Cromomicina A5 induz alterações fenotípicas de morte celular imunogênica em linhagem de melanoma humano com mutação BRAF

Abstract

Immunogenic cell death (ICD) represents a specialized form of regulated cell death characterized not only by the elimination of tumor cells but also by the induction of an adaptive immune response against the tumor itself. This form of cell death is marked by phenotypic events such as autophagy, endoplasmic reticulum stress and apoptosis. The exposure of calreticulin (CRT) on the plasma membrane, extracellular release of HMGB1 protein and ATP, act as danger signals and promote immune recruitment. The search for pharmacological agents capable of triggering ICD has emerged as a promising alternative, particularly for aggressive and therapy-resistant neoplasms such as metastatic melanoma. Chromomycin A5 (CA5), isolated from the marine bacterium Streptomyces sp. BRA-384, has demonstrated, in preclinical studies using melanoma murine models, the ability to induce ICD. In this context, the present study aimed to investigate the immunogenic effects of CA5 in human melanoma models using the SK-MEL-28 cell line (BRAF V600E mutation) to assess its translational potential. The molecule was obtained through the cultivation of the Streptomyces sp. BRA-384 strain, followed by extraction and purification according to previously standardized protocols, with a focus on maximizing yield and compound purity. The cytotoxic activity of CA5 was assessed using the sulforhodamine B (SRB) assay. Flow cytometry was employed to analyze morphological parameters, plasma membrane integrity, CRT exposure on the cell surface, and HMGB1 release, both considered classical ICD markers. Additionally, the expression of genes associated with apoptosis, endoplasmic reticulum stress, and autophagy was evaluated by quantitative polymerase chain reaction (qPCR) at two time points: 6 and 24 hours after exposure to CA5. The results revealed that CA5 exhibits concentration-dependent cytotoxic and cytostatic activity, with a total growth inhibition (TGI) concentration of approximately 4 nM. Flow cytometry analysis showed increased cell granularity, reduced cell volume, and loss of membrane integrity—alterations consistent with cellular stress and regulated cell death. Gene expression analysis indicated the modulation of nine genes associated with regulated cell death pathways, including apoptosis, endoplasmic reticulum stress, and autophagy. CRT exposure on the cell surface was also observed, suggesting the initiation of an immunogenic cell death process. However, HMGB1 release was not detected at the analyzed time point, which may be attributed to the late occurrence of this event in the ICD cascade. The findings demonstrate that CA5 induces features consistent with ICD in vitro in human melanoma cells, partially reproducing effects previously observed in murine models. These results support the translational potential of CA5 as a promising therapeutic prototype in cancer immunotherapy, especially for the treatment of aggressive melanomas.