Gentian Violet Inhibits Cell Proliferation through Induction of Apoptosis in Ovarian Cancer Cells

Abstract

Gentian violet (GV) is known to have antibacterial and antifungal effects, but recent studies have demonstrated its inhibitory effects on the growth of several types of cancer cells. Here, we investigated the anticancer efficacy of GV in ovarian cancer cells. GV significantly reduced the proliferation of OVCAR8, SKOV3, and A2780 cells. Results of transferase dUTP nick and labeling (TUNEL) assay and Western blot assay indicated that the inhibitory effect of GV on ovarian cancer cells was due to the induction of apoptosis. Moreover, GV significantly increased reactive oxygen species (ROS) and upregulated the expression of p53, PUMA, BAX, and p21, critical components for apoptosis induction, in ovarian cancer cells. Our results suggest that GV is a novel antiproliferative agent and is worthy of exploration as a potential therapeutic agent for ovarian cancer.

Keywords: apoptosis; gentian violet; ovarian cancer.

Figure 1

Gentian violet (GV) inhibited the proliferation of ovarian cancer (OC) cells. (A) OVCAR8, (B) SKOV3, or (C) A2780 cells were seeded onto 96-well tissue culture plates and treated with the indicated concentrations of GV for 72 h. (D) OVCAR8, (E) SKOV3, or (F) A2780 cells were treated with 0, 0.33, 1, or 3 μm of GV for 0, 24, 48, or 72 h. Relative cell proliferation rates were determined by WST-1 assay monitoring the mitochondrial succinate reductase activity at 450 nm. Data are shown as mean ± SEM duplicates from one of two independent experiments. The p-value was calculated by Student t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 2

GV-induced apoptosis in OC cells. Apoptotic cells were examined using the TUNEL assay for DNA fragmentation. (A) OVCAR8 and (B) SKOV3 were treated with 0, 1, or 3 μm of GV for 48 h and imaged by confocal microscopy. TUNEL-positive nuclei are shown in green and total nuclei stained with Hoechst 33342 are shown in blue (upper). Relative TUNEL intensities versus negative control were quantified in three randomly selected microscopic fields (lower). Data are shown as mean ± SEM from one of two independent experiments. Scale bar = 100 μm.

Figure 3

GV induced the cleavage of PARP and caspase-3 in OC cells. (A) OVCAR8 and (B) SKOV3 were treated with 0, 1, or 3 μm of GV for 24 h and whole-cell lysates were immunoblotted with antibodies against caspase-3, cleaved-caspase-3, PARP, cleaved-PARP, or β-actin. Results are representative of at least three independent experiments.

Figure 4

GV increased ROS level in OC cells. (A) OVCAR8 and (B) SKOV3 cells were treated with the indicated concentrations of GV for 24 h and stained with 10 μm H₂DCFDA. ROS levels were measured with a microplate reader at 485/530 nm. Data are shown as mean ± SEM duplicates from one of two independent experiments. The p-value was calculated by Student’s t-test (* p < 0.05 and ** p < 0.01).

Figure 5

GV upregulated p53, PUMA, BAX, and p21 in OC cells. (A) OVCAR8 and (B) SKOV3 were treated with 0, 1, or 3 μm of GV for 24 h and whole-cell lysates were immunoblotted with antibodies against p53, PUMA, BAX, p21, or β-actin. Results are representative of at least three independent experiments.

Figure 6

Schematic illustration of the mechanism by which GV induces apoptosis in OC cells. GV increases ROS and upregulates p53, which increases PUMA, BAX, and p21. Increased PUMA and BAX release cytochrome c from mitochondria and consequently induce cleavage of caspase-3 and PARP, inducing apoptosis.