Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines

Abstract

In patients with malignancy, the major barrier to achieving complete response is emergence of resistance to current chemotherapeutic agents. One of the major mechanisms by which tumour cells become resistant to therapies is by altering cellular drug targets through mutations and/or deletions. Resistance by this mechanism is achieved more easily if the drug has limited cellular targets and/or processes. We hypothesized that as Pseudomonas aeruginosa exotoxin T (ExoT) targets six proteins that are required for cancer cell survival and proliferation, it is highly unlikely for cancer cells to develop resistance to this toxin. We assessed ExoT's cytotoxicity against multiple invasive and highly resistant tumour cell lines in order to evaluate its potential as a chemotherapeutic agent. Our data demonstrated that ExoT induced potent cytotoxicity in all tumour cell lines that we examined. Collectively, our data highlighted the potential of ExoT as a possible chemotherapeutic candidate for the treatment of cancer.

Fig. 1.

Infection with ExoT-expressing P. aeruginosa can cause cytotoxicity in a number of cancer cell lines. The indicated cancer cells were either infected with ExoT-expressing (ΔU) or ExoT-defective T3SS mutant (pscJ) P. aeruginosa isogenic strains at m.o.i.~10, or cultured in media alone. Media were collected at 10 h intervals over a 30 h period and the amount of LDH released into media was analysed using the LDH release assay. The per cent LDH release was determined by the ratio of experimental LDH release to the total LDH release in the presence of Triton X-100. Analysis of LDH release of infected cancer cells, especially at later time points, indicated that infection with ExoT-expressing ΔU resulted in significantly more cytotoxicity compared with cells treated with the pscJ strain or left uninfected. *P<0.05, n = 6, Student’s t-test.

Fig. 2.

Kinetics of ExoT-induced cytotoxicity in cancer cell lines. (a) The indicated cancer cell lines were treated with ExoT-expressing (ΔU) or ExoT-defective (pscJ) P. aeruginosa strains at m.o.i.~10, or left untreated. Cells were observed by time-lapse fluorescence video microscopy in the presence of PI (×100 magnification, 15 min intervals). Cytotoxicity was assessed by total PI uptake at 15 min intervals (cytotoxicity is shown as the mean of three fields of view). (b) Cytotoxicity analysis of B16 melanoma cells as assessed by PI staining (red). (c) Representative frames from B16 time-lapse video microscopy.

Fig. 3.

ExoT is sufficient to cause cytotoxicity in cancer cells in vitro. Indicated cancer cells were transfected with pGFP vector control or pExoT-GFP and cytotoxicity was assessed by time-lapse fluorescence video microscopy in the presence of PI. (a) Tabulated data for A375, MCF-7, EMT6 and Calu-3. (b) Representative frames of B16 transfected with pExoT-GFP or pGFP. (c) Tabulated data for B16. *P<0.0001; n = 3 independent experiments each with ~100 events counted; Student’s t-test.

Fig. 4.

Assessment of ExoT-induced cytotoxicity in cancer by Viability Dye. Cancer cells were transfected with pGFP vector control or pExoT-GFP. At 17 h after transfection, cells were stained with Viability Dye and analysed by flow cytometry. (a) Representative FACS plots for B16 show ExoT was sufficient to cause cytotoxicity in transfected cells. SSC, side scatter; FSC, forward scatter. (b) FACS analysis of transfected B16 cells indicated that pExoT-GFP resulted in significantly more cytotoxicity compared with pGFP-transfected cells. *P<0.0001; n = 7; Student’s two-tailed t-test.

Fig. 5.

ExoT slows B16 tumour burden in vivo. (a) B16 tumour cells were transfected with pGFP or pExoT ex vivo. At 15 h after transfection, 1×10⁵ pGFP- or pExoT-GFP-transfected B16 tumour cells were transferred subcutaneously by injection in the flank of C57BL/6 mice. Tumour size was measured daily by use of callipers until tumours reached 150 mm². Tumour sizes are shown as mean±sem from the time of injection to the first animal death. n = 5 mice per group, *P<0.05. (b) Individual tumour growth curves for the experiment performed in (a).