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Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this stud...
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Hinokitiol Inhibits Cell Growth through Induction of S‑Phase Arrest and Apoptosis in Human Colon Cancer Cells and Suppresses Tumor Growth in a Mouse Xenograft Experiment Youn-Sun Lee,† Kyeong-Mi Choi,‡ Wonkyun Kim,‡ Young-Soo Jeon,‡ Yong-Moon Lee,‡ Jin-Tae Hong,‡ Yeo-Pyo Yun,‡ and Hwan-Soo Yoo*,‡ †

Department of Biology Education, College of Education, Chungbuk National University, Cheongju 361-763, Korea College of Pharmacy and Center for Innovative Cancer Therapeutics, Chungbuk National University, Cheongju 361-763, Korea



ABSTRACT: Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this study, the inhibitory effects of 1 were investigated on human colon cancer cell growth and tumor formation of xenograft mice. HCT-116 and SW-620 cells derived from human colon cancers were found to be similarly susceptible to 1, with IC50 values of 4.5 and 4.4 μM, respectively. Compound 1 induced S-phase arrest in the cell cycle progression and decreased the expression levels of cyclin A, cyclin E, and Cdk2. Conversely, 1 increased the expression of p21, a Cdk inhibitor. Compound 1 decreased Bcl-2 expression and increased the expression of Bax, and cleaved caspase-9 and -3. The effect of 1 on tumor formation when administered orally was evaluated in male BALB/c-nude mice implanted intradermally separately with HCT-116 and SW-620 cells. Tumor volumes and tumor weights in the mice treated with 1 (100 mg/kg) were decreased in both cases. These results suggest that the suppression of tumor formation by compound 1 in human colon cancer may occur through cell cycle arrest and apoptosis.

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proteins.13 Expression of p21 is mediated by p53-dependent and by p53-independent mechanisms and is essential for DNAdamage-induced cell cycle arrest.14−16 p21 can also suppress the expression of antiapoptotic genes such as Bcl-2 and Bcl-XL.17,18 Bax protein controls cell death through activation of caspase-9 and -3.19 Hinokitiol is a tropolone-based phenolic component of the essential oil of the Japanese cypress [Chamaecyparis obtusa Siebold & Zucc. (Cupressaceae)].20 Hinokitiol (1) used in this study was isolated from Thujopsis dolabrata Siebold & Zucc. var. hondai Makino (Cupressaceae). Compound 1 is known also as β-thujaplicin and has the chemical structure 2-hydroxy4-isopropylcyclohepta-2,4,6-trien-1-one. Hinokitiol induces apoptosis and has potential anticancer activity, having been reported to induce apoptosis of teratocarcinoma F9 cells through the activation of caspase-3.21 This compound suppresses cell growth through disruption of androgen receptor-mediated signaling in LNCaP cells.22 The mechanism of action of 1 involves accumulation of p27, downregulation of Skp2, and impairment of Cdk2 function in FEM human melanoma cells.23 However, there has been no previous study regarding the inhibition of 1 on the growth of colon cancer cells. Reduced

olon cancer is the third most common cancer in the world and has a higher prevalence in Western countries than elsewhere, with diet considered to be the most important factor leading to its incidence.1 Chemotherapeutic agents for colon cancer have limited efficacy and often produce unpleasant side effects. Combination therapy protocols, such as FOLFIRI (folinic acid, fluorouracil, and irinotecan) and FOLFOX (folinic acid, fluorouracil, and oxaliplatin), are used currently for treating colon cancer.2−4 Recently, several newer drugs for colon cancer, including bevacizumab, cetuximab, and panitumumab, have been developed.5,6 These drugs are monoclonal antibodies specific for the epidermal growth factor receptor, which signals through the Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt pathways.7 Other drugs for inhibiting intracellular signaling transduction molecules, such as mTOR and MAPK, have also been developed.8,9 Cell cycle progression is regulated cooperatively by cyclin/ cyclin-dependent kinase (Cdk) complexes. Cyclin E/Cdk2 and cyclin A/Cdk2 complexes play key roles in the initiation and progression of the S-phase, respectively.10 S-phase arrest is associated with activation of the Cdk2-cyclin E/cyclin A and caspase family in ovarian cancer cells.11 A CdK inhibitor, p21, is known to be mediated in the regulation of cellular events such as cell cycle arrest, senescence, and apoptosis.12 The functional activity of p21 is regulated through phosphorylation and dephosphorylation of the Cdk © 2013 American Chemical Society and American Society of Pharmacognosy

Received: July 4, 2013 Published: December 5, 2013 2195

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cells treated with 10 μM was inhibited by about 63%. Viability slopes of these two types of colon cancer cells indicated that HCT-116 cells appeared to be more sensitive than SW-620 cells between 0.1 and 2 μM concentrations of 1. However, CCD-112CoN cells were not sensitive to 1 up to 10 μM. The IC50 values of 1 for the cell growth of HCT-116 and SW-620 cells were 4.5 and 4.4 μM, respectively. HCT-116 cells treated with 10 μM 1 for 24 h inhibited cell viability by approximately 27% compared to that of CCD-112CoN cells, and the maximal inhibition of cell growth occurred at 48 h and was maintained until 72 h (Figure 1B). SW-620 cells also showed a similar pattern of growth inhibition to that of HCT-116 cells. Other studies have shown that 1 at concentrations of 10− 100, 20−80, and 60 μM suppressed cell growth in human prostate carcinoma LNCaP cells, FEM human melanoma cells, and teratocarcinoma F9 cells, respectively.21−23 Inhibition of cell growth by 1 in colon cancer cells appears to be much more sensitive than that in other cancer cell types, suggesting that hinokitiol may be a specific candidate for the treatment of colon cancer. Hinokitiol (1) Induces S-Phase Cell Cycle Arrest and Inhibits the Expression of S-Phase Regulatory Proteins. The effect of 1 on the various phases of cell cycle progression was determined, and its mechanism elucidated. Accumulation of human colon cancer cells at the S-phase occurred in a concentration-dependent manner after addition of 0−10 μM 1 (Figures 2A and B). The percentages of 1-treated HCT-116 cells at 0, 5, and 10 μM in the S-phase were 36.3 ± 1.7%, 43.5

rates of apoptosis in colorectal mucosa have been reported to be associated closely with a high risk of adenomas and tumorigenesis.24 Thus, apoptosis appears to be an effective strategy for screening chemotherapeutic agents for colon cancer. In this study, the inhibitory effect of 1 was investigated on human colon cancer cell growth and its mechanism of action probed.



RESULTS AND DISCUSSION Hinokitiol (1) Inhibits Cell Growth of Human Colon Cancer Cells. To determine whether 1 inhibits human colon cancer cell growth, the sensitivity of HCT-116 and SW-620 cells to increasing concentrations of 1 using a cell counting assay was examined. Compound 1 inhibited the growth of HCT-116 and SW-620 cells in a concentration- and timedependent manner (Figures 1A and B). Significant inhibition of HCT-116 cell viability from CCD-112CoN cells, a normal human colon cell line, began to occur at 0.5 μM 1 after 48 h, and the maximal inhibition was approximately 58% at 10 μM (Figure 1A). In comparison, the cell death of SW-620 cells occurred with 2 μM 1 after 48 h, and the cell growth of SW-620

Figure 1. Inhibitory effects of hinokitiol (1) on cell viability in human colon cancer cells. (A) CCD-112CoN, HCT-116, and SW-620 cells were cultured in six-well plates to near confluence and treated with varying concentrations of 1 (0−10 μM) for 48 h. (B) Cells were treated with 10 μM 1 for 0−72 h. The cells were then trypsinized, and the numbers were counted using a hemocytometer. At 48 h after treatment with 1, the morphological changes of the cells were observed under a phase-contrast microscope (magnification 200×). Data are expressed by the percentage of control cells as means ± SD of three experiments conducted in triplicate (*p < 0.05 and ***p < 0.001 vs control group). 2196

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Figure 3. Effect of hinokitiol (1) on expression of cell cycle regulatory proteins. The human colon cancer cells were exposed to 1 at 0−10 μM for 12−24 h. (A) HCT-116 and (B) SW-620 cells were then lysed, and Cdk2, cyclin E, cyclin A, β-actin, and (C) p21 were analyzed using 10−12.5% SDS-PAGE and immunoblotting.

phase arrest in the cell cycle may occur through inhibiting the expression of Cdk2, cyclin A, and cyclin E. The factor p21 plays a critical role in the regulation of the cell cycle in cancer cells. There are several reports that upregulation of p21 in human cancer cells inhibits the expression of cell cycle regulatory proteins such as cyclin A, cyclin D, cyclin E, Cdk2, Cdk4, and Cdk6.25 Cladosporol A reduced the expression of cyclin D1, cyclin E, Cdk2, and Cdk4 by upregulation of p21 in human colon carcinoma HT-29 cells.26 Honokiol, a biphenyl plant lignan, was found to downregulate the expression of cyclin D1, cyclin D2, Cdk2, Cdk4, and Cdk6 by activation of p21 in human epidermoid A431 cells.27 2,6-Bis(2-chloroacetamido)anthraquinone induces cell cycle arrest through the downregulation of cyclin D1 and the upregulation of p21 in lung adenocarcinoma A549 cells.28 To clarify the mechanism of 1-induced cell cycle arrest, the expression of p21 was determined by immunoblotting. As shown in Figure 3C, treatment of HCT-116 and SW-620 cells with 0−10 μM 1 for 24 h exhibited elevation of p21 expression (Figure 3C). Therefore, cell cycle arrest by 1 in human colon cancer cells was not p53-dependent. Since the p53 gene is deleted in SW-620 cells, it is most likely that the induction of p21 is mediated through a p53-independent pathway. However, the expression of p27 and p57, and other Cdk inhibitors, was not changed after exposure to hinokitiol (data not shown). Taken together, these results indicate that cell cycle arrest by 1 in HCT-116 and SW-620 cells may occur through the activation of p21 expression. Hinokitiol (1) Causes Apoptosis through the Expression of Apoptotic Proteins. To assess whether cell cycle arrest resulted in the induction of apoptosis, flow cytometric analysis was used (Figure 4). HCT-116 and SW-

Figure 2. Effect of hinokitiol (1) on colon cancer cell cycle progression. (A) HCT-116 and (B) SW-620 cells were treated with 1 at 0−10 μM for 48 h and harvested by trypsinization. The cells were stained with PI solution, and 10000 events per experiment were analyzed using a flow cytometer. The cell cycle progression was determined using the Modifit LT program. Data are expressed as means ± SD of three different experiments conducted in triplicate (*p < 0.05, **p < 0.01, and ***p < 0.001 vs control group).

± 1.4%, and 53.8 ± 1.2%, respectively. The percentages of the S-phase in SW-620 cells treated with 0, 5, and 10 μM 1 were 43.1 ± 1.0%, 51.9 ± 0.8%, and 87.0 ± 0.6%, respectively. These results demonstrate that 1 induces S-phase arrest in cell cycle progression in human colon cancer cells. The effect of 1 on cell cycle-regulatory proteins specific for the S-phase was then determined (Figure 3). Treatment of HCT-116 and SW-620 cells with 1 at 5 and 10 μM for 24 h showed decreased expression of Cdk2, cyclin E, and cyclin A in a concentration-dependent manner compared to control cells (Figure 3A,B). Thus, these results indicated that 1-induced S2197

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Compound 1 increased the expression of cleaved caspase-9 and -3 significantly in a concentration-dependent manner (Figures 5A and B). The expression of anti- and proapoptotic proteins can be modulated by p21.14 Hinokitiol (1) induced apoptosis through the activation of cleaved caspase-3 in teratocarcinoma F9 cells.21 It was found that 1 decreased Bcl-2 expression and increased the expression of Bax and cleaved caspase-9 and -3 in human colon cancer HCT-116 and SW-620 cells. Expression of Bax and Bcl-2 in apoptosis induction is associated with the regulation of p21 signaling.17,18 Hinokitiol-induced apoptosis in HCT-116 and SW-620 human colon cancer cells occurred following the elevation of the Bax/Bcl-2 expression ratio through the increased expression of p21. Knockdown of p21 Reverses the Inhibition of Cell Growth and the Apoptotic Signaling Pathway by Hinokitiol (1). To elucidate whether p21 signaling may regulate 1-induced apoptosis in HCT-116 and SW-620 cells, its effect of p21 knockdown was investigated on cell growth inhibition of these colon cancer cells. p21-specific siRNA was used to confirm that p21 may play a critical role in cell death. HCT-116 and SW-620 cells were transfected with either scrambled siRNA or p21 siRNA. Inhibition of cell growth by 1 was effectively attenuated in the cells transfected with p21 siRNA, and morphological observation showed the increased confluency (Figures 6A and B). Thus, knockdown of p21 appears to reverse 1-induced cell growth inhibition as well as apoptotic cell death. p21 siRNA transfection abolished the 1induced increase in the expression of cleaved caspase-9 and -3 (Figure 6C). These results demonstrated that inhibition of cell growth as well as apoptosis by 1 occurs through the activation of the p21 signaling pathway. Most human cancers result from the inactivation of p21 tumor suppressor gene.17 It has been well established that p21 is involved in the inhibition of cancer cell growth through regulation of cell cycle arrest or apoptosis when the cells are exposed to chemical stress.28 Increased expression of p21 by therapeutic agents has been observed in prostate, ovarian, and breast cancer and esophageal squamous cell carcinoma.29−32 An effective strategy for cancer treatment is to activate p21 signaling and then either induce apoptosis or inhibit cancer cell growth. In this study, p21-dependent cell growth inhibition and apoptosis were induced by hinokitiol (1) in colon carcinoma HCT-116 and SW-620 cells. In addition, knockdown of p21 attenuated both hinokitiol-induced cell growth inhibition and apoptotic cell death. Proteins named death receptors (DR), the upstream signaling of p21, are classified as FasL, tumor necrosis factor receptor (TNFR)-1, and TNF-related apoptosis-inducing ligand (TRAIL) receptor.33 The DR are coupled to caspase cascades essential for the induction of apoptosis.34 Thus, the possible molecular target of hinokitiol (1) in human colon cancer cells may be either p21 or the DR signaling pathway. Hinokitiol (1) Inhibits Tumor Formation in Xenograft Mice. The effect of 1 on tumor formation was determined in xenografted mice inoculated with HCT-116 and SW-620 cells. Compound 1 was administered orally to the mice every other day starting from day 2 to day 21. Tumor growth in mice treated with 1 (100 mg/kg, n = 10) was compared to that in the control group (n = 10). Compound 1 had a significant inhibitory effect on tumor growth in xenografted mice (Figure 7A). Tumor volume and weight were significantly decreased by oral administration of 1

Figure 4. Apoptosis induction of hinokitiol (1) in human colon cancer cells. (A) HCT-116 and (B) SW-620 cells were treated with 1 at 0−10 μM for 48 h and then double-stained with Annexin V-FITC and PI. Samples were analyzed by flow cytometry using excitation/emission wavelengths of 488/525 and 488/675 nm for Annexin V-FITC and PI, respectively. Data are expressed as means ± SD of three different experiments conducted in triplicate (***p < 0.001 vs control group).

620 cells were treated with varying concentrations of 1 (0−10 μM) for 48 h, and a significant increase in the number of apoptotic cells was observed at 5 and 10 μM 1. The percentages of apoptosis in 0, 5, and 10 μM 1-treated HCT-116 cells were 7.6 ± 3.0%, 15.6 ± 2.6%, and 24.7 ± 3.2%, respectively (Figure 4A). The percentages of apoptosis in 0, 5, and 10 μM 1exposed SW-620 cells were 3.8 ± 2.1%, 53.1 ± 0.8%, and 80.3 ± 0.7%, respectively (Figure 4B). Under the same culture condition, S-phase arrest of cell cycle progression in the colon cancer cells occurred at 5 and 10 μM of 1 (Figure 2). Bcl-2 and Bax play a central role in apoptosis. HCT-116 and SW-620 cells were treated with 1 at 0−10 μM for 24 h, and the expression of Bcl-2 and Bax was measured (Figure 5). Expression of the antiapoptotic protein Bcl-2 was decreased significantly by treatment with hinokitiol. In contrast, 1 increased the expression of the proapoptotic protein Bax in a concentration-dependent manner. The increased expression ratio of Bax/Bcl-2 led to activation of caspase signaling. 2198

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Figure 5. Effect of hinokitiol (1) on expression of apoptotic proteins in HCT-116 and SW-620 cells. Human colon cancer cells were treated with various concentrations of 1 (0−10 μM) for 24 h. (A) HCT-116 and (B) SW-620 cells were then lysed, and Bcl-2, Bax, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, and β-actin were analyzed using 15% SDS-PAGE and immunoblotting. After densitometric quantification, data are expressed as means ± SD of three different experiments conducted in duplicate (**p < 0.01 and ***p < 0.001 vs control group or treated group). from Cell Signaling Technology (Beverly, MA, USA). Other reagents were of the highest purity available. Cell Culture. Human colon cancer HCT-116 and SW-620 cells and the non-neoplastic human colon cell line CCD-112CoN were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine in a CO2 incubator at 37 °C. Compound 1 was dissolved in ethanol and made up to a maximum final concentration of 0.01% (v/v) in the culture medium. Cell Viability Assay. Cells were seeded into six-well culture plates at 105 cells per well and were grown in RPMI 1640 containing 10% FBS at 37 °C. Subconfluent cells were subsequently treated with 1, either at 0, 0.1, 0.5, 1, 2, 5, and 10 μM for 48 h or at 10 μM for 24, 48, and 72 h. The cells were harvested by trypsin−EDTA treatment, and then the cell numbers were counted using a hemocytometer. Cell Cycle Analysis. Cell cycle progression was determined by flow cytometric analysis, in which subconfluent cells were treated with 1 at 0, 0.1, 0.5, 1, 2, 5, and 10 μM for 48 h. A suspension of cells with a density of 105 cells per well was fixed overnight with 70% ethanol at 4 °C and then incubated overnight with PI staining reagent. The population of cells was analyzed using FACS Calibur, and cell cycle progression was determined with the Modifit LT program (Verity Software House; Topsham, ME, USA). Annexin-V and PI Double Staining Assay. Annexin-V and PI labeling for the detection of apoptotic or necrotic cell death was performed using an Annexin-V-Flous staining kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). Briefly, HCT-116 and SW-620 cells were seeded in six-well plates and grown in RPMI 1640 medium containing 10% FBS at 37 °C. Subconfluent cells were treated with 1 at 0, 0.1, 0.5, 1, 2, 5, and 10 μM for 48 h. The

compared to the control (Figures 7B and C). Compound 1 reduced the tumor volume in HCT-116 and SW-620 xenograft mice at day 21 (Figure 7B). The mean weights of the tumors in HCT-116 and SW-620 xenograft mice treated with 1 were 245 and 252 mg, respectively, while those of the corresponding control mice were approximately 469 and 417 mg (Figure 7C). Differences in the body weights of the control vs the groups treated with 1 were significant at day 21, possibly due to the formation of tumor tissues (Figure 7D). These results supported that hinokitiol (1) suppressed tumor formation by inhibiting colon cancer cell growth. In conclusion, the present study has demonstrated that hinokitiol (1) suppressed tumor growth in human colon cancer through cell cycle arrest and apoptosis in a p21-mediated upregulation, suggesting that this compound is worthy of further investigation as a potential colon cancer treatment agent.



EXPERIMENTAL SECTION

General Experimental Procedures. Hinokitiol (1) was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and exhibited 99% purity when analyzed by GC. Propidium iodide (PI) was obtained from Becton-Dickinson Co. (San Diego, CA, USA), and RPMI 1640 and OPTI-MEM media were purchased from Gibco-BRL (Grand Island, NY, USA). Primary antibodies against cyclin A, cyclin E, Cdk2, Bax, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and p21 antibodies were obtained from Upstate Biotechnology (Lake Placid, NY, USA). Bcl-2, caspase-9, cleaved caspase-9, caspase-3, and cleaved caspase-3 were purchased 2199

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Figure 6. Effect of p21 knockdown on hinokitiol (1)-induced cell viability and apoptotic protein inhibition. HCT-116 and SW-620 cells were transfected with either scrambled siRNA or p21 siRNA as described in the Experimental Section for 4 h, grown for 24 h in complete medium, and treated with 10 μM 1 for 24 h. (A) Cell viability was measured by direct counting after trypan blue staining. (B) The morphological changes of the cells were observed under a phase-contrast microscope (magnification 200×). (C) p21, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, and β-actin were analyzed using 15% SDS-PAGE and immunoblotting. After densitometric quantification, data are expressed as means ± SD of three different experiments conducted in triplicate (*p < 0.05 and **p < 0.01 vs sc siRNA-treated group). and 1 mM NaF) at 4 °C, followed by the BCA protein assay. Protein samples were separated electrophoretically by 10−15% SDS-PAGE and were then transferred to a polyvinylidene difluoride membrane (GE Healthcare Life Sciences; Piscataway, NJ, USA). The membranes were incubated with primary and secondary antibodies and then developed with enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA) before exposure to X-ray film (Eastman-Kodak, Rochester, NY, USA). The primary antibodies and their dilution factors were the following: cyclin A (1:500), cyclin E (1:500), Cdk2 (1:500), Bcl-2 (1:1000), Bax (1:500), caspase-9 (1:1000), cleaved caspase-9 (1:1000), caspase-3 (1:1000), cleaved caspase-3 (1:1000), and β-actin (1:1000). Tumor Xenograft Studies. This study was approved by the ethical committee of animal experiment in Chungbuk National University (protocol number: CBNUA-611-13-01), and all animal procedures were performed in accordance with the Public Health Service policy. Male BALB/c-nude mice (n = 50, 6 weeks old) were

harvested cells were incubated with FITC-labeled annexin-V/PI solution (50 μg/mL) for 10 min and analyzed using the FACS Calibur equipment (Becton-Dickinson Co., Palo Alto, CA, USA). siRNA Transfection. HCT-116 and SW-620 cells (5 × 104 cells/ well) were plated in 24-well plates and transfected with 100 nM of nontargeting control siRNA and p21 siRNA, using WelFect-EX Plus transfection reagent (WelGENE, Seoul, Korea) prepared in OPTIMEM medium at 37 °C. After 4 h, the complete medium (RPMI 1640 medium containing 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine) was added, and the cells were further cultured for 24 h. Western Blot Analysis. For the analysis of cyclin A, cyclin E, Cdk2, Bcl-2, Bax, caspase-9, cleaved caspase-9, caspase-3, and cleaved caspase-3 expression, cells were resuspended in a protein lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 1% Triton X100) containing proteinase inhibitors (1 mM aprotinin, 1 mM leupeptin, and 1 mM PMSF) and protease inhibitors (1 mM NaOV3 2200

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Figure 7. Effect of hinokitiol (1) on tumor growth in human tumor xenograft mice. HCT-116 and SW-620 cells were implanted intradermally separately into the flank of BALB/c-nude mice on day 0. Compound 1 (100 mg/kg) was administered orally to mice every other day for 3 weeks. (A) Representative photographs are shown. (B) Tumor dimensions were measured using a caliper, and tumor volume was calculated by the equation [(width)2 × (length)/2]. (C) Tumor weight was recorded at the end of the study. (D) Body weight was measured every three days and expressed as grams. All values are expressed as means ± SE (n = 10, *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group). tumor. After 21 days, all mice were sacrificed, and the tumors removed, weighed, and photographed. The tumors were rinsed with physiological saline solution and stored at −80 °C. Statistical Analysis. All values are expressed as means ± SD or means ± SE. One-way analysis of variance was used for multiple comparisons with the Newman−Keuls multiple comparison test, with p-values less than 0.05 considered to be significant statistically.

purchased from Orient Bio (Gyeonggi-Do, Republic of Korea). All mice were housed in a room with controlled temperature (21−23 °C), humidity (55−60%), and lighting (12 h light/dark cycle) and were supplied with water. After being acclimated for 1 week, nude mice were randomly distributed in five groups: normal (n = 10), HCT-116 xenograft (n = 10), HCT-116 xenograft−hinokitiol (1) treatment (n = 10), SW-620 xenograft (n = 10), and SW-620 xenograft−hinokitiol (1) treatment (n = 10). The mice were inoculated with HCT-116 or SW620 cells by intradermal injection (2 × 106 tumor cells in 0.2 mL of PBS/mouse) with a 26-gauge needle into the flank. After 2 days, mice were orally administered hinokitiol (100 mg/kg, every other day for 3 weeks). Mouse body weights and tumor volumes were periodically measured. The tumor dimensions were measured with vernier calipers, and tumor volumes were calculated with the following formula: (A × B2)/2, where A is the larger and B is the smaller dimension of the



AUTHOR INFORMATION

Corresponding Author

*Tel: +82-43-261-3215. Fax: +82-43-268-2732. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2201

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0013320), the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (MRC, 2008-0062275), the Ministry of Trade, Industry & Energy (MOTIE, 1415126993) through the fostering project of Osong Academy-Industry Convergence (BAIO), and the “Leaders INdustry-university Cooperation” Project funded by the Ministry of Education, a research grant from Chungbuk National University in 2013.



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