Molecular determinants of etoposide resistance in HL60 cells.

Chemotherapy resistance is the main reason for treatment failure in acute myeloid leukemia (AML) and the major cause of its mortality. Etoposide is a DNA topoisomerase-II inhibitor that is used either as a single agent or in combination with cytarabine, azacytidine, vinca alkaloids, and anthracyclines for the treatment of relapsed /refractory AML. In this study, we sought to determine and understand the mechanism of etoposide resistance in AML using the HL60 cell line.HL60 cells were treated with incremental doses of etoposide and resistant colonies were isolated by culturing the resistant cells in semi-solid culture media. Three clones were selected for etoposide resistance namely, HL60-EtopR H1A, HL60-EtopR H1B, and HL60-EtopR H1C which demonstrated 4.78, 2.39, and 4.42-fold higher resistance to etoposide compared with the parental cells. To determine molecular differences between the etoposide-resistant HL60-EtopR cells and the parental cells, microarray-based gene expression profiling was performed. We found up regulation of members of the src tyrosine kinase family genes in the etoposide resistant cells. Further studies are required to evaluate the role of Src inhibitors in targeting etoposide resistant cells.

LDL-R promoting activity of peptides derived from human PCSK9 catalytic domain (153-421): design, synthesis and biochemical evaluation.

BACKGROUND: High level of Low Density Lipoprotein-Cholesterol (LDL-C) in circulation in the blood is associated with an elevated risk of cardiovascular disease (CVD) and stroke. Currently the statin drugs which inhibit the enzyme HMG-CoA reductase responsible for cholesterol synthesis in the liver are very effective in lowering LDL-cholesterol. However these drugs are often associated with serious side effects particularly for ∼10-12% of cases. Therefore there is a need to develop non-statin based cholesterol reducing agents. Recently it was revealed that the secreted Proprotein Convertase Subtilisin Kexin 9 (PCSK9) binds with LDL-receptor (LDL-R) causing its degradation in the lysosome with the result of LDL-C accumulating in the blood. Thus PCSK9 has become an alternative target for development of non-statin cholesterol reducing agents. It is established that the catalytic domain of PCSK9 (aa153-421) and the EGF-A domain of LDL-R (aa314-355) are involved in the above bind leading to the reduction of LDL-R level and accumulation of LDL-C. OBJECTIVE: The major goal of this study is to identify peptide/s from the catalytic domain of hPCSK9 that can block the binding of hPCSK9 and LDL-R and therefore can reduce LDL-R degradation leading to the clearance of LDL-C from the plasma. RESULTS: Using 51 synthetic linear peptides (P1-P51) of 15aa long with 10 amino acids overlapping sequences spanning the entire catalytic segment of hPCSK9 (aa153-421), we identified two domains of hPCSK9 namely (aa323-358) and (aa365-384) that exhibited strong binding affinity towards synthetic EGF-A peptide. The results were based on mass spectrometry, fluorescence spectroscopy and native gel electrophoresis. Thus peptides containing the above segments in part (P35-P39 and P42-P47) exhibited LDL-R promoting activity when added exogenously to culture medium of growing human hepatic cells like HepG2 and HuH7. The effects were particularly significant with peptides P36, P37, P46 and P47. Interestingly, the first two peptides are present within the disulphide loop Cys(323)-Cys(358) and contain the key gain of function mutation D(374)/Y site while the last two peptides contain another disulphide bridge loop Cys(375)-Cys(378) and the second most potent gain of function mutation R(357)/H. Further studies revealed that S-S bridged cyclic loop peptide hPCSK9(365-384) exhibited the highest (∼3.5-fold) LDL-R promoting activity in both HepG2 and HuH7 when applied at 5 µM concentration level. This effect is completely abrogated when one of the Cys residues is substituted by Ala thereby preventing any S-S bond formation. This suggested its critical role in the bioactivity. It is proposed that LDL-R promoting activity of this and other selected PCSK9 catalytic peptides such as P36, P37, P46 and P47 are most likely mediated via intervention of PCSK9:LDL-R complex formation. Our findings may find useful application in future development of small molecule PCSK9 inhibitors for intervention of hypercholesterolemia and associated cardiovascular disease.