| BackgroundsFree calcium ion (Ca2+)is a ubiquitous intracellular second messenger,participating in the regulation of multiple pHysioloGqcal and patholoGqcal processes. The calcium sensitive receptor (CaSR) belongs to the G protein coupled receptor C family, as an important regulator of calcium balance in the body. The CaSR is expressed in the whole digestive system and plays an important role in gastrointestinal pHysiology and pathopHysiology. The CaSR can mediate insulin and gastric acid secretions as well as intestinal fluid transports. High dietary Ca2+ may inhibit tumor development and increase the chemotherapeutic sensitivity of colonic cancer cells through CaSR activtion. Moreover, the CaSR is also correlated withdiarrheal diseases and stone associated pancreatitis. However, little is known about the pHysioloGqcal and pathopHysioloGqcal roles of the CaSR in the upper gastrointestinal tract Peptic ulcers and gastrointestinal tumors are the common diseases in digestive system, and the gastrointestinal tumors have a high incidence, higher invasion and metastasis potentials. Although the mechanisms of tumor cell transformation, invasion and metastasis have been studied for many years, the long-term efficacy is still not satisfactory. In addition, the recurrence of peptic ulcers is also a problem inclinical treatment. The CaSR has different roles in different types of cancers. It was reported as a tumor suppressor in colorectal cancer, but tumor promotor in breast cancer. Moreover, it was reported that the CaSR could regulate pancreatic bicarbonate secretion Therefore, this study was aim to investigate the roles of of the CaSR in gastric cancer development and the duodenal bicarbonate secretion and also the underlying mechanisms. This study may provide the theoretical foundation for the potential new drug development to treat digestive diseases.ResultsPart 11. CaSR expression in both transcripts and proteins are higher in cancer tissues and cell lines than in normal tissues and cell lines. CaSR expression is significantly increased in bigger size, moderate differentiation and late stage of the cancer.(p<0.05)2. A positive correlation exists between CaSR expression levels and serum calcium concentrations. (p=0.0015＜＜0.05) We also found a decreased survival rate for the patients with higher CaSR expression compared to those with lower expression3. CaSR activation by CaCl2 led to proliferation in cancer cell lines (SGC-7901 and MKN45 cells) but not in normal cell line (GES-1 cells). However,1.2 mM CaCl2 induced migration and invasion of both normal cell line GES and cancer cells MKN45. (p<0.05) The proliferation, migration and invasion of cancer cells induced by CaSR activation could be attenuated by the CaSR calcilytics (NPS 2143) and CaSR shRNA. (p<0.05)4. We established subcutaneously xenografted gastric tumor models in nude mice. CaSR activation was found to increase tumor volumes, which was attenuated by NPS2143. However, NPS2143 itself did not affect tumor volumes. (p<0.05)5. We examine the involvement of intracellular calcium ([Ca2+]cyt) signaling which were induced by CaSR activity in normal and cancer cells. Interestingly, CaSR activity induced a slight rise in [Ca2+]cyt in normal cells, but induced a marked rise in [Ca2+]cyt in cancer cells.(p<0.01)This is specific CaSR-mediated [Ca2+]cyt signaling because it was attenuated by CaSR shRNA, and abolished by pHarmacoloGqcal inhibition of the CaSR (NPS2143).(p<0.05)6. To test which pathway are involved in the rise of [Ca2+]cyt in cancer cells by CaSR activation, cells were superfused with spermine in the presenceor the absence of 2 mM CaC12. spermine induced a significant increase in intracellular calcium inextracellular calcium containing solutions, but not in extracellular caclium-free solution, and the significant rise in [Ca2+]cyt was also inhibited by PLC inhibitor U73122and SOC/ROC inhibitor 2-APB. p<0.05)7. CaSR activation induced ERK1/21/2 and JNK phosphorylation in 5-10 min and the pHospHorylation in both normal cell and cancer cells. However, β-catenin (Ser675) and PI3K/AKT(Ser473) pHospHorylation was found to decrease in normal cells but increase in cancer cells when the CaSR is activated.8. The intracellular calcium chelator, BAPTA-AM, could reverse CaSR activation induced β-catenin(Ser675) and PI3K/AKT(Ser473) pHospHorylationin MKN45 cells; however, CaSR-mediated cancer cell proliferation and migration were abolished by the inhibitors of PI3K/AKT, Wnt/β-catenin and BAPTA-AM.9. Overexpression of CaSR gene in GES cell increase intracellular calcium concentration and reversed tendency of β-catenin(Ser675) and PI3K/AKT(ser473) pHospHorylation in normal cells. (p<0.05)10. Consistent with previous reports, both CaCi2 and spermine stimulated acid secretion in normal GES-1 cells, but reduced acid secretion in SGC-7901 and MKN45 cancer cells. (p<0.05)Part 21. Western blot and immunohistochemistry experiment showed CaSR expressionin duodenum mucosa tissue, SCBN cell lines and the human colon carcinoma SW480, Caco-2cell Lines. Intense CaSR immunoreactivity was noted on both apical and basolateral membranes of the villous and crypt epithelial cells, and on the membrane and cytoplasm.2. CaSR activators, CaCl2 and spermine dose-dependently increased[Ca2+]cyt in SCBN cells, (p<0.01) which was inhibited markedly by the selective CaSR antagonist calhex 231.(p<0.05)3. Spermine induced a significant increase in intracellular calcium inextracellular calcium containing solutions, but not in extracellular caclium-free solution. The significant rise in [Ca2+]cyt was also inhibited by SOC/ROC inhibitors,2-APB and SKF. (p<0.05)4. CaSR activator spermine and CFTR activator genistein induced HCO3-fluxes across SCBN cells, but CFTR blocker CFTRinh-173 and SOC/ROC inhibitor 2-APB could reverse spermine-and genistein-induced HCO3- fluxes across the cells. (p<0.01)5. CaSR activator spermine and Gd3+ markedly stimulated duodenal bicarbonate secretion in CFTR wild-type mice(p<0.01), but not in the CFTR knockout mice(p>0.05). Selective PLC inhibitor U73122significantly inhibited CaSR activator-induced duodenal bicarbonate secretion. However, the CaSR activator did not significantly affect duodenal basal Isc. (p<0.05)6. IKca inhibitor clotrimazole but not the KCNQ1 inhibitor chromanol 293 B significant blocked the CaSR-mediated duodenal bicarbonate secretion. (p<0.05) There was no difference in CaSR-mediated duodenal bicarbonate secretion in KCNQlwild type mice and KCNQ1 knockout mice.(p>0.05)7. Intestinal HCO3- and Cl- secretion may be riggered independently by different cell signaling, and the CaSR/Ca2+ signal pathway mainly affects the HCO3- secretion.Conclusion1. Here we provide the first evidence for the CaSR as a promoter of human gastric cancer. CaSR activation significantly increased proliferation, migration and invasion of gastric cancer cells likely through Ca2+and downstream PI3K/AKT and β-catenin signaling pathways. CaSR expression level and the range of [Ca2+]cytmay affect the cancer cell behavior and the action of downstream signal pathways. These findings provide a possible interpretation for the patients with hypercalcemia usually have poor prognosis in their gastric cancer outcome, led us to suggest that low dietary calcium intake reduces the risk of developing gastric cancer and the CaSR could be a novel therapeutic strategy for gastric cancer.2. On the basis of this study, we conclude that dietary calcium and spermine could activate the CaSR in duodenal epithelial cells to specifically trigger Ca2+dependent DBS that protects mucosa, CaSR activation-induced Ca2+entry through ROC is critical to trigger DBS, and Ca2+signaling regulates DBS, likely through activation of IKCa and CFTR channels. This study not only reveals that [Ca2+]cyt signaling is critical for CaSR-induced DBS but also provides novel insights into the molecular mechanisms of [Ca2+]cyt signaling-mediated transepithelial HCO3- secretion. |
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