Mechanisms for 2-methoxyestradiol-induced apoptosis of prostate cancer cells

Prostate and breast carcinomas are sex hormone-related carcinomas, which are known to be associated with an over-expression of the proto-oncogene Bcl-2. Here, we report that 2-methoxyestradiol (2-ME), an endogenous metabolite of estrogen that does not bind to nuclear estrogen receptors, effectively induces apoptosis in Bcl-2-expressing human prostate and breast carcinoma cells in vitro and in a rat prostate tumor model in vivo. In several cell lines derived from prostate, breast, liver and colorectal carcinomas, 2-ME treatment led to an activation of c-Jun N-terminal kinase (JNK) and phosphorylation of Bcl-2, which preceded the induction of apoptosis. In summary, our data suggest that 2-ME induces apoptosis in epithelial carcinomas by causing phosphorylation of JNK, which appears to be correlated with phosphorylation of Bcl-2.     

Phosphorylation of Bcl-2 and activation of caspase-3 via the c-Jun N-terminal kinase pathway in ursolic acid-induced DU145 cells apoptosis

There is currently no successful therapy for androgen-independent prostate cancer. Ursolic acid (UA), a pentacyclic triterpenoid compound, has been shown to have an anti-proliferative effect on various tumors. We investigated the effect of UA on cell viability in the human hormone-refractory prostate cancer cell line DU145, as well as the molecular mechanisms underlying its growth inhibiting effect. We demonstrated that UA induces apoptosis and the activation of caspase-3 in DU145 cells. UA also causes the activation of c-Jun N-terminal kinase (JNK), but has no effect on extracellular signal-regulated protein kinases (ERK1/2) and p38 MAP kinases (p38). UA-induced JNK activation could result in Bcl-2 phosphorylation (Ser70) and degradation in DU145 cells, which may be one of the molecular mechanisms by which it induces apoptosis. Although further evaluation, such as in vivo testing, is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer.     

Oestrogen-mediated suppression of tumour necrosis factor alpha-induced apoptosis in MCF-7 cells: subversion of Bcl-2 by anti-oestrogens

In oestrogen receptor (ER)-positive breast carcinoma cells, 17β-oestradiol suppresses a dose-dependent induction of cell death by tumour necrosis factor alpha (TNF). The ability of oestrogens to promote cell survival in ER-positive breast carcinoma cells is linked to a coordinate increase in Bcl-2 expression, an effect that is blocked with the pure anti-oestrogen ICI 182,780. The role of Bcl-2 in MCF-7 cell survival was confirmed by stable overexpression of Bcl-2 which resulted in suppression of apoptosis induced by doxorubicin (DOX), paclitaxel (TAX) and TNF as compared to vector-control cells. The pure anti-oestrogen ICI 182,780 in combination with TNF, DOX or TAX potentiated apoptosis in vector-transfected cells. Interestingly, pre-treatment with ICI 182,780 markedly enhanced chemotherapeutic drug- or TNF-induced apoptosis in Bcl-2 expressing cells, an effect that was correlated with ICI 182,780 induced activation of c-Jun N-terminal kinase. Our results suggest that the effects of oestrogens/anti-oestrogens on the regulation of apoptosis may involve coordinate activation of signalling events and Bcl-2 expression.     

Bcl-2 enhances neurite extension via activation of c-Jun N-terminal kinase

Recent studies suggest that Bcl-2 may play an active role in neuronal differentiation. Here, we showed a marked neurite extension in MN9D dopaminergic neuronal cells overexpressing Bcl-2 (MN9D/Bcl-2) or Bcl-X(L) (MN9D/Bcl-X(L)). We found a specific increase in phosphorylation of c-Jun N-terminal kinase (JNK) accompanied by neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells. Consequently, neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells was suppressed by treatment with SP600125, a specific inhibitor of JNK. Inhibition of other mitogen-activated protein kinases-including p38 and extracellular signal-regulated kinase-did not affect Bcl-2-mediated neurite extension in MN9D cells. While the expression levels of such protein markers of maturation as SNAP-25, phosphorylated NF-H, and neuron-specific enolase were increased in MN9D/Bcl-2 cells, only upregulation of SNAP-25 was inhibited after treatment with SP600125. Thus, the JNK signal activated by Bcl-2 seems to play an important role during morphological and certain biochemical differentiation in cultured dopaminergic neurons.     

Induction of apoptosis by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac)(5)-GP, was produced by acetylation of a glycoside, isolated from an extract of Gardenia fructus. Previously, we have reported that C6 glioma cells could be inhibited in culturing as well as in bearing rats by treating with (Ac)(5)-GP. In this study, the effect and action of (Ac)(5)-GP on inducing cell death was examined in rat C6 glioma cells. Treatment of C6 glioma cells with (Ac)(5)-GP caused cell death, chromatin condensation, and internucleosomal DNA ladder. Also, cell cycle arrest at G(0)/G(1) phase revealed that (Ac)(5)-GP-induced cell death appears to be mediated by apoptosis. In addition, the results also showed that p53 and c-Myc increased due to treatment of (Ac)(5)-GP in a dose-response and time-dependent manner. Concomitant with the expression of p53 and c-Myc, decreased level of Bcl-2 and increased level of Bax protein were observed. These results suggest that cell death caused by (Ac)(5)-GP through apoptosis and cell cycle arrest at G(0)/G(1) may be associated with the induction of p53, c-Myc and may be mediated with apoptosis-related Bcl-2 family proteins.     

MiR-136 promotes apoptosis of glioma cells by targeting AEG-1 and Bcl-2

MicroRNAs have the capacity to coordinately repress multiple target genes and interfere with biological functions of the cell, such as proliferation and apoptosis. Here we report that miR-136 is downregulated in human glioma, and that the miRNA promotes apoptosis of glioma cells induced by chemotherapy. Two anti-apoptotic genes, AEG-1 and Bcl-2, are identified as targets of miR-136, and restoration of AEG-1 or Bcl-2 expression suppresses miR-136-enhanced apoptosis. Therefore, miR-136 might play a tumor-suppressive role in human glioma and thereby might represent a potential therapeutic strategy.     

FGF2 stimulates the proliferation of human mesenchymal stem cells through the transient activation of JNK signaling

FGF2 has been shown to enhance proliferation and maintain differentiation potential in hMSCs during in vitro propagation. In this study, we investigated the role of mitogen-activated protein kinase in the functions of FGF2 in hMSCs. We demonstrated that FGF2 induces the transient activation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 protein kinase. SP600125 and a dominant negative JNK1 significantly reduced the FGF2-enhanced proliferation of hMSCs. Treatment with SP600125 also diminished the activity of FGF2 in the maintenance of adipogenic and osteogenic differentiation potential. These results suggest that JNK signaling is involved in the FGF2-induced stimulation of the proliferation and the maintenance of differentiation potential in hMSCs.     

Induction of apoptosis in rat C6 glioma cells by panaxydol

Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. Previous studies have shown that panaxydol inhibited the growth of various kinds of malignant cell lines. To date, there has been no report concerning the effect of panaxydol on cell growth inhibition in glioma cells. In this paper, we examined panaxydol's antiproliferation and proapoptotic effects on rat C6 glioma cells and investigated its mechanism. Cell growth inhibition of panaxydol was determined by MTT reduction assay. Apoptosis of cells was measured by both Hoechst 33258 staining and Annexin V analysis. It was found that panaxydol markedly inhibited proliferation of C6 cells in a dose-dependent manner with ID(50) of 40 microM. The cell apoptosis was observed at 48 h in the presence of panaxydol. In concert with these findings, Western blot analysis showed a decreased expression of bcl-2 and increased levels of Bax and caspase-3 in C6 cells treated by panaxydol. In conclusion, panaxydol has profound effects on growth and apoptosis of C6 cells, suggesting that panaxydol may be a potential candidate for the treatment of malignant gliomas.     

Enzastaurin-induced apoptosis in glioma cells is caspase-dependent and inhibited by BCL-XL

The novel protein kinase C-beta inhibitor enzastaurin (ENZA) induced apoptosis in LNT-229 and T98G cells whereas A172 cells were resistant. Further, ENZA reduced proliferation in glioblastoma-initiating cells T 269 and T 323 but did not induce apoptosis. ENZA-induced apoptosis involved cleavage of caspases 3, 8, and 9 and led to mitochondrial cytochrome c release and was strongly suppressed by the broad spectrum caspase inhibitor zVAD-fmk but only slightly by the expression of the viral caspase 1/8 inhibitor cytokine response modifier-A. ENZA did not reduce the phosphorylation of protein kinase B (Akt), but of p70 S6 kinase and of its substrate S6 protein in T98G cells. Inhibition of the phosphatidylinositol 3 kinase signaling pathway did not restore sensitivity of A172 cells towards ENZA, and constitutively active Akt did not protect LNT-229 and T98G cells from ENZA-induced apoptosis. Dephosphorylation of glycogen synthase kinase 3beta, a biomarker of ENZA action, and cell death induction by ENZA were separately regulated. Inhibition or activation of Akt only weakly modulated ENZA-induced dephosphorylation of glycogen synthase kinase 3beta. In ENZA-resistant A172 cells, apoptosis ligand 2 (Apo2L.0)-induced cleavage of caspases 3, 8, and 9 was increased by ENZA, resulting in synergistic activity of ENZA and Apo2L.0.     

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (?)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [³H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c-Jun mutant, also prevented stimulation of [³H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.     

Ultrastructure of nuclei of cisplatin-treated C6 glioma cells undergoing apoptosis

C6 glioma cells, treated with a cytostatic dose of cisplatin (1.66 x 10(-5) M) ceased dividing by 24 h and, most of them had undergone apoptosis by 72-96 h. The reactive cells were classified into 5 types (T-I to V), according to the ultrastructure of nuclei. At 4 h, 20.4% of cells (T-I) showed minute condensation and margination of chromatin. The nuclear envelope (NE) formed slim and deep invaginations consisting of the inner or both membranes. The later kind of NE invaginations often extended to the enlarged nucleoli and contained nucleolus-like material at its cytoplasmic side. Some nuclear pores were covered with a dome-shaped "cap" formed by fine filamentous material. The number of T-I cells increased to 53.3% by 72 h. In T-II cells, which appeared at 24 h, the chromatin was condensed into dense irregular masses separated from the NE by a lucent space with filamentous structures preventing complete margination of chromatin. Nucleoli of T-II cells were small and showed partial segregation of their components. The "capped" pores were absent in these apparently more damaged cells. From 24 h, cells with large and lobulated nuclei (T-III) started to increase in number and peaked at 72 h (6.6%). Except for some small lobules, the chromatin of T-III cells was moderately aggregated and the NE was well preserved. Typical apoptotic cells with highly condensed and marginated chromatin (T-IV) peaked at 48-72 h (2.4-4.8%). They appeared in 2 varieties, including cells with wrinkled nuclei with less condensed and incompletely marginated chromatin or more lobulated forms with highly condensed marginated chromatin suggesting their origin from T-II or T-III cells. T-IV cells, as well as their fragments, underwent phagocytosis and secondary necrosis (T-V cells, 48.6% at 96 h). Two alternative routes of nuclear changes leading to cisplatin-triggered apoptosis, as represented by the sequence T-I --> T-III --> T-IV/V or T-I --> T-II --> T-IV/V, may explain the initially less or more damaged cells. These alternatives, together with progressive recruitment of reactive cells, suggest intrapopulation differences in the sensitivity of cells or in the cell cycle perturbations induced by cisplatin. Except for the T-IV and T-V cells, observed alterations of cytoplasmic organelles, including mitochondria, were fewer than reported in previous studies on cisplatin.     

Growth inhibition and differentiation of C6 glioma cells on treatment with hmba.

HMBA, a differentiation inducer belonging to the class of hybrid polar compounds, is known to induce terminal differentiation of a number of leukemic and solid tumour cell lines. In this report we have shown that HMBA markedly inhibits growth of C6 glioma cells at non-cytotoxic concentrations ranging from 2.5 m m to 10 m m in a dose-dependent manner. The growth inhibitory effect can be detected as early as 18--24 h. By the sixth day the growth inhibition decreases at all the concentrations tested. Treatment with HMBA results in an accumulation of C6 cells in G0/G1 phase along with a decrease in the number of cells in S phase. HMBA induces morphological differentiation of C6 cells and increases expression of glial fibriliary acidic protein (GFAP), a marker for mature astrocytes. HMBA induces c-fos and represses cycloheximide-induced c-jun and fra-1 expression. HMBA-induced growth inhibition of C6 cells is accompanied by a decrease in Cdk4 protein levels. However, HMBA fails to sustain low Cdk4 levels, which may be responsible for HMBA's failure to sustain the growth inhibitory effect.     

USP49 inhibits ischemia–reperfusion-induced cell viability suppression and apoptosis in human AC16 cardiomyocytes through DUSP1–JNK1/2 signaling

Dual-specificity protein phosphatases (DUSP) also known as mitogen-activated protein kinase (MAPK) phosphatases (MKPs) can dephosphorylate MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38. DUSP1-mediated JNK dephosphorylation has been found to play an antiapoptotic role against cardiac ischemia–reperfusion (I/R) injury. However, the regulation of DUSP1–JNK pathway remains unclear. In the current study, ubiquitin-specific peptidase 49 (USP49) expression in human AC16 cardiomyocytes following I/R injury was measured by real-time polymerase chain reaction and western blot analysis. Cell viability, apoptosis, the Bax, Bcl-2, and DUSP1 expression, and the activity of MAPKs in AC16 cardiomyocytes following indicated treatment was measured by CCK-8, flow cytometry, and western blot analysis. The direct interaction between USP49 and DUSP1 was measured by coimmunoprecipitation and ubiquitination analysis. The effect of USP49 on apoptosis and JNK activity in rat cardiomyocytes following I/R injury was also measured by TUNEL and western blot analysis. Here, we found that USP49 expression was time-dependently increased in AC16 cardiomyocytes following I/R. I/R-induced cell apoptosis and JNK1/2 activation both in in vivo and in vitro reversed by USP49 overexpression in AC16 cardiomyocytes. Inhibiting JNK1/2 activation significantly inhibited USP49 knockdown-induced the cell viability inhibition, apoptosis and the JNK1/2 activation in AC16 cardiomyocytes. Moreover, USP49 positively regulated DUSP1 expression through deubiquitinating DUSP1. Overall, our findings establish USP49 as a novel regulator of DUSP1–JNK1/2 signaling pathway with a protective role in cardiac I/R injury.     

2‐Hexadecenal inhibits growth of C6 glioma cells

2‐Hexadecenal (2HD) formation in the organism occurs via irreversible enzymatic degradation of sphingosine‐1‐phosphate or nonenzymatic γ‐, UV‐, or HOCl‐induced destruction of a number of sphingolipids including S1P. The current research focuses on the study of 2HD effects on C6 glioma cells growth. The results obtained show that 2HD causes a dose‐dependent decrease in proliferative and mitotic indices. The change in the mitotic index is due to the redistribution of cells in the different phases of mitosis. These processes are accompanied by cytoskeleton rearrangement and changes in cell morphology, which are expressed in F‐actin redistribution, change in the number and type of filopodia and fibrils, leading to cell shape changes, decrease in intercellular contacts and monolayer rarefaction. Cells treatment with 2HD leads to apoptosis induction and signalling pathways modification, including activation of JNK, p38, and ERK1/2 MAPK but not PI3K. The effects observed are not related to the cytotoxicity of 2HD. Significance of the study: 2HD—an unsaturated aldehyde, which level can rise under conditions of oxidative stress as a result of nonenzymatic sphingolipids' destruction. The mechanisms of 2HD action on various cell types have not been sufficiently studied. Therefore, the study on functional role of this aldehyde in different cell types that may be its target is relevant. This study demonstrated that 2HD inhibits growth of C6 glioma cells due to modification of intracellular processes of signal transduction, cytoskeleton rearrangement, change in the mitotic regimen and apoptosis induction.     

Detection of apoptosis and phagocytosis in vitro in C6 rat glioma cells treated with tiazofurin

Tiazofurin, an anticancer drug which inhibits IMP dehydrogenase activity and decreases GTP concentration in various malignant cells, induced inhibition of growth and apoptosis in C6 rat glioma in vitro. The effects of tiazofurin were significantly blocked by addition of exogenous guanosine, suggesting the role of decreased GTP in triggering specific signal transduction pathways involved in apoptosis of C6 cells. The most interesting result of this study was the evidence of phagocytosis of apoptotic cells in vitro by neighbouring cells, a phenomenon considered to occur only in apoptosis in vivo. The possibility of observing phagocytosis in C6 glioma cells suggests that this cell system could be a good model for studying mechanisms of phagocytosis in vitro.     

The effect of C-terminal fragment of JNK2 on the stability of p53 and cell proliferation

The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramatically inhibited 24 h later. However, the expresion of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21^waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.     

Effects of the Ca ionophore A23187 on zinc-induced apoptosis in C6 glioma cells

Zinc ions are essential, but at elevated concentrations, they also have toxic effects on mammalian cells. Zinc plays a crucial role in cell proliferation and differentiation and it even protects cells against apoptosis caused by various reagents. On the other hand, zinc at high concentrations causes cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, and breakdown of the mitochondrial membrane potential. In the present work, a clone of rat C6 glioma cells that was resistant to toxic effects of ZnCl₂ up to 250 μM was employed to study the effect of the ionophore A23187 on zinc-induced apoptosis. Neither 150 μM Zn²⁺ nor 100 nM A23187 alone caused apoptosis as measured by internucleosomal DNA fragmentation. However, combined exposure of C6 cells to 100 nM A23187 and 150 μM Zn²⁺ for 48 h was effective in inducing apoptosis. Because the so-called calcium ionophore A23187 is not specific for Ca²⁺ ions but also transports Zn²⁺ with high selectivity over Ca²⁺, we investigated whether this substance promoted the uptake of Zn²⁺ ions into C6 cells. Employing the zinc-specific fluorescence probe Zinquin, we observed that the very low concentration of 1.9 nM A23187 significantly and rapidly raised the intracellular mobile Zn²⁺ content. Analysis by atomic absorption spectroscopy revealed that incubation with 1.9 nM A23187 caused a doubling of the total intracellular zinc level within 60 min. We conclude that the apoptosis evoked by the combined action of Zn²⁺ and A23187 was the result of enhanced Zn²⁺ influx evoked by the ionophore, resulting in higher intracellular zinc levels.