Synergistic effect of granzyme B-azurin fusion protein on breast cancer cells

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• Breast cancer is a prevalent malignancy with limitations in current therapies, including surgery, chemotherapy, and antibodies.
• Existing treatments face challenges such as adverse effects, drug resistance, and suboptimal efficacy, necessitating novel therapeutic platforms.
• Granzyme B (GrB) is an immune protein that induces apoptosis, while azurin targets cancer cells via a p53-dependent pathway.

Purpose of the Study:

• To develop and characterize a novel Granzyme B-Azurin (GrB-Azurin) fusion protein as a potential biotherapeutic for breast cancer.
• To evaluate the selective toxicity and apoptosis-inducing capabilities of the GrB-Azurin fusion protein on various breast cancer cell lines and normal breast cells.

Main Methods:

• Overexpression and purification of the GrB-Azurin fusion protein in HEK293T cells using metal chromatography.
• Confirmation of protein expression and purification via SDS-PAGE, Western blotting, and ELISA.
• Assessment of apoptosis induction using quantitative real-time RT-PCR for pro-apoptotic genes (p21, Fas, DR5), DNA fragmentation assays, and WST-1 cell viability assays.

Main Results:

• Successful expression and purification of the GrB-Azurin fusion protein were confirmed.
• Treatment with GrB-Azurin significantly induced apoptosis in breast cancer cell lines (MDA-MB-231, MCF7, SK-BR-3) by upregulating p21, Fas, and DR5.
• Insignificant cytotoxicity was observed in normal breast cells (MCF 10A), indicating selective targeting.

Conclusions:

• The GrB-Azurin fusion protein demonstrates potent and selective anti-cancer activity against breast cancer cells.
• This novel fusion protein represents a promising candidate for the development of a new generation of anti-cancer biodrugs.
• The selective effectiveness highlights the potential of engineered fusion proteins in targeted cancer therapy.