Emptying of Intracellular Calcium Pool and Oxidative Stress Imbalance Are Associated with the Glyphosate-Induced Proliferation in Human Skin Keratinocytes HaCaT Cells

Abstract

We demonstrated that glyphosate possesses tumor promoting potential in mouse skin carcinogenesis and SOD 1, calcyclin (S100A6), and calgranulin B (S100A9) have been associated with this potential, although the mechanism is unclear. We aimed to clarify whether imbalance in between [Ca(2+)] i levels and oxidative stress is associated with glyphosate-induced proliferation in human keratinocytes HaCaT cells. The [Ca(2+)] i levels, ROS generation, and expressions of G1/S cyclins, IP3R1, S100A6, S100A9, and SOD 1, and apoptosis-related proteins were investigated upon glyphosate exposure in HaCaT cells. Glyphosate (0.1 mM) significantly induced proliferation, decreases [Ca(2+)] i , and increases ROS generation in HaCaT cells, whereas antioxidant N-acetyl-L-cysteine (NAC) pretreatment reverts these effects which directly indicated that glyphosate induced cell proliferation by lowering [Ca(2+)] i levels via ROS generation. Glyphosate also enhanced the expression of G1/S cyclins associated with a sharp decrease in G0/G1 and a corresponding increase in S-phases. Additionally, glyphosate also triggers S100A6/S100A9 expression and decreases IP3R1 and SOD 1 expressions in HaCaT cells. Notably, Ca(2+) suppression also prevented apoptotic related events including Bax/Bcl-2 ratio and caspases activation. This study highlights that glyphosate promotes proliferation in HaCaT cells probably by disrupting the balance in between [Ca(2+)] i levels and oxidative stress which in turn facilitated the downregulation of mitochondrial apoptotic signaling pathways.

Figure 1

(a) Proliferation effects of glyphosate on HaCaT cells. Cells were exposed to glyphosate (0.01–1 mM) and positive control, and TPA (10 nM) for 24, 48, and 72 h. (b) Inhibition of glyphosate (0.1 mM) induced cell proliferation after 72 h by a prior treatment of NAC (10 mM and 20 mM). OD values from three separate experiments are shown as mean ± SD. *Increase over vehicle control cells (P < 0.01); **decrease over glyphosate alone treated cells (P < 0.01).

Figure 2

PCNA and BrdU staining of glyphosate and TPA-treated HaCaT cells. HaCaT cells were treated with glyphosate and TPA and were incubated for 72 h. (a) Cells were fixed, and PCNA was detected by immunofluorescence with an anti-PCNA antibody and FITC-labeled secondary antibody (green color). Pictures with green and blue colors were merged. DNA was stained with DAPI (blue color). (b) Cells were pulsed for 4 h with BrdU, fixed, and detected with an anti-BrdU antibody and rhodamine-labeled secondary antibody (red color). DNA was stained with DAPI (blue color). Pictures with red and blue colors were merged. Histogram represents the relative fluorescence intensity of PCNA and BrdU positive cells expressed as a ratio between control and treated HaCaT cells. For all panels, representative data from mean ± SD of at least three independent experiments are shown. P < 0.05 versus control cells.

Figure 3

Measurement of glyphosate-induced changes in intracellular Ca²⁺ level in HaCaT cells by spectrofluorometry with Fura-2/AM dye. (a) Ca²⁺ level was measured after treatment with NAC (20 mM), 0.1 mM glyphosate, and 10 nM TPA at 0–6 h. (b) Ca²⁺ level was measured after a prior treatment with 20 mM NAC. Results are mean ± SD from three independent experiments. *Decrease over control cells (P < 0.01); **increase over control cells (P < 0.01).

Figure 4

Measurement of glyphosate-induced generation of ROS in HaCaT cells. (a) Generation of ROS was measured using the fluorescent dye (5 (and 6)-chloromethyl-20, 70-dichlorodihydro-fluorescein diacetate acetyl ester) after treatment of HaCaT cells with vehicle alone, NAC (20 mM), (0.01–0.1 mM) glyphosate, and 10 nM TPA. (b) Inhibition of ROS generation after a prior treatment of NAC. Data are expressed as mean ± SD of three independent experiments. *Increase in ROS generation versus control cells (P < 0.05); **decrease in ROS generation versus control cells (P < 0.05).

Figure 5

Effect of glyphosate on cell-cycle progression and G1/S-phase proteins in HaCaT cells. Cultured cells were treated with (a) vehicle, glyphosate (0.1 mM), and TPA (10 nM). 24 h later, cell cycle was analyzed by flow cytometry. (b) Quantitative assessment of the percentage of HaCaT cells in G1/S-phases, as indicated by propidium iodide (PI). (c) Immunoblot of G1-S-phase cell-cycle-associated proteins in HaCaT cells after treatment with glyphosate and TPA for 24, 42, and 72 h. All results represent the average of three independent experiments ± S.D. *P < 0.01 compared with the vehicle control.

Figure 6

Western blots showing the proliferative effect of glyphosate (0.1 mM) and TPA (10 nM) on the expression levels of (a) Ca²⁺ binding and oxidative stress and (b) apoptosis-related proteins in HaCaT cells for 24, 42, and 72 h. The bands shown here are from a representative experiment repeated three times with similar results. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. Quantitative fold change was calculated in respect to control on the basis of pixel density measured by UNSCAN-IT software.

Figure 7

Proposed itinerary for glyphosate-induced proliferation in human skin keratinocytes HaCaT cells.