Exploring the mechanisms of early acquired resistance to doxorubicin in melanoma spheroids

This study investigated the mechanisms underlying early settlement of doxorubicin (DOX) resistance in B16.F10 murine melanoma spheroids, following repeated exposure to a subinhibitory concentration of the drug. Melanoma spheroids were twice treated with DOX for 48h with a 48h recovery period, and changes in viability, growth, gene/protein expression, and enzyme activity were assessed using RNA-seq, RT-qPCR, western blot, protein array, and gelatinase assays. DOX exposure triggered a biphasic response in melanoma spheroids, with the initial exposure downregulating transcripts involved in cell cycle, DNA damage and repair responses, and suppressing of TNF-α via NF-κB and mTORC1 stress response-related signaling pathways, indicating cell cycle arrest, enhanced DNA damage, and apoptosis resistance. Concurrently, upregulation of Notch1 , and of angiogenic, adhesion, and ECM remodeling genes and proteins indicated early DOX-adaptive responses aimed at evading checkpoint arrest and increasing cell aggressiveness. A second DOX exposure amplified these responses in melanoma spheroids, leading to upregulation of some genes involved in cell cycle progression, DNA repair damage responses, along with increased Aqp1 , VEGF , Ackr3 , MMP-2 expression, as well as elevated MMP-9 activity. Our results offer valuable insights into the molecular drivers of chemoresistance, revealing that early DOX-resistance in melanoma arises from adaptive mechanisms that support cell survival through enhanced angiogenesis and cell migration and motility capacity.