Werner Sebastian, Mendoza Arnulfo, Hilger Ralf A, Erlacher Miriam, Reichardt Wilfried, Lissat Andrej, Konanz Claudia, Uhl Marcus, Niemeyer Charlotte M, Khanna Chand, Kontny Udo
Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, Albert-Ludwigs-University, Mathildenstr. 1, 79106, Freiburg, Germany.
Cancer Chemother Pharmacol. 2008 Jun;62(1):19-31. doi: 10.1007/s00280-007-0566-9. Epub 2007 Sep 1.
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.
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.
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.
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.
使用烷化剂白消安进行的大剂量化疗已广泛应用于高危尤因肉瘤患者的治疗。由于存在毒性风险，白消安和放疗不能同时应用，导致遗漏了一种有效的治疗成分。曲奥舒凡是白消安的衍生物，其副作用比白消安小，可与放疗联合使用。我们研究了曲奥舒凡对一组尤因肉瘤细胞系的细胞存活、细胞周期和体外凋亡的影响，并将其与白消安进行比较。此外，还在原位尤因肉瘤小鼠异种移植模型中研究了曲奥舒凡的抗肿瘤作用。
通过MTT法测定细胞存活率，通过流式细胞术分析细胞周期。通过检测DNA片段化、Hoechst 33258染色、膜联蛋白V以及半胱天冬酶-3和9的裂解来分析凋亡。在NOD/SCID小鼠的尤因肉瘤异种移植模型中评估曲奥舒凡和白消安对原发性肿瘤生长的影响（每组10只小鼠），在裸鼠中分析曲奥舒凡的药代动力学。
在体内可达到的浓度下，曲奥舒凡在所有细胞系中均能将细胞生长抑制至少70%。与白消安相比，曲奥舒凡在等效浓度下对细胞生长的抑制作用更大。曲奥舒凡在低浓度下的生长抑制作用主要是由于G2期细胞周期阻滞，而在高浓度下则是由于凋亡。与白消安相比，曲奥舒凡在较低浓度下即可诱导凋亡。与白消安不同，曲奥舒凡在体内可达到的浓度下能诱导凋亡缺陷细胞系发生细胞死亡。在小鼠中，曲奥舒凡在2500和3000mg/kg剂量下可抑制肿瘤生长。曲奥舒凡在小鼠中的药代动力学暴露情况与先前在人类患者中的报道相似。在最大耐受剂量下，曲奥舒凡的抗肿瘤活性高于白消安。
我们的结果表明，曲奥舒凡在体外和小鼠体内对尤因肉瘤细胞均有效。因此，有必要进行对照试验来研究曲奥舒凡在尤因肉瘤患者中的作用。
