CNS Tumor Cell Research Articles

LGG-55. Autophagy sensitizes CNS tumors to targeted therapy by lowering their apoptotic threshold

Autophagy inhibition improves the effectiveness and overcomes RAF pathway inhibition (RAFi) resistance across multiple CNS tumors and molecularly distinct resistance mechanisms. Mechanistic links between autophagy and apoptotic cell death may explain this ability to improve RAFi response and reverse resistance. RAFi sensitive (MAF 794, AM38) and resistant (MAF 794R, MAF 905-3, AM38R, B76) BRAFV600E CNS tumor cell lines were analyzed at baseline, following RAFi (vemurafenib), autophagy inhibition (chloroquine or shRNAs), and combination therapy. Growth assays and caspase activation were monitored by Incucyte Zoom. qRT-PCR evaluated key pro-apoptotic BH3-only members of the BCL-2 family. Broad BH-3 profiling was completed using the Letai JC-1 Plate-Based protocol. Western blot analysis assessed protein levels. Combination pharmacologic treatment caused alterations in key pro-apoptotic BH3-only proteins including an increase in BNIP3L and PUMA. Genetically inhibiting autophagy with shRNAs for ATG5 and ATG7 (proteins required for formation of the autophagosome) produced similar results with increases in both protein and mRNA levels of BNIP3L and PUMA following RAFi treatment. This suggested autophagy-mediated regulation of BH3 proteins functions to determine cellular apoptotic threshold. Caspase activation demonstrated increased effectiveness of combined RAFi and autophagy inhibition overcoming the apoptotic threshold compared to single drug treatment. BH3 profiling demonstrated a dependence on BCL-2 to inhibit apoptosis. BH3 mimetics competitively bind to pro-survival BCL-2 family members, blocking their protective effects and pushing tumor cells towards apoptosis. Autophagy inhibition can also improve treatment response by overcoming the apoptotic threshold in RAFi resistant cells and magnifying the apoptotic response in sensitive cells. BH3 profiling reveals CNS BRAFV600E are BCL-2 dependent cells, unprimed for apoptosis, which may be good candidates for additional treatment with BH3 mimetics such as venetoclax. This presents an attractive treatment for MAPK activated CNS tumors by enhancing apoptotic cell death by targeting the MAPK pathway, autophagy and BH3.

Design, synthesis, in vitro antiproliferative evaluation and GSK-3β kinase inhibition of a new series of pyrimidin-4-one based amide conjugates

A new series of novel amide conjugates of pyrimidin-4-one and aromatic/heteroaromatic /secondary cyclic amines has been synthesized and their in vitro antiproliferative activities against a panel of 60 human cancer cell lines of nine different cancer types were tested at NCI. Among the synthesized compounds, compound (4i) showed significant anti-proliferative activity. Compound (4i) displayed most potent activity against the breast tumor cell line T-47D and CNS tumor cell line SNB-75 exhibiting a growth of 1.93 % and 14.63 %, respectively. ADMET studies of the synthesized compounds were also performed and they were found to exhibit good drug like properties. Compound (4i) was found to exhibit potential inhibitory effect over GSK-3β with IC50 value of 71 nM. The molecular docking studies revealed that (4i) showed good binding affinity to GSK-3β and revealed multiple H-bonding and p-cation interactions with important amino acid residues on the receptor site. Compound (4i) may thus serve as a potential candidate for further development of novel anticancer therapeutics.

Effect of Serial Systemic and Intratumoral Injections of Oncolytic ZIKVBR in Mice Bearing Embryonal CNS Tumors.

The Zika virus (ZIKV) has shown a promising oncolytic effect against embryonal CNS tumors. However, studies on the effect of different administration routes and the ideal viral load in preclinical models are highly relevant aiming for treatment safety and efficiency. Here, we investigated the effect and effectiveness of different routes of administration, and the number of ZIKVBR injections on tumor tropism, destruction, and side effects. Furthermore, we designed an early-stage human brain organoid co-cultured with embryonal CNS tumors to analyze the ZIKVBR oncolytic effect. We showed that in the mice bearing subcutaneous tumors, the ZIKVBR systemically presented a tropism to the brain. When the tumor was located in the mice’s brain, serial systemic injections presented efficient tumor destruction, with no neurological or other organ injury and increased mice survival. In the human cerebral organoid model co-cultured with embryonal CNS tumor cells, ZIKVBR impaired tumor progression. The gene expression of cytokines and chemokines in both models suggested an enhancement of immune cells recruitment and tumor inflammation after the treatment. These results open new perspectives for virotherapy using the ZIKVBR systemic administration route and multiple doses of low virus load for safe and effective treatment of embryonal CNS tumors, an orphan disease that urges new effective therapies.

Abstract 2869: Zika virus is a potent oncolytic agent against aggressive human ependymoma

Abstract Ependymoma is a metastatic CNS tumor responsible for 10% of all pediatric brain tumors. Displaying a poor survival rate of only 32.5%, the conventional ependymoma treatment is surgery and radiation therapy associated to significant morbidity. Previous studies found significantly increased expression of NESTIN, a neural progenitor cell (NPC) marker, in aggressive ependymoma. Zika virus (ZIKV) has recently emerged as an oncolytic therapy against adult and pediatric CNS tumors due to its ability to infect NPCs and neural stem-like tumor cells. We showed for the first time that Brazilian Zika virus strain efficiently destroyed pediatric CNS tumor cells in vitro and in vivo, resulting in significantly mice longer survival and fewer metastasis. Once tumor cells overexpressing NPCs markers are more susceptible to ZIKV infection, here we evaluated the oncolytic properties of Brazilian ZIKV against human ependymoma. A patient-derived ependymoma cell line (USP21-EPE) has been established and found to abnormally express the pluripotency markers CD133, SOX2 and NESTIN. USP21-EPE monolayer culture showed massive cell death three days after ZIKV infection in four tested MOIs: 0.01, 0.1, 1 and 2. At MOI 0,1 50% of cell population decreased viability while at higher MOIs was observed 100% of cell death 72 hours post ZIKV infection. Moreover, ZIKV significantly disrupted CNS ependymoma tumorspheres reinforcing its oncolytic effect against the ependymoma tumor cells. Considering the poor effectiveness and severe side effects of available treatments for ependymoma and that most ZIKV infections are associated to very mild or no symptoms, our findings open new avenues for novel therapies against this aggressive pediatric tumor. Citation Format: Carolini Kaid, Patrícia Semedo-Kuriki, Ernesto Goulart, Luiz Caires-Júnior, Renato Astray, Oswaldo Keith Okamoto, Mayana Zatz. Zika virus is a potent oncolytic agent against aggressive human ependymoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2869.

IMPS-13IMMUNOLOGIC PROFILING OF PEDIATRIC CNS MALIGNANCIES FOR THE RATIONAL SELECTION OF IMUNOTHERAPEUTIC TRIALS

BACKGROUND: Although clinical trials have resulted in significant improvements in childhood brain cancer treatments, further progress using strategically targeted, potentially less toxic modalities are needed. Immunotherapy is one such emerging treatment approach. The degree of tumor-infiltrating cytotoxic T-lymphocytes (CTLs) directly correlates with prognosis for several cancer types. MHC Class I is down-regulated in 40-90% of adult cancers, rendering them resistant to CTL attack, correlating with poorer prognosis. MHC I expression in medulloblastomas is associated with poor prognostic factors, a pattern opposite of most other cancer-MHC I associations described. Natural Killer (NK)-cell based therapies, dependent upon low MHC I expression, are therefore proposed as an immunotherapeutic approach to medulloblastomas. Very little literature exists classifying MHC I expression in other pediatric brain cancers, despite several ongoing trials using MHC I-dependent strategies. This lack of data raises uncertainty of which immunotherapies are optimal for these diseases. Toward this end, we classified key immunologic characteristics of pediatric brain tumors. METHODS: We interrogated pre-clinical and clinical mRNA microarray databases to identify tumor types which exhibit MHC I and the associated b-2-microglobulin molecule expression. We up-regulated MHC I expression in vitro in pediatric CNS tumor cell lines and identified intra-tumoral innate and adaptive immunologic cellular infiltrates within pediatric CNS tumor samples through immunohistochemistry. RESULTS: We found that atypical teratoid/rhabdoid tumors (AT/RT) and ependymomas exhibit decreased, whereas medulloblastomas and glioblastomas express higher MHC I mRNA levels than most other pediatric cancer types. We discovered MHC I can be up-regulated in vitro in medulloblastomas and AT/RT's, indicating a reversible epigenetic mechanism for down-regulation. There were decreased NK and regulatory T-cell, while increased CTL infiltrates in recurred versus diagnostic pediatric glioblastoma tumor samples. CONCLUSIONS: The immunologic profiling of pediatric brain cancers will guide rational selection of immune-based therapies, serving as an important biomarker for patient inclusion in immunotherapeutic trials.

Sorafenib/regorafenib and lapatinib interact to kill CNS tumor cells.

The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3-GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, BCL-XL, or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy.

PDE5 inhibitors enhance the lethality of standard of care chemotherapy in pediatric CNS tumor cells

We determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill medulloblastoma cells. In medulloblastoma cells PDE5 inhibitors interacted in a greater than additive fashion with vincristine/etoposide/cisplatin to cause cell death. Knockdown of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of dominant negative caspase 9 did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with the PDE5 inhibitor sildenafil. Overexpression of BCL-XL and c-FLIP-s suppressed individual and combination drug toxicities. Knockdown of CD95 or FADD suppressed drug combination toxicity. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy which was maximal at ~12 h post-treatment, and in a cell type-dependent manner knockdown of Beclin1 or ATG5 either suppressed or enhanced drug combination lethality. PDE5 inhibitors enhanced the induction of chemotherapy-induced DNA damage in a nitric oxide synthase-dependent fashion. In conclusion, our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for medulloblastoma represents a possible novel modality for future treatment of this disease.

Sorafenib and HDAC inhibitors synergize to kill CNS tumor cells

The present studies were designed to determine whether the multi-kinase inhibitor sorafenib (Nexavar) interacted with histone deacetylase inhibitors to kill glioblastoma and medulloblastoma cells. In a dose-dependent fashion sorafenib lethality was enhanced in multiple genetically disparate primary human glioblastoma isolates by the HDAC inhibitor sodium valproate (Depakote). Drug exposure reduced phosphorylation of p70 S6K and of mTOR. Similar data to that with valproate were also obtained using the HDAC inhibitor vorinostat (Zolinza). Sorafenib and valproate also interacted to kill medulloblastoma and PNET cell lines. Treatment with sorafenib and HDAC inhibitors radio-sensitized both GBM and medulloblastoma cell lines. Knock down of death receptor (CD95) expression protected GBM cells from the drug combination, as did overexpression of c-FLIP-s, BCL-XL and dominant negative caspase 9. Knock down of PDGFRα recapitulated the effect of sorafenib in combination with HDAC inhibitors. Collectively, our data demonstrate that the combination of sorafenib and HDAC inhibitors kills through activation of the extrinsic pathway, and could represent a useful approach to treat CNS-derived tumors.

Abstract 2269: Lapatinib and obatoclax kill cns tumor cells through blockade of erbb1 / 3 / 4 and through inhibition of bcl-xl and mcl-1

Abstract Previous studies in breast cancer cells have shown that Lapatinib (Lap) and Obatoclax (GX) interact in a greater than additive manner to cause cell death. The present studies sought to expand our analyses to CNS (Glioblastoma & Medulloblastoma) tumor cells and to further advance our understanding of the mechanisms of drug action. Combinational treatment with Lap / GX resulted in enhanced tumor cell death in multiple CNS tumor types. Moreover, it was discovered that cells lacking PTEN function were resistant to drug combination lethality and re-expression of PTEN in PTEN null cells restored drug sensitivity. Furthermore, PTEN expressing cancer cells, sensitive to Lap / GX treatment, demonstrated drug resistance upon knockdown of PTEN. Additionally, we established that inhibition of ERBB1 / 3 / 4 receptors was most significant for enhancing GX lethality, and that knockdown of BCL-XL and particularly MCL-1, interacted to facilitate Lap lethality. Pre-treatment of tumor cells in vitro and in vivo with GX enhanced the lethality of Lap to a greater extent than concomitant treatment and suppressed tumor growth. Treatment of animals carrying orthotopic CNS tumor isolates and those implanted with anoikis resistant breast tumors in the brain with Lap / GX prolonged survival. Collectively our data argues that combinational Lap / GX therapy could be of use in the treatment of tumors located in the CNS. Further studies must be carried out in order to elucidate whether autophagy or apoptosis plays the most predominant role in cell death in response to Lap / GX treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2269. doi:1538-7445.AM2012-2269

Bioactive acylphloroglucinols from Hypericum densiflorum

Three acylphloroglucinol derivatives have been isolated from the hexane and acetone extracts of the aerial parts of Hypericum densiflorum Pursch. The compounds were characterized by NMR spectroscopy and mass spectrometry and identified as 4-geranyloxy-2,6-dihydroxybenzophenone (1), 4-geranyloxy-1-(2-methylpropanoyl)- phloroglucinol (2) and 4-geranyloxy-1-(2-methylbutanoyl)-phloroglucinol (3). Compounds 1-3 were evaluated for in vitro cell proliferation inhibitory activity against human breast (MCF-7), lung (NCI H460), CNS (SF-268), stomach (AGS) and colon (HCT-116) tumor cell lines; antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA); inhibition of cyclooxygenase (COX-1 and -2) enzymes; and antioxidant activity in the lipid peroxidation (LPO) assay. All three compounds showed moderate to strong antitumor, antibacterial, antioxidant and inhibition of COX-2 activities. Also, this is the first reported occurrence of compound 3 in the Hypericum genus.

New Benzophenone O-Glucoside from Hypericum Ellipticum

Examination of the acetone extract of the aerial parts of Hypericum ellipticum afforded a new acetylated benzophenone glucoside (3'-O-beta-D-3",4",6"-triacetylglucopyranosyl-2,4,5',6-tetrahydroxybenzophenone) together with catechin and epicatechin. The structure of the benzophenone glucoside was determined by 2D NMR spectroscopic data. The compound inhibited the proliferation of CNS tumor cell line (SF-268) and lipid peroxidation in in vitro assays.

Identification, characterization and potent antitumor activity of ECO-4601, a novel peripheral benzodiazepine receptor ligand

ECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER technology, Thallion's proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. In the work presented here, ECO-4601 was further evaluated against brain tumor cell lines. Preliminary mechanistic studies as well as in vivo antitumor evaluation were performed. Since ECO-4601 has a benzodiazepinone moiety, we first investigated if it binds the central and/or peripheral benzodiazepine receptors. ECO-4601 was tested in radioligand binding assays on benzodiazepine receptors obtained from rat hearts. The ability of ECO-4601 to inhibit the growth of CNS cancers was evaluated on a panel of mouse, rat and human glioma cell lines using a standard MTT assay. Antitumor efficacy studies were performed on gliomas (rat and human), human breast and human prostate mouse tumor xenografts. Antitumor activity and pharmacokinetic analysis of ECO-4601 was evaluated following intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) bolus administrations. ECO-4601 was shown to bind the peripheral but not the central benzodiazepine receptor and inhibited the growth of CNS tumor cell lines. Bolus s.c. and i.p. administration gave rise to low but sustained drug exposure, and resulted in moderate to significant antitumor activity at doses that were well tolerated. In a rat glioma (C6) xenograft model, ECO-4601 produced up to 70% tumor growth inhibition (TGI) while in a human glioma (U-87MG) xenograft, TGI was 34%. Antitumor activity was highly significant in both human hormone-independent breast (MDA-MB-231) and prostate (PC-3) xenografts, resulting in TGI of 72 and 100%, respectively. On the other hand, i.v. dosing was followed by rapid elimination of the drug and was ineffective. Antitumor efficacy of ECO-4601 appears to be associated with the exposure parameter AUC and/or sustained drug levels rather than C (max). These in vivo data constitute a rationale for clinical studies testing prolonged continuous administration of ECO-4601.

Lipid peroxidation, cyclooxygenase enzyme and tumor cell proliferation inhibitory compounds in Cornus kousa fruits

The genus Cornus is well known for its medicinal properties. Bioassay-guided isolation and characterization of C. kousa fruits afforded kaempferol 3-O-rhamnoside (1), myricetin 3-O-rhamnoside (2), kaempferol 3-O-glucoside (3), cornin (4) and stenophyllin (5) in addition to ursolic acid and beta-sitosterol. These compounds are isolated for the first time from C. kousa. Compounds 1-5 inhibited Fe(2+) catalyzed lipid peroxidation by 63%, 57%, 61%, 53%, and 51%, at 23, 22, 23, 129, and 108 microM, respectively. Similarly, they inhibited COX-1 and -2 enzymes activities by 24% and 47%, 40% and 37%, 20% and 37%, 52% and 63%, and 48% and 55% respectively, at 231, 215, 226, 258, and 217 microM, respectively. At 129 microM, compound 4 displayed growth inhibition of HCT-116 (colon), MCF-7 (breast), NCI-H460 (lung), SF-268 (central nervous system CNS), and AGS (stomach) human tumor cell lines by 31%, 29%, 40%, 9%, and 28%, respectively. Similarly, compound 5 inhibited the growth of colon, breast, lung, CNS, and stomach tumor cell lines by 0%, 27%, 35%, 16%, and 27%, respectively, at 108 microM.

Acylphloroglucinol Derivatives from Hypericum prolificum

Three new acylphloroglucinol derivatives have been isolated from the hexane extract of the aerial parts of Hypericum prolificum L.: prolificin A (1), prolifenone A (2), and prolifenone B (3). The structures were elucidated on the basis of extensive 2D NMR and MS data. All three compounds were evaluated for in vitro cell proliferation inhibitory activity against human breast (MCF-7), lung (NCI-H460), CNS (SF-268), stomach (AGS), and colon (HCT-116) tumor cell lines. Prolificin A showed growth inhibition of all cell lines with IC50 values ranging from 23 to 36 microM. Prolifenones A and B were inactive at the concentrations tested.

Ectopic expression of Par-4 leads to induction of apoptosis in CNS tumor cell lines

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein originally identified as a gene product upregulated in prostate tumor cells undergoing apoptosis. Down-regulation of Par-4 has been linked to several cancers. Since Par-4 also plays a crucial role in neuronal apoptosis, we investigated the expression of Par-4 in tumor cell lines derived from representative tumor types of the CNS, including primitive neuroectodermal tumor (PNET), medulloblastoma, neuroblastoma and glioma of human, rat and murine origin. We show that Par-4 is frequently down-regulated, either transcriptionally or post-transcriptionally in the CNS tumor cell lines. Moreover, we demonstrate that ectopic expression of Par-4 is sufficient to directly induce apoptosis in these CNS tumor cells, in contrast to other cancer cells where replenishment of Par-4 levels only sensitizes the cells to apoptotic stimuli. Induction of apoptosis by Par-4 in the neural tumor cell lines is independent of endogenous Bcl-2 levels and PKCzeta activity, although it has been proposed that Par-4 can exert its pro-apoptotic function by down-modulation of Bcl2 expression and inhibition of PKCzeta. Co-expression of Par-4 and a dominant-negative mutant of FADD resulted in a slight reduction of apoptosis in some tumor cell lines, indicating that Par-4 may partially induce apoptosis via the Fas death pathway. Furthermore, these data suggested that the pro-apoptotic function of Par-4 involves (an)other yet unidentified apoptotic pathway(s) in the CNS tumor cell lines. Since Par-4 by itself is not sufficient to induce apoptosis in non-tumor cells, reintroduction of Par-4 into primary CNS tumors or reactivation of the pathways of Par-4-mediated apoptosis represent promising targets in anti-tumor therapy.

The human ATP binding cassette gene ABCA13, located on chromosome 7p12.3, encodes a 5058 amino acid protein with an extracellular domain encoded in part by a 4.8-kb conserved exon

The ABCA subfamily of ATP-binding cassette (ABC) transporters includes eleven members to date. In this study, we describe a new, unusually large gene on chromosome 7p12.3, ABCA13. This gene spans over 450 kb and is split into 62 exons. The predicted ABCA13 protein consists of 5,058 ami- no acid residues making it the largest ABC protein described to date. Like the other ABCA subfamily members, ABCA13 contains a hydrophobic, predicted transmembrane segment at the N-terminus, followed by a large hydrophilic region. In the case of ABCA13, the hydrophilic region is unexpectedly large, more than 3,500 amino acids, encoded by 30 exons, two of which are 4.8 and 1.7 kb in length. These two large exons are adjacent to each other and are conserved in the mouse Abca13 gene. Tissue profiling of the major transcript reveals the highest expression in human trachea, testis, and bone marrow. The expression of the gene was also determined in 60 tumor cell lines and the highest expression was detected in the SR leukemia, SNB-19 CNS tumor and DU-145 prostate tumor cell lines. ABCA13 has high similarity with other ABCA subfamily genes which are associated with human inherited diseases: ABCA1 with the cholesterol transport disorders Tangier disease and familial hypoalphalipoproteinemia, and ABCA4 with several retinal degeneration disorders. The ABCA13 gene maps to chromosome 7p12.3, a region that contains an inherited disorder affecting the pancreas (Shwachman-Diamond syndrome) as well as a locus involved in T-cell tumor invasion and metastasis (INM7), and therefore is a positional candidate for these pathologies.

Cytotoxic effects of pyridino[2,3-f]indole-4,9-diones on human tumor cell lines.

The cytotoxicities of pyridino[2,3-f]indole-4,9-dione derivatives were examined against human lung tumor cell lines (A 549), human ovarian tumor cell lines (SK-OV-3), human melanoma tumor cell lines (SK-MEL-2), human CNS tumor cell lines (XF 498) and human colon tumor cell lines (HCT 15) in vitro using a Sulforhodamine B assay. 3-Ethoxycarbonyl-1-(2-methoxyethyl)-2-methyl-1H-pyridino[2,3-f]ind ole-4,9-dione (5) showed excellent cytotoxicity against XF 498 and HCT 15. The ED50 values of 5 were 0.006 microg/ml against XF 498 and 0.073 microg/ml against HCT 15, while those of doxorubicin were 0.012 and 0.264 microg/ml, respectively. 1-Benzyl-3-ethoxycarbonyl-2-methyl-1H-pyridino[2,3-f]indole-4,9-di one (7) (ED50 value 0.065 microg/ml) was also significantly more cytotoxic against HCT 15 compared with doxorubicin.

Cerebrospinal fluid of newborn infants contains a deglycosylated form of the intermediate filament nestin.

Nestin is an intermediate filament protein found in CNS progenitor cells. Nestin reappears in CNS tumor cells and reactive astrocytes after CNS injury. In this study we investigated whether nestin could be detected in the cerebrospinal fluid (CSF) of newborn infants and whether expression levels change with gestational age (GA) and/or brain injury. Using Western blot analysis, we examined the expression of nestin in the CSF of newborn infants (GA 25-42 wk) with asphyxia (n = 14), periventricular leukomalacia and peri(intra)ventricular hemorrhage (n = 7), and in a control group (n = 11). Protein extract from the periventricular brain tissue of a 1-wk-old infant was also analyzed. Nestin was detected in all the CSF samples and in the protein extract from the periventricular brain tissue. Although the CSF levels of nestin expression did not change with increasing GA, the asphyxia group had significantly lower levels of nestin in the CSF. An unexpected finding was that brain-derived nestin had an apparent molecular mass of approximately 240 kD, whereas all analyzed CSF samples contained two nestin-immunoreactive proteins at 200 and 220 kD. Experimental deglycosylation of the 240-kD form reduced the molecular mass to 220 kD, indicating that nestin undergoes a specific deglycosylation upon release into the CSF.

Synthesis and Antitumor Activity of Polyanion-Pt-Complexes Containing Alicyclic Amines as Ligands

Four polyanion-Pt(II)-complexes, PEE-cis-DACH-Pt, PEE- cis- (ACP)2-Pt, PCR- cis -DACH-Pt, and PCR- cis-(ACP)2-Pt, were synthesized by the reactions of polyanions such as poly[(2-ethoxytetrahydropyran-5,6-diyl)(1,2- dicarboxyethylene)](PEE) and poly[(3,4-dicarboxytetrahydropyran-2,6-diyl)- (1,2-dicarboxypropylene)](PCP) with cis-[Pt(amine)2)2](NO3) 2, where the amine was 1,2-diaminocyclohexane (DACH) or aminocyclopropane (ACP). The biologi cal activities of the complexes were measured in vitro against A-459 nonsmall cell lung, Sk-OV-3 ovarian, SK-Mel-2 melanoma, XF-498 CNS, and HCT-15 colon tumor cell lines. The increased life span was measured against L1210 leukemia and found to be 223% for PCP- cis-(ACP)2-Pt(II) at the dose of 25 mg/Kg.

Tumor angiogenesis factor (TAF) in cultured cells derived from central nervous system tumors in humans.

The angiogenic activity in the supernatants of cultured cells derived from human CNS tumors was examined by a bioassay method using chick chorioallantoic membrane (CAM). A quantitative assessment was designed to obtain a more accurate evaluation of angiogenesis on CAM. Three out of five glioma cell lines, three out of four primary cultures of meningiomas and one metastatic tumor cell line showed angiogenic activity in the culture supernatants. The angiogenic activity was destroyed by ribonuclease or heating at 80°C for 30 minutes but remained intact with exposure to trypsin, deoxyribonuclease or heating at 56°C for 30 minutes. TAF samples obtained from glioma cells which had been irradiated (2, 500 R) before TAF extraction, also showed angiogenic activity. TAF was present in the supernatants of long-term cultured human CNS tumor cell lines and primary cultures of meningiomas. Thus, the nature of TAF may related to RNA of the tumor cells and the TAF producing ability of these cells is apparently not inhibited by irradiation.