1067

The diagnosis of glioblastoma, the most malignant and prevalent brain tumor, is considered as a death sentence in humans because all current therapeutic strategies remain so far ineffective in controlling the malignant growth of glioblastoma. Chemotherapy, which is almost always used at some point in the treatment regimen for glioblastoma, has shown little promise. For our current investigation, we used all-trans retinoic acid (ATRA) and taxol (TXL) alone and also in combination for controlling the growth of human malignant glioblastoma U87MG cells xenografted in athymic nude mice. For xenotransplantation of glioblastoma, 6 weeks-old athymic nude mice (Charles Rivers) were subcutaneously (sc) injected with the exponentially growing U87MG (5 million cells/mouse). Animals with 3 weeks-old glioblastoma xenografts were randomly divided into 4 groups: control, ATRA, TXL, and ATRA plus TXL. Animals in control group did not receive any therapy. Each animal in other group received intraperitoneally (ip) a daily dose of ATRA (1.5 μg/kg), or TXL (45 μg/kg), or ATRA (1.5 μg/kg) plus 4 h later TXL (45 μg/kg) for 7 days. Histopathological examination of H&E stained tumor sections showed that control group maintained characteristic growth of glioblastoma, ATRA alone inhibited tumor cell proliferation and caused astrocytic differentiation, TXL alone induced notable amounts of apoptosis, and ATRA plus TXL caused significant amounts of differentiation with apoptosis. In situ immunofluorescent labeling detected increased calpain expression in apoptosis. Also, in situ TUNEL and double immunofluorescent labeling demonstrated cell death with increased calpain expression in tumor sections treated with TXL, or ATRA plus TXL. Western blot analyses showed changes in expression of Bax and Bcl-2 proteins leading to increased Bax:Bcl-2 ratio, cytochrome c release from mitochondria, activation of calpain and caspase-3 for degradation of 270 kD α-spectrin at specific sites to generate correspondingly 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, also activation of caspase-12, and increased fragmentation of inhibitor-of-caspase-3-activated-DNase (ICAD) for completion of apoptotic death in tumors treated with TXL, or ATRA plus TXL. Western blot analyses further showed alterations in levels of expression of p65 NFκB, IκBα, JNK, p-JNK, and p-p38 proteins so as to favor the apoptotic process. Taken together, our investigation in animal model revealed that treatment of xenografted glioblastoma with ATRA plus TXL effectively controlled malignant growth. Further studies need to be conducted to see the efficacy of this combination therapy in brain itself using rats with allografted glioblastoma. This investigation was supported in part by the R01 grants from the NCI and NINDS of the NIH (Bethesda, MD), and also a grant from the State of SC.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]