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. 2014 Aug 8;9(8):e104203.
doi: 10.1371/journal.pone.0104203. eCollection 2014.

Hinokitiol induces DNA damage and autophagy followed by cell cycle arrest and senescence in gefitinib-resistant lung adenocarcinoma cells

Lan-Hui Li  1 , Ping Wu  2 , Jen-Yi Lee  2 , Pei-Rong Li  2 , Wan-Yu Hsieh  2 , Chao-Chi Ho  3 , Chen-Lung Ho  4 , Wan-Jiun Chen  5 , Chien-Chun Wang  6 , Muh-Yong Yen  7 , Shun-Min Yang  8 , Huei-Wen Chen  2
Affiliations

Hinokitiol induces DNA damage and autophagy followed by cell cycle arrest and senescence in gefitinib-resistant lung adenocarcinoma cells

Lan-Hui Li et al. PLoS One. .

Abstract

Despite good initial responses, drug resistance and disease recurrence remain major issues for lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutations taking EGFR-tyrosine kinase inhibitors (TKI). To discover new strategies to overcome this issue, we investigated 40 essential oils from plants indigenous to Taiwan as alternative treatments for a wide range of illnesses. Here, we found that hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, exhibited potent anticancer effects. In this study, we demonstrated that hinokitiol inhibited the proliferation and colony formation ability of lung adenocarcinoma cells as well as the EGFR-TKI-resistant lines PC9-IR and H1975. Transcriptomic analysis and pathway prediction algorithms indicated that the main implicated pathways included DNA damage, autophagy, and cell cycle. Further investigations confirmed that in lung cancer cells, hinokitiol inhibited cell proliferation by inducing the p53-independent DNA damage response, autophagy (not apoptosis), S-phase cell cycle arrest, and senescence. Furthermore, hinokitiol inhibited the growth of xenograft tumors in association with DNA damage and autophagy but exhibited fewer effects on lung stromal fibroblasts. In summary, we demonstrated novel mechanisms by which hinokitiol, an essential oil extract, acted as a promising anticancer agent to overcome EGFR-TKI resistance in lung cancer cells via inducing DNA damage, autophagy, cell cycle arrest, and senescence in vitro and in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The effects of hinokitiol on cell proliferation.
(A) The chemical structure of hinokitiol. (B) The effect of a 72-h hinokitiol treatment on H1975 and PC9-IR cell proliferation, as assayed through trypan blue staining. (C) The effect of hinokitiol on the colony formation ability of H1975 cells. (D) The effect of hinokitiol on the colony formation ability of PC9-IR cells. In (B), (C), and (D), the results are representative of three different experiments and are expressed as the mean ± SD and as % of control. *, **, and *** indicate a significant difference at the level of p<0.05, p<0.01, and p<0.001, respectively.
Figure 2
Figure 2. The effects of hinokitiol on gene expression.
(A) Microarray profiling of H1975 cells and PC9-IR cells treated with 5 µM hinokitiol for 48 h. (B) Q-PCR array validation of the expression of genes related to DNA damage and autophagy in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment for 24 h. The results are representative of those obtained in three different experiments and are expressed as the fold change compared with control. * and ** indicate a significant difference at the level of p<0.05 and p<0.01, respectively.
Figure 3
Figure 3. The effects of hinokitiol on the expression of DNA damage regulatory proteins.
(A) The effect of hinokitiol (5 µM) or cisplatin (25 µM) on the level of γ-H2AX phosphorylation and total p53 expression in H1975 cells, as assayed using western blots. (B) Assessment of hinokitiol-induced DNA damage in H1975 cells through an immunofluorescence γ-H2AX focus assay. (C) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation and total p53 expression in lung stromal fibroblasts, as assayed using western blots. (D) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation in H1299 cells. (E) The effect of hinokitiol (25 µM) or cisplatin (CDDP, 25 µM) on the phosphorylation and total level of ATM, SMC3, and p53 in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control.
Figure 4
Figure 4. The effects of hinokitiol on apoptosis and autophagy.
(A) Apoptosis was assessed using an annexin-V/PI binding assay in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment. Western blot analysis of PARP in H1975 cells and lung stromal fibroblasts (B), LC3, p62 and ATG5 expression in (C) H1975 cells and (F) lung stromal fibroblasts. The treatment of 100 nM rapamycin for 48 h was used as a positive control for LC3 expression. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (D) The formation of AVOs was quantified by flow-cytometry after acridine orange staining in H1975 cells treated with 5 µM hinokitiol for 8 h. (E) H1975 cells were pretreated with 2.5 mM of 3-MA for 1 h, followed by exposure to 5 µM hinokitiol for 48 h. Cell proliferation was analyzed through a trypan blue staining assay. The results are representative of three different experiments and are expressed as the mean ± SD. ** indicates a significant difference at the level of p<0.01.
Figure 5
Figure 5. The effect of hinokitiol on cell cycle distribution.
H1975 cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A), (B) and (C), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p<0.05, p<0.01 and <0.001, respectively. In (F), the histogram shows the quantification expressed as the mean ± SD of ratio in 5-10 fields per coverslip. * indicates significant differences at the level of p<0.05.
Figure 6
Figure 6. Hinokitiol induced cellular senescence in H1975 cells and lung stromal fibroblasts.
(A) The senescent cells were quantified at 200× magnification under a standard light microscope. (B) Hinokitiol induced cellular senescence was attenuated by autophagy inhibitors in H1975 cells. (C) Hinokitiol induced cellular senescence was attenuated by transfection of siRNA against ATG5 in H1975 cells. Corresponding protein expression was detected by western blot. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. In (A), (B) and (C), each value is the mean ± SD of 3-5 fields of three different experiments. * and ** indicate a significant difference at the level of p<0.05 and p<0.01, respectively.
Figure 7
Figure 7. In vivo antitumor activity of hinokitiol.
(A) The growth curves of subcutaneous xenografts of H1975 are shown. (B) The excised tumors were weighed and imaged. All results are given as the mean ± SD; n = 5 - 7 for each group. *indicates a significant difference at the level of p<0.05 compared with the control group. (C) Hematoxylin and eosin-stained tumor sections at days 14 or 21 from each group were analyzed. Arrow heads indicate the atypical nuclei or abnormal mitosis. Immunohistochemically stained tumor sections at days 14 or 21 from each group were analyzed to assess γ-H2AX and LC3 expression (D). The atypical nuclei, abnormal mitosis, and positive cells were quantified at 400× magnification under a standard light microscope (Olympus BX51, Japan). Each value is the mean ± SD of 5–10 fields of triplicate tumor sections. *, ** and *** indicate a significant difference compared with its' own control at the level of p<0.05, p<0.01, and p<0.001, respectively.
Figure 8
Figure 8. A schematic representation of the hypothetical mechanisms for the role of hinokitiol in suppressing lung adenocarcinomas.
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Grants and funding

This work was supported by the grants from Ministry of Science and Technology (102-2325-B-002-046-) and National Taiwan University Cutting-Edge Steering Research Project (NTU CESRP-10R71602C2 and 100R705057). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.