Shiga-like toxin I exerts specific and potent anti-tumour efficacy against gastric cancer cell proliferation when driven by tumour-preferential Frizzled-7 promoter.

OBJECTIVES: Tumour-targeted gene therapy is a promising approach for effective control of gastric cancer cell proliferation. Our study aims to develop a cancer therapy which combines tumour-targeting promoters with cytotoxins. METHODS: The expression of globotriaosylceramide (Gb3), which is a Shiga-like toxin I (Stx1) receptor, was verified in gastric cancer compared with normal stomach tissues as assessed by flow cytometry and immunohistochemical analysis. We therefore constructed the recombinant pFZD7-Stx1 plasmid vectors with tumour-preferential Frizzled-7 promoter and Stx1. pFZD7-Stx1 was used to treat gastric cancer in vitro and in vivo. The gastric cancer cell proliferation and tumour growth were identified after the transfection with the pFZD7-Stx1. RESULTS: Globotriaosylceramide was obviously increased in gastric cancer compared with normal stomach. The gastric cancer cell proliferation and tumour growth decreased significantly after the transfection with the pFZD7-Stx1. CONCLUSION: Frizzled-7 promoter is preferentially active, and Gb3 is abundant in gastric cancer cells. Frizzled-7 promoter and Stx1 may be used to determine a novel and relatively specific and potent gastric cancer therapeutic strategy.

Cisplatin-induced expression of Gb3 enables verotoxin-1 treatment of cisplatin resistance in malignant pleural mesothelioma cells.

BACKGROUND: A major problem with cisplatin treatment is the development of acquired-drug resistance of the tumour cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the membrane glycolipid globotriasosylceramide (Gb3), a molecule associated with drug resistance. Cisplatin- and VT-1-induced apoptosis involves mitogen-activated protein kinase (MAPK) activation, and deactivation of MAPKs is associated with cisplatin resistance. This study aimed to investigate whether a sub-toxic concentration of VT-1 could enhance cisplatin-induced apoptosis and overcome acquired-cisplatin resistance in cultured cancer cell lines. METHOD: P31 and H1299 cells with corresponding cisplatin-resistant sub-lines (P31res/H1299res) were incubated with VT-1 and/or cisplatin followed by determination of Gb3 expression, cell viability, apoptosis, and signalling pathways. RESULTS: Cells from the resistant sub-lines had elevated Gb3 expression compared with the parental cell lines, and cisplatin further increased Gb3 expression, whereas VT-1 reduced the percentage of Gb3-expressing cells. Combination of cisplatin and sub-toxic concentrations of VT-1 led to a super-additive increase of cytotoxicity and TUNEL staining, especially in the cisplatin-resistant sub-lines. Blockade of Gb3 synthesis by a Gb3 synthesis inhibitor not only led to eradicated TUNEL staining of P31 cells, but also sensitised P31res cells to the induction of apoptosis by cisplatin alone. Cisplatin- and VT-1-induced apoptosis involved the MAPK pathways with increased C-Jun N-terminal kinase and MAPK kinase-3 and -6 phosphorylation. CONCLUSIONS: We show the presence of Gb3 in acquired-cisplatin resistance in P31res and H1299res cells. Cisplatin up-regulated Gb3 expression in all cells and thus sensitised the cells to VT-1-induced cytotoxicity. A strong super-additive effect of combined cisplatin and a sub-toxic concentration of VT-1 in cisplatin-resistant malignant pleural mesothelioma cells were observed, indicating a new potential clinical-treatment approach.

The treatment of malignant meningioma with verotoxin.

Malignant meningiomas (MMs) are aggressive intracranial neoplasms with a 75% 5-year recurrence rate. Verotoxin 1 (VT1) is an Escherichia coli toxin, which has recently been shown to have anti-neoplastic action by targeting the globotriosylceramide (Gb(3)) glycolipid on tumor cells and tumor neovasculature. To investigate the potential use of VT1 as a clinical agent for MM, we initially tested 16 meningiomas for Gb(3) expression. Nine of 11 MMs (82%), but only one of five benign meningiomas (20%), were positive for Gb(3). An orthotopic xenograft model was used to test the efficacy of VT1 treatment for MM. We first demonstrated that Gb(3) was highly expressed by the MM cell line, IOMM-Lee, and that this cell line was highly sensitive to VT1 treatment in vitro. A single intratumoral injection of VT1 significantly improved survival in nude mice harboring intracranial tumours (P<.0001). Factor-eight immunostaining of tumours harvested from VT1-treated animals revealed a marked reduction in the tumour microvascular density. In addition, the tumors of VT1-treated animals displayed increased apoptosis by TUNEL analysis and showed a significant decrease in cell proliferation, as determined by MIB-5 immunostaining. VT1 treatment of MM is effective in our orthotopic xenograft model, and warrants further exploration as a potential treatment for these highly anaplastic and aggressive neoplasms.

The use of Shiga-like toxin 1 in cancer therapy.

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1, SLT-I, Verotoxin 1, VT1) targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. The frequent occurrence of SLT-1 receptors on tumor cells derived from patients with hematological cancers (follicular lymphoma, multiple myeloma, chronic lymphocytic leukemia) and their absence on human CD34(+) hematopoietic stem cells suggest the ex vivo use of Shiga-like toxin-1 in purging CD77(+) tumor cells from autologous stem cell transplants. SLT-1 receptors are also commonly expressed on breast cancer, ovarian cancer and astrocytoma cells. In particular, the sensitivity of astrocytoma cell lines to this toxin provides an opportunity for using SLT-1 in vivo in the context of treating patients afflicted by this common form of brain tumor. Finally, the known structural features of SLT-1 allow one to contemplate altering its receptor specificity in an effort to target CD77(-) tumor cell populations.

CD40 ligand, Bcl-2, and Bcl-xL spare group I Burkitt lymphoma cells from CD77-directed killing via Verotoxin-1 B chain but fail to protect against the holotoxin.

Owing to its lineage and differentiation stage-restricted expression, CD77 has been mooted as a therapeutic target in Burkitt lymphoma (BL). The recognition that the globotriaosyl moiety of this neutral glycosphingolipid is a receptor for Escherichia coli-derived Verotoxin-1 (Shiga-Like Toxin-1) offers a potential delivery system for the attack. Here we show that CD77-expressing Group I BL cells which are normally susceptible to activation-induced death on binding Verotoxin-1 B chain are protected in the presence of CD40 ligand. Ectopic expression of either bcl-2 or bcl-xL also afforded resistance to the actions of the B chain. In total contrast, neither of the survival genes nor a CD40 signal - even when acting in concert - protected against killing mediated by the holotoxin. These findings indicate that while therapeutic modalities for CD77-expressing B cell tumors (which include follicular lymphoma) based on the use of Verotoxin-1 B chain might be compromised by the activation of endogenous or exogenous survival pathways, those exploiting the holotoxin should be left unscathed.