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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 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.
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pone-0104203-g005: 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.

Mentions: We observed that hinokitiol reduced the proliferation of cancer cells, but this was not due to cytotoxicity (Fig. 4A & B). As such, we examined the effect of hinokitiol treatment on the cell cycle distribution of H1975 cells and found that the ratio of cells in S phase significantly increased after hinokitiol treatment. Concomitantly, the percentage of cells in the G1 phase was reduced compared with control cells. This result indicated that hinokitiol induced the accumulation of cancer cells in the S phase of the cell cycle (Fig. 5A). Interestingly, this effect on cell cycle distribution was not significantly observed in human lung stromal fibroblasts treated with hinokitiol (Fig. 5B). Furthermore, we used the BrdU flow assay to corroborate the S-phase arrest data in response to hinokitiol exposure in H1975 cells. In Fig. 5C, the percentage of BrdU-negative cells in S-phase was higher in the hinokitiol exposure group; whereas the newly incorporated BrdU-labeled cells in S-phase were lower in H1975 cells. Moreover, both cancer and stromal fibroblasts in the sub-G1 phase were unaffected by the treatment with hinokitiol (Fig. 5A & B); these results were associated with the lack of apoptosis in H1975 cells, as shown in Fig. 4A & B. To investigate the underlying mechanism by which hinokitiol treatment induced cell-cycle arrest at S phase, we examined the key regulators during cell cycle progression. We found that the protein levels of cyclin D1, p21, cyclin A2, and cyclin B1 were down-regulated and that the levels of cyclin E2 were 1.9 times up-regulated in response to a 72-h treatment with hinokitiol compared with control (Fig. 5D). In addition, we found that the phosphorylation levels of EGFR and ERK, the up-stream signaling regulators of cyclin D1 [21], were significantly reduced after long-term treatment with hinokitiol (5 µM hinokitiol, 72 h; Fig. 5E). The nuclear staining in H1975 cells revealed that the proportion of abnormal mitosis was reduced after 5 µM hinokitiol exposure for 72 h (Fig. 5F).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104203-g005: 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.
Mentions: We observed that hinokitiol reduced the proliferation of cancer cells, but this was not due to cytotoxicity (Fig. 4A & B). As such, we examined the effect of hinokitiol treatment on the cell cycle distribution of H1975 cells and found that the ratio of cells in S phase significantly increased after hinokitiol treatment. Concomitantly, the percentage of cells in the G1 phase was reduced compared with control cells. This result indicated that hinokitiol induced the accumulation of cancer cells in the S phase of the cell cycle (Fig. 5A). Interestingly, this effect on cell cycle distribution was not significantly observed in human lung stromal fibroblasts treated with hinokitiol (Fig. 5B). Furthermore, we used the BrdU flow assay to corroborate the S-phase arrest data in response to hinokitiol exposure in H1975 cells. In Fig. 5C, the percentage of BrdU-negative cells in S-phase was higher in the hinokitiol exposure group; whereas the newly incorporated BrdU-labeled cells in S-phase were lower in H1975 cells. Moreover, both cancer and stromal fibroblasts in the sub-G1 phase were unaffected by the treatment with hinokitiol (Fig. 5A & B); these results were associated with the lack of apoptosis in H1975 cells, as shown in Fig. 4A & B. To investigate the underlying mechanism by which hinokitiol treatment induced cell-cycle arrest at S phase, we examined the key regulators during cell cycle progression. We found that the protein levels of cyclin D1, p21, cyclin A2, and cyclin B1 were down-regulated and that the levels of cyclin E2 were 1.9 times up-regulated in response to a 72-h treatment with hinokitiol compared with control (Fig. 5D). In addition, we found that the phosphorylation levels of EGFR and ERK, the up-stream signaling regulators of cyclin D1 [21], were significantly reduced after long-term treatment with hinokitiol (5 µM hinokitiol, 72 h; Fig. 5E). The nuclear staining in H1975 cells revealed that the proportion of abnormal mitosis was reduced after 5 µM hinokitiol exposure for 72 h (Fig. 5F).

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