曲古抑菌素A对结肠癌细胞细胞周期影响的机制研究

目的 研究组蛋白去乙酰化酶（HDAC）的抑制剂曲古抑菌素A（TSA）对结肠癌细胞株SW480细胞周期、凋亡的影响,初步探讨TSA作用细胞周期的可能机制,为HDAC抑制剂用于结肠癌治疗提供理论依据.方法 培养人结肠癌细胞系SW480,采用HDAC抑制刺TSA干预细胞,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用Western blot对细胞周期相关的基因进行检测.结果 TSA处理细胞后,流式细胞计数分析显示,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyelinE、cyclinA聚集,而对凋亡无明显的影响.Western blot显示,TSA能够上调p21wafl/Cipl、p27Kipl的表达,下调CDK2、cyclinE以及cyclinA的表达.结论 在结肠癌细胞中,TSA能够通过上调p21Wafl/Cip1、p27Kip1的表达以及下调CDK2、cyclinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长.     

Kynurenic acid enhances expression of p21 Waf1/Cip1 in colon cancer HT-29 cells

BackgroundKynurenic acid (KYNA), a tryptophan metabolite, was found in the mucus of rat small intestine. However, its role in the gastrointestinal tract is still not fully elucidated.MethodsTo verify whether KYNA affects cell cycle regulators, the protein expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 was investigated in colon adenocarcinoma HT-29 cells exposed to KYNA. MTT, BrdU assay and siRNA technology were used to evaluate the effect of KYNA on cancer cell proliferation.ResultsKYNA significantly enhanced the expression of p21 Waf1/Cip1. Importantly, the overexpression of this protein was involved in inhibition of proliferation and DNA synthesis in HT-29 cells.ConclusionsKYNA may be considered as a potential chemoprevention agent against colon cancer.     

Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy.

The partial cross-utilization of molecules and pathways involved in opposing processes like cell survival, proliferation and cell death, assures that mutations within one signaling cascade will also affect the other opposite process at least to some extent, thus contributing to homeostatic regulatory circuits. This review highlights some of the connections between opposite-acting pathways. Thus, we discuss the role of cyclins in the apoptotic process, and in the regulation of cell proliferation. CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), and the Cip1/Waf1/Kip1-2-family (p21(Cip1/Waf1), p27(Kip1), p57(Kip2)) are shown both in the context of proliferation regulators and as contributors to the apoptotic machinery. Bcl2-family members (i.e. Bcl2, Bcl-X(L) Mcl-1(L); Bax, Bok/Mtd, Bak, and Bcl-X(S); Bad, Bid, Bim(EL), Bmf, Mcl-1(S)) are highlighted both for their apoptosis-regulating capacity and also for their effect on the cell cycle progression. The PI3-K/Akt cell survival pathway is shown as regulator of cell metabolism and cell survival, but examples are also provided where aberrant activity of the pathway may contribute to the induction of apoptosis. Myc/Mad/Max proteins are shown both as a powerful S-phase driving complex and as apoptosis-sensitizers. We also discuss multifunctional proteins like p53 and Rb (RBL1/p107, RBL2/p130) both in the context of G1-S transition and as apoptotic triggers. Finally, we reflect on novel therapeutic approaches that would involve redirecting over-active survival and proliferation pathways towards induction of apoptosis in cancer cells.     

Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21Waf1/Cip1 expression: involvement of p38 mitogen-activated protein kinase

Flavonoids are a broadly distributed class of plant pigments, universally present in plants. They are strong anti-oxidants that can inhibit carcinogenesis in rodents. Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammatory, anti-oxidant properties, promotes cell death, and perturbing cell cycle progression. However, the mechanism by which chrysin inhibits cancer cell growth remains poorly understood. Therefore, we developed an interest in the relationship between MAPK signaling pathways and cell growth inhibition after chrysin treatment in rat C6 glioma cells. Cell viability assay and flow cytometric analysis suggested that chrysin exhibited a dose-dependent and time-dependent ability to block rat C6 glioma cell line cell cycle progression at the G1 phase. Western blotting analysis showed that the levels of Rb phosphorylation in C6 glioma cells exposed to 30 microM chrysin for 24h decreased significantly. We demonstrated the expression of cyclin-dependent kinase inhibitor, p21(Waf1/Cip1), to be significantly increased, but the p53 protein level did not change in chrysin-treated cells. Both cyclin-dependent kinase 2 (CDK2) and 4 (CDK4) kinase activities were reduced by chrysin in a dose-dependent manner. Furthermore, chrysin also inhibited proteasome activity. We further showed that chrysin induced p38-MAPK activation, and using a specific p38-MAPK inhibitor, SB203580, attenuated chrysin-induced p21(Waf1/Cip1) expression. These results suggest that chrysin exerts its growth-inhibitory effects either through activating p38-MAPK leading to the accumulation of p21(Waf1/Cip1) protein or mediating the inhibition of proteasome activity.     

Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs

Antitumorigenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. However, the mechanisms driving these processes are not understood in all details. In our study, we observed significant differences in sensitivity of cancer epithelial cell lines to COX-independent antiproliferative effects of NSAIDs. The prostate cancer cell line LNCaP, lacking both critical enzymes in the negative control of PKB/Akt activation, PTEN and SHIP2, was the most sensitive to these effects, as assessed by analysing the cell cycle profile and expression of cell cycle regulating proteins. We found that p53 protein and its signalling pathway is not involved in early antiproliferative action of the selected NSAID—indomethacin. RNAi provided evidence for the involvement of p21^Cip1/Waf1, but not GDF-15, in antiproliferative effects of indomethacin in LNCaP cells. Interestingly, we also found that indomethacin activated PKB/Akt and induced nuclear localisation of p21^Cip1/Waf1 and Akt2 isoform. Our results are in agreement with other studies and suggest that maintaining of the p21^Cip1/Waf1 level and its intracellular localisation might be influenced by Akt2. Knock-down of SHIP2 by RNAi in PTEN negative prostate and colon cancer cell lines resulted in higher sensitivity to antiproliferative effects of indomethacin. Our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.     

Cyclooxygenase-2 in cancer cells and macrophages induces colon cancer cell growth by cigarette smoke extract

Cigarette smoking, cyclooxygenase-2 (COX-2) and macrophages are independently associated with colorectal cancer. In the present study, cigarette smoke ethanol extract was applied to colon cancer cells (SW1116) or indirectly via activated macrophages (THP-1 cells) to attest their effects on cancer cell proliferation and tumor growth both in vitro and in vivo. Ethanol extract induced COX-2 expression in SW1116 and THP-1 cells. Combination of THP-1 pre-incubated medium and ethanol extract further potentiated COX-2 expression and proliferation of SW1116 cells. Tumor growth in nude mice was positively associated with the medium and/or ethanol extract treatments, together with the up-regulation of cell proliferation and angiogenesis, and down-regulation of apoptosis. Application of a COX-2 inhibitor (SC236) reduced tumor growth as well as cell proliferation and angiogenesis. These actions are partially depended on the decrease of COX-2 expression. Taken together, inhibition of COX-2 activity may have significant implication to prevent colon cancer in smokers.     

Differential roles of p21(Waf1) and p27(Kip1) in modulating chemosensitivity and their possible application in drug discovery studies

In this study, the differential role of the cyclin-dependent kinase (CDK) inhibitors p21(Waf1) and p27(Kip1) in cell cycle regulation was proposed for use in screening natural or synthetic compounds for cell cycle-dependent (particularly M phase-dependent) antineoplastic activity. p21(Waf1) or p27(Kip1) was ectopically expressed with an ecdysone-inducible mammalian expression system in a human colon adenocarcinoma cell line. Induction of p21(Waf1) or p27(Kip1) expression inhibited the activities of CDK2 and completely arrested cells at G(1) phase of the cell cycle by p27(Kip1) and at G(1) and G(2) phases by p21(Waf1). We examined the sensitivity of these cells to several antineoplastic agents known to be cell cycle-dependent or -independent. Substantially increased resistance to cell cycle-dependent antineoplastic agents was found in the cells when the expression of p21(Waf1) or p27(Kip1) was induced. In contrast, only a desensitization to cell cycle-independent antineoplastic agents was found in the cells arrested by p21(Waf1) or p27(Kip1). Because p21(Waf1) induces an additional block at G(2) phase that inhibits cell entry into M phase, we further examined the difference between p21(Waf1)- and p27(Kip1)-induced cells in their sensitivity to D-24851, a novel M phase-dependent compound. We found that induction of p21(Waf1) after exposure of the cells to D-24851 conferred stronger resistance than did induction of p27(Kip1). Taken together, our results suggest that the differential effect of p21(Waf1) and p27(Kip1) on cell cycle regulation may be advantageous for screening chemical libraries for novel antineoplastic candidates that are cell cycle-dependent, and M phase-dependent in particular.     

Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27-dependent G1/S cell cycle arrest in conjunction with NF-κB activation

2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21^Cip1/Waf1 and up-regulating p16^INK4A. Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27^Kip1. Bcl-2 and p27^Kip1 were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-κB activity and p50 levels were increased by 2-ME2 and suppression of NF-κB signaling reduced p27^Kip1 expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27^Kip1 in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27^Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.     

Proteasome inhibitor inhibits proliferation and induces apoptosis in renal interstitial fibroblasts

BackgroundThe ubiquitin proteasome pathway plays a pivotal role in controlling cell proliferation, apoptosis and differentiation in a variety of normal and tumor cells. This study aimed to investigate the role of a proteasome inhibitor on proliferation, apoptosis and related proteins in renal interstitial fibroblasts (NRK-49F).MethodsNRK-49F cells were induced using transforming growth factor-β1 (TGF-β1) and pretreated with the proteasome inhibitor MG-132. Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cell cycle and apoptosis were analyzed using flow cytometry. Apoptosis was also analyzed using a DNAladder. The protein expression of p53, p27, p21, caspase-3, Bcl-2 and Bax was examined using western blots.ResultsThe results showed that TGF-β1 (5 ng/ml) can stimulate the proliferation of NRK-49F cells.MG-132 (0.25–5 μM) inhibited TGF-β1-induced proliferation in a dose-dependent manner through G1-arrest; TGF-β1 alone did not induce apoptosis (3.8 ± 0.4% vs. 4.7 ± 1.6%). However, pretreatment with MG-132 significantly induced apoptosis in TGF-β1-stimulated NRK-49F cells in a dosedependentmanner. AtypicalDNAladderwas also confirmed in these two groups.Western blot analysis showed thatMG-132 activated p53, p21, caspase-3 and Bax, and inhibited Bcl-2 in a dose-dependent manner, while p27 expression remained unchanged.ConclusionsA proteasome inhibitor inhibited proliferation and induced apoptosis in renal interstitial fibroblasts stimulated by TGF-β1. The mechanism may relate to the p53, p21, caspase-3, Bcl-2 and Bax pathways. Our results suggest that a proteasome inhibitor could be a new strategy to treat renal interstitial fibrosis.     

微小 RNA-4478下调鼠双微体 -2对结肠癌细胞增殖、迁移、侵袭的影响

目的 探讨微小RNA-4478(miR-4478)对结肠癌细胞增殖、迁移、侵袭的作用并阐明相关机制.方法 实时定量聚合酶链式反应(PCR)检测miR-4478在结肠癌组织中的表达.结肠癌SW1116细胞转染miR-4478 mimics及阴性对照后,分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)和小室(Transwell)法检测细胞的增殖、迁移及侵袭.预测miR-4478的靶基因,双荧光素酶报告基因实验验证靶基因.结肠癌SW1116细胞转染miR-4478 mimics或抑制物后,蛋白质免疫印迹(Western blot)法检测细胞靶基因表达.靶基因过表达验证细胞增殖、迁移及侵袭.Western blot法检测细胞周期蛋白D1(Cy-clin D1)、基质金属蛋白酶2(MMP-2)、P21、钙粘附蛋白E(E-cadherin)蛋白表达.结果 相对于癌旁组织,miR-4478在结肠癌组织中的表达显著降低[(0.84±0.07)比(0.26±0.02),t=48.461,P<0.05];转染miR-4478 mimics可显著抑制细胞增殖、迁移及侵袭(P<0.001).miR-4478可负向调控MDM2表达(P<0.001).miR-4478过表达可抑制由MDM2过表达导致的结肠癌细胞增殖、迁移及侵袭(P<0.001).结论 miR-4478通过抑制MDM2表达而抑制结肠癌细胞增殖、迁移及侵袭.     

The effects of natural ligands of hormone receptors and their antagonists on telomerase activity in the androgen sensitive prostatic cancer cell line LNCaP

The effects of the 17beta-estradiol, dihydrotestosterone and hormone antagonists tamoxifen and bicalutamide on telomerase activity and expression of cell cycle related proteins in the androgen-sensitive prostatic cancer cell line LNCaP were studied. The cell line was grown in RPMI supplemented with 2.5% charcoal-stripped FBS for 72 hr. The IC(50) of tamoxifen and bicalutamide and the optimal stimulatory concentrations of 17beta-estradiol and dihydrotestosterone were determined by means of the cell-viability assay, the activity of telomerase was measured by the telomere repeat amplification protocol (TRAP) and the expression of proteins was analysed by the Western blot technique. 17beta-estradiol stimulated cell growth more effectively than dihydrotestosterone whereas hormone antagonists tamoxifen and bicalutamide caused a significant decrease in cell viability. The treatment of cells by a combination of low doses of 17 beta-estradiol and dihydrotestosterone stimulated cells stronger than treatment by a single hormone. Only 17beta-estradiol, in concentration of 10nM, increased strongly the expression of p21(Waf1/Cip1) and increased slightly telomerase activity in the LNCaP cells. 50 microM of bicalutamide down-regulated the levels of the androgen receptor, the proliferating cell nuclear antigen and telomerase activity, and up-regulated the expression of p27(Kip1). We hereby describe the first observation of the influence of bicalutamide on telomerase activity and a positive correlation between the effect of 17beta-estradiol and the induction of both the endogenous cyclin-dependent kinase inhibitor, p21(Waf1/Cip1), and telomerase activity in a prostatic cancer cell line LNCaP. These findings can shed a new light on the steroid-signaling pathway in prostate cancer cells.     

环状 RNA肌球蛋白轻链激酶靶向微小 RNA-497-5p调控结肠癌细胞的增殖、迁移和侵袭

目的探讨环状 RNA肌球蛋白轻链激酶( circMYLK)对结肠癌细胞增殖、迁移和侵袭的影响和可能机制。方法本研究 2019年 3月至 2020年 1月进行。实时荧光定量 PCR(RT-qPCR)检测检测正常结肠上皮细胞( NCM460)和结肠癌细胞(SW480、SW620、Caco-2)中 circMYLK和微小 RNA-497-5p(miR-497-5p)表达水平。将 circMYLK小干扰 RNA(si-circMYLK)、 miR-497-5p模拟物分别转染 SW480细胞,采用细胞计数试剂盒(CCK-8)、 Transwell实验分别检测干扰 circMYLK或过表达 miR-497-5p对 SW480细胞增殖、迁移和侵袭的影响。双荧光素酶报告基因实验和 RT-qPCR确定 circMYLK对 miR-497-5p的调控作用。结果与 NCM460细胞比较,结肠癌细胞中 circMYLK表达( 1.00±0.06比 3.39±0.23、2.31±0.24、2.98±0.18)显著升高, miR-497-5p表达( 1.00±0.08比 0.36±0.04、0.63±0.05、0.54±0.05)显著降低( P<0.05)。干扰 circMYLK表达后 SW480、细胞活力、迁移和侵袭细胞数显著降低( P<0.05)。过表达 miR-497-5p后 SW480细胞活力、迁移和侵袭细胞数显著降低( P<0.05)。 circMYLK靶向负性调控 miR-497-5p表达。抑制 miR-497-5p表达能够逆转干扰 circMYLK对 SW480细胞增殖、迁移和侵袭的影响,恢复细胞活力、迁移和侵袭能力( P<0.05)。结论结肠癌细胞中 circMYLK呈高表达,干扰 circMYLK通过靶向 miR-497-5p能够抑制结肠癌细胞增殖、迁移和侵袭。     

Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation

Aim:

Probucol, an anti-hyperlipidemic drug, has been reported to exert antitumor activities at various stages of tumor initiation, promotion and progression. In this study we examined whether the drug affected glioma cell growth in vitro and the underlying mechanisms.

Methods:

Human glioma U87 and glioblastoma SF295 cell lines were used. Cell proliferation was accessed using the cell proliferation assay and BrdU incorporation. The phosphorylation of AMPK, liver kinase B1 (LKB1) and p27^Kip1 was detected by Western blot. The activity of 26S proteasome was assessed with an in situ fluorescent substrate. siRNAs were used to suppress the expression of the relevant signaling proteins.

Results:

Treatment of U87 glioma cells with probucol (10–100 μmol/L) suppressed the cell proliferation in dose- and time dependent manners. Meanwhile, probucol markedly increased the ROS production, phosphorylation of AMPK at Thr172 and LKB1 at Ser428 in the cells. Furthermore, probucol significantly decreased 26S proteasome activity and increased p27^Kip1 protein level in the cells in an AMPK-dependent manner. Probucol-induced suppression of U87 cell proliferation could be reversed by pretreatment with tempol (a superoxide dismutase mimetic), MG132 (proteasome inhibitor) or compound C (AMPK inhibitor), or by gene silencing of LKB1, AMPK or p27^Kip1. Similar results were observed in probucol-treated SF295 cells.

Conclusion:

Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation, which reduces 26S proteasome-dependent degradation of p27^Kip1.     

MK801对结肠癌细胞增殖、凋亡和迁移的影响

摘要： 目的 探究 N-甲基-D-天冬氨酸受体亚型 1（NMDAR1）在结肠癌细胞 HT29 和 SW116 中的表达, 以及 NMDAR1 拮抗剂 MK801 对 HT-29 和 SW116 细胞生长抑制、 凋亡和迁移的影响。方法 采用免疫组织化学法检测 结肠癌细胞 HT-29 和 SW116 细胞表面 NMDAR1 的表达; 应用噻唑蓝（MTT）比色法测定 62.5、 125.0、 250.0、 500.0、 1 000.0、 2 000.0 μmol/L 的 MK801 对于 HT-29 和 SW116 细胞增殖作用的影响; 应用流式细胞术检测 2 000 μmol/L 的 MK801 对 HT29 和 SW116 细胞凋亡的影响; 应用细胞划痕实验检测 50 μmol/L MK801 对于结肠癌细胞 HT-29 和 SW116 迁移能力的影响。结果 结肠癌细胞 HT-29 和 SW116 均表达 NMDAR1, 且主要表达于细胞质中; 各浓度的 MK801 对 HT-29 细胞, 以及浓度为 500.0、 1 000.0、 2 000.0 μmol/L 的 MK801 对 SW116 细胞的生长抑制作用具有时 间效应关系, 24、 48 及 72 h 各 MK801 浓度组对 HT-29 和 SW116 细胞的抑制率随浓度升高整体呈增强趋势, 但抑制 率不呈明显的剂量效应关系; MK801 具有促进 HT-29 和 SW116 细胞凋亡的作用, 且主要表现诱导细胞早期凋亡; MK801 可抑制 HT-29 和 SW116 细胞迁移。结论 NMDAR1 在结肠癌细胞胞质中表达, 且 NMDAR1 拮抗剂 MK801 具有抑制肿瘤细胞生长、 迁移, 促进其早期凋亡的作用, 有望成为新一代抗肿瘤药物。     

TGF-β1 induction of p21<Superscript>WAF1/cip1</Superscript> requires smad-independent protein kinase C signaling pathway

Although it has been demonstrated that p21WAF1/Cip1 could be induced by transforming growth factor-beta1 (TGF-beta1) in a Smad-dependent manner, the cross-talk of Smad signaling pathway with other signaling pathways still remains poorly understood. In this study, we investigated a possible role of protein kinase C (PKC) signaling pathway in TGF-beta1 induction of p21WAF1/Cip1 in human keratinocytes HaCaT cells. Our data show that PKC is required for TGF-beta1 induction of p21WAF1/Cip1, as evidenced by the fact that specific inhibition of PKC leads to a decrease in p21WAF1/Cip1 protein and mRNA expression induced by TGF-beta1. And this notion is further supported by the observation that activation of p21WAF1/Cip1 promoter activity is dramatically attenu ated by treatment with PKC inhibitor. However, PKC signaling pathway is not associated with TGF-beta1 activation of Smad signaling pathway, because inhibition of PKC signaling pathway does not affect nuclear translocation of Smads induced by TGF-beta1. Taken together, our data suggest that PKC signaling pathway is required for p21WAF1/Cip1 expression by TGF-beta1, which is independent of Smad signaling pathway.     

苦参碱和氧化苦参碱抗大肠癌药理作用研究进展

苦参碱和氧化苦参碱能防治氧化偶氮甲烷/葡聚糖硫酸钠诱发小鼠原发性结直肠癌和CT26细胞或SW480-EGFP细胞移植瘤在大鼠或小鼠体内生长。体外实验发现苦参碱和氧化苦参碱能浓度相关地抑制大肠癌SW480细胞、SW480-EGFP细胞、SW480/M5细胞、SW620细胞、SW1116细胞、LoVo细胞、HT-29细胞、Colon26细胞、HCT116细胞、HCT-8细胞、HCL细胞和LS174t细胞增殖,并诱导凋亡。苦参碱能增强奥沙利铂抗SW480细胞、SW620细胞和SW1116细胞的作用。氧化苦参碱能增强5-氟尿嘧啶抗LoVo细胞的作用。     

Proteasome inhibitor MG-132 induces C6 glioma cell apoptosis via oxidative stress

Aim:

Proteasome inhibitors have been found to suppress glioma cell proliferation and induce apoptosis, but the mechanisms are not fully elucidated. In this study we investigated the mechanisms underlying the apoptosis induced by the proteasome inhibitor MG-132 in glioma cells.

Methods:

C6 glioma cells were used. MTT assay was used to analyze cell proliferation. Proteasome activity was assayed using Succinyl-LLVY-AMC, and intracellular ROS level was evaluated with the redox-sensitive dye DCFH-DA. Apoptosis was detected using fluorescence and transmission electron microscopy as well as flow cytometry. The expression of apoptosis-related proteins was investigated using Western blot analysis.

Results:

MG-132 inhibited C6 glioma cell proliferation in a time- and dose-dependent manner (the IC₅₀ value at 24 h was 18.5 μmol/L). MG-132 (18.5 μmol/L) suppressed the proteasome activity by about 70% at 3 h. It induced apoptosis via down-regulation of antiapoptotic proteins Bcl-2 and XIAP, up-regulation of pro-apoptotic protein Bax and caspase-3, and production of cleaved C-terminal 85 kDa PARP). It also caused a more than 5-fold increase of reactive oxygen species. Tiron (1 mmol/L) effectively blocked oxidative stress induced by MG-132 (18.5 μmol/L), attenuated proliferation inhibition and apoptosis in C6 glioma cells, and reversed the expression pattern of apoptosis-related proteins.

Conclusion:

MG-132 induced apoptosis of C6 glioma cells via the oxidative stress.