EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL PROLIFERATION IN PANCREATIC CANCER CELLS
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1 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
2 Pancreatic Cancer 2016 statistics: 53,070 Americans will be diagnosed 41,780 Americans will die There was an increase in 7% for estimated deaths 5-year survival rate of just 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
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 AC/cAMP pathway Vasoactive Intestinal polypeptide (VIP) VPAC1/2R Secretin Secretin R EGF EGFR Somatostatin ss2r β Gαs γ β Gαs γ β Gαi γ Gαi Gαs Gαi ACs Forskolin (FSK) ATP camp
6 Only Forskolin stimulates camp formation in pancreatic adenocarcinoma HPAC cells 200 * camp (pmol/ug) Control VIP Secretin FSK (n=3) (n=3) (n=3) (n=3) Credit: Sierra Quinn
7 AC/cAMP pathway inhibits cell proliferation in pancreatic cancer cell HPAC line 0.6 rate of cell proliferation ( Abs/h) * 0.2 Control FSK Credit: Sierra Quinn
8 Both AC1 and AC3 are highly expressed in pancreatic tumor tissue qpcr Western Blotting kda 250 AC3 AC1 AC7 tubulin healthy tissue tumor tissue Credit: Sierra Quinn
9 AC1 knockdown impairs the inhibitory effect of FSK on the rate of cell proliferation in both pancreatic cancer cell lines Credit: Sierra Quinn
10 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
11 Hypothesis Because PKA and EPAC are known effectors of camp, our hypothesis is that: both PKA and EPAC are involved in the inhibitory effect of FSK/AC1/cAMP pathway on cell proliferation of pancreatic cancer cells.
12 Objective To identify the downstream mediators implicated in effect of the FSK/AC1/cyclic AMP pathway, given that this particular pathway cause an inhibition on proliferation of pancreatic adenocarcinoma epithelial HPAC cell line.
13 Methods Plasmid: pcmv-sport6 ADCY1 Transfection reagent: Lipofectamine P3000 ACs Forskolin (FSK) Western Blotting To test exogen AC1 expression ATP camp determination using EIA kit from Cayman To test the enzymatic activity of exogen AC1 Vybrant MTT Cell Proliferation Assay Kit To determine the cell proliferation camp H-89 FSK (20 µm) To activate AC isoforms, except AC9 Inhibitors: H89 (10 µm) and ESI-09 (15 nm) To inhibit PKA and EPAC activity, respectively. ESI EPAC PKA
14 Results
15 AC1 is over expressed in HPAC cells using ADCY1 plasmid Western Blotting camp determination ** AC1 tubulin control ADCY1 * 6-well plate
16 PKA is the downstream effector implicated in the FSK/AC1/cAMP inhibitory effect on HPAC cells Rate of Cell Proliferation Fold of increase
17 Conclusion AC1 was overexpressed successfully using ADCY1 plasmid FSK inhibited cell proliferation in HPAC cells as previously found. 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. 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
18 Acknowledgements Dr. Sabbatini American Physiological Society (APS) Department of Biological Sciences Center for Undergraduate Research
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EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL MIGRATION IN PANCREATIC CANCER CELLS
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 Pancreatic
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