A new hexapeptide from the leader peptide of rMnSOD enters cells through the oestrogen receptor to deliver therapeutic molecules

Abstract

A 24-amino acid leader peptide of a new human recombinant manganese superoxide dismutase can enter cells and carry molecules. Here, we demonstrated that six of the 24 amino acids penetrate cells through a particular gate represented by a specific amino acid sequence of the oestrogen receptor (ER). We analysed the internalization of the synthetic hexapeptide and the cytotoxic activity of the hexapeptide conjugated to cisplatin on a cell line panel. In most cell lines, the hexapeptide delivered an amount of cisplatin that was 2 to 8 times greater than that released by cisplatin when the drug was used alone. This increased delivery increases the therapeutic index of cisplatin and reduces side effects caused by a high dosage or long-term treatment times. We may consider this hexapeptide a new molecular carrier to deliver molecules with therapeutic activity into ER(+) cells for diagnostic purposes and clinical or immune therapy.

Figure 1. Representative confocal internal plane images of cell lines.

(a) confocal images of the internalization of the hexapeptide in the cytoplasm. In the first column respectively PaTu-8902, A2780, MCF-7, HuH-7 and in the second column the control of each adjacent cell line; in the third column respectively A375, DU145, MCF-10A and in the fourth column the control of each adjacent cell line. Note the green fluorescence dispersed in the cytoplasm. No labelling of the controls was observed. This figure is a merged image of green and blue fluorescence images (exc. 488 nm Argon Laser/em. BP500–550 filter). Scale bar = 14 μm for all images except for A375 and its control = 25 μm. (b) confocal images of the internalization of the scrambled hexapeptide in the cytoplasm of MCF-7 and MCF-10A cell lines. Scrambled does not enter both cells. It is detectable outside the cell membrane. Controls are not shown.

Figure 2. Clonogenic assay.

Figure 2 shows the cell surviving fraction observed after drug treatment with LP-CC6 ranging from 0% (MCF-7, A2780 cell lines) to 58% (A375 cell line) and, for LP-CC24, from 7% (HuH-7) to 71.8% (A375) while the surviving fraction of tumour cells in the presence of CisPt alone at the same concentration was approximately 60%. The plating efficiency and the surviving fraction for each cell line after each treatment were calculated according to the method proposed by Franken et al. and averaged approximately 80% for all cell lines. Survival was calculated compared with non-treated samples using an average of three determinations of the same dose rate of cells (±SE). The average values with a standard deviation were determined from three independent experiments using different LP-CC6, LP-CC24 and CisPt preparations.

Figure 3. Real-time RT-PCR analysis.

Figure 3 shows analysis of mRNA levels of TP53, BAX, BCL2 in all cells treated with LP-CC6, LP-CC24 and CisPt alone by Real-time RT-PCR. We observed increased mRNA levels of TP53 and BAX after 3 hours of treatment with LP-CC6. The expression of these genes was lower in the presence of LP-CC24 in all cell lines except for the melanoma (A375) and pancreatic carcinoma (PaTu- 8902) cell lines. In these cells, all three drugs exhibited little effect on the expression of the transcripts examined. Also evident is a downregulation of the transcript for BCL2, well represented in almost all control cell lines, after treatment with LP-CC6 and LP-CC24. Each sample was normalized to human GAPDH. The bars indicate the mean values of three independent experiments (the bars indicate the s.d.; **p-value < 0.005; *p-value < 0.05; Student’s t-test).

Figure 4. ER expression analysis by Real-time RT-PCR.

Figure 4 shows analysis of mRNA levels of ESR in all cells examined by Real-time RT-PCR. We observed higher level of ESR in MCF-7 and a moderate and/or low expression in the other cell lines. Each sample was normalized to human GAPDH. The bars indicate the mean values of three independent experiments (the bars indicate the s.d.; **p-value < 0.005; *p-value < 0.05; Student’s t-test).