Carbonyl-Substituted Titanocenes as New Cytostatic Agents Against Lymphoma and Leukemia in Childhood.

Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood. Despite a relatively good prognosis approximately one fourth of the patients suffers from relapse and, consequently, a worse prognosis. Only 40% of these children with relapsed ALL will survive. Patients who fail to achieve a complete remission in chemotherapy do not survive. This explains the need for new compounds that may overcome resistance against clinically employed cytostatic drugs. In the present study we examined novel titan-containing agents concerning their cytotoxic potential with great success in vitro, ex vivo in primary leukemia cells from children with primary and relapsed ALL and in vivo in a lymphoma model in mice. We could show anti-leukemic and anti-lymphoma activity in various established cell lines, like Nalm6, Reh, SupB15 and BJAB. By Casy Counter we detected a decrease of proliferation and vitality in cell culture. That the cell death is effected by apotosis induction we investigated by flowcytometric measurement of DNA-fragmentation after propidiumiodid staining (LC₅₀ = 5–10 μM) and phosphatidylserin-staining on cell membrane surface. Via western blotting we could show titanocene-induced caspase-processing and PARP cleavage. To characterize the titanocene apoptosis pathway we used cellular model systems with deficiencies in different molecular parts of the apoptosis cascade. The induced apoptosis reveals to be independent of CD95 receptor, the pro-apoptotic factor SMAC and the anti-apoptotic protein Bcl-2. Even in mamma carcinoma cells (MCF-7) with Caspase 3- deficiency titanocenes were able to induce apoptosis significantly. We also investigated a loss of mitochondrial membrane potential, which indicates the involvement of the mitochondrial apoptosis machinery in titanocene-induced cell death. Unspecific cytotoxic effects, like necrosis, could be excluded by lactatdehydrogenase-release assay. Ex vivo we tested isolated primary lymphoblasts of children with primary and relapsed ALL. We could demonstrate, that even lymphoblasts with resistance against doxorubicin (p < 0.005) and cytarabine (p < 0.01) are sensitive to titanocenes. In vivo tests in a mouse model with human lymphoma cells (BJAB) show significant reduce of tumor progression effected by oral treatment with carbonyl-substituted titanocenes compared with a control group. These experiments in mice show a good tolerability of titanocenes. Taken together, carbonyl-substituted titanocenes comprise a totally new, very promising class of cytostatic agents for lymphoma and leukemia therapy, especially for the therapy of relapsed ALL in childhood.