Heart-specific Small Subunit of Myosin Light Chain Phosphatase Activates Rho-associated Kinase and Regulates Phosphorylation of Myosin Phosphatase Target Subunit 1

Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction, and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2), and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, whereas the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M₂₁) that increased the sensitivity to Ca²⁺ in muscle contraction. In this study, we investigated the role of hHS-M₂₁ in the regulation of MLCP phosphorylation. Two isoforms of hHS-M₂₁, hHS-M₂₁A and hHS-M₂₁B, preferentially bound the C-terminal one-third region of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser-852, impaired the binding of MYPT1 and hHS-M₂₁. The hHS-M₂₁ increased the phosphorylation level of MYPT1 at Thr-696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M₂₁ bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M₂₁ is a heart-specific effector of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr-696 by ROCK.     

RhoA/ROCK/PTEN signaling is involved in AT-101-mediated apoptosis in human leukemia cells in vitro and in vivo

R-(−)-gossypol acetic acid (AT-101) is a natural cottonseed product that exhibits anticancer activity. However, the molecular mechanism behind the antileukemic activity of AT-101 has not been well characterized. In this study, we investigated how AT-101 induces apoptosis in human leukemia cells. Exposure to AT-101 significantly increased apoptosis in both human leukemia cell lines and primary human leukemia cells. This increase was accompanied by the activation of caspases, cytochrome c release, Bcl2-associated X protein (Bax) translocation, myeloid cell leukemia-1 (Mcl-1) downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, Akt inactivation, and RhoA/Rho-associated coiled-coil containing protein kinase 1/phosphatase and tensin homolog (RhoA/ROCK1/PTEN) activation. RhoA, rather than caspase-3 cleavage, mediated the cleavage/activation of ROCK1 that AT-101 induced. Inhibiting RhoA and ROCK1 activation by C3 exoenzyme (C3) and Y27632, respectively, attenuated the ROCK1 cleavage/activation, PTEN activity, Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and apoptosis mediated by AT-101. Knocking down ROCK1 expression using a ROCK1-specific siRNA also significantly abrogated AT-101-mediated apoptosis. Constitutively active Akt prevented the AT-101-induced Mcl-1 downregulation, Bad dephosphorylation, and apoptosis. Conversely, AT-101 lethality was potentiated by the phosphatidylinositol 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. In vivo, the tumor growth inhibition caused by AT-101 was also associated with RhoA/ROCK1/PTEN activation and Akt inactivation in a mouse leukemia xenograft model. Collectively, these findings suggest that AT-101 may preferentially induce apoptosis in leukemia cells by interrupting the RhoA/ROCK1/PTEN pathway, leading to Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and Bax translocation, which culminate in mitochondrial injury and apoptosis.     

Biphasic Regulation of Myosin Light Chain Phosphorylation by p21-activated Kinase Modulates Intestinal Smooth Muscle Contractility

Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.     

The 2-arachidonoylglycerol effect on myosin light chain phosphorylation in human platelets

In human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) stimulates some important pathways leading to thromboxane B₂ formation, calcium intracellular elevation, ATP secretion and actin polymerisation. The aim of the present study was to examine the 2-AG effect on myosin light chain (MLC) phosphorylation and to investigate the mechanisms involved. We demonstrated that 2-AG induced a rapid MLC phosphorylation, stimulating both the RhoA kinase (ROCK) and MLC kinase (MLCK) in a dose and time-dependent manner. In addition MLC phosphorylation was strengthened through the MLC phosphatase inhibition. MLC phosphatase inhibition was accomplished through the RhoA/ROCK and protein kinase C mediated phosphorylation of MLC phosphatase inhibiting subunits MYPT1 and CPI-17. The presence of CB1 receptor in human platelets and the involvement of CB1 receptor in MLC phosphorylation and MLC phosphatase inhibition was shown.     

Autophagy in pancreatic cancer: An emerging mechanism of cell death

Pancreatic cancer, the fourth leading cause of cancer-related death in the United States, is resistant to current chemotherapies. Therefore, identification of different pathways of cell death is important to develop novel therapeutics. Our previous study has shown that triptolide, a diterpene triepoxide, inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. However, the mechanism by which triptolide kills pancreatic cancer cells was not known, hence, this study aimed at elucidating it. Our study reveals that triptolide kills diverse types of pancreatic cancer cells by two different pathways; it induces caspase-dependent apoptotic death in some cell lines and death via a caspase-independent autophagic pathway in the other cell lines tested. Triptolide-induced autophagy requires autophagy-specific genes, atg5 or beclin 1 and its inhibition results in cell death via the apoptotic pathway, whereas inhibition of both autophagy and apoptosis rescues triptolide-mediated cell death. Our study shows for the first time that induction of autophagy by triptolide has a pro-death role in pancreatic cancer cells. Since triptolide kills diverse pancreatic cancer cells by different mechanisms, it makes an attractive chemotherapeutic agent for future use against a broad spectrum of pancreatic cancers.Key words: pancreatic cancer, triptolide, apoptosis, caspase-3Pancreatic adenocarcinoma is one of the most lethal human malignancies. It is the fourth leading cause of cancer-related death in the United States. The five-year survival rate for pancreatic cancer is estimated to be <5% due to its aggressive growth, metastasis and resistance to radiation and most systemic chemotherapies. Hence, efforts are ongoing to understand the pathobiology of pancreatic cancer to develop innovative and effective therapies against it. A promising candidate for future therapeutic use against pancreatic cancer is a diterpene triepoxide, triptolide. Our previous studies show that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. Since the mechanism by which triptolide kills pancreatic cancer cells was not known, we decided to elucidate it.The K-ras, p53, p16 and DPC4 genes are the most frequently altered genes in pancreatic adenocarcinoma. In this study we have used diverse pancreatic cancer cell lines, MiaPaCa-2, Capan-1, S2-013 and S2-VP10 cells, which have mutations in all the above-mentioned genes and BxPC-3 and Hs766T cells, which have mutations in the p53, p16 and DPC4 genes, but have a wild-type K-ras gene. The treatment of all the cell lines with triptolide results in a significant time- and dose-dependent decrease in cell viability, independent of cell cycle arrest. After treatment with triptolide, only MiaPaCa-2, Capan-1 and BxPC-3 cells show an increase in the apoptosis parameters: cytochrome c release from mitochondria into the cytosol, caspase-3 activation and phosphatidylserine externalization. In contrast to this, S2-013, S2-VP10 and Hs766T cells show an induction of autophagy: an increase in LC3-II levels (by immunoblotting and immufluorescence), increase in acridine orange-positive cells, inhibition of the PtdIns3K/Akt/mTOR pathway and induction of the ERK1/2 pathway. Also, none of the cell lines tested show necrosis as evidenced by the absence of the release of lactate dehydrogenase. These results indicate that triptolide induces apoptosis in MiaPaCa-2, Capan-1 and BxPC-3 cells, whereas it induces autophagy in S2-013, S2-VP10 and Hs766T cells.Since the role of autophagy in cancer was controversial we investigated whether triptolide-induced autophagy has a prosurvival or a pro-death role. As autophagy-associated cell death is independent of caspase-3, we tested the effect of triptolide on pancreatic cancer cells in the absence of caspase-3. Treatment of cells with triptolide post-caspase-3 knockdown shows a significant rescue of cell viability only in MiaPaCa-2, but not S2-013 or S2-VP10 cells. This indicates that in contrast to MiaPaCa-2, triptolide-mediated cell death in S2-013 and S2-VP10 cells is independent of caspase-3. Next, we tested the role of autophagy in triptolide-mediated cell death in pancreatic cancer cells. In spite of a knockdown of autophagy-specific genes (atg5 and beclin 1), treatment of S2-013 and S2-VP10 cells with triptolide show a significant decline in cell viability, which is comparable to the cells treated with triptolide in the presence of autophagy genes. Subsequently we show that death in the absence of autophagy-specific genes is due to the utilization of an alternate cell death pathway, apoptosis. Furthermore, in the absence of both autophagy-specific and apoptosis-specific genes, triptolide-mediated cell death is rescued in S2-013 and S2-VP10 cells. Thus, these results confirm that triptolide-induced autophagy has a pro-death role in S2-013 and S2-VP10 cells and that these cells do not have a defect in the apoptotic machinery; however, they respond to triptolide by activating the autophagic pathway instead of the apoptotic pathway. Our studies also reveal the presence of a crosstalk between the two cell death pathways, apoptosis and autophagy, in pancreatic cancer cells.In conclusion, our study shows for the first time that triptolide induces autophagy in pancreatic cancer cells. It sheds light on the fundamental question as to whether autophagy is protective or causes cell death, proving convincingly that induction of autophagy causes cell death of some pancreatic cancer cells. Although a basal level of autophagy is necessary to maintain cellular homeostasis, its prosurvival role can be switched into a cell death mechanism if the amplitude of autophagy increases above a threshold level which is incompatible with viability, as seen in S2-013, S2-VP10 and Hs766T cells after triptolide treatment. Furthermore, there exists a crosstalk between apoptosis and autophagy in S2-013 and S2-VP10 cells; either both pathways function independently to kill the cells, with autophagy being the preferred pathway or autophagy antagonizes apoptosis and hence apoptosis is seen only after inhibiting autophagy. Although there is no direct correlation between the selection of cell death pathway in response to triptolide and the genotype of the cell lines, the choice of autophagic cell death pathway could depend on the metastatic potential of the cells; S2-013, S2-VP10 and Hs766T cell lines being more metastatic than the others, which merits further investigation. In conclusion, the ability of triptolide to induce cell death in diverse pancreatic cancer cells by either mechanism makes it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

RhoA- and PKC-alpha-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-alpha. Presently, we examined the cross talk between RhoA and PKC-alpha in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-alpha, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21+/-3.52 and 42.19+/-3.85% nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12+/-46.60% and 290.59+/-22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-alpha by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-alpha pathways, suggesting cross talk between RhoA and PKC-alpha resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.     

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).     

Eupatorin-Induced Cell Death in Human Leukemia Cells Is Dependent on Caspases and Activates the Mitogen-Activated Protein Kinase Pathway

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G₂-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation.     

Cordycepin induces cell cycle arrest and apoptosis by inducing DNA damage and up-regulation of p53 in Leukemia cells

Cordycepin, an adenosine analog derived from Cordyceps militaris has been shown to exert anti-tumor activity in many ways. However, the mechanisms by which cordycepin contributes to the anti-tumor still obscure. Here our present work showed that cordycepin inhibits cell growth in NB-4 and U937 cells by inducing apoptosis. Further study showed that cordycepin increases the expression of p53 which promotes the release of cytochrome c from mitochondria to the cytosol. The released cytochrome c can then activate caspase-9 and trigger intrinsic apoptosis. Cordycepin also blocks MAPK pathway by inhibiting the phosphorylation of ERK1/2, and thus sensitizes the apoptosis. In addition, our results showed that cordycepin inhibits the expression of cyclin A2, cyclin E, and CDK2, which leads to the accumulation of cells in S-phase. Moreover, our study showed that cordycepin induces DNA damage and causes degradation of Cdc25A, suggesting that cordycepin-induced S-phase arrest involves activation of Chk2-Cdc25A pathway. In conclusion, cordycepin-induced DNA damage initiates cell cycle arrest and apoptosis which leads to the growth inhibition of NB-4 and U937 cells.     

Pramanicin induces apoptosis in Jurkat leukemia cells: A role for JNK,p38 and caspase activation

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells.     

Activation of protein kinase C delta following cerebral ischemia leads to release of cytochrome C from the mitochondria via bad pathway

Background

The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.

Methods/Findings

We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.

Conclusions

We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.     

Interruption of the MEK/ERK signaling cascade promotes dihydroartemisinin-induced apoptosis in vitro and in vivo

Artemisinin, the active principle of the Chinese medicinal herb Artemisia annua, and its derivatives (i.e. dihydroartemisinin, DHA) were reported to exhibit anti-tumor activity both in vitro and in vivo. The purpose of the present study was to investigate the functional role of Mitogen-Activated Protein Kinase (MEK)/Extracellular signal-regulated protein Kinase (ERK) signaling cascade in dihydroartemisinin (DHA)-induced apoptosis in human leukemia cells in vitro and anti-leukemic activity in vivo. Human leukemia cells were treated with DHA in dose- and time-dependent manners, after which apoptosis, caspase activation, Mcl-1 expression, and cell signaling pathways were evaluated. Parallel studies were performed in AML and ALL primary human leukemia cells. In vivo anti-leukemic activity mediated by DHA was also investigated using U937 xenograft mouse model. Exposure of DHA resulted in a pronounced increase in apoptosis in both transformed and primary human leukemia cells but not in normal peripheral blood mononuclear cells. DHA-induced apoptosis was accompanied by caspase activation, cytochrome c release, Mcl-1 down-regulation, as well as MEK/ERK inactivation. Pretreatment with MEK inhibitor PD98059, which potentiated DHA-mediated MEK and ERK inactivation, intensified DHA-mediated apoptosis. Conversely, enforced expression of a constitutively active MEK1 attenuated DHA-induced apoptosis. Furthermore, DHA-mediated inhibition of tumor growth of mouse U937 xenograft was associated with induction of apoptosis and inactivation of ERK. The findings in the present study showed that DHA-induced apoptosis in human leukemia cells in vitro and exhibited an anti-leukemic activity in vivo through a process that involves MEK/ERK inactivation, Mcl-1 down-regulation, culminating in cytochrome c release and caspase activation.     

Delay in Apoptosome Formation Attenuates Apoptosis in Mouse Embryonic Stem Cell Differentiation

Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs.     

Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release

Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors—imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.     

Inhibition of apoptosome formation by suppression of Hsp90beta phosphorylation in tyrosine kinase-induced leukemias

Constitutively active tyrosine kinases promote leukemogenesis by increasing cell proliferation and inhibiting apoptosis. However, mechanisms underlying apoptotic inhibition have not been fully elucidated. In many settings, apoptosis occurs by mitochondrial cytochrome c release, which nucleates the Apaf-1/caspase-9 apoptosome. Here we report that the leukemogenic kinases, Bcr-Abl, FLT3/D835Y, and Tel-PDGFRβ, all can inhibit apoptosome function. In cells expressing these kinases, the previously reported apoptosome inhibitor, Hsp90β, bound strongly to Apaf-1, preventing cytochrome c-induced Apaf-1 oligomerization and caspase-9 recruitment. Hsp90β interacted weakly with the apoptosome in untransformed cells. While Hsp90β was phosphorylated at Ser 226/Ser 255 in untransformed cells, phosphorylation was absent in leukemic cells. Expression of mutant Hsp90β (S226A/S255A), which mimics the hypophosphorylated form in leukemic cells, conferred resistance to cytochrome c-induced apoptosome activation in normal cells, reflecting enhanced binding of nonphosphorylatable Hsp90β to Apaf-1. In Bcr-Abl-positive mouse bone marrow cells, nonphosphorylatable Hsp90β expression conferred imatinib (Gleevec) resistance. These data provide an explanation for apoptosome inhibition by activated leukemogenic tyrosine kinases and suggest that alterations in Hsp90β-apoptosome interactions may contribute to chemoresistance in leukemias.     

Rsk-mediated phosphorylation and 14-3-3ɛ binding of Apaf-1 suppresses cytochrome c-induced apoptosis

Many pro‐apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen‐activated protein kinase (MAPK) cascade, promote cell viability by impeding mitochondrial cytochrome c release and by inhibiting subsequent caspase activation. Here, we describe a mechanism for the inhibition of cytochrome c‐induced caspase activation by MAPK signalling, identifying a novel mode of apoptotic regulation exerted through Apaf‐1 phosphorylation by the 90‐kDa ribosomal S6 kinase (Rsk). Recruitment of 14‐3‐3ε to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14‐3‐3ε binding to Apaf‐1 and rendered the cells insensitive to cytochrome c, suggesting a potential role for Rsk signalling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. Collectively, these results identify a novel locus of apoptosomal regulation wherein MAPK signalling promotes Rsk‐catalysed Apaf‐1 phosphorylation and consequent binding of 14‐3‐3ε, resulting in decreased cellular responsiveness to cytochrome c.     

Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing

Increased phosphorylation of myosin light chain (MLC) is necessary for the dynamic membrane blebbing that is observed at the onset of apoptosis. Here we identify ROCK I, an effector of the small GTPase Rho, as a new substrate for caspases. ROCK I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. ROCK proteins are known to regulate MLC-phosphorylation, and apoptotic cells exhibit a gradual increase in levels of phosphorylated MLC concomitant with ROCK I cleavage. This phosphorylation, as well as membrane blebbing, is abrogated by inhibition of caspases or ROCK proteins, but both processes are independent of Rho activity. We also show that expression of active truncated ROCK I induces cell blebbing. Thus, activation of ROCK I by caspase-3 seems to be responsible for bleb formation in apoptotic cells.     

The coffee diterpene kahweol induces apoptosis in human leukemia U937 cells through down-regulation of Akt phosphorylation and activation of JNK

Kahweol, the coffee-specific diterpene, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity. The mechanism by which kahweol initiates apoptosis remains poorly understood. In the present study, we investigated the effect of kahweol on the apoptotic pathway in U937 human promonocytic cells. We show that kahweol induces apoptosis in association with the activation of caspase 3 and cytochrome c release from the mitochondria to the cytosol, as well as down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and XIAP). Kahweol altered the phosphorylation state of members of the MAPKs and Akt. Ectopic expression of Bcl-2 or constitutive active Akt (myr-Akt) in U937 cells attenuates kahweol-induced apoptosis. In addition, we have also shown that JNK and Akt signal pathway plays a crucial role in kahweol-induced apoptosis in U937 cells. Taken together, our results show the activity of kahweol to modulate multiple components in apoptotic response of human leukemia cells and raise the possibility a novel therapeutic strategy in hematological malignancies.     

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.