Gramicidin, a Bactericidal Antibiotic, Is an Antiproliferative Agent for Ovarian Cancer Cells

Abstract

Background and Objectives: Gramicidin, a bactericidal antibiotic used in dermatology and ophthalmology, has recently garnered attention for its inhibitory actions against cancer cell growth. However, the effects of gramicidin on ovarian cancer cells and the underlying mechanisms are still poorly understood. We aimed to elucidate the anticancer efficacy of gramicidin against ovarian cancer cells. Materials and Methods: The anticancer effect of gramicidin was investigated through an in vitro experiment. We analyzed cell proliferation, DNA fragmentation, cell cycle arrest and apoptosis in ovarian cancer cells using WST-1 assay, terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL), DNA agarose gel electrophoresis, flow cytometry and western blot. Results: Gramicidin treatment induces dose- and time-dependent decreases in OVCAR8, SKOV3, and A2780 ovarian cancer cell proliferation. TUNEL assay and DNA agarose gel electrophoresis showed that gramicidin caused DNA fragmentation in ovarian cancer cells. Flow cytometry demonstrated that gramicidin induced cell cycle arrest. Furthermore, we confirmed via Western blot that gramicidin triggered apoptosis in ovarian cancer cells. Conclusions: Our results strongly suggest that gramicidin exerts its inhibitory effect on cancer cell growth by triggering apoptosis. Conclusively, this study provides new insights into the previously unexplored anticancer properties of gramicidin against ovarian cancer cells.

Keywords: apoptosis; drug repositioning; gramicidin; ovarian cancer.

Figure 1

Gramicidin inhibited the proliferation of ovarian cancer (OC) cells. (A) OVCAR8, (B) SKOV3, and (C) A2780 cells were treated with the indicated concentrations of gramicidin for 72 h. (D) OVCAR8, (E) SKOV3, and (F) A2780 cells were treated with 0, 0.33, 1, or 3 μM gramicidin for 0, 24, 48, or 72 h. Relative cell proliferation rates were assessed using the WST-1 assay, which monitors mitochondrial succinate reductase activity at 450 nm. The data represent the mean ± SEM of duplicates from two independent experiments.

Figure 2

Gramicidin-induced apoptosis, as indicated by DNA fragmentation in OC cells. Apoptotic cells were detected by DNA fragmentation using the TUNEL assay and DNA agarose gel electrophoresis. (A) OVCAR8 and (B) SKOV3 were treated with 0, 0.1, 0.3, or 1 μM gramicidin for 48 h and imaged using confocal microscopy. TUNEL-positive nuclei are shown in green, and total nuclei stained with Hoechst 33,342 are presented in blue (left panel). Relative TUNEL intensities, normalized to the negative control, were quantified in five randomly selected microscopic fields (right panel). (C) OVCAR8 and SKOV3 were treated with 0, 0.1, or 0.3 μM of gramicidin for 48 h, and genomic DNA was isolated. DNA fragmentation was assessed by agarose gel electrophoresis with ethidium bromide staining. The data are presented as mean ± SEM of a minimum of three independent experiments. Statistical significance was calculated using one-way ANOVA (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 3

Gramicidin increased sub-G1 in OC cells. (A) OVCAR8 and (B) SKOV3 were treated with 0, 0.1, 0.3, or 1 μM of gramicidin for 48 h and subsequently stained with propidium iodide (PI) for cell cycle analysis using flow cytometry. The distribution of cells at different cell cycle phases was determined, and the percentages of cells in each phase relative to the total number of cells were calculated. The data are presented as mean ± SEM of three independent experiments.

Figure 4

Gramicidin-induced cleavage of caspase-3 and PARP in OC cells. (A) OVCAR8 and (B) SKOV3 cells were treated with 0, 0.1, 0.3, or 1 μM of gramicidin for 24 h. Whole-cell lysates were analyzed via immunoblotting against caspase-3, cleaved caspase-3, PARP, cleaved PARP, or β-actin. The results represent a minimum of three independent experiments.