BackgroundGlioblastoma multiforme (GBM) is the most lethal primary brain tumors which remains difficult to cure despite advances in surgery, radiotherapy and chemotherapy. Therefore, the development of new drug is urgently needed. α-carboline derivatives were usually isolated from marine animals such as Britannia marine tunicate Dendrodoa grossularia and Indonesian ascidian Polycarpa aurata. In this study, we have synthesized several α-carboline compounds and examined their anti-glioma activities.ResultsWe report that among α-carboline derivatives TJY-16 (6-acetyl-9-(3,4,5-trimethoxybenzyl)-9H-pyrido[2,3-b] indole) is the most potent α-carboline analog to induce glioma cell death with IC50 value of around 50 nM. TJY-16 decreased cell viability of glioma cells in a concentration- and time-dependent manner. Trypan blue exclusion assay showed that the reduction of cell viability was due to both cell growth inhibition and cell death. Flow cytometric analysis showed that TJY-16 induced G2/M cell cycle arrest followed by induction of sub-G1 phase. Hoechst staining detected the apoptotic features such as nuclear shrinkage and DNA condensation. Western blot analysis showed the increased level of cleaved caspase-3. The activation of caspase-8 and depolarization of mitochondrial membrane potential (ΔΨm) indicated that both extrinsic and intrinsic apoptotic pathways were involved in TJY-16-induced apoptosis. TJY-16 effectively inhibited tumor growth and induced caspase-3 activation in the xenograft tumor model of U87 glioma cells.ConclusionsOur results suggest that TJY-16 may kill glioma cells by inducing G2/M cell cycle arrest followed by apoptosis. Thus, TJY-16 is a promising agent for the treatment of malignant gliomas.
Abstract. The hepatitis B virus X protein (HBx) critically modulates cell growth by inducing apoptosis or proliferation. We sought to clarify whether HBx-mediated apoptosis in a CCL13 stable cell line (Chang-HBx) with inducible HBx expression proceeds through the extrinsic (death receptormediated) and/or intrinsic (mitochondrial-mediated) pathways of apoptosis. We used western blotting, cell viability assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, caspase activity assays, JC-1 staining and DNA fragmentation analysis to study the role of HBx in apoptosis. The expression of the pro-apoptotic proteins Bax and Bad and the release of cytochrome c also increased slightly upon HBx induction. JC-1 staining showed a loss of mitochondrial membrane potential upon HBx induction. Additionally, induction of HBx increased the levels of cleaved caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway) and the common effector caspase-3 as measured by western blotting. This elevation of cleaved caspase-8 or caspase-3 and caspase-9 or caspase-3 decreased in the presence of caspase-8 inhibitor Z-IETD-FMK or caspase-9 inhibitor Z-LEHD-FMK, respectively. Both inhibitors also rescued cell growth, and the caspase-8 inhibitor Z-IETD-FMK prevented apoptotic phenomena including the TUNEL signal. DNA fragmentation analysis showed that these phenomena were not detected in the presence of higher concentration of inhibitors. Our data suggest that HBx induces apoptosis through both extrinsic and intrinsic pathways.
A novel synthetic compound 6-acetyl-9-(3,4,5-trimetho-xybenzyl)-9H-pyrido[2,3-b]indole (HAC-Y6) demonstrated selective anticancer activity. In the present study, COLO 205 cells were treated with HAC-Y6 to investigate the molecular mechanisms underlying its effects. HAC-Y6 induced growth inhibition, G2/M arrest and apoptosis in COLO 205 cells with an IC50 of 0.52±0.035 µM. Annexin V/PI double staining demonstrated the presence of apoptotic cells. JC-1 staining analysis showed that HAC-Y6 decreased mitochondrial membrane potential in support of apoptosis. An immunostaining assay revealed that HAC-Y6 depolymerized microtubules. Treatment of COLO 205 cells with HAC-Y6 resulted in increased expression of BubR1 and cyclin B1 and decreased expression of aurora A, phospho-aurora A, aurora B, phospho-aurora B and phospho-H3. HAC-Y6 treatment increased protein levels of active caspase-3, caspase-9, Endo G, AIF, Apaf-1, cytochrome c and Bax, but treatment with the compound caused reduced levels of procaspase-3, procaspase-9, Bcl-xL and Bcl-2. Overall, our results suggest that HAC-Y6 exerts anticancer effects by disrupting microtubule assembly and inducing G2/M arrest, polyploidy and apoptosis via mitochondrial pathways in COLO 205 cells.
Abstract. The present data showed that a novel synthesized compound, 6-acetyl-9-(3,4,5-trimethoxybenzyl)-9H-pyrido [2,3-b]indole (HAC-Y6), exhibited potent antitumor activity against human hepatocellular carcinoma (HCC) cells in vitro. Western blot and immunofluorescence experiments showed that HAC-Y6 depolymerized microtubules similarly to the effects of colchicine. HAC-Y6-treatment in Hep3B cells resulted in the accumulation of the G2/M phase and induced apoptosis. In addition, HAC-Y6-treatment influenced the expression of cell cycle and apoptosis related proteins in Hep3B cells. HAC-Y6 exposure increased caspases-3, -8, -9 and Bax protein levels, while reducing levels of Bcl-2 family proteins. Moreover, Bid, a substrate of caspase-8, was also activated by HAC-Y6. Treatment of cells caused the upregulation of the death receptor 4 (DR4) and phosphorylation of p38. Taken together, we show that HAC-Y6 exhibited its antitumor activity by disrupting microtubule assembly, causing cell cycle arrest and apoptosis through both extrinsic and intrinsic pathways in Hep3B cells. Therefore, the novel compound HAC-Y6 is a promising microtubule inhibitor that has great potential for treatment of HCC.
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