engleski

Utilizing cell penetrating peptides to enhance the potency of a thermally responsive c-Myc inhibitory polypeptide

Current cancer therapy is limited by the extreme toxicity of chemotherapeutic drugs, which can cause extensive damage to non-cancerous tissues when used in doses required to eradicate cancer cells. The side effect profile and the efficacy of chemotherapeutics can be improved by two mechanisms: 1) developing drugs which are specifically toxic to the cancer cells and 2) developing methods to deliver drugs to the tumor site. In an attempt to combine these approaches, we developed a thermally responsive polypeptide inhibitor of the c-Myc oncogene. This polypeptide is based on the thermally responsive Elastin-like polypeptide (ELP). When injected systemically, ELP-fused drugs will aggregate and accumulate at the tumor site where local hyperthermia is applied. For this study, ELP was fused to a peptide which blocks c-Myc / Max dimerization (H1), thereby inhibiting transcription activation by c-Myc. In a previous study, the H1 peptide was fused to ELP and the penetratin peptide (Pen-ELP-H1), and it was shown that Pen-ELP-H1 was capable of entering MCF-7 breast cancer cells, binding and sequestering c-Myc to the cytoplasm, preventing expression of c-Myc target genes, and inhibiting cell proliferation. Here, the ELP-H1 polypeptide is fused to two different cell penetrating peptides (CPPs), the Tat peptide from HIV-1 and the Bac peptide from the antimicrobial bactenecin 7 peptide. Both CPPs improved cellular uptake relative to ELP-H1 alone, but neither produced intracellular levels equivalent to those seen with Pen-ELP-H1. However, Bac-ELP-H1 was a more potent inhibitor of MCF-7 cell proliferation than Pen-ELP-H1. Bac-ELP-H1 localized to the nucleus in a percentage of the cells, and the nuclear targeting was proportional to increases in both polypeptide concentration and time after treatment. Although more Pen-ELP-H1 entered the cell, it functioned through a mechanism of binding and sequestering newly translated c-Myc to the cytoplasm. The improved potency of Bac-ELP-H1 is likely due to its ability to deliver the H1 peptide to the nucleus, where the c-Myc / Max interaction naturally occurs. The potency of Bac-ELP-H1 was also tested against four cell lines with varying c-Myc expression levels. Bac-ELP-H1 was most potent in the cancer cell lines with high c-Myc expression, HeLa and MCF-7, and less potent in the low c-Myc expressing non-cancerous cell lines MCF-10A and WI-38. This data demonstrates that ELP can be targeted to the desired cellular compartment simply by choice of the CPP used. Also, when targeted to the nucleus by Bac, the c-Myc inhibitor was most potent in the cell lines with the highest c-Myc expression levels. The ELP-based c-Myc inhibitor employs both design strategies described above, creating a therapeutic molecule with two levels of target specificity. The polypeptide can be targeted to the tumor site using focused hyperthermia, and the polypeptide is most toxic to the cancerous cells.

ELP; targeted drug delivery; solid tumors

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