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Proapoptotic effect of endocannabinoids in prostate cancer cells.

 

1
Physiology and Biophysics Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago 8389100, Chile.
2
Pathological Anatomy Service, Clinic Hospital of the University of Chile, Santiago 8389100, Chile.
3
Urology Service, Clinic Hospital of the University of Chile, Santiago 8389100, Chile.
4
Laboratory of Nutrition and Metabolic Regulation, INTA, University of Chile, Santiago 8389100, Chile.

Abstract

In the early stages, prostate cancer is androgen‑ dependent; therefore, medical castration has shown significant results during the initial stages of this pathology. Despite this early effect, advanced prostate cancer is resilient to such treatment. Recent evidence shows that derivatives of Cannabis sativa and its analogs may exert a protective effect against different types of oncologic pathologies. The purpose of the present study was to detect the presence of cannabinoid receptors (CB1 and CB2) on cancer cells with a prostatic origin and to evaluate the effect of the in vitro use of synthetic analogs. In order to do this, we used a commercial cell line and primary cultures derived from prostate cancer and benign prostatic hyperplasia. The presence of the CB1 and CB2 receptors was determined by immunohistochemistry where we showed a higher expression of these receptors in later stages of the disease (samples with a high Gleason score). Later, treatments were conducted using anandamide, 2-arachidonoyl glycerol and a synthetic analog of anandamide, methanandamide. Using the MTT assay, we proved that the treatments produced a cell growth inhibitory effect on all the different prostate cancer cultures. This effect was demonstrated to be dose-dependent. The use of a specific CB1 receptor blocker (SR141716) confirmed that this effect was produced primarily from the activation of the CB1 receptor. In order to understand the MTT assay results, we determined cell cycle distribution by flow cytometry, which showed no variation at the different cell cycle stages in all the cultures after treatment. Treatment with endocannabinoids resulted in an increase in the percentage of apoptotic cells as determined by Annexin V assays and caused an increase in the levels of activated caspase-3 and a reduction in the levels of Bcl-2 confirming that the reduction in cell viability noted in the MTT assay was caused by the activation of the apoptotic pathway. Finally, we observed that endocannabinoid treatment activated the Erk pathway and at the same time, produced a decrease in the activation levels of the Akt pathway. Based on these results, we suggest that endocannabinoids may be a beneficial option for the treatment of prostate cancer that has become nonresponsive to common therapies.

Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: implication of epidermal growth factor receptor down-regulation and ceramide production.

Institut de Chimie Pharmaceutique Albert Lespagnol, 3 Rue du Professeur Laguesse, BP83, Lille, France.

Abstract

BACKGROUND:

Anandamide (ANA) is an endogenous lipid which acts as a cannabinoid receptor ligand and with potent anticarcinogenic activity in several cancer cell types.

METHODS:

The inhibitory effect of ANA on the epidermal growth factor receptor (EGFR) levels expressed on the EGF-stimulated prostatic cancer cells LNCaP, DU145, and PC3 was estimated by ELISA tests. The anti-proliferative and cytotoxic effects of ANA were also evaluated on these human prostatic cancer cell lines by growth tests, flow cytometric analyses, trypan blue dye exclusion assays combined with the Papanicolaou cytological staining method.

RESULTS:

ANA induced a decrease of EGFR levels on LNCaP, DU145, and PC3 prostatic cancer cells by acting through cannabinoid CB(1) receptor subtype and this leaded to an inhibition of the EGF-stimulated growth of these cells. Moreover, the G(1) arrest of metastatic DU145 and PC3 growth was accompanied by a massive cell death by apoptosis and/or necrosis while LNCaP cells were less sensitive to cytotoxic effects of ANA. The apoptotic/necrotic responses induced by ANA on these prostatic cancer cells were also potentiated by the acidic ceramidase inhibitor, N-oleoylethanolamine and partially inhibited by the specific ceramide synthetase inhibitor, fumonisin B1 indicating that these cytotoxic actions of ANA might be induced via the cellular ceramide production.

CONCLUSIONS:

The potent anti-proliferative and cytotoxic effects of ANA on metastatic prostatic cancer cells might provide basis for the design of new therapeutic agents for effective treatment of recurrent and invasive prostatic cancers.

Non-THC cannabinoids inhibit prostate carcinoma growth in vitro and in vivo: pro-apoptotic effects and underlying mechanisms.

 

Abstract

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Serum deprivation or modification of serum content differently affects the effect of cannabinoids (CBD, CBG, CBC) on the viability of human PCCs in the MTT assay. (A) Cells were grown in presence of 10% FBS in six-well dishes. After adhesion, cells were serum-deprived (with or without 0.5%BSA) for 16 h and subsequently treated with compounds for 24 h. Cell viability was assessed by the MTT assay. Data shown are means ± SEM of % inhibition of MTT-reducing activity, calculated from three independent experiments. ***P < 0.001, FBS-free vs. plus 0.5% BSA; anova followed by Bonferroni’s test. (B) Cells grown in presence of 10% FBS were incubated, after adhesion, under different conditions (a–c) in six-well dishes for 72 h: (a) cells were incubated in presence of 10% FBS; (b) cells were incubated with 10% FBS that had been protein-depleted; (c) cells were incubated again in 10% FBS that had been protein-depleted, but supplemented with 0.5%BSA. Cell viability was assessed by the MTT assay. Data shown are means ± SEM of % inhibition of MTT-reducing activity calculated from three independent experiments. ###P < 0.001 protein-depleted serum [b] vs. 10% FBS [a] and protein-depleted serum plus 0.5% BSA [c]; $$P < 0.01 protein-depleted serum plus 0.5% BSA [c] vs. 10% FBS [a]; anova followed by Bonferroni’s test. (C) Cells were grown in 10% FBS in six-well dishes. After adhesion, cells were serum-deprived for 16 h and then treated with compounds for 24 h. Cell viability was assessed by the MTT assay. Data shown are means ± SEM of % inhibition of MTT-reducing activity calculated from three independent experiments.

BACKGROUND AND PURPOSE:

Cannabinoid receptor activation induces prostate carcinoma cell (PCC) apoptosis, but cannabinoids other than Δ(9) -tetrahydrocannabinol (THC), which lack potency at cannabinoid receptors, have not been investigated. Some of these compounds antagonize transient receptor potential melastatin type-8 (TRPM8) channels, the expression of which is necessary for androgen receptor (AR)-dependent PCC survival.

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Effect of CBD-BDS on the growth of xenograft tumours from LNCaP and DU-145 cells in athymic mice, per se or co-administered with docetaxel (taxotere) or bicalutamide. Effect of increasing doses of CBD-BDS (i.p.) or docetaxel (i.v.) or combinations thereof on the growth of LNCaP (A) and DU-145 (B) cell xenografts. N= 10 mice were used for each group. In panel A, *P= 0.0008, **P < 0.0001 versus vehicle; ***P < 0.0001 versus both vehicle and CBD–BDS + docetaxel; in panel B, *P < 0.0001 versus vehicle; #P < 0.0001 versus docetaxel alone; two-way anova. (C) Effect of increasing doses of CBD-BDS (i.p.) or bicalutamide (p.o.) or in combination, on the growth of LNCaP cell xenografts. A higher dose of bicalutamide (50 mg·kg−1) was studied, but the effect was not different from that of the 25 mg·kg−1 dose. *P= 0.0005 (group 5 vs. group 1); #P = 0.001 (group 5 vs. group 2); two-way anova. (D) Kaplan–Meier survival plots for the study described in (C). Statistical significance was assessed by the Log rank-Wilcoxon analysis.

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Effect of cannabinoids and on the release of caspase 3/7 from various PCC lines. Cells (10 000 per data point) were treated under the conditions shown, and caspase 3/7 activity was assessed with the luminescence assay. Other compounds tested that exhibited no activity are not shown. Effect of various compounds, at various concentrations, in LNCaP (A) and DU-145 (DU) (B) cells incubated in the presence of serum for 24 h, and of the positive control, anti-FAS + camptothecin (campto + AFAS). Note how the TRPM8 channel antagonist OMDM233 stimulates caspase 3/7 activity and how the TRPM8 channel agonist icilin inhibits the effect of CBD in LNCaP cells. (C) Effect of varying duration of SDP before and after treatment (12 h + 8 h treatment, 12 h + 12 h treatment, 24 h + 12 h treatment) with varying doses of the TRPM8 antagonist OMDM233, or of the TRPM8 agonist icilin, or of CBD, with and without icilin, on caspase 3/7 activity in LNCaP cells. (D) Effect of various durations of SDP before and after treatment (12 h + 12 h treatment, 12 h + 24 h of treatment or 24 h + 12 h treatment) with varying doses of CBG and CBC on caspase 3/7 activity in LNCaP cells. (E) Effect of BSA (0.5%), or testosterone (T, 50 µM), or subsequent addition of FBS, or of the use of charcoal-stripped FBS (strip) on the effect of CBD on caspase 3/7 activity in LNCaP cells. These experiments were carried out under different conditions of pre-treatment serum deprivation (12 or 24 h), whereas the treatment with CBD (10 µM) was always carried out for 12 h, thus leading to total durations of experiments of 24 or 36 h. (F) Effect of CBD on caspase 3/7 activity in SDP DU-145 cells. Experiments were carried out with either varying doses of CBD for 18 h, after a previous serum deprivation of 6 h, or with 10 µM CBD for a total of 24 h with different combinations of previous serum deprivation (0, 2 and 4 h) and treatment (24, 22 and 20 h). Finally, the effect of CBC and CBG were studied in 22RV1 (G) and PC3 (H) cells. In both cases, the experiment lasted for 24 h, with a previous serum deprivation of 4 h in panel G and 6 h in panel H. Data are means ± SEM of at least n= 3 experiments. Means were compared by anova followed by Bonferroni’s test. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective control (first bar in each panel, which represents the baseline level of caspase 3/7, which, for a given cell line, did not vary significantly regardless of the duration of the experiment and the presence of serum in the 12–36 h range). In panels C and E, #P < 0.01 versus SDP 24 + 12 CBD 10 µM.

EXPERIMENTAL APPROACH:

We tested pure cannabinoids and extracts from Cannabis strains enriched in particular cannabinoids (BDS), on AR-positive (LNCaP and 22RV1) and -negative (DU-145 and PC-3) cells, by evaluating cell viability (MTT test), cell cycle arrest and apoptosis induction, by FACS scans, caspase 3/7 assays, DNA fragmentation and TUNEL, and size of xenograft tumours induced by LNCaP and DU-145 cells.

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Effect of cannabinoids on intracellular Ca2+ and ROS in prostate carcinoma cells. (A) Typical dose-dependent effects for cannabinoids on intracellular Ca2+ in PCCs, with either efficacy or potency being higher in cells serum-deprived (SDP) for 24 h. The effect of CBG in LNCaP and DU-145 cells is shown. See Table S6 for the full data in the four PCCs with CBD, CBG and CBC. (B,C) Involvement of ROS in the effect of CBD on different PCCs. Time course of ROS production by PCC cells as measured by spectrofluorometric analysis as described in Methods. Fluorescence detection was carried out after the incubation of either 100 µM H2O2 or CBD (10 µM) at different times (0–30–60–120 min) in cells grown in normal medium (B) or in cells kept without serum prior to treatment (C). The fluorescence measured at time 0 was considered as basal ROS production and subtracted from the fluorescence at different times (Δ1). Data are reported as Δ2 (i.e. Δ1 values at different doses minus the Δ1 values of cells incubated with vehicle), and are mean ± SEM of at least n= 3 experiments. Note how the effect of both CBD and H2O2 becomes significant only in the absence of serum (C) and only in LNCaP cells.

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Effects of CBD on neuroendocrine-like LNCaP cells. Cells were differentiated with db-cAMP + IBMX for 36 h in serum-deprived medium, in the presence or absence of CBD and various other compounds. (A) Effect on caspase 3/7 activity of just serum deprivation for 36 h, alone or with CBD (10 µM) for 12 h, or with db-cAMP + IBMX for 36 h, or with db-cAMP + IBMX for 24 h followed by 12 h CBD, or with db-cAMP + IBMX followed by 12 h icilin (1 µM) or OMDM233 (2 µM), or with db-cAMP + IBMX followed by 12 h CBD + icilin. *, **, ***P < 0.05, 0.01, 0.001 versus SDP 36. (B) NSE mRNA in differentiated LNCaP cells. Cells were cultured in the presence of serum (CTR), in serum-deprived medium for 36 h in the presence of db-cAMP and IBMX (SDP) and in the presence of 10 µM CBD for 12 h during db-cAMP + IBMX treatment (SDP + CBD). qRT-PCR was performed using 20 ng of cDNA per assay. The expression levels of NSE mRNA, normalized respect to the reference gene, were scaled to the lowest expression value condition, considered as 1; i.e., SDP + CBD (28.67 cq vs. background >40 cq). PUMA (C), p27kip (D), AR (E) and TRPM8 (F) mRNA levels in LNCaP cells following various treatments in serum-deprived (SDP) cells. Cell were cultured in presence of serum (CTR), in serum-deprived medium for 36 h in presence of db-cAMP + IBMX (dbcAMP) and in presence of 10 µM CBD for 12 h during db-cAMP + IBMX treatment (dbcAMP + CBD). For all the targets, the expression levels normalized respect to the reference gene were scaled to the lowest expression value condition, considered as 1; i.e. CTR (28.68 cq vs. background at 37.53) for PUMA and p27kip (24.75 cq vs. background at 38.40cq); and db-cAMP + CBD for AR (29.04 vs. background >40 cq) and TRPM8 (29.50 vs. background at 35.80 cq). In panels B–F, qRT-PCR was performed using 20 ng of cDNA per assay, and a typical experiment (R.I.N. > 8.5) is shown. Standard deviations were calculated by the gene expression module of iQ5 real-time PCR. All differences indicated in the graph (*) were significant (P < 0.05) versus CTR as evaluated according to Pfaffl, 2010 (see Supporting information). # denotes P < 0.05 versus CTR.

KEY RESULTS:

Cannabidiol (CBD) significantly inhibited cell viability. Other compounds became effective in cells deprived of serum for 24 h. Several BDS were more potent than the pure compounds in the presence of serum. CBD-BDS (i.p.) potentiated the effects of bicalutamide and docetaxel against LNCaP and DU-145 xenograft tumours and, given alone, reduced LNCaP xenograft size. CBD (1-10 µM) induced apoptosis and induced markers of intrinsic apoptotic pathways (PUMA and CHOP expression and intracellular Ca(2+)). In LNCaP cells, the pro-apoptotic effect of CBD was only partly due to TRPM8 antagonism and was accompanied by down-regulation of AR, p53 activation and elevation of reactive oxygen species. LNCaP cells differentiated to androgen-insensitive neuroendocrine-like cells were more sensitive to CBD-induced apoptosis.

CONCLUSIONS AND IMPLICATIONS:

These data support the clinical testing of CBD against prostate carcinoma.


 

Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation of p16 INK4a /pRB pathway

Abstract

It has been suggested that the helix–loop–helix protein Id-1 plays an important role in tumourigenesis in certain types of human cancer. Previously, we reported that Id-1 was up-regulated during sex hormone-induced prostate carcinogenesis in a Noble rat model (Ouyang et al . (2001) Carcinogenesis , 22 , 965–973). In the present study, we investigated the direct effect of Id-1 expression on human prostate cancer cell proliferation by transfecting an Id-1 expression vector into a prostate cancer cell line LNCaP. Ten stable transfectant clones were isolated and the ectopic Id-1 expression resulted in both increased DNA synthesis rate and the percentage of S phase cells. To study the possible mechanisms involved in the Id-1 induced prostate cancer cell growth, we examined the expression of several factors responsible for G 1 to S phase progression. We found that Id-1 expression induced phosphorylation of RB and down-regulation of p16 INK4a but not p21 Waf1 or p27 Kip1 . Our results indicate that the Id-1 induced inactivation of p16 INK4a /pRB pathway may be responsible for the increased cell proliferation in prostate cancer cells. Given the fact that both Id-1 over-expression and inactivation of p16 INK4a /pRB are common events in prostate cancer, our results provide a possible mechanism on the molecular basis of prostate carcinogenesis.

Introduction

Id proteins (inhibitor of differentiation or DNA binding) are a group of helix–loop–helix (HLH) transcription factors that lack the DNA-binding domain. Therefore, their function is mainly to act as dominant inhibitors of basic HLH proteins by forming non-functional Id-bHLH heterodimers. Since most of the bHLH proteins positively activate genes in cell differentiation, the Id proteins are considered to be the negative regulators of differentiation ( 1 , 2 ). The fact that Id proteins can stimulate DNA synthesis and immortalize mammalian cells, either alone or incorporated with additional oncogenes ( 35 ), indicates that they may function as potential oncogenes. Increased Id-1 expression has been found in several types of primary tumours including breast ( 6 ), pancreatic ( 7 , 8 ), prostate ( 9 ) and head and neck ( 10 ). Recently ectopic expression of Id-1 induced increased aggressiveness and metastasis in breast cancer cells ( 6 ), and up-regulation of Id-1 has also been correlated with increased tumour stage in several human cancers ( 8 , 10 ). In addition, in Id-1+/–Id3–/– knockout mice, a significantly reduced metastatic ability of tumour xenografts has been reported ( 11 ). These lines of evidence strongly suggest that Id proteins play important roles not only in tumourigenesis but also in tumour progression.

Previously, using cDNA array technique, we reported an up-regulation of Id-1 during sex hormone-induced prostate carcinogenesis in a Noble rat model ( 9 ) and increased Id-1 expression was also correlated with progression of human prostate cancer ( 12 ). Although it has been suggested that Id-2 and Id-4 promote cell proliferation through direct inactivation of pRB in human osteosarcoma and glioma cells ( 13 ), there is little evidence on the mechanisms involved in the function of Id-1. Recently, Id-1 has been shown to facilitate the bypass of replicative senescence by directly inhibiting p16INK4a expression in mouse and young human diploid fibroblasts ( 14 , 15 ). To study the direct effect of Id-1 on human prostate cancer cell growth and the possible mechanisms involved, in the present study we transfected an Id-1 expression vector into a prostate cancer cell line LNCaP, which showed undetectable levels of Id-1 in the absence of fetal calf serum (FCS), and isolated 10 stable transfectant clones. Here we report that ectopic Id-1 expression stimulated serum independent prostate cancer cell proliferation through inactivation of p16 INK4a /pRB pathway.

Materials and methods

Cell lines and cell culture conditions

Human prostate cancer cell line LNCaP was obtained from American Tissue Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 medium supplemented with 10% FCS and penicillin (50 units/ml) and streptomycin (50 mg/ml) 37°C.

Generation of Id-1 transfectants

The retroviral vector containing full length Id-1 cDNA (pBabe-Id-1) ( 6 ) or pBabe-puro was transfected into the PG13 packaging cell line (obtained from ATCC) using the calcium phosphate method. After one-week’s selection in 4 μg/ml puromycin, the culture medium containing infectious viruses was harvested for retroviral infection of LNCaP cells. Briefly, the virus-containing supernatant was mixed with an equal volume of fresh medium containing 8 μg/ml polybrene and then added to LNCaP cells. Puromycin (1 μg/ml), which killed all of the parental cells, was added 24 h later and ten Id-1 stable transfectant clones were isolated ~14 days after drug selection to generate LNCaP-pBabe-Id-1 C1 to C10 clones. Vector control was generated from a pool of >20 individual clones transfected with pBabe.

Measurement of cell growth

Two thousand cells were plated in each well in 24-well plates in medium containing 5% fetal calf serum (FCS). Serum free medium replaced the FCS containing medium 24 h after plating and the cells were counted every day using trypan blue assay. Each data point was tested on triplicate wells and each experiment was repeated at least three times. Cell growth curves were drawn using the means of each experiment and the error bars represent the standard error of the means.

Cell cycle analysis

Cells (5 × 10 5 ) were trypsinized and washed once in PBS. They were then fixed in cold 70% ethanol and stored at 4°C. Before testing, the ethanol was removed and the cells were resuspended in PBS. The fixed cells were then washed with PBS and treated with RNase (1 μg/ml) and stained with propidium iodide (50 μg/ml) for 30 min at 37°C. Cell cycle analysis was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter Electronics, Hialeah, FL).

5′-Bromo-2′-deoxyuridine (BrdU) incorporation

Cells grown on 4 mm Chamber slides (ICN, Biomedicals, Aurora, OH) were treated with BrdU (10 μM) for 2 h and then washed once with PBS. The cells were then fixed in cold methanol/acetone (1:1) for 5 min at room temperature and washed in PBS. The cells were incubated with monoclonal antibody against BrdU (1:10, Roche) for 1 h at 37°C and detailed procedures were described in the protocols provided in Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Each experiment was repeated three times and at least 1000 cells were evaluated in each experiment. The error bars represent the standard deviation (SD) from three independent experiments.

Western blotting

Cell lysate was prepared by suspending the cells in a modified radioimmunoprecipitation (RIPA) buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS) including proteinase inhibitors (1mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM PMSF), and protein concentrations were measured using the protein assay kit (Bio-Rad). Equal amounts of proteins (50 μg) were separated by electrophoresis on a 12.5% SDS–polyacrylamide gel (SDS–PAGE) and blotted onto the nitrocellulose membrane (Amersham). After blocking with 5% non-fat dry milk/2% BSA in TBS for 1 h, the blots were incubated with primary antibodies for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Amersham) for another 1 h. The immunoreactive signals were detected by ECL Plus western blot detection reagents (Amersham) following the manufacturer’s instructions. Antibodies against Id-1 (1:200, C20, Santa Cruz Biotechnology), p16 INK4a (1:500, N20, Santa Cruz Biotechnology), CDK4 (1:250, Transduction Laboratories), p21 Waf1 (1:1000, N20, Santa Cruz Biotechnology), p27 Kip1 (1:1000, Santa Cruz Biotechnology), CDK2 (1:2000, Transduction Laboratories) and pRB (1:500, Ab-1, Oncogene) were used. The relative amounts of each protein were quantitated as ratios to Actin (1:500, Amersham).

Results

Introduction of ectopic Id-1 expression and its effect on prostate cancer growth

The effect of FCS on Id-1 expression in LNCaP cells was studied using western blotting analysis. As shown in Figure 1A , Id-1 expression was high when cultured in 10% FCS and reduced with decreased FCS concentrations (10% to 0%). In the absence of FCS for 24 to 48 h, Id-1 was undetectable in LNCaP cells. To study the effect of ectopic Id-1 expression on prostate cancer cells, a retroviral vector containing full-length human Id-1 cDNA (pBabe-Id-1) ( 6 ) was transfected into LNCaP cells and ten stable transfectants clones were selected in puromycin (1 mg/ml). Vector control was generated using a pool of multiple clones transfected with the control vector pBabe. As shown in Figure 1B , in the absence of FCS, seven out of the ten transfectant clones expressed Id-1 at different levels. The effect of Id-1 on prostate cancer cell growth was studied on these transfectant clones and additional controls, including the parental LNCaP cells and LNCaP-pBabe (vector control) under the same culture conditions.

It has been shown that when cultured in serum free medium and drug selective conditions, LNCaP cells sometimes show unstable morphology, however, we did not observe any significant morphological changes in LNCaP cells after introduction of Id-1 or cultured in serum free medium for up to 72 h (Figure 2A ). However, introduction of Id-1 resulted in an increase in cell growth which was also correlated with the expression levels of Id-1 (Figure 2B ).

Effect of Id-1 expression on DNA synthesis and cell cycle distribution in LNCaP cells

Next we studied if the Id-1 induced cell growth was due to its ability to initiate DNA synthesis in prostate cancer cells in serum-free medium. Cell cycle analysis showed that in the absence of FCS, there was 5.89% of S phase cells in the control LNCaP-pBabe cells but the percentage of S phase cells was significantly increased (11–19%) in the Id-1 expressing transfectants (Id-1-C2-7 and C10) (Figure 3A ). The number of S phase cells present in these transfectants was comparable with the vector control LNCaP-pBabe cultured in 10% FCS (14%). However, there was no significant increase in S phase cells in Id-1-C1, 8 and 9 (6–8%), which showed undetectable levels of Id-1 under the same culture conditions, compared with LNCaP-pBabe. The Id-1 induced DNA synthesis was also evident when measured by BrdU incorporation (Figure 3B ). After 48 h in serum-free medium, all the Id-1 expressing clones showed increased BrdU incorporation (25–80% increase) compared with the vector control (LNCaP-pBabe) or the Id-1 negative clones (C1, C8, C9). The level of increment was correlated with the levels of Id-1 expression, as C4 and C5 showed both higher Id-1 expression and BrdU incorporation compared with Id-1-C2 (Figures 1B and 3B ). The DNA synthesis rate in the clones with higher Id-1 levels was similar to the control LNCaP-pBabe cultured in 10% FCS.

Effect of Id-1 expression on RB/p16 INK4a pathway

To investigate the mechanisms involved in Id-1-induced cell proliferation in prostate cancer cells, we studied the expression levels of p16 INK4a , CDK4, p21 Waf1 , p27 Kip1 , CDK2 and RB in the Id-1 expressing clones and compared with the controls. As shown in Figure 4B , p16 INK4a was much lower or undetectable in all of the Id-1 expressing clones (Id-1-C2-7 and C10), while ~2 to 3-fold increase in p16 INK4a levels was observed in the controls and the Id-1 negative clones (C1, C8 and C9). In the presence of 10% FCS, LNCaP-pBabe also showed decreased p16 INK4a levels compared with the controls cultured in serum free medium. These results clearly demonstrate that expression of Id-1 reduced p16 INK4a protein levels in LNCaP cells. We also found that the phosphorylated form of CDK4 (upper band) (Figure 4B ) and CDK2 (lower band) (Figure 4C ) was apparent in all of the Id-1 expressing clones but not in the controls or the Id-1 negative clones (Figure 4B ). However, we did not observe any significant changes in p21 Waf1 or p27 Kip1 levels in the Id-1 expressing clones (Figure 4C ). As shown in Figure 4D , in the Id-1 expressing transfectants, phosphorylated RB (upper band) was found in all of the clones while there was no evidence of RB phosphorylation in the controls or Id-1 negative transfectants.

Discussion

In this study, we have demonstrated the significance of Id-1 expression in serum independent proliferation of prostate cancer cells. In addition, our results indicate that inactivation of RB pathway may be responsible for its action. Our evidence may provide a possible novel mechanism on the molecular basis of prostate carcinogenesis.

After transfection of Id-1, LNCaP cells showed an increase in serum independent growth (Figure 2B ) which was accompanied with increased percentage of cell cycle S phase cells (Figure 3A ) and BrdU incorporation rate (Figure 3B ). Previously, it was reported that ectopic Id-1 expression led to cell cycle G 1 to S progression in mouse 3T3 cells and human fibroblasts ( 17 , 18 ) and inactivation of Id-1 by antisense oligonucleotides resulted in decreased cell proliferation ( 16 , 19 ). Our results are consistent with previous findings on mouse cells and human breast cancer cells that ectopic Id-1 expression stimulated DNA synthesis and induced cell cycle progression from G 1 to S phase ( 16 , 18 ). Our evidence further confirms the function of Id-1 as a promoter of cell proliferation in human cancers including prostate cancer.

Ectopic Id-1 expression induced RB phosphorylation in human keratinocytes ( 4 ) and down-regulation of p16 INK4a in mouse and human young primary fibroblasts ( 14 , 15 ). In the present study, ectopic Id-1 expression resulted in down-regulation of p16 INK4a in LNCaP cells (Figure 4B ). One of the functions of p16 INK4a is to inhibit the function of cyclin dependent kinases such as CDK4 and prevents phosphorylation of RB. We also found an increase in the expression of phosphorylated CDK4 (Figure 4B , upper band). This indicates that activation of CDK4 by phosphorylation was associated with down-regulation of p16 INK4a in the Id-1 transfectants. One of the pathways that regulates RB phosphorylation and controls cell cycle from G 1 to S progression is through cyclinD and CDK4/6 complex. The activated cyclinD/CDK4 complex can phosphorylate RB and prevents its binding to E2F, resulting in the entry from G 1 to S progression ( 20 ). In the Id-1 transfectants, phosphorylated RB (upper band) was evident in all of the Id-1 expressing clones but absent in the Id-1 negative clones or the controls (Figure 4D ). These lines of evidence indicate that Id-1 expression resulted in the phosphorylation of RB protein possibly through down-regulation of p16 INK4a . The decreased p16 INK4a and increased RB phosphorylation in Id-1 transfectants also correlated with the increased S phase fraction (Figure 3A ) and BrdU incorporation rate (Figure 3B ) in these cells. These results suggest that the effect of Id-1 on growth stimulation on prostate cancer cells may be due to the decreased p16 INK4a , in turn the inactivation of RB. Previously, partial inhibition of p16 INK4a by ectopic Id-1 expression was observed in human keratinocytes, but no significant changes were found in CDK4 and RB levels ( 5 ). Our evidence, however, agrees with a separate study showing that Id-1 expression induced RB phosphorylation through inactivation of p16 INK4a ( 4 ).

Like p16 INK4a , p21 Waf1 and p27 Kip1 are other kinase inhibitors that have been shown to promote de-phosphorylation of RB by inhibiting CDK2 ( 21 ). In mouse 3T3 cells, overexpression of Id-1 leads to inhibition of p21 Waf1 at both mRNA and protein levels which correlate with the increased cell growth ( 22 ). However, there was no evidence of p21 Waf1 involvement in a separate study on human keratinocytes transfected with Id-1, even though the Id-1 induced cell growth was also observed ( 5 ). This indicates that interaction between Id-1 and p21 Waf1 may be cell type specific. In the present study, we did not observe any significant changes in p21 Waf1 or p27 Kip1 levels in the Id-1 expressing clones (Figure 4C ). However, phosphorylated CDK2 levels were found to be increased in these cells, indicating the involvement of additional factors in the activation of CDK2. It is possible that increased CDK2 phosphorylation or possible activation of CDK2 is independent of either p21 Waf1 or p27 Kip1 and the mechanisms involved in this process are currently under investigation. Nevertheless, activation of CDK2 may facilitate the phosphorylation of RB observed in the Id-1 transfectants.

In summary, we provide evidence for the first time on Id-1 induced cell proliferation in prostate cancer cells. The evidence that decreased p16 INK4a expression and increased CDK and pRB phosphorylation were observed in Id-1 expressing transfectants indicates that Id-1 may stimulate prostate cancer growth through inactivation of p16 INK4a /pRB pathway. Both Id-1 overexpression ( 12 ) and inactivation of p16 INK4a and RB ( 23 , 24 ) are common events in prostate cancer, and our results provide a possible mechanism on the molecular basis of prostate carcinogenesis.

Fig. 1.

( A ) Serum-dependent Id-1 expression in LNCaP cells. LNCaP cells were cultured in medium (RPMI 1640, Sigma) containing different concentrations of FCS for 24 to 48 h before being analyzed by western blotting. Note that Id-1 expression decreases with decreased FCS concentrations. ( B ) Id-1 expression levels in stable transfectant clones (Id-1-C1-10), vector control (pBabe) and parental LNCaP cells cultured in serum free medium for 48 h. Note that seven out of 10 clones express different levels of Id-1 protein while Id-1 is undetectable in the controls. Expression of actin was tested as an internal control.

Fig. 1.

( A ) Serum-dependent Id-1 expression in LNCaP cells. LNCaP cells were cultured in medium (RPMI 1640, Sigma) containing different concentrations of FCS for 24 to 48 h before being analyzed by western blotting. Note that Id-1 expression decreases with decreased FCS concentrations. ( B ) Id-1 expression levels in stable transfectant clones (Id-1-C1-10), vector control (pBabe) and parental LNCaP cells cultured in serum free medium for 48 h. Note that seven out of 10 clones express different levels of Id-1 protein while Id-1 is undetectable in the controls. Expression of actin was tested as an internal control.

Fig. 2.

Cellular morphology and cell growth rate in LNCaP cells and the Id-1 transfectants. ( A ) Morphological changes before and after introduction of Id-1 in LNCaP cells. ( 1 ): pBabe cultured in 5% FCS; ( 2 ): pBabe cultured in serum-free (SF) medium for 48 h; ( 3 ) and ( 4 ): Id-1-C2 and C7 cultured in SF medium for 48 h. Photos were taken under 200× magnification. Note that there are no significant morphological changes before and after introduction of Id-1 . ( B ) Growth curves of the Id-1 transfectants and pBabe. Each time point was derived from three independent experiments and the error bars represent standard deviation. Note that increased cell growth rate is correlated with the increased levels of Id-1 expression.

Fig. 2.

Cellular morphology and cell growth rate in LNCaP cells and the Id-1 transfectants. ( A ) Morphological changes before and after introduction of Id-1 in LNCaP cells. ( 1 ): pBabe cultured in 5% FCS; ( 2 ): pBabe cultured in serum-free (SF) medium for 48 h; ( 3 ) and ( 4 ): Id-1-C2 and C7 cultured in SF medium for 48 h. Photos were taken under 200× magnification. Note that there are no significant morphological changes before and after introduction of Id-1 . ( B ) Growth curves of the Id-1 transfectants and pBabe. Each time point was derived from three independent experiments and the error bars represent standard deviation. Note that increased cell growth rate is correlated with the increased levels of Id-1 expression.

Fig. 3.

Induction of prostate cancer cell proliferation in Id-1 expressing transfectants. ( A ) Cell cycle distribution in the cells cultured in SF medium for 48 h, unless indicated. Flow cytometric analysis was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter). Note that there is an increased number of S phase cells in the cells expressing Id-1. ( B ) BrdU incorporation in Id-1 transfectants and controls. At least 500 cells were counted in each experiment and the percentage of BrdU positive cells was calculated and compared with the controls. All cells were cultured in SF medium for 48 h before testing, unless otherwise indicated. Note that increased BrdU incorporation rate is found in Id-1 expressing clones.

Fig. 3.

Induction of prostate cancer cell proliferation in Id-1 expressing transfectants. ( A ) Cell cycle distribution in the cells cultured in SF medium for 48 h, unless indicated. Flow cytometric analysis was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter). Note that there is an increased number of S phase cells in the cells expressing Id-1. ( B ) BrdU incorporation in Id-1 transfectants and controls. At least 500 cells were counted in each experiment and the percentage of BrdU positive cells was calculated and compared with the controls. All cells were cultured in SF medium for 48 h before testing, unless otherwise indicated. Note that increased BrdU incorporation rate is found in Id-1 expressing clones.

Fig. 4.

Western blotting analysis of p16 INK4a , CDK4, p21 Waf1 , p27 Kip1 , CDK2 and RB expression in Id-1 transfectants and controls. Cells were cultured in SF medium for 48 h before harvesting, unless otherwise indicated. Results represent three independent experiments. ( A ) Id-1 expression levels in the transfectants and the controls; ( B ) Decreased p16 INK4a expression and the presence of CDK4 phosphorylation are found in the cells expressing Id-1; ( C ) No significant changes in p21 Waf1 and p27 Kip1 levels after Id-1 transfection but there is an increase in the phosphorylation of CDK2; ( D ) Increased RB phosphorylation is present in the cells expressing Id-1 but absent in the controls.


Inhibition of colon carcinogenesis by a standardized Cannabis sativa extract with high content of cannabidiol.

1
Department of Pharmacy, University of Naples Federico II, Naples, Italy; Endocannabinoid Research Group, Italy; School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom.
2
Department of Pharmacy, University of Naples Federico II, Naples, Italy; Endocannabinoid Research Group, Italy.
3
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom.
4
Department of Pharmacy, University of Naples Federico II, Naples, Italy; Endocannabinoid Research Group, Italy. Electronic address: aaizzo@unina.it.

Abstract

PURPOSE:

Colon cancer is a major public health problem. Cannabis-based medicines are useful adjunctive treatments in cancer patients. Here, we have investigated the effect of a standardized Cannabis sativa extract with high content of cannabidiol (CBD), here named CBD BDS, i.e. CBD botanical drug substance, on colorectal cancer cell proliferation and in experimental models of colon cancer in vivo.

METHODS:

Proliferation was evaluated in colorectal carcinoma (DLD-1 and HCT116) as well as in healthy colonic cells using the MTT assay. CBD BDS binding was evaluated by its ability to displace [(3)H]CP55940 from human cannabinoid CB1 and CB2 receptors. In vivo, the effect of CBD BDS was examined on the preneoplastic lesions (aberrant crypt foci), polyps and tumours induced by the carcinogenic agent azoxymethane (AOM) as well as in a xenograft model of colon cancer in mice.

RESULTS:

CBD BDS and CBD reduced cell proliferation in tumoral, but not in healthy, cells. The effect of CBD BDS was counteracted by selective CB1 and CB2 receptor antagonists. Pure CBD reduced cell proliferation in a CB1-sensitive antagonist manner only. In binding assays, CBD BDS showed greater affinity than pure CBD for both CB1 and CB2 receptors, with pure CBD having very little affinity. In vivo, CBD BDS reduced AOM-induced preneoplastic lesions and polyps as well as tumour growth in the xenograft model of colon cancer.

CONCLUSIONS:

CBD BDS attenuates colon carcinogenesis and inhibits colorectal cancer cell proliferation via CB1 and CB2 receptor activation. The results may have some clinical relevance for the use of Cannabis-based medicines in cancer patients.


Induction of apoptosis by cannabinoids in prostate and colon cancer cells is phosphatase dependent.

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.

Abstract

AIM:

We hypothesized that the anticancer activity of cannabinoids was linked to induction of phosphatases.

MATERIALS AND METHODS:

The effects of cannabidiol (CBD) and the synthetic cannabinoid WIN-55,212 (WIN) on LNCaP (prostate) and SW480 (colon) cancer cell proliferation were determined by cell counting; apoptosis was determined by cleavage of poly(ADP)ribose polymerase (PARP) and caspase-3 (Western blots); and phosphatase mRNAs were determined by real-time PCR. The role of phosphatases and cannabinoid receptors in mediating CBD- and WIN-induced apoptosis was determined by inhibition and receptor knockdown.

RESULTS:

CBD and WIN inhibited LNCaP and SW480 cell growth and induced mRNA expression of several phosphatases, and the phosphatase inhibitor sodium orthovanadate significantly inhibited cannabinoid-induced PARP cleavage in both cell lines, whereas only CBD-induced apoptosis was CB1 and CB2 receptor-dependent.

CONCLUSION:

Cannabinoid receptor agonists induce phosphatases and phosphatase-dependent apoptosis in cancer cell lines; however, the role of the CB receptor in mediating this response is ligand-dependent.


Chemopreventive effect of the non-psychotropic phytocannabinoid cannabidiol on experimental colon cancer.

Department of Experimental Pharmacology, Endocannabinoid Research Group, University of Naples Federico II, Naples, Italy.

Abstract

Colon cancer affects millions of individuals in Western countries. Cannabidiol, a safe and non-psychotropic ingredient of Cannabis sativa, exerts pharmacological actions (antioxidant and intestinal antinflammatory) and mechanisms (inhibition of endocannabinoid enzymatic degradation) potentially beneficial for colon carcinogenesis. Thus, we investigated its possible chemopreventive effect in the model of colon cancer induced by azoxymethane (AOM) in mice. AOM treatment was associated with aberrant crypt foci (ACF, preneoplastic lesions), polyps, and tumour formation, up-regulation of phospho-Akt, iNOS and COX-2 and down-regulation of caspase-3. Cannabidiol-reduced ACF, polyps and tumours and counteracted AOM-induced phospho-Akt and caspase-3 changes. In colorectal carcinoma cell lines, cannabidiol protected DNA from oxidative damage, increased endocannabinoid levels and reduced cell proliferation in a CB(1)-, TRPV1- and PPARγ-antagonists sensitive manner. It is concluded that cannabidiol exerts chemopreventive effect in vivo and reduces cell proliferation through multiple mechanisms.

In Vitro Anticancer Activity of Plant-Derived Cannabidiol on Prostate Cancer Cell Lines

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DOI: 10.4236/pp.2014.58091    4,858 Downloads   13,536 Views   Citations

ABSTRACT

<span “=””>Cannabinoids, the active components of Cannabis sativa Linnaeus, have received renewed interest in recent years due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory effects and tumor regression, but their use in chemotherapy is limited by their psychotropic activity. To date, cannabinoids have been successfully used in the treatment of nausea and vomiting, two common side effects that accompany chemotherapy in cancer patients. Most non-THC plant cannabinoids e.g. cannabidiol and cannabigerol, seem to be devoid of psychotropic properties. However, the precise pathways through which these molecules produce an antitumor effect have not yet been fully characterized. We therefore investigated the antitumor and anti-inflammatory activities of cannabidiol (CBD) in human prostate cancer cell lines LNCaP, DU145, PC3, and assessed whether there is any advantage in using cannabis extracts enriched in cannabidiol and low in THC. Results obtained in a panel of prostate cancer cell lines clearly indicate that cannabidiol is a potent inhibitor of cancer cell growth, with significantly lower potency in non-cancer cells. The mRNA expression level of cannabinoid receptors CB1 and CB2, vascular endothelial growth factor (VEGF), PSA (prostate specific antigen) are significantly higher in human prostate cell lines. Treatment with Cannabis extract containing high CBD down regulates CB1, CB2, VEGF, PSA, pro-inflammatory cytokines/chemokine IL-6/IL-8. Our overall findings support the concept that cannabidiol, which lacks psychotropic activity, may possess anti-inflammatory property and down regulates both cannabinoid receptors, PSA, VEGF, IL-6 and IL-8. High CBD cannabis extracts are cytotoxic to androgen responsive LNCaP cells and may effectively inhibit spheroid formation in cancer stem cells. This activity may contribute to its anticancer and chemosensitizing effect against prostate cancer. Cannabidiol and other non-habit forming cannabinoids could be used as novel therapeutic agents for the treatment of prostate cancer.

 
 
 

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