Expression of verotoxin-1 receptor Gb3 in breast cancer tissue and verotoxin-1 signal transduction to apoptosis.

ABSTRACT

BACKGROUND: The prerequisite for the potential use of the bacterial toxin verotoxin-1 in the treatment of breast cancer was investigated by first determining the expression of its receptor Gb3 (CD77) in clinical breast cancer tissue specimens. We then examined the cytotoxicity and mechanism of apoptosis induction of Escherichia coli verotoxin-1 (VT-1) in two human breast cancer cell lines. METHODS: Immunohistochemistry for Gb3 expression was performed on cryostat section from 25 breast cancer specimens. The human breast cancer cell lines T47D and MCF-7 were screened for Gb3 expression by flow cytometry. Fluorescein diacetate and LDH release was used to determine cell viability after VT-1 exposure. Apoptosis was studied by measuring caspase activity and DNA-fragmentation. Signal transduction studies were performed on T47D cells with immunoblotting. RESULTS: Gb3 expression was detected in the vascular endothelial cells of all tumours specimens, and in tumour cells in 17 of the specimens. We found no associations between tumour cell Gb3-expression and age, tumour size, TNM-classification, histological type, hormone receptor expression, or survival time. T47D cells strongly expressed Gb3 and were sensitive to the cytotoxicity, caspase activation and DNA fragmentation by VT-1, whereas MCF-7 cells with faint Gb3-expression were insensitive to VT-1. VT-1 (0.01 - 5 microg/L) exposure for 72 h resulted in a small percentage of viable T47D cells whereas the cytotoxicity of cells pre-treated with 2 micromol/L D, L-treo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, an inhibitor of glucosylceramide synthesis) was eliminated (< or = 0.1 microg/L VT-1) or reduced (0.5 - 5 microg/L VT-1). VT-1 did not cause cellular LDH-release or cell cycle arrest. VT-1 induction of caspase-3 (0.1, 1, and 5 microg/L VT-1), -8, and -9 (1 and 5 microg/L VT-1) activity and DNA fragmentation of T47D cells was blocked by PPMP. Key components of MAP kinase signalling pathways that control mitochondrial function were investigated. VT-1 0.1 - 5 microg/L induced phosphorylation of JNK as well as MKK3/6 suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. CONCLUSION: The high specificity and apoptosis-inducing properties of verotoxin-1 indicates that the toxin potentially may be used for treatment of Gb3-expressing breast cancer.