PARP‑1 may be involved in hydroquinone‑induced apoptosis by poly ADP‑ribosylation of ZO‑2

Abstract

Hydroquinone (HQ), a major reactive metabolite of benzene, contributes to benzene‑induced leukemia. The molecular mechanisms that underlie this activity remain to be elucidated. Poly ADP‑ribosylation (PARylation) is a type of reversible posttranslational modification that is performed by enzymes in the PAR polymerase (PARP) family and mediates different biological processes, including apoptosis. Zona occludens 2 (ZO‑2) is a tight junction scaffold protein, which is involved in cell proliferation and apoptosis. The present study investigated the activity and mechanisms regulated by PARP‑1 during HQ‑induced apoptosis using TK6 lymphoblastoid cells and PARP‑1‑silenced TK6 cells. The results revealed that exposure to 10 µM HQ for 72 h induced apoptosis in TK6 cells and that apoptosis was attenuated in PARP‑1‑silenced TK6 cells. In cells treated with HQ, inhibition of PARP‑1 increased the expression of B cell leukemia/lymphoma 2 (Bcl‑2), increased ATP production and reduced reactive oxygen species (ROS) production relative to the levels observed in cells treated with HQ alone. Co‑localization of ZO‑2 and PAR (or PARP‑1 protein) was determined using immunofluorescence confocal microscopy. The findings of the present study revealed that ZO‑2 was PARylated via an interaction with PARP‑1, which was consistent with an analysis of protein expression that was performed using western blot analysis, which determined that ZO‑2 protein expression was upregulated in HQ‑treated control cells and downregulated in HQ‑treated PARP‑1‑silenced TK6 cells. These findings indicated that prolonged exposure to a low dose of HQ induced TK6 cells to undergo apoptosis, whereas inhibiting PARP‑1 attenuates cellular apoptosis by activating Bcl‑2 and energy‑saving processes and reducing ROS. The present study determined that PARP‑1 was involved in HQ‑induced apoptosis by PARylation of ZO‑2.

Figure 1.

Efficiency of PARP-1 silencing in TK6 cells. (A) Representative images of western blot analysis. Compared to the control cells, PARP-1 was silenced in shPARP-1 cells. (B) Densitometric analysis of PARP-1 protein expression levels. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. control cells. PARP-1, poly (ADP-ribose) polymerase-1; shPARP-1, short hairpin PARP-1.

Figure 2.

PARP-1 attenuated HQ-induced apoptosis. Empty TK6 cells were used as control cells. Cells were treated with PBS or 10 µM HQ for 72 h. (A) Representative images of flow cytometry using Annexin V-FITC and PI double-stained cells. (B) Cell apoptosis rates are presented as the mean ± standard deviation for at least three independent experiments. (C) Number of cells was counted using Countstar (IC1000). (D) Protein expression levels of the Bcl-2, caspase-3 and Bax proteins were detected using western blot analysis. (E) Caspase-3/7 luminescence values, which indicate caspase-3/7 activity, were detected using Caspase-Glo-3/7 assay kits. (F) ATP luminescence values, which indicate ATP level, were measured using CellTiter-Glo Luminescent Cell Viability assay kits. (G) Reactive oxygen species production was measured using DCFDA. *P<0.05. HQ, hydroquinone; PARP-1, poly (ADP-ribose) polymerase-1; shPARP-1, short hairpin PARP-1; Bcl2, B cell leukemia/lymphoma 2; Bax, BCL2 associated X; DCFDA, 2′,7′-dichlorofluorescin diacetate; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 3.

PARP-1 protein is required for the abundance of the ZO-2 protein. (A) Venn diagram showing the direct targets of PARP-1 that were identified by Carter-O'Connell et al (17). (B) PARP-1 and ZO-2 protein expression levels were detected using western blot analysis in control cells and shPARP-1 cells that were treated with 10 µM HQ for 72 h. (C) ZO-2 mRNA expression was analyzed and normalized to GAPDH in reverse transcription-quantitative polymerase chain reaction. *P<0.05. HQ, hydroquinone; PARP-1, poly (ADP-ribose) polymerase-1; shPARP-1, short hairpin PARP-1; ZO-2, zona occludens 2.

Figure 4.

ZO-2 may be modified by PARylation. Cells were treated with 10 µM HQ for 72 h and then stained using indirect immunofluorescence for ZO-2 (green), PAR (red, upper panel), PARP-1 (red, lower panel) and DAPI (blue) and the ZO-2 protein was modified by PARylation (upper panel) via an interaction with PARP-1 (lower panel). Magnification, ×1,000. ZO-2, zona occludens 2; DIC, differential interference contrast; HQ, hydroquinone; PARP-1, poly (ADP-ribose) polymerase-1; shPARP-1, short hairpin PARP-1; PAR, poly (ADP-ribose).