The oncoprotein Bcl-2 protects cells against apoptosis, but the exact molecular mechanism that underlies this function has not yet been identified. Studying H2O2-induced cell injury in Rat-1 fibroblast cells, we observed that Bcl-2 had a protective effect against the increase in cytosolic calcium concentration and subsequent cell death. Furthermore, overexpression of Bcl-2 resulted in an alteration of cellular glutathione status: the total amount of cellular glutathione was increased by about 60% and the redox potential of the cellular glutathione pool was maintained in a more reduced state during H2O2 exposure compared with non-Bcl-2-expressing controls. In our cytotoxicity model, disruption of cellular glutathione homoeostasis closely correlated with the pathological elevation of cytosolic calcium concentration. Stabilization of the glutathione pool by Bcl-2, N-acetylcysteine or glucose delayed the cytosolic calcium increase and subsequent cell death, whereas depletion of glutathione by DL-buthionine-(S,R)-sulphoximine, sensitized Bcl-2-transfected cells towards cytosolic calcium increase and cell death. We therefore suggest that the protection exerted by Bcl-2 against H2O2-induced cytosolic calcium elevation and subsequent cell death is secondary to its effect on the cellular glutathione metabolism.

The study demonstrates that proteolytic enzymes can alter the glycoprotein composition of the surface at different tumor cell lines. This membrane modulation measured by lectin binding, is being considered as a prerequisite for the main target, namely the individualized therapeutic use of proteases for adjuvant cancer treatment. The treatment of a human bladder carcinoma cell line and the murine melanoma cell line B16 as well as its variant F1 with bromelain and ficin resulted in a reduced binding of Concanavalin A (ConA). No difference to the controls could be seen when the B16 variant F10 was further examined, whereas a human pancreas carcinoma cell line showed an elevated ConA binding. The experiments with wheat germ agglutinin (WGA) and the lectins from elder bark (SNA) and from Maackia amurensis (MAA) with specifity for N-acetylneuraminic acid also showed a strongly reduced binding after protease treatment. Finally a panel of different proteases showed similar binding effects with the exception of actinidin that revealed no detectable changes in lectin binding. Plasmin and elastase, assayed in physiological concentrations, also showed no remarkable effect. Not unexpected but still surprising, further purification of pure bromelain resulted in a loss of surface-modulating capacity. Concerning the protease concentrations a dose dependence could not be noted in the range from 0.17-2.04 U per ml. Altogether the findings support the advanced use of enzymes as an approach for the successful adjuvant cancer treatment. This is due to the fact that the metastatic potential is modulated by enzymes as the recognition signals are being altered enzymatically.

The ability of malignant tumor cells to form secondary lesions depends on multiple adhesive steps during invasion and metastasis. Details are given that a proteolytic treatment of the tumor cell surface causes a decreased attachment of the cells to matrix components. The attachment of murine B16 melanoma cells to the complete matrix (ECM) showed 40% inhibition after a bromelain treatment in comparison to control cells. A reduced binding rate to the isolated matrix components laminin and fibronectin was also found for the melanoma cell line as well as for a human bladder carcinoma (BC) after a proteolytic treatment with bromelain or ficin. Furthermore, the haptotactic migration of pancreatic carcinoma cells (DAN-G cell line) to laminin and fibronectin and that of B16-F1 cells to fibronectin was also influenced by ficin and bromelain, as shown in an in vitro invasion system. Other experiments were performed with arg-gly-asp-containing synthetic peptides (RGD polymer) as attachment protein mimicking the cell-binding domain of many adhesive proteins, which is recognized by several cell surface receptors. A bromelain treatment reduced the RGD attachment of B16 melanoma cells and that of their B16-F1 and B16-F10 variants, which differ in their capacity to colonize the lung. A panel of other proteases, including human elastase and plasmin, assayed in a physiological concentration, showed a similiar adhesion-reducing effect. The chromatogra-phic purification of bromelain resulted in fractions which only influenced the cell binding to the adhesive sequence in a lower extent than the starting material. The ability of the tested proteases to reduce the cell binding to the RGD polymer indicates an influence of the proteolytic treatment on the RGD-dependent integrin system. The findings support the possibility for a therapeutic use of proteases for adjuvant cancer treatment.