Expression of anti-apoptotic factors modulates Apo2L/TRAIL resistance in colon carcinoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) selectively induces apoptosis in transformed cells. Normal cells and certain tumor cells can evade Apo2L/TRAIL induced cell death, but the determinants of Apo2L/TRAIL sensitivity are poorly understood. To better understand the factors that contribute to Apo2L/TRAIL resistance, we characterized two colon carcinoma lines with pronounced differences in Apo2L/TRAIL sensitivity. Colo205 cells are highly sensitive to Apo2L/TRAIL whereas Colo320 cells are unresponsive. Components of the DISC (death inducing signaling complex) could be immunoprecipitated from both cell lines in response to Apo2L/TRAIL. Sensitizing agents including a proteasome inhibitor conferred Apo2L/TRAIL sensitivity in Colo320 cells, indicating that the apoptotic machinery was intact and functional. We specifically suppressed the expression of Bcl-2, FLIP or XIAP in Colo320 cells. Downregulation of either FLIP or XIAP but not Bcl-2 restored sensitivity of Colo320 cells to Apo2L/TRAIL. Moreover, stable knockdown of XIAP expression in Colo320 subcutaneous tumors resulted in suppression of tumor growth and sensitivity to Apo2L/TRAIL in vivo. Our results indicate that only a specific subset of anti-apoptotic proteins can confer resistance to Apo2L/TRAIL in Colo320 cells. Elucidation of the factors that contribute to Apo2L/TRAIL resistance in tumor cells may provide insight into combination therapies with Apo2L/TRAIL in a clinical setting.     

Sensitization of prostate carcinoma cells to Apo2L/TRAIL by a Bcl-2 family protein inhibitor

Overexpression of anti-apoptotic Bcl-2 family proteins may play an important role in the aggressive behavior of prostate cancer cells and their resistance to therapy. The Bcl-2 homology 3 domain (BH3) is a uniquely important functional element within the pro-apoptotic class of the Bcl-2-related proteins, mediating their ability to dimerize with other Bcl-2-related proteins and promote apoptosis. The BH3 inhibitors (BH3Is) function by disrupting the interactions mediated by the BH3 domain between pro- and anti-apoptotic members of the Bcl-2 family and liberating more Bax/Bak to induce mitochondrial membrane permeabilization. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to non-tagged, human recombinant soluble Apo2 ligand [Apo2L, also Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL], a tumor specific drug that is now in clinical trials. However, when Apo2L/TRAIL was combined with the Bcl-xL inhibitor, BH3I-2′, it induced apoptosis synergistically through activation of Caspase-8 and the proapoptotic Bcl-2 family member Bid, resulting in the activation of effector Caspase-3 and proteolytic cleavage of Poly(ADP-ribose) polymerase, events that were blocked by the pan-caspase inhibitor zVAD-fmk. Our data indicate that, in combination with the BH3 mimetic, BH3I-2′, Apo2L/TRAIL synergistically induces apoptosis in C4-2 human prostate cancer cells through both the extrinsic and intrinsic apoptotic pathways.     

Rapid and profound potentiation of Apo2L/TRAIL-mediated cytotoxicity and apoptosis in thoracic cancer cells by the histone deacetylase inhibitor Trichostatin A: the essential role of the mitochondria-mediated caspase activation cascade

Apo2L/TRAIL is actively investigated as a novel targeted agent to directly induce apoptosis of susceptible cancer cells. Apo2L/TRAIL-refractory cells can be sensitized to the cytotoxic effect of this ligand by cytotoxic chemotherapeutics. The aim of this study was to evaluate the in vitro tumoricidal activity of the Apo2L/TRAIL + Trichostatin A in cultured thoracic cancer cells and to elucidate the molecular basis of the synergistic cytotoxicity of this combination. Concurrent exposure of cultured cancer cells to sublethal concentrations of Apo2L/TRAIL and Trichostatin A resulted in profound enhancement of Apo2L/TRAIL-mediated cytotoxicity in all cell lines regardless of their intrinsic susceptibility to this ligand. This combination was not toxic to primary normal cells. While Apo2L/TRAIL alone or Trichostatin A alone mediated < 20% cell death, 60 to 90% of cancer cells were apoptotic following treatment with TSA + Apo2L/TRAIL combinations. Complete translocation of Bax from the cytosol to the mitochondria compartment was mainly observed in combination-treated cells and this was correlated with robust elevation of caspase 9 proteolytic activity indicative of activation of the mitochondria apoptogenic effect. Profound TSA + Apo2L/TRAIL–mediated cytotoxicity and apoptosis were completely abrogated by either Bcl2 over-expression or by the selective caspase 9 inhibitor, highlighting the essential role of mitochondria-dependent apoptosis signaling cascade in this process. Moreover, increased caspase 8 activity observed in cells treated with the TSA + Apo2L/TRAIL combination was completely suppressed by Bcl-2 over-expression or by the selective caspase 9 inhibitor indicating that the elevated caspase 8 activity in combination-treated cells was secondary to a mitochondria-mediated amplification feedback loop of caspase activation. These finding form the basis for further development of HDAC inhibitors + Apo2L/TRAIL combination as novel targeted therapy for thoracic malignancies. R.M. Reddy and W.-S. Yeow contributed equally to this work. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH.     

Death to the bad guys: Targeting cancer via Apo2L/TRAIL

All higher organisms consist of an ordered society of individual cells that must communicate to maintain and regulate their functions. This is achieved through a complex but highly regulated network of hormones, chemical mediators, chemokines and other cytokines, acting as ligands for intra or extra-cellular receptors. Ligands and receptors of the tumor necrosis factor (TNF) superfamilies are examples of signal transducers, whose integrated actions influence the development, homeostasis and adaptive responses of many cells and tissue types. Apo2L/TRAIL is one of several members of the tumour necrosis factor superfamily that induce apoptosis through the engagement of death receptors. Apo2L/TRAIL interacts with an unusually complex receptor system, which in humans comprises two death receptors and three decoy receptors. This molecule has received considerable attention recently because of the finding that many cancer cell types are sensitive to Apo2L/TRAIL-induced apoptosis, while most normal cells appear to be resistant to this action of Apo2L/TRAIL. In this review, we specifically emphasise on the actions of Apo2L/TRAIL with respect to its apoptotic signaling pathways and summarise what is known about its physiological role. The potential therapeutic usefulness of Apo2L/TRAIL, especially in combination with chemotherapeutic agents, is also discussed in some detail.     

1'-Acetoxychavicol acetate induces apoptosis of myeloma cells via induction of TRAIL

A component of a traditional Thai condiment, 1'-acetoxychavicol acetate (ACA), is a natural compound, and it is obtained from rhizomes of the ethno-medicinal plant Languas galanga (Zingiberaceae). Our previous studies showed that ACA dramatically inhibited cellular growth of multiple myeloma cells in vivo and in vitro through the induction of apoptosis in association with the activation of caspase-8, inactivation of NF-kappaB, and down-regulation of anti-apoptotic proteins. Subsequently, we investigated the detailed apoptotic pathway of ACA and further demonstrated that ACA up-regulates the expression of both TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) and TRAIL receptor death receptor 5 (DR5). In addition, TRAIL/R-Fc chimera neutralizes the ACA-induced apoptosis. These results suggest that the death signaling of TRAIL is involved in the ACA-induced apoptosis of myeloma cells, and provide a rationale for the induction of TRAIL/Apo2L by ACA, which could potentially be used as a novel therapeutic agent in patients with multiple myeloma.     

Induction of necrotic tumor cell death by TRAIL/Apo-2L

A great deal of enthusiasm is being generated for TRAIL (TNF-related apoptosis-inducing ligand)/Apo-2L as a tumor therapeutic agent because it is cytotoxic to a variety of tumor cell types but not normal cells. Moreover, it is well documented that TRAIL/Apo-2L-induced tumor cell death is a caspase-dependent apoptotic process. Through the use of a transfected cell line expressing murine TRAIL/Apo-2L and a recombinant adenovirus encoding the murine TRAIL/Apo-2L cDNA (Ad5-mTRAIL) against two murine tumor cell lines [TRAMP-C2 (prostate adenocarcinoma) and Renca (renal adenocarcinoma)], we found that mTRAIL/Apo-2L also can kill tumor cells by inducing necrosis. Specifically, we observed the default method of mTRAIL/Apo-2L-induced death in TRAMP-C2 cells was via a necrotic process, characterized by the complete lack of an annexin V⁺/PI^– population, SAPK/JNK phosphorylation, caspase activation, Bid cleavage, or cytochrome c release. Moreover, the inclusion of zVAD-fmk, an inhibitor of caspase activation, markedly enhanced mTRAIL/Apo-2L-mediated killing of TRAMP-C2. In contrast, apoptosis was induced in TRAMP-C2 using TNF, as measured by the criteria listed above, as was Renca by mTRAIL/Apo-2L. These results demonstrate the natural occurrence of both TRAIL/Apo-2L-induced apoptotic and necrotic signaling mechanisms within tumor cells.     

Wnt-expressing rat embryonic fibroblasts suppress Apo2L/TRAIL-induced apoptosis of human leukemia cells

Wnt signaling enhances cell proliferation and the maintenance of hematopoietic cells. In contrast, cytotoxic ligand Apo2L/TRAIL induces the apoptosis of various transformed cells. We observed that co-culture of human pre-B leukemia cells KM3 and REH with Wnt1- or Wnt3a-producing rat embryonic fibroblasts efficiently suppressed Apo2L/TRAIL-induced apoptosis of the lymphoid cells. This suppression occurs at the early stages of the Apo2L/TRAIL apoptotic cascade and, interestingly, the activation of the Wnt pathway alone in human leukemia cells is not sufficient for their full anti-apoptotic protection. We hypothesize that a stimulus emanating specifically from Wnt1- or Wnt3a-expressing rat fibroblasts is responsible for the observed resistance to Apo2L/TRAIL. This anti-apoptotic signaling was significantly hampered by the inhibition of the MEK1/ERK1/2 or NFκB pathways in KM3 and REH cells. Our results imply that paracrine Wnt-related signals could be important for the survival of pre-B cell-derived malignancies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. L. Doubravská and Š. Šímová are joint first authors. V. Kořínek and L. Anděra are senior co-authors.     

Blockade of preS2 down-regulates the apoptosis of HepG2.2.15 cells induced by TRAIL

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Our previous studies showed that both the whole HBV genome and its HBx protein enhanced TRAIL-induced hepatocyte apoptosis. We report here that preS2-containing viral proteins are also potent regulators of TRAIL-induced apoptosis. HBV-transfected hepatoma cell line, HepG2.2.15, pretreated with antisense oligonucleotide against preS2 gene, showed a lower sensitivity towards TRAIL-induced apoptosis. However, this effect might not be related with the expression level of TRAIL receptors. These results establish that besides HBx, preS2 viral proteins are also involved in enhancing TRAIL-induced hepatocyte apoptosis. The novel role of preS2 would be useful to further unravel the mechanisms of imbalanced apoptosis during HBV infection and provides a potential therapeutic target for HBV-related diseases.     

Multiple effects of TRAIL in human carcinoma cells: induction of apoptosis, senescence, proliferation, and cytokine production

TRAIL is a death ligand that induces apoptosis in malignant but not normal cells. Recently the ability of TRAIL to induce proliferation in apoptosis-resistant normal and malignant cells was reported. In this study, we analyzed TRAIL effects in apoptosis sensitive MCF7, OVCAR3 and H460 human tumor cell lines. TRAIL at low concentrations preferentially induced cell proliferation. At 100 ng/ml, apoptotic death was readily observed, however surviving cells acquired higher proliferative capacity. TRAIL-stimulated production of several cytokines, IL-8, RANTES, MCP-1 and bFGF, and activation of caspases 1 and 8 was essential for this effect. Antibodies to IL-8, RANTES, and bFGF blocked TRAIL-induced cell proliferation and further stimulated apoptosis. For the first time, we report that high TRAIL concentrations induced cell senescence as determined by the altered morphology and expression of several senescence markers: SA-beta-gal, p21Waf1/Cip1, p16INK4a, and HMGA. Caspase 9 inhibition protected TRAIL-treated cells from senescence, whereas inhibition of caspases 1 and 8 increased the yield of SLP cells. In conclusion, in cultured human carcinoma cells, TRAIL therapy results in three functional outcomes, apoptosis, proliferation and senescence. TRAIL-induced proapoptotic and prosurvival responses correlate with the strength of signaling. TRAIL-induced cytokine production is responsible for its proliferative and prosurvival effects.     

ALS2CR7 (CDK15) attenuates TRAIL induced apoptosis by inducing phosphorylation of survivin Thr34

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising agent for medical applications because it induces apoptosis selectively in a variety of cancer cells without toxicity to normal human cells. However, its therapeutic potential has been limited by the existence of several cancer cells with TRAIL resistance. TRAIL resistance results from a variety of mechanisms, which occur at various points in the cellular signaling pathways. In this study, we demonstrate that ALS2CR7 (CDK15) can mediate resistance to TRAIL. We also demonstrate that cell viability of TRAIL sensitive HCT116 and MDA-MB-231 cells increased after TRAIL treatment in ALS2CR7 transfected cancer cells compared with vector transfected cancer cells. Furthermore, cell viability was decreased by TRAIL treatment after knockdown with ALS2CR7 siRNA in TRAIL resistant HT29 and MCF-7 cells. We also show that the activated form of apoptotic proteins such as caspase-3, -8 and -9 and PARP increased after TRAIL treatment in the control group, but decreased in the ALS2CR7 transfected group. The expression of survival proteins such as bcl2 and survivin in TRAIL sensitive cancer cells increased in the ALS2CR7 transfected group, but decreased in TRAIL resistant cancer cells treated with ALS2CR7 siRNA. Other survival proteins such as FLIP and XIAP were not affected. ALS2CR7 appears to bind with only survivin, and not bcl2. The phospho-survivin (Thr34) critical in drug resistance was increased by transfection with ALS2CR7, but the expression of death receptors such as DR4 and DR5 was not affected. ALS2CR7 did not bind with any of the death receptors in our study. In summary, our results suggest that ALS2CR7 confers TRAIL resistance to cancer cells via phosphorylation of survivin.     

Membrane expression of DR4, DR5 and caspase-8 levels, but not Mcl-1, determine sensitivity of human myeloma cells to Apo2L/TRAIL

The improved recombinant form of the death ligand Apo2L/TRAIL (Apo2L/TRAIL.0) is not cytotoxic for normal human cells and is a good candidate for the therapy of multiple myeloma (MM), a B-cell neoplasia that remains incurable. We have analyzed the molecular determinants of myeloma sensitivity to Apo2L/TRAIL.0 in a number of MM cell lines, the mechanisms of resistance and a possible way of overcoming it. Expression of one death receptor for Apo2L/TRAIL (DR4 or DR5) is sufficient to transduce death signals, though DR5 was more efficient when both receptors were present. Membrane expression of decoy receptors (DcR1, DcR2) and intracellular levels of c-FLIP(L), XIAP and Mcl-1 were not predictive of resistance to Apo2L/TRAIL. Inhibition of Mcl-1 degradation did not prevent Apo2L/TRAIL-induced apoptosis. In IM-9 cells, resistance was associated to a reduced caspase-8 expression. U266 cells, though expressing significant levels of DR4 and caspase-8, were nevertheless resistant to Apo2L/TRAIL. This resistance could be overcome by co-treatment with valproic acid (VPA), a histone deacetylase inhibitor. VPA caused the redistribution of DR4 to plasma membrane lipid rafts and restored DR4 signaling. Overexpression of Mcl-1 in U266 cells did not prevent Apo2L/TRAIL cytotoxicity in VPA-sensitized cells. These results, taken together, support the possible use of Apo2L/TRAIL.0 in the treatment of MM.     

Curcumin inhibits interferon-alpha induced NF-kappaB and COX-2 in human A549 non-small cell lung cancer cells

The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-alpha treatment. The IFN-alpha-treated A549 cells showed increase in protein expression levels of NF-kappaB and COX-2. IFN-alpha induced NF-kappaB binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-alpha-induced COX-2 expression in A549 cells. Within 10 min, IFN-alpha rapidly induced the binding activity of a gamma-(32)P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-alpha-induced activations of NF-kappaB and COX-2 were inhibited by the addition of curcumin in A549 cells.     

TRAIL包涵体在细胞内重折叠现象的初步研究

肿瘤坏死因子相关凋亡诱导配体（Ap02L／TRAIL）是一种新型的抗肿瘤蛋白。但在大肠杆菌表达系统中,表达的TRAIL蛋白往往容易聚集形成包涵体。本文对重组TRAIL包涵体在胞内重折叠转化成可溶性TRAIL蛋白作了初步探索,结果表明：当蛋白表达后,迅速降低温度至30℃并添加50μg／mL氯霉素继续培养4h,可溶性TRAIL产量可提高至16．7％。这一结果在3．7L罐上也得到了证实,可溶性TRAIL表达量达到14．5％。     

Sulindac-derived reactive oxygen species induce apoptosis of human multiple myeloma cells via p38 mitogen activated protein kinase-induced mitochondrial dysfunction

Non-steroidal anti-inflammatory drugs are well known to induce apoptosis of cancer cells independent of their ability to inhibit cyclooxygenase-2, but the molecular mechanism for this effect has not yet been fully elucidated. The purpose of this study was to elucidate the potential signaling components underlying sulindac-induced apoptosis in human multiple myeloma (MM) cells. We found that sulindac induces apoptosis by promoting ROS generation, accompanied by opening of mitochondrial permeability transition pores, release of cytochrome c and apoptosis inducing factor from mitochondria, followed by caspase activation. Bcl-2 cleavage and down-regulation of the inhibitor of apoptosis proteins (IAPs) family including cIAP-1/2, XIAP, and survivin, occurred downstream of ROS production during sulindac-induced apoptosis. Forced expression of survivin and Bcl-2 blocked sulindac-induced apoptosis. Most importantly, sulindac-derived ROS activated p38 mitogen-activated protein kinase and p53. SB203580, a p38 mitogen-activated protein kinase inhibitor, and RNA inhibition of p53 inhibited the sulindac-induced apoptosis. Furthermore, p53, Bax, and Bak accumulated in mitochondria during sulindac-induced apoptosis. All of these events were significantly suppressed by SB203580. Our results demonstrate a novel mechanism of sulindac-induced apoptosis in human MM cells, namely, accumulation of p53, Bax, and Bak in mitochondria mediated by p38 MAPK activation downstream of ROS production.     

Plasminogen activator urokinase expression reveals TRAIL responsiveness and supports fractional survival of cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10/Apo2L) holds promise for cancer therapy as it induces apoptosis in a large variety of cancer cells while exerting negligible toxicity in normal ones. However, TRAIL can also induce proliferative and migratory signaling in cancer cells resistant to apoptosis induced by this cytokine. In that regard, the molecular mechanisms underlying the tumor selectivity of TRAIL and those balancing apoptosis versus survival remain largely elusive. We show here that high mRNA levels of PLAU, which encodes urokinase plasminogen activator (uPA), are characteristic of cancer cells with functional TRAIL signaling. Notably, decreasing uPA levels sensitized cancer cells to TRAIL, leading to markedly increased apoptosis. Mechanistic analyses revealed three molecular events taking place in uPA-depleted cells: reduced basal ERK1/2 prosurvival signaling, decreased preligand decoy receptor 2 (DcR2)-death receptor 5 (DR5) interaction and attenuated recruitment of DcR2 to the death-inducing signaling complex upon TRAIL challenge. These phenomena were accompanied by increased FADD and procaspase-8 recruitment and processing, thus guiding cells toward a caspase-dependent cell death that is largely independent of the intrinsic apoptosis pathway. Collectively, our results unveil PLAU mRNA levels as marker for the identification of TRAIL-responsive tumor cells and highlight a key role of uPA signaling in ‘apoptosis versus survival'' decision-making processes upon TRAIL challenge.     

Identification of a potential HIV-induced source of bystander-mediated apoptosis in T cells: Upregulation of TRAIL in primary human macrophages by HIV-1 tat

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.     

The combination of TRAIL and luteolin enhances apoptosis in human cervical cancer HeLa cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for cancer therapeutics. However, some tumor cells are resistant to TRAIL-induced apoptosis. Our previous studies have shown that luteolin, a naturally occurring flavonoid, induces the up-regulation of death receptor 5 (DR5), which is a receptor for TRAIL. Here, we show for the first time that luteolin synergistically acts with exogenous soluble recombinant human TRAIL to induce apoptosis in HeLa cells, but not in normal human peripheral blood mononuclear cells. The combined use of luteolin and TRAIL induced Bid cleavage and the activation of caspase-8. Also, human recombinant DR5/Fc chimera protein, caspase inhibitors, and DR5 siRNA efficiently reduced apoptosis induced by co-treatment with luteolin and TRAIL. These results raise the possibility that this combined treatment with luteolin and TRAIL might be promising as a new therapy against cancer.