Original Research Article Anti proliferative and pro apoptotic effects of flavonoid quercetin are mediated by CB1 receptor in human colon cancer cell lines† Maria Grazia Refolo1*, Rosalba D’Alessandro1*, Natascia Malerba2, Chiara Laezza3,4, Maurizio Bifulco5, Caterina Messa1, Maria Gabriella Caruso2, Maria Notarnicola2 and Valeria Tutino2# 1
Laboratory of Cellular and Molecular Biology and 2 Nutritional Biochemistry, National Institute for Digestive Diseases S. de Bellis, via Turi 27, 70013 Castellana Grotte, Bari, Italy; 3 Institute of Endocrinology and Experimental Oncology−CNR, via Pansini 5, 80131 Napoli, Italy; 4 Department of Biology and Cellular Pathology, University Federico II, via Pansini, 80131 Napoli, Italy; 5 Department of Medicine and Surgery, University of Salerno, via Allende, 84081 Baronissi, Salerno, Italy * The authors have equally contributed #
Corresponding author at: Valeria Tutino, Laboratory of Nutritional Biochemistry, National Institute for Digestive Diseases S. de Bellis, via Turi 27, 70013 Castellana Grotte, BariItaly, Tel. +39 080 4994342 e-mail: [email protected] Running title: Quercetin effects mediated by CB1-R induction Keywords: quercetin CB1 receptor Wnt/β-catenin pathway colon cancer intracellular signaling
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.25026]
Received 3 November 2014; Revised 31 March 2015; Accepted 16 April 2015 Journal of Cellular Physiology This article is protected by copyright. All rights reserved DOI 10.1002/jcp.25026
This article is protected by copyright. All rights reserved
1
Abstract Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1-R) after quercetin treatment. CB1-R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1-R leading to the regulation of cell growth. In order to clarify the contribution of the CB1-R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β-catenin was modified by flavonoid both directly and through activated CB1-R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (MetF-AEA), a CB1-R agonist, and partially counteracted by SR141716, a CB1-R antagonist. These findings open new perspectives for anticancer therapeutic strategies. This article is protected by copyright. All rights reserved
This article is protected by copyright. All rights reserved
2
Introduction Flavonoids are a class of polyphenolic plant compounds widely present in human diet. Quercetin, the major representative of the flavonols, is distributed in fresh onions, fruit and vegetables (Sampson et al., 2002; Ross et al., 2002). At non toxic concentrations in organism, quercetin possesses a variety of biological activities, although the precise mechanisms underlying these processes are still unknown (Ferraresi et al., 2005; Shih et al., 2000). Epidemiological studies have demonstrated that dietary constituents are closely associated with the risk for several types of cancers. It has been shown that quercetin specifically exerts anti proliferative and anti neoplastic activities influencing proliferation, differentiation and apoptosis in a variety of tumor cell types including colon cancer (Murakami et al., 2008; Kuo, 1996; Granado-Serrano et al., 2006). Yoshida et al. (1990) demonstrated that this polyphenolic compound is able to modulate the cell growth inducing the block of the cell cycle in human cancer cell lines. To explain the anticancer effects of quercetin, several possible mechanisms have been proposed including inhibition of various tyrosine kinases (Kandaswami et al., 2005), alteration of signal transduction pathways associated with the process of carcinogenesis, interaction with receptors and other proteins (Shih et al., 2000; Granado-Serrano et al., 2006; Nuyen et al., 2004). Park et al. (2005) showed that quercetin arrests the carcinogenic process by inhibiting the β-catenin/Tcf signaling. This pathway normally regulates the processes of embryonic development including cell proliferation, differentiation and epithelial-mesenchymal interactions (Woll et al., 2008). Deregulation of this signaling cascade is crucial in the development of colorectal carcinogenesis. Mutations in the adenomatous polyposis coli (APC) and/or β-catenin genes lead to pathway activation and to the accumulation of β-catenin in the nucleus where it actives the expression of target genes such as cyclin D1, c-jun and c-myc that are increased in This article is protected by copyright. All rights reserved
3
colon cancer (He et al., 1998; Mann et al., 1999). Recent findings reported that natural substances inhibit Wnt signaling preventing cancer and its metastases (Sebio et al., 2014). Moreover, studies by Park et al. (2005) demonstrated that quercetin has an inhibitory effect on the binding of β-catenin to the Tcf/Lef transcriptional complex, leading to a block of transcription of downstream genes and a decrease of β-catenin levels in the nucleus. Quercetin, as phytoestrogen, may also regulate the expression of cellular receptors known as "estrogen responsive", such as cannabinoid CB1 receptor (CB1-R) (Notarnicola et al., 2008) that exerts an inhibitory tone on cell growth (Bifulco et al., 2008; Di Leo et al., 2001; De Petrocellis et al., 1998). The cannabinoid receptors (CB1-R and CB2-R), the endogenous cannabinoids and the enzymes involved in their inactivation, constitute the Endocannabinoid
System
(Grotenhermen,
2005).
CB1-R,
a
G-protein-coupled
cannabinoid receptor, is particularly expressed in several brain regions but it is also present in peripheral areas including normal colonic epithelium, smooth muscle, and carries out various activities (Pertwee, 1997). Plant-derived, synthetic or endogenous cannabinoids are skilled to activate CB1-R controlling colorectal cancer cell proliferation (De Petrocellis et al., 1998; Patsos et al., 2005; Blázquez et al., 2004). Several studies have demonstrated antitumor action of cannabinoids receptor agonists mostly via CB1 activation in several tumor cells. In fact, the anti neoplastic effects of cannabinoids are explicated through different mechanisms that may involve induction of apoptosis and inhibition of proliferation in cancer growth in vitro (Pacher et al., 2006; Ligresti et al., 2003; Joseph et al., 2004). Previously, we have demonstrated that anandamide (Met-F-AEA), an endogenous agonist for CB1-R, inhibits the proliferation of colon cancer cell lines. This anti proliferative effect was not related to toxicity or to apoptosis of cells, but it was in correlation with This article is protected by copyright. All rights reserved
4
reduction of cells in the S phase of cell cycle as a consequence of a decreased polyamines levels due to the induction of the receptor (Linsalata et al., 2010). We have also demonstrated an estrogenic induction of CB1-R at mRNA and protein level in human colon cancer cells. In effect, an up-regulation of the receptor expression mediated by 17βestradiol was detected, suggesting a possible role of CB1-R and its ligands as cell proliferation mediators (Notarnicola et al., 2008). Recent evidences have also shown that the induction of CB1-R by Met-F-AEA leads to the activation of the Wnt/β-catenin pathway. Laezza et al. (2012) demonstrated that the synthetic cannabinoid reduces nuclear β-catenin levels and inhibits transcription of target genes involved in the cell cycle and proliferation. In the present study, we investigated for the first time the molecular mechanisms that support the anti neoplastic effects of quercetin in colon cancer cells through the modulation of estrogen responsive receptor CB1-R.
This article is protected by copyright. All rights reserved
5
Materials and Methods Cell culture conditions The human colon adenocarcinoma derived cell lines caracterized by different grade of differentation, Caco2 and DLD-1, were obtained from the ICLC (IST, Genoa, Italy). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% non essential
amino
acids
(NEAA),
2mM
glutamine,
100U/ml
penicillin,
100mg/ml
streptomycin, in monolayer cultures, and incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. At confluence, the grown cells were harvested and serially subcultured. All cell culture components were purchased from Sigma Aldrich, Milan, Italy.
Cell treatment The quercetin was purchased from MP Biomedicals, LCC (Solon, Ohio, USA); the SR141716 also known as N-(piperidino)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4methylpyrazole-3-carboxyamide from Sanofi Aventis (Montpellier, France) and the Met-FAEA from Cayman Chemicals (Ann Arbor, Michigan, USA). The cells were allowed to grow for established time and then harvested. The cell pellet was used for subsequent analysis. Each experiment included an untreated control and a control with the equivalent concentration of dimethyl sulfoxide (DMSO) or absolute ethanol as had been used for adding drugs. The solvent reached a concentration not higher than 0.3% in all experiments.
Assessment of cell proliferation Cells were treated with increasing concentrations of quercetin (10, 20 and 50µM that corresponds to the IC50) or SR141716 (0.1µM) dissolved in DMSO and Met-F-AEA (10µM) dissolved in absolute ethanol, alone or in combination. The proliferative response This article is protected by copyright. All rights reserved
6
was estimated by colorimetric 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium-bromide (MTT) test (Sigma Aldrich; Milan, Italy), as previously described (Linsalata et al., 2010), after 24 and 48h. Cell viability, determined using the trypan blue exclusion test, always exceeded 90%.
Assessment of cell apoptosis Caco2 cells were cultured with quercetin (50µM) and SR141716 (0.1µM) for 48h. The Muse Annexin V and Dead Cell Assay Kit (Millipore, Darmstadt, Germany) for quantitative analysis of live, early/late apoptotic and dead cells was used with a Muse Cell Analyzer (Millipore). The cells were then processed as described in the user’s guide.
Migration assay Caco2 cells were growing until confluence in RPMI 1640 containing 10% FBS and then the assay was assessed as previously described using quercetin (10µM) or SR141716 (0.1µM) alone or in combination (Liang et al., 2007; Carr et al., 2013).
Protein expression levels Cells treated for 48h with quercetin (10, 20 or 50µM), Met-F-AEA (10μM) and SR141716 (0.1μM) alone or in combination, were washed with cold PBS, scraped, and then centrifuged at 1000rpm for 10min at 4°C. Total cell extracts were obtained by collecting and suspending cell pellet in lyses buffer (Xu et al., 2003). After centrifugation at 14000rpm for 15min at 4°C, protein concentration was measured by a standard Bradford assay (Bio Rad Laboratories, Milan, Italy). The nuclear fraction was separated from the cytoplasmic extracts resuspending the cell pellet in buffer containing 10mM HEPES/KOH (pH 7.9), 10mM KCl, 1.5mM MgCl2, H2O, 0.1% NP-40 and protease and phosphatase This article is protected by copyright. All rights reserved
7
inhibitors. After incubation, the cell suspension was homogenized, centrifuged at 2800rpm for 5min and the supernatant was used to measure cytoplasmic protein concentration. The pellet was resuspended in buffer containing 20mM HEPES/KOH (pH 7.9), 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 25% Glycerol, H2O and protease and phosphatase inhibitors. After incubation, the pellet was centrifuged at 13000rpm for 10min and the supernatant was used to determine nuclear protein concentration. Aliquots of 50μg of total, cytoplasmic and nuclear proteins were separated by SDS PAGE and transferred onto a PVDF membrane (Bio Rad Laboratories, Milan, Italy). The primary antibodies were directed against the following proteins: β-catenin and phospho-β-catenin (P-β-catenin), PI3K and phospho-PI3K (P-PI3K), Akt and phospho-Akt (P-Akt, T308 and S473), S6 and phospho-S6 (P-S6), 4EBP1, GSK3β and phospho-GSK3β (P-GSK3β), STAT3 and phospho-STAT3 (P-STAT3, S727), JNK and phospho-JNK (P-JNK), c-jun and phospho-cjun (P-c-jun), c-myc and phospho-c-myc (P-c-myc), β-actin and histone 3 (H3) (Cell Signaling, Beverly, MA, USA). The immunoreactive bands were visualized and analyzed using chemiluminescence detection system (ChemiDoc XRS apparatus and software, Bio Rad, Milan, Italy). The β-actin and histone 3 were used as a loading controls for cytoplasmic and nuclear extracts respectively.
Cell cycle analysis Caco2 cells were synchronized by using 0.2M thymidine added to the medium. After 18h of incubation, the medium containing thymidine was replaced from fresh medium for 9h, then cells were treated with thymidine for an additional 17h. Cells were separated into two groups: one group was collected for cell cycle analysis and the other one continued culturing; quercetin (50µM) and SR141716 (0.1µM) were added and after 6h of treatment cells were collected to be processed, according to the user’s guide, with the Muse Cell This article is protected by copyright. All rights reserved
8
Cycle Kit (Millipore, Darmstadt, Germany) which determines the percentage of cells in the G0/G1, S and G2/M phases of cell cycle with the Muse Cell Analyzer.
Gene expression analysis Cells were cultured with quercetin (10, 20 and 50μM) for 48h in order to evaluate CB1-R mRNA expression and then processed as previously described (Notarnicola et al., 2008). The used primer sequences were: sense 5’-GGAGAACATCCAGTGTGGGG-3’ and antisense
5’-CATTGGGGCTGTCTTTACGG-3’
AAAGACCTGTACGCCAACACAGTGCTGTCTGG-3′
for
CB1-R; and
sense
antisense
5′5′-
CGTCATACTCCTGCTTGCTGATCCACATCTGC-3’ for β-actin.
Statistical analysis Data were evaluated by one way ANOVA and appropriate post-test. P values
Laboratory of Cellular and Molecular Biology and 2 Nutritional Biochemistry, National Institute for Digestive Diseases S. de Bellis, via Turi 27, 70013 Castellana Grotte, Bari, Italy; 3 Institute of Endocrinology and Experimental Oncology−CNR, via Pansini 5, 80131 Napoli, Italy; 4 Department of Biology and Cellular Pathology, University Federico II, via Pansini, 80131 Napoli, Italy; 5 Department of Medicine and Surgery, University of Salerno, via Allende, 84081 Baronissi, Salerno, Italy * The authors have equally contributed #
Corresponding author at: Valeria Tutino, Laboratory of Nutritional Biochemistry, National Institute for Digestive Diseases S. de Bellis, via Turi 27, 70013 Castellana Grotte, BariItaly, Tel. +39 080 4994342 e-mail: [email protected] Running title: Quercetin effects mediated by CB1-R induction Keywords: quercetin CB1 receptor Wnt/β-catenin pathway colon cancer intracellular signaling
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.25026]
Received 3 November 2014; Revised 31 March 2015; Accepted 16 April 2015 Journal of Cellular Physiology This article is protected by copyright. All rights reserved DOI 10.1002/jcp.25026
This article is protected by copyright. All rights reserved
1
Abstract Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1-R) after quercetin treatment. CB1-R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1-R leading to the regulation of cell growth. In order to clarify the contribution of the CB1-R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β-catenin was modified by flavonoid both directly and through activated CB1-R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (MetF-AEA), a CB1-R agonist, and partially counteracted by SR141716, a CB1-R antagonist. These findings open new perspectives for anticancer therapeutic strategies. This article is protected by copyright. All rights reserved
This article is protected by copyright. All rights reserved
2
Introduction Flavonoids are a class of polyphenolic plant compounds widely present in human diet. Quercetin, the major representative of the flavonols, is distributed in fresh onions, fruit and vegetables (Sampson et al., 2002; Ross et al., 2002). At non toxic concentrations in organism, quercetin possesses a variety of biological activities, although the precise mechanisms underlying these processes are still unknown (Ferraresi et al., 2005; Shih et al., 2000). Epidemiological studies have demonstrated that dietary constituents are closely associated with the risk for several types of cancers. It has been shown that quercetin specifically exerts anti proliferative and anti neoplastic activities influencing proliferation, differentiation and apoptosis in a variety of tumor cell types including colon cancer (Murakami et al., 2008; Kuo, 1996; Granado-Serrano et al., 2006). Yoshida et al. (1990) demonstrated that this polyphenolic compound is able to modulate the cell growth inducing the block of the cell cycle in human cancer cell lines. To explain the anticancer effects of quercetin, several possible mechanisms have been proposed including inhibition of various tyrosine kinases (Kandaswami et al., 2005), alteration of signal transduction pathways associated with the process of carcinogenesis, interaction with receptors and other proteins (Shih et al., 2000; Granado-Serrano et al., 2006; Nuyen et al., 2004). Park et al. (2005) showed that quercetin arrests the carcinogenic process by inhibiting the β-catenin/Tcf signaling. This pathway normally regulates the processes of embryonic development including cell proliferation, differentiation and epithelial-mesenchymal interactions (Woll et al., 2008). Deregulation of this signaling cascade is crucial in the development of colorectal carcinogenesis. Mutations in the adenomatous polyposis coli (APC) and/or β-catenin genes lead to pathway activation and to the accumulation of β-catenin in the nucleus where it actives the expression of target genes such as cyclin D1, c-jun and c-myc that are increased in This article is protected by copyright. All rights reserved
3
colon cancer (He et al., 1998; Mann et al., 1999). Recent findings reported that natural substances inhibit Wnt signaling preventing cancer and its metastases (Sebio et al., 2014). Moreover, studies by Park et al. (2005) demonstrated that quercetin has an inhibitory effect on the binding of β-catenin to the Tcf/Lef transcriptional complex, leading to a block of transcription of downstream genes and a decrease of β-catenin levels in the nucleus. Quercetin, as phytoestrogen, may also regulate the expression of cellular receptors known as "estrogen responsive", such as cannabinoid CB1 receptor (CB1-R) (Notarnicola et al., 2008) that exerts an inhibitory tone on cell growth (Bifulco et al., 2008; Di Leo et al., 2001; De Petrocellis et al., 1998). The cannabinoid receptors (CB1-R and CB2-R), the endogenous cannabinoids and the enzymes involved in their inactivation, constitute the Endocannabinoid
System
(Grotenhermen,
2005).
CB1-R,
a
G-protein-coupled
cannabinoid receptor, is particularly expressed in several brain regions but it is also present in peripheral areas including normal colonic epithelium, smooth muscle, and carries out various activities (Pertwee, 1997). Plant-derived, synthetic or endogenous cannabinoids are skilled to activate CB1-R controlling colorectal cancer cell proliferation (De Petrocellis et al., 1998; Patsos et al., 2005; Blázquez et al., 2004). Several studies have demonstrated antitumor action of cannabinoids receptor agonists mostly via CB1 activation in several tumor cells. In fact, the anti neoplastic effects of cannabinoids are explicated through different mechanisms that may involve induction of apoptosis and inhibition of proliferation in cancer growth in vitro (Pacher et al., 2006; Ligresti et al., 2003; Joseph et al., 2004). Previously, we have demonstrated that anandamide (Met-F-AEA), an endogenous agonist for CB1-R, inhibits the proliferation of colon cancer cell lines. This anti proliferative effect was not related to toxicity or to apoptosis of cells, but it was in correlation with This article is protected by copyright. All rights reserved
4
reduction of cells in the S phase of cell cycle as a consequence of a decreased polyamines levels due to the induction of the receptor (Linsalata et al., 2010). We have also demonstrated an estrogenic induction of CB1-R at mRNA and protein level in human colon cancer cells. In effect, an up-regulation of the receptor expression mediated by 17βestradiol was detected, suggesting a possible role of CB1-R and its ligands as cell proliferation mediators (Notarnicola et al., 2008). Recent evidences have also shown that the induction of CB1-R by Met-F-AEA leads to the activation of the Wnt/β-catenin pathway. Laezza et al. (2012) demonstrated that the synthetic cannabinoid reduces nuclear β-catenin levels and inhibits transcription of target genes involved in the cell cycle and proliferation. In the present study, we investigated for the first time the molecular mechanisms that support the anti neoplastic effects of quercetin in colon cancer cells through the modulation of estrogen responsive receptor CB1-R.
This article is protected by copyright. All rights reserved
5
Materials and Methods Cell culture conditions The human colon adenocarcinoma derived cell lines caracterized by different grade of differentation, Caco2 and DLD-1, were obtained from the ICLC (IST, Genoa, Italy). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% non essential
amino
acids
(NEAA),
2mM
glutamine,
100U/ml
penicillin,
100mg/ml
streptomycin, in monolayer cultures, and incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. At confluence, the grown cells were harvested and serially subcultured. All cell culture components were purchased from Sigma Aldrich, Milan, Italy.
Cell treatment The quercetin was purchased from MP Biomedicals, LCC (Solon, Ohio, USA); the SR141716 also known as N-(piperidino)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4methylpyrazole-3-carboxyamide from Sanofi Aventis (Montpellier, France) and the Met-FAEA from Cayman Chemicals (Ann Arbor, Michigan, USA). The cells were allowed to grow for established time and then harvested. The cell pellet was used for subsequent analysis. Each experiment included an untreated control and a control with the equivalent concentration of dimethyl sulfoxide (DMSO) or absolute ethanol as had been used for adding drugs. The solvent reached a concentration not higher than 0.3% in all experiments.
Assessment of cell proliferation Cells were treated with increasing concentrations of quercetin (10, 20 and 50µM that corresponds to the IC50) or SR141716 (0.1µM) dissolved in DMSO and Met-F-AEA (10µM) dissolved in absolute ethanol, alone or in combination. The proliferative response This article is protected by copyright. All rights reserved
6
was estimated by colorimetric 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium-bromide (MTT) test (Sigma Aldrich; Milan, Italy), as previously described (Linsalata et al., 2010), after 24 and 48h. Cell viability, determined using the trypan blue exclusion test, always exceeded 90%.
Assessment of cell apoptosis Caco2 cells were cultured with quercetin (50µM) and SR141716 (0.1µM) for 48h. The Muse Annexin V and Dead Cell Assay Kit (Millipore, Darmstadt, Germany) for quantitative analysis of live, early/late apoptotic and dead cells was used with a Muse Cell Analyzer (Millipore). The cells were then processed as described in the user’s guide.
Migration assay Caco2 cells were growing until confluence in RPMI 1640 containing 10% FBS and then the assay was assessed as previously described using quercetin (10µM) or SR141716 (0.1µM) alone or in combination (Liang et al., 2007; Carr et al., 2013).
Protein expression levels Cells treated for 48h with quercetin (10, 20 or 50µM), Met-F-AEA (10μM) and SR141716 (0.1μM) alone or in combination, were washed with cold PBS, scraped, and then centrifuged at 1000rpm for 10min at 4°C. Total cell extracts were obtained by collecting and suspending cell pellet in lyses buffer (Xu et al., 2003). After centrifugation at 14000rpm for 15min at 4°C, protein concentration was measured by a standard Bradford assay (Bio Rad Laboratories, Milan, Italy). The nuclear fraction was separated from the cytoplasmic extracts resuspending the cell pellet in buffer containing 10mM HEPES/KOH (pH 7.9), 10mM KCl, 1.5mM MgCl2, H2O, 0.1% NP-40 and protease and phosphatase This article is protected by copyright. All rights reserved
7
inhibitors. After incubation, the cell suspension was homogenized, centrifuged at 2800rpm for 5min and the supernatant was used to measure cytoplasmic protein concentration. The pellet was resuspended in buffer containing 20mM HEPES/KOH (pH 7.9), 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 25% Glycerol, H2O and protease and phosphatase inhibitors. After incubation, the pellet was centrifuged at 13000rpm for 10min and the supernatant was used to determine nuclear protein concentration. Aliquots of 50μg of total, cytoplasmic and nuclear proteins were separated by SDS PAGE and transferred onto a PVDF membrane (Bio Rad Laboratories, Milan, Italy). The primary antibodies were directed against the following proteins: β-catenin and phospho-β-catenin (P-β-catenin), PI3K and phospho-PI3K (P-PI3K), Akt and phospho-Akt (P-Akt, T308 and S473), S6 and phospho-S6 (P-S6), 4EBP1, GSK3β and phospho-GSK3β (P-GSK3β), STAT3 and phospho-STAT3 (P-STAT3, S727), JNK and phospho-JNK (P-JNK), c-jun and phospho-cjun (P-c-jun), c-myc and phospho-c-myc (P-c-myc), β-actin and histone 3 (H3) (Cell Signaling, Beverly, MA, USA). The immunoreactive bands were visualized and analyzed using chemiluminescence detection system (ChemiDoc XRS apparatus and software, Bio Rad, Milan, Italy). The β-actin and histone 3 were used as a loading controls for cytoplasmic and nuclear extracts respectively.
Cell cycle analysis Caco2 cells were synchronized by using 0.2M thymidine added to the medium. After 18h of incubation, the medium containing thymidine was replaced from fresh medium for 9h, then cells were treated with thymidine for an additional 17h. Cells were separated into two groups: one group was collected for cell cycle analysis and the other one continued culturing; quercetin (50µM) and SR141716 (0.1µM) were added and after 6h of treatment cells were collected to be processed, according to the user’s guide, with the Muse Cell This article is protected by copyright. All rights reserved
8
Cycle Kit (Millipore, Darmstadt, Germany) which determines the percentage of cells in the G0/G1, S and G2/M phases of cell cycle with the Muse Cell Analyzer.
Gene expression analysis Cells were cultured with quercetin (10, 20 and 50μM) for 48h in order to evaluate CB1-R mRNA expression and then processed as previously described (Notarnicola et al., 2008). The used primer sequences were: sense 5’-GGAGAACATCCAGTGTGGGG-3’ and antisense
5’-CATTGGGGCTGTCTTTACGG-3’
AAAGACCTGTACGCCAACACAGTGCTGTCTGG-3′
for
CB1-R; and
sense
antisense
5′5′-
CGTCATACTCCTGCTTGCTGATCCACATCTGC-3’ for β-actin.
Statistical analysis Data were evaluated by one way ANOVA and appropriate post-test. P values
