The expression of pluripotency factors, and their associations with clinicopathological parameters and drug response have been described in various cancers, including gastric cancer. This study investigated the association of pluripotency factor expression with the clinicopathological characteristics of gastric cancer patients, as well as changes in the expression of these factors upon the stem cell-enriching spheroid culture of gastric cancer cells, regulation of sphere-forming capacity, and response to cisplatin and TRAIL treatments by Nanog and KLF4. Nanog expression was significantly associated with the emergence of a new tumor and a worse prognosis in gastric cancer patients. The expression of the pluripotency factors varied among six gastric cancer cells. KLF4 and Nanog were expressed high in SNU-601, whereas SOX2 was expressed high in SNU-484. The expression of KLF4 and SOX2 was increased upon the spheroid culture of SNU-601 (KLF4/Nanog-high) and SNU-638 (KLF4/Nanog-low). The spheroid culture of them enhanced TRAIL-induced viability reduction, which was accompanied by the upregulation of death receptors, DR4 and DR5. Knockdown and overexpression of Nanog in SNU-601 and SNU-638, respectively, did not affect spheroid-forming capacity, however, its expression was inversely correlated with DR4/DR5 expression and TRAIL sensitivity. In contrast, KLF4 overexpression in SNU-638 increased spheroid formation, susceptibility to cisplatin and TRAIL treatments, and DR4/DR5 expression, while the opposite was found in KLF4-silenced SNU-601. KLF4 is supposed to play a critical role in DR4/DR5 expression and responses to TRAIL and cisplatin, whereas Nanog is only implicated in the former events only. Direct regulation of death receptor expression and TRAIL response by KLF4 and Nanog have not been well documented previously, and the regulatory mechanism behind the process remains to be elucidated.

Shear stress induces atrial Ca²⁺ waves via connexin43 (Cx43)-mediated ATP release. Here, we examined whether ventricular myocytes release ATP under shear stress, and the underlying and regulatory mechanisms. A bioluminescence assay and the ‘sniffer patch’ were used to measure ATP release from multiple and single murine ventricular myocytes, respectively, in combination with laminar flow or micro-puffing. Shear stress (∼16 dyn/cm²) induced transient ATP release from myocyte batches, peaking at ~7 mol/cm²-membrane within 2 s. This response was abolished by the application of La³⁺ but was not affected by Gap19 treatment or zero external Ca²⁺. In addition, shear-induced ATP release was not different between control and cardiac-specific Cx43-conditional knockout (Cx43-cKO) ventricular cells, suggesting no role for Cx43. Shear-induced ATP release was suppressed by probenecid, low concentrations of carbenoxolone, or the pannexin (Panx) 1 inhibitory peptide ¹⁰Panx1 by 60−75%, suggesting a major role for Panx. Pharmacological screening further revealed a partial (30−40%) role of 9‐anthracenecarboxylic acid-sensitive, tamoxifen-insensitive Cl^- channels in ATP release. The sniffer patch, using a P2X7 receptor-overexpressing HEK293 cell positioned at a local region of the ventricular cell, showed a rapid development of shear-induced currents, approximated to be ∼1−10 μM ATP. The Cx43-cKO did not affect these sniffer cell currents. This shear response was suppressed by mitochondrial uncoupling or inhibition of the electron transport chain by 70-80% but was rather enhanced by Mito-TEMPO. Our data suggest that shear stress induces ATP release from murine ventricle cells mainly via Panx and that mitochondrial oxidative phosphorylation may be required for this response.