scholarly activities. Commonly cited barriers include lack of knowledge about institutional research programs, lack of confidence regarding medical writing skills, lack of time, and failure to understand the value of research. The residency directors at Ochsner initiated the Consolidated Academic and Research Exposition (CARE) program to teach basic research skills and encourage residents' interest and productivity in research. METHODS: The CARE program includes 4 core components: house staff mentoring and the Resident Career Development Program, a journal club, medical writing instruction, and research engagement. Particular emphasis is given to projects that could be completed within a 1-month period and result in publication, enabling residents to use a 1-month elective rotation during their first postgraduate year. The sessions are mandatory for residents, except for those on specified rotations, including the critical care service and the night float rotation and those who are postcall. RESULTS: In 2010-2011, 6 residents submitted abstracts to the Louisiana Chapter of the American College of Physicians Associates meeting; 2 abstracts were accepted for presentation. In 2011-2012, there were 14 submissions, 4 of which were accepted for presentation. In 2010-2011, there were 4 submissions to the Southern Hospitalist Conference, which increased to 7 submissions in 2011-2012. The second best presentation award at the Southern Hospitalist Conference was also earned by a resident of this institution. The program saw a 110% total increase in scholarly activity from 2010-2011 to 2011-2012. DISCUSSION: The CARE program has been in existence for approximately 1 year. Preliminary results were tabulated based on research proposals, posters, abstracts, case reports, and presentations submitted and/or accepted at leading medical conferences over the past year as compared to the same period 1 year ago. Residents, based on the Accreditation Council for Graduate Medical Education Resident Survey responses, were more satisfied with the opportunities provided to them to participate in research or scholarly activities. Our preliminary results suggest that an organized, structured research curriculum in internal medicine residency programs is critical to promoting, initiating, and completing scholarly activity during a residency program. CONCLUSION: Ochsner's CARE program has appreciably enhanced internal medicine residents' interest in research-related activity, resulting in a significant increase in resident-authored research papers, abstracts, posters, and case reports being accepted at leading national medical conferences.

Next-generation sequencing and de novo assembly, genome organization, and comparative genomic analyses of the genomes of two Helicobacter pylori isolates from duodenal ulcer patients in India.

The prevalence of different H. pylori genotypes in various geographical regions indicates region-specific adaptations during the course of evolution. Complete genomes of H. pylori from countries with high infection burdens, such as India, have not yet been described. Herein we present genome sequences of two H. pylori strains, NAB47 and NAD1, from India. In this report, we briefly mention the sequencing and finishing approaches, genome assembly with downstream statistics, and important features of the two draft genomes, including their phylogenetic status. We believe that these genome sequences and the comparative genomics emanating thereupon will help us to clearly understand the ancestry and biology of the Indian H. pylori genotypes, and this will be helpful in solving the so-called Indian enigma, by which high infection rates do not corroborate the minuscule number of serious outcomes observed, including gastric cancer.

Statins modulate transcriptional activity of heme-oxygenase-1 promoter in NIH 3T3 Cells.

Statins, inhibitors of HMG CoA reductase, have pleiotropic effects independent of their capacity to lower cholesterol. Heme-oxygenase-1(HO-1) plays an important role as an anti-oxidant and anti-inflammatory enzyme. In the present study, we used NIH 3T3 cells which express HO-1 to investigate the molecular mechanisms of HO-1 induction by statins. Simvastatin or fluvastatin induced a significant increase in HO-1 protein expression and mRNA levels. Both statins stimulated activity of a mouse HO-1 promoter (-1,287 to +73 bp)/luciferase reporter gene, 3.25 ± 0.23 (Mean ± S.E.M., n = 15, P < 0.001, t-test) and 3.13 ± 0.33 (Mean ± S.E.M., n = 6, P < 0.001, t-test), respectively. This effect was more pronounced in the short proximal promoter than the full promoter of HO-1. Gel retardation experiments for C/EBP and upstream stimulatory factor (USF) DNA-binding activities using simvastatin- or fluvastatin-treated cells showed significant nuclear protein-DNA complexes which were supershifted with antibodies specific for C/EBP ß and δ or USF-1 and USF-2. Point mutations of the proximal HO-1 promoter (-149 to +73 bp) for the myc/max which binds USF or the C/EBP binding sequences showed a reduction in statin-induced reporter activity whereas no role of the distal C/EBP binding elements located at -4 kb was observed. Moreover, overexpression of mutated C/EBP ß and USF factor or the siRNA for both factors supported a role of these transcription factors in statin-dependent induction of HO-1, with a clearer effect for C/EBP.

Significant association of the dupA gene of Helicobacter pylori with duodenal ulcer development in a South-east Indian population.

A novel virulence factor, duodenal ulcer-promoting gene A (dupA), in Helicobacter pylori has been found to be associated with disease in certain populations but not in others. This study analysed a South-east Indian population as part of the debate about the relevance of dupA for the prediction of clinical outcomes. A total of 140 H. pylori strains isolated from duodenal ulcer (DU) (n = 83) and non-ulcer dyspepsia (NUD) patients (n = 57) were screened by PCR and dot-blot hybridization to determine the presence of the ORFs jhp0917 and jhp0918. Part of jhp0917-jhp0918 was sequenced to search for the C/T insertion that characterizes dupA and the levels of dupA transcripts were also assessed. The PCR and dot-blot results indicated the presence of jhp0917 and jhp0918 in 37.3 % (31/83) and 12.2 % (7/57) of H. pylori strains isolated from DU and NUD patients, respectively. Sequencing analysis showed insertion of a C at nt 1386 in the 3' region of jhp0917, forming the dupA gene in 35 strains. RT-PCR analysis detected the dupA transcript in 28 of these 35 strains. The expression level of the dupA transcript varied from strain to strain, as shown by real-time PCR. The results demonstrated that analysis based on PCR only for dupA may produce an erroneous interpretation. The prevalence of dupA was significantly greater among strains isolated from patients with DU than from patients with NUD in this population (P = 0.001, odds ratio = 4.26, confidence interval = 1.60-11.74). Based on these findings, dupA can be considered a biomarker for DU patients in India. The reported discrepancies for this putative virulence marker in different populations may be due to the genome plasticity of H. pylori.

Gαo potentiates estrogen receptor α activity via the ERK signaling pathway.

The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17ß-estradiol (E(2)). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gα(o) protein subunit potentiated ERα activity in the absence and presence of E(2). Transient transfection of the human breast cancer cell line MCF-7 showed that Gα(o) augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gα(o) revealed that Gα(o) stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gα(o), through activation of the MAPK pathway, plays a role in the regulation of ERα activity.

Distinct repeat motifs at the C-terminal region of CagA of Helicobacter pylori strains isolated from diseased patients and asymptomatic individuals in West Bengal, India.

BACKGROUND: Infection with Helicobacter pylori strains that express CagA is associated with gastritis, peptic ulcer disease, and gastric adenocarcinoma. The biological function of CagA depends on tyrosine phosphorylation by a cellular kinase. The phosphate acceptor tyrosine moiety is present within the EPIYA motif at the C-terminal region of the protein. This region is highly polymorphic due to variations in the number of EPIYA motifs and the polymorphism found in spacer regions among EPIYA motifs. The aim of this study was to analyze the polymorphism at the C-terminal end of CagA and to evaluate its association with the clinical status of the host in West Bengal, India. RESULTS: Seventy-seven H. pylori strains isolated from patients with various clinical statuses were used to characterize the C-ternimal polymorphic region of CagA. Our analysis showed that there is no correlation between the previously described CagA types and various disease outcomes in Indian context. Further analyses of different CagA structures revealed that the repeat units in the spacer sequences within the EPIYA motifs are actually more discrete than the previously proposed models of CagA variants. CONCLUSION: Our analyses suggest that EPIYA motifs as well as the spacer sequence units are present as distinct insertions and deletions, which possibly have arisen from extensive recombination events. Moreover, we have identified several new CagA types, which could not be typed by the existing systems and therefore, we have proposed a new typing system. We hypothesize that a cagA gene encoding higher number EPIYA motifs may perhaps have arisen from cagA genes that encode lesser EPIYA motifs by acquisition of DNA segments through recombination events.

Antiestrogenic activity of flavonoid phytochemicals mediated via the c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha.

Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling independent of direct receptor binding. Here we demonstrate both chalcone and flavone function as cell type-specific selective ER modulators. In MCF-7 breast carcinoma cells chalcone and flavone suppress ERα activity through stimulation of the stress-activated members of the mitogen-activated protein kinase (MAPK) family: c-Jun N-terminal kinase (JNK)1 and JNK2. The use of dominant-negative mutants of JNK1 or JNK2 in stable transfected cells established that the antiestrogenic effects of chalcone and flavone required intact JNK signaling. We further show that constitutive activation of the JNK pathway partially suppresses estrogen (E2)-mediated gene expression in breast, but not endometrial carcinoma cells. Our results demonstrate a role for stress-activated MAPKs in the cell type-specific regulation of ERα function.

Cell-penetrating peptides corresponding to the angiotensin II Type 1 receptor reduce receptor accumulation and cell surface expression and signaling.

BACKGROUND: Our previous published studies have established the γ-aminobutyric acid (GABA) receptor-associated protein (GABARAP) as a trafficking protein for the angiotensin II type 1A receptor (AT(1)R). GABARAP overexpression increases both AT(1)R protein accumulation and translocation to the plasma membrane. The present study examined the inhibitory effects of decoy peptides on receptor expression and plasma membrane accumulation. The decoy peptides correspond to the AT(1)R cytoplasmic domain located immediately proximal to the 7th transmembrane domain, a region implicated in GABARAP binding. This competitive binding study was designed as a first step toward evaluating the GABARAP:AT(1)R binding interface as a target for reducing AT(1)R trafficking to the plasma membrane. METHODS: AT(1)R and GABARAP plasmids were transfected into mammalian cell lines simultaneously with cell-penetrating peptides (CPPs). CPP-1 and CPP-2 consist of the penetratin (pANT(43-58)) CPP with downstream fusions of GKKFKKYFLQL (AT(1)R) and GKKFEEAFLQL (AT(1)R-mutant) amino acids, respectively. CPP-3 consists of the HIV TAT(48-60) CPP with GKKFKKYFLQL (AT(1)R) fused downstream. Western blotting, signal transduction studies, and 3D deconvolution microscopy experiments were employed. RESULTS: Immunoblot analyses and live cell deconvolution microscopy demonstrated that inhibitory (but not control) peptides completely blocked GABARAP-induced intracellular AT(1)R accumulation and cell surface accumulation. GABARAP also stimulated angiotensin II-mediated phospho-ERK1/2 induction by ~ fivefold. This activation was, similarly, quantitatively blocked by the inhibitory peptides. CONCLUSIONS: Cell-penetrating decoy peptides which were designed to block the AT(1)R:GABARAP interaction, effectively reduced AT(1)R intracellular accumulation and cell-surface trafficking and signaling. The binding interaction site between AT(1)R and GABARAP represents a potential therapeutic target.

Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.

C-terminal processing of GABARAP is not required for trafficking of the angiotensin II type 1A receptor.

OBJECTIVE: GABARAP, a small (117 aa) trafficking protein, binds to the C-terminal, cytoplasmic domain of rat angiotensin type-1A receptor (AT(1)R), the predominant effector of the octapeptide angiotensin II (Ang II) (Cook et al., Circ. Res. 2008;102:1539-47). The objectives of this study were to map the interaction domains of GABARAP and AT(1)R, to determine the effect of GABARAP association on AT(1)R signaling activity, and to determine the importance of post-translational processing of GABARAP on accumulation of AT(1)R on the plasma membrane and its signaling function. RESULTS: Deletion analysis identified two regions within GABARAP necessary for interaction with AT(1)R in yeast two-hybrid assays: 1) a domain comprised of residues 32-51 that is nearly identical to that involved in binding and intracellular trafficking of the GABA(A) receptor and 2) a domain encompassing the C-terminal 21 aa. The GABARAP interaction domain of AT(1)R was delimited to the 15 aa immediately downstream of the last membrane spanning region. Overexpression of GABARAP in rat adrenal pheochromocytoma PC-12 cells increased the cell-surface expression of AT(1)R and Ang II-dependent activation of the cAMP signaling pathway. Residues within AT(1)R necessary for these responses were identified by mutational analysis. In PC-12 cells, GABARAP was constitutively and quantitatively cleaved at the C-terminus peptide bond and this cleavage was prevented by mutation of Gly(116). Wild-type GABARAP and the G116A mutant were, however, equally effective in stimulating AT(1)R surface expression and signaling activity. CONCLUSIONS: GABARAP and AT(1)R interact through discrete domains and this association regulates the cell-surface accumulation and, consequently, ligand-induced function of the receptor. Unlike that observed with the GABA(A) receptor, this regulation is not dependent on C-terminal processing and modification of GABARAP.