A Functional Signature Ontology (FUSION) screen detects an AMPK inhibitor with selective toxicity toward human colon tumor cells

Abstract

AMPK is a serine threonine kinase composed of a heterotrimer of a catalytic, kinase-containing α and regulatory β and γ subunits. Here we show that individual AMPK subunit expression and requirement for survival varies across colon cancer cell lines. While AMPKα1 expression is relatively consistent across colon cancer cell lines, AMPKα1 depletion does not induce cell death. Conversely, AMPKα2 is expressed at variable levels in colon cancer cells. In high expressing SW480 and moderate expressing HCT116 colon cancer cells, siRNA-mediated depletion induces cell death. These data suggest that AMPK kinase inhibition may be a useful component of future therapeutic strategies. We used Functional Signature Ontology (FUSION) to screen a natural product library to identify compounds that were inhibitors of AMPK to test its potential for detecting small molecules with preferential toxicity toward human colon tumor cells. FUSION identified 5′-hydroxy-staurosporine, which competitively inhibits AMPK. Human colon cancer cell lines are notably more sensitive to 5′-hydroxy-staurosporine than are non-transformed human colon epithelial cells. This study serves as proof-of-concept for unbiased FUSION-based detection of small molecule inhibitors of therapeutic targets and highlights its potential to identify novel compounds for cancer therapy development.

Ethanol and C2 ceramide activate fatty acid oxidation in human hepatoma cells

“…In some cases, the gels were cut and strips from multiple gels were transferred together 41 . Membranes were blocked with 5% milk in TBST and incubated overnight at 4 °C with antibodies recognizing PLIN2 (1:1000, Abcam, Cambridge, MA) 7 , GAPDH (1:1000, Millipore) 42 , phosphorylated or total AMPK 43 , 44 and ACC 45 , 46 (Cell Signaling, Beverly, MA). Signal was detected with enhanced chemiluminescence (GE Healthcare, Piscataway, NJ) following incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz Technology).…”

Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2

“…180 This approach has been successfully utilized to link natural products to their mechanisms of action 180 and to identify a marine-derived natural product that inhibits AMPK kinase activity in colon tumor cells. 181…”

<p>miR-4999-5p Predicts Colorectal Cancer Survival Outcome and Reprograms Glucose Metabolism by Targeting PRKAA2</p>

“…29 In addition, PRKAA2 has been shown to act as a tumor repressor in CRC. 30,31 As a subunit of AMPK, PRKAA2 has been shown to inhibit cell proliferation through the p53/p21 pathway, and through modulation of the expression of p27. 32,33 Previous studies have shown that PRKAA2/mTOR signaling was crucial to cancer cell proliferation.…”