Bevacizumab and carboplatin increase survival and asymptomatic tumor volume in a glioma model.

To evaluate efficacy and MRI findings after intravenous bevacizumab and/or carboplatin in a human glioma animal model, we randomized male nude rats with intracerebral UW28 human glioma xenografts to four groups: (1) controls (n = 9), (2) bevacizumab 10 mg/kg (n = 6), (3) carboplatin 200 mg/m(2) (n = 6), and (4) bevacizumab + carboplatin (n = 6). MRI was performed on the day of treatment (day 7-10) and 1 week later, and rats were followed for survival. Dynamic MRI was done in three controls and three rats treated with bevacizumab with or without carboplatin before and 24 h after treatment. Median overall survival (OS) was as follows: group 1, 16 days; group 2, 23 days; group 3, 22 days; group 4, 36 days. OS was significantly longer in group 4 than in group 1 (p = 0.0011), group 2 (p = 0.0014), and group 3 (p = 0.0015), and rats had significantly larger tumors. No objective tumor responses were observed on MR images at 1 week after treatment; however, after bevacizumab, dynamic MRI showed reduced gadolinium enhancement intensity and increased time to peak, consistent with decreased vascular permeability. Carboplatin + bevacizumab is effective and superior over bevacizumab or carboplatin monotherapy in this animal model. Increased survival concomitant with increased asymptomatic tumor volume is suggestive that vascular targeting with reduced peritumoral edema and mass effect contributes to the efficacy of bevacizumab. The promising survival data warrant future clinical trials using bevacizumab + carboplatin.

Efficacy and MRI of rituximab and methotrexate treatment in a nude rat model of CNS lymphoma.

To determine the efficacy of methotrexate and/or rituximab in a CNS lymphoma model and to evaluate MRI modalities for monitoring efficacy, we inoculated female athymic nude rats (rnu/rnu) intracerebrally with human MC116 B-lymphoma cells. Between days 16 and 26, rats were randomized to receive intravenous (IV) treatment with (1) saline (controls, n = 15), (2) methotrexate 1,000 mg/m(2) (n = 6), (3) rituximab 375 mg/m(2) (n = 6), or (4) rituximab plus methotrexate (n = 6). T2/fluid-attenuated inversion recovery (FLAIR) and gadolinium contrast-enhanced T1 MRI sequences were performed prior to and 1 week after treatment. IV rituximab gave an objective tumor response in four of six animals (>50% reduction in tumor volume comparing pre- and posttreatment T2/FLAIR MRI) and resulted in stable disease (50%-125% of baseline) in another animal. The percent change in tumor volume on T2/FLAIR images was significantly different in the control versus rituximab group (p = 0.0051). IV methotrexate slowed tumor growth, compared to controls, but only one of six animals had an objective response. In untreated controls, tumor histological volumes correlated well with T2/FLAIR or contrast-enhanced T1 images (r = 0.877). In the treatment groups, T2/FLAIR correlation was good, but the gadolinium-enhanced T1 MRI was not significantly correlated with histology (r = 0.19). The MC116 CNS lymphoma model seems valuable for preclinical testing of efficacy and toxicity of treatment regimens. IV rituximab was highly effective, but methotrexate was only minimally effective. T2/FLAIR was superior to contrast-enhanced T1 for monitoring efficacy.

Identification of novel small molecule Beclin 1 mimetics activating autophagy.

Anti-apoptotic proteins Bcl-2 and Bcl-xL could block autophagy by binding to Beclin 1 protein, an essential inducer of autophagy. Compounds mimicking Beclin 1 might be able to disrupt Bcl-xL/2-Beclin 1 interaction, free out Beclin 1, and thus trigger autophagy. In order to identify small molecule Beclin 1 mimetics, a fluorescence polarization-based high-throughput screening of 50,316 compounds was carried out with a Z' score of 0.82 ± 0.05, and an outcome of 58 hits. After the structure analysis, three acridine analogues were unveiled and confirmed using the fluorescence polarization assay and the surface plasmon resonance assay. Moreover, a set of 17 additional acridine analogues was prepared and tested. Compound 7 showed selectivity for Bcl-xL (KD = 6.5 µM) over Bcl-2 (KD = 160 µM) protein, and potent cytotoxicity (nanomolar scale) in PC-3, PC-3a and DU145 prostate cancer cells. Furthermore, induction of autophagy was also demonstrated in PC-3 and PC-3a cells treated with some acridine compounds by LC3 conversion immunoblotting and LC3 fluorescence microscopy. These Beclin 1 mimetics will be invaluable tools for developing novel autophagy inducers, better understanding the roles of autophagy in cancer, and will contribute to cancer therapy.

Ewing sarcoma ewsa protein regulates chondrogenesis of Meckel's cartilage through modulation of Sox9 in zebrafish.

Ewing sarcoma is the second most common skeletal (bone and cartilage) cancer in adolescents, and it is characterized by the expression of the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in mitosis, splicing and transcription. We have previously shown that EWS/FLI1 interacts with EWS, and it inhibits EWS activity in a dominant manner. Ewing sarcoma is a cancer that specifically develops in skeletal tissues, and although the above data suggests the significance of EWS, its role in chondrogenesis/skeletogenesis is not understood. To elucidate the function of EWS in skeletal development, we generated and analyzed a maternal zygotic (MZ) ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appeared to be normal and fertile. Compared with wt/wt, the Meckel's cartilage of MZ ewsa/ewsa mutants had a higher number of craniofacial prehypertrophic chondrocytes that failed to mature into hypertrophic chondrocytes at 4 days post-fertilization (dpf). Ewsa interacted with Sox9, which is the master transcription factor for chondrogenesis. Sox9 target genes were either upregulated (ctgfa, ctgfb, col2a1a, and col2a1b) or downregulated (sox5, nog1, nog2, and bmp4) in MZ ewsa/ewsa embryos compared with the wt/wt zebrafish embryos. Among these Sox9 target genes, the chromatin immunoprecipitation (ChIP) experiment demonstrated that Ewsa directly binds to ctgfa and ctgfb loci. Consistently, immunohistochemistry showed that the Ctgf protein is upregulated in the Meckel's cartilage of MZ ewsa/ewsa mutants. Together, we propose that Ewsa promotes the differentiation from prehypertrophic chondrocytes to hypertrophic chondrocytes of Meckel's cartilage through inhibiting Sox9 binding site of the ctgf gene promoter. Because Ewing sarcoma specifically develops in skeletal tissue that is originating from chondrocytes, this new role of EWS may provide a potential molecular basis of its pathogenesis.

Imaging and therapy with rituximab anti-CD20 immunotherapy in an animal model of central nervous system lymphoma.

PURPOSE: To evaluate the effect of rituximab monoclonal antibody (mAb) on MRI tumor volumetrics and efficacy in a rat model of central nervous system (CNS) lymphoma when delivery to the brain was optimized with osmotic blood-brain barrier disruption (BBBD). EXPERIMENTAL DESIGN: Female nude rats with intracerebral MC116 human B-cell lymphoma xenografts underwent baseline MRI and were randomized into 5 groups (n = 6 per group): (i) BBBD saline control; (ii) methotrexate with BBBD; (iii) rituximab with BBBD; (iv) rituximab and methotrexate with BBBD; and (v) intravenous rituximab. Tumor volumes were assessed by MRI at 1 week, and rats were followed for survival. RESULTS: BBBD increased delivery of yttrium-90-radiolabeled mAb in the model of CNS lymphoma. Control rats showed 201 ± 102% increase in tumor volume on MRI 1 week after entering the study and median 14-day survival (range: 6-33). Tumor growth on MRI was slowed in the methotrexate treatment group, but survival time (median: 7 days; range: 5-12) was not different from controls. Among 17 evaluable rats treated with rituximab, 10 showed decreased tumor volume on MRI. All rituximab groups had increased survival compared with control, with a combined median of 43 days (range: 20-60, P < 0.001). There were no differences by route of delivery or combination with methotrexate. CONCLUSIONS: Rituximab was effective at decreasing tumor volume and improving survival in a model of CNS lymphoma and was not affected by combination with methotrexate or by BBBD. We suggest that rituximab warrants further study in human primary CNS lymphoma.

Cyclophosphamide enhances human tumor growth in nude rat xenografted tumor models.

The effect of the immunomodulatory chemotherapeutic agent cyclophosphamide (CTX) on tumor growth was investigated in primary and metastatic intracerebral and subcutaneous rat xenograft models. Nude rats were treated with CTX (100 mg/kg, intraperitoneally) 24 hours before human ovarian carcinoma (SKOV3), small cell lung carcinoma (LX-1 SCLC), and glioma (UW28, U87MG, and U251) tumor cells were inoculated subcutaneously, intraperitoneally, or in the right cerebral hemisphere or were infused into the right internal carotid artery. Tumor development was monitored and recorded. Potential mechanisms were further investigated. Only animals that received both CTX and Matrigel showed consistent growth of subcutaneous tumors. Cyclophosphamide pretreatment increased the percentage (83.3% vs 0%) of animals showing intraperitoneal tumors. In intracerebral implantation tumor models, CTX pretreatment increased the tumor volume and the percentage of animals showing tumors. Cyclophosphamide increased lung carcinoma bone and facial metastases after intra-arterial injection, and 20% of animals showed brain metastases. Cyclophosphamide transiently decreased nude rat white blood cell counts and glutathione concentration, whereas serum vascular endothelial growth factor was significantly elevated. Cyclophosphamide also increased CD31 reactivity, a marker of vascular endothelium, and macrophage (CD68-positive) infiltration into glioma cell-inoculated rat brains. Cyclophosphamide may enhance primary and metastatic tumor growth through multiple mechanisms, including immune modulation, decreased response to oxidative stress, increased tumor vascularization, and increased macrophage infiltration. These findings may be clinically relevant because chemotherapy may predispose human cancer subjects to tumor growth in the brain or other tissues.