Roles of the Akt/mTOR/p70S6K and ERK1/2 Signaling Pathways in Curcumin-Induced Autophagy

Curcumin has a potent anticancer effect and is a promising new therapeutic strategy. We previously demonstrated that curcumin induced non-apoptotic autophagic cell death in malignant glioma cells in vitro and in vivo. This compound inhibited the Akt/mammalian target of rapamycin/p70 ribosomal protein S6 kinase pathway and activated the extracellular signal-regulated kinases 1/2 thereby inducing autophagy. Interestingly, activation of the first pathway inhibited curcumin-induced autophagy and cytotoxicity, whereas inhibition of the latter pathway inhibited curcumin-induced autophagy and induced apoptosis, thus augmenting the cytotoxicity of curcumin. These results imply that these two autophagic pathways have opposite effects on curcumin’s cytotoxicity. However, inhibition of nuclear factor κB, which is the main target of curcumin for its anticancer effect, was not observed in malignant glioma cells. These results suggest that autophagy but not nuclear factor κB plays a central role in curcumin anticancer therapy and warrant further investigation toward application in patients with malignant gliomas. Here, we discuss the therapeutic role of two autophagic pathways influenced by curcumin.

Addendum to:

Evidence That Curcumin Suppresses the Growth of Malignant Gliomas in Vitro and in Vivo through Induction of Autophagy: Role of Akt and Extracellular Signal-Regulated Kinase Signaling Pathways

H. Aoki, Y. Takada, S. Kondo, R. Sawaya, B. B. Aggarwal and Y. Kondo

Mol Pharmacol 2007; 72:29-39     

Roles of the Akt/mTOR/p70S6K and ERK1/2 signaling pathways in curcumin-induced autophagy

Curcumin has a potent anticancer effect and is a promising new therapeutic strategy. We previously demonstrated that curcumin induced non-apoptotic autophagic cell death in malignant glioma cells in vitro and in vivo. This compound inhibited the Akt/mammalian target of rapamycin/p70 ribosomal protein S6 kinase pathway and activated the extracellular signal-regulated kinases 1/2 thereby inducing autophagy. Interestingly, activation of the first pathway inhibited curcumin-induced autophagy and cytotoxicity, whereas inhibition of the latter pathway inhibited curcumin-induced autophagy and induced apoptosis, thus augmenting the cytotoxicity of curcumin. These results imply that these two autophagic pathways have opposite effects on curcumin's cytotoxicity. However, inhibition of nuclear factor kappaB, which is the main target of curcumin for its anticancer effect, was not observed in malignant glioma cells. These results suggest that autophagy but not nuclear factor kappaB plays a central role in curcumin anticancer therapy and warrant further investigation toward application in patients with malignant gliomas. Here, we discuss the therapeutic role of two autophagic pathways influenced by curcumin.     

Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

The role of autophagy and lysosomal degradation pathway in the regulation of skeletal muscle metabolism was previously studied. However, underlying molecular mechanisms are poorly understood. L-lactate which is utilized as an energetic substrate by skeletal muscle can also augment genes expression related to metabolism and up-regulate those being responsive to reactive oxygen species (ROS). Since ROS is the most important regulator of autophagy in skeletal muscle, we tested if there is a link between cellular lactate metabolism and autophagy in differentiated C2C12 myotubes and the gastrocnemius muscle of male wistar rats. C2C12 mouse skeletal muscle was exposed to 2, 6, 10, and 20 mM lactate and evaluated for lactate autophagic effects. Lactate dose-dependently increased autophagy and augmented ROS generation in differentiated C2C12 myotubes. The autophagic effect of lactate deterred in N-acetylcysteine presence (NAC, a ROS scavenger) indicated lactate regulates autophagy with ROS participation. Lactate-induced up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) through ROS was required to regulate the autophagy by lactate. Further analysis about ERK1/2 up- and downstream indicated that lactate regulates autophagy through ROS-mediated the activation of ERK1/2/mTOR/p70S6K pathway in skeletal muscle. The in vitro effects of lactate on autophagy also occurred in the gastrocnemius muscle of male Wistar rats. In conclusion, we provided the lactate-associated regulation evidence of autophagy in skeletal muscle by activating ROS-mediated ERK1/2/mTOR/p70S6K pathway. Since the increase in cellular lactate concentration is a hallmark of energy deficiency, the results provide insight into a skeletal muscle mechanism to fulfill its enhanced energy requirement.     

Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms

The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.     

Mollugin induces tumor cell apoptosis and autophagy via the PI3K/AKT/mTOR/p70S6K and ERK signaling pathways

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L., has shown preclinical anticancer efficacy in various cancer models. However the effects of mollugin in regulating cancer cell survival and death remains undefined. In the present study we found that mollugin exhibited cytotoxicity on various cancer models. The suppression of cell viability was due to the induction of mitochondria apoptosis. In addition, the presence of autophagic hallmarks was observed in mollugin-treated cells. Notably, blockade of autophagy by a chemical inhibitor or RNA interference enhanced the cytotoxicity of mollugin. Further experiments demonstrated that phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) and extracellular regulated protein kinases (ERK) signaling pathways participated in mollugin-induced autophagy and apoptosis. Together, these findings support further studies of mollugin as candidate for treatment of human cancer cells.     

Total ERK1/2 activity regulates cell proliferation

Regulating ERK activity is essential for normal cell proliferation to occur. In mammals and most vertebrates ERK activity is provided by ERK1 and ERK2 that are highly similar, ubiquitously expressed and share activators and substrates. By combining single and double silencings of ERK1 and ERK2 we recently demonstrated that the apparent dominant role of ERK2 to regulate cell proliferation was due to its markedly higher expression level than ERK1. The contribution of ERK1 was revealed when ERK2 activation was clamped to avoid compensating over-activation of ERK2. We found no evidences in the literature for insulated isoform-specific modules in the Ras/Raf/MEK signaling cascade that could activate specifically ERK1 or ERK2. Obviously in frogs all signal integration and fine modulation provided by three Ras and three Raf isoforms is conducted by only one MEK and one ERK isoform. In mammals, ERK1 and ERK2 display similar specific activities and are activated respectively to their expression levels. After integrating signals from Ras, Raf and MEK isoforms, ERK1 and ERK2 regulate positively cell proliferation according to their expression levels.     

Contraction-mediated mTOR, p70S6k, and ERK1/2 phosphorylation in aged skeletal muscle.

With age, skeletal muscle experiences substantial atrophy and weakness. Although resistance training can increase muscle size and strength, the myogenic response to exercise and the capacity for muscle hypertrophy in older humans and animals is limited. In the present study, we assessed the ability of muscle contractile activity to activate cellular pathways involved in muscle cell growth and myogenesis in adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats. A single bout of rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla) and tibialis anterior (TA) muscles were assayed for mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70(S6K)), and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and total protein either at baseline, immediately after, or 6 h after HFES. mTOR phosphorylation was elevated in Pla (1.3 +/- 0.3-fold, P < 0.05) immediately after HFES and to a lesser extent 6 h after HFES (0.6 +/- 0.1-fold, P < 0.05) in O rats. Post-HFES, p70(S6K) phosphorylation increased 1.2 +/- 0.3-fold in TA (P < 0.05) and remained elevated 6 h later (0.6 +/- 0.2-fold, P < 0.05) in O rats. ERK phosphorylation was lower in O rats immediately after exercise in both TA (11.1 +/- 2.9 vs. 2.1 +/- 0.5-fold, P < 0.05) and Pla (6.5 +/- 1.5 vs. 1.8 +/- 0.5-fold, P < 0.05) and returned to baseline by 6 h in both Y and O rats. Phosphorylation of mTOR, p70(S6K), and ERK1/2 are increased in skeletal muscle after a single bout of in situ muscle contractile activity in aged animals, and the response is less than that observed in adult animals. These observations suggest that the anabolic response to a single bout of contraction is attenuated with aging and may help explain the reduced capacity for hypertrophy in aged animals.     

PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1

BACKGROUND: The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation via the downstream targets ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). We have identified phosphatidic acid (PA) as a mediator of mitogenic activation of mTOR signaling. In this study, we set out to test the hypotheses that phospholipase D 1 (PLD1) is an upstream regulator of mTOR and that the previously reported S6K1 activation by Cdc42 is mediated by PLD1. RESULTS: Overexpression of wild-type PLD1 increased S6K1 activity in serum-stimulated cells, whereas a catalytically inactive PLD1 exerted a dominant-negative effect on S6K1. More importantly, eliminating endogenous PLD1 by RNAi led to drastic inhibition of serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation in both HEK293 and COS-7 cells. Knockdown of PLD1 also resulted in reduced cell size, suggesting a critical role for PLD1 in cell growth control. Using a rapamycin-resistant S6K1 mutant, Cdc42's action was demonstrated to be through the mTOR pathway. When Cdc42 was mutated in a region specifically required for PLD1 activation, its ability to activate S6K1 in the presence of serum was hindered. However, when exogenous PA was used as a stimulus, the PLD1-inactive Cdc42 mutant behaved similarly to the wild-type protein. CONCLUSIONS: Our observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA.     

Arsenite induces cell transformation by reactive oxygen species, AKT, ERK1/2, and p70S6K1

Arsenic is naturally occurring element that exists in both organic and inorganic formulations. The inorganic form arsenite has a positive association with development of multiple cancer types. There are significant populations throughout the world with high exposure to arsenite via drinking water. Thus, human exposure to arsenic has become a significant public health problem. Recent evidence suggests that reactive oxygen species (ROS) mediate multiple changes to cell behavior after acute arsenic exposure, including activation of proliferative signaling and angiogenesis. However, the role of ROS in mediating cell transformation by chronic arsenic exposure is unknown. We found that cells chronically exposed to sodium arsenite increased proliferation and gained anchorage-independent growth. This cell transformation phenotype required constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. We also observed these cells constitutively produce ROS, which was required for the constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. Suppression of ROS levels by forced expression of catalase also reduced cell proliferation and anchorage-independent growth. These results indicate cell transformation induced by chronic arsenic exposure is mediated by increased cellular levels of ROS, which mediates activation of AKT, ERK1/2, and p70S6K1.     

Cross-talk between the ERK and p70 S6 kinase (S6K) signaling pathways. MEK-dependent activation of S6K2 in cardiomyocytes.

The alpha(1)-adrenergic agonist phenylephrine (PE) and insulin each stimulate protein synthesis in cardiomyocytes. Activation of protein synthesis by PE is involved in the development of cardiac hypertrophy. One component involved here is p70 S6 kinase 1 (S6K1), which lies downstream of mammalian target of rapamycin, whose regulation is thought to involve phosphatidylinositol 3-kinase and protein kinase B (PKB). S6K2 is a recently identified homolog of S6K1 whose regulation is poorly understood. Here we demonstrate that in adult rat ventricular cardiomyocytes, PE and insulin each activate S6K2, activation being 3.5- and 5-fold above basal, respectively. Rapamycin completely blocked S6K2 activation by either PE or insulin. Three different inhibitors of MEK1/2 abolished PE-induced activation of S6K2 whereas expression of constitutively active MEK1 activated S6K2, without affecting the p38 mitogen-activated protein kinase and JNK pathways, indicating that MEK/ERK signaling plays a key role in regulation of S6K2 by PE. PE did not activate PKB, and expression of dominant negative PKB failed to block activation of S6K2 by PE, indicating PE-induced S6K2 activation is independent of PKB. However, this PKB mutant did partially block S6K2 activation by insulin, indicating PKB is required here. Another hypertrophic agent, endothelin 1, also activated S6K2 in a MEK-dependent manner. Our findings provide strong evidence for novel signaling connections between MEK/ERK and S6K2.     

p130Cas promotes invasiveness of three-dimensional ErbB2-transformed mammary acinar structures by enhanced activation of mTOR/p70S6K and Rac1

ErbB2 over-expression is detected in approximately 25% of invasive breast cancers and is strongly associated with poor patient survival. We have previously demonstrated that p130Cas adaptor is a crucial mediator of ErbB2 transformation. Here, we analysed the molecular mechanisms through which p130Cas controls ErbB2-dependent invasion in three-dimensional cultures of mammary epithelial cells. Concomitant p130Cas over-expression and ErbB2 activation enhance PI3K/Akt and Erk1/2 MAPK signalling pathways and promote invasion of mammary acini. By using pharmacological inhibitors, we demonstrate that both signalling cascades are required for the invasive behaviour of p130Cas over-expressing and ErbB2 activated acini. Erk1/2 MAPK and PI3K/Akt signalling triggers invasion through distinct downstream effectors involving mTOR/p70S6K and Rac1 activation, respectively. Moreover, in silico analyses indicate that p130Cas expression in ErbB2 positive human breast cancers significantly correlates with higher risk to develop distant metastasis, thus underlying the value of the p130Cas/ErbB2 synergism in regulating breast cancer invasion. In conclusion, high levels of p130Cas favour progression of ErbB2-transformed cells towards an invasive phenotype.     

Akirin2 could promote the proliferation but not the differentiation of duck myoblasts via the activation of the mTOR/p70S6K signaling pathway

The Akirin gene family normally contains two members that are essential to myoblast differentiation. Noticeably, the avian Akirin gene family comprises only one gene (Akirin2), However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation. Interestingly, the unexpected functions of duck Akirin2 were revealed in the present study. The Real-time PCR results showed that between 12 and 48 h during the process of duck myoblasts differentiation, the overexpression of Akirin2 did not significantly increase the expression of myogenic regulatory factors. Flow cytometry analysis revealed that the cell cycle transition was accelerated by Akirin2 overexpression. Moreover, the overexpression of Akirin2 did not influence the myotube formation. Strikingly, when duck myoblasts were cultured in the growth medium, the overexpression of Akirin2 significantly enhanced cell viability. Although the expression of cyclin-dependent proteins did not significantly increase after transfection, the expression of the mammalian targets of rapamycin (mTOR) and p70 S6 kinase (p70S6K) increased. Furthermore, the protein expression of phospho-p70S6K (Ser 417) also increased. However, when rapamycin and pEGFP-N1-Akirin2 plasmids were added together to the growth medium, the positive impact of Akirin2 on cell viability and the mRNA expression of mTOR and p70S6K were significantly blocked. Furthermore, the expression of phospho-mTOR (Ser 2448) and phospho-p70S6K (Ser 417) were also blocked. Taken together, these results could suggest that duck Akirin2 could promote myoblast proliferation via the activation of the mTOR/p70S6K signaling pathway.     

Programmed cell death 6 (PDCD6) inhibits angiogenesis through PI3K/mTOR/p70S6K pathway by interacting of VEGFR-2

Programmed cell death 6 (PDCD6) was originally found as a pro-apoptotic protein, but its molecular mechanism is not well understood. In this study, we have attempted to investigate the effects of PDCD6 on the inhibition of angiogenesis-mediated cell growth as a novel anti-angiogenic protein. Purified recombinant human PDCD6 inhibited cell migration in a concentration-time-dependent manner. We also found that overexpressed PDCD6 suppressed vascular endothelial growth factor (VEGF)-induced proliferation, invasion, and capillary-like structure tube formation in vitro. PDCD6 suppressed phosphorylation of signaling regulators downstream from PI3K, including Akt, mammalian target of rapamycin (mTOR), glycogen synthase kinase-3β(GSK-3β), ribosomal protein S6 kinase (p70S6K), and also decreased cyclin D1 expression. We found binding PDCD6 to VEGFR-2, a key player in the PI3K/mTOR/P70S6K signaling pathway. Taken together, these data suggest that PDCD6 plays a significant role in modulating cellular angiogenesis.     

Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway

Dysregulation of apoptotic and autophagic function are characterized as the main pathogeneses of diabetic nephropathy (DN). It has been reported that Karyopherin Alpha 2 (KPNA2) contributes to apoptosis and autophagy in various cells, but its role in DN development remains unknown. The purpose of present study was to explore the function and underling mechanisms of KPNA2 in development of DN. In this study, 30 mM high glucose (HG)-evoked podocytes were used as DN model. The expression of KPNA2 was detected by qRT-PCR and Western blot assays. The cell viability was tested by CCK-8 kit, the apoptosis was measured using flow cytometry assay, the apoptotic and the autophagy related genes was detected by Western blot. Our results indicated that KPNA2 was significantly increased after HG stimulation. Knockdown of KPNA2 inhibited apoptosis, and promoted cell viability and autophagy in HG-treated podocytes. In addition, silencing of KPNA2 deactivated mTORC1/p70S6K pathway activation via regulating SLC1A5. Further results demonstrated that activating mTORC1/p70S6K pathway strongly ameliorated the effect of KPNA2 on cell viability, apoptosis and autophagy. Therefore, our study suggested that knockdown of KPNA2 rescued HG-induced injury via blocking activation of mTORC1/p70S6K pathway by mediating SLC1A5.     

Abl regulates smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 activation

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.