Regulation of GSK-3 activity by curcumin, berberine and resveratrol: Potential effects on multiple diseases.

This study published here

1. Introduction
The PI3K/PTEN/AKT/mTORC1/GSK-3 pathway is a central signaling axis involved in many biological processes. This pathway plays pivotal roles in cell growth, inflammation, diabetes as well as neurological disorders such as Alzheimer’s disease (AD). GSK-3 is an important kinase involved in many biochemical properties including: neurological disorders, diabetes, cancer and signaling pathways such as WNT/beta-catenin and NOTCH as well as others. The biological and biochemical effects of GSK-3 have been recently reviewed (McCubrey JA. et al., 2014a; McCubrey et al., 2014b; McCubrey et al., 2014c; Chappell et al., 2016; McCubrey et al., 2106a; McCubrey 2016b; McCubrey et al., 2017a) GSK-3 often serves to negatively regulate certain molecules by phosphorylation. Phosphorylation of proteins by GSK-3 can result in their targeting to the proteasome and subsequent degradation. Since some of the target proteins of GSK-3 are often associated with apoptosis, cancer and development, GSK- 3 has been proposed to have tumor-suppressor effects. Although some studies have indicated that GSK-3 may have tumor promoter effects.

GSK-3 is phosphorylated and activated by AKT phosphorylation. AKT is a central kinase that is activated by cytokines and other stimulatory signals. The PI3K/PTEN/AKT/mTORC1/GSK-3 pathway is often associated with proliferation and prevention of apoptosis. Thus, GSK-3 is fundamentally linked with PTEN, AKT, mTORC1 and other signaling and metabolic pathways. This review will focus on some of the effects of nutraceuticals on GSK-3 often in relation to the PI3K/PTEN/AKT/mTORC1 pathway.
2. Effects of Curcumin on GSK-3 Activity
The effects of CUR on the OCT-4 transcription factor and GSK-3beta activation have been examined in NCCIT human embryonic carcinoma cells. CUR was determined to inhibit OCT-4 transcription and increase the total level of GSK-3beta which resulted in the induction of apoptosis (Yun et al., 2015).

The abilities of CUR to inhibit GSK-3 have been investigated. By stimulated molecular docking experiments, CUR was determined to fit into the binding pocket of GSK-3. CUR inhibited GSK-3 activity with an IC50 of approximately 66.3 nM.

CUR treatment of mice resulted in an increase of glycogen in the liver potentially because glycogen synthase (GS) is a target of GSK-3, and after CUR treatment GS was not phosphorylated by GSK3 and thus was active (Bustanji et al., 2019). A diagram of the effects of CUR on GSK-3 and various diseases is presented in Figure 1.
3. Effects of CUR on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway Important in Neurological Diseases
Often various signaling pathways are aberrantly regulated in various neurological diseases including: AZ, Amyotrophic lateral sclerosis and others (Tomita et al., 2015; Bradshaw et al., 2015; Shamseddine et al., 2015; Tu-Sekine et al., 2015, Aditi et al., 2016; Rohacs T. 2016; Giudichi et al., 2016; Yang et al., 2016; Kang et al., 2016; Hayashi et al., 2016 Scarlata et al., 2016; Ghim et al., 2016) CUR has been linked with anti-amyloidogenic properties. Injection of the beta-amyloid (A-beta) peptide into rats resulted in learning developmental abilities.

The animals had activated astrocytes and microglial cells. The activity of the AKT/GSK-3 pathways was altered as well as the expression of brain-derived neurotrophic factor (BDNF). CUR was determined to prevent some of the effects of A-beta injection. CUR-lipid core nanoparticles (CUR-LNC) were demonstrated to induce protective effects against the signaling disturbance induced by A-beta at 20-fold lower levels than administration of CUR by itself. Thus, CUR in LNC may represent an alternative means to treat AD patients which can have alterations in GSK- 3beta signaling (Hoppe et al., 2013).

The effects of CUR on human SH-SY5Y neuroblastoma cells were examined. CUR was determined to inhibit Abeta-induced tau hyperphosphorylation in the SH-SY5Y cells. Hyperphosphorylated tau and accumulated A-beta are important in AD disease. CUR inhibited tau phosphorylation at T231 and S396 and decreased phosphorylation of GSK-3beta at S9. CUR treatment prevented A-beta peptide induced downregulation of Akt phosphorylation at T308 and S473. CUR also had effects on PTEN levels as CUR depressed A-beta peptide-induced up- regulation of PTEN. These results point to the interactions between the Abeta-induced tau phosphorylation in AD which may be affected by CUR treatment (Huang et al., 2012; Huang et al., 2014)

CUR has been shown to have protective effects against the mitochondrial dysfunction induced by A-beta in AD. CUR protected neuroblastoma SH-SY5Y cells against the harmful effects of energy metabolism. CUR prevents many of the A-beta effects on mitochondrial dysfunction as well as the levels of total and phosphorylated GSK-3beta. CUR was determined to suppress the expression of proteins associated with the apoptosis induced by mitochondrial dysfunction including, BAX, Caspase-3 and cytochrome C which can be activated by A-beta. Thus, CUR treatment could provide protection against A-beta-stimulated stimulated oxidative stress and mitochondrial metabolic deficiency. GSK-3beta maybe a target of CUR in these events (Huang et al., 2012).
Presenilin-1 (PS1) is a substrate of GSK-3beta. PS1 is important in gamma-secretase activity. Gamma and beta secretases process amyloid precursor protein (APP) into A-beta. CUR treatment reduced the production of A-beta, PS1 and GSK-3beta. CUR was proposed to decrease A-beta production by suppressing the GSK-3beta induced activation of PS1 (Xiong et al., 2011).

CUR can activate the WNT/beta-catenin pathway by suppressing GSK-3beta in SH- SY5Y neuroblastoma cells. In contrast, the levels of beta-catenin and Cyclin D increased. The levels of beta-catenin were higher in the nucleus after CUR treatment (Zhang et al., 2011).

Effects of CUR on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Obesity/Diabetes/Cardiovascular Diseases
The PI3K/PTEN/Akt/mTORC1/GSK-3 pathway is very important in obesity, diabetes
and cardiovascular diseases (Lupieri et al., 2015; Guidetti et al., 2015; Beretta et al., 2015;
Mikoshiba, 2015; Huang et al., 2015). CUR has previously been shown to ameliorate
inflammation and diabetes in obese mice. CUR suppresses adipogenic differentiation. This was
accompanied by activation of the WNT/beta-catenin signaling pathway. CUR inhibited ERK, JNK and p38
adipocyte differentiation, CUR restored the nuclear translocation of beta-catenin. In contrast, CUR treatment reduced the expression of casein kinase alpha (CK1-alpha), GSK-3beta, Axin and other components of the beta-catenin destruction complex. Thus CUR suppressed the adipogenic process of 3T3-L1 cells by increasing levels of WNT targets such as: c-Myc, cyclin D1, WNT10b, FZZ and LRP5 and decreased CK1-alpha, GSK-3beta, Axin and stabilized beta- catenin. (Ahn J et al., 2010).

CUR has been shown to alleviate diabetic cardiomyopathy which can be experimentally induced in rats by streptozoticin treatment. CUR had protective effects on the induction of apoptosis, cardiac fibrosis, inflammation, myocardial dysfunction and oxidative stress in the diabetic rat hearts. CUR also effected the phosphorylation of AKT and GSK-3beta in this
phosphorylation in the 3T3-L1 pre-adipocyte line. During the process of model. The authors postulated that AKT/GSK-3beta may be important for mediating the effects of CUR in this diabetic cardiomyopathy model (Yu et al., 2012).

CUR has been shown to suppress GSK-3 and other signaling pathways in nephrectomized rats. CUR prevented cardiac remodeling by suppression of hypertrophic signaling including GSK-3beta phosphorylation which is normally induced in nephrectomized mice. CUR may prove useful in suppressing hypertrophy in patients with chronic renal therapy (Ghosh et al., 2010).

CUR was shown to protect rats in regional myocardial ischemic/reperfusion (I/R) injury.
CUR reduced the infarct size. CUR enhanced the phosphorylation of AKT, ERK1/2 and GSK-
3beta. In contrast, the phosphorylation of p38 and JNK were reduced after CUR treatment.
The phosphorylation of GSK-3beta that was induced by CUR was suppressed by the PI3K inhibitor Wortmannin and the MEK inhibitor UO126. Treatment with the GSK-3 inhibitor SB216763 suppressed the infarct size and increased the levels of GSK-3beta phosphorylation which results in its inactivation (Jeong et al., 2012). These studies indicate that in some experimental systems CUR can have opposite effects on various biological processes.

Effects of CUR on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Cancer
The PI3K/PTEN/Akt/mTORC1/GSK-3 pathway is very important in cancer (Kriplani et al., 2015; Fitzgerald et al., 2015; Baer et al., 2015; Kriplani et al., 2015; Jhanwar. 2015; Mollinedo et al., 2015; Shears SB. 2015; McCubrey JA et al., 2015, Dusaban et al., 2015; Olayanju et al., 2015; Spinelli et al., 2015; Jhanwar-Uniyal et al., 2015; Carroll et al., 2015; Schurmans et al., 2015 Jahan et al., 2015; Barker et al., 2015 CJ, Li L, Köhler & Berggrenm, 2015; Scoumanne et al., 2016; Geck & Tocker, 2016; Fields et al., 2016; Anderson et al., 2016; Maczis et al., 2016; Falasca & Ferro. 2016; Cocco et al., 2016; Tanaka et al., 2016; Perdios et al., 2016; Erneux et al., 2016; Banfic et al., 2016; Pyne et al., 2016; McCubrey et al., 2017a; McCubrey et al., 2017b). The spleen tyrosine kinase (SYK) is constitutively-activated in B- lymphomas. CUR down-regulated SYK and AKT activity. Some of the effects of CUR may be mediated by inhibition of GSK-3 phosphorylation which is normally stimulated by AKT (Gururajan et al., 2007).

Combining CUR with the PI3K inhibitor LY294002 increased apoptosis in MCF-7 breast cancer cells. In these studies, increased levels of phosphorylated AKT (activated) and GSK-3 (inactivated) were detected after CUR treatment (Kizhakkayil et al., 2010).

CUR can act as an anti-oxidant and synergize with other drugs. When CUR is exposed to hypoxic conditions, it a free radical which can cause apoptosis. This may affect aldehyde dehydrogenase 1 (ALDH1A1) and GSK-3 activity in drug-resistant breast cancers. (Kesharwani et al., 2015).

CUR regulated the expression of miR-9 in oral squamous cell carcinoma (OSCC). The levels of miR-9 were significantly lower in clinical OSCC patient samples than in adjacent non- tumor specimens. CUR upregulated the expression of miR-9 and suppressed WNT/beta-catenin expression. The expression of GSK-3beta was increased in CUR-treated cells as was phosphorylated GSK-3beta and beta-catenin while the levels of Cyclin D1 were decreased. Suppression of miR-9 by anti-miR-9 oligonucleotides prevented the effects of CUR on growth suppression and re-activated WNT/beta-catenin signaling (Xiao et al., 2014).

The curcumin derivative tetrahydrocurcumin (THC) has been determined to be more effective than curcumin in suppressing colon carcinogenesis that can be experimentally induced by azoxymethane treatment. CUR and THC have been shown to suppress WNT1, beta-catenin protein levels in colonic tissue. Thus, CUR and THC may suppress WNT1 and beta-catenin protein levels as well as phosphorylation of GSK-3beta. Thus CUR and THC may suppress the WNT3/beta-catenin/GSK-3 signaling which is important in AOM-induced colon tumorigenesis (Lai et al., 2011).
One of the major therapeutic problems with administration of CUR to cancer patients is the low solubility of CUR. Lipid based nanoparticles have shown promise in increasing the effectiveness of CUR in various cancer scenarios. A recent report deals with the delivery of CUR in lipid based nanoparticles to ovarian cancer cells. (Bondi et al., 2017).

Effects of berberine on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Diabetes
Berberine can decrease blood glucose by elevating GS. GSK-3 normally phosphorylates and inactivates GS. Berberine can lower the high blood glucose in alloxan-induced diabetic mice. Decreased phosphorylated AKT was detected in diabetic mice but berberine inhibited this from occurring. Berberine also inhibited the increase in GSK-3beta phosphorylation which would decrease GSK-3beta activity and increase GS activity. berberine lead to lowering the high glucose in the alloxan-induced diabetic mice (Xie et al., 2011).

Effects of berberine on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Cardiac Function

Berberine reduced GSK-3beta activation of streptozotocin-induced type-II diabetic rats. This resulted in improved cardiac function which is normally a major cause of death and type-II diabetics. berberine treatment of diabetic rats increased 5’adenosine monophosphate-activated protein kinase (AMPK) localized in the heart as well as AKT activation and decreased the levels of GSK-3beta. The AMPK inhibitor compound C was determined to block the effects of berberine (Chang et al., 2015). A diagram of the effects of berberine on various diseases is presented in Figure 2.
8. Effects of berberine on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Neurology berberine isolated from Coptis chinensis Franch, has been shown to improve cognitive ability in an AD transgenic mouse model (TgCRND8). berberine improved beta-amyloid pathology and gliosis. berberine treatment reduced the level of both detergent-soluble and -insoluble beta-amyloid in mouse brain homogenates. GSK-3beta is a major kinase involved in amyloid precursor protein (APP) and tau phosphorylation. berberine inhibited GSK-3. This resulted in decreased levels of C- terminal APP fragments and phosphorylation of APP and tau. Thus berberine may negatively regulate GSK-3 and APP processing which are important in AD (Durairajan et al., 2012).

Berberine treatment has been shown to result in increased phosphorylation of AKT (activation) and GSK-3beta (inactivation) in the ischemic cerebral cortex of rat brains after permanent middle cerebral artery occlusion (pMCAO). berberine treatment decreased neurological memory problems, brain water content and infarct sizes. berberine treatment increased the expression of phosphorylated AKT, GSK-3beta, CREB and claudin-5 and decreased the levels of NF- kappaB (Zhang et al., 2012).

In a model with hippocampal specimens isolated form 6-8 day old male rats, berberine was shown to evoke neuroprotection to oxygen and glucose deprivation (OGD). Western blot protein analysis suggested roles for AKT, GSK-3beta, ERK and JNK in berberine-mediated neuroprotection after ischemia. AKT and ERK1/2 were postulated to be involved in protective survival while JNK, Caspase-3 and GSK-3beta were inhibited (Simões Pires et al., 2014).
GSK-3beta is involved in WNT signaling in neurons. berberine may regulate lipoprotein receptor expression in cerebellar granule neurons (CGN). berberine increased very-low-density lipoprotein receptor (VLDLR) expression in CGN. VLDLR upregulation is associated with stabilization of hypoxia-induced factor-1 alpha (HIF-1alpha) and suppressed beta-catenin levels which inhibited WNT signaling. This occurs via GSK-3beta phosphorylation (Kysenius et al., 2016).
8. Effects of berberine on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Osteoarthritis
berberine can have effects on the WNT/beta-catenin pathways by promoting sodium nitoroprusside-mediated proliferation of rat chondrocytes in a rat osteoarthritic (OA) model. In this model, berberine treatment increased the percentage of cells in S-phase and decreased the percentage of cells in G0/G1 phase. This resulted in the proliferation of sodium nitroprusside- stimulated rat chondrocytes. In this model, berberine treatment increased beta-catenin, c-MYC and Cyclin D1 but down regulated GSK-3beta and MMP-7 expression (Zhou et al., 2016).

Effects of berberine on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Cancer
Combining berberine with the dual EGFR/HER2 inhibitor lapatinib can suppress lapatinib- resistance of HER2+ breast cancer. This was shown to occur by increasing the levels of ROS. Lapatinib activates the c-MYC/pro-NRF2 and GSK-3beta signaling pathways. This results in stabilization of NRF2 and a low level of ROS in lapatinib-resistant cells. BBB increased ROS production by decreasing c-MYC expression and lapatinib-resistance was decreased (Zhang et al., 2016).

Berberine has been shown to inhibit the effects of the alpha melanocyte stimulating hormone (alpha MSH) on melanoma cells. berberine prevented the induction microphtalia-associated transcription factor (MITF) and tyrosinase normally mediated by alpha MSH treatment. berberine prevented the phosphorylation of PI3K, AKT and GSK-3beta and ERK1,2 which are normally stimulated by alpha-MSH treatment. berberine inhibited melanin synthesis and tyrosinase activity. berberine may be useful in the treatment of certain skin pigmentation disorders (Song et al., 2015). 10. Effects of RES on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Aging The effects of RES on Sirtuin-1 (SIRT1) activity were examined on rat somatotrophs anterior pituitary cells in vivo and in vitro. SIRT1 activation by RES suppressed GSK-3beta acetylation on K205. This leads to GKS3 activation, which is an important step in Sirt1 induced CREB inhibition. CREB inhibition compromises growth hormone (GH) synthesis. Inhibition of GH synthesis increases lifespan and improves metabolic profile in obese humans. Thus, RES might reduce aging-related pathologies in part by regulating GH level through Sirt1 and GSK3 pathway (Monteserin-Garcia et al., 2013).

A diagram of the effects of RES is presented in Figure 3.
11. Effects of RES on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Inflammation
RES has been shown to inhibit the inflammation induced by heat-killed Listeria monocytogenes (HKLM). HKLM stimulated phosphorylation of ERK1,2 and GSK-3. The GSK- 3 inhibitor potentiated and The MEK inhibitor suppressed the inflammatory effects of HKLM. Treatment with RES blocked phosphorylation of the two kinases. These authors have proposed that the anti-inflammatory effects of RES may be mediated in part by ERK1,2 and GSK-3 (Park et al., 2012).

Effects of RES on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Neurological Disorders

The neuroprotective effects of RES have been examined in an in vitro model of ischemia which employed oxygen-glucose deprivation of hippocampal samples. RES was shown to induce the phosphorylation of AKT, GSK-3 and ERK1,2. The neuroprotective effects of RES were suppressed by PI3K but not MEK inhibitors (Zamin et al., 2006).
RES will activate AMPK to stimulate insulin signaling and glucose uptake in skeletal muscle cells. RES treatment of Neuro-2a (N2A) cells resulted in AKT and GSK-3beta phosphorylation. AMPK was activated after RES treatment which could be inhibited by treatment with the AMPK inhibitor compound C. Compound C also abolished AKT and GSK- 3beta phosphorylation, glucose uptake and insulin signaling (Patel et al., 2011).

RES protects against cerebral ischemia by its anti-oxidant and oxygen free radicals scavenging properties. In the four-vessel occlusion model of global cerebral ischemia, GSK- 3beta and CREB are believed to play roles in RES-evoked neuroprotection (Simão et al., 2012).

The effects of delivery of RES in lipid core nanocapsules has been determined to improve neuroprotective roles after A-beta administration to rats. The use of lipid core nanoparticles might be a more effective means to deliver RES and alter JNK and GSK-3 signaling pathways altered in AD due to A-beta peptide (Frozza et al., 2013).
RES treatment can result in GSK-3beta phosphorylation. Dietary supplementation of RES resulted in increased levels of phosphorylation GSK-3beta and protein levels of debrin and transthyretin, two important proteins involved in protection against neurogenerative diseases (Varamini et al., 2013).

RES has neuroprotective effects on astrocytes after traumatic brain injury. This was shown to occur by inhibiting both apoptotic and autophagic cell death. In a traumatic brain injury model, both apoptotic and autophagic cell death were induced in CTX TNA2 astrocytes by glutamate-mediated GSK-3beta activation. Suppression of GSK-3beta activity resulted in increased survival, while overexpression of GSK-3beta increase glutamate-mediated excitotoxicity. RES suppressed ROS-mediated GSK-3beta activation. The ROS/GSK-3beta pathway was also determined to induce various damage processes associated with the mitochondria (Lin et al., 2014). RES has been determined to induce its neuroprotective effects against brain ischemia reperfusion in rats by decreasing the DJ-1 protein and activating the PI3K/AKT which suppressed GSK-3beta activity (Abdel-Aleem et al., 2016).

Effects of RES on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Diabetes

Treatment of diabetic rats with RES regulated the levels of lactadherin. Lactadherin is detected at higher levels in the aorta of diabetic rats. Lactadherin may play a role in advanced glycation end products (AGE)-induced apoptosis which may be important in endothelial cell (EC) dysfunction and the development of diabetes. Overexpression of lactadherin reduced GSK- 3beta phosphorylation. RES protected against AGE-mediated EC apoptosis by targeting lactadherin (Li et al., 2011).

RES protected slow-twitch muscle in streptozotocin-induced diabetic rats. Phosphorylation of AKT and GSK-3beta were decreased in the diabetic rats in both their fast- and slow twitch muscles (Chang et al., 2014).

In a porcine model of metabolic syndrome, REV supplementation led to improved glucose control. REV significantly elevated the expression of several proteins regulating glucose metabolism in skeletal muscles and liver, among them phospho-AKT (residue T308), GLUT-4, peroxisome proliferating activation receptor γ coactivator 1α (PGC-1α) and insulin receptor substrate 1. phospho-AKT inhibits GSK-3 thus enhances GS and reduces hyperglycemia.
Therefore, RES prevents insulin resistance induced by a high-calorie, high-fat diet in part acting through GSK-3 (Burgess et al., 2011).
14. Effects of RES on chronic skeletal muscle impairment.

In C2C12 cells, REV stimulated AKT and ERK1,2 activation and AMPK protein levels. These proteins, by activating downstream signaling pathways, have positive effect on myoblasts differentiation and muscle hypertrophy. These authors conclude that REV might be used in treatment of chronic functional and morphological muscle impairment (Montesano et al., 2013). AKT promoted skeletal myotubes hypertrophy in part by inhibiting GSK-3 (Rommel et al., 2001). Thus, some of the effects of REV on muscle hypertrophy might be mediated by regulation of GSK-3 activity.
15. Effects of RES on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Cardiovascular Disorders.

Isorhapontigenin (ISO) is a RES analog. ISO blocks oxidative stress and associated signaling pathways in cardiomyocytes. This inhibits cardiac hypertrophy. ISO was determined to suppress the effects of protein kinase C (PKC) on the PI3K/PTEN/AKT/GSK-3 pathway. ISO was shown to suppress the development of cardiac hypertrophy via various signaling pathways (Li et al., 2005).

RES has been determined to reduce cardiac mitochondrial swelling and infarct size at reperfusion. This can result in cardiac protection. RES was determined to enhance GSK-3beta inhibitory phosphorylation. RES may target the mitochondrial permeability transition pore (mPTP) by inducing the translocation of GSK-3 from cytosol to mitochondrial which allows GSK-3beta to interact with cyclophilin D to regulate mPTP (Xi et al., 2009).
GSK-3 inhibition has been shown to stimulate muscle fructose-1,6-bisphosphatase (FBP2) interaction with cardiac mitochondria. FBP2 protects mitochondria against swelling interacting with proteins involved in formation of mPTP. Thus, this might be another part of the mechanism of RES-induced reduction cardiac mitochondria swelling (Gizak et al., 2012).

Five micromolar RES treatment can activate the Raf/MEK/ERK and PI3K/PTEN/AKT/mTORC1 pathways which will result in phosphorylation of GSK-3beta in human umbilical vein endothelial cells (HUVEC) cells. RES at concentrations between one to ten micromolar increased the expression of VEGF and stimulated angiogenesis. In contrast, at high RES concentrations, twenty mircomolar and higher, it had the reverse effects. The increase in VEGF expression was dependent on nuclear accumulation of beta-catenin (Wang et al., 2010).

16. Effects of RES on PI3K/PTEN/Akt/mTORC1/GSK-3 Pathway in Cancer
The effects of 3,5,4′-trimethoxystilbene (MR-3), a methoxylated derivative of RES, has been examined in MCF-7 breast cancer cells. MR-3 treatment increased E-cadherin expression, while decreasing SNAIL, SLUG and vimentin expression. This treatment also resulted in decreased invasion and migration. These effects were linked with decreased expression and nuclear translocation of beta-catenin, as well as, decreased transcription of beta-catenin target genes. MR-3 treatment also restored GSK-3beta activity and suppressed AKT phosphorylation. Thus, EMT and invasion were blocked in MR-3-treated MCF-7 cells (Tsai et al., 2013).

Ten micromolar RES can affect the PI3K pathway in an estrogen-receptor (ER)-alpha dependent fashion in breast cancer cells. In contrast, fifty micromolar RES treatment resulted in decreased activity. The anti-estrogen ICI 182,780 blocked the ER-alpha dependent effects. RES induced the proteasomal degradation of ER-alpha. Thus, RES could suppress survival and proliferation of ER+ breast cancer cells by regulating ER-alpha-associated PI3K/PEN/AKT/mTORC/GSK-3beta pathway (Pozo-Guisado et al., 2004).

RES has been shown to stimulate endoplasmic reticulim (ER) stress mediated apoptosis by interfering with glycosylation. RES inhibited the hexosamine biosynthetic pathways and activated GSK-3beta. This resulted in interrupted protein glycosylation. Inhibitors of GSK-3 suppressed the effects of RES on protein glycosylation (Gwak et al., 2016).

The effects of RES on apoptosis regulated by beta-arrestin-2 have been shown to be dependent on the PI3K/PTEN/AKT/mTORC1/GSK-3 pathway in endometrial cancer (EC). Transfection with beta-arrestin and treatment with RES resulted in reduced levels of phosphorylated AKT and GSK-3beta (Sun et al., 2010).
Suppression of GSK-3beta by siRNA knockdown increased the levels of ferritin H mRNA induction by RES. Suppression of AMPK-alpha by compound C or siRNA knockdown suppressed the protective effects of RES in CD3+ T cells exposed to oxidative stress. AMPK- alpha was shown to play a role in the expression of the ferritin H gene which is induced by RES via an anti-oxidant responsive element present in the promotor regions of certain genes (Iwasaki et al., 2013).

RES will suppress the development of experimental N-nitrosbis (2-oxopropyl)amine pancreatic cancers in hamsters. RES treatment decreased PI3K/PTEN/AKT/mTORC1/GSK- 3beta and RAF/MEK/ERK signaling. Decreased levels of phosphorylated GSK-3beta at S9 and ERK1,2 were detected in the nucleus (Kato et al., 2015).
The relationship between RES and VEGF-B was examined in pancreatic cancer cells. RES was determined to suppress growth and induce apoptosis in Capan-2 pancreatic cancer cells. RES induced BAX and VEGFR-B expression. siRNA specific for VEGF-B increased the apoptotic rate and suppressed GSK-3beta but did not affect BAX. Thus, RES exerted two disparate effects, it increased the anti-cancer BAX levels and it increased the pro-cancer VEGFR-B levels. The authors proposed that the anti-cancer effects of RER were stronger than the pro-cancer effects (Yang et al., 2014).

GSK-3beta will phosphorylate cyclin D1 at T286 which results in its targeting for ubiquitination and proteasomal degradation. Knock down of quinone reductase 2 (NQO2) in CWR22Rv-1 prostate cancer cells resulted in slower proliferation and decreased phosphorylation of RB and Cyclin D1. Increased AKT and decreased GSK-3beta activity and cyclin D1 phosphorylation were observed in the cells after NQO2 knockdown. Knockdown of NQO2 prevented the ability of RES to downregulated Cyclin D1. These results suggest that RES may exert inhibitory effects on prostate cancer cells by NQO2, GSK3-beta and Cyclin D1 (Hsieh et al., 2012).
RES has also been shown to down regulate Cyclin D1 in ovarian cancer cells by affecting AKT and GSK-3beta phosphorylation. RES also reduced the levels of ERK1,2 phosphorylation. PI3K and MEK inhibitors increased the effects of RES (Vergara et al., 2012).

RES can protect mitochondria against ROS-induced oxidative stress. This occurred by inhibition of GSK-3beta. RES can exert many of its effects by modulating the activities of SIRT1, LKB1 and AMPK. RES treatment suppressed the apoptosis, glutathione depletion and ROS production that are normally induced by arachidonic acid (AA) and iron in HEPG2 liver cells. RES increased phosphorylation of GSK-3beta which lies downstream of AMPK. Suppression of LKB1 with a siRNA resulted in inhibition of AMPK and prevented the ability of RES to protect the HEPG2 cells from mitochondrial dysfunction. These studies indicate a role of RES in the activation of AMPK and inhibition of GSK-3beta. This results in protection of liver cancer cells from AA- and iron-induced ROS production and mitochondrial dysfunction (Shin et al., 2009).

The synthetic RES aliphatic derivative (R6A) has been shown to inhibit Toll-mediated apoptosis in HEK 293 cells. R6A inhibited Toll like receptor-2 (TLR2) expression. Overexpression of TLR2 resulted in a decrease in apoptosis after treatment with R6A. The effects of R6A on TLR2 expression were determined to involve the AKT/GSK-3beta signaling axis (Chen et al., 2009).

RES has been shown to induce apoptosis in the MOLT-4 T lymphoblastic leukemia cell line. RES inhibited the NOTCH signaling pathway. RES also inhibited the PI3K/PTEN/AKT pathway which resulted in GSK-3beta activation. RES was shown to induce apoptosis in MOLT- 4 cells by suppressing the NOTCH signaling pathway which has interactions with GSK-3 (Cecchinato et al., 2007).

RES has been shown to regulate the activity of the androgen and estrogen receptors in prostate cancer cells. RES was shown to inhibit PI3K activity in LNCaP and PC3 prostate cancer cells. RES treatment resulted in decreased GSK-3 phosphorylation which resulted in its activation and phosphorylation of Cyclin D1. Thus, RES was inducing GSK-3 activity which was suppressing Cyclin D1 (Benitez et al., 2007).
17. Conclusions
Natural products/nutraceuticals such as CUR, BER and RES may serve to regulate GSK- 3 activity which can often have suppressive effects on various diverse biochemical processes. Understanding the effects of these natural compounds on various signaling pathways implicated in different diseases may enhance their usefulness as dietary supplements and drugs.