Suicide cancer gene therapy using pore-forming toxin, streptolysin O

Abstract

We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH 2 -terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell -derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus -injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents. [Mol Cancer Ther 2006;5(6):1610 -9]

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors

“…Similar observations were made for streptolysin O gene transfer, where intracellular toxin expression leads to perforation of cell membranes verified by LDH release. 16 We also showed that intracellularly accumulated CPE causes membrane disruption associated with LDH relase in HCT116 and MCF-7 cells (Figure 4). More interestingly, we detected released biologically active CPE in the medium of transfected cells, although no signal peptide sequence was attached to CPE.…”

“…Comparable effects were described for streptolysin O-transfected cells. 16 Only in HCT116 cells, delayed (48 h post transfection) release of capases 3 and 7 was detected. This can be attributed to the late action of liberated CPE on non-transfected HCT116 cells in a bystander effect.…”

“…The two latter toxins are both pore-forming proteins, of which streptolysin O is rather unspecific, whereas CPE possesses specificity in action. [15][16][17] The targeted action of CPE is mediated by binding to a subset of claudins, mainly to claudin-3 and -4, which are frequently overexpressed in epithelial tumors. 22,24,25 This binding leads to disintegration of plasma membrane in association with rapid cytolysis of treated cells.…”

“…This approach is advantageous over other toxins used in cancer therapy, as they act on more ubiquitous, non-tumorspecific targets, such as E2F for diphteria toxin or membranous cholesterol for streptolysin O. 4,16 For proper tumor targeting, modification of these toxins is required, for example, by creating fusion proteins as immunotoxins. 4 CPE has also been used to generate fusion proteins.…”

“…[5][6][7][8][10][11][12][13][14] Alternatively, the pore-forming bacterial toxins streptolysin O (Streptococcus pyrogenes) and Clostridium perfringens enterotoxin (CPE) came into focus as promising cancer therapeutics. [15][16][17][18] CPE is produced by the anaerobic gram-positive Clostridium perfringens type A strain, known to cause food poisoning. 19 CPE is a 35-kDa protein, which increases cell membrane permeability by formation of large prepore complexes.…”