Antitumor Activity of Spicatoside A by Modulation of Autophagy and

Apr 11, 2016 - Won Kyung Kim†, Yuna Pyee†, Hwa-Jin Chung†, Hyen Joo Park†, Ji-Young Hong†, Kun Ho Son‡, and Sang Kook Lee†. † College ...
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Antitumor Activity of Spicatoside A by Modulation of Autophagy and Apoptosis in Human Colorectal Cancer Cells Won Kyung Kim,† Yuna Pyee,† Hwa-Jin Chung,† Hyen Joo Park,† Ji-Young Hong,† Kun Ho Son,‡ and Sang Kook Lee*,† †

College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742, Republic of Korea Department of Food Science and Nutrition, Andong National University, Andong 760-749, Republic of Korea



ABSTRACT: The antitumor activity of spicatoside A (1), a steroidal saponin isolated from the tuber of Liriope platyphylla, and its underlying mechanisms were investigated in HCT116 human colorectal cancer cells. Compound 1 induced autophagy and apoptotic cell death and inhibited tumor growth in a nude mouse xenograft model implanted with HCT116 cells. Treatment with 1 for 24 h enhanced the formation of acidic vesicular organelles in the cytoplasm, indicating the induction of the onset of autophagy. This event was associated with the regulation of autophagic markers including microtubule-associated protein 1 light chain 3 (LC3)-II, p62, beclin 1, lysosomal-associated membrane protein 1 (LAMP 1), and cathepsin D by inhibiting the PI3K/Akt/mTOR signaling pathway, regulating mitogen-activated protein kinase (MAPK) signaling, and increasing p53 levels. However, a prolonged exposure to 1 resulted in apoptosis characterized by the accumulation of a sub-G1 cell population and an annexin V/propidium iodide (PI)-positive cell population. Apoptosis induced by 1 was associated with the regulation of apoptotic proteins including Bcl-2, Bax, and Bid, the release of cytochrome c into the cytosol, and the accumulation of cleaved poly-ADP-ribose polymerase (PARP). Further study revealed that cleavage of beclin 1 by caspases plays a critical role in the 1-mediated switch from autophagy to apoptosis. Taken together, these findings highlight the significance of 1 in the modulation of crosstalk between autophagy and apoptosis, as well as the potential use of 1 as a novel candidate in the treatment of human colorectal cancer cells.

chemotherapy, which is associated with preservation of cell homeostasis by blocking uncontrolled proliferation in cancer cells. Apoptosis, type I PCD, is a cellular process characterized as a suicide program, and it is a major cytotoxic mechanism of anticancer agents.14 Apoptotic cell death, characterized by cell shrinkage, chromatin condensation, DNA fragmentation, and apoptotic body formation, is known to be induced by two main molecular signaling pathways, namely, the death receptor extrinsic pathway and the mitochondrial intrinsic pathway.15 Furthermore, these pathways can be regulated by caspases.16 Activation of caspase-8 and downstream effector caspase-3 is mostly involved in the extrinsic pathway, leading to cleavage of cellular substrates including poly-ADP-ribose polymerase (PARP) and inhibitors of caspase-activated DNase.16 In contrast, the intrinsic pathway is initiated by changes in the mitochondrial permeability, which is regulated mainly by the Bcl-2 family of proteins, such as Bcl-2 and Bax. These changes function to promote the release of cytochrome c from mitochondria, leading to activation of caspase-9 and then eventually apoptosis.17 Furthermore, Bid cleavage by caspase-8 is found to be the linkage of the extrinsic and intrinsic pathway through its translocation to mitochondria followed by the release of cytochrome c.18

Liriope platyphylla (Liliaceae), a medical plant distributed mainly in China, Taiwan, and Korea, has been used traditionally for treatment of cough, sputum, asthma, and neurodegenerative diseases.1−3 L. platyphylla also exhibits various biological effects on inflammation, atopic dermatitis, and cardiovascular diseases.4,5 A phytochemical study revealed that L. platyphylla contains various steroidal saponins as major secondary metabolites.6 Steroidal saponins, which are the main bioactive constituents in a variety of medicinal plants, have been considered to be a potential class of compounds in the development of anticancer agents.7 Indeed, steroidal saponins, including timosaponin A-III from Anemarrhena asphodeloides, terrestrosin D from Tribulus terrestris, and macrostemonoside A from Allium macrostemon, show potent antiproliferative and antitumor activities against several human cancer cells. Their reported mechanisms have been associated with the induction of cell cycle arrest and apoptosis.8−10 Of the known steroidal saponins isolated from the tubers of L. platyphylla, spicatoside A (1) has been reported to have antiosteoarthritic activity, memory enhancement activity, and alleviation effects of inflammatory pulmonary diseases.11−13 However, the antitumor potential and its underlying mechanism of action of 1 against human cancer cells have not been elucidated yet. Accumulating evidence has suggested that the regulation of programmed cell death (PCD) is an important target in cancer © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 3, 2016

A

DOI: 10.1021/acs.jnatprod.6b00006 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Autophagy is an intercellular process for catabolic degradation to keep cellular homeostasis during metabolic stress, and it is involved in the formation of autophagosomes, which are further fused with lysosomes to form acidic vesicular organelles including autolysosomes. It initially involves signal transduction mediated by phosphoinositide 3-kinase (PI3K), mitogenactivated protein kinases (MAPKs), and transcription factors such as p53 and p21.19 Prolonged stress induces excessive autophagy, which causes the cell death by cellular overconsumption. This autophagic cell death is implicated as type II PCD.20 Because cancer cells often exhibit defective autophagic capacities, autophagic cell death is considered as a tumor suppressor.21 However, emerging evidence indicates that autophagy is not only a death pathway but also a survival pathway exploited by cancer cells to endure metabolic stress.22 In addition, the inhibition of autophagy leads to apoptotic cell death as a result of the failure to adapt to stress. Therefore, autophagy inhibitors are also considered as an attractive strategy to enhance the sensitivity of cancer cells toward anticancer drugs by manipulating the autophagic process.23 The interconnection of molecular mechanisms and the functional relationship between apoptosis and autophagy have been clarified.24 However, the apoptotic and autophagic response machineries are complex, controversial, and difficult to distinguish because they can occur simultaneously, sequentially, or exclusively.25 It is possible that different sensitivity thresholds can determine whether apoptosis or autophagy will be induced. Among the overlapping signaling molecules, beclin 1 is considered an important regulator of crosstalk between apoptosis and autophagy.26 Beclin 1 functions as a scaffold for the formation of a multiprotein complex by binding to PI3K class-III/Vps34a, and it is known to be a key autophagy signaling molecule.27 Recently, autophagy has been shown to be inhibited by caspase-mediated cleavage of beclin 1. Therefore, beclin 1 cleavage by caspases can be a strategic target for the switch from cytoprotective autophagy to apoptotic cell death in cancer cells.28 In the present study, the antitumor activity of 1 and its underlying molecular mechanism of action with the modulation of crosstalk between autophagy and apoptosis were investigated in HCT116 human colon cancer cells under cell culture conditions and in a nude mouse xenograft model.

Table 1. Effects of Spicatoside A (1) on the Cell Proliferation of Human Cancer Cells and Normal Cells IC50 (μM)

a

cell line

classification

1

ellipticinea

A549 HCT116 MDA-MB-231 SNU-638 SK-HEP-1 MRC5

lung cancer cells colon cancer cells breast cancer cells stomach cancer cells liver cancer cells lung epithelial normal cells

26.0 ± 4.9 13.8 ± 1.3 29.6 ± 2.8 14.8 ± 0.4 15.5 ± 1.9 82.0 ± 5.6

0.4 ± 0.1 1.0 ± 0.2 0.1 ± 0.1 2.7 ± 0.2 0.4 ± 0.1 1.3 ± 0.8

Ellipticine was used as a positive control.

activity of 1 was found against the tested human cancer cell lines, compound 1 did not show considerable growth inhibition in cultured MRC-5 human lung normal epithelial cells (IC50 > 80 μM), indicating that 1 selectively inhibits the proliferation of cancer cells compared to normal cells. In addition, based on the sensitivity of the HCT116 human colon cancer cells to 1, the plausible mechanisms of action of 1 were further elucidated using HCT116 cells. Spicatoside A (1) Induces Autophagy in HCT116 Cells. In addition to the 72 h growth inhibitory activity of 1 in HCT116 cells, a short-term exposure of the cells to 1 was also investigated to characterize the cell growth features. After a 24 h treatment with 1, cell growth was inhibited in a concentration-dependent manner with an IC50 value of 28.9 μM (Figure 1A). In addition, analysis of morphological changes using a phase-contrast microscope showed the formation of vacuoles in the cytoplasm in cells treated with 1 compared with cells treated with the vehicle control (Figure 1B). To confirm if these vacuoles were acidic vesicular organelles (AVOs), which are indicative of autophagy, HCT116 cells were treated with 1 for 24 h and then were stained with acridine orange (AO). AO moves freely into biological membranes and accumulates in acidic compartments, resulting in a bright red fluorescence in AVOs. 29 As demonstrated in Figure 1C, treatment with 1 (20 μM) significantly enhanced the strength of fluorescence compared to the vehicle control, and the intensity was blocked by bafilomycin A1 (BaF), an autophagy inhibitor, indicating that 1 induced the formation of AVOs. To further confirm 1-induced autophagy, Western blot analysis was used to detect autophagy biomarkers. Accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II and beclin 1 was detected in HCT116 cells after 24 h treatment with 1 in a concentration-dependent manner. Treatment with 1 also up-regulated the expression levels of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin D, and it down-regulated the expression of p62 in HCT116 cells, indicating the presence of autophagy induced by 1 (Figure 1D). In addition, LC3-II accumulation was further enhanced by cotreatment with 1 and BaF, indicating that 1 induced autophagosome accumulation by increasing autophagic flux, rather than by blocking autophagic degradation (Figure 1E).30 Taken together, these findings suggested that treatment with 1 for 24 h induces autophagy in HCT116 cells. Spicatoside A (1) Induces Autophagy via Regulation of the PI3K/Akt/mTOR, MAPK, and p53 Signaling Pathways. To further explore the signaling pathway involved in the regulation of autophagy in 1-treated HCT116 cells, the role of the PI3K-Akt-mTOR pathway in the modulation of autophagy was investigated by Western blot analysis. Accumulating evidence has suggested that activation of the PI3K/Akt/ mTOR signaling pathway is critical for cell proliferation,



RESULTS AND DISCUSSION Spicatoside A (1) Exhibits Antiproliferative Activity against Several Human Cancer Cell Lines. To evaluate the effects of 1 on the growth of human cancer cells, the growth inhibitory potential of 1 for 72 h was determined in a panel of cancer cell lines and a normal cell line (Table 1). As shown in Table 1, compound 1 inhibited cancer cell growth with IC50 values ranging from 13.8 to 29.6 μM. Although antiproliferative B

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Figure 1. Effect of spicatoside A (1) on the induction of autophagy in HCT116 cells. (A) HCT116 cells were treated with various concentrations of 1 for 24 h, and cell viability was measured using the SRB assay. Data are expressed as the means ± SD (n = 3) and are representative of three separate determinations (*p < 0.05 or **p < 0.01). (B) Morphological changes in the presence or absence of 20 μM 1 were observed under an inverted phasecontrast microscope (100× magnification) and photographed. Arrows indicate cells containing the formation of vacuoles. (C) Cells were treated with 20 μM 1 or 2.5 nM BaF or cotreated with 20 μM 1 and 2.5 nM BaF for 24 h, and the cells were then observed and quantified using acridine orange staining and an Operetta high content imaging system using Harmony High Content Imaging and Analysis software. The data are presented as the index of acridine orange-positive cells compared to vehicle-treated control cells. The values are expressed as the means ± SD of triplicate tests (**p < 0.01). (D) HCT116 cells were cultured with the indicated concentrations of 1 for 24 h. The protein expression levels of autophagy markers were determined by Western blot analysis. (E) Cells were treated with 20 μM 1 or 2.5 nM BaF or cotreated with 20 μM 1 and 2.5 nM BaF for 24 h. The protein expression levels of LC3-II were detected by Western blot analysis. β-Actin was used as an internal standard. The images shown are representative of three separate determinations.

Figure 2. Effect of spicatoside A (1) on the PI3K/Akt/mTOR signaling pathway and the p53-mediated mitogen-activated protein kinase (MAPK) signaling in HCT116 cells. (A−C) Cells were lysed after treatment with indicated concentrations of 1 for 24 h. The protein expression levels of PI3K, PTEN, Akt, and mTOR were determined by Western blot analysis (A). Western blotting was also performed for MAPK proteins including p-ERK, ERK, p-p38, p38 (B), p53, and p21 (C). β-Actin was used as an internal standard. The images shown are representative of three separate determinations.

metastasis, and acquired drug resistance of cancer cells. In addition, negative regulation of the mTOR pathway is also considered to induce autophagy.31 As shown in Figure 2A, treatment with 1 for 24 h resulted in the up-regulation of PTEN

and down-regulation of PI3K in HCT116 cells. Furthermore, the expression levels of phosphorylated Akt and its downstream effector, mTOR, were also suppressed by 1, demonstrating the C

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Figure 3. Effect of spicatoside A (1) on the induction of apoptotic cell death in HCT116 cells. (A−C) HCT116 cells were treated with indicated concentrations of 1 for 48 h. Cell viability was measured using the SRB assay (A). The presence of cells with sub-G1 DNA content was analyzed by flow cytometry, and data are presented as the percentage of sub-G1 cell population (B). Flow cytometric analysis of annexin V/PI-positive cells was also performed to evaluate apoptotic cells (C). Data are expressed as the means ± SD (n = 3) and are representative of three separate determinations (**p < 0.01).

Figure 4. Effect of spicatoside A (1) on the expression of apoptosis-related proteins in HCT116 cells. (A−C) HCT116 cells were treated with indicated concentrations of 1 for 48 h. The activation of caspase-8, caspase-9, caspase-3, and PARP was determined by Western blot analysis (A). Western blotting was also performed to detect the expression levels of pro-apoptotic/anti-apoptotic proteins (B) and cytosolic cytochrome c (C). β-Actin or α-tubulin was used as an internal standard. COX IV was used to confirm the purity of subcellular fractions. The images shown are representative of three separate determinations.

p38 and JNK contribute to the accumulation of LC3-II.33,34 To investigate the role of MAPK signaling pathways in 1-induced autophagy, the activation of ERK1/2, p38 MAPK, and JNK was determined by Western blot analysis. As shown in Figure 2B, 1 inhibited the phosphorylation of ERK1/2 and activated the expression of p-p38 in a concentration-dependent manner. However, the activation of JNK was not detected in 1-treated HCT116 cells (data not shown). These results suggested that 1-

potential role of 1 in the regulation of the PI3K/Akt/mTOR signaling pathway. MAPK signaling pathways consisting of extracellular signalregulated kinase (ERK), p38 MAPK, and c-jun NH2-termianl kinase (JNK) have also been considered as chemotherapeutic targets for sensitizing cancer cells to autophagy.32 Inactivation of ERK1/2 induces mTOR inhibition through phosphorylation of the tumor suppressor tuberous sclerosis-2 (TSC2), and activated D

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Figure 5. Effect of spicatoside A (1) on the modulation of crosstalk between autophagy and apoptosis. (A) HCT116 cells were treated with 30 μM 1 for 6, 12, 24, 36, and 48 h. The protein expression levels of autophagy markers (LC3-II and beclin 1) and apoptosis-related proteins (cleaved caspase-8 and cleaved PARP) were determined by Western blot analysis. (B) Cells were treated with 2 mM 3-MA for 1 h and then co-incubated with 1 for an additional 24 h. Western blotting was performed for LC3-II and cleaved PARP. β-Actin was used as an internal standard. The images shown are representative of three separate determinations. (C) HCT116 cells were incubated with the indicated concentrations of 1 for 48 h in the presence or absence of 2 mM 3MA. Cell viability was measured using the SRB assay. (D) HCT116 cells were cultured with 30 μM 1 in the presence or absence of 20 μM z-IETD-FMK for 48 h. The expression levels of cleaved caspase-8, beclin 1, cleaved beclin 1, and LC3-II were analyzed by Western blot analysis. Data are expressed as the means ± SD (n = 3) and are representative of three separate determinations (*p < 0.05, **p < 0.01).

induced autophagy in HCT116 cells is, in part, associated with the inhibition of ERK1/2 and activation of p38 MAPK. The effect of 1 on the expression levels of p53 was further examined because p53 is linked to autophagy via inhibition of mTOR by AMPK, TSC1, and TSC2.35 p53 also plays a central role in the transcriptional regulation of autophagy-related target genes, such as PTEN, Sestrin 1/2, and damage-regulated autophagy modulator.36−38 As shown in Figure 2C, compound 1 enhanced the expression levels of p53 and its downstream effector, p21, in a concentration-dependent manner. These data indicated that 1 induces autophagy via inhibition of the PI3K/ Akt/mTOR signaling pathway, modulation of ERK, regulation of p38, and activation of p53. Apoptotic Cell Death Is Induced after Prolonged Treatment with Spicatoside A (1) in HCT116 Cells. To evaluate if 1 can induce autophagy for an extended time in HCT116 cells, cells were treated with 1 for 48 h. As shown in Figure 3A, 1 exhibited an inhibitory effect on HCT116 cell proliferation in a concentration-dependent manner with an IC50 value of 20.9 μM. Interestingly, morphological changes, such as cell rounding and shrinkage, were observed in HCT116 cells treated with 1 for 48 h (data not shown). In addition, the populations of sub-G1 phase cells were increased to 5.52%, 12.56%, and 45.37% after a 48 h treatment with 10, 20, and 30 μM 1, respectively, suggesting that 1 may induce apoptosis in HCT116 cells (Figure 3B). To further confirm the induction of apoptosis by 1, flow cytometric analysis was conducted to detect annexin V-positive cells. As shown in Figure 3C, the apoptotic (annexin V-positive) cell population was increased to 13.42%,

18.58%, and 24.58% after treatment of HCT116 cells with 10, 20, and 30 μM 1, respectively. These data suggested that the longer treatment of 1 induces apoptotic cell death in HCT116 cells. Induction of Apotosis by Spicatoside A (1) Is Associated with Caspase-Mediated Pathway. In general, apoptosis occurs via either the mitochondrial (intrinsic) pathway with the activation of caspase-3 and -9 or the death receptor (extrinsic) pathway with the activation of caspase-8. To further explore the molecular mechanism involved in the 1-mediated apoptosis of HCT116 cells, the effect of 1 on the activation of caspases was determined by Western blot analysis. The active cleavage forms of caspase-3, -8, and -9 were observed after a 48 h treatment with 1. Furthermore, PARP cleavage was also evoked by 1, indicating that 1 may induce apoptotic cell death by activation of caspases (Figure 4A). Enhanced mitochondrial membrane permeability, which is mediated by the induction of pro-apoptotic proteins, the suppression of anti-apoptotic proteins, and the activation of BID cleavage by active caspase-8, plays a central role in apoptotic cell death. Mitochondrial membrane permeabilization induces the release of cytochrome c, which leads to caspase-dependent apoptosis. Because the induction of apoptosis by 1 may be associated with activation of the caspase cascade, the expression of mitochondria-mediated proteins after a 48 h treatment with 1 was examined by Western blot analysis. As shown in Figure 4B, the pro-apoptotic protein, Bax, was up-regulated, and the antiapoptotic protein, Bcl-2, was down-regulated by 1. In addition, the expression of cleaved BID, known as t-BID, was increased in a concentration-dependent manner, which was correlated with the E

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Figure 6. Antitumor activity of spicatoside A (1) in the HCT116 xenograft model. (A) HCT116 cells (2 × 106 cells) were injected subcutaneously into the right flank of nude mice. Treatment was initiated when tumor volumes reached approximately ∼200 mm3. Compound 1 (2.5 or 5 mg/kg body weight) was administered intraperitoneally three times per week for 28 days. Tumor volumes were measured with a caliper every 3−4 days (*p < 0.05, **p < 0.01). (B) Body weights of the mice were monitored during the experiments for toxicity. (C) Tumors were excised from animals on day 28 after treatment, and tumor weights were measured. (D) Immunohistochemical analysis of Ki-67 in tumor tissue sections. Sections were counterstained with hematoxylin and observed under an inverted phase-contrast microscope (200× magnification). (E) Small portions of tumors from each group were homogenized in Complete Lysis Buffer (Active Motif). The expression levels of cleaved PARP were determined by Western blot analysis. β-Actin was used as an internal standard. The images shown are representative of three separate determinations.

activation of caspase-8 by 1. The cytosolic levels of cytochrome c were also increased in a concentration-dependent manner (Figure 4C). These findings suggested that the induction of apoptosis by 1 might be associated with the modulation of apoptotic regulatory proteins by triggering the caspase cascade. Spicatoside A (1) Induces the Switch from Autophagy to Apoptosis in HCT116 cells. Because treatment with 1 seems to induce autophagy in an early stage and apoptosis in a late stage in HCT116 cells, the time-dependent expression of autophagy- and apoptosis-specific proteins after treatment with 30 μM 1 was determined to confirm whether 1 induces autophagy and apoptosis sequentially. As a result, the induction of autophagy by 1, as indicated by the expression of LC3-II, LAMP1, and beclin 1, was gradually increased and reached a peak at 24 h, but the activation of apoptosis by 1, as indicated by the levels of cleaved caspase-8 and cleaved PARP, continued up to 48 h simultaneously (Figure 5A). These data suggest that the induction of autophagy and apoptosis by 1 is not associated with a successive process but a simultaneous approach in an early stage, and the prolonged treatment with 1 induces the switch from autophagy to apoptotic cell death.39 To confirm the role of autophagy in 1-induced cell death, 3-methyladenine (3-MA), a specific autophagy inhibitor, was used to block acidic vacuole formation. HCT116 cells were pretreated with 2 mM 3-MA for 1 h followed by a 24 h treatment with 30 μM 1. Although the 1induced LC3-II accumulation was suppressed by 3-MA, the levels of cleaved PARP were increased in cells cotreated with 1 and 3-MA compared to cells treated with 1 alone (Figure 5B). These data were consistent with the cell proliferation assay using 3-MA, which showed that 3-MA significantly enhanced the

inhibitory effect of 1 on HCT116 cell proliferation compared to treatment with 1 alone (Figure 5C). These findings suggested that inhibition of autophagy by 3-MA potentiates 1-mediated apoptotic cell death in human colorectal cancer cells, indicating that 1-induced autophagy may play a survival role during cellular stress. Accumulating evidence has suggested that autophagy and apoptosis can be interconnected with overlapping molecular regulators. A recent study has shown that caspase-8-mediated cleavage of beclin 1, which can enhance the release of cytochrome c, is a key mechanism of the switch from autophagy to apoptosis.26 To further elucidate the interconnection between autophagy and apoptosis in 1-associated cell death, a caspase-8 inhibitor, z-IETD-FMK, was employed to block the cleavage of beclin 1. As a result, the activated cleavage of caspase-8 and beclin 1 by 1 was blocked in the presence of z-IETD-FMK. Moreover, the LC3-II level was higher in cells cotreated with 1 and z-IETDFMK compared with 1 alone (Figure 5D). These findings suggested that the longer treatment of 1 may induce the switch from cytoprotective autophagy to apoptotic cell death by regulating caspase-8-mediated cleavage of beclin 1. Spicatoside A (1) Exhibits Antitumor Activity in a Tumor Xenograft Mouse Model. The in vivo efficacy of 1 was evaluated in a nude mouse xenograft model implanted with HCT116 human colorectal cancer cells (2 × 106 cells/mouse). When the tumor volume reached approximately 200 mm3, compound 1 (2.5 or 5 mg/kg) and irinotecan (10 mg/kg), a positive control, were administered intraperitoneally three times a week until the tumor volumes in the vehicle-treated control group were approximately 1900 mm3 on day 28 after treatment. F

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SRB in 1% acetic acid. The unbound dye was washed out, and the stained cells were dried and resuspended in 10 mM Tris (pH 10.0). The absorbance (515 nm) was measured, and cell proliferation was determined as follows: cell proliferation (%) = (average absorbancecompound − average absorbanceday zero)/(average absorbancecontrol − average absorbanceday zero) × 100. IC50 values were calculated by nonlinear regression analysis using TableCurve 2D v5.01 (Systat Software Inc., Richmond, CA, USA). Acridine Orange Staining. Cells (1 × 105 cells/well) were seeded in 12-well plates, incubated for 24 h, treated with various concentrations of 1 in the presence or absence of BaF, and cultured for an additional 24 h. To quantify the development of AVOs, the tumor cells were stained with acridine orange (2 μg/mL) for 15 min, washed with Dulbecco’s phosphate-buffered saline, observed, and quantified with an Operetta high content imaging system using Harmony High Content Imaging and Analysis software (PerkinElmer, Waltham, MA, USA). Western Blot Analysis. Total cell lysates were prepared in 2× sample loading buffer [250 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% β-mercaptoethanol, 50 mM sodium fluoride, and 5 mM sodium orthovanadate] and boiled for 5−20 min at 100 °C. The protein concentration of cell lysates was determined by the BCA method. Equal amounts (10−30 μg) of protein samples were subjected to 6−15% SDS-PAGE. Separated proteins were electrically transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were blocked with 5% BSA in PBS containing 0.1% Tween-20 for 1 h at room temperature and probed with the indicated antibodies. The blots were detected with an enhanced chemiluminescence detection kit (GE Healthcare, Little Chalfont, UK). Flow Cytometric Analysis of Cell Cycle Distribution. The analysis of cell cycle dynamics was performed by flow cytometry as described previously.40 Briefly, HCT116 cells were seeded at a density of 1 × 105 cells per 100 mm culture dish and cultured for 24 h. After treatment with 1 for 48 h, adherent and floating cells were harvested, washed twice with PBS, fixed with 100% methanol, and incubated with a staining solution [0.2% NP-40, 50 μg/mL propidium iodide, and 50 μg/ mL RNase A in phosphate-citrate buffer (pH 7.2)] for 30 min at room temperature. Fluorescence intensity was analyzed by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The percentages of the distributions in distinct cell cycle phases were determined using ModFIT LT V2.0 software. Annexin V-FITC/Propidium Iodide (PI) Staining. HCT116 cells were treated with 1 for 48 h and then stained with annexin V-FITC and PI using an annexin V-FITC apoptosis detection kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s recommendation. Briefly, cells were resuspended with 1× binding buffer at a density of 1 × 106 cells/mL. Annexin V-FITC and PI (5 μL) were added to cell suspensions (100 μL) and further incubated for 20 min at room temperature in the dark. Stained cells were diluted with 1× binding buffer and immediately analyzed by flow cytometry. In Vivo Tumor Xenograft Model. All animal use and care followed the guidelines approved by the Seoul National University Institutional Animal Care and Use Committee (IACUC; permission number: SNU130128-3). Male nude mice (4−6 weeks old, BALB/c-nu) were purchased from Central Laboratory Animal, Inc. (Seoul, Korea) and housed in the animal care facility at Seoul National University under pathogen-free conditions with a 12 h light−dark schedule. HCT116 cells were injected subcutaneously into the flanks of mice (2 × 106 cells in 200 μL of medium) using 27 gauge needles. Tumors were allowed to grow, and when the tumor volume reached approximately 200 mm3, the mice were randomized into vehicle control and treatment groups of five animals per group. Compound 1 (2.5 or 5 mg/kg body weight) was dissolved in 200 μL of vehicle solution (normal saline with 0.5% Tween 80) and administered intraperitoneally three times a week for 4 weeks. Irinotecan (10 mg/kg body weight), a positive reference control, was administered two times a week. The control group was treated with an equal volume of vehicle solution. Tumor volume was measured using a digital slide caliper and estimated according to the following formula: Tumor volume (mm3) = L × W × H/2, where L is the length, W is the width, and H is the height of the tumor. The experiment was terminated when the average tumor volume of the control group reached

Compared to the vehicle-treated control group, the tumor volumes in 1-administered groups with 2.5 and 5 mg/kg were significantly inhibited by 38.8% and 57.5%, respectively (p < 0.05; Figure 6A). No overt toxicity or change in body weight was observed in the treatment groups compared to the vehicletreated control group (Figure 6B). The inhibition rates of tumor weight with 2.5 and 5 mg/kg 1 compared to the vehicle control group were 43.0% and 56.6%, respectively (Figure 6C). In addition, an immunohistochemical analysis of tumor tissues with Ki-67 antibody showed that 1 inhibited the expression of Ki-67, a proliferation biomarker, in xenograft tumor cells (Figure 6D). The protein levels of cleaved PARP, which is associated with apoptotic cell death, were also increased in the tumors of the 1treated group (Figure 6E). These results confirmed that 1 effectively inhibits the tumor growth of colorectal cancer cells in vivo and that the antitumor activity of 1 may be, in part, associated with the induction of apoptosis. In summary, the present study demonstrated that spicatoside A (1) has antiproliferative and antitumor activities against human colorectal cancer cells both in vitro and in vivo. The underlying mechanisms by which 1 induces cancer cell death are associated with the modulation of crosstalk between autophagy and apoptosis. Further study revealed that the switch from autophagy to apoptosis by 1 is mediated by the cleavage of beclin 1 via activation of caspase-8 and that 1-induced autophagy acts as a prosurvival mechanism following subsequent 1-induced apoptosis. Taken together, spicatoside A (1) may be a promising chemotherapeutic agent for the management of human colon cancer.



EXPERIMENTAL SECTION

General Experimental Procedures. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, Lglutamine, antibiotic/antimycotic solution, and trypsin-EDTA were purchased from Invitrogen (Grand Island, NY, USA). Sulforhodamine B (SRB), acridine orange (AO), 3-methyladenine (3-MA), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, goat anti-goat IgG-HRP, p62, β-actin, ERK1/2, p-ERK1/2, p53, Bcl-2, Bax, and α-tubulin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against LC3, beclin 1, LAMP1, cathepsin D, PI3K (p85), PI3K (p110), PTEN, Akt, pAkt, mTOR, p38, p-p38, p-mTOR, caspase-3, caspase-8, caspase-9, BID, and COX IV were purchased from Cell Signaling Technology (Beverly, MA, USA). The annexin V-fluorescein isothiocyante (FITC) apoptosis detection kit and antibodies for p21, BIM, cytochrome c, and cleaved PARP were acquired from BD Biosciences (San Diego, CA, USA). The Ki-67 antibody was purchased from Abcam (Cambridge, MA, USA). ZIETD-FMK was purchased from Merck Millipore (Bedford, MA, USA). Spicatoside A (1; purity >98% by HPLC analysis) was isolated from a CH2Cl2-soluble extract of the roots of Liriope platyphylla as described previously.13 Cell Culture. Human lung cancer (A549), colorectal cancer (HCT116), breast cancer (MDA-MB-231), stomach cancer (SNU638), liver cancer (SK-HEP-1), and lung epithelial normal (MRC5) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in medium (RPMI 1640 for A549, HCT116, and SNU-638 cells; DMEM medium for MDA-MB231, SK-HEP-1, and MRC5 cells) supplemented with 10% heatinactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. Cells were incubated at 37 °C with 5% CO2 in a humidified atmosphere. Cell Proliferation Assay. Cells (6 × 103 cells/well) were seeded in 96-well plates, incubated for 24 h, and fixed (for day zero controls) or treated with the test compounds for 24 and 48 h. After incubation, cells were fixed with 10% trichloroacetic acid, dried, and stained with 0.4% G

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approximately 2000 mm3. The mice were euthanized, and the tumors were excised, weighed, and frozen for further biochemical analysis. Toxicity was assessed based on the lethality and body weight loss exhibited by the nude mice. Immunohistochemistry of Tumors. Excised tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Serial sections of the embedded specimens were deparaffinized, rehydrated, and subjected to antigen retrieval. The slides were incubated with an antiKi67 antibody, which was detected using the LSAB+ System-HRP kit (Dako, Glostrup, Denmark) and counterstained with hematoxylin. Stained sections were observed and photographed using an inverted phase-contrast microscope. Ex Vivo Biochemical Analysis of Tumors. A portion of the frozen excised tumor was thawed on ice and homogenized using a hand-held homogenizer in Complete Lysis Buffer (Active Motif, Carlsbad, CA, USA). The protein concentrations of the tumor lysates were determined, and aliquots were stored at −80 °C. Statistics. Data are presented as the means ± SD for the indicated number of independently performed experiments. Data are representative of at least three independent experiments. Statistical significance (**p < 0.01, *p < 0.05) was assessed using Student’s t-test or one-way analysis of variance (ANOVA) coupled with Dunnett’s t-test.



(15) Debatin, K. M. Cancer Immunol. Immunother. 2004, 53, 153−159. (16) Thornberry, N. A.; Lazebnik, Y. Science 1998, 281, 1312−1316. (17) Kothakota, S.; Azuma, T.; Reinhard, C.; Klippel, A.; Tang, J.; Chu, K.; McGarry, T. J.; Kirschner, M. W.; Koths, K.; Kwiatkowski, D. J.; Williams, L. T. Science 1997, 278, 294−298. (18) Li, H.; Zhu, H.; Xu, C. J.; Yuan, J. Cell 1998, 94, 491−501. (19) Sun, H.; Wang, Z.; Yakisich, J. S. Anti-Cancer Agents Med. Chem. 2013, 13, 1048−1056. (20) Bhutia, S. K.; Mukhopadhyay, S.; Sinha, N.; Das, D. N.; Panda, P. K.; Patra, S. K.; Maiti, T. K.; Mandal, M.; Dent, P.; Wang, X. Y.; Das, S. K.; Sarkar, D.; Fisher, P. B. Adv. Cancer Res. 2013, 118, 61−95. (21) Gozuacik, D.; Kimchi, A. Oncogene 2004, 23, 2891−2896. (22) White, E.; DiPaola, R. S. Clin. Cancer Res. 2009, 15, 5308−5316. (23) Amaravadi, R. K.; Yu, D.; Lum, J. J.; Bui, T.; Christophorou, M. A.; Evan, G. I.; Thomas-Tikhonenko, A.; Thompson, C. B. J. Clin. Invest. 2007, 117, 326−336. (24) Maiuri, M. C.; Zalckvar, E.; Kimchi, A.; Kroemer, G. Nat. Rev. Mol. Cell Biol. 2007, 8, 741−752. (25) Eisenberg-Lerner, A.; Bialik, S.; Simon, H. U.; Kimchi, A. Cell Death Differ. 2009, 16, 966−975. (26) Li, H.; Wang, P.; Sun, Q.; Ding, W. X.; Yin, X. M.; Sobol, R. W.; Stolz, D. B.; Yu, J.; Zhang, L. Cancer Res. 2011, 71, 3625−3634. (27) Funderburk, S. F.; Wang, Q. J.; Yue, Z. Trends Cell Biol. 2010, 20, 355−362. (28) Wirawan, E.; Vande Walle, L.; Kersse, K.; Cornelis, S.; Claerhout, S.; Vanoverberghe, I.; Roelandt, R.; De Rycke, R.; Verspurten, J.; Declercq, W.; Agostinis, P.; Vanden Berghe, T.; Lippens, S.; Vandenabeele, P. Cell Death Dis. 2010, 1, e18. (29) Stankiewicz, M.; Jonas, W.; Hadas, E.; Cabaj, W.; Douch, P. G. Int. J. Parasitol. 1996, 26, 445−446. (30) Mizushima, N.; Yoshimori, T. Autophagy 2007, 3, 542−545. (31) Rosen, N.; She, Q. B. Cancer Cell 2006, 10, 254−256. (32) Sun, Y.; Zou, M.; Hu, C.; Qin, Y.; Song, X.; Lu, N.; Guo, Q. Food Chem. Toxicol. 2013, 51, 53−60. (33) Dodd, K. M.; Tee, A. R. Am. J. Physiol. Endocrinol. Metab. 2012, 302, e1329−1342. (34) Sui, X.; Kong, N.; Ye, L.; Han, W.; Zhou, J.; Zhang, Q.; He, C.; Pan, H. Cancer Lett. 2014, 344, 174−179. (35) Feng, Z.; Zhang, H.; Levine, A. J.; Jin, S. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 8204−8209. (36) Stambolic, V.; MacPherson, D.; Sas, D.; Lin, Y.; Snow, B.; Jang, Y.; Benchimol, S.; Mak, T. W. Mol. Cell 2001, 8, 317−325. (37) Milner, J.; Allison, S. J. Cell Cycle 2011, 10, 3049. (38) Crighton, D.; Wilkinson, S.; O’Prey, J.; Syed, N.; Smith, P.; Harrison, P. R.; Gasco, M.; Garrone, O.; Crook, T.; Ryan, K. M. Cell 2006, 126, 121−134. (39) Panda, P. K.; Mukhopadhyay, S.; Das, D. N.; Sinha, N.; Naik, P. P.; Bhutia, S. K. Semin. Cell Dev. Biol. 2015, 39, 43−55. (40) Hong, J. Y.; Chung, H. J.; Lee, H. J.; Park, H. J.; Lee, S. K. J. Nat. Prod. 2011, 74, 2102−2108.

AUTHOR INFORMATION

Corresponding Author

*Tel (S. K. Lee): +82-2-880-2475. Fax: +82-2-762-8322. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 20120004939 and 2009-0083533) and a grant (12172MFDS989) from the Ministry of Food and Drug Safety in 2012.



REFERENCES

(1) Hur, J.; Lee, P.; Moon, E.; Kang, I.; Kim, S. H.; Oh, M. S.; Kim, S. Y. Eur. J. Pharmacol. 2009, 620, 9−15. (2) Lee, Y. C.; Lee, J. C.; Seo, Y. B.; Kook, Y. B. J. Ethnopharmacol. 2005, 101, 144−152. (3) Hur, J.; Lee, P.; Kim, J.; Kim, A. J.; Kim, H.; Kim, S. Y. Biol. Pharm. Bull. 2004, 27, 1257−1260. (4) Kwak, M. H.; Kim, J. E.; Hwang, I. S.; Lee, Y. J.; An, B. S.; Hong, J. T.; Lee, S. H.; Hwang, D. Y. J. Ethnopharmacol. 2013, 148, 880−889. (5) Tsai, Y. C.; Chiang, S. Y.; El-Shazly, M.; Wu, C. C.; Beerhues, L.; Lai, W. C.; Wu, S. F.; Yen, M. H.; Wu, Y. C.; Chang, F. R. Food Chem. 2013, 140, 305−14. (6) Sun, K.; Cao, S.; Pei, L.; Matsuura, A.; Xiang, L.; Qi, J. Int. J. Mol. Sci. 2013, 14, 4461−4475. (7) Man, S.; Gao, W.; Zhang, Y.; Huang, L.; Liu, C. Fitoterapia 2010, 81, 703−714. (8) Sy, L. K.; Yan, S. C.; Lok, C. N.; Man, R. Y.; Che, C. M. Cancer Res. 2008, 68, 10229−10237. (9) Wei, S.; Fukuhara, H.; Chen, G.; Kawada, C.; Kurabayashi, A.; Furihata, M.; Inoue, K.; Shuin, T. Pathobiology 2014, 81, 123−132. (10) Wang, Y.; Tang, Q.; Jiang, S.; Li, M.; Wang, X. Biochem. Biophys. Res. Commun. 2013, 441, 825−830. (11) Lim, H.; Min, D. S.; Kang, Y.; Kim, H. W.; Son, K. H.; Kim, H. P. Arch. Pharmacal Res. 2015, 38, 1108−1116. (12) Kwon, G.; Lee, H. E.; Lee, D. H.; Woo, H.; Park, S. J.; Gao, Q.; Ahn, Y. J.; Son, K. H.; Ryu, J. H. Neurosci. Lett. 2014, 572, 58−62. (13) Park, S. H.; Lee, H. J.; Ryu, J.; Son, K. H.; Kwon, S. Y.; Lee, S. K.; Kim, Y. S.; Hong, J. H.; Seok, J. H.; Lee, C. J. Phytomedicine 2014, 21, 172−176. (14) Adams, J. M.; Cory, S. Oncogene 2007, 26, 1324−1337. H

DOI: 10.1021/acs.jnatprod.6b00006 J. Nat. Prod. XXXX, XXX, XXX−XXX