Impact of cellular ATP levels on cell viability in response to fluorouracil through lysophosphatidic acid (LPA) receptor-4 (LPA4) and LPA6 in colon cancer cells

Abstract

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA₁ to LPA₆) mediates various aspects of cancer cell behaviors. This study aimed to investigate the variation in intracellular ATP levels and its impact on cell viability in response to fluorouracil (5-FU) through LPA₄ and LPA₆ in colon cancer DLD-1 cells. LPA₄ and LPA₆ are linked to Gs and Gi proteins. Gs protein stimulates the activity of adenylyl cyclase, which catalyzes the conversion of ATP to cAMP, whereas Gi protein inhibits this activity. In cell survival assay, cells were treated with 5-FU every 24 h for 3 days. The viability in response to 5-FU in DLD-1 cells was enhanced by LPA₄ and LPA₆ knockdowns. Furthermore, LPA₄ and LPA₆ knockdowns reduced the expression of cleaved-PARP1 protein when cells were treated with 5-FU. Since ethidium bromide (EtBr) reduces mitochondrial DNA level in cultured cells, EtBr-treated (DLD-EtBr) cells were generated from DLD-1 cells. The viability to 5-FU in DLD-EtBr cells was higher than that of DLD-1 cells. Additionally, culturing DLD-1 cells in a low glucose-containing medium led to increased viability to 5-FU. LPAR4 and LPAR6 expressions were reduced in both DLD-EtBr and low glucose-treated cells. The cellular ATP levels were significantly decreased in DLD-1 cells following EtBr treatment and exposure to low glucose conditions. Conversely, in the presence of LPA, LPA₄ and LPA₆ knockdowns resulted in a marked elevation of ATP levels. These results suggest that cell viability to 5-FU is negatively regulated via the activation of LPA₄-and LPA₆-Gs protein pathways in DLD-1 cells rather than Gi protein.