Proapoptotic effect of Cancer Marijuana (endocannabinoids) in prostate cancer cells
- Physiology and Biophysics Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago 8389100, Chile.
- Pathological Anatomy Service, Clinic Hospital of the University of Chile, Santiago 8389100, Chile.
- Urology Service, Clinic Hospital of the University of Chile, Santiago 8389100, Chile.
- Laboratory of Nutrition and Metabolic Regulation, INTA, University of Chile, Santiago 8389100, Chile.
Abstract Cancer Marijuana
In the early stages of Cancer Marijuana treatments in 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.
Anandamide (ANA) is an endogenous lipid which acts as a cannabinoid receptor ligand and with potent anticarcinogenic activity in several cancer cell types.
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.
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.
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.
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.
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.
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
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 ( 3 – 5 ), 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.
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).
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.
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.