Apoptosis In C6 Cells Research Articles

Synergism of 4HPR and SAHA increases anti-tumor actions in glioblastoma cells.

Glioblastoma is the most malignant and prevalent brain tumor in adults. It can grow and spread quickly causing harm to the brain health. One of the major challenges in treatment of glioblastoma is drug resistance. Use of synergistic combination of two drugs with different anti-tumor effects is nowadays highly considered in the development of effective therapeutic strategies for many malignancies. In the present study, we showed synergistic therapeutic efficacies of two chemical compounds, N-(4-hydroxyphenyl) retinamide (4HPR) and suberoylanilide hydroxamic acid (SAHA), for significant reduction in cell viability of rat C6 and human T98G glioblastoma cells. These compounds (4HPR and SAHA)were used alone or in synergistic combination for evaluating their various anti-tumor effects. The results showed that combination of 4HPR and SAHA significantly induced morphological and molecular features of astrocytic differentiation in C6 and T98G glioblastoma cells. Combination of 4HPR and SAHA proved to be an important therapeutic strategy for inhibiting cell growth and inducing differentiation in glioblastoma cells. Furthermore, combination of the two drugs showed more efficacies than either dug alone in reducing in vitro cell invasion (transwell assay), cell migration (wound healing assay), and angiogenesis (tube formation assay) due to down regulation of the molecules involved in these processes. The ultimate of goal of using this combination of drugs was induction of apoptosis. The results showed that these drugs in synergistic combination contributed highly to increases in morphological and molecular features of apoptotic death in the tumor cells. The results from molecular studies indicated that cell death occurred via activation of the extrinsic and intrinsic pathways of apoptosis in both C6 and T98G cells. The drugs in combination also contributed to dramatic inhibition of histone deacetylase 1, an important epigenetic player in promoting growth in glioblastoma cells. This novel combination of drugs should also be considered as a promising therapeutic strategy for the treatment of glioblastoma in vivo.

MicroRNA-181a-5p Down-Regulation Presents Neuroprotective Effect in 1-Methyl-4-Phenylpyridinium-Induced Parkinson’s Disease: An In Vitro Study

Background: Parkinson’s disease (PD) is a long-term degenerative disease that mainly affects the central nervous system of the motor system. MicroRNA-181a-5p (miR-181a-5p) is well studied a variety of malignancies. But, till now, there is no report of the role of miR-181a-5p in PD. Methods: miR-181a-5p level was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blotting analysis/qRT-PCR was used to measure the level of Sirtuin 1 (SIRT1). We evaluated cell viability and apoptosis using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry (FCM), and used enzyme linked immunosorbent assay (ELISA) assay to examine the releases of inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Commercially available kits was applied to assess the lactate dehydrogenase (LDH) activity. Western blotting analyse was used to detect nuclear factor-κB p65 (NF-κB p65) and phosphorylated (p)-p65 protein expression. TargetScan and Dual luciferase reporter assay were carried out to determine the interaction between miR-181a-5p and SIRT1. Results: Results showed that miR-181a-5p was up-regulated and SIRT1 was down-regulated in MMP+-treated C6 cells. miR-181a-5p knockdown attenuated the neurotoxicity induced by MMP+-treatment in C6 cells, evidenced by the enhancement of C6 cell viability, and the suppression of C6 cell apoptosis, LDH activity and inflammation. SIRT1 was found as a direct target of miR-181a-5p, and all these effects of miR-181a-5p inhibition on MMP+-treated C6 cells were reversed by SIRT1 knockdown. Conclusion: Our data suggested that miR-181a-5p down-regulation presents neuroprotective effect in MMP+-induced PD in vitro model by targeting SIRT1.

Acquisition of temozolomide resistance by the rat C6 glioma cell line increases cell migration and side population phenotype.

Cancer stem cells are reportedly associated with drug resistance in glioma, but there are conflicting findings on the effects of cancer stem cells on drug resistance. The aim of the present study was to identify the underlying mechanisms of drug resistance in rat C6 glioma cells, through the use of Transwell invasion assays, flow cytometric and western blot analyses as well as immunohistochemical staining. The results revealed that acquisition of drug resistance by C6 cells enhanced migration ability in vivo and in vitro. Notably, drug resistance did not depend on the cancer stem cells of C6 cells, but on the increase of a side population phenotype. Blockade of the ABC transporter could increase sensitivity to temozolomide and temozolomide-induced apoptosis in C6 cells. Collectively, these data indicated that drug resistance of C6 cells was mediated by the side population phenotype rather than by cancer stem cells.

Mechanisms of the mixed umbilical cord blood-derived mesenchymal stem cells in inhibiting proliferation and inducing apoptosis of glioma cells

Objective To investigate the possible mechanism of mixed umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in vitro to inhibit proliferation and induce apoptosis of C6 glioma cells. Methods The expression of B cell lymphoma/leukemia-2 (bcl-2) and cysteinyl aspartate-specific protease (Caspase)-3 proteins in C6 cells was detected by immunohistochemistry and Western blotting imprinting method, and C6 cell cycle was detected by flow cytometry. Results Different sources of UCB-MSCs have a tendency to increase the expression of apoptosis protein Caspase-3 in C6 cells and reduce the expression of apoptosis protein bcl-2. Different sources UCB-MSCs induce C6 cell apoptosis, E/T=0∶1, 5∶1, (2.5+ 2.5)∶1, 10∶1, (5+ 5)∶1 C6 cells acting on, The percentages of G0/G1 were (65.49±1.83)%, (64.83±2.19)%, (70.30±3.21)%, (69.03±2.19)%, (77.63±3.10)%, and the percentage of C6 cells in G0/G1 increased with the increase of the E/T ratio, showing a proportional dependence. Conclusion After phytohemagglutinin (PHA) stimulation, large doses of UCB-MSCs and mixed UCB-MSCs have inhibitory effects on proliferation of C6 cells in vitro, and this inhibition may be related to apoptosis induction Key words: Mesenchymal stem cells; Induce; Apoptosis

Photodynamic therapy combined with temozolomide inhibits C6 glioma migration and invasion and promotes mitochondrial-associated apoptosis by inhibiting sodium-hydrogen exchanger isoform 1.

As a targeted therapeutic technique for glioma inhibition, photodynamic therapy (PDT) has gradually become a focus of basic research related to glioma treatment. The capacity of PDT to kill glioma cells involves varieties of pathways. In glioma cells, activated sodium-hydrogen exchanger isoform 1 (NHE1) can inhibit the cytotoxic effect of temozolomide (TMZ), promote cell migration and invasion, and inhibit cell apoptosis by changing the acid-base equilibrium. The purpose of our study was to explore if PDT combined with TMZ can effectively inhibit glioma cells by influencing NHE1 in vitro. We analyzed the expression levels of proteins such as NHE1, ezrin, vimentin, Bcl-2, and Bax by Western blot analysis, we assessed the migration and invasion of rat C6 glioma cells by Transwell assay, and we evaluated C6 cell apoptosis in vitro by flow cytometry. Western blot results indicated that NHE1, ezrin and vimentin were downregulated after cotreatment of C6 cells, and intracellular acidification was detected by a fluorometric intracellular pH assay. The migration and invasion capacities of C6 cells were significantly hindered after cotreatment, as shown by the Transwell assay. Experimental data also revealed a significant increase in cell apoptosis after cotreatment, as detected by flow cytometry; corresponding proapoptotic changes in Bcl-2, Bax and caspase-3 were also observed in vitro. These results demonstrate that PDT combined with TMZ can inhibit C6 cell migration and invasion and promote mitochondrial-associated apoptosis by inhibiting NHE1. Therefore, this study provides supporting evidence for a potential method for the treatment of glioma.

Mechanisms of mixed umbilical cord blood-derived mesenchymal stem cells inhibiting proliferation and inducing apoptosis of C6 glioma cells in vitro

Objective To study the mechanisms of the mixed umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) inhibiting proliferation and inducing apoptosis of C6 glioma cells in vitro. Methods Cysteinyl aspartate-specific protease (Caspase)-8, B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2) and Caspase-3 protein expression in C6 cells was analyzed by immunohistochemistry and Western blotting, respectively. Caspase-8, bax, bcl-2 and Caspase-3 mRNA levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Cell cycle was detected by flow cytometry. Apoptosis of C6 cells was examined by Annexin V-propidium iodide (PI) staining and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) methods, respectively. Results The mixed UCB-MSCs had a tendency to increase the expression of Caspase-8, bax, Caspase-3 mRNA, and decrease the expression of bcl-2 mRNA. The expression of apoptosis protein and the number of apoptosis cells increased with the increase of the ratio of E/T (E: effect cells namely UCB-MSCs; T: target cells namely C6 cells), showing a proportional dependence relationship; E∶T=(5+ 5)∶1. The relative expression of Caspase-8, bax, bcl-2, and Caspase-3 mRNA in C6 cells was 0.78±0.13, 0.28±0.03, 0.70±0.08, and 0.76±0.12, respectively. When E/T=0∶1, 5∶1, (2.5+ 2.5)∶1, 10∶1, (5+ 5)∶1, the number of apoptosis C6 cells was (2.9±0.6), (3.1±0.5), (14.5±1.8), (14.3±2.2), and (22.8±4.5) cells, respectively. Conclusion After phytohemagglutinin (PHA) stimulation, the mixed UCB-MSCs could inhibit proliferation of C6 cells in vitro, which was related to induction of apoptosis of glioma cells. Key words: Glioma; Umbilical cord blood; Mesenchymal stem cells; Apoptosis; Proliferation

Quercetin and Sodium Butyrate Synergistically Increase Apoptosis in Rat C6 and Human T98G Glioblastoma Cells Through Inhibition of Autophagy.

This study investigated the efficacy of quercetin (QCT) in combination with sodium butyrate (NaB) in enhancing apoptosis in rat C6 and human T98G glioblastoma cells though blockage of autophagy under nutrient-starvation. The most synergistic doses of the drugs were determined to be 25µM QCT and 1mM NaB in both cell lines. After QCT and QCT + NaB treatments, autophagy quantification with acridine orange staining showed a drastic decrease in protective autophagy in the cells under nutrient-starvation. Decrease in autophagy was correlated with decreases in expression of Beclin-1 and LC3B II. Combination treatment increased the morphological signs of apoptosis including membrane blebbing, nuclear fragmentation, and chromatin condensation. Annexin V staining was also performed for detection and quantification of increases in apoptosis. Western blotting results showed that combination of QCT and NaB increased apoptosis by decreasing anti-apoptotic Bcl-2 and increasing pro-apoptotic Bax, decreasing survivin, activating caspase-3, and degrading poly (ADP-ribose) polymerase (PARP). This study demonstrated the therapeutic potentials of a novel combination therapy in inhibiting protective autophagy to enhance apoptosis in rat C6 and human T98G glioblastoma cells.

Cigarette smoke extracts and cadmium induce COX-2 expression through γ-secretase-mediated p38 MAPK activation in C6 astroglia cells.

Exposure to cigarette smoke has been implicated in the progression of cerebrovascular and neurological disorders like stroke through inflammation and blood-brain barrier disruption. In this study, we investigated the signaling cascade activated by cigarette smoke extracts (CSE) and cadmium (Cd) resulting in the COX-2 induction in C6 rat astroglia cells. CSE or Cd induced Notch1 cleavage and activated p38 MAPK and CREB signaling pathways in C6 astroglia cells. Knockdown of nicastrin using siRNA or γ-secretase inhibitors, DAPT and L-685,486, reduced Notch1 cleavage and phosphorylation of p38 MAPK and CREB, while phosphorylation of ERK and JNK remained unaffected. Additionally, the blockage of γ-secretase activity did not show any effect on the phosphorylation of AKT, another upstream activator of CREB, indicating that γ-secretase-mediated CREB activation occurs via p38 MAPK. γ-secretase inhibitor also inhibited the CSE and Cd-mediated increase in the expression of COX-2. Furthermore, recombinant overexpression of Notch1 intracellular domain resulted in an increase in the expression of COX-2. Notch signaling induced by CSE and Cd induced apoptosis in C6 cells. Our results demonstrate that CSE exposure activated the p38 MAPK and CREB-mediated induction in COX-2 expression in astrocytes via γ-secretase-mediated Notch1 signaling. Our data provides novel insights into the potential mechanism of pro-inflammatory response activated by exposure to cigarette smoke.

Caspase‑dependent apoptotic death by gadolinium chloride (GdCl3) via reactive oxygen species production and MAPK signaling in rat C6 glioma cells.

Gadolinium (Gd) compounds serve as magnetic resonance imaging contrast agents and exert certain anticancer activities. Yet, the molecular signaling underlying the antitumor effect of Gd chloride (GdCl3) on glioma remains unclear. In the present study, we aimed to ascertain the apoptotic mechanisms of GdCl3 on rat glioma C6 cells. Our results demonstrated that GdCl3 significantly reduced cell viability and shrunk cell morphology of C6 cells in a concentration‑dependent manner. GdCl3 led to apoptotic C6 cell death as detected by TUNEL staining. An increase in cleaved caspase‑3, cleaved caspase‑8 and cleaved caspase‑9 occurred in GdCl3‑treated C6 cells as detected by immunoblotting analysis. The activities of caspase‑3, caspase‑8 and caspase‑9 were increased, and the specific inhibitors of caspase‑3/‑8/‑9 individually reversed cell viability, which caused apoptotic death in C6 cells prior to GdCl3 exposure. GdCl3 also caused an elevation in the cytoplasmic Ca2+ level and reactive oxygen species (ROS) production, as well as the loss of mitochondrial membrane potential (ΔΨm) as shown by flow cytometric analysis in C6 cells. The results from the immunoblotting analysis demonstrated that there were upregulated protein levels of cytochromec and Bax but a downregulated protein level of Bcl‑2 in C6 cells after GdCl3 treatment. Additionally, GdCl3 decreased the protein levels of phosphorylated‑extracellular signal‑regulated kinases, phosphorylated‑c‑Jun N‑terminal kinase and phosphorylated‑p38 mitogen‑activated protein kinases in C6 cells. In conclusion, ROS production and MAPKs signaling pathways contribute to GdCl3‑induced caspase cascade‑mediated apoptosis in C6 cells. Our findings provide a better understanding of the molecular mechanisms underlying the role of GdCl3 in rat glioma C6 cells.

Synergistic Anticancer Effects of Formononetin and Temozolomide on Glioma C6 Cells

Temozolomide (TMZ) is currently the first-line drug used for clinical postoperative or non-surgical chemotherapy for glioma, but acquired and intrinsic resistance to TMZ limits its application. The anti-proliferative effect of formononetin on human glioma cells had been confirmed. To improve therapeutic effects of TMZ, we studied the effect of formononetin in combination with TMZ on C6 glioma cells. The anti-proliferative effect of C6 cells was tested by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. The synergy was evaluated by Chou-Talalay method. Morphological changes were observed by hematoxylin-eosin (HE) staining. The effect of formononetin in combination with TMZ on apoptosis of C6 cells was investigated by flow cytometry. The effect of formononetin in combination with TMZ on migration of cells was investigated by wound healing assay and transwell assay. The expression of proteins related to apoptosis and migration were detected by Western blot. These results showed that formononetin or TMZ alone could inhibit the growth of C6 cells in dose-dependent manner and formononetin in combination with TMZ had synergy effect on C6 cells. Further changes in cell morphology could be observed in drug combination by HE staining. Drug combination enhanced the expression of Bax, Cleaved Caspase-3, Cleaved Caspase-9, decreased the expression of Bcl-2, and promoted tumor cells apoptosis. In addition, the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were down-regulated via drug combination which resulted into inhibiting migration of C6 cell. In conclusion, formononetin in combination with TMZ can play a synergistic role in anti-C6 cells, the mechanisms of synergy depended on multiple pathways.

An in vitro analysis of the rat C6 glioma cells to elucidate the linear alkylbenzene sulfonate induced oxidative stress and consequent G2/M phase cell cycle arrest and cellular apoptosis

Linear alkylbenzene Sulphonate (LAS) is the anionic surfactant component of globally consumed detergents. Exposure of sub-inhibitory fractions viz., 1/10th (T1), 1/5th (T2), and 1/2.5th (T3) of IC50 for 48 h, of LAS (5 μM, 10 μM, and 20 μM, respectively) to viable C6 glioma cells of rat, besides imparting morphological alterations leads to gross cytotoxicity. Expression of the damaged DNA coupled with cleaved PARP (p < 0.05; p < 0.01 and p < 0.001) were recorded for T1, T2 and T3, respectively. Subsequently, the cell cycle at G2/M check point was significantly arrested (p < 0.05 for T1 and T2; p < 0.01 for T3). The flow cytometric analysis reveals the initiation of apoptosis in C6 cells as is evident by a significant increase (p < 0.01 for T1, p < 0.001 for T2, and T3) in the intake of annexin-V, the calcium dependent apoptotic phospholipid binding protein. Moreover, significantly increased reactive oxygen species (ROS) (p < 0.05; p < 0.01 and p < 0.001) after 6 h of exposure for all the three sets, registered a declining trend (P < 0.001) when T3 cells were co-treated with N-acetyl cysteine (NAC). Furthermore, the significant attenuation (p < 0.01) of expression of the cleaved PARP and a consequent decrease (p < 0.05) in the cell cycle arrest at G2/M phase after scavenging ROS induced oxidative stress by treating C6 cells with NAC clearly evinces that LAS induced apoptosis is mediated by intracellular ROS. Thus, these findings provide a tangible basis for further investigations including in vivo studies, to unravel the molecular mechanism involved in ROS mediated and LAS induced cytotoxic manifestations.

AG‐1031 induced autophagic cell death and apoptosis in C6 glioma cells associated with Notch‐1 signaling pathway

Glioma is the most common primary brain tumor with high morbidity and poor prognosis. The effect of AG-1031, which is developed as an antineoplastic drug, on C6 glioma cells is still not clear. The aim of this research was to explore the effect of AG-1031 on C6 cells, and tried to find out its potential mechanism on cytotoxicity of C6 cells. The 3-(4,5-dimethylthiazol -2-yl) -2,5- diphenyltetrazolium bromide (MTT) assay showed that AG-1031 inhibited cell viability in a concentration-dependent manner, whereas 3-methyadenine (3-MA) reduced this effect. Results from hoechst 33342 staining and Western blot assay indicated that AG-1031 induced C6 cell apoptosis. Western blot assay presented that AG-1031 notably increased the LC3-II/LC3-I ratio and decreased the expression of P62. Besides, our results showed that bafilomycin A1 increased the expression of LC3-II in cells treated with AG-1031, which indicated that AG-1031 can increase autophagy in C6 cells. Meanwhile, Western blot assay showed that AG-1031 can inhibit Notch-1 signaling by testing relative protein expressions. The expression of the intracellular domain of Notch (NICD) also decreased according to immunofluorescence assay. Additionally, the activation of Notch-1 signaling alleviated AG-1031-induced autophagic cell death and apoptosis. Furthermore, phosphorylated Akt and its downstream effector mechanistic target of rapamycin (mTOR) were reduced with AG-1031. These results suggest that AG-1031 may induce autophagic cell death through the inhibition of Akt-mTOR signaling pathway by down-regulating Notch-1 signaling pathway and activating apoptosis in C6 cells via Notch-1 signaling, which develops a new target spot to treat glioma in the future.

Hyperthermia with different temperatures inhibits proliferation and promotes apoptosis through the EGFR/STAT3 pathway in C6 rat glioma cells.

Malignant gliomas are a group of aggressive neoplasms among human cancers. The curative effects of current treatments are finite for improving the prognosis of patients. Hyperthermia (HT) is an effective treatment for cancers; however, the effects of HT with different temperatures in treatment of MG and relevant mechanisms remain unclear. MTT assay and Annexin V-fluorescein isothiocyanate/propidium iodide staining were used for investigating the proliferation and apoptosis of C6 cells, respectively. Western blotting was applied to detect the expression of proteins. Ultrasonography was employed to evaluate the tumor formation rate, growth rate, angiogenesis rate and degree of hardness of tumors in vivo. The authors certified that HT with 42–46°C × 1 h, 1 t could inhibit proliferation, promote apoptosis, reduce tumor formation rate, growth rate, angiogenesis rate, degree of hardness of tumors, ischemic tolerance and anoxic tolerance, and have synergy with temozolomide in C6 cells. Long-term HT (43°C × 1 h, 1 t/5 d, 90 d) did not cut down the sensitivity of C6 cells to HT, and sustainably inhibited the proliferation of C6 cells. Furthermore, the authors proved HT produced these effects primarily through inhibition of the EGFR/STAT3/HIF-1A/VEGF-A pathway.

MiR-1298 affects cell proliferation and apoptosis in C6 cells by targeting SET domain containing 7.

Our previous high-throughput sequencing indicated that rno-miR-1298 was down-regulated in ischemia-reperfusion model of rat. However, little is known about the function and molecular mechanism of rno-miR-1298 in rat tumor cell. In this study, rno-miR-1298 was detected to be significantly down-regulated in rat tumor C6 cells. Moreover, overexpression of rno-miR-1298 obviously inhibited the proliferation and induced apoptosis in C6 cells. SET domain containing 7 (SETD 7) was identified to be a target of rno-miR-1298 using bioinformatics and luciferase reporter assays. Overexpression of rno-miR-1298 markedly reduced the expression of SETD 7 at protein level. Knockdown of SETD 7 also suppressed proliferation and promoted apoptosis in C6 cells. It was indicated that rno-miR-1298 affected cell proliferation and apoptosis of rat tumor cells by targeting SETD 7. Thus, the newly identified miR-1298/SETD 7 expands the elaboration of the mechanisms of the development and progression of tumors and may provide therapeutic target for tumors of nervous system.

Application of luteolin nanomicelles anti-glioma effect with improvement in vitro and in vivo.

Glioblastoma multiforme (GBM) is one of the most common and malignant tumor. Luteolin, a polyphenolic compound, has been proposed to have anti-tumor activity against various cancers. However, the greatest obstacle in the administration of luteolin is its hydrophobicity as well as the low oral bioavailability. In this study, we formulated luteolin-loaded MPEG-PCL (Luteolin/MPEG-PCL) micelles aiming to improve its solubility in aqueous solution and investigate the anti-tumor effect on glioma in vitro and in vivo. The spherical Luteolin/MPEG-PCL micelles were completely dispersible in normal saline and could release luteolin in a sustained manner in vitro. We demonstrated that Luteolin/MPEG-PCL micelles had stronger cytotoxicity and induced a higher percentage of apoptosis in C6 and U87 cells than free luteolin in vitro. The immunohistochemical study revealed that Luteolin/MPEG-PCL micelles induced more glioma cell apoptosis than free luteolin and inhibited neovascularization in tumor tissues. The Pro-caspase9 and Bcl-2 down-regulation and cleaved-caspase9 and Bax up-regulation suggested that luteolin induced apoptosis via the mitochondrial pathway in vitro. What is more, we found the drug could cumulated much more in the nano-drug group than free drug group through imaging in vivo. In conclusion, the Luteolin/MPEG-PCL micelles have the potential clinical application in glioma chemotherapy.

Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells.

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

A study of radiation injury in rat C6 glioma cell line by 1H-nuclear magnetic resonance spectroscopy

Objective To study the radiation injury of rat C6 glioma cell line by high resolution, 1H-nuclear magnetic resonance (1H NMR) spectroscopy, and to preliminarily investigate its mechanism. Methods Metabolite concentrations in C6 cells were determined by 1H NMR spectroscopy. Comet assay was used to evaluate DNA damage. Flow cytometry was used to determine the cell cycle and apoptosis rate. Colony-forming assay was used to measure the colony-forming rate and preliminarily investigate the mechanism of radiation injury. The results were analyzed by one-way analysis of variance and Pearson correlation analysis. Results With the increase in radiation dose from 0 Gy to 1, 5, 10, and 15 Gy, DNA damage was enhanced in a dose-dependent manner (P=0.000-0.690); the percentage of cells in G1 phase increased (P=0.026-0.749); the apoptosis rate significantly increased (all P=0.000); the colony-forming rate significantly declined (P=0.000-0.004); the Lac/Cr ratio significantly decreased (P=0.000-0.015), which had a negative linear correlation with DNA damage parameters (tail length, r=-0.971; %DNA in the tail, r=-0.998; tail moment, r=-0.995) and apoptosis rate (r=0.978). Conclusions 1H NMR spectroscopy reveals that the change in the Lac/Cr ratio is associated with injury and apoptosis of C6 cells after radiation. 1H NMR spectroscopy has the potential to predict radiation injury of glioma. Key words: Deoxyribonucleic acid damage; Proton magnetic resonance; Cell line, glioma; Apoptosis; Cell cycle arrest

Sea Buckthorn Leaf Extract Inhibits Glioma Cell Growth by Reducing Reactive Oxygen Species and Promoting Apoptosis.

Hippophae rhamnoides L., also known as sea buckthorn (SBT), possesses a wide range of biological and pharmacological activities. However, the underlying mechanism is largely unknown. The present study examined whether SBT leaf extract could inhibit proliferation and promote apoptosis of rat glioma C6 cells. The results revealed that the treatment with SBT leaf extract inhibited proliferation of rat C6 glioma cells in a dose-dependent manner. SBT-induced reduction of C6 glioma cell proliferation and viability was accompanied by a decrease in production of reactive oxygen species (ROS), which are critical for the proliferation of tumor cells. SBT treatment not only significantly upregulated the expression of the pro-apoptotic protein Bcl-2-associated X (Bax) but also promoted its localization in the nucleus. Although increased expression and nuclear translocation of Bax were observed in SBT-treated C6 glioma cells, the induced nuclear morphological change was distinct from that of typical apoptotic cells in that most of SBT-treated cells were characterized by convoluted nuclei with cavitations and clumps of chromatin. All of these results suggest that SBT leaf extract could inhibit the rapid proliferation of rat C6 glioma cells, possibly by inducing the early events of apoptosis. Thus, SBT may serve as a potential therapeutic candidate for the treatment of glioma.

54 Anti-glioma activity of Rana temporaria chensinensis egg protein hydrolysate

Objectives Rana temporaria chensinensis only live in the Changbai mountain area in the northeast of China. The eggs of R.temporaria chensinensis contain many special ingredients that modulate incretion. This study aimed to investigate the effects of R.temporaria chensinensis egg protein hydrolysate on human glioma C6 cell proliferation and apoptosis. Methods We extracted R.temporaria chensinensis egg protein (500 mg/mL) and investigated its effects on human glioma C6 cell cultures, and subjected it to an MTT assay, colony forming assay, Western blot assay and flow cytometry analysis of apoptosis. We further investigated the effects of R. temporaria chensinensis egg protein hydrolysate (1.5 g/kg) on glioma development and progression in vivo using a mouse model of glioma. Results R.temporaria chensinensis egg protein hydrolysate inhibited the proliferation of glioma cells; these effects were mediated by the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway. Conclusions This study suggested that R.temporaria chensinensis egg protein hydrolysate promotes apoptosis of glioma cells both in vitro and in vivo. Acknowledgments This research was financially supported by Jilin Province Science and Technology Development Program (Grant Nos. 20150309008YY and 20150309009YY).

Synthesis and Evaluation of New Benzodioxole- Based Thiosemicarbazone Derivatives as Potential Antitumor Agents

New benzodioxole-based thiosemicarbazone derivatives were synthesized and evaluated for their cytotoxic effects on A549 human lung adenocarcinoma, C6 rat glioma and NIH/3T3 mouse embryonic fibroblast cells. In order to examine the correlation between anticancer activity and cholinesterases, the compounds were evaluated for their inhibitory effects on AChE and BuChE. The most effective anticancer agents were investigated for their effects on DNA synthesis, apoptosis and mitochondrial membrane potential. 4-(1,3-Benzodioxol-5-yl)-1-([1,1′-biphenyl]-4-ylmethylene)thiosemicarbazide (5) was identified as the most promising anticancer agent against C6 and A549 cell lines due to its inhibitory effects on C6 and A549 cells and low toxicity to NIH/3T3 cells. Compound 5 increased early and late apoptosis in A549 and C6 cells. Compound 5 also caused disturbance on mitochondrial membrane potential and showed DNA synthesis inhibitory activity in A549 and C6 cells. Compound 5 was investigated for SIRT1 inhibitory activity to provide mechanistic insight and for that purpose docking studies were also performed for this compound on SIRT1. On the other hand, compound 5 did not show any inhibitory activity against AChE and BuChE. This outcome pointed out that there is no relationship between anticancer activity of compound 5 and cholinesterases.