Different Cell Responses to Hinokitiol Treatment Result in Senescence or Apoptosis in Human Osteosarcoma Cell Lines

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.


Introduction
Osteosarcoma is the most common type of primary malignant bone tumor, mainly identifying in adolescents and young adults. Surgical resection is the major treatment for osteosarcoma, but survival rate of the patients treated with surgery alone is lower than 20%. In the 1970s, effective chemotherapeutic agents, including adriamycin, cisplatin, epirubicin, and methotrexate, were applied as adjuvant treatments to facilitate surgical

Hinokitiol Reduces Viability and Proliferation of Osteosarcoma Cell Lines
The cytotoxic effect of hinokitiol on human osteosarcoma U-2 OS and MG-63 cells was determined by MTT assay. The data showed that the treatment with hinokitiol decreased the viability of U-2 OS or MG-63 cells at concentration of 10 to 80 µM after treatment for 48 h, and U-2 OS cells were more sensitive to this drug ( Figure 1A). The half maximal inhibitory concentration (IC50) for U-2 OS cells were 44 µM at 24 h and 25 µM at 48 h, and those for MG-63 cells were 36 µM at 48 h. Furthermore, a colony formation assay was performed to demonstrate the influence of hinokitiol on the proliferative potential of a single cell. The data showed that hinokitiol exposure reduced colony formation ability in a dose-dependent manner ( Figure 1B). Overall, the data indicate that hinokitiol exposure decreases viability and proliferation of osteosarcoma cell lines. A dosage range of 20 to 80 µM was chosen for the current study. These doses were less cytotoxic in human lung fibroblast MRC-5 cells ( Figure 1C) and other normal cells [22]. The IC50 for MRC-5 was 72 µM at 48 h.

Hinokitiol Reduces Viability and Proliferation of Osteosarcoma Cell Lines
The cytotoxic effect of hinokitiol on human osteosarcoma U-2 OS and MG-63 cells was determined by MTT assay. The data showed that the treatment with hinokitiol decreased the viability of U-2 OS or MG-63 cells at concentration of 10 to 80 μM after treatment for 48 h, and U-2 OS cells were more sensitive to this drug ( Figure 1A). The half maximal inhibitory concentration (IC50) for U-2 OS cells were 44 μM at 24 h and 25 μM at 48 h, and those for MG-63 cells were 36 μM at 48 h. Furthermore, a colony formation assay was performed to demonstrate the influence of hinokitiol on the proliferative potential of a single cell. The data showed that hinokitiol exposure reduced colony formation ability in a dose-dependent manner ( Figure 1B). Overall, the data indicate that hinokitiol exposure decreases viability and proliferation of osteosarcoma cell lines. A dosage range of 20 to 80 μM was chosen for the current study. These doses were less cytotoxic in human lung fibroblast MRC-5 cells ( Figure 1C) and other normal cells [22]. The IC50 for MRC-5 was 72 μM at 48 h. (B) Hinokitiol inhibits colony formation of osteosarcoma cell line. After administration of hinokitiol for 6 h, U-2 OS cells were washed by PBS, trypsinized, and then five thousand cells were cultured onto 35 mm dishes with drug-free complete medium for 10 days to allow colony formation. Colonies were stained by 1% crystal violet solution before counting. Quantitative data of colony number/dish were estimated as below. Data are expressed as mean ± SD of three independent experiments. (C) The effect of hinokitiol on human lung fibroblast MRC-5 cells. MRC-5 cells were incubated with different doses of hinokitiol for 24 or 48 h. MTT assays were performed to measure cell viability. * p < 0.001, and ** p = 0.001. (B) Hinokitiol inhibits colony formation of osteosarcoma cell line. After administration of hinokitiol for 6 h, U-2 OS cells were washed by PBS, trypsinized, and then five thousand cells were cultured onto 35 mm dishes with drug-free complete medium for 10 days to allow colony formation. Colonies were stained by 1% crystal violet solution before counting. Quantitative data of colony number/dish were estimated as below. Data are expressed as mean ± SD of three independent experiments. (C) The effect of hinokitiol on human lung fibroblast MRC-5 cells. MRC-5 cells were incubated with different doses of hinokitiol for 24 or 48 h. MTT assays were performed to measure cell viability. * p < 0.001, and ** p = 0.001.

Hinokitiol Induces S-Phase Arrest of Cell Cycle and DNA Damage Response
To demonstrate whether an antiproliferative effect of hinokitiol in osteosarcoma cell lines was associated with abnormal cell-cycle progression, cell-cycle analysis was performed by flow cytometry. The data showed that the proportion of cells in the S phase was significantly increased after hinokitiol exposure. At the same time, the proportion of cells in the G1 phase was decreased compared with untreated group. The results indicate that hinokitiol exposure resulted in an increased proportion of S-phase cells in U-2 OS and MG-63 cells (Figure 2A,B). Furthermore, we performed an Edu incorporation experiment to verify the effect of hinokitiol on cell-cycle arrest. As shown in Figure 2C, the newly incorporated Edu-labeled cells were largely down-regulated in hinokitiol-exposed cells ( Figure 2C). Overall, our data indicate that hinokitiol exposure induces a cell-cycle arrest at S phase in U-2 OS and MG-63 cells.
MG-63 cells (Figure 2A,B). Furthermore, we performed an Edu incorporation experiment to verify the effect of hinokitiol on cell-cycle arrest. As shown in Figure 2C, the newly incorporated Edu-labeled cells were largely down-regulated in hinokitiol-exposed cells ( Figure 2C). Overall, our data indicate that hinokitiol exposure induces a cell-cycle arrest at S phase in U-2 OS and MG-63 cells.
A DNA damage response has been shown to have an effect on inhibition of cell proliferation and S-phase arrest of the cell cycle [18]. To assess the mechanism involved in hinokitiol-induced S phase arrest in U-2 OS and MG-63 cell lines, we investigated the expression of DNA damage response-related proteins by Western blotting. The data revealed that hinokitiol exposure resulted in rapid phosphorylation of ATM (at serine 1981), ATR (at ser 428), Chk1 (at serine 345), Chk2 (at threonine 68), and γ-H2AX ( Figure 3A). In addition, we found that γ-H2AX formed distinct nuclear foci after hinokitiol treatment in both cell lines ( Figure 3B). These data suggest that the DNA damage response may contribute to hinokitiol-induced S phase arrest of the cell cycle in U-2 OS and MG-63 cells.  A DNA damage response has been shown to have an effect on inhibition of cell proliferation and S-phase arrest of the cell cycle [18]. To assess the mechanism involved in hinokitiol-induced S phase arrest in U-2 OS and MG-63 cell lines, we investigated the expression of DNA damage response-related proteins by Western blotting. The data revealed that hinokitiol exposure resulted in rapid phosphorylation of ATM (at serine 1981), ATR (at ser 428), Chk1 (at serine 345), Chk2 (at threonine 68), and γ-H2AX ( Figure 3A). In addition, we found that γ-H2AX formed distinct nuclear foci after hinokitiol treatment in both cell lines ( Figure 3B). These data suggest that the DNA damage response may contribute to hinokitiol-induced S phase arrest of the cell cycle in U-2 OS and MG-63 cells.

Hinokitiol Induces Senescence in U-2 OS Cells
As U-2 OS cells were exposed to hinokitiol, we observed a flattened and e morphologies in the treated cells. These characteristics were similar to the senescen notype. To evaluate the effect of hinokitiol exposure on senescence, the staining o cence-associated β-galactosidase (SA-β-gal) was performed in treated U-2 OS ce data showed that the treated-U-2 OS cells were strongly stained blue compared w treated cells, suggesting that hinokitiol treatment could trigger cellular senescenc OS cells ( Figure 4A). Furthermore, we found that the tumor suppressor p53 and its downstream ge could be activated in hinokitiol-exposed U-2 OS cells ( Figure 4B). To examine the p53 in hinokitiol-induced senescence, the stable Tet-on U2OS-shp53 line [15], wh sesses a Tet operator-driven short hairpin RNA (shRNA) that targets p53, was The results showed, in the shp53-expressing cells (with tetracycline), that the prote of p53 was efficiently attenuated in hinokitiol-treated U-2 OS cells. Once p53 wa

Hinokitiol Induces Senescence in U-2 OS Cells
As U-2 OS cells were exposed to hinokitiol, we observed a flattened and enlarged morphologies in the treated cells. These characteristics were similar to the senescence phenotype. To evaluate the effect of hinokitiol exposure on senescence, the staining of senescence-associated β-galactosidase (SA-β-gal) was performed in treated U-2 OS cells. The data showed that the treated-U-2 OS cells were strongly stained blue compared with untreated cells, suggesting that hinokitiol treatment could trigger cellular senescence in U-2 OS cells ( Figure 4A).
We also assessed the influence of hinokitiol on apoptosis in U-2 OS cells by staining with Annexin V and propidium iodide (PI). The data shows that there were a few increases in Annexin V-positive cells as U-2 OS cells were incubated with hinokitiol (40 or 80 μm) for 48 h ( Figure 4C). Overall, the results indicate that hinokitiol exposure triggers a p53-dependent senescence in U-2 OS cells.

Hinokitiol Triggers Apoptosis in MG-63 Cells
As MG-63 cells were exposed to hinokitiol, we also observed a flattened morphology in the treated cells, with some round cells in higher dose (80 μm) ( Figure 5A). To analyze whether there was the same senescent response that occurred in U-2 OS cells, the staining of SA-β-gal was performed in treated MG-63 cells. As shown in Figure 5A, the stained blue color in cells was weaker, and the percentage of the stained cells was not greatly increased compared to untreated cells ( Figure 5A). Based on this finding, we tested the Furthermore, we found that the tumor suppressor p53 and its downstream gene, p21, could be activated in hinokitiol-exposed U-2 OS cells ( Figure 4B). To examine the roles of p53 in hinokitiol-induced senescence, the stable Tet-on U2OS-shp53 line [15], which possesses a Tet operator-driven short hairpin RNA (shRNA) that targets p53, was applied. The results showed, in the shp53-expressing cells (with tetracycline), that the protein level of p53 was efficiently attenuated in hinokitiol-treated U-2 OS cells. Once p53 was down-regulated, the level of p21 and the senescence triggered by hinokitiol exposure were significantly decreased ( Figure 4A,B). In addition, the level of the senescence marker, plasminogen activator inhibitor-1 (PAI-1) [23], was increased in hinokitiol-treated U-2 OS cells as well, and may be regulated by p53 in these cells ( Figure 4B).
We also assessed the influence of hinokitiol on apoptosis in U-2 OS cells by staining with Annexin V and propidium iodide (PI). The data shows that there were a few increases in Annexin V-positive cells as U-2 OS cells were incubated with hinokitiol (40 or 80 µm) for 48 h ( Figure 4C). Overall, the results indicate that hinokitiol exposure triggers a p53dependent senescence in U-2 OS cells.

Hinokitiol Triggers Apoptosis in MG-63 Cells
As MG-63 cells were exposed to hinokitiol, we also observed a flattened morphology in the treated cells, with some round cells in higher dose (80 µm) ( Figure 5A). To analyze whether there was the same senescent response that occurred in U-2 OS cells, the staining of SA-β-gal was performed in treated MG-63 cells. As shown in Figure 5A, the stained blue color in cells was weaker, and the percentage of the stained cells was not greatly increased compared to untreated cells ( Figure 5A). Based on this finding, we tested the effect of hinokitiol on apoptosis in MG-63 cells. The data showed that hinokitiol exposure induced expression of cleaved PARP and caspase 3, and effectively increased the ratio of

The Suppression of Autophagy Enhances Hinokitiol-Induced Response
We examined whether autophagy was involved in hinokitiol-induced response in osteosarcoma cell lines. The results revealed that hinokitiol exposure up-regulated expression of LC3-II and enhanced LC3 puncta formation in U-2 OS and MG-63 cells ( Figures  6A,B and 7A,B), indicating autophagy is induced in both cell lines after treatment. Furthermore, as autophagy was suppressed by chloroquine in U-2 OS cells, SA-β-gal positive cells ( Figure 6C, D) and p53 expression ( Figure 6E) were increased, indicating suppression of autophagy enhances p53-dependent senescence in hinokitiol-exposed U-2 OS cells. In addition, co-treatment of chloroquine in MG-63 cells increased the levels of cleaved PARP and caspase 3 ( Figure 7C), and decreased cell viability ( Figure 7D), indicating that atten-

The Suppression of Autophagy Enhances Hinokitiol-Induced Response
We examined whether autophagy was involved in hinokitiol-induced response in osteosarcoma cell lines. The results revealed that hinokitiol exposure up-regulated expression of LC3-II and enhanced LC3 puncta formation in U-2 OS and MG-63 cells ( Figures 6A,B  and 7A,B), indicating autophagy is induced in both cell lines after treatment. Furthermore, as autophagy was suppressed by chloroquine in U-2 OS cells, SA-β-gal positive cells ( Figure 6C,D) and p53 expression ( Figure 6E) were increased, indicating suppression of autophagy enhances p53-dependent senescence in hinokitiol-exposed U-2 OS cells. In addition, co-treatment of chloroquine in MG-63 cells increased the levels of cleaved PARP and caspase 3 ( Figure 7C), and decreased cell viability ( Figure 7D), indicating that attenuation of autophagy up-regulates apoptosis in hinokitiol-exposed MG-63 cells.

Discussion
Cellular senescence could be induced primarily through p53 and p16 pathways. The literature has shown that hinokitiol treatment induces senescence in lung adenocarcinoma H1975 cells. Although induced γ-H2AX was observed after hinokitiol treatment for 24 or 48 h, p53 was not activated in H1975 cells that possessed a wild type p53 [24]. In our report, hinokitiol exposure resulted in rapid phosphorylation of ATM (at serine 1981), ATR (at ser 428), Chk1 (at serine 345), Chk2 (at threonine 68), and γ-H2AX, and induced expressions of p53 and p21 in U-2 OS cells. Knockdown of p53 could suppress hinokitiol-induced senescence in U-2 OS cells. It was suggested that the mechanisms involved in regulating hinokitiol-induced senescence may be different between the two cell lines.
DNA damage response (DDR) starts with the recognition of DNA lesions by sensor proteins. This action recruits and activates the most upstream DDR kinases, and thus turns on a cellular signaling cascade to arrest the cell cycle, activate DNA repair, and to remove cells with unrepairable genomes. ATM (ataxia-telangiectasia mutated), ATR (ATMand Rad3-Related), and DNA-PKcs (DNA-dependent protein kinase) kinases are the most upstream DDR kinases. ATM and DNA-PKcs are activated by double-stranded DNA breaks, whereas ATR is activated by a broad spectrum of genotoxic stresses that induces single-stranded DNA (ssDNA) [25,26]. Our data indicated that hinokitiol exposure resulted in rapid phosphorylation of ATM and ATR. However, it was uncertain how hinokitiol induced DDR, and what kinds of DNA lesions were induced.
Autophagy is a lysosome-dependent self-digestive program. It deals with damaged or useless proteins, organelles, or other cytoplasmic components to restore energy balance. The role of autophagy in regulating cancer cell death or survival remains divergent, and may depend on cellular contents [27]. The literature shows that suppression of autophagy inhibits hinokitiol-induced senescence in lung adenocarcinoma H1975 cells [24]. Suppression of autophagy increased cell viability that inhibited by hinokitiol treatment in murine breast and colorectal cancer cell lines [28] and human hepatocellular carcinoma cell lines [8].
In this study, we found that autophagy was activated as a protective mechanism to mediate hinokitiol-induced responses in osteosarcoma cell lines. Suppression of autophagy up-regulated hinokitiol-induced senescence in U-2 OS cells, and enhanced apoptosis in hinokitiol-treated MG-63 cells.
The initial demonstration of cell senescence considers senescence as an important mechanism of tumor suppression. Senescence could arrest the proliferation of cancer cells and facilitate the clearance of affected cells through immunosurveillance. Recent studies show most senescent cells secrete diverse inflammatory cytokines, chemokines and proteases, which are referred to as the senescent associated secretory phenotype (SASP). The SASP factors could either stimulate or suppress tumor growth and progression depending on their exact compositions. The compositions are determined by different cell types and drug treatment, and may change with time [13,29]. Our data showed that hinokitiol exposure inhibited growth of osteosarcoma cell lines through senescence or apoptosis. However, the limitation of this study is that all results were from in vitro experiments. Recent studies have shown anticancer activity of hinokitiol against different tumors in a mouse model. In these studies, the effective dose was ranged from 2 mg to 100 mg/kg/day in mice, and the data shows that hinokitiol is relatively nontoxic to the animals even at higher doses [8,11,24]. Whether hinokitiol could inhibit the growth of osteosarcoma and what is effective dose in in vivo remains to be demonstrated. In addition, that literature has shown anti-inflammatory properties of hinokitiol in MG-63 cells [30]. Inflammation plays a critical role in tumor progression, and anti-inflammation has been considered a good strategy for cancer therapy [31]. Whether this property is involved in anticancer activity of hinokitiol against osteosarcoma should be further addressed in an in vivo study.
In conclusion, our data showed that hinokitiol exposure suppressed cell viability, and induced S-phase arrest of the cell cycle and the DNA damage response in osteosarcoma cell lines. p53-dependent senescence was observed in osteosarcoma U-2 OS cells, and apoptotic cells were found in osteosarcoma MG-63 cells that possess mutated p53 in response to hinokitiol treatment. Suppression of autophagy largely enhanced senescence in U-2 OS cells and apoptosis in MG-63 cells (Figure 8). We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors may produce more specific and effective therapies in hinokitiol-related trials for treating osteosarcoma. and induced S-phase arrest of the cell cycle and the DNA damage response in osteosarcoma cell lines. p53-dependent senescence was observed in osteosarcoma U-2 OS cells, and apoptotic cells were found in osteosarcoma MG-63 cells that possess mutated p53 in response to hinokitiol treatment. Suppression of autophagy largely enhanced senescence in U-2 OS cells and apoptosis in MG-63 cells (Figure 8). We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors may produce more specific and effective therapies in hinokitiol-related trials for treating osteosarcoma.

Cell Culture and Reagents
Human osteosarcoma U-2 OS, MG-63 cells, and human lung fibroblast MRC-5 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). U-2 OS cells were cultured in McCoy's 5A medium, and MG-63 cells and MRC-5 were maintained in Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin/streptomycin, and maintained at 37 °C in a humidified atmosphere of 5% CO2. The passage 97th to 112nd of MG-63 cells, and the passage 28th to 33rd of MCR-5 were used in this study. The stable Tet-on U-2 OS-shp53 cell line was generated

Cell Culture and Reagents
Human osteosarcoma U-2 OS, MG-63 cells, and human lung fibroblast MRC-5 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). U-2 OS cells were cultured in McCoy's 5A medium, and MG-63 cells and MRC-5 were maintained in Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin/streptomycin, and maintained at 37 • C in a humidified atmosphere of 5% CO 2 . The passage 97th to 112nd of MG-63 cells, and the passage 28th to 33rd of MCR-5 were used in this study. The stable Tet-on U-2 OS-shp53 cell line was generated as previously described [18]. Hinokitiol were purchased from Cayman Chemical (Ann Arbor, MI, USA), and the purity was greater than 98%.

Colony Formation Assay
U-2 OS cells (2 × 10 5 cells/dish) were seeded in 35 mm dish for overnight and then incubated with different doses of hinokitiol for 6 h. The treated cells were washed by PBS, trypsinized, and then five thousand cells were cultured onto 35 mm dishes with drug-free complete medium for 10 days to allow colony formation. Colonies were stained by 1% crystal violet solution before counting.

Flow Cytometry Analysis
U-2 OS (2 × 10 5 cells/dish) and MG-63 cells (1.5 × 10 5 cells/dish) were seeded in 35 mm dish for overnight and then exposed to different doses of hinokitiol for 24, 48, or 72 h. The treated cells were collected to analyze DNA content. The details are described in previous report [32].

Apoptosis Assay
U-2 OS (2 × 10 5 cells/well) and MG-63 cells (1.5 × 10 5 cells/well) were seeded in 35 mm dish for overnight, and then exposed to different doses of hinokitiol for 48 h. Cell apoptosis was determined by flow cytometry using the Annexin-V-FITC staining kit (Becton Dickinson, San Jose, CA, USA) according to the manufacturer's instructions as described in a previous report [32].

EdU Incorporation Assay
Edu (5-ethynyl-20-deoxyuridine) was applied to detect newly DNA synthesis. The cells were seeded on glass coverslips with poly-l-lysine coating. The cells were incubated with hinokitiol (40 µM) for 48 h, and then incubated with EdU (10 µM) for 30 min. The cells were harvested and analyzed by Click-iT EdU imaging kit (Invitrog en, Carlsbad, CA, USA) according to the manufacturer's protocol.

Statistical Analysis
All experiments were repeated at least three times independently. Statistical significance was assessed by GraphPad Prism 4 (GraphPad Software; San Diego, CA, USA) using the Student's t-test. p values of 0.05 were considered significant.