Abstract
Abstract In order to identify the patient population who will benefit the most from treatment with c-Myc inhibitors, we have examined and compared the effects that our construct (Bac-ELP-H1) has on different breast cancer cell lines.This construct is a genetically engineered polypeptide composed of the c-Myc inhibitory peptide H1, our macromolecular drug carrier Elastin-like polypeptide (ELP), and the cell penetrating peptide (CPP) Bac. ELP is a thermally responsive macromolecule. Below its transition temperature (Tt), ELP is soluble in aqueous solution; but, when the temperature is raised above the Tt, ELP forms aggregates. Due to its thermal responsiveness, ELPs may be used as a thermally targeted drug delivery carrier. To penetrate tissues and cell plasma membrane, its coding sequence is modified by the addition of a CPP such as Bac, a peptide derived from bovine antimicrobial bactenecin peptide Bac7. When fused to the N-terminus of ELP, Bac facilitates uptake of the CPP-ELP conjugate and its localization to the cytoplasm and the nucleus. Since ELP is genetically encoded and has an easily modified sequence, it can be successfully used for therapeutic peptide (TP) delivery. In this study, we modified the Bac-ELP sequence with the H1 peptide that is derived from helix 1 of the basic helix-loop-helix-zipper domain of c-Myc, a transcriptional regulator that has been shown to be dysregulated in many cancers. H1 inhibits c-Myc and Max dimerization, a crucial step in the c-Myc pathway, thus inhibiting cancer cell proliferation. We tested the antiproliferative activity of Bac-ELP-H1 by MTT in four breast cancer cell lines: MDA-MB-231 (triple negative), MCF-7 (estrogen receptor positive), SK-BR-3 (overexpresses the Her2 gene product) and MCF-7/VP (multidrug resistant). Based on the data obtained from proliferation assays, MCF-7 was the most sensitive to the Bac-ELP-H1 treatment. At 30 μM, the highest concentration used, Bac-E1-H1 resulted in more than 75% inhibition of cell proliferation in MCF-7 cells, while the inhibition of cell proliferation in the other three cell lines, MDA-MB-231, SK-BR-3 and MCF-7/VP, was approximately 50, 60 and 50%, respectively. In order to examine whether inhibition of cell proliferation is a result of cells undergoing apoptosis, an assay using propidium iodide (PI) and Annexin-V-Alexa 488 staining was done. There was no increase in apoptotic cells due to the treatment, indicating that apoptosis is not the mechanism of Bac-ELP-H1 antiproliferative activity. We have also compared c-Myc mRNA levels in these cell lines after the treatment with Bac-ELP-H1 using qPCR. Bac-ELP-H1 treatment lowered the endogenous c-Myc levels in examined cell lines. Further studies are planned to correlate levels of c-Myc mRNA with antiproliferative activity of Bac-ELP-H1. Citation Format: Marija Kuna, Drazen Raucher. Comparison of c-Myc inhibitor effectiveness in various cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2015-5524
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