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

Li LH, Wu P, Lee JY, Li PR, Hsieh WY, Ho CC, Ho CL, Chen WJ, Wang CC, Yen MY, Yang SM, Chen HW - PLoS ONE (2014)

Bottom Line: Here, we found that hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, exhibited potent anticancer effects.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan; Department of Laboratory, Kunming Branch, Taipei City Hospital, Taipei, Taiwan.

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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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.
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pone-0104203-g001: 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.

Mentions: Hinokitiol is the major active compound in the essential oil of Calocedrus formosana heart wood [16], and its chemical structure is shown in Figure 1A. To investigate the potential anticancer activity of hinokitiol on human lung adenocarcinoma cells, six different human lung adenocarcinoma cell lines with different EGFR status, A549 (EGFRwt), PC9 (EGFRdel19), H1299 (EGFRwt), H3255 (EGFRL858R), PC9-IR (EGFRdel19, with resistance to gefitinib) and H1975 (EGFRL858R+T790M, with resistance to gefitinib) cells, were treated with hinokitiol (5 and 10 µM) for 48 and 72 h. Then, cell proliferation was evaluated by directly counting cells after trypan blue staining. As shown in Table 2, hinokitiol inhibited the proliferation of all cells in a time- and concentration-dependent manner. Interestingly, the gefitinib-resistant cell lines, H1975 and PC9-IR, were inhibited by hinokitiol at a dose similar to that required for the gefitinib-sensitive cell lines PC9 and H3255. We further focused on the effects and underlying mechanisms of the action of hinokitiol on the gefitinib-resistant cells, H1975 and PC9-IR [17], [18]. We found that hinokitiol had IC50 values of 1.57 and 1.87 µM (72 h) in H1975 and PC9-IR cells, respectively (Fig. 1B). In addition, Figure 1C and 1D show that hinokitiol inhibited the colony formation ability of H1975 and PC9-IR cells in a concentration-dependent manner with an IC50 <1 µM. These results indicated that hinokitiol potently reduced the proliferation and colony formation potential of H1975 and PC9-IR cells.


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

Li LH, Wu P, Lee JY, Li PR, Hsieh WY, Ho CC, Ho CL, Chen WJ, Wang CC, Yen MY, Yang SM, Chen HW - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126702&req=5

pone-0104203-g001: 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.
Mentions: Hinokitiol is the major active compound in the essential oil of Calocedrus formosana heart wood [16], and its chemical structure is shown in Figure 1A. To investigate the potential anticancer activity of hinokitiol on human lung adenocarcinoma cells, six different human lung adenocarcinoma cell lines with different EGFR status, A549 (EGFRwt), PC9 (EGFRdel19), H1299 (EGFRwt), H3255 (EGFRL858R), PC9-IR (EGFRdel19, with resistance to gefitinib) and H1975 (EGFRL858R+T790M, with resistance to gefitinib) cells, were treated with hinokitiol (5 and 10 µM) for 48 and 72 h. Then, cell proliferation was evaluated by directly counting cells after trypan blue staining. As shown in Table 2, hinokitiol inhibited the proliferation of all cells in a time- and concentration-dependent manner. Interestingly, the gefitinib-resistant cell lines, H1975 and PC9-IR, were inhibited by hinokitiol at a dose similar to that required for the gefitinib-sensitive cell lines PC9 and H3255. We further focused on the effects and underlying mechanisms of the action of hinokitiol on the gefitinib-resistant cells, H1975 and PC9-IR [17], [18]. We found that hinokitiol had IC50 values of 1.57 and 1.87 µM (72 h) in H1975 and PC9-IR cells, respectively (Fig. 1B). In addition, Figure 1C and 1D show that hinokitiol inhibited the colony formation ability of H1975 and PC9-IR cells in a concentration-dependent manner with an IC50 <1 µM. These results indicated that hinokitiol potently reduced the proliferation and colony formation potential of H1975 and PC9-IR cells.

Bottom Line: Here, we found that hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, exhibited potent anticancer effects.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan; Department of Laboratory, Kunming Branch, Taipei City Hospital, Taipei, Taiwan.

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.

Show MeSH
Related in: MedlinePlus