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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 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.
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pone-0104203-g004: 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.

Mentions: To gain further insight into the mode of action by which hinokitiol limited cancer cell proliferation, the effect of hinokitiol on apoptosis was examined by flow cytometry with annexin V-FITC/PI staining in H1975 cells. We found that hinokitiol treatment for 72 h did not significantly affect the percentage of cells in early or late apoptosis (Fig. 4A). Hinokitiol also did not induce apoptosis in human stromal fibroblasts (Fig. 4A). In addition, hinokitiol treatment did not induce detectable PARP cleavage in H1975 cells or human stromal fibroblasts (Fig. 4B). These results prompted us to investigate whether hinokitiol induced autophagy in H1975 cells. We found that the expression of LC3-II, p62 and ATG5 proteins, which are markers of autophagosome formation [19], [20], increased after the hinokitiol treatment (Fig. 4C). Figure 4E provides additional evidence that hinokitiol induces cell autophagy, showing that 3-MA, an autophagy inhibitor, partially rescued the inhibition of cell growth induced by hinokitiol. In addition, we confirmed the autophagic response to hinokitiol by the analysis of the formation of AVOs. The flow cytometry analysis showed that the number of acidic vesicles in the H1975 cells slightly increased after hinokitiol exposure (Fig. 4D). Interestingly, hinokitiol did not induce significant levels of autophagy in human stromal fibroblasts (Fig. 4F), and this result correlated with the expression of genes related to autophagy shown in Figure 2.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104203-g004: 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.
Mentions: To gain further insight into the mode of action by which hinokitiol limited cancer cell proliferation, the effect of hinokitiol on apoptosis was examined by flow cytometry with annexin V-FITC/PI staining in H1975 cells. We found that hinokitiol treatment for 72 h did not significantly affect the percentage of cells in early or late apoptosis (Fig. 4A). Hinokitiol also did not induce apoptosis in human stromal fibroblasts (Fig. 4A). In addition, hinokitiol treatment did not induce detectable PARP cleavage in H1975 cells or human stromal fibroblasts (Fig. 4B). These results prompted us to investigate whether hinokitiol induced autophagy in H1975 cells. We found that the expression of LC3-II, p62 and ATG5 proteins, which are markers of autophagosome formation [19], [20], increased after the hinokitiol treatment (Fig. 4C). Figure 4E provides additional evidence that hinokitiol induces cell autophagy, showing that 3-MA, an autophagy inhibitor, partially rescued the inhibition of cell growth induced by hinokitiol. In addition, we confirmed the autophagic response to hinokitiol by the analysis of the formation of AVOs. The flow cytometry analysis showed that the number of acidic vesicles in the H1975 cells slightly increased after hinokitiol exposure (Fig. 4D). Interestingly, hinokitiol did not induce significant levels of autophagy in human stromal fibroblasts (Fig. 4F), and this result correlated with the expression of genes related to autophagy shown in Figure 2.

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