The global disparity in cancer incidence remains a major public health problem. We focused on prostate cancer since microscopic disease in men is common, but the incidence of clinical disease varies more than 100 fold worldwide. Ca2+ signaling is a central regulator of cell proliferation, but has received little attention in cancer prevention. We and others have reported a strong dose-dependent reduction in the incidence of prostate and lung cancer within populations exposed to boron (B) in drinking water and food; and in tumor and cell proliferation in animal and cell culture models.
We examined the impact of B on Ca2+ stores using cancer and non-cancer human prostate cell lines, Ca2+ indicators Rhod-2 AM and Indo-1 AM and confocal microscopy. In DU-145 cells, inhibition of Ca2+ release was apparent following treatment with Ringers containing RyR agonists cADPR, 4CmC or caffeine and respective levels of BA (50 µM), (1, 10 µM) or (10, 20, 50,150 µM). Less aggressive LNCaP cancer cells required 20 µM BA and the non-tumor cell line PWR1E required 150 µM BA to significantly inhibit caffeine stimulated Ca2+ release. BA (10 µM) and the RyR antagonist dantroline (10 µM) were equivalent in their ability to inhibit ER Ca2+ loss. Flow cytometry and confocal microscopy analysis showed exposure of DU-145 cells to 50 µM BA for 1 hr decreased stored [Ca2+] by 32%.
We show B causes a dose dependent decrease of Ca2+ release from ryanodine receptor sensitive stores. This occurred at BA concentrations present in blood of geographically disparate populations. Our results suggest higher BA blood levels lower the risk of prostate cancer by reducing intracellular Ca2+ signals and storage.
Citation: Henderson K, Stella SL Jr, Kobylewski S, Eckhert CD (2009) Receptor Activated Ca2+ Release Is Inhibited by Boric Acid in Prostate Cancer Cells. PLoS ONE4(6): e6009. https://doi.org/10.1371/journal.pone.0006009
Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong
Received: November 17, 2008; Accepted: May 20, 2009; Published: June 23, 2009
Copyright: © 2009 Henderson 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.
Funding: The work for this grant was funded by personal funds (CE) and in part by the University of California Toxic Substances Research and Training Program (TSR&TP) for partial support of KH and SK. CE is the doctoral mentor for KH and SK. TSR&TP funding was for student fellowships restricted to student support and had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
One of the ways cells respond to environmental stimuli is by opening channels between sites of stored calcium, such as the endoplasmic reticulum (ER), Golgi, and mitochondria (mt), which contain high free Ca2+ concentrations (500 µM), and the cytoplasm, which contains low free Ca2+ concentrations (100 nM) . A rapid rise in cytoplasmic Ca2+ can be achieved by capacitative calcium entry (CCE) involving release of stored Ca2+ by the ryanodine receptor (RyR) and the IP3 receptor (IP3R) into the cytoplasm followed by an influx of extracellular Ca2+. CCE activates Ca2+ binding proteins that regulate numerous cell functions including gene transcription, cell proliferation, vesicle secretion, and apoptosis , . Cytoplasmic Ca2+ concentrations are returned to normal as Ca2+ is removed by transporters such as the Na2+- Ca2+ exchanger in the plasma membrane, the sarcoplasmic endoplasmic ATPase (SERCA) in the ER membrane, Ca2+ uniporter in mitochondria, and by binding to high affinity binding proteins , . In this report we present evidence that physiological levels of boric acid (BA) inhibit stored Ca2+ release from RyR agonist sensitive sites.
Boron (B) is the 9th most abundant element in seawater (425 µM B) and until recently dismissed as biologically irrelevant . B is bound to oxygen in nature and in physiological fluids 98.4% is present in the form of B(OH)3 boric acid and 1.6% as B(OH)4− borate. Biology has used this element in the structure of several molecules including: antibiotics in fungi ; quorum sensing auto inducer 2 in bacteria ; and the rhamnogalacturonan-II dimer in plants . In plants, B is required for cell elongation, flowering and seed formation and is an integral component of food crops. Non-charged BA crosses the plasma membrane of root epidermal cells into the cytosol by passive diffusion and this is facilitated by NIP5; 1 transporters . BA is partially converted to borate in the cytosol (pKa 9.6) and transported into the xylem using the export transporter BOR1 , . A human homolog of BOR1 named NaBC1has been reported to be present in mammalian cell lines and to enhance cell proliferation at low BA concentrations , but this has yet to be confirmed by another laboratory. The effect of BA on growth and cell proliferation in trout, zebrafish and the mammalian HEK and HeLa cells follows an inverted U-shape with higher concentrations causing cell growth inhibition –. Concentrations of 60 to 100 µM BA inhibit cell proliferation in prostate cancer cells whereas high concentrations of 500 to 1000 µM BA are required to inhibit the proliferation of non-tumor prostate epithelial cells over the same time frame .
Human blood levels of BA reflect the local geology, water quality, and plants in the diet with a world-wide range from 2 to 120 µM (21 to 1232 ng B/g wet blood) in free living healthy men and women , . Several human studies have observed a decrease in the risk of prostate and lung cancer, and abnormal cervical cytopathology in proportion to the amount of B ingested from food and water –. One study did not observe a protective effect, but differed from other studies in using a different B food database and estimated B content of some foods , .
The biological plausibility for the chemopreventative effect of BA is supported by several lines of investigation. In immunocompromised mice, BA supplementation reduced the growth of transplanted human prostate tumors, decreased IGF-1 tissue concentrations, and lowered serum prostate specific antigen levels . In cell culture, BA reduced the proliferation of human cancer prostate cell lines in a dose dependent manner and inhibited cell migration and invasion , , . We discovered the relationship between BA's ability to inhibit prostate cancer cell proliferation and calcium signaling after mass spectrometry studies showed the affinity of BA for NAD+ was reduced by phosphorylation and therefore potentially subject to biological regulation , . BA was also shown to be a non-competitive inhibitor of ADP-ribosyl cyclase . This led us to study its impact on the NAD+/cADPR Ca2+ release pathway. We showed that pharmacological levels of BA (250 and 1000 µM), but not methylboronic acid, decreased NAD+ stimulated release of Ca2+ without affecting calcium release when applied on its own . It was not clear from these studies if BA was inhibiting Ca2+ release by interfering with NAD+ conversion to cADPR, blocking release of Ca2+ stores, or interfering with CCE. It also raised the possibility that BA concentrations in the normal blood range might be able to inhibit Ca2+ release from ryanodine receptor (RyR) sensitive stores. Here we demonstrate that physiological levels of BA inhibit Ca2+ release from RyR responsive stores in human prostate epithelial cells and lower luminal Ca2+ levels.
Experiments used prostate cancer cell lines DU-145 and LNCaP and the non-tumor cell line PWR1E. Cells were grown and maintained in cell culture plates at 37°C in 95% air and 5% CO2 humidified incubator. For confocal experiments, cells were cultured on glass cover slips for 24 hours prior to performing assays and studied at a confluency of less than 80%. DU-145 and PWR1E cells were acquired from American Type Culture Collection (ATCC, Manassas, VA) and LNCaP was a gift from Dr Allen Pantuck of the Department of Urology, Geffen School of Medicine at the University of California, Los Angeles. RPMI cell culture media was supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (200 mM). PWR1E non-tumor cells were maintained in Keratinocyte media containing streptomycin (100 µg/ml), L-glutamine (200 mM), 2.5 µg human recombinant EGF, and 25 mg of bovine pituitary extract (Gibco, Carlsbad, CA). Boron depleted media was prepared using the boron specific ionic exchange resin, Amberlite IRE-743 (Sigma) as previously described  and modified as follows. Nine grams of autoclaved IRA-743 ion exchange resin were added to 500 ml media and mixed on an Orbit rotator at 75 to 100 rpm for 15 to 20 hrs at 9°C.
Measurement of Ca2+ Transients
Storage Ca2+ changes were monitored using the calcium sensitive dye Rhod-2, AM ester (Biotium, Hayward, CA) which accumulates in organelles. Rhod-2, AM ester has a Kd of 1 mM and has been shown to compartmentalize, particularly in the mitochondria, but as we show, in other organelles as well (Fig. 1). We also used ER Tracker green and Mito Tracker green (Molecular Probes, Carlsbad, CA) in conjunction with Rhod-2, Am ester. They are highly specific fluorescent labels for the endoplasmic reticulum and mitochondria, respectively. We analyzed Ca2+ changes in response to BA and various agonists by selecting regions of interest in cells that overlapped the red Rhod-2 Ca2+ label with the green ER tracker (Fig. 1A) –. Rhod-2, AM ester was prepared as a 1 mM stock solution in DMSO and diluted in either complete RPMI media or complete Keratinocyte media to 3 µM. Cells were incubated with Rhod-2 AM ester (5 µM) and ER or Mt Tracker (0.5 µM, 250 nM respectively) in RPMI or Keratinocyte media for 30 minutes at 37°C. Ca2+ release was stimulated using agonists in combination with different concentrations of BA in Ringers solution. Images were collected with a Zeiss 510 LSM 5 Pascal mounted to an upright microscope (Zeiss Axioplan 2) equipped with an Axoplan X63 (NA 0.95) water immersion objective. A HeNe laser was used to excite Rhod-2 at 543 nm. 488 nm from a laser diode was used to excite ER or Mt Tracker. The emission was collected on a photomultiplier tube through a 560 nm LP filter for Rhod-2 and a 505 LP filter for ER and Mito trackers. Additional magnification, time series, and background subtraction were controlled using Zeiss LSM acquisition software. All images were acquired as 12 bit.
A. Two DU-145 cells labeled with red fluorescent intracellular calcium fluorophore, Rhod-2, and green fluorescent endoplasmic reticulum label, ER tracker. Yellow indicates overlap of Ca2+ and ER and the circle is the region of interest (ROI) analyzed in these experiments. Arrows indicate organelles: nucl (nucleolus), nuc (nucleus), ER (endoplasmic reticulum) B. Two DU-145 cells labeled with red fluorescent intracellular Ca2+ fluorophore, Rhod-2, and green fluorescent mitochondrial label, Mito tracker. Yellow indicates overlap of calcium and mitochondria. Arrows indicate organelles: nucl (nucleolus), nuc (nucleus), mt (mitochondria).
Analysis of Ca2+ release using confocal microscopy
BA treatments were applied in Ca2+ free Ringer's solution prepared using ultrapure water to remove B , . Ca2+ release from the RyR was activated by the addition of either 25 µM cADPR to 10 µM digitonin permeabilized cells, or 20 mM caffeine or 100 µM 4-chloro-m-cresol (4cmc) in intact cells , . Inhibition of Ca2+ release was achieved using 10 µM dantrolene or varying concentrations of BA in the presence of an agonist . SERCA was inhibited using 10 µM cyclopiazonic acid (CPA) . All drug applications were for a 30 second duration in calcium free Ringer's solution (143 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, 1 mM EGTA-2H2O, 1 mM EGTA) in order to view Ca2+ release from the stores without input of external Ca2+. Drug application was followed by a three minute wash in Ringer's solution containing calcium (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES). Solutions were delivered to the perfusion chamber at a rate of 1 ml/min by using a single-pass, gravity-feed perfusion system. Confocal images were taken every second and began 2 minutes prior to the first treatment application in order to establish a base line of fluorescence intensity. The change in fluorescence intensity (f) caused by the drug application was compared to an averaged baseline measurement which consisted of three measurements prior to the application of the drug (fo). All measurements were normalized to the base line in this manner using the ratio f-fo/fo. Typically 6–10 cells were analyzed in one field of view and all experiments were performed on a minimum of three independent preparations. The order of drug application was based on an initial experiment involving consecutive agonist-only applications. If the 2nd application of agonist caused a lower Ca2+ release than the 1st, cells were treated with agonist plus BA first. This avoided the possibility that measured inhibition of Ca2+ release due to BA was in part due to refractory cells made so by prior treatment with agonist.
Analysis of stored calcium levels
DU-145 prostate cancer cells were treated with BA for 1 to 72 hrs in media stripped of boron using Amberlite IRA-743 ion-exchange resin (Sigma-Aldrich, St. Louis, MO) as previously described. Just prior to analysis cells were removed from the plate using 1% trypsin digestion, centrifuged, and labeled for 45 minutes in standard media with 3 µM Rhod-2 AM ester for storage Ca2+ analysis. In order to monitor cytoplasmic Ca2+ levels, cells were labeled with 5 µM Indo-1 AM (Invitrogen, Carlsbad, CA) for 45 minutes. Labeled cells were then washed and resuspended in 1 mL of normal media. Fluorescence was analyzed using a Beckton Dickinson BD-LSR I analytic flow cytometer. Rhod-2 excitation was achieved with a 514 nm argon laser and analyzed in the (581 nm) FL-2 channel. Indo-1 was excited with a UV laser (351 nm) and analyzed on 400 nm (FL5) and 510 nm (FL4) band-pass filters. Results for Indo-1 labeled cells are given as ratios of fluorescence (FL5/FL4). Forward and side scatter were used to gate out cellular fragments . Raw flow cytometry data was analyzed using FLOWJO software (Treestar Software, San Carlos, CA, USA). Results are presented as% Ca2+ levels compared to untreated cells. Storage Ca2+ levels were also analyzed in BA treated and non-treated cells by comparing Ca2+ storage emptying in response to 100 µM thapsigargin using confocal microscopy .
Statistical analysis of the data
Data are presented as mean±SD and were analyzed using Student's paired or unpaired t-test or one-way ANOVA for multiple comparisons with Dunnett's multiple comparison post-hoc test using Graphpad Prism 4.0. A p value<0.05 was considered to be statistically significant. The equation for non-linear one site binding (hyperbola) Y = Bmax*X/(Kd+X) was used to calculate Kd and Bmax values using Graphpad software.
Chemicals and supplies
B was removed from water and media using methods previously described . Caffeine, 4-CmC, ATP, CPA, dantrolene, thapsigargin, cADPR, digitonin and boric acid were obtained from Sigma (St. Louis, MO) diluted in calcium free Ringer's Solution to ensure that if DMSO was present levels were no greater than 0.1%. All solutions were prepared fresh prior to perfusion. Superfusion with 0.1% DMSO in calcium free Ringer's solution did not affect ER Ca2+ levels or imaging responses.
The objective of the study was to determine if physiological concentrations of BA inhibit the release of Ca2+ from RyR sensitive stores in human prostate cancer and non-tumor epithelial cells. DU-145 cells were loaded with Rhod-2 and ER tracker or Mito tracker to determine if Rhod-2 compartmentalized in one or both compartments. It was not possible to delineate Ca2+ signaling from the ER versus mitochondria in live cells using confocal microscopy. Rhod-2 labeled Ca2+ in mitochondria, ER, nucleolus, and other areas in the cell (Fig. 1A–B). In our experiments, a combination of ER Tracker and Rhod-2 were used to define our regions of interest on sites of stored Ca2+ (Fig. 1A). This area included Ca2+ stores located in the ER and mitochondria (Fig. 1A–B). Application of BA from 0.1–1000 µM alone did not demonstrate an immediate measurable effect on storage Ca2+ using confocal microscopy (data not shown).
Boric acid inhibits Ca2+ release in response to ryanodine receptor agonists
To determine if BA inhibited Ca2+ release from the RyR, we treated DU145 cells with three different RyR agonists in combination with BA. Competitive ligand binding analysis showed BA was a single site reversible competitive inhibitor of cADPR, an endogenous agonist of RyR (Fig. 2A–C). The equilibrium dissociation constant, (KD) for cADPR was 15.10, but in the presence of BA increased to 49.39. Inhibition by BA was reversed by increasing the concentration of cADPR and the presence of BA did not change the maximum number of binding sites (Bmax) (Table 1).
A. 50 µM BA inhibited 25 µM cADPR induced Ca2+ release (n = 6; ***p<0.001). B. Competitive ligand binding study showed BA was a surmountable competitive antagonist of cADPR in a single site model. Concentrations of BA were held at 50 µM. At low cADPR concentrations BA shifted the Ca2+ release response to the right, but this was reversed at higher cADPR concentrations and the maximum response (Bmax) was not changed. Each data point is the mean of n = 6. R2 is 0.9155 and 0.8606 for cADPR without and with BA, respectively.
We then analyzed the effect of BA on other RyR agonists in intact cells. We first used caffeine [20 mM] an agonist that potentiates RyR sensitivity to its native ligand, Ca2+. In response to two consecutive applications of caffeine, the second release was slightly greater than the first (Fig. 3A). This sequence was then repeated with BA added in combination with caffeine in the second application. The results of these experiments show that BA reduced Ca2+ release in a dose dependent manner from 10 to 150 µM BA (Fig. 3B–F). We then examined the effect of BA on 4CmC, a direct agonist of the RyR. Consecutive applications of 50 µM 4CmC resulted in equivalent Ca2+ release (Fig. 4A). As observed with caffeine, BA decreased 4CmC stimulated Ca2+ release in a dose dependent manner (Fig. 4B–E). However, inhibition began at 1 µM BA and reached a maximum at 10 µM BA.
A. Ca2+ release in DU-145 cells in response to consecutive applications of 20 mM caffeine (n = 6, *p = 0.0168). B. 10 µM BA significantly inhibited Ca2+ release (n = 6, **p<0.01). C. 20 µM M BA significantly inhibited Ca2+ release (n = 6, *p = 0.0163). D. 50 µM BA significantly inhibited Ca2+ release (n = 6, **p = 0.0042). E. 150 µM BA significantly inhibited Ca2+ release, n = 6, ***p = 0.0001. F. combined data analysis showing inhibitory dose response effect of BA on caffeine stimulated Ca2+ release, (n = 6 per concentration, **p<0.01). Figures 2–9, each bar represents the mean response of 6 experiments (n = 6) replicated 3 times. The line scans on the right of each bar graph are representative responses from a single experiment.
A. Consecutive applications of 4-CmC did not alter the calcium release response (n = 6, NS). B. 0.1 µM BA did not inhibit Ca2+ release. C. 1.0 µM BA inhibited Ca2+ release (n = 6, *p<0.05). D. 10 µM BA inhibited Ca2+ release (n = 6, ***p = 0.0005). E. Combined data analysis showing a dose dependent Ca2+ release response (n = 6 per concentration (**p<0.01).
Previous studies have shown that the inhibitory effect of BA on cell proliferation at the IC50 for DU-145 cells was approximately 10% less effective on the less aggressive LNCaP prostate cancer cell line. In addition, BA was not able to achieve a 50% reduction in proliferation in non-tumor PWR1E prostate epithelial cells in experiments using up to 4 times the IC50 for DU145 . We examined these cell lines to determine if BA was also less effective in inhibiting caffeine sensitive Ca2+ release. Inhibition of Ca2+ release in response to caffeine in LNCaP prostate tumor cells occurred over 20 µM-150 µM BA (Fig. 5A–D). Thus, LNCaP cells were less sensitive to BA compared to DU-145. LNCaP cells were not responsive to 4CmC treatment (not shown). The PWR1E non-tumor, hyperplasic prostate cell line required 150 µM BA to inhibit caffeine stimulated Ca2+ release (Fig. 6A–D). PWR1E cells were non-responsive to 4CmC treatment (not shown).
A. 20 µM BA inhibition of calcium release (n = 6, *p = 0.0226). B. 50 µM BA inhibition of Ca2+ release (n = 6, **p = 0.0019). C. 150 µM BA inhibition of caffeine induced Ca2+ release (n = 6, ***p<0.0001). D. Combined data showing concentration dependent inhibition of caffeine stimulated release (n = 6, **p<0.01).
A. Consecutive caffeine applications in PWR1E prostate cell. (n = 6, NS). B. 50 µM BA and caffeine induced Ca2+ release shows no inhibition (n = 6, NS). C. 150 µM BA inhibited caffeine induced Ca2+ release (n = 6, **p = 0.0029). D. Combined data showing no dose response and inhibition only at 150 µM (n = 6, *p<0.05).
Boric acid inhibits cyclopiazonic acid induced Ca2+ release from the ER
The release of ER Ca2+ stores in some non-excitable cells can be stimulated using cyclopiazonic acid (CPA) which inhibits SERCA in a manner similar to thapsigargin, but can be washed out . BA (0.1–1000 µM) by itself did not alter storage calcium levels during the time course of the experiment (not shown). Responses to consecutive applications of CPA [10 µM] did not decay (Fig. 7A). Simultaneous application of CPA and 1 µM BA caused a significant decrease in release and 10 µM BA caused near complete inhibition (Fig. 7B–D). We then tested the effect of dantrolene, a known inhibitor of the RyR on Ca2+ release by CPA. We found that 10 µM dantrolene inhibited CPA stimulated Ca2+ release at a level equivalent to 10 µM BA (Fig. 8A–B).
Cells were tested with CPA+BA followed by CPA. A. Consecutive applications of CPA did not alter Ca2+ release (n = 6, NS). B. 0.1 µM M BA did not inhibit CPA stimulated Ca2+ release (n = 6, NS). C. 1.0 µM BA inhibited CPA stimulated Ca2+ release (n = 6, **p = 0.0037). D. 10 µM BA inhibited CPA stimulated Ca2+ release (n = 6, ***p<0.0001).
A. Cells were tested with CPA (10 µM)+dantrolene (10 µM) followed by CPA (n = 6, ***p<0.0001. B. BA inhibited CPA stimulated Ca2+ release in a concentration dependent manner. The inhibitory effect of dantroline and BA were equivalent at 10 µM, CPA (n = 6, ** p<0.01).
Boric acid treatment lowers storage [Ca2+] with no effect on cytoplasmic [Ca2+]
Our results showing BA inhibited Ca2+ release lead us to analyze relative [Ca2+]st levels using Rhod-2 stained cells and relative [Ca2+ ]cyt levels using Indo-1 stained cells and flow cytometry. Exposure of DU-145 cells to BA [10–50 µM] for 24 hours did not affect [Ca2+ ]cyt (Fig. 9A). However, reductions [Ca2+]st (22%) occurred with 10 µM BA resulted in a 32% reduction in [Ca2+]st by 50 µM BA at 1 hour (Fig. 9B). Neither higher BA concentrations nor treatment with 10 µM BA up to 72 hours resulted in further reduction in [Ca2+ ]st (Fig. 9BC). We then conducted confocal measurements of thapsigargin stimulated Ca2+ release after DU-145 cells were pretreated with BA (50 µM) for 24 hrs and observed a 35% decrease in Ca2+ release (Fig. 9D).
A. Relative [Ca2+]cyt following exposure to 0, 10 and 50 µM BA for 24 hrs (n = 5, NS). B. [Ca2+]st as percent of 0 treatment in DU-145 cells treated with 0–250 µM BA for 24 hrs. Boric acid [10–50 µM] reduced ER calcium levels by more than 22%–32% compared to untreated cells (n = 5,*p<0.05, **p<0.01, ***p<0.001). C. Reduction of [Ca2+]st in DU145 cells treated with 50 µM BA from 1–72 hours (n = 5, **p<0.01). D. Confocal measurement showed pretreatment of cells with BA for 24 hrs lowered thapsigargin stimulated Ca2+ release compared to untreated cells (n = 6, ***p<0.0001).
This study reports the unexpected finding that stored Ca2+ release and luminal levels can be modulated by physiologically relevant levels of BA in DU-145 prostate cancer epithelial cells. This is relevant to our understanding of cancer risk since blood levels of BA are determined by the consumption of B in drinking water and plant derived foods . It is likely to be the first cellular response to B exposure and thus a starting point for understanding how the risk of prostate and lung cancer is reduced by B in a dose dependent manner , , . BA exhibited the attributes of a classic antagonist in that it did not have an immediate effect on Ca2+ release when applied by itself and its effects were agonist dependent. BA dose dependently inhibited Ca2+ release in response to cADPR, an endogenous agonist of the RyR, and agonists caffeine and 4-CmC (Fig. 2–5). In our competitive ligand binding analysis, BA displayed the characteristic of a single site antagonist in that it was reversible at higher concentrations of cADPR (fig. 2B). BA increased Kd, from 15.1 to 49.4, but did not affect BMax (Table 1). BA has been shown to bind to cADPR at high concentrations, however, BA's ability to inhibit cADPR, caffeine and 4CmC induced Ca2+ release indicates that at physiological concentrations BA may bind to the cADPR binding site on the RyR stabilizing the Ca2+ channel in its inactive state .
Proliferation of LNCaP cells have been reported to be about 10% less sensitive and PWR1E non-tumor cells are more than 4 fold less sensitive to BA than DU-145 cells . We also observed this pattern of sensitivity in BA's ability to inhibit caffeine stimulated Ca2+ release. The lowest effective concentration for DU-145 was 10 µM BA, LNCaP was 20 µM BA, and PWR1E was 150 µM BA (Fig. 3F, 5D & 6D). In addition to the decreased responsiveness to BA, both LNCaP and PWR1E were non-responsive to 4CmC treatment. RyR isoforms 1 and 2, but not 3 are known to be activated by 4CmC in some cell lines . It is possible that cell line differences were due to differences in RyR isoforms or their expression. LNCaP cell lines have been shown to express RyR 1 and 2, but not 3 . We were unable to locate studies in the literature that identified RyR isoforms in DU-145 or PWR1E cells in any prostate cell line.
Renal function tests have shown BA is reabsorbed by the kidney in non-pregnant and pregnant women . The discovery of an electrogenic, voltage-regulated bicarbonate sodium-coupled borate co-transporter (NaBC1) in rat kidney tubules may explain this observation, but it has not yet been confirmed by another laboratory. NaBC1 expression induced proliferation in HEK and Hela cells by activation of the MAPK pathway 16 hours post-treatment . It is unknown at this time whether NaBC1 is expressed in prostate tumor and non-tumor cell lines, but differences in its expression has been raised as a possible explanation for the variation in cellular sensitivity to BA between prostate tumor and non-tumor cells .
To further explore BA's ability to inhibit stored Ca2+ release we tested its effects in the presence of CPA (Fig. 7–8). CPA inhibits the SERCA channel resulting in emptying of Ca2+ stores . Our study showed that BA dose dependently blocked CPA mediated Ca2+ release in DU-145 cells. We also observed a reduction of storage Ca2+ levels by 22% to 32% in 10 to 50 µM BA treated DU-145 cells compared to untreated cells. STIM proteins are involved in triggering Ca2+ influx into the ER –. If BA reduces Ca2+ leakage through RyR channels the major loss by leakage may occur through presenilins and other non-channel proteins that do not stimulate STIM proteins. This would result in decreased stored Ca2+ levels that are unable to signal refilling.
The importance of Ca2+ in cell cycle control and proliferation is a well established area of cancer research, but it has not been studied as a mode of action in cancer prevention –. BA's ability to modulate cell proliferation has been linked to changes in the expression of cyclins and the MAPK pathway in DU-145, HEK293 and HeLa cells , , . However, it is possible these effects occur in response to reductions in Ca2+ release or storage. In the prostate cancer LNCaP cell line, Humez observed that IGF (5 ng/ml), which increases cell growth, increased ER [Ca2+]ER, whereas TNF alpha (1 ng/ml), which reduces cell proliferation, reduced [Ca2+]ER .
In conclusion, BA has previously been reported to reduce the risk of prostate cancer in humans and reduce tumor growth and prostate cancer cell proliferation, migration, and invasion. Our results demonstrate that physiological levels of BA inhibit agonist stimulated release of stored Ca2+ in a dose dependent manner and lower stored Ca2+ storage levels. This suggests that BA's ability to dampen Ca2+ signaling in cancer cells underlies its ability to reduce clinical cancer risk.
Conceived and designed the experiments: KH CDE. Performed the experiments: KH SK. Analyzed the data: KH CDE. Contributed reagents/materials/analysis tools: SLSJ. Wrote the paper: KH CDE.
- 1. Berridge M, Bootman M, Roderick H (2003) Calcium signaling: dynamics, homeostasis and remodeling. Mol Cell Bio 4: 517–529.M. BerridgeM. BootmanH. Roderick2003Calcium signaling: dynamics, homeostasis and remodeling.Mol Cell Bio4517529
- 2. Golovina V, Mordecai B (1997) Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum. Science 275: 1643–1648.V. GolovinaB. Mordecai1997Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum.Science27516431648
- 3. Golovina VA (1999) Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes. Am J Physiol Cell Physiol 277: C343–349.VA Golovina1999Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes.Am J Physiol Cell Physiol277C343349
- 4. Szabsdkai G, Muchen M (2007) Mitochondria: The hub of cellular Ca2+ signaling. Physiology 23: 84–94.G. SzabsdkaiM. Muchen2007Mitochondria: The hub of cellular Ca2+ signaling.Physiology238494
- 5. Watson W, Facchina S, Grimaldi M, Verma A (2003) Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system. J Neurochem 87: 30–43.W. WatsonS. FacchinaM. GrimaldiA. Verma2003Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system.J Neurochem873043
- 6. Corey E (2003) Boron. Chemical and Engineering News 81: 40.E. Corey2003Boron.Chemical and Engineering News8140
- 7. Woods W (1994) An introduction to boron: history, sources, uses, and chemistry. Environ Health Perspect 102: 5–11.W. Woods1994An introduction to boron: history, sources, uses, and chemistry.Environ Health Perspect102511
- 8. Chen X, Schauder S, Potier N, Dorsselaer A, Pelczer I, et al. (2002) Structural identification of a bacterial quorum sensing signal containing boron. Nature 415: 545–549.X. ChenS. SchauderN. PotierA. DorsselaerI. Pelczer2002Structural identification of a bacterial quorum sensing signal containing boron.Nature415545549
- 9. O'Neill MA, W D, Kates K, Pellerin P, Doco T, Darvill AG, Albersheim P (1996) Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. J Biol Chem 271: 22923–22930.MA O'NeillD. WK. KatesP. PellerinT. DocoAG DarvillP. Albersheim1996Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester.J Biol Chem2712292322930
- 10. Takano J, Miwa K, Fujiwara T (2008) Boron transport mechanisms: collaboration of channels and transporters. Trends in Plant Science 13: 451–457.J. TakanoK. MiwaT. Fujiwara2008Boron transport mechanisms: collaboration of channels and transporters.Trends in Plant Science13451457
- 11. Sutton T, B U, Hayes J, Collins NC, Shi B-J, Schnurbusch T, Hay A, Mayo G, Pallotta M, Tester M, Langridge P (2007) Boron toxicity tolerance in barley arising from efflux transporter amplification. Science 318: 1446–1449.T. SuttonU. BJ. HayesNC CollinsB-J ShiT. SchnurbuschA. HayG. MayoM. PallottaM. TesterP. Langridge2007Boron toxicity tolerance in barley arising from efflux transporter amplification.Science31814461449
- 12. Tankano J, Kyoko Miwa K, Yuan L, von Wiren N, Fujiwara T (2005) Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability. Proc Natl Acad Sci U S A 102: 12276–12281.J. TankanoK. Kyoko MiwaL. YuanN. von WirenT. Fujiwara2005Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability.Proc Natl Acad Sci U S A1021227612281
- 13. Park M, L Q, Shcheynikov N, Zeng W, Mualiem S (2004) NaBC1 is a ubiquitous electrogenic Na+-coupled borate transporter essential for cellular boron homeostasis and cell growth and proliferation. Mol Cell Bio 16: 331–341.M. ParkQ. LN. ShcheynikovW. ZengS. Mualiem2004NaBC1 is a ubiquitous electrogenic Na+-coupled borate transporter essential for cellular boron homeostasis and cell growth and proliferation.Mol Cell Bio16331341
- 14. Eckhert C (1998) Boron stimulates embryonic trout growth. J Nutr 128: 2488–2493.C. Eckhert1998Boron stimulates embryonic trout growth.J Nutr12824882493
- 15. Rowe R, Eckhert C (1999) Boron is required for zebrafish embryogenesis. Journal of Experimental Biology 202: 1649–1654.R. RoweC. Eckhert1999Boron is required for zebrafish embryogenesis.Journal of Experimental Biology20216491654
- 16. Barranco W, Eckhert C (2004) Boric acid inhibits prostate cancer cell proliferation. Cancer Letters 216: 21–29.W. BarrancoC. Eckhert2004Boric acid inhibits prostate cancer cell proliferation.Cancer Letters2162129
- 17. Barr R, Clarke W, Clarke R, Venturelli J, Norman G, et al. (1993) Regulation of lithium and boron levels in normal human blood: Environmental and genetic considerations. J Lab Clin Med 121: 614–619.R. BarrW. ClarkeR. ClarkeJ. VenturelliG. Norman1993Regulation of lithium and boron levels in normal human blood: Environmental and genetic considerations.J Lab Clin Med 121614619
- 18. IPCS (1998) Boron, Environmental Health Criteria. WHO. pp. 61–65.IPCS1998Boron, Environmental Health CriteriaWHO6165
- 19. Barranco W, Hudak P, Eckhert C (2007) Evaluation of ecological and in vitro effects of boron on prostate cancer risk. Cancer Causes Control 18: 583–584.W. BarrancoP. HudakC. Eckhert2007Evaluation of ecological and in vitro effects of boron on prostate cancer risk.Cancer Causes Control18583584
- 20. Barranco W, Hudak P, Eckhert C (2007) Erratum: Evaluation of ecological and in vitro effects of boron on prostate cancer risk. Publisher Erratum Figure 1. Cancer Causes Control 18: 583–584.W. BarrancoP. HudakC. Eckhert2007Erratum: Evaluation of ecological and in vitro effects of boron on prostate cancer risk. Publisher Erratum Figure 1.Cancer Causes Control18583584
- 21. Mahabir S, Spitza M, Barrera S, Dong Y, Eastham C, et al. (2008) Dietary boron and hormone replacement therapy as risk factors for lung cancer in women. Am J Epidem 167: 1070–1080.S. MahabirM. SpitzaS. BarreraY. DongC. Eastham2008Dietary boron and hormone replacement therapy as risk factors for lung cancer in women.Am J Epidem16710701080
- 22. Cui Y, Winton M, Zhang Z-F, Rainey C, Marshall J, et al. (2004) Dietary boron intake and prostate cancer risk. Oncology Reports 11: 887–892.Y. CuiM. WintonZ-F ZhangC. RaineyJ. Marshall2004Dietary boron intake and prostate cancer risk.Oncology Reports11887892
- 23. Gonzalez A, Peters U, Lampe J, White E (2007) Boron intake and prostate cancer risk. Cancer Causes Control 18: 1131–1140.A. GonzalezU. PetersJ. LampeE. White2007Boron intake and prostate cancer risk.Cancer Causes Control1811311140
- 24. Hunt C, Meacham S (2001) Aluminum, boron, calcium, copper, iron, magnesium, manganese, molybdenum, phosphorus, potassum, sodium, and zinc: Concentrations in common Western foods and estimated daily intakes by infants; toddlers; and male and female adolescents, adults, and seniors in the United States. Journal of the American Dietetic Association 101: 1058–1060.C. HuntS. Meacham2001Aluminum, boron, calcium, copper, iron, magnesium, manganese, molybdenum, phosphorus, potassum, sodium, and zinc: Concentrations in common Western foods and estimated daily intakes by infants; toddlers; and male and female adolescents, adults, and seniors in the United States.Journal of the American Dietetic Association10110581060
- 25. Gallardo-Williams M, Chapin R, King P, Moser G, Goldsworthy T, et al. (2004) Boron supplementation inhibits the growth and local expression of IGF-1 in human prostate adenocarcinoma (LNCaP) tumors in nude mice. Tox Path 32: 73–78.M. Gallardo-WilliamsR. ChapinP. KingG. MoserT. Goldsworthy2004Boron supplementation inhibits the growth and local expression of IGF-1 in human prostate adenocarcinoma (LNCaP) tumors in nude mice.Tox Path327378
- 26. Barranco W, Eckhert C (2006) Cellular changes in boric acid-treated DU-145 prostate cancer cells. Brit J Cancer 94: 884–890.W. BarrancoC. Eckhert2006Cellular changes in boric acid-treated DU-145 prostate cancer cells.Brit J Cancer94884890
- 27. Barranco W, Kim D, Stella S, Eckhert C (2008) Boric Acid inhibits stored Ca2+ release in DU-145 prostate cancer cells. Cell Bio Tox. W. BarrancoD. KimS. StellaC. Eckhert2008Boric Acid inhibits stored Ca2+ release in DU-145 prostate cancer cells.Cell Bio Tox10.1007/s10565-008-9085-7. 10.1007/s10565-008-9085-7.
- 28. Kim D, Faull K, Norris A, Eckhert C (2004) B orate-nucleotide complex formation depends on charge and phosphorylation state. J Mass Spectrometry 39: 743–451.D. KimK. FaullA. NorrisC. Eckhert2004B orate-nucleotide complex formation depends on charge and phosphorylation state.J Mass Spectrometry39743451
- 29. Kim D, Marbois B, Faull K, Eckhert C (2003) Esterification of borate with NAD+ and NADH as studied by electrospray ionization mass spectrometry and 11B NMR spectroscopy. J Mass Spectrometry 38: 632–640.D. KimB. MarboisK. FaullC. Eckhert2003Esterification of borate with NAD+ and NADH as studied by electrospray ionization mass spectrometry and 11B NMR spectroscopy.J Mass Spectrometry38632640
- 30. Kim D, Que Hee S, Norris A, Faull K, Eckhert C (2006) Boric acid inhibits ADP-ribosyl cyclase non-competitively. J Chromatography A 1115: 246–252.D. KimS. Que HeeA. NorrisK. FaullC. Eckhert2006Boric acid inhibits ADP-ribosyl cyclase non-competitively.J Chromatography A1115246252
- 31. Chameau P, Vrede Y, Fossier P, Baux G (2001) Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release. European Journal of Physiology 443: 289–296.P. ChameauY. VredeP. FossierG. Baux2001Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release.European Journal of Physiology443289296
- 32. Minta A, Kao J, Tsien R (1989) Fluorescent indicators for cytosolic calcium based rhodamine and fluorescein chromophores. J Bio Chem 264: 8171–8178.A. MintaJ. KaoR. Tsien1989Fluorescent indicators for cytosolic calcium based rhodamine and fluorescein chromophores.J Bio Chem26481718178
- 33. Mironov S, Ivannikov M, Johansson M (2005) [Ca2+]i Signaling between mitochondria and endoplasmic reticulum in neuron is regulated by microtubules. J Bio Chem 250: 715–721.S. MironovM. IvannikovM. Johansson2005[Ca2+]i Signaling between mitochondria and endoplasmic reticulum in neuron is regulated by microtubules.J Bio Chem250715721
- 34. Bennett A, Rowe R, Soch N, Eckhert C (1999) Boron Stimulates Yeast (Saccharomyces cerevisiae) Growth. J Nutr 129: 2236–2238.A. BennettR. RoweN. SochC. Eckhert1999Boron Stimulates Yeast (Saccharomyces cerevisiae) Growth.J Nutr12922362238
- 35. Kong H, Jones PP, Koon A, Zhang L, Duff HJ, et al. (2008) Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor. Biochemistry Journal 414: 441–452.H. KongPP JonesA. KoonL. ZhangHJ Duff2008Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor.Biochemistry Journal414441452
- 36. Morita K, Kitayama T, Kitayama S, Hohi T (2006) Cyclic ADP-ribose requires FK506-binding protein to regulate intracellular Ca2+ dynamics and cateholamine release in acetylcholine stimulated bovine adrenal chromaffin cells. Journal of Pharmacological Sciences. K. MoritaT. KitayamaS. KitayamaT. Hohi2006Cyclic ADP-ribose requires FK506-binding protein to regulate intracellular Ca2+ dynamics and cateholamine release in acetylcholine stimulated bovine adrenal chromaffin cells.Journal of Pharmacological Sciences
- 37. Kaufman A, Kraft B, Michalek-Sauberer A, Weigl L (2008) Novel ryanodine receptor mutation that may cause malignant hyperthermia. Anesthesiology 109: 457–464.A. KaufmanB. KraftA. Michalek-SaubererL. Weigl2008Novel ryanodine receptor mutation that may cause malignant hyperthermia.Anesthesiology109457464
- 38. Xia T, Kovochich M, Brant J, Hotze M, Sempf J, et al. (2006) Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Letters 6: 1794–1807.T. XiaM. KovochichJ. BrantM. HotzeJ. Sempf2006Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm.Nano Letters617941807
- 39. Li N, Lin P, Danielpour D, Pan Z, Ma J (2008) Overexpression of bax induces down-regulation of store operated calcium entry in prostate cancer cells. Journal of Cellular Physiology 216: 172–179.N. LiP. LinD. DanielpourZ. PanJ. Ma2008Overexpression of bax induces down-regulation of store operated calcium entry in prostate cancer cells.Journal of Cellular Physiology216172179
- 40. Matsumoto H, Takeuchi R, Arai T, Sato N, Akimoto Y, et al. (2003) Cyclopiazonic acid discharges intracellular Ca2+ stores and stimulates Ca2+ influx in cultured human ginvival fibroblasts. Journal of Oral Science 45: 139–144.H. MatsumotoR. TakeuchiT. AraiN. SatoY. Akimoto2003Cyclopiazonic acid discharges intracellular Ca2+ stores and stimulates Ca2+ influx in cultured human ginvival fibroblasts.Journal of Oral Science45139144
- 41. Fessenden J, Feng W, Pessah I, Allen P (2006) Amino acid residue Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol. J Biol Chem 281: 21022–21031.J. FessendenW. FengI. PessahP. Allen2006Amino acid residue Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol.J Biol Chem2812102221031
- 42. Mariot P, Prevarskaya N, Roudbaraki M, Le Bourhis X, Van Coppenolle F, et al. (2000) Evidence of functional ryanodine receptor involved in apoptosis of prostate cancer (LNCaP) cells. The Prostate 43: 205–214.P. MariotN. PrevarskayaM. RoudbarakiX. Le BourhisF. Van Coppenolle2000Evidence of functional ryanodine receptor involved in apoptosis of prostate cancer (LNCaP) cells.The Prostate43205214
- 43. Pahl MV, C BD, Strong PL, Murray FJ, Vaziri ND (2001) The effect of pregnancy on renal clearance of boron in humans: a study based on normal dietary intake of boron. Toxicological Sciences 60: 252–256.MV PahlBD CPL StrongFJ MurrayND Vaziri2001The effect of pregnancy on renal clearance of boron in humans: a study based on normal dietary intake of boron.Toxicological Sciences60252256
- 44. Wayman C, McFadzean I, Gibson A, Tucker J (1996) Two distinct membrane currents activated by cyclopiazonic acid-induced calcium store depletion in single smooth muscle cells of the mouse anococcygeus. British Journal of Pharmacology 117: 566–572.C. WaymanI. McFadzeanA. GibsonJ. Tucker1996Two distinct membrane currents activated by cyclopiazonic acid-induced calcium store depletion in single smooth muscle cells of the mouse anococcygeus.British Journal of Pharmacology117566572
- 45. Brandman O, Liou J, Park W, Meyer T (2007) STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Cell 131: 1327–1339.O. BrandmanJ. LiouW. ParkT. Meyer2007STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels.Cell13113271339
- 46. Liou J, Kim M, Heo W, Myers J, Ferrell J, et al. (2005) STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Current Bio 1235–1241.J. LiouM. KimW. HeoJ. MyersJ. Ferrell2005STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx.Current Bio12351241
- 47. Lopez J, Jardin I, Bobe R, Pariente J, Enouf J, et al. (2008) STIM1 regulates acidic Ca2+ store refilling by interaction with SERCA3 in human platelets. Biochem Pharm 75: 2157–2164.J. LopezI. JardinR. BobeJ. ParienteJ. Enouf2008STIM1 regulates acidic Ca2+ store refilling by interaction with SERCA3 in human platelets.Biochem Pharm7521572164
- 48. Tu H, Nelson O, Bezprozvanny A, Wang Z, Lee S-F, et al. (2006) Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell 126: 981–993.H. TuO. NelsonA. BezprozvannyZ. WangS-F Lee2006Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations.Cell126981993
- 49. Whitfield J (1995) Calcium in Cell Cycles and Cancer Boca Raton, Florida: CRC Press. 1–214.J. Whitfield1995Calcium in Cell Cycles and CancerBoca Raton, FloridaCRC Press1214
- 50. Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL (1993) Intracellular Ca2+ pool content is linked to control of cell growth. Proc Natl Acad Sci U S A 90: 4986–4990.AD ShortJ. BianTK GhoshRT WaldronSL Rybak1993Intracellular Ca2+ pool content is linked to control of cell growth.Proc Natl Acad Sci U S A9049864990
- 51. Legrand G, Humez S, Slomianny C, Dewailly E, Abeele FV, et al. (2001) Ca2+ Pools and Cell Growth. Evidence for sarcoendplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control. J Biol Chem 276: 47608–47614.G. LegrandS. HumezC. SlomiannyE. DewaillyFV Abeele2001Ca2+ Pools and Cell Growth. Evidence for sarcoendplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control.J Biol Chem2764760847614
- 52. Humez S, Legrand G, Vanden-Abeele F, Monet M, Marchetti P, et al. (2004) Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha. Journal of Cellular Physiology 201: 201–213.S. HumezG. LegrandF. Vanden-AbeeleM. MonetP. Marchetti2004Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha.Journal of Cellular Physiology201201213