Food & Function View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
PAPER
View Journal | View Issue
Antitumor activity of the dietary diterpene carnosol against a panel of human cancer cell lines† Cite this: Food Funct., 2014, 5, 1261
Daniele Vergara,ab Pasquale Simeone,cd Simona Bettini,e Andrea Tinelli,f Ludovico Valli,e Carlo Storelli,a Silvana Leo,g Angelo Santino‡h and Michele Maffia‡*ab Dietary phytochemicals found in vegetables and fruits consist of a wide variety of biologically active compounds with anti-carcinogenic activity. The aim of this study was to evaluate the antigrowth activity of carnosol, a dietary diterpene, as a single agent or in combination with other dietary phytochemicals or chemotherapeutic drugs against a panel of tumor cell lines. Carnosol decreased cell viability in human breast, ovarian, and intestinal tumor cell lines, and inhibited cancer cell adhesion on fibronectin and growth of cancer cells in suspension. Carnosol also inhibited EGF-induced epithelial mesenchymal transition in ovarian cancer cells. The combination treatment with other dietary phytochemicals increased the anti-proliferative activity of carnosol. The combination with curcumin resulted in a synergistic reduction of vitality in SKOV-3 and MDA-231 cells and potently inhibited viability of primary
Received 14th January 2014 Accepted 9th March 2014
cancer cells isolated from the pleural fluid or ascites of patients with metastatic cancers. These results
DOI: 10.1039/c4fo00023d
provide additional evidence about the anticancer role of carnosol and its potential in blocking the
www.rsc.org/foodfunction
growth of tumor cells.
1. Introduction The Mediterranean diet has long been reported to be protective against the occurrence of several different diseases. The health effects of such a dietary prole have been recognized in many different studies and associated with a reduced risk for chronic diseases and some types of cancer.1–3 Multiple mechanisms have been proposed to explain the benecial effects of the Mediterranean diet. In principle, the Mediterranean diet plan involves a high consumption of fruits, vegetables, legumes, sh, olive oil and a moderate amount of red wine during meals, limiting the intake of red meat and avoiding processed meat. The active components from some of
a
Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, Via per Monteroni, 73100, Lecce, Italy. E-mail: michele.maffi[email protected]; Fax: +39 0832 324200
b
Laboratory of Clinical Proteomics, “Giovanni Paolo II” Hospital, ASL-Lecce, Italy
Unit of Cancer Pathology, CeSI, Foundation University “G. d'Annunzio”, ChietiPescara, Italy
c
Department of Neuroscience, Imaging and Clinical Sciences, University “G. d'Annunzio”, Chieti-Pescara, Italy
d
e
Department of Engineering for Innovation, University of Salento, Via per Monteroni, 73100, Lecce, Italy
f
Department of Obstetrics and Gynaecology, Vito Fazzi Hospital, Lecce, Italy
g
Department of Geriatric Oncology, Vito Fazzi Hospital, Lecce, Italy
h
Institute of Science of Food Production, CNR, Unit of Lecce, Via per Monteroni, 73100, Lecce, Italy † Electronic supplementary 10.1039/c4fo00023d
information
(ESI)
‡ Equal contribution.
This journal is © The Royal Society of Chemistry 2014
available.
See
DOI:
these foods were characterised in a large number of in vitro and animal studies for their ability to target specic cellular molecules including various signal transduction pathways and proteins involved in cell cycle progression.4–6 One example of plant-derived compounds currently under investigation for their health benets is Mediterranean herbs7 such as rosemary, basil, sage, marjoram and oregano. They represent a rich source of a specic class of molecules known as diterpenes. Carnosol and carnosic acid are the two major dietary diterpenes present in herbal extracts where they are found in considerable quantities.8–10 The possible benecial effects of these two compounds against inammation and cancer prevention were described in vitro and in vivo.11–19 Carnosol and carnosic acid are able to inhibit prostaglandin PGE(2) formation by selectively targeting microsomal PGE(2) synthase (mPGES-1).11 Both phytochemicals also reduced the expression of pro-inammatory cytokines interleukin-1 beta (IL-1b) and tumor necrosis factor alpha (TNF-a) and selectively inhibited cyclooxygenase 2 (COX-2) in vivo.12 In vitro treatment with carnosol of cell lines derived from oral leukoplakia (MSK-Leuk1), skin (HaCaT) and human bronchial epithelium (BEAS-2B) resulted in decrease of benzo[a]pyrene (B[a]P) mediated induction of cytochrome P450 (CYP) 1A1 and CYP1B1, induction of glutathione-S-transferase and inhibition of mutagenesis.13,14 Carnosol also induced the phase II enzyme heme oxygenase-1 (HO-1) through a pathway that involves phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt).15 Singletary and coworkers found that dietary administration of rosemary
Food Funct., 2014, 5, 1261–1269 | 1261
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
extract for two weeks can prevent 7,12-dimethylbenz[a] anthracene (DMBA)-induced DNA damage and tumor formation in the rat mammary gland.16 The inhibitory effect of topically applied carnosol on tumor promotion by 12-Otetradecanoylphorbol-13-acetate (TPA) was also described.17 Carnosol and carnosic acid were also reported to inhibit cell growth and proliferation of tumor cells by arresting the cell cycle at the G2/M phase18 and inducing apoptosis in B-lineage leukaemia cells through the down-regulation of the antiapoptotic protein Bcl-2.19 In spite of increasing interest in the anticancer activity of carnosol during initiation and promotion phases of cancer development, few studies have proved the efficacy of diterpenoids in monotherapy or when paired with other phytochemicals or chemotherapeutics. To achieve this, we used a panel of human cancer cell lines and primary tumor-derived cell lines to demonstrate the ability of carnosol to inhibit cell viability, cell adhesion, epithelial to mesenchymal transition (EMT) and to induce anoikis. We further demonstrated that carnosol can be combined with other phytochemicals or commonly used chemotherapeutic drugs in order to increase their antiproliferative properties.
2. 2.1
Materials and methods Chemicals
Capsaicin, carnosol, curcumin, piperin, quercetin, resveratrol and rosmarinic acid were purchased from Sigma-Aldrich. Capsaicin, carnosol, curcumin, quercetin, and rosmarinic acid were all dissolved in dimethyl sulfoxide (DMSO), and resveratrol was dissolved in ethanol 70% and immediately used. The following reagents were obtained from GE Healthcare: Hybond ECL membrane and ECL western blotting detection reagents. Fibronectin was from BD Biosciences. Polyhydroxyethylmethacrylate (Poli-HEMA) was from Santa Cruz. ZVAD-fmk was from Abcam. Primary antibodies against Bcl-2 (sc-492), Colin (sc-33779), Colin (Ser3) (sc-21867-R), Cyclin D1 (sc-20044), E-cadherin (sc-71009), N-cadherin (sc-59987), p27 (sc-527), Rock (sc-5560), Survivin (sc-8808), b-catenin (sc-1496), a-Tubulin (sc-23948), and secondary antibodies (HRP-conjugated) were all from Santa Cruz. Anti-extracellular-signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204) (4695), anti-Myosin light chain 2 (Ser19) (3671), anti-STAT3 (Tyr705) (9138), and anti-Akt (Ser473) (4051) were from Cell Signaling. All other reagents were from standard commercial sources and were of the highest grade available. 2.2
Cell cultures and ascites-derived tumor cells
Human tumor cells were purchased from the American Type Culture Collection (ATCC) or from Banca Biologica and Cell Factory (IRCCS Azienda Ospedaliera Universitaria San MartinoIST Istituto. Nazionale per la ricerca sul cancro, Genova, Italy). Normal Human Dermal Fibroblasts (NHDF) were kindly provided by Prof. T. Cocco and Dr A. Gaballo. The human ovarian cancer cell lines OVCAR-3 and SKOV-3, the colorectal adenocarcinoma cell line Caco-2, and the human
1262 | Food Funct., 2014, 5, 1261–1269
Paper
breast cancer cell lines HBL-100, MCF-7, MDA-231, MDA-361 and MDA-435 were all cultured in DMEM medium supplemented with 10% FBS, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin at 37 C in an atmosphere of 5% CO2. The cells were kept in an exponential growth phase during experiments. All research studies involving human subjects were approved by the institutional review board (Human Bioethic Center of the University of Salento and “Vito Fazzi” Hospital). All patients provided written informed consent. Ascites were collected from ve chemotherapy-na¨ıve ovarian cancer patients, who ranged in age from 51 to 72 years, diagnosed with serous adenocarcinoma and presented as stage III (FIGO classication) at the time of clinical intervention. Primary human normal broblasts were isolated from omental tissues of patients undergoing surgery for benign ovarian cysts as described in ref. 20. Epithelial cells were veried by cytokeratin 8-, 18- and 19-expression, whereas broblasts were veried by vimentin positive but cytokeratin 8-negative expression. Lung cancer cells were isolated from the pleural uid of a patient with non-small cell lung cancer maintained in DMEM with 20% FBS. Endometrial cancer cells were isolated from the peritoneal washing of endometrial cancer patients and maintained in DMEM with 20% FBS. Briey, ascites or pleural uids (2–3 liters) were transferred to 50 ml Falcon tubes and centrifuged at 1200 rpm for 10 minutes to pellet the cells. The nal cell pellets were washed twice with a phosphate-buffered saline (PBS) solution, pooled, and resuspended in normal growth medium. All primary cells isolated for this study were used aer two passages. 2.3
Cell viability assay
Cells were seeded at a density of 5 103 cells per well in a 96-well plate containing 100 ml of complete medium and allowed to adhere to the plate overnight. For determining cell viability, the MTT assay was used. Aer treatment, the culture medium was aspirated and 100 ml of RPMI-phenol free medium containing 10 ml of MTT stock solution, 5 mg ml 1 in PBS solution, was added. Two hours later, 100 ml DMSO was added to each well to dissolve the resulting formazan crystals. Absorbance of the converted dye was measured at a wavelength of 570 nm. The relative cell viability was expressed as a percentage of the untreated control wells. 2.4
Cell adhesion assay
Cells were treated with carnosol, maintained in a suspension for 1 hour, and adhered onto 96-well plates coated with bronectin (10 mg ml 1) for 1 hour at 37 C. Briey, non-adherent cells were removed by gentle washing with PBS. Attached cells were xed with 70% methanol for 10 minutes. Cells were stained with 0.02% crystal violet in 0.2% ethanol solution. The incorporated dye was dissolved in 10% acetic acid, and the absorbance was measured at 560 nm. Each experiment was done in triplicate. 2.5
Adhesion-independent cell survival (anoikis assay)
12-well cell culture dishes were coated with 500 ml of poly-HEMA (12 mg ml 1 in 95% ethanol). The solution was le to dry for
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Paper
Food & Function
24 h at room temperature. Cells were then seeded in the polyHEMA-coated wells and cultured in a medium supplemented with 10% serum, with or without natural compounds for the indicated period of time. At the end of the indicated culture period the cells in the suspension were collected, treated with trypsin or disaggregated by vigorous pipetting to separate single cells from aggregated cells, resuspended in complete DMEM, seeded in 96-well plates and cultured overnight. Cell viability was determined by MTT. In another way, suspended cells were collected by aspiration and the percentage of viable cells was determined immediately aer disaggregation by trypan blue staining. 2.6
Reverse transcriptase-PCR (RT-PCR)
RT-PCR was performed as described in ref. 4. Briey, total cellular RNA was isolated using an Illustra TriplePrep extraction kit following manufacturer's instruction and reverse transcribed into cDNA using a High Capacity RNA-to-cDNA Master Mix (Applied Biosystem). PCR was carried out using the following conditions: denaturation at 95 C for 30 s, annealing at 60 C for 30 s, and extension at 72 C for 45 s. PCR samples were loaded onto a 1.2% agarose gel containing ethidium bromide and analysed. The primers used for PCR amplication were designed using the Primer blast program and listed elsewhere.5 2.7
Western blotting analysis
Whole proteins were extracted in RIPA buffer (Cell Signaling) and quantied by the Bradford protein assay (BIORAD). Samples were separated by 12% SDS-PAGE and transferred to the Hybond ECL nitrocellulose membranes. The membranes were blocked overnight in Blotto A (Santa Cruz) at 4 C, and subsequently probed by the appropriately diluted primary antibodies for 2 h at room temperature as previously described.4 2.8
Statistical analysis
Data were analyzed with GraphPad Prism statistical soware. Experiments were carried out three times and statistical evaluation was carried out using Student's t-test. Data are presented as mean values standard deviation. P values 1.2 indicates antagonism.
3.
Results and discussion
3.1 Effects of carnosol on viability of human cancer cells under monolayer and suspension conditions In the preliminary stage of investigation, we tested the sensitivity of different human tumor cell lines to different concentrations of carnosol. Two different cell culture conditions (cells grown as a monolayer or maintained in a suspension) were
This journal is © The Royal Society of Chemistry 2014
used. The experiment was conducted using human breast cancer models (HBL-100, MDA-231, MDA-361, MDA-435, MCF7), human ovarian cancer models (OVCAR-3, SKOV-3), and the human colon cancer cell line CACO-2. Results from MTT assay indicated that the viability of all tested tumor cell lines under monolayer culture conditions was signicantly inhibited by carnosol (Fig. 1a). Treatment of these human cancer models at different time points and different carnosol concentrations resulted in a dose- and time-dependent inhibition of cell growth with an IC50 (the concentration causing 50% cell death) greater than 50 mM. As shown in Fig. 1a, at the concentrations of 100 mM and 200 mM, cell viability was completely abrogated in all cell lines tested while no effect on cell viability was observed at concentrations lower than 25 mM. To test whether the concentrations of carnosol used to inhibit the growth of cancer cells were also toxic for normal cells, we investigated the effects of carnosol on the viability of the normal dermal broblast cell line NHDF. Carnosol induced a modest reduction of cell viability only at the concentration of 200 mM (Fig. 1b). This implies a selective effect of carnosol on tumor cells. Previous studies have suggested that carnosol may have antiproliferative and apoptotic activities.19 Here, we have shown that exposure of SKOV-3 cells to a concentration of carnosol of 70 mM for 24 h reduced the phosphorylation state of ERK 1/2 and Akt. No difference in STAT3 expression was found (Fig. 1c). Moreover, carnosol treatment reduced Cyclin D1 expression and induced the cleavage of ROCK1 with a concomitant increase in the phosphorylation of Cof and Mlc2.22 Pre-incubation with caspase inhibitors z-VAD-fmk abolished ROCK cleavage, Cof and Mlc2 phosphorylation, thus suggesting that the molecule had an apoptotic effect on cultured human tumor cells. Anchorage-independent growth is a hallmark in the metastatic cascade. In fact, following intravasation, circulating cells are deprived of ECM interactions. The ability of tumor cells to survive independent of anchorage is an essential prerequisite for the metastatic spread of cancer. For this reason, we were interested in studying the effects of carnosol on human cancer cells cultured in a suspension. With this aim, we examined the effects of carnosol on the viability of cancer cells cultured in poly-HEMA coated multiwell plates, in which the adherence of cells to the well is prevented. As shown in Fig. 1d, when placed in suspension culture, tumor cells tend to form large and compact aggregates in the absence of a matrix attachment. Control and carnosol-treated cells were maintained in the suspension for 48 h before being harvested for further analyses. Cell viability was then determined by the MTT test. Carnosol inhibited the viability of all cancer cell lines investigated in a concentration-dependent manner. As cell adhesion is one of the essential steps involved in cancer metastasis, we investigated the effect of carnosol on cancer cell adhesion to bronectin, a major extracellular matrix (ECM) component. As shown in Fig. 1e, carnosol was able to suppress dose dependently the adhesion of all the four different cancer cell lines used in this experiment to bronectin. Food Funct., 2014, 5, 1261–1269 | 1263
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
Paper
Fig. 1 Effect of carnosol on human tumor cell lines. (a) Human cancer cell lines were treated with the indicated doses of carnosol (mM) for 24 h (black column), 48 h (grey column), and 72 h (white column). Control cells (CTR) were treated with vehicle (DMSO). The MTT test was used to measure cell viability. ***p < 0.001 compared with the untreated group. (b) Human normal dermal fibroblasts (NHDF) were treated with carnosol as indicated for 24 h (black column), 48 h (grey column), and 72 h (white column). (c) SKOV-3 cells were treated with carnosol at the concentration of 70 mM with or without pre-treatment of 50 mM ZVAD-fmk for 30 min prior to exposure to carnosol for 24 h. After treatment, whole lysates were probed with specific antibodies as described. (d) Effect of carnosol treatment on anchorage-independent growth of MCF-7, MDA-231, and SKOV-3 cells. Representative images (top panel) of MCF-7 cells maintained in a suspension and treated with carnosol at the indicated concentrations (4 magnification). Cell viability (bottom panel) after carnosol treatment was assessed by MTT assay. (e) Carnosol reduces cancer cell adhesion on fibronectin-coated plates. Human cancer cells were harvested and pretreated with carnosol for 1 h as indicated and then seeded onto 10 mg ml 1 fibronectin-coated 96-well plates. Top images show cell spreading on the fibronectin-coated surface; bottom image is the quantitative data based on at least three independent experiments. Columns in the plot are the mean of three independent experiments expressed as percentage comparing with the control without treatment.
3.2 Carnosol inhibits the EGF-induced EMT in human ovarian cancer cells The ability of cancer cells to undergo invasion and metastasis is regulated through the activation of a particular cellular program named EMT. During EMT, epithelial cells lose their compact organization, acquire a spindle shaped morphology and transform themselves in a more motile and invasive counterpart acquiring migratory behaviour.23–25 The identication of factors affecting this cellular program could be a logical approach for preventing spreading of tumor cells. The extracellular signal induced by the epidermal growth factor (EGF) is strongly implicated in EMT.25 To test whether carnosol inhibits EGFinduced EMT, cancer cells were isolated from the ascitic uid of
1264 | Food Funct., 2014, 5, 1261–1269
a patient with ovarian cancer and maintained in culture under normal growth conditions up to two passages (Fig. 2aI). RT-PCR analysis conrmed that these cells were positive for the expression of the epithelial markers cytokeratins 8, 18 and 19 (Fig. 2b). To induce the transition to a mesenchymal phenotype, cells were treated with 100 ng ml 1 of EGF for 48 h. As shown in Fig. 2aIII, EGF-treated cells exhibited typical EMT morphological changes including an elongated morphology, an increased expression of the mesenchymal markers Vimentin, Snail, and N-cadherin, with the concomitant down-regulation of the epithelial marker E-cadherin (Fig. 2c). In contrast, pre-treatment with 50 mM carnosol for 6 h followed by the addition of EGF resulted in the inhibition of the EGF-induced EMT program. Indeed, cells treated with carnosol maintained a
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Paper
Carnosol inhibits EGF-induced EMT in ascites cells isolated from a patient with ovarian cancer. (a) A phase contrast microscope image of ascites cells isolated from a patient with ovarian adenocarcinoma (magnification 10). Control cells were cobblestone shaped, demonstrating an epithelial-like morphology (I). Ascites cells were treated with EGF with (II) or without pre-treatment with carnosol (III). (b) mRNA expression of cytokeratins 8, 18, and 19 in ascites tumor cells. (c and d) Protein and mRNA expression of epithelial/mesenchymal markers in ascites tumor cells treated with carnosol and EGF as indicated.
Fig. 2
typical epithelial morphology (Fig. 2aII) and no differences were observed in the expression of epithelial and mesenchymal markers between control and carnosol-plus EGF-treated cells (Fig. 2c and d). Moreover, pre-treatment with carnosol decreased phosphorylation of ERK suggesting that carnosol may inhibit EGF-induced EMT through an ERK-related signaling pathway (Fig. 2d). The induction of the MET (mesenchymal to epithelial transition) phenotype in aggressive tumor cells is associated with a reduction of their aggressive behaviour.26 We evaluated whether carnosol induces MET in the mesenchymal breast cancer cell line MDA-231, which we have previously characterized for the expression of epithelial and mesenchymal markers.26 However, aer treatment of MDA-231 cells with carnosol at the concentrations of 25 mM or 50 mM up to 7 days we did not observe the reversion to an epithelial morphology (data not shown). 3.3
Combination treatment of carnosol and phytochemicals
Several studies have clearly shown that specic combinations of phytochemicals can be more effective in cancer protection than single compounds. The actions of phytochemicals administered as dietary supplements alone do not explain completely the observed health benets of a complete diet. For this reason, we combined carnosol with other plant nutrients and evaluated their
This journal is © The Royal Society of Chemistry 2014
Food & Function
potential synergy. As shown in Fig. 3a, these combinations were associated with a signicant difference in efficacy between single agents and combinations mainly at higher concentrations of each agent. This result is well illustrated, for instance, for capsaicin, quercetin, and rosmarinic acid that show a very limited anticancer activity when administered alone but a stronger effect when combined with carnosol (Fig. 3a). When phytochemical combinations were statistically assessed, the addition of curcumin to carnosol was synergistic in SKOV-3 cells (CI value at ED50: 0.69; CI value at ED75: 0.73; CI value at ED90: 0.78; CI value at ED95: 0.81) (ESI† Fig. 1), whereas other combinations did not produce a synergistic interaction. The treatment in combination with carnosol and curcumin was also synergistic in reducing tumor cell attachment on bronectin in comparison with the compounds administered individually (Fig. 3b). To investigate whether the possible mechanisms underlying the observed synergy resulted from the inhibition of distinct signalling pathways, the phosphorylation status of different kinases and cell cycle regulatory proteins was investigated by western blotting. In MDA-231 and SKOV-3 cells, we observed that the treatment with curcumin and carnosol alone inhibited STAT3 and Akt phosphorylation, respectively. Combination treatment did not induce an additive inhibition of these two kinases in either cell types (Fig. 3c). Western blot data also showed that exposure of cells to the combination reduced Cyclin D1, Bcl2, and Survivin and increased p27 levels better than single treatment. These results suggest that carnosol and curcumin act together to reduce the cell cycle and anti-apoptosis protein expression by acting on different signalling pathways. Treatment with a 1 : 2 drug combination (curcumin : carnosol) was tested in a panel of primary tumor cells isolated from patients with lung, ovarian and endometrial cancer and associated with a greater reduction of cell viability in all the tumor cell lines used, when compared with carnosol and curcumin given as single treatment (Fig. 4a). It is noteworthy that the drug concentrations of carnosol and curcumin used for these experiments did not affect the viability of normal human broblasts (Fig. 4b). The failure to respond to a specic therapy is the main obstacle to successful treatment of cancer. The combination of carnosol and curcumin demonstrated to be effective in reducing cancer cell viability also in primary tumor cells. We intend now to (i) explore the potential use of these phytochemicals in cancer cell lines that are resistant to standard chemotherapy; (ii) investigate potential combinations between carnosol, curcumin and chemotherapeutics. These results may have implications in future preclinical and clinical study. In our study, cancer cells were isolated from chemotherapyna¨ıve patients and for this reason no information was available about the sensitivity of these cells to standard treatments. The only exception was the cell line named as LuCA (Lung CAncer), which was, in fact, isolated from the pleural uid of a patient with lung adenocarcinoma who was treated with and became refractory to Erlotinib, an EGFR inhibitor. We rst tested the in vitro resistance of our isolate to Erlotinib and we conrmed that these cells still continued to be resistant to this drug (data not shown). Then, we tested the effects of carnosol, curcumin or
Food Funct., 2014, 5, 1261–1269 | 1265
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
Paper
Combination treatment of carnosol and phytochemicals inhibits the growth of human cancer cell lines. (a) SKOV-3 cells were treated with different concentrations of capsaicin, curcumin, quercetin, and rosmarinic acid as indicated, alone or in combination with carnosol at the concentration of 50 mM for 24 h, 48 h, and 72 h. Control cells (CTR) were treated with vehicle (DMSO). (b) Adhesion of SKOV3 cells to fibronectin was reduced after treatment with carnosol, curcumin, and their combination. **p < 0.01, ***p < 0.001 compared with treated and untreated groups. (c) SKOV-3 and MDA-231 cells were treated with carnosol, curcumin alone or in combination for 4 h. After treatment, whole lysates were probed with specific antibodies as described.
Fig. 3
Fig. 4 (a) Effects of carnosol 50 mM, curcumin 25 mM and both drugs in combination (ratio curcumin : carnosol 1 : 2) on the growth of primary human cancer cells after 48 h of treatment. Control cells were treated with vehicle. LuCa, Lung Cancer isolate; OvCa, Ovarian Cancer isolate; EnCa, Endometrial Cancer isolate. (b) Human fibroblasts were treated with carnosol 50 mM and curcumin 12.5 mM for 24 h, 48 h, and 72 h. Control cells (CTR) were treated with vehicle (DMSO).
combination of both compounds towards these cells. Our results showed that this Erlotinib resistant isolate was sensitive to these phytochemicals (Fig. 4a). These results may have implications in the therapy of patients who are refractory to the therapy with Erlotinib. 3.4 Combination of carnosol and curcumin with chemotherapeutic drugs The effects of carnosol and curcumin alone or in combination with chemotherapeutic drugs were evaluated by the MTT test
1266 | Food Funct., 2014, 5, 1261–1269
(Fig. 5a). The drugs used are multikinase inhibitors directed towards specic intracellular targets including EGFR and VEGFR (vandetanib), c-MET (crizotinib), EGFR (erlotinib), and VEGFR (axitinib). Of special note is that, the effects on cell viability of carnosol or curcumin administered alone with the drugs were not signicantly different from the effects displayed by the drugs individually. In contrast, the combination of carnosol and curcumin increased the anti-proliferative effects of these chemotherapeutics (Fig. 5a). Among the tested combination, carnosol + curcumin + vandetanib was particularly
This journal is © The Royal Society of Chemistry 2014
View Article Online
Food & Function
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Paper
Fig. 5 Carnosol and curcumin reduced cell viability in combination with chemotherapeutic drugs. (a) SKOV3 cells were treated with carnosol, curcumin, vandetanib, erlotinib, axitinib, and crizotinib for 48 h as indicated, cell viability was then determined by the MTT test. (b) Representative images of SKOV3 cells treated with carnosol, curcumin, and vandetanib alone or in combination. Images were taken with an Olympus IX51 inverted microscope (10 magnification) after 48 h of treatment. For every treatment, 5 random fields of vision were acquired. (c) Western blot analysis on whole-cell lysates of SKOV3 cells treated for 24 h with carnosol, curcumin, and vandetanib alone or in combination as indicated. Western blot data are presented as a relative expression of selected proteins compared to tubulin or total Akt and total Erk and normalised to control. Error bars indicate the standard deviation of three independent experiments. **p < 0.01, ***p < 0.001 compared with the untreated group.
efficient in reducing cell viability (Fig. 5 a and b). In order to investigate the mechanism underlying the synergistic cytotoxicity in SKOV-3 cells, we next investigated changes in the expression of proteins involved in cell cycle regulation, apoptosis and cell proliferation. As shown in Fig. 5c, the combination reduced Cyclin D1, Bcl2, phospho-ERK1/2, phospho-Akt, and phospho-STAT3 expression. By contrast, b-catenin levels were unchanged aer treatment. Treatment of SKOV3 cells with carnosol for 24 h reduced phosphorylation of Akt but had no effects on STAT3. Conversely, treatment with curcumin reduced phosphorylation of STAT3. Both molecules reduced the
This journal is © The Royal Society of Chemistry 2014
cyclin D1 level and reduced phosphorylation of ERK1/2. Treatment with vandetanib reduced the phosphorylation status of ERK1/2 but increased p-Akt and p-STAT3 levels. Taken together, these results support at the biochemical level the enhanced efficacy in reducing cell viability.
4. Conclusions Cancer metastasis is commonly dened as the diffusion of cancer cells from the primary tumor site to distal organs where secondary tumors start to be formed. Metastasis is a complex
Food Funct., 2014, 5, 1261–1269 | 1267
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
multistep process beginning with the entrance of primary tumor cells into the vasculature, migration to distal organs, adhesion to endothelial cells and extravasation into the underlying tissue. Scientic evidence that natural compounds may have a potential inhibitory effect on cancer invasion and metastasis is recently increasing. Curcumin, resveratrol, and their related derivatives are the most studied compounds in this context. In this study, we demonstrated that carnosol, a dietary diterpene isolated from herbs such as rosemary, sage and oregano, displays potent antimetastatic effects in various cancer cells and primary tumors cells. Carnosol was able to inhibit cancer cell viability, cellular adhesion on bronectin and inhibits the EGF-induced EMT program. Moreover, we observed that cancer cells maintained in a suspension showed greater sensitivity to carnosol, this result may have particular importance for the development of strategies aimed at targeting circulating tumor cells with natural substances. Carnosol shows enhanced anti-proliferative activity when combined with other dietary compounds, an aspect of great relevance considering that the health benets of food are mainly due to the synergy of a variety of nutrients. In particular, the combination of carnosol and curcumin found a signicant effect also on primary human cancer cells isolated from patients with metastatic cancers. Our results indicate that inhibition of different signalling pathways may account for the observed synergy. Carnosol acts on Akt whereas curcumin reduces STAT3 phosphorylation. The combination of these two molecules with a drug, vandetanib, that reduces signicantly the phosphorylation of another kinase, ERK1/2, resulted in a greater reduction of cell viability. This is a classical mechanism of drug synergy in which molecules with different cellular targets can be combined together to switch off multiple kinases. In conclusion, results from the present study provide new evidence about the anticancer activity of carnosol. As also already performed with curcumin,27 the development of structural carnosol analogues could be a strategy for designing more effective compounds, improving carnosol efficacy/stability and translational potential.
Acknowledgements We thank Dr Antonio Danieli and Dr Raffaele Acierno for their technical support. This work was supported by the PS105 ARTI strategic project “Development and realization of bio-chip for molecular diagnostic and typization of human pathogenic viruses (HPV, HCV)” of Apulia Region, by the PON project 254/ Ric. “Implementation of human and environment health research center” cod. PONa3_00334, and by the Prin project n. 2010FPTBSH_006 "NANO Molecular Technologies for Drug delivery - NANOMED".
References 1 J. Salas-Salvado, M. A. Martinez-Gonzalez, M. Bullo and E. Ros, Nutr., Metab. Cardiovasc. Dis., 2011, 21(suppl. 2), B32–B48.
1268 | Food Funct., 2014, 5, 1261–1269
Paper
2 F. So, R. Abbate, G. F. Gensini and A. Casini, Am. J. Clin. Nutr., 2010, 92, 1189–1196. 3 L. J. Dominguez, M. Bes-Rastrollo, C. de la Fuente-Arrillaga, E. Toledo, J. J. Beunza, M. Barbagallo and M. A. MartinezGonzalez, Nutr., Metab. Cardiovasc. Dis., 2013, 23, 451– 458. 4 D. Vergara, P. Simeone, D. Toraldo, P. Del Boccio, V. Vergaro, S. Leporatti, D. Pieragostino, A. Tinelli, S. De Domenico, S. Alberti, A. Urbani, M. Salzet, A. Santino and M. Maffia, Mol. Biosyst., 2012, 8, 1078–1087. 5 D. Vergara, C. M. Valente, A. Tinelli, C. Siciliano, V. Lorusso, R. Acierno, G. Giovinazzo, A. Santino, C. Storelli and M. Maffia, Cancer Lett., 2011, 310, 1–8. 6 Y. J. Surh, Nat. Rev. Cancer, 2003, 3, 768–780. 7 C. Fortes, F. Forastiere, S. Farchi, S. Mallone, T. Trequattrinni, F. Anatra, G. Schmid and C. A. Perucci, Nutr. Cancer, 2003, 46, 30–37. 8 E. Vagi, E. Rapavi, M. Hadolin, K. Vasarhelyine Peredi, A. Balazs, A. Blazovics and B. Simandi, J. Agric. Food Chem., 2005, 53, 17–21. 9 M. J. del Bano, J. Lorente, J. Castillo, O. Benavente-Garcia, J. A. del Rio, A. Ortuno, K. W. Quirin and D. Gerard, J. Agric. Food Chem., 2003, 51, 4247–4253. 10 N. Bai, K. He, M. Roller, C. S. Lai, X. Shao, M. H. Pan and C. T. Ho, J. Agric. Food Chem., 2010, 58, 5363–5367. 11 J. Bauer, S. Kuehnl, J. M. Rollinger, O. Scherer, H. Northoff, H. Stuppner, O. Werz and A. Koeberle, J. Pharmacol. Exp. Ther., 2012, 342, 169–176. 12 E. S. Mengoni, G. Vichera, L. A. Rigano, M. L. RodriguezPuebla, S. R. Galliano, E. E. Cafferata, O. H. Pivetta, S. Moreno and A. A. Vojnov, Fitoterapia, 2011, 82, 414– 421. 13 A. Mohebati, J. B. Guttenplan, A. Kochhar, Z. L. Zhao, W. Kosinska, K. Subbaramaiah and A. J. Dannenberg, Cancer Prev. Res., 2012, 5, 593–602. 14 E. A. Offord, K. Mace, C. Ruffieux, A. Malnoe and A. M. Pfeifer, Carcinogenesis, 1995, 16, 2057–2062. 15 D. Martin, A. I. Rojo, M. Salinas, R. Diaz, G. Gallardo, J. Alam, C. M. De Galarreta and A. Cuadrado, J. Biol. Chem., 2004, 279, 8919–8929. 16 K. Singletary, C. MacDonald and M. Wallig, Cancer Lett., 1996, 104, 43–48. 17 M. T. Huang, C. T. Ho, Z. Y. Wang, T. Ferraro, Y. R. Lou, K. Stauber, W. Ma, C. Georgiadis, J. D. Laskin and A. H. Conney, Cancer Res., 1994, 54, 701–708. 18 J. M. Visanji, D. G. Thompson and P. J. Padeld, Cancer Lett., 2006, 237, 130–136. 19 J. Dorrie, K. Sapala and S. J. Zunino, Cancer Lett., 2001, 170, 33–39. 20 M. Wintzell, E. Hjerpe, E. Avall Lundqvist and M. Shoshan, BMC Cancer, 2012, 12, 359. 21 T. C. Chou, Pharmacol. Rev., 2006, 58, 621–681. 22 M. Sebbagh, C. Renvoize, J. Hamelin, N. Riche, J. Bertoglio and J. Breard, Nat. Cell Biol., 2001, 3, 346–352. 23 D. C. Radisky, J. Cell Sci., 2005, 118, 4325–4326. 24 J. P. Thiery and J. P. Sleeman, Nat. Rev. Mol. Cell Biol., 2006, 7, 131–142.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
G. Giannelli, P. Sacchetta and M. Maffia, Mol. Biosyst., 2013, 9, 1127–1138. 27 T. Esatbeyoglu, P. Huebbe, I. M. Ernst, D. Chin, A. E. Wagner and G. Rimbach, Angew. Chem., Int. Ed., 2012, 51, 5308–5332.
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
25 D. Vergara, B. Merlot, J. P. Lucot, P. Collinet, D. Vinatier, I. Fournier and M. Salzet, Cancer Lett., 2010, 291, 59–66. 26 D. Vergara, P. Simeone, P. del Boccio, C. Toto, D. Pieragostino, A. Tinelli, R. Acierno, S. Alberti, M. Salzet,
Food & Function
This journal is © The Royal Society of Chemistry 2014
Food Funct., 2014, 5, 1261–1269 | 1269
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
PAPER
View Journal | View Issue
Antitumor activity of the dietary diterpene carnosol against a panel of human cancer cell lines† Cite this: Food Funct., 2014, 5, 1261
Daniele Vergara,ab Pasquale Simeone,cd Simona Bettini,e Andrea Tinelli,f Ludovico Valli,e Carlo Storelli,a Silvana Leo,g Angelo Santino‡h and Michele Maffia‡*ab Dietary phytochemicals found in vegetables and fruits consist of a wide variety of biologically active compounds with anti-carcinogenic activity. The aim of this study was to evaluate the antigrowth activity of carnosol, a dietary diterpene, as a single agent or in combination with other dietary phytochemicals or chemotherapeutic drugs against a panel of tumor cell lines. Carnosol decreased cell viability in human breast, ovarian, and intestinal tumor cell lines, and inhibited cancer cell adhesion on fibronectin and growth of cancer cells in suspension. Carnosol also inhibited EGF-induced epithelial mesenchymal transition in ovarian cancer cells. The combination treatment with other dietary phytochemicals increased the anti-proliferative activity of carnosol. The combination with curcumin resulted in a synergistic reduction of vitality in SKOV-3 and MDA-231 cells and potently inhibited viability of primary
Received 14th January 2014 Accepted 9th March 2014
cancer cells isolated from the pleural fluid or ascites of patients with metastatic cancers. These results
DOI: 10.1039/c4fo00023d
provide additional evidence about the anticancer role of carnosol and its potential in blocking the
www.rsc.org/foodfunction
growth of tumor cells.
1. Introduction The Mediterranean diet has long been reported to be protective against the occurrence of several different diseases. The health effects of such a dietary prole have been recognized in many different studies and associated with a reduced risk for chronic diseases and some types of cancer.1–3 Multiple mechanisms have been proposed to explain the benecial effects of the Mediterranean diet. In principle, the Mediterranean diet plan involves a high consumption of fruits, vegetables, legumes, sh, olive oil and a moderate amount of red wine during meals, limiting the intake of red meat and avoiding processed meat. The active components from some of
a
Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, Via per Monteroni, 73100, Lecce, Italy. E-mail: michele.maffi[email protected]; Fax: +39 0832 324200
b
Laboratory of Clinical Proteomics, “Giovanni Paolo II” Hospital, ASL-Lecce, Italy
Unit of Cancer Pathology, CeSI, Foundation University “G. d'Annunzio”, ChietiPescara, Italy
c
Department of Neuroscience, Imaging and Clinical Sciences, University “G. d'Annunzio”, Chieti-Pescara, Italy
d
e
Department of Engineering for Innovation, University of Salento, Via per Monteroni, 73100, Lecce, Italy
f
Department of Obstetrics and Gynaecology, Vito Fazzi Hospital, Lecce, Italy
g
Department of Geriatric Oncology, Vito Fazzi Hospital, Lecce, Italy
h
Institute of Science of Food Production, CNR, Unit of Lecce, Via per Monteroni, 73100, Lecce, Italy † Electronic supplementary 10.1039/c4fo00023d
information
(ESI)
‡ Equal contribution.
This journal is © The Royal Society of Chemistry 2014
available.
See
DOI:
these foods were characterised in a large number of in vitro and animal studies for their ability to target specic cellular molecules including various signal transduction pathways and proteins involved in cell cycle progression.4–6 One example of plant-derived compounds currently under investigation for their health benets is Mediterranean herbs7 such as rosemary, basil, sage, marjoram and oregano. They represent a rich source of a specic class of molecules known as diterpenes. Carnosol and carnosic acid are the two major dietary diterpenes present in herbal extracts where they are found in considerable quantities.8–10 The possible benecial effects of these two compounds against inammation and cancer prevention were described in vitro and in vivo.11–19 Carnosol and carnosic acid are able to inhibit prostaglandin PGE(2) formation by selectively targeting microsomal PGE(2) synthase (mPGES-1).11 Both phytochemicals also reduced the expression of pro-inammatory cytokines interleukin-1 beta (IL-1b) and tumor necrosis factor alpha (TNF-a) and selectively inhibited cyclooxygenase 2 (COX-2) in vivo.12 In vitro treatment with carnosol of cell lines derived from oral leukoplakia (MSK-Leuk1), skin (HaCaT) and human bronchial epithelium (BEAS-2B) resulted in decrease of benzo[a]pyrene (B[a]P) mediated induction of cytochrome P450 (CYP) 1A1 and CYP1B1, induction of glutathione-S-transferase and inhibition of mutagenesis.13,14 Carnosol also induced the phase II enzyme heme oxygenase-1 (HO-1) through a pathway that involves phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt).15 Singletary and coworkers found that dietary administration of rosemary
Food Funct., 2014, 5, 1261–1269 | 1261
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
extract for two weeks can prevent 7,12-dimethylbenz[a] anthracene (DMBA)-induced DNA damage and tumor formation in the rat mammary gland.16 The inhibitory effect of topically applied carnosol on tumor promotion by 12-Otetradecanoylphorbol-13-acetate (TPA) was also described.17 Carnosol and carnosic acid were also reported to inhibit cell growth and proliferation of tumor cells by arresting the cell cycle at the G2/M phase18 and inducing apoptosis in B-lineage leukaemia cells through the down-regulation of the antiapoptotic protein Bcl-2.19 In spite of increasing interest in the anticancer activity of carnosol during initiation and promotion phases of cancer development, few studies have proved the efficacy of diterpenoids in monotherapy or when paired with other phytochemicals or chemotherapeutics. To achieve this, we used a panel of human cancer cell lines and primary tumor-derived cell lines to demonstrate the ability of carnosol to inhibit cell viability, cell adhesion, epithelial to mesenchymal transition (EMT) and to induce anoikis. We further demonstrated that carnosol can be combined with other phytochemicals or commonly used chemotherapeutic drugs in order to increase their antiproliferative properties.
2. 2.1
Materials and methods Chemicals
Capsaicin, carnosol, curcumin, piperin, quercetin, resveratrol and rosmarinic acid were purchased from Sigma-Aldrich. Capsaicin, carnosol, curcumin, quercetin, and rosmarinic acid were all dissolved in dimethyl sulfoxide (DMSO), and resveratrol was dissolved in ethanol 70% and immediately used. The following reagents were obtained from GE Healthcare: Hybond ECL membrane and ECL western blotting detection reagents. Fibronectin was from BD Biosciences. Polyhydroxyethylmethacrylate (Poli-HEMA) was from Santa Cruz. ZVAD-fmk was from Abcam. Primary antibodies against Bcl-2 (sc-492), Colin (sc-33779), Colin (Ser3) (sc-21867-R), Cyclin D1 (sc-20044), E-cadherin (sc-71009), N-cadherin (sc-59987), p27 (sc-527), Rock (sc-5560), Survivin (sc-8808), b-catenin (sc-1496), a-Tubulin (sc-23948), and secondary antibodies (HRP-conjugated) were all from Santa Cruz. Anti-extracellular-signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204) (4695), anti-Myosin light chain 2 (Ser19) (3671), anti-STAT3 (Tyr705) (9138), and anti-Akt (Ser473) (4051) were from Cell Signaling. All other reagents were from standard commercial sources and were of the highest grade available. 2.2
Cell cultures and ascites-derived tumor cells
Human tumor cells were purchased from the American Type Culture Collection (ATCC) or from Banca Biologica and Cell Factory (IRCCS Azienda Ospedaliera Universitaria San MartinoIST Istituto. Nazionale per la ricerca sul cancro, Genova, Italy). Normal Human Dermal Fibroblasts (NHDF) were kindly provided by Prof. T. Cocco and Dr A. Gaballo. The human ovarian cancer cell lines OVCAR-3 and SKOV-3, the colorectal adenocarcinoma cell line Caco-2, and the human
1262 | Food Funct., 2014, 5, 1261–1269
Paper
breast cancer cell lines HBL-100, MCF-7, MDA-231, MDA-361 and MDA-435 were all cultured in DMEM medium supplemented with 10% FBS, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin at 37 C in an atmosphere of 5% CO2. The cells were kept in an exponential growth phase during experiments. All research studies involving human subjects were approved by the institutional review board (Human Bioethic Center of the University of Salento and “Vito Fazzi” Hospital). All patients provided written informed consent. Ascites were collected from ve chemotherapy-na¨ıve ovarian cancer patients, who ranged in age from 51 to 72 years, diagnosed with serous adenocarcinoma and presented as stage III (FIGO classication) at the time of clinical intervention. Primary human normal broblasts were isolated from omental tissues of patients undergoing surgery for benign ovarian cysts as described in ref. 20. Epithelial cells were veried by cytokeratin 8-, 18- and 19-expression, whereas broblasts were veried by vimentin positive but cytokeratin 8-negative expression. Lung cancer cells were isolated from the pleural uid of a patient with non-small cell lung cancer maintained in DMEM with 20% FBS. Endometrial cancer cells were isolated from the peritoneal washing of endometrial cancer patients and maintained in DMEM with 20% FBS. Briey, ascites or pleural uids (2–3 liters) were transferred to 50 ml Falcon tubes and centrifuged at 1200 rpm for 10 minutes to pellet the cells. The nal cell pellets were washed twice with a phosphate-buffered saline (PBS) solution, pooled, and resuspended in normal growth medium. All primary cells isolated for this study were used aer two passages. 2.3
Cell viability assay
Cells were seeded at a density of 5 103 cells per well in a 96-well plate containing 100 ml of complete medium and allowed to adhere to the plate overnight. For determining cell viability, the MTT assay was used. Aer treatment, the culture medium was aspirated and 100 ml of RPMI-phenol free medium containing 10 ml of MTT stock solution, 5 mg ml 1 in PBS solution, was added. Two hours later, 100 ml DMSO was added to each well to dissolve the resulting formazan crystals. Absorbance of the converted dye was measured at a wavelength of 570 nm. The relative cell viability was expressed as a percentage of the untreated control wells. 2.4
Cell adhesion assay
Cells were treated with carnosol, maintained in a suspension for 1 hour, and adhered onto 96-well plates coated with bronectin (10 mg ml 1) for 1 hour at 37 C. Briey, non-adherent cells were removed by gentle washing with PBS. Attached cells were xed with 70% methanol for 10 minutes. Cells were stained with 0.02% crystal violet in 0.2% ethanol solution. The incorporated dye was dissolved in 10% acetic acid, and the absorbance was measured at 560 nm. Each experiment was done in triplicate. 2.5
Adhesion-independent cell survival (anoikis assay)
12-well cell culture dishes were coated with 500 ml of poly-HEMA (12 mg ml 1 in 95% ethanol). The solution was le to dry for
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Paper
Food & Function
24 h at room temperature. Cells were then seeded in the polyHEMA-coated wells and cultured in a medium supplemented with 10% serum, with or without natural compounds for the indicated period of time. At the end of the indicated culture period the cells in the suspension were collected, treated with trypsin or disaggregated by vigorous pipetting to separate single cells from aggregated cells, resuspended in complete DMEM, seeded in 96-well plates and cultured overnight. Cell viability was determined by MTT. In another way, suspended cells were collected by aspiration and the percentage of viable cells was determined immediately aer disaggregation by trypan blue staining. 2.6
Reverse transcriptase-PCR (RT-PCR)
RT-PCR was performed as described in ref. 4. Briey, total cellular RNA was isolated using an Illustra TriplePrep extraction kit following manufacturer's instruction and reverse transcribed into cDNA using a High Capacity RNA-to-cDNA Master Mix (Applied Biosystem). PCR was carried out using the following conditions: denaturation at 95 C for 30 s, annealing at 60 C for 30 s, and extension at 72 C for 45 s. PCR samples were loaded onto a 1.2% agarose gel containing ethidium bromide and analysed. The primers used for PCR amplication were designed using the Primer blast program and listed elsewhere.5 2.7
Western blotting analysis
Whole proteins were extracted in RIPA buffer (Cell Signaling) and quantied by the Bradford protein assay (BIORAD). Samples were separated by 12% SDS-PAGE and transferred to the Hybond ECL nitrocellulose membranes. The membranes were blocked overnight in Blotto A (Santa Cruz) at 4 C, and subsequently probed by the appropriately diluted primary antibodies for 2 h at room temperature as previously described.4 2.8
Statistical analysis
Data were analyzed with GraphPad Prism statistical soware. Experiments were carried out three times and statistical evaluation was carried out using Student's t-test. Data are presented as mean values standard deviation. P values 1.2 indicates antagonism.
3.
Results and discussion
3.1 Effects of carnosol on viability of human cancer cells under monolayer and suspension conditions In the preliminary stage of investigation, we tested the sensitivity of different human tumor cell lines to different concentrations of carnosol. Two different cell culture conditions (cells grown as a monolayer or maintained in a suspension) were
This journal is © The Royal Society of Chemistry 2014
used. The experiment was conducted using human breast cancer models (HBL-100, MDA-231, MDA-361, MDA-435, MCF7), human ovarian cancer models (OVCAR-3, SKOV-3), and the human colon cancer cell line CACO-2. Results from MTT assay indicated that the viability of all tested tumor cell lines under monolayer culture conditions was signicantly inhibited by carnosol (Fig. 1a). Treatment of these human cancer models at different time points and different carnosol concentrations resulted in a dose- and time-dependent inhibition of cell growth with an IC50 (the concentration causing 50% cell death) greater than 50 mM. As shown in Fig. 1a, at the concentrations of 100 mM and 200 mM, cell viability was completely abrogated in all cell lines tested while no effect on cell viability was observed at concentrations lower than 25 mM. To test whether the concentrations of carnosol used to inhibit the growth of cancer cells were also toxic for normal cells, we investigated the effects of carnosol on the viability of the normal dermal broblast cell line NHDF. Carnosol induced a modest reduction of cell viability only at the concentration of 200 mM (Fig. 1b). This implies a selective effect of carnosol on tumor cells. Previous studies have suggested that carnosol may have antiproliferative and apoptotic activities.19 Here, we have shown that exposure of SKOV-3 cells to a concentration of carnosol of 70 mM for 24 h reduced the phosphorylation state of ERK 1/2 and Akt. No difference in STAT3 expression was found (Fig. 1c). Moreover, carnosol treatment reduced Cyclin D1 expression and induced the cleavage of ROCK1 with a concomitant increase in the phosphorylation of Cof and Mlc2.22 Pre-incubation with caspase inhibitors z-VAD-fmk abolished ROCK cleavage, Cof and Mlc2 phosphorylation, thus suggesting that the molecule had an apoptotic effect on cultured human tumor cells. Anchorage-independent growth is a hallmark in the metastatic cascade. In fact, following intravasation, circulating cells are deprived of ECM interactions. The ability of tumor cells to survive independent of anchorage is an essential prerequisite for the metastatic spread of cancer. For this reason, we were interested in studying the effects of carnosol on human cancer cells cultured in a suspension. With this aim, we examined the effects of carnosol on the viability of cancer cells cultured in poly-HEMA coated multiwell plates, in which the adherence of cells to the well is prevented. As shown in Fig. 1d, when placed in suspension culture, tumor cells tend to form large and compact aggregates in the absence of a matrix attachment. Control and carnosol-treated cells were maintained in the suspension for 48 h before being harvested for further analyses. Cell viability was then determined by the MTT test. Carnosol inhibited the viability of all cancer cell lines investigated in a concentration-dependent manner. As cell adhesion is one of the essential steps involved in cancer metastasis, we investigated the effect of carnosol on cancer cell adhesion to bronectin, a major extracellular matrix (ECM) component. As shown in Fig. 1e, carnosol was able to suppress dose dependently the adhesion of all the four different cancer cell lines used in this experiment to bronectin. Food Funct., 2014, 5, 1261–1269 | 1263
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
Paper
Fig. 1 Effect of carnosol on human tumor cell lines. (a) Human cancer cell lines were treated with the indicated doses of carnosol (mM) for 24 h (black column), 48 h (grey column), and 72 h (white column). Control cells (CTR) were treated with vehicle (DMSO). The MTT test was used to measure cell viability. ***p < 0.001 compared with the untreated group. (b) Human normal dermal fibroblasts (NHDF) were treated with carnosol as indicated for 24 h (black column), 48 h (grey column), and 72 h (white column). (c) SKOV-3 cells were treated with carnosol at the concentration of 70 mM with or without pre-treatment of 50 mM ZVAD-fmk for 30 min prior to exposure to carnosol for 24 h. After treatment, whole lysates were probed with specific antibodies as described. (d) Effect of carnosol treatment on anchorage-independent growth of MCF-7, MDA-231, and SKOV-3 cells. Representative images (top panel) of MCF-7 cells maintained in a suspension and treated with carnosol at the indicated concentrations (4 magnification). Cell viability (bottom panel) after carnosol treatment was assessed by MTT assay. (e) Carnosol reduces cancer cell adhesion on fibronectin-coated plates. Human cancer cells were harvested and pretreated with carnosol for 1 h as indicated and then seeded onto 10 mg ml 1 fibronectin-coated 96-well plates. Top images show cell spreading on the fibronectin-coated surface; bottom image is the quantitative data based on at least three independent experiments. Columns in the plot are the mean of three independent experiments expressed as percentage comparing with the control without treatment.
3.2 Carnosol inhibits the EGF-induced EMT in human ovarian cancer cells The ability of cancer cells to undergo invasion and metastasis is regulated through the activation of a particular cellular program named EMT. During EMT, epithelial cells lose their compact organization, acquire a spindle shaped morphology and transform themselves in a more motile and invasive counterpart acquiring migratory behaviour.23–25 The identication of factors affecting this cellular program could be a logical approach for preventing spreading of tumor cells. The extracellular signal induced by the epidermal growth factor (EGF) is strongly implicated in EMT.25 To test whether carnosol inhibits EGFinduced EMT, cancer cells were isolated from the ascitic uid of
1264 | Food Funct., 2014, 5, 1261–1269
a patient with ovarian cancer and maintained in culture under normal growth conditions up to two passages (Fig. 2aI). RT-PCR analysis conrmed that these cells were positive for the expression of the epithelial markers cytokeratins 8, 18 and 19 (Fig. 2b). To induce the transition to a mesenchymal phenotype, cells were treated with 100 ng ml 1 of EGF for 48 h. As shown in Fig. 2aIII, EGF-treated cells exhibited typical EMT morphological changes including an elongated morphology, an increased expression of the mesenchymal markers Vimentin, Snail, and N-cadherin, with the concomitant down-regulation of the epithelial marker E-cadherin (Fig. 2c). In contrast, pre-treatment with 50 mM carnosol for 6 h followed by the addition of EGF resulted in the inhibition of the EGF-induced EMT program. Indeed, cells treated with carnosol maintained a
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Paper
Carnosol inhibits EGF-induced EMT in ascites cells isolated from a patient with ovarian cancer. (a) A phase contrast microscope image of ascites cells isolated from a patient with ovarian adenocarcinoma (magnification 10). Control cells were cobblestone shaped, demonstrating an epithelial-like morphology (I). Ascites cells were treated with EGF with (II) or without pre-treatment with carnosol (III). (b) mRNA expression of cytokeratins 8, 18, and 19 in ascites tumor cells. (c and d) Protein and mRNA expression of epithelial/mesenchymal markers in ascites tumor cells treated with carnosol and EGF as indicated.
Fig. 2
typical epithelial morphology (Fig. 2aII) and no differences were observed in the expression of epithelial and mesenchymal markers between control and carnosol-plus EGF-treated cells (Fig. 2c and d). Moreover, pre-treatment with carnosol decreased phosphorylation of ERK suggesting that carnosol may inhibit EGF-induced EMT through an ERK-related signaling pathway (Fig. 2d). The induction of the MET (mesenchymal to epithelial transition) phenotype in aggressive tumor cells is associated with a reduction of their aggressive behaviour.26 We evaluated whether carnosol induces MET in the mesenchymal breast cancer cell line MDA-231, which we have previously characterized for the expression of epithelial and mesenchymal markers.26 However, aer treatment of MDA-231 cells with carnosol at the concentrations of 25 mM or 50 mM up to 7 days we did not observe the reversion to an epithelial morphology (data not shown). 3.3
Combination treatment of carnosol and phytochemicals
Several studies have clearly shown that specic combinations of phytochemicals can be more effective in cancer protection than single compounds. The actions of phytochemicals administered as dietary supplements alone do not explain completely the observed health benets of a complete diet. For this reason, we combined carnosol with other plant nutrients and evaluated their
This journal is © The Royal Society of Chemistry 2014
Food & Function
potential synergy. As shown in Fig. 3a, these combinations were associated with a signicant difference in efficacy between single agents and combinations mainly at higher concentrations of each agent. This result is well illustrated, for instance, for capsaicin, quercetin, and rosmarinic acid that show a very limited anticancer activity when administered alone but a stronger effect when combined with carnosol (Fig. 3a). When phytochemical combinations were statistically assessed, the addition of curcumin to carnosol was synergistic in SKOV-3 cells (CI value at ED50: 0.69; CI value at ED75: 0.73; CI value at ED90: 0.78; CI value at ED95: 0.81) (ESI† Fig. 1), whereas other combinations did not produce a synergistic interaction. The treatment in combination with carnosol and curcumin was also synergistic in reducing tumor cell attachment on bronectin in comparison with the compounds administered individually (Fig. 3b). To investigate whether the possible mechanisms underlying the observed synergy resulted from the inhibition of distinct signalling pathways, the phosphorylation status of different kinases and cell cycle regulatory proteins was investigated by western blotting. In MDA-231 and SKOV-3 cells, we observed that the treatment with curcumin and carnosol alone inhibited STAT3 and Akt phosphorylation, respectively. Combination treatment did not induce an additive inhibition of these two kinases in either cell types (Fig. 3c). Western blot data also showed that exposure of cells to the combination reduced Cyclin D1, Bcl2, and Survivin and increased p27 levels better than single treatment. These results suggest that carnosol and curcumin act together to reduce the cell cycle and anti-apoptosis protein expression by acting on different signalling pathways. Treatment with a 1 : 2 drug combination (curcumin : carnosol) was tested in a panel of primary tumor cells isolated from patients with lung, ovarian and endometrial cancer and associated with a greater reduction of cell viability in all the tumor cell lines used, when compared with carnosol and curcumin given as single treatment (Fig. 4a). It is noteworthy that the drug concentrations of carnosol and curcumin used for these experiments did not affect the viability of normal human broblasts (Fig. 4b). The failure to respond to a specic therapy is the main obstacle to successful treatment of cancer. The combination of carnosol and curcumin demonstrated to be effective in reducing cancer cell viability also in primary tumor cells. We intend now to (i) explore the potential use of these phytochemicals in cancer cell lines that are resistant to standard chemotherapy; (ii) investigate potential combinations between carnosol, curcumin and chemotherapeutics. These results may have implications in future preclinical and clinical study. In our study, cancer cells were isolated from chemotherapyna¨ıve patients and for this reason no information was available about the sensitivity of these cells to standard treatments. The only exception was the cell line named as LuCA (Lung CAncer), which was, in fact, isolated from the pleural uid of a patient with lung adenocarcinoma who was treated with and became refractory to Erlotinib, an EGFR inhibitor. We rst tested the in vitro resistance of our isolate to Erlotinib and we conrmed that these cells still continued to be resistant to this drug (data not shown). Then, we tested the effects of carnosol, curcumin or
Food Funct., 2014, 5, 1261–1269 | 1265
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
Paper
Combination treatment of carnosol and phytochemicals inhibits the growth of human cancer cell lines. (a) SKOV-3 cells were treated with different concentrations of capsaicin, curcumin, quercetin, and rosmarinic acid as indicated, alone or in combination with carnosol at the concentration of 50 mM for 24 h, 48 h, and 72 h. Control cells (CTR) were treated with vehicle (DMSO). (b) Adhesion of SKOV3 cells to fibronectin was reduced after treatment with carnosol, curcumin, and their combination. **p < 0.01, ***p < 0.001 compared with treated and untreated groups. (c) SKOV-3 and MDA-231 cells were treated with carnosol, curcumin alone or in combination for 4 h. After treatment, whole lysates were probed with specific antibodies as described.
Fig. 3
Fig. 4 (a) Effects of carnosol 50 mM, curcumin 25 mM and both drugs in combination (ratio curcumin : carnosol 1 : 2) on the growth of primary human cancer cells after 48 h of treatment. Control cells were treated with vehicle. LuCa, Lung Cancer isolate; OvCa, Ovarian Cancer isolate; EnCa, Endometrial Cancer isolate. (b) Human fibroblasts were treated with carnosol 50 mM and curcumin 12.5 mM for 24 h, 48 h, and 72 h. Control cells (CTR) were treated with vehicle (DMSO).
combination of both compounds towards these cells. Our results showed that this Erlotinib resistant isolate was sensitive to these phytochemicals (Fig. 4a). These results may have implications in the therapy of patients who are refractory to the therapy with Erlotinib. 3.4 Combination of carnosol and curcumin with chemotherapeutic drugs The effects of carnosol and curcumin alone or in combination with chemotherapeutic drugs were evaluated by the MTT test
1266 | Food Funct., 2014, 5, 1261–1269
(Fig. 5a). The drugs used are multikinase inhibitors directed towards specic intracellular targets including EGFR and VEGFR (vandetanib), c-MET (crizotinib), EGFR (erlotinib), and VEGFR (axitinib). Of special note is that, the effects on cell viability of carnosol or curcumin administered alone with the drugs were not signicantly different from the effects displayed by the drugs individually. In contrast, the combination of carnosol and curcumin increased the anti-proliferative effects of these chemotherapeutics (Fig. 5a). Among the tested combination, carnosol + curcumin + vandetanib was particularly
This journal is © The Royal Society of Chemistry 2014
View Article Online
Food & Function
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Paper
Fig. 5 Carnosol and curcumin reduced cell viability in combination with chemotherapeutic drugs. (a) SKOV3 cells were treated with carnosol, curcumin, vandetanib, erlotinib, axitinib, and crizotinib for 48 h as indicated, cell viability was then determined by the MTT test. (b) Representative images of SKOV3 cells treated with carnosol, curcumin, and vandetanib alone or in combination. Images were taken with an Olympus IX51 inverted microscope (10 magnification) after 48 h of treatment. For every treatment, 5 random fields of vision were acquired. (c) Western blot analysis on whole-cell lysates of SKOV3 cells treated for 24 h with carnosol, curcumin, and vandetanib alone or in combination as indicated. Western blot data are presented as a relative expression of selected proteins compared to tubulin or total Akt and total Erk and normalised to control. Error bars indicate the standard deviation of three independent experiments. **p < 0.01, ***p < 0.001 compared with the untreated group.
efficient in reducing cell viability (Fig. 5 a and b). In order to investigate the mechanism underlying the synergistic cytotoxicity in SKOV-3 cells, we next investigated changes in the expression of proteins involved in cell cycle regulation, apoptosis and cell proliferation. As shown in Fig. 5c, the combination reduced Cyclin D1, Bcl2, phospho-ERK1/2, phospho-Akt, and phospho-STAT3 expression. By contrast, b-catenin levels were unchanged aer treatment. Treatment of SKOV3 cells with carnosol for 24 h reduced phosphorylation of Akt but had no effects on STAT3. Conversely, treatment with curcumin reduced phosphorylation of STAT3. Both molecules reduced the
This journal is © The Royal Society of Chemistry 2014
cyclin D1 level and reduced phosphorylation of ERK1/2. Treatment with vandetanib reduced the phosphorylation status of ERK1/2 but increased p-Akt and p-STAT3 levels. Taken together, these results support at the biochemical level the enhanced efficacy in reducing cell viability.
4. Conclusions Cancer metastasis is commonly dened as the diffusion of cancer cells from the primary tumor site to distal organs where secondary tumors start to be formed. Metastasis is a complex
Food Funct., 2014, 5, 1261–1269 | 1267
View Article Online
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
Food & Function
multistep process beginning with the entrance of primary tumor cells into the vasculature, migration to distal organs, adhesion to endothelial cells and extravasation into the underlying tissue. Scientic evidence that natural compounds may have a potential inhibitory effect on cancer invasion and metastasis is recently increasing. Curcumin, resveratrol, and their related derivatives are the most studied compounds in this context. In this study, we demonstrated that carnosol, a dietary diterpene isolated from herbs such as rosemary, sage and oregano, displays potent antimetastatic effects in various cancer cells and primary tumors cells. Carnosol was able to inhibit cancer cell viability, cellular adhesion on bronectin and inhibits the EGF-induced EMT program. Moreover, we observed that cancer cells maintained in a suspension showed greater sensitivity to carnosol, this result may have particular importance for the development of strategies aimed at targeting circulating tumor cells with natural substances. Carnosol shows enhanced anti-proliferative activity when combined with other dietary compounds, an aspect of great relevance considering that the health benets of food are mainly due to the synergy of a variety of nutrients. In particular, the combination of carnosol and curcumin found a signicant effect also on primary human cancer cells isolated from patients with metastatic cancers. Our results indicate that inhibition of different signalling pathways may account for the observed synergy. Carnosol acts on Akt whereas curcumin reduces STAT3 phosphorylation. The combination of these two molecules with a drug, vandetanib, that reduces signicantly the phosphorylation of another kinase, ERK1/2, resulted in a greater reduction of cell viability. This is a classical mechanism of drug synergy in which molecules with different cellular targets can be combined together to switch off multiple kinases. In conclusion, results from the present study provide new evidence about the anticancer activity of carnosol. As also already performed with curcumin,27 the development of structural carnosol analogues could be a strategy for designing more effective compounds, improving carnosol efficacy/stability and translational potential.
Acknowledgements We thank Dr Antonio Danieli and Dr Raffaele Acierno for their technical support. This work was supported by the PS105 ARTI strategic project “Development and realization of bio-chip for molecular diagnostic and typization of human pathogenic viruses (HPV, HCV)” of Apulia Region, by the PON project 254/ Ric. “Implementation of human and environment health research center” cod. PONa3_00334, and by the Prin project n. 2010FPTBSH_006 "NANO Molecular Technologies for Drug delivery - NANOMED".
References 1 J. Salas-Salvado, M. A. Martinez-Gonzalez, M. Bullo and E. Ros, Nutr., Metab. Cardiovasc. Dis., 2011, 21(suppl. 2), B32–B48.
1268 | Food Funct., 2014, 5, 1261–1269
Paper
2 F. So, R. Abbate, G. F. Gensini and A. Casini, Am. J. Clin. Nutr., 2010, 92, 1189–1196. 3 L. J. Dominguez, M. Bes-Rastrollo, C. de la Fuente-Arrillaga, E. Toledo, J. J. Beunza, M. Barbagallo and M. A. MartinezGonzalez, Nutr., Metab. Cardiovasc. Dis., 2013, 23, 451– 458. 4 D. Vergara, P. Simeone, D. Toraldo, P. Del Boccio, V. Vergaro, S. Leporatti, D. Pieragostino, A. Tinelli, S. De Domenico, S. Alberti, A. Urbani, M. Salzet, A. Santino and M. Maffia, Mol. Biosyst., 2012, 8, 1078–1087. 5 D. Vergara, C. M. Valente, A. Tinelli, C. Siciliano, V. Lorusso, R. Acierno, G. Giovinazzo, A. Santino, C. Storelli and M. Maffia, Cancer Lett., 2011, 310, 1–8. 6 Y. J. Surh, Nat. Rev. Cancer, 2003, 3, 768–780. 7 C. Fortes, F. Forastiere, S. Farchi, S. Mallone, T. Trequattrinni, F. Anatra, G. Schmid and C. A. Perucci, Nutr. Cancer, 2003, 46, 30–37. 8 E. Vagi, E. Rapavi, M. Hadolin, K. Vasarhelyine Peredi, A. Balazs, A. Blazovics and B. Simandi, J. Agric. Food Chem., 2005, 53, 17–21. 9 M. J. del Bano, J. Lorente, J. Castillo, O. Benavente-Garcia, J. A. del Rio, A. Ortuno, K. W. Quirin and D. Gerard, J. Agric. Food Chem., 2003, 51, 4247–4253. 10 N. Bai, K. He, M. Roller, C. S. Lai, X. Shao, M. H. Pan and C. T. Ho, J. Agric. Food Chem., 2010, 58, 5363–5367. 11 J. Bauer, S. Kuehnl, J. M. Rollinger, O. Scherer, H. Northoff, H. Stuppner, O. Werz and A. Koeberle, J. Pharmacol. Exp. Ther., 2012, 342, 169–176. 12 E. S. Mengoni, G. Vichera, L. A. Rigano, M. L. RodriguezPuebla, S. R. Galliano, E. E. Cafferata, O. H. Pivetta, S. Moreno and A. A. Vojnov, Fitoterapia, 2011, 82, 414– 421. 13 A. Mohebati, J. B. Guttenplan, A. Kochhar, Z. L. Zhao, W. Kosinska, K. Subbaramaiah and A. J. Dannenberg, Cancer Prev. Res., 2012, 5, 593–602. 14 E. A. Offord, K. Mace, C. Ruffieux, A. Malnoe and A. M. Pfeifer, Carcinogenesis, 1995, 16, 2057–2062. 15 D. Martin, A. I. Rojo, M. Salinas, R. Diaz, G. Gallardo, J. Alam, C. M. De Galarreta and A. Cuadrado, J. Biol. Chem., 2004, 279, 8919–8929. 16 K. Singletary, C. MacDonald and M. Wallig, Cancer Lett., 1996, 104, 43–48. 17 M. T. Huang, C. T. Ho, Z. Y. Wang, T. Ferraro, Y. R. Lou, K. Stauber, W. Ma, C. Georgiadis, J. D. Laskin and A. H. Conney, Cancer Res., 1994, 54, 701–708. 18 J. M. Visanji, D. G. Thompson and P. J. Padeld, Cancer Lett., 2006, 237, 130–136. 19 J. Dorrie, K. Sapala and S. J. Zunino, Cancer Lett., 2001, 170, 33–39. 20 M. Wintzell, E. Hjerpe, E. Avall Lundqvist and M. Shoshan, BMC Cancer, 2012, 12, 359. 21 T. C. Chou, Pharmacol. Rev., 2006, 58, 621–681. 22 M. Sebbagh, C. Renvoize, J. Hamelin, N. Riche, J. Bertoglio and J. Breard, Nat. Cell Biol., 2001, 3, 346–352. 23 D. C. Radisky, J. Cell Sci., 2005, 118, 4325–4326. 24 J. P. Thiery and J. P. Sleeman, Nat. Rev. Mol. Cell Biol., 2006, 7, 131–142.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
G. Giannelli, P. Sacchetta and M. Maffia, Mol. Biosyst., 2013, 9, 1127–1138. 27 T. Esatbeyoglu, P. Huebbe, I. M. Ernst, D. Chin, A. E. Wagner and G. Rimbach, Angew. Chem., Int. Ed., 2012, 51, 5308–5332.
Published on 11 March 2014. Downloaded by Queens University - Kingston on 26/10/2014 21:00:00.
25 D. Vergara, B. Merlot, J. P. Lucot, P. Collinet, D. Vinatier, I. Fournier and M. Salzet, Cancer Lett., 2010, 291, 59–66. 26 D. Vergara, P. Simeone, P. del Boccio, C. Toto, D. Pieragostino, A. Tinelli, R. Acierno, S. Alberti, M. Salzet,
Food & Function
This journal is © The Royal Society of Chemistry 2014
Food Funct., 2014, 5, 1261–1269 | 1269
