The highly cytotoxic marine
natural product callyspongiolide holds great promise as a warhead of
antibody-drug conjugate in
cancer therapeutics; however, the mechanism underlying its cytotoxicity remains unclear. To elucidate how
callyspongiolide kills cells, we employed label-free target identification with thermal stability-shift-based fluorescence difference in two-dimensional (2-D) gel electrophoresis (TS-FITGE), which allowed observation of a unique phenomenon of
protein-spot separation on 2-D
gels upon treatment with
callyspongiolide at increasing temperatures. During our exploration of what
proteins were associated with this phenomenon as well as why it happens, we found that
callyspongiolide induces mitochondrial/lysosomal dysfunction and autophagy inhibition. Moreover, molecular biology studies revealed that
callyspongiolide causes lysosomal dysfunction, which induces cellular
iron depletion and leads to
mitochondrial dysfunction and subsequent cytotoxicity. Notably, these effects were rescued through
iron supplementation. Although our approach was unable to reveal the direct
protein targets of
callyspongiolide, unique phenomena observed only by TS-FITGE provided critical insight into the mechanism of action of
callyspongiolide and specifically its cytotoxic activity via induction of
mitochondrial dysfunction through cellular
iron depletion caused by lysosomal deacidification, which occurred independent of known programmed cell death pathways.