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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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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.
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pone-0104203-g006: 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.

Mentions: Taken together, our results showed that hinokitiol inhibited cell proliferation by inducing DNA damage, autophagy, and cell cycle arrest in lung adenocarcinoma cells but not in human lung stromal fibroblasts. Because apoptosis and autophagy were not observed in hinokitiol-treated fibroblasts, we sought to evaluate whether cellular senescence could be triggered by hinokitiol treatment. The effect of hinokitiol on cellular senescence was assessed through SA-β-Gal staining, and we found that hinokitiol treatment (5 µM, 72 h) induced cellular senescence in H1975 cells and, more significantly, in human lung stromal fibroblasts (Fig. 6A). Next, we further clarify whether autophagy induced senescence in the H1975 cells after hinokitiol treatment. Thus, we used the autophagy inhibitors 3-MA and chloroquine and transfected the cells with siRNA against ATG5 to evaluate the hinokitiol-induced senescence. In Figure 6B & C, hinokitiol-induced senescence was attenuated by cotreatment with 3-MA (2.5 mM), chloroquine (10 µM), and transfected with ATG5 siRNA plasmid (2 µg). Accordingly, we conclude that hinokitiol inhibited cell proliferation in normal and tumor cells through different mechanisms, including modulating cell autophagy, cell cycle regulation, the p53-independent DNA damage response, and senescence.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104203-g006: 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.
Mentions: Taken together, our results showed that hinokitiol inhibited cell proliferation by inducing DNA damage, autophagy, and cell cycle arrest in lung adenocarcinoma cells but not in human lung stromal fibroblasts. Because apoptosis and autophagy were not observed in hinokitiol-treated fibroblasts, we sought to evaluate whether cellular senescence could be triggered by hinokitiol treatment. The effect of hinokitiol on cellular senescence was assessed through SA-β-Gal staining, and we found that hinokitiol treatment (5 µM, 72 h) induced cellular senescence in H1975 cells and, more significantly, in human lung stromal fibroblasts (Fig. 6A). Next, we further clarify whether autophagy induced senescence in the H1975 cells after hinokitiol treatment. Thus, we used the autophagy inhibitors 3-MA and chloroquine and transfected the cells with siRNA against ATG5 to evaluate the hinokitiol-induced senescence. In Figure 6B & C, hinokitiol-induced senescence was attenuated by cotreatment with 3-MA (2.5 mM), chloroquine (10 µM), and transfected with ATG5 siRNA plasmid (2 µg). Accordingly, we conclude that hinokitiol inhibited cell proliferation in normal and tumor cells through different mechanisms, including modulating cell autophagy, cell cycle regulation, the p53-independent DNA damage response, and senescence.

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