Magnetic hyperthermia therapy in glioblastoma tumor on-a-Chip model / Terapia de magneto-hipertermia no modelo de tumor de glioblastoma on-a-Chip

ABSTRACT Objective: To evaluate the magnetic hyperthermia therapy in glioblastoma tumor-on-a-Chip model using a microfluidics device. Methods: The magnetic nanoparticles coated with aminosilane were used for the therapy of magnetic hyperthermia, being evaluated the specific absorption rate of the magnetic nanoparticles at 300 Gauss and 305kHz. A preculture of C6 cells was performed before the 3D cells culture on the chip. The process of magnetic hyperthermia on the Chip was performed after administration of 20μL of magnetic nanoparticles (10mgFe/mL) using the parameters that generated the specific absorption rate value. The efficacy of magnetic hyperthermia therapy was evaluated by using the cell viability test through the following fluorescence staining: calcein acetoxymethyl ester (492/513nm), for live cells, and ethidium homodimer-1 (526/619nm) for dead cells dyes. Results: Magnetic nanoparticles when submitted to the alternating magnetic field (300 Gauss and 305kHz) produced a mean value of the specific absorption rate of 115.4±6.0W/g. The 3D culture of C6 cells evaluated by light field microscopy imaging showed the proliferation and morphology of the cells prior to the application of magnetic hyperthermia therapy. Fluorescence images showed decreased viability of cultured cells in organ-on-a-Chip by 20% and 100% after 10 and 30 minutes of the magnetic hyperthermia therapy application respectively. Conclusion: The study showed that the therapeutic process of magnetic hyperthermia in the glioblastoma on-a-chip model was effective to produce the total cell lise after 30 minutes of therapy.

RESUMO Objetivo: Avaliar a terapia de magneto-hipertermia em modelo de tumor de glioblastoma on-a-Chip. Métodos: As nanopartículas magnéticas recobertas com aminosilana foram utilizadas para a terapia da magneto-hipertermia, sendo avaliada a taxa de absorção específica das nanopartículas magnéticas em 300 Gauss e 305kHz. Uma pré-cultura de células C6 foi realizada e, seguidamente, foi feito o cultivo das células 3D no chip. O processo de magneto-hipertermia no chip foi realizado após administração de 20μL de nanopartículas magnéticas (10mgFe/mL), utilizando os parâmetros que geraram o valor da taxa de absorção específica. A eficácia da terapia de magneto-hipertermia foi avaliada pela viabilidade celular por meio dos corantes fluorescentes acetoximetiléster de calceína (492/513nm), para células vivas, e etídio homodímero-1 (526/619nm), para células mortas. Resultados: As nanopartículas magnéticas, quando submetidas ao campo magnético alternado (300 Gauss e 305kHz), produziram um valor médio da taxa de absorção específica de 115,4±6,0W/g. A cultura 3D das células C6 avaliada por imagem de microscopia de campo claro mostrou a proliferação e a morfologia das células antes da aplicação da terapia de magneto-hipertermia. As imagens de fluorescência mostraram diminuição da viabilidade das células cultivadas no organ-on-a-Chip em 20% e 100% após 10 e 30 minutos, respectivamente, da aplicação da terapia de magneto-hipertermia. Conclusão: O processo terapêutico da magneto-hipertermia no modelo de tumor glioblastoma on-a-chip foi eficaz para produzir lise total das células após 30 minutos de terapia.

Study on preparation of curcumin-PLGA nanoparticles and their effect on apoptosis in glioma cells / 中国药理学通报

Aim To prepare curcumin-PLGA nanoparticles [curcumin poly(lactic-co-glycolic acid) nanoparticles, Cur-PLGA-NPs ] and evaluate their effects on glioma cells in vitro. Methods C6 cells and U251 cells were subcultured and randomly divided into control group, curcumin group and curcumin-PLGA group. The morphology of Cur-PLGA-NPs was observed by inverted fluorescence microscope and transmission electron microscopy. The particle size of Cur-PLGA-NPs was detected by Malvem particle size potentiometer. The encapsulation efficiency and drug loading rate of Cur-PLGA-NPs were detected by a multi-function microplate reader. The uptake of Cur-PLGA-NPs by C6 cells and U251 cells was observed by fluorescence microscopy. The effect of Cur-PLGA-NPs on the viability of C6 cells and U251 cells was detected by CCK-8. The effect of Cur-PLGA-NPs on apoptosis was assessed by flow cytometry. Apoptosis was detected by Hoechst staining. Results The Cur-PLGA-NPs had an average particle size of (284. 6 ± 9. 0) nm and were spherical. The encapsulation efficiency was (70.712 ±2.615)%, and the drug loading rate was (2.828 ±0.105) %. Compared with control group and curcumin group, C6 cells and U251 cells significantly increased the uptake of Cur-PLGA-NPs (P < 0. 05). Compared with control group and curcumin group, the cell viability of C6 cells and U251 cells in curcumin-PLGA group significantly decreased (P < 0. 05). Compared with control group and curcumin group, the Cur-PLGA-NPs induced apoptosis significantly in C6 cells and U251 cells(P <0. 05). Conclusion Compared with curcumin, Cur-PLGA-NPs can enhance the uptake of drugs by tumor cells, promoting tumor cell apoptosis.

The anti-proliferative effects of rHSG gene on glioblastoma cells through P53-P21cip1 pathway / 实用医学杂志

Objective To explore the mechanism of the rat hyperplasia suppressor gene (rHSG) inhibited proliferation in C6 rat glioma cells. Methods C6 cells were cultured in vitro and transduced with the adenovirus vector which carried rHSG gene (Adv-rHSG-GFP). The transduction efficiency of adenovirus vector was measured by inverted microscope and flow cytometry in C6 cells. Flow cytometry was used to analyze the C6 cells cycle. Western blot was adopted to test the change of rHSG protein expression, the protein of cancer suppressor gene P53, cell cycle control protein of P21cip1, phosphorylation and non-phosphorylation retinoblastoma proteins (p-Rb, Rb). Results The adenovirus can insert the target gene into the genome of C6 target cell efficiently. The expression level of rHSG protein of Adv-rHSG-GFP group is obviously higher than that of PBS Group and Adv-GFP group. Meanwhile, the over-expressed C6 cells of rHSG that arrest in G0/G1 phase are largely increased (P < 0.01). Besides, there is a large increase in the protein expression of P53 and P21cip1 (P < 0.01), decrease in the expression of p-Rb (P < 0.01) and no significant change in the expression of Rb (P < 0.05). Conclusion rHSG might inhibit the proliferation of C6 rat glioma cells through P53-P21cip1 pathway.

Study on the apoptosis of rat glioma cells induced by defective interfering particles of Sendai virus strain Tianjin / 中华微生物学和免疫学杂志

Objective To investigate the apoptosis of rat glioma C 6 cells induced by defective in-terfering( DI) particles of Sendai virus strain Tianjin .Methods Rat glioma C6 cells were treated with dif-ferent titers of DI particles of Sendai virus strain Tianjin in vitro with culture media as negative control and intact virus as positive control .At different time point , cells were collected and their apoptosis was detected by DNA gel electrophorsis , TUNEL assay and AnnexinⅤ/PI double-labeled flow cytometry .The C6 glioma-bearing rat model was established and then treated with three intratumoral injections of DI particles , intact virus or saline three times at interval of two days .The antitumor effects of ID particles were evaluated through daily measuring of the tumor size .Hematoxylin-eosin( HE) staining was used to observe the patho-logical changes in tumor tissues .TUNEL assay was performed to detect the apoptosis of tumor tissues .Re-sults Rat glioma C6 cells treated with DI particles or intact virus in vitro showed typical DNA ladder pattern in agarose gel electrophoresis in a time-and dose-dependent manner .With the intervention of DI particles , the apoptosis rate of C6 cells showed a time-and dose-dependent manner and was significantly higher than that of the control group (P0.05).Conclusion The DI particles of Sendai virus strain Tianjin could induce apoptosis of rat glioma C 6 cells in a time-and dose-dependent manner both in vitro and in vivo, suggesting that the DI particles might be applicable for the treatment of neurogliocytoma in the future.

Fluoxetine Increases the Expression of NCAM140 and pCREB in Rat C6 Glioma Cells

OBJECTIVE: Dysfunction of neural plasticity in the brain is known to alter neural networks, resulting in depression. To understand how fluoxetine regulates molecules involved in neural plasticity, the expression levels of NCAM, NCAM140, CREB and pCREB, in rat C6 glioma cells after fluoxetine treatment were examined. METHODS: C6 cells were cultured after 20 min or after 6, 24 or 72 h treatments with 10 microM fluoxetine. Immunocytochemistry was used to determine the effect of fluoxetine on the expression of NCAM. Western blot analysis was used to measure the expression levels of NCAM140 and CREB and the induction of pCREB after fluoxetine treatment. RESULTS: NCAM expression following 72-h fluoxetine treatment was significantly increased around cell membranes compared to control cells. Cells treated with fluoxetine for 6 and 72 h showed a significant increase in NCAM140 expression compared to cells treated for 20 min. The level of pCREB in the cells treated with fluoxetine for 72 h not only increased more than 60%, but was also significantly different when compared with the other treatment times. The 72-h fluoxetine treatment led to the increase of NCAM140 and the phosphorylation of CREB in C6 cells. CONCLUSION: Our findings indicate that fluoxetine treatment regulates neuronal plasticity and neurite outgrowth by phosphorylating and activating CREB via the NCAM140 homophilic interaction-induced activation of the Ras-MAPK pathway.

Natural Growth Characteristics in C6 Glioma Model / 中国康复理论与实践

@#Objective To establish C6 glioma model in rat brain and to study its biological behavior(such as the incidence of tumor development, the process of cell invasion pathological characteristics of C6 and neoangiogenesis, the spontaneous regression of experimental gliomas and the best experimental time window).Methods C6 tumor cells and DMEM were implanted into the right caudate of 50 male Wistar rats. 9 rats implanted DMEM is the control group. The animals were examined by MRI and pathological staining at postoperative day (POD) 3, 7, 14, 21,28, 35, and 50. Matrix metalloproteinases-2 (MMP-2) and CD31 immunohistochemistry staining were used to study the histopathological features of the developed tumor. Methodology, physical findings and biological behavior were also discussed. Results 45 Wistar rats survived after surgery and tolerated MRI procedures well. On POD 7, there was a focal signal at the implantation site. The C6 cells sprout to the surroundings along the nerve fiber. During the day 14～28, the tumor exhibited a marked increase in size with focal mass effect, and immunohistochemical-staining shows MMP-2 and CD31 is overexpression; C6 cells were aggregated and blood brain barrier were destroyed greatly. Most of the tumor bearing rats died within 30 days. But, C6 cells in the two rats retrogress spontaneously after more leucocytes rounded 28 days. HE staining shows tumors.Conclusion The characteristics of rat C6 brain tumor model mimicked the human tumor with respect to its development, progression, and invasion. Although, part of C6 tumor spontaneously regressed, it is a useful animal model of glioblastoma for pre-clinical evaluation of various therapeutic strategies for the management of glioblastoma. The best experimental time window is 14 to 28 days.

Experimental nodel of C6 brain tumors in athymic rats / Modelo experimental de tumor cerebral C6 em ratos atímicos

Malignant brain tumor experimental models tend to employ cells that are immunologically compatible with the receptor animal. In this study, we have proposed an experimental model of encephalic tumor development by injecting C6 cells into athymic Rowett rats, aiming at reaching a model which more closely resembles to the human glioma tumor. In our model, we observed micro-infiltration of tumor cell clusters in the vicinity of the main tumor mass, and of more distal isolated tumor cells immersed in normal encephalic parenchyma. This degree of infiltration is superior to that usually observed in other C6 models.

Modelos experimentais de tumores cerebrais malignos geralmente utilizam células imunologicamente compatíveis com o animal receptor. Neste estudo apresentamos um modelo experimental baseado na inoculação de células C6 em ratos atímicos Rowett, visando obter um tumor que se assemelhe mais àqueles observados nos seres humanos. Neste modelo observamos microinfiltração de ilhotas de células na periferia da massa tumoral principal e nas áreas mais distantes, células tumorais isoladas no tecido cerebral normal. Este grau de infiltração é superior àquele observado em outros modelos utilizando as células C6.

Effect of quercetin on proliferation of rat glioma C6 cells / 吉林大学学报(医学版)

Objective To study the effects of quercetin(QUE) on proliferation of rat glioma C6 cell line in vitro.Methods The cells were divided into 5 treatment groups(10,25,50,75 and 100 ?mol?L~(-1) QUE),blank control and menstruum control group.The rat C6 cells were cultivated to 1?10~6?mL~(-1) in the RPMI 1640 medium,then added into 96 holes board with various doses of QUE by 3 holes per group,and MTT assay was used to observe the proliferation of the cells treated for 24,48 and 72 h.The change of cell cycle was also observed by flow cytometry(FCM) after the cells were treated with 50 and 100 ?mol?L~(-1) QUE for 48 h.The changes of the protein P53 and Bcl-2 of C6 cells treated with 50 ?mol?L~(-1) QUE for 48 h were detected by immunocytochemical methods.(Results With) the augmentation of QUE and the extension of the treated time,the C6 cell growth was inhibited,the A values decreased and the cell number in G_0/G_l phase was increased,the cell numbers in S and G_2/M phases were cut down,and the decreased expression of Bcl-2 protein and the increased expression of P53 protein were also observed after treatment with QUE.Conclusion Inhibitory effect of QUE on C6 cell line is proved to be dependent on the treated time of the drug and the dose of QUE,and the induced apoptosis of C6 cells is implemented by the means of up-regulation of P53 protein expression and down-regulation of Bcl-2 protein expression.

Signal transduction of rapid inhibitory effect of corticosterone on glycine uptake in C6 cells / 中国病理生理杂志

AIM: To investigate the signal transduction mechanism of rapid effect of corticosterone. METHODS: With liquid scintillation technique, the effect of corticosterone on glycine uptake in C6 cells was examined. RESULTS: The bovine serum album in coupled with corticosterone had the same effect as corticosterone 21-sulfate(B); Neomycin partially blocked the effect of B in C6 cells; GDP-?-S attenuated the effects of B on glycine uptake in C6 cells; Chelerythrine chloride (Chelery) partially blocked the effect of B in C6 cells; H 89 had no influence on the effect of B in C6 cells. The Km and Vmax of effect of B on glycine uptake in C6 cells were different from those in SK-N-SH cells. CONCLUSION: PKC, instead of PKA, was involved in the signal transduction of the rapid effect of B on glycine uptake in C6 cells. The converse effects of B on C6 cells and SK-N-SH cells resulted from different transportation system in two kinds of cells.