Biphasic ROS production, p53 and BIK dictate the mode of cell death in response to DNA damage in colon cancer cells

Abstract

Necrosis, apoptosis and autophagic cell death are the main cell death pathways in multicellular organisms, all with distinct and overlapping cellular and biochemical features. DNA damage may trigger different types of cell death in cancer cells but the molecular events governing the mode of cell death remain elusive. Here we showed that increased BH3-only protein BIK levels promoted cisplatin- and UV-induced mitochondrial apoptosis and biphasic ROS production in HCT-116 wild-type cells. Nonetheless, early single peak of ROS formation along with lysosomal membrane permeabilization and cathepsin activation regulated cisplatin- and UV-induced necrosis in p53-null HCT-116 cells. Of note, necrotic cell death in p53-null HCT-116 cells did not depend on BIK, mitochondrial outer membrane permeabilization or caspase activation. These data demonstrate how cancer cells with different p53 background respond to DNA-damaging agents by integrating distinct cell signaling pathways dictating the mode of cell death.

Fig 1. BIK is involved in DNA damage-induced apoptosis in HCT-116 wt cells, but not in HCT-116 p53 -/- cells.

(A) HCT-116 wt and HCT-116 p53 -/- cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 0-12h and BIK protein levels were detected by immunoblot analysis. Actin was probed as a loading control. (B) HCT-116 wt and HCT-116 p53 -/- cells were transiently transfected with BIK siRNA or scrambled siRNA for 48h. The efficiency of knockdown was monitored by immunoblots. (C) BIK siRNA-transfected or scrambled siRNA-transfected cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 48h and cell viability was evaluated by CellTiterGlo assay and expressed as % of untreated control (mean±SEM, n = 3, **P<0.01). (D) BIK siRNA-transfected or scrambled siRNA-transfected cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 48h and M30 (apoptosis) and M65 (apoptosis + necrosis) was evaluated by M30/M65 Apoptosense ELISA (mean±SEM, n = 4, **P<0.01). (E) BIK siRNA-transfected or scrambled siRNA-transfected cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 24h and caspase-3 and caspase-9 activities were determined by fluorometric caspase assays (mean±SEM, n = 3, *P<0.05, **P<0.01). (F) Cells were pretreated with Nec-1 (20 μM), Q-VD-OPh (20 μM), 4-ANI (2 μM) for 2h and then treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 48h. Cell viability was evaluated by CellTiterGlo assay and expressed as % of untreated control (mean±SEM, n = 3, **P<0.01).

Fig 2. BIK mediates cisplatin-induced cell death in HCT-116 wt cellular spheroids, but not in HCT-116 p53 -/- spheroids.

(A) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were subsequently grown in 24-well 3D Algimatrix plates in the presence of RNAi duplexes. Microscopic evaluation of spheroids was done to verify that siRNA treatments did not interfere with the 3D growth of HCT-116 cells. (B) The efficiency of BIK depletion by RNA interference in HCT-116 wt and HCT-116 p53 -/- was determined by immunoblot analysis. Actin was probed as loading control. (C) Spheroids were treated with cisplatin (200 μM) for 48h and cell viability was assessed by using alamarBlue assay (mean±SEM, n = 3, *P<0.05).

Fig 3. BIK/BCL-2 and BIK/BCL-XL interactions were increased in HCT-116 wt cells in response to DNA damage.

HCT-116 wt and HCT-116 p53 -/- cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 4h and the interaction of BIK with BCL-2, BCL-XL, and MCL-1 was detected by coimmunoprecipitation assays. Inputs for coimmunoprecipitation experiments were also subjected to immunoblot analysis and actin was probed as loading control.

Fig 4. Depletion of BIK led to decreased mitochondrial cell death priming in HCT-116 wt cells.

(A) HCT-116 wt and (B) HCT-116 p53 -/- cells were untreated (upper panels) or transiently transfected with BIK siRNA (lower panels) and mitochondrial depolarization was measured following incubation of cells with DMSO, BIM, BID, BAD, PUMA, BMF and NOXA peptides at 100 μM and FCCP at 10 μM. Graphs are shown as mean±SEM, n = 3 and sample mitochondrial potential (ΔΨm) kinetic tracings are provided. (C) Microplate-based BH3 profiles were confirmed by ELISA-based cytochrome c release assays following incubation of isolated mitochondria with DMSO, BIM, BID, BAD, PUMA, BMF and NOXA peptides at 100 μM (mean±SEM, n = 3).

Fig 5. DNA damage induces biphasic ROS production in HCT-116 wt cells.

HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. (A) Untransfected and transfected cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) and total ROS formation was monitored by using DCF-DA probe. Kinetic tracing was processed by measurements in every 15 min for 12h (mean RFU±SEM, n = 3) (B) Untransfected and transfected cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) and superoxide formation was monitored by MitoSOX Red probe. Kinetic tracing was processed by measurements in every 15 min for 12h (mean RFU±SEM, n = 3). (C) Cells were pretreated with NAC (10 mM) or TIRON (10 mM) for 2h and then treated with cisplatin for 48h. Cell viability was evaluated by CellTiterGlo assay and expressed as % of untreated control (mean±SEM, n = 3, *P<0.05, **P<0.01).

Fig 6. Cisplatin induces early LMP in HCT-116 p53 -/- cells.

(A) HCT-116 wt and HCT-116 p53-/- cells were treated with cisplatin (20 μM) for 1h. HCT-116 p53 -/- cells were TIRON (10 mM) for 2h and then treated with cisplatin for 1h to examine the effect of TIRON on cisplatin-induced LMP. Cells were stained for galectin-3 to evaluate the lysosomal membrane permeabilization. (B) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 1h and the Cathepsin B/L activity was measured in untransfected and transfected cells (mean RFU±SEM, n = 3). (C) HCT-116 p53 -/- cells were pretreated with CA-074Me (100 μM) or Z-FA-FMK (10 μM) for 2h and then treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 48h. Cell viability was determined by CellTiterGlo assay and expressed as % of untreated control (mean±SEM, n = 3, *P<0.05, **P<0.01).