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25380 Cholecystokinin-B Receptor -Mediates Growth of Hepatocellular Carcinoma
- Martha Dee Gay, Anita Safronenka, Hong Cao, Robin Tucker, Narayan Shivapurkar, Jill P. Smith
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- Journal:
- Journal of Clinical and Translational Science / Volume 5 / Issue s1 / March 2021
- Published online by Cambridge University Press:
- 30 March 2021, p. 10
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ABSTRACT IMPACT: Cholecystokinin-B Receptor -Mediates Growth of Hepatocellular Carcinoma with the use proglumide. Proglumide is a non-selective antagonistic drug therefore, strategies that block signaling at the CCK-BR may provide to be a novel therapeutic option for Hepatocellular Carcinoma treatment OBJECTIVES/GOALS: Cholecystokinin (CCK)and gastrin mediate the growth of Hepatocellular Carcinoma (HCC) through CCK-R and interruption of this signaling pathway could decrease HCC. CCK-Receptors are overexpressed in HCC and proliferation may be mediated through CCK-B. Blockade of the CCK-BR with proglumide decreased both growth in vitro and tumor growth in vivo. METHODS/STUDY POPULATION: RNA was extracted from murine Hepa1-6, RIL-175 and human HepG2 cells and was evaluated by qRT-PCR for expression of CCK-AR, CCK-BR and gastrin. CCK-R protein expression was analyzed by flow cytometry. HCC cells were treated in vitro with CCK peptide, the CCK-AR antagonist or the CCK-BR antagonist. Proliferation of selective CCK-R KO cells was compared to that of wild-type cells. To determine the effect of a CCK-R antagonist on tumor growth in vivo two cohorts of mice bearing subcutaneous Hepa1-6 or RIL-175 HCC tumors were treated with an oral bioavailable CCK-R antagonist proglumide or untreated water for 3-4 weeks. The mice bearing Hepa1-6 tumors were placed on a high-fat diet to raise blood CCK levels. Mice bearing RIL-175 tumors were fed standard chow to determine if proglumide could block autocrine growth by gastrin. RESULTS/ANTICIPATED RESULTS: The mRNA expression of CCK-AR, CCK-BR and gastrin were increased 80-90-fold in all HCC cell lines compared to that of normal liver. CCK-BRs were detected on >85% of the cells by flow cytometry. CCK peptide (1nM) stimulated HCC growth in vitro in both wild-type cells and in CCK-AR KO cells but not in CCK-BR KO cells. CCK-BR antagonist blocked CCK-stimulated growth in vitro but the CCK-AR antagonist did not, suggesting that the CCK-BR was responsible for mediating proliferation. In vivo tumor growth was significantly reduced with proglumide treatment by 70% (p<0.05) in Hepa1-6 and by 73% (p<0.001) in RIL-75 tumors, respectively. DISCUSSION/SIGNIFICANCE OF FINDINGS: CCK-Rs are overexpressed in HCC and proliferation appears to be mediated through the CCK-BR. Downregulation with CRISPR Cas9 or blockade of the CCK-BR with an antagonist decreases growth in vitro and proglumide therapy decreases tumor growth in vivo. Strategies that block signaling at the CCK-BR maybe a novel therapeutic option for HCC treatment.
4121 The beneficial, anti-fibrotic effects of chemokine receptor 2 and 5 antagonists on fat-exposed mouse primary hepatic stellate cells (pHSCs)
- Annie J. Kruger, Kruger Bergman, Martha Gay, Hong Cao, Robin Tucker, Narayan Shivapurkar, Jill P. Smith
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- Journal:
- Journal of Clinical and Translational Science / Volume 4 / Issue s1 / June 2020
- Published online by Cambridge University Press:
- 29 July 2020, pp. 102-103
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OBJECTIVES/GOALS: Non-alcoholic steatohepatitis (NASH) is a leading cause of cirrhosis in the world for which no anti-fibrotic therapies exist. We hypothesized that BMS-22 and maraviroc (MVC), chemokine receptor 2 (CCR2) and 5 (CCR5) antagonists, respectively, would diminish the fibrogenic activity of "fat-exposed" murine pHSCs. METHODS/STUDY POPULATION: pHSCs were isolated from livers of 6 week old male mice following 4 weeks on a NASH-inducing choline-deficient high fat diet (CDAHFD, “fat-exposed”) or standard diet (SD) and passaged in vitro. Early passage (6-12) pHSCs were plate-adhered and TGF-b-treated (10ng/mL) to maximally activate their pro-fibrogenic genes, collagen 1α1 (Col1A1), tissue inhibitor of metalloproteinase 1 (TIMP1), or α-smooth muscle actin (ACTA2). CDAHFD and SD pHSCs were then treated for 48 hours with increasing doses of BMS-22 or MVC (range: 0.3-120ng/mL) to determine (1) the degree of attenuation of the pro-fibrogenic response as measured by qPCR of fibrogenic genes (Col1A1, TIMP1,ACTA2); (2) enhancement of a fibrolytic response as measured by qPCR of matrix metalloproteinases (MMP) 2, 9 and 13 genes; and (3) pHSC migration using the scratch assay. Cell viability and CCR2 and CCR5 gene expression in response to escalating doses of antagonists were also measured. RESULTS/ANTICIPATED RESULTS: Plate- and TGF-b activated CDAHFD pHSCs had a 2-fold greater, dose-dependent attenuation of their pro-fibrogenic activity in response to BMS-CCR2-22 and MVC, when compared with plate- and TGF-b activated SD pHSCs, as measured by reductions in collagen 1α1 (Col1A1) and α-smooth muscle actin (ACTA2) gene expression. TIMP1 gene expression was unaffected by drug treatment for 48 hours. Cell viability was not affected up to doses of 30ng/mL of each drug. pHSCs also demonstrated a dose-dependent increase in CCR2, CCR5 and MMP-9 gene expression in response to surface receptor antagonism. Migration assays comparing CDAHFD and SD pHSCs in response to escalating doses of MVC and BMS-22 are ongoing and expected to demonstrate a significantly decreased migratory capacity of CDAHFD pHSCs than SD pHSCs in response to therapy, reflecting the increased susceptibility of the “fat-exposed” pHSCs to anti-fibrotic therapy than normal pHSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: Anti-fibrotic drugs that dampen pro-fibrogenic activities of “fat-exposed” pHSCs are urgently needed. CCR2 and CCR5 antagonists, BMS-22 and MVC, respectively, can selectively dampen the pro-fibrogenic response of fat-exposed pHSCs, and must be considered for future trials in human NASH. CONFLICT OF INTEREST DESCRIPTION: Dr. Jill Smith has a patent licensing agreement with Immune Therapeutics, Inc.
4119 Cholecystokinin (CCK) Receptor Antagonist Reverses Nonalcoholic Steatohepatitis (NASH) by Reducing Hepatic Macrophages and Inflammatory Cytokines
- Martha Gay, Anita Safronenka, Hong Cao, Robin Tucker, Narayan Shivapurkar, Annie Kruger, Jill Smith
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- Journal:
- Journal of Clinical and Translational Science / Volume 4 / Issue s1 / June 2020
- Published online by Cambridge University Press:
- 29 July 2020, pp. 93-94
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OBJECTIVES/GOALS: NASH increases the risk of cirrhosis and liver cancer. High-fat diets increase CCK levels and CCK receptors have been identified on fibroblasts and immune cells. We hypothesized that CCK receptor blockade could prevent NASH by altering the hepatic microenvironment and macrophage activation. METHODS/STUDY POPULATION: Female mice were fed a Choline Deficient Ethionine supplemented (CDE) saturated fat diet or control high-fat diet for 18 weeks. Mice in each group were treated with a CCK receptor antagonist, proglumide (0.1 mg/ml) in the drinking water or regular water. Resected livers were stained for H&E for features of NASH and F4/80 for macrophages analysis. Liver RNA was evaluated for the expression of cytokines and chemokines using an 84-gene Profiler array (Qiagen). Oxidative stress was analyzed by qRT-PCR for heat shock proteins (HSPs) 27, 60, 70 and 90 and for glutathione by a fluorometric assay. Differences in CDE fed and CDE/proglumide-treated mouse livers were evaluated. RESULTS/ANTICIPATED RESULTS: Livers from mice on the CDE diet displayed histologic features of NASH that were prevented by proglumide. Cytokines and chemokines expression, especially CCL20 and CCL2, were increased in the CDE fed mice and these levels were reduced greater than 20-fold with proglumide. Infiltration of F4/80+ macrophages was markedly increased in the CDE livers and these were reduced by > 50% (p < 0.0001) with proglumide. RNA expression of HSP70 (p = 0.006) and HSP27 (p = 0.011) were reduced with proglumide. Hepatic glutathione concentration more than doubled in the CDE/proglumide treated mice compared to CDE mice. CCK-B receptor expression increased in the CDE-fed mouse livers compared to controls. DISCUSSION/SIGNIFICANCE OF IMPACT: CCK receptor blockade decreases NASH by reducing hepatic macrophages, oxidative stress, and blocking inflammatory cytokines and chemokines. This data supports our novel hypothesis that CCK receptors play a role in NASH and proglumide may provide an innovative treatment for this condition.
2466 MicroRNA-451: A potential key player in the development of diabetic nephropathy in an insulin resistant mouse model
- Maurice B Fluitt, Lijun Li, Narayan Shivapurkar, Carolyn Ecelbarger
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- Journal:
- Journal of Clinical and Translational Science / Volume 2 / Issue S1 / June 2018
- Published online by Cambridge University Press:
- 21 November 2018, p. 20
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OBJECTIVES/SPECIFIC AIMS: MicroRNAs (miRNA) affect transcription of a number of genes involved in the development and progression of diabetic nephropathy (DN), and have become attractive therapeutic targets and biomarkers. Elevated renal gluconeogenesis, fibrosis, and albuminuria are early markers of incipient DN. Recent studies report that renal miRNA-451 may protect against DN and reduce renal gluconeogenesis in rodent models. MiRNA-451 is thought to act by targeting select factors resulting from disrupted insulin and growth factor signaling and the mechanistic-target of rapamycin (mTOR) in early DN. This study aimed to elucidate the role of miRNA-451 in the development and progression of DN. METHODS/STUDY POPULATION: To further elucidate the role of miRNA-451 in DN, we placed male insulin-resistant, TALLYHO/Jng mice on a high-fat diet (60% kCal). The mice were divided into 2 treatment groups and received 8 consecutive weekly intraperitoneal injections of locked nucleic acid (LNA) miR-451-inhibitor or LNA-scrambled compound (2 mg/kg·bw; n=8/treatment). Mice were euthanized after 12 weeks (4 weeks sans injections) and kidneys, liver, pancreas and abdominal adipose tissue were harvested for analysis. RESULTS/ANTICIPATED RESULTS: Renal homogenate expression of miRNA-451 was drastically reduced in inhibitor-treated mice (~6-fold) in comparison with scramble-treated mice. Western blotting of cortex homogenates for indicators of fibrosis and targets of miRNA-451 revealed a significant reduction in collagen IV (marker of epithelial integrity) in inhibitor-treated mice. In addition, metalloproteinase type 9 (MMP9, a known type IV collagenase), YWHAZ (a scaffolding protein and known target of miR-451), mTOR, and fructose bisphosphatase (FBP1, a rate-limiting gene in gluconeogenesis) were significantly increased in this group in comparison to scramble-treated mice. However, no differences were found in protein levels for glucose-6-phosphatase (G-6-Pase) or phosphoenolpyruvate (PEPCK), 2 additional gluconeogenic rate-limiting genes. MiRNA-451 antagonist did not significantly affect final body weight or blood glucose; however, mean blood sodium concentrations were slightly, but significantly higher (2%) in the LNA-inhibitor treated group (when compared with the scramble-treated group). No differences in blood potassium or chloride were found. Anion gap was 90% higher in the LNA-inhibitor treated group when compared with scramble-treated mice. No differences in urinary albumin to creatinine ratio were found between the two treatment groups. However, Masson Trichrome scoring revealed a 59% increase in fibrosis in inhibitor-treated mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Collectively, these findings support a potentially protective role of miRNA-451 in attenuating signaling via mTOR that may alter both renal gluconeogenic potential (contributing to the diabetic phenotype) and activation and progression of renal fibrosis. Therapies to enhance miRNA-451 signaling may be beneficial to reduce renal pathology associated with DN.