Jingyu Xu

Expression and functional role of vacuolar H+-ATPase in human hepatocellular carcinoma

Tumor cells often exist in a hypoxic microenvironment, which produces acidic metabolites. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. Vacuolar H(+)-adenosine triphosphatase (V-ATPase) is considered to play an important role in the regulation of the acidic microenvironment of some tumors. In this study, we made an investigation on the expression and functional role of V-ATPase in native human hepatocellular carcinoma (HCC). The results showed that the messenger RNA and protein expression levels of V-ATPase subunit ATP6L in native human HCC tissues were markedly increased, compared with normal liver tissues. Immunohistochemical analysis further confirmed the enhanced expression of V-ATPase ATP6L in human HCC cells and revealed that V-ATPase ATP6L was distributed in the cytoplasm and plasma membrane of HCC cells. The results from immunofluorescence and biotinylation of cell surface protein showed that V-ATPase ATP6L was conspicuously located in the plasma membrane of human HCC cells. Bafilomycin A1, a specific V-ATPase inhibitor, markedly slowed the intracellular pH (pHi) recovery after acid load in human HCC cells and retarded the growth of human HCC in orthotopic xenograft model. These results demonstrated that V-ATPase is up-regulated in human HCC and involved in the regulation of pHi of human HCC cells. The inhibition of V-ATPase can effectively retard the growth of HCC, indicating that V-ATPase may play an important role in the development and progression of human HCC, and targeting V-ATPase may be a promising therapeutic strategy against human HCC.

The P2Y2 nucleotide receptor mediates the proliferation and migration of human hepatocellular carcinoma cells induced by ATP.

Background: Hepatocarcinogenesis is a complex process that involves various modifications to a number of molecular pathways. Results: The P2Y2 receptor (P2Y2R) mediates the effect of ATP on the cellular behavior of hepatocellular carcinoma (HCC) cells through store-operated calcium channel (SOCs)-mediated Ca2+ signaling. Conclusion: P2Y2R is involved in the development and progression of HCC. Significance: P2Y2R may be a promising therapeutic target against human HCC. ATP is an abundant biochemical component of the tumor microenvironment and a physiologic ligand for the P2Y2 nucleotide receptor (P2Y2R). In this study, we investigated the effect of ATP on the cellular behavior of human hepatocellular carcinoma (HCC) cells and the role of P2Y2R in ATP action and aimed to find a new therapeutic target against HCC. The experiments were performed in native isolated human HCC cells, normal hepatocytes, human HCC cell lines, and nude mice. We found that the mRNA and protein expression levels of P2Y2R in native human HCC cells and the human HCC cell lines HepG2 and BEL-7404 were enhanced markedly compared with human normal hepatocytes and the normal hepatocyte line LO2, respectively. ATP induced intracellular Ca2+ increases in HCC cells and promoted the proliferation and migration of HCC cells and the growth of HCC in nude mice. The P2Y receptor antagonist suramin, P2Y2R-specific shRNA, the store-operated calcium channel inhibitors 2-aminoethoxydiphenyl borate (2-APB) and 1-(β-3-(4-methoxy-phenyl) propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride (SKF96365), and stromal interaction molecule (STIM1)-specific shRNA inhibited the action of ATP on HCC cells. In conclusion, P2Y2R mediated the action of ATP on the cellular behavior of HCC cells through store-operated calcium channel-mediated Ca2+ signaling, and targeting P2Y2R may be a promising therapeutic strategy against human HCC.

Na+/H+ exchanger 1, Na+/Ca2+ exchanger 1 and calmodulin complex regulates interleukin 6-mediated cellular behavior of human hepatocellular carcinoma

Interleukin 6 (IL6) is a key cytokine involved in the development and progression of inflammation-associated hepatocellular carcinoma (HCC). However, the mechanisms of IL6 action on HCC remain largely unknown. Proton and Ca(2+) are two intracellular messenger ions, which are believed to play a central role in tumorigenesis and tumor progression. In this study, we found that IL6 stimulation markedly increased intracellualr pH recovery rates of human HCC cells, Huh7 and HepG2, after NH4Cl acidification, and the NH4Cl acidification induced transient intracellular Ca(2+) increases in the HCC cells. The inhibition of Na(+)/H(+) exchanger 1 (NHE1), Na(+)/Ca(2+) exchanger 1 (NCX1) and calmodulin (CaM) inhibited the IL6 stimulation-induced intracellular pH recovery increases and NH4Cl acidification-induced intracellular Ca(2+) increases. IL6 stimulation also induced the structural interaction of NHE1, NCX1 and CaM proteins. The protein expression levels of NHE1, NCX1 and CaM in native human HCC tissues were markedly higher than those in normal liver tissues. IL6 upregulated the expressions of NHE1, NCX1 and CaM in Huh7 and HepG2 cells. NHE1, NCX1 and CaM mediated the promotion of IL6 on the proliferation, migration and invasion of Huh7 and HepG2 cells and the growth of HCC in nude mice. In conclusion, IL6 activates the functional activity of NHE1 and induces the functional and structural interaction of NHE1, NCX1 and CaM. The interaction of NHE1, NCX1 and CaM mediates the effects of IL6 on human HCC.

Estrogen potentiates prostaglandin E2-stimulated duodenal mucosal HCO3− secretion in mice

The cause of lower prevalence of duodenal ulcer in young women compared with men is largely unknown. We recently found that sex difference in duodenal mucosal HCO₃⁻ secretion existed in humans and mice, but the mechanisms are not clear. Prostaglandin E₂ (PGE₂) is an important endogenous mediator that plays an important role in the regulation of duodenal HCO₃⁻ secretion. Therefore, in the present study, we investigated the effect of estrogen on PGE₂-stimulated duodenal HCO₃⁻ secretion and the underlying mechanisms. The results showed that 17β-estradiol at the physiological concentration (1 nM) had no significant effects on duodenal mucosal HCO₃⁻ secretion or short-circuit current (I(sc)) in mice. However, the pretreatment of 17β-estradiol (1 nM) markedly potentiated PGE₂-stimulated duodenal HCO₃⁻ secretion and I(sc) (P < 0.01 and P < 0.05). Global estrogen receptor (ER) antagonist ICI-182,780 and ERα-specific antagonist MPP, but not the ERβ-specific antagonist PHTPP, abolished estrogen-potentiated PGE₂-stimulated duodenal HCO₃⁻ secretion and I(sc). 17β-Estradiol and PGE₂ additively increased phosphatidylinositol 3-kinase (PI3K) activity and Akt phosphorylation. Wortmannin, a specific PI3K inhibitor, inhibited estrogen-potentiated PGE₂-stimulated duodenal HCO₃⁻ secretion and I(sc). In conclusion, estrogen at the physiological concentration potentiates PGE₂-stimulated duodenal mucosal HCO₃⁻ secretion through the activation of ERα and the PI3K-dependent mechanism, which may contribute to the sex difference in duodenal mucosal HCO₃⁻ secretion and the lower prevalence of duodenal ulcer in young women.

Calcium Promotes Human Gastric Cancer via a Novel Coupling of Calcium-Sensing Receptor and TRPV4 Channel

Although dietary calcium intake has long been recommended for disease prevention, the influence of calcium in development of cancer in the upper gastrointestinal tract has not been explored. Here, we assess the roles of calcium and calcium-sensing receptor (CaSR) in gastric cancer development. CaSR expression was enhanced in gastric cancer specimens, which positively correlated with serum calcium concentrations, tumor progression, poor survival, and male gender in gastric cancer patients. CaSR and transient receptor potential cation channel subfamily V member 4 (TRPV4) were colocalized in gastric cancer cells, and CaSR activation evoked TRPV4-mediated Ca2+ entry. Both CaSR and TRPV4 were involved in Ca2+-induced proliferation, migration, and invasion of gastric cancer cells through a Ca2+/AKT/β-catenin relay, which occurred only in gastric cancer cells or normal cells overexpressing CaSR. Tumor growth and metastasis of gastric cancer depended on CaSR in nude mice. Overall, our findings indicate that calcium may enhance expression and function of CaSR to potentially promote gastric cancer, and that targeting the novel CaSR/TRPV4/Ca2+ pathway might serve as preventive or therapeutic strategies for gastric cancer. Cancer Res; 77(23); 6499-512. ©2017 AACR.

The NCX1/TRPC6 complex mediates TGFβ-driven migration and invasion of human hepatocellular carcinoma cells

TGFβ plays an important role in the progression and metastasis of hepatocellular carcinoma (HCC), yet the cellular and molecular mechanisms underlying this role are not completely understood. In this study, we investigated the roles of Na+/Ca2+ exchanger 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) in regulating TGFβ in human HCC. In HepG2 and Huh7 cells, TGFβ-stimulated intracellular Ca2+ increases through NCX1 and TRPC6 and induced the formation of a TRPC6/NCX1 molecular complex. This complex-mediated Ca2+ signaling regulated the effect of TGFβ on the migration, invasion, and intrahepatic metastasis of human HCC cells in nude mice. TGFβ upregulated TRPC6 and NCX1 expression, and there was a positive feedback between TRPC6/NCX1 signaling and Smad signaling. Expression of both TRPC6 and NCX1 were markedly increased in native human HCC tissues, and their expression levels positively correlated with advancement of HCC in patients. These data reveal the role of the TRPC6/NCX1 molecular complex in HCC and in regulating TGFβ signaling, and they implicate TRPC6 and NCX1 as potential targets for therapy in HCC.Significance: TGFβ induces the formation and activation of a TRPC6/NCX1 molecular complex, which mediates the effects of TGFβ on the migration, invasion, and intrahepatic metastasis of HCC. Cancer Res; 78(10); 2564-76. ©2018 AACR.

Effects of Helicobacter pylori Infection on the Expressions and Functional Activities of Human Duodenal Mucosal Bicarbonate Transport Proteins

The mechanisms for Helicobacter pylori (H. pylori)‐induced duodenal ulcerogenesis are not fully understood. In this study, we investigated the effects of H. pylori infection on the expressions and functional activities of human duodenal mucosal bicarbonate transport proteins and hope to further clarify the pathogenesis of H. pylori‐associated duodenal ulcer.

Oestrogen upregulates the expression levels and functional activities of duodenal mucosal CFTR and SLC26A6

What is the central question of this study? Duodenal ulcer is a common disease. A sex‐based difference in the incidence of duodenal ulcer has long been observed clinically, but the cause is unclear. What is the main finding and its importance? Duodenal mucosal bicarbonate secretion is the most important protective factor in duodenal mucosa against acid‐induced damage. The cystic fibrosis transmembrane conductance regulator (CFTR) and the solute‐linked carrier 26 gene family A6 (SLC26A6) are two key bicarbonate transport proteins that mediate duodenal mucosal bicarbonate secretion. We demonstrate that endogenous oestrogen upregulates the expression levels and functional activities of duodenal mucosal CFTR and SLC26A6, which contributes to the sex difference in the prevalence of duodenal ulcer.

Novel insights into ion channels in cancer stem cells (Review)

Cancer stem cells (CSCs) are immortal cells in tumor tissues that have been proposed as the driving force of tumorigenesis and tumor invasion. Previously, ion channels were revealed to contribute to cancer cell proliferation, migration and apoptosis. Recent studies have demonstrated that ion channels are present in various CSCs; however, the functions of ion channels and their mechanisms in CSCs remain unknown. The present review aimed to focus on the roles of ion channels in the regulation of CSC behavior and the CSC-like properties of cancer cells. Evaluation of the relationship between ion channels and CSCs is critically important for understanding malignancy.

Helicobacter pylori infection downregulates duodenal CFTR and SLC26A6 expressions through TGFβ signaling pathway

BackgroundThe pathogenesis of Helicobacter pylori (H. pylori) infection-induced duodenal ulcer remains to be elucidated. Duodenal mucosal bicarbonate secretion is the most important protective factor against acid-induced mucosal injury. We previously revealed that H. pylori infection downregulated the expression and functional activity of duodenal mucosal cystic fibrosis transmembrane conductance regulator (CFTR) and solute linked carrier 26 gene family A6 (SLC26A6) which are the two key duodenal mucosal epithelial cellular bicarbonate transporters to mediate duodenal bicarbonate secretion. In this study, we investigated the mechanism of H. pylori infection-induced duodenal CFTR and SLC26A6 expression downregulation.ResultsWe found that H. pylori infection induced the increase of serum transforming growth factor β (TGFβ) level and duodenal mucosal TGFβ expression and the decrease of duodenal mucosal CFTR and SLC26A6 expressions in C57 BL/6 mice. The results from the experiments of human duodenal epithelial cells (SCBN) showed that H. pylori increased TGFβ production and decreased CFTR and SLC26A6 expressions in SCBN cells. TGFβ inhibitor SB431542 reversed the H. pylori-induced CFTR and SLC26A6 expression decreases. The further results showed that TGFβ directly decreased CFTR and SLC26A6 expressions in SCBN cells. TGFβ induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and P38 MAPK inhibitor SB203580 reversed the TGFβ-induced CFTR and SLC26A6 expression decreases.ConclusionsH. pylori infection downregulates duodenal epithelial cellular CFTR and SLC26A6 expressions through TGFβ-mediated P38 MAPK signaling pathway, which contributes to further elucidating the pathogenesis of H. pylori-associated duodenal ulcer.