Cytotoxic effect and uptake of estramustine in a rat glioma model

The cytotoxic effect of estramustine-phosphate (EMP) and the uptake in tumor tissue were investigated in a rat glioma model in vitro and in vivo. EMP, a combination of nornitrogen mustard and 17beta-estradiol, is a cytotoxic drug which main target is assumed to be the microtubule system. EMP and its metabolite estramustine (EaM) have a demonstrated anti-tumorous effect on human glioma cells in vitro. The drug uptake in tumor tissue and subsequently also the cytotoxic effect, is believed to depend, at least partially, on a specific estramustine-binding protein (EMBP) which is present in human glioma tissue. In this study we have examined the effects and pharmacokinetics of EaM in the nitrosourea induced BT4C rat glioma model. The tumor was characterized by infiltrative growth with a histopathological picture resembling gliosarcoma. The presence of EMBP was demonstrated by immunohistology. In vitro EMP caused a dose-related inhibition of BT4C-cell growth. In vivo, in the rat model, a significant inhibition of tumor growth was obtained after administration of EaM 20 mg/kg/d i.p. The pharmacokinetics of EaM resembled that found in the human clinical situation with EaM as the main metabolite accumulating in tumor tissue. The mean concentration ratio of EaM was 15.6 in tumor versus serum, and 1.8 in tumor versus normal brain of 1.8. The cytotoxic effect demonstrated in the rat glioma model justifies further evaluation of EMP/EaM in the treatment of malignant gliomas.     

Anaplastic ependymoma simulating glioblastoma in the cerebrum of an adult

A case of anaplastic ependymoma of the cerebral hemisphere in which the histopathological features closely simulated those of glioblastoma is reported. The patient was a 72-year-old woman with a large, well-demarcated tumor in the left temporal lobe. The tumor was totally extirpated, but recurred 18 months later, and the patient died after 4 months. The extirpated tumor was well circumscribed from the surrounding brain tissue and consisted of a sheet-like, dense proliferation of atypical, short spindle or polygonal cells. Extensive geographic necrosis with nuclear pseudopalisading was seen. Although perivascular pseudorosettes were observed in many areas, true ependymal rosettes were absent. Immunohistochemistry for glial fibrillary acidic protein and epithelial membrane antigen and ultrastructural study confirmed the ependymal nature of tumor cells. The histopathological spectrum of anaplastic ependymoma is very wide and reflects the basically dual characteristics of ependymal cells: epithelial and glial phenotypes. The present case indicates that some anaplastic ependymomas strongly express the glial phenotype and also show remarkable anaplastic cytological features, thus closely simulating glioblastoma. The diagnostic criteria for anaplastic ependymoma, and the nosological position of highly anaplastic ependymoma and its possible clinical implications, are briefly discussed.     

Immunohistochemical analysis of SOX6 expression in human brain tumors

We previously demonstrated that the developmentally regulated gene,SOX6, is strongly expressed in glioma cells and in the fetal brain, but only faintly in the normal adult brain. Recent studies have indicated that brain tumor cells may share antigens, signaling systems, and behavior with neural stem/progenitor cells. To test the validity of this proposition, we analyzed the expression of SOX6 in various human central nervous system (CNS) tumors. Immunohistochemical analysis revealed that astrocytic and oligodendroglial tumors expressed SOX6; neuronal-glial cell tumors (central neurocytoma) and embryonal tumors (medulloblastoma), which arise from multipotential stem cell precursors, also showed a high intensity of SOX6 staining. In contrast, ependymal tumors (ependymoma and subependymoma), meningioma, and schwannoma, which are all well differentiated tumors, showed either no staining or only faint staining for SOX6. These results suggest that SOX6 may be expressed in bipotential or multipotential cells capable of neuronal and glial differentiation, but not in fully differentiated cells. SOX6 may be a useful marker for the diagnosis of tumors arising from immature bipotential cells that may differentiate into neuronal and glial cells.     

Invasiveness in vitro and biological markers in human primary glioblastomas

Invasion of spheroids from 20 human primary glioblastomas into precultured fetal rat brain tissue in culture has been studied and quantified. Between 30 and 98 percent of the normal brain tissue was destroyed by invading glioma cells within 4 days. The degree of invasion did not correlate with patient survival. A slightly higher invasiveness and shorter survival was seen in tumors with EGF receptor overexpression, and the opposite pattern was found for tumors with a TP53 mutation.The degree of invasiveness in vitro was far higher than would be expected from the dynamics of clinically observed tumor spread. This suggests that mechanisms suppressing invasion may be operative in the normal brain; alternatively the differences may be due to a higher permissiveness of the fetal brain tissue for invasion in vitro.     

CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells

Human brain tumor stem cells have been enriched using antibodies against the surface protein CD133. An antibody recognizing CD133 also served to isolate normal neural stem cells from fetal human brain, suggesting a possible lineage relationship between normal neural and brain tumor stem cells. Whether CD133-positive brain tumor stem cells can be derived from CD133-positive neural stem or progenitor cells still requires direct experimental evidence, and an important step toward such investigations is the identification and characterization of normal CD133-presenting cells in neurogenic regions of the embryonic and adult brain. Here, we present evidence that CD133 is a marker for embryonic neural stem cells, an intermediate radial glial/ependymal cell type in the early postnatal stage, and for ependymal cells in the adult brain, but not for neurogenic astrocytes in the adult subventricular zone. Our findings suggest two principal possibilities for the origin of brain tumor stem cells: a derivation from CD133-expressing cells, which are normally not present in the adult brain (embryonic neural stem cells and an early postnatal intermediate radial glial/ependymal cell type), or from CD133-positive ependymal cells in the adult brain, which are, however, generally regarded as postmitotic. Alternatively, brain tumor stem cells could be derived from proliferative but CD133-negative neurogenic astrocytes in the adult brain. In the latter case, brain tumor development would involve the production of CD133.     

Tumor imprint cytology of ovarian ependymoma. A case report.

BACKGROUND: Ependymoma is a tumor that usually develops in the central nervous system and is extremely rare in the ovary. The first case of ovarian ependymoma was reported by Kleinman et al. (Kleinman GM, Young RH, Scully RE. Ependymoma of the ovary: report of three cases. Hum Pathol 1984;15:632-8.) in 1984, and only eight cases have been reported since then. Criteria for the histopathologic diagnosis of ependymoma are already established, but there has been no investigation of the cytologic diagnosis of ovarian ependymoma. METHODS: An imprint cytologic specimen was obtained from a recurrent ovarian ependymoma. The imprint cytologic features were compared with the findings of histologic examination, immunostaining, and electron microscopy. RESULTS: Imprint cytology revealed clusters of small cells with tapering cytoplasmic processes and a round nucleus. On the basis of these features, a neurogenic tumor could be included in the differential diagnosis. Furthermore, many rosette-like collections of cells that were suggestive of ependymal rosettes or perivascular pseudorosettes, characteristic of ependymoma, were found. The presence of ependymal rosettes and perivascular pseudorosettes also were confirmed by the histopathologic examination. Together with positive immunostaining for glial fibrillary acidic protein, this led to the diagnosis of ependymoma, which also was supported by the electron microscopic findings. CONCLUSIONS: Careful observation of the imprint cytologic specimen of an ovarian ependymoma should reveal numerous rosette-like collections of cells that were suggestive of ependymal rosettes or perivascular pseudorosettes. In addition, if we remember that ependymoma can develop in the ovary and find cells with tapering processes that suggest a neurogenic tumor, it may be possible to detect histologic features characteristic of ependymoma by the imprint cytology. To our knowledge, this is the first report on the imprint cytologic diagnosis of ependymoma originating in the ovary.     

Uptake and retention of estramustine and the presence of estramustine binding protein in malignant brain tumours in humans.

Estraumustine phosphate (EMP), a cytotoxic drug used in the treatment of prostatic carcinoma, has been shown to exert cytotoxic effects on glioma cells in vitro. The drug uptake is assumed to depend on a specific estramustine binding protein (EMBP). One of the main difficulties in achieving cytotoxic effect in malignant brain tumours is believed to be due to the poor penetration of cytotoxic drugs into tumour tissue. In patients with malignant supratentorial brain tumours we have analysed the uptake of EMP metabolites in tumour tissue after oral administration and demonstrated EMBP in the same tissue specimens. Sixteen patients were given 280 mg EMP orally 14 h prior to surgery. Specimens from brain tumour tissue, cystic fluid, and serum were collected during surgery. Using gas chromatography the metabolites of EMP, estramustine (EaM) and estromustine (EoM), were quantified, EMBP was demonstrated by immunohistochemistry. The mean concentrations of EaM and EoM, expressed in ng g-1, were 60.3 and 38.4 in tumour tissue and 3.5 and 56.3 in serum, respectively. An accumulation of EaM in tumour tissue was found with a mean concentration gradient of 16.1 versus serum, while the gradient for EoM was 0.76. EMBP was demonstrated with a high degree of staining in all but one tumour. The high concentrations of EaM and EoM found in malignant brain tumour tissue correspond to potentially cytotoxic levels. The present results as well as the earlier in vitro demonstrated cytotoxic effects on glioma cells strengthen the possibility of a therapeutic effect of EMP in the treatment of malignant brain tumours.     

The Expression and Activation of Matrix Metalloproteinase-2 in Rat Brain After Implantation of C6 Rat Glioma Cells

It has been reported that matrix metalloproteinases (MMPs) are highly expressed in malignant glioma cells and that this increased expression may facilitate the invasiveness of tumor cells. The authors investigated the expression and enzymatic activity of MMPs in rat brain during the growth of malignant gliomas at different time intervals. C6 rat glioma cells were unilaterally implanted into rat cerebral hemispheres. After 7 or 14 days, these brain tissues were prepared for SDS-PAGE zymography, Western blotting, immunohistochemistry, and in situ zymography. SDS-PAGE zymography and Western blotting revealed that the expression of proMMP-2 in rat brains with C6 glioma cells was significantly higher than that in normal or the sham-operated rat brains, and that the activated form of MMP-2 was detected only in the former but not in the latter. On immunohistochemistry, C6 glioma cells presenting invasive growth into the rat brain parenchyma and vessels demonstrated MMP-2 immunoreactivity. On in situ zymography, foci of invasive C6 glioma cells in rat brain tissue showed gelatinolytic activity. These results suggest that expression and activation of MMP-2 may be one of the crucial steps for glioma cell invasion into the brain parenchyma in vivo.     

Immunocytochemical detection of 14-3-3 in primary nervous system tumors

Summary 14-3-3 proteins have attracted much recent interest in the etiopathogenesis of human cancers owing to their involvement in the prevention of apoptosis. However, the expression of 14-3-3 in primary nervous system tumors has not been previously characterized. In this paper, Immunohistochemistry using a specific anti-14-3-3 antibody was performed on formalin-fixed, paraffin embedded archival tissue from 124 primary human nervous system tumors and 10 normal brain tissues. In the normal control brains, 14-3-3 immunoreactivity was localized mainly in the neuronal somata and processes, and some glial cells showed only weak immunoreactivity. However, 14-3-3 immunoreactivity was seen in the majority of astrocytomas [grade I (9/11), II (16/21), III (13/17), IV (17/21)]. There was no difference between the positive expression rates of 14-3-3 in different grades of astrocytomas (P = 0.968). But the intensity and degree of 14-3-3 immunoreactivity in diffuse astrocytomas, anaplastic astrocytoma, and glioblastoma multiformes showed trends with tumor grade, with glioblastomas having the highest positivity (P = 0.048). The 14-3-3 immunoreactivity was also seen in the majority of other gliomas [oligodendroglioma (2/3), anaplastic oligodendroglioma (4/4), ependymoma (1/2), anaplastic ependymoma (2/2), choroid plexus papilloma (3/3), pineocytoma (2/2), medulloblastoma (5/8)]. All meningiomas [syncytical (3), fibrous/fibroblastic (4), angiomatous (4), transitional/mixed (3)] were intensely and diffusely positive. All schwannomas (4), neurofibromas (2), pituitary adenomas (6) and craniopharyngiomas(4) also showed intense positive staining. These results showed that 14-3-3 is expressed in the majority of the primary human nervous system tumors. The up-regulated expression of 14-3-3 may be a common mechanism for evading apoptosis in most primary human nervous system tumors, and targeting 14-3-3 may be a novel promising strategy for the treatment of these tumors, especially for malignant tumors.     

Tanycytic ependymoma of the filum terminale with pleomorphic giant cells

A tanycytic ependymoma measuring 1.5 cm in maximal dimension, which involved the filum terminale and conus medullaris of a 55-year-old woman, is reported. The tumor consisted of a compact fascicular proliferation of spindle cells having long bipolar cytoplasmic processes, and immunohistochemical and ultrastructural studies demonstrated the ependymal features of neoplastic cells. The most prominent finding was an appearance of many atypical and pleomorphic, often monstrous, giant cells, which was not associated with an increase in proliferative activity. Remarkable nuclear atypism and pleomorphism with formation of giant cells have not been documented previously in tanycytic ependymoma. These nuclear changes are considered essentially degenerative in nature and probably do not portend a worse prognosis despite their worrisome appearance. This tumor could be appropriately termed “giant cell tanycytic ependymoma.”     

Ependymoma with cartilaginous metaplasia might have more aggressive behavior: a case report and literature review

Ependymoma with cartilaginous metaplasia with or without bone formation is exceedingly rare. Only eight cases have been reported in the literature. We report a case of ependymoma with cartilaginous and osseous metaplasia in a 5-year-old boy. Microscopically, the tumor was composed of neoplastic ependymal tissue and mature cartilage and bone. Immunohistochemically, glial fibrillary acidic protein and epithelial membrane antigen were positive for ependymoma cells but negative for cartilage and bone. Recurrence occurred after 15-month follow-up. The patient deteriorated rapidly and died after 1 month. Reviewing 8 reported cases and our latest case, we found that 3 cases of ependymoma with cartilaginous metaplasia were treated with radiotherapy. Six cases had recurrence from 6 months to 8 years and 2 cases died on the day of operation. These findings suggest that ependymoma with cartilaginous metaplasia might have more aggressive clinical behavior.     

Radiosensitizing effect of estramustine in malignant gliomain vitro andin vivo

Summary Estramustine-phosphate (EMP), a combination of nornitrogen mustard and 17-estradiol, has been demonstrated to exert specific antiproliferative effects on human glioma cellsin vitro. The cytotoxic effect is, at least partially, mediated by inhibiting microtubule function. In this study the combined effect of EMP and radiation was evaluated in the human glioma cell-lines, 251-MG and 105-MG,in vitro, and in the rat glioma BT4Cin vitro andin vivo. In all cell-lines an additive effect of EMP and radiation was obtainedin vitro. Assuming equal effect of EMP is obtained in subsequent radiation fractions, the cell kill will be increased from 2–3 to 5–10 logs if delivering 30 fractions of 2 Gy combined with EMP. In the BT4C rat model the combined effect was found to be synergistic. Flow cytometry demonstrated an arrest in G2/M phase in all cell-lines after EMP treatment. This block in G2/M phase in addition to the previously demonstrated induction of free oxygen radicals, and the increase of blood flow with an assumed subsequent increase of oxygenation, might provide an explanation for the observed radiosensitizing effect of estramustine.     

Pathology of brain parenchyna in meningeal carcinomatosis; Immunohistochemical study with astroprotein (GFAP) and tubulin

Effects of leptomeningeal tumor on the brain parenchyma was studied by the immunohistochemical method with astroprotein (GFAP) and tubulin in a rat model of meningeal carcinomatosis. Thickening of subpial glial lining (external glial layer) and hypertrophy of subpial astrocytes, detected by the antiserum to GFAP, was the early sign of parenchymal involvement. The glial lining was continuous as far as the tumor cells were confined to the subarachnoid space, however, penetration of tumor cells into subpial brain was associated with disruption of the glial lining. Speculative role of this lining in preventing the tumor cell to infiltrate into brain tissue was discussed. In contrast to the prominent immunohistochemical changes in astrocytes, neuronal tubulin immunoreactivity was not altered even in the late stage of the disease. The present study demonstrated that the leptomeningeal dissemination of tumor cells did cause pathologic change in brain parenchyma as was evidenced by the reactive change of astrocytes. However, the preserved immunoreaction for tubulin suggested that the nerve cell damage was not severe even at the advanced stage of the disease. address for offprints     

Pre-B-cell leukemia homeobox interacting protein 1 is overexpressed in astrocytoma and promotes tumor cell growth and migration

Background

Glial brain tumors cause considerable mortality and morbidity in children and adults. Innovative targets for therapy are needed to improve survival and reduce long-term sequelae. The aim of this study was to find a candidate tumor-promoting protein, abundantly expressed in tumor cells but not in normal brain tissues, as a potential target for therapy.

Methods

In silico proteomics and genomics, immunohistochemistry, and immunofluorescence microscopy validation were performed. RNA interference was used to ascertain the functional role of the overexpressed candidate target protein.

Results

In silico proteomics and genomics revealed pre-B-cell leukemia homeobox (PBX) interacting protein 1 (PBXIP1) overexpression in adult and childhood high-grade glioma and ependymoma compared with normal brain. PBXIP1 is a PBX-family interacting microtubule-binding protein with a putative role in migration and proliferation of cancer cells. Immunohistochemical studies in glial tumors validated PBXIP1 expression in astrocytoma and ependymoma but not in oligodendroglioma. RNAi-mediated PBXIP1-knockdown in glioblastoma cell lines strongly reduced proliferation and migration and induced morphological changes, indicating that PBXIP1 knockdown decreases glioma cell viability and motility through rearrangements of the actin cytoskeleton. Furthermore, expression of PBXIP1 was observed in radial glia and astrocytic progenitor cells in human fetal tissues, suggesting that PBXIP1 is an astroglial progenitor cell marker during human embryonic development.

Conclusion

PBXIP1 is a novel protein overexpressed in astrocytoma and ependymoma, involved in tumor cell proliferation and migration, that warrants further exploration as a novel therapeutic target in these tumors.     

HUlip, a human homologue of unc-33-like phosphoprotein of Caenorhabditis elegans; Immunohistochemical localization in the developing human brain and patterns of expression in nervous system tumors

HUlip is a human homologue of a C. elegans gene, unc-33, that is developmentally regulated during maturation of the nervous system. HUlip is highly expressed only in the fetal brain and spinal cord, and is undetected in the adult brain. The purpose of this study was to investigate the pattern of hUlip expression in the developing human brain and nervous system tumors. Ten human brains at different developmental stages and 118 cases of nervous system tumor tissues were examined by immunohistochemistry. Twelve related tumor cell lines were also analyzed by northern blotting and immunoblotting. HUlip was expressed in late fetal and early postnatal brains; strongly in the neurons of the brain stem, basal ganglia/thalamus, and dentate gyrus of the hippocampus, and relatively weakly in the cerebral and cerebellar cortex. Among tumors, hUlip expression was easily detected in tumor cells undergoing neuronal differentiation such as ganglioneuroblastomas and ganglioneuromas. Furthermore, hUlip immunoreactivity was also found in various brain tumors showing neuronal differentiation: central neurocytomas (6 of 6 cases were positive), medulloblastomas (5/11), atypical teratoid rhabdoid tumor (1/1) and gangliogliomas (4/7). Some astrocytic tumors also showed weak positivity: astrocytomas (1 of 5 cases), anaplastic astrocytomas (2/5), and glioblastomas (3/11). Subependymal giant cell astrocytomas and subependymomas, which are of controversial histogenetic origin, showed strong hUlip immunoreactivity. The results of this study indicate that the expression of hUlip protein is distinctly restricted to the late fetal and early postnatal periods of human nervous system development and to certain subsets of nervous system tumors. The exact function of hUlip needs to be further clarified; yet the results of our study strongly imply that hUlip function is important in human nervous system development and its aberrant expression in various types of nervous system tumors suggests a role of hUlip as an oncofetal neural antigen.     

PARP inhibition sensitizes childhood high grade glioma, medulloblastoma and ependymoma to radiation

Poly ADP-ribose polymerase (PARP) is a protein involved in single strand break repair. Recently, PARP inhibitors have shown considerable promise in the treatment of several cancers, both in monotherapy and in combination with cytotoxic agents. Synthetic lethal action of PARP inhibitors has been observed in tumors with mutations in double strand break repair pathways. In addition, PARP inhibition potentially enhances sensitivity of tumor cells to DNA damaging agents, including radiotherapy. Aim of this study is to determine the radiosensitizing properties of the PARP inhibitor Olaparib in childhood medulloblastoma, ependymoma and high grade glioma (HGG). Increased PARP1 expression was observed in medulloblastoma, ependymoma and HGG, as compared to non-neoplastic brain tissue. Pediatric high grade glioma, medulloblastoma and ependymoma gene expression profiling revealed that high PARP1 expression is associated with poor prognosis. Cell growth inhibition assays with Olaparib resulted in differential sensitivity, with IC50 values ranging from 1.4 to 8.4 μM, irrespective of tumor type and PARP1 protein expression. Sensitization to radiation was observed in medulloblastoma, ependymoma and HGG cell lines with subcytotoxic concentrations of Olaparib, which coincided with persistence of double strand breaks. Combining PARP inhibitors with radiotherapy in clinical studies in childhood high grade brain tumors may improve therapeutic outcome.     

Tanycytic ependymoma of the spinal cord with anaplastic cytological features

In a 43-year-old man, an intramedullary spinal cord tumor spreading from the level of the T2 to T5 vertebrae was subtotally resected. The tumor predominantly consisted of a fascicular proliferation of spindle cells having bland nuclei and bipolar, long cytoplasmic processes, and a few perivascular pseudo-rosettes were found. Although there were no true ependymal rosettes, intracytoplasmic dot-like immunoreactivity for epithelial membrane antigen (EMA) was found in a few cells. In some areas, a dense and diffuse proliferation of anaplastic, short-spindled cells having hyperchromatic nuclei and scant cytoplasm was noted, and the Ki-67 labeling index was remarkably higher (18.2%) in these areas. Neither microvascular proliferation nor necrosis was observed. In the boundary region, these two areas showed gradual transition from one to the other. The patient has remained free from recurrence for 10 months postoperatively. This is the first documentation of tanycytic ependymoma in which tumor cells showed anaplastic cytological features.     

Localization of endostatin in rat and human gliomas

BACKGROUND: Endostatin is a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. Its occurrence and localization has not yet been examined in human brain tumors. The authors report the production of a monoclonal antibody and detection of endostatin in rat and human gliomas by immunohistochemistry. METHODS: The authors analyzed localization and tissue distribution of endostatin in 41 paraffin embedded glioma samples (18 glioblastoma multiforme, 7 WHO Grade III astrocytomas, 13 fibrillary, and 3 protoplasmic WHO Grade II astrocytomas) of human origin and 21 rat C6 gliomas by immunohistochemistry. Double labeling experiments confirmed the origin of endostatin-labeled cells. RESULTS: Endostatin immunoreactivity was detected in tumor cells, endothelial cells, macrophages, and lymphocytes of both rat and human gliomas. The percentage of cells labeled with the endostatin antibody was significantly lower (P = 0.0126) in the tumor parenchyma of human glioblastomas than in WHO Grade II astrocytomas. CONCLUSIONS: Endostatin was present in various cell types in rat and human gliomas in vivo. Lower levels in glioblastomas than in WHO Grade II astrocytomas might have reflected the shift of a probable regulatory balance between promoters and inhibitors of angiogenesis towards facilitation of neovascularization.     

Ependymal and choroid plexus tumors. Cytokeratin and GFAP expression

Twenty-six ependymal and 15 choroid plexus tumors were examined with monoclonal antibody against cytokeratin using the avidin-biotin-peroxidase complex (ABC) technique. Serial sections were examined with antisera to glial fibrillary acidic protein (GFAP). In five ependymal tumors (one ependymoma, two papillary ependymomas, and two primitive neuroectodermal tumors [PNET] with ependymal cells), a variable number of cytokeratin-positive cells were present. Most tumor cells (except two PNET) were positive with GFAP antisera. Many cytokeratin-positive cells were present in all choroid plexus tumors. GFAP-positive cells were present focally in six of 11 papillomas and in one of four carcinomas. Although their staining patterns and distribution were clearly different, focal coexistence of cytokeratin and GFAP was observed in six papillomas and two ependymal tumors. Thus, some ependymal tumors (especially papillary ependymomas and occasional PNET) and many choroid plexus tumors have demonstrable positivity with antibody to cytokeratin, suggesting a transitional cell type with features of both ependyma and choroid plexus.