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. 2012:2012:818261.
doi: 10.1155/2012/818261. Epub 2011 Jun 20.

Terpinen-4-ol Induces Apoptosis in Human Nonsmall Cell Lung Cancer In Vitro and In Vivo

Chieh-Shan Wu  1 , Yun-Ju Chen, Jeremy J W Chen, Jeng-Jer Shieh, Chia-Hsin Huang, Pei-Shan Lin, Gee-Chen Chang, Jinghua-Tsai Chang, Chi-Chen Lin
Affiliations

Terpinen-4-ol Induces Apoptosis in Human Nonsmall Cell Lung Cancer In Vitro and In Vivo

Chieh-Shan Wu et al. Evid Based Complement Alternat Med. 2012.

Abstract

Terpinen-4-ol, a monoterpene component of the essential oils of several aromatic plants, exhibits antitumor effects. In this study, the antitumor effects of terpinen-4-ol and the cellular and molecular mechanisms responsible for it were evaluated and studied, respectively on human nonsmall cell lung cancer (NSCLC) cells. Our results indicated that terpinen-4-ol elicited a dose-dependent cytotoxic effect, as determined by MTT assay. Increased sub-G1 population and annexin-V binding, activation of caspases 9 and 3, cleavage of poly(ADPribose) polymerase (PARP), and a decrease of mitochondrial membrane potential (MMP) indicated involvement of the mitochondrial apoptotic pathway in terpinen-4-ol-treated A549 and CL1-0 cells. Elevation of the Bax/Bcl-2 ratio and a decrease in IAP family proteins XIAP and survivin were also observed following terpinen-4-ol treatment. Notably, terpinen-4-ol was able to increase p53 levels in A549 and CL1-0 cells. Diminution of p53 by RNA interference induced necrosis instead of apoptosis in A549 cells following terpinen-4-ol treatment, indicating that terpinen-4-ol-elicited apoptosis is p53-dependent. Moreover, intratumoral administration of terpinen-4-ol significantly suppressed the growth of s.c. A549 xenografts by inducing apoptosis, as confirmed by TUNEL assay. Collectively, these data provide insight into the molecular mechanisms underlying terpinen-4-ol-induced apoptosis in NSCLC cells, rendering this compound a potential anticancer drug for NSCLC.

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Figures

Figure 1
Figure 1
Effects of terpinen-4-ol on viability and morphology of A549 and CL1-0 cells. (a) A549 and CL1-0 cells were seeded at 5 × 104 cells/well and then treated with the indicated concentrations of terpinen-4-ol for 24 hours following attachment. The cell viability was determined by the MTT assay. Each point on the graph represents the mean ± SD of triplicate tests. *P value <.05 compared with the untreated control group. (b) The morphology of A549 and CL1-0 cells treated with various concentrations of terpinen-4-ol for 24 hours were observed by phase-contrast microscopy and photographed (400×).
Figure 2
Figure 2
Effects of terpinen-4-ol on cell-cycle distribution in A549 and CL1-0 cells. (a) Cell-cycle analysis of terpinen-4-ol-treated cells. Cells were treated with the indicated concentrations of terpinen-4-ol for 24 hours, and then, cell-cycle distributions were determined by propidium iodide (PI) staining and subsequent flow cytometry analysis (Gating for each cell-cycle phase M1: <2N; M2: G0-G1; M3: S; M4: G2-M). Data are representative of three independent experiments with similar results. (b) Flow cytometry analysis of terpinen-4-ol-induced apoptosis in A549 and CL1-0 cells. The cells were treated with the indicated concentrations of terpinen-4-ol for 24 hours, followed by labeling for phosphatidylserine externalization with FITC-annexin-V and cell membrane integrity with PI. The lower right quadrant (annexin-V+/PI-) represents early apoptosis, while the upper right quadrant (annexin V+/PI+) represents late apoptosis and necrosis. Data are representative of three independent experiments with similar results.
Figure 3
Figure 3
Effects of terpinen-4-ol on caspase activation in A549 and CL1-0 cells. (a) Cells were treated with the indicated concentrations of terpinen-4-ol for 24 hours. Total cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies to detect the cleaved forms of caspase-8, caspase-9, caspase-3, and PARP. Data are representative of three independent experiments with similar results. (b) Cells were treated with terpinen-4-ol and/or the indicated caspase inhibitor for 24 hours, and cell viability was determined using the MTT assay. Data are mean ± SD of three independent experiments.
Figure 4
Figure 4
Effects of terpinen-4-ol on mitochondrial membrane potential and cytochrome c release in A549 and CL1-0 cells. (a) The cells were stained with JC-1 fluorescence dye and the change in mitochondrial membrane potential (ΔΨm) was examined by flow cytometry. Data are representative of three independent experiments with similar results. (b) Cytosolic lysates were prepared and subjected to SDS-PAGE followed by Western blotting with anticytochrome c antibody. Data are representative of three independent experiments showing similar results.
Figure 5
Figure 5
Effects of terpinen-4-ol on the expression of Bcl-2 family proteins and IAPs in A549 and CL1-0 cells. Cells were treated with the indicated concentrations of terpinen-4-ol for 24 hours. Total cell lysates were prepared, subjected to SDS-PAGE, and immunoblotted with antibodies to detect Bcl-2, Bcl-xl, Bax, XIAP, and survivin. Data are representative of three independent experiments with similar results.
Figure 6
Figure 6
Effect of terpinen-4-ol on cell viability, caspase 3 activation, and PARP cleavage in A549 and p53-silenced A549 cells. (a) A549 and p53-silenced A549 cells were seeded at 5 × 104 cells/well and then treated with the indicated concentrations of terpinen-4-ol for 24 hours following attachment. Cell viability was determined using the MTT assay. Each point on the graph represents the mean ± SD of triplicate tests. *P value <.05 compared with the untreated control group. (b) A549 and p53-silenced A549 cells were treated with the indicated concentrations of terpinen-4-ol for 24 hours. Total cell lysates were prepared, resolved by SDS-PAGE and immunoblotted with the indicated antibodies to detect the cleaved forms of caspase-3 and PARP. Data are representative of three independent experiments with similar results. (c) A549 and p53-silenced A549 cells were treated with 0 or 0.06% terpinen-4-ol for 6 hours and subjected to flow cytometry analysis after staining with annexin V-FITC and propidium iodide (PI). The lower right quadrant (annexin V+/PI−) represents early apoptosis, and the upper right quadrant (annexin V+/PI+) represents late apoptosis and necrosis. Data are a representative of three independent experiments showing similar results.
Figure 7
Figure 7
Effects of terpinen-4-ol on tumor growth in vivo. (a) 1 × 107 A549 cells were s.c. injected into the right flank of BALB/c nu/nu mice (n = 5), followed by terpinen-4-ol therapy, as described in the Materials and Methods. The mean tumor volume was measured every three days from the onset of terpinen-4-ol treatment and plotted. Experiments were repeated two times and provided similar results. (b) TUNEL labeling of apoptotic DNA fragmentation in A549 tumor sections from PBS-treated control animals and animals treated with terpinen-4-ol. Apoptotic cells were identified as dark brown nuclei using light microscopy. The number of apoptotic cells was counted under high magnification (400×) in five randomly chosen fields for each sample. The data are presented as the mean ± SD of groups of three samples pooled from two independent experiments.
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