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The Effect of Small Molecule Pharmacological Agents on the Triterpenoid Saponin Induced Endolysosomal Escape of Saporin and a Saporin-Based Immunotoxin in Target Human Lymphoma Cells.

ABSTRACT: Triterpenoid saponins augment the cytotoxicity of saporin based immunotoxins. It is postulated that this results from a saponin-mediated increase in the endolysosomal escape of the toxin to the cytosol, but this remains to be confirmed. To address this issue, we used a number of pharmacological inhibitors of endocytic processes as probes to investigate the role played by saponin in the endolysosomal escape of fluorescently labeled saporin and a saporin based immunotoxin targeted against CD38 on human lymphoma and leukemia cell lines. Endolysosomal escape of the toxin was measured by flow cytometric pulse shape analysis. These results were compared to the effects of the various inhibitors on the saponin-mediated augmentation of toxin and immunotoxin cytotoxicity. Inhibitors of clathrin-mediated endocytosis, micropinocytosis, and endosomal acidification abrogated the saponin-induced increase in the endolysosomal escape of the toxin into the cytosol, suggesting that these processes may be involved in the internalization of saponin to the same endolysosomal vesicle as the toxin. Alternatively, these processes may play a direct role in the mechanism by which saponin promotes toxin escape from the endolysosomal compartment to the cytosol. Correlation with the effects of these inhibitors on the augmentation of cytotoxicity provides additional evidence that endolysosomal escape is involved in driving augmentation.

SUBMITTER: Wensley HJ

PROVIDER: S-EPMC8000476 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDevelopment of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma.Methods5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin.ResultsThe 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population.ConclusionOur findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

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The Effect of Small Molecule Pharmacological Agents on the Triterpenoid Saponin Induced Endolysosomal Escape of Saporin and a Saporin-Based Immunotoxin in Target Human Lymphoma Cells.

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