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.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.     

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.     

Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection

Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an anti-apoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.     

A polysaccharide of the marine alga Capsosiphon fulvescens induces apoptosis in AGS gastric cancer cells via an IGF-IR-mediated PI3K/Akt pathway

Because seaweed extracts have recently been found to have antioxidant and anti-tumor activities, we analyzed a hot-water-soluble polysaccharide (PS) of the marine alga Capsosiphon fulvescens for its potential as a functional foodstuff by determining its effects on cell growth and DNA synthesis. MTS assays showed that the C. fulvescens PS (Cf-PS) significantly inhibited the proliferation of cultured human cancer cells in a dose-dependent manner. Cf-PS-treated AGS cells exhibited a marked increase in caspase-3 activation and a decrease in Bcl-2 expression. In addition, phosphorylation of insulin-like growth factor-I receptor (IGF-IR) was decreased in Cf-PS-treated AGS cells as compared to non-treated control cells, which is consistent with PI3-kinase (PI3K)/Akt activation. Cf-PS also decreased IGF-I-stimulated recruitment of p85 to IGF-IR and IRS-1. These results indicate that Cf-PS inhibits cell proliferation and induces apoptosis by inhibiting IGF-IR signaling and the PI3K/Akt pathway.     

HER2 signaling downregulation by trastuzumab and suppression of the PI3K/Akt pathway: an unexpected effect on TRAIL-induced apoptosis

We investigated whether HER2 downregulation by trastuzumab modulates the responsiveness of breast cancer cells to TNF-related apoptosis-inducing ligand (TRAIL). Interestingly, in contrast to increased response to TRAIL in SKBr3 cells, trastuzumab decreased the susceptibility of BT474 cells to TRAIL. This decrease was also observed after exogenous inhibition of PI3-K/Akt kinase, but not MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK). In BT474 cells, but not SKBr3 cells, inhibition of the HER2/phosphatidylinositol 3' kinase (PI3K)/Akt pathway resulted in downregulation of the pro-apoptotic receptors TRAIL-receptor 1 (TRAIL-R1) and TRAIL-R2. TRAIL-induced caspase-8 activation, Bid processing, drop of DeltaPsi(m), and poly ADP-ribose polymerase (PARP) cleavage but not in caspase-9 activation, and these events were inhibited in HER2/PI3K/Akt-suppressed BT474 cells, which on the other hand exhibited downregulation of Bcl-xL and increased response to mitomycin C. We show that HER2/PI3K/Akt pathway may play a specific pro-apoptotic role in certain cell type by inducing TRAIL-R1 and -R2 expression and thereby enhancing responsiveness to TRAIL.     

Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: dependency on reactive oxygen species, Akt, Bid, cytochrome and caspase pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-cysteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and-3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia.     

Photodynamic therapy induces caspase-3 activation in HL-60 cells

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.     

The Early Activation of PI3K Strongly Enhances the Resistance of Cortical Neurons to Hypoxic Injury via the Activation of Downstream Targets of the PI3K Pathway and the Normalization of the Levels of PARP Activity, ATP, and NAD+

Phosphatidylinositol 3-kinase (PI3K) plays several important roles in neuronal survival. Activation of the pathway is essential for the neuroprotective mechanisms of materials that shield neuronal cells from many stressful conditions. However, there have been no reports to date about the effect of the direct activation of the pathway in hypoxic injury of neuronal cells. We investigated whether the direct activation of the PI3K pathway inhibits neuronal cell death induced by hypoxia. Primary cultured cortical neurons (PCCNs) were exposed to hypoxic conditions (less than 1 mol% O₂) and/or treated with PI3K activator. Hypoxia reduced the viability of PCCNs in a time-dependent manner, but treatment with PI3K significantly restored viability in a concentration-dependent manner. Among the signaling proteins involved in the PI3K pathway, those associated with survival, including Akt and glycogen synthase kinase-3β, were decreased shortly after exposure to hypoxia and those associated with cell death, including BAX, apoptosis-induced factor, cytochrome c, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP), were increased. However, treatment with PI3K activator normalized the expression levels of those signaling proteins. PARP activity and levels of ATP and NAD⁺ altered by hypoxia were also normalized with direct PI3K activation. All these findings suggest that direct and early activation is important for protecting neuronal cells from hypoxic injury.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

Effects of Chronic and Acute Zinc Supplementation on Myocardial Ischemia-Reperfusion Injury in Rats

A ginseng polysaccharide was extracted, purified, and modified by nitric acid-selenious acid (HNO₃-H₂SeO₃) method to yield one selenylation-modified polysaccharide (sGP). We reported for the first time the anticancer potential of sGP on the human promyelocytic leukemia HL-60 cell line and evaluated its relevant underlying mechanism. Our results showed that sGP markedly inhibited the growth of HL-60 cells via induction of apoptosis. The event of apoptosis was accompanied by the formation of apoptotic bodies; the release of cytochrome c; loss of mitochondrial membrane potential; and activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. In addition, western blot analysis showed that sGP inhibited antiapoptotic Bcl-2 protein expression and increased proapoptotic Bax protein expression in cells under identical conditions. Together, our study suggests that sGP induces apoptosis of HL-60 cells through the mitochondrial-dependent pathway.     

Inhibition of the PI3K pathway sensitizes fludarabine-induced apoptosis in human leukemic cells through an inactivation of MAPK-dependent pathway

In the present study, we have investigated the effects of PI3K/Akt pathway on the response of human leukemia cells to fludarabine. Inhibition of PI3K/Akt pathway with a selective inhibitor (e.g., LY294002, or wortmannin) in leukemic cells markedly potentiated fludarabine-induced apoptosis. Inhibition of the PI3K/Akt downstream target mTOR by rapamycin also significantly enhanced fludarabine-induced apoptosis. The co-treatment of fludarabine/LY294002 resulted in significant attenuation in the levels of both phospho-Erk1/2 and phospho-Akt, as well as a marked increase in the level of phospho-JNK. The broad spectrum caspase inhibitor BOC-D-fmk markedly blocked fludarabine/LY-induced apoptosis, had no effect on cytochrome c release to the cytosol, and did abrogate caspase and PARP cleavage. This indicates that mitochondrial dysfunction is upstream of the caspase cascade. Moreover, constitutive activation of the MEK/Erk pathway completely blocked apoptosis induced by the combination of fludarabine/LY294002. Additionally, either constitutive activation of Akt or blockage of the JNK pathway significantly diminished apoptosis induced by the combination. Collectively, these findings demonstrate that inactivation of MAPK, Akt, and activation of the JNK pathway contributes to the induction of apoptosis induced by fludarabine/LY. Comparatively, MAPK inactivation plays a crucial role in fludarabine/LY-induced apoptosis. These results also strongly suggest that combining fludarabine with an inhibitor of the PI3K/Akt/mTOR pathway may represent a novel therapeutic strategy for hematological malignancies.     

Phenethyl isothiocyanate exhibits antileukemic activity in vitro and in vivo by inactivation of Akt and activation of JNK pathways

Effects of phenethyl isothiocyanate (PEITC) have been investigated in human leukemia cells (U937, Jurkat, and HL-60) as well as in primary human acute myeloid leukemia (AML) cells in relation to apoptosis and cell signaling events. Exposure of cells to PEITC resulted in pronounced increase in the activation of caspase-3, -8, -9, cleavage/degradation of PARP, and apoptosis in dose- and time-dependent manners. These events were accompanied by the caspase-independent downregulation of Mcl-1, inactivation of Akt, as well as activation of Jun N-terminal kinase (JNK). Inhibition of PI3K/Akt by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 significantly enhanced PEITC-induced apoptosis. Conversely, enforced activation of Akt by a constitutively active Akt construct markedly abrogated PEITC-mediated JNK activation, Mcl-1 downregulation, caspase activation, and apoptosis, and also interruption of the JNK pathway by pharmacological or genetically (e.g., siRNA) attenuated PEITC-induced apoptosis. Finally, administration of PEITC markedly inhibited tumor growth and induced apoptosis in U937 xenograft model in association with inactivation of Akt, activation of JNK, as well as downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Akt/JNK/Mcl-1 pathway potentiate PEITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of U937 xenograft model.     

Reversal of boswellic acid analog BA145 induced caspase dependent apoptosis by PI3K inhibitor LY294002 and MEK inhibitor PD98059

PI3K/Akt and ERK pathways are important for growth and proliferation of many types of cancers. Therefore, PI3K inhibitor LY294002 (LY) and MEK1/2 inhibitor PD98059 (PD) are used to sensitize many types of cancer cell lines to chemotherapeutic agents, where AKT and ERK pathways are over activated. However, in this study, we show for the first time that PD could protect the leukemia cells independent of ERK pathway inhibition, besides, we also report a detailed mechanism for antiapoptotic effect of LY in HL-60 cells against the cytotoxicity induced by a boswellic acid analog BA145. Apoptosis induced by BA145 is accompanied by downregulation of PI3K/Akt and ERK pathways in human myelogenous leukemia HL-60 cells, having activating N-Ras mutation. Both LY and PD protected the cells against mitochondrial stress caused by BA145, and reduced the release of cytochrome c and consequent activation of caspase-9. LY and PD also diminished the activation of caspase-8 without affecting the death receptors. Besides, LY and PD also reversed the caspase dependent DNA damage induced by BA145. Further studies revealed that LY and PD significantly reversed the inhibitory effect of BA145 on cell cycle regulatory proteins by upregulating hyperphosphorylated retinoblastoma, pRB (S795) and downregulating p21 and cyclin E. More importantly, all these events were reversed by caspase inhibition by Z-VAD-fmk, suggesting that both LY and PD act at the level of caspases to diminish the apoptosis induced by BA145. These results indicate that inhibitors of PI3K/Akt and ERK pathways can play dual role and act against chemotherapeutic agents.     

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.     

Quercetin-induced apoptosis of HL-60 cells by reducing PI3K/Akt

To explore the effect and mechanism of quercetin on proliferation and apoptosis of leukemia cells, and provide a theoretical basis for its clinical application. HL-60 leukemia cell lines was treated with different dose quercetin, the proliferation activity of leukemia cells was assessed by MTT method; the morphological changes of apoptosis of HL-60 cells, including nuclear condensation and DNA fragmentation, were observed by Hoechst 33258 fluorescence staining, the apoptosis rate and caspase 2,3 activation were assessed by flow cytometry, and the cell signal pathway including phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), Bcl-2, Bax were detected by western blotting. Quercetin could significantly decrease the proliferation activity of HL-60 cells through the blockade of G(0)/G(1) phase, and induce the apoptosis of HL-60 cells in a time- and dose-dependent manner. Quercetin caused leukemia cells apoptosis by decreasing the protein expression of PI3K and Bax, the inhibitory phosphorylation of Akt, the decreased levels of Bcl-2 protein and increased activations of caspase-2 and -3, and increased poly(ADP-ribose) polymerase cleavage. Our results indicate that the apoptotic processes caused by quercetin are mediated by the decrease of pAkt and Bcl-2 levels, the increase of Bax level, and the activation of caspase families in HL-60 cells.     

Inhibition of autophagy enhances effects of PF-04691502 on apoptosis and DNA damage of lung cancer cells

Autophagy modulation has been considered as a potential therapeutic strategy for lung diseases. The PI3K-Akt-mTOR pathway may be one of the main targets for regulation of autophagy. We previously reported that a PI3 K/mTOR dual inhibitor PF-04691502 suppressed hepatoma cells growth in vitro. However, it is still unclear whether PF-04691502 induces autophagy and its roles in DNA damage and cell death in human lung cancer cells. In this study, we investigate the effects of PF-04691502 on the autophagy and its correlation with cell apoptosis and DNA damage in non-small-cell lung cancer (NSCLC) cell lines. PF-04691502 efficiently inhibited the phosphorylation of Akt and showed dose-dependent cytotoxicity in A549 and H1299 cells. PF-04691502 also triggered apoptosis and the cleavage of caspase-3 and PARP. Phosphorylated histone H2AX (γ-H2AX), a hallmark of DNA damage response, was dramatically induced by PF-04691502 treatment. By exposure to PF-04691502, A549 cells acquired a senescent-like phenotype with an increase in the level of β-galactosidase. Furthermore, PF-04691502 enhanced the expression of LC3-II in a concentration-dependent manner. More interestingly, effects of PF-04691502 on toxicity and DNA damage were remarkably increased by co-treatment with an autophagy inhibitor, chloroquine (CQ), in human lung cancer cells. These data suggest that a strategy of blocking autophagy to enhance the activity of PI3 K/mTOR inhibitors warrants further attention in treatment of NSCLC cells.