Accepted Manuscript Title: Diterpenes from Rosemary (Rosmarinus officinalis): Defining their potential for anti-cancer activity Author: Sakina M. Petiwala, Jeremy J. Johnson PII: DOI: Reference:
S0304-3835(15)00446-2 http://dx.doi.org/doi:10.1016/j.canlet.2015.07.005 CAN 12462
To appear in:
Cancer Letters
Received date: Revised date: Accepted date:
28-4-2015 1-7-2015 6-7-2015
Please cite this article as: Sakina M. Petiwala, Jeremy J. Johnson, Diterpenes from Rosemary (Rosmarinus officinalis): Defining their potential for anti-cancer activity, Cancer Letters (2015), http://dx.doi.org/doi:10.1016/j.canlet.2015.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Diterpenes from Rosemary (Rosmarinus officinalis): Defining their potential for anti-cancer activity Running Title: Anti-cancer activity of rosemary diterpenes
Sakina M. Petiwala 1 and Jeremy J. Johnson 1,2 1
University of Illinois at Chicago, College of Pharmacy, and 2 University of Illinois Cancer Center, Chicago, IL 60612
Corresponding Author: Jeremy J. Johnson, PharmD, PhD University of Illinois at Chicago Chicago, IL 60612 Tel: 312-996-4368 Fax: 312-996-0379 Email:
[email protected]
Highlights
Diterpenes from rosemary interact with the cell signaling machinery of cancer cells leading to decreased cell viability and proliferation The pharmacokinetic properties of diterpenes suggest they are well absorbed and can achieve plasma concentrations that are physiologically relevant Standardized rosemary extracts are approved as a food preservative in the European Union increasing the likelihood of human exposure to these phytochemicals
ABSTRACT
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Recently, rosemary extracts standardized to diterpenes (e.g. carnosic acid and carnosol) have been approved by the European Union (EU) and given a GRAS (Generally Recognized as Safe) status in the United States by the Food and Drug Administration (FDA).
Incorporation of
rosemary into our food system and through dietary selection (e.g. Mediterranean Diet) has increased the likelihood of exposure to diterpenes in rosemary. In consideration of this a more thorough understanding of rosemary diterpenes is needed to understand its potential for a positive impact on human health. Three agents in particular have received the most attention that includes carnosic acid, carnosol, and rosmanol with promising results of anti-cancer activity. These studies have provided evidence of diterpenes to modulate deregulated signaling pathways in different solid and blood cancers. Rosemary extracts and the phytochemicals therein appear to be well tolerated in different animal models as evidenced by the extensive studies performed for approval by the EU and the FDA as an antioxidant food preservative. This mini-review reports on the pre-clinical studies performed with carnosic acid, carnosol, and rosmanol describing their mechanism of action in different cancers.
KEYWORDS Carnosol, carnosic acid, rosmanol, rosemary, cancer
Keywords: rosemary, rosmarinus officinalis, carnosic acid, carnosol, rosmanol, diterpene, nrf2, chemoprevention
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INTRODUCTION: Rosemary (Rosmarinus officinalis) is an aromatic evergreen herb from the Lamiaceae mint family. It is native to the Mediterranean region growing up to 5ft tall. Rosemary is an abundant herb in the Mediterranean region making it one of many herbs that are an important component of the Mediterranean diet as a spice and flavoring agent.
More recently, rosemary extracts
standardized to diterpenes (e.g. carnosic acid and carnosol) derived primarily from its leaves are increasingly being incorporated into the food processing industry to preserve oxygensensitive foods. In traditional medicine, rosemary has been used for the treatment of a variety of disorders [1] and has been well-known for its antimicrobial [2], anti-inflammatory [3] and especially for its antioxidant activities [2,4]. In addition to its antioxidant property, rosemary extract has also been shown to have anticancer activity in different in vitro [5-8] and in vivo tumor models [9,10]. Recently we have shown that rosemary extract standardized to carnosic acid decreases cellular viability and induces apoptosis in prostate cancer cells, LNCaP and 22Rv1. Moreover, standardized rosemary extract promoted androgen receptor (AR) degradation via the modulation of endoplasmic reticulum (ER) stress proteins, immunoglobulin heavy-chain binding protein (BiP) and C/EBP homologous protein (CHOP). ER stress dependent AR degradation was also observed in vivo along with 46% prostate tumor suppression when mice were treated with standardized rosemary extract [11]. We have also reported the apoptotic effect and upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) in colon cancer cells HCT116 and SW480 upon treatment with rosemary extract. Furthermore, rosemary extract significantly inhibited the growth of HCT116 tumor cells compared to control mice [12].
Rosemary extract constitutes many different compounds namely volatile oils, flavonoids, polyphenols and terpenoids. Terpenoids or terpenes are recognized for various physiological functions especially in the prevention and treatment of different cancers [13-16]. Terpenes are
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composed of two five-carbon (C5) building blocks also called isoprenoids, based on which they are classified as monoterpenes (C10), diterpenes (C20) and triterpenes (C30).
Rosemary
extract’s highest antioxidant properties are primarily due to the presence of phenolic diterpenes which constitute an important class of terpene superfamily [17,18]. The most common diterpenes present in rosemary are carnosol, carnosic acid, rosmanol, epirosmanol, isorosmanol and 7-methyl-epirosmanol (Figure 1). However, more than 90% of rosemary’s antioxidant activity is attributed to its principal components carnosic acid and carnosol [17]. This review herein focuses on rosemary diterpenes carnosic acid, carnosol and rosmanol and summarizes the mechanisms by which these dietary components prevent carcinogenesis. To date no antitumorogenic activity has been reported with epirosmanol, isorosmanol and 7methyl-epirosmanol and hence these diterpenes have been excluded from this review.
CARNOSIC ACID: Carnosic
acid
(5,6-dihydroxy-1,1-dimethyl-7-propan-2-yl-2,3,4,9,10,10a-
hexahydrophenanthrene-4a-carboxylic acid), a natural benzenediol abietane diterpene, constitutes 1.5 to 2.5% of dried leaves of rosemary. Carnosic acid with an empirical formula C20H28O4 was first discovered in sage in 1962 followed by its discovery in Rosmarinus officinalis by Wenkert et al. in 1965 [19]. Chemical structure of carnosic acid consists of three sixmembered rings, including a dihydric polyphenolic ring and a free carboxylic acid. Because of its typical o-diphenol structure carnosic acid is unstable in solution and can get converted to carnosol upon oxidation [20].
In vitro studies Being one of the most potent antioxidant agents of rosemary, carnosic acid is known to exhibit effective anti-cancer activity against various cancer cell lines derived from human leukemia, breast, prostate, lung and liver malignant tissues at different half maximal inhibitory 2 Page 5 of 33
concentration (IC50) values (Figure 2) [21]. Carnosic acid inhibits growth of human neuroblastoma, IMR-32 cells, at an IC50 value of 30µM [22]. This cytotoxic effect is accompanied by downregulation of B-cell lymphoma 2 (Bcl-2) protein and an increased expression of cleaved caspase-9, -3 and poly ADP ribose polymerase (PARP) proteins suggestive of apoptotic cell death via the mitochondrial pathway. Carnosic acid also increased levels of reactive oxygen species (ROS) in IMR-32 cells along with p38 protein activation and downregulation of extracellular-signal-regulated kinase 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK) proteins. Moreover, individual treatment with N-acetylcysteine (NAC) and p38siRNA inhibited activation of caspase 3 proposing that carnosic acid-induced apoptosis in IMR32 cells was due to ROS-mediated p38 mitogen-activated protein kinase (MAPK) activation [22].
Inhibitory effect of carnosic acid can vary depending on the concentration used. Low concentrations of carnosic acid (<7.5µM) did not significantly affect the cell viability of human myeloid leukemia HL-60 cells nor induced apoptotic cell death. Instead the inhibitory effect of 7.5µM carnosic acid on HL-60 and U937 was due to accumulation of these cells at G0/G1 phase of cell division accompanied by upregulation of cyclin-dependent kinase (CDK) inhibitors p21WAF1 and p27Kip1 [23]. On the other hand, carnosic acid at 10-20µM, induced apoptosis as well as G1 cell cycle arrest in HL-60 cells. Apoptosis was associated with modulation of phosphatase and tensin homolog/protein kinase B (PTEN/Akt) pathway with increased expression of cleaved caspase-9 and PTEN protein along with reduced expression of pAkt. Cell cycle arrest in HL-60 cells was associated with increase in p27 protein [24,25].
Moreover
carnosic acid significantly enhanced G1 cell cycle arrest and apoptosis in HL60 cells when treated in combination with low concentrations of arsenic trioxide [24,25].
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The anticancer activity of carnosic acid is cell line dependent. Inhibitory effect of carnosic acid was 2 fold higher in MDA-MB-231 breast cancer cells compared to estrogen receptor positive MCF-7 breast cancer cells [21]. Similarly, carnosic acid inhibited cell growth along with G1 cell cycle arrest in MDA-MB-453 and MDA-MB-468 cells at an IC50 concentration of 7µM and 6µM respectively. MDA-MB-453 cells overexpressing human epidermal growth factor receptor 2 (HER2) were found to be more sensitive to carnosic acid treatment suggesting activity of carnosic acid is increased in the presence of HER2. In contrast, low HER2 expressing MCF7 cells were inhibited at an IC50 of 75µM [26]. Moreover low concentrations of carnosic acid at 20µg/ml (60µM) significantly increased the percent of MDA-MB-453 cells in the G1 phase whereas a higher dose of 40µg/ml resulted in slight increase of cells in the G 2 phase of cell cycle. In the same way microarray analysis using MDA-MB-468 triple negative breast cancer cells revealed that at 5µg/ml carnosic acid activated expression of three genes involved in glutathione (GSH) biosynthesis [cytochrome P450, family 4, subfamily F, polypeptide 3 (CYP4F3), glutamate—cysteine ligase catalytic subunit (GCLC)] and transport [solute carrier family 7A11 (SLC7A11)] in comparison to 124 genes activated when treated with 20µg/ml (60µM) carnosic acid. At 20µg/ml, carnosic acid upregulated expression of anti-inflammation, xenobiotic metabolism, antioxidant, transport, GSH biosynthesis, signaling and apoptosis related genes and downregulated expression of transcription inhibitors and cell cycle genes. Furthermore this study showed that carnosic acid enhanced the growth inhibitory effect of curcumin on MDA-MB-468 triple negative breast cancer cells [26].
Dose and time dependent growth inhibitory effect of carnosic acid was also observed with prostate cancer cells, another hormone dependent cancer besides breast cancer. Antiproliferative effect of carnosic acid was more on PC3 prostate cancer cells (IC50 of 41.1µM) compared to DU145 cells. The anti-proliferative effect of carnosic acid was accompanied by both intrinsic as well as extrinsic pathway of apoptosis as evident by activation of caspases -8, 4 Page 7 of 33
9, -3, -7 and PARP cleavage, downregulation of inhibitor of apoptosis (IAP) family of proteins (XIAP, cIAP1 and cIAP2), upregulation of BCL2-associated X protein (Bax): Bcl-2 ratio and cytochrome c release. Carnosic acid induced apoptosis was mediated by inhibition of Akt/ inhibitory subunit of NF-κB (IκB) kinaseIKK/nuclear factor-kB (Akt/IKK/Nf-ĸB) signaling pathway via activation of serine/threonine phosphatase PP2A in PC3 cells [27].
Recently, our lab has reported on the anticancer activity of a standardized rosemary extract and carnosic acid on HCT116 and SW480 colon cancer cells [12]. Carnosic acid decreased the cell viability and induced apoptosis in a dose and time dependent manner in these cells by activating caspase 3 cleavage. Nrf2 is a transcription factor that regulates the expression of antioxidant proteins and has been shown to be a substrate for protein kinase RNA-like endoplasmic reticulum kinase (PERK). Carnosic acid induced the expression of Nrf2 via induction of ER stress by activating PERK as evidenced by increase in phosphorylation at site threonine 980 (Thr980). Research suggests that continued oxidative stress can lead to chronic inflammation which in turn can cause cancer and hence it is important to consider modulation of oxidative stress as a possible mechanism in anticancer activity [28]. Correspondingly, carnosic acid increased the expression of sestrin-2 protein that helps in regulating oxidative stress in HCT116 cells via Nrf2-ARE (antioxidant response element) pathway [12]. Likewise, another study by Barni et al. studied the role of cyclooxygenase-2 (COX-2) pathway in carnosic acid induced cell inhibition. COX-2 enzyme causes inflammation and hence inhibiting COX-2 facilitates anticancer activity in cells [29]. Carnosic acid downregulated the expression of COX-2 protein by 2-3 fold in human colonic adenocarcinoma Caco-2 cells. The study further showed that carnosic acid inhibited adhesion of Caco-2 cells to type I collagen and fibronectin surfaces along with Caco-2 cell migration suggesting a role for carnosic acid in inhibiting tumor invasion and migration. Treatment with carnosic acid decreased the activity of extracellular matrix (ECM)-degrading enzymes urokinase plasminogen activator (uPA) and metalloproteinases 5 Page 8 of 33
MMP-9 and MMP-2 [29]. Anti-proliferative effect of carnosic acid on Caco-2 cells was studied by Visanji et al. In this study 50µM of carnosic acid had no cytotoxic effect on Caco-2 cells but exerted cytostatic effect by accumulating the cells predominantly at G2/M phase of cell cycle accompanied with reduced cyclin A levels [30]. Similarly, 12.5µg/ml (38µM) of carnosic acid had no cytotoxic but a cytostatic effect on HT-29 cells by inhibiting cell cycle progression from G1 to S phase [31]. Furthermore, this study used a Foodomics (i.e. genomic, transcriptomic, proteomic, and metabolomics) approach to identify transcriptional and metabolic changes occurring in carnosic acid treated HT-29 cells [32]. At the transcriptome level carnosic acid increased the activation of detoxifying enzyme genes with the most prominent being monoamine oxidase B (MAOB), glutathione peroxidase 3 (GPX3), glutathione peroxidase 8 (GPX8) and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1) genes degrading amines, hydrogen peroxide and aldehydes respectively. A modulation of the expression of genes linked to transport and biosynthesis of terpenoids was also observed. Additionally, this study revealed activation of ROS metabolism upon treatment with carnosic acid and alteration of several genes involved in oxidative degradation pathways. Metabolomics study showed increase in reduced intracellular GSH levels in treated HT-29 cells further demonstrating the chemopreventive response of these cells to carnosic acid treatment [31]. In a different study 1µM and 10µM of carnosic acid inhibited growth of HT-29 cells by exhibiting significant accumulation of cells in the S phase with decreased expression of cyclinD1 and CDK4 along with apoptosis induction [33]. Interestingly, this study highlights the use of carnosic acid in alleviating adiposity related acceleration of colon cancer formation. In vitro, carnosic acid suppressed the growth of adipocytes followed by reduced triacylglycerol (TG) accumulation and adipokine release. Upon co-culturing HT-29 cells with 3T3-L1 adipocytes the authors found increased proliferation of HT-29 cells mediated by activation of Akt, ERK and leptin receptor (Ob-R) proteins and increased production of leptin and interleukin-6 (IL-6). The accelerated growth of HT-29 cells in presence of adipocytes, however, was reversed upon treatment with 6 Page 9 of 33
carnosic acid through cell cycle arrest and enhanced apoptosis by inhibiting Ob-R signaling and inhibition of Akt/ERK pathway. In another study by Gonzalez Vallinas et al the authors tested five different rosemary extracts with different carnosic acid and carnosol concentrations in vitro on colon cancer (DLD-1 and SW620) and pancreatic cancer (PANC-1 and MIA-PaCa-2) cell lines. The extract with high amounts of carnosic acid was more effective in inhibiting cell viability and inducing apoptosis as measured by PARP1 cleavage. This effect was more prominent in colon cancer cell lines as compared to pancreatic cell lines. The effect of carnosic acid however was significantly enhanced by addition of minimal concentrations of carnosol. The antitumor activity of carnosic acid in this study was associated with upregulation of glucosaminyl (Nacetyl) transferase 3, mucin type (GCNT3) expression and downregulation of microRNA 15b (miR-15b) [34].
Recently, antitumorigenic effect of carnosic acid was shown in human renal carcinoma Caki cells [35]. Antitumorigenic activity was accompanied by apoptosis as seen by induction of PARP cleavage and activation of caspase 3. Induction of apoptosis was also found to be dependent on ROS production and on expression of ER stress proteins, activating transcription factor 4 (ATF4) and CHOP. Apoptotic effect of carnosic acid was not observed in normal mouse kidney epithelial TMCK-1 cells or in normal human skin fibroblast cells suggestive of its safe use [35].
Besides apoptosis, autophagy has recently been recognized as another cell death mechanism. A study by Gao et al. has shown that carnosic acid induced autophagic cell death in human hepatoma HepG2 cells via inhibition of Akt/mTOR (mammalian target of rapamycin) pathway. The autophagic effect was accompanied by decrease in levels of pAkt and mTOR proteins without any change in phosphoinositide 3-kinase (PI3K) and PTEN protein levels [36].
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In vivo studies In addition to its in vitro activity (Table I) carnosic acid is also known to exhibit in vivo anticarcinogenic effects (Table II). Corresponding to the in vitro use of carnosic acid in alleviating adiposity related acceleration of colon cancer formation, colonic expression levels of Ob-R, pAkt, p-ERK, insulin and leptin was also suppressed in vivo in mice fed with a high fat diet (HFD) supplemented with carnosic acid compared to HFD alone. Correspondingly, the number of colon tumors in mice nurtured with HFD supplemented with carnosic acid reduced to 6.7-8.4 in comparison to 12.7 tumors in HFD mice [33]. This study thus highlights the anti-adipogenic activity of carnosic acid in addition to its anti-carcinogenic activity. Similarly, using different rosemary extracts differing in their carnosic acid and carnosol concentrations Gonzalez-Vallinas et al. has shown 24-27% tumor inhibition in vivo against SW620 colon cancer xenografts with extracts containing higher amounts of carnosic acid [34].
Manoharan et al have demonstrated that oral administration of carnosic acid at 10mg/kg body weight/day completely abrogated the formation of oral carcinoma in hamsters treated with 7,12dimethylbenz(a)anthracene (DMBA), a potent carcinogen [37]. There were no signs of any tumor incidence, tumor frequency, and tumor burden or tumor volume in DMBA + carnosic acid treated hamsters compared to 100% tumor formation in hamsters induced with DMBA alone. Histopathological changes were mild in the buccal mucosa of DMBA +carnosic acid treated hamsters compared to severe histopathological features observed in hamsters induced with DMBA alone [37]. Inhibition of tumor formation by carnosic acid was associated with restoration of normal levels of detoxification phase I and phase II enzymes. The chemoprotective effect of carnosic acid against oral carcinogenesis was also evident in another study conducted on Syrian hamsters wherein tumor volume of DMBA+carnosic acid treated animals was 1.05mm3 compared to 137.74mm3 in DMBA control hamsters [38].
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Using a mouse leukemia model Sharabani et al. has shown that treatment with carnosic acidrich rosemary extract in combination with 1α,25-dihydroxyvitamin D3
(1,25D3) resulted in
significant 55% reduction in tumor incidence and tumor formation compared to mice treated with 1,25D3 alone [39]. Compounds such as arsenic trioxide and 1,25-D3 have been effective in treatment of myeloid leukemia however they are toxic when used at high concentrations. These studies provide evidence that carnosic acid can be used as an adjuvant in myeloid leukemia therapy.
CARNOSOL: Carnosol (4aR- (4aalpha, 9alpha, 10abeta))- 1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1,1dimethyl-7-(1-methylethyl)-2H-9,4a-(epoxymethano)phenanthren-12-one), a dietary phenolic diterpene with an empirical formula C20H28O4, is a derivative of carnosic acid containing a lactone ring. First isolated in 1942 from sage, its chemical structure was elucidated by Brieskorn et al. in the year 1964 [40]. Carnosol possesses numerous pharmacological properties including anti-inflammatory, antioxidant and antitumor activities.
In vitro studies Carnosol has been proposed as an anti-cancer agent in several different in vitro studies [41]. Decrease in Bcl-2 and pro-caspase 8 protein, along with increased expression of Bax was observed when prostate cancer cell line, PC3, was treated with carnosol (Figure 3) [42]. Nonetheless, in this study apoptosis was not the only mechanism responsible for PC3 cell death. This study reflects on how carnosol can target multiple signaling pathways that play an important role in growth and survival of cancer cells. While carnosol inhibited the PI3K/Akt pathway it simultaneously activated the adenosine monophosphate-activated protein kinase (AMPK) pathway leading to inhibition of mTOR phosphorylation thereby inhibiting growth of cancer cells [42]. AMPK protein was upregulated by 365% in carnosol treated cells compared to 9 Page 12 of 33
control cells in an antibody array performed to understand the mechanism underlying carnosolinduced PC3 cell death.
Carnosol-mediated apoptotic cell death was also observed in human colon cancer HCT116 cells. Carnosol reduced the viability of HCT116 cells in a dose and time dependent manner and was found to be associated with apoptosis. Carnosol treatment activated levels of caspase -9 and -3, induced PARP cleavage, increased levels of Bax protein and decreased levels of Bcl-2 protein in these cells. Since p53 and signal transducer and activator of transcription 3 (STAT3) proteins can modulate apoptosis, this study determined the effect of carnosol on these proteins. As per the results carnosol treatment induced generation of ROS which further led to activation of p53 expression. Moreover carnosol mediated apoptosis was also found to be associated with inactivation of STAT3 signaling pathway [43].
Treatment of B-lineage acute lymphoblastic leukemia (ALL) cell lines and pre-B leukemia cell lines with carnosol induced apoptotic cell death in a dose-dependent manner. Apoptosis, in these cell lines, was characterized by disruption of mitochondrial membranes and downregulation of cellular Bcl-2. Carnosol at 6µm concentration caused cell death in B-cell leukemic cell lines; however it was not toxic to normal peripheral blood mononuclear cells (PBMCs) [44]. Carnosol is also known to exert its apoptotic effect in adult T-cell leukemia (ATL) cell lines, ED and S1T cells. The anticancer activity in ATL cells was accompanied by increase in expression of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductases and enzymes of the glycolytic and pentose phosphate pathways. In addition the protein levels of GSH were decreased in ATL cells when treated with 40µM carnosol for 3 or 6hrs [45].
In contrast, inhibitory effect of carnosol on human hepatoma HepG2 cells was due to increased levels of GSH [46]. 12hr treatment with carnosol at 5µmol/L increased the production of GSH to 10 Page 13 of 33
near 160% of basal levels. However, at this concentration carnosol was non-cytotoxic on HepG2 cells. Moreover, 5µmol/L carnosol increased the expression of glutamate-cysteine ligase (consisting of GCLC and GCLM subunits) enzyme involved in the synthesis of GSH via activation of Nrf-2 pathway in HepG2 cells [46]. These results illustrate that differential effects of carnosol exist depending on the concentration of the compound and the cell line used.
Carnosol treatment does not always evoke a cytotoxic response. In certain cells, depending on the concentrations used, treatment with carnosol can lead to accumulation of cells at G 2/M phase of cell cycle. Carnosol induced G2 cell cycle arrest in both Caco-2 and PC3 cells [30,42]. Cell cycle arrest in PC3 cells was associated with increased expression of p21 and p27 cell cycle regulatory proteins, decrease in levels of cyclins A, D1, D2 and CDK proteins -2 and -6 [42]. On the other hand, carnosol treatment in Caco-2 cells caused a concentration-dependent increase in levels of cyclin B1 with no effect on cyclin A protein [30]. This discrepancy in cyclin A levels could either be cell-specific related or maybe due to a difference in duration of carnosol treatment. While Caco-2 cells were treated with carnosol for only 12hrs, PC3 cells were treated for 48hrs. Further studies are required to evaluate the effect of carnosol on different cell lines.
Cancer is a multistage disease divided into three stages: initiation, promotion and progression. An effective chemopreventive agent would be the one that can efficiently block one or more stages of cancer. Carnosol, in addition to inhibiting tumor formation, has also been shown to have anti-metastatic activity. In a study by Huang et al., the authors have demonstrated using the in vitro transwell assay, how carnosol inhibits the migration and invasion of highly metastatic B16/F10 melanoma cells by 19% compared to control cells. The anti-metastatic activity was associated with reduced MMP-9 protein levels mediated by inhibition of NF-kB and activator protein 1 (AP-1) binding activity and downregulation of p38, AKT and JNK proteins [47]. Another study by Vergara et al. also tested the anti-metastatic activity of carnosol on human breast 11 Page 14 of 33
cancer cell lines (HBL-100, MDA-231, MDA-361, MDA-435, MCF-7), human ovarian cancer cells (OVCAR-3, SKOV03) and human colon cancer cell line Caco-2 [48]. This study tested the effect of carnosol on different cancer cell lines cultured under monolayer conditions as well as in suspension. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) assay revealed inhibition of cell growth in all cells under monolayer conditions at IC50 greater than 50µM. Moreover carnosol treatment of SKOV-3 cells resulted in reduction of pERK1/2 and Akt with no difference found in levels of STAT3 protein expression. Additionally, carnosol treatment inhibited the growth of all cancer cell lines when cultured in suspension using poly- 2-hydroxyethyl methacrylate (pHEMA) coated plates which prevented the adherence of the cells to the wells. This test was a used as a measure to estimate the viability of circulating tumor cells that survive independent of adherent growth. Concomitantly, treatment with carnosol suppressed the adhesion of Caco-2, SKOV-3, OVCAR-3, MCF-7 and MDA-361 cancer cell lines to fibronectin, a component of ECM. Furthermore, carnosol inhibited the epidermal growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) program, a program which regulates the ability of cells to undergo invasion and metastasis, in ovarian cancer cells [48]. Fascinatingly, this study also showed that carnosol enhanced the anti-proliferative activity of phytochemicals capsaicin, quercetin and rosmarinic acid that have limited anticancer activity when given alone and had a synergistic effect when used in combination with curcumin. In addition, the results show that combination of carnosol and curcumin increased the anti-proliferative effects of many different chemotherapeutic drugs, the most significant one observed with vandetanib [48].
A recent study evaluated the effectiveness of carnosol in triple negative breast cancer MDA-MB231 cells [49]. Carnosol reduced the growth of MDA-MB-231 cells in a time and dose dependent manner with an IC50 value of 83µM and 25µM after 24 and 48hrs respectively. The anticancer activity of carnosol was accompanied by G2/M phase of cell cycle arrest along with induction of both intrinsic and extrinsic apoptotic pathways as evident by cleavage of PARP and caspases 3, 12 Page 15 of 33
8 and 9, reduced Bcl2 levels, increase in Bax expression and mitochondrial membrane depolarization. Interestingly, carnosol induced cell death of MDA-MB-231 cells was also associated with autophagy as evident by the presence of autophagic vacuoles and modulation of autophagic markers microtubule-associated protein 1A/1B-light chain 3 I (LC3 I) to microtubule-associated protein 1A/1B-light chain 3 II (LC3 II) and p62. Furthermore, the authors illustrated that carnosol-mediated activation of ROS triggered the induction of both apoptosis and autophagic mediated cell death [49].
In vivo studies Corresponding to the in vitro studies (Table I) carnosol has been proposed as an anti-cancer agent in several different in vivo studies for prostate, colon, skin, breast and other leukemic cancers [41]. We have previously shown that oral administration of carnosol (30mg/kg) inhibited the growth of prostate cancer in athymic nude mice by 36% along with a 26% decrease in serum prostate-specific antigen (PSA) levels compared to control animals [50]. Our study also revealed that carnosol binds antagonistically to AR and estrogen receptor α (ERα) by interacting with its ligand-binding domain resulting in reduced protein levels of both AR and ERα in mice treated with carnosol as well as in carnosol treated prostate cancer cell lines, LNCaP and 22Rv1[50]. Both androgen and estrogen hormones are required for inducing prostate carcinogenesis by binding to their receptors, AR and ERα, respectively. Thus the ability of carnosol to dually disrupt both receptors suggest a great potential for it to be used as a chemopreventive and possibly a chemotherapeutic agent against hormone-induced cancers such as prostate cancer and breast cancer. In fact, carnosol also decreased protein expression of AR and ERα in MCF-7 breast cancer cells [50] and has been effectively used to inhibit initiation of mammary tumor formation in female rats. Intraperitoneal (IP) administration of carnosol decreased the formation of DMBA-induced tumors by an average of 35% and also inhibited the in vivo formation of DMBA-DNA adducts by 44% compared to control animals [9]. 13 Page 16 of 33
Carnosol prevented the formation of DMBA-induced tumors by activating liver detoxification enzymes, glutathione S-transferase (GST) and quinone reductase (QR).
Besides prostate and breast cancer, carnosol has been shown to reduce intestinal tumors and skin tumors in mouse models. In C57BL/6J/Min/+, colonic tumorogenesis mouse model, oral administration of 0.1% carnosol reduced the incidence of intestinal tumors by 46% compared to control mice [51]. Similarly, carnosol at 10µm concentration when applied to the back of mice inhibited the initiation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of DMBA-induced tumors to 77% compared to 93% tumor formation in control mice [10]. A recent study by Rahnama et al. has studied not only the anti-cancer effects of carnosol but also its immunomodulatory effects in a mouse fibrosarcoma model. In this study IP injection of carnosol at 5 or 10mg/kg daily to Balb/c mice bearing fibrosarcoma tumors significantly suppressed the growth of tumors compared to control mice. In addition, carnosol depleted splenic and tumorassociated regulatory T cells (Treg cells), decreased production of interleukin-4 (IL-4) and interleukin-10 (IL-10) and increased levels of interferon-ϒ (IFNϒ) compared to control mice [52].
ROSMANOL: Rosmanol, a phenolic diterpene was first isolated from the leaves of rosemary by Inatani et al. in the year 1982 [53]. Ethanol extract of rosemary leaves contain less than 0.5% rosmanol, which can be increased by further thermal or oxidative treatment [54].
In vitro studies Rosmanol possesses high antioxidant, anti-inflammatory and anti-tumorogenic activity (Figure 4). 99% pure fraction of rosmanol, isolated from rosemary, was found to be a more potent cytotoxic agent compared to carnosol and rosmarinic acid when treated with human colon adenocarcinoma COLO 205 cells. Dose-dependent decrease in cell viability of COLO 205 cells 14 Page 17 of 33
was characterized by DNA fragmentation, cell shrinkage and chromatin condensation, which are all key features of apoptotic cell death [54]. Cheng et al. further establish that rosmanol-induced apoptosis of COLO 205 cells occurs via both the mitochondrial and receptor-mediated pathways. Fas and FasL protein expression was upregulated whereas procaspase-8 and BH3 interacting-domain (Bid) proteins were downregulated upon treatment with 50µM rosmanol, indicating rosmanol induced apoptosis of COLO 205 adenocarcinoma cells through the receptor-mediated pathway [54]. Rosmanol did not affect Bcl-2 and Bcl-XL protein expression in these cells; however it decreased cytosolic Bax and increased mitochondrial Bax expression, which help regulate the mitochondrial pathway of apoptosis. The translocation of Bax from the cytosol to the mitochondria was further shown to disrupt the mitochondrial membrane and release cytochrome c which in turn activated downstream caspases leading to cleavage of PARP and DNA fragmentation factor-45 (DFF-45) [54].
Besides inhibiting tumor development, rosmanol also acts as a strong anti-inflammatory agent. Inflammation has been linked to several human cancers and transcription factor NF- ĸB plays a central role in transformation of inflammation to cancer [55]. Numerous other pro-inflammatory cytokines and enzymes like the tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and COX-2 also contribute to pathogenesis of inflammation and carcinogenesis. Rosmanol inhibits iNOS and COX-2 mRNA and protein expression in lipopolysaccharide (LPS)induced RAW264.7 murine macrophage cell line [56]. Many different transcription factors like NF-ĸB, AP-1, interferon-regulatory factor 1(IRF1), STATs and CCAAT/enhancer-binding protein(C/EBP) bind and regulate the promoter region of iNOS and COX-2 genes [57]. In this study, Lai et al., have demonstrated that rosmanol downregulated iNOS and COX-2 expression via multiple signaling pathways including inhibition of STAT3 and C/EBP proteins and inhibiting NF- ĸB activity by blocking the activation of upstream kinases PI3/AKt/IKK and p38 and ERK1/2 MAPK kinases[56]. 15 Page 18 of 33
CLINICAL SAFETY AND EFFICACY: The necessity to develop safe, non-toxic cancer therapeutics for mankind has led researchers focus on using natural herbs and spices as the new chemopreventive and chemotherapeutic measures to fight against cancer. Rosemary (Rosmarinus officinalis) extracts are well-known plant derived natural antioxidants commonly used in the food processing industry to preserve oxygen-sensitive foods like beef [58], poultry [59] and pork [60]. Rosemary has been used as a natural medicinal agent for centuries for treatment of digestive, dermal, respiratory, cardiovascular, rheumatism, joint and muscle pain related illnesses [61].
Numerous studies in animal models demonstrate the non-toxicity of rosemary extract and its principal components, carnosic acid and carnosol. Some of these studies illustrate that rosemary and its components are protective against environmental toxins in experimental animal models of hepatotoxicity [62], bronchial cells [63] and may assist in inducing phase II detoxification enzymes. Rosemary extract was also found to be radioprotective against gamma radiation induced disorders in mice [64,65].
A single dose of rosemary extract (2,000mg/kg) administered by oral gavage to Wistar rats has been shown to be tolerable over a 2 week observation period [66]. The median lethal dose (LD50) is unknown for rosemary extract but can be reasonably determined to be greater than 2,000 mg/kg. At the conclusion of the study no changes were observed in behavior, body weight, hematological endpoints, serum chemistry, gross histological examination and in food and water consumption. We [50] as well as others [51] have observed that carnosol administered orally on a daily basis to be tolerable in mice.
Recently, in 2008 the European Food Safety Authority Panel on Food Activities approved the use of rosemary extract standardized to carnosic acid and carnosol for its use in food 16 Page 19 of 33
preservation [67]. Rosemary extract and purified carnosic acid and carnosol were tested for mutagenicity in bacterial Ames test using different Salmonella tester strains and also for genotoxicity in vitro using human lymphocyte assay and in vivo using a mouse micronucleus test. The panel concluded that rosemary extract and its components carnosic acid and carnosol did not pose a safety issue as far as genotoxicity and mutagenicity is concerned. Moreover the extracts, given orally, were found to be well tolerated in male and female rats with no signs of low acute and sub-chronic toxicity. In addition the panel estimated the daily normal dietary exposure values for adults and preschool children to be 0.04 and 0.11mg of carnosol + carnosic acid/kgbw/day.
PHARMACOKINETICS Rosemary extract and its principal components, carnosic acid and carnosol, have been shown to be safe and well tolerated in animal studies, however to better associate with human clinical trials studies pertaining to pharmacokinetics and bioavailability of these compounds is a requisite. Three different pharmacokinetic studies have been reported describing the pharmacokinetic properties of carnosic acid, all within the rat species, in an attempt to establish the translational potential of carnosic acid as a pharmacological agent.
In male Sprague-
Dawley rats (190-220g) carnosic acid was administered intragastrically (90mg/kg) [68]. Oral administration of carnosic acid had the following parameters: time of maximum concentration (Tmax) (125.6min), maximum plasma concentration (Cmax) (126μM), area under the curve (AUC0t)
(21755.3mg/L/min), and oral bioavailability (65.09%). A second study with male Sprague-
Dawley rats (200-330g) administered carnosic acid intragastrically (64.3mg/kg) achieving the following pharmacokinetic parameters: Tmax (136.6min), Cmax (105μM), AUC0-t (7.05mg/mL/min), and oral bioavailability (40.1%) [69]. A third pharmacokinetic evaluation was performed using a rosemary extract standardized to 40% carnosic acid [70]. Female Zucker rats (174.8g +/- 11.3) were administered 571mg/kg of rosemary extract (containing 230mg/kg of carnosic acid). A 17 Page 20 of 33
total of 26 diterpenes with the primary metabolites were identified with the most abundant being carnosic acid, carnosic acid 12-methyl ether, rosmariquinone, carnosic acid glucuronide, carnosol, carnosol glucuronide. The Tmax of carnosic acid was 24mins with a Cmax of 26.6μM and AUCall of 17.8μM h. This study also determined the pharmacokinetic properties for carnosol that was a metabolic conversion from carnosic acid. The Tmax of carnosol was 13.3mins with a Cmax of 18.2μM and AUCall of 137.4μM h [70].
There appears to be some differences in the pharmacokinetic profile of pure carnosic acid versus carnosic acid from a standardized rosemary extract, specifically, the T max and the Cmax. The maximum concentration of carnosic acid was 105 to 126μM while rosemary extract with ~2.5-3.5 times the amount of carnosic acid achieved a Cmax of only 26.6μM. Regardless, in all three studies plasma levels were well within the range that elicits a pharmacological response in cell culture models. Performing a dose translation from animals to humans with the following parameters: animal dose, mass of animal, body surface area, Km factor (i.e. surface area to weight ratio), the human equivalent dose of pure carnosic acid could range from 630mg to 875mg of carnosic acid in a 60kg adult [71,72]. This dose could easily be achieved in two capsules to achieve plasma levels that are significantly higher than a variety of other dietary phytochemicals that have been evaluated in animal models and clinical trials [73,74]. The other parameter that was different between rosemary extract and pure carnosic acid was the Tmax. Absorption of rosemary derived carnosic acid was faster compared to pure carnosic acid with Tmax decreasing from 2 to 0.4hrs when administered as an extract. One possible explanation is that within the extract protein, carbohydrates and dietary fiber were present and may contribute to an overall lower Cmax and decrease in Tmax. Another consideration that cannot be ruled out is the potential of other phytochemicals in rosemary modulating the absorption or metabolism profile of carnosic acid. A third explanation could be the difference of vehicle in the different studies. A final consideration is the difference in animal model used. The rosemary extract 18 Page 21 of 33
study utilized female Zucker rats, while the pure carnosic acid studies utilized Sprague-Dawley males. One apparent advantage of rosemary diterpenes such as carnosic acid is the high bioavailability with estimates ranging from 40 to 65% which is significantly higher than other phytochemicals which could be as low as 1 to 5% [68,69,73,74].Taken together, the pharmacokinetic profile of carnosic acid appears to be well within the concentrations that have been used to report a variety of pharmacological actions in different cell culture models.
CONCLUSION: The concept that there exists a close relationship between dietary constituents and the risk of developing specific cancers is widely accepted. Many scientists are now focusing on using natural herbs and spices as the new generation of chemopreventive and perhaps even chemotherapeutic drugs. In this review we have summarized the antitumorogenic activities of rosemary diterpenes, carnosic acid, carnosol and rosmanol. Dietary constituents of rosemary are multifunctional in nature inhibiting tumor formation by inducing cell cycle arrest, activation of apoptosis and autophagy and inhibiting signal transduction pathways (Table I). In addition phenolic components of rosemary are known to possess anti-invasive and anti-metastatic activity (Table I). Many in vivo studies have been done with carnosic acid and carnosol that further confirm the results obtained from the in vitro studies done using these compounds (Table II). Evidently rosmanol demands many more studies to further elucidate its role in cancer prevention [68]. Although many studies have been carried out with carnosol and carnosic acid both in vivo and in vitro, further mechanistic studies are needed to delineate the underlying molecular events in detail and to see if there is any cross-talk occurring between more than two signaling pathways. Future studies that deconstruct the rosemary extract to individual phytochemicals will help future investigators understand the synergistic action of rosemary diterpenes for their anti-cancer activity.
19 Page 22 of 33
Conflicts of Interest Statement None
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Table I. In vitro activity of rosemary diterpenes
Anticancer activity
Carnosic acid
Carnosol
Rosmanol
Reference
Apoptotic
-Downregulation of Bcl-2 and IAP proteins (XIAP, cIAP1, cIAP2). -Increased expression of cleaved caspase -8, -9, -3, -7, PARP and PTEN proteins. Increase in Bax protein expression and cytohrome c release.
-Downregulation of Bcl-2. -Increased expression of cleaved caspase -8, -9, -3, and PARP. Increase in Bax protein and mitochondrial membrane depolarization.
-DNA fragmentation, cell shrinkage and chromatin condensation. -Downregulation of procaspase 8 and Bid. -Upregulation of Fas and FasL, cytohrome c release, PARP cleavage
[12],[22],[24],[25],[27],[35],[49],[44], [42],[43],[54]
Autophagic
Inhibition of Akt/mTOR pathway.
Modulation of autophagic markers LC3 I to LC3 II and p62.
[36],[49]
Cytostatic
-Reduced levels of cyclin A, cyclin D1 and CDK4. -Upregulation of CDK inhibitors p21WAF1 and p27Kip1.
-Reduced levels of cyclins A, D1, D2, CDK-2 and CDK-6. -Increased levels of cyclin B1, p21 and p27 cell cycle proteins.
[23],[30],[33],[42]
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Signaling pathway
-Activation of p38 and downregulation of ERK1/2 and JNK proteins. -Decrease in pAKT and mTOR protein levels with no change in PI3K and PTEN -Inhibition of Akt/IKK/Nf-ĸB pathway via activating ser/thr phosphatase PP2A
Hormone signaling Anti-metastatic activity
-Inhibits adhesion to type 1 collagen and fibronectin. -Inhibits cell migration by decreasing activity of ECM-degrading enzymes like uPA, MMP-9 and MMP-2
Antiinflammatory
-Downregulation of COX-2 protein. -Upregulation of antiinflammatory genes
-Downregulation of pERK1/2, p38, Akt, JNK with no difference in STAT3 levels. -Inhibition of PI3K/Akt pathway but activation AMPK pathway leading to inhibition of mTOR phosphorylation -Inactivation of STAT3 signaling pathway
-Inhibition of STAT3, C/EBP proteins and NFĸB activity -Inhibition of PI3/AKt/IKK, p38 and ERK1/2 MAPK kinases
[22],[25],[27],[33],[42],[43],[47],[48], [56]
Decreased expression of AR and ERα protein levels
[50]
-Inhibits adhesion of cancer cells to fibronectin. Inhibits EGF-induced EMT program which regulates invasion and metastasis. -Inhibition of NF-kB and AP-1 binding activity leads to reduced MMP-9 levels.
[29],[47],[48]
Downregulation of COX-2 protein and iNOS
[29],[56]
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Anti-oxidative
-Increased expression of ROS. -Tanscriptional activation of detoxifying enzyme genes. -Increased expression of 3 genes involved in glutathione synthesis (CYP4F3, GCLC) and transport (SLC7A11). -Increase in reduced glutathione (GSH) levels. -Increased expression of Nrf2 and sestrin-2.
Imunomodulatory
ER stress activation
-Activation of ROS and p53. -Activation of liver detoxifying enzymes GST and QR. -Increase in expression of of NADPH-dependent reductases and enzymes of glycolytic and pentose phosphate pathway. At 40µM GSH levels reduced but at 5µmol/L GSH levels increased along with increased expression of GCL. -Activation of Nrf-2 pathway
[12],[22],[26],[31],[35],[9],[49],[43],[45], [46]
-Depletion of splenic and tumor associated Treg cells. -Decreased production of IL-4 and IL-10 with increase in IFN-ϒ levels
[52]
Increased expression of ER stress proteins ATF4, PERK and CHOP
[12],[35]
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Table II. In vivo activity of rosemary diterpenes Rosemary Dose and Route Diterpene
Animal Model (age in weeks) -Male golden Syrian hamsters (810) -Male Syrian hamsters (20)
Diet fed
Standard powdered rodent diet plus autoclaved water ad libitum Different groups fed with normal fat diet (NFD) or High Fat Diet (HFD)
Myeloid leukemia developed from WEHI-3B Dcells Adiposity related colon cancer
Significantly reduced the number of colon tumors at both doses by 47% and 34% in comparison to HFD treated mice
[33]
Autoclaved diet ad libitum
22Rv1 prostate cancer xenograft
Significant suppression of tumor growth by 36% along with decrease in PSA, AR and ERα protein expression
[50]
Carnosic acid
-10mg/kg body weight/day orally -0.075mg in 0.1 ml saline solution given orally
Carnosic acid
Oral administration of 1% carnosic acid-rich rosemary extract mixed in diet
Female Balb/c mice (6-8)
Carnosic acid
0.01% or 0.02% carnosic acid supplemented with HFD
Male A/J mice (4)
Carnosol
30mg/kg carnosol for 5 days/week for 28 days by oral gavage
Athymic nu/nu male nude mice (7)
Cancer type
Standard DMBApellet diet and induced oral water ad carcinoma libitum
In vivo Efficacy -Complete abrogation of tumor incidence and retained near normal levels of phase I and II enzymes in liver -Significant 99% reduction in tumors when treated in combination with DMBA Reduced tumor incidence and formation significantly by 55% in synergy with vitamin D3 derivative
Reference
[37],[38]
[39]
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Carnosol
IP administration of 200mg/kg of for 5 days
Female SpragueDawley rats
Carnosol
Topical application of 1,3 or 10µM carnosol
Female CD-1 mice (7)
Carnosol
Dietary administration of 0.1% carnosol for 10 weeks
Carnosol
IP injection of 5 or 10mg/kg daily for 7 days
Control diet consisting of vitamin-free casein, methionine, sucrose, cellulose, AIN-76A vitamin mix, AIN-76 mineral mix, choline dihydrogen citrate, corn oil and cornstarch Purina Lab Chow 5001 diet ad libitum plus water ad libitum
DMBAinduced mammary tumor
Significantly inhibited DMBA-DNA adduct formation by 40% and significantly suppressed the growth of adenocarcinoma by 65%.
[9]
TPA-induced promotion of DMBAinduced skin cancer
Together with 5nmol TPA carnosol inhibited number of skin tumors by 38, 63 or 78%
[10]
Female AIN-76A diet C57Bl/6J/Min/+ and tap water mice (5) ad libitum
Intestinal tumors
Significantly reduced tumor multiplicity by 46%
[51]
Balb/c mice
Fibrosarcoma Significantly suppressed growth of fibrosarcoma tumors at both doses and depleted splenic and Treg cells
--
[52]
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Figure 1. Chemical Structures of rosemary diterpenes with an abietane scaffold (Carnosic Acid, Carnosol, Rosmanol)
Figure 2. Anticancer molecular mechanisms of Carnosic acid
Figure 3. Anticancer molecular mechanisms of Carnosol
Figure 4. Anticancer molecular mechanisms of Rosmanol
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