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Recombinant RGD-Apoptins Decrease Human Melanoma Cell Viability
Dmitriy Shirokov1,2, Daria Lepekhina1, Valentin Manuvera1,3
1Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia.
Abstract:
Cutaneous melanoma is an extremely dangerous tumor disease with poor prognosis at advanced stages. Accounting for a small percentage of all skin tumors, malignant melanoma leads the mortality rate in this group of cancers. Clearly, the search for new drugs and therapeutic approaches for the treatment of cutaneous melanoma is a highly pressing issue in modern medicine. In this study, novel recombinant proteins with anti-melanoma activity, called RGD-apoptins, were produced in an E. coli expression system, and their properties were evaluated in human cell models. These chimeric proteins consist of two parts, each tumor-specific. One part of the chimeric molecule is the RGD peptide, which binds to αVβ3 integrins widely expressed on the surface of malignant melanocytes. The other part is the viral protein apoptin, known to induce programmed cell death in tumor cells but not in normal cells. This molecular design aims to enhance the specificity of potential therapeutic agent toward malignant melanoma cells while reducing cytolytic effects on healthy tissue. In a resazurin assay, RGD-apoptins decreased the viability of MeWo human melanoma cells and did not affect the viability of HaCaT human keratinocyte cell line and primary skin fibroblasts. Using an annexin V assay, we confirmed that malignant melanocytes death occurs via apoptosis. Transcriptomic analysis allowed us to dynamically evaluate the spectrum of differentially expressed genes 24 and 48 h after treating melanoma cells with recombinant RGD-apoptin.
Insights
Novel RGD-apoptin proteins show promise for treating melanoma. These engineered proteins target cancer cells specifically, inducing apoptosis while sparing healthy tissues, offering a potential new therapeutic strategy for this dangerous skin cancer.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Cutaneous melanoma is a dangerous skin cancer with poor prognosis in advanced stages.
- Malignant melanoma accounts for a small fraction of skin tumors but has a high mortality rate.
- Developing novel therapeutic strategies for melanoma is a critical medical need.
Purpose of the Study:
- To produce and evaluate novel recombinant proteins, RGD-apoptins, for anti-melanoma activity.
- To assess the specificity and efficacy of RGD-apoptins in human cell models.
- To investigate the mechanism of cell death induced by RGD-apoptins.
Main Methods:
- Production of RGD-apoptins using an E. coli expression system.
- Evaluation of RGD-apoptin cytotoxicity using resazurin and annexin V assays in melanoma cells, keratinocytes, and fibroblasts.
- Transcriptomic analysis to study gene expression changes post-treatment.
Main Results:
- RGD-apoptins significantly reduced the viability of MeWo human melanoma cells.
- RGD-apoptins did not affect the viability of HaCaT human keratinocytes or primary skin fibroblasts, indicating specificity.
- Cell death in melanoma cells was confirmed to occur via apoptosis.
- Transcriptomic analysis provided dynamic insights into gene expression alterations.
Conclusions:
- RGD-apoptins demonstrate specific anti-melanoma activity by inducing apoptosis in cancer cells.
- The RGD peptide and apoptin components contribute to targeted delivery and tumor cell death.
- These findings support the potential of RGD-apoptins as a targeted therapy for cutaneous melanoma.
