Curcumin Complex

The mTOR Signalling Pathway in Cancer and the Potential mTOR Inhibitory Activities of Natural Phytochemicals

The mammalian target of rapamycin (mTOR) kinase plays an important role in regulating cell growth and cell cycle progression in response to cellular signals. It is a key regulator of cell proliferation and many upstream activators and downstream effectors of mTOR are known to be deregulated in various types of cancers.

Since the mTOR signalling pathway is commonly activated in human cancers, many researchers are actively developing inhibitors that target key components in the pathway and some of these drugs are already on the market. Numerous preclinical investigations have also suggested that some herbs and natural phytochemicals, such as curcumin, resveratrol, timosaponin III, gallic acid, diosgenin, pomegranate, epigallocatechin gallate (EGCC), genistein and 3,3’-diindolylmethane inhibit the mTOR pathway either directly or indirectly. Some of these natural compounds are also in the clinical trial stage.

The mTOR pathway is a key regulator of cell proliferation and several upstream activators and downstream effectors of mTOR are known to be deregulated in some cancers such as renal cell carcinoma, non-small cell lung cancer, breast cancer, sarcomas, colorectal and gastrointestinal tumors (Law, 2005; Tokunaga et al., 2008; Li et al., 2013; Takahashi et al., 2014; Wang and Zhang, 2014). The mTOR signalling is constitutively activated in many tumor types, suggesting that mTOR is an attractive target for cancer drug development and therapy (Yu et al., 2001; Chan, 2004; Shor et al., 2009; Han et al., 2013; Pandurangan, 2013). The mTOR signalling network consists of a number of tumor suppressor genes and proto-oncogenes, thereby explains that aberrant activities of these genes will promote the formation of cancerous cells.

Numerous important anticancer drugs in the market are either obtained from natural sources, by structural modification of natural compounds, or by synthesis of new compounds using natural compound as lead (Cragg et al., 1997; da Rocha et al., 2001). Therefore, sourcing out new drugs and the continuous interest in using natural compounds for cancer therapy is a global effort. Numerous preclinical investigations have shown that some herbs and natural phytochemicals, such as curcumin, resveratrol, timosaponin III, gallic acid, diosgenin, pomegranate, epigallocatechin gallate (EGCC), genistein, and 3,3’-diindolylmethane inhibit mTOR pathway either directly or indirectly. Some of them are undergoing clinical trials as chemotherapeutic agents, chemopreventive compounds and/or combination therapy to improve the efficacy of the standard chemotherapy. These natural phytochemicals with mTOR inhibitory activities have great potential in cancer prevention. This is in view that higher consumption of fruits and vegetables was associated with lower risk of cancer (Gullett et al., 2010).

Curcumin, a polyphenol natural compound extracted from the plant Curcuma longa L., is commonly used as spice in India and Southeast Asia. It is used as food additive and traditional Indian medicine for the treatment of various diseases such as biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism and sinusitis (Shishodia et al., 2007). Curcumin has shown exceptional chemopreventive and anti-tumor activities in some pre-clinical studies. In HCT116 colorectal cancer cells, curcumin downregulates protein and mRNA expression of mTOR, Raptor and Rictor, suggesting that curcumin exerts its anti-proliferative effects by inhibiting the mTOR signalling pathway and thus may represent a novel class of mTOR inhibitor (Johnson et al., 2009).

In human Rh1 and Rh30 rhabdomyosarcoma cells, DU145 prostate cancer cells, MCF-7 breast cancer cells and Hela cervical cancer cells, curcumin rapidly inhibits the phosphorylation of mTOR and its downstream effector molecules such as p70S6K and 4E-BP1, indicating that curcumin may execute its anticancer activity primarily by blocking mTOR-mediated signalling pathways in these tumor cells (Beevers et al., 2006). Furthermore, curcumin induces apoptosis, inhibits cell growth and inhibits the basal or type I insulin-like growth factor-induced motility of the Rh1 and Rh30 cells (Beevers et al., 2006). Curcumin is found to dissociate Raptor, at low concentration, and Rictor, at high concentration, from mTOR complex. However, it is unclear if curcumin disrupts the mTOR complex by direct binding to mTOR or to a component of the mTOR complexes (Beevers et al., 2009).

In human PC3 prostate cancer cells, curcumin suppresses murine double minute 2 (MDM2) oncogene expression through the erythroblastosis virus transcription factor 2 (EST2) by modulating PI3K/mTOR/ETS2 signalling pathway (Li et al., 2007a). In both human U87-MG and U373-MG malignant glioma cells, curcumin inhibits the Akt/mTOR/ p70S6K pathway and activates the extracellular signal- regulated kinase (ERK) pathway, resulting in the induction of autophagy. On the other hand, activation of Akt pathway by recombinant full-length human active Akt1 protein (rAkt1) inhibited curcumin-induced autophagy and decreased curcumin-inhibited phosphorylation of Akt and p70S6K, suggesting that curcumin-induced inactivation of Akt/mTOR/p70S6K pathway plays a role in induction of autophagy (Aoki et al., 2007). As combined treatment, curcumin and dual PI3K/Akt and mTOR inhibitor induce apoptosis through p53-dependent Bcl-2 mRNA down- regulation at the transcriptional level and Mcl-1 protein down-regulation at the post-transcriptional level in human renal carcinoma Caki cells (Seo et al., 2014).

No

Natural compounds

Target

Natural Source

Clinical trial phase

1

Curcumin

Akt and mTOR

Curcuma longa L.

In Phase I-II for pancreatic cance1r, colorectal cancer2, colon cancer3, rectal cancer4, advanced cancer5. In clinical trial for familial adenom-a tous polyposis6

2

Resveratrol

PI3K, Akt and mTOR

Grapes and red wine

In clinical trial for neuroendocrine tumo7r

3

Pomegranate

PI3K, Akt and mTOR

Punica granatum L.

In Phase II for prostate cancer8,9,10

4

Genistein

Akt and mTOR

Glycine max (L.) Merr. and several plants

In Phase I-II for prostate cancer11,12, breast cancer13, 16, pancreatic cancer14, bladder cancer15, endometrial cancer16

5

3, 3-diindolylmethane

PI3K, Akt and mTOR

Cruciferous vegetables

In Phase II-III for breast cancer17 and prostate cancer18

6

Green tea extract or polyphenon E

Akt and mTOR

Green tea

In Phase I-II for breast cancer19,20, leukemia21, monoclonal gammopathy of undetermined significance and/or smoldering multiple myeloma22, prostatic hyperplasia23, premalignant lesions of the head and neck24

7

Epigallocatechin gallate (EGCG)

Akt and mTOR

Green tea

In preclinical study for human hepatoma cells and keloid fibroblast

8

Timosaponin AIII

Akt and mTOR

Anemarrhena asphodeloides Bunge

In pre-clinical study for BT-549 and MDAM231 breast cancer cells

9

Gallic acid

Akt and mTOR

Phaleria macrocarpa(Scheff.) Boerl.

In pre-clinical study for TE-2 esophageal cancer cells

10

Diosgenin

Akt and mTOR

Dioscorea spp.

In pre-clinical study for AU565 breast adenocarcinoma cells

Source
Tan HK, Moad AIH, Tan ML. Asian Pacific Journal of Cancer Prevention, Vol 15, 2014. DOI:http://dx.doi.org/10.7314/APJCP.2014.15.16.6463

Curcumin, a major constituent of the spice turmeric, suppresses expression of the enzyme cyclooxygenase 2 (Cox-2) and has cancer chemopreventive properties in rodents. It possesses poor systemic availability. Marczylo et al., (2007) explored whether formulation with phosphatidylcholine increases the oral bioavailability or affects the metabolite profile of curcumin. Their results suggest that curcumin formulated with phosphatidylcholine furnishes higher systemic levels of parent agent than unformulated curcumin.

Curcuminoids are poorly water-soluble compounds and to overcome some of the drawbacks of curcuminoids, Aditya et al., (2012) explored the potential of liposomes for the intravenous delivery of curcuminoids. The curcuminoids-loaded liposomes were formulated from phosphatidylcholine (soy PC). Curcumin/curcuminoids were encapsulated in phosphatidylcholine vesicles with high yields. Vesicles in the size range around 200 nm were selected for stability and cell experiments.

Liposomal curcumin were found to be twofold to sixfold more potent than corresponding curcuminoids. Moreover, the mixture of curcuminoids was found to be more potent than pure curcumin in regard to the antioxidant and anti-inflammatory activities (Basnet et al., 2012). Results suggest that the curcumin-phosphatidylcholine complex improves the survival rate by increasing the antioxidant activity (Inokuma et al., 2012). Recent clinical trials on the effectiveness of phosphatidylcholine formulated curcumin in treating eye diseases have also shown promising results, making curcumin a potent therapeutic drug candidate for inflammatory and degenerative retinal and eye diseases (Wang et al., 2012).

Data demonstrate that treatment with curcumin dissolved in sesame oil or phosphatidylcholine curcumin improves the peripheral neuropathy of R98C mice by alleviating endoplasmic reticulum stress, by reducing the activation of unfolded protein response (Patzkó et al., 2012).

Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup. They are a major component of biological membranes and can be easily obtained from a variety of readily available sources, such as egg yolk or soybeans, from which they are mechanically or chemically extracted using hexane. Phosphatidylcholine is a major constituent of cell membranes and pulmonary surfactant, and is more commonly found in the exoplasmic or outer leaflet of a cell membrane. It is thought to be transported between membranes within the cell by phosphatidylcholine transfer protein (PCTP) (Wirtz, 1991) Phosphatidylcholine also plays a role in membrane-mediated cell signaling and PCTP activation of other enzymes.

Reference
Aditya NP, Chimote G, Gunalan K, et al. (2012) Curcuminoids-loaded liposomes in combination with arteether protects against Plasmodium berghei infection in mice. Exp Parasitol. 2012 Jul;131(3):292-9. doi: 10.1016/j.exppara.2012.04.010.
Basnet P, Hussain H, Tho I, Skalko-Basnet N. (2012) Liposomal delivery system enhances anti-inflammatory properties of curcumin. J Pharm Sci. 2012 Feb;101(2):598-609. doi: 10.1002/jps.22785.
Inokuma, T., Yamanouchi, K., Tomonaga, T., et al. (2012) Curcumin improves the survival rate after a massive hepatectomy in rats. Hepatogastroenterology. 2012 Oct;59(119):2243-7. doi: 10.5754/hge10650.
Kanno K, Wu MK, Agate DA, et al. (2007) Interacting proteins dictate function of the minimal START domain phosphatidylcholine transfer protein/StarD2. J. Biol. Chem. 282 (42): 30728–36. doi:10.1074/jbc.M703745200.
Marczylo TH, Verschoyle RD, Cooke DN, et al. (2007) Comparison of systemic availability of curcumin with that of curcumin formulated with phosphatidylcholine. Cancer Chemother Pharmacol. 2007 Jul;60(2):171-7.
Patzkó A, Bai Y, Saporta MA, et al. (2012) Curcumin derivatives promote Schwann cell differentiation and improve neuropathy in R98C CMT1B mice. Brain. 2012 Dec;135(Pt 12):3551-66. doi: 10.1093/brain/aws299.
Wang, L.L., Sun, Y., Huang, K., Zheng, L. (2012) Curcumin, a potential therapeutic candidate for retinal diseases. Mol Nutr Food Res. 2013 Sep;57(9):1557-68. doi: 10.1002/mnfr.201200718.
Wirtz KW (1991). Phospholipid transfer proteins. Ann. Rev. Biochem. 60 (13): 73–99. doi:10.1146/annurev.bi.60.070191.000445.

The essential oil from Curcuma longa L. was analysed by GC/MS.

The major components of the oil were ar-turmerone (33.2%), α -turmerone (23.5%) and β -turmerone (22.7%).

J Med Food. 2012 Mar;15(3):242-52. doi: 10.1089/jmf.2011.1845. Epub 2011 Dec 19.

The role of turmerones on curcumin transportation and P-glycoprotein activities in intestinal Caco-2 cells.

Yue GG1, Cheng SW, Yu H, Xu ZS, Lee JK, Hon PM, Lee MY, Kennelly EJ, Deng G, Yeung SK, Cassileth BR, Fung KP, Leung PC, Lau CB.

Abstract
The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases.

Biofactors. 2013 Mar-Apr;39(2):221-32. doi: 10.1002/biof.1054. Epub 2012 Dec 11.

Curcumin combined with turmerones, essential oil components of turmeric, abolishes inflammation-associated mouse colon carcinogenesis.

Murakami A1, Furukawa I, Miyamoto S, Tanaka T, Ohigashi H.

Abstract
Curcumin (CUR), a yellow pigment in turmeric, has marked potential for preventing colon cancer. We recently reported that ar-turmerone (ATM) suppressed nitric oxide (NO) generation in macrophages. In the present study, we explored the molecular mechanisms by which ATM attenuates NO generation and examined the anti-carcinogenesis activity of turmerones (TUR, a mixture of 5 sesquiterpenes including ATM). Both CUR and ATM inhibited lipopolysaccharide (LPS)-induced expression of inducible forms of both nitric oxide synthase and cyclooxygenase (iNOS and COX-2, respectively). A chase experiment using actinomycin D revealed that ATM accelerated the decay of iNOS and COX-2 mRNA, suggesting a post-transcriptional mechanism. ATM prevented LPS-induced translocation of HuR, an AU-rich element-binding protein that determines mRNA stability of certain inflammatory genes. In a colitis model, oral administration of TUR significantly suppressed 2% dextran sulfate sodium (DSS)-induced shortening of the large bowel by 52-58%. We also evaluated the chemopreventive effects of oral feeding of TUR, CUR, and their combinations using a model of dimethylhydradine-initiated and DSS-promoted mouse colon carcinogenesis. At the low dose, TUR markedly suppressed adenoma multiplicity by 73%, while CUR at both doses suppressed adenocarcinoma multiplicity by 63-69%. Interestingly, the combination of CUR and TUR at both low and high doses abolished tumor formation. Collectively, our results led to our hypothesis that TUR is a novel candidate for colon cancer prevention. Furthermore, we consider that its use in combination with CUR may become a powerful method for prevention of inflammation-associated colon carcinogenesis.
Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Li Y, Shi X, Zhang J, Zhang X, Martin RC. Hepatic protection and anticancer activity of curcuma: a potential chemopreventive strategy against hepatocellular carcinoma. Int J Oncol. 2014 Feb;44(2):505-13. doi: 10.3892/ijo.2013.2184.  

Malignant transformation of hepatocellular carcinoma (HCC) occurs through repetitive liver injury in a context of inflammation and oxidative DNA damage. A spectrum of natural sesquiterpenoids from curcuma oil has displayed antioxidant, anti-inflammatory and anti-carcinogenic properties. The aim of the study was to investigate the hepatoprotective and anti-HCC effects of curcuma oil in vivo and in vitro. Mice were pretreated with curcuma oil (100 mg/kg) for 3 days, then treated with Concanavalin A (30 mg/kg). The hepatic tissue was evaluated for histology, CD4+ cell, interferon-γ, apoptosis, lipid peroxidation, 8-hydroxy-deoxyguanosine and MnSOD. C57L/J mice were treated with curcuma oil and 107 Hepa1-6 cells directly inoculated into liver lobes. The effects of curcuma oil on cell growth and cell death were evaluated. In addition, MnSOD, HSP60, catalase, NF-κB and caspase-3 were also investigated in the Hepa1-6 cells treated with curcuma oil. Pretreatment with curcuma oil significantly attenuates inflammation and oxidative damage by Concanavalin A. Treatment with curcuma oil can decrease the incidence of HCC. Curcuma oil inhibits cell growth and induces cell death in Hepa1-6 cells. Curcuma protected mice with hepatic injury from inflammatory and oxidative stress. Curcuma oil can inhibit hepatoma cell growth in vivo and in vitro.

Nwozo SO, Osunmadewa DA, Oyinloye BE. Anti-fatty liver effects of oils from Zingiber officinale and Curcuma longa on ethanol-induced fatty liver in rats. J Integr Med. 2014 Jan;12(1):59-65. doi: 10.1016/S2095-4964(14)60006-6.
The present study is aimed at evaluating the protective effects of oils from Zingiber officinale (ginger) and Curcuma longa (turmeric) on acute ethanol-induced fatty liver in male Wistar rats.

METHODS:
Ferric reducing antioxidant power activity and oxygen radical absorbance capacity of the oils were evaluated ex vivo. Rats were pretreated for 28 d with standard drug (Livolin Forte) and oils from Z. officinale and C. longa before they were exposed to 45% ethanol (4.8 g/kg) to induce acute fatty liver. Histological changes were observed and the degree of protection was measured by using biochemical parameters such as alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities. Serum triglyceride (TG) level, total cholesterol (TC) level and the effects of both oils on reduced gluthatione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD) and hepatic malondialdehyde (MDA) levels were estimated.

RESULTS:
Oils from Z. officinale and C. longa at a dose of 200 mg/kg showed hepatoprotection by decreasing the activities of serum enzymes, serum TG, serum TC and hepatic MDA, while they significantly restored the level of GSH as well as GST and SOD activities. Histological examination of rats tissues was related to the obtained results.

CONCLUSION:
From the results it may be concluded that oils from Z. officinale and C. longa (200 mg/kg) exhibited hepatoprotective activity in acute ethanol-induced fatty liver and Z. officinale oil was identified to have better effects than C. longa oil.

Bagad AS, Joseph JA, Bhaskaran N, Agarwal A. Comparative Evaluation of Anti-Inflammatory Activity of Curcuminoids, Turmerones, and Aqueous Extract of Curcuma longa. Adv Pharmacol Sci. 2013;2013:805756. doi: 10.1155/2013/805756. 
Curcuma longa is widely known for its anti-inflammatory activity in traditional system of medicine for centuries and has been scientifically validated extensively. The present study was conducted to evaluate the anti-inflammatory activity of curcuminoids and oil-free aqueous extract (COFAE) of C. longa and compare it with that of curcuminoids and turmerones (volatile oil), the bioactive components of C. longa that are proven for the anti-inflammatory potential. The activity against inflammation was evaluated in xylene-induced ear edema, cotton pellet granuloma models in albino Swiss mice and albino Wistar rats, respectively. The results showed that COFAE of C. longa at three dose levels significantly (P ≤ 0.05) inhibited inflammation in both models, as evidenced by reduction in ear weight and decrease in wet as well as dry weights of cotton pellets, when compared to the vehicle control. The COFAE of C. longa showed considerable anti-inflammatory effects against acute and chronic inflammation and the effects were comparable to those of curcuminoids and turmerones.

Dias Ferreira F, Mossini SA, Dias Ferreira FM, Arrotéia CC, da Costa CL, Nakamura CV, Machinski M Jr. The inhibitory effects of Curcuma longa L. essential oil and curcumin on Aspergillus flavus link growth and morphology. ScientificWorldJournal. 2013 Dec 3;2013:343804. doi: 10.1155/2013/343804. eCollection 2013.
The essential oil from Curcuma longa L. was analysed by GC/MS. The major components of the oil were ar-turmerone (33.2%), α -turmerone (23.5%) and β -turmerone (22.7%). The antifungal activities of the oil were studied with regard to Aspergillus flavus growth inhibition and altered morphology, as preliminary studies indicated that the essential oil from C. longa inhibited Aspergillus flavus Link aflatoxin production. The concentration of essential oil in the culture media ranged from 0.01% to 5.0% v/v, and the concentration of curcumin was 0.01-0.5% v/v. The effects on sporulation, spore viability, and fungal morphology were determined. The essential oil exhibited stronger antifungal activity than curcumin on A. flavus. The essential oil reduced the fungal growth in a concentration-dependent manner. A. flavus growth rate was reduced by C. longa essential oil at 0.10%, and this inhibition effect was more efficient in concentrations above 0.50%. Germination and sporulation were 100% inhibited in 0.5% oil. Scanning electron microscopy (SEM) of A. flavus exposed to oil showed damage to hyphae membranes and conidiophores. Because the fungus is a plant pathogen and aflatoxin producer, C. longa essential oil may be used in the management of host plants. 

Chainani-Wu N. Safety and anti-inflammatory activity of curcumin: a component of tumeric (Curcuma longa). The Journal of Alternative & Complementary Medicine 2003; 9: 161-8 
Kuttan R, et al. Potential anticancer activity of tumeric. Cancer Lett 1985;29.197-202.  
Lin CC, Lin HY, Chi MH, Shen CM, Chen HW, Yang WJ, Lee MH. Preparation of curcumin microemulsions with food-grade soybean oil/lecithin and their cytotoxicity on the HepG2 cell line. Food Chem. 2014 Jul 1;154:282-90. doi: 10.1016/j.foodchem.2014.01.012. 
Rachmawati H, Budiputra DK, Mauludin R. Curcumin nanoemulsion for transdermal application: formulation and evaluation. Drug Dev Ind Pharm. 2014 Feb 7.
El-Bahr SM. Curcumin regulates gene expression of insulin like growth factor, B-cell CLL/lymphoma 2 and antioxidant enzymes in streptozotocin induced diabetic rats. BMC Complement Altern Med. 2013 Dec 24;13:368. doi: 10.1186/1472-6882-13-368.