EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL MIGRATION IN PANCREATIC CANCER CELLS
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1 EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL MIGRATION IN PANCREATIC CANCER CELLS VIDYA MEDEPALLI ADVISOR: Maria Eugenia Sabbatini, PhD PHI KAPPA PHI RESEARCH CONFERENCE MARCH 8 th, 2017
2 Pancreatic Cancer Cancer is the second leading cause of death in the United States statistics: 53,670 estimated new cases 43,090 estimated deaths Increase in 3% for estimated deaths 5-year survival rate- 8% Risk factors: Smoking Poor diet Exposure to pesticides, dyes, and chemicals
3 Cyclic AMP Second messenger generated from ATP, reaction catalyzed by adenylyl cyclases Implicated in the differentiation and progression of several normal and malignant cell types Has controversial effects on cell proliferation in cancer cells In pancreatic cancer cell lines, an increase in camplevels by Forskolinreduces cell proliferation and migration. Nothing is known about the upstream mechanism or downstream effectors involved in the inhibitory effect of camp.
4 Adenylyl Cyclase Adenylyl cyclases(ac) are enzymes which increase the levels of camp when activated. Ten isoforms of AC exist, nine are transmembrane and only one is soluble. Gαs Gαi ACs Forskolin (FSK) ATP camp
5 Pathways implicated in the effects of camp There are currently two pathways implicated in the effect of camp: Protein Kinase A (PKA) Exchange protein directly activated by camp (EPAC) ATP ACs camp Forskolin (FSK) EPAC PKA
6 Preliminary Data FSK inhibited cell proliferation in HPAC cells. The overexpression of AC1 enhanced the inhibitory effect of FSK on cell proliferation. The inhibitory effect of FSK/AC1/cAMP pathway was counteracted in presence of the PKA inhibitor H-89. The inhibitory effect of FSK/AC1/cAMP pathway was not modified in presence of the Epac inhibitor ESI-09. Through the overexpression of the ADCY1 plasmid and the use of chemical inhibitors, we are able to conclude that Protein Kinase A is the main mediator of the inhibitory effect of FSK/AC1/cAMP pathway on proliferation of pancreatic cancer cells. FSK AC1 camp PKA HPAC cell proliferation
7 Hypothesis Because PKA was found to be the effector of campin cell proliferation of HPAC cells, our hypothesis is that: PKA is involved in the inhibitory effect of FSK/AC1/cAMP pathway on migration of pancreatic cancer cells.
8 Objective To identify the downstream mediators implicated in effect of the FSK/AC/cyclic AMP pathway, given that this particular pathway inhibits the migration of pancreatic adenocarcinoma epithelial HPAC cells.
9 Methods Plasmid: pcmv-sport6 ADCY1 Transfection reagent: Lipofectamine p3000 Western Blotting To test exogenac1 expression ATP ACs Forskolin (FSK camp determination using EIA kit from Cayman To test the enzymatic activity of exogen AC1 Cytoselect 24-Well Cell Migration Assay Kit (8 µm, Colorimetric Format) To determine the cell migration camp H-89 FSK (20 µm) To activate transmembrane AC isoforms, except AC9 Inhibitors: H89 (10 µm) and ESI-09(15 nm) To inhibit PKA and EPAC activity, respectively. EPAC PKA ESI
10 Cell Migration Procedure Image taken from Cell BioLabs, Inc website.
11 Result
12 Confirmation that the insert is in the plasmid Gel Electrophoresis 4.4 kb 2 kb * Ladder ADCY1 insert in the pcmv-sport 6 treated with EcoRI and Hind III
13 AC1 is over expressed in HPAC cells using ADCY1 plasmid Western Blotting camp determination AC1 ** tubulin control ADCY1 6-well plate Non-transfected HPAC cells ADCY1- transfected HPAC cells *
14 Neither PKA nor EPAC are implicated in the FSK/AC1/cAMP inhibitory effect on cell migration in HPAC cells n=3
15 Neither PKA nor EPAC are implicated in the FSK/AC1/cAMP inhibitory effect on cell migration in HPAC cells Control AC1 AC1 + FSK H-89 + FSK + AC1 ESI-09 + FSK + AC1
16 Conclusion Through the overexpression of the ADCY1 plasmid and the use of chemical inhibitors, we are able to conclude that neither effector is a mediator in the inhibitory effect of FSK/AC/cAMP pathway on migration of pancreatic cancer cells. PKA, as an effector of AC/cAMP pathway, is likely to be associated with signaling changes dependent on gene expression, including cell proliferation. The inhibitory effect of forskolin on cell migration does not require any camp effector. It may require the physical interaction with proteins involved in migration, such as G-actin.
17 Acknowledgements Dr. Sabbatini American Physiological Society (APS) Department of Biological Sciences Center for Undergraduate Research Augusta University Honors Program
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EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL PROLIFERATION IN PANCREATIC CANCER CELLS
EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL PROLIFERATION IN PANCREATIC CANCER CELLS VIDYA MEDEPALLI ADVISOR: Maria Eugenia Sabbatini, PhD PHI KAPPA PHI RESEARCH CONFERENCE MARCH 18 TH, 2016
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