Induction of Cell Cycle Arrest, Apoptosis, and Reducing the Expression of MCM Proteins in Human Lung Carcinoma A549 Cells by Cedrol, Isolated from Juniperus chinensis

Abstract

Proteins related to DNA replication have been proposed as cancer biomarkers and targets for anticancer agents. Among them, minichromosome maintenance (MCM) proteins, often overexpressed in various cancer cells, are recognized both as notable biomarkers for cancer diagnosis and as targets for cancer treatment. Here, we investigated the activity of cedrol, a single compound isolated from Juniperus chinensis, in reducing the expression of MCM proteins in human lung carcinoma A549 cells. Remarkably, cedrol also strongly inhibited the expression of all other MCM protein family members in A549 cells. Moreover, cedrol treatment reduced cell viability in A549 cells, accompanied by cell cycle arrest at the G1 phase, and enhanced apoptosis. Taken together, this study broadens our understanding of how cedrol executes its anticancer activity while demonstrating that cedrol has potential application in the treatment of human lung cancer as an inhibitor of MCM proteins.

Keywords: Apoptosis; Juniperus chinensis; cedrol; cell cycle arrest; lung carcinoma A549; minichromosome maintenance proteins.

Fig. 1. Isolation and identification of cedrol from J. chinensis.

(A) Extraction and fractionation procedure of J. chinensis to obtain cedrol. (B) GC-Mass, ¹H-NMR, and ¹³C-NMR spectra of cedrol. (C) Molecular structure of cedrol.

Fig. 2. Cedrol inhibits the expression of MCM proteins and cell proliferation in human lung carcinoma A549 cells.

(A) Western blot analysis was performed after treatment with cedrol at the indicated doses (i.e., 0, 5, 10, 15, 20, and 25 μg/ ml) for 48 h. Actin was used as an internal control. (B) After treatment with indicated doses of cedrol for 48 h, cell viability was measured using an EZ-Cytox Cell Viability Assay Kit and expressed as a percentage of control. Data are presented as mean ± SD of three independent experiments. *p < 0.05 vs. each control.

Fig. 3. Cedrol induces G1 arrest of the cell cycle in A549 cells.

(A) After treatment with indicated doses of cedrol for 48 h, cell cycle profiles were evaluated using a Muse Cell Cycle Kit with a Muse Cell Analyzer. (B) Percentages of cell populations in G0/G1, S, and G2/M phases are calculated for each dose of cedrol. Data are presented as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 vs. each control. (C) Western blot analysis was performed after treatment with indicated doses of cedrol for 48 h to determine whether the expression of cell cycle-related proteins was altered by cedrol treatment. Actin was used as an internal control.

Fig. 4. Cedrol induces apoptosis in A549 cells.

(A) After treatment with indicated doses of cedrol for 48 h, cells were collected and stained with annexin V and 7-AAD. Induction of apoptosis by cedrol treatment was measured using a Muse Annexin V and Dead Cell Assay Kit with a Muse Cell Analyzer. (B) Percentages of early and late apoptotic cells were then calculated for each dose of cedrol. Data are presented as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 vs. each control. (C) Effects of cedrol on the expression of apoptosis-related proteins were confirmed by western blot analysis after treatment with indicated doses of cedrol for 48 h. Actin was used as an internal control.

Fig. 5. Proposed molecular mechanism of cedrol through G1 arrest and apoptosis association with MCM downregulation in A549 cells.

Cedrol induces G1 arrest through the upregulation of p53 and p21, and downregulation of cyclin, CDK, and phosphorylated pRb. At the same time, cedrol induces apoptosis through the regulation of Bcl-2 family proteins and activation of caspase. Induction of G1 arrest and apoptosis by cedrol may be linked by its strong MCM inhibitory activity in A549 cells.