Abstract
Background: Bicalutamide (BIC) is an alternative treatment to castration for advanced prostate cancer. Breast events are common adverse effects which can be effectively prevented by the concurrent administration of tamoxifen, a selective estrogen receptor modulator. Materials and Methods: We investigated the effects of BIC, 4-hydroxy Tamoxifen (4OHT), the active metabolite of tamoxifen, and their combination on the expression of a panel of genes implicated in prostate cancer development and progression in LNCaP cells stimulated with dihydrotestosterone. Results: Our findings confirm the anti-proliferative activity of BIC on LNCaP cell growth but also show the down-regulating function of this anti-androgen on the expression of genes involved in tumor proliferation and invasion [cyclins, caspases, epidermal growth factor (EGF)]. The combination with 4OHT exerts a synergistic effect on the downregulation of some genes involved in prostate cancer progression. Conclusion: The observation that the expression of several genes [such as B-cell lymphoma-2 (BCL2), myelocytomatosis oncogene (MYC), caspases] is modulated midly-to-moderately, after 4OHT addition suggests that this combined approach in the clinical setting should be further investigated through appropriate trials.
Monotherapy of bicalutamide (BIC) at 150 mg daily is an established treatment for selected patients with prostate cancer who wish to avoid the side-effects of androgen deprivation. However gynaecomastia and breast pain are common adverse events and result in treatment discontinuation in up to 20% of patients (1, 2). We have previously shown that the co-medication of BIC with tamoxifen, an anti-estrogen commonly used to manage breast cancer, is highly effective in preventing both events without inducing unpleasant side-effects or interfering with the antitumor activity of BIC (3, 4). In fact no differences in prostate-specific antigen (PSA) behaviour were demonstrated between patients treated with BIC-alone and those treated with BIC and tamoxifen (3). Moreover, circulating plasma concentrations of BIC were not influenced by the co-administration of tamoxifen (5). To better-define any putative interaction between these drugs at the cellular level, we subsequently performed several experiments using the androgen-responsive prostate cancer cell line LNCaP (6). This cell line expresses the androgen-receptor (AR), exhibits androgen-regulated expression of androgen-responsive genes such as the PSA encoding gene, grows in an androgen-regulated manner in cell culture conditions and forms androgen-dependent tumours in xenograft models (7, 8). LNCaP cells express a mutation in the hormone-binding domain of AR which causes the stimulation by anti-androgens, with the exception of BIC, which acts as an AR antagonist and causes both cell proliferation inhibition and cell apoptosis (9, 10). Our previous experiments confirmed that LNCaP growth is sustained by dihydrotestosterone (DHT) and is inhibited by BIC in a dose-dependent manner, exactly as occurs in androgen-dependent prostate cancer in humans, and showed that no substantial interference with inhibitory effects of BIC on cell proliferation is induced by the co-administration of 4-hydroxy tamoxifen (4OHT), one of the more potent tamoxifen metabolites in humans (6). In view of these findings, we extended our studies in LNCaP cells by investigating the effects of BIC, 4OHT and their combination on the mRNA expression levels of a selected panel of genes implicated in prostate cancer progression.
Materials and Methods
Cell culture conditions. LNCaP fast-growing colony (FGC) prostate cancer cell line was purchased from ATCC/LGC (Promochem s.r.l. Milan, Italy). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 nM L-glutamine and 1% penicillin/streptomycin (PS) at 37°C and in an atmosphere of 5% CO2 in air. The medium was changed two times a week.
Cell growth and experiments. Our experiments were performed in white RPMI (no phenol red) medium, supplemented with 10% charcoal-treated FCS, 2 nM L-glutamine and 1% PS, in presence of DHT 0.1 nM. Log-phase growing cells were harvested from 75-cm2 flasks by trypsinization and loaded onto a 6-multiwell plate at a final density of 2.5×105 cells for cell growth experiments, while for gene expression experiments, cells were loaded into 25-cm2 flasks at a final density of 1×106 cells. After 48 h, the medium was changed and control cells were incubated with fresh white medium, containing 0.1 nM DHT-only, for 72 h. Subsequently, the cells were incubated for a further 72 h with 10 μM BIC, or 10 nM 4OHT, or a combination of the two, again in the presence of DHT. In each case, the total incubation time was 144 h. Cell viability was assessed through the trypan blue exclusion test. All experiments were run in triplicate and results were expressed as mean±standard error (SE).
Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR). Total RNA isolation from cells was achieved using Trizol reagent (Invitrogen Dynal, Irvine, CA, USA), according to the manufacturer's instructions. First-strand cDNA was synthesized using SuperScript™ II Reverse Transcriptase kit (Invitrogen) and oligo dT primers in a final volume of 20 μl.
qRT–PCR was performed on an I-Cycler (Bio-Rad, Hercules, CA, USA) using Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen) supplemented with 10 nM fluorescein (Bio-Rad), 3 pmol sense and antisense primers in a final reaction volume of 25 μl. All primers for the tested genes were designed using primer3 software (http://frodo.wi.mit.edu/primer3/input.htm) with an optimum temperature of 60°C and a product length of 80-150 nucleotides. After an initial denaturation step of 3 min, 50 cycles of 15 s at 95°C, followed by 30 s at 60°C, were performed. Fluorescence was measured during the annealing step in each cycle. After amplification, melting curves, with 80 steps of 15 s and 0.5°C increase, were determined to monitor amplicon identity.
Expression data were normalized using glyceraldehyde-3-phosphate dehydrogenase and RNA polymerase II as housekeeping genes. Relative expression values with standard errors were obtained using Qgene software. All the experiments were run in triplicate for each gene. Normalized data were imported into TMeV program (version 4.4.1; http://www.tm4.org/mev/), an application which includes various algorithms for the analysis, visualization and data mining of large-scale data. Unsupervised hierarchical clustering with Euclidean distance and average linkage was performed.
Statistical analysis. One-way ANOVA and Student's t-test were used to assess the statistical significance of differences between means. Differences between means were considered significant when p<0.05. All statistical analyses were carried out with the GraphPad Prism 4 software (GraphPad Software, Inc., La Jolla, CA, USA).
Results
Effects of treaments on LNCaP cell growth and PSA expression. The LNCaP cell line used in our study was the FGC subline, which is androgen-dependent for growth and does not express estrogen receptors. The proliferation of LNCaP cells is regulated by androgens in a biphasic manner: it is enhanced by low concentrations of DHT, whereas higher androgen doses induce a progressive decline in cellular proliferation (11). In this study, LNCaP cell proliferation, sustained with a low concentration of DHT (0.1 nM), was inhibited after BIC or 4OHT addition (BIC: −45%, p<0.01; 4OHT: −28%, p<0.05). No overt interaction between 4OHT and BIC was evident in comparison to the cells exposed to BIC-alone (Figure 1A). Since plasma PSA levels in patients with prostate cancer are considered a measure of tumour growth and disease progression, we evaluated the PSA gene expression and, as expected, the BIC treatment resulted in a significant suppression of PSA mRNA levels (-21%, p<0.05), compared to the basal condition. The same effect was observed after 4OHT treatment and when the two drugs were used in combination (Figure 1B).
Modulation of LNCaP cell growth and prostate-specific antigen (PSA) expression levels after treatments. To evaluate cell growth, LNCaP cells were treated either with 10 μM bicalutamide (BIC), or 10 nM 4-hydroxy tamoxifen (4OHT) or with the two compounds simultaneously (B+T), as indicated in the Materials and Methods section. Cell viability was assessed through the trypan blue exclusion test (A). The PSA gene expression levels were evaluated by quantitative real-time PCR. The LNCaP cells were treated with the same concentrations of BIC and 4OHT used for cell growth analysis. The bar graphs show the means±SE of quantitative real-time PCR from three independent experiments. The PSA expression levels are also represented as a colour scheme where green means down-regulated and red up-regulated (B). The experiments were repeated in triplicate and results are expressed as mean±SE. * And ** indicate p<0.05 and p<0.01 compared to the basal condition (CTR).
Panel of the selected genes implicated in prostate cancer development and progression.
Effects of BIC on gene expression. Genes selected for the present study are listed in Table I. The mRNA expression levels under different culture conditions were subjected to unsupervised hierarchical clustering analysis and results are summarized in Figure 2. The cluster analysis was used to identify the relationship between gene expression and treatments and the possible association between the genes implicated in prostate cancer progression. Two major clusters showing a different gene expression profiling were generated. In the upper cluster the stimulatory effect of BIC compared with the other two conditions is apparent. In particular, this effect involved all the caspase genes, but was statistically significant only for the pro-apoptotic CASP6 and CASP8 genes (p<0.05) (Figure 3). Another antiproliferative action of BIC treatment was confirmed by the moderate activation of phosphatase and tensin homolog (PTEN) expression (Figure 2), whose loss can promote cancer progression. Although the BIC concentration used in these experiments was significantly able to inhibit the LNCAP cell growth and to down-regulate PSA expression, it did not affect the AR expression. However, as can be seen in the lower cluster (Figure 2), the negative modulation of LNCAP cell growth was confirmed by the significant decrease of the CCNA2 mRNA level (p<0.01), while that for CCND1 showed a decreasing tendency that was not statistically significant (Figure 3). BIC treatment negatively affected genes with a mitogenic effect as epidermal growth factor (EGF) (p<0.05) or involved in invasion and metastasis such as matrix metallopeptidase 13 (MMP13) (p<0.01) (Figure 3). A moderate decrease in insulin-like growth-factor 1 (IGF1), telomerase reverse transcriptase (h-TERT) and BCL2-like 1 (BCL2L1) was also observed (Figure 2).
Effects of 4OHT on gene expression. The inhibitory effect of 4OHT was also confirmed on both cyclins, IGF1, platelet-derived growth factor (PDGF) and topoisomerase (DNA)-II beta (TOP2B) (p<0.05) (Figure 3). In contrast to BIC, 4OHT exerted a pro-apoptotic effect through BCL2L1 modulation (Figure 3). No significant changes in the AR gene expression were evident. Anti-estrogen administration was also associated with a moderate decrease in the expression of PTEN, h-TERT, EGF, MMP9, transforming growth factor beta 1 (TGFB1), MYC and E-Cadherin (CDH1) genes (Figure 2).
Unsupervised hierarchical clustering analysis of gene expression in LNCaP cells. Tumour cells were treated either with 10 μM bicalutamide (BIC), or 10 nM 4-hydroxy tamoxifen (4OHT) or with the two compounds simultaneously (B+T), as indicated in the Materials and Methods section. Up-regulated genes are shown in red, down-regulated genes in green, non-changing genes in black. CTR: Basal condition. See Table I for full gene names.
Effects of BIC and 4OHT combination on gene expression. The evaluation of BIC administration effects in combination with 4OHT may provide guidance to the potential value of this therapeutic approach. Taking into account the heat map represented in Figure 2, we evaluated three possible actions performed on the genes by the co-administration of 4OHT: no antagonistic effect, synergistic effect and antagonistic effect. As can be seen in Table II, while there was no 4OHT additive effect in the cell-cycle regulation, or, better still a synergistic effect on the down-regulation of the genes stimulating tumor progression and invasion was observed, a mild-to-moderate interaction of 4OHT on the remaining BIC modulated genes was found. Interestingly, the behaviour of some genes (TOP2A, BCL2, MYC and h-TERT) in the presence of the two drugs was opposite to what was observed when BIC and 4OHT were individually administered.
Genes in LNCaP cells modulated by bicalutamide and affected in different ways by the co-administration of 4-hydroxy tamoxifen.
Discussion
Androgens are the primary regulators of prostate cancer cell growth and proliferation. When androgens are ablated or withdrawn, apoptosis is observed in a proportion of cells, while those that survive are arrested in the G1 phase of the cell-cycle (12, 13). However, castration-resistant prostate cancer cells do not undergo apoptosis, suggesting that AR-mediated survival signalling is reactivated even at low androgen concentrations or that prostate cancer cells can develop other signalling pathways which are not AR-driven (14). Therefore, we investigated the effects of BIC, an anti-androgen frequently used to treat prostate cancer, on a selected panel of genes that are key regulators of cell cycle, proliferation, apoptosis, invasiveness and metastasis. In particular, we assessed whether the possible therapeutic utility of adding an anti-estrogen to counteract common adverse events such as gynaecomastia and breast pain, would be supported without a negative effect on BIC modulation.
Our results demonstrated that exposure to BIC resulted in a marked anti-proliferative and pro-apototic effect through the down-regulation of both cyclins and the induction of CASP6 and CASP8. This effect was not affected by the presence of 4OHT. Anti-apoptotic BCL2 family factors (BCL2 and BCL2L1) were affected differently under hormone-depleted conditions. In fact, we found that BCL2L1 was down-regulated after exposure to BIC-whereas the opposite happened following treatment with 4OHT. On the other hand, BCL2 expression was not affected by administration of BIC alone, but the presence of both drugs led to evident down-regulation. This suggests a different modulation of programmed cell death by these two drugs. Androgen-induced cell proliferation is also mediated by peptide growth factors produced by stromal or epithelial cells themselves, such as IGF1, EGF and TGFB1 (15). Namely, there is growing evidence that IGF1 and IGFBP3 may play a major role in prostate cancer promotion and progression (16). In the present study, BIC induced a moderate down-regulation of IGF1 expression levels that was statistically significant when the anti-androgen and 4OHT were co-administrated. The tumor suppressor gene PTEN is another key regulatory gene known to be involved in the regulation of cell proliferation and survival in human prostate cancer (17). In our study, PTEN expression was increased by treatment with BIC. By contrast, treatment of LNCaP cells with 4OHT, both alone and in combination with BIC, prevented the increase of PTEN mRNA expression in comparison to the basal condition. Results from several in vitro studies using established PC3 prostate cancer cell lines demonstrated that PTEN is a negative modulator of AR transcription activity (18). Under experimental conditions, PTEN and AR exert opposite effects on cell growth and apoptosis. Moreover, a recent study demonstrated that conditional expression of PTEN enhances in vitro sensitivity of LNCaP to BIC (19). The hTERT gene is also a potential target of androgens essential for tumour growth (20). h-TERT is the rate-limiting component of the telomerase complex, and its expression correlates with telomerase activity; both h-TERT expression and telomerase activity are elevated in most human tumours (21). MYC is also up-regulated by androgens in prostate epithelial cells and MYC family proteins are implicated in the autoregulation of AR (22). Moreover, it has been recently reported that androgen withdrawal after three days of treatment of LNCaP cells with BIC results in the decrease of both h-TERT and MYC expression (23). In our findings, MYC and h-TERT genes are included in the same cluster having a similar expression profile with a prevalent inhibitory effect after 4OHT admnistration (Figure 2). While the single administration of BIC or 4OHT was associated with a decrease in the mRNA levels of both genes, the growth inhibition of LNCaP cells after the co-administration of BIC and 4OHT did not result in a further negative modulation of h-TERT and MYC expression. The inhibitory effects of BIC were also confirmed on MMPs, a class of proteolytic enzymes that digest the extracellular matrix of the tumour-surrounding tissues (24). However, a possible synergistic effect between the two drugs was confirmed only on MMP9 gene expression. Indeed, MMP13 gene expression, significantly inhibited by BIC, was stimulated after co-administration with 4OHT.
Histogram representation of gene expression. A graphic representation of the modulation of genes implicated in cell cycle progression, apoptosis and invasion in LNCaP cells following treatment either with 10 μM bicalutamide (BIC), or 10 nM 4-hydroxy tamoxifen (4OHT) or with the two compounds simultaneously (B+T). The bar graphs show the means±SE of quantitative real-time PCR from three independent experiments. * And ** indicate p<0.05 and p<0.01 compared to the basal condition (CTR). See Table I for full gene names.
Our findings confirm the antiproliferative activity of BIC on DHT-substained prostate cancer cell growth, but also show the down-regulating function of this antiandrogen on the expression of genes involved in tumour proliferation and invasion. The addition of 4OHT does not appear to interfere with the inhibitory effects of BIC on cell proliferation: in fact, this combination appears to exert a synergistic effect on the down-regulation of some key genes involved in prostate cancer progression. However, although the simultaneous treatment with BIC and tamoxifen allows patients with prostate cancer to avoid unpleasant side-effects, such as gynecomastia or breast pain, the observation that several genes were affected mildly-to-moderately after 4OHT addition suggests that the safety of this combined approach in the clinical setting should be further investigated through appropriate trials.
Acknowledgements
This work was supported by the Italian Health Ministry (2005 conv. 93; 2005 conv. 65) and Compagnia di San Paolo (4820SD/cv2005/0265).
Footnotes
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Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received October 10, 2012.
- Revision received October 25, 2012.
- Accepted October 26, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
