Mebendazole Exerts Anticancer Activity in Ovarian Cancer Cell Lines via Novel Girdin-Mediated AKT/IKKα/β/NF-κB Signaling Axis

Abstract

Mebendazole (MBZ), a benzimidazole anthelmintic and cytoskeleton-disrupting compound, exhibits antitumor properties; however, its action on ovarian cancer (OC) is not clearly understood. This study evaluates the effect of MBZ on OC cell lines OVCAR3 and OAW42, focusing on cell proliferation, migration, invasion, and cancer stemness. The underlying mechanisms, including cytoskeletal disruption, epithelial-mesenchymal transition (EMT), and signaling pathways, were explored. MBZ inhibited OVCAR3 and OAW42 cell proliferation in a dose- and time-dependent manner. Additionally, MBZ significantly impedes migration, spheroid invasion, colony formation, and stemness. In addition, it reduced actin polymerization and down-regulated CSC markers (e.g., CD24, CD44, EpCAM). Moreover, MBZ suppressed MMP-9 activity and inhibited the EMT marker as judged by decreased N-Cadherin and Vimentin and increased E-Cadherin. Furthermore, MBZ induced G2/M cell cycle arrest by modulating Cyclin B1, CDC25C, and WEE1. Also, it triggered apoptosis by disrupting mitochondrial membrane potential. Mechanistic studies revealed a significant downregulation of Girdin, an Akt modulator, along with reduced p-Akt, p-IKKα/β, and p-NF-κB, indicating MBZ's novel mechanism of action through the Girdin-mediated Akt/IKKα/β/NF-κB signaling axis. Thus, by targeting Girdin, MBZ presents a promising repurposed therapeutic strategy to inhibit cancer cell proliferation and metastasis in ovarian cancer.

Keywords: EMT; MMP-9; girdin; mebendazole; metastasis; ovarian cancer.

Figure 1

Mebendazole (MBZ) eloquently obstructs the cell proliferation of OVCAR3 and OAW42 cells in a dose- and time-dependent manner. (A,B) Cells were treated with MBZ at varying concentrations (0.156, 0.312, 0.625, 1.25, and 2.5 µM) and DMSO vehicle control (VC) for different time points (24, 48, and 72 h). Cell proliferation was assessed using the MTT assay. The half-maximal inhibitory concentration (IC₅₀) of MBZ was determined for each time point, and a quantitative plot was generated using GraphPad Prism software. Data represent the mean ± standard error of the mean of two independent experiments in triplicate. Additionally, Mebendazole (MBZ) significantly impedes cell migration, spheroid invasion, and colony formation in both cell lines. (C,E,G,I,K,M) Represent microscopic images of cell migration (magnification 10×, scale bar = 100 μm), spheroid invasion (magnification 10×, scale bar = 100 μm), and colony formation assay (digital camera, 2× Zoom) after 48 h of treatment at the indicated concentration of MBZ studied. Further, (D,F,H,J,L,N) represent the quantitative plots of percentage (%) values of areas of cell migration, spheroid invasion, and number of colonies formation. The bars represent the mean ± standard error of the mean of three independent experiments. Statistical significance was determined by one-way ANOVA, with * p < 0.05, ** p < 0.01, and *** p < 0.001 considered statistically significant and ns: not significantcompared to the DMSO-treated vehicle control (VC).

Figure 2

MBZ significantly hinders the actin polymerization in OC cell lines in a dose-dependent manner. OVCAR3 and OAW42 cells were treated with different concentrations of MBZ and DMSO vehicle control (VC) for 48 h and were determined by staining the cells with Phalloidin-FITC and analyzed using a flow cytometer. (A,C) signify the histogram overlays shift plot and (B,D) show a graphical representation of the quantitative change in the percentage of mean fluorescence intensity (MFI) shifts, which indicate waning in actin polymerization. (E,F) denote the confocal microscopic images of Phalloidin-FITC staining of actin filaments of OVCAR3 and OAW42 cells and counter staining with DAPI. The red arrow in the zoomed image of DMSO (vehicle control (VC)) indicates the intact actin filamentous network of individual cells with organized cytoskeleton while the yellow arrow shows the disrupted actin filamentous network with disordered cytoskeleton arrangement in MBZ (IC₅₀)-treated OVCAR3 and OAW42 cells (scale bar = 10 µm). (G,H) indicate the quantitative plots of the percentage of mean fluorescence intensity (MFI) shifts, which signify decrease in actin polymerization in both the cell lines compared to DMSO (vehicle control (VC)). The bars represent the mean ± standard error of the mean of three independent experiments. Level of significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001, and ns: not significant) compared to DMSO (vehicle control (VC)), performed using 1-way ANOVA test or t-test.

Figure 3

(A–F) Mebendazole (MBZ) ardently disrupts the cancer stemness characteristics of both OVCAR3 and OAW42 cells in a dose-dependent manner, analyzed by flow cytometry. (A) CD24 vs. CD44 and (B) CD44 vs. EPCAM dot plot showing a decrease in the percentage of CD24-, CD44-, and EPCAM-expressing cells with increasing concentration of mebendazole in the OVCAR3 cell line. In the OAW42 cell line, (C) CD24 vs. CD44 dot plot showed a similar pattern of decrease in % of expression as OVCAR3 cells. However, (D) CD44 vs. EPCAM dot plot of OAW42 cell line shows no dose-dependent decrease pattern in % of EPCAM-expressing cells. (E,F) show a graphical representation of change in CD24, CD44, and EPCAM expression with increasing concentration of Mebendazole in OVCAR and OAW42 cell lines, respectively. (G–N) MBZ also thwarts gelatinolytic activity and modulates the metastasis-related proteins in OC cell lines in a dose-responsive way. (G,I) represent the gelatin zymography of MMP-9 activity alterations in OVCAR3 and OAW42 cells, and supernatant was collected after treatment with different concentrations of MBZ and DMSO vehicle control for 48 h. (H,J) The gelatinolytic activity of MMP-9 at different MBZ concentrations was calculated and graphed using GraphPad Prism software. (K,M) The expression of proteins involved in MBZ-dissuaded metastasis potential was analyzed by Western blot, with GAPDH as the loading control. (L,N) represent the quantitative graphs of the expression level of proteins in fold change. Western blot and quantitative analysis revealed that as the MBZ concentration increases, the expression of N-cadherin, vimentin, and MMP-9 reduces while the expression of E-cadherin increases significantly compared to DMSO vehicle control (VC). The bars represent the mean ± standard error mean of three independent experiments. Level of significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001, and ns: not significant) compared to DMSO (vehicle control (VC)), performed using 1-way or 2-way ANOVA test.

Figure 4

Mebendazole induces G2/M cell cycle arrest in ovarian cancer cell lines dose-dependently. (A,C) show the histogram plots of cell cycle alterations in OVCAR3 and OAW42 cells treated with indicated concentrations of the MBZ and DMSO vehicle control for 48 h. (B,D) indicate the quantitative plots of the percentage of cells in each phase of the cell cycle at different MBZ concentrations. (E,G) The expression level of proteins involved in MBZ-mediated cell cycle arrest was analyzed by Western blot, with GAPDH as the loading control. (F,H) denote the quantitative plots of expression of proteins in fold change. Western blot and quantitative analysis revealed that as the MBZ concentration increases it reduces the expression of Cdc25C, and cyclin B1 while increasing the expression of Wee1 substantially compared to the DMSO vehicle control (VC). The bars represent the mean ± standard error mean of three independent experiments in triplicate. Level of significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001, and ns: not significant) compared to DMSO (vehicle control (VC)), performed using 1-way or 2-way ANOVA test.

Figure 5

Mebendazole notably disrupts mitochondrial membrane potential and induces apoptosis in ovarian cancer cell lines in a dose-dependent manner. OVCAR3 and OAW42 cells were treated with different concentrations of MBZ and DMSO vehicle control for 48 h and were determined by staining the cells with Rhodamine123 and analyzed using a flow cytometer. (A,C) signify the histogram overlays shift plot and (B,D) shows a graphical representation of quantitative change in the percentage of mean fluorescence intensity (MFI) shifts, which indicates depolarization of mitochondrial membrane potential. (E,G) show the dot plot of apoptosis in OVCAR3 and OAW42 cells treated with different concentrations of the MBZ or DMSO vehicle control for 48 h and was analyzed by flow cytometry using FITC-Annexin V/PI. (F,H) indicate the quantitative plots of the percentage of cells of live (LV), early apoptosis (EA), late apoptosis (LA), and necrosis (NR). (I,K) represent the blot image of the expression of proteins involved in MBZ-induced apoptosis, analyzed using Western blot, with GAPDH as the loading control. (J,L) denote the quantitative plots of the level of expression of proteins in fold change. Western blot analysis showed that as the MBZ concentration increases, it increases the apoptotic protein expression (Cytochrome C, Bax, cleaved-PARP, cleaved-Caspase 3, cleaved-Caspase 7, and cleaved-Caspase 9) while reducing the expression of an anti-apoptotic protein (Bcl2) significantly compared to DMSO (vehicle) control. The bars represent the mean ± standard error of the mean of three independent experiments in triplicate. Level of significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001, and ns: not significant) compared to DMSO (vehicle control (VC)), performed using 1-way or 2-way ANOVA test. LV: live; EA: early apoptosis; LA: late apoptosis; and NR: necrosis.

Figure 6

MBZ significantly fettered the expression of p-Nf-κB and its translocation from cytoplasm to the nucleus in both the cell lines after the treatment of MBZ (IC₅₀) for 48 h and the images were acquired through a confocal microscope (scale bar = 10 µm). (A,C) denote the confocal microscopic images of OVCAR3 and OAW42 cells, after immunofluorescence (IF) staining with antibody against p-Nf-κB (p65) and Alexa 488-tagged secondary antibody and counter-stained with DAPI. (B,D) indicate the quantitative plots of the percentage of MFI of nucleus vs. cytoplasm ratio, which signifies a decrease in translocation of p-Nf-κB (p65) expression compared to DMSO (vehicle control (VC) in both the cell lines. MBZ substantially exerts its anticancer effects on OVCAR3 and OAW42 cells through a novel Girdin-mediated Akt/IKKα/β/NF-κB signaling axis. (E,G) show the blot image of Girdin, p-Akt, p-IKKα/β, and p-Nf-κB determined by Western blot with GAPDH as the loading control. (F,H) represent the quantitative plots of expression of proteins. Western blot and quantitative analysis revealed that as the MBZ concentration increases, it reduces the expression of Girdin, p-Akt, p-IKKα/β, and p-Nf-κB substantially compared to DMSO (vehicle control (VC)) in both the cell lines. The bars represent the mean ± standard error of the mean of three independent experiments. Level of significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001, and ns: not significant) compared to DMSO (vehicle control (VC)), performed using a 2-way ANOVA test or t-test.