Antitumor effect of Onconase in A375 melanoma cell line. A proteomic analysis.

Antitumor effect of Onconase in A375 melanoma cell line. A proteomic analysis.

1_{Department of Neurosciences, Biomedicine and Movement Sciences, Biochemistry Section,}

University of Verona, Verona, Italy; 2_{Department of Biotechnology, Proteomics and Mass}

Spectrometry Laboratory, University of Verona, Verona, Italy; 3_{ISALIT s.r.l., Novara, Italy;} 4_{Department of Sciences and Technological Innovation, University of Eastern Piedmont,}

Alessandria, Italy; 5_{Center for Translational Research on Autoimmune & Allergic Diseases –}

CAAD, Novara, Italy; 6_{Department of Translational Medicine, University of Eastern Piedmont,}

Novara, Italy.

Onconase (ONC) is a 104 residues basic enzyme that is extracted from the Rana Pipiens frog oocytes and belongs to the “pancreatic-type” ribonuclease super-family. Contrarily to most monomeric RNases, ONC evades the cytosolic RNase inhibitor and exerts remarkable cytostatic/cytotoxic activities against cancer cells which display more negatively membranes than the normal ones.

ONC cytotoxicity has already been tested against some cancer cell lines and also exploited in clinical trials against non-small-lung cancer and unresectable malignant mesothelioma[1].

Human melanoma, an aggressive malignancy characterized by rapidly growing incidence and high mortality rate, is resistant to radiation therapy and cytotoxic chemotherapy[2]. Hence, we evaluated here the effects of ONC against melanoma, registering a remarkable cytotoxic activity in the A375 melanoma cell line, while almost negligible in normal melanocytes (NHEM).

In order to explain the biological effects caused by the drug, the proteomic analysis offer the possibility to find the differential protein expression elicited by ONC treatment in melanoma. A375 cells were cultured in DMEM supplemented with 10% FBS and treated with 2 µM ONC for 48 h. Afterwards, cells were harvested and the total protein extract was analyzed with MS/MS spectrometry by a SWATH label-free proteomic approach. After 48 h treatment with ONC, proteomic analysis showed that 45 proteins were upregulated and 52 downregulated by the drug (fold change >1.5; < 0.667; p value < 0.05). GO enriched biological processes were: telomere regulation, ribosome small subunit biogenesis, mesenchymal cell development and cellular response to ionized radiation. Again, after 48 h, ONC induced a reduction of about 50% cell viability and cell proliferation rate, measured by Crystal Violet assay and BrdU incorporation, respectively. The BrdU result is in agreement with the protein expression reduction of the cell proliferation marker Ki67. Indeed, a down regulation of Ki67 emerged from our proteomic data (FC<0.076) and subsequently validated by western blot analysis.

These preliminary results can represent a useful tool to deepen the study of the proteins involved in the phenomena elicited by the antitumor action exerted by ONC.

References

1.Ardelt et al. (2009). Ribonucleases as potential modalities in anticancer therapy. Eur J Pharmacol. 625, 181-9.

2.Uhara (2018). Recent advances in therapeutic strategies for unresectable or metastatic melanoma and real-world data in Japan. Int J Clin Oncol. doi: 10.1007/s10147-018-1246-y.