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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 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.
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pone-0104203-g003: 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.

Mentions: According to our genome-wide transcriptomic analysis and Q-PCR validation, we found that the DNA damage-related genes ERCC1, XPC, and CRY1 were up-regulated in hinokitiol-treated lung cancer cells. To further investigate whether hinokitiol can cause DNA damage, the levels of phosphorylated γ-H2AX and total and phosphorylated p53 were examined. Figure 3A shows that the levels of phosphorylated γ-H2AX were augmented after 48 h of hinokitiol treatment in H1975 cells, whereas total p53 was unchanged (Fig. 3A). The effect of hinokitiol on γ-H2AX phosphorylation was confirmed by immunostaining, which showed γ-H2AX protein accumulation in the nucleus of H1975 cells treated with hinokitiol (Fig. 3B), indicating that hinokitiol induced DNA damage in H1975 cells. Interestingly, hinokitiol did not induce DNA damage in human lung stromal fibroblasts (Fig. 3C), and this result correlated with the expression of genes related to DNA damage shown in Figure 2. To confirm whether hinokitiol-induced DNA damage occurred independent of p53, we treated p53- H1299 cells with hinokitiol and found that hinokitiol still induced DNA damage in these cells (Fig. 3D). Furthermore, we detected the major regulatory pathway of DNA damage response in the H1975 cells, such as the levels of phosphorylated and total ATM and SMC3. Additionally, we further detected the phosphorylated p53 to corroborate the DNA damage response is independent of p53 status evidenced by the phosphorylated or total p53 were unchanged by hinokitiol treatment (25 µM hinokitiol; Fig. 3E).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104203-g003: 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.
Mentions: According to our genome-wide transcriptomic analysis and Q-PCR validation, we found that the DNA damage-related genes ERCC1, XPC, and CRY1 were up-regulated in hinokitiol-treated lung cancer cells. To further investigate whether hinokitiol can cause DNA damage, the levels of phosphorylated γ-H2AX and total and phosphorylated p53 were examined. Figure 3A shows that the levels of phosphorylated γ-H2AX were augmented after 48 h of hinokitiol treatment in H1975 cells, whereas total p53 was unchanged (Fig. 3A). The effect of hinokitiol on γ-H2AX phosphorylation was confirmed by immunostaining, which showed γ-H2AX protein accumulation in the nucleus of H1975 cells treated with hinokitiol (Fig. 3B), indicating that hinokitiol induced DNA damage in H1975 cells. Interestingly, hinokitiol did not induce DNA damage in human lung stromal fibroblasts (Fig. 3C), and this result correlated with the expression of genes related to DNA damage shown in Figure 2. To confirm whether hinokitiol-induced DNA damage occurred independent of p53, we treated p53- H1299 cells with hinokitiol and found that hinokitiol still induced DNA damage in these cells (Fig. 3D). Furthermore, we detected the major regulatory pathway of DNA damage response in the H1975 cells, such as the levels of phosphorylated and total ATM and SMC3. Additionally, we further detected the phosphorylated p53 to corroborate the DNA damage response is independent of p53 status evidenced by the phosphorylated or total p53 were unchanged by hinokitiol treatment (25 µM hinokitiol; Fig. 3E).

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