The role of the renin-angiotensin system in thoracic aortic aneurysms: clinical implications

Thoracic aortic aneurysms (TAAs) are a potential life-threatening disease with limited pharmacological treatment options. Current treatment options are aimed at lowering aortic hemodynamic stress, predominantly with β-adrenoceptor blockers. Increasing evidence supports a role for the renin-angiotensin system (RAS) in aneurysm development. RAS blockade would not only lower blood pressure, but might also target the molecular pathways involved in aneurysm formation, in particular the transforming growth factor-β and extracellular signal-regulated kinase 1/2 pathways. Indeed, the angiotensin II type 1 (AT₁) receptor blocker losartan was effective in lowering aortic root growth in mice and patients with Marfan's syndrome. RAS inhibition (currently possible at 3 levels, i.e. renin, ACE and the AT₁ receptor) is always accompanied by a rise in renin due to interference with the negative feedback loop between renin and angiotensin II. Only during AT₁ receptor blockade will this result in stimulation of the non-blocked angiotensin II type 2 (AT₂) receptor. This review summarizes the clinical aspects of TAAs, provides an overview of the current mouse models for TAAs, and focuses on the RAS as a new target for TAA treatment, discussing in particular the possibility that AT₂ receptor stimulation might be crucial in this regard. If true, this would imply that AT₁ receptor blockers (and not ACE inhibitors or renin inhibitors) should be the preferred treatment option for TAAs.     

Effects of dichloroacetate and ubiquinone infusions on glycolysis activity and thermal sensitivity during sepsis

Energy-metabolism disturbances during sepsis are characterized by enhanced glycolytic fluxes and reduced mitochondrial respiration. However, it is not known whether these abnormalities are the result of a specific mitochondrial alteration, decreased pyruvate dehydrogenase (PDH) complex activity, depletion of ubiquinone (CoQ(10); electron donor for the mitochondrial complex III), or all 3. In this study we sought to specify metabolism disturbances in a murine model of sepsis, using either a PDH-activator infusion (dichloroacetate, DCA) or CoQ(10) supplementation. After anesthesia, Sprague-Dawley rats received intravenous saline solution (control; n = 5), DCA (n = 5; 20 mg/100 g), or CoQ(10) (n = 5; 1 mg/100 g), before the induction of sepsis. Increased plasma lactate levels and increased muscle glucose content were observed after 4 hours in the control group. In the DCA group, a decrease in the muscle content of lactate (P <.05) and an increase in muscle glucose content (P <.05) were observed at 4 hours, but no lactatemia variation was noted. In the CoQ(10) group, only increased plasma lactate levels were observed. Increased muscle glycolysis fluxes were observed after 4 hours in the control group, but to a slighter degree in both the DCA and CoQ(10) groups. Only DCA restored a normal temperature sensitivity in the hyperthermia range, but we noted no differences in survival time. In conclusion, only DCA infusion restores normal glycolysis function.     

Differential roles of hydrogen peroxide and hydroxyl radical in cisplatin-induced cell death in renal proximal tubular epithelial cells

Reactive oxygen species (ROS) have been suggested as important mediators of cisplatin-induced acute renal failure in vivo. However, our previous studies have shown that cisplatin-induced cell death in vitro could not be prevented by scavengers of hydrogen peroxide and hydroxyl radical in rabbit renal cortical slices. This discrepancy may be attributed to differential roles of ROS in necrotic and apoptotic cell death. We therefore examined, in this study, the roles of ROS in necrosis and apoptosis induced by cisplatin in primary cultured rabbit proximal tubule. Cisplatin induced necrosis at high concentrations over a few hours and apoptosis at much lower concentrations over longer periods. Necrosis induced by high concentration of cisplatin was prevented by a cell-permeable superoxide scavenger (tiron), hydrogen peroxide scavengers (catalase and pyruvate), and antioxidants (Trolox and deferoxamine), whereas hydroxyl radical scavengers (dimethythiourea and thiourea) did not affect the cisplatin-induced necrosis. However, apoptosis induced by lower concentration of cisplatin was partially prevented by tiron and hydroxyl radical scavengers but not by hydrogen peroxide scavengers and antioxidants. Cisplatin-induced apoptosis was mediated by the signaling pathway that is associated with cytochrome c release from mitochondria and caspase-3 activation. These effects were prevented by tiron and dimethylthiourea but not by catalase. Dimethylthiourea produced a significant protection against cisplatin-induced acute renal failure, and the effect was associated with an inhibition of apoptosis. These results suggest that hydrogen peroxide is involved in the cisplatin-induced necrosis, whereas hydroxyl radical is responsible for the cisplatin-induced apoptosis. The protective effects of hydroxyl radical scavengers are associated with an inhibition of cytochrome c release and caspase activation.     

Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes.

Mice deficient in various mismatch repair (MMR) enzymes were examined to determine whether this repair pathway is involved in antibody class switch recombination. Splenic B cells from mice deficient in Msh2, Mlh1, Pms2, or Mlh1 and Pms2 were stimulated in culture with lipopolysaccharide (LPS) to induce immunoglobulin (Ig)G2b and IgG3, LPS and interleukin (IL)-4 to induce IgG1, or LPS, anti-delta-dextran, IL-4, IL-5, and transforming growth factor (TGF)-beta1 to induce IgA. After 4 d in culture, cells were surface stained for IgM and non-IgM isotypes and analyzed by FACS((R)). B cells from MMR-deficient mice show a 35-75% reduction in isotype switching, depending on the isotype and on the particular MMR enzyme missing. IgG2b is the most affected, reduced by 75% in Mlh1-deficient animals. The switching defect is not due to a lack of maturation of the B cells, as purified IgM(+)IgD(+) B cells show the same reduction. MMR deficiency had no effect on cell proliferation, viability, or apoptosis, as detected by [(3)H]thymidine incorporation and by propidium iodide staining. The reduction in isotype switching was demonstrated to be at the level of DNA recombination by digestion-circularization polymerase chain reaction (DC-PCR). A model of the potential role for MMR enzymes in class switch recombination is presented.     

Integrin alphaIIbbeta3 and shear-dependent action of glycoprotein Ibalpha stimulate platelet-dependent thrombin formation in stirred plasma

Under conditions of arterial-wall shear rates, platelets bind to von Willebrand factor (vWf) by way of the glycoprotein Ib (GP Ib) complex and integrin alpha(IIb)beta(3). Both adhesive receptors may also play roles in the development of procoagulant activity of platelets. Here, we investigated the effect of shear stress, as provided by a rotating cylinder, on GP Ib- and integrin alpha(IIb)beta(3)-dependent thrombin generation in coagulating platelet-rich plasma (PRP). We measured thrombin continuously with the use of fluorometry from the cleavage rate of a fluorescent low-affinity substrate. The integrin alpha(IIb)beta(3) antagonist abciximab progressively reduced the peak of thrombin formation up to 43% when rate of stirring and shear stress were increased (estimated shear rates of 105-420 s(-1)). Abciximab did not lower the peak of thrombin formation in stirred PRP from patients with Glanzmann's thrombasthenia lacking alpha(IIb)beta(3) but, surprisingly, shortened the time until onset. In PRP from control subjects, antibodies specifically directed against vWf-binding epitopes on GP Ibalpha reduced thrombin formation, with 25% to 30% at the high but not at the low stirring rate. In combination with the anti-GP Ib antibody, abciximab retained its strong inhibitory effect only at the high stirring rate. We conclude that thrombin formation and coagulation in stirred PRP depend, to a large extent, on platelet adhesion to integrin alpha(IIb)beta(3) and, in a shear-dependent way, on GP Ib.     

Effect of quercetin on hypoxic injury in freshly isolated rat proximal tubules

The bioflavonoid quercetin, which has antioxidant properties, protects renal tubular epithelial cells from oxidant-induced injury by inhibiting lipid peroxidation. We examined the effect of quercetin on hypoxia-induced injury in freshly isolated rat renal proximal tubules. Hypoxia induced rapid loss of cellular ATP, followed by functional and structural alterations measured as a decrease in tubular potassium content and sequentially by an increase in lactate dehydrogenase release. Furthermore, hypoxia increased lipid peroxidation, measured as thiobarbituric acid-reactive substances. Quercetin significantly inhibited hypoxia-induced functional and structural tubular injury in addition to lipid peroxidation but did not alter hypoxia-induced ATP depletion. These results demonstrate the potency of the bioflavonoid quercetin in protecting proximal tubules from hypoxic injury, which is independent of tubular energy metabolism and may be related to the inhibition of lipid peroxidation.     

In vitro and in vivo antipseudomonal activity, acute toxicity, and mode of action of a newly synthesized fluoroquinolonyl ampicillin derivative

Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the α-amino-group of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity. Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both. The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa. Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule. The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration. Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA. However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity.     

AT2 receptors: functional relevance in cardiovascular disease

The renin angiotensin system (RAS) is intricately involved in normal cardiovascular homeostasis. Excessive stimulation by the octapeptide angiotensin II contributes to a range of cardiovascular pathologies and diseases via angiotensin type 1 receptor (AT1R) activation. On the other hand, tElsevier Inc.he angiotensin type 2 receptor (AT2R) is thought to counter-regulate AT1R function. In this review, we describe the enhanced expression and function of AT2R in various cardiovascular disease settings. In addition, we illustrate that the RAS consists of a family of angiotensin peptides that exert cardiovascular effects that are often distinct from those of Ang II. During cardiovascular disease, there is likely to be an increased functional importance of AT2R, stimulated by Ang II, or even shorter angiotensin peptide fragments, to limit AT1R-mediated overactivity and cardiovascular pathologies.     

Precursor B cell receptor-dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment

The capacity of precursor B (pre-B) I cells from fetal liver and bone marrow to proliferate and differentiate into surface immunoglobulin-positive immature B cells in vitro was analyzed. Both fetal liver- and bone marrow-derived progenitors do so in a pre-B cell receptor (pre-BCR)-dependent manner in tissue culture medium alone, without addition of other cells or cytokines. Approximately 20% of the initial pre-B I cells enter more than one division. Analyses at the single-cell level show that approximately 15% divide two to five times. Coculture of pre-B I cells with stromal cells did not enhance proliferation or differentiation, whereas the presence of interleukin 7, especially in combination with stromal cells, resulted mainly in the expansion of pre-B I cells and prevented their further differentiation. Thus, the environment of fetal liver or bone marrow is not required for the pre-BCR to exert its function, which is to select and expand cells that have undergone an inframe V(H)-D(H)J(H) rearrangement that produces a pre-BCR-compatible muH chain. It appears unlikely that a ligand for the pre-BCR drives this pre-B cell proliferation.     

Tolerability profile of tasosartan, a long-acting angiotensin II AT1 receptor blocker, in the treatment of patients with essential hypertension

This paper summarizes tolerability data for 1420 patients with essential hypertension who were enrolled in eight double-masked, controlled clinical trials and who received tasosartan, a long-acting, nonpeptidic angiotensin II AT₁ receptor blocker. A total of 2084 patients were included in all treatment groups in these studies. Patients were treated with tasosartan at doses ranging from 10 to 600 mg, over periods of 3 to 16 weeks, except for one study with a 6-day treatment period. Tasosartan was administered once daily (seven studies) or twice daily (one study). No adverse events occurred at a significantly greater frequency in tasosartan-treated patients than in patients who received placebo. Headache, the most frequently reported adverse event, occurred significantly less frequently among tasosartan-treated patients than among those taking placebo (19% and 28%, respectively). The following adverse events (tasosartan and placebo groups) were reported by at least 3% of tasosartan-treated patients: asthenia (7% in each group), pharyngitis (7% in each group), dizziness (7% and 5%, respectively), infection (6% and 7%, respectively), rhinitis (4% and 6%, respectively), pain (4% in each group), diarrhea (4% in each group), nausea (3% in each group), and dyspepsia (3% and 2%, respectively). Cough occurred with a similar frequency in the tasosartan group and in the placebo group (2% and 3%, respectively). Peripheral edema occurred in 2.7% of tasosartan-treated patients and 3.4% of the patients who received placebo. Hyperglycemia occurred in <1% of patients in both groups. Potentially clinically significant laboratory values occurred with comparable frequency in the tasosartan and placebo groups. Discontinuation rates because of adverse events were similar in the tasosartan and placebo groups (2.8% and 2.6%, respectively). Discontinuations because of other medical events occurred less frequently in the tasosartan group than in the placebo group (2.5% vs 4.6%, respectively). Tasosartan had a smooth onset of action without clinical evidence of first-dose hypotension. Blood pressure gradually returned to baseline after tasosartan withdrawal. No safety concerns were identified in these studies. The excellent tolerability profile of tasosartan administered once daily makes it an attractive choice for the clinical management of essential hypertension.     

Increased susceptibility of fat-laden Zucker-rat hepatocytes to bile acid-induced oncotic necrosis: an in vitro model of steatocholestasis

Metabolic liver disorders cause chronic liver disease and liver failure in childhood. Many of these disorders share the histologic features of steatosis and cholestasis, or steatocholestasis. In this study we sought to (1) develop an in vitro model of steatocholestasis, (2) determine the mechanisms of cell death in this model, and (3) determine the role of mitochondrial disturbances in this model. Methods: Hepatocytes were isolated from 8-week-old obese (fa/fa) and lean Zucker rats. Cell suspensions were treated with glycochenodeoxycholic acid (GCDC), after which reactive oxygen species (ROS) generation, oncotic necrosis, apoptosis, and ATP content were assessed. Isolated liver mitochondria were exposed to GCDC and analyzed for ROS generation, mitochondrial membrane-permeability transition (MPT), and cytochrome c release. Oncotic necrosis was significantly increased and apoptosis reduced in fa/fa hepatocytes exposed to GCDC compared with that in lean hepatocytes. Necrosis occurred by way of an ROS- and MPT-dependent pathway. Basal and dynamic ATP content did not differ between fa/fa and lean hepatocytes. GCDC stimulated ROS generation, MPT, and cytochrome c release to a similar extent in purified mitochondria from both fa/fa and lean rats. These findings suggest that fat-laden hepatocytes favor a necrotic rather than an apoptotic cell death when exposed to low concentrations of bile acids. The protective effects of antioxidants and MPT blockers suggest novel therapeutic strategies for the treatment of steatocholestatic metabolic liver diseases.     

Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation

Lycopene is a natural carotenoid antioxidant that is present in tomatoes and tomato products. The pharmacologic function of lycopene in platelets is not yet understood. Therefore, in this study we sought to systematically examine the effects of lycopene in the prevention of platelet aggregation and thrombus formation. We found that lycopene concentration-dependently (2-12 micromol/L) inhibited platelet aggregation in human platelets stimulated by agonists. Lycopene (6 and 12 micromol/L) inhibited phosphoinositide breakdown in platelets labeled with tritiated inositol, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane B2 formation stimulated by collagen. In addition, lycopene (6 and 12 micromol/L) significantly increased the formations of cyclic GMP and nitrate but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of 47,000 Da (P47), a marker of protein kinase C activation, was triggered by PDBu (60 nmol/L). This phosphorylation was markedly inhibited by lycopene (12 micromol/L) in phosphorus-32-labeled platelets. In an in vivo study, thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Lycopene (5, 10, and 20 mg/kg) significantly prolonged the latency period for the induction of platelet-plug formation in mesenteric venules. These results indicate that the antiplatelet activity of lycopene may involve the following pathways: (1) Lycopene may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane B2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization. (2) Lycopene also activated the formations of cyclic GMP/nitrate in human platelets, resulting in the inhibition of platelet aggregation. The results may imply that tomato-based foods are especially beneficial in the prevention of platelet aggregation and thrombosis.     

Central muscarinic receptor subtypes and carrageenin-induced paw oedema in rats

In an earlier report from this laboratory, it was demonstrated that the central cholinergic system exerted a modulatory pro-inflammatory effect on carrageenin-induced paw inflammation in rats. In this study, the role of the central muscarinic receptors in cholinergic modulation of peripheral inflammation was investigated by using several M₁ and M₂ receptor agonists and antagonists. The M₁ receptor agonists aceclidine and arecholine and the nonspecific muscarinic receptor agonist oxotremorine augmented carrageenin oedema, an effect attenuated by the M₁ receptor antagonist scopolamine. Physostigmine behaved like a M₁ receptor agonist at all dose levels. However, the other M₂ receptor agonist, carbachol, produced a dose-dependent dual effect, with lower doses attenuating the oedema and higher doses augmenting the inflammation. While the former action appeared to be due to M₂ receptor stimulation, because it was blocked by AF-DX 116—a M₂ receptor antagonist, the latter action appeared to be induced by M₁ receptor stimulation, because it was inhibited by scopolamine. The pro-inflammatory effect of the M₂ receptor antagonists AF-DX 116 and gallamine appeared to be induced by enhanced neuronal release of acetyl-choline, because the effects were not evident following pretreatment with hemicholinium, which attenuates synthesis of the amine. Muscarinic receptor binding studies with (³H)-QNB indicated that the corpus striatum has substantially higher population of M₁ receptors compared with the cerebellum. In the corpus striatum, (³H)-QNB binding indicated initial up-regulation followed by down-regulation of M₁ receptors during peak inflammation, which appeared to persist even after a decrease in the inflammation. In contrast, the M₁ receptors in the cerebellum appeared to be down-regulated very transiently during the early phase of the inflammation. While these receptor alterations may be due to the inflammation, it is equally possible that they represent changes induced by the stress of pain and inflammation induced by carrageenin.     

A Pilot Study of Gene/Gene and Gene/Environment Interactions in Alzheimer Disease

Background: Although some genes associated with increased risk of Alzheimer Disease (AD) have been identified, few data exist related to gene/gene and gene/environment risk of AD. The purpose of this pilot study was to explore gene/gene and gene/environment associations in AD and to obtain data for sample size estimates for larger, more definitive studies of AD.Methods: The effect of gene/gene and gene/environment interaction related to late onset Alzheimer Disease (LOAD) was investigated in 153 subjects with LOAD and 302 gender matched controls enrolled in the Personalized Medicine Research Project, a population-based bio-repository. Genetic risk factors examined included APOE, ACE, OLR1,and CYP46 genes, and environmental factors included smoking, total cholesterol, LDL, HDL, triglycerides, C-reactive protein, blood pressure, statin use, and body mass index.Results: The mean age of the cases was 78.2 years and the mean age of the controls was 87.2 years. APOE4 was significantly associated with LOAD (OR=3.55, 95%CL=1.70, 7.45). Cases were significantly more likely to have ever smoked cigarettes during their life (49.3% versus 38.4%, p=0.03). The highest recorded blood pressure and pulse pressure measurements were significantly higher in the controls than the cases (all P<0.005). Although not statistically significant in this pilot study, the relationship of the following factors was associated in opposite directions with LOAD based on the presence of an APOE4 allele: obesity at the age of 50, ACE, OLR1, and CYP46.Conclusions: These pilot data suggest that gene/gene and gene/environment interactions may be important in LOAD, with APOE, a known risk factor for LOAD, affecting the relationship of ACE and OLR1 to LOAD. Replication with a larger sample size and in other racial/ethnic groups is warranted and the allele and risk factor frequencies will assist in choosing an appropriate sample size for a definitive study.     

Molecular and Biological Characterization of the Murine Leukotriene B4 Receptor Expressed on Eosinophils

The movement of leukocytes into tissues is regulated by the local production of chemical mediators collectively referred to as chemoattractants. Although chemoattractants constitute a diverse array of molecules, including proteins, peptides, and lipids, they all appear to signal leukocytes through a related family of seven transmembrane–spanning G protein–coupled receptors. The eosinophil is a potent proinflammatory cell that is attracted into tissues during allergic inflammation, parasitic infection, and certain malignancies. Since the molecular mechanisms controlling eosinophil recruitment are incompletely understood, we performed a degenerate polymerase chain reaction on cDNA isolated from murine eosinophils to identify novel chemoattractant receptors. We report the isolation of a cDNA that encodes a 351–amino acid glycoprotein that is 78% identical to a human gene that has been reported to be a purinoceptor (P2Y₇) and a leukotriene B₄ (LTB₄) receptor (BLTR). Chinese hamster ovary (CHO) cells transfected with this cDNA specifically bound [³H]LTB₄ with a dissociation constant of 0.6 ± 0.1 nM. Furthermore, LTB₄ induced a dose-dependent intracellular calcium flux in transfected CHO cells. In contrast, [³⁵S]dATP did not specifically bind to these transfectants. This mRNA was expressed at high levels in interleukin 5–exposed eosinophils, elicited peritoneal macrophages and neutrophils, and to a lesser extent interferon γ stimulated macrophages. Low levels of expression were detected in the lung, lymph node, and spleen of unchallenged mice. Western blot analysis detected the mBLTR protein in murine eosinophils and alveolar macrophages as well as human eosinophils. In addition, elevated levels of mBLTR mRNA were found in the lungs of mice in a murine model of allergic pulmonary inflammation in a time course consistent with the influx of eosinophils. Our findings indicate that this murine receptor is an LTB₄ receptor that is highly expressed on activated leukocytes, including eosinophils, and may play an important role in mediating eosinophil recruitment into inflammatory foci.     

Feedback Control of the Arachidonate Cascade in Osteoblastic Cells by 15-deoxy-Δ12,14-Prostaglandin J2

15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) and an anti-diabetic thiazolidinedione, troglitazone (TRO) are peroxisome proliferator-activated receptor (PPAR)-γ ligands, which regulate immuno-inflammatory reactions as well as adipocyte differentiation. We previously reported that 15d-PGJ₂ can suppress interleukin (IL)-1β-induced prostaglandin E₂ (PGE₂) synthesis in synoviocytes of rheumatoid arthritis (RA). IL-1 also stimulates PGE₂ synthesis in osteoblasts by regulation of cyclooxygenase (COX)-2 and regulates osteoclastic bone resorption in various diseases such as RA and osteoporosis. In this study, we investigated the feedback mechanism of the arachidonate cascade in mouse osteoblastic cells, MC3T3-E1 cells, which differentiate into mature osteoblasts. Treatment with 15d-PGJ₂ led to a significant increase in IL-1α-induced COX-2 expression and PGE₂ production in a dose dependent manner. The effect of 15d-PGJ₂ was stronger than that of TRO. However, it did not affect the expression of COX-1. In addition, cell viability of MC3T3-E1 cells was not changed in the condition we established. This means that 15d-PGJ₂ exerts a positive feedback regulation of the arachidonate cascade of PGE₂ in osteoblastic cells. These results may provide important information about the pathogenesis and treatment of bone resorption in a variety of diseases such as RA and osteoporosis.     

Menthol Enhances an Antiproliferative Activity of 1α,25-Dihydroxyvitamin D3 in LNCaP Cells

1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], the most active form of vitamin D₃, and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1α,25(OH)₂D₃ for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1α,25(OH)₂D₃ in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1α,25(OH)₂D₃-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1α,25(OH)₂D₃ does not induce acute Ca²⁺ response, whereas menthol evokes an increase in [Ca²⁺]_i, which suggests that cross-talks of menthol-induced Ca²⁺ signaling with 1α,25(OH)₂D₃-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1α,25(OH)₂D₃ and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1α,25(OH)₂D₃-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1α,25(OH)₂D₃.