Preclinical studies of treosulfan demonstrate potent activity in Ewing's sarcoma.
OBJECTIVE
High-dose chemotherapy with the alkylating agent busulfan has been widely used in the treatment of patients with high-risk Ewing's sarcoma. Because of risks for toxicity, busulfan and radiotherapy can not be applied together, leading to the omission of one effective therapy component. Treosulfan is a derivative of busulfan which has a lower side effect profile than busulfan and which can be used together with radiotherapy. We investigated the effect of treosulfan in a panel of Ewing's sarcoma cell lines on cell survival, cell cycle and apoptosis in vitro and compared it to busulfan. Furthermore, the anti-tumor effect of treosulfan was studied in an orthotopic Ewing's sarcoma mouse xenograft model.


METHODS
Cell survival was measured by MTT assay and cell cycle analysis by flow cytometry. Apoptosis was analyzed via detection of DNA fragmentation, Hoechst 33258 staining, Annexin V, and cleavage of caspases-3 and 9. The effect of treosulfan and busulfan on primary tumor growth was assessed in Ewing's sarcoma xenografts in NOD/SCID mice (10 mice per group), pharmacokinetics of treosulfan were analyzed in nude mice.


RESULTS
Treosulfan inhibited cell growth to at least 70% in all cell lines at concentrations achievable in vivo. Treosulfan had a greater effect on the inhibition of cell growth at equivalent concentrations compared to busulfan. The growth inhibitory effect of treosulfan at low concentrations was mainly due to a G2 cell cycle arrest, whereas at higher concentrations it was due to apoptosis. Apoptosis was induced at lower concentrations compared to busulfan. In contrast to busulfan, treosulfan induced cell death in an apoptosis-deficient cell line at concentrations achievable in vivo. In mice, treosulfan suppressed tumor growth at dosages of 2,500 and 3,000 mg/kg. Pharmacokinetic exposures of treosulfan in mice were similar to previous reports in human patients. At maximal tolerated dosages treosulfan had a higher anti-tumor activity than busulfan.


CONCLUSIONS
Our results suggest that treosulfan has efficacy against Ewing's sarcoma cells in vitro and in mice. Therefore, controlled trials examining the role of treosulfan in patients with Ewing's sarcoma are warranted.
Werner
Sebastian
S
Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, Albert-Ludwigs-University, Mathildenstr. 1, 79106, Freiburg, Germany.
Mendoza
Arnulfo
A
Hilger
Ralf A
RA
Erlacher
Miriam
M
Reichardt
Wilfried
W
Lissat
Andrej
A
Konanz
Claudia
C
Uhl
Marcus
M
Niemeyer
Charlotte M
CM
Khanna
Chand
C
Kontny
Udo
U
D000818:Animals; D017304:Annexin A5 / Q000502:physiology; D018906:Antineoplastic Agents, Alkylating / Q000493:pharmacokinetics / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D006690:Bisbenzimidazole; D015153:Blotting, Western; D002066:Busulfan / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000627:therapeutic use; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D016193:G1 Phase / Q000187:drug effects; D007202:Indicators and Reagents; D007274:Injections, Intraperitoneal; D051379:Mice; D008819:Mice, Nude; D012512:Sarcoma, Ewing / Q000188:drug therapy*; D013778:Tetrazolium Salts; D013844:Thiazoles; D023041:Xenograft Model Antitumor Assays