The proteasome inhibitor MG132 potentiates TRAIL receptor agonist-induced apoptosis by stabilizing tBid and Bik in human head and neck squamous cell carcinoma cells

Head and neck squamous cell carcinoma (HNSCC) is often resistant to conventional chemotherapy and thus requires novel treatment regimens. Here, we investigated the effects of the proteasome inhibitor MG132 in combination with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAIL receptor 1 (DR4)-specific monoclonal antibody, AY4, on sensitization of TRAIL- and AY4-resistant human HNSCC cell lines. Combination treatment of HNSCC cells synergistically induced apoptotic cell death accompanied by caspase-8, caspase-9, and caspase-3 activation and Bid cleavage into truncated Bid (tBid). Generation and accumulation of tBid through the cooperative action of MG132 with TRAIL or AY4 and Bik accumulation through MG132-mediated proteasome inhibition are critical to the synergistic apoptosis. In HNSCC cells, Bak was constrained by Mcl-1 and Bcl-X(L), but not by Bcl-2. Conversely, Bax did not interact with Mcl-1, Bcl-X(L), or Bcl-2. Importantly, tBid plays a major role in Bax activation, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-X(L), pointing to the synergistic mechanism of the combination treatment. In addition, knockdown of both Mcl-1 and Bcl-X(L) significantly sensitized HNSCC cells to TRAIL and AY4 as a single agent, suggesting that Bak constraint by Mcl-1 and Bcl-X(L) is an important resistance mechanism of TRAIL receptor-mediated apoptotic cell death. Our results provide a novel molecular mechanism for the potent synergy between MG132 proteasome inhibitor and TRAIL receptor agonists in HNSCC cells, suggesting that the combination of these agents may offer a new therapeutic strategy for HNSCC treatment.     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

p53-Independent up-regulation of a TRAIL receptor DR5 by proteasome inhibitors: a mechanism for proteasome inhibitor-enhanced TRAIL-induced apoptosis

Gliomas are the most common brain tumors in adults and account for more than half of all brain tumors. Despite intensive clinical investigations, average survival for the patients harboring the malignancy has not been significantly improved. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), shown to have potent and cancer-selective killing activity, has drawn considerable attention as a promising anti-cancer therapy. In an attempt to develop TRAIL as an anti-cancer therapy for gliomas, tumor suppressor activity of TRAIL was assessed using human glioma cell lines such as U373MG, U343MG, U87MG and LN18. U343MG, U87MG and LN18 cells were susceptible to TRAIL; however, U373MG cells were completely refractory to TRAIL. Resistance to the applied therapies is a key issue in cancer treatment; thus, various combination treatments were evaluated using U373MG cells to identify a better regimen. Unlike Doxorubicin, Etoposide, Actinomycin D and Wortmannin, a proteasome inhibitor MG132 significantly enhanced TRAIL-induced apoptosis. Similarly, other proteasome inhibitors, including Lactacystin, Proteasome inhibitor I and Velcade (Bortezomib), also enhanced apoptotic activity of TRAIL. Among these proteasome inhibitors, Velcade, the only approved drug, was as effective as MG132 in enhancing TRAIL-induced apoptosis. Both Velcade and MG132 increased the protein levels of DR5, a TRAIL receptor known to be up-regulated by p53, in U373MG cells where p53 is mutated. Our data indicate that proteasome inhibitors up-regulate DR5 in a p53-independent manner and a combination therapy comprising TRAIL and Velcade become a better treatment regimen for gliomas.     

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p < 0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p < 0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p = 0.3), 30% (p = 0.03) and 37% (p = 0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.     

Upregulation of c-FLIP-short in response to TRAIL promotes survival of NSCLC cells,which could be suppressed by inhibition of Ca2+/calmodulin signaling

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for killing tumor cells. However, a number of studies have demonstrated that different cancer cells resist TRAIL treatment and, moreover, TRAIL can promote invasion and metastasis in resistant cells. Here we report that TRAIL rapidly activates caspase-8 in a panel of non-small-cell lung carcinomas (NSCLCs). Adenocarcinomas derived from the lung in addition to high caspase-8 expression are characterized by increased expression of DR4 compared with adjacent non-neoplastic tissues. Blocking DR4 or lowering caspase-8 expression significantly reduced apoptosis in NSCLC cell lines, indicating the importance of DR4 and signifying that higher levels of caspase-8 in lung adenocarcinomas make them more susceptible to TRAIL treatment. Despite rapid and robust initial responsiveness to TRAIL, surviving cells quickly acquired resistance to the additional TRAIL treatment. The expression of cellular-FLIP-short (c-FLIP_S) was significantly increased in surviving cells. Such upregulation of c-FLIP_S was rapidly reduced and TRAIL sensitivity was restored by treatment with cycloheximide. Silencing of c-FLIP_S, but not c-FLIP-long (c-FLIP_L), resulted in a remarkable increase in apoptosis and significant reduction of clonogenic survival. Furthermore, chelation of intracellular Ca²⁺ or inhibition of calmodulin caused a rapid proteasomal degradation of c-FLIP_S, a significant increase of the two-step processing of procaspase-8, and reduced clonogenicity in response to TRAIL. Thus, our results revealed that the upregulation of DR4 and caspase-8 expression in NSCLC cells make them more susceptible to TRAIL. However, these cells could survive TRAIL treatment via upregulation of c-FLIP_S, and it is suggested that blocking c-FLIP_S expression by inhibition of Ca²⁺/calmodulin signaling significantly overcomes the acquired resistance of NSCLC cells to TRAIL.     

Caspase-8 dependent osteosarcoma cell apoptosis induced by proteasome inhibitor MG132

Many researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells, such as breast cancer cell, lung cancer cell, and lymphoma cell. However, the effect of proteasome inhibitors on osteocsarcoma cells and the mechanisms are seldom studied. In this study, we found proteasome inhibitor MG132 was an effective inducer of apoptosis in human osteosarcoma MG-63 cells. On normal human diploid fibroblast cells, MG132 did not show any apoptosis-inducing effects. Apoptotic changes such as DNA fragment and apoptotic body were observed in MG132-treated cells and MG132 mostly caused MG-63 cell arrest at G(2)-M-phase by cell cycle analysis. Increased activation of caspase-8, accumulation of p27(Kip1), and an increased ratio of Bax:Bcl-2 were detected by RT-PCR and Western blot analysis. Activation of caspase-3 and caspase-9 were not observed. This suggests that the apoptosis induced by MG132 in MG63 cells is caspase-8 dependent, p27 and bcl-2 family related.     

Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.     

Individual caspase-10 isoforms play distinct and opposing roles in the initiation of death receptor-mediated tumour cell apoptosis

The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin–proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.     

蛋白酶体抑制剂MG132诱导K562和HeLa细胞凋亡程度存在明显差异

蛋白酶体抑制剂MG132诱导人白血病细胞K562和宫颈癌细胞HeLa凋亡,用3个不同浓度的蛋白酶体抑制剂MG132处理人白血病细胞K562和宫颈癌细胞HeLa,通过MTT检测、annexin Ⅴ/ PI 双染法、流式细胞术、酶标仪和Western 印迹分别检测MG132对K562细胞和HeLa细胞的生长效应、细胞凋亡率、细胞内活性氧(ROS)水平和caspase-3活性变化的影响.蛋白酶体抑制剂MG132诱导K562细胞凋亡明显,对HeLa细胞诱导凋亡不明显.结果表明,蛋白酶体抑制剂MG132特异性诱导不同肿瘤细胞凋亡的程度存在明显差异.     

The changes of reactive oxygen species and glutathione by MG132, a proteasome inhibitor affect As4.1 juxtaglomerular cell growth and death

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth and death of As4.1 juxtaglomerular cells in relation to ROS and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells with an IC₅₀ of approximately 0.3–0.4 μM at 48 h and induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, activation of caspase-3 and -8, and PARP cleavage. MG132 increased intracellular ROS levels including O₂^? and GSH depleted cell numbers. N-acetyl cysteine (NAC, a well-known antioxidant) significantly decreased ROS level and GSH depleted cell numbers in MG132-treated As4.1 cells, along with the prevention of cell growth inhibition, cell death and MMP (ΔΨ_m) loss. NAC also decreased the caspase-3 activity of MG132. l-Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) did not affect cell growth, death, ROS and GSH levels in MG132-treated As4.1 cells. Conclusively, MG132 reduced the growth of As4.1 cells via apoptosis. The changes of ROS and GSH by MG132 were involved in As4.1 cell growth and death.     

Programmed cell death 4 (PDCD4) mediates the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by down-regulation of FLIP expression

Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL.     

XIAP-targeting drugs re-sensitize PIK3CA-mutated colorectal cancer cells for death receptor-induced apoptosis

Mutations in the oncogenic PIK3CA gene are found in 10–20% of colorectal cancers (CRCs) and are associated with poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic TRAIL death receptor antibodies emerged as promising anti-neoplastic therapeutics, but to date failed to prove their capability in the clinical setting as especially primary tumors exhibit high rates of TRAIL resistance. In our study, we investigated the molecular mechanisms underlying TRAIL resistance in CRC cells with a mutant PIK3CA (PIK3CA-mut) gene. We show that inhibition of the constitutively active phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway only partially overcame TRAIL resistance in PIK3CA-mut-protected HCT116 cells, although synergistic effects of TRAIL plus PI3K, Akt or cyclin-dependent kinase (CDK) inhibitors could be noted. In sharp contrast, TRAIL triggered full-blown cell death induction in HCT116 PIK3CA-mut cells treated with proteasome inhibitors such as bortezomib and MG132. At the molecular level, resistance of HCT116 PIK3CA-mut cells against TRAIL was reflected by impaired caspase-3 activation and we provide evidence for a crucial involvement of the E3-ligase X-linked inhibitor of apoptosis protein (XIAP) therein. Drugs interfering with the activity and/or the expression of XIAP, such as the second mitochondria-derived activator of caspase mimetic BV6 and mithramycin-A, completely restored TRAIL sensitivity in PIK3CA-mut-protected HCT116 cells independent of a functional mitochondrial cell death pathway. Importantly, proteasome inhibitors and XIAP-targeting agents also sensitized other CRC cell lines with mutated PIK3CA for TRAIL-induced cell death. Together, our data suggest that proteasome- or XIAP-targeting drugs offer a novel therapeutic approach to overcome TRAIL resistance in PIK3CA-mutated CRC.Colorectal cancer (CRC) is among the three most common malignancies worldwide.¹ Pathophysiologically, CRC development been linked to the acquisition of oncogenic mutations such as alterations in the phosphoinositide-3 kinase (PI3K)/Akt pathway. PI3K converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, thereby generating a docking site for pleckstrin homology domain containing proteins such as Akt/PKB. In CRC, approximately 10–20% of tumors exhibit mutations in the p110α catalytic subunit (predominantly H1047R and E545K substitutions in the PIK3CA gene), causing constitutive PI3K/Akt activation² and worsening clinical outcome.³Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) emerged as a promising anti-cancer agent, capable of selectively inducing cell death in tumor cells.⁴ TRAIL binding to TRAIL receptor 1 (TRAIL-R1) or TRAIL-R2 induces formation of a chain-like death-inducing signaling complex (DISC). This allows stepwise caspase-8 activation and initiates a cascade of proteolytic cleavage events finally activating caspase-3 and triggering the execution phase of apoptosis.In so-called type I cells, initial caspase-8-mediated cleavage of caspase-3 efficiently triggers further autocatalytic caspase-3 processing to the mature heterotetrameric p12-p17 molecule. In type II cells, however, X-linked inhibitor of apoptosis protein (XIAP) inhibits processing of the caspase-3 p19 intermediate to the p17 subunit of the mature enzyme. Death receptor-induced apoptosis in these cells therefore relies on a mitochondria-dependent amplification loop that is triggered by caspase-8-mediated cleavage of the BH3-interacting domain death agonist (Bid) to tBid.⁵ tBid activates Bcl2-associated X protein (Bax) and Bcl2-antagonist/killer (Bak), enabling pore-formation in the outer mitochondrial membrane and release of apoptogenic factors such as cytochrome c and second mitochondria-derived activator of caspase (SMAC).⁶ The pro-apoptotic effect is at least twofold: cytochrome c associates with apoptotic protease-activating factor 1 (Apaf-1), forming a molecular scaffold for caspase-9 activation (‘apoptosome''), which in turn boosts downstream effector caspase activation. Synergistically, SMAC neutralizes cytosolic inhibitors of apoptosis proteins (IAPs), such as cIAP1, cIAP2 and especially XIAP.⁷High levels of IAPs or deregulated expression of Bcl2 family proteins are common in human cancers and often confer apoptosis resistance. This hampers efficacy of TRAIL-based therapies and to date, the therapeutic benefit of TRAIL in clinical trials is indeed rather limited.⁸We have recently found that mutant PIK3CA licensed TRAIL and CD95L to induce an amoeboid morphology in CRC cells, which is associated with increased invasiveness in vitro.⁹ Here, we show that targeting of the aberrantly active PI3K/Akt signaling pathway in TRAIL resistant, PIK3CA-mutated CRC cells only partially restored death receptor-triggered apoptosis induction. We identified impaired caspase-3 maturation by XIAP as the underlying molecular mechanism of TRAIL resistance in HCT116 PIK3CA-mut cells. Inhibition of XIAP or the proteasome efficiently restored TRAIL sensitivity irrespective of mitochondria-dependent death signal amplification. Together, our results indicate that targeting XIAP or the proteasome in CRC with PIK3CA mutations may offer a promising strategy to exploit the therapeutic potential of TRAIL in cancer therapy.     

Proteasome inhibitors and immunosuppressive drugs promote the cleavage of eIF4GI and eIF4GII by caspase-8-independent mechanisms in Jurkat T cell lines

Previously, we have shown that translation eukaryotic initiation factor (eIF) 4GI is cleaved during anti-Fas-mediated apoptosis. Here, we have investigated the effects of the proteasome inhibitors, MG132 and lactacystin, and the immunosuppressants, 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol (FTY720) and cyclosporin A, on the integrity of eIF4GI and eIF4GII in T cells. Using wild-type Jurkat T cells, we show that the proteasome inhibitors MG132 and lactacystin promote the cleavage of eIF4G, activate caspase-8 and caspase-3-like activities and decrease cell viability. Furthermore, MG132 also promotes the cleavage of eIF4G and the activation of caspase-3-like activity in a caspase-8-deficient Jurkat cell line which is resistant to anti-Fas-mediated apoptosis. Using specific anti-peptide antisera, we show that both eIF4GI and eIF4GII are cleaved in either cell line in response to MG132 and lactacystin. In response to such treatments, we demonstrate that the fragments of eIF4GI generated include those previously observed with anti-Fas antiserum together with a novel product which lacks the ability to interact with eIF4E. In contrast, cells treated with the immunosuppressants FTY720 and cyclosporin A appear to contain only the novel cleavage fragment of eIF4GI and to lack those characteristic of cells treated with anti-Fas antiserum. These data suggest that caspase-8 activation is not required for apoptosis and eIF4G cleavage mediated by proteasome inhibitors and immunosuppressants in human T cells.     

Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

DNA damage activated by Adriamycin (ADR) promotes ubiquitin–proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7^ADR) cells, protein ubiquitylation is significantly reduced compared to the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the ubiquitylation pattern observed in MCF7 or MCF7^ADR cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7^ADR cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7^ADR cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or β-tubulin. Only the proteasome inhibitor MG132, but not the autophagy inhibitor chloroquine partially recovers the levels of total protein ubiquitylation in MCF7^ADR cells. p53 ubiquitylation is markedly increased in MCF7^ADR cells after proteasome inhibition or a short treatment with the isopeptidase inhibitor PR619, suggesting an active role of these enzymes in the regulation of this tumor suppressor. Notably, MG132 alone increases apoptosis of MCF7^ADR and multidrug resistant ovarian cancer A2780DR1 and A2780DR2 cells. Altogether, our results highlight the use of ubiquitylation defects to predict resistance to ADR and underline the potential of proteasome inhibitors to treat these chemoresistant cells.     

Neobavaisoflavone sensitizes apoptosis via the inhibition of metastasis in TRAIL-resistant human glioma U373MG cells

Aims

Neobavaisoflavone (NBIF), an isoflavone isolated from Psoralea corylifolia (Leguminosae), has striking anti-inflammatory and anti-cancer effects. NBIF inhibits the proliferation of prostate cancer in vitro and in vivo.

Main methods

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a key endogenous molecule that selectively induces apoptosis in cancer cells with little or no toxicity in normal cells. However, some cancer cells, including U373MG cells, are resistant to TRAIL-mediated apoptosis. We demonstrated that the cell viability, migration and invasion assay were used in U373MG glioma cells.

Key findings

In this study, we found that NBIF sensitizes human U373MG glioma cells to TRAIL-mediated apoptosis. Co-treatment of TRAIL and NBIF effectively induced Bid cleavage and activated caspases 3, 8, and 9. Importantly, DR5 expression was upregulated by NBIF. We also observed that the combination NBIF and TRAIL increased expression of BAX. We further demonstrate that NBIF induced TRAIL-mediated apoptosis in human glioma cells by suppressing migration and invasion, and by inhibiting anoikis resistance.

Significance

Taken together, our results suggest that NBIF reduces the resistance of cancer cells to TRAIL and that the combination of NBIF and TRAIL may be a new therapeutic strategy for treating TRAIL-resistant glioma cells.     

Lamotrigine Attenuates Proteasome Inhibition-Induced Apoptosis by Suppressing the Activation of the Mitochondrial Pathway and the Caspase-8- and Bid-Dependent Pathways

Proteasome impairment has been shown to be involved in neuronal degeneration. Antiepileptic lamotrigine has been demonstrated to have a neuroprotective effect. However, the effect of lamotrigine on the proteasome inhibition-induced neuronal cell death has not been studied. Therefore, we assessed the effect of lamotrigine on the proteasome inhibition-induced neuronal cell apoptosis in relation to cell death process using differentiated PC12 cells and SH-SY5Y cells. The proteasome inhibitors MG132 and MG115 induced a decrease in the levels of Bid and Bcl-2 proteins, an increase in the levels of Bax and p53, loss of the mitochondrial transmembrane potential, cytochrome c release and activation of caspases (-8, -9 and -3). The addition of lamotrigine reduced the proteasome inhibitor-induced changes in the apoptosis-related protein levels, production of reactive oxygen species, depletion and oxidation of glutathione (GSH), and cell death in both cell lines. Lamotrigine and N-acetylcysteine alone did not affect the levels of 26S proteasome and activity of 20S proteasome. MG132 did not alter the levels of 26S proteasome but decreased activity of 20S proteasome. Lamotrigine and N-acetylcysteine attenuated MG132-induced decrease in the activity of 20S proteasome. The results show that lamotrigine appears to suppress the proteasome inhibitor-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The suppressive effect of lamotrigine appears to be associated with its inhibitory effect on the production of reactive oxygen species, the depletion and oxidation of GSH and the activity reduction of 20S proteasome.     

Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins

We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.