Paracetamol Induces Apoptosis, Reduces Colony Formation, and Increases PTPRO Gene Expression in Human Embryonic Kidney HEK 293 Cells

Abstract

Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. Although many studies are available showing that paracetamol causes hepatic damage, few studies exist on renal damage. Protein tyrosine phosphatase receptor type O (PTPRO) protein has tyrosine phosphatase activity and plays a role in the regulation of the renal glomerular pressure/filtration rate relationship in podocyte structure. The PTPRO gene also exhibits characteristics of a candidate tumor suppressor gene. In this study, apoptosis, cell cycle, colony formation, and PTPRO gene expression of the human embryonic kidney (HEK 293) cells were assessed by treating with paracetamol at different concentrations for 24 and 26 h in at least three independent trials. Paracetamol induced dose-dependent apoptosis at all concentrations (5, 10, 15, 20, and 30 mM) for both 24 and 26 h (p < 0.05-p < 0.001). Paracetamol decreased dose-dependent colony-forming potential of cells at 10.000 cells seeding in HEK 293 cells at all concentrations (5, 10, 15, 20, and 30 mM) (p < 0.05-p < 0.001). In HEK 293 cells, paracetamol also increased cell population in the G0-G1 phase at low 5 mM concentration for 26 h (p < 0.05), while reducing cell population in the G2-M phase of the cell cycle at 10 mM and increasing at 30 mM concentration for 24 h (p < 0.05 and p < 0.05, respectively). Paracetamol at high concentration (30 mM) increased PTPRO gene expression in HEK 293 cells for both 24 and 26 h (p < 0.05). In conclusion, our results may indicate that paracetamol may suppress the development of renal cancer by inducing apoptosis and reducing colony formation in a dose-dependent manner, and upregulating PTPRO gene expression in HEK 293 cells at high concentration, in addition to its renal-damaging role. Further research is necessary to determine the potential role or detrimental effects of paracetamol on the development of renal cancer, as the mechanism underlying its effects on renal damage and cancer formation remains unclear.

Keywords: HEK 293 cells; PTPRO; acetaminophen (paracetamol); apoptosis; cell cycle; colony formation; renal cancer.

FIGURE 1

Cell index curves of HEK 293 cells were obtained from four different cell seeding densities for 96 h of culture with the iCELLigence system. Red: 10.000 cells; Green: 20.000 cells; Blue: 30.000 cells; Pink: 40.000 cells.

FIGURE 2

Normalized cell index curves of HEK 293 cells obtained by the iCELLigence system. (A) HEK 293 cells treated with seven different paracetamol concentrations at the 24th hour after cell seeding for 48 and 50‐h cultures. Red: 0.5 mM, Green: 1 mM, Blue: 5 mM, Pink: 10 mM, Turquoise: 20 mM, Purple: 40 mM, Brown: 60 mM. (B) HEK 293 cells treated with five different paracetamol concentrations at the 20th hour after cell seeding for 60 h of culture. Red: 1 mM; Green: 10 mM; Blue: 20 mM; Pink: 60 mM; Turquoise: 75 mM.

FIGURE 3

(A) Analysis of apoptotic cells after different paracetamol treatments of 24 and 26 h by flow cytometry. (B) Total apoptotic cell percentages in HEK 293 cells after 24 h of treatment at different paracetamol concentrations. (C) Total apoptotic cell percentages in HEK 293 cells after 26 h of treatment at different paracetamol concentrations. *p < 0.05, **p < 0.01, and ***p < 0.001 when concentrations were compared to the control.

FIGURE 4

(A) Analysis of cell cycle profiles after different paracetamol treatments of 24 and 26 h by Muse Cell analyzer. (B) Cell cycle phase percentages in HEK 293 cells after 24 h of treatment at different paracetamol concentrations. (C) Cell cycle phase percentages in HEK 293 cells after 26 h of treatment at different paracetamol concentrations. *p < 0.05 when concentrations were compared to the control.

FIGURE 5

Effects of different paracetamol concentrations on colony formation in HEK 293 cells at 2.500, 5.000, and 10.000 cell densities. *p < 0.05, **p < 0.01, and ***p < 0.001 when concentrations were compared to the control.

FIGURE 6

PTPRO gene expression levels in HEK 293 cells after 24 and 26 h of treatment at different paracetamol concentrations. *p < 0.05 when concentrations were compared to the control.