Effects of Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine on intracellular calcium, mitochondrial function, and viability in human nasal epithelial cells

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that produces phenazine metabolites pyocyanin and 1-hydroxyphenazine that have been suggested to have detrimental effects on mitochondrial function and reactive oxygen species (ROS) production. Prior studies have suggested activation of Ca2+ signaling by pyocyanin in an airway cell line, while others have shown apoptotic effects on cancer cells. Ca2+ is tightly linked to both normal mitochondrial function as well as mitochondrial ROS and apoptosis during mitochondrial Ca2+ overload. We found that pyocyanin but not 1-hydroxyphenazine induced both cytosolic and mitochondrial Ca2+ increases in RPMI 2650 and primary human nasal epithelial cells (HNEC). Similar results were seen in HNEC air-liquid interface (ALI) cells, but these cells did not display a cytosolic Ca2+ response after treatment with pyocyanin. In RPMI 2650, activation of PKC, ER Ca2+ release, and PLC inhibition, indicated potential GPCR activation from pyocyanin. Similarly, ROS production increased after treatment with pyocyanin, but not 1-hydroxyphenazine in all 3 cell types, but with stark differences in CF and non-CF ALIs. In HNEC ALI, pyocyanin reduced ciliary beat frequency (CBF) after 4 hours, while 1-hydroxpyhenazine did not. Despite this, both pyocyanin and 1-hydroxyphenazine decreased in cell viability in RPMI 2650 nasal carcinoma cells but not in HNEC at 24 hours. However, in both RPMI 2650 and HNEC, mitochondrial membrane potential acutely decreased after treatment with either pyocyanin or 1-hydroxyphenazine. Finally, 24-hour pyocyanin treatment decreased expression of ER stress genes in some cancer cells, but not in non-cancerous HNEC. Our data suggest that Ca2+ signaling is not required for acute effects of 1-hydroxyphenazine or pyocyanin on mitochondrial function. The greater sensitivity of RPMI 2650 cells to pyocyanin-induced and 1-hydroxyphenzine-induced cytotoxicity compared with primary cells suggests that these compounds might have some applicability in treating nasal squamous carcinomas or other types of head and neck squamous carcinomas. Although the exact mechanisms of pyocyanin induced apoptosis remains uncertain, the downregulation of the ER stress response may play a role.