The fine-tuning of TRAF2–GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K_d=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K_d≥2.6 μM) or is inactivated by oxidation (K_d=1.7 μM). This indicates that GSTP1-1''s TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex''s binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1''s association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex''s drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH''s intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.     

BRCA1-induced apoptosis involves inactivation of ERK1/2 activities

Mutation in the BRCA1 gene is associated with an increased risk of breast and ovarian cancer. Recent studies have shown that the BRCA1 gene product may be important in mediating responses to DNA damage and genomic instability. Previous studies have indicated that overexpression of BRCA1 can induce apoptosis or cell cycle arrest at the G(2)/M border in various cell types. Although the activation of JNK kinase has been implicated in BRCA1-induced apoptosis, the role of other members of the mitogen-activated protein kinase family in mediating the cellular response to BRCA1 has not yet been examined. In this study, we monitored the activities of three members of the MAPK family (ERK1/2, JNK, p38) in MCF-7 breast cancer cells and U2OS osteosarcoma cells after their exposure to a recombinant adenovirus expressing wild type BRCA1 (Ad.BRCA1). Overexpression of BRCA1 in MCF-7 cells resulted in arrest at the G(2)/M border; however, BRCA1 expression in U2OS cells induced apoptosis. Although BRCA1 induced JNK activation in both cell lines, there were marked differences in ERK1/2 activation in response to BRCA1 expression in these two cell lines. BRCA1-induced apoptosis in U2OS cells was associated with no activation of ERK1/2. In contrast, BRCA1 expression in MCF-7 cells resulted in the activation of both ERK1/2 and JNK. To directly assess the role of ERK1/2 in determining the cellular response to BRCA1, we used dominant negative mutants of MEK1 as well as MEK1/2 inhibitor PD98059. Our results indicate that inhibition of ERK1/2 activation resulted in increased apoptosis after BRCA1 expression in MCF-7 cells. Furthermore, BRCA1-induced apoptosis involved activation of JNK, induction of Fas-L/Fas interaction, and activation of caspases 8 and 9. The studies presented in this report indicate that the response to BRCA1 expression is determined by the regulation of both the JNK and ERK1/2 signaling pathways in cells.     

A strong glutathione S-transferase inhibitor overcomes the P-glycoprotein-mediated resistance in tumor cells. 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) triggers a caspase-dependent apoptosis in MDR1-expressing leukemia cells

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoprotein-positive tumors.     

NALP1 基因与骨肉瘤细胞的相关研究

目的:探讨NALP1基因在骨肉瘤细胞株MG-63、U-2OS中的表达,以及高表达NALP1基因对于骨肉瘤细胞体外凋亡的影响。方法:使用RT-PCR、Western-blot法检测骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平并与人成骨细胞株hFOB1.19比较。将NALP1基因转染质粒PcDNA3.1,将重组质粒转染骨肉瘤细胞,分成高表达基因组、空质粒组及对照组,加入抗肿瘤药物顺铂及甲氨蝶呤促使肿瘤细胞凋亡,使用流式细胞仪测定各组肿瘤细胞凋亡率。结果:通过统计分析,骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平均低于人成骨细胞株hFOB1.19(P<0.05),NALP1高表达组的肿瘤细胞凋亡率明显高于空白质粒组及对照组。结论:上调骨肉瘤细胞株MG-63、U-2OS中的NALP1的表达量可以促进肿瘤细胞凋亡。     

Apoptotic effect and cell arrest of deoxyshikonin in human osteosarcoma cells through the p38 pathway

Osteosarcoma is the most common primary bone cancer that affects adolescents with early metastatic potential and drastically reduces their long-term survival rate if pulmonary metastases are detected at diagnosis. The natural naphthoquinol compound deoxyshikonin exhibits anticancer properties, so we hypothesized that it has an apoptotic effect on osteosarcoma U2OS and HOS cells and studied its mechanisms. After deoxyshikonin treatment, dose-dependent decreases in cell viability, induction of cell apoptosis and arrest in the sub-G1 phase of U2OS and HOS cells were observed. The increases in cleaved caspase 3 expression and the decreases in X-chromosome-linked IAP (XIAP) and cellular inhibitors of apoptosis 1 (cIAP-1) expressions after deoxyshikonin treatment in the human apoptosis array were identified in HOS cells, and dose-dependent expression changes of IAPs and cleaved caspase 3, 8 and 9 were verified by Western blotting in U2OS and HOS cells. Phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38 expressions in U2OS and HOS cells was also increased by deoxyshikonin in a dose-dependent manner. Subsequently, cotreatment with inhibitors of ERK (U0126), JNK (JNK-IN-8) and p38 (SB203580) was performed to show that p38 signalling is responsible for deoxyshikonin-induced apoptosis in U2OS and HOS cells, but not via the ERK and JNK pathways. These discoveries demonstrate that deoxyshikonin may be a possible chemotherapeutic candidate to induce cell arrest and apoptosis by activating extrinsic and intrinsic pathways through p38 for human osteosarcoma.     

The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.     

Viability inhibition effect of gambogic acid combined with cisplatin on osteosarcoma cells via mitochondria-independent apoptotic pathway

We previously demonstrated that gambogic acid (GA) is a promising chemotherapeutic compound for human osteosarcoma treatment. The aim of this study was to detect whether the combination of lower-dose GA (0.3 mg/L) and cisplatin (CDDP) (1 mg/L) could perform a synergistic effect on inhibiting tumor in four osteosarcoma cell lines. Our results showed that the combination between GA at lower dose and CDDP significantly exerts a synergistic effect on inhibiting the cellular viability in MG63, HOS, and U2OS cells. In contrast, an antagonistic character was detected in SAOS2 cells exposed to the combined use of lower-dose GA (0.3 mg/L) and CDDP (1 mg/L). Then, analysis of cell cycle showed the combination of both drugs significantly induced the G₂/M phase arrest, without any difference relative to GA treatment alone, in MG63 cells. Flow-cytometric analysis of cell apoptosis displayed that the apoptotic rate in the combination group is higher than that in GA treatment alone in MG63, HOS, and U2OS cells. The combined use of both drugs had no effect on mitochondrial membrane potential, but promoted the apoptosis-inducing function through triggering of CDDP in the three cell lines. By measurement of mitochondrial membrane potential, the activity of caspase-3 and the expressions of caspase-8 and caspase-9, it was showed that the apoptosis-promoting effect of the combined use of both drugs could be dependent on the death receptor apoptosis pathway, not dependent on the mitochondria apoptosis mechanism. This research, for the first time, demonstrates that GA could increase the chemotherapeutic effect of CDDP in human osteosarcoma treatment through inducing the cell cycle arrest and promoting cell apoptosis.     

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.     

7-Nitro-2,1,3-benzoxadiazole derivatives, a new class of suicide inhibitors for glutathione S-transferases. Mechanism of action of potential anticancer drugs

Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK.GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a sigma complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the sigma complex was the favored event. Docking studies demonstrated the likely localization of the sigma complex in the GST active sites and provide a structural explanation for its strong stabilization.     

Cantharidin inhibits osteosarcoma proliferation and metastasis by directly targeting miR-214-3p/DKK3 axis to inactivate β-catenin nuclear translocation and LEF1 translation

Background: As the leading primary bone cancer in adolescents and children, osteosarcoma patients with metastasis show a five-year-survival-rate of 20-30%, without improvement over the past 30 years. Wnt/β-catenin is important in promoting osteosarcoma development. DKK3 is a Wnt/β-catenin antagonist and predicted to have the specific binding site in 3′-UTR with miR-214-3p.Methods: miR-214-3p and DKK3 levels were investigated in human osteosarcoma tissues and cells by RT-qPCR; the prognostic importance of DKK3 level in osteosarcoma patients was determined with Log-rank test; direct binding between DKK3 with miR-214-3p was identified with targetscan; anti-osteosarcoma mechanism of cantharidin was investigated by miR-214-3p silence/over-expression with or without cantharidin treatment, and nuclear/cytoplasmic protein assay in osteosarcoma cells.Results: Down-regulated DKK3 indicated poor prognosis of osteosarcoma patients. Up-regulated miR-214-3p promoted proliferation and migration, while suppressed apoptosis of osteosarcoma cells by increasing β-catenin nuclear translocation and LEF1 translation via degradation of DKK3. Cantharidin suppressed viabilities, migration and invasion, while promoted cell cycle arrest and apoptosis in 143B and U-2 OS cells via down-regulating miR-214-3p to up-regulate DKK3, thus inhibited p-GSK-3β expression, β-catenin nuclear translocation and LEF1 translation. Meanwhile, cantharidin inhibited tumor growth in xenograft-bearing mice with 143B cell injection in tibia.Conclusion: miR-214-3p mediated Wnt/β-catenin/LEF1 signaling activation by targeting DKK3 to promote oncogenesis of osteosarcoma; cantharidin inhibited proliferation and metastasis of osteosarcoma cells via down-regulating miR-214-3p to up-regulate DKK3 and decrease β-catenin nuclear translocation, indicating that cantharidin may be a prospective candidate for osteosarcoma treatment by targeting miR-214-3p/DKK3/β-catenin signaling.     

Dioscin inhibits the growth of human osteosarcoma by inducing G2/M-phase arrest,apoptosis, and GSDME-dependent cell death in vitro and in vivo

Pyroptosis is a form of programmed cell death (PCD) that plays a vital role in immunity and diseases. Although it was recently reported that chemotherapy drugs can induce pyroptosis through caspase-3-dependent cleavage of gasdermin E (GSDME), the role of pyroptosis in osteosarcoma (OS) with dioscin is less understood. In this study, we explored the effects of dioscin on OS in vitro and in vivo and further elucidated the underlying molecular mechanisms and found that dioscin-triggered pyroptosis in GSDME-dependent cell death and that GSDME-N was generated by caspase-3. Furthermore, dioscin inhibited cancer cell growth by inducing G2/M arrest and apoptosis through the JNK/p38 pathway. In vivo, dioscin significantly inhibited OS proliferation. Taken together, our results demonstrate that dioscin can induce apoptosis through the JNK/p38 pathway and GSDME-dependent pyroptosis in OS, identifying it as a potential therapeutic drug for treatment of this disease.     

Cantharidin decreased viable cell number in human osteosarcoma U-2 OS cells through G2/M phase arrest and induction of cell apoptosis

ABSTRACT

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G₂/M phase arrest. CTD increased the production of reactive oxygen species and Ca²⁺, and elevated the activities of caspase-3 and ?9, but decreased the level of mitochondrial membrane potential. Furthermore, CTD increased the ROS- and ER stress-associated protein expressions and increased the levels of pro-apoptosis-associated proteins, but decreased that of anti-apoptosis-associated proteins. Based on these observations, we suggested that CTD decreased cell number through G₂/M phase arrest and the induction of cell apoptosis in U-2 OS cells and CTD could be a potential candidate for osteosarcoma treatments.     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.     

Co-localization of GSTP1 and JNK in transitional cell carcinoma of urinary bladder

Transitional cell carcinoma (TCC) of urinary bladder belongs to glutathione S-transferase P1 (GSTP1) overexpressing tumors. Upregulated GSTP1 in TCC is related to apoptosis inhibition. This antiapoptotic effects of GSTP1 might be mediated through protein:protein interaction with c-Jun NH(2) -terminal kinase (JNK). Herein, we analyzed whether a direct link between GSTP1 and JNK exists in TCC. The presence of GSTP1/JNK complexes was analyzed by immunoprecipitation and Western blotting in 20 TCC specimens, obtained after surgery. Co-localization of GSTP1 and JNK was also investigated in the 5637 TCC cell line by immunofluorescence confocal microscopy. By means of immunoprecipitation we show for the first time the presence of GSTP1/JNK complexes in all TCC samples studied. A co-localization of GSTP1 and JNK was also demonstrated in the 5637 TCC cell line by means of confocal microscopy. Protein-protein interactions, together with co-localization between GSTP1 and JNK provide evidence that GSTP1 most probably inhibits apoptosis in TCC cells by non-covalent binding to JNK.     

Targeting glutathione-S transferase enzymes in musculoskeletal sarcomas: a promising therapeutic strategy

Recent studies have indicated that targeting glutathione-S-transferase (GST) isoenzymes may be a promising novel strategy to improve the efficacy of conventional chemotherapy in the three most common musculoskeletal tumours: osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma. By using a panel of 15 drug-sensitive and drug-resistant human osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma cell lines, the efficay of the GST-targeting agent 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) has been assessed and related to GST isoenzymes expression (namely GSTP1, GSTA1, GSTM1, and MGST). NBDHEX showed a relevant in vitro activity on all cell lines, including the drug-resistant ones and those with higher GSTs levels. The in vitro activity of NBDHEX was mostly related to cytostatic effects, with a less evident apoptotic induction. NBDHEX positively interacted with doxorubicin, vincristine, cisplatin but showed antagonistic effects with methotrexate. In vivo studies confirmed the cytostatic efficay of NBDHEX and its positive interaction with vincristine in Ewing's sarcoma cells, and also indicated a positive effect against the metastatisation of osteosarcoma cells. The whole body of evidence found in this study indicated that targeting GSTs in osteosarcoma, Ewing's sarcoma and rhabdomyosarcoma may be an interesting new therapeutic option, which can be considered for patients who are scarcely responsive to conventional regimens.