Biological Effects of a De Novo Designed Myxoma Virus Peptide Analogue: Evaluation of Cytotoxicity on Tumor Cells

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A bioactive peptide analogue for myxoma virus protein with a targeted cytotoxicity for human skin cancer <it>in vitro</it>

Abstract Background Cancer is an international health problem, and the search for effective treatments is still in progress. Peptide therapy is focused on the development of short peptides with strong tumoricidal activity and low toxicity. In this study, we investigated the efficacy of a myxoma virus peptide analogue (RRM-MV) as a candidate for skin cancer therapy. RRM-MV was designed using the Resonant Recognition Model (RRM) and its effect was examined on human skin cancer and normal human skin cells in vitro. Methods Cell cultures were treated with various concentrations of the peptides at different incubation intervals. Cellular morphological changes (apoptosis and necrosis) were evaluated using confocal laser scanning microscopy. The cytotoxic effects of RRM-MV on human skin cancer and normal human skin cells were quantitatively determined by cytotoxicity and cell viability assays. The effect on human erythrocytes was also determined using quantitative hemolysis assay. DNA fragmentation assay was performed to detect early apoptotic events in treated cancer cells. Furthermore, to investigate the possible cell signalling pathway targeted by the peptides treatment, the levels of p-Akt expression in skin cancer and normal cells were detected by immunoblotting. Results Our results indicate that RRM-MV has a dose-dependent toxic effect on cancer cells only up to 18 h. The immunoblotting results indicated that the RRM-MV slightly increased p-Akt expression in melanoma and carcinoma cells, but did not seem to affect p-Akt expression in normal skin cells. Conclusions RRM-MV targets and lethally harms cancer cells and leaves normal cells unharmed. It is able to reduce the cancer cell viability, disrupting the LDH activity in cancer cells and can significantly affect cancer progression. Further investigation into other cell signalling pathways is needed in the process leading to the in vivo testing of this peptide to prove its safety as a possible effective treatment for skin cancer.</p

CLSM micrographs for human squamous carcinoma COLO 16 cell line.

<p>Cell cultures were treated with 50 ng/ml, 100 ng/ml and 200 ng/ml of RRM-MV for 3 h in <b>A</b>, <b>B</b>, and <b>C</b> respectively. Cell culture in <b>D</b> was treated with 200 ng/ml of RRM-C, while cell cultures in <b>E</b> were similarly incubated without any treatment. More necrotic cells and detachment can be seen in B and C as compared with A indicating dose-dependent cytotoxic effect of RRM-MV. No cellular detachment can be seen in the cell culture treated with 200 ng/ml of the negative control RRM-C in D or in the non-treated cell culture in E.</p

CLSM micrographs for apoptosis/necrosis assay with annexin V-Alexa Fluor 488 (green fluorescence) and propidium iodide (red fluorescence) in mouse melanoma cell line (B16F0).

<p>After 3 h incubation with DMEM only in <b>A</b> (blank), 3 h incubation with 800 ng/ml RRM-C in <b>B</b>, and with 800 ng/ml RRM-MV in C. Cytotoxic changes including detachment of confluent layer, apoptotic cells (green) and necrotic cells (red) and are obvious in <b>C</b> when compared with A and B. Longer treatment periods 6 h; 9 h; and 18 h in (<b>D–F</b> respectively) with increased levels of necrosis and cellular detachment when B16F0 cell cultures were treated with (800 ng/ml) of RRM-MV. (200× magnification).</p

Cytotoxic effect of RRM peptide analogues on normal and cancer cells measured by LDH assay.

<p>Cells (3×10⁵) were incubated for 3 h with control peptide (RRM-C), or with RRM-MV at 400 ng/ml (for COLO16) and 1600 ng/ml (for CHO, J774A.1 and B16-F0). Cells without treatment were similarly incubated for 3 h (blank). Each bar represents mean ± standard errors of 3 separate experiments in triplicate. Data values that are significantly altered (ANOVA and Dunnett's post-hoc analysis) are indicated by <b>*</b> (when compared to control treated cells) and <b>+</b> (when compared to untreated cells) at a significant level of <i>p</i><0.05.</p

Cellular viability of mammalian cell lines treated with a single dose of different concentrations of the RRM peptides up to 72 h after treatment.

<p><b>A.</b> CHO, <b>B.</b> B16F0 and <b>C.</b> COLO16 cells. Cell cultures (1×10⁵) were incubated for 4 h, 8 h, 16 h, 24 h, 48 h and 72 h with (50 ng/ml, 100 ng/ml, 200 ng/ml, 400 ng/ml and 800 ng/ml) of RRM-MV and 400 ng/ml of RRM-C. A blank (no treatment control) and a positive control (treated with 60% DMSO) were included in all assays. OD was measured at 570 nm after addition of the Prestoblue™ reagent and incubation for 30 min. Cell viability was calculated and is shown relative to that of untreated (blank) sample (set to 100%). Each bar represents mean ± standard errors of 3 separate experiments in triplicate. Data values that are significantly altered (ANOVA and Dunnett's post hoc analysis) when compared to the untreated cells are indicated by the star symbol (<i>p</i><0.05).</p

Multiple cross-spectral function of myxoma virus proteins (10 sequences alignment).

<p>Multiple cross-spectral function of myxoma virus proteins (10 sequences alignment).</p

The effect of RRM peptide treatment on total Akt and p- Akt in B16F0 cell line.

<p><b>A.</b> Western blots for total Akt (pan) Rabbit MAB in <b>I</b> and p-Akt (Thr308) Rabbit MAB in <b>II</b> without Akt inhibitor. <b>B.</b> Western blots for B16F0 cells treated with 50 µM LY294002 (PI3 kinase inhibitor) prior to immunoblotting with total Akt rabbit MAB in <b>III</b> and p-Akt (Thr308) rabbit MAB in <b>IV</b>. In <b>A</b>, Cells were grown in DMEM only in lane 1; DMEM and 800 ng/ml RRM-C in lane 2; and DMEM with 800 ng/ml RRM-MV in lane 3. In <b>B</b>, cells were either grown in DMEM in lane 1, or in DMEM with 50 µM LY294002 for 1 h in lane 2. Similar intensities of the 60 kDa immune bands for total Akt and p-Akt in treated and non treated cells in A indicate the lack of effect of the RRM-designed peptides on p-Akt activity as compared with the inhibitory effect of the Akt inhibitor on p-Akt activity in B.</p

CLSM micrographs for the apoptosis/necrosis assay in three normal cell lines after 3 h incubation with 800 ng/ml of RRM-MV; or 800 ng/ml of RRM-C.

<p>Mouse skin fibroblasts in <b>A</b>; mouse macrophages J774 in <b>B</b>; and CHO in <b>C</b>. No significant cytotoxic effects (apoptosis, necrosis and cellular detachment) were detected in all cell cultures treated with RRM-MV or RRM-C as compared with the non-treated cell cultures similarly incubated in DMEM, indicating the minimal cytotoxic effect of RRM-MV on the 3 normal cell lines.</p