Rat Glioma Cells Research Articles

Antiproliferative activity of Juglone derivatives on rat glioma

Malignant gliomas are aggressive and life-threatening tumours that still show a poor prognosis: the current therapeutic approach based on surgical resection and chemotherapy combined with radiotherapy does not provide a satisfactory chance of long-term survival to patients. Natural bioactive compounds represent a precious source of molecules with antiproliferative activity, potentially effective also against glioma cells. Among these, Juglone is a known allelopathic compound extracted from the eastern black walnut (Juglans nigra) whose antimitotic effect has been extensively described in mammalian cells. We investigated the antiproliferative effect of a synthetic derivative of this natural compound, 2-(2,4-dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone (DiNAF), in rat glioma cells. We compared this molecule and its effect with the natural reference compound and with newly synthesised derivatives to build a preliminar structure–activity relationship. Biological assays and NMR-based redox experiments confirmed that DiNAF is a promising lead and supported the hypothesis of a redox mechanism underlying its cytotoxic activity.

Biological evaluation of new [18F]F‐labeled synthetic amino acid derivatives as oncologic radiotracers

The present study evaluated the tumoral uptake of the novel synthetic amino acid positron emission tomography (PET) tracers (S)-2-amino-3-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)propanoic acid (AMC-101), (S)-2-amino-4-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)butanoic acid (AMC-102), and (S)-2-amino-5-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)pentanoic acid (AMC-103), all of which are (S)-2-amino-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)alkyl acids. In vitro cellular uptake was investigated using the rat glioma cell lines 9L and C6. In vitro competitive inhibition tests were performed to identify the involvement of specific amino acid transporters. In vivo dynamic PET images of 9L xenograft tumor-bearing model mice were acquired over 2 h after AMC administration. [(18) F]FDOPA PET studies were performed with and without S-carbidopa pretreatment for comparison. All three AMCs exhibited good in vitro cell uptake through the L and alanine-serine-cysteine transporters and enabled clear tumor visualization on PET, leaving the brain devoid of the tracer. Thirty minutes after injection, the mean tumor standardized uptake values were 1.59 ± 0.05, 1.89 ± 0.27, and 1.74 ± 0.13 for AMC-101, AMC-102, and AMC-103, respectively. Although the tumor uptake values of AMCs were lower than that of [(18) F]FDOPA with S-carbidopa pretreatment, AMCs enabled higher contrast images with lower background activity compared with [(18) F]FDOPA with S-carbidopa pretreatment. Our results indicate the potential uses of these new synthetic amino acids as oncologic radiotracers.

Antitumor activity of combined endostatin and thymidine kinase gene therapy in C6 glioma models.

The combination of Endostatin (ES) and Herpes Simplex Virus thymidine kinase (HSV‐TK) gene therapy is known to have antitumor activity in bladder cancer. The potential effect of ES and TK therapy in glioma has not yet been investigated. In this study, pTK‐internal ribosome entry site (IRES), pIRES‐ES, and pTK‐IRES‐ES plasmids were constructed; pIRES empty vector served as the negative control. The recombinant constructs were transfected into human umbilical vein endothelial cells (HUVECs) ECV304 and C6 rat glioma cell line. Ganciclovir (GCV) was used to induce cell death in transfected C6 cells. We found that ECV304 cells expressing either ES or TK‐ES showed reduced proliferation, decreased migration capacity, and increased apoptosis, as compared to untransfected cells or controls. pTK‐IRES‐ES/GCV or pTK‐IRES/GCV significantly suppressed cell proliferation and induced cell apoptosis in C6 cells, as compared to the control. In addition, the administration of pIRES‐ES, pTK‐IRES/GCV, or pTK‐IRES‐ES/GCV therapy improved animal activity and behavior; was associated with prolonged animal survival, and a lower microvessel density (MVD) value in tumor tissues of C6 glioma rats. In comparison to others, dual gene therapy in form of pTK‐IRES‐ES/GCV had a significant antitumor activity against C6 glioma. These findings indicate combined TK and ES gene therapy was associated with a superior antitumor efficacy as compared to single gene therapy in C6 glioma.

Radiosensitizing effect of irisquinone on glioma through the downregulation of HIF-1α evaluated by 18F-FDG and 18F-FMISO PET/CT.

The aim of this study was to elucidate the radiosensitizing mechanism of irisquinone (IQ) and evaluate the utility of F-fluorodeoxyglucose (F-FDG) and F-fluoromisonidazole (F-FMISO) PET/computed tomography (CT) in assessing the radiosensitizing effect of IQ. In an in-vitro experiment, C6 rat glioma cells were treated with IQ, radiation, or both. The viability and radiosensitivity of C6 cells were detected using the MTT assay and clonogenic survival assay. The expression of hypoxia-inducible factor-1α (HIF-1α) was evaluated by real-time PCR and western blot. In an in-vivo experiment, C6 rat glioma cells were implanted into the right flank of rats and treated with IQ, radiation, both, or no treatment. F-FDG and F-FMISO PET/CT images were obtained before and after treatment. The expression of HIF-1α was detected by immunohistochemistry staining. In the in-vitro experiment, the results of the MTT assay showed that the half-inhibition concentration (IC50) of IQ for normoxic and hypoxic C6 tumor cells was 17.2 and 21.0 nmol/l, respectively. Clonogenic survival assay showed that IQ could improve the radiosensitivity of both normoxic and hypoxic C6 tumor cells. When the concentration of irradiation was 20% IC50 (4.2 nmol/l), the sensitive enhancement ratio of normoxic and hypoxic C6 tumor cells was 1.18 and 1.33, respectively. The mRNA and protein expression levels of HIF-1α decreased significantly when treated with IQ plus radiation compared with the other groups.In the in-vivo experiment, 24 or 48 h after different treatments, the maximum standardized uptake values (SUVmax) of F-FDG or F-FMISO uptake decreased in the radiation group and the IQ plus radiation group, whereas these values increased in the control and IQ groups. The SUVmax of F-FDG or F-FMISO uptake in IQ plus radiation group were lower than those of the radiation group (t=3.28, 2.62, P<0.05). However, there was no significant decrease in tumor volumes in the radiation group and the IQ plus radiation treatment group early after treatment.Immunohistochemistry staining showed that there were significant differences in the expression of HIF-1α in the four groups (F=87.1, P<0.01). The SUVmax of both F-FDG and F-FMISO uptake showed a significant correlation with the expression of HIF-1α. F-FMISO provided a higher correlation coefficient with HIF-1α than F-FDG (r=0.93, 0.82, P<0.01). The present experiments indicated that IQ enhanced the radiosensitivity of C6 rat glioma cells both in vitro and in vivo. The primary mechanism of this radiosensitizing effect involves the downregulation of HIF-1α. F-FDG and F-FMISO PET/CT were sensitive and noninvasive for monitoring the early radiosensitizing effect of IQ. Meanwhile, F-FMISO PET/CT provided more information on the changes in tumor hypoxic status.

Resistance to DNA Damaging Agents Produced Invasive Phenotype of Rat Glioma Cells-Characterization of a New in Vivo Model.

Chemoresistance and invasion properties are severe limitations to efficient glioma therapy. Therefore, development of glioma in vivo models that more accurately resemble the situation observed in patients emerges. Previously, we established RC6 rat glioma cell line resistant to DNA damaging agents including antiglioma approved therapies such as 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ). Herein, we evaluated the invasiveness of RC6 cells in vitro and in a new orthotopic animal model. For comparison, we used C6 cells from which RC6 cells originated. Differences in cell growth properties were assessed by real-time cell analyzer. Cells’ invasive potential in vitro was studied in fluorescently labeled gelatin and by formation of multicellular spheroids in hydrogel. For animal studies, fluorescently labeled cells were inoculated into adult male Wistar rat brains. Consecutive coronal and sagittal brain sections were analyzed 10 and 25 days post-inoculation, while rats’ behavior was recorded during three days in the open field test starting from 25th day post-inoculation. We demonstrated that development of chemoresistance induced invasive phenotype of RC6 cells with significant behavioral impediments implying usefulness of orthotopic RC6 glioma allograft in preclinical studies for the examination of new approaches to counteract both chemoresistance and invasion of glioma cells.

Effects of propofol on invasion and migration of glioma cells in rats and the role of ADAR2-AMPA receptor GluR2 pathway

Objective To evaluate the effects of propofol on the invasion and migration of glioma cells in the rats and the role of adenosine deaminase acting on RNA 2 (ADAR2)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit glutamate 2 (GluR2) pathway. Methods C6 glioma cells were subcultured and randomly divided into 4 groups (n=24 each) using a random number table: control group (group C); propofol group (group P); negative siRNA transfection + propofol group (group NP); ADAR2-siRNA transfection + propofol group (group AP). The cells were cultured in the common culture medium in group C. In NP and AP groups, negative siRNA and ADAR2-siRNA were transfected into the cells, respectively, and 48 h later the other procedures were similar to those previously described in group P. Propofol with the final concentration of 1.2 μg/ml was added, the cells were cultured for 6 h and then were cultured in the common culture medium for another 18 h in group P. The cells were selected to detect the cell viability by MTT colorimetric assay.The invasion of cells was determined by Transwell invasion assay, and the invaded cells were counted.The migration of cells was determined by cell scratch test, and cell migration rates were calculated.The expression of ADAR2 in the nucleus of cells and GluR2 in the cytomembrane was detected by Western blot. Results Compared with group C, the cell viability, the number of invaded cells and cell migration rates were significantly decreased, and the expression of ADAR2 in the nucleus of cells and GluR2 in the cytomembrane was significantly up-regulated in P and NP groups (P<0.05). Compared with group P, the cell viability, the number of invaded cells and cell migration rates were significantly increased, and the expression of ADAR2 in the nucleus of cells and GluR2 in the cytomembrane was significantly down-regulated in group AP (P<0.05). Compared with group NP, the cell viability, the number of invaded cells and cell migration rates were significantly increased, and the expression of ADAR2 in the nucleus of cells and GluR2 in the cytomembrane was significantly down-regulated in group AP (P<0.05). Conclusion Propofol can inhibit the invasion and migration of glioma cells in the rats, and the mechanism is associated with activation of ADAR2-AMPA receptor GluR2 pathway. Key words: Propofol; Glioma; Cell line, tumor; Neoplasm invasiveness; Neoplasm metastasis

TSPO Ligand-Methotrexate Prodrug Conjugates: Design, Synthesis, and Biological Evaluation.

The 18-kDa translocator protein (TSPO) is a potential mitochondrial target for drug delivery to tumors overexpressing TSPO, including brain cancers, and selective TSPO ligands have been successfully used to selectively deliver drugs into the target. Methotrexate (MTX) is an anticancer drug of choice for the treatment of several cancers, but its permeability through the blood brain barrier (BBB) is poor, making it unsuitable for the treatment of brain tumors. Therefore, in this study, MTX was selected to achieve two TSPO ligand-MTX conjugates (TSPO ligand α-MTX and TSPO ligand γ-MTX), potentially useful for the treatment of TSPO-rich cancers, including brain tumors. In this work, we have presented the synthesis, the physicochemical characterizations, as well as the in vitro stabilities of the new TSPO ligand-MTX conjugates. The binding affinity for TSPO and the selectivity versus central-type benzodiazepine receptor (CBR) was also investigated. The cytotoxicity of prepared conjugates was evaluated on MTX-sensitive human and rat glioma cell lines overexpressing TSPO. The estimated coefficients of lipophilicity and the stability studies of the conjugates confirm that the synthesized molecules are stable enough in buffer solution at pH 7.4, as well in physiological medium, and show an increased lipophilicity compared to the MTX, compatible with a likely ability to cross the blood brain barrier. The latter feature of two TSPO ligand-MTX conjugates was also confirmed by in vitro permeability studies conducted on Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) monolayers. TSPO ligand-MTX conjugates have shown to possess a high binding affinity for TSPO, with IC50 values ranging from 7.2 to 40.3 nM, and exhibited marked toxicity against glioma cells overexpressing TSPO, in comparison with the parent drug MTX.

Synthesis, characterization, and self-assembly with plasmid DNA of a quaternary ammonium derivative of pectic galactan and its fluorescent labeling for bioimaging applications

Quaternized derivatives of pectic galactan (QPG) were synthesized by a reaction of pectic galactan (PG) with 3-chloro-2-hydroxypropyl trimethyl ammonium chloride (CHPTAC) in the presence of aqueous sodium hydroxide solution under mild reaction conditions. The results showed that the concentration of CHPTAC and NaOH has great impact on the quaternization reaction. QPG was found to interact electrostatically with plasmid DNA in aqueous solution to form complexes in globular condensed morphology in a nanometer scale size ranging from 60 to 160nm. Complexes formed with QPG fluorescently labeled with 5-DTAF (QPG-5-DTAF) were introduced to the C6 rat glioma cell line, and were found to be able to enter the cell and approach the nucleus within 24h. The results suggest that this type of modified natural polysaccharide may have an advantage as a biocompatible and biodegradable gene delivery carrier and furthermore may serve as a cell specific carrier.

A biased mu‐opioid receptor positive allosteric modulator that does not alter receptor regulation

Currently used opioid analgesics compete with endogenous opioid peptide ligands by binding to the orthosteric site of the mu opioid receptor (MOPr). Activation of this G protein‐coupled receptor (GPCR) affords analgesia but also causes respiratory depression, sedation, constipation, and euphoria leading to a high addictive liability. Our lab is currently pursuing the idea that positive allosteric modulators of MOPr (mu‐PAMs) may be a way to treat pain with a better therapeutic profile. Such compounds, including the lead ligand BMS‐986122, enhance the affinity, potency, and efficacy of opioid agonists to bind MOPr and activate G protein. On the other hand, BMS‐986122 does not stimulate G protein activation, as measured by [35S]GTPγS binding in the absence of an orthosteric ligand. Here, we sought to determine if BMS‐986122 activates non‐G protein mediated pathways downstream of MOPr and to investigate the long‐term consequences of BMS‐986122 exposure on the regulation of MOPr in vitro using C6 rat glioma cells expressing rat MOPr. The results demonstrate that BMS‐986122 initiates phosphorylation of ERK1/2 in a pertussis‐toxin insensitive manner supporting a G protein‐independent mechanism. BMS‐986122 alone did not cause any long‐term adaptations in MOPr signaling. Moreover, in contrast to its ability to enhance agonist‐mediated activation of G protein, BMS‐986122 failed to enhance agonist‐induced desensitization of MOPr as measured by receptor downregulation, receptor phosphorylation, and loss of receptor‐mediated signaling following long‐term agonist exposure. Development of biased allosteric ligands at GPCRs represents a novel avenue for analgesic drug development. Future work will determine the physiological consequences of this biased PAM in mouse models of pain.Support or Funding InformationFunded by DA039997

Anticancer and antiangiogenic effects of methanol extracts of Lonicera caprifolium L. on C6 rat glioma cells

SUMMARY Objective: Gliomas are brain tumors with high morbidity and mortality. For the treatment of gliomas, it is important to develop new and powerful treatments that could complement existing clinical treatment. Lonicera caprifolium L. ( L. caprifolium ) has various uses in herbal traditional medicine. This study was conducted to determine the phenolic acid levels and DNA damage protection potential of L. caprifolium extract, and to explore the antitumor effect of the extract by investigating its toxicity on C6 rat glioma cell lines and normal L929 mouse fibroblast cell lines. We also aimed to investigate the antiangiogenic potential of the extract. Method: Phenolic acid content was determined by HPLC analysis. DNA damage protection potential was evaluated on pBR322 plasmid DNA. The effect of extracts on the proliferation of cancer cells was evaluated by XTT assay. Antiangiogenic effect was determined with Chorioallantoic membrane model. Results: The extract was found rich in vanillic acid (273.003 µg/g); while the amount of chlorogenic acid was almost at negligible level (0.028 µg/g). 0.005-0.05 mg / ml extract protected against the hazardous effects of UV and H2O2 in all DNA bands. The presence of the extract significantly reduced C6 cell proliferation compared to control (p<0.05). The extract had antiproliferative effect with a half maximum inhibition of concentration (IC 50 ) value of 0.45 mg/ml. L. caprifolium extract in 10 -6 , 10 -5 and 10 -4 M concentrations caused antiangiogenic effect. Antiangiogenic scores of L. caprifolium were 0.6, 0.73 and 1.6, respectively. Conclusions: These results show that L. caprifolium has potential cytotoxic and antiangiogenic effect on C6 rat glioma cells and that the phenolic acid content of the plant may partially influence these activities. Keywords: Glioma, Lonicera caprifolium, angiogenesis, cytotoxic effect, phenolic acid OZET Amac: Gliomolar yuksek morbitite ve mortalite orani olan beyin tumorleridir. Gliomalarin tedavisi icin, mevcut klinik tedaviye tamamlayici olabilecek yeni ve guclu tedavi cozumleri gelistirmek onemlidir. Lonicera caprifolium L.’nin ( L. caprifolium ) bitkisel geleneksel tipta cesitli kullanimlari vardir. Bu calisma, Lonicera caprifolium L. (Bahce Hanimelisi) ekstraktinin fenolik asit duzeylerini ve DNA hasari koruma potansiyelini belirlemek ve C6 sican glioma hucre hatlari ve normal L929 fare fibroblast hucre hatlari uzerindeki toksisitesinin inceleyerek Lonicera caprifolium ekstraktinin anti-tumor etkisini arastirmak icin gerceklestirilmistir. Bu calismada ayrica ekstraktin anti-anjiyogenik potansiyelinin arastirilmasi amaclanmistir. Yontem: Ekstraktlarin fenolik asit icerigi, HPLC analizi ile tespit edildi. DNA hasari koruma potansiyeli pBR322 plazmid DNA uzerinde degerlendirildi. Ekstraktin kanser hucrelerinin proliferasyonu uzerindeki etkisi XTT testi ile degerlendirildi. Anti-anjiyogenik etki koryoallonterik membran modeli ile tespit edildi. Bulgular: Ekstrakt vanillik asitten (273,003 µg/g), zengin bulundu; klorogenik asit miktari hemen hemen goz ardi edilebilir seviyede (0,028 µg/g) tespit edildi. 0.005-0.05 mg/ml araligindaki ekstrakt, tum DNA bantlarini UV ve H 2 O 2’ nin tehlikeli etkisine karsi korudu. Kontrol grubu ile karsilastirildiginda ekstraktin varligi onemli olcude C6 hucre cogalmasini azaltti (p<0.05). Ekstraktin, 0.45 mg / ml yari maximum inhibisyon konsantrasyon (IC50) degerinde antiproliferatif etkisi gozlendi. Lonicera caprifolium ekstrakti 10 -6 , 10 -5 ve 10 -4 M konsantrasyonlarda anti-anjiyogenik etkiye neden oldu. Lonicera caprifolium ekstraktinin anti-anjiyogenik skorlari sirasiyla 0.6, 0.73 ve 1.6 idi. Sonuc: Bu sonuclar, Lonicera caprifolium’un C6 sican glioma hucreleri uzerinde potansiyel sitotoksik ve anti-anjiogenik etkisi oldugunu gostermektedir ve bu aktivitelerde bitki fenolik asit iceriginin, kismen, etkisi olabilir. Anahtar sozcukler : Glioma; Lonicera caprifolium; Anjiogenez; Sitotoksik etki; Fenolik asit

Intracellular PK/PD Relationships of Free and Liposomal Doxorubicin: Quantitative Analyses and PK/PD Modeling.

Nanomedicines are widely studied for intracellular delivery of cancer drugs. However, the relationship between intracellular drug concentrations and drug responses are poorly understood. In this study, cellular and nuclear concentrations of doxorubicin were quantified with LC/MS after cell exposure with free and liposomal doxorubicin (pH-sensitive and pegylated liposomes). Cellular uptake of pegylated liposomes was low (∼3-fold extracellular concentrations) compared with doxorubicin in free form and pH-sensitive liposomes (up to 280-fold extracellular concentrations) in rat glioma (BT4C) and renal clear cell carcinoma (Caki-2) cells. However, after the cell exposure with pegylated liposomes, intracellular doxorubicin was distributed into the nuclear compartment in both cell types. Despite high drug concentrations in the nuclei, Caki-2 cells showed strong resistance toward doxorubicin. A model was successfully built to describe PK/PD relationship between drug concentrations in nucleus and cytotoxic responses in BT4C cells. This model is the first step to link target site concentration of doxorubicin into its effect and can be a useful part of more comprehensive future in vivo PK/PD models.

Bioactive lignan constituents from the twigs of Sambucus williamsii

As part of our ongoing search for bioactive constituents of natural Korean medicinal plants, three new lignan derivatives, sambucasinol A–C (1–3), together with 7 known compounds (4–10) were isolated from the twigs of Sambucus williamsii. The structures of these new compounds were determined by a combination of 1D and 2D NMR spectroscopic data analysis, as well as circular dichroism (CD) spectroscopy studies. Here, we evaluated the anti-inflammatory effects of 1–10 in lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cells. Compounds 1–3 exhibited significant inhibitory effects on nitric oxide production in LPS-activated BV-2 cells, with IC50 values of 6.82, 7.04, and 14.70μM, respectively. Additionally, we evaluated the effects of compounds 1–10 on NGF induction in a C6 rat glioma cell line. Compounds 1–3 upregulated NGF secretion to 183.95±2.63%, 153.99±5.15%, and 155.96±5.15%, respectively, without any significant cell toxicity. Moreover, all isolates were evaluated for their cytotoxicity against A549, SK-OV-3, SK-MEL-2, and XF498 cell lines. Compounds 1–3 showed consistent cytotoxicity against the four human cell lines, with IC50 values in the range of 11.07–19.62μM.

Temozolomide in combination with metformin act synergistically to inhibit proliferation and expansion of glioma stem-like cells.

Glioblastoma is the most common and most aggressive brain tumor in adults. The introduction of temozolomide (TMZ) has advanced chemotherapy for malignant gliomas, but it is not curative. The difficulties in treating glioblastoma may be as a result of the presence of glioma stem cells (GSCs), which are a source of relapse and chemoresistance. Another reason may be that endogenous Akt kinase activity may be activated in response to clinically relevant concentrations of TMZ. Akt activation is correlated with the increased tumorigenicity, invasiveness and stemness of cancer cells and overexpression of an active form of Akt increases glioma cell resistance to TMZ. Mounting evidence has demonstrated that cancer stem cells are preferentially sensitive to an inhibitor of Akt and down-regulation of the PI3K/Akt pathway may enhance the cytotoxicity of TMZ. Metformin (MET), the first-line drug for treating diabetes, it has been proved that it reduces AKT activation and selectively kills cancer stem cells, but whether it can potentiate the cytotoxicity of TMZ for GSCs remains unknown. In the present study, the GSCs isolated from human glioma cell line U87 and Rat glioma cell line C6, in vitro treatment with TMZ either alone or with MET. The present study demonstrates that MET acts synergistically with TMZ in inhibiting GSCs proliferation and generating the highest apoptotic rates when compared to either drug alone. These findings implicate that GSCs cytotoxicity mediated by TMZ may be stimulated by MET, have a synergistic effect, but the definite mechanisms remain elusive.

Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

IntroductionGlioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43.Materials and MethodsF98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.ResultsE2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.DiscussionThese findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

Additive antiangiogenesis effect of ginsenoside Rg3 with low-dose metronomic temozolomide on rat glioma cells both in vivo and in vitro.

BackgroundGlioblastoma is the most common and deadly primary brain tumor in adults. Low-dose,metronomic (LDM) temozolomide (TMZ) displays improved efficacy in the treatment of glioblastoma by targeting angiogenesis, but has a limited effect on recurrence. The antiangiogenesis drug ginsenoside Rg3 (RG3) is the main active ingredient of ginseng, a popular herbal medicine.MethodsUsing an in vitro and a rat model of an orthotopic glioma allograft, this study was to determine whether RG3 enhanced the antiangiogenesis activity of LDM TMZ in the treatment of glioblastoma.ResultsOur results showed that combined use of TMZ with RG3 displayed additive inhibition on proliferation of both human umbilical vein endothelial cells (HUVEC) and rat C6 glioma cells in vitro. They additively arrested cell cycle, increased apoptosis, and decreased VEGF-A and BCL-2 expression in HUVEC. Antiangiogenesis effect was also evaluated in the rat model of orthotopic glioma allograft, based upon markers including relative cerebral blood volume (rCBV) by magnetic resonance imaging (MRI), VEGF levels and microvessel density (MVD)/CD34 staining. LDM TMZ alone was potent in suppressing angiogenesis and tumor growth, whereas RG3 alone only had modest antiangiogenesis effects. Combined treatment significantly and additively suppressed angiogenesis, without additive inhibitory effects on allografted tumor growth.ConclusionsThese data provide evidence showing the efficacy of LDM TMZ on glioma treatment. The combined additive antiangiogenesis effect suggests that RG3 has the potential to further increase the efficacy of LDM TMZ in the treatment of glioblastoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0274-y) contains supplementary material, which is available to authorized users.

A Minimalistic in Vitro 3D Model to Study F98 Rat Brain Tumor Growth

Classical in vitro methods for studying cell behavior, mainly 2D cell cultures on Petri dishes, suffer from many biases (simple dimensionality, absence of biochemical gradients, rigidity⋯). On the other hand, in vivo studies involve very complex systems and do not allow separating the role of each component. Alternative strategies inspired from in vivo tissue's architecture and composition, have been under development for years, in order to mimic extracellular 3D matrix (ECM). Among them, synthetic hydrogels are good in vitro minimalistic models, allowing fine tuning of their composition as well as their structural and functional features.In this context, we manufactured an original PEG-based hydrogel grafted with poly-L-lysine moieties in order to: i) validate this substrate as an artificial 3D ECM; ii) follow the growth (proliferation, migration) of a population of the F98 rat glioma cell line in this minimalistic tissue-like structure.First, we studied the hydrogel physical properties when varying the size and concentration of PEG and poly-L-lysine, using rheology and FRAP. In particular, we explored a range of rigidities, including that of brain ECM (1kPa), and diffusivities following different polymer mesh size. In a second step, we examined how the gel properties and its composition (PEG and poly-L-lysine) influence the organization and growth rate of the cell population.In our 3D substrate, cells have rounded shape different from 2D form. They organize as aggregates for which growth rate and shape varies as a function of hydrogel composition: the higher is the density of PEG and poly-L-lysine, the smaller is the size of the aggregates.Finally, our data clearly give an insight on how spatial constraints in the 3D hydrogel architecture limit tumor's progression into the polymer network. Correlation with in vivo characteristics would be further investigated.

An Investigation of In vitro Cytotoxicity of Silver Nanoparticles (Ag-NPs) on Cancerous Cell Lines

An Investigation of In vitro Cytotoxicity of Silver Nanoparticles (Ag-NPs) on Cancerous Cell Lines Cytotoxicity and apoptotic effect of silver nanoparticles (Ag-NPs) was estimated in cancerous Hepatocellular carcinoma (HepG2), Human breast cancer (MCF-7), Human neuroblastoma (SK-N-SH) and Rat glioma (C6) cell lines, which were produced from Corynebacterium glutamicum. The studies of cytotoxicity were carried out under normoxia and hypoxia conditions. The cell lines of Ag-NPs cytotoxicity were demonstrated by MTT assay and half maximum inhibitory concentrations (IC50) were evaluated. Ag-NPs were exhibited good cytotoxic effects in C6 followed by HepG2, MCF-7 and SK-N-SH. Reactive Oxygen Species (ROS) production in all cell lines was examined. Analysis of apoptosis and necrosis was carried out by annexin-V/ PI staining method using flow cytometry. The present study was designed to recognize the anticancer property of Ag-NPs against HepG2, MCF-7, C6 and SK-N-SH and to the best of our knowledge this is the first report on cytotoxic activity of Ag-NPs synthesized from Corynebacterium glutamicum.

ParamagneticGd2O3Nanoparticle-Based Targeting Theranostic Agent for C6 Rat Glioma Cell

This study aimed to synthesize theranostic agent targeting C6 rat glioma cell, which was based on the dextran coated paramagnetic gadolinium oxide nanoparticles (D-PGONs) conjugated with folic acid (FA) or paclitaxel (PTX). The D-PGONs were synthesized by thein situcoprecipitation method, and the average value of the size distribution was 2.9 nm. FTIR spectroscopy was fulfilled to confirm the conjugations of FA or PTX with D-PGONs. The bioprotective effects of dextran coating and chemotherapeutic effect of PTX in the C6 glioma cell were evaluated by the MTT assay. The differences in uptakes between the synthesized theranostic agents into C6 cells were observed by confocal laser scanning microscopy. In addition, the magnetic contrast enhancement with different concentration of the synthesized agent was compared by theT1-weighted MRI imaging. It was experimentally shown that the synthesized theranostic agent targets C6 cells due to the ligand-receptor-mediated endocytosis and provides enhancement in MR contrast depending on the concentration due to the paramagnetic property of gadolinium nanoparticle. In addition, it was shown by the results of MTT assay that the synthesized nanocomposites were more effective in reducing cell viability than bare gadolinium nanoparticles. In conclusion, it was shown that FA and PTX conjugated D-PGONs could be used as the theranostic agent with paramagnetism and chemotherapeutic property.

Synergistic Anti-glioma Effects in Vitro and in Vivo of Enediyne Antibiotic Neocarzinostatin and Paclitaxel via Enhanced Growth Delay and Apoptosis-Induction.

Neocarzinostatin (NCS) is a member of enediyne antibiotics with high anticancer potential. Our study was performed to explore the synergistic anti-glioma effects of NCS and paclitaxel (PTX) in vitro and in vivo. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicities of the drugs to human glioma cells U87MG and rat glioma cells C6 were evaluated. The results showed that the combinations of NCS and PTX can synergistically inhibit glioma cells survival. Cell apoptosis was detected by flow cytometry, and the results showed that the combinations of NCS and PTX synergistically enhanced apoptosis ratio of glioma cells. Western blot revealed that the cell signaling pathways of proliferation and apoptosis were synergistically regulated, in which Akt was synergistically inactivated, p53 was up-regulated with down-regulation of bcl-2. Meanwhile, with the subcutaneous model of U87MG cells and intracerebral implantation model of C6 cells, the combination strategy could synergistically delay the glioma growth and significantly prolong the survival of rats bearing orthotopic glioma. This study demonstrates that the combination of NCS and PTX can potentiate the effect on survival and apoptosis of glioma cells via suppression of Akt, bcl-2, and activations of p53; Meanwhile, the in vivo studies also confirmed that the combination of NCS and PTX synergistically inhibit the gliom growth. Our data about the combinational effects of NCS with PTX may provide an alternative strategy for glioma therapy.

Hsp70 chaperone rescues C6 rat glioblastoma cells from oxidative stress by sequestration of aggregating GAPDH

The Hsp70 chaperone is known to elicit cytoprotective activity and this protection has a negative impact in anti-tumor therapy. In cancer cells subjected to oxidative stress Hsp70 may bind damaged polypeptides and proteins involved in apoptosis signaling. Since one of the important targets of oxidative stress is glyceraldehyde-3-phospate dehydrogenase (GAPDH) we suggested that Hsp70 might elicit its protective effect by binding GAPDH. Microscopy data show that in C6 rat glioma cells subjected to hydrogen peroxide treatment a considerable proportion of the GAPDH molecules are denatured and according to dot ultrafiltration data they form SDS-insoluble aggregates. Using two newly developed assays we show that Hsp70 can bind oxidized GAPDH in an ATP-dependent manner. Pharmacological up- or down-regulation of Hsp70 with the aid of U133 echinochrome or triptolide, respectively, reduced or increased the number of C6 glioma cells containing GAPDH aggregates and dying due to treatment with hydrogen peroxide. Using immunoprecipitation we found that Hsp70 is able to sequester aggregation-prone GAPDH and this may explain the anti-oxidative power of the chaperone. The results of this study led us to conclude that in cancer cells constantly exposed to conditions of oxidative stress, the protective power of Hsp70 should be abolished by specific inhibitors of Hsp70 expression.