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Breast cancer remains a significant global health challenge, necessitating the development of innovative therapeutic strategies. This study aims to integrate immunotherapy and suicide gene therapy for breast cancer treatment. The cytokines IL-2 and GM-CSF, which are crucial in orchestrating immune responses against cancer, are the focal points of investigation. This study focused on the co-expression of these cytokines alongside apoptin, a promising protein derived from chicken anemia virus, via the bidirectional survivin promoter (pAIG). The constructed vectors were transfected into MCF-7 cells, which resulted in a significant increase in gene expression. Compared with individual cytokine and apoptin vectors (pIG and pA), the pAIG vector exhibited superior efficacy, reducing cell viability and inducing apoptosis (31.7%) at 72 h post-transfection. Exploration of the interaction of apoptin revealed its association with the human PIK3R1 protein, which interacts with both cytokines, contributing to the inhibition of cell viability. Leveraging the survivin promoter for co-expression presents a promising avenue for targeted breast cancer gene therapy, potentially disrupting intricate signaling networks involved in cancer progression. This study provides valuable insights into the synergistic effects of IL-2, GM-CSF, and apoptin co-expression, offering a compelling approach to advancing breast cancer treatment. These findings contribute to the broader landscape of cancer gene therapy, emphasizing the potential of combining immunomodulatory agents and gene-directed approaches to improve therapeutic outcomes.