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The mechanism of action of a potential anti-cancer therapeutic: new technique to monitor subcellular environments

Black tube sponges

Callyspongiolide (CSG), derived from marine sponges, is highly toxic to human cancer cell lines from a variety of origin tissues and presents a potential cell-killing agent to be included in antibody-drug conjugates for anti-cancer therapeutics. CSG-mediated cell death doesn’t appear to occur via the canonical apoptosis pathway because cell death is not blocked by caspase inhibitors. CSG has been shown to inhibit vacuolar ATPase in yeast, but the exact mechanism of CSG-mediated cell death remains unclear.

In a recent Communications Biology publication, Ha and Park report that CSG kills cells by inducing lysosomal dysfunction, leading to cellular iron depletion and dysfunction ultimately resulting in cell death. The authors initially set out to identify proteins that interact directly with CSG using a label-free method of target identification called thermal stability shift–based fluorescence difference in two-dimensional gel electrophoresis (TS-FITGE). TS-FITGE identifies proteins whose thermal stability changes upon binding to a ligand. Ha and Park applied TS-FITGE to CSG-treated lung cancer cells.

The Sapphire Biomolecular Imager was used to capture fluorescent images of 2D gels comparing proteins isolated from CSG-treated and control cells (labeled with Cy5 and Cy3 respectively) before and after heat shock. CSG-related increases in thermal stability were expected to appear as spots becoming more red with increased temperature. However, no proteins were found to have increased thermal stability. In contrast, several protein spots were observed to “split” on the 2D gels. The authors hypothesized the isoelectric points of these proteins were changing in a temperature-dependent way in the presence of CSG.

Figure 2 from Ha J and Park SB. (2021) Callyspongiolide kills cells by inducing mitochondrial dysfunction via cellular iron depletion. Licensed under CC BY 4.0. The Azure Sapphire was used to capture images of proteins from cells treated with callyspongiolide (CSG) (labeled with Cy5, red) or from control cells (labeled with Cy3, green) and separated by 2D electrophoresis. The overlaid images reveal that some proteins migrate differently after treatment with CSG and heat shock.

The spot splits were observed to be dependent on the presence of intact subcellular compartments, and 10 proteins whose migration was altered by CSG-treatment were isolated from gels and identified, revealing that most of the proteins were mitochondrial and five were components of the electron transport chain, implicating mitochondrial involvement.

Through a broad range of additional experiments, Ha and Park demonstrated that CSG did not act directly on mitochondria and that cell death did not involve the known apoptosis, parthanatos, or ferroptosis pathways. Rather, CSG blocked acidification of lysosomes, possibly through action on the vacuolar ATPase. The resulting loss of lysosomal acidification decreased the amount of iron available to the cells and iron supplementation was found to rescue the cells. The authors also conclude the TS-FITGE assay can be used to monitor changes in cellular environments after drug treatment in addition to identifying proteins that bind a specific ligand.

In addition to multichannel fluorescence imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry and near-infrared, phosphor, and white light imaging of blots, gels, tissues, and more.

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