Synergistic cytotoxicity of topoisomerase I inhibitors with alkylating agents and etoposide in human brain tumor cell lines.

To evaluate potential synergistic interactions between topoisomerase I (Topo I) inhibitors, i.e. camptothecin (CPT) and topotecan (TPT), and chemotherapeutic agents known to be active in treatment of brain tumors, in vitro studies were conducted with human glioma and medulloblastoma cell lines. Tumor cells were exposed to CPT or TPT alone or in combination with cisplatin, 4-hydroperoxycyclophosphamide (4-HC), BCNU or etoposide (VP-16). Cytotoxicity was assessed by colony formation assays. Drug interactions were evaluated by means of a novel analytical model which permits statistical evaluation over a range of dose combination. Experimental results were corroborated by published models of drug interaction. Our findings indicate that in vitro cytotoxic interactions in brain tumor cells between Topo I inhibitors and alkylating agents or etoposide depend on drug dose, dose schedule and tumor cell line. Treatment of DAOY medulloblastoma cells with CPT and either cisplatin, 4-HC or VP-16 produced significant synergistic cytotoxicity over a wide range of dose combinations. When VP-16 was administered after CPT, synergy was reduced to a narrow range of dose combinations. For U251 glioma cells, incubation with CPT and cisplatin or 4HC also produced synergistic cytotoxicity over a broad range of dose combinations. By contrast, antagonistic interactions were observed after administration of CPT with BCNU or VP-16. Treatment of U251 cells with CPT and cisplatin produced synergistic or antagonistic cytotoxicity depending on the dose combination used. These findings support a role for pharmacokinetic analysis in multiagent phase 11 trials involving Topo I inhibitors and have important implications for clinical trial design strategies.

Caffeine and staurosporine enhance the cytotoxicity of cisplatin and camptothecin in human brain tumor cell lines.

Caffeine and staurosporine have been shown to attenuate G2 delay produced by DNA-damaging agents and to augment the cytotoxicity of these agents in a number of cell lines in vitro. Studies in rodent brain tumor cell lines suggest that modulation of the G2/M transition may not contribute to the enhanced cytotoxicity produced by caffeine in brain tumor cells. To evaluate the impact of agents that decrease G2 delay on the cytotoxicity of chemotherapy in human brain tumor cells, we examined the ability of caffeine and staurosporine to modulate the G2 delay and cytotoxicity produced by cisplatin (CDDP) and camptothecin (CPT) in U251 glioma and DAOY medulloblastoma cells. Synchronized U251 were incubated with 20 microM CDDP in the presence or absence of 2 mM caffeine. DAOY cells were incubated with 100 nM CPT in the presence or absence of 2 nM staurosporine. Caffeine and staurosporine attenuated G2 delay produced by CDDP and CPT, respectively. Clonogenic assays indicated that continuous exposure to 2 mM caffeine substantially lowered the ID50 and ID90 of CDDP in U251 cells without significantly altering plating efficiency. Twenty-four-hour exposure to 2 nM staurosporine lowered the ID50 and ID90 of CPT in DAOY cells without significantly altering plating efficiency. Evaluation of programmed cell death using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay indicated that one mechanism for synergistic cytotoxicty of caffeine with CDDP and staurosporine with CPT in U251 and DAOY cells, respectively, is to promote apoptosis. These results underscore the importance of understanding regulation of G2/M transition in brain tumor cells. Such an understanding may lead to novel therapies that target G2 check points to augment the efficacy of currently available treatments for brain tumors.

Transforming growth factor-alpha-Pseudomonas exotoxin fusion protein (TGF-alpha-PE38) treatment of subcutaneous and intracranial human glioma and medulloblastoma xenografts in athymic mice.

Epidermal growth factor receptor (EGFR) is amplified or overexpressed in many malignant gliomas and other primary brain tumors but is low or undetectable in normal brain. In the present study, this differential expression has been exploited for targeted brain tumor therapy using a TGF-alpha-Pseudomonas exotoxin recombinant toxin, TGF-alpha-PE38. In vitro experiments demonstrate that the cytotoxicity of this fusion protein is primarily determined by tumor EGFR expression and that TGF-alpha-PE38 cytotoxicity is abolished by pretreatment with excess epidermal growth factor. Treatment with i.p. TGF-alpha-PE38 in nude mice bearing glioblastoma or medulloblastoma s.c. xenografts produced tumor regression and growth delay. For intracranial xenograft implants treated with i.p. TGF-alpha-PE38, significant increases in median survival were noted only for tumors with the highest EGFR expression. However, intracranial tumors treated with a single intratumoral injection of TGF-alpha-PE38 showed increased survival in all xenografts tested. These results indicate that TGF-alpha-PE38 is active against primary human brain tumors ranging from moderate to high EGFR expression. For intracranial tumors, however, the higher survival rates produced by intracranial injection of TGF-alpha-PE38 than by continuous i.p. administration suggest that increased drug clearance or impaired drug delivery reduces the efficacy of systemic TGF-alpha-PE38. Direct delivery of TGF-alpha-PE38 into brain tumors by controlled-release biodegradable polymers or intratumoral implanted catheters, or intrathecal administration into the colony stimulating factor of patients with leptomeningeal metastasis, may represent clinically useful applications of recombinant toxin therapy in tumors with high EGFR expression.