jshs

Journal of Steroids & Hormonal Science

ISSN - 2157-7536

Research Article - (2010) Volume 1, Issue 1

Liposomal 1,25-dihydroxyvitamin D3-3β-bromoacetate is a Stronger Growth-inhibiting Agent than its un-encapsulated Counterpart in Prostate Cancer Cells

Kelly S. Persons, Shwetha Hareesh, Vikram J. Eddy and Rahul Ray*
Boston University School of Medicine, 85 East Newton Street, Boston, MA 02118, USA
*Corresponding Author: Rahul Ray, Boston University School of Medicine, Boston, MA, USA, Tel: +617-638-8199, Fax: +617-638-8194 Email:

Abstract

Encapsulating cytotoxic drugs in liposomal vehicles allows for the targeting of tumors while protecting the drugs from premature degradation. 1,25-Dihydroxyvitamin D3-3?-bromoacetate (1,25(OH)2D3-3-BE), a vitamin D receptor-alkylating agent inhibits the growth of prostate cancer cells. The aim of the study was to evaluate the efficacy of a liposomal preparation of 1,25(OH)2D3-3-BE versus 1,25(OH)2D3-3-BE in modulating the growth of prostate cancer cells. Results demonstrate that liposomal 1,25(OH)2D3-3-BE is significantly better than 1,25(OH)2D3-3-BE in inhibiting the growth. In addition, liposomal 1,25(OH)2D3-3-BE was found to be stable in human serum. Taken together, results of the studies delineated here suggest a therapeutic potential of liposomal 1,25(OH)2D3-3-BE in prostate cancer.

Keywords: Liposomal 1,25-dihydroxyvitamin D3-3?-bromoacetate, Vitamin D receptor alkylating agent, Prostate cancer.

Introduction

Liposomes are recognized as important vehicles for cytotoxic drugs because they can protect the drugs from degradation in circulation, thereby protecting healthy cells and tissues from exposure to lethal drug doses. Additionally, liposomes can extravasate through leaky tumor vasculature selectively over normal tissues and release drugs into the tumor [1-4].

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is an antiproliferative and anti-cancer agent [5]. But its clinical applicability has been limited by hyper-calcemia and related toxicity, brought in by high therapeutic doses [6-8]. Paradoxically high doses are required to counter its rapid catabolic degradation. 1α,25-dihydroxyvitamin D3-3β-bromoacetate (1,25(OH)2D3-3-BE) was developed in our laboratory to counter this problem by covalently attaching 1,25(OH)2D3 deep inside the ligandbinding pocket of vitamin D receptor (VDR), the transcriptional factor that regulates the biological activities of 1,25(OH)2D3 [9-14]. In recent publications it was demonstrated that 1,25(OH)2D3-3- BE and 25-hydroxyvitamin D3-3-bromoacetate, the counterpart of 1,25(OH)2D3-3-BE without the 1-hydroxyl group, are considerably stronger antiproliferative agents than 1,25(OH)2D3 in prostate and pancreatic cancer cells [12-15], as well as high-risk neuroblastoma cells [16].

In the present communication we report that a liposomal preparation of 1,25(OH)2D3-3-BE has a better growth-inhibitory property in three prostate cancer cell lines than the un-encapsulated compound (1,25(OH)2D3-3-BE) and the parent hormone, 1,25(OH)2D3 (un-encapsulated and liposomal). We also report that liposomal 1,25(OH)2D3-3-BE is stable in human serum, thereby attesting therapeutic potential of liposomal 1,25(OH)2D3-3-BE in prostate cancer.

Materials and Methods

All chemicals were obtained from Sigma-Aldrich, Milwaukee, WI, unless mentioned otherwise. Cell-lines were obtained from ATCC (Manasas, VA).

Preparation of liposomes

A solution of cholesterol (1 µg), dimethylphosphotidyl choline (DMPC) (20 µg) and 1,25(OH)2D3 (1 µg, a kind gift from Dr. Milan Uskokovic, Hoffman La-Roche, Nutley, NJ) or 1,25(OH)2D3-3-BE (1 µg, synthesized in our laboratory, reference [17]) in chloroform was dried in a stream of argon. Phosphated saline (PBS, 2.5 ml) was added to the solid residue followed by mixing by brief vortexing and the mixture was sonicated for 15 min. The milky solution was incubated at 50°C for 50 min and frozen at -77°C for 20 min. This heating and freezing cycle was repeated once and the preparation was stored at 4°C for use in assays. Prior to each assay the liposomal preparation was sonicated and vortexed briefly for proper mixing.

In a separate experiment a chloroform solution of cholesterol, DMPC and 1,25(OH)2D3 was spiked with 3H-1,25(OH)2D3 (100,000 cpm, sp. activity 120 Ci/mM, Amersham, GE Healthcare), followed by mixing (in PBS) and sonication etc. The preparation was centrifuged at 4°C in an ultracentrifuge (Beckman Ultracentrifuge L7-65) using a Beckman 50.2 Ti rotor at 35,000 rpm for 60 min. The supernatant and pellet (dispersed in one ml of PBS) were mixed with scintillation fluid and counted. We routinely obtained >90% incorporation of radioactivity in the pellet.

Antiproliferation assays

We tested antiproliferative activity of 1,25(OH)2D3, liposomal 1,25(OH)2D3 (1,25(OH)2D3)L, 1,25(OH)2D3-3-BE and liposomal 1,25(OH)2D3-3-BE (1,25(OH)2D3-3-BE)L in LNCaP, PC-3 and DU-145 prostate cancer cells by MTT assay (according to manufacturer’s procedure (Trevigen, Gaithersburg, MD) or 3H-thymidine incorporation assay (see below).

3H-thymidine-incorporation assay: In a typical assay cells were grown to 50-60% confluence in 24-well plates in DMEM media containing 10% FBS, serum-starved for 20 hours, followed by treatment with various concentrations of 1,25(OH)2D3 or 1,25(OH)2D3- 3-BE (as 0.1% ethanolic solution) or ethanol (vehicle) or (1,25(OH)2D3) L or (1,25(OH)2D3-3-BE)L or blank liposome in serum-containing medium for 16 hr. After the treatment, media was removed from the wells and replaced with media containing 3H-thymidine (0.1 µ Ci, Sigma-Aldrich, Milwaukee, WI) per well. Plates were incubated for 3 hr at 37°C followed by the following sequence of steps. Liquid was removed by aspiration, cells washed thoroughly (3X0.5 ml) with PBS, ice-cold 5% perchloric acid solution (0.5 ml/well) added, incubated on ice for 20 min, perchloric acid removed by aspiration, replaced with 0.5 ml of fresh perchloric acid, incubated at 70°C for 20 minutes. Finally, solution from each well was mixed with scintillation fluid and counted in a liquid scintillation counter. There were six (6) wells per sample and statistics was carried out by Student’s t test.

Growth assay: DU-145 cells were treated with various doses (as indicated in Figure 4) of 1,25(OH)2D3-3-BE,(1,25(OH)2D3-3-BE) L, ethanolor blank liposomeon 1st, 3rd and 5th days, followed by harvesting of the cells (by trypsinization) on 7th day and counting the cells in a hemocytometer. There were three (3) wells per sample and statistics was carried out by Student’s t test.

Serum-stability study of (1,25(OH)2D3-3-BE)L

One ml of pooled human serum was incubated at 37°C for 60 minutes with a sample of (1,25(OH)2D3-3-BE)L (10µg) followed by extraction with 5 x 0.5ml of ethyl acetate. The organic layer was dried in a stream of nitrogen and the residue was analyzed by HPLC (Agilent 1100 series, Thermo-Scientific, Waltham, MA, 5% H2O-MeOH mobile phase, 1.5 ml/min, 265 nm detection, Agilent C18 column). A standard sample of 1,25(OH)2D3-3-BE was also analyzed by HPLC under same conditions.

Results

(1,25(OH)2D3-3-BE)L has the strongest growth-inhibitory property in comaprison with 1,25(OH)2D3-3-BE, 1,25(OH)2D3 and (1,25(OH)2D3)L in prostate cancer cells

In growth-inhibition studies with androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU-145 prostate cancer cells, 10-7-6M of 1,25(OH)2D3 or (1,25(OH)2D3)L failed to show any significant effect (Figures 1A, 2A and 3A respectively). But, in all these cell-lines 10-7M of 1,25(OH)2D3-3-BE and (1,25(OH)2D3-3-BE)L displayed considerable antiproliferative effect (Figures 1B-3B). Importantly, in each case (1,25(OH)2D3-3-BE)L showed a stronger antiproliferative effect than naked 1,25(OH)2D3-3-BE. For example, in LNCaP, PC-3 and DU-145 cells, 10-7M of 1,25(OH)2D3-3-BE inhibited growth by approximately 35%, 20% and 15% respectively, while an equivalent dose of (1,25(OH)2D3-3- BE)L inhibited growth by approximately 55%, 45% and 55% respectively (Figures 1B-3B). Growth of LNCaP and DU-145 cells was particularly sensitive to 10-6M of 1,25(OH)2D3-3-BE and (1,25(OH)2D3-3-BE)L and their growth was almost completely inhibited (Figures 1B and 3B) In comparison, PC-3 cells were less sensitive to these treatments (Figure 2B). A 10-6M dose may be considered supra-physiological, but we employed this dose simply to compare the effects between 1,25(OH)2D3 and 1,25(OH)2D3-3-BE and their liposomal formulations. Collectively these results showed that 1,25(OH)2D3-3-BE, particularly in liposomal formulation has strong growth-inhibitory effect in prostate cancer cells.

steroids-hormonal-science-reagent-treated-cells

Figure 1: Cytotoxicity of 1,25(OH)2D3, (1,25(OH)2D3)L, 1,25(OH)2D3-3-BE or (1,25(OH)2D3-3-BE)L in LNCaP cells. Cells were treated with ethanol (control), blank liposome (control), various doses of 1,25(OH)2D3, (1,25(OH)2D3) L, 1,25(OH)2D3-3-BE or (1,25(OH)2D3-3-BE)L for 16 hr. Metabolic activity in reagent-treated cells relative to controls was quantitated using a tetrazoliumbased (MTT) chromogenic assay. OD was measured spectrophotometrically at 450 nm. There were six replicates for each sample. Statistics was done by student’s t test: ***p<0.001, **p<0.01.

steroids-hormonal-science-each-sample-Statistics

Figure 2: Cytotoxicity of 1,25(OH)2D3, (1,25(OH)2D3)L, 1,25(OH)2D3-3-BE or (1,25(OH)2D3-3-BE)L in PC cells. Cells were treated with ethanol (control), blank liposome (control), various doses of 1,25(OH)2D3, (1,25(OH)2D3)L, 1,25(OH)2D3-3-BE or (1,25(OH)2D3-3-BE)L for 16 hr followed by 3H-Thymidine incorporation assay by a procedure described in Materials and Methods section. There were six replicates for each sample. Statistics was done by student’s t test: ***p<0.001, **p<0.01.

steroids-hormonal-science-liposome-various-doses

Figure 3: Cytotoxicity of 1,25(OH)2D3, (1,25(OH)2D3)L, 1,25(OH)2D3-3-BE or (1,25(OH)2D3-3-BE)L in DU-145 cells. Cells were treated with ethanol (control), blank liposome (control), various doses of 1,25(OH)2D3, (1,25(OH)2D3)L, 1,25(OH)2D3-3-BE or (1,25(OH)2D3-3-BE)L for 16 hr followed by 3H-Thymidine incorporation assay by a procedure described in Materials and Methods section. There were six replicates for each sample. Statistics was done by student’s t test: ***p<0.001, **p<0.01.

(1,25(OH)2D3-3-BE)L has stronger anti-growth effect than 1,25(OH)2D3-3-BE in a chronic and long-term growth assay in DU-145 cells

In the next study DU-145 cells were treated three times in a week-long growth assay to mimic chronic administration of drugs in in vivo studies. Results of this assay, shown in Figure 4, demonstrate that (1,25(OH)2D3-3-BE)L has a stronger anti-growth effect than 1,25(OH)2D3-3-BE in a dose-dependent manner.

(1,25(OH)2D3-3-BE)L is stable in human serum

Serum-stability assay of (1,25(OH)2D3-3-BE)L by HPLC produced a sharp peak with a retention time of 6.68 min and a small shoulder at approximately 6 min (Figure 5B, Bottom Panel). Most importantly, 1,25(OH)2D3-3-BE did not produce a peak for 1,25(OH)2D3 (indicated by arrow). These results strongly suggested that 1,25(OH)2D3-3-BE is stable in human serum for at least up to 60 min at 37°C.

Discussion

1,25(OH)2D3 inhibits growth of many cancer cells, suggesting its potential as a cancer therapeutic agent. However, its clinical use has been limited by its toxicity in pharmacological doses [8]. Requirement of high clinical dose is related to its rapid catabolic degradation (warranting higher doses) and lack of selectivity for tumor cells over normal cells. As discussed earlier, these problems can potentially be alleviated by encapsulating 1,25(OH)2D3 in liposomes/nanosomes. It would be even better if an analog of 1,25(OH)2D3 with a better therapeutic index (than 1,25(OH)2D3) is subjected to the same process. In previous publications we have demonstrated that 1,25(OH)2D3- 3-BE possesses considerably stronger antiproliferative activity than 1,25(OH)2D3 in prostate and pancreatic cancer cells [12,15], underscoring therapeutic potential of 1,25(OH)2D3-3-BE. However, evaluation of such a translational potential requires development of an effective formulation of the compound.

Liposomes have been touted as tumor-specific and effective carriers of cytotoxic drugs [1]. However, they are not devoid of significant problems including premature destruction to cause toxicity to healthy tissues or in the other extreme, undesirably long stability to prevent effective delivery to the tumor cells [1]. These problems have been addressed to some extent by preparing sterically hindered pegylated liposomes [18,19] and incorporation of spore proteins [20-23].

As a prelude to more elaborate studies we have developed a simple yet highly efficient method to encapsulate 1,25(OH)2D3 and 1,25(OH)2D3-3-BE in liposomes and evaluated anti-growth properties of these formulations in prostate cancer cells.

Results of our in vitro studies show that (1,25(OH)2D3-3-BE) L has stronger growth-inhibitory property than 1,25(OH)2D3-3-BE, 1,25(OH)2D3 and (1,25(OH)2D3)L (Figure 1, 2 and 3) in all three prostate cancer cells. In addition, (1,25(OH)2D3-3-BE)L is significantly more efficacious as an antiproliferative agent than 1,25(OH)2D3-3-BE in a chronic growth assay with DU-145 cells (Figure 4).

steroids-hormonal-science-each-sample-Statistics

Figure 4: Growth assay of DU-145 cells, as they were treated with 1,25(OH)2D3-3-BE or (1,25(OH)2D3-3-BE)L. Cells were with ethanol (control), blank liposome (control), various doses of 1,25(OH)2D3-3-BE or (1,25(OH)2D3- 3-BE)L on 1st, 3rd and 5th days, harvested on 7th day and counted in a hemocytometer. There were three replicates for each sample. Statistics was done by student’s t test: ***p<0.001, **p<0.01.

1,25(OH)2D3-3-BE is an ester of 1,25(OH)2D3 and its hydrolysis in vivo would produce equivalent amounts 1,25(OH)2D3 and bromoacetic acid, thereby reducing its efficacy (Figure 5, Inset). HPLC-analysis indicates that (1,25(OH)2D3-3-BE)L is stable in human serum for at least up to one hour as denoted by the absence of 1,25(OH)2D3 peak in the HPLC of the organic-extract of (1,25(OH)2D3-3-BE)L (Figure 5).

steroids-hormonal-science-Evaluation-serum-stability

Figure 5: Evaluation of the serum-stability of (1,25(OH)2D3-3-BE)L. One ml of human serum was incubated at 37°C for 60 minutes with a sample of (1,25(OH)2D3-3-BE)L (10 µg) followed by extraction with ethyl acetate and HPLC-analysis of the organic-extract. A standard sample of 1,25(OH)2D3-3-BE was also analyzed by HPLC under same conditions. Inset: Graphic depiction of hydrolytic production of equimolar amount of 1,25(OH)2D3 and bromoacetic acid from 1,25(OH)2D3-3-BE.

In summary, results, delineated in this study demonstrate that (1,25(OH)2D3-3-BE)L has a stronger growth-inhibitory effect than 1,25(OH)2D3-3-BE in prostate cancer cells. This information, coupled with its serum-stability strongly suggests a therapeutic potential of (1,25(OH)2D3-3-BE)L in androgen-sensitive and androgen-insensitive prostate cancer.

Acknowledgement

Authors acknowledge research-support by grants from National Cancer Institute (1R21CA127629-01A2, 1R43CA12617-01A1) and Department of Defense (PC 051136).

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Citation: Persons KS, Hareesh S, Eddy VJ, Ray R (2010) Liposomal 1,25-dihydroxyvitamin D3-3?-bromoacetate is a Stronger Growth-inhibiting Agent than its un-encapsulated Counterpart in Prostate Cancer Cells. J Steroids Hormon Sci 1:101.

Copyright: © 2010 Persons KS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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