Antimicrobial peptides of the Cecropin-family show potent antitumor activity against bladder cancer cells

Abstract

Background: This study evaluated the cytotoxic and antiproliferative efficacy of two well-characterized members of the Cecropin-family of antimicrobial peptides against bladder tumor cells and benign fibroblasts.

Methods: The antiproliferative and cytotoxic potential of the Cecropins A and B was quantified by colorimetric WST-1-, BrdU- and LDH-assays in four bladder cancer cell lines as well as in murine and human fibroblast cell lines. IC50 values were assessed by logarithmic extrapolation, representing the concentration at which cell viability was reduced by 50%. Scanning electron microscopy (SEM) was performed to visualize the morphological changes induced by Cecropin A and B in bladder tumor cells and fibroblasts.

Results: Cecropin A and B inhibit bladder cancer cell proliferation and viability in a dose-dependent fashion. The average IC50 values of Cecropin A and B against all bladder cancer cell lines ranged between 73.29 mug/ml and 220.05 mug/ml. In contrast, benign fibroblasts were significantly less or not at all susceptible to Cecropin A and B. Both Cecropins induced an increase in LDH release from bladder tumor cells whereas benign fibroblasts were not affected. SEM demonstrated lethal membrane disruption in bladder cancer cells as opposed to fibroblasts.

Conclusion: Cecropin A and B exert selective cytotoxic and antiproliferative efficacy in bladder cancer cells while sparing targets of benign murine or human fibroblast origin. Both peptides may offer novel therapeutic strategies for the treatment of bladder cancer with limited cytotoxic effects on benign cells.

Figure 1

Impact of Cecropin A and B on bladder cancer cells and fibroblasts. The four bladder cancer cell lines RT4, 647V, J82 and 486P as well as human (ZF07) and murine (3T6) fibroblasts were coincubated with increasing concentrations of up to 400 μg/ml Cecropin A and B. Cecropin A (A) and B (B) both demonstrate significant inhibitory activity on the viability of all four bladder cancer cell lines as measured by the WST-1 cell viability assay. In contrast, benign fibroblasts are less susceptible to Cecropin (p < 0,05). Additionally, Cecropin A (C) and B (D) both demonstrate significant inhibitory activity on the proliferation of all four bladder cancer cell lines as measured by the BrdU proliferation assay. In contrast, benign fibroblasts are not susceptible to Cecropin (p < 0,05). Cecropin A (E) and B (F) both exert significant cytotoxicity against all bladder cancer cell lines as measured by the LDH release assay. In contrast, benign fibroblasts are not susceptible to Cecropin-mediated cytolysis (p < 0,05). Experiments were performed in triplicate with results shown as mean ± standard deviation.

Figure 2

Effect of Cecropin B on cell membranes of 486P bladder cancer cells and ZF07 fibroblasts as visualized by scanning electron microscopy (SEM). (A) Representative example of an untreated 486P bladder cancer cell showing a smooth surface. (B) 486P cells treated with 65 μM Cecropin B reveal a disrupted cell membrane with only small islands of intact surface left (arrow). In contrast, untreated (C) ZF07 fibroblasts and (D) fibroblasts after incubation with 65 μM Cecropin B do not display any changes in cell morphology with no observable damage to the cell membrane.