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ABSTRACT: Cholesterol-lowering treatment has been suggested to delay progression of prostate cancer by decreasing serum LDL. We studied in vitro the effect of extracellular LDL-cholesterol on the number of prostate epithelial cells and on the expression of key regulators of cholesterol metabolism. Two normal prostatic epithelial cell lines (P96E, P97E), two in vitro immortalized epithelial cell lines (PWR-1E, RWPE-1) and two cancer cell lines (LNCaP and VCaP) were grown in cholesterol-deficient conditions. Cells were treated with 1-50 µg/ml LDL-cholesterol and/or 100 nM simvastatin for seven days. Cell number relative to control was measured with crystal violet staining. Changes in mRNA and protein expression of key effectors in cholesterol metabolism (HMGCR, LDLR, SREBP2 and ABCA1) were measured with RT-PCR and immunoblotting, respectively. LDL increased the relative cell number of prostate cancer cell lines, but reduced the number of normal epithelial cells at high concentrations. Treatment with cholesterol-lowering simvastatin induced up to 90% reduction in relative cell number of normal cell lines but a 15-20% reduction in relative number of cancer cells, an effect accompanied by sharp upregulation of HMGCR and LDLR. These effects were prevented by LDL. Compared to the normal cells, prostate cancer cells showed high expression of cholesterol-producing HMGCR but failed to express the major cholesterol exporter ABCA1. LDL increased relative cell number of cancer cell lines, and these cells were less vulnerable than normal cells to cholesterol-lowering simvastatin treatment. Our study supports the importance of LDL for prostate cancer cells, and suggests that cholesterol metabolism in prostate cancer has been reprogrammed to increased production in order to support rapid cell growth.
PLoS ONE 01/2012; 7(6):e39445. · 4.09 Impact Factor
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ABSTRACT: Epidemiological studies have linked statin use with a decreased risk of advanced prostate cancer and an improved recurrence-free survival after radical therapy. It is unclear, however, whether statins could have direct effects against prostate cancer in a clinical setting, as their growth-inhibiting effects on prostate cancer cells have been demonstrated at drug concentrations which exceed the level in plasma during standard clinical dosing. We compared responses to high-dose and therapeutic-dose simvastatin in normal and cancerous prostate epithelial cells. Simvastatin was more effective at inhibiting the growth of normal prostate epithelial cells than of cancer cells. At therapeutic 100 nM concentration simvastatin had a cytostatic effect on normal cells: apoptosis was only slightly induced, but a decrease in cell cycle activity and an increase in senescence were observed. At therapeutic concentrations, lipophilic simvastatin caused a stronger growth inhibition than did hydrophilic rosuvastatin. In contrast, 10 μM simvastatin had a cytotoxic effect both on normal and cancer cells. Addition of LDL-cholesterol effectively reversed the cytostatic effect in all cell lines, but overcoming the cytotoxicity of 10 μM simvastatin required a combination of LDL-cholesterol and mevalonate. As LDL-cholesterol completely prevented the growth-inhibiting effect of therapeutic-dose simvastatin already at low, subphysiological concentrations it is unlikely that statins have direct effects on growth of prostate epithelial cells in vivo. Statins' possible benefits against prostate cancer could be due to systemic cholesterol-lowering, as suggested by epidemiological studies. Future clinical studies evaluating the effects of statins on prostate cancer prevention should monitor serum LDL and should probably administer statins at higher concentrations than those currently used in the treatment of hypercholesterolemia.
European journal of pharmacology 12/2011; 673(1-3):96-100. · 2.59 Impact Factor
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ABSTRACT: The vitamin D endocrine system is essential for calcium and bone homeostasis. Vitamin D deficits are associated with muscle weakness and osteoporosis, whereas vitamin D supplementation may improve muscle function, body sway and frequency of falls, growth and mineral homeostasis of bones. The loss of muscle strength and mass, as well as deficits in bone formation, lead to poor balance. Poor balance is one of the main causes of falls, and may lead to dangerous injuries. Here we examine balance functions in vitamin D receptor deficient (VDR-/-) mice, an animal model of vitamin D-dependent rickets type II, and in 1alpha-hydroxylase deficient (1alpha-OHase-/-) mice, an animal model of pseudovitamin D-deficiency rickets. Recently developed methods (tilting box, rotating tube test), swim test, and modified accelerating rotarod protocol were used to examine whether the absence of functional VDR, or the lack of a key vitamin D-activating enzyme, could lead to mouse vestibular dysfunctions. Overall, VDR-/- mice, but not 1alpha-OHase-/- mice, showed shorter latency to fall from the rotarod, smaller fall angle in the tilting box test, and aberrant poor swimming. These data suggest that VDR deficiency in mice is associated with decreased balance function, and may be relevant to poorer balance/posture control in humans with low levels of vitamin D.
The Journal of steroid biochemistry and molecular biology 05/2009; 114(3-5):161-6. · 2.66 Impact Factor
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ABSTRACT: Non-steroidal anti-inflammatory drugs and cholesterol-lowering statins have been reported to inhibit prostate cancer cell growth suggesting their chemopreventive potential within the prostate. However, the effect has been demonstrated only with advanced prostate cancer cell lines and with drug concentrations above the clinical therapeutic range. In this study we compared the effect of therapeutic concentrations of acetylsalicylic acid, simvastatin and rosiglitazone on the growth of a set of prostatic primary cultures and various prostate epithelial cell lines.
Two primary epithelial cell lines isolated from surgical resecates of normal prostate tissue (P96E, P97E), a primary cell line isolated from untreated prostate carcinoma (ESTO1), two transformed prostate epithelial cell lines (PWR1-E, RWPE-1) and advanced cancer cell lines LNCaP and VCaP were used in the study. Cells were treated for seven days with therapeutic concentrations of acetylsalisylic acid, simvastatin, rosiglitazone or their combination. Cellular growth rate was measured by crystal violet staining method.
Acetylsalicylic acid (0.5 mM) and simvastatin (10 nM) inhibited the growth of prostate epithelial cells of normal and primary cancer origin, whereas advanced cancer cell lines were resistant to the effect. Rosiglitazone at the therapeutic level of 1 microM did not reduce the growth of any cell type studied.
Our results demonstrate that acetylsalicylic acid and simvastatin inhibit prostate epithelial cell growth at clinically relevant doses. This should be acknowledged when designing possible prostate cancer chemopreventive trials.
The Prostate 04/2009; 69(9):1017-23. · 3.48 Impact Factor
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ABSTRACT: Statins are cholesterol-lowering drugs that are widely used to prevent and treat atherosclerotic cardiovascular disease. Recent research from both in vitro and in vivo studies suggests that there is an association between the use of statins and a reduction in the incidence of and mortality from prostate cancer. Several mechanisms of action that might bring about these beneficial effects of statins have been proposed, most of which include direct effects of statins on intracellular signaling. In this Review we discuss the current knowledge on the use of statins to prevent prostate cancer. We will also look at future directions for clinical research on this topic.
Nature Clinical Practice Urology 08/2008; 5(7):376-87. · 4.07 Impact Factor
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ABSTRACT: Factors related to the metabolic syndrome and low levels of vitamin D have been implicated as risk factors for prostate cancer. Insofar, no studies have assessed their joint effects on prostate cancer risk.
We studied (a) the associations of vitamin D with the metabolic syndrome factors body mass index, systolic and diastolic blood pressure, and high-density lipoprotein cholesterol (HDL-C) and (b) the prostate cancer risk associated with these factors and especially their joint effects with vitamin D on risk of prostate cancer. We did a longitudinal nested case-control study on 132 prostate cancer cases and 456 matched controls from a cohort of 18,939 Finnish middle-aged men from the Helsinki Heart Study. The odds ratios (OR) of prostate cancer were assessed via conditional logistic regression analysis.
Apart from HDL-C, there was no linear association between the metabolic syndrome factors and vitamin D levels. In univariate analysis, men in the highest quartiles of body mass index (>28 kg/m(2)) and systolic blood pressure (>150 mmHg) showed a modest increase in risks of prostate cancer, with ORs of 1.37 (P = 0.16) and 1.53 (P = 0.05) when compared with the three lower quartiles, but low HDL-C entailed no prostate cancer risk. However, with all three factors present, the OR was 3.36 (P = 0.02), and jointly with low vitamin D (<or=40 nmol/L), the OR was 8.03 (P = 0.005) compared with those with no metabolic syndrome factors and intermediate levels of vitamin D. There was an interaction between vitamin D and the metabolic syndrome factors so that a clustering of these factors entailed high risk of prostate cancer but only if vitamin D level was low (<or=40 nmol/L). If it was at intermediate levels, the metabolic syndrome factors entailed no prostate cancer risk.
We conclude that the prostate cancer risk associated with factors related to the metabolic syndrome is strongly conditioned by levels of vitamin D.
Cancer Epidemiology Biomarkers & Prevention 03/2007; 16(2):302-7. · 4.12 Impact Factor
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ABSTRACT: According to the present paradigm, 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] is a biologically active hormone; whereas 25-hydroxyvitamin D3 (25OHD3) is regarded as a prohormone activated through the action of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase). Although the role of vitamin D3 in the regulation of growth and differentiation of prostatic epithelial cells has been well studied, its action and metabolism in prostatic stroma are still largely unknown. We investigated the effects of 25OHD3 and 1alpha,25-(OH)2D3 on two human stromal primary cultures termed P29SN and P32S. In a cell proliferation assay, 25OHD3 was found at physiological concentrations of 100-250 nM to inhibit the growth of both primary cultures, whereas 1alpha,25-(OH)2D3 at a pharmacological concentration of 10 nM exhibited the growth-inhibitory effects on P29SN cells but not on P32S cells. Quantitative real-time RT-PCR analysis revealed that both 25OHD3 and 1alpha,25-(OH)2D3 induced 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) mRNA in a dose- and time-dependent manner. By inhibiting 1alpha-hydroxylase and/or 24-hydroxylase enzyme activities, the induction of 24-hydroxylase mRNA by 250 nM 25OHD3 was clearly enhanced, suggesting that 1alpha-hydroxylation is not a prerequisite for the hormonal activity of 25OHD3. Altogether our results suggest that 25OHD3 at a high but physiological concentration acts as an active hormone with respect to vitamin D3 responsive gene regulation and suppression of cell proliferation.
The FASEB Journal 03/2004; 18(2):332-4. · 5.71 Impact Factor
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ABSTRACT: Vitamin D and its analogues are potent regulators of cell growth and differentiation both in vivo and in vitro. We studied the effects of 25-hydroxyvitamin D(3) [25(OH)D(3)], 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and vitamin D analogue, EB 1089, on the growth of a human ovarian cancer cell line, OVCAR-3. We also studied the expression of vitamin D metabolising enzymes 24-hydroxylase (24OHase) and 1alpha-hydroxylase (1alphaOHase). Our results showed that high concentrations (10 and 100 nM) of 1,25(OH)(2)D(3) inhibited a cell proliferation, whereas low concentration (0.1 nM) stimulated growth of the OVCAR-3 cells. In the concentration range of 10-500 nM a prohormone, 25(OH)D(3), stimulated growth. An amount of 1 nM EB 1089 and 100 nM 1,25(OH)(2)D(3) inhibited growth with an equal magnitude. The expression of 24OHase was strongly induced by 1,25(OH)(2)D(3) and EB 1089 in OVCAR-3 cells, and analysis of vitamin D metabolites showed the functionality of 24OHase. An inhibition of 24OHase activity with a novel 24OHase inhibitor enhanced growth-inhibiting effects of 1,25(OH)(2)D(3) and suppressed the growth stimulation of 100 nM 25(OH)D(3). We also report the expression of a vitamin D activating enzyme, 1alphaOHase, in 7 ovarian cancer cell lines. The production of 1,25(OH)(2)D(3) in OVCAR-3 cells was low, possibly due to an extensive activity of 24OHase or a low 1alphaOHase activity. These results suggest that in ovarian cancer cells vitamin D metabolizing enzymes might play a key role in modulating the growth response to vitamin D. The possible mitogenic effects of vitamin D should be considered when evaluating treatment of ovarian cancer with vitamin D.
International Journal of Cancer 02/2004; 108(3):367-73. · 5.44 Impact Factor
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ABSTRACT: Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.
The Journal of Steroid Biochemistry and Molecular Biology 04/2002; 80(3):307-13. · 3.05 Impact Factor
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ABSTRACT: Bisphosphonates inhibit the development of experimental atherosclerosis and decrease the intima-media thickness of human carotid artery. Since arterial macrophages have a key role in atherogenesis, we studied whether clodronate, an antiatherogenic bisphosphonate, will suppress the appearance of macrophages generated by atheromatous process in the rabbit aorta. The atherosclerosis was caused in rabbits by means of a high-cholesterol (1%) diet, and the animals were treated simultaneously with saline (n = 11) or 25 mg/kg of clodronate disodium (n= 12) intravenously twice a week for 6 to 12 weeks. The cholesterol diet for 6 weeks caused no visible atheromatous plaques in the aorta, but feeding for 6 more weeks produced progressively atheromatous lesions. Immunohistochemistry with specific antimacrophage antibody showed an intensive accumulation of macrophages in the subendothelial layer of the aorta in cholesterol-fed rabbits treated with saline or clodronate for 6 weeks. In the aorta of rabbits treated with cholesterol diet + saline for 12 weeks, the area of immunoreactive macrophages extended from the internal elastic lamina up to the luminal surface of the aorta. However, far less immunoreactive macrophages were present in the atheromatous regions of the aorta of rabbits medicated with clodronate for 12 weeks; in the clodronate-treated animals the macrophages were located closer to the luminal surface of the aorta than in controls on saline. No atheromatous lesions and macrophages appeared in the aorta of rabbits on standard diet (n = 7). The results suggest that clodronate suppresses the appearance of cholesterol-phagocyting macrophages in arterial walls during atherogenesis.
Pharmacology & Toxicology 04/2002; 90(3):139-43.
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ABSTRACT: During the past few years, it has become apparent that vitamin D may play an important role in malignant transformation. Epidemiological studies suggest that low vitamin D serum concentration increases especially the risk of hormone-related cancers. Experimentally, vitamin D suppresses the proliferation of normal and malignant cells and induces differentiation and apoptosis. In the present review we discuss the mechanisms whereby vitamin D regulates cell proliferation and whether it could be used in prevention and treatment of hyperproliferative disorders like cancers.
Vitamins & Hormones 02/2002; 64:357-406. · 2.19 Impact Factor
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ABSTRACT: With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.
European Journal of Biochemistry. 10/2001; 268(20):5337 - 5342.
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ABSTRACT: Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed
to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in
normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the
most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct
cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland,
nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression
of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian
gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the
receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor
in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.
The Histochemical Journal 01/1996; 28(7):477-483.
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ABSTRACT: We raised a polyclonal antibody, αD, against a synthetic peptide (amino acids 522–535) of chichen progesterone receptor (PR). The Sequence is located between the DNA-binding domain and the hormone-binding domain in the refion within the sequences required for stability of the oligomeric form of PR. In the immunoblot, αD reacted with both A and B forms of PR. in the sucrose gradient and dot-blot the antibody did bot recognize the so-called 8S form of PR, which is an oligomeric complex of PR and other proteins, When the oligomeric complex was dissociated by salt treatment, the antibody recognized the resulting 4S form of PR. This would suggest that the epitope is masked in the 8S form of PR and exposed in the 45 form. To study whether a similar Complex exists in vivo, we used the antibody for immunohistochemistry. Two different fixation techniques were employed, Freeze-drying-vapor fixation and liquid fixation. In the animals not treated with progesterone, intensive nuclear Staining was Detected independent of the fixation technique. when receptor from similarly treated animals was analyzed by sucrose gradient, all of the receptor molecules were in the oligomeric complex (85). Ligand binding is known to promote a dissociation of this complex. Thus progesterone treatment should lead to an incerased immunodetection of the epitope; however, progesterone treatment decreased the intensity of PR immunostaining. These Results Suggest that the oligomeric complex (85), Present in tissue extracts, does not exist in intact cell nuclei. They also Call into question the propesed role of hsp90 in regulating progesterone receptor function. © Wiley-Liss, Inc.
Journal of Cellular Biochemistry 04/1995; 58(1):95 - 104. · 2.87 Impact Factor
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ABSTRACT: Genomic actions of progesterone are mediated via A and B isoforms of the progesterone receptor (PR). One major factor controlling PR level is progesterone causing negative autoregulation (down-regulation) of the receptor protein. In this work we studied the mechanism whereby progesterone exerts its effects on PR level in the chicken oviduct. We found that progesterone does not markedly regulate PR mRNA expression. Furthermore, we demonstrate here for the first time that PR is a target for ubiquitylation and that the proportion of ubiquitylated PR is increased by progesterone treatment. Our data suggest that ligand-induced down-regulation of PR involves enhanced degradation of receptor protein by ubiquitin-proteasome system in vivo.
Life Sciences.
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ABSTRACT: In hypotonic cell extract (cytosol), unliganded progesterone receptor (PR) is known to form an oligomeric complex with heat shock protein 90 (hsp90), and this complex does not bind to DNA. Since ligand binding has been shown to render the complex less stable in vitro, it has been proposed that ligand binding regulates DNA binding and receptor activity in vivo by altering the stability of the oligomeric complex. However, there is no direct evidence as to whether this oligomeric complex is present in vivo. The present study addressed this problem. First, we used an immunoelectron-microscopic technique and monoclonal antibodies to ascertain the location of PR and hsp90 in chick oviduct cells. Hsp90 was found in the cytoplasm and PR in the nucleus. To study the relative affinities of the PR and hsp90 antibodies, we then constructed a chimeric protein (PR-hsp90), which was expressed in the HeLa cells. Both hsp90 and PR antigens of the chimera were detected in the nuclei with the same intensity, which indicates that the antibodies have equal sensitivities in detecting their antigens. This suggests that if significant amounts of nuclear hsp90 were present in intact cells, it should have been detected by our method. Our results indicate that the PR does not exist in vivo as an oligomeric, nonDNA-binding form in the cell nuclei and that the oligomeric form found in tissue extracts is possibly formed during tissue processing.
The Journal of Steroid Biochemistry and Molecular Biology.
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ABSTRACT: Keratinocyte growth factor (FGF-7/KGF) is a secreted member of the fibroblast growth factor family, which functions primarily as an important paracrine mediator of cell growth and differentiation. Inhibitory pathways of vitamin D may also involve participation of some growth factors. To determine whether vitamin D may play a role in the expression of FGF-7, we investigated FGF-7 expression in human breast cancer cells treated with 1,25-dihydroxyvitamin D3, which inhibited the growth of the cells. By means of cDNA microarray, RT-PCR, and Western blot analysis, we have shown an increase in expression of FGF-7 on both mRNA and protein levels after vitamin D exposure. This is the first demonstration of vitamin D regulation of FGF-7 expression and its possible involvement in mediating growth and differentiation by vitamin D.
Biochemical and Biophysical Research Communications.
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ABSTRACT: Epidemiological studies have linked statin use with a decreased risk of advanced prostate cancer and an improved recurrence-free survival after radical therapy. It is unclear, however, whether statins could have direct effects against prostate cancer in a clinical setting, as their growth-inhibiting effects on prostate cancer cells have been demonstrated at drug concentrations which exceed the level in plasma during standard clinical dosing. We compared responses to high-dose and therapeutic-dose simvastatin in normal and cancerous prostate epithelial cells. Simvastatin was more effective at inhibiting the growth of normal prostate epithelial cells than of cancer cells. At therapeutic 100 nM concentration simvastatin had a cytostatic effect on normal cells: apoptosis was only slightly induced, but a decrease in cell cycle activity and an increase in senescence were observed. At therapeutic concentrations, lipophilic simvastatin caused a stronger growth inhibition than did hydrophilic rosuvastatin. In contrast, 10 μM simvastatin had a cytotoxic effect both on normal and cancer cells. Addition of LDL-cholesterol effectively reversed the cytostatic effect in all cell lines, but overcoming the cytotoxicity of 10 μM simvastatin required a combination of LDL-cholesterol and mevalonate. As LDL-cholesterol completely prevented the growth-inhibiting effect of therapeutic-dose simvastatin already at low, subphysiological concentrations it is unlikely that statins have direct effects on growth of prostate epithelial cells in vivo. Statins' possible benefits against prostate cancer could be due to systemic cholesterol-lowering, as suggested by epidemiological studies. Future clinical studies evaluating the effects of statins on prostate cancer prevention should monitor serum LDL and should probably administer statins at higher concentrations than those currently used in the treatment of hypercholesterolemia.
European Journal of Pharmacology. 673:96-100.
