Methoxylated Cinnamic Esters with Antiproliferative and Antimetastatic Effects on Human Lung Adenocarcinoma Cells

Abstract

Lung cancer is the leading cause of cancer mortality worldwide, and malignant melanomas are highly lethal owing to their elevated metastatic potential. Despite improvements in therapeutic approaches, cancer treatments are not completely effective. Thus, new drug candidates are continuously sought. We synthesized mono- and di-methoxylated cinnamic acid esters and investigated their antitumor potential. A cell viability assay was performed to identify promising substances against A549 (non-small-cell lung cancer) and SK-MEL-147 (melanoma) cells. (E)-2,5-dimethoxybenzyl 3-(4-methoxyphenyl)acrylate (4m), a monomethoxylated cinnamic acid derivative, was identified as the lead antitumor compound, and its antitumor potential was deeply investigated. Various approaches were employed to investigate the antiproliferative (clonogenic assay and cell cycle analysis), proapoptotic (annexin V assay), and antimigratory (wound-healing and adhesion assays) activities of 4m on A549 cells. In addition, western blotting was performed to explore its mechanism of action. We demonstrated that 4m inhibits the proliferation of A549 by promoting cyclin B downregulation and cell cycle arrest at G2/M. Antimigratory and proapoptotic activities of 4m on A549 were also observed. The antitumor potential of 4m involved its ability to modulate the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway once phosphorylated-ERK expression was considerably reduced in response to treatment. Our findings demonstrate that 4m is a promising anticancer drug candidate.

Keywords: antiproliferative activity; monomethoxylated cinnamic acid derivatives; non-small-cell lung cancer.

Figure 1

Structures of cinnamic acid and its derivatives found in nature.

Scheme 1

Preparation of cinnamic ester derivatives 4a–4o and 5a–5o.

Scheme 2

Formation of cinnamides 4p and 5p from cinnamic acids 1 and 2.

Figure 2

Monomethoxylated compound 4m is cytotoxic to lung adenocarcinoma A549 cells but not to primary dermal fibroblast. (A) Cell viability was determined by sulforhodamine B (SRB) after 48 h of treatment; 1 and 2 cinnamic acid. *** p < 0.001 compared to negative control DMSO according to ANOVA followed by Dunnet post-test (B) Illustrative images of the cell cultures at 48 h treatment with 40 μM 4m (60× manignification). (C) IC₅₀ (µM) values were determined from viability assay data. Cell cultures were treated with 4m or cisplatin for 48 h. Growth curves determined by SRB assay after 48 h of treatment with compound 4m against A549 cells, SK-MEL-147 cells, and dermal fibroblast cells. DMSO (0.1% v/v) was used as the negative control and cisplatin as the positive control. Non-tumor cell line: dermal fibroblast (CCD-1059Sk). ND: not determined once cell viability was not sufficiently reduced to determine IC₅₀ values. (D) Clonogenic assay of A549 cells treated with 20 or 40 µM 4m for 24 h and recovered in fresh medium for additional 12 days. *** p < 0.001 compared to control group according to ANOVA followed by Dunnet post-test.

Figure 3

Compound 4m induces cell cycle arrest and apoptosis in A549 cells. (A) Illustrative histograms showing cell populations distributed in different phases of the cell cycle after 48 h of treatment with 4m. Brown, pink, light green, blue, and dark green bars represent, respectively, sub-G1, G0/G1, S, G2/M, and hypertretraploid populations (DNA content higher than 4C). (B) Cell cycle analysis. ** p < 0.01 and *** p < 0.001 compared to control group according to ANOVA followed by Dunnet post-test. The hypertetraploid population was included in the analysis. (C) Relative protein levels of Cyclin B assessed by immunoblot in A549 cells treated for 24 and 48 h with 40 µM 4m. α-tubulin was used as a loading control. ** p < 0.01 and *** p < 0.001 compared to control groups according to Student’s t-test. (D,E) Representative dot plots and analysis of annexin V/7-AAD assays performed in A549 cultures after 48 h treatment with 4m. Viable cells (lower left quadrants), early apoptosis (lower right quadrants), late apoptosis (upper right quadrants), and necrotic cells (upper left quadrants). *** p < 0.001 compared to control group according to ANOVA followed by Dunnet post-test. (F) pERK expression determined by immunoblot in A549 cells treated for 24 or 48 h with 40 µM 4m. ERK-1 was used as a loading control. ** p < 0.01 compared to control group according to Student’s t-test.

Figure 4

Compound 4m reduces the metastatic behavior of A549 cells. (A) Photomicrography represents the wound taken at 0 and 36 h (methanol for 30 min and crystal violet (1%) for 20 min) after treatment with 5, 10, and 20 μM 4m (40× magnification). (B) The bar graphs represent migrative cell number that was treated with 5, 10 and 20 μM of compound 4m after 18 and 36 h. (C) Photomicrography represents the cells invasion through matrigel coating (100× magnification). (D) The bar graphs represent invasive cell number that was treated with 5, 10, and 20 μM 4m after 24 h. (E) Photomicrography represents the cells adhered on matrigel coating (40× magnification). (F) The bar graph represents the adhered cell number that was treated with with 5, 10, and 20 μM 4m after 6 h. Statistical significance was determined by ANOVA and Dunnet post-test, ** p ˂ 0.01 and *** p ˂ 0.001 compared to control groups.