| Abstract [eng] |
Carbonic anhydrase IX (CAIX) is a transmembrane enzyme overexpressed in many solid tumours, where it supports cancer cell survival under hypoxic conditions by regulating pH homeostasis. While hypoxia is a well-established inducer of CAIX via HIF-1α, recent evidence suggests that CAIX expression can also be upregulated by other cellular stresses, including nutrient deprivation such as riboflavin (RF) depletion and culture in low serum (1% FBS). Selective inhibition of CAIX is a promising strategy for cancer therapy, but the metabolic plasticity of cancer cells often limits the efficacy of such inhibitors. This study investigates whether metabolic reprogram-ming-via manipulation of glucose and galactose availability and RF deprivation can sensitize osteosarcoma cells (143B) to CAIX inhibition, and characterizes the resulting proteomic changes. 143B human osteosarcoma cells were subjected to metabolic reprogramming using RF deprivation or galactose, then treated with two CAIX inhibitors (VD11-4-2 and AZ19-3-2). Mitochondrial respiration was assessed using the Seahorse XF Cell Mito Stress Test, while cell viability was measured by flow cytometry, AlamarBlue and SRB assays. CRISPR/Cas9 was used to generate knockout CA9 gene cell lines. Protein profiling was conducted to identify oncoprotein expression changes in response to CAIX inhibition and metabolic stress. Metabolic reprogramming by RF deprivation reduced mitochondrial respiratory capacity in 143B cells. Both CAIX inhibitors decreased cell viability in a dose-dependent manner, with en-hanced sensitivity observed under metabolic stress conditions. Notably, CAIX expression in 143B cells can induced not only by hypoxia but also by RF depletion and low serum concentration, highlighting the CAIX role as a cellular stress responder. Proteomic profiling revealed significant alterations in proteins related to energy metabolism, stress response, and cell survival pathways following CAIX inhibition. These findings support the combined use of metabolic interventions and CAIX inhibitors as a therapeutic strategy in cancer. |
