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

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

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 [...

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

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

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

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

Chitosan-Coated Poly(lactic-<i>co</i>-glycolic acid) Nanoparticles Loaded with Ursolic Acid for Breast Cancer Therapy

Ursolic acid (UA), a pentacyclic triterpenoid found in various fruits and herbs, has the potential as an anticancer agent against multiple cancer types. Nevertheless, its clinical use was limited by its poor water solubility. To overcome this drawback, several nanocarriers were proposed to increase the bioavailability and efficacy of UA. However, the insights into the cellular targets and mechanisms of UA and UA nanoparticles (NPs) remain limited. In this study, chitosan-coated poly(lactic-co-glycolic acid) (PLGA/CS) NPs were loaded with UA. The obtained (UA)-PLGA/CS NPs were spherical with an approximate size of 250 nm and an encapsulation efficiency of 25%. Owing to their promising potential as drug carriers, the NPs were successfully delivered into breast cancer cells (MCF-7 and MDA-MB-231). Moreover, (UA)-PLGA/CS NPs enhanced the anticancer activity of UA, as evidenced by the IC50 values of 26.74 and 40.67 μM in MCF-7 and MDA-MB-231 cells, respectively. These values were lower than those of free UA (90.25 and 85.63 μM in MCF-7 and MDA-MB-231 cells, respectively). The improved cytotoxicity induced by (UA)-PLGA/CS NPs can be attributed to apoptosis induction, collective cell migration and invasion inhibition, and cell proliferation pathway disruption. These findings led to a better understanding of the anticancer effects and molecular mechanisms of (UA)-PLGA/CS NPs and their potential targets for breast cancer therapy

Chitosan-Coated Poly(lactic-<i>co</i>-glycolic acid) Nanoparticles Loaded with Ursolic Acid for Breast Cancer Therapy

Ursolic acid (UA), a pentacyclic triterpenoid found in various fruits and herbs, has the potential as an anticancer agent against multiple cancer types. Nevertheless, its clinical use was limited by its poor water solubility. To overcome this drawback, several nanocarriers were proposed to increase the bioavailability and efficacy of UA. However, the insights into the cellular targets and mechanisms of UA and UA nanoparticles (NPs) remain limited. In this study, chitosan-coated poly(lactic-co-glycolic acid) (PLGA/CS) NPs were loaded with UA. The obtained (UA)-PLGA/CS NPs were spherical with an approximate size of 250 nm and an encapsulation efficiency of 25%. Owing to their promising potential as drug carriers, the NPs were successfully delivered into breast cancer cells (MCF-7 and MDA-MB-231). Moreover, (UA)-PLGA/CS NPs enhanced the anticancer activity of UA, as evidenced by the IC50 values of 26.74 and 40.67 μM in MCF-7 and MDA-MB-231 cells, respectively. These values were lower than those of free UA (90.25 and 85.63 μM in MCF-7 and MDA-MB-231 cells, respectively). The improved cytotoxicity induced by (UA)-PLGA/CS NPs can be attributed to apoptosis induction, collective cell migration and invasion inhibition, and cell proliferation pathway disruption. These findings led to a better understanding of the anticancer effects and molecular mechanisms of (UA)-PLGA/CS NPs and their potential targets for breast cancer therapy

The role of FeCl₃ on calcium transport across Caco-2 monolayer pre-treated with 1,25(OH)₂D₃ and ascorbic acid (Asc).

(A) Experimental timeline (please see text for detail). (B–E) Transepithelial calcium transport and epithelial electrical parameters (PD, Isc, and TER) in Caco-2 monolayers with or without 10 nM 1,25(OH)2D3, 200 μM FeCl3, and 0.5 mM Asc. PD values were the magnitudes of potential difference (the apical side being negative with respect to the basolateral side), and glucose made the apical side more negative. (n = 10; *P P ††P †††P 2D3-treated group (blue bar).</p