Xerophilusin B Induces Cell Cycle Arrest and Apoptosis in

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Xerophilusin B Induces Cell Cycle Arrest and Apoptosis in Esophageal Squamous Cell Carcinoma Cells and Does Not Cause Toxicity in Nude Mice Ran Yao,† Zhaoli Chen,† Chengcheng Zhou,† Mei Luo,† Xuejiao Shi,† Jiagen Li,† Yibo Gao,† Fang Zhou,† Jianxin Pu,*,‡ Handong Sun,‡ and Jie He*,† †

Department of Thoracic Surgery, Cancer Institute and Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, People’s Republic of China ‡ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China S Supporting Information *

ABSTRACT: Esophageal cancer is the eighth most common cancer in the world and ranks as the sixth leading cause of cancer-related mortality. Esophageal cancer has a poor prognosis partially due to its low sensitivity to chemotherapy agents, and the development of new therapeutic agents is urgently needed. Here, the antitumor activity of a natural entkaurane diterpenoid, xerophilusin B (1), which was isolated from Isodon xerophilus, a perennial herb frequently used in Chinese folk medicine for tumor treatment, was investigated. Compound 1 exhibited antiproliferative effects against esophageal squamous cell carcinoma (ESCC) cell lines in a time- and dose-dependent manner with lower toxicity against normal human and murine cell lines. In vivo studies demonstrated that 1 inhibited tumor growth of a human esophageal tumor xenograft in BALB/c nude mice without significant secondary adverse effects, indicating its safety in treating ESCC. Furthermore, 1 induced G2/M cell cycle arrest and promoted apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway in ESCC cell lines. In conclusion, the observations herein reported showed that 1 is a potential chemotherapeutic agent for ESCC and merits further preclinical and clinical investigation for cancer drug development.

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become a promising source for the discovery and development of novel chemotherapeutic or chemopreventive agents against a variety of malignant diseases, including esophageal carcinoma, stomach cancer, lung cancer, and leukemia.16−19 Ponicidin is a diterpenoid compound isolated from I. xerophilus that has significant pro-apoptotic, cell cycle arrest, and antiangiogenic activities in cancer cells. In recent years, ponicidin has been used effectively alone or in combination with other drugs for the treatment of acute myeloid leukemia (AML), lung adenocarcinoma, hepatocellular carcinoma, and breast cancer.18,20−22 There is a growing interest in exploring natural herbal medicines that can reduce the adverse effects, especially hepatic and renal damages, of clinical treatments toward cancer. In this regard, the natural chemical constituents isolated from I. xerophilus were tested to find more effective anticancer compounds with low toxicity. Xerophilusin B (1) was isolated from I. xerophilus and showed antitumor activity in human ESCC cell lines. This compound was isolated initially from I. xerophilus and had been

sophageal cancer is the eighth most common cancer and the sixth leading cause of cancer death worldwide.1−3 Unlike Western countries, esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype and accounts for nearly 90% of the esophageal cancer cases in China, and China has more than half of the ESCC patients of the world.4−6 Despite improvements in ESCC diagnosis, staging, and treatment in recent years, its prognosis remains poor, with an overall five-year survival rate below 10%.7,8 Among the factors related to the failure of ESCC treatment, a major one is resistance to chemotherapy.9,10 The most widely used first-line chemotherapeutic agent for ESCC is cisplatin, which increases median survival time but causes severe side effects, including hepatotoxicity and renal damage.11,12 Therefore, exploring novel, effective therapeutic agents with low toxicity remains a major task for the treatment of ESCC. Isodon xerophilus (C. Y. Wu et H. W. Li) H. Hara is a perennial herb of the Isodon genus. It is mainly distributed in western China and has a wide spectrum of important pharmacological activities such as antitumor, anti-inflammatory, and antibacterial effects.13−15 The plants of the Isodon genus have attracted considerable attention in recent years and have © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 12, 2014

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(p < 0.001). A similar inhibitory effect was observed in the xenograft model after 7.5 or 15 mg/kg 1 treatment per day (p < 0.001). Cisplatin (DDP), a chemotherapeutic agent that causes cell death through apoptotic pathways and is used as the conventional treatment for ESCC, was administered as a positive control compound.25 The treatments of compound 1 at 15 mg/kg per day and DDP at 2 mg/kg every 2 days caused similar proliferation inhibition profiles (Figure 2A−C). At the

reported to exert strong antiproliferative activity and induce cell death by inhibiting telomerase activity in the K562 human leukemic cell line and exhibit anti-inflammatory effect through the reduction of NF-κB activation in LPS-stimulated RAW 264.7 murine macrophages.13,23,24 However, the in vivo safety and antitumor activity of 1 have not been investigated as yet. In the current work, the effect of 1 on tumor growth and its toxicity were evaluated in an ESCC xenograft tumor model. Moreover, the mechanism of 1-induced cancer cell death was investigated. The results indicated that 1 induced G2/M phase cell cycle arrest and promoted cell apoptosis through the mitochondrial cytochrome c-dependent pathway in ESCC cell lines.

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RESULTS AND DISCUSSION Xerophilusin B (1) Inhibits ESCC Cell Proliferation in Vitro and in Vivo. To further study the cytotoxicity of 1, four ESCC cells lines (KYSE-140, KYSE-150, KYSE-450, and KYSE510) were treated with 1 at various concentrations (0−10 μM) for 0−72 h, and cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay. The inhibition effect of 1 on the proliferation of ESCC cells was dose- and time-dependent (Figure 1A). The concentration of 1 causing a 50% inhibition

Figure 2. Antitumor effect of xerophilusin B (1) was assessed in vivo using tumor-bearing BALB/c nude mice. Cisplatin (DDP) (2 mg/kg every 2 days) was used as a positive control. The treatments began at the eighth day after tumor cell inoculation. (A) Tumor volume of different groups of KYSE-150 and KYSE-450 xenograft mice. (B) Tumor weights of KYSE-150 and KYSE-450 xenograft tumors in mice. P values were determined by one-way ANOVA analysis compared with the negative control (1% Pluronic F68; *p < 0.05, **p < 0.01, and ***p < 0.001). (C) Tumors obtained from the KYSE-150 and KYSE450 tumor-bearing mice. (D) Histochemical analysis of tumors. Sections of paraffin-embedded tumors from each group of KYSE-150 (upper panel) and KYSE-450 (lower panel) xenograft mice were stained with H&E. The images of each group are depicted at 100× and 400× magnifications.

Figure 1. Growth inhibition effects of xerophilusin B (1) on human esophageal cancer cell lines. (A) Growth inhibition rates of normal cell lines and esophageal cancer cell lines treated with 1 at the indicated concentrations for 72 h. Each column represents the mean ± SD for six trials in three independent experiments. (B) IC50 results obtained from the CCK-8 assay for the normal and esophageal cancer cell lines treated with 1.

of cell growth (IC50) of ESCC cell lines was calculated as follows: KYSE-140, 2.8 μM; KYSE-150, 1.2 μM; KYSE-450, 1.7 μM; and KYSE-510, 2.6 μM (Figure 1B). Two cell lines (KYSE-150 and KYSE-450) with lower IC50 values for 1 were used for further in vivo studies and mechanism investigation. Next, BALB/c nude mice were subcutaneously injected with KYSE-150 or KYSE-450 cells and used as a model to investigate the antitumor effect of 1. Compound 1 administerd at 7.5 or 15 mg/kg per day slowed the growth of KYSE-150 and KYSE-450 tumors, resulting in significantly lighter tumor masses on day 20 in the animals treated with 1 relative to negative control mice

end of the treatment, the xenograft tumors were collected, and the formalin-fixed paraffin-embedded (FFPE) tumor samples were prepared. Hematoxylin and eosin (H&E) staining showed large vacuolization and cell debris in 1-treated samples, while only several focal cavities were observed in DDP-treated samples. In contrast, xenograft tumor tissues from negative controls displayed a high density of capillaries surrounding the B

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2.84% and 1.34%; KSYE-450, 62.05% vs 1.75% and 1.56%, p < 0.001), suggesting that 5 μM 1 led to a notable level of cell apoptosis (Figure 3A). Apoptosis is a tightly controlled pattern of cell death that destroys abnormal and injured cells in multicellular organisms. In cancer cells, various mechanisms have evolved to avoid apoptosis; however, the induction of apoptosis in cancer cells is considered crucial in cancer prevention and treatment.28,29 Therefore, FITC-annexin V/PI staining and flow cytometry assays were used to detect cell apoptosis after 1 treatment. Following 24 or 48 h of treatment, the proportion of apoptotic cells was significantly increased in cells exposed to 2 μM 1 compared with vehicle (KYSE-150, 21.67% vs 5.13% at 24 h, 35.17% vs 6.07% at 48 h, p < 0.001, Figure 4A,C; KYSE-450, 17.5% vs 4.07% at 24 h, 39.87% vs 4.53% at 48 h, p < 0.001, Figure 4B,C). Furthermore, a drastic increase in the apoptotic cell population was observed in cells exposed to 5 μM 1 compared with the negative control (KYSE-150, 88.47% vs 5.13% at 24 h, 94% vs 6.07% at 48 h, p < 0.001, Figure 4A, C;

nests of confluent tumor cells without notable cell death. The typical apoptotic appearance, such as cell shrinkage and pyknosis, was observed in both 1- and DDP-treated samples, indicating that 1 may cause ESCC cell death by inducing cell apoptosis (Figure 2D). Xerophilusin B (1) Induces G2/M Phase Cell Cycle Arrest and Promotes Apoptosis of ESCC Cells. Uncontrolled cell division or propagation of damaged DNA can contribute to genomic instability and tumorigenesis. Blocking passage through checkpoints can inhibit cancer cell growth and division.26 Oridonin, the most representative diterpenoid compound, has been reported to have significant effects on inducing G2/M cell cycle arrest in ESCC cells (Figure S1).27 Herein, propidium iodide (PI) was used to evaluate whether 1 could perturb the cell cycle of ESCC cells. To investigate the mechanism of the antiproliferative effect of 1 on ESCC cells, KYSE-150 and KYSE-450 cells were treated with 2 or 5 μM 1 for 24 h. As shown in Figure 3B, the percentage of cells in G2

Figure 3. Xerophilusin B (1) induces the accumulation of G2/M and apoptotic cell populations in KYSE-150 and KYSE 450 cells. Cells were treated with 1 at various concentrations (0, 2, or 5 μM) for 24 h, stained with PI, and analyzed by flow cytometry. The histograms indicate the percentage of cells in the apoptotic population (blue fraction), G1 (left red fraction), S (gray fraction), and G2/M (right red fraction) phases of the cell cycle. (A) The populations of KYSE-150 and KYSE 450 cells in G2/M phase increased after treatment with 2 μM 1. The apoptotic population increased after treatment with 5 μM 1. (B) The cell cycle distributions of KYSE-150 and KYSE 450 cells after treatment with 1 are presented as a histogram graph. The experiments were repeated three times, and the data are shown as the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4. Xerophilusin B (1) induces apoptosis in KYSE-150 and KYSE-450 cells. (A, B) Cells were treated with 1 at various concentrations (0, 2, and 5 μM) for 24 or 48 h, stained with annexin V/PI, and tested by flow cytometry. Treatment with 1 caused an increase in the annexin V+/PI− (lower right quadrant) and annexin V+/PI+ (upper right quadrant) percentages in both KYSE-150 and KYSE-450 cells. (C) The percentages of annexin V (+) cells of KYSE150 and KYSE 450 cells after treatment with 1 at various concentrations are presented as a histogram. The experiments were repeated three times, and the data are shown as the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.

was significantly increased after treatment with 2 μM 1 for 24 h compared with the negative control (KYSE-150, 31.82% vs 22.27%; KYSE-450, 31.78% vs 19.65%, p < 0.001), suggesting that 2 μM 1 arrested cells in G2/M phase. The proportion of apoptotic cells was significantly increased in cells exposed to 5 μM 1 compared with 2 μM 1 or vehicle (KYSE-150, 39.67% vs C

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KYSE-450, 90.97% vs 4.07% at 24 h, 96.57% vs 4.53% at 48 h, p < 0.001, Figure 4B,C). These results indicated that the antiproliferation effect of 1 was mediated mainly by inducing G2/M cell cycle arrest at low concentrations, whereas promoting apoptosis played leading roles at higher concentrations. Xerophilusin B (1) Induces Apoptosis in ESCC Cells through Mitochondrial Cytochrome c-Dependent Activation of the Caspase-9 and Caspase-3 Cascade. The mechanisms of apoptosis are highly complex, and there are two vital apoptotic pathways: the intrinsic or mitochondrial pathway and the extrinsic pathway. The caspases are a family of intracellular cysteine proteases with proteolytic activity to cleave proteins at aspartic acid residues, which triggers apoptosis.30 All caspases play key roles in drug-induced apoptosis in several types of cancer cells.31 In most cells, caspases are widely expressed in an inactive zymogen form and, once activated, can activate downstream procaspases, allowing for the initiation of a proteolytic cascade. Once one caspase activates other caspases, the apoptotic signal is spread, leading to programmed cell death.32 Many diterpenoid compounds, such as oridonin and ponicidin, have been reported to possess apoptosis-inducing activities against a variety of cancer cells through the classic intrinsic pathways (Figure S1).18,33 Thus, to further determine the molecular mechanisms involved in 1-induced apoptosis in ESCC cells, the proapoptotic factor cytochrome c was detected, which is known to be frequently involved in chemically induced apoptotic signaling pathways and the subsequent activation of the caspase cascade involving caspase-9, caspase-3, and caspase7, leading to poly(ADP-ribose) polymerase (PARP) degradation.34 In the present study, most of the KYSE-150 and KYSE450 cells died after treatment with 5 μM 1, resulting in insufficient amounts of protein, and the protein bands were too dim to be identified by the imaging system for Western blot detection (Figure S6, Supporting Information). Therefore, 1 was tested at concentrations of 0, 0.5, 1, and 2 μM. After 1 treatment, the level of released mitochondrial cytochrome c increased in a concentration-dependent manner. Meanwhile, caspase-9 was proteolytically degraded from the inactive zymogen (47 kDa) into the active form (37 kDa), and caspase-3 was degraded from its zymogen (35 kDa) into its active form (19 kDa). Accordingly, caspase-7 levels also decreased. As a downstream target of active caspase-3 and caspase-7, PARP, the hallmark of apoptosis, significantly decreased after the 1 treatment (Figure 5A). It has been clearly demonstrated that mitochondrial membrane permeability is regulated by Bcl-2 family members, which are the central regulators of caspase activation.35,36 The Bcl-2 family can be classified into two subgroups: those that promote cell death (e.g., Bax) and those involved in prosurvival members (e.g., Bcl-2). Bax has been considered as the final step of cytochrome c release, and its homo-oligomerization on the mitochondrial membranes is crucial for membrane permeability, while Bcl-2 inhibits mitochondrial protein release by binding with and inhibiting Bax.37 In the present study, a concentration-dependent increase of the pro-death protein Bax was observed following 1 treatment (0−2 μM), while the level of Bcl-2 decreased in an opposite trend (Figure 5B). The decrease in the ratio of Bcl-2/Bax can cause the release of cytochrome c and the cleavage of caspases-3, -7, and -9 (Figure 5), suggesting that 1-induced apoptosis in KYSE-150 and KYSE-450 cells involves the intrinsic apoptotic pathway.38,39

Figure 5. Effects of xerophilusin B (1) on changes of apoptosis-related proteins. KYSE-150 and KYSE-450 cells were treated with 1 at various concentrations (0, 0.5, 1, and 2 μM) for 24 h before analysis. β-Actin was used as a loading control. (A) Western blot bands for cytochrome c, cleaved caspase-9, pro-caspase-9, cleaved caspase-3, pro-caspase-3, pro-caspase-7, and pro-PARP proteins. (B) Expression of Bcl-2 and Bax proteins. (C) The Bcl-2/Bax ratio was down-regulated by 1 treatment in both KYSE-150 and KYSE 450 cells. The intensity of the bands was quantified by optical density (OD) and normalized to the OD of β-actin. Significance values were determined by one-way ANOVA (*p < 0.05, **p < 0.01, and ***p < 0.001).

Evaluation of the Safety of Xerophilusin B (1) in Vitro and in Vivo. DDP is one of the most common anticancer drugs used in chemotherapy. Nevertheless, the clinical utility of DDP is often limited by its serious side effects such as hepatotoxicity.40−42 Thus, selective toxicity for tumor cells is essential in the development of novel cancer chemical therapeutics.43 To evaluate the potential of 1 as a chemotherapeutic drug, the toxicity of 1 to nontumor cells was assessed both in vitro and in vivo. To detect the cytotoxicity of 1 on nontumor cells, human embryo kidney cells (HEK-293) and mouse embryo fibroblast cells (NIH-3T3) were treated with various concentrations of 1, and cell viability was assessed using the CCK-8 assay (Figure 1A). The IC50 value of 1 for HEK-293 (3.24 μM) and NIH-3T3 (2.61 μM) was higher than the median IC50 value of 1 for the four ESCC cell lines tested (2.06 μM), indicating a certain degree of selectivity of 1 against cancer cells and lower toxicity toward the nontumor cells (Figure 1B). The hepatic and renal toxicity of 1 were further evaluated. Peripheral blood samples from all mice bearing a xenograft tumor were collected at sacrifice, and 16 common serum D

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parameters routinely assessed in Chinese hospitals for diagnosing patients, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urine nitrogen (UN), and creatinine (CREA), were detected to evaluate the chemical toxicity and side effects.44 As shown in Figure 6 and

Figure 7. Histopathological analysis of liver and renal tissue structures in xenograft mice. (A) Tissue sections of the vehicle control and the 1treated group of KYSE-150 (upper panel) and KYSE-450 (lower panel) xenograft mice exhibited normal liver structures. The liver tissue of cisplatin (DDP)-treated mice showed distinct hepatotoxicity. (B) No significant architectural or pathologic changes in the kidney were found in 1-treated mice. The images of each group are depicted at 100× and 400× magnifications.

Figure 6. Measurement of serum biochemical parameters of the xenograft mice. Blood biochemical parameters included the following: (A) ALT (alanine aminotransferase); (B) AST (aspartate aminotransferase); (C) UN (urea nitrogen); and (D) CREA (creatinine) of KYSE-150 and KYSE-450 xenograft mice. P values were determined by one-way ANOVA (***p < 0.001 relative to control).

supplemental Table 1, no significant alterations in these biochemical parameters were observed after 1 treatment. Additionally, no significant histological structure and pathologic alterations were found in histological examination of mouse organs, including heart, lung, liver, spleen, and kidney after 1 treatment (Figures 7, S7, S8, Supporting Information). In addition, no adverse consequences were observed in gross measurements after 1 treatment such as ruffling of fur, behavior, or feeding (data not shown). However, DDP treatment led to a significant increase in serum ALT and AST levels in mice with a KYSE-150 xenograft tumor compared with the negative control mice (Figure 6A,B). Typical pathological hepatic damage was also found in DDPtreated mice including sinusoidal congestion, extensive disorganization of hepatocytes, expanded periportal areas, and significant fibrosis around central venules, which is in accordance with previous studies of DDP-induced hepatotoxicity (Figure 7A). DDP treatment led to significant weight loss in mice with xenograft tumors compared with 1 treatment (Figure 8). These results indicated that 1 is specific for tumor cells; however, the target and mechanism require further exploration. Taken together, the present data showed that compound 1 caused ESCC cells to undergo cell cycle arrest followed by cell death via apoptosis and that these effects are due predominantly to induced mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway. Additionally, 1 exhibited antineoplastic activity on ESCC cells in vivo in a dose-dependent manner with relative safety. To the best of our knowledge, this is the first report on the in vivo antitumor efficacy of 1. Accordingly, 1 has the potential to be used as a lead compound for further development as a

Figure 8. Effects of xerophilusin B (1) on the body weight of xenograft nude mice. The treatments began at the eighth day after tumor cells inoculation. (A, B) The weights of the KYSE-150 and KYSE-450 xenograft mice were measured daily and calculated in millimeters by the formula 4π/3 × (width/2)2 × (length/2). Significance values were determined by one-way ANOVA (*p < 0.05, **p < 0.01, and ***p < 0.001).

chemotherapy agent in human ESCC patients, and more extensive mechanism studies on this new compound are ongoing.

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EXPERIMENTAL SECTION

Extraction and Isolation. Xerophilusin B (1) was isolated from the leaves of Isodon xerophilus as described previously.15 Briefly, the milled, dried plant material (8 kg) was soaked with acetone (3 × 12 L, every 3 days) at room temperature. Then, the crude extract was filtered and evaporated in vacuo to yield a residue. The residue was dissolved in H2O (4 L) and then extracted with petroleum ether (3 × 3 L) and ethyl acetate (3 × 4 L), consecutively. The extract (504 g) was decolorized using MCI gel and further eluted with 90% MeOH− H2O, to yield a yellow gum (420 g). The gum was subjected to a silica gel column and eluted with a CHCl3−Me2CO (1:0 → 0:1) gradient system. Seven fractions were obtained, A−G, and fraction B (33 g) was separated by silica gel column chromatography and eluted with a petroleum ether−acetone (6:1 → 2:1) gradient system to yield 1 (104 mg). The purity of the isolated compound 1 was at least 98%. E

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Reagents. The powder of compound 1 was dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) to form a 10−2 M solution and stocked at −20 °C. DDP was obtained from Qilu Pharma (Qilu Pharmaceutical, Inc., Jinan, China), dissolved in 0.9% w/v NaCl solution, and stocked at 4 °C. 10% Pluronic F68 was purchased from Gibco (Life Technologies Corporation, Carlsbad, CA, USA). Monoclonal rabbit antibodies against cytochrome c, pro-caspase-3, cleaved caspase-3, pro-caspase-7, pro-caspase-9, cleaved caspase-9, poly(ADP-ribose) polymerase, Bax, and Bcl-2 were purchased from CST (Cell Signaling Technology, Danvers, MA, USA). An anti-β-actin antibody and horseradish-peroxidase-conjugated secondary antibodies (goat-anti-rabbit) were purchased from Bioss (Biosynthesis Biotechnology, Beijing, China). Bovine serum albumin was supplied with the BCA protein assay kit (Themo, Rockford, IL, USA). All experiments described herein, except for the in vivo antitumor assay, were repeated at least three times. Animals and Cell Lines. Female BALB/c nude mice (SPF, 4 weeks old), weighing 15−19 g, were purchased from HFK Bioscience Co., Ltd. (Certificate No. 11027182, Beijing, China). The animals were fed a standard commercial diet produced by the experimental animal center of the Chinese Academy of Medical Sciences and maintained in specific pathogen-free conditions with a 12 h light−dark schedule. Human ESCC cell lines KYSE-140, KYSE-150, KYSE-450, and KYSE-510 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium (Hyclone, Logan, UT, USA). The normal NIH-3T3 and HEK-293 cell lines were purchased from ATCC and maintained in DMEM (Hyclone, Logan, UT, USA) and MEM (Hyclone) medium, respectively. All the culture media included 10% v/v fetal bovine serum (Gibco, New York, NY, USA), 100 UI/mL penicillin, and 100 UI/mL streptomycin (Hyclone), and the cells were cultured in a 37 °C incubator (Themo) supplied with 5% CO2. Cell Viability Assay. Cell viability was assessed using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) assay. Cell suspensions were plated into 96-well plates (Corning, New York, NY, USA) at a density of 2 × 103 cells/well and were preincubated in a humidified incubator at 37 °C and 5% CO2. After 24 h, the media were changed to fresh, and the cells were treated with the indicated concentrations of compound or with the vehicle DMSO for 0, 6, 12, 24, 48, and 72 h. The DMSO concentration was kept under 0.1% in the cell culture and caused no detectable effect on cell proliferation. A 10 μL amount of the CCK-8 solution was added to each well in the plate and incubated for 2 h before measuring the optical density (OD) value at 450 nm in a microplate spectrophotometer (SpectraMax190, Molecular Device, Sunnyvale, CA, USA). Each time point was tested in six wells for each concentration. The inhibition rates were determined as follows: inhibition rate (%) = (1 − ODcompound treatment/ODDMSO) × 100. The half-maximal inhibitory concentration values (IC50) were calculated by nonlinear regression analysis using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Cell Cycle Analysis. The cell cycle analysis was performed using a cell cycle detection kit (KGA, Beijing, China), according to the manufacturer’s instructions. Briefly, KYSE-150 and KYSE-450 cells were treated with compound 1 at concentrations of 0, 2, and 5 μM for 24 h. Then, the cells were harvested and fixed with 70% alcohol at 4 °C for 12 h. DNA was stained with propidium iodide in the presence of 1% DNase-free RNase A at 37 °C for 30 min before flow cytometer analysis (Becton Dickinson FACSCanto II, NJ, USA). The distribution of cells in distinct cell cycle phases was determined using ModFit LT3.2 (Verity Software House Co., USA) cell cycle analysis software. Cell Apoptosis Detection. Cell apoptosis was assessed using a BD Pharmingen FITC annexin V apoptosis detection kit I (BD Biosciences, NJ, USA) according to the manufacturer’s instructions. Briefly, KYSE-150 and KYSE-450 cells were treated with 1 at various concentrations (0, 2, and 5 μM) for 24 and 48 h, and then, both adherent and floating cells were collected, washed with binding buffer, and stained with annexin V-fluorescein isothiocyanate (FITC) and PI. After incubation at 15 °C for 15 min in the dark, the samples were quantified by flow cytometry (Becton Dickinson FACSCanto II).

Western Blot Analysis. Western blots were performed to detect the presence of cytochrome c, pro-caspase-9 cleaved-caspase-9, procaspase-3, cleaved-caspase-3, pro-caspase-7, PARP, Bax, and Bcl-2. Briefly, KYSE-150 and KYSE-450 cells were cultured at a density of 2 × 106 cells/mL in a 100 mm × 20 mm Cell Culture Dish (Corning) and treated with various concentrations of 1 (0, 0.5, 1, 2, and 5 μM) for 24 h. Subsequently, the cells were collected and incubated in RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The cell lysates were centrifuged and used as protein extracts. The concentration of each extract was determined by a BCA protein assay, and the maximum amount of protein in one sample well of the gel was 200 μg. The proteins were subjected to a 10% SDS-PAGE gel electrophoresis. Then proteins were transferred onto a nitrocellulose transfer membrane (Whatman GmbH, Maidstone, Kent, UK) and blocked for 2 h using 5% (w/v) nonfat dried milk at room temperature. After incubating in TBS/0.1% Tween-20, primary antibodies against cytochrome c, pro-caspase-9, cleaved-caspase-9, pro-caspase-3, cleaved-caspase-3, caspase-7, PARP, Bax, Bcl-2, and βactin were added at a ratio of 1:1000 (v/v). After overnight incubation, the primary antibodies were washed away and the appropriate secondary antibodies (diluted 1:5000) were added. Then, immunoreactive protein bands were visualized by chemiluminescence using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) and scanned by Q-IMAGING gel-imaging system (Sage Creation, Halifax, NS, CAN). The optical density of the bands was measured by Quantity One version 4.1.1 software (BioRad, Hercules, CA, USA) with normalization to β-actin. The Bcl-2/Bax ratio is the optical density ratio of the Bcl-2 and Bax proteins. In Vivo Antitumor Experiment. Antitumor activity against a solid tumor xenograft model was evaluated using BALB/c nude mice (female, 4 weeks old). The mice used in this study received humane care, and the experiments were performed according to the guidelines approved by the Cancer Institute and Hospital of Chinese Academy of Medical Sciences Institutional Animal Care and Use Committee (IACUC; permission number: NCC2013A058). Briefly, 400 μL of 2 × 107 cells/mL KYSE-150 or KYSE-450 cells diluted with 1% Pluronic F68 vehicle was inoculated through subcutaneous injection in the right flank of each nude mouse to establish a xenograft. Seven days after inoculation, 20 mice were divided randomly into four groups (5 mice per group) and were injected intraperitoneally with a dose volume of 200 μL daily for 19 and 20 days for KYSE-150 and KYSE-450 xenograft mice, respectively, with one of the following treatments: (1) vehicle control (1% Pluronic F68 per day); (2) low-dose 1 (7.5 mg/kg per day); (3) high-dose 1 (15 mg/kg per day); and (4) the positive control drug DDP (2 mg/kg, every 2 days). Mouse weights were measured daily using a digital slide caliper and calculated in millimeters by the formula 4π/3 × (width/2)2 × (length/2), representing the volume of an oblate spheroid. All animals were sacrificed 19 or 20 days after the first treatment, and the tumors were weighed. The inhibition ratio (%) was estimated using the following equation: IR% = [1 − Wtumor (treated)/Wtumor (vehicle control)] × 100%. Measurement of Serum Biochemical Parameters. At the end of the treatment period, blood samples were collected from the retroorbital plexus of the mice under light ether anesthesia, left to stand at room temperature for 1 h, and then centrifuged (3500 rpm, 10 min at 4 °C) to obtain the serum. The biochemical parameters tested on the mouse serum included the following: (1) alanine aminotransferase (ALT); (2) aspartate aminotransferase (AST); (3) total protein (TP); (4) albumin (ALB); (5) globulin (GLOB); (6) totalbilirubin (TBIL); (7) alkaline phosphatase (ALP); (8) gamma-glutamyl transpeptidase (GGT); (9) glucose (GLU); (10) urea nitrogen (UN); (11) creatinine (CREA); (12) uric acid (UA); (13) cholesterol (CHO); (14) triglyceride (TG); (15) lactate dehydrogenase (LDH); and (16) the albumin/globulin ratio (A/G). All biochemical assays were performed using a CL-7200 spectrophotometer (Shimadzu, Shanghai, China) and commercially available kits (Randox, Schwyz, Switzerland). The data were analyzed using one-way ANOVA. Histology and Pathology Assessment. The tumor, heart, lung, liver, spleen, and kidney were excised, fixed in 10% neutral formalin, and embedded in paraffin. Histopathological changes in the tumor and F

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organ structure were assessed in at least 10 randomly selected tissue sections from each group studied. The tissues were cut into 4-μm-thick sections and stained with hematoxylin and eosin (H&E) to examine tissue and cell structure. The sections were examined by light microscopy (Nikon, Tokyo, Japan) and analyzed by NIS-Elements F3.0 software (Nikon, Tokyo, Japan). Statistical Analyses. Statistical analyses of the data were performed using GraphPad Prism 5.0 software. The data were expressed as the mean ± SD for in vitro studies and as the mean ± SEM for in vivo studies. One-way ANOVA followed by Student’s t test was used for multiple comparisons. The results with a value of *p < 0.05 were considered significant, **p < 0.01 more significant, and ***p < 0.001 highly significant.

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data of xerophilusin B (1); chemical structures of oridonin and ponicidin; Western blot bands for β-actin at various concentrations (0−5 μM) of 1; images of H&E-stained murine organic tissues; and table of results of serum biochemical parameters. The information is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(J. He) Tel: +86 10 87788798. Fax: +86 10 67709698. E-mail: [email protected]. *(J. Pu) Tel: +86 87 15223251. Fax: +86 87 15216343. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Chinese National Natural Science Joint Foundation of Yunnan Province (Grant U1302223) and the National Natural Science Foundation of China (Grants 21322204, 81172939, and 81172336). The authors thank L. Jiang (Institute of Laboratory Animal Sciences, CAMS & PUMC, Beijing) and X. Zeng (WuXi AppTec, Shanghai) for their help and support.

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DOI: 10.1021/np500429w J. Nat. Prod. XXXX, XXX, XXX−XXX