ERK1/2 and Akt pathway activated during (3R,6R)-bassiatin(1)-induced apoptosis in MCF-7 cells

(3R,6R)-bassiatin(1) was isolated from the endogenous fungus, Fusarium oxysporum J8-1-2. Previous studies showed that (3R,6R)-bassiatin(1) has anti-oestrogen properties which make it cytotoxic to ER (oestrogen receptor)-positive breast cancer cells (MCF-7) by cell cycle arrest and induction of apoptosis. (3R,6R)-bassiatin(1) suppresses mRNA and protein expression of the ERα and oestrogen responsive genes of cyclin D1 and PR. We have investigated the interaction between (3R,6R)-bassiatin(1) and ERα and followed the roles of ERK (extracellular-signal-regulated kinase), Akt and GSK3β (glycogen synthase kinase 3β) during (3R,6R)-bassiatin(1)-induced apoptosis of MCF-7 cells. (3R,6R)-bassiatin(1) competed with E2 (17β-estradiol) for ERα active sites to inhibit ERα activation. However, while ERK1/2 and Akt were activated, GSK3β was inactivated during (3R,6R)-bassiatin(1)-induced apoptosis, suggesting that this compound is indeed an anti-oestrogen agent that can also activate the survival signalling pathway. Apoptosis caused by (3R,6R)-bassiatin(1) may be related to activation of ERK.     

Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells

There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer.In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells.Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells.     

Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser⁴⁷³ phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.     

Overexpression of cyclin D1 is associated with elevated levels of MAP kinases, Akt and Pak1 during diethylnitrosamine-induced progressive liver carcinogenesis

Hepatocellular carcinoma (HCC) is a multi-factorial and multi-step process. However, the molecular mechanisms, which play a pivotal role during progressive development of HCC, are not known. Accordingly Sprague-Dawley rats were administered diethylnitrosamine (DEN) for one to three months in order to understand the molecular alterations during progressive development of liver tumor. In this study involvement of G1/S regulatory proteins, MAP kinases and cell survival factors were analyzed using RT-PCR, western blotting and in vitro kinase assays. The data showed overexpression of cyclin D1 and increased expression and activation of ERK1/2, p38 kinase and JNK1/2 with progression of tumor suggesting that MAP kinases play an important role during tumorigenesis. These molecular alterations were supported by Akt upregulation and increase in the levels of inactive GSK3beta with progression of liver tumor. Further, p21-actived kinase1 (Pak1) was found to be upregulated with tumor progression, which is a novel observation during progressive liver carcinogenesis. These results indicate that elevated levels of all the three MAP kinases (ERK1/2, p38 and JNK1/2), Akt/GSK3beta and Pak1 are associated with cyclin D1 upregulation, which helps in the disruption of the G1/S regulatory point of the cell cycle and leads to abnormal cell proliferation during progressive hepatocarcinogenesis.     

The role of mitochondrial reactive oxygen species in cartilage matrix destruction

According to a compelling body of evidence anesthetic preconditioning (APC) attenuates the deleterious consequences of ischemia–reperfusion and protects the heart through a mechanism similar to ischemic preconditioning. The present study was purported to investigate the intracellular signaling pathways activated in human myocardium in response to a preconditioning protocol with two different volatile anesthetics, namely isoflurane and sevoflurane. To this aim, phosphorylation of PKCα and -δ, ERK1/2, Akt, and GSK3β was determined at the end of the APC protocol, in human atrial samples harvested from patients undergoing open-heart surgery. The results demonstrate that preconditioning with volatile anesthetics triggers the activation of PKCδ and -α isoforms and of prosurvival kinases, ERK1/2, and Akt, while inhibiting their downstream target GSK3β during the memory phase.     

L-leucine increases [3H]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.     

Enhancement of insulin-induced PI3K/Akt/GSK-3beta and ERK signaling by neuronal nicotinic receptor/PKC-alpha/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells treated with nicotine (10 µm for 24 h), phosphorylation of Akt, glycogen synthase kinase‐3β (GSK‐3β) and extracellular signal‐regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by ～ 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)‐ and concentration (EC₅₀ 3.6 and 13 µm )‐dependent increases in insulin receptor substrate (IRS)‐1 and IRS‐2 levels by ～ 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin‐induced tyrosine phosphorylation of IRS‐1/IRS‐2 and recruitment of phosphoinositide 3‐kinase (PI3K) to IRS‐1/IRS‐2 were augmented by ～ 63%. The increase in IRS‐1/IRS‐2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis‐acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS‐1 and IRS‐2 mRNA levels by ～ 57 and ～ 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC‐α, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC‐α by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up‐regulated expression of IRS‐1/IRS‐2 via Ca²⁺‐dependent sequential activation of cPKC‐α and ERK, and enhanced insulin‐induced PI3K/Akt/GSK‐3β and ERK signaling pathways.     

Role of PI3K in myocardial ischaemic preconditioning: mapping pro‐survival cascades at the trigger phase and at reperfusion

The Reperfusion Injury Salvage Kinase (RISK) pathway is considered the main pro‐survival kinase cascade mediating the ischaemic preconditioning (IPC) cardioprotective effect. To assess the role of PI3K‐Akt, its negative regulator PTEN and other pro‐survival proteins such as ERK and STAT3 in the context of IPC, C57BL/6 mouse hearts were retrogradely perfused in a Langendorff system and subjected to 4 cycles of 5 min. ischaemia and 5 min. reperfusion prior to 35 min. of global ischaemia and 120 min. of reperfusion. Wortmannin, a PI3K inhibitor, was administered either at the stabilization period or during reperfusion. Infarct size was assessed using triphenyl tetrazolium staining, and phosphorylation levels of Akt, PTEN, ERK, GSK3β and STAT3 were evaluated using Western blot analyses. IPC reduced infarct size in hearts subjected to lethal ischaemia and reperfusion, but this effect was lost in the presence of Wortmannin, whether it was present only during preconditioning or only during early reperfusion. IPC increased the levels of Akt phosphorylation during both phases and this effect was fully abrogated by PI3K, whilst its downstream GSK3β was phosphorylated only during the trigger phase after IPC. Both PTEN and STAT3 were phosphorylated during both phases after IPC, but this was PI3K independent. IPC increases ERK phosphorylation during both phases, being only PI3K‐dependent during the IPC phase. In conclusion, PI3K‐Akt plays a major role in IPC‐induced cardioprotection. However, PTEN, ERK and STAT3 are also phosphorylated by IPC through a PI3K‐independent pathway, suggesting that cardioprotection is mediated through more than one cell signalling cascade.     

Biphasic estradiol-induced AKT phosphorylation is modulated by PTEN via MAP kinase in HepG2 cells

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1-S transition by the parallel stimulation of both PKC-alpha and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17beta-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1-S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1-S phase transition.     

Signal transduction of MEK/ERK and PI3K/Akt activation by hypoxia/reoxygenation in renal epithelial cells

The extracellular signal-regulated kinase (ERK) and Akt have been reported to be activated by ischemia/reperfusion in vivo. However, the signaling pathways involved in activation of these kinases and their potential roles were not fully understood in the postischemic kidney. In the present study, we observed that these kinases are activated by hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion, in opossum kidney (OK) cells and elucidated the signaling pathways of these kinases. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-12h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of ERK upstream MAPK/ERK kinase (MEK), but not by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), whereas Akt activation was blocked by LY294002, but not by U0126. Inhibitors of epidermal growth factor receptor (EGFR) (AG 1478), Ras and Raf, as well as antioxidants inhibited activation of ERK and Akt, while the Src inhibitor PP2 had no effect. PI3K/Akt activation was shown to be associated with up-regulation of X chromosome-linked inhibitor of apoptosis (XIAP), but not survivin. Reoxygenation following 4-h hypoxia-stimulated cell proliferation, which was dependent on ERK and Akt activation and was also inhibited by antioxidants and AG 1478. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt/XIAP survival signaling pathways through the reactive oxygen species-dependent EGFR/Ras/Raf cascade. Activation of these kinases may be involved in the repair process during ischemia/reperfusion.     

Regulation of protein kinases in insulin,growth factor and Wnt signalling

Protein kinase cascades provide the regulatory mechanisms for many of the essential processes in eukaryotic cells. Recent structural and biochemical work has revealed the basis of phosphorylation regulation of three consecutive protein kinases - phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (PKB)/Akt and glycogen synthase kinase 3beta (GSK3beta) - which transduce signals generated by insulin and/or growth factors binding to cell surface receptors. PDK1 and PKB are both AGC family kinases. Whereas PKB is positively regulated via its phosphorylated C-terminal hydrophobic motif, the activity and specificity of PDK1 are determined by equivalent hydrophobic motifs of substrate AGC kinases. In a contrasting mechanism, GSK3beta is negatively regulated by competitive autoinhibition by its phosphorylated N terminus. GSK3beta also functions in the developmental Wnt signalling pathway, but without cross-talk with the PDK1-PKB/Akt pathway. Structural studies of GSK3beta complexes are contributing to our understanding of the phosphorylation-independent mechanism that insulates the Wnt and insulin/growth factor pathways.     

Cross-talk between insulin and Wnt signaling in preadipocytes: role of Wnt co-receptor low density lipoprotein receptor-related protein-5 (LRP5)

Disturbed Wnt signaling has been implicated in numerous diseases, including type 2 diabetes and the metabolic syndrome. In the present study, we have investigated cross-talk between insulin and Wnt signaling pathways using preadipocytes with and without knockdown of the Wnt co-receptors LRP5 and LRP6 and with and without knock-out of insulin and IGF-1 receptors. We find that Wnt stimulation leads to phosphorylation of insulin signaling key mediators, including Akt, GSK3β, and ERK1/2, although with a lower fold stimulation and slower time course than observed for insulin. These Wnt effects are insulin/IGF-1 receptor-dependent and are lost in insulin/IGF-1 receptor double knock-out cells. Conversely, in LRP5 knockdown preadipocytes, insulin-induced phosphorylation of IRS1, Akt, GSK3β, and ERK1/2 is highly reduced. This effect is specific to insulin, as compared with IGF-1, stimulation and appears to be due to an inducible interaction between LRP5 and the insulin receptor as demonstrated by co-immunoprecipitation. These data demonstrate that Wnt and insulin signaling pathways exhibit cross-talk at multiple levels. Wnt induces phosphorylation of Akt, ERK1/2, and GSK3β, and this is dependent on insulin/IGF-1 receptors. Insulin signaling also involves the Wnt co-receptor LRP5, which has a positive effect on insulin signaling. Thus, altered Wnt and LRP5 activity can serve as modifiers of insulin action and insulin resistance in the pathophysiology of diabetes and metabolic syndrome.     

Study of the pathways involved in apoptosis induced by PI3K inhibition in cerebellar granule neurons

In the present study we focused in the PI3K/Akt pathway which plays a key role in neuronal survival. Here we show that inhibition of PI3K/Akt by means of LY294002 induces apoptosis via a caspase-dependent and calpain-independent pathway in cerebellar granule neurons (CGNs). This finding was confirmed using zVAD-fmk, a widely caspase inhibitor that prevents apoptosis. For this purpose, we compared two models of apoptosis in CGNs, namely inhibition of PI3K/Akt, and serum potassium deprivation (S/K deprivation). In contrast to the S/K deprivation model, caspase-3 was not activated when PI3K is inhibited. Likewise, CDK5 activation was not involved in this apoptotic process, because calpain activation is responsible for the formation of CDK5/p25 neurotoxic form. However, S/K deprivation activated calpain, as it is shown by α-spectrin breakdown, and favoured the formation of CDK5/p25. Moreover, although PI3K/Akt inhibition enhanced pRbser780 phosphorylation, no increase in the expression of cell-cycle proteins, namely: cyclin D, cyclin E, CDK2 or CDK4, was detected. Furthermore, BrdU incorporation assay did not shown any increase in DNA synthesis. Likewise, PI3K/Akt inhibition increased GSK3β activity and c-Jun phosphorylation, which implicates these two pathways in this apoptotic route. Although previous reports suggest that apoptosis induced in CGNs by LY294002 and S/K deprivation causes PI3K inhibition and increases GSK3β activity and c-Jun phosphorylation activation, our results demonstrate substantial differences between them and point to a key role of GSK3β in the apoptosis induced in CGNs in the two models tested.     

PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells

Lithium is a well‐established non‐competitive inhibitor of glycogen synthase kinase‐3β (GSK‐3β), a kinase that is involved in several cellular processes related to cancer progression. GSK‐3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK‐3β‐independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment. J. Cell. Biochem. 117: 458–469, 2016. © 2015 Wiley Periodicals, Inc.     

Regulation of PI3K/Akt/GSK-3 pathway by cannabinoids in the brain

Delta9-tetrahydrocannabinol (THC), the main psychoactive component in Cannabis sativa preparations, exerts its central effects mainly through the G-protein coupled receptor CB1, a component of the endocannabinoid system. Several in vitro and in vivo studies have reported neuroprotective effects of cannabinoids in excitotoxicity and neurodegeneration models. However, the intraneuronal signaling pathways activated in vivo by THC underlying its central effects remain poorly understood. We report that THC acute administration (10 mg/kg, i.p.) increases the phosphorylation of Akt in mouse hippocampus, striatum, and cerebellum. This phosphorylation was mediated by CB1 receptors as it was blocked by the selective CB1 antagonist rimonabant. Moreover, PI3K inhibition by wortmannin abrogated THC-induced phosphorylation of Akt, but blockade of extracellular signal-regulated protein kinases by SL327 did not modify this activation/phosphorylation of Akt. Moreover, administration of the dopaminergic D1 (SCH 23390) and D2 (raclopride) receptor antagonists did not block the activation of PI3K/Akt pathway induced in the striatum by cannabinoid receptor stimulation, suggesting that this effect is independent of the dopaminergic system. In addition, THC increased the phosphorylation of glycogen synthase kinase 3 beta. Therefore, activation of the PI3K/Akt/GSK-3 signaling pathway may be related to the in vivo neuroprotective properties attributed to cannabinoids.     

Stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1alpha treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1alpha induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important "cross-talk" between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.