Quercetin sensitizes pancreatic cancer cells to TRAIL-induced apoptosis through JNK-mediated cFLIP turnover

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that can selectively kill cancer cells. Nonetheless, many cancers are resistant to TRAIL, and the molecular mechanisms of TRAIL resistance in cancer, particularly pancreatic cancer, are still unclear. In this study, we tested the hypothesis that quercetin, a flavonoid, induces apoptosis in TRAIL-resistant pancreatic cancer cells. Although quercetin alone had no significant cytotoxic effect, when combined with TRAIL, it promoted TRAIL-induced apoptosis that required mitochondrial outer membrane permeabilization. A BH3-only protein BID knockdown dramatically attenuated TRAIL/quercetin-induced apoptosis. The expression levels of cellular FLICE-like inhibitory protein (cFLIP) decreased in a dose-dependent manner in the presence of quercetin, and overexpression of cFLIP was able to robustly rescue pancreatic cancer cells from TRAIL/quercetin-induced apoptosis. Additionally, quercetin activated c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which in turn induced the proteasomal degradation of cFLIP, and JNK activation also sensitized pancreatic cancer cells to TRAIL-induced apoptosis. Thus, our results suggest that quercetin induces TRAIL-induced apoptosis via JNK activation-mediated cFLIP turnover.     

TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects.     

Acrolein sensitizes human renal cancer Caki cells to TRAIL-induced apoptosis via ROS-mediated up-regulation of death receptor-5 (DR5) and down-regulation of Bcl-2

TRAIL resistance in many cancer cells is one of the major problems in TRAIL-based cancer therapy. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are strictly needed for the improvement of anti-cancer effect of TRAIL. Acrolein is a byproduct of lipid peroxidation, which has been involved in pulmonary, cardiac and neurodegenerative diseases. We investigated whether acrolein, an α,β-unsaturated aldehyde, can potentiate TRAIL-induced apoptosis in human renal cancer cells. The combined treatment with acrolein and TRAIL significantly induced apoptosis, and stimulated of caspase-3 activity, DNA fragmentation, and cleavage of PARP. We found that acrolein down-regulated the protein level of Bcl-2 and Bcl-2 overexpression inhibited the cell death induced by the combined treatment with acrolein and TRAIL. In addition, acrolein up-regulated C/EBP homologous protein (CHOP) and TRAIL death receptor 5 (DR5) and down-regulation of CHOP or DR5 expression using the respective small interfering RNA significantly attenuated the apoptosis induced by acrolein plus TRAIL. Interestingly, pretreatment with an antioxidant, N-acetylcysteine (NAC), inhibited not only CHOP and DR5 up-regulation but also the cell death induced by acrolein plus TRAIL. Taken together, our results demonstrated that acrolein enhances TRAIL-induced apoptosis in Caki cells through down-regulation of Bcl-2 and ROS dependent up-regulation of DR5.     

Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

Phenethyl isothiocyanate sensitizes glioma cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Here we show that in combination with phenethyl isothiocyanate (PEITC), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Subtoxic concentrations of PEITC significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. PEITC dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. PEITC enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-induced-p53.     

Caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) offers promising therapeutic potential based on its ability to induce apoptosis in various cancer cell lines without obvious adverse effect to normal cells. However, the mechanism of the differential sensitivity towards TRAIL-induced apoptosis remains unclear. Here, we demonstrate that caveolin-1 directly regulated TRAIL-induced apoptosis in HepG2 cells. ShRNA-mediated caveolin knockdown sensitized TRAIL-induced apoptosis and disruption of caveolae structure by the cholesterol-extracting reagent, methyl-β-cyclodextrin (MCD), enhanced TRAIL-induced apoptosis. Over-expression of caveolin-1 partially blocked TRAIL-induced apoptosis. The engagement of TRAIL with its receptor DR4 reduced the localization of DR4 in caveolae and resulted in its internalization. Blockade of caveolae-mediated internalization of DR4 by filipin III effectively enhanced TRAIL-induced apoptosis. Collectively, our results reveal a new mechanism by which caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells.     

2-Deoxyglucose sensitizes melanoma cells to TRAIL-induced apoptosis which is reduced by mannose

While melanoma cell lines use aerobic glycolysis, addition of a competitive inhibitor such as 2-deoxyglucose (2DG) by itself achieved only modest killing. To overcome high levels of pro-survival proteins in melanoma cells, 2DG or glucose deprivation (GD) was combined with tumor necrosis factor-related apoptosis inducing-ligand (TRAIL). TRAIL treatment by itself also only induced modest killing, but combining TRAIL with 2DG or GD triggered a synergistic pro-apoptotic response in melanoma lines but not melanocytes. In melanoma cells, there was cleavage of caspases 3, 8 and Bid. Killing by combination treatments was completely blocked by a pan-caspase inhibitor, z-VAD. Mechanistically, 2DG and GD enhanced surface levels for both death receptors (DR4 and DR5); which was accompanied by reductions in levels of Mcl-1, Bcl-2 and survivin. Mannose pre-treatment reduced enhanced killing by combination treatments, accompanied by reduced DR5 levels. These results indicate melanoma cells in which there is altered glucose-related metabolomics can be exploited by interfering with glucose metabolism in combination with TRAIL; thereby overcoming the notorious death resistance of melanoma. Thus, a new therapeutic window is open for future clinical trials using agents targeting the glucose-related metabolome, in combination with agents triggering death receptors in patients with melanoma.     

Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) shows promise as a chemotherapeutic agent. However, many human cancer cells are resistant to killing by TRAIL. We have previously demonstrated that reovirus infection increases the susceptibility of human lung (H157) and breast (ZR75-1) cancer cell lines to TRAIL-induced apoptosis. We now show that reovirus also increases the susceptibility of human ovarian cancer cell lines (OVCAR3, PA-1 and SKOV-3) to TRAIL-induced apoptosis. Reovirus-induced increases in susceptibility of OVCAR3 cells to TRAIL require virus uncoating and involve increased activation of caspases 3 and 8. Reovirus infection results in the down-regulation of cFLIP (cellular FLICE inhibitory protein) in OVCAR3 cells. Down-regulation of cFLIP following treatment of OVCAR3 cells with antisense cFLIP oligonucleotides or PI3 kinase inhibition also increases the susceptibility of OVCAR3 cells to TRAIL-induced apoptosis. Finally, over-expression of cFLIP blocks reovirus-induced sensitization of OVCAR3 cells to TRAIL-induced apoptosis. The combination of reovirus and TRAIL thus represents a promising new therapeutic approach for the treatment of ovarian cancer.     

ROS-dependent phosphorylation of Bax by wortmannin sensitizes melanoma cells for TRAIL-induced apoptosis

The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive. Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways. Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression. The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns. Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced. Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here. Characteristically, production of ROS was seen early in response to wortmannin and MK-2206. Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation. This correlated with reduced Bax phosphorylation at threonine-167. The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation. The pathway may be considered for proapoptotic, anticancer strategies.     

Triptolide sensitizes TRAIL-induced apoptosis in prostate cancer cells via p53-mediated DR5 up-regulation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy. However, a number of prostate cancer cells exhibit high resistance to TRAIL effect. In this study, we found that Triptolide, a Chinese medicine, significantly sensitizes prostate cancer cells to TRAIL-mediated cellular apoptosis by up-regulating DR5 expression. Triptolide treatment can suppress Akt/Hdm2 signaling pathway, and lead to p53 accumulation, thereby up-regulating DR5 expression. Taken together, all evidences indicate that Triptolide may become a promising therapeutic agent that prevents the progression of prostate cancer.     

Targeting glutamine metabolism sensitizes melanoma cells to TRAIL-induced death

Targeting specific metabolic pathways has emerged for cancer therapeutics. For melanoma, metabolic studies have solely focused on high glucose uptake. By contrast, little is known regarding addiction to glutamine. Using five melanoma lines and two normal cell types, addition of aminooxyacetate (AOA), an inhibitor of glutamate-dependent transaminase regulating glutaminolytic pathway, two lines underwent low levels of apoptosis (>30%), while the other three lines were resistant, as were normal cells to AOA. However, three resistant lines (but not normal cells), became sensitized to undergoing apoptosis when TRAIL was combined with AOA. TRAIL by itself had minimal effects on all cell lines and normal cells, and did not augment AOA-induced killing in the two sensitive melanoma lines. AOA plus TRAIL induced a caspase-dependent apoptotic response. AOA did not influence TRAIL DR4 or DR5 cell surface death receptor levels, but AOA enhanced pro-apoptotic protein levels of Noxa, while reducing pro-survival protein Mcl-1. To verify AOA was targeting glutamine pathway, depletion of glutamine produced similar results, because absence of glutamine sensitized three melanoma lines, but not fibroblasts to killing by TRAIL. Glutamine depletion also led to Noxa induction. These results indicate some lines are addicted to glutamine, and treatment with AOA or glutamine depletion sensitizes melanoma to TRAIL-mediated killing, while sparing normal cells. Future studies are indicated to translate these discoveries to metastatic melanoma as there is currently no treatment available to prolong survival.     

RAC3 down-regulation sensitizes human chronic myeloid leukemia cells to TRAIL-induced apoptosis

The nuclear receptor coactivator RAC3 plays important roles in many biological processes and tumorigenesis. We found that RAC3 is over-expressed in human chronic myeloid leukemia cells K562, which are normally resistant to TRAIL-induced apoptosis. RAC3 down-regulation by siRNA rendered these cells sensitive to TRAIL-induced cell death. In addition to the up-regulation of TRAIL receptors, the process involves Bid, caspases and PARP activation, loss of mitochondrial membrane potential, and release of AIF, cytochrome c and Smac/DIABLO to the cytoplasm. We conclude that RAC3 is required for TRAIL resistance and that this anti-apoptotic function is independent of its role in hormone receptor signaling.     

Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis

In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

Sub-toxic dose of apigenin sensitizes HepG2 cells to TRAIL through ERK-dependent up-regulation of TRAIL receptor DR5

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a promising candidate for anticancer therapy due to its selective toxicity to cancer cells. Nevertheless, because of TRAIL resistance in some cancer cells, combined treatment with sensitizing agents is required to enhance the anticancer potential of TRAIL. In this study, we investigated the underlying mechanism of apigenin-induced sensitization of HepG2 cells to TRAIL-induced cell death. Synergistic induction of apoptosis by combination was confirmed by examining the typical morphology changes of apoptosis, PARP-cleavage, and activation of effector caspases. Z-VAD-fmk, a pan-caspase inhibitor, inhibited the enhanced cell death by combined treatment of apigenin and TRAIL, demonstrating that a caspase-dependent pathway is involved in apigenin/TRAIL-mediated apoptosis. In addition, we found that apigenin/ TRAIL co-treatment up-regulates DR5 cell surface expression. The synergistic induction of cell death by the apigenin/ TRAIL combination was significantly attenuated by DR5 blocking chimera antibody. Next, using pharmacological inhibitors, we found that ERK activation is involved in the induction of DR5 expression. Inhibition of ERK1/2 by U0126 significantly decreased the apigenin/TRAIL-induced DR5 expression and apoptosis. Taken together, our results indicate that apigenin can enhance the apoptotic effect of TRAIL via ERK-induced up-regulation of DR5.