Establishment of cisplatin treated cells and the sensitivity to Pladienolide B.

<p><i>A</i>, efficacy of cisplatin in SW1271 and H1048 parental cells. <i>B</i>, efficacy of Pladienolide B in parental cells and cisplatin-treated cells. Percent growth was calculated relative to DMSO-treated control.</p

MYC expression in SCLC cell lines by IHC.

<p>Representative MYC IHC staining patterns are shown: <i>A</i>, negative (H196 / H-score 0); <i>B</i>, low MYC (H1963 / H-score 55); <i>C</i>, high MYC (H146 / H-score 140); and <i>D</i>, high MYC (N417 / H-score 230). <i>E</i>, summary of MYC IHC staining by positive cell ratio. The red star indicates cells with <i>MYC</i> gene amplification.</p

Efficacy of a spliceosome inhibitor, Pladienolide B, on SCLC cell lines.

<p>Cells were treated with Pladienolide B for 72 hours and the percent growth was calculated relative to the DMSO-treated control. <i>A</i>, cell lines were classified based on the IHC MYC expression status. <i>B</i>, cell lines were classified based on the presence / absence of a previous chemotherapy at the time of cell line establishment.</p

Inhibitory Activity of Extracts of Top Botanical Candidates.

<p>Inhibitory Activity of Extracts of Top Botanical Candidates.</p

HPLC chromatograms of extracts of top botanical candidates.

<p>(A) <i>Angelica archangelica</i>, (B) <i>Scutellaria baicalensis</i>, (C) <i>Petroselinum crispum</i>, and (D) <i>Garcinia mangostana</i>. The arrows indicate the marker compounds. A1: osthenol, A2: osthole, B1: baicalin, C1: apiin, and D1: α-/γ-mangostin.</p

HPLC gradient conditions.

<p>HPLC gradient conditions.</p

Cell viability after exposure to top botanical candidates.

<p>MTT assay was utilized to determine % cell viability of Hep G2 cells after 72 hr exposure to increasing doses of the top four botanical extracts (Lot #1). (A) <i>Angelica archangelica</i>, (B) <i>Scutellaria baicalensis</i>, (C) <i>Petroselinum crispum</i>, and (D) <i>Garcinia mangostana</i>.</p

3-Step recombinant ketohexokinase enzymatic assay.

<p>KHKC Ketohexokinase C isoform. PK pyruvate kinase. LDH lactate dehydrogenase.</p

Inhibitory KHKC Activity of Candidate Botanical Extracts.

<p>Inhibitory KHKC Activity of Candidate Botanical Extracts.</p

Counteracting Roles of AMP Deaminase and AMP Kinase in the Development of Fatty Liver

<div><p>Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation –either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.</p> </div