Ixocarpalactone A isolated from the Mexican tomatillo shows potent antiproliferative and apoptotic activity in colon cancer cells Juliana K. Choi 1 , Genoveva Murillo 2 , Bao-Ning Su 3 , John M. Pezzuto 4 , A. D. Kinghorn 3 and Rajendra G. Mehta 2 1 Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, IL, USA 2 Carcinogenesis and Chemoprevention Division, Life Sciences Group, IIT Research Institute, Chicago, IL, USA 3 Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, OH, USA 4 Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue University, West Lafayatte, IN, USA As the third leading cause of cancer deaths, colon cancer continues to be a major cause of mortality in the United States [1]. Several epidemiological studies have indicated a correlation between diet and colon cancer risk [2–4]. Diet is considered one of the most important environmental factors in colon cancer development, particularly those characterized by decreased consumption of fruits and vegetables and increased intake of meats and fats [5–7]. Westerniza- tion of diets, or greater intake of meats and fats, has been linked with an increased incidence of colon can- cer, providing support for the influence of diet on colon cancer development [6,8,9]. Therefore, the dis- covery of novel chemopreventive agents of natural origin has been targeted, with fruits and vegetables being of key interest. Keywords apoptosis; colon cancer; ixocarpalactone A; Physalis philadelphica; tomatillo Correspondence R. G. Mehta, Carcinogenesis and Chemoprevention Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616, USA Fax: +1 312 567 4931 Tel: +1 312 567 4970 E-mail: RMehta@iitri.org (Received 20 September 2006, accepted 27 October 2006) doi:10.1111/j.1742-4658.2006.05560.x Physalis philadelphica Lam, commonly known as a tomatillo, is a staple of the Mesoamerican cuisine. In our laboratory, an ethyl acetate-soluble extract and four withanolides [ixocarpalactone A (IxoA), ixocarpalac- tone B, philadelphicalactone B, and withaphysacarpin] were isolated. Stud- ies conducted on Hepa-1c1c7 hepatoma cells revealed that withanolides were potent inducers of quinone reductase, suggesting possible cancer chemo- protective activity. Here we evaluated the antiproliferative properties of the withanolides in SW480 human colon cancer cells. IxoA, which is present in the edible part of the tomatillo, was selected for further evaluation. SW480 cells treated with IxoA showed cell cycle arrest in the G2⁄ M phase, up-regu- lation of hyper-phosphorylated retinoblastoma, and down-regulation of E2F-1 and DP-1. On the basis of flow cytometry analysis, ethidium bro- mide ⁄ acridine orange, and 4¢,6-diamidino-2-phenylindole staining, it was found that IxoA induces apoptosis in SW480 cells. Moreover, increased concentrations of the pro-apoptotic protein, BIM ⁄ BOD, were found by western blot analysis and immunocytochemistry. Morphological examina- tion revealed vacuole formation in cells treated with IxoA, and Oil Red O staining showed that the vacuole content was nonlipid. Furthermore, immunocytochemistry demonstrated increased concentrations of mucin 3 in IxoA-treated SW480 cells. These findings suggest that chemicals present in tomatillos (e.g. IxoA) may have cancer chemopreventive properties. Abbreviations DAPI, 4¢,6-diamidino-2-phenylindole; IxoA, ixocarpalactone A; IxoB, ixocarpalactone B; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide; PhilB, philadelphicalactone B; pRb, hyperphosphorylated retinoblastoma; Rb, retinoblastoma; Withpc, withaphysacarpin. 5714 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS The beneficial effects of fruits and vegetables have been attributed among other things to the high content of bioactive compounds [10]. Studies conducted in the last two decades have shown that these bioactive compounds have important roles in the prevention of chronic diseases, including cancer, diabetes and hypercholesterolemia [11]. Noteworthy examples of plant-derived substances that have been shown to reduce experimental colon carcinogenesis are indole-3- carbinol from cruciferous vegetables such as brussel sprouts and broccoli [12], curcumin from the root of Curcuma [13], and epigallocatechin gallate from tea [14]. Some of these agents are currently being investi- gated in clinical trials for the prevention or treatment of cancer [15]. The use of plant-derived agents to prevent the onset or delay progression of the carcinogenic process has attracted considerable interest, with much attention aimed at understanding the mode of action by which they function. Several cellular signaling pathways involved in apoptosis, proliferation, cell cycle, and angiogenesis, all processes implicated in many cancers, have been shown to be modulated by chemopreventive agents. Natural agents derived from dietary sources, unlike conventional single-site agents, offer the ability to exhibit multisite mechanisms of action. Moreover, a role for these compounds in combinatorial therapy with more traditional chemotherapeutics has been suggested, with the aim of lowering the toxicity and enhancing the efficacy of treatments of more advanced cancers. As part of our continuing search for novel, plant- derived cancer chemopreventive agents [16,17], we have evaluated a number of plants originating from differ- ent parts of the world. Physalis philadelphica is an example of such a plant. The fruit of P. philadelphica (Fig. 1A), commonly known as tomatillos, husk toma- toes, ground cherries, jamberries or fresadillas [18], are everyday components of the Mexican and Guatemalan diet [19]. Several medicinal properties have been attrib- uted to P. philadelphica, e.g. antibacterial properties against respiratory infections caused by Staphylococ- cus aureus, Streptococcus pneumoniae, and Streptococ- cus pyogenes [20]. Moreover, in Guatemala, the tomatillo was believed to have health benefits against gastrointestinal disorders [21]. Previously in our laboratory, an ethyl acetate-soluble extract and four withanolides [ixocarpalactone A (IxoA)], ixocarpalactone B (IxoB), philadelphicalac- tone B (PhilB), and withaphysacarpin (Withpc)] were isolated in pure form. All four have been shown to be present in the leaves and stems of P. philadelphica [22]. Furthermore, IxoA and Withpc have been found in the fresh fruits of P. philadelphica [23]. Earlier studies demonstrated that IxoA possessed quinone reductase activity with an IC 50 (concentration that produces 50% inhibition) of 7.54 lm in Hepa-1c1c7 mouse hepatoma cells. IxoA was also shown to inhibit the transformation of the murine epidermal JB6 cell with an IC 50 of 0.26 lm [23]. On the basis of these results, we selected IxoA (Fig. 1B) for further investigations. Results Treatment with P. philadelphica extract and withanolide isolates inhibits growth of human colon cancer cells The antiproliferative effects of ethyl acetate-soluble extract from P. philadelphica were evaluated in a human colon cancer cell line (SW480). For these stud- ies, cells were treated at a concentration range of 1– 20 lgÆmL )1 for 2–7 days. As shown in Fig. 2A, extract treatment demonstrated significant growth inhibition in treated SW480 cells, with 85% inhibition at 5 lgÆmL )1 and 100% at doses ‡ 10 lgÆmL )1 . Next the effects of the four isolates, IxoA, IxoB, PhilB and A B OH OH O O OH O OH O Fig. 1. (A) Tomatillo fruit. (B) Structure of IxoA. J. K. Choi et al. Tomatillo and colon carcinogenesis FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5715 Withpc, were evaluated using the same cell line. As illustrated in Fig. 2B, all four compounds significantly suppressed cell proliferation in a dose-dependent man- ner ranging from 80% to 99%, at 1 lm and 10 lm, respectively. Similar findings were observed in the HT- 29 and SW620 human colon cancer cell lines (data not shown). IxoA was selected for further evaluation for the fol- lowing reasons: it is found in the edible fruit of the tomatillo plant, it has previously been reported to have potent quinone reductase activity in hepatoma cells, and because it has been shown to inhibit the transfor- mation of murine epidermal JB6 cells with an IC 50 of 0.26 lm [23]. Therefore, cell growth studies with IxoA were conducted in three additional human colon can- cer cell lines (HT-29, Caco-2 and HCT116 in addition to SW480). As shown in Fig. 2C, dose-dependent inhi- bition was evident in all four cell lines studied after 5 days of treatment. IxoA showed equal or greatest inhibition in SW480 cells, with percentage inhibitions ranging from 19.0 to 100% at concentrations of 0.1– 10.0 lm, respectively. Subsequently, the time-dependent effects of IxoA were evaluated in the SW480 cells. For these experi- ments, cells were treated for 1–7 days with doses ran- ging from 250 nm to 10.0 lm. As shown in Fig. 2D, by day 2 after treatment, > 60%, > 83%, and 100% growth inhibition was noted for cells treated with IxoA at 1.0 lm, 2.5 lm, and 7.5 lm, respectively. The growth inhibition remained evident until day 7 after treatment. SW480 cells treated with IxoA for 1 day also showed a large percentage of growth inhibition; how- ever, consistent with a time-dependent pattern, the per- centage inhibition was not as great as observed at longer time points. After 1 day of IxoA treatment, 58.1%, 67.6%, 87.1% and 90.3% inhibition was observed at 2.5, 5.0, 7.5 and 10.0 lm IxoA, respect- ively. The IC 50 for 1 day of IxoA treatment was 1.66 lm. To ensure a minimum of 50% inhibition of SW480 proliferation at shorter time points (1 day), a concentration of 5.0 lm IxoA was used for subsequent studies. IxoA treatment induces G2/M cell cycle arrest in SW480 cells To determine whether the antiproliferative actions of IxoA were mediated by an arrest in the cell cycle, SW480 cells were treated with 5 lm IxoA for 12–24 h and analyzed by flow cytometric analysis. Cell cycle analysis demonstrated that 5 lm IxoA treatment resul- ted in an accumulation of cells in the G2 ⁄ M phase of the cell cycle, as shown in Fig. 3A,B. At 12 h, a 20.0% increase in SW480 cells arrested in G2 ⁄ M was observed, and at 24 h a 20.2% increase (Fig. 3C). Flow cytometric analysis was repeated in HT-29 cells, and similar results were obtained (data not shown). AB CD Fig. 2. Percentage growth inhibition of human colon cancer cells treated with P. philadelphica extract and ⁄ or withanolide isolates. (A) Effect of ethyl acetate-soluble extract from P. philadelphica on SW480 human colon cancer cells. Cells were seeded in 96-well plates as described in Experimental procedures and treated with the indicated concentrations of treatment or vehicle (Me 2 SO). Cell proliferation was determined by MTT assay at 2, 3, 4, 5 and 7 day time points by measuring the absorb- ance of formazan at 570 nm. The data are mean ± SD from triplicate wells. (B) The effects of IxoA, IxoB, PhilB and Withpc on SW480 cells were measured at 5 days. (C) The effect of IxoA on HT29, Caco-2, HCT116 and SW480 human colon cancer cell lines was measured at 5 days. (D) Effect of IxoA on SW480 cells at 1, 2, 4, 5, 6 and 7 days. The experimental procedures for (B), (C) and (D) were the same as in (A). Tomatillo and colon carcinogenesis J. K. Choi et al. 5716 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS Hyperphosphorylated retinoblastoma (pRb) is up-regulated, whereas E2F-1 and DP-1 are down- regulated in SW480 cells treated with IxoA Given that IxoA induced G2 ⁄ M cell cycle arrest, west- ern blot analysis was used to examine the effects of this compound in G2-related proteins. These studies revealed an increased expression of pRb with a simul- taneous decrease in the expression of retinoblastoma (Rb) in SW480 cells exposed to 5 lm IxoA for 24– 72 h. Densitometric analysis revealed 28.5–51.9% increase in pRb and 4.5–41.4% reduction in Rb com- pared with the control band (b-actin). Western blot analysis demonstrated that E2F-1 expres- sion was down-regulated by 7.3–54.3% when com- pared with b-actin. DP-1 expression varied from 29.3% up-regulated at 24 h to 51.8% down-regulated at 48 h (compared with b-actin). No significant chan- ges in cyclin A and cdk1 concentrations were observed (Fig. 4). IxoA induces apoptosis in SW480 cells The effects of IxoA on apoptosis were measured by four independent assays. Initially, acridine orange ⁄ ethi- dium bromide staining was used to evaluate apoptosis in SW480 cells treated with IxoA. SW480 cells were treated with 5 lm IxoA for 24 h, stained with acridine orange ⁄ ethidium bromide and examined by fluorescent microscopy. Morphological changes characteristic of apoptosis, including fragmented nuclei, blebbing, and irregular cytoplasmic membranes, were evident in the nuclei of IxoA-treated cells. Treatment with IxoA for 24 h revealed 54% of the SW480 cells were orange in color (late apoptosis), 36% were observed to be AC B Fig. 3. Effect of IxoA on cell cycle distribu- tion in SW480 cells. Cells were prepared for flow cytometry analysis as described in Experimental procedures. (A) SW480 cells were treated with vehicle (Me 2 SO) as con- trol for 12 h. (B) or with IxoA 5 l M (C) Percentages of cells in each cell cycle phase at 12, 18 and 24 h. An increase in the num- ber of cells arrested in the G2 ⁄ M phase of the cell cycle is observed at each time point. Fig. 4. Western blot analyses of G2-related proteins. SW480 cells were treated with 5 l M IxoA for 24-72 h. As described in Experi- mental procedures, cell lysate was collected, and western blot ana- lysis was conducted to determine the protein expression of pRb and Rb, E2F-1, DP-1, cdk1 and cyclin A. Cell lysate was also collec- ted from untreated (Untxd) SW480 cells at each time point, and protein expression was compared between untreated and treated SW480 cells. All bands were compared with b-actin bands using densitometric analysis. The percentage change for each protein compared with b -actin bands is indicated as up-regulation (+) or down-regulation (–). J. K. Choi et al. Tomatillo and colon carcinogenesis FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5717 blebbing (early apoptosis), and 9% were a green color (live cells) (Fig. 5B–D) The control cells, treated with vehicle (Me 2 SO) were 19% orange, 7% blebbing, and 73% green in color (Fig. 5A). To better evaluate nuclear fragmentation, a feature of apoptotic cells, the fluorescent DNA-binding dye, 4¢,6-diamidino-2-phenylindole (DAPI) was used. As shown in Fig. 6, cells treated with 5 lm IxoA for 24 h displayed the typical morphological features, con- densed and fragment nuclei, of apoptotic cells. Members of the BH3 domain-only pro-apoptotic proteins, including BIM ⁄ BOD, have been shown to have a critical role in initiating the apoptotic program by antagonizing the function of the antiapoptotic BCL-2 and activating BAX and BAK [24]. Therefore, the expression of BIM ⁄ BOD was evaluated by western blot analysis. As shown in Fig. 7A, exposure of SW480 cells to IxoA increased expression of BIM ⁄ BOD. SW480 cells treated with 5 lm IxoA for 24, 48 and 72 h revealed a 20%, 35%, and 64% up-regula- tion, respectively, in BIM ⁄ BOD compared with the control band (b-actin) upon evaluation by densito- metry. After these studies, the effects of IxoA on BIM ⁄ BOD were examined by immunocytochemistry (Fig. 7B,C). Treatment with 5 lm IxoA for 24 h increased BIM ⁄ BOD protein staining. This result complements those obtained by western blot analysis. Vacuole content detection Treatment of SW480 cells with 5 lm IxoA for 24 h induced the formation of multiple vacuoles within each cell. To determine the content of these vacuoles, Oil Red O, a red stain specific for lipids, was used to stain the SW480 cells. Figure 8A,B show that the vacuole content was not positive for the presence of A C E B D Fig. 5. Cell apoptosis and morphological changes in the nuclei of SW480 cells trea- ted with or without IxoA were identified by fluorescent staining with acridine orange ⁄ ethidium bromide. Non-viable cells had orange-stained nuclei, and viable cells had green-stained nuclei under fluorescent microscopy. (A) Control SW480 cells were treated with Me 2 SO. The green color indi- cates viability. (B–D) SW480 cells were trea- ted with 5 l M IxoA for 24 h. Blebbing of the membrane, chromatin aggregation, and nuc- lear condensation (B and C) were criteria used to identify apoptotic cells. (D) The orange color indicates non-viable cells. Ori- ginal magnification, 40·. (E) Percentage distribution is presented for control and treatment. AB Fig. 6. Morphological evidence of apoptosis in SW480 cells stained with DAPI. (A) Con- trol cells treated with Me 2 SO had intact nuclei. (B) After 24 h, the nuclei of SW480 cells treated with 5 l M IxoA showed nuclear fragmentation and chromatin condensation characteristic of apoptosis. Magnification, 40·. Tomatillo and colon carcinogenesis J. K. Choi et al. 5718 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS lipid, as no red color was detected in the IxoA-treated cells. Increased concentrations of mucin 3, however, were observed by immunocytochemical analysis (Fig. 8C,D). Increased mucin 3 protein staining was observed in SW480 cells treated for 24 h with 5 lm IxoA, suggesting that the vacuole content includes mucin. Discussion This study was part of a large-scale investigation of the efficacy of natural products as chemopreventive agents, particularly those found in the diet [25]. Thus far, over 200 active compounds have been identified as chemopreventive agents, including resveratrol [26,27], brassinin [28,29], and deguelin [30,31]. Resveratrol is present in grapes, red wine and peanuts, brassinin is from Chinese cabbage, and deguelin is from an Afri- can plant, Mundule sericea. The success of these nat- ural products as anticancer agents led us to evaluate an additional plant, P. philadelphica or more com- monly known as the tomatillo, to determine its efficacy as a chemopreventive agent. Because of the efficacy of fruits and vegetables against colon cancer [2–4,8,9], we elected to study the effects of tomatillos against colon cancer in vitro. A BC Fig. 7. BIM ⁄ BOD, a BH3-region only pro-apoptotic protein, was investigated to further characterize the apoptosis observed in IxoA-treated SW480 cells. (A) SW480 cells were treated with 5 l M IxoA for 24–72 h. Cell lysate was collected (also from untreated SW480 cells at each time point), and western blot analysis was conducted to determine the protein expression of BIM ⁄ BOD. Protein expression was compared between untreated and treated SW480 cells, and BIM ⁄ BOD expression was shown to increase at each time point. All bands were com- pared with b-actin bands using densitometric analysis. (B) Immunocytochemistry was also performed on SW480 cells to confirm BIM ⁄ BOD expression. Control cells were treated with Me 2 SO for 24 h. (C) SW480 cells treated with 5 lM IxoA for 24 h confirmed up-regulation of BIM ⁄ BOD. Magnification, 40·. AB C D Fig. 8. Identification of vacuole content in treated SW480 cells. The formation of mul- tiple vacuoles within the cells was observed after treatment of SW480 cells with 5 l M IxoA for 24 h. SW480 cells were plated, treated and fixed as described in Experimen- tal procedures. Oil Red O staining counter- stained with hematoxylin was then performed to distinguish the vacuole con- tent as lipid or non-lipid. (A) SW480 cells treated with Me 2 SO for 24 h served as controls. (B) SW480 cells treated with 5 l M IxoA for 24 h did not stain red, indicating a non-lipid vacuole content. Also, immunocyto- chemistry was performed on SW480 cells to investigate mucin 3 expression. (C) Me 2 SO-treated SW480 cells served as con- trols. (D) SW480 cells treated with 5 l M IxoA for 24 h showed increased mucin 3 protein staining. Magnification, 40·. J. K. Choi et al. Tomatillo and colon carcinogenesis FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5719 Our rationale for selecting tomatillos for evaluation has been described previously [16,17,23]. Briefly, plants are selected on the basis of information about its expected antiproliferative activity, nontoxic nature, and from information received from the population that uses the plant for medicinal purposes. Then, selec- ted plant parts are extracted with ethyl acetate and evaluated for activity using select in vitro bioassays, including induction of quinone reductase with Hepa 1c1c7 cells and inhibition of transformation with JB6 cells [23,32]. After in vitro bioassays, the mouse mam- mary gland organ culture (MMOC) model is used to select active agents for further evaluation [33]. Chosen extracts are then fractionated using an HPLC solvent system, and all fractions are evaluated in in vitro bioas- says specific to the extract. Pure compounds are then isolated from active fractions and the structure of the compound is determined. The activity of the chemo- preventive agent is then confirmed in experimental carcinogenesis models as shown in this study. Using the above screening process, we have identi- fied and evaluated a novel chemopreventive agent. Upon fractionation of the ethyl acetate-soluble toma- tillo extract, we discovered four compounds with chemopreventive potential. Although all four com- pounds showed significant antiproliferative activity in colon cancer cells, we elected to focus on IxoA because of its abundance in the leaves and stems of the plant and its presence in the fruits. To examine the mechanism that might account for the effects of IxoA in colon cancer cells, we investi- gated its effects on cell cycle distribution. A noticeable accumulation of colon cancer cells in the G2 ⁄ M phase of the cell cycle occurred, with a concomitant decrease of cells in the G0 ⁄ G1 phase. This suggests that IxoA has a pronounced effect on colon cancer proliferation which is due to cell cycle arrest. In support of this observation, SW480 cells cultured with IxoA (5 lm) for 24–72 h showed an increased level of expression of pRb and a decreased level of expression of E2F-1 and DP-1 (at 48 h). E2F-1 and DP-1 are known to exist in a complex and act synergistically in E2F site-depend- ent transcriptional activation [34], and pRb can inhibit transcriptional activation of E2F [35]. Therefore, IxoA-induced pRb inhibition of E2F-1 and DP-1 may account for the accumulation of cells at the G2 ⁄ M phase of the cell cycle. We show here that sustained G2 ⁄ M arrest induced by IxoA may be E2F-1-depend- ent and involves an increase in expression of the mito- tic regulator, pRb. In addition, IxoA was shown for the first time to induce apoptosis in SW480 human colon cancer cells. To investigate the effects of IxoA on apoptosis, we used acridine orange ⁄ ethidium bromide staining and DAPI staining and found a marked increase in the per- centage of apoptotic cells in SW480 cells exposed to IxoA for 24 h. Additional apoptotic studies focused on BIM ⁄ BOD, a BH3 region-only pro-apoptotic Bcl-2 family member [36–38]. Pro-apoptotic proteins are divi- ded into two subgroups, those that possess BH1, BH2 and BH3 regions and those that only possess the BH3 region [36,39]. Pro-apoptotic proteins induce the release of cytochrome c from the mitochondria, and their abil- ity to achieve this depends on a hydrophobic pocket and an amphipathic a-helix. The BH1, BH2 and BH3 regions form a hydrophobic pocket, which binds to a BH3 region of another protein, and the BH3 region consists of an amphipathic a-helix. Furthermore, some Bcl-2 family members have exposed BH3 regions, whereas other members have buried BH3 regions that require cleavage to expose the BH3 region and activate cytochrome c release. BH3 region-only proteins with exposed BH3 regions, such as BIM ⁄ BOD, appear to represent a death ligand, which can neutralize certain pro-survival members of the Bcl-2 family [38,39]. On the basis of immunoblotting and immunocytochemistry results obtained to date, the mechanism of IxoA- induced apoptosis appears to involve the interaction of IxoA with BIM ⁄ BOD death receptors. Treatment of SW480 cells with IxoA also caused the formation of numerous vacuoles. To identify the con- tent of these vacuoles, we began by staining for lipids using Oil Red O. We were interested in lipid build-up because several studies have indicated that fatty acids such as linoleic acid may hold anticancer properties [40,41], and perhaps a build-up of fatty acids was triggering apoptosis or cell cycle arrest. However, Oil Red O staining revealed that the vacuole content was not lipid. Next, the vacuole content was tested for mucin formation. A common feature of colonic neoplasia is altered concentrations of mucin. Compared with normal colon, colon cancers have been reported to express decreased concentrations of mucin 3 [42,43]. Secreted isoforms of mucin 3 have been reported to protect the colonic epithelial surface [44], and immuno- cytochemical analysis performed on SW480 cells treated with IxoA demonstrated increased mucin 3 concentrations compared with untreated cells. This suggests that the vacuole content may include mucin 3, which may play a protective role. In summary, the data reveal that IxoA shows potent antiproliferative and apoptosis activity in human colon cancer cells. The evidence presented here suggests for the first time that IxoA present in tomatillos may have chemopreventive or therapeutic value in the manage- ment of colon cancer. Tomatillo and colon carcinogenesis J. K. Choi et al. 5720 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS Experimental procedures Physalis philadelphica extract and withanolide isolates An ethyl acetate-soluble extract of P. philadelphica as well as four withanolides, IxoA, IxoB, PhilB, and Withpc, were isolated as previously described [23,32]. Antibodies The mouse monoclonal antibodies used for these studies included Rb (clone 1F8), E2F-1 Ab-7 (clone KH129), cdk1 ⁄ p34cdc2 Ab-3 (clone A17.1.1 + POH-1), cyclin A (clone CYA06), and mucin 3 Ab-1 (clone M3.1) as well as rabbit polyclonal antibody against Bcl-2-related ovarian death gene (BIM ⁄ BOD; clone 1F8), which were purchased from NeoMarkers (Fremont, CA, USA). Rabbit polyclonal DP-1 (clone K-20) sc-610 antibody and goat polyclonal b-actin (clone I-19) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell lines and culture conditions SW480, SW620, HT-29, Caco-2 and HCT116 human colon cancer cells were obtained from the American Tissue Cul- ture Collection (Manassas, VA, USA). The cells were cul- tured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mml-glutamine and 1% antibiotic ⁄ antimycotic solution (10 UÆlL )1 penicillin, 10 lgÆlL )1 streptomycin and 25 lgÆmL )1 amphotericin B) at 37 °Cina 5% CO 2 humidified atmosphere. Growth inhibition assay The antiproliferative effects of IxoA, IxoB, PhilB, Withpc, and ethyl acetate-soluble extract were evaluated in human colon cancer cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay (TACS MTT cell proliferation assay kit; Trevigen, Gaithersburg, MD, USA). Cells were seeded in 96-well plates at a density of 5 · 10 2 per well. Cell viability was analyzed at various time points between 1 and 7 days after treatment with extract or vehicle (Me 2 SO). After treatment with extract, cells were incubated with MTT tetrazolium reagent for 2 h at 37 °C, and the absorbance of formazan was then measured at 570 nm. Each treatment was performed in triplicate, and the percentage cell growth inhibition was calculated by comparison of the absorbance readings of the control and treated cells. Fluorescence-activated cell sorter analyses SW480 cells were treated with or without IxoA for 12, 18 and 24 h, harvested with trypsin, and washed with NaCl ⁄ P i . After the final wash, the cells were resuspended in 1.0 mL NaCl ⁄ P i and 9.0 mL ice-cold 70% ethanol. The samples were stored at )20 °C until staining. In preparation for staining, cells were washed three times with NaCl ⁄ P i and resuspended in 0.3 mL citrate buffer [250 mm sucrose, 40 mm trisodium citrate, 0.05% (v ⁄ v) Me 2 SO, pH 7.6]. The samples were then stained with propidium iodide using a previously described method [45]. Apoptosis studies Acridine orange ⁄ ethidium bromide staining SW480 cells with or without IxoA treatment were centri- fuged and suspended in NaCl ⁄ P i , followed by the addition of the acridine ⁄ ethidium mixture. Fluorescent microscopy was used to distinguish nonviable cells with orange-stained nuclei from viable cells with green-stained nuclei, which do not absorb ethidium bromide. The percentage of apoptotic cells and those with highly condensed or fragmented nuclei was determined quantitatively. DNA-binding dye, DAPI staining SW480 cells with or without IxoA treatment were also eval- uated by DAPI staining. For this, cells were grown on glass microscope slides, fixed in formalin and methanol, and stained with DAPI. Stained nuclei were visualized using a fluorescent microscope. Blebbing of the membrane, chroma- tin aggregation, and nuclear condensation were used as cri- teria to identify cells undergoing apoptosis. Western blot analysis Treated and control SW480 cells were lysed in freshly pre- pared extraction buffer (20 mm Hepes, pH 7.9, 400 mm NaCl, 0.1% Nonidet P-40, 10% glycerol, 1 mm sodium vanadate, 1 mm NaF, 1 mm dithiothreitol, 1 mm phenyl- methanesulfonyl fluoride, 10 lgÆmL )1 aprotinin, 10 lgÆmL )1 leupeptin) for 45 min on ice. Lysate was centrifuged at 15 000 g for 10 min using the Eppendorf 5417R centrifuge, supernatant collected, and protein concentration was deter- mined using a modified Lowry method (Bio-Rad, Hercules, CA, USA). Samples were separated using 7.5–12.0% poly- acrylamide gels and ⁄ or ready-made gradient gels from Bio-Rad, and transferred to nitrocellulose membranes. The membranes were blocked in 5% milk followed by incuba- tion with appropriate primary antibodies for 2 h at room temperature. The membranes were then washed and incuba- ted for 45 min at room temperature with the corresponding secondary antibodies. The chemiluminescence reaction was performed using the ECL system and protocol from Amer- sham Pharmacia Biotech (Piscataway, NJ, USA). Using Un-Scan-It Image Digitizing Software (Silk Scientific; Orem, UT, USA), the bands of interest were compared with those of b-actin, and the relative intensity ratios were calculated. J. K. Choi et al. Tomatillo and colon carcinogenesis FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5721 Immunocytochemistry SW480 cells were plated on coverslips and allowed to adhere for 24 h before treatment with IxoA or vehicle for appropriate time points. The cells were washed with NaCl ⁄ P i , and then fixed with 10% buffered formalin and cold methanol. Staining was then conducted using a BIM ⁄ BOD rabbit polyclonal antibody or a mucin 3 mouse monoclonal antibody. The immunoperoxidase reaction was performed using the Dako LSAB2 System kit (Dako Cor- poration, Carpinteria, CA, USA). Briefly, the biotinylated link IgG was applied for 10 min, followed by incubation of horseradish peroxidase-linked streptavidin. After the sec- tions had been washed with NaCl ⁄ P i , 3-amino-9-ethyl- carbazole (AEC) substrate ⁄ chromogen solution was applied. The cells were then counterstained with hematoxy- lin and examined for antibody localization. Oil Red O staining Levels of lipid accumulation, a classic differentiation mar- ker, were measured in IxoA-treated SW480 cells by histo- chemical analysis using Oil Red O staining. Briefly, SW480 cells were plated, treated, and fixed as previously described for immunocytochemistry studies. The samples were then placed in propylene glycol for 2 min, followed by 1 h incu- bation in Oil Red O at room temperature. Counterstaining was performed with hematoxylin. 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