Dayami Lopez

Purified human chorionic gonadotropin induces apoptosis in breast cancer

Agents that induce apoptosis in breast cancer cells have great potential to facilitate chemotherapeutic intervention and improve patient outcomes. In this study, the effects of injecting purified human chorionic gonadotropin (hCG) directly into human breast cancer xenografts grown in nude mice were examined. It was shown that intratumoral injection of purified hCG increased the apoptotic index in breast cancer xenografts. These results were supported by the findings that exposure of breast cancer cells to purified hCG decreased cell viability in five different breast cancer cell lines. In some of these cell lines, the effects of hCG in cell viability appear to correlate with activation/expression of the hCG/luteinizing hormone receptor. Preoperative apoptotic induction by factors such as purified hCG may improve local control or work synergistically with neoadjuvant chemotherapy to improve complete pathologic response of locally advanced breast cancer. [Mol Cancer Ther 2008;7(9):2837–8]

Having excess levels of PCSK9 is not sufficient to induce complex formation between PCSK9 and the LDL receptor

Proprotein convertase subtilisin/kexin-9 (PCSK9) acts mainly by forming complexes with the LDL receptor at the cell surface, which are then degraded in the lysosome. Studies were performed to determine whether excess levels of PCSK9 was sufficient to induce PCSK9/LDL receptor complex formation in human hepatocyte-like C3A cells. It was demonstrated using ELISA that instead of considering the overall levels of PCSK9 protein that is produced in response to certain treatment, what is critical is how much PCSK9 is actually capable of forming complexes. Despite the high levels, most of the PCSK9 produced as a result of incubating cells with a medium supplemented with BD™ MITO+ serum extender (MITO+ medium) appeared to be inhibited by a secreted factor. Having lower levels of PCSK9/LDL receptor complexes did not prevent an increase in the degradation rate of LDL receptors in MITO+ medium as compared to fetal bovine serum (FBS) containing medium (Regular medium), an effect that did not correlate with an increase in protein levels of the inducible degrader of LDL receptors (IDOL), as demonstrated using Western blotting analysis. Additional studies are required to determine the exact mechanism(s) for the degradation of the LDL receptor and/or to identify the secreted inhibitor of PCSK9.

Distribution of the LDL receptor within clathrin-coated pits and caveolae in rat and human liver.

Several findings suggest that the low-density lipoprotein (LDL) receptor may internalize different lipoprotein particles via diverse pathways. Using a combination of discontinuous sucrose gradients and Triton solubilization studies, we demonstrated that the LDL receptor could be located simultaneously in clathrin-coated pits and caveolae in rat and human liver and in human hepatocyte-like C3A cells. Treatment with the cholesterol biosynthesis inhibitor, zaragozic acid A, shifted the distribution of the LDL receptor to clathrin containing fractions, whereas treatment with cholesterol or LDL shifted the receptor distribution towards caveolin-1 containing fractions. The LDL-dependent shift of the LDL receptor to caveolae coincided with a reduction in internalization of Bodipy-LDL. Redistribution within plasma membrane microdomains in response to specific treatments resulting in changes in LDL receptor function represents a novel paradigm that could be exploited in the development of a new class of therapeutic drugs.

Hypercholesterolemia: The role of PCSK9

Heart disease ends the life of more people than any other disease in the United States. High levels of low density lipoprotein (LDL)-cholesterol cause heart diseases by increasing the formation of atherosclerotic plaques. Proprotein convertase subtilisin/kexin-9 (PCSK9) indirectly regulates plasma LDL levels by controlling the LDL receptor expression at the plasma membrane. PCSK9 also appears to modulate glucose intolerance, insulin resistance, abdominal obesity, inflammation, and hypertension. The magnitude of PCSK9s involvement in the onset of these metabolic abnormalities appears to be associated with age, sex, and ethnic background. Another regulator, the inducible degrader of the LDL receptor (IDOL), works by enhancing the ubiquitination of the LDL receptor. Herein, we will review the functions and regulatory mechanisms of PCSK9. The effects of PCSK9 on the LDL receptor, the relationship of this convertase with IDOL, and treatments currently available against hypercholesterolemia are also discussed.

Osmotic Stress Modulates the Expression and Function of the Human LDL Receptor

The purpose of this study was to determine whether the Low-Density Lipoprotein (LDL) receptor could be directly regulated by high glucose (HG) in human hepatocyte-like C3A cells. C3A cells were cultured in a medium supplemented with BD™ MITO+ serum extender (MITO+ medium), a serum-free, cholesterol deficient medium. We found that HG reduced receptor mRNA levels without significantly affecting overall or plasma membrane receptor protein expression. Interestingly, these effects were also seen in the presence of low glucose + high mannitol (LG +HM). LDL receptor protein synthesis, protein degradation, and receptor function (LDL internalization) were increased by HG and LG+HM. However, no changes in protein expression of proprotein convertase subtilisin kexin type 9 (PCSK9) or the inducible degrader of LDL receptors (IDOL), the known degraders of the LDL receptor, were seen under the same conditions. These results implied that the effects of HG and LG+HM on the expression/ function of the LDL receptor were mostly due to an osmotic stress induced by the high levels of these monosaccharides. Further studies are required to determine how other factors found in diabetic patients, such as high cholesterol and/or high fatty acid levels, may influence the osmotic- dependent regulation of the LDL receptor expression/function due to hyperglycemia.

Abstract 1289: Cooperation between beta-catenin, Snail, Slug, and Twist is required for maximum repression of the human vascular endothelial-cadherin promoter

Vascular endothelial (VE)-cadherin is a key protein in the adherens junctions. This protein forms homophilic intercellular interactions and is required for the integrity of the endothelial monolayer, endothelial permeability, and control of cell growth. We have previously demonstrated that exposure of endothelial cells to breast cancer cell-conditioned media results in upregulation of Twist, Slug, and Snail expression, which in turn downregulate the activity of the human VE-cadherin promoter through at least two E-box motifs. Since these repressors have been linked to the β-catenin signaling pathway, we chose to determine whether β-catenin could regulate the activity of the human VE-cadherin promoter. Herein, we demonstrate that β-catenin represses the VE-cadherin promoter either alone or in coordination with the repressors Twist, Slug, and Snail. Electrophoretic mobility shift assays showed that the VE-cadherin promoter contains four β-catenin/LEF-1 binding motifs (designated Site-1-4) that appear to be involved in this regulatory process. In addition, we identified TGFβ as one of the factors released by breast cancer cells that could be involved in the regulation of the VE-cadherin gene when HDMEC are exposed to breast cancer cell conditioned medium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1289. doi:1538-7445.AM2012-1289

Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T3

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp − 612 and − 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the − 612 and − 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.