Activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death

Despite extensive investigation, the molecular mechanism of anticancer activity of sphingolipid metabolites remains to be clarified. Here we demonstrate that sphingosine induces mitochondrial cell death via Lck-mediated conformational activation of Bak in Jurkat T cell lymphoma. Treatment of cells with sphingosine rapidly induced mitochondrial membrane potential loss, cytochrome c release from mitochondria, and apoptotic cell death. Sphingosine also induced conformational activation of Bak, but not Bax. siRNA targeting of Bak effectively attenuated sphingosine-induced mitochondrial cell death, indicating that Bak is involved in sphingosine-induced mitochondrial cell death. Sphingosine also induced activation of tyrosine kinase Lck. Inhibition of Lck by treatment of PP2, a Lck inhibitor or siRNA targeting of Lck suppressed sphingosine-induced conformational activation and oligomerization of Bak, mitochondrial membrane potential loss, and apoptotic cell death, implying that activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death. The results elucidated in this study provide a novel cellular mechanism for the anticancer activity of sphingolipid metabolites.     

Induction of apoptosis by Meretrix lusoria through reactive oxygen species production, glutathione depletion, and caspase activation in human leukemia cells

Apoptosis-induced directed fractionation and purification was used to identify the bioactive components of hard clams (HC), Meretrix lusoria. Two stereoisomers of epidioxysterol were previously identified as the active compounds in the ethyl acetate fraction (HC-EA). The molecular mechanism of HC-EA-induced apoptosis was also investigated in this study. Dissipation of mitochondrial membrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of pro-caspase-9 and -3 processing preceded apoptosis in HL-60 cells, confirmed by DNA fragmentation, chromatin condensation, changes in the cell membrane and the appearance of a sub-G1 DNA peak. Furthermore, treatment with HC-EA caused a rapid loss of intracellular glutathione content and stimulation of reactive oxygen species (ROS). Antioxidants such as catalase, N-acetylcysteine, pyrrolidine dithiocarbamate, and superoxide dismutase, but not allopurinol and diphenylene iodonium, significantly inhibited HC-EA-induced cell death. Apoptosis was completely prevented by a pan-caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK). The induction of apoptosis by M. lusoria may prove to be a pivotal mechanism for its cancer chemopreventive action.     

The antimicrobial peptide, psacotheasin induces reactive oxygen species and triggers apoptosis in Candida albicans

Previously, the antimicrobial effects and membrane-active action of psacotheasin in Candida albicans were investigated. In this study, we have further found that a series of characteristic cellular changes of apoptosis in C. albicans can be induced by the accumulation of intracellular reactive oxygen species, specifically hydroxyl radicals, the well-known important regulators of apoptosis. Cells treated with psacotheasin showed diagnostic markers in yeast apoptosis at early stages: phosphatidylserine externalization from the inner to the outer membrane surface, visualized by Annexin V-staining; mitochondrial membrane depolarization, observed by DiOC₆(3) staining; and increase of metacaspase activity, measured using the CaspACE FITC-VAD-FMK. Moreover, DNA fragmentation and condensation also revealed apoptotic phenomena at late stages through the TUNEL assay staining and DAPI staining, respectively. Taken together, our findings suggest that psacotheasin possess an antifungal property in C. albicans via apoptosis as another mode of action.     

Induction of yeast apoptosis by an antimicrobial peptide, Papiliocin

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC₆(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

Rapamycin sensitive mTOR activation mediates nerve growth factor (NGF) induced cell migration and pro-survival effects against hydrogen peroxide in retinal pigment epithelial cells

Patients with age related macular degeneration (AMD) have a loss of vision in the center of the visual field. Oxidative stress plays an important role in this progress. Nerve growth factor (NGF) is important for the survival and maintenance of sympathetic and sensory neurons and NGF eye drops improve visual acuity and electro-functional activity in patients with AMD. However, the molecular mechanisms and signaling events involved in this have not been fully investigated. Using cultured human retinal pigment epithelial (RPE) cells, we demonstrate here that NGF protects RPE cells against hydrogen peroxide (H₂O₂)-induced cell apoptosis. NGF also induces RPE cell migration, the latter is important for retinal regeneration and the recovery from AMD. H₂O₂ decreases S6 phosphorylation and cell viability, which is restored by NGF. Rapamycin, the pharmacologic inhibitor of mammalian target of rapamycin (mTOR), diminished NGF-induced S6 phosphorylation, cell migration and protective effects against oxidative stress. Collectively, we conclude that activation of rapamycin sensitive mTOR signaling mediates NGF induced cell migration and pro-survival effects in H₂O₂ treated RPE cells.     

Formononetin potentiates epirubicin-induced apoptosis via ROS production in HeLa cells in vitro

The frequent development of multidrug resistance (MDR) hampers the efficacy of available anticancer drugs in treating cervical cancer. In this study, we aimed to use formononetin (7-hydroxy-4′-methoxyisoflavone), a potential herbal isoflavone, to intensify the chemosensitivity of human cervical cancer HeLa cells to epirubicin, an anticancer drug. The reactive oxygen species (ROS) levels were correlated with MDR modulation mechanisms, including the transporter inhibition and apoptosis induction. Our results revealed that formononetin significantly enhanced the cytotoxicity of epirubicin. Co-incubation of epirubicin with formononetin increased the ROS levels, including hydrogen peroxide and superoxide free radicals. Epirubicin alone markedly increased the mRNA expression of MDR1, MDR-associated protein (MRP) 1, and MRP2. In contrast, formononetin alone or combined treatment decreased the mRNA expression of MRP1 and MRP2. This result indicates that efflux transporter-mediated epirubicin resistance is inhibited at different degrees by the addition of formononetin. This isoflavone significantly intensified epirubicin uptake into HeLa cells. Apoptosis was induced by formononetin and/or epirubicin, as signified by nuclear DNA fragmentation, chromatin condensation, increased sub-G1 and G2/M phases. The cotreatment triggered the mitochondrial apoptotic pathway indicated by increased Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, and significant activation of caspase-9 and -3. In addition, extrinsic/caspases-8 apoptotic pathway was also induced by the cotreatment. N-acetyl cysteine abrogated these events induced by formononetin, supporting the involvement of ROS in the MDR reversal mechanism. This study pioneered in demonstrating that formononetin may potentiate the cytotoxicity of epirubicin in HeLa cells through the ROS-mediated MRP inhibition and concurrent activation of the mitochondrial and death receptor pathways of apoptosis. Hence, the circumvention of pump and non-pump resistance using formononetin and epirubicin may pave the way for a powerful chemotherapeutic regimen for treating human cervical cancer.     

p38 MAPK is involved in CB2 receptor-induced apoptosis of human leukaemia cells

Cannabinoids have been shown to inhibit the growth of a broad spectrum of tumour cells. However, the molecular mechanisms involved in that effect have not been completely elucidated. Here, we investigated the possible involvement of mitogen-activated protein kinases (MAPKs) in CB2 receptor-induced apoptosis of human leukaemia cells. Results show that stimulation of the CB2 receptor leads to p38 MAPK activation and that inhibition of this kinase attenuates CB2 receptor-induced caspase activation and apoptosis. These findings support a role for p38 MAPK in CB2 receptor-induced apoptosis of human leukaemia cells.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

Glutathione selectively inhibits Doxorubicin induced phosphorylation of p53Ser, caspase dependent ceramide production and apoptosis in human leukemic cells

Glutathione (GSH) is the most abundant non-protein antioxidant in mammalian cells. It has been implicated in playing an important role in different signal transduction pathways, and its depletion is an early hallmark in the progression of apoptosis in response to a number of proapoptotic stimuli. We have selectively investigated the role of GSH in cytotoxic response of Jurkat and Molt-4 human leukemic cells to the anti-cancer drug Doxorubicin. In this study, we have shown that extracellular supplementation of GSH to human leukemic cells renders them a resistant phenotype to Doxorubicin treatment. Glutathione pre-treatment inhibits Doxorubicin-induced p53Ser¹⁵ phosphorylation, caspase dependent ceramide (Cer) generation, Poly (ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. Taken together, these results indicate that the major cellular antioxidant GSH influences the chemotherapeutic efficacy of Doxorubicin towards human leukemic cells.     

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.     

Inhibitory effects of interleukin-6 on release of PGI2 by cultured human pulmonary artery smooth muscle cells

We evaluated the effect of interleukin-6 (IL-6) on the production of prostacyclin (PGI₂) by cultured human pulmonary artery smooth muscle cells (HPASMC). Incubation of these cells for up to 48 h with IL-6 led to a dose- and time-dependent decrease in the concentration of PGI₂ in the culture medium. The incubation of HPASMC with 10 μg/ml of lipopolysaccharide (LPS), 200 U/ml of IL-1β or 500 U/ml of TNFα for 24 hr significantly increased the concentration of PGI₂ in the medium. However, the addition of IL-6 to a medium containing LPS, IL-1β, or TNFα significantly inhibited the stimulatory effect of those substances on PGI₂ production. Such inhibition was closely related to the concentration of IL-6. IL-6 may counteract the roles of LPS and of other cytokines on the regulation of pulmonary vascular tension in endotoxin- and cytokine-mediated disorders such as sepsis and the acute respiratory distress syndrome (ARDS).     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

SAHA treatment overcomes the anti-apoptotic effects of Bcl-2 and is associated with the formation of mature PML nuclear bodies in human leukemic U937 cells

Bcl-2 protects tumor cells from the apoptotic effects of various antineoplastic agents. Increased expression of Bcl-2 has been associated with poor response to chemotherapy in various malignancies, including leukemia. Therefore, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. We undertook this study to examine whether SAHA (suberoylanilide hydroxamic acid) overcomes the resistance by Bcl-2 in human leukemic cells, with a specific focus on the involvement of PML-NBs. Experiments were conducted with Bcl-2-overexpressing human leukemic U937 cells. Since we previously demonstrated that overexpression of Bcl-2 attenuates resveratrol-induced apoptosis in human leukemic U937 cells, resveratrol-treated U937 cells were used as a negative control. The present study indicates that SAHA at 1-7 μM, the dose range known to induce apoptosis in various cancer cells, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2-overexpressing human leukemic U937 cells. Notably, we observed that SAHA-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in human leukemic U937 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that could help bypass the resistance to apoptosis conferred by Bcl-2. Elucidating exactly how PML regulates Bcl-2 will require further work.     

Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells

Ceramide-1-phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro-apoptotic ceramide. To examine the role of endogenous C1P, expression of ceramide kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that ceramide kinase determines the balance between pro-apoptotic ceramide and anti-apoptotic C1P to regulate cell fate, reminiscent of its function in plants.     

Synergism of Rana catesbeiana ribonuclease and IFN-gamma triggers distinct death machineries in different human cancer cells

Rana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-gamma. However, it is unclear how RC-RNase and RC-RNase/IFN-gamma induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-gamma-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-gamma induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-gamma can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from Psacothea hilaris

Psacotheasin is a 34-mer knottin-type peptide that is derived from Psacothea hilaris larvae. In this study, the antifungal activity and mechanism(s) by which psacotheasin affects human fungal pathogens were investigated. Psacotheasin shows remarkable antifungal properties without hemolytic activity against human erythrocytes. To understand the antifungal mechanism(s) of psacotheasin in Candida albicans, flow cytometric analysis with DiBAC₄(3) and PI was conducted. The results showed that psacotheasin depolarized and perturbed the plasma membrane of the C. albicans. Three-dimensional (3D)-flow cytometric contour-plot analysis, accompanied by decreased forward scatter (FS), which indicates cell size, confirmed that psacotheasin exerted antifungal effects via membrane permeabilization. The membrane studies, using a single GUV and FITC-dextran (FD) loaded liposomes, indicate that psacotheasin acts as a pore-forming peptide in the model membrane of C. albicans and the radius of pores were presumed to be anywhere from 2.3 to 3.3 nm. Therefore, the current study suggests that the mechanism(s) of psacotheasin’s antifungal properties function within the membrane.     

D2/D3 receptor agonist ropinirole protects dopaminergic cell line against rotenone-induced apoptosis through inhibition of caspase- and JNK-dependent pathways

Ropinirole, a D2/D3 receptor agonist has been reported to have neuroprotective effects. We showed that ropinirole can prevent rotenone-induced apoptosis in dopaminergic cell line SH-SY5Y through D3 receptor. We found that ropinirole can block the rotenone-induced phosphorylation of JNK, P38 and p-c-Jun, but promote the phosphorylation of ERK1/2. Furthermore, we demonstrated that ropinirole can reduce the rotenone-induced cleavages of caspase 9, caspase 3 and PARP and elevate the expression of anti-apoptotic proteins of p-Akt and bcl-2. These results provide a basis for neuroprotection by this drug for the treatment of Parkinson disease.