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Clinical Cancer Research Vol. 12, 2812-2816, May 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Clinical

Effect of Calcitriol on Prostate-Specific Antigen In vitro and in Humans

Tomasz M. Beer1, Mark Garzotto2, Byung Park3, Motomi Mori3, Anne Myrthue1, Nicole Janeba2, David Sauer4 and Kristine Eilers1

Authors' Affiliations: 1 Division of Hematology and Medical Oncology, Oregon Health and Science University; 2 Division of Urology, Portland Veterans Affairs Medical Center; 3 Biostatistics Shared Resource, Oregon Health and Science University Cancer Institute; and 4 Department of Pathology, Oregon Health and Science University, Portland, Oregon

Requests for reprints: Tomasz M. Beer, Division of Hematology and Medical Oncology, Oregon Health and Science University, CR-145, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239-3098. Phone: 503-494-0365; Fax: 503-494-6197; E-mail: beert{at}ohsu.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Background: Calcitriol, the natural ligand for the vitamin D receptor, has significant potential in prostate cancer treatment. Measurement of its antineoplastic activity in prostate cancer clinical trials may be complicated by effects of calcitriol on prostate-specific antigen (PSA) production. We examined the effects of calcitriol at similar concentration on cell proliferation, androgen receptor (AR) expression, and PSA production in vitro and on PSA concentrations in prostate cancer patients.

Experimental Design: LNCaP prostate cancer cell proliferation was examined by cell counts 6 days after exposure to a range of concentrations of calcitriol. AR and PSA protein was quantified in LNCaP cells over 96 hours after exposure to 1 nmol/L calcitriol. Serum PSA and free PSA was serially measured by immunoassay over a period of 8 days in patients with hormone-naïve prostate cancer after a single dose of 0.5 µg/kg calcitriol.

Results: Calcitriol treatment resulted in dose-dependent growth inhibition of LNCaP with ~50% growth inhibition at the clinically achievable concentration of 1 nmol/L. Time-dependent up-regulation of AR expression and of PSA production in LNCaP cells was shown at the same concentration. No significant change in serum PSA or free PSA over 8 days was seen in eight subjects treated with a single dose of 0.5 µg/kg calcitriol. The analysis was powered to detect a 1.23-fold change between the baseline and day 8 serum PSA.

Conclusions: At clinically achievable concentrations, calcitriol inhibits growth and induces AR and PSA expression in LNCaP cells. We did not detect similar changes in serum PSA or free PSA in patients exposed to similar concentrations of calcitriol. Thus, a PSA flare, predicted by preclinical systems, is unlikely to occur in patients and therefore unlikely to complicate interpretation of clinical trial outcomes.

There is ample preclinical evidence supporting the investigation of vitamin D receptor ligands in prostate cancer therapy. Both tissue culture systems (1–6) and mouse models (6–8) show the inhibitory activity of vitamin D receptor ligands in prostate cancer. Physiologic levels of calcitriol range between 0.05 and 0.16 nmol/L; however, significant growth inhibition requires calcitriol concentrations of ≥1 nmol/L (416.7 pg/mL) in most studies (4, 5).

Encouraging preclinical data have led to the development of several clinical trials of calcitriol in prostate cancer, all of which sought to escalate the doses of calcitriol in the hope of achieving therapeutic concentrations. A modest degree of dose escalation was possible with daily dosing before hypercalcemia and/or hypercalcuria was encountered (9, 10). Weekly dosing of calcitriol allowed significant dose escalation and peak serum concentrations of calcitriol above 1 nmol/L were achievable with this approach (11, 12).

Activity of new agents in prostate cancer is routinely assessed by measuring changes in serum prostate-specific antigen (PSA) levels (13). Indeed, recent data suggest that in androgen-independent prostate cancer, posttherapy PSA changes can be used as a surrogate for overall survival (14). However, before accepting PSA reduction as a measure of clinical activity, it is essential to evaluate the direct effect of the agent under study on PSA production and secretion. The use of serum PSA to monitor the effects of calcitriol on human prostate cancer has been questioned due to the possibility that calcitriol may affect PSA production independently of its effect on tumor proliferation.

Differentiating agents like calcitriol can stimulate PSA production in vitro. Indeed, several investigators have shown vitamin D receptor ligand–induced increases in PSA in LNCaP cells, the most well-characterized PSA-producing in vitro model of prostate cancer (3, 4, 15–19).

Miller et al. (15) showed an ~4-fold increase in PSA secretion 72 hours after treating LNCaP cells with calcitriol. Similar results were reported by Skowronski and other investigators (3, 4, 18, 19). Hsieh et al. (16, 17) showed increases in both intracellular and secreted PSA with calcitriol therapy of LNCaP cells.

The mechanism by which calcitriol stimulates PSA production is not completely understood. Hsieh et al. (16) showed that PSA induction is preceded by up-regulation of androgen receptor (AR) expression, suggesting a possible indirect mechanism for calcitriol induction of PSA. Calcitriol-mediated induction of translocation of the AR to the nucleus has also been shown (17). Hsieh et al. (17) has suggested that the increase in AR in the nucleus leads to increased transcription of PSA mRNA by allowing a more efficient and productive interaction with the upstream regulatory region of the PSA gene.

Taken together, these preclinical data bring to light the need for a critical evaluation of the effects of calcitriol therapy on PSA production in prostate cancer patients. In this study, we sought to examine the acute effects of calcitriol on PSA regulation in vitro and in human subjects with androgen-responsive prostate cancer.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. LNCaP cells were obtained from American Type Tissue Culture Collection (Manassas, VA). Cells were subcultured every 48 to 72 hours in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Cells were incubated at 37°C with standard 5% CO2 content in a humidified incubator.

Cell proliferation. 1,25-Dihydroxyvitamin D3 (calcitriol) was a kind gift from Dr. Milan Uskokovic, Hoffmann-La Roche (Nutley, NJ). LNCaP cells (100,000 per well in six-well plates) were treated with indicated concentrations of calcitriol for 24 hours and again 96 hours after plating. Cells were harvested by trypsinization 6 days after treatment and resuspended in medium containing 0.4% trypan blue (Invitrogen). Live cells were counted under a microscope using a hemocytometer (Hausser Scientific, Horsham, PA).

Immunoblots. Log-phase LNCaP were treated with 1 nmol/L calcitriol and harvested at 0, 24, 48, 72, and 96 hours after treatment. Cells were lysed using radioimmunoprecipitation assay buffer (1% NP40, 0.1% SDS, and 0.5% deoxycholic acid) containing protease inhibitors (Sigma, St. Louis, MO). Protein concentration was determined using a BCA protein assay method (Pierce Biotechnology, Rockford, IL). Proteins (30 µg) were separated on a 12% SDS-PAGE gel then transferred to a nitrocellulose membrane. The membrane was probed with polyclonal rabbit anti-human PSA (DakoCytomation, Carpinteria, CA) at a dilution of 1:10,000, polyclonal rabbit anti-human AR (N-20; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:2,000, and monoclonal mouse anti-ß-tubulin (Sigma) at a dilution of 1:1,000. The immunoblot was developed using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). Densitometry was done using Opti-Quant software (Perkin-Elmer, Boston, MA). Results are expressed as a ratio of the loading control.

To further explore the effect of shorter-term calcitriol treatment on PSA, LNCaP cells were incubated with 1 nmol/L calcitriol for 24 and 48 hours harvested after 48 hours and interrogated for PSA production as above.

Patients. Detailed eligibility criteria were previously described (12). Briefly, patients had adenocarcinoma of the prostate with an increasing serum PSA after either prostatectomy or radiation therapy; Eastern Cooperative Oncology Group performance status ≤3; adequate cardiac, hepatic, renal, and bone marrow function; no history of cancer-related hypercalcemia; no prior systemic treatment for prostate cancer; and no kidney stones within 5 years. The study was approved by the institutional review boards of the Oregon Health and Science University and Portland Veterans Affairs Medical Center. Signed, informed patient consent was obtained before any procedures. Patients received 0.5 µg/kg calcitriol (Rocaltrol, 0.5 µg capsules, Roche Pharmaceuticals, Nutley, NJ) given orally at time 0. Each dose was divided into four doses and taken orally during each hour of a 4-hour period. Detailed calcitriol pharmacokinetics was measured in the same patients that are included in this analysis and have been previously described (12). Briefly, mean peak calcitriol concentrations were 1.9 nmol/L (95% confidence interval, 1.39-2.36 nmol/L) and the mean elimination half-life was 10.9 hours (95% confidence interval, 7.7-14.1 hours).

Measurment of serum PSA and free PSA. Serum samples from eight subjects were obtained immediately before calcitriol administration and at 0.25, 0.5, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 10, 15, 24, 48, 72, 96, 120, 144, and 192 hours following calcitriol administration. Samples were available for analysis for all subjects through the first 24 hours, six of the eight subjects at 72 hours, seven of the eight subjects at 96 and 120 hours, and five of the eight subjects at 144 and 192 hours. Free PSA was measured using the Elecsys 2010 free PSA immunoassay (Roche Diagnostics, Mannheim, Germany) with a lower detection limit of 0.01 ng/mL. Total PSA was measured using the Elecsys 2010 total PSA immunoassay (Roche Diagnostics), with a lower detection limit of 0.002 ng/mL. Both assays were run according to published instructions from the manufacturer.

Statistical analyses. We characterized the effects of calcitriol on PSA over 8 days. Because the distributions of PSA and free PSA were positively skewed, we applied log2 transformation. Because PSA and free PSA data from the same patient are likely to be correlated, we used the mixed-effects model to evaluate changes in PSA and free PSA over time. Based on the Bayesian information criterion (20), we selected a compound symmetrical structure as the variance-covariance matrix (also called the random intercept model). Post hoc comparisons with the baseline were done using Dunnett's multiple comparison procedure (21). P < 0.05 was considered statistically significant. All analyses were done using Statistical Analysis System version 9.1 (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
In vitro experiments. In Fig. 1A , we show dose-dependent growth inhibition of LNCaP cells by calcitriol. Notably, at clinically achievable concentrations of 1 nmol/L, ~50% inhibition is observed. In Fig. 1B, we show time-dependent up-regulation of AR expression and of PSA production in LNCaP cells after exposure to 1 nmol/L calcitriol. Androgen receptor expression is increased 1.31-fold at 24 hours, 2-fold at 48 hours, 1.93-fold at 72 hours, and 1.6-fold at 96 hours. No significant change in PSA expression is seen after 24 hours. PSA expression is increased 2.75-fold at 48 hours, 2.87-fold at 72 hours, and 2.3-fold at 96 hours. Thus, we show that in vitro exposure to calcitriol concentrations similar to those studied in our patients produces significant growth inhibition, with divergent results on AR and PSA expression. At a clinically achievable dose, calcitriol induces AR expression and increases PSA expression nearly 3-fold within 48 hours. PSA induction measured at 48 hours was similar after either continuous or 24-hour "pulse" exposure to calcitriol (data not shown).


Figure 1
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  		Fig. 1. Effect of calcitriol on prostate cancer cell growth inhibition, AR signaling, and PSA production. A, log-phase LNCaP cells were treated with specified concentration of calcitriol and harvested 6 days later for assessment of cell number. Columns, mean of triplicate samples; bars, SD. B, immonoblot assay showing effect of 1 nmol/L calcitriol on PSA production and AR expression levels in LNCaP cells over 96 hours.

 
PSA results in human subjects. Baseline serum total and free PSA (untransformed) for the eight subjects is shown in Table 1 . To visualize a change in PSA and free PSA after calcitriol administration, we plotted total PSA and free PSA (log2 scale) for each individual patient (see Fig. 2A and B ). In a log2 scale, a change in b unit implies 2b-fold change in PSA from the baseline. These figures suggest that, overall, there is very little change in PSA during an 8-day period. Table 1 shows the descriptive statistics for total PSA and free PSA at baseline.


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  		Table 1. Descriptive statistics for total PSA and free PSA at baseline

 

Figure 2
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  		Fig. 2. A, individual profile of total PSA over time (log2 scale). B, individual profile of total PSA over time (log2 scale).

 
The analyses based on the mixed-effects model failed to show a significant time trend for either total PSA (P = 0.4444) or free PSA (P = 0.3854). Further, none of the changes from the baseline was significant in total PSA or free PSA using Dunnett's multiple comparison procedure (Table 2 ). We then analyzed the ratio of free to total PSA. Again, no effect of time on the ratio of free to total PSA was observed (data not shown).


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  		Table 2. Comparison of log2(total PSA) and log2(free PSA) at baseline versus subsequent time points using Dunnett's multiple comparison procedure

 
Finally, we examined both total and free PSA during the first 24 hours following calcitriol administration only. The mixed-effects analysis failed to show a significant change during the first 24-hour period for either total PSA (P = 0.30) or free PSA (P = 0.38; data not shown).

Because a lack of statistical significance could be due to the small sample size (n = 8), we did a post hoc power analysis to determine a fold change that could be detected given the present sample size and SD. The study would have been able to detect 1.23-fold change between the baseline and 192 hours with 80% power. Therefore, even with this modest sample size, we had adequate power to detect a clinically meaningful change in total or free PSA.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Calcitriol and other vitamin D receptor ligands have shown significant promise in the prevention and treatment of prostate cancer in both preclinical and clinical investigations. Inhibition of cellular growth is thought to occur through a number of downstream molecular targets (22). These growth-inhibitory effects have also been shown in several murine models of prostate cancer. Currently, the use of calcitriol in combination with chemotherapy is under intense study for patients with advanced disease (23).

As changes in serum PSA are routinely used to screen for drug activity and calcitriol has been reported to increase PSA production in vitro, we did parallel analyses of acute affects of calcitriol in vitro and in patients at similar concentrations. Interestingly, these studies showed discordant effects of calcitriol on cell growth and PSA production in cell culture.

Weekly dosing of oral calcitriol has made it possible to achieve peak blood concentrations of calcitriol of nearly 2 nmol/L. We show that significant growth inhibition of vitamin D receptor expressing LNCaP prostate cancer cells occurs after exposure to 1 nmol/L calcitriol (Fig. 1A), a concentration rendered clinically relevant by the development of weekly intermittent dosing strategies. At the same concentration, we observed up-regulation of AR expression and PSA production (Fig. 1B). Although LNCaP is the most extensively studied androgen-responsive prostate cancer cell line, these observations would be strengthened by confirmation in additional cell lines. These divergent effects suggest that in clinical trials, successful treatment of prostate cancer with calcitriol may not be associated with PSA reduction and raises the possibility that a PSA increase or flare could occur and complicate interpretation of study outcomes.

This effect of calcitriol on PSA has been previously reported. In many, but not all, of the previous experiments, this effect was shown at higher, clinically unachievable calcitriol concentrations. Thus, our findings confirm those of others and show that calcitriol at concentrations similar to those measured in our patients induces PSA production in vitro. The effect of calcitriol on AR expression observed here is similar to that reported by Zhao et al. (24). Bao et al. (25) also reported that calcitriol up-regulates AR expression in LNCaP and CWR22R cells; however, these experiments were carried out using 100 nmol/L calcitriol. Studies by Hsieh et al. (16, 17) showed up-regulation of AR expression as well as AR translocation to the nucleus in LNCaP cells exposed to calcitriol. These experiments were carried out using 10 to 100 nmol/L calcitriol. Thus, our results confirm those of Zhao et al. and show that calcitriol induces AR expression in LNCaP cells exposed to clinically relevant concentrations of calcitriol.

The preclinical effects of calcitriol on PSA could complicate the clinical assessment of the efficacy of calcitriol in prostate cancer patients. This is because serum PSA monitoring is an important component of efficacy evaluation of prostate cancer therapies. Indeed, in some settings, such as that of an increasing PSA after surgery or radiation (12), serum PSA is the only clinical marker available to measure the effect of therapy on prostate cancer. In this setting, an increase in the PSA due to a differentiation effect would likely be misinterpreted as evidence of tumor progression. Thus, we sought to examine in detail the effect of a single dose of 0.5 mg/kg calcitriol on serum PSA and free PSA.

With weekly repeat administration in a Phase II study of 22 patients with hormone-naive prostate cancer, no patients met the primary efficacy end point of a confirmed 50% reduction in the PSA. Lesser reductions in the PSA (confirmed reductions of 47%, 28%, and 10%) were seen in three patients. In these three patients, PSA remained below baseline for 4.5, 19.3, and 22.6+ months, respectively. In three additional patients, PSA doubling time increased significantly (559%, 353%, and 143% of pretreatment). The remaining 16 patients had no statistically detectable change in the PSA doubling time.

The median PSA doubling time for the entire study population increased from 7.8 to 10.3 months (P = 0.03 by Wilcoxon signed rank test).

In an analysis that was powered to detect a 1.23-fold change, we did not detect any significant changes in serum PSA, serum free PSA, or the ratio of free to total PSA monitored for up to 8 days after a dose of calcitriol. As we previously reported, longer-term therapy with weekly administration of calcitriol produced reductions in serum PSA that ranged from 10% to 47% in 3 of 22 patients. Several additional patients experienced slowing in the rate of increase in their serum PSA with weekly calcitriol therapy. This and similar doses of calcitriol are being examined in additional clinical trials for prostate cancer. Our results show that acute increases in serum PSA production predicted by preclinical experiments are unlikely to complicate the interpretation of these clinical trials. At the same time, the preclinical finding of divergent effects on proliferation and PSA production suggests that if calcitriol therapy is effective in androgen-sensitive prostate cancer, we should not expect to detect this activity by reductions in serum PSA.

The induction of AR signaling as measured by PSA production after calcitriol therapy raises several important questions. Prostate cancer cells are known to express PSA and other markers of differentiation in less aggressive clones. Thus, PSA induction due to calcitriol therapy may be a marker of cellular transformation to a more indolent phenotype. Further, the observed PSA increases may be due to a transient stress response seen in this and other therapies such as docetaxel (26). Last, more recent data have shown that the PSA protein has significant biological properties that may reduce tumor invasiveness. PSA is a member of the tissue kallikrien family and it has significant serine protease activity. This activity may play a role in reduced tumor cell angiogenesis. Further study is needed in this area to help define the significance of PSA production in tumor cell progression.

In summary, we carried out parallel analyses of the effect of calcitriol at clinically achievable concentrations on tumor proliferation, AR expression, and PSA production in vitro and on serum PSA and free PSA in patients. In the in vitro model, we showed inhibition of proliferation as well as AR induction and a significant increase in PSA production. Similar increases in PSA production were not seen in humans treated with similar concentrations of calcitriol.


   Acknowledgments
 
We thank Dr. Milan Uskokovic for providing calcitriol for in vitro experiments.


   Footnotes
 
Grant support: USPHS grants 5 M01 RR00334-33S2 and 5 R21 CA85585-02 and Roche Pharmaceuticals.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/24/05; revised 12/27/05; accepted 2/28/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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