Shile Huang

The targets of curcumin.

Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-kB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimers disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here.

Curcumin inhibits the mammalian target of rapamycin-mediated signaling pathways in cancer cells.

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Here we show that curcumin inhibited growth of rhabdomyosarcoma cells (Rh1 and Rh30) (IC50 = 2–5 μM) and arrested cells in G1 phase of the cell cycle. Curcumin also induced apoptosis and inhibited the basal or type I insulin‐like growth factor‐induced motility of the cells. At physiological concentrations (˜2.5 μM), curcumin rapidly inhibited phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream effector molecules, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E‐BP1), in a panel of cell lines (Rh1, Rh30, DU145, MCF‐7 and Hela). Curcumin also inhibited phosphorylation of Akt in the cells, but only at high concentrations (>40 μM). The data suggest that curcumin may execute its anticancer activity primarily by blocking mTOR‐mediated signaling pathways in the tumor cells.

Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.

Curcumin disrupts the mammalian target of rapamycin-raptor complex.

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Recently, we have shown that curcumin inhibits phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), two downstream effector molecules of the mammalian target of rapamycin complex 1 (mTORC1) in numerous cancer cell lines. This study was designed to elucidate the underlying mechanism. We observed that curcumin inhibited mTORC1 signaling not by inhibition of the upstream kinases, such as insulin-like growth factor 1 receptor (IGF-IR) and phosphoinositide-dependent kinase 1 (PDK1). Further, we found that curcumin inhibited mTORC1 signaling independently of protein phosphatase 2A (PP2A) or AMP-activated protein kinase AMPK-tuberous sclerosis complex (TSC). This is evidenced by the findings that curcumin was able to inhibit phosphorylation of S6K1 and 4E-BP1 in the cells pretreated with PP2A inhibitor (okadaic acid) or AMPK inhibitor (compound C), or in the cells expressing dominant-negative (dn) PP2A, shRNA to PP2A-A subunit, or dn-AMPKalpha. Curcumin did not alter the TSC1/2 interaction. Knockout of TSC2 did not affect curcumin inhibition of mTOR signaling. Finally, we identified that curcumin was able to dissociate raptor from mTOR, leading to inhibition of mTORC1 activity. Therefore, our data indicate that curcumin may represent a new class of mTOR inhibitor.

Hydrogen peroxide-induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation of MAPK pathway

Oxidative stress-induced neuronal apoptosis is a prominent feature found in neurodegenerative disorders. However, how oxidative stress induces neuronal apoptosis is not well understood. To address this question, undifferentiated and differentiated neuronal cell lines (PC12 and SH-SY5Y) were exposed to hydrogen peroxide (H(2)O(2)), a major oxidant generated when oxidative stress occurs. We observed that H(2)O(2) induced generation of reactive oxygen species (ROS), leading to apoptosis of the cells in a concentration- and time-dependent manner. H(2)O(2) rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2, JNK or p38 with kinase inhibitors (U0126, SP600125 or PD169316, respectively), downregulation of Erk1/2 or p38 using RNA interference, or expression of dominant negative c-Jun partially prevented H(2)O(2)-induced apoptosis. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged H(2)O(2)-induced ROS, blocking activation of MAPKs and cell death. Furthermore, we found that H(2)O(2)-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented H(2)O(2)-activation of Erk/12, JNK and p38, as well as cell death. Similar results were observed in primary murine neurons as well. The results suggest that H(2)O(2)-induction of ROS inhibit PP2A and PP5, leading to activation of Erk1/2, JNK and p38 pathways thereby resulting in neuronal apoptosis. Our findings suggest that inhibitors of MAPKs (JNK, Erk1/2 and p38), activators of phosphatases (PP2A and PP5) or antioxidants may have potentials to prevent and treat oxidative stress-induced neurodegenerative diseases.

Hydrogen peroxide inhibits mTOR signaling by activation of AMPKα leading to apoptosis of neuronal cells

Oxidative stress results in apoptosis of neuronal cells, leading to neurodegenerative disorders. However, the underlying molecular mechanism remains to be elucidated. Here, we show that hydrogen peroxide (H2O2), a major oxidant generated when oxidative stress occurs, induced apoptosis of neuronal cells (PC12 cells and primary murine neurons), by inhibiting the mammalian target of rapamycin (mTOR)-mediated phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), blocked H2O2 inhibition of mTOR signaling. Ectopic expression of wild-type (wt) mTOR, constitutively active S6K1 or downregulation of 4E-BP1 partially prevented H2O2 induction of apoptosis. Furthermore, we identified that H2O2 induction of ROS inhibited the upstream kinases, Akt and phosphoinositide-dependent kinase 1 (PDK1), but not the type I insulin-like growth factor receptor (IGFR), and activated the negative regulator, AMP-activated protein kinase α (AMPKα), but not the phosphatase and tensin homolog (PTEN) in the cells. Expression of a dominant negative AMPKα or downregulation of AMPKα1 conferred partial resistance to H2O2 inhibition of phosphorylation of S6K1 and 4E-BP1, as well as cell viability, indicating that H2O2 inhibition of mTOR signaling is at least in part through activation of AMPK. Our findings suggest that AMPK inhibitors may be exploited for prevention of H2O2-induced neurodegenerative diseases.

Updates of mTOR Inhibitors

Mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation, metabolism and angiogenesis. mTOR signaling is often dysregulated in various human diseases and thus attracts great interest in developing drugs that target mTOR. Currently it is known that mTOR functions as two complexes, mTOR complex 1/2 (mTORC1/2). Rapamycin and its analogs (all termed rapalogs) first form a complex with the intracellular receptor FK506 binding protein 12 (FKBP12) and then bind a domain separated from the catalytic site of mTOR, blocking mTOR function. Rapalogs are selective for mTORC1 and effective as anticancer agents in various preclinical models. In clinical trials, rapalogs have demonstrated efficacy against certain types of cancer. Recently, a new generation of mTOR inhibitors, which compete with ATP in the catalytic site of mTOR and inhibit both mTORC1 and mTORC2 with a high degree of selectivity, have been developed. Besides, some natural products, such as epigallocatechin gallate (EGCG), caffeine, curcumin and resveratrol, have been found to inhibit mTOR as well. Here, we summarize the current findings regarding mTOR signaling pathway and review the updated data about mTOR inhibitors as anticancer agents.

Calcium Signaling Is Involved in Cadmium-Induced Neuronal Apoptosis via Induction of Reactive Oxygen Species and Activation of MAPK/mTOR Network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+]i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+]i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+]i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+]i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+]i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Role of mTOR Signaling in Tumor Cell Motility, Invasion and Metastasis

Tumor cell migration and invasion play fundamental roles in cancer metastasis. The mammalian target of rapamycin (mTOR), a highly conserved and ubiquitously expressed serine/threonine (Ser/Thr) kinase, is a central regulator of cell growth, proliferation, differentiation and survival. Recent studies have shown that mTOR also plays a critical role in the regulation of tumor cell motility, invasion and cancer metastasis. Current knowledge indicates that mTOR functions as two distinct complexes, mTORC1 and mTORC2. mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, survival and motility. mTORC2 phosphorylates Akt, protein kinase C α (PKCα) and the focal adhesion proteins, and controls the activities of the small GTPases (RhoA, Cdc42 and Rac1), and regulates cell survival and the actin cytoskeleton. Here we briefly review recent knowledge of mTOR complexes and the role of mTOR signaling in tumor cell migration and invasion. We also discuss recent efforts about the mechanism by which rapamycin, a specific inhibitor of mTOR, inhibits cell migration, invasion and cancer metastasis.

MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis.

Cadmium (Cd) may be accumulated in human body through long‐term exposure to Cd‐polluted environment, resulting in neurodegeneration and other diseases. To study the mechanism of Cd‐induced neurodegeneration, PC12 and SH‐SY5Y cells were exposed to Cd. We observed that Cd‐induced apoptosis in the cells in a time‐ and concentration‐dependent manner. Cd rapidly activated the mitogen‐activated protein kinases (MAPK) including extracellular signal‐regulated kinase 1/2 (Erk1/2), c‐Jun N‐terminal kinase (JNK) and p38. Inhibition of Erk1/2 and JNK, but not p38, partially protected the cells from Cd‐induced apoptosis. Consistently, over‐expression of dominant negative c‐Jun or down‐regulation of Erk1/2, but not p38 MAPK, partially prevented Cd‐induced apoptosis. To our surprise, Cd also activated mammalian target of rapamycin (mTOR)‐mediated signaling pathways. Treatment with rapamycin, an mTOR inhibitor, blocked Cd‐induced phosphorylation of S6K1 and eukaryotic initiation factor 4E binding protein 1, and markedly inhibited Cd‐induced apoptosis. Down‐regulation of mTOR by RNA interference also in part, rescued cells from Cd‐induced death. These findings indicate that activation of the signaling network of MAPK and mTOR is associated with Cd‐induced neuronal apoptosis. Our results strongly suggest that inhibitors of MAPK and mTOR may have a potential for prevention of Cd‐induced neurodegeneration.