Background Aerolysin is a pore-forming toxin produced by Aeromonas hydrophila and interacts with targets through binding to glycosylphosphatidyl inositol-anchored proteins, subsequently forming a pore in the membrane. Perforin (PFP), a prototype of pore-forming proteins, is the central element of cell-mediated cytotoxicity and induces apoptosis through granule-exocytosis.
Aims Since PFP is pivotal to cell-mediated cytotoxicity in short term and due to similarities between PFP and aerolysin action, we investigated the correlation between aerolysin cytotoxicity and short-term lymphokine activated killer (LAK) cell-mediated cytotoxicity against human tumor cells.
Methods We studied the effect of active aerolysin (2 × 10-10M) against 8 tumor cell lines and 5 acute myeloid leukemia (AML) patients. AML cells K562, U937, CMK, Meg-01, HL-60 and resistant central nervous system tumor cells (HTB-11, HTB-12 and HTB-14) were included in this study. Tumor cell type selection was based on their sensitivity against LAK cell-mediated cytotoxicity. Aerolysin cytotoxicity was assessed at 7 different time points up to 4 hours of incubation consistent with the known optimal cell kill period mediated by PFP. Drug and aerolysin cytotoxicity was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6(3) /PI staining and further quantification of fluorosphere-adjusted events. LAK cell-mediated cytotoxicity against tumor cell lines and patient samples was measured by our established method flow cytometric cell-mediated cytotoxicity assay.
Results In timing studies, aerolysin treatment reached peak cytotoxicity around 2 hours of incubation similar to LAK cytotoxicity. U937 was the most sensitive cell type to both LAK cells and aerolysin. Some cells were very resistant to LAK killing and aerolysin at the concentration used. There was significant correlation between aerolysin cytotoxicity and LAK cell-mediated kill at 2 hours in cell lines and patient samples (r = .99, p < .001; r = .85, p = .035, respectively). However, there was no correlation between drug cytotoxicity and LAK cell-mediated cytotoxicity or aerolysin-induced cell kill.
Conclusion Aerolysin sensitivity may be used as a surrogate marker for the assessment of in vitro LAK cell-mediated tumor cell kill caused by granule/exocytosis pathway.
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