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.
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) 2 D 3 effectively induced intestinal cell calcium uptake, but it did not affect intestinal cell iron uptake or iron transporter expression. The 1,25(OH) 2 D 3 -induced intestinal cell calcium uptake was abolished by 12 mM CaCl 2 ; however, hepcidin could not rescue intestinal cell calcium uptake suppression by CaCl 2 . 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) 2 D 3 .
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