Fe3+ opposes the 1,25(OH)2D3-induced calcium transport across intestinal epithelium-like Caco-2 monolayer in the presence or absence of ascorbic acid.

Although iron is an essential element for hemoglobin and cytochrome synthesis, excessive intestinal iron absorption-as seen in dietary iron supplementation and hereditary disease called thalassemia-could interfere with transepithelial transport of calcium across the intestinal mucosa. The underlying cellular mechanism of iron-induced decrease in intestinal calcium absorption remains elusive, but it has been hypothesized that excess iron probably negates the actions of 1,25-dihydroxyvitamin D [1,25(OH)2D3]. Herein, we exposed the 1,25(OH)2D3-treated epithelium-like Caco-2 monolayer to FeCl3 to demonstrate the inhibitory effect of ferric ion on 1,25(OH)2D3-induced transepithelial calcium transport. We found that a 24-h exposure to FeCl3 on the apical side significantly decreased calcium transport, while increasing the transepithelial resistance (TER) in 1,25(OH)2D3-treated monolayer. The inhibitory action of FeCl3 was considered rapid since 60-min exposure was sufficient to block the 1,25(OH)2D3-induced decrease in TER and increase in calcium flux. Interestingly, FeCl3 did not affect the baseline calcium transport in the absence of 1,25(OH)2D3 treatment. Furthermore, although ascorbic acid is often administered to maximize calcium solubility and to enhance intestinal calcium absorption, it apparently had no effect on calcium transport across the FeCl3- and 1,25(OH)2D3-treated Caco-2 monolayer. In conclusion, apical exposure to ferric ion appeared to negate the 1,25(OH)2D3-stimulated calcium transport across the intestinal epithelium. The present finding has, therefore, provided important information for development of calcium and iron supplement products and treatment protocol for specific groups of individuals, such as thalassemia patients and pregnant women.

Activation of cannabinoid receptors in breast cancer cells improves osteoblast viability in cancer-bone interaction model while reducing breast cancer cell survival and migration.

The endocannabinoid system has been postulated to help restrict cancer progression and maintain osteoblastic function during bone metastasis. Herein, the effects of cannabinoid receptor (CB) type 1 and 2 activation on breast cancer cell and osteoblast interaction were investigated by using ACEA and GW405833 as CB1 and CB2 agonists, respectively. Our results showed that breast cancer cell (MDA-MB-231)-derived conditioned media markedly decreased osteoblast-like UMR-106 cell viability. In contrast, media from MDA-MB-231 cells pre-treated with GW405833 improved UMR-106 cell viability. MDA-MB-231 cells were apparently more susceptible to both CB agonists than UMR-106 cells. Thereafter, we sought to answer the question as to how CB agonists reduced MDA-MB-231 cell virulence. Present data showed that co-activation of CB1 and CB2 exerted cytotoxic effects on MDA-MB-231 cells by increasing apoptotic cell death through suppression of the NF-κB signaling pathway in an ROS-independent mechanism. ACEA or GW405833 alone or in combination also inhibited MDA-MB-231 cell migration. Thus, it can be concluded that the endocannabinoid system is able to provide protection during breast cancer bone metastasis by interfering cancer and bone cell interaction as well as by the direct suppression of cancer cell growth and migration.

Mild-intensity physical activity prevents cardiac and osseous iron deposition without affecting bone mechanical property or porosity in thalassemic mice.

Thalassemia causes anemia, ineffective erythropoiesis, bone loss and iron accumulation in several tissues, e.g., liver, bone and heart, the last of which leads to lethal cardiomyopathy and arrhythmia. Although exercise reportedly improves bone density in thalassemic mice, exercise performance is compromised and might pose risk of cardiovascular accident in thalassemic patients. Therefore, we sought to explore whether mild-intensity physical activity (MPA) with 30-50% of maximal oxygen consumption was sufficient to benefit the heart and bone. Herein, male hemizygous ß-globin knockout (BKO) mice and wild-type littermates were subjected to voluntary wheel running 1 h/day, 5 days/week for 3 months (MPA group) or kept sedentary (SDN; control). As determined by atomic absorption spectroscopy, BKO-MPA mice had less iron accumulation in heart and bone tissues compared with BKO-SDN mice. Meanwhile, the circulating level of fibroblast growth factor-23-a factor known to reduce serum iron and intestinal calcium absorption-was increased early in young BKO-MPA mice. Nevertheless, MPA did not affect duodenal calcium transport or body calcium retention. Although MPA restored the aberrant bone calcium-phosphorus ratio to normal range, it did not change vertebral calcium content or femoral mechanical properties. Microstructural porosity in tibia of BKO-MPA mice remained unaltered as determined by synchrotron radiation X-ray tomographic microscopy. In conclusion, MPA prevents cardiac and bone iron accumulation, which is beneficial to thalassemic patients with limited physical fitness or deteriorated cardiac performance. However, in contrast to moderate-intensity exercise, MPA does not improve bone mechanical properties or reduce bone porosity.

Correction: Lertsuwan et al. CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism. Cancers 2018, 10, 283.

Authors would like to make a correction to the previous article [...].

Hepcidin induces intestinal calcium uptake while suppressing iron uptake in Caco-2 cells.

Abnormal calcium absorption and iron overload from iron hyperabsorption can contribute to osteoporosis as found in several diseases, including hemochromatosis and thalassemia. Previous studies in thalassemic mice showed the positive effects of the iron uptake suppressor, hepcidin, on calcium transport. However, whether this effect could be replicated in other conditions is not known. Therefore, this study aimed to investigate the effects of hepcidin on iron and calcium uptake ability under physiological, iron uptake stimulation and calcium uptake suppression. To investigate the potential mechanism, effects of hepcidin on the expression of iron and calcium transporter and transport-associated protein in Caco-2 cells were also determined. Our results showed that intestinal cell iron uptake was significantly increased by ascorbic acid together with ferric ammonium citrate (FAC), but this phenomenon was suppressed by hepcidin. Interestingly, hepcidin significantly increased calcium uptake under physiological condition but not under iron uptake stimulation. While hepcidin significantly suppressed the expression of iron transporter, it had no effect on calcium transporter expression. This indicated that hepcidin-induced intestinal cell calcium uptake did not occur through the stimulation of calcium transporter expression. On the other hand, 1,25(OH)2D3 effectively induced intestinal cell calcium uptake, but it did not affect intestinal cell iron uptake or iron transporter expression. The 1,25(OH)2D3-induced intestinal cell calcium uptake was abolished by 12 mM CaCl2; however, hepcidin could not rescue intestinal cell calcium uptake suppression by CaCl2. Taken together, our results showed that hepcidin could effectively and concurrently induce intestinal cell calcium uptake while reducing intestinal cell iron uptake under physiological and iron uptake stimulation conditions, suggesting its therapeutic potential for inactive calcium absorption, particularly in thalassemic patients or patients who did not adequately respond to 1,25(OH)2D3.

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance.

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl- and HCO3 - across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO3 - efflux through CFTR often required the intracellular H+/HCO3 - production and/or the Na+-dependent basolateral HCO3 - uptake, the intracellular pH (pHi) balance might be disturbed, especially as a consequence of increased endogenous H+ and HCO3 - production. However, measurement of pHi by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO3 - transport. In addition, although the plasma membrane Na+/K+-ATPase (NKA) is normally essential for basolateral HCO3 - uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pHi or NKA abundance on cell membrane.

Overexpression of Pyruvate Carboxylase Is Correlated With Colorectal Cancer Progression and Supports Growth of Invasive Colon Cancer HT-29 Cell Line.

BACKGROUND/AIM: Pyruvate carboxylase (PC) is a major anaplerotic enzyme for generating oxaloacetate for the TCA cycle and also a key enzyme in gluconeogenesis, de novo fatty acid and amino acid synthesis in normal cells. Recent studies have identified PC overexpression in different cancers, such as breast and lung. However, the involvement of PC in colorectal cancer (CRC) is unclear. Our purpose was to investigate the PC expression levels and its correlations with potentially relevant clinical-pathological parameters in CRC. MATERIALS AND METHODS: PC expression levels in tissues from 60 Thai CRC patients were investigated by immunohistochemistry while a clonogenic assay was performed for determining cell growth of HT-29 cells with PC knockdown. RESULTS: Our results showed for the first time that high PC expression levels were significantly correlated with late stage of the cancer, perineural invasion and lymph node metastasis. The overexpression of PC was also significantly associated with poor overall and disease-free survival times of CRC patients. In addition, suppression of cancer cell growth was found in PC-deficient cell lines using CRISPR-Cas9. CONCLUSION: The overexpression levels of PC were correlated with CRC progression and survival times. Therefore, PC might serve as a potential clinical prognostic marker for colorectal cancer.

Differential effects of Fe2+ and Fe3+ on osteoblasts and the effects of 1,25(OH)2D3, deferiprone and extracellular calcium on osteoblast viability under iron-overloaded conditions.

One of the potential contributing factors for iron overload-induced osteoporosis is the iron toxicity on bone forming cells, osteoblasts. In this study, the comparative effects of Fe3+ and Fe2+ on osteoblast differentiation and mineralization were studied in UMR-106 osteoblast cells by using ferric ammonium citrate and ferrous ammonium sulfate as Fe3+ and Fe2+ donors, respectively. Effects of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and iron chelator deferiprone on iron uptake ability of osteoblasts were examined, and the potential protective ability of 1,25(OH)2D3, deferiprone and extracellular calcium treatment in osteoblast cell survival under iron overload was also elucidated. The differential effects of Fe3+ and Fe2+ on reactive oxygen species (ROS) production in osteoblasts were also compared. Our results showed that both iron species suppressed alkaline phosphatase gene expression and mineralization with the stronger effects from Fe3+ than Fe2+. 1,25(OH)2D3 significantly increased the intracellular iron but minimally affected osteoblast cell survival under iron overload. Deferiprone markedly decreased intracellular iron in osteoblasts, but it could not recover iron-induced osteoblast cell death. Interestingly, extracellular calcium was able to rescue osteoblasts from iron-induced osteoblast cell death. Additionally, both iron species could induce ROS production and G0/G1 cell cycle arrest in osteoblasts with the stronger effects from Fe3+. In conclusions, Fe3+ and Fe2+ differentially compromised the osteoblast functions and viability, which can be alleviated by an increase in extracellular ionized calcium, but not 1,25(OH)2D3 or iron chelator deferiprone. This study has provided the invaluable information for therapeutic design targeting specific iron specie(s) in iron overload-induced osteoporosis. Moreover, an increase in extracellular calcium could be beneficial for this group of patients.

MALAT1 Decreases the Sensitivity of Head and Neck Squamous Cell Carcinoma Cells to Radiation and Cisplatin.

BACKGROUND/AIM: Two-thirds of head and neck squamous cell carcinoma (HNSCC) patients present with locally advanced (LA) stages and have a poor survival rate. The aim of this study was to investigate the roles of the long non-coding RNAs MALAT1 on radiation and cisplatin sensitivity of HNSCC cells. MATERIALS AND METHODS: Clonogenic, cell viability, and apoptosis assays were performed in cells following MALAT1 knockdown using CRISPR/Cas9 system. RESULTS: MALAT1 was overexpressed in HNSCC cell lines as compared to a non-tumorigenic cell line. The number of colonies formed after radiation was significantly reduced in MALAT1 knockdown cells. The IC50 value of cisplatin in MALAT1 knockdown cells was lower than that of the control cells. MALAT1 knockdown resulted in cell cycle arrest at G2/M phase, DNA damage and apoptotic cell death. CONCLUSION: MALAT1 knockdown enhanced the sensitivity of HNSCC cells to radiation and cisplatin partly through the induction of G2/M cell cycle arrest resulting in DNA damage and apoptosis.

Adenosine Suppresses Cholangiocarcinoma Cell Growth and Invasion in Equilibrative Nucleoside Transporters-Dependent Pathway

Cholangiocarcinoma (CCA) is a lethal disease with increasing incidence worldwide. Previous study showed that CCA was sensitive to adenosine. Thereby, molecular mechanisms of CCA inhibition by adenosine were examined in this study. Our results showed that adenosine inhibited CCA cells via an uptake of adenosine through equilibrative nucleoside transporters (ENTs), instead of activation of adenosine receptors. The inhibition of ENTs by NBTI caused the inhibitory effect of adenosine to subside, while adenosine receptor antagonists, caffeine and CGS-15943, failed to do so. Intracellular adenosine level was increased after adenosine treatment. Also, a conversion of adenosine to AMP by adenosine kinase is required in this inhibition. On the other hand, inosine, which is a metabolic product of adenosine has very little inhibitory effect on CCA cells. This indicates that a conversion of adenosine to inosine may reduce adenosine inhibitory effect. Furthermore, there was no specific correlation between level of proinflammatory proteins and CCA responses to adenosine. A metabolic stable analog of adenosine, 2Cl-adenosine, exerted higher inhibition on CCA cell growth. The disturbance in intracellular AMP level also led to an activation of 5' AMP-activated protein kinase (AMPK). Accordingly, we proposed a novel adenosine-mediated cancer cell growth and invasion suppression via a receptor-independent mechanism in CCA.

Parathyroid hormone increases CFTR expression and function in Caco-2 intestinal epithelial cells.

Parathyroid hormone (PTH) enhances cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion secretion by the human intestinal epithelial cell line Caco-2. With the patch-clamp and Ussing chamber techniques, we investigated how PTH stimulates CFTR activity in Caco-2 cells. Cell-attached recordings revealed that PTH stimulated the opening of CFTR-like channels, while impedance analysis demonstrated that PTH increased apical membrane capacitance, a measure of membrane surface area. Using ion substitution experiments, the PTH-stimulated increase in short-circuit current (Isc), a measure of transepithelial ion transport, was demonstrated to be Cl-- and HCO3--dependent. However, the PTH-stimulated increase in Isc was unaffected by the carbonic anhydrase inhibitor acetazolamide, but partially blocked by the intermediate-conductance Ca2+-activated K+ channel (IKCa) inhibitor clotrimazole. TRAM-34, a related IKCa inhibitor, failed to directly inhibit CFTR Cl- channels in cell-free membrane patches, excluding its action on CFTR. In conclusion, PTH enhances CFTR-mediated anion secretion by Caco-2 monolayers by increasing the expression and function of CFTR in the apical membrane and IKCa activity in the basolateral membrane.

Differential expression of Sox9 protein and proteoglycans in the epiphyseal cartilage of bromocriptine-treated pregnant and lactating rats.

Several investigations have shown that pregnancy and lactation are able to induce elongation of long bone by altering epiphyseal cartilage function in a prolactin-dependent manner. Since the transcription factor Sox9 is of utmost importance for chondrocyte proliferation and differentiation and since bromocriptine, a dopaminergic D2 agonist widely used to suppress milk production, is known to disrupt the production and release of prolactin, we herein aimed to investigate whether pregnancy and lactation as well as bromocriptine could alter the expression of Sox9. Our immunohistochemical analysis showed that the Sox9 expression levels were markedly upregulated in the tibial proliferative zone of day 21 pregnant rats. In day 8 (early) and day 14 (mid) lactating rats, the Sox9 expression was enhanced only in the proliferative zone, but not in the resting and hypertrophic zones. There was no change in Sox9 expression in day 21 (late) lactating rats. Postweaning rats manifested a decreased Sox9 expression in the hypertrophic zone. Bromocriptine had no effect on Sox9 expression in the proliferative zone of day 21 pregnant rats; however, it completely prevented the Sox9 upregulation in those of early and mid-lactating rats. A differential response was observed in the proliferative and hypertrophic zones of late lactating rats, in which bromocriptine enhanced Sox9 expression. Further investigation of cartilaginous matrix revealed no change in proteoglycans accumulation in lactating rats. In conclusion, the upregulated Sox9 expression predominantly occurred in the proliferative zone during late pregnancy and early and mid-lactation, while the bromocriptine effects depended on the periods and epiphyseal zones.

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism.

Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α' catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings.

Ferrous and ferric differentially deteriorate proliferation and differentiation of osteoblast-like UMR-106 cells.

The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, ß-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.

Intestinal calcium transport and its regulation in thalassemia: interaction between calcium and iron metabolism.

Osteoporosis and derangement of calcium homeostasis are common complications of thalassemia. Despite being an important process for bone and calcium metabolism, little is known about intestinal calcium transport in thalassemia. Recent reports of decreases in both intestinal calcium transport and bone mineral density in thalassemic patients and animal models suggested that defective calcium absorption might be a cause of thalassemic bone disorder. Herein, the possible mechanisms associated with intestinal calcium malabsorption in thalassemia are discussed. This includes alterations in the calcium transporters and hormonal controls of the transcellular and paracellular intestinal transport systems in thalassemia. In addition, the effects of iron overload on intestinal calcium absorption, and the reciprocal interaction between iron and calcium transport in thalassemia are elaborated. Understanding the mechanisms underlining calcium malabsorption in thalassemia would lead to development of therapeutic agents and mineral supplements that restore calcium absorption as well as prevent osteoporosis in thalassemic patients.

CFTR-mediated anion secretion across intestinal epithelium-like Caco-2 monolayer under PTH stimulation is dependent on intermediate conductance K+ channels.

Parathyroid hormone (PTH), a pleiotropic hormone that maintains mineral homeostasis, is also essential for controlling pH balance and ion transport across renal and intestinal epithelia. Optimization of luminal pH is important for absorption of trace elements, e.g., calcium and phosphorus. We have previously demonstrated that PTH rapidly stimulated electrogenic [Formula: see text] secretion in intestinal epithelial-like Caco-2 monolayers, but the underlying cellular mechanism, contributions of other ions, particularly Cl- and K+, and long-lasting responses are not completely understood. Herein, PTH and forskolin were confirmed to induce anion secretion, which peaked within 1-3 min (early phase), followed by an abrupt decay and plateau that lasted for 60 min (late phase). In both early and late phases, apical membrane capacitance was increased with a decrease in basolateral capacitance after PTH or forskolin exposure. PTH also induced a transient increase in apical conductance with a long-lasting decrease in basolateral conductance. Anion secretion in both phases was reduced under [Formula: see text]-free and/or Cl--free conditions or after exposure to carbonic anhydrase inhibitor (acetazolamide), CFTR inhibitor (CFTRinh-172), Na+/H+ exchanger (NHE)-3 inhibitor (tenapanor), or K+ channel inhibitors (BaCl2, clotrimazole, and TRAM-34; basolateral side), the latter of which suggested that PTH action was dependent on basolateral K+ recycling. Furthermore, early- and late-phase responses to PTH were diminished by inhibitors of PI3K (wortmannin and LY-294002) and PKA (PKI 14-22). In conclusion, PTH requires NHE3 and basolateral K+ channels to induce [Formula: see text] and Cl- secretion, thus explaining how PTH regulated luminal pH balance and pH-dependent absorption of trace minerals.

Purinergic Receptor Expression and Cellular Responses to Purinergic Agonists in Human Prostate Cancer Cells.

BACKGROUND: Anticancer activity of extracellular nucleotides has been investigated in many types of cancer. Herein, the effects of extracellular nucleotides and the receptor profile for these nucleotides on prostate cancer (PCa) were elaborated. MATERIALS AND METHODS: PCa cell lines representing different stages of PCa were used. The effects of ATP and adenosine on PCa growth and migration on different extracellular matrix proteins were examined by MTT and wound-healing assays. Purinergic receptor profiling was carried out by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A growth-inhibitory effect of ATP and adenosine was observed on all PCa cell lines tested. Several ATP-recognized P2 receptors and adenosine receptors were commonly expressed in PCa cell lines. Neither ATP nor adenosine had any significant effect on PCa migration. CONCLUSION: ATP and adenosine had an antiproliferative effect on PCa cells without affecting their motility, indicating their potential as a novel therapy for PCa.

Identification of Fibulin-1 as a Human Bone Marrow Stromal (HS-5) Cell-Derived Factor That Induces Human Prostate Cancer Cell Death.

BACKGROUND: Previous studies showed that human bone marrow stromal HS-5 cells secreted unidentified factor(s) inducing PCa cell death. Herein, the HS-5-derived factor (HS-5 DF) was characterized and identified. METHODS: Conditioned media from confluent HS-5 cells were collected and modified for biochemical characteristic testing of HS-5 DF. Cell survival was measured by apoptosis assay and live/dead assay. Fibulin-1 was identified from gel electrophoresis and mass spectrometry. The validation of Fibulin-1 as a HS-5 DF was done by immunoprecipitation (IP) and genetic knockdown by CRISPR/Cas9 system. RESULTS: HS-5 DF was trypsin and heat sensitive, but pH stable. The tentative size of the factor fell between 30 kDa and 100 kDa. TGF-ß1 treatment led to a suppression of HS-5 DF activity, a property consistent with bone metastasis in prostate cancer. Examination of TGF-ß1 down regulated proteins led to identification of fibulin-1 as a candidate for the DF. IP of Fibulin-1 from HS-5 CM and CRISPR knockdown of Fibulin-1 showed a significant reduction of HS-5 CM-derived PCa cell death. These results strongly support a role for fibulin-1 in HS-5 bone marrow stromal cell induction of PCa cell death. CONCLUSION: Our data indicate that Fibulin-1 functions as a HS-5 bone marrow stromal cell-derived factor inducing prostate cancer cell death. Prostate 77:729-742, 2017. © 2016 Wiley Periodicals, Inc.

Na+/H+ exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous ß-globin knockout thalassemic mice.

Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous ß-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe2+ and H+ cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na+/H+ exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na+/Ca2+ exchanger (NCX1) and plasma membrane Ca2+-ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in ß-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

Na+/H+ exchanger 3 inhibitor diminishes the amino-acid-enhanced transepithelial calcium transport across the rat duodenum.

Na+/H+ exchanger (NHE)-3 is important for intestinal absorption of nutrients and minerals, including calcium. The previous investigations have shown that the intestinal calcium absorption is also dependent on luminal nutrients, but whether aliphatic amino acids and glucose, which are abundant in the luminal fluid during a meal, similarly enhance calcium transport remains elusive. Herein, we used the in vitro Ussing chamber technique to determine epithelial electrical parameters, i.e., potential difference (PD), short-circuit current (Isc), and transepithelial resistance, as well as 45Ca flux in the rat duodenum directly exposed on the mucosal side to glucose or various amino acids. We found that mucosal glucose exposure led to the enhanced calcium transport, PD, and Isc, all of which were insensitive to NHE3 inhibitor (100 nM tenapanor). In the absence of mucosal glucose, several amino acids (12 mM in the mucosal side), i.e., alanine, isoleucine, leucine, proline, and hydroxyproline, markedly increased the duodenal calcium transport. An inhibitor for NHE3 exposure on the mucosal side completely abolished proline- and leucine-enhanced calcium transport, but not transepithelial transport of both amino acids themselves. In conclusion, glucose and certain amino acids in the mucosal side were potent stimulators of the duodenal calcium absorption, but only amino-acid-enhanced calcium transport was NHE3-dependent.