Effects of low-density lipoprotein docosahexaenoic acid nanoparticles on cancer stem cells isolated from human hepatoma cell lines.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid with anti-cancer properties. Recently, DHA packaged within low-density lipoprotein (LDL) nanoparticles (LDL-DHA) was demonstrated to be effective in a murine model of hepatocellular carcinoma (HCC). Cancer stem cells (CSCs) are a subpopulation of tumor cells that are resistant to most cancer therapies and thereby, contribute to tumor recurrences. To determine whether LDL-DHA is effective against CSCs derived from human HCC cell lines and tumor bearing rats. Epithelial cellular adhesion molecule positive and CD133 negative (EpCAM+CD133-) CSCs were isolated from HuH-7 and HepG2 human HCC lines and exposed to varying concentrations (1-60 µM) of LDL-DHA nanoparticles for 0-72 h. HCC tumor bearing rats were treated with 2 mg/kg LDL-DHA nanoparticles for 3 days. Regardless of the cell line employed, LDL-DHA nanoparticles achieved 70-100% killing of EpCAM+CD133- CSCs at a concentration of 40 µM after 48 h of exposure while DHA and LDL alone had minimal or no cytotoxic effects. Similar results were obtained with LDL-DHA nanoparticle treatment of EpCAM-CD133- adult cancer cells (ACCs). In keeping with these findings were similar levels of low density lipoprotein receptor expression and LDL-DHA nanoparticle induced lipid peroxidation activity and reactive oxygen species in the CSC and ACC populations. However, differences in sensitivity to LDL-DHA treatment were observed in vivo experiments where LDL-DHA nanoparticle treated tumors had a higher percent of surviving CSCs relative to ACCs. Further research on LDL-DHA nanoparticle therapy for human HCC is warranted.

Heteromultimeric Kv1.2-Kv1.5 channels underlie 4-aminopyridine-sensitive delayed rectifier K(+) current of rabbit vascular myocytes.

The molecular identity of vascular delayed rectifier K(+) channels (K(DR)) is poorly characterized. Inhibition by 4-aminopyridine (4-AP) of K(DR) of rabbit portal vein (RPV) myocytes was studied by patch clamp and compared with that of channels composed of Kv1.5 and/or Kv1.2 subunits cloned from the RPV and expressed in mammalian cells. 4-AP block of K(DR) was pulse-frequency dependent, required channel activation, and was associated with a positive shift in voltage dependence of activation. 4-AP caused a voltage-dependent reduction in mean open time of K(DR). Relief of 4-AP block of whole cell currents during washout required channel activation and was unaffected by voltage. Homotetrameric Kv1.5 channels did not exhibit the shift in voltage dependence of activation exhibited by the native channels. In contrast, Kv1.2 channels displayed a shift in voltage dependence of activation, and this characteristic was also evident during 4-AP treatment when Kv1.2 was coexpressed with Kv1.5 or coupled to Kv1.5 in a tandem construct to produce heterotetrameric [Kv1.5/Kv1.2](2) channels. K(DR) currents were not sensitive to charybdotoxin, which blocks homotetrameric Kv1.2 channels. The findings of this study (1) indicate that vascular K(DR) are inhibited by 4-AP via an open-state block mechanism and trapping of the drug within the pore on channel closure and (2) provide novel evidence based on a comparison of functional characteristics that indicate the dominant form of vascular K(DR) channel complex in RPV involves the heteromultimeric association of Kv1.2 and Kv1.5 subunits.

Overexpression of FGF-2 increases cardiac myocyte viability after injury in isolated mouse hearts.

We generated transgenic (TG) mice overexpressing fibroblast growth factor (FGF)-2 protein (22- to 34-fold) in the heart. Chronic FGF-2 overexpression revealed no significant effect on heart weight-to-body weight ratio or expression of cardiac differentiation markers. There was, however, a significant 20% increase in capillary density. Although there was no change in FGF receptor-1 expression, relative levels of phosphorylated c-Jun NH(2)-terminal kinase and p38 kinase as well as of membrane-associated protein kinase C (PKC)-alpha and total PKC-epsilon were increased in FGF-2-TG mouse hearts. An isolated mouse heart model of ischemia-reperfusion injury was used to assess the potential of increased endogenous FGF-2 for cardioprotection. A significant 34-45% increase in myocyte viability, reflected in a decrease in lactate dehydrogenase released into the perfusate, was observed in FGF-2 overexpressing mice and non-TG mice treated exogenously with FGF-2. In conclusion, FGF-2 overexpression causes augmentation of signal transduction pathways and increased resistance to ischemic injury. Thus, stimulation of endogenous FGF-2 expression offers a potential mechanism to enhance cardioprotection.

Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle cells.

The effect of protein kinase C (PKC) activation on 4-aminopyridine (4-AP)-sensitive delayed rectifier current (IdK) was studied in isolated rabbit portal vein smooth muscle cells by use of standard whole cell voltage clamp. The effects of the phorbol ester, 4 beta-phorbol 12,13-dibutyrate (PdBu, 100 nM) and diacylglycerol analogues, 1,2-dioctanoyl-sn-glycerol (1,2-diC8, 10 microM) and 1,3-dioctanoyl-sn-glycerol (1,3-diC8, 10 microM), on macroscopic whole cell IdK were assessed in myocytes dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 5 mM ATP (20-22 degrees C). Activation of PKC by 1,2-diC8 or PdBu caused a decline in IdK that was reversed with washout of drug. 1,2-diC8 had no effect on outward current present after exposure to 4-AP (20 mM). The inactive analogue, 1,3-diC8, did not affect IdK, but subsequent exposure to the active analogue, 1,2-diC8, caused a marked depression of the current. The inhibition of IdK by 1,2-diC8 was significantly reduced by intracellular dialysis with the inhibitors of PKC, chelerythrine (50 microM) and calphostin C (1 microM). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 mM intracellular BAPTA did not affect the suppression of IdK by 1,2-diC8, indicating the involvement of a Ca(2+)-independent isoform of PKC. This study suggests a novel signal transduction mechanism for inhibition of 4-AP-sensitive IdK involving a phosphotransferase reaction catalyzed by PKC in vascular smooth muscle myocytes.