Anticancer Effects and Molecular Mechanisms of Apigenin in Cervical Cancer Cells

Abstract

Cervical cancer is the fourth most frequent malignancy in women. Apigenin is a natural plant-derived flavonoid present in common fruit, vegetables, and herbs, and has been found to possess antioxidant and anti-inflammatory properties as a health-promoting agent. It also exhibits important anticancer effects in various cancers, but its effects are not widely accepted by clinical practitioners. The present study investigated the anticancer effects and molecular mechanisms of apigenin in cervical cancer in vitro and in vivo. HeLa and C33A cells were treated with different concentrations of apigenin. The effects of apigenin on cell viability, cell cycle distribution, migration potential, phosphorylation of PI3K/AKT, the integrin β1-FAK signaling pathway, and epithelial-to-mesenchymal transition (EMT)-related protein levels were investigated. Mechanisms identified from the in vitro study were further validated in a cervical tumor xenograft mouse model. Apigenin effectively inhibited the growth of cervical cancer cells and cervical tumors in xenograft mice. Furthermore, the apigenin down-regulated FAK signaling (FAK, paxillin, and integrin β1) and PI3K/AKT signaling (PI3K, AKT, and mTOR), inactivated or activated various signaling targets, such as Bcl-2, Bax, p21^cip1, CDK1, CDC25c, cyclin B1, fibronectin, N-cadherin, vimentin, laminin, and E-cadherin, promoted mitochondrial-mediated apoptosis, induced G2/M-phase cell cycle arrest, and reduced EMT to inhibit HeLa and C33A cancer cell migration, producing anticancer effects in cervical cancer. Thus, apigenin may act as a chemotherapeutic agent for cervical cancer treatment.

Keywords: EMT; PI3K/AKT; apigenin; apoptosis; cervical cancer; integrin β1-FAK.

Figure 1

Apigenin inhibits human cervical cancer cell viability and induces cell cycle arrest. HeLa (A) and C33A (B) cells were treated with increasing doses of apigenin for 24 h. Cell viability was determined using PrestoBlue™ cell viability reagent. HeLa (C) and C33A (D) cells were treated with or without 50 μM of apigenin for 24 h, and an estimation of the cell cycle phase distribution (G0/G1, S, and G2/M) was determined by PI staining via flow cytometry, followed by quantification. Data are presented as the mean ± SD of at least three independent experiments. * p < 0.05 indicates a significant difference as compared with the corresponding control. CON, 0.1% DMSO; API (50 μM), 50 μM apigenin.

Figure 2

Proposed mechanism and signaling pathways of the apoptosis and cell cycle arrest induced by apigenin in cervical cancer cells. Cell cycle G2/M phase-related proteins CDK1, CDC25C, cyclin B1, and p21, and apoptosis-related proteins, Bcl-2 and Bax, were detected in HeLa (A) and C33A (B) cells with or without 50 μM apigenin treatment for 24 h via Western blotting and quantified. Data are presented as the mean ± SD of at least three independent experiments. * p < 0.05 indicates a significant difference as compared with the corresponding control. CON, 0.1% DMSO; Api 50, 50 μM apigenin.

Figure 3

Effects of apigenin on the PI3K/AKT/mTOR signaling pathway in cervical cancer cells. Proteins p-PI3K (p85), PI3K (p85), p-AKT, AKT, p-mTOR, and mTOR were detected in HeLa (A) and C33A (B) cells with or without 50 μM apigenin treatment for 24 h via Western blotting and quantified. Data are presented as the mean ± SD of at least three independent experiments. * p < 0.05 indicates a significant difference as compared with the corresponding control. CON, 0.1% DMSO; Api 50, 50 μM apigenin.

Figure 4

Apigenin inhibits cancer cell migration and inactivates the integrin β1-FAK signaling pathway. Wound-healing assays were performed with or without 50–100 μM apigenin in HeLa (A) and C33A (B) cells for 0, 24, and 48 h. Left: representative images of scratches and recovery of wounded areas on cell monolayers at 0, 24, and 48 h after wounding. Right: semi-quantitative analysis of relative cell migration was performed according to the cells moving towards the scratched area at a certain time. Cell migration-related proteins p-FAK, paxillin, and integrin β1 were detected in HeLa (C) and C33A (D) cells with or without 50 μM apigenin treatment for 24 and 48 h via Western blotting and quantified. Data are presented as the mean ± SD of at least three independent experiments. * and † p < 0.05 indicate significant differences as compared with the corresponding control or Api 50-treated groups. CON, 0.1% DMSO; Api 50, 50 μM apigenin; Api 100, 100 μM apigenin; Api50 24 h, 50 μM apigenin at 24 h; Api50 48 h, 50 μM apigenin at 48 h.

Figure 4

Apigenin inhibits cancer cell migration and inactivates the integrin β1-FAK signaling pathway. Wound-healing assays were performed with or without 50–100 μM apigenin in HeLa (A) and C33A (B) cells for 0, 24, and 48 h. Left: representative images of scratches and recovery of wounded areas on cell monolayers at 0, 24, and 48 h after wounding. Right: semi-quantitative analysis of relative cell migration was performed according to the cells moving towards the scratched area at a certain time. Cell migration-related proteins p-FAK, paxillin, and integrin β1 were detected in HeLa (C) and C33A (D) cells with or without 50 μM apigenin treatment for 24 and 48 h via Western blotting and quantified. Data are presented as the mean ± SD of at least three independent experiments. * and † p < 0.05 indicate significant differences as compared with the corresponding control or Api 50-treated groups. CON, 0.1% DMSO; Api 50, 50 μM apigenin; Api 100, 100 μM apigenin; Api50 24 h, 50 μM apigenin at 24 h; Api50 48 h, 50 μM apigenin at 48 h.

Figure 5

Apigenin disrupts cancer cell metastasis and inhibits epithelial-to-mesenchymal transition. Proteins fibronectin, N-cadherin, vimentin, laminin, and E-cadherin were detected in HeLa (A) and C33A (B) cells with or without 50 μM apigenin treatment for 24 and 48 h via Western blotting and quantified. Data are presented as the mean ± SD of at least three independent experiments. * and † p < 0.05 indicate significant differences as compared with the corresponding control or Api 50-treated groups. CON, 0.1% DMSO; Api50 24 h, 50 μM apigenin at 24 h; Api50 48 h, 50 μM apigenin at 48 h.

Figure 6

Apigenin suppresses the growth of C33A xenograft tumors in vivo. C33A human cervical cancer cells (1 × 10⁷ cells) were implanted into the right flank of BALB/c nude mice. When the subcutaneous tumor volume reached ~200 mm³, mice were treated with the solvent control (10% DMSO) or apigenin (IP, 50 mg/kg/day) for 16 days. (A) Schematic representation of the experiment. (B) Representative image of a tumor, and average tumor volume and body weight. Tumor tissue samples were analyzed by hematoxylin, eosin staining (C), and immunohistochemistry (D,E) to examine the histopathology and expression levels of ki67, Bcl-2, cyclin B1, phospho-FAK, paxillin, integrin β1, fibronectin, N-cadherin, vimentin, laminin, and E-cadherin (shown as brown staining) (H&E, 400×, bar = 20 μm; IHC, 400×, bar = 20 μm). Values represent the mean ± SD (n = 6); * p < 0.01, *** p < 0.001 indicate significant differences as compared with the corresponding control. CON, control; API, apigenin.

Figure 6

Apigenin suppresses the growth of C33A xenograft tumors in vivo. C33A human cervical cancer cells (1 × 10⁷ cells) were implanted into the right flank of BALB/c nude mice. When the subcutaneous tumor volume reached ~200 mm³, mice were treated with the solvent control (10% DMSO) or apigenin (IP, 50 mg/kg/day) for 16 days. (A) Schematic representation of the experiment. (B) Representative image of a tumor, and average tumor volume and body weight. Tumor tissue samples were analyzed by hematoxylin, eosin staining (C), and immunohistochemistry (D,E) to examine the histopathology and expression levels of ki67, Bcl-2, cyclin B1, phospho-FAK, paxillin, integrin β1, fibronectin, N-cadherin, vimentin, laminin, and E-cadherin (shown as brown staining) (H&E, 400×, bar = 20 μm; IHC, 400×, bar = 20 μm). Values represent the mean ± SD (n = 6); * p < 0.01, *** p < 0.001 indicate significant differences as compared with the corresponding control. CON, control; API, apigenin.

Figure 6

Apigenin suppresses the growth of C33A xenograft tumors in vivo. C33A human cervical cancer cells (1 × 10⁷ cells) were implanted into the right flank of BALB/c nude mice. When the subcutaneous tumor volume reached ~200 mm³, mice were treated with the solvent control (10% DMSO) or apigenin (IP, 50 mg/kg/day) for 16 days. (A) Schematic representation of the experiment. (B) Representative image of a tumor, and average tumor volume and body weight. Tumor tissue samples were analyzed by hematoxylin, eosin staining (C), and immunohistochemistry (D,E) to examine the histopathology and expression levels of ki67, Bcl-2, cyclin B1, phospho-FAK, paxillin, integrin β1, fibronectin, N-cadherin, vimentin, laminin, and E-cadherin (shown as brown staining) (H&E, 400×, bar = 20 μm; IHC, 400×, bar = 20 μm). Values represent the mean ± SD (n = 6); * p < 0.01, *** p < 0.001 indicate significant differences as compared with the corresponding control. CON, control; API, apigenin.

Figure 7

Schematic representation of the anticancer molecular mechanism of apigenin in cervical cancer. Apigenin down-regulated FAK signaling (FAK, paxillin, and integrin β1) and PI3K/AKT signaling (PI3K, AKT, and mTOR), which inactivated or activated various signaling targets, such as Bcl2, Bax, p21^cip1, CDK1, CDC25c, cyclin B1, fibronectin, N-cadherin, vimentin, laminin, and E-cadherin, leading to mitochondrial-mediated apoptosis, G2/M-phase arrest, and reduced EMT to induce anticancer effects in cervical cancer.