Brain Tumor Cell Lines Research Articles

Transferrin conjugated nontoxic carbon dots for doxorubicin delivery to target pediatric brain tumor cells.

Among various cancers, pediatric brain tumors represent the most common cancer type in children and the second most common cause of cancer related deaths. Anticancer drugs and therapies, such as doxorubicin (Dox), have severe side effects on patients during chemotherapy, especially for children as their bodies are still under development. These side effects are believed to be due to the lack of a delivery system with high efficacy and targeting selectivity, resulting in serious damages of normal cells. To improve the efficacy and selectivity, the transferrin (Trans) receptor mediated endocytosis can be utilized for drug delivery system design, as transferrin receptors are expressed on the blood brain barrier (BBB) and often over expressed in brain tumor cells. Carbon dots (C-Dots) have recently emerged as benign nanoparticles in biomedical applications owing to their good water solubility, tunable surface functionalities and excellent biocompatibility. The unique characteristics of C-Dots make them promising candidates for drug delivery development. In this study, carbon dots-transferrin-doxorubicin covalent conjugate (C-Dots-Trans-Dox) was synthesized, characterized by different spectroscopic techniques and investigated for the potential application as a drug delivery system for anticancer drug doxorubicin to treat pediatric brain tumors. Our in vitro results demonstrate greater uptake of the C-Dots-Trans-Dox conjugate compared to Dox alone presumably owing to the high levels of transferrin receptors on these tumor cells. Experiment showed that C-Dots-Trans-Dox at 10 nM was significantly more cytotoxic than Dox alone, reducing viability by 14-45%, across multiple pediatric brain tumor cell lines.

Antiproliferative activities of several plant extracts from Turkey on rat brain tumor and human cervix carcinoma cell lines

Turkey has a wide range of flora and fauna due to its climatic diversity. Medicinal plants from Turkey have been used since ancient times for their primary health care. In this study, we examined antiproliferative activities of the extracts from Crataegus monogyna, Vitis vinifera, Glycrrhiza glabra, Alnus glutinosa L. gaertn, and Alcea rosea against rat brain tumor (C6) and human cervical cancer (HeLa) cell lines. The results were compared with the standard anticancer drugs 5-Flurouracil (5-FU) and Cisplatin. C. monogyna, V. vinifera and A. rosea exhibited better antiproliferative activity than 5-FU and cisplatin at 100-75 µg/mL concentrations, against C6 cell lines. On the other hand, C. monogyna and V. vinifera extracts showed considerable antiproliferative activity against HeLa cells compared with 5-FU and cisplatin at 100-75 µg/mL. It can be suggested that, C. monogyna, A. glutinosa L. gaertn, V. vinifera and A. rosea extracts could be developed as an anticancer drug.

ATPS-36ETHNOMEDICAL SURVEY AND SCIENTIFIC VALIDATION OF ANTICANCER ACTIVITIES OF SOME NIGERIAN HERBAL PLANTS

Cancer is the second leading cause of death in many countries and there is no cure for different forms of the disease. In Africa, traditional medicine practitioners (TMP) claim to have effective treatments for what they diagnose as “cancers”. This study used tissue culture and cancer cell viability techniques to validate the rationale for the folkloric use of herbal medications by TMP. We worked with a well-established TMP (Engineer Ali Musa Audu) to collect medicinal plants used in the treatment of cancers (daji) in the North-east geopolitical zone of Nigeria. We identified 42 plants that are used in the folkloric treatment of cancers and the botanical names were authenticated by a Taxonomist. The plant parts used in folkloric medicine were defatted with petroleum ether and further extracted with ethanol. The dried ethanol extracts were screened for anticancer cell viability activities against well-characterized brain tumour (A171, U87 and U1242) and breast cancer (231) cell lines. We screened 42 extracts and established robust anticancer cell viability activities of 8 herbal plants with concentration that inhibited 50% cell viability of less than 62.0 mg/ml. Three of the plant extracts were also tested on normal human astrocytes and showed toxicity only at higher concentration of 250.0 mg/ml. This is the first report of anticancer activities of medicinal plants from the North-east geopolitical zone of Nigeria.

Abstract 2528: Vardenafil is superior to idarubicin in treatment of glioma

Abstract Idarubicin (IDA) is a very potent second generation anthracycline that has shown high efficacy against glioma in vitro. However, this agent has failed to exert satisfactory therapeutic response in patients with brain tumors. Phosphodiesterase (PDE-5), an enzyme which hydrolyzes intracellular cyclic guanosine monophosphate (cGMP), is highly expressed in many brain tumor cell lines. We proposed that increasing cGMP in the brain by the potent PDE-5 inhibitor vardenafil (VAR) will augment the antitumor efficacy of IDA against glioma. We also proposed that VAR will induce apoptosis of glioma cells. Here we show for the first time that VAR (10 mg/kg) enhanced the antitumor activity of IDA (0.1mg/kg). VAR alone inhibited tumor growth and prolonged survival of glioma bearing rats. Histopathological examination revealed that tumors from glioma bearing rats treated with VAR alone or VAR+ IDA presented a significant reduction in mitotic index, indicating less proliferative tumors, as compared to those which received PBS (control) or IDA. Real time PCR results demonstrated that VAR inhibited mRNA expression of PDE-5 in the brains of animals treated with VAR, compared to control-treated group, implicating an important role for the inhibition of PDE-5 in the antitumor activity of VAR. Interestingly, the antitumor activity of VAR was similar to that of IDA. Moreover, animals receiving either VAR alone or VAR+IDA demonstrated statistically significant improvement in overall survival when compared with control (PBS) or IDA groups. In order to explain such an unexpected finding, we examined the antiproliferative, pro-apoptotic and anti-invasive activities VAR in vitro. MTT assay results showed that VAR significantly inhibited the growth of rat C6 glioma cells. Annexin V propidium iodide assay results revealed that the inhibition of C6 cell growth is mediated at least in part by inducing C6 apoptosis. Wound healing and soft agar colony formation assays showed that VAR inhibited the migration and anchorage-independent growth of C6 cells, respectively. This suggests that the enhanced antitumor effect of VAR could be due to a combined antiproliferative, pro-apoptotic and anti-invasive activities. Together, our data support anti-glioma properties of VAR, and provide a proof-of-concept basis for further development of VAR as potential adjuvant for treatment of brain tumors. Citation Format: Abdelkader E. Ashour, Abdulrahman Z. Alzahrani, Hala E. Abdel-Hamied, Khairy M. Zoheir, Sheikh F. Ahmed, Salem S. Al-Rejaie, Adel R. Abd-Allah. Vardenafil is superior to idarubicin in treatment of glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2528. doi:10.1158/1538-7445.AM2015-2528

Cannabinoid receptor CB1 regulates STAT3 activity and its expression dictates the responsiveness to SR141716 treatment in human glioma patients' cells.

Herein we show that a majority of human brain tumor samples and cell lines over-expressed cannabinoid receptor CB1 as compared to normal human astrocytes (NHA), while uniformly expressed low levels of CB2. This finding prompted us to investigate the therapeutic exploitation of CB1 inactivation by SR141716 treatment, with regard to its direct and indirect cell-mediated effects against gliomas. Functional studies, using U251MG glioma cells and primary tumor cell lines derived from glioma patients expressing different levels of CB1, highlighted SR141716 efficacy in inducing apoptosis via G1 phase stasis and block of TGF-β1 secretion through a mechanism that involves STAT3 inhibition. According to the multivariate role of STAT3 in the immune escape too, interestingly SR141716 lead also to the functional and selective expression of MICA/B on the surface of responsive malignant glioma cells, but not on NHA. This makes SR141716 treated-glioma cells potent targets for allogeneic NK cell-mediated recognition through a NKG2D restricted mechanism, thus priming them for NK cell antitumor reactivity. These results indicate that CB1 and STAT3 participate in a new oncogenic network in the complex biology of glioma and their expression levels in patients dictate the efficacy of the CB1 antagonist SR141716 in multimodal glioma destruction.

Overexpression of the ∆Np73 isoform is associated with centrosome amplification in brain tumor cell lines.

The p73 protein is a member of the p53 family, and this protein is known to be essential for the maintenance of genomic stability, DNA repair, and apoptosis regulation. Transcription from two promoters leads to two main N-terminal isoforms: the TAp73 isoform is reported to have tumor suppressor function, whereas the ΔNp73 isoform likely has oncogenic potential. The present study is focused on the investigation of a possible role of both these p73 N-terminal isoforms in the process of centrosome amplification. HGG-02 and GM7 glioblastoma cell lines and the Daoy medulloblastoma cell line were used in this study. The cells were analyzed using indirect immunofluorescence to determine TAp73 and ΔNp73 expression patterns and possible co-localization with the BubR1 protein, as well as the number of centrosomes. A transiently transfected GM7 cell line was used to verify the results concerning the N-terminal isoforms in relation to centrosome amplification. We found that increased immunoreactivity for the ΔNp73 isoform is associated with the occurrence of an abnormal number of centrosomes in particular cells. Using the transiently transfected GM7 cell line, we confirmed that centrosome amplification is present in cells with overexpression of the ΔNp73 isoform. In contrast, the immunoreactivity for the TAp73 isoform was weak or medium in most of the cells with an aberrant number of centrosomes. To determine the putative counterpart of the p73 N-terminal isoforms among spindle assembly checkpoint (SAC) proteins, we also evaluated possible interactions between the N-terminal isoforms and BubR1 protein, but no co-localization of these proteins was observed.

Magnetic resonance spectroscopy for detection of choline kinase inhibition in the treatment of brain tumors.

Abnormal choline metabolism is a hallmark of cancer and is associated with oncogenesis and tumor progression. Increased choline is consistently observed in both preclinical tumor models and in human brain tumors by proton magnetic resonance spectroscopy (MRS). Thus, inhibition of choline metabolism using specific choline kinase inhibitors such as MN58b may be a promising new strategy for treatment of brain tumors. We demonstrate the efficacy of MN58b in suppressing phosphocholine production in three brain tumor cell lines. In vivo MRS studies of rats with intracranial F98-derived brain tumors showed a significant decrease in tumor total choline concentration after treatment with MN58b. High-resolution MRS of tissue extracts confirmed that this decrease was due to a significant reduction in phosphocholine. Concomitantly, a significant increase in poly-unsaturated lipid resonances was also observed in treated tumors, indicating apoptotic cell death. MRI-based volume measurements demonstrated a significant growth arrest in the MN58b-treated tumors in comparison with saline-treated controls. Histologically, MN58b-treated tumors showed decreased cell density, as well as increased apoptotic cells. These results suggest that inhibition of choline kinase can be used as an adjuvant to chemotherapy in the treatment of brain tumors and that decreases in total choline observed by MRS can be used as an effective pharmacodynamic biomarker of treatment response.

Distinctive responses of brain tumor cells to TLR2 ligands:

Malignant brain tumor mass contains significant numbers of infiltrating glial cells that may intimately interact with tumor cells and influence cancer treatments. Understanding of characteristic discrepancies between normal GLIA and tumor cells would, therefore, be valuable for improving anticancer therapeutics. Here, we report distinct differences in toll-like receptors (TLR)-2-mediated responses between normal glia and primary brain tumor cell lines. We found that tyrosine phosphorylation of STAT1 by TLR2 ligands and its downstream events did not occur in mouse, rat, or human brain tumor cell lines, but were markedly induced in normal primary microglia and astrocytes. Using TLR2-deficient, interferon (IFN)-γ-deficient, and IFNγ-receptor-1-deficient mice, we revealed that the impaired phosphorylation of STAT1 might be linked with defective TLR2 system in tumor cells, and that a TLR2-dependent pathway, not IFNγ-receptor machinery, might be critical for tyrosine STAT1 phosphorylation by TLR2 ligands. We also found that TLR2 and its heterodimeric partners, TLR1 and 6, on brain tumor cells failed to properly respond to TLR2 ligands, and representative TLR2-dependent cellular events, such as inflammatory responses and cell death, were not detected in brain tumor cells. Similar results were obtained in in vitro and in vivo experiments using orthotopic mouse and rat brain tumor models. Collectively, these results suggest that primary brain tumor cells may exhibit a distinctive dysfunction of TLR2-associated responses, resulting in abnormal signaling and cellular events. Careful targeting of this distinctive property could serve as the basis for effective therapeutic approaches against primary brain tumors.

Pediatric brain tumor cell lines.

Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research.

CB-19 * PID1, A NEW GROWTH-INHIBITORY GENE, SENSITIZES MEDULLOBLASTOMA AND GLIOMA CELL LINES TO CHEMOTHERAPY

BACKGROUND: Phosphotyrosine Interaction Domain containing protein-1 (PID1) was discovered in 2006. We recently showed that PID1 inhibits growth of brain tumor cell lines and its mRNA level is directly correlated with survival in glioma and medulloblastoma patients (Erdreich-Epstein et al, Clin Cancer Res). The growth-inhibitory effect of PID1 was due to decreased proliferation and increased cell death. METHODS/RESULTS: We hypothesized that PID1 sensitizes brain tumors to therapy, thus accounting for the longer survival in patients with higher PID1 mRNA. Indeed, while cisplatin (10 µg/ml) or transient PID1 overexpression each increased apoptosis of glioma and medulloblastoma cell lines, combining cisplatin with PID1 resulted in markedly higher apoptosis. Moreover, knockdown of PID1 by siRNA inhibited the cisplatin-induced mitochondrial membrane depolarization and apoptosis, suggesting that PID1 is partially required for cisplatin-induced apoptosis. Taken together, this suggests that PID1 sensitizes both glioma and medulloblastoma cell lines to cisplatin. Intriguingly, PID1 mRNA increased in response to chemotherapy in a time- and dose-dependent manner in brain tumor cell lines. The chemotherapy-induced increase in PID1 mRNA was blocked by inhibitors of NFkB, suggesting that regulation of PID1 mRNA increase may be an NF-kB-dependent process. Consistent with this, cisplatin increased activity of an NF-kB promoter reporter. Surprisingly, despite the chemotherapy-induced increase in PID1 mRNA, PID1 protein decreased in response to cisplatin, suggesting post-translational modification(s) of PID1 induced by cisplatin. Ongoing work is focusing on the mechanism and role of PID1 in response to chemotherapy, and examining the effect of PID1 on the response of tumors to chemotherapy in vivo. CONCLUSIONS: Our data show that PID1 sensitizes glioma and medulloblastoma cell lines to chemotherapy, possibly explaining the correlation between higher PID1 mRNA and longer patient survival. This suggests also that PID1 mimetics may act as chemotherapy sensitizers in brain tumor treatment.

Abstract 3975: PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy

Abstract BACKGROUND: Phosphotyrosine Interaction Domain containing protein-1 (PID1) is a gene discovered in 2006 in the context of obesity, which we recently showed inhibits growth of brain tumor cell lines and its mRNA level is directly correlated with survival of patients with gliomas and medulloblastomas (Erdreich-Epstein et al, Clin Cancer Res, In Press). PID1 mRNA was lower in more aggressive medulloblastomas and gliomas, and patients whose tumor had higher PID1 mRNA had longer survival. Moreover, overexpression of PID1 markedly decreased 2D colony formation in cell lines of medulloblastomas, GBM and atypical teratoid rhabdoid tumors (ATRT). The growth-inhibitory effect was due to both decreased proliferation and increased cell death. METHODS/RESULTS: COSMIC showed no mutations of PID1 in brain tumors, suggesting that PID1 was not a formal tumor suppressor gene. Therefore, we hypothesized that PID1 may sensitize brain tumor cells to therapy, and thus account for the longer survival in patients with higher PID1 mRNA. Indeed, while both cisplatin (10µg/ml) or transient PID1 overexpression increased apoptosis of glioma and medulloblastoma cell lines (indicated by increased AnnexinV and mitochondrial depolarization), combining cisplatin with PID1 caused a markedly higher apoptosis. Moreover, knockdown of PID1 by siRNA inhibited the cisplatin-induced mitochondrial membrane depolarization and apoptosis (AnnexinV), suggesting that PID1 may be required for cisplatin-induced apoptosis. Taken together, this suggests a sensitizing effect of PID1 to chemotherapy in both glioma and medulloblastoma cell lines. Intriguingly, PID1 mRNA and protein both increased in response to etoposide (5µg/ml), vincristine (50ng/ml) or cisplatin (5µg/ml) in a time- and dose-dependent manner in the brain tumor cell lines. This chemotherapy-induced increase in PID1 mRNA was blocked by inhibitors of NFKB, suggesting that regulation of PID1 may be an NFKB-dependent mechanism. Ongoing work seeks to decipher the mechanism of these effects and test them in vivo in mouse models. CONCLUSIONS: Our data suggest that PID1 acts to sensitize glioma and medulloblastoma cells to chemotherapy, possibly explaining the correlation between higher PID1 mRNA and survival in patients. Citation Format: Jingying Xu, Xiuhai Ren, Anthony Tran, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3975. doi:10.1158/1538-7445.AM2014-3975

Altered expression of apoptotic genes in response to OCT4B1 suppression in human tumor cell lines.

OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1.

PID1 IS A NOVEL SENSITIZER OF BRAIN TUMOR CELLS TO CHEMOTHERAPY

BACKGROUND: PID1 is a phosphotyrosine binding domain-containing protein of unknown function in cancer. We recently provided the first report of PID1 in brain tumors (and in cancer). PID1 inhibits growth and proliferation and induces cell death, apoptosis and mitochondrial depolarization in glioblastoma, medulloblastoma and ATRT cell lines. PID1 siRNA had the opposite effect on mitochondrial depolarization. PID1 mRNA level was directly correlated with survival of patients with medulloblastoma or glioma: PID1 mRNA was lowest in tumor subgroups with the poorest prognosis and highest in the more favorable prognosis groups within each diagnosis. The mechanism by which PID1 affects these tumors is currently unknown, and is the focus of studies in our laboratory. METHODS: Experiments were conducted in tissue culture in primary and established cell lines using established methods to assess mRNA, proliferation and signal transduction. RESULTS: Since lower PID1 mRNA was associated with poorer outcome in medulloblastoma and gliomas and higher PID1 was associated with improved outcome, we hypothesized that PID1 level may affect responsiveness of these brain tumors to therapy: higher responsiveness in tumors with high PID1 and resistance when it is low. Indeed, while both cisplatin (10 µg/ml) or transient PID1 overexpression increased apoptosis of glioma and medulloblastoma cell lines (indicated by increased AnnexinV, caspase-3 cleavage and mitochondrial depolarization), combining cisplatin with PID1 caused a markedly higher effect than each alone. Moreover, knockdown of PID1 by siRNA inhibited the cisplatin-induced mitochondrial membrane depolarization and apoptosis (AnnexinV and caspase-3 cleavage), suggesting that PID1 may be required for cisplatin-induced apoptosis. This supports our hypothesis that PID1 may sensitize brain tumor cells to chemotherapy. Intriguingly, PID1 mRNA increased in response to cisplatin (5 µg/ml) as well as to etoposide (5 µg/ml) and vincristine (50ng/ml), in a time- and dose-dependent manner in the brain tumor cell lines. Further, etoposide and cisplatin also increased PID1 luciferase promoter activity. Interestingly, the increase in chemotherapy-induced PID1 promoter reporter activity as well as the chemotherapy-induced increase in PID1 mRNA were both blocked by inhibitors of NFκB, consistent with presence of an NBκB recognition site in the promoter of PID1, and indicating that regulation of PID1 may be an NFκB-dependent mechanism. CONCLUSIONS: Our data suggest that PID1 mediates glioma and medulloblastoma cell response to chemotherapy in an NFκB-dependent manner. Moreover, these data also suggest that PID1 sensitizes medulloblastoma and glioma to chemotherapy, providing the initial mechanistic step to understand the correlation between higher PID1 mRNA and survival in patients. SECONDARY CATEGORY: Pediatrics.

Kinetics of porphyrin fluorescence accumulation in pediatric brain tumor cells incubated in 5-aminolevulinic acid

Fluorescence-guided surgery with 5-aminolevulinic acid (5-ALA) enables more complete resections of tumors in adults. 5-ALA elicits accumulation of fluorescent porphyrins in various cancerous tissues, which can be visualized using a modified neurosurgical microscope with blue light. Although this technique is well established in adults, it has not been investigated systematically in pediatric brain tumors. Specifically, it is unknown how quickly, how long, and to what extent various pediatric tumors accumulate fluorescence. The purpose of this study was to determine utility and time course of 5-ALA-induced fluorescence in typical pediatric brain tumors in vitro. Cell cultures of medulloblastoma [DAOY and UW228], cPNET [PFSK] atypical teratoid rhabdoid tumor [BT16] and ependymoma [RES196] were incubated with 5-ALA for either 60 minutes or continuously. Porphyrin fluorescence intensities were determined using a fluorescence-activated cell sorter (FACS) after 1, 3, 6, 9, 12 and 24 hours. C6 and U87 cells served as controls. All pediatric brain tumor cell lines displayed fluorescence compared to their respective controls without 5-ALA (p < 0.05). Sixty minutes of incubation resulted in peaks between 3 and 6 hours, whereas continuous incubation resulted in peaks at 12 hours or beyond. 60 minute incubation peak levels were between 52 and 91 % of maxima achieved with continuous incubation. Accumulation and clearance varied between cell types. We demonstrate that 5-ALA exposure of cell lines derived from typical pediatric central nervous system (CNS) tumors induces accumulation of fluorescent porphyrins. Differences in uptake and clearance indicate that different application modes may be necessary for fluorescence-guided resection, depending on tumor type.

Reduced phosphorylation of Stat3 at Ser-727 mediated by casein kinase 2 — Protein phosphatase 2A enhances Stat3 Tyr-705 induced tumorigenic potential of glioma cells

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival and proliferation and has been found to be persistently activated in most human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3 Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition was observed. Overexpression of CK2 (α, α′ or β subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasion which are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the Stat3 WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2–PP2A pathway.

Vaccination with dendritic cells loaded with allogeneic brain tumor cells for recurrent malignant brain tumors induces a CD4+IL17+ response

BackgroundWe tested the hypothesis that a novel vaccine developed from autologous dendritic cells (DC) loaded with cells from a unique allogeneic brain tumor cell line (GBM6-AD) would be well-tolerated and would generate an immune response.MethodPatients with recurrent primary brain tumors underwent vaccination with GBM6-AD/DC vaccine. Subjects were treated at escalating DC cell doses: 5 × 106 (one patient), 10 × 106 (one patient) and 15 × 106 (6 patients). Subcutaneous injections were planned for days 0, 14, 28, 42, 56, and monthly thereafter. The primary endpoint was the safety of the GBM6-AD/DC vaccination. The secondary endpoints were immune response, measured by flow cytometry, and the clinical outcome of tumor response defined by time to progression and overall survival.ResultsEight patients were treated. The first three patients were treated in the dose escalation phase of the trial; the remaining five patients received the maximum dose of 15 × 106 DC. No dose limiting toxicity was observed. The best response per modified McDonald criteria was partial response in one patient. Flow cytometric immune profiling revealed significant differences in CD4+IL17+ lymphocytes and myeloid derived suppressor cell populations between patients characterized as having stable vs. non-stable disease.ConclusionThis first-in-human study shows that the GBM6-AD/DC vaccine was well tolerated and was associated with an immune response in a subset of patients. No MTD was achieved in this trial. This small-scale pilot provides information for larger scale investigations into the use of this allogeneic vaccine source.

Brain tumor cell line authentication, an efficient alternative to capillary electrophoresis by using a microfluidics-based system

The current method for cell line authentication is genotyping based on short tandem repeat (STR)-PCR involving coamplification of a panel of STR loci by multiplex PCR and downstream fragment length analysis (FLA), usually performed by capillary electrophoresis. FLA by capillary electrophoresis is time-consuming and can be expensive, as the facilities are generally not accessible for many research laboratories. In the present study, a microfluidic electrophoresis system, the Agilent 2100 Bioanalyzer, was used to analyze the STR-PCR fragments from 10 human genomic loci of a number of human cell lines, including 6 gliomas, 1 astrocyte, 1 primary lung cancer, 1 lung brain metastatic cancer, and 1 rhabdomyosarcoma; and this was compared with the standard method, that is, capillary electrophoresis, using the Applied Biosystems 3130xl Genetic Analyzer. The microfluidic electrophoresis method produced highly reproducible results with good sensitivity in sizing of multiple PCR fragments, and each cell line demonstrated a unique DNA profile. Furthermore, DNA fingerprinting of samples from 5 different passage numbers of the same cell line showed excellent reproducibility when FLA was performed with the Bioanalyzer, indicating that no cross-contamination had occurred during the culture period. This novel application provides a straightforward and cost-effective alternative to STR-based cell line authentication. In addition, this application would be of great value for cell bank repositories to maintain and distribute precious cell lines.

Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage

The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O(6)-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl2-DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O(6)-benzylguanine; nevertheless, the 24-36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF.

Biological assays on the effects of Acra3 peptide from Turkish scorpion Androctonus crassicauda venom on a mouse brain tumor cell line (BC3H1) and production of specific monoclonal antibodies

Constitutes of the venom scorpion are a rich source of low molecular mass peptides which are toxic to various organisms, including man. Androctonus crassicauda is one of the scorpions from the Southeastern Anatolia of Turkey with public health importance. This work is focused on the investigation of biological effects of Acra3 peptide from Androctonus crassicauda. For this purpose, Acra3 isolated from crude venoms was tested for its cytotoxicity on BC3H1 mouse brain tumor cells using tetrazolium salt cleavage and lactate dehydrogenase activity assays. To determine whether the cytotoxic effects of Acra3 was related to the induction of apoptosis, the morphology of the cells and the nuclear fragmentation was examined by using Acridin Orange staining and DNA fragmentation assay, respectively. Caspase 3 and caspase 9 activities were measured spectrophotometrically and flow cytometric assay was performed using Annexin-V FITC and Propidium Iodide staining. Furthermore toxic peptide Acra3 was used as an antigen for immunological studies.Results showed that Acra3 exerted very strong cytotoxic effect on BC3H1 cells with an IC50 value of 5 μg/ml. Exposure of the cells to 0.1 and 0.5 μg/ml was resulted in very strong appearance of the apoptotic morphology in a dose dependent manner. On the other side, not any DNA fragmentation was observed after treatment of the cells. Caspase 3 and 9 activities were slightly decreased with Acra3. Results from flow cytometry and lactate dehydrogenase activity assays indicate that Acra3 exerts its effects by inducing a stronger necrosis than apoptosis in BC3H1 cells. To evaluate its immunogenicity, monoclonal antibody (MAb) specific for Acra3 antigen (5B9) was developed by hybridoma technology using spleen and lymph nodes of mice and immunoglobulin type of antibody was found to be IgM.We suggest that Acra3 may exert its effects by inducing both necrotic and apoptotic pathway in some way on mouse brain tumor cells. These findings will be useful for understanding the mechanism of cell death caused by venom in vitro. Anti-Acra3 monoclonal antibody can be further used as a bioactive tools for exploring the structure/function relationship and the pharmacological mechanism of scorpion peptide neurotoxins.

Cancer Prognosis

Cancer Prognosis