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.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.     

Crosstalk from survival to necrotic death coexists in DU-145 cells by curcumin treatment

Curcumin as an anticancer agent was investigated in regards to its ability to regulate the switching of cancer cells from survival to necrotic cell death. At higher concentrations, curcumin induced ROS production leading to JNK and p38 phosphorylation in DU-145 prostate cancer cells. Of the MAP kinases, ERK or p38/JNK were phosphorylated earlier during curcumin treatment, and were responsible for curcumin-induced cell survival at early time of treatment with the help of phosphorylated Akt, while significant amounts of ROS production in later periods stimulated cell death with caspase degradation. In addition to autophagic signaling, necrosis was dominant with little apoptotic cell death. Caspase activation was completely prohibited by procaspase degradation, which contributed to curcumin-induced early necrosis. At the later incubation period (24 h), cytotoxicity caused by curcumin peaked, at which time survival or proliferation signals, such as phosphorylated Akt and phosphorylated ERK, was almost completely diminished. Curcumin-induced ROS were shown to function, biphasically depending on the incubation period; facilitating survival, in the earlier incubation period, and necrotic death in the later. Based on all of these results, we concluded that curcumin contributes to a complex signaling network, affecting cell survival and necrotic cell death, which in turn could inhibit apoptotic cell death.     

Curcumin induces small cell lung cancer NCI-H446 cell apoptosis via the reactive oxygen species-mediated mitochondrial pathway and not the cell death receptor pathway

Curcumin (diferuloylmethane), an active component of the spice turmeric, induces apoptosis in several types of malignancies. However, little is known about its anticancer activity in small cell lung cancer (SCLC). SCLC represents a highly malignant and particularly aggressive form of cancer, with early and widespread metastases and a poor prognosis. In this study, we found that curcumin does not activate caspase-8 cleavage or alter the expression of apoptotic receptors FAS and TRAIL in NCI-H446 cells, suggesting that curcumin-induced apoptosis is not associated with death receptor-mediated pathways in these cells. Instead, curcumin caused apoptosis by increasing Bax expression while decreasing the expression of Bcl-2 and Bcl-xL. Curcumin induced a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into the cytosol, followed by activation of caspase-9 and caspase-3. In addition, curcumin-induced apoptosis was accompanied by an increase of intracellular reactive oxygen species (ROS) level. These results indicated that a ROS-mediated mitochondrial pathway played an important role in the process of curcumin-induced apoptosis of human SCLC NCI-H446 cells.     

Spleen tyrosine kinase (Syk), a novel target of curcumin, is required for B lymphoma growth

Curcumin (diferuloylmethane), a component of dietary spice turmeric (Curcuma longa), has been shown in recent studies to have therapeutic potential in the treatment of cancer, diabetes, arthritis, and osteoporosis. We investigated the ability of curcumin to modulate the growth of B lymphomas. Curcumin inhibited the growth of both murine and human B lymphoma in vitro and murine B lymphoma in vivo. We also demonstrate that curcumin-mediated growth inhibition of B lymphoma is through inhibition of the survival kinase Akt and its key target Bad. However, in vitro kinase assays show that Akt is not a direct target of curcumin. We identified a novel target for curcumin in B lymphoma viz spleen tyrosine kinase (Syk). Syk is constitutively activated in primary tumors and B lymphoma cell lines and curcumin down-modulates Syk activity accompanied by down-regulation of Akt activation. Moreover, we show that overexpression of Akt, a target of Syk, or Bcl-x(L), a target of Akt can overcome curcumin-induced apoptosis of B lymphoma cells. These observations suggest a novel growth promoting role for Syk in lymphoma cells.     

Effect of curcumin on glioblastoma cells

Curcumin is a polyphenolic compound derived from Curcumin longa L. There are growing bodies of evidence revealing the antitumor effect of curcumin in different tumors; although the molecular mechanism behind this inhibition in glioblastoma multiform (GBM) still remains unclear. Here we investigated the antitumor activity of nano micelles curcumin compared with erlotinib in U-373 cells in monolayer cell cultures and spheroids models. Furthermore, we characterized affecting cell cycle perturbation, as well as apoptosis induction in GBM cells. The antiproliferative activity of nano micelles curcumin and erlotinib were assessed in monolayer and spheroid models. The influence of the cell cycle and expression levels of nuclear factor κB (NF-κB) and Wnt/β-catenin pathway was checked. Nano micelles curcumin suppressed cell growth in U-373 cells via modulation of Wnt and NF-κB pathways. Moreover, cells developed an early G2/M cell cycle arrest followed by sub-G1 apoptosis and apoptotic bodies formation posttreatment with nano micelles curcumin and erlotinib. In the core signaling pathways of GBM, nano micelles curcumin either significantly influences the NF-κB pathway by decreasing p-65 expression or significantly inhibits the Wnt/β-catenin pathway by declining cyclin D1 expression. In conclusion, we have shown that nano micelles curcumin effectively prevent proliferation, and invasion of GBM cells through perturbation of Wnt/β-catenin and NF-κB pathways, suggesting further investigations on the therapeutic application of this novel anticancer drug in in vivo models.     

Curcumin Attenuates gp120-Induced Microglial Inflammation by Inhibiting Autophagy via the PI3K Pathway

Microglial inflammation plays an essential role in the pathogenesis of HIV-associated neurocognitive disorders. A previous study indicated that curcumin relieved microglial inflammatory responses. However, the mechanism of this process remained unclear. Autophagy is a lysosome-mediated cell content-dependent degradation pathway, and uncontrolled autophagy leads to enhanced inflammation. The role of autophagy in curcumin-attenuating BV2 cell inflammation caused by gp120 was investigated with or without pretreatment with the autophagy inhibitor 3-MA and blockers of NF-κB, IKK, AKT, and PI3K, and we then detected the production of the inflammatory mediators monocyte chemoattractant protein-1 (MCP-1) and IL17 using ELISA, and autophagy markers ATG5 and LC3 II by Western Blot. The autophagic flux was observed by transuding mRFP-GFP-LC3 adenovirus. The effect of the blockers on gp120-induced BV2 cells was examined by the expression of p-AKT, p-IKK, NF-κB, and p65 in the nuclei and LC3 II and ATG5. gp120 promoted the expression of MCP-1 and IL-17, enhanced autophagic flux, and up-regulated the expression of LC3 II and ATG5, while the autophagy inhibitor 3-MA down-regulated the phenomena above. Curcumin has similar effects with 3-MA, in which curcumin inhibited NF-κB by preventing the translocation of NF-κB p65. Curcumin also inhibited the phosphorylation of p-PI3K, p-AKT, and p-IKK, which leads to down-regulation of NF-κB. Curcumin reduced autophagy via PI3K/AKT/IKK/NF-κB, thereby reducing BV2 cellular inflammation induced by gp120.     

Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway

Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.     

Hirudin inhibits glioma growth through mTOR-regulated autophagy

Glioma is the most common primary malignant brain tumour, and survival is poor. Hirudin has anticancer pharmacological effects through suppression of glioma cell progression, but the molecular target and mechanism are poorly understood. In this study, we observed that hirudin dose- and time-dependently inhibited glioma invasion, migration and proliferation. Mechanistically, hirudin activated LC3-II but not Caspase-3 to induce the autophagic death of glioma cells by decreasing the phosphorylation of mTOR and its downstream substrates ULK1, P70S6K and 4EBP1. Furthermore, hirudin inhibited glioma growth and induced changes in autophagy in cell-derived xenograft (CDX) nude mice, with a decrease in mTOR activity and activation of LC3-II. Collectively, our results highlight a new anticancer mechanism of hirudin in which hirudin-induced inhibition of glioma progression through autophagy activation is likely achieved by inhibition of the mTOR signalling pathway, thus providing a molecular basis for hirudin as a potential and effective clinical drug for glioma therapy.     

Curcumin inhibits the migration and invasion of mouse hepatoma Hca-F cells through down-regulating caveolin-1 expression and epidermal growth factor receptor signaling

Mouse hepatoma cellular carinoma cell line (Hca-F) cells have highly invasive and lymphatic metastasis potential in vitro and in vivo. Curcumin (diferuloylmethane) is an active component of the spice turmeric and has a diversity of antitumor activities. However, there is no available information to address the effects of curcumin on migration and invasion of mouse hepatoma Hca-F cells. In this study, we found that curcumin exerted a concentration- and time-dependent inhibitory effect on the migration and invasion of Hca-F cells in vitro. Curcumin inhibited the expression of the tumor promoter caveolin-1 (Cav-1) in Hca-F cells. Up-regulation of Cav-1 expression by pcDNA3.1/Cav-1 plasmid was able to reverse the curcumin-induced antimigration and anti-invasion effects in vitro. Curcumin down-regulated the expression of cluster of differntiation (CD)147, matrix metalloproteinase 2, and matrix metalloproteinase 9 and inhibited the phosphorylation of epidermal growth factor receptor (EGFR), the phosphoinositilde 3-kinase (PI3K)/protein kinase B (Akt), p38 mitogen-activated protein kinase (MAPK), and p44/42MAPK in Hca-F cells. Taken together, our findings suggest that curcumin can suppress the migratory and invasive ability of mouse hepatoma Hca-F cells, and this action is mediated through a novel mechanism involving inactivation of Cav-1 and EGFR signaling pathways.     

Autophagy suppression sensitizes glioma cells to IMP dehydrogenase inhibition-induced apoptotic death

We investigated the role of autophagy, a process of controlled self-digestion, in the in vitro anticancer action of the inosine monophosphate dehydrogenase (IMPDH) inhibitor ribavirin. Ribavirin-triggered oxidative stress, caspase activation, and apoptotic death in U251 human glioma cells were associated with the induction of autophagy, as confirmed by intracellular acidification, appearance of autophagic vesicles, conversion of microtubule associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, and degradation of autophagic target p62/sequestosome 1. Ribavirin downregulated the activity of autophagy-inhibiting mammalian target of rapamycin complex 1 (mTORC1), as indicated by a decrease in phosphorylation of the mTORC1 substrate ribosomal p70S6 kinase and reduction of the mTORC1-activating Src/Akt signaling. Guanosine supplementation inhibited, while IMPDH inhibitor tiazofurin mimicked ribavirin-mediated autophagy induction, suggesting the involvement of IMPDH blockade in the observed effect. Autophagy suppression by ammonium chloride, bafilomycin A1, or RNA interference-mediated knockdown of LC3 sensitized glioma cells to ribavirin-induced apoptosis. Ribavirin also induced cytoprotective autophagy associated with Akt/mTORC1 inhibition in C6 rat glioma cells. Our data demonstrate that ribavirin-triggered Akt/mTORC1-dependent autophagy counteracts apoptotic death of glioma cells, indicating autophagy suppression as a plausible therapeutic strategy for sensitization of cancer cells to IMPDH inhibition.     

Induction of apoptotic death by curcumin in human tongue squamous cell carcinoma SCC-4 cells is mediated through endoplasmic reticulum stress and mitochondria-dependent pathways

Curcumin from the rhizome of the Curcuma longa plant has been noted for its chemo-preventative and chemo-therapy activities, and it inhibits the growth of many types of human cancer cell lines. In this study, the mechanisms of cell death involved in curcumin-induced growth inhibition, including cell cycle arrest and induction of apoptosis in human tongue cancer SCC-4 cells, were investigated. Herein, we observed that curcumin inhibited cell growth of SCC-4 cells and induced cell death in a dose-dependent manner. Treatment of SCC-4 cells with curcumin caused a moderate and promoted the G(2) /M phase arrest, which was accompanied with decreases in cyclin B/CDK1 and CDC25C protein levels. Moreover, curcumin significantly induced apoptosis of SCC-4 cells with a decrease of the Bcl-2 level, reduction of mitochondrial membrane potential (ΔΨ(m) ), and promoted the active forms of caspase-3. Curcumin also promoted the releases of AIF and Endo G from the mitochondria in SCC-4 cells by using confocal laser microscope. Therefore, we suggest that curcumin induced apoptosis through a mitochondria-dependent pathway in SCC-4 cells. In addition, we also found that curcumin-induced apoptosis of SCC-4 cells was partly through endoplasmic reticulum stress. In conclusion, curcumin increased G(2) /M phase arrest and induced apoptosis through ER stress and mitochondria-dependent pathways in SCC-4 cells.     

Versatile role of curcumin and its derivatives in lung cancer therapy

Lung cancer is a main cause of death all over the world with a high incidence rate. Metastasis into neighboring and distant tissues as well as resistance of cancer cells to chemotherapy demand novel strategies in lung cancer therapy. Curcumin is a naturally occurring nutraceutical compound derived from Curcuma longa (turmeric) that has great pharmacological effects, such as anti-inflammatory, neuroprotective, and antidiabetic. The excellent antitumor activity of curcumin has led to its extensive application in the treatment of various cancers. In the present review, we describe the antitumor activity of curcumin against lung cancer. Curcumin affects different molecular pathways such as vascular endothelial growth factors, nuclear factor-κB (NF-κB), mammalian target of rapamycin, PI3/Akt, microRNAs, and long noncoding RNAs in treatment of lung cancer. Curcumin also can induce autophagy, apoptosis, and cell cycle arrest to reduce the viability and proliferation of lung cancer cells. Notably, curcumin supplementation sensitizes cancer cells to chemotherapy and enhances chemotherapy-mediated apoptosis. Curcumin can elevate the efficacy of radiotherapy in lung cancer therapy by targeting various signaling pathways, such as epidermal growth factor receptor and NF-κB. Curcumin-loaded nanocarriers enhance the bioavailability, cellular uptake, and antitumor activity of curcumin. The aforementioned effects are comprehensively discussed in the current review to further direct studies for applying curcumin in lung cancer therapy.     

Role of protein kinase C delta in curcumin-induced antioxidant response element-mediated gene expression in human monocytes

The Nrf2/antioxidant response element (ARE) signaling pathway plays a key role in activating cellular antioxidants, including heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase-1 (NQO1), and glutathione. Protein kinase C (PKC) may also regulate these antioxidants, as PKC phosphorylates Nrf2 in vitro. This study examined the role of PKC in ARE-mediated gene regulation in human monocytes by curcumin, a potent inducer of the Nrf2/ARE pathway. Curcumin increased HO-1 and glutamyl cysteine ligase modulator (GCLM) expression and stimulated Nrf2 binding to the ARE. Curcumin also rapidly stimulated PKC phosphorylation and Ro-31-8220, a pan-PKC inhibitor, decreased curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Rottlerin (a PKC delta inhibitor) and PKC delta antisense oligonucleotides significantly inhibited curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Furthermore, a p38 MAP kinase inhibitor reduced GCLM and HO-1 expression and rottlerin inhibited curcumin-induced p38 phosphorylation. In summary, curcumin activates ARE-mediated gene expression in human monocytes via PKC delta, upstream of p38 and Nrf2.     

Curcumin Induces Apoptosis in Human Melanoma Cells through a Fas Receptor/Caspase-8 Pathway Independent of p53

In this study, we investigated the molecular pathways targeted by curcumin during apoptosis of human melanoma cell lines. We found that curcumin caused cell death in eight melanoma cell lines, four with wild-type and four with mutant p53. We demonstrate that curcumin-induced apoptosis is both dose- and time-dependent. We found that curcumin did not induce p53, suggesting that curcumin activates other apoptosis pathways. Our data show that curcumin activates caspases-3 and -8 but not caspase-9, supporting the rationale that apoptosis occurs via a membrane-mediated mechanism. Both a caspase-8 and broad-based caspase inhibitor, but not a caspase-9 specific inhibitor, suppressed curcumin-induced cell death. To further support our hypothesis that curcumin induces activation of a death receptor pathway, we show that curcumin induces Fas receptor aggregation in a FasL-independent manner and that low-temperature incubation, previously shown to inhibit receptor aggregation, prevented curcumin-induced cell death. Moreover, we demonstrate that expression of dominant negative FADD significantly inhibited curcumin-induced cell death. In addition, our results indicate that curcumin also blocks the NF-kappaB cell survival pathway and suppresses the apoptotic inhibitor, XIAP. Since melanoma cells with mutant p53 are strongly resistant to conventional chemotherapy, curcumin may overcome the chemoresistance of these cells and provide potential new avenues for treatment.