Antitumor Activity of Americanin A Isolated from the ... - ACS Publications

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Antitumor Activity of Americanin A Isolated from the Seeds of Phytolacca americana by Regulating the ATM/ATR Signaling Pathway and the Skp2−p27 Axis in Human Colon Cancer Cells Cholomi Jung, Ji-Young Hong, Song Yi Bae, Sam Sik Kang, Hyen Joo Park, and Sang Kook Lee* College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742, Korea ABSTRACT: The antiproliferative and antitumor activities of americanin A (1), a neolignan isolated from the seeds of Phytolacca americana, were investigated in human colon cancer cells. Compound 1 inhibited the proliferation of HCT116 human colon cancer cells both in vitro and in vivo. The induction of G2/ M cell-cycle arrest by 1 was concomitant with regulation of the ataxia telangiectasia-mutated/ATM and Rad3-related (ATM/ ATR) signaling pathway. Treatment with 1 activated ATM and ATR, initiating the subsequent signal transduction cascades that include checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and tumor suppressor p53. Another line of evidence underlined the significance of 1 in regulation of the S phase kinase-associated protein 2 (Skp2)−p27 axis. Compound 1 targeted selectively Skp2 for degradation and thereby stabilized p27. Therefore, compound 1 suppressed the activity of cyclin B1 and its partner cell division cycle 2 (cdc2) to prevent entry into mitosis. Furthermore, prolonged treatment with 1 induced apoptosis by producing excessive reactive oxygen species. The intraperitoneal administration of 1 inhibited the growth of HCT116 tumor xenografts in nude mice without any overt toxicity. Modulation of the ATM/ATR signaling pathway and the Skp2−p27 axis might be plausible mechanisms of action for the antiproliferative and antitumor activities of 1 in human colon cancer cells.

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olon cancer is one of the best-understood cancers, yet almost one million patients are diagnosed with colon cancer and half a million deaths from this neoplasm occur annually worldwide.1 The statistics imply that while the current understanding of colon cancer provides a basis for improved prognosis, treatment of colon cancer remains a challenge, and adverse effects and the emergence of resistance also remain major concerns.2,3 Therefore, the ongoing exploration of natural products as leads is indispensable in the development of novel approaches to prevent and treat colon cancer. In this study, naturally occurring americanin A (1) isolated from the seeds of Phytolacca americana (Phytolaccaceae) was first identified as a promising antitumor agent, and its underlying mechanisms of action were investigated in HCT116 human colon cancer cells.4 P. americana, a herbaceous perennial plant native to North and South America, has a history of medicinal use in treating breast and gastrointestinal problems, inflammation, and skin rashes.5,6 In earlier reports, emphasis was confined chiefly to the identification of active compounds or biological activities of the extracts of P. americana. The extracts of P. americana exhibit antibacterial, anticarcinogenic, anti-inflammatory, antioxidant, and antiviral activities.7−10 Only a few studies have noted that 1 possesses acetyltransferase, anti-inflammatory, antimelanogenic, and antioxidant activities.11−15 However, the antitumor activity of 1 and its molecular mechanisms have not yet been reported. The cell cycle is a highly ordered sequence of events involving the growth and division of a eukaryotic cell. The mechanisms that © XXXX American Chemical Society and American Society of Pharmacognosy

monitor this orderly progression are called cell-cycle checkpoints.16 Of the checkpoints, the DNA damage-induced G2/M checkpoint serves to ensure the fidelity of genomic information.17 Although defects in this checkpoint may result in genomic instability, cell death, and carcinogenesis, they are often employed as a potential therapeutic intervention to evaluate the efficacy of anticancer agents from natural products against cancer cells.18 The initiation of a G2/M checkpoint that requires activation of ataxia telangiectasia (ATM) and ATM- and Rad3related (ATR) kinase in response to DNA damage may be an important target in cancer chemotherapy.19 Upon DNA damage, ATM and ATR are activated and elicit the subsequent signal transduction cascades that include checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2).20 While Chk1 and Chk2 regulate the activity of their shared downstream substrate, cell division cycle 25C (cdc25C), Chk2 regulates stabilization and Received: August 19, 2015

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DOI: 10.1021/acs.jnatprod.5b00743 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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activity of 1 was additionally evaluated in other colon cancer cell lines (SW480, HCT-15, and RKO). As shown in Table 2, 1 exerted potential growth inhibition against all of the tested colon cancer cells (IC50; 9.0 to 27.4 μM).

activation of both tumor suppressor p53 and its negative regulator mouse double minute 2 (MDM2), which are associated with aberrant proliferation of cancer cells.21,22 Impact on the activity of cdc25C results in the induction of G2/M cell-cycle arrest through its direct effects on the activities of its corresponding cyclins and cyclin-dependent kinases (CDKs).23,24 The ubiquitin-proteasome system (UPS) is the mainstay of protein turnover control, which serves as a central node of regulation of cell proliferation, survival, and differentiation.25 The rate-limiting step of UPS is based on substrate recognition by highly selective E3 ubiquitin ligases, and abnormal activities may contribute to cancer development and progression.26,27 In particular, the Skp1-Cullin-1-F-box-protein (SCF) E3 ubiquitin ligase plays crucial roles both in normal cell-cycle control and in cancer development and progression.28 The F-box protein S phase kinase-associated 2 (Skp2) is a substrate-specific component of the SCF E3 ubiquitin ligase and is of considerable interest as an oncogene that governs G2/M transition via regulation of CDK inhibitor p27.29−32 Overexpression of Skp2 is well-correlated not only with tumor malignancy in human cancers, particularly in breast and colon cancer, but also with loss of CDK inhibitor p27 levels.33−35 It is, therefore, necessary to gain a better understanding of Skp2 and its role in cell-cycle control and cell proliferation, because this will eventually lead to the development of a novel class of anticancer agents with enhanced specificity. In the present study, the antiproliferative and antitumor potential of 1 and its underlying molecular mechanisms against HCT116 human colon cancer cells were investigated under both in vitro and in vivo conditions.

Table 2. Inhibitory Effects of Americanin A (1) on the Proliferation of Human Colon Cancer Cells IC50 (μM)a

IC50 (μM)a A549 Caki-1 K562 HCT116 MRC-5

etoposidec

39.1 66.4 12.9 9.0 >100

0.8 1.1 6.7 3.3 >20

HCT116 SW480 HCT-15 RKO

9.0 27.4 12.5 24.4

2.0 12.3 8.5 6.2

In addition, the isolated compounds (Chart 1) from the seeds of P. americana were evaluated for antiproliferative activity in HCT116 cells.36,37 Compounds 4 and 5 showed promising antiproliferative activities with IC50 values of 4.0 and 5.6 μM, respectively. However, 2 and 3 moderately suppressed the growth of HCT116 cells, whereas 6 and 7 did not possess any significant antiproliferative activities against the HCT116 cell line (Table 3). Although 4 and 5 were more sensitive than was 1 to the HCT116 cell line, 1 was the most abundant constituent of P. americana. Therefore, further investigation on the potential mechanisms of action of 1 in the regulation of cancer cell proliferation was carried out using the HCT116 cell line. Since the antiproliferative activity of 1 was observed against the HCT116 cell line for 72 h, inhibitory effects of 1 on HCT116 cell growth for 24 and 48 h were also investigated. As a result, 1 inhibited the growth of HCT116 cells in a concentration- and time-dependent manner (Figure 1A). After 24 h of treatment with 1, a morphological change of HCT116 cells was detected under phase-contrast microscopy (Figure 1B). The untreated cells maintained their original morphology, whereas HCT116 cells exposed to 30 μM 1 became rounded and lost contact with adjacent cells and with the surface of the culture dish. These morphological changes became even more pronounced after 48 h of treatment with 1 (data not shown). To verify that the antiproliferative activity of 1 in HCT116 cells corresponds to regulation of the cell cycle, cell-cycle analysis using propidium iodide (PI) staining was performed. In response to 24 h treatment with 1 (30 μM), about 38.9% of the cells were in G2/M, characterized by 4N DNA content. In contrast, only 20.6% of untreated cells were found in G2/M (Figure 2A). Even though the proportion of sub-G1 cells, an indicator of cellular apoptosis, increased in a concentration-dependent manner, it was not a significant event for 24 h treatment with 1. This finding suggests that 1 induces G2/M cell-cycle arrest without obvious apoptotic cell death. Cyclins are a family of proteins that control the passage of a cell through the cell cycle via activation of their corresponding CDKs, and putative biomarkers for the G2/M phase are cyclins A and B1, cdc2, and CDK2.38,39 Therefore, inhibitory effects of 1 on these biomarkers’ expression were determined by Western blot analysis. As shown in Figure 2B, treatment with 1 led to a significant suppression of these proteins. Furthermore, the expression of phosphorylated cyclin B1 at Ser147 was also

Table 1. Inhibitory Effects of Americanin A (1) on the Proliferation of Human Cancer and Normal Cells americanin A (1)

irinotecanb

Results are expressed as the calculated half-maximal inhibitory concentration of 1 and irinotecan (μM). Data represent mean values ± SD from three independent studies. bIrinotecan was used as a positive control.

RESULTS AND DISCUSSION To determine whether americanin A (1) exhibits the potential growth inhibition of human cancer cells, the antiproliferative activity of 1 was primarily evaluated in a panel of human cancer and normal cells. As summarized in Table 1, 1 inhibited the

cell line

americanin A (1)

a

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b

cell line

a

Results are expressed as the calculated half-maximal inhibitory concentration of 1 and etoposide (μM). Data represent mean values ± SD from three independent studies. bHuman cancer and normal cell lines were A549 (lung), Caki-1 (renal), K562 (erythromyelobalsoid leukemia), HCT116 (colon), and MRC-5 (fetal lung fibroblast). c Etoposide was used as a positive control.

growth of human cancer cells with IC50 values ranging from 9.0 to 66.4 μM. While the considerable growth-inhibitory activity of 1 was found against these human cancer cell lines, 1 did not exhibit a potential growth inhibition against MRC-5 human normal lung epithelial cells (IC50 > 100 μM), suggesting that 1 might selectively inhibit the proliferation of human cancer cells. Since compound 1 exhibited the most growth inhibitory activity against human HCT-116 colon cancer cells, the antiproliferative B



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Chart 1. Chemical Structures of the Isolates

Table 3. Inhibitory Effects of Americanin A (1) and the Isolates (2−7) on the Proliferation of HCT116 Cells compound

IC50 (μM)a

1 2 3 4 5 6 7 etoposideb

7.6 10.9 10.4 4.0 5.6 >100 >100 0.4

a

Results are expressed as the calculated half-maximal inhibitory concentration of 1 or etoposide (μM). Data represent mean values ± SD from three independent studies. bEtoposide was used as a positive control. Figure 1. Effects of americanin A (1) on the growth of HCT116 human colon cancer cells. (A) HCT116 cells were cultivated in a 96-well plate and treated with the indicated concentrations of 1 for 24−72 h. Cell proliferation was then determined by SRB assay. (B) The cells were treated with the indicated concentrations of 1 for 24 h. Cell morphology was observed and photographed under a phase-contrast microscope.

suppressed by 1. Several studies have reported that activation of the cyclin B1−cdc2 complex, whose role is to promote nuclear entry of cyclin B1, first requires phosphorylation of cyclin B1 on serine residues. However, there is still a considerable debate over whether phosphorylation of cyclin B1 at Ser147 and/or Ser133 is important for the nuclear translocation of cyclin B1 during prophase.40,41 The role of these individual sites of cyclin B1 still remains unclear, but these findings suggest that 1 prompts G2/M cell-cycle arrest by modulating cell-cycle regulators in HCT116 cells. An increasing body of evidence has reported that G2/M cellcycle arrest is often accompanied by DNA damage.42 Thus, the expressions of DNA damage-responsive histone proteins were determined by Western blot analysis. As shown in Figure 3A, histone H2A.X, which is the most sensitive biomarker detected in the presence of DNA damage, was exceedingly phosphorylated at Ser139 by treatment with 1.43 The expression of the acetylated histone H3 at Lys9 was decreased significantly by 1,

demonstrating the potential role of 1 in causing damage to DNA.44 The regulation of DNA damage surveillance pathways was, therefore, explored to elucidate the molecular mechanisms of 1. Of the DNA damage surveillance pathways, the ATM/ATR signaling pathway acts in an early signal transmission through a G2/M checkpoint by trans- or autophosphorylation on serine residues.45−47 On the basis of this information, a time-course analysis of the expression of ATM, ATR, and their activated forms was performed by Western blot analysis. The expression of ATM, ATR, and their activated forms was found to be upregulated at 1 h, but expression was time-dependently suppressed C



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Figure 2. Effect of 1 on the regulation of cell-cycle progression. HCT116 cells were treated with 1 for 24 h. (A) After being fixed with 70% cold ethanol overnight, the cells were then incubated with RNase A and PI for 30 min, and the cell-cycle distribution was analyzed by flow cytometry. (B) The expression of cell-cycle regulatory proteins was determined by Western blot analysis. β-Actin was used as an internal control.

Figure 3. Effect of 1 on the regulation of the ATM/ATR signaling pathway. (A) HCT116 cells were treated with 1 for 24 h, and the expression of DNA damage-responsive histone proteins was determined by Western blot analysis. (B) The cells were treated with 1 for 0−24 h, and the expression of ATM and ATR was determined by Western blot analysis. (C) The cells were treated with 1 for 24 h, and the expression of the downstream targets of ATM and ATR was determined by Western blot analysis.

after 1 h (Figure 3B). To this end, the effect of 1 on the expressions of ATM, ATR, and their activated forms was investigated after 1 h treatment with 1. These expressions were increased by 1 in a concentration-dependent manner. Further study was conducted to correlate effects of 1 with the regulation of the downstream targets Chk1 and Chk2. Chk1 and Chk2 are

largely responsible for the induction of the G2/M checkpoint and, thus, for the regulation of cancer cell proliferation.48,49 In response to DNA damage, ATM and ATR sequentially activate Chk1 and Chk2 by phosphorylation on specific residues, leading to the suppression of cdc25C.50,51 As shown in Figure 3C, compound 1 enhanced phosphorylation of Chk1 and Chk2 on D



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Figure 4. Effect of 1 on the regulation of the p53-MDM2 feedback loop and dependence on p53. (A) HCT116 cells were treated with 1 for 24 h, and the expressions of p53 and MDM2 were determined by Western blot analysis. (B) p53 wild-type and p53-null HCT116 cells were treated with 1 for 24 h, and the expressions of p53 and MDM2 were determined by Western blot analysis. (C) HCT116 cells were treated with 1 for 24 h, and total cell lysates were immunoprecipitated with an anti-p53 antibody. Protein expression was determined by Western blot analysis. (D) p53 wild-type and p53-null HCT116 cells were cultivated in a 96-well plate and treated with the indicated concentrations of 1 for 72 h. Cell proliferation was then determined by SRB assay.

inhibits cell-cycle progression mostly through p53-independent ATM/ATR signaling. Since down-regulation of cyclins and their CDKs was detected previously, effects of 1 on CDK inhibitors p21 and p27 were investigated further by Western blot analysis. As shown in Figure 5A, compound 1 up-regulated the expressions of p21 and p27 in

serine and threonine residues, respectively, and down-regulated the activity of cdc25C in a concentration-dependent manner. These data indicate that DNA damage induced by 1 might lead to activation of ATM and ATR and initiate the subsequent signal transduction cascade associated with the G2/M checkpoint. Tumor suppressor p53 is another important substrate of Chk2 that controls a G2/M transition.52 Because the significance of p53 in cancer development and progression has been accepted widely, p53 is, therefore, considered an important therapeutic target. However, it is negatively regulated by MDM2 either through the direct binding to p53 to prohibit the transcriptional activity of p53 or through the degradation of p53 by UPS.53−57 As shown in Figure 4A, compound 1 up-regulated the expressions of both p53 and MDM2. This conflicting result has prompted further assessment of the role of MDM2 in the inhibition of cancer cell growth. Regardless of p53 status, compound 1 up-regulated the expression of MDM2 (Figure 4B). This result suggests that MDM2 might inhibit the proliferation of HCT116 cells independently of p53, but further studies are still needed to obtain a full understanding of the underlying molecular mechanisms of MDM2. In addition, the binding of p53 to MDM2 was analyzed by immunoprecipitation assay. As shown in Figure 4C, treatment with 1 resulted in a disrupted interaction of p53 and MDM2, thereby stabilizing p53. On the basis of these findings, the inhibitory effect on p53 wild-type and p53-null HCT116 cells was determined. The cells were treated with 1 for 72 h, and cell viability was measured by SRB staining. The IC50 values of 1 were 8.3 μM for p53 wild-type and 7.8 μM for p53 null, suggesting a negligible difference (Figure 4D). These data demonstrate that while stabilization of p53 might contribute partly to the inhibition of HCT116 cell growth, 1

Figure 5. Effect of 1 on the regulation of the Skp2−p27 axis. HCT116 cells were treated with 1 for 24 h prior to analysis by Western blot for the expression of the CDK inhibitors p21 and p27. (A) and components of the SCF E3 ubiquitin ligase (B). Total cell lysates were immunoprecipitated with an anti-Cul-1 antibody (C) or an anti-Skp2 antibody (D), and protein expression was determined by Western blot analysis. E



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Figure 6. Effect of 1 on the induction of apoptosis. HCT116 cells were treated with 1 for 48 h, fixed with 70% cold ethanol overnight, and incubated with RNase A and PI for 30 min (A) or annexin V/FITC and PI (B). The cell-cycle distribution was then analyzed by flow cytometry. (C) The expressions of apoptosis-related proteins was determined by Western blot analysis.

Instead, compound 1 may act as a small-molecule inhibitor to down-regulate selectively the expression of Skp2 to stabilize p27 and prevent the cells from entering mitosis. Several studies have reported that cells tend to allow time for DNA repair prior to resuming mitosis.64 Hence, a cell-cycle analysis using PI staining was performed to evaluate whether 1 elicits cellular apoptosis, which might correlate with the inhibition of cancer cell growth. While there was not any specific cell-cycle arrest in response to 48 h treatment with 1, an accumulation of cells in sub-G1 was observed. The proportion of sub-G1 cells was increased from 6.5% to 52.0% compared to the untreated cells (Figure 6A). In addition, flow cytometry using annexin V/flourescein isothicyanate (annexin V/FITC) and PI staining showed that the cells that were treated with 1 were detected in early and late apoptotic stages (Figure 6B). Based on these flow cytometric data, 1 may induce apoptosis. Activation of caspases is considered a required step for the execution of apoptosis, thereby serving as a potential cancer chemotherapeutic target for colon cancer.65 As shown in Figure 6C, the caspase family members and poly(ADP-ribose) polymerase (PARP) were present in cleaved forms after a 48 h treatment with 1. Moreover, compound 1 modulated the expressions of the B cell lymphoma 2 (Bcl-2) family members. The expressions of antiapoptotic proteins such as Bcl-2 and BclxL were down-regulated, while that of Bcl-2 interacting mediator (Bim) was increased in a concentration-dependent manner. In previous findings, intracellular reactive oxygen species (ROS) have long been implicated in the induction of apoptosis.66 Thus, flow cytometry using DCFH-DA staining was performed to verify whether 1 promotes ROS-dependent apoptosis. Compound 1 produced intracellular ROS in a concentrationdependent manner (Figure 7A). In particular, 30 μM 1 resulted in a significantly increased intracellular ROS level that was

a concentration-dependent manner. In addition, the decrease in phosphorylated p27 at Thr187 was also partially associated with the regulation of UPS. Since the turnover rate of p27 is determined predominantly by Skp2,58 additional experiments were conducted to determine whether 1 regulates the expression of Skp2. A decrease in the expression of Skp2 was observed after treatment with 1 (Figure 5B). In addition, the expression of other key components of the SCF E3 ubiquitin ligase was affected by 1. The expression of the Cul-1 backbone was increased, while that of Skp1 was decreased by treatment with 1. The relationship between the structure of the SCF E3 ubiquitin ligase and its E3 activity is that the rigidity of the Cul-1 scaffold is required.59 Thus, it is plausible that 1 may interfere with the interaction of Cul-1 and Skp1 to restore p27 levels, because Cul-1 physically interacts with Skp1.60 Nonetheless, compound 1 showed no change in the interaction of Cul-1 and Skp1 (Figure 5C). Recent studies demonstrated that Cul-1 may function as a tumor suppressor by regulating Polo-like kinase 4 (PLK4) protein and thereby restrain centriole multiplication.61,62 Therefore, the present findings with the increase of Cul-1 expression by 1 might be in part associated with the induction of G2/M cell-cycle arrest by suppressing centriole multiplication, but the detailed mechanism of action needs to be further clarified. Recent studies have documented that novel inhibitors of the Skp1−Skp2 interface have been identified and that these inhibitors induce accumulation of p27 to promote cell-cycle arrest.63 However, the binding of Skp2 to Skp1 was not affected by 1 (Figure 5D). Therefore, the inhibitory effect of 1 on the interaction of Skp2 and p27 was explored. However, treatment with 1 had no effect on the inhibition of the Skp2−p27 interaction. These results suggest that 1 might increase the stability of p27 independently of physically manipulating the assembly of the components of the SCF E3 ubiquitin ligase. F



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Figure 7. Effect of 1 on the production of ROS. HCT116 cells were treated with 1 and/or NAC for 48 h. The cells were stained with DCFH-DA (A) or annexin V/FITC and PI (B) and then analyzed by a flow cytometry. (C) The expression of apoptosis-related proteins was determined by Western blot analysis.

elevated by a factor of approximately 10 compared to that of the untreated cells. However, cotreatment with 1 and Nacetylcysteine (NAC) reduced intracellular ROS levels. NAC, a free radical scavenger, was used to evaluate further the significance of 1 in the induction of apoptosis.67 While treatment with 1 resulted in an increased population of apoptotic cells, cotreatment with 1 and NAC attenuated this effect and rescued the cells from apoptosis (Figure 7B). On the basis of Western blot analysis, activation of the apoptotic cascade manifested by caspases and PARP was blocked by cotreatment with 1 and NAC (Figure 7C). These results demonstrate that a plausible mechanism of action for 1-mediated apoptosis involves excessive production of intracellular ROS. To demonstrate the in vivo efficacy of 1, a tumor xenograft model was engaged. Nude mice bearing HCT116 tumor xenografts were randomized into groups and received the intraperitoneal (ip) administration of either the vehicle or 1 (5 or 10 mg/kg). Irinotecan, a widely used clinical chemotherapeutic agent, was used as a reference compound.47 As shown in Figure 8A and B, the tumor volumes in 1-administered groups with 5 and 10 mg/kg were significantly reduced by 45.0% and 55.7%, respectively, at the termination of the experiment on day 41. The difference in the tumor volumes was statically significant throughout the experiment. There was neither overt toxicity nor noticeable change in the body weight of nude mice seen in 1administered groups compared to the vehicle group (Figure 8C). These results confirm that 1 inhibits the tumor growth of human colon cancer cells in vivo.

In addition, biochemical analyses of extirpated tumor xenografts were further conducted to substantiate the role of 1 in the Skp2−p27 axis. These experiments revealed an increase in the expression of CDK inhibitors p21 and p27, whereas a decrease in the expression of Skp2 was detected in the tumors of mice administered 1 (Figure 8D). Consistent with previous observations, immunohistochemical analyses showed that staining of p27 was much pronounced, while staining of Skp2 and of a proliferation biomarker Ki-67 was deminished in the tumors of mice that received 1 compared to the tumors of control mice (Figure 8E). Collectively, these results suggest that 1 suppresses the tumor growth of HCT116 tumor xenografts in vivo in association with the induction of p27 in a Skp2-dependent manner. In summary, the present study demonstrates the potent antiproliferative and antitumor activities of americanin A (1) against human colon cancer cells both in vitro and in vivo. While the use of high concentrations of available anticancer drugs is limited because of their cytotoxicity against the surrounding tissues, 1 may selectively inhibit the proliferation of cancer cells, particularly of colon cancer cells, via the induction of G2/M cellcycle arrest followed by apoptosis. The mechanisms by which 1 induces DNA damage to activate ATM and ATR and stabilize p27 in a Skp2-dependent manner may act in synergy to facilitate the specific targeting of colon cancer cells (Scheme 1). To date, there has been only one report of an FDA-approved smallmolecule inhibitor (bortezomib) that regulates the expression of Skp2 in human colon cancer cells.68,69 Therefore, this study is G



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Figure 8. Effect of 1 on the growth of HCT116 tumor xenografts in vivo. Nude mice were challenged subcutaneously with HCT116 cells. As the tumor xenografts reached a volume of approximately 120 mm3, they were randomly assigned to receive ip administration of the vehicle, irinotecan, or 1. (A) The tumor volume was monitored and measured twice a week. (B) The volume of each tumor xenograft was measured at the termination of the experiment on day 41. (C) The body weight was monitored and measured twice a week. (D) Excised tumor xenografts were homogenized, and the expressions of p21, p27, and Skp2 were determined by Western blotting. β-Actin was used as an internal control. (E) Formalin-fixed, paraffin-embedded tumor sections were probed with the desired antibodies and photographed under an inverted phase-contrast microscope.

significant not only because it is the first report of the antitumor activity of 1 and its molecular mechanisms proposed for the first time but also because the Skp2−p27 axis may open up a new avenue of cancer treatment. Taken together, americanin A (1) might be a promising chemotherapeutic agent in colon cancer treatment.

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Anti-histone H3, anti-acetyl-histone H3 (K9), anti-p-histone H2A.X (Ser139), anti-ATM, anti-p-ATM (Ser1981), anti-ATR, anti-p-ATR (Ser428), anti-Chk1, anti-p-Chk1 (Ser345), anti-Chk2, anti-p-Chk2 (Thr68), anti-cdc25C, anti-p-cyclin B1 (Ser147), anti-Skp1, anti-Skp2, anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-cleaved caspase-3, and anti-Bid were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p53, anti-MDM2, anti-cyclin A, anti-cyclin B1, antiCDK2, anti-cdc2 p34, anti-p21, anti-p27, anti-p-p27 (Thr187), anti-Cul1, anti-Bcl-2, anti-Bcl-xL, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Technology (Santa Cruz, CA, USA). AntiPARP, anti-cleaved PARP, anti-Bim, and annexin V/FITC apoptosis detection kit I were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-Ki-67 was purchased from Abcam (Cambridge, MA, USA). Americanin A (1; purity >94.1% by HPLC analysis) and the isolates (2− 7) were isolated from the seeds of Phytolacca americana (Phytolaccaceae) as previously described and were kindly provided by Prof. Sam Sik Kang of Seoul National University, Korea.4,36,37 The stock solutions were dissolved in 100% DMSO. Cell Culture. HCT116, A549, K562, Caki-1, SW480, HCT-15, RKO, and MRC-5 cells were purchased from American Type Culture Collection (Manassas, VA, USA). HCT116, A549, Caki-1, K562, HCT15, SW480, and RKO cells were maintained as a monolayer in RPMI 1640 medium, while MRC-5 cells were cultured in MEM medium supplemented with 10% FBS and antibiotic−antimycotic solution (PSF;

EXPERIMENTAL SECTION

General Experimental Procedures. Roswell Park Memorial Institute (RPMI) 1640 medium, minimal essential medium (MEM), fetal bovine serum (FBS), antibiotic−antimycotic solution (100×), trypsin-EDTA solution (1×), and phosphate-buffered saline (PBS; 1×) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Laemmli sample buffer (2×) and 2-mercaptomethanol were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), copper(II) sulfate solution, bovine serum albumin (BSA), trichloroacetic acid (TCA), SRB, Bradford reagent, 2′,7′-dichlrodihydrofluorescein diacetate (DCFH-DA), NAC, ribonuclease A (Rnase A), irinotecan, and camptothecin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein G sepharose beads were purchased from GE Healthcare Life Sciences (Little Chalfront, UK). Complete lysis buffer from nuclear extract kit was purchased from Active Motif, Inc. (Carlsbad, CA, USA). H



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Tween 20 (TBST; 1×) for half an hour at RT and incubated with the desired anti-antibodies diluted in 2.5% BSA in TBST overnight at 4 °C. The membranes were washed three times for 10 min each with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at RT. The membranes were washed three times for 10 min each with TBST and then detected with an enhanced chemiluminscence detection kit from GE Healthcare and an LAS-4000 Imager (Fuji Film Corp., Tokyo, Japan). Immunoprecipitation. HCT116 cells were treated with 1 for 24 h and lysed in immunoprecipitation (IP) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40; pH 7.4). The quantitative determination of total protein concentration of each group was done by the Bradford protein assay. Equivalent levels of protein from each group were incubated with the corresponding secondary antibodies on a rocking platform for an hour at 4 °C and were incubated with washed Protein G sepharose beads on a rocking platform for half an hour at 4 °C, and incubated with the desired anti-antibodies overnight on a rocking platform at 4 °C. Protein G sepharose beads were then washed in IP lysis buffer several times, and the desired protein was eluted from the beads by adding a mixture of Laemmli sample buffer and 2-mercaptoethanol and boiling in a heat block for 5 min. The elution samples were then subjected to Western blotting. Annexin V/FITC and PI Staining. HCT116 cells were treated with test compounds for 48 h in complete medium. The cells were then harvested and washed twice in cold PBS. The cells were in 1× binding buffer containing annexin V/FITC and PI in the dark for 15 min. The stained cells were analyzed by a FACSCalibur flow cytometer from BD Biosciences. Annexin V/FITC and PI staining of 20 000 cells from each group was analyzed, and the results are demonstrated as quadrants using CELLQuest 3.0.1 software from BD Biosciences. DCFH-DA Staining. HCT116 cells were cultivated in a flat six-well plate overnight and treated with test compounds for 48 h in complete medium. The cells were then incubated with 20 μM DCFH-DA for 30 min. The stained cells were analyzed by a FACSCalibur flow cytometer from BD Biosciences. Cellular ROS production in 20 000 cells from each group was analyzed, and the results are demonstrated as histograms using CELLQuest 3.0.1 software from BD Biosciences. In Vivo Tumor Xenograft Model. The use and care of animals was carried out in strict accordance with the guidelines by Seoul National University Institutional Animal Care and Use Committees (IACUC; permission number SNU-140928-2). Four- to 6-week-old BALB/c-nu female nude mice were purchased from Central Laboratory Animal, Inc. (Seoul, Korea) and housed in the animal care facility at Seoul National University under specific pathogen-free (SPF) conditions with a 12 h light−dark schedule. After the mice were acclimated for a week, HCT116 cells (2 × 106 cells in 200 μL of medium) were injected subcutaneously into the flanks of mice using 27-gauge needles, and tumors were allowed to develop until they reached approximately 120 mm3. Mice were randomly sorted into the vehicle group or treatment groups of six mice per group, and administration was initiated. The 1administered groups received the ip administration at doses of 5 and 10 mg/kg body weight in a volume of 200 μL three times a week. The irinotecan-administered group received ip administration at a dose of 10 mg/kg body weight in a volume of 200 μL once a week. All compounds were dissolved in 0.5% Tween 80 in normal saline. Tumor growth was measured using a digital slide caliper, and the volumes were 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. At the termination of the experiment on day 41, tumors were excised, weighed, and frozen for further analyses. Toxicity was assessed by body weight loss. Ex Vivo Biochemical Analysis. A portion of frozen tumors excised from nude mice was thawed on ice and homogenized using a hand-held homogenizer in complete lysis buffer from Active Motif. Aliquots were stored at −80 °C, while tumor lysates were subjected to Bradford protein assay and Western blotting. Immunohistochemical Analysis. Excised tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Serial section slides of the embedded specimens were deparaffinized, rehydrated, and subjected to antigen retrieval. Slides were incubated with the desired

Scheme 1. Schematic Representation of the Mechanisms of Action of 1 against Human Colon Cancer Cells

100 units/mL penicillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL amphothericin B). Cell Proliferation Assay. Cells were cultivated in a flat 96-well plate with test compounds for the desired treatment times (24−72 h). The cells were then fixed with 10% TCA solution for half an hour at 4 °C and stained in 0.4% SRB dissolved in 1% acetic acid solution for an hour at RT. After unbound SRB was removed by washing with 1% acetic acid solution, the stained cells were air-dried and dissolved in 10 mM Tris buffer (pH 10.0). Absorbance was measured at 515 nm, and cell viability was expressed as a percentage of solvent-treated control (100%). The IC50 values were calculated by a nonlinear regression analysis using TableCurve 2D v5.01 software from Systat Software Inc. (Richmond, CA, USA). Cell-Cycle Analysis. HCT116 cells were treated with 1 for either 24 or 48 h in complete medium. The cells were harvested, washed with cold PBS twice, and then fixed in 70% cold ethanol overnight at −20 °C. The fixed cells were washed with cold PBS and suspended in a staining solution containing 100 μg/mL of Rnase A and 50 μg/mL of PI in the dark for half an hour. The stained cells were sorted, and cellular DNA content was analyzed by a FACSCalibur flow cytometer from BD Biosciences. The DNA content of 15 000 cells from each group was analyzed, and the results are presented as histograms using CELLQuest 3.0.1 software from BD Biosciences. Western Blot Analysis. The cells were treated with test compounds for the desired treatment times (0−24 h) in complete medium. The cells were then washed with PBS and lysed in RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS; pH 8.0), and the quantitative determination of total protein in each group was accomplished by BCA assay. Equivalent levels of proteins from each group were subjected to 8−13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then electrotransferred onto polyvinylidene difluoride (PVDF) membranes from EDM Millipore (Bedford, MA, USA). The membranes were blocked with 5% BSA in a mixture of Tris-buffered saline and 0.1% I



Article

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anti-antibodies and detected using an LSAB System/HRP kit from Dako (Glostrup, Denmark) and counterstained with hematoxylin and eosin (H&E) stain. The stained sections were observed and photographed under an inverted phase-contrast microscope. Statistical Analysis. Data were expressed as means ± SD for the incubated number of independently performed experiments. Statistical significance was analyzed using the Student’s t test or one-way analysis of variance (ANOVA) coupled with the Dunnett’s t test. Differences were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-880-2475. Fax: +82-2-762-8322. E-mail: sklee61@ snu.ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (No. 20120004939) and a grant from procurement and development of biological resources funded by the Ministry of Education, Science and Technology of the Korean government (2011-00499).

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