TRAIL promotes membrane blebbing, detachment and migration of cells displaying a dysfunctional intrinsic pathway of apoptosis

Recently, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to be a potential candidate for cancer therapy. TRAIL induces apoptosis in various cancer cells but not in normal tissues. Here we show that HCT116 and SW480 cells with a deficient mitochondrial apoptotic pathway were resistant to TRAIL-induced apoptosis, whereas HCT116 and SW480 cells with a functional mitochondrial apoptotic pathway underwent apoptosis upon exposure to TRAIL. Surprisingly, TRAIL induced phenotypic changes in cells with a dysfunctional mitochondrial apoptotic pathway, including membrane blebbing and a transient loss of adhesion properties to the substratum. Accordingly, TRAIL stimulated the ability of these cells to migrate. This behavior was the consequence of a transient TRAIL-induced ROCK1 cleavage. In addition, we report that Bax-deficient HCT116 cells exposed to TRAIL for a prolonged period lost their sensitivity to TRAIL as a result of downregulation of TRAIL receptor expression, and became resistant to combination of TRAIL and other drugs such as MG-132 and bortezomib. These findings may have important consequences for TRAIL anti-cancer therapy.     

RelA,p50 and Inhibitor of kappa B alpha are Elevated in Human Metastatic Melanoma Cells and Respond Aberrantly to Ultraviolet Light B

Metastatic melanomas are typically resistant to radiation and chemotherapy. The underlying basis for this phenomenon may result in part from defects in apoptotic pathways. Nuclear factor kappa B (NFκB) has been shown to control apoptosis in many cell types and normally functions as an immediate stress response mechanism that is rigorously controlled by multiple inhibitory complexes. We have previously shown that NFκB binding is elevated in metastatic melanoma cells relative to normal melanocytes. In the current study, Western blot analysis showed that, compared with normal melanocytes, melanoma cell lines have higher nuclear levels of the NFκB subunits p50 (7‐fold) and RelA (5–10‐fold). In response to tumor necrosis factor‐alpha (TNFα), both melanocytes and melanoma cells showed increased nuclear p50 and RelA levels, but levels in melanoma cells remained higher than in melanocytes. We also found that melanoma cells expressed higher cytoplasmic levels of RelA, p105/p50 and the inhibitory protein, inhibitor of kappa B alpha (IκBα) than melanocytes. To directly test whether RelA expression has an impact on melanoma cell survival, we used antisense RelA phosphorothioate oligonucleotides and found that melanoma cell viability was significantly decreased compared with untreated or control cultures. The constitutive activation of NFκB in metastatic melanoma cell cultures may, therefore, support an inappropriate cell survival pathway that can be therapeutically manipulated.     

Astrocytic metabolic and inflammatory changes as a function of age

This study examines age‐dependent metabolic‐inflammatory axis in primary astrocytes isolated from brain cortices of 7‐, 13‐, and 18‐month‐old Sprague–Dawley male rats. Astrocytes showed an age‐dependent increase in mitochondrial oxidative metabolism respiring on glucose and/or pyruvate substrates; this increase in mitochondrial oxidative metabolism was accompanied by increases in COX3/18SrDNA values, thus suggesting an enhanced mitochondrial biogenesis. Enhanced mitochondrial respiration in astrocytes limits the substrate supply from astrocytes to neurons; this may be viewed as an adaptive mechanism to altered cellular inflammatory–redox environment with age. These metabolic changes were associated with an age‐dependent increase in hydrogen peroxide generation (largely ascribed to an enhanced expression of NOX2) and NFκB signaling in the cytosol as well as its translocation to the nucleus. Astrocytes also displayed augmented responses with age to inflammatory cytokines, IL‐1β, and TNFα. Activation of NFκB signaling resulted in increased expression of nitric oxide synthase 2 (inducible nitric oxide synthase), leading to elevated nitric oxide production. IL‐1β and TNFα treatment stimulated mitochondrial oxidative metabolism and mitochondrial biogenesis in astrocytes. It may be surmised that increased mitochondrial aerobic metabolism and inflammatory responses are interconnected and support the functionality switch of astrocytes, from neurotrophic to neurotoxic with age.     

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV‐2 Microglial Cells

Naegleria fowleri, a free‐living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV‐2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV‐2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL‐1α and TNF‐α. NfESP‐induced IL‐1α and TNF‐α expression in BV‐2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP‐induced IL‐1α and TNF‐α production in BV‐2 cells were effectively downregulated by inhibition of NF‐kB and AP‐1. These results collectively suggest that NfESP stimulates BV‐2 cells to release IL‐1α and TNF‐α via NF‐kB‐ and AP‐1‐dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.     

Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor

Although chemotherapeutic drugs could theoretically target all metastatic sites, current treatments do not provide complementary therapeutics. Therefore, the development of an alternative approach replacing the traditional therapy is urgently needed. To assess the killing efficiency of the functionally identified apoptosis-related protein (APR)-1 in melanoma cells, we established a system for the regulated expression of APR-1. The induction of APR-1 expression caused apoptosis of melanoma cells via the interaction with the juxtamembrane region of p75 neurotrophin receptor (p75NTR), and possible also via the competition with tumour necrosis factor receptor-associated factor-6 (TRAF6) and the catalytic receptor of neurotrophin (Trk) for the same p75NTR interacting site. The accumulation of APR-1 in melanoma cells may block the physical association of p75NRT with TRAF6 and/or Trk, leading to the disruption of both NF-κB and extracellular signal-regulated kinase (ERK) pathways. Also, accumulation of APR-1 protein enhanced the activity of both c-Jun-N-terminal kinase (JNK) and p38 pathways. However, the analysis of APR-1-modulated pathways demonstrated the involvement of apoptosis-regulating kinase 1-JNK/p38 pathway in the induction of Bax expression leading to both mitochondrial dysregulation [as demonstrated by the loss of mitochondrial membrane potential, the release of both cytochrome c and apoptosis-inducing factor into cytoplasm, and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP)] and endoplasmic reticulum stress as demonstrated by the increase of intracellular Ca(2+) release. Thus, besides the analysis of its pro-apoptotic function, our data provide insight into the molecular mechanism of APR-1-induced apoptosis of melanoma cells.     

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.     

IL‐27 Rα+ cells promoted allorejection via enhancing STAT1/3/5 phosphorylation

Recently, emerging evidence strongly suggested that the activation of interleukin‐27 Receptor α (IL‐27Rα) could modulate different inflammatory diseases. However, whether IL‐27Rα affects allotransplantation rejection is not fully understood. Here, we investigated the role of IL‐27Rα on allorejection both in vivo and in vitro. The skin allotransplantation mice models were established, and the dynamic IL‐27Rα/IL‐27 expression was detected, and IL‐27Rα⁺ spleen cells adoptive transfer was performed. STAT1/3/5 phosphorylation, proliferation and apoptosis were investigated in mixed lymphocyte reaction (MLR) with recombinant IL‐27 (rIL‐27) stimulation. Finally, IFN‐γ/ IL‐10 in graft/serum from model mice was detected. Results showed higher IL‐27Rα/IL‐27 expression in allografted group compared that syngrafted group on day 10 (top point of allorejection). IL‐27Rα⁺ spleen cells accelerated allograft rejection in vivo. rIL‐27 significantly promoted proliferation, inhibited apoptosis and increased STAT1/3/5 phosphorylation of alloreactive splenocytes, and these effects of rIL‐27 could be almost totally blocked by JAK/ STAT inhibitor and anti‐IL‐27 p28 Ab. Finally, higher IL‐27Rα⁺IFN‐γ⁺ cells and lower IL‐27Rα⁺IL‐10⁺ cells within allografts, and high IFN‐γ/low IL‐10 in serum of allorejecting mice were detected. In conclusion, these data suggested that IL‐27Rα⁺ cells apparently promoted allograft rejection through enhancing alloreactive proliferation, inhibiting apoptosis and up‐regulating IFN‐γ via enhancing STAT pathway. Blocking IL‐27 pathway may favour to prevent allorejection, and IL‐27Rα may be as a high selective molecule for targeting diagnosis and therapy for allotransplantation rejection.     

Cytokine induction of tumor necrosis factor receptor 2 is mediated by STAT3 in colon cancer cells

The IL-6/STAT3 and TNFα/NFκB pathways are emerging as critical mediators of inflammation-associated colon cancer. TNF receptor (TNFR) 2 expression is increased in inflammatory bowel diseases, the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer, and by combined interleukin (IL) 6 and TNFα. The molecular mechanisms that regulate TNFR2 remain undefined. This study used colon cancer cell lines to test the hypothesis that IL-6 and TNFα induce TNFR2 via STAT3 and/or NFκB. Basal and IL-6 + TNFα-induced TNFR2 were decreased by pharmacologic STAT3 inhibition. NFκB inhibition had little effect on IL-6 + TNFα-induced TNFR2, but did inhibit induction of endogenous IL-6 and TNFR2 in cells treated with TNFα alone. Chromatin immunoprecipitation (ChIP) revealed cooperative effects of IL-6 + TNFα to induce STAT3 binding to a -1,578 STAT response element in the TNFR2 promoter but no effect on NFκB binding to consensus sites. Constitutively active STAT3 was sufficient to induce TNFR2 expression. Overexpression of SOCS3, a cytokine-inducible STAT3 inhibitor, which reduces tumorigenesis in preclinical models of colitis-associated cancer, decreased cytokine-induced TNFR2 expression and STAT3 binding to the -1,578 STAT response element. SOCS3 overexpression also decreased proliferation of colon cancer cells and dramatically decreased anchorage-independent growth of colon cancer cells, even cells overexpressing TNFR2. Collectively, these studies show that IL-6- and TNFα-induced TNFR2 expression in colon cancer cells is mediated primarily by STAT3 and provide evidence that TNFR2 may contribute to the tumor-promoting roles of STAT3.     

Tumour necrosis factor‐α promotes liver ischaemia‐reperfusion injury through the PGC‐1α/Mfn2 pathway

Tumour necrosis factor (TNF)‐α has been considered to induce ischaemia‐reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor‐γ co‐activator (PGC)‐1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC‐1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF‐α had increased TNF‐α levels and resulted in down‐regulation of PGC‐1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF‐α by neutralizing antibody reversed PGC‐1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC‐1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF‐α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral‐Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF‐α. However, there was no up‐regulation of PGC‐1α. These findings suggest that PGC‐1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF‐α‐induced hepatic IRI. Inhibition of the TNF‐α or PGC‐1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI.     

Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis

Melanoma cells are relatively resistant to Apo2L/TRAIL (TNF-related apoptosis-inducing ligand). We postulated that resistance might result from higher expression of inhibitors of apoptosis including Bcl-2, FLIP (FLICE-like inhibitory protein) or IAPs such as XIAP (X-linked inhibitor of apoptosis) or survivin. Compared to scrambled or mismatch controls, targeting individual inhibitors with siRNA (si-Bcl-2, si-XIAP, si-FLIP or si-Surv), followed by Apo2L/TRAIL resulted in marked increase in apoptosis in melanoma cells. Compared to Bcl-2 or FLIP, siRNAs against XIAP and survivin were most potent in sensitizing melanoma cells. A similar substantial increase in apoptosis was seen in renal carcinoma cells (SKRC-45, Caki-2), following the inhibition of either XIAP or survivin by siRNAs. Apo2L/TRAIL treatment in IAP-targeted cells resulted in cleavage of Bid, activation of caspase-9 and cleavage of PARP (poly ADP-ribose polymerase). Thus, Apo2L/TRAIL resistance can be overcome by interfering with expression of inhibitors of apoptosis regulating both extrinsic (death receptor) or intrinsic (mitochondrial) pathways of apoptosis in melanoma cells.     

IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis

All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.     

Identification of molecular targets for selective elimination of TRAIL‐resistant leukemia cells. From spots to in vitro assays using TOP15 charts

The resistance of malignant cells to chemotherapy calls for the development of novel anti‐cancer drugs. TNF‐related apoptosis‐inducing ligand (TRAIL) is a pro‐apoptotic cytokine, which selectively induces apoptosis in malignant cells. We derived two TRAIL‐resistant HL‐60 subclones, HL‐60/P1 and HL‐60/P2, from a TRAIL‐sensitive HL‐60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable “weaknesses” of the TRAIL‐resistant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2‐DE) analysis and compared both TRAIL‐resistant subclones with the original TRAIL‐sensitive HL‐60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so‐called “TOP15” proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL‐60/P1 cells, and the marked down‐regulation of enzyme adenosine deaminase in HL‐60/P2 cells, suggests increased sensitivity of these cells to DNA‐interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL‐60/P1 cells, and adenosine and vidarabine to HL‐60/P2, compared with TRAIL‐sensitive HL‐60 cells.     

Interleukin‐6 contributes to chemoresistance in MDA‐MB‐231 cells via targeting HIF‐1α

Chemoresistance is a critical challenge in the clinical treatment of triple‐negative breast cancer (TNBC). It has been well documented that inflammatory mediators from tumor microenvironment are involved in the pathogenesis of TNBC and might be related to chemoresistance of cancer cells. In this study, the contribution of interleukin‐6 (IL‐6), one of the principal oncogenic molecules, in chemoresistance of a TNBC cell line MDA‐MB‐231 was first investigated. The results showed that IL‐6 treatment could induce upregulation of HIF‐1α via the activation of STAT3 in MDA‐MB‐231 cells, which consequently contributed to its effect against chemotherapeutic drug‐induced cytotoxicity and cell apoptosis. However, knockdown of HIF‐1α attenuated such effect via affecting the expressions of apoptosis‐related molecules as Bax and Bcl‐2 and drug transporters as P‐gp and MRP1. This study indicated that targeting at IL‐6/HIF‐1α signaling pathway might be an effective strategy to overcome chemoresistance in TNBC therapy.     

Cytoskeleton inhibitors combined with TRAIL induce apoptosis in HeLa carcinoma cells overexpressing antiapoptotic protein Bcl-2

TRAIL (Apo2L), a cytokine from the family of tumor necrosis factors (TNF), causes apoptosis in various types of tumor cells but is not toxic for normal cells. Recombinant TRAIL obtained using an original method stimulates the release of cytochrome c from mitochondria into the cytoplasm and apoptosis in HeLa carcinoma cells. Expression of oncoprotein Bcl-2 in these cells blocks both processes. The microtubule inhibitors taxol, nocodazole, and colcemid, as well as an inhibitor of actin microfilaments cytochalasin D, enhance the action of TRAIL and allow it to overcome protection caused by overexpression of Bcl-2. This effect is not associated with enhancement of early steps of TRAIL-dependent apoptosis leading to activation of caspase-8 and Bid protein. The inactivation of Bcl-2 also does not define the effect of cytoskeleton inhibitors. It is supposed that destruction of cytoskeleton alters the mechanism of the TRAIL- (or TNF)-dependent cytochrome c release from mitochondria by making it resistant to Bcl-2. The combined use of cytoskeleton inhibitors, which are antitumor drugs, with the recombinant TRAIL preparations may be efficient in therapy of tumors resistant to traditional chemotherapy.     

Potential mechanisms of resistance to TRAIL/Apo2L-induced apoptosis in human promyelocytic leukemia HL-60 cells during granulocytic differentiation

Human promyelocytic leukemia HL-60 cells are well known to differentiate into granulocytes or monocytes in the presence of some agents such as DMSO or PMA, respectively. Differentiated HL-60 cells become resistant to some apoptotic stimuli including anticancer drugs or irradiation though undifferentiated cells significantly respond to these stimuli. TRAIL (TNF-related apoptosis-inducing ligand) which is also known as Apo2 ligand (Apo2L), a new member of TNF family, can induce apoptosis in some tumor cells but not in many normal cells. We show here that apoptosis is well induced in HL-60 cells by TRAIL, but susceptibility to TRAIL is reduced during granulocytic differentiation by DMSO. We also suggest some possible mechanisms by which granulocytic differentiated cells become resistant to TRAIL-induced apoptosis. First, in granulocytic differentiated cells, expression of antagonistic decoy receptors for TRAIL (TRAIL-R3/TRID/DcR1/LIT and TRAIL-R4/TRUNDD/DcR2) were enhanced. In addition, expression of Toso, a cell surface apoptosis regulator, seemed to block activation of caspase-8 by TRAIL via enhanced expression of FLIPL in granulocytic differentiated cells. These findings suggest that differentiated cells are resistant using plural mechanisms against various apoptosis-inducing stimuli rather than undifferentiated cells.