Cyclooxygenase-2 positively regulates Akt signalling and enhances survival of erythroleukemia cells exposed to anticancer agents

Cyclooxygenase-2 (COX-2) has been found to be highly expressed in many types of cancers and to contribute to tumorigenesis via the inhibition of apoptosis, increased angiogenesis and invasiveness. In hematological malignancies, COX-2 expression was found to correlate with poor patient prognosis. However, the exact role of COX-2 expression in these malignancies, and particularly in erythroleukemias, remains unclear. The aim of this work was to describe and understand the relationships between COX-2 expression and apoptosis rate in erythroleukemia cells after apoptosis induction by several anticancer agents. We used three different erythroleukemia cell lines in which COX-2 expression was modulated by transfection with either COX-2 siRNA or COX-2 cDNA. These cellular models were then treated with apoptosis inducers and apoptosis onset and intensity was followed. Cell signalling was evaluated in unstimulated transfected cells or after apoptosis induction. We found that COX-2 inhibition rendered erythroleukemia cells more sensitive to apoptosis induction and that in cells overexpressing COX-2 apoptosis induction was reduced. We demonstrated that COX-2 inhibition decreased the pro-survival Akt signalling and activated the negative regulator of Akt signalling, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Conversely, in COX-2 overexpressing cells, Akt signalling was activated and PTEN was inhibited. In these last cells, inhibition of casein kinase 2 or Akt signalling restored sensitivity to apoptotic agents. Our findings highlighted that COX-2 can positively regulate Akt signalling mostly through PTEN inhibition, partly via casein kinase 2 activation, and enhances survival of erythroleukemia cells exposed to anticancer agents.     

Interplay between PI3K/Akt and MAPK signaling pathways in DNA-damaging drug-induced apoptosis

In order to elucidate the role of the mitogen-activated protein kinases, including JNK, p38 MAPK and ERK, as well as the survival-associated PI3K/Akt signaling pathway, in the response to chemotherapy, we have conducted a comparative study regarding the effects of doxorubicin on these pathways. Doxorubicin was determined to elicit the apoptosis of NIH3T3 cells in a dose-dependent manner. Prior to cell death, both Akt and p38 MAPK were transiently activated, and subsequently inactivated almost wholly, whereas ERK and JNK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and p38 MAPK both accelerated and enhanced doxorubicin-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. The modulation of PI3K/Akt activation by treatment of LY294002 or expression of Akt mutants such as Akt-DN or Myr-Akt exerted a significant effect on the activation of ERK1/2. We also observed that PI3K/Akt and sustained ERK activation were associated intimately with the etoposide-induced apoptosis. Taken together, our results clearly suggest that the differential regulation of the PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways are crucial in the context of DNA-damaging drug-induced apoptosis, and this has compelled us to propose that the sustained activation of ERK1/2 pathway may be generally involved in the apoptosis induced by anticancer DNA-damaging drugs, including doxorubicin and etoposide.     

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.     

Cbl-b promotes chemotherapy-induced apoptosis in rat basophilic leukemia cells by suppressing PI3K/Akt activation and enhancing MEK/ERK activation

The ubiquitin ligase Cbl-b is a negative regulator of the PI3K/Akt pathway, the survival pathway implicated in chemotherapy resistance. However, it remains unclear whether Cbl-b can regulate chemosensitivity through modulating Akt activation. In this study, VP-16-induced RBL-2H3 cells apoptosis was accompanied by the activation of Akt and ERK. The PI3K inhibitor LY294002, not the ERK inhibitor PD98059, enhanced the apoptosis. In addition, down-regulation of Cbl-b was also detected. Over expression of Cbl-b significantly enhanced VP-16-induced cell apoptosis with inhibition of Akt activity, while a dominant negative (DN) RING Finger domain mutation completely abolished this enhancement. On the other hand, ERK activity was enhanced by Cbl-b, and the ERK inhibitor PD98059 reversed Cbl-b-enhanced apoptosis. The consistent results were also showed in the process of Ara-c treatment. These observations indicate that Cbl-b promotes RBL-2H3 apoptosis induced by VP-16 or Ara-c, probably through inhibition of Akt and activation of ERK.     

Regulation of rheumatoid synovial cell growth by ceramide

Overgrowth of rheumatoid synoviocytes, which results in joint destruction, is due to impaired balance between cell proliferation and cell death (apoptosis). Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. We investigated the effects of ceramide on growth-promoting anti-apoptotic signals in rheumatoid synovial cells. Human synovial cells isolated from patients with rheumatoid arthritis (RA) were stimulated with platelet-derived growth factor (PDGF) in the presence or absence of C2-ceramide. The kinase activity of Akt, MEK, and ERK1/2 was analyzed in PDGF-stimulated synovial cells by Western blot analysis. Pretreatment with C2-ceramide completely inhibited PDGF-induced cell cycle progression of rheumatoid synovial cells. PDGF stimulation induced phosphorylation and activation of Akt, MEK, and ERK1/2 in rheumatoid synovial cells. C2-ceramide inhibited the activation of Akt, MEK and ERK1/2 in PDGF-stimulated synovial cells. Our data demonstrated that inhibition of anti-apoptotic kinases, such as Akt and ERK1/2, may play an important role in ceramide-mediated apoptosis of rheumatoid synovial cells.     

Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis

Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NFkappaB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFkappaB signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.     

PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent.MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation.Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.     

Apoptosis induced by troglitazone is both peroxisome proliferator-activated receptor-gamma- and ERK-dependent in human non-small lung cancer cells

The role of the peroxisome proliferator-activated receptor-gamma (PPARgamma) in cell differentiation, cell-cycle arrest, and apoptosis has attracted increasing attention. We have recently demonstrated that PPARgamma ligands-troglitazone (TGZ) induced apoptosis in lung cancer cells. In this report, we further studied the role of ERK1/2 in lung cancer cells treated by TGZ. The result demonstrated that TGZ induced PPARgamma and ERK1/2 accumulation in the nucleus, in which the co-localization of both proteins was found. The activation of ERK1/2 resulted in apoptosis via a mitochondrial pathway. Both PPARgamma siRNA and U0126, a specific inhibitor of ERK1/2, were able to block these effects of TGZ, suggesting that apoptosis induced by TGZ was PPARgamma and ERK1/2 dependent. Inhibition of ERK1/2 by U0126 also led to a significant decrease in the level of PPARgamma, indicating a positive cross-talk between PPARgamma and ERK1/2 or an auto-regulatory feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and apoptosis. In addition to ERK1/2, TGZ also activated Akt. Interestingly, inhibition of ERK1/2 prevented the activation of Akt whereas the suppression of Akt had no effect on ERK1/2, suggesting that Akt was not necessary for TGZ-PPARgamma-ERK pathway. However, the inhibition of Akt promoted the release of cytochrome c, suggesting the activation of Akt may have a negative effect on apoptosis induced by TGZ. In conclusion, our study has demonstrated that TGZ, a synthetic PPARgamma ligand, induced apoptosis in NCI-H23 lung cancer cells via a mitochondrial pathway and this pathway was PPARgamma and ERK1/2 dependent.     

Signaling mechanisms leading to the regulation of differentiation and apoptosis of articular chondrocytes by insulin-like growth factor-1

Cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes. Differentiated chondrocytes in articular cartilage undergo dedifferentiation and apoptosis during arthritis, in which NO production plays a critical role. Here, we investigated the roles and mechanisms of action of insulin-like growth factor-1 (IGF-1) in the chondrogenesis of mesenchymal cells and the maintenance and survival of differentiated articular chondrocytes. IGF-1 induced chondrogenesis of limb bud mesenchymal cells during micromass culture through the activation of phosphatidylinositol 3-kinase (PI3K) and Akt. PI3K activation is required for the activation of protein kinase C (PKC)-alpha and p38 kinase and inhibition of ERK1/2. These events are necessary for chondrogenesis. The growth factor additionally blocked NO-induced dedifferentiation and apoptosis of primary culture articular chondrocytes. NO production in chondrocytes induced down-regulation of PI3K and Akt activities, which was blocked by IGF-1 treatment. Stimulation of PI3K by IGF-1 resulted in blockage of NO-induced activation of p38 kinase and ERK1/2 and inhibition of PKCalpha and PKCzeta, which in turn suppressed dedifferentiation and apoptosis. Our results collectively indicate that IGF-1 regulates differentiation, maintenance of the differentiated phenotype, and apoptosis of articular chondrocytes via a PI3K pathway that modulates ERK, p38 kinase, and PKC signaling.     

Cytoprotective roles of ERK and Akt in endoplasmic reticulum stress triggered by subtilase cytotoxin

Subtilase cytotoxin (SubAB) is the prototype of a distinct AB₅ toxin family produced by Shiga toxigenic Escherichia coli. Recent reports disclosed pro-apoptotic pathways triggered by SubAB, whereas its anti-apoptotic signals have not been elucidated. In the present study, we investigated pro-survival signaling elicited by SubAB, especially focusing on extracellular signal-regulated kinase (ERK) and Akt. We found that SubAB activated ERK and Akt, and inhibition of individual kinases enhanced SubAB-triggered apoptosis. SubAB induced endoplasmic reticulum (ER) stress, and other ER stress inducers mimicked the stimulatory effects of SubAB on ERK and Akt. Attenuation of ER stress reduced SubAB-induced phosphorylation of these kinases, suggesting involvement of the unfolded protein response (UPR). SubAB induced activation of protein kinase-like ER kinase (PERK) and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and phosphorylation of eIF2α by salubrinal caused activation of ERK and Akt, leading to cell survival. Dominant-negative inhibition of PERK enhanced SubAB-induced apoptosis and reduced phosphorylation of ERK and Akt. Furthermore, the anti-apoptotic effect of eIF2α was significantly reversed by inhibition of ERK and Akt. These results suggest cytoprotective roles of ERK and Akt in SubAB-triggered, ER stress-mediated apoptosis.     

Wogonin induces cross-regulation between autophagy and apoptosis via a variety of Akt pathway in human nasopharyngeal carcinoma cells

Autophagy as well as apoptosis is an emerging target for cancer therapy. Wogonin, a flavonoid compound derived from the traditional Chinese medicine of Huang‐Qin, has anticancer activity in many cancer cells including human nasopharyngeal carcinoma (NPC). However, the involvement of autophagy in the wogonin‐induced apoptosis of NPC cells was still uninvestigated. In this study, we found wogonin‐induced autophagy had interference on the process of apoptosis. Wogonin‐induced autophagy formation evidenced by LC3 I/II cleavage, acridine orange (AO)‐stained vacuoles and the autophagosome/autolysosome images of TEM analysis. Activation of autophagy with rapamycin resulted in increased wogonin‐mediated autophagy via inhibition of mTOR/P70S6K pathway. The functional relevance of autophagy in the antitumor activity was investigated by annexin V‐positive stained cells and PARP cleavage. Induction of autophagy by rapamycin ameliorated the wogonin‐mediated apoptosis, whereas inhibition of autophagy by 3‐methyladenine (3‐MA) or bafilomycin A1 increased the apoptotic effect. Interestingly, this study also found, in addition the mTOR/P70S6K pathway, wogonin also inhibited Raf/ERK pathway, a variety of Akt pathways. Inactivation of PI₃K/Akt by their inhibitors significantly induced apoptosis and markedly sensitized the NPC cells to wogonin‐induced apoptosis. This anticancer effect of Akt was further confirmed by SH6, a specific inhibitor of Akt. Importantly, inactivation of its downstream molecule ERK by PD98059, a MEK inhibitor, also induced apoptosis. This study indicated wogonin‐induced both autophagy and apoptosis through a variety of Akt pathways and suggested modulation of autophagy might provide profoundly the potential therapeutic effect. J. Cell. Biochem. 113: 3476–3485, 2012. © 2012 Wiley Periodicals, Inc.     

Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1

Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.     

Long chain n-3 polyunsaturated fatty acids increase the efficacy of docetaxel in mammary cancer cells by downregulating Akt and PKCε/δ-induced ERK pathways

Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.     

Sphingosine-1-phosphate promotes mouse melanocyte survival via ERK and Akt activation

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation.     

Cyclin-dependent kinase-5 prevents neuronal apoptosis through ERK-mediated upregulation of Bcl-2

Cyclin-dependent kinase-5 (Cdk5) is required for neuronal survival, but its targets in the apoptotic pathways remain unknown. Here, we show that Cdk5 kinase activity prevents neuronal apoptosis through the upregulation of Bcl-2. Treatment of SH-SY5Y cells with retinoid acid (RA) and brain-derived neurotrophic factor (BDNF) generates differentiated neuron-like cells. DNA damage triggers apoptosis in the undifferentiated cells through mitochondrial pathway; however, RA/BDNF treatment results in Bcl-2 upregulation and inhibition of the mitochondrial pathway in the differentiated cells. RA/BDNF treatment activates Cdk5-mediated PI3K/Akt and ERK pathways. Inhibition of Cdk5 inhibits PI3K/Akt and ERK phosphorylation and Bcl-2 expression, and thus sensitizes the differentiated cells to DNA-damage. Inhibition of ERK, but not PI3K/Akt, abrogates Cdk5-medidated Bcl-2 upregulation and the protection of the differentiated cells. This study suggests that ERK-mediated Bcl-2 upregulation contributes to BDNF-induced Cdk5-mediated neuronal survival.     

Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

Roles of ERK, PI3 kinase, and PLC-γ pathways induced by overexpression of translationally controlled tumor protein in HeLa cells

We reported previously that translationally controlled tumor protein (TCTP) is a cytoplasmic repressor of Na,K-ATPase in HeLa cells. In the current study, we showed that TCTP overexpression using adenovirus as vehicle, induced partial inhibition of Na,K-ATPase; phosphorylation of EGFR tyrosine residues 845, 992, 1068, and 1148; activation of Ras/Raf/ERK pathway; activation of PI3K/Akt pathway; and phosphorylation of PLC-γ in HeLa cells. Specific inhibition of PI3K/Akt pathway in contrast to the inhibition of ERK, significantly decreased TCTP overexpression-induced survival signal. Inhibition of PLC-γ pathway significantly decreased TCTP overexpression-induced cell migration but inhibition of ERK had less effect. These results suggest that TCTP plays a key physiological role in cell survival through Akt pathway and migration through PLC-γ pathway.     

肠道病毒A71型致人扁桃体上皮细胞系UT-SCC-60B凋亡和S期阻滞的研究

肠道病毒A71型(Enterovirus A71,EV-A71)是手足口病的重要病原体,为研究EV-A71感染人扁桃体上皮细胞后对细胞凋亡和细胞周期的影响,确定ERK1/2、JNK1/2、PI3K/Akt和含半胱氨酸的天冬氨酸蛋白水解酶(Cysteinyl aspartate specific proteinase,Caspase)的作用,本文以人扁桃体上皮细胞系UT-SCC-60B为细胞模型,CCK-8试剂盒检测EV-A71对UT-SCC-60B的抑制率、流式细胞仪检测EV-A71感染组和抑制剂处理组的凋亡和细胞周期、Caspase活力检测试剂盒测定Caspase-3,Caspase-8,Caspase-9活力。EV-A71以感染剂量和感染时间依赖方式抑制UT-SCC-60B增殖;EV-A71感染致UT-SCC-60B发生细胞凋亡,抑制ERK1/2、JNK1/2和PI3K/Akt能够降低UT-SCC-60B细胞凋亡比例;EV-A71感染UT-SCC-60B后发生S期阻滞,抑制ERK1/2、JNK1/2、PI3K/Akt和Caspase阻止UT-SCC-60B发生S期阻滞;EV-A71感染UT-SCC-60B能够活化Caspase-3,Caspase-8,Caspase-9且ERK1/2、JNK1/2和PI3K/Akt调控Caspase-3,Caspase-8,Caspase-9活力。因此,EV-A71能够导致人扁桃体上皮细胞UT-SCC-60B发生凋亡和S期阻滞,并且ERK1/2、JNK1/2、PI3K/Akt和Caspase参与凋亡和S期阻滞的调控。     

The hVps34‐SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumour growth

We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT‐474 breast cancer cell‐derived tumours in a xenograft model. Finally, we present the kinome‐wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34‐SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.     

Cytochrome P-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-alpha via MAPK and PI3K/Akt signaling pathways

Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-alpha induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-alpha. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-alpha-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-alpha. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-alpha. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.