Anticancer activity of caffeic acid n‑butyl ester against A431 skin carcinoma cell line occurs via induction of apoptosis and inhibition of the mTOR/PI3K/AKT signaling pathway

Abstract

Skin cancer is one of the primary causes of mortality worldwide. With an increasing frequency of skin cancers, there is an urgent requirement for the development of numerous treatment options. The present study investigated the anticancer activity of caffeic acid n‑butyl ester (CAE) against the A431 skin cancer cell line. Antiproliferative effects were investigated using an MMT assay. Apoptosis was examined by DAPI and Annexin V/fluorescein isothiocyanate and propidium iodide staining. Reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and cell cycle analyses were performed via flow cytometry. Protein expression was determined by western blotting. The findings of the present study demonstrated that among a variety of cancer cell lines, CAE exhibited significant anticancer activity against the A431 skin cancer cell line with a half‑maximal inhibitory concentration of 20 µM. CAE was associated with apoptosis and cell cycle arrest of A431 cells, and induced ROS‑mediated alterations in MMP. In addition, CAE considerably suppressed the expression of some of the important proteins of the phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) cascade. The results of the present study indicated that CAE exerted anticancer effects on the A431 skin carcinoma cell line via the induction of apoptosis and suppression of the PI3K/AKT/mTOR signaling pathway. Therefore, CAE may be beneficial for the development of chemotherapy for skin cancers.

Figure 1.

Effects of indicated concentrations of CAE on (A) cell viability and (B) colony formation. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.001, **P<0.001 and ***P<0.0001 vs. control. CAE, caffeic acid n-butyl ester.

Figure 2.

Induction of apoptosis by caffeic acid n-butyl ester at indicated concentrations as depicted by DAPI staining. All experiments were carried out in triplicate.

Figure 3.

Estimation of apoptotic cell populations at indicated concentrations of caffeic acid n-butyl ester by Annexin V/propidium iodide staining followed by flow cytometry. Experiments were carried out in triplicate.

Figure 4.

Effect of CAE on the protein expression of Bax and Bcl-2 was determined using western blot analysis. The experiments were carried out in triplicates. *P<0.001, **P<0.001 and ***P<0.0001 vs. control. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X; CAE, caffeic acid n-butyl ester.

Figure 5.

Effects of indicated concentration of CAE on (A) ROS generation and (B) MMP. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.001, **P<0.001 and ***P<0.0001 vs. control.MMP, mitochondrial membrane potential; ROS, reactive oxygen species. CAE, caffeic acid n-butyl ester.

Figure 6.

CAE triggers G2/M cell cycle arrest at indicated doses of CAE. Experiments were performed in triplicate. CAE, caffeic acid n-butyl ester.

Figure 7.

Effects of CAE on the migration of A431 cells at indicated concentrations. Experiments were performed in triplicate. *P<0.001, **P<0.001 and ***P<0.0001 vs. control.

Figure 8.

Western blot analysis demonstrated the effect of indicated concentrations of CAE on expression of mTOR/PI3K/AKT signaling pathway proteins. The experiments were performed in triplicates. *P<0.001, **P<0.001 and ***P<0.0001 vs. control. p, phosphorylated; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase.