title: Combination therapy with irinotecan and protein kinase C inhibitors in malignant glioma.
abstract: The topoisomerase-I inhibitor irinotecan (CPT-11) is currently used in Phase I/II trials for the treatment of patients with recurrent malignant gliomas. Protein kinase C (PKC) inhibitors such as high-dose tamoxifen and hypericin also have been used in the treatment of malignant gliomas. The current study examined the role of PKC inhibitors as chemosensitizers for CPT-11 and their proposed mechanism of action. Two glioma cell lines (A-172 and U-87) and one primary glioma cell culture (LA-567) were used. Proliferation ((3)H-thymidine) and cytotoxicity (methylthiotetrazole) studies were performed using CPT-11 (0-100 microM) alone, 7-ethyl-10-hydroxy camptothecin (SN-38) (0-1000 nM) alone or in the presence of a PKC inhibitor, tamoxifen (10 microM), hypericin (10 microM), calphositin C (400 nM), or staurosporine (10 nM). The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assay was used to determine apoptosis as the mechanism of cytotoxicity; alterations in bcl-2 and bax expression were determined using Western blot analysis. Conversion of CPT-11 to SN-38 by glioma cells was determined using high-performance liquid chromatography (HPLC) analysis. Increasing CPT-11 and SN-38 concentrations induced cytotoxic morphologic changes, decreased proliferation, and increased cytotoxicity on all glioma cell lines tested. These changes were increased in the presence of a PKC inhibitor. The mechanism of the cytotoxicity was determined to be apoptosis by the TUNEL assay. The combination of a PKC inhibitor with CPT-11 or SN-38 led to decreased expression of the antiapoptotic protein bcl-2, and increased expression of the proapoptotic protein bax. HPLC analysis demonstrated conversion of CPT-11 to SN-38 by glioma cells. A combination of CPT-11 or SN-38 with a PKC inhibitor was found to lead to a decrease in proliferation and an increase in apoptosis in malignant glioma cells. The induction of apoptosis was secondary to a decrease in bcl-2 and an increase in bax expression. Glioma cells are capable of converting CPT-11 to SN-38 by intrinsic tumor carboxylesterases.
authors.affiliation: Department of Neurosurgery, University of Southern California, School of Medicine, Los Angeles, California 90033, USA. tcchen@hsc.usc.edu
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Chen
authors.forename: Susan
authors.initials: S
authors.lastname: Su
authors.forename: David
authors.initials: D
authors.lastname: Fry
authors.forename: Leonard
authors.initials: L
authors.lastname: Liebes
mesh_terms: D000873:Anthracenes; D000972:Antineoplastic Agents, Phytogenic / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000473:pathology; D002166:Camptothecin / Q000008:administration & dosage* / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D002460:Cell Line; D005909:Glioblastoma / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D000077146:Irinotecan; D009281:Naphthalenes / Q000008:administration & dosage; D010569:Perylene / Q000008:administration & dosage / Q000031:analogs & derivatives*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D019311:Staurosporine / Q000008:administration & dosage; D013629:Tamoxifen / Q000008:administration & dosage; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
chemical_list: D000873:Anthracenes; D000972:Antineoplastic Agents, Phytogenic; C494826:BAX protein, human; D009281:Naphthalenes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C061465:calphostin complex; D013629:Tamoxifen; D010569:Perylene; D000077146:Irinotecan; C004965:hypericin; D011493:Protein Kinase C; D019311:Staurosporine; D002166:Camptothecin