Oxaliplatin enhances TRAIL-induced apoptosis in gastric cancer cells by CBL-regulated death receptor redistribution in lipid rafts

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family that selectively induces apoptosis in cancer cells. However, gastric cancer cells are insensitive to TRAIL. In the present study, we show that oxaliplatin enhanced TRAIL-induced apoptosis of MGC803, BGC823, and SGC7901 cells. Oxaliplatin promoted death receptor 4 (DR4) and death receptor 5 (DR5) clustering into aggregated lipid rafts, while the cholesterol-sequestering agent nystatin partially prevented lipid raft aggregation, DR4 and DR5 clustering, and reduced apoptosis. Furthermore, the expression of the casitas B-lineage lymphoma (Cbl) family was downregulated by oxaliplatin. Transfection of c-Cbl or Cbl-b partially reversed oxaliplatin-induced lipid raft aggregation. These results indicated that oxaliplatin enhanced TRAIL-induced gastric cancer cell apoptosis at least partially through Cbl-regulated death receptor redistribution in lipid rafts.     

A triterpenediol from <Emphasis Type="Italic">Boswellia serrata</Emphasis> induces apoptosis through both the intrinsic and extrinsic apoptotic pathways in human leukemia HL-60 cells

A triterpenediol (TPD) comprising of isomeric mixture of 3α, 24-dihydroxyurs-12-ene and 3α, 24-dihydroxyolean-12-ene from Boswellia serrata induces apoptosis in cancer cells. An attempt was made in this study to investigate the mechanism of cell death by TPD in human leukemia HL-60 cells. It inhibited cell proliferation with IC₅₀ ∼ 12 μg/ml and produced apoptosis as measured by various biological end points e.g. increased sub-G0 DNA fraction, DNA ladder formation, enhanced AnnexinV-FITC binding of the cells. Further, initial events involved massive reactive oxygen species (ROS) and nitric oxide (NO) formation, which were significantly inhibited by their respective inhibitors. Persistent high levels of NO and ROS caused Bcl-2 cleavage and translocation of Bax to mitochondria, which lead to loss of mitochondrial membrane potential (Δψ_m) and release of cytochrome c, AIF, Smac/DIABLO to the cytosol. These events were associated with decreased expression of survivin and ICAD with attendant activation of caspases leading to PARP cleavage. Furthermore, TPD up regulated the expression of cell death receptors DR4 and TNF-R1 level, leading to caspase-8 activation. These studies thus demonstrate that TPD produces oxidative stress in cancer cells that triggers self-demise by ROS and NO regulated activation of both the intrinsic and extrinsic signaling cascades.     

An essential oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cytochrome c and apical death receptors in human leukaemia HL-60 cells

An essential oil from a lemon grass variety of Cymbopogon flexuosus (CFO) and its major chemical constituent sesquiterpene isointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation of apoptosis is the hallmark of cancer cells. CFO and ISO inhibited cell proliferation with 48 h IC50 of approximately 30 and 20 microg/ml, respectively. Both induced concentration dependent strong and early apoptosis as measured by various end-points, e.g. annexinV binding, DNA laddering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6h period of study. This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expression of mitochondrial cytochrome c protein with its concomitant release to cytosol leading to caspase-9 activation, suggesting thereby the involvement of both the intrinsic and extrinsic pathways of apoptosis. Further, Bax translocation, and decrease in nuclear NF-kappaB expression predict multi-target effects of the essential oil and ISO while both appeared to follow similar signaling apoptosis pathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highly useful in the development of anti-cancer therapeutics.     

Amplification loop cascade for increasing caspase activity induced by docetaxel

The hierarchy of events accompanying induction of apoptosis by the microtubule inhibitor docetaxel was investigated in HL-60 human leukemia cells. Treatment of HL-60 cells with docetaxel resulted in the production of reactive oxygen species (ROS), activation of caspase-3 (-like) protease, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation, bcl-2 phosphorylation and apoptosis. Docetaxel elicited ROS production from NADPH oxidase as demonstrated by specific oxidase inhibitor diphenylene iodonium (DPI). ROS mediated the caspase-3 activation and apoptosis in HL-60 cells. The caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) effectively inhibited JNK/SAPK activation, bcl-2 phosphorylation and partially attenuated the ROS production induced by docetaxel. Docetaxel-induced bcl-2 phosphorylation was completely blocked by expression of dominant negative JNK or the JNK/SAPK inhibitor SP600125. Overexpression of bcl-2 partially prevented docetaxel-mediated ROS production and subsequent caspase-3 activation, thereby inhibiting apoptotic cell death. It is thus conferred that such sequent events as ROS production, caspase activation, JNK/SAPK activation, bcl-2 phosphorylation and the further generation of ROS should be parts of an amplification loop to increase caspase activity, thereby facilitating the apoptotic cell death process.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

Nitric oxide-enhanced caspase-3 and acidic sphingomyelinase interaction: a novel mechanism by which airway epithelial cells escape ceramide-induced apoptosis

Reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. The molecular mechanisms and signaling events involved in lung cell injury by RNS are still poorly understood. In the current study, we observe a novel anti-apoptotic response to nitric oxide (NO) exposure (via the NO donors 3-morpholine-syndnonimine (SIN1) or papa-NONOate) of human airway epithelial (HAE) cells. NO exposure via the NO donors increased cellular ceramide levels via ceramide synthase but did not trigger an apoptotic response. Rather, exposure to the NO donors promoted an increase in the protein-protein interaction between acidic sphingomyelinase (aSMase) and caspase-3, with aSMase sequestering caspase-3 and preventing its cleavage. In contrast, when aSMase was silenced in HAE cells or was knocked out in mice, an increase in cleaved caspase-3 was observed. This elevated caspase-3 cleavage was further augmented upon NO exposure (via SIN1 or papa-NONOate) of HAE cells and could be prevented by an inhibitor to ceramide synthase. These results demonstrate a novel mechanism of NO modulation of apoptosis, in which HAE cells exposed to NO via an NO donor induces ceramide generation via ceramide synthase. However, this ceramide induction does not lead to apoptosis unless aSMase is knocked down, allowing the release of caspase-3, its activation and execution of apoptosis.     

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.     

In vitro cytotoxic potential of Polyalthia longifolia on human cancer cell lines and induction of apoptosis through mitochondrial-dependent pathway in HL-60 cells

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. We are reporting first time the anticancer potential of P. longifolia leaves extract (A001) and its chloroform fraction (F002). Both inhibited cell proliferation of various human cancer cell lines in which colon cancer cells SW-620 showed maximum inhibition with IC(50) value 6.1 microg/ml. Furthermore, F002 induce apoptosis in human leukemia HL-60 cells as measured by several biological end points. F002 induce apoptotic bodies formation, DNA ladder, enhanced annexin-V-FITC binding of the cells, increased sub-G(0) DNA fraction, loss of mitochondrial membrane potential (DeltaPsi(mt)), release of cytochrome c, activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. All the above parameters revealed that F002-induced apoptosis through the mitochondrial-dependent pathway in HL-60 cells.     

Lupulone, a hop bitter acid, activates different death pathways involving apoptotic TRAIL-receptors, in human colon tumor cells and in their derived metastatic cells

Our study aimed to compare death signalling pathways triggered by lupulone in TRAIL-sensitive human colon cancer cells (SW480) and in their derived TRAIL-resistant metastatic cells (SW620). Lupulone (40 μg/ml) up-regulated expression of TRAIL DR4/DR5 death receptors at the cell surface of both cell lines, even in the absence of exogenous TRAIL ligand. Cell death induced by lupulone was inhibited in SW480 and SW620 cells exposed to blocking anti-DR4/DR5 antibodies. In SW480 cells, lupulone triggered cell death through a cross-talk between TRAIL-DR4/DR5 and the mitochondrial (intrinsic) pathways involving caspase-8 activation and Bid protein cleavage. As a consequence mitochondrial cytochrome c was released into the cytosol and activation of caspases-9 and -3 was observed. In the metastatic SW620 cells, lupulone restored the sensibility of these cells to TRAIL ligand and activated the extrinsic apoptotic pathway via DR4/DR5 death receptors and the involvement of the caspase-8/caspase-3 cascade. The demonstration that lupulone is able to activate TRAIL-death signalling pathways even in TRAIL resistant cancer cells highlights the potential of this natural compound for cancer prevention and therapy.     

A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells.

AP9-cd, a standardized lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by flow cytometry using specific fluorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant effect of AP9-cd. Further, caspase-3 activation correlated with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of pro-oxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-cd.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Caspase inhibition and N6-benzyladenosine-induced apoptosis in HL-60 cells.

As an extension of our recently published work (Mlejnek and Kuglík [2000] J. Cell. Biochem. 77:6-17), the role of caspases in N(6)-benzylaminopurine riboside (BAPR)-induced apotosis in HL-60 cells was evaluated in this study. Here, BAPR-induced apoptosis was accompanied by activation of caspase-3 and caspase-9. However, when these caspases were selectively inhibited, the progression of BAPR-induced apoptosis was not markedly affected. Besides that, activation of caspase-3 and caspase-9 was found to be rather late event in apoptotic process. These results suggested that other caspases might be critically implicated. Indeed, pan-specific caspase inhibitor, Z-VAD-FMK, completely prevented DNA cleavage and apoptotic bodies formation. However, Z-VAD-FMK failed to prevent cell death and it was incapable to fully counteract the main apoptotic hallmark-chromatin condensation. Finally, our data indicate that cellular decision between apoptosis and necrosis is made upon the availability of both caspase proteases and intracellular ATP.     

Phyllanthus urinaria induces the Fas receptor/ligand expression and ceramide-mediated apoptosis in HL-60 cells

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect on human myeloid leukemia cells in this study. The water extract of P. urinaria induced the apoptosis of HL-60 cells as demonstrated by morphological change, DNA fragmentation and increased caspase-3 activity. However, normal human peripheral mononuclear cells remained viable under the same treatment. The P. urinaria-induced apoptosis of HL-60 cells was associated with the increased Bax gene expression and decreased Bcl-2 gene expression. In addition, the gene expressions of Fas receptor and Fas ligand, but not p53, were also induced in HL-60 cells dose- and time-dependently. The inhibitor of ceramide synthase, fumonisin B1, completely suppressed the apoptosis induced by P. urinaria and this inhibitory effect of fumonisin B1 could be eliminated by the addition of ceramide. It indicated that the activity of ceramide synthase is critical for the P. urinaria-induced apoptosis in HL-60 cells. The P. urinaria-induced apoptosis in HL-60 cells is mediated through a ceramide-related pathway.     

Targeting AML through DR4 with a novel variant of rhTRAIL

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.     

α-Tocopherol impairs 7-ketocholesterol-induced caspase-3-dependent apoptosis involving GSK-3 activation and Mcl-1 degradation on 158N murine oligodendrocytes

In important and severe neurodegenerative pathologies, 7-ketocholesterol, mainly resulting from cholesterol autoxidation, may contribute to dys- or demyelination processes. On various cell types, 7-ketocholesterol has often been shown to induce a complex mode of cell death by apoptosis associated with phospholipidosis. On 158N murine oligodendrocytes treated with 7-ketocholesterol (20 μg/mL corresponding to 50 μM, 24–48 h), the induction of a mode of cell death by apoptosis characterised by the occurrence of cells with condensed and/or fragmented nuclei, caspase activation (including caspase-3) and internucleosomal DNA fragmentation was observed. It was associated with a loss of transmembrane mitochondrial potential (ΔΨm) measured with JC-1, with a dephosphorylation of Akt and GSK3 (especially GSK3β), and with degradation of Mcl-1. With α-tocopherol (400 μM), which was capable of counteracting 7-ketocholesterol-induced apoptosis, Akt and GSK3β dephosphorylation were inhibited as well as Mcl-1 degradation. These data underline that the potential protective effects of α-tocopherol against 7-ketocholesterol-induced apoptosis do not depend on the cell line considered, and that the cascade of events (Akt/GSK3β/Mcl-1) constitutes a link between 7-ketocholesterol-induced cytoplasmic membrane dysfunctions and mitochondrial depolarisation leading to apoptosis.     

Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells.