Korean J Physiol Pharmacol.  2021 May;25(3):189-195. 10.4196/kjpp.2021.25.3.189.

Fluoxetine affects cytosolic cAMP, ATP, Ca²⁺ responses to forskolin, and survival of human ovarian granulosa tumor COV434 cells

• Nguyen TMD
• Klett D
• Combarnous Y

• KMID: 2514944
• DOI: http://doi.org/10.4196/kjpp.2021.25.3.189

Abstract

Fluoxetine (FLX), a selective serotonin reuptake inhibitor antidepressant, exhibits various other mechanisms of action in numerous cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The aim of this study was to determine the off-target effects of the anti-depressant drug FLX, on the human ovarian granulosa tumor COV434 cells stimulated by forskolin (FSK), by measuring the real-time kinetics of intracellular cyclic AMP (cAMP), ATP level, cytoplasmic calcium ([Ca ²⁺ ] _cyt ) and survival of COV434 cells. We show that incubating COV434 cells with FLX (between 0.6 and 10 ㎛) induces a decrease in intracellular cAMP response to FSK, a drop in ATP content and stimulates cytoplasmic Ca ²⁺ accumulation in COV434 cells. Only the highest concentrations of FLX (5–10 ㎛) diminished cell viability. The present report is the first to identify an action mechanism of FLX in human tumor ovarian cells COV434 cells and thus opening the way to potential use of fluoxetine as a complementary tool, in granulosa tumor treatments.

Keyword

Cyclic AMP; Fluoxetine; Forskolin; Ovary; Tumor

Figure

• 

  Fig. 1 Effect of fluoxetine (FLX) on intracellular cAMP response of COV434 cells to 10 µM forskolin (FSK). Cells were treated with the indicated concentrations of fluoxetine for 2 h (A, B) or 24 h (C, D). (A, C) Real-time recording of luminescence under stimulation of COV434 cells by 10 µM FSK in the presence of fluoxetine; (B, D) Dose-dependent response to fluoxetine determined by the area under curve (AUC) of individual kinetics in (A, C). Different letters (a–e) in each incubation time indicate significant differences between control and treatment at p < 0.05.

• 

  Fig. 2 Effect of fluoxetine (FLX) on ATP levels in COV434 cells and the number of viable COV434 cells. ATP level (A) and the viability of COV434 cells (B) were measured after incubation with various concentrations of FLX for 2 h or 24 h. The experiments were repeated four times; values are mean ± SEM. Different letters (a–e) in each incubation time indicate significant differences between control and treatment at p < 0.05.

• 

  Fig. 3 Effect of fluoxetine (FLX) on [Ca2+]cyt in Fluo4-AM-loaded COV434 cells. Cells were incubated with Fluo4-AM as described in experimental procedures. The intracellular Ca2+ levels were measured spectrofluorimetry using an excitation wavelength of 494 nm and an emission wavelength of 516 nm. The [Ca2+]cyt measurements were made every 52 sec. (A) Baseline levels of [Ca2+]cyt were monitored for 156 sec prior to adding Ca2+ at 2 mM final concentration, changes in [Ca2+]cyt were monitored for an additional 800 sec. (B) Dose-dependent response to fluoxetine determined by the area under curve (AUC) of individual kinetics in (A). (C) Signal of Ca2+ responses induced by FLX (10 µM) in a Ca2+-free medium and Control (Ctrl) (absence of FLX). (D) Cells loaded with Fluo4-AM were incubated first with 10 µM FLX in Ca2+-free medium, and external Ca2+ was added after 1,000 sec. Asterisks in each concentration indicate significant differences between control and treatment at p < 0.05.

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