Physangulidine A, a Withanolide from Physalis angulata, Perturbs the

Dec 27, 2012 - It was determined also that 1 produces programed cell death by apoptosis, ... Yan-Wei Yang , Lei Yang , Chao Zhang , Cai-Yun Gao , Ting...
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Physangulidine A, a Withanolide from Physalis angulata, Perturbs the Cell Cycle and Induces Cell Death by Apoptosis in Prostate Cancer Cells E. Merit Reyes-Reyes,† Zhuang Jin,‡ Abraham J. Vaisberg,§ Gerald B. Hammond,‡ and Paula J. Bates*,†,⊥ †

Department of Medicine, University of Louisville, Louisville, Kentucky 40202, United States Department of Chemistry, University of Louisville, Louisville, Kentucky 40292, United States § Departamento de Ciencias Celulares y Moleculares y Laboratorios de Investigacion y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Perú ⊥ Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40202, United States ‡

S Supporting Information *

ABSTRACT: Recently, our group reported the discovery of three new withanolides, physangulidines A−C, from Physalis angulata. In this study, the biological effects of physangulidine A (1), which was the most active and abundant of the three new constituents, are described. It was found that 1 significantly reduces survival in clonogenic assays for two hormone-independent prostate cancer cell lines. Flow cytometry and confocal microscopy studies in DU145 human prostate cancer cells indicated that 1 induces cell cycle arrest in the G2/M phase and causes defective mitosis. It was determined also that 1 produces programed cell death by apoptosis, as evidenced by biochemical markers and distinct changes in cell morphology. These results imply that the antimitotic and proapoptotic effects of 1 may contribute significantly to the biological activities and potential medicinal properties of its plant of origin.

Physalis angulata L. (Solanaceae) is a plant used widely as popular medicine in several countries. Extracts or infusions from this plant have been used in the treatment of different illnesses such as malaria, asthma, hepatitis, dermatitis, liver problems, and rheumatism, as well as for their diuretic, antimycobacterial, antipyretic, and immunomodulatory properties.1−5 This plant has also been reported to exhibit potential anticancer properties, with P. angulata extracts found to exert strong antiproliferative effects and induce cell death by apoptosis in human oral squamous carcinoma and breast cancer cell lines.6,7 Furthermore, its ethyl acetate extract was reported to inhibit the metastatic and angiogenic ability of a highly metastatic human oral squamous carcinoma cell line.6 Currently, many cancer chemotherapeutic agents in clinical use are derived from natural products. Thus, P. angulata promises to be a rich source for the discovery of potential bioactive agents, and there is significant interest in identifying and evaluating the components responsible. Various antiproliferative compounds have been isolated from this plant, several of which are withanolides. Withanolides are a group of naturally occurring steroids built on an intact or modified ergostane skeleton, in which the C-26 and C-22, or C-26 and C-23, positions are oxidized in order to form a γ- or δ-lactone.8 Examples of withanolides isolated from P. angulata include physalin, withangulatin, and physangulin, which have been © 2012 American Chemical Society and American Society of Pharmacognosy

reported to show antitumor, immunosuppressive, and antiinflammatory activities.9−14

As part of a program aimed at the discovery of novel bioactive natural products from Amazonian rainforest plants, we have previously reported three new withanolides with an unusual carbon skeleton isolated from P. angulata, namely, physangulidines A−C (1−3).15 In the current work, we have investigated the cytotoxicity and mechanism of action for 1 in hormone-independent prostate cancer cells.

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RESULTS AND DISCUSSION Physangulidine A (1) Inhibits Clonogenic Survival of Prostate Cancer Cells. In previous work where we identified Received: June 29, 2012 Published: December 27, 2012 2

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resulting in cells with ca. 8n DNA. It was found that 1, at a concentration of 10 μM, was also able to cause G2/M arrest in PC3 cells (Figure S3A, Supporting Information), indicating that the activity of 1 was not just DU145 cell type-specific. It is concluded from these results that physangulidine A is a strong inhibitor of cell cycle progression at the G2/M phase. Physangulidine A (1) Promotes Cell Death by Apoptosis. Since prolonged cell cycle arrest frequently causes induction of cell death, the viability of prostate cancer cells treated with 1 was investigated. To begin, we used the trypan blue exclusion assay, which is based on the principle that live cells will exclude membrane-impermeable dyes such as trypan blue, whereas trypan blue will penetrate inside dead cells and stain them. This test showed that almost 90% of DU145 cells were dead after treatment with 1 (10 μM) for 72 h (Figure 3). To determine whether apoptosis (programed cell death) is the cause of cell death induced by 1, cells were examined for biochemical and morphological markers of apoptosis. Cells undergoing apoptosis specifically translocate phosphatidylserine (PS) phospholipid from the inner face of the plasma membrane to the cell surface; therefore, apoptotic cells can be identified by the presence of PS on the cell surface.18 Detection of PS is achieved by staining with a fluorescent conjugate of annexin V, a protein that has a high affinity for PS, followed by flow cytometry analysis. Cells are stained in parallel with propidium iodide (PI), which can enter the cell only when the plasma membrane is damaged. This allows early apoptotic cells (positive for PS, but negative for PI) to be distinguished from late apoptotic and necrotic cells (positive for both PS and PI). Figure 4A and Figure S2B (Supporting Information) show that DU145 prostate cancer cells receiving control treatments (untreated or DMSO) possess a baseline apoptotic cell population of less than 4%. Treatment of DU145 cells with 1 at a concentration of 5 μM for 24, 48, and 72 h resulted in small increases in the apoptotic population of 5.2%, 9.2%, and 11.5%, respectively (Figure S2B, Supporting Information). After 24 h, DU145 cells treated with 1 at a concentration of 10 μM showed a small increase in the apoptotic population (7.4%). However, after 48 and 72 h, 1 induced a drastic increase in the apoptotic cell population to 32.3% and 46.4%, respectively. Furthermore, a large population of late apoptotic/necrotic cells (28.4%) was also observed after 72 h treatment with 1 (10 μM) (Figure 4A). Next, to further confirm that 1 induces apoptosis in prostate cancer cells, key apoptotic molecular markers were evaluated. Caspase-3 activation and poly(ADP-ribose) polymerase (PARP-1) cleavage into fragments of 89 and 24 kDa are considered as hallmarks of apoptosis.18 Caspase-3 activity is essential for the proteolytic cleavage of many key proteins during apoptosis, including PARP-1. Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated 17/19 and 12 kDa fragments.18 Therefore, it was examined whether 1 might induce caspase-3 activation (i.e., caspase-3 cleavage) and PARP-1 cleavage. Protein extracts from DU145 cells were collected at different time points after treatment with 1, and equal amounts of protein extract were examined by immunoblotting with anti-cleaved caspase-3 and anti-PARP-1 antibody. Cell extracts from control cells (untreated or DMSO) showed no detectable activation of caspase3 (cleaved caspase-3), and PARP-1 existed predominantly as the full-length product (116 kDa) (Figure 4B). However, activated caspase-3 and cleavage of PARP-1 protein (additional band at 89 kDa) were detected in extracts from cells treated with 1 (10 μM) for 48 and 72 h. Caspase-3 activation and

physangulidine A (1), the compound was found to inhibit proliferation of DU145 prostate cancer cells in a sulforhodamine B (SRB) assay with a GI50 value of 3.6 μM (1.8 μg/ mL).15 To determine if 1 can also compromise the colonyforming capacity of prostate cancer cells, its activity was measured at 2.5, 5, and 10 μM in both DU145 and PC-3 cells using clonogenic assays. Substantial inhibitory effects were seen at 2.5 μM, and 1 completely blocked the capacity of single cancer cells to generate colonies for both prostate cancer cell lines at a concentration of 5 μM or higher (Figure 1). Since 1

Figure 1. Clonogenic assays in prostate cancer cells treated with physangulidine A (1). Prostate cancer cells, DU145 (A) or PC3 (B), were plated at very low density in six-well plates and incubated 18 h at 37 °C. Cells were treated by addition of various concentrations of 1 or with an equivalent volume of vehicle (DMSO), or were left untreated (medium). After 10 days of treatment, cells were washed with cold PBS, fixed with paraformaldehyde, and stained with crystal violet solution.

was found to have the maximum antiproliferative activity at a concentration of 10 μM on two prostate cancer cell lines (DU145 and PC3) after three days of treatment (Figure S1B, Supporting Information), this concentration was chosen to investigate the mechanism of 1. Physangulidine A (1) Induces G2/M Cell Cycle Arrest. It has been reported that extracts of P. angulata have the ability to cause cell cycle arrest in the G2/M phase and to induce apoptosis in cancer cells.6,7,16,17 To investigate whether 1 could induce cell cycle perturbations in prostate cancer cells, flow cytometric analyses of propidium iodide stained nuclei cells were performed. Cell cycle parameters were compared for DU145 cells that had been incubated for 24 or 48 h with 1 (10 or 5 μM), with vehicle as control (DMSO), or without treatment (untreated). Cell cycle parameters were not perturbed in DU145 cells treated with 1 at a concentration of 5 μM (Figure S2A, Supporting Information). However, as shown in Figure 2, following 24 h of treatment with 1 at a concentration of 10 μM, there was a decrease in the fraction of cells in the G1 phase (8% compared to 39% in untreated cells) and an increase in the proportion of cells in the S phase (37% compared to 28%) and the G2/M phase (49% compared to 31%). By 48 h, the percentage of cells in the S phase had decreased in samples treated with 1, and there was a concomitant increase in the fraction of cells in the G2/M phase (66% compared to 36% in untreated cells). An additional peak to the right of the G2/M peak was also observed, consistent with a population of aneuploid cells that contain more than 4n DNA. This peak may represent a population of cells that has escaped mitotic arrest and continued to replicate as multinuclear cells without dividing (i.e., endoreduplication), or it may be that there was a small fraction of tetraploid cells already present in the culture, which undergo G2/M arrest, 3

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Figure 2. Flow cytometric analysis of cell cycle parameters. DU145 prostate cancer cells were incubated for 24 h (A) or for 48 h (B) in the presence of 10 μM physangulidine A (1) or vehicle (DMSO), or without additive (untreated). Cells were then harvested by trypsinization, fixed, and stained with propidium iodide for analysis by flow cytometry. Each histogram indicates the percent of cells in G1 (blue fraction), S (red fraction), and G2/M (gray fraction) phases of the cell cycle. Data were gated to exclude apoptotic cells for these calculations. A small population of aneuploid cells with >4n DNA content, which became apparent in the cells treated with 1, is also indicated (percentage underlined).

(to detect microtubules) and DAPI (to detect DNA). The results (Figure 5) showed that, after 24 h treatment with 1 (10 μM), there was an increased number of DU145 cells that displayed evidence of aberrant mitosis (white arrows). These were characterized by condensed chromosomes (blue) and mitotic spindles (green), but in most case both the chromosomes and microtubules were different from what would be expected for normal mitosis. After 48 h treatment with physangulidine A, almost all of the cells showed evidence of abnormal mitosis (white arrows) or apoptosis (white asterisks). In conclusion, the present results indicate that physangulidine A (1) causes cell cycle arrest followed by cell death via apoptosis and that these effects are due predominantly to induced defects in the mitotic process. Thus, 1 likely contributes significantly to the cytotoxicity and other biological properties attributed to P. angulata.

PARP-1 cleavage could not be detected in protein extracts from cells treated for time periods shorter than 48 h (Figure 4B). Physangulidine A (1) was also able to induce the activation of caspase-3 and the cleavage of PARP-1 in PC3 cells (Figure S3B, Supporting Information). For further evidence of apoptosis, we tested whether 1 might induce the cleavage of chromatin into oligonucleosome-length DNA fragments, which appear as a “DNA ladder” on agarose gels, because this is another biochemical hallmark of apoptosis.18 For both DU145 cells (Figure 4C) and PC3 cells (Figure S3C, Supporting Information), the characteristic “DNA ladder” was seen only when cells were treated with 1 (10 μM) for the time indicated in the figures, but not in the control cells (DMSO treated), verifying that 1 induces apoptosis. Finally, to demonstrate that the cytometric analyses and biochemical findings correlated with the morphological appearance of cells, confocal microscopy was performed to examine cells that had been fluorescently stained for α-tubulin 4

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Figure 5. Fluorescent microscopy of DU145 prostate cancer cells. DU145 cells were plated in culture dishes designed for confocal microscopy and allowed to adhere for 18 h at 37 °C. They were then for incubated for a further 24 and 48 h with 10 μM physangulidine (1) or 48 h with vehicle (DMSO). After that, cells were washed with cold PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton 100X. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), which binds to DNA, and with anti-α-tubulin antibody labeled with Alexa Fluor 488 (green) to show the location of microtubules. Scale bars indicate a length of 10 μm, arrows indicate cells with evidence of aberrant mitosis, and asterisks indicate cells with apoptotic morphology.

Figure 3. Trypan blue exclusion assay to determine cell death. DU145 prostate cancer cells were left untreated or treated with 10 μM physangulidine (1) or with vehicle (DMSO) for 72 h. After treatment, all cells (both adherent and floating) were harvested, stained with trypan blue, and counted immediately to determine the percentage of nonviable cells (stained blue). Columns represent the mean of three independent experiments, bars represent standard error, and numbers above each column represent the percentage of blue cells in the sample.

Figure 4. Assays to detect apoptosis in DU145 cells. Prostate cancer cells were left untreated (−) or treated with 10 μM physangulidine (1) or with vehicle (DMSO) for the time indicated in the figure. (A) After treatment, cells were harvested by trypsinization, washed with PBS, and then incubated with FITC-conjugated annexin V and propidium iodide. Flow cytometric analysis was performed. The histogram for each sample was split into four quadrants to indicate viable cells (lower left quadrant), apoptotic cells (lower right quadrant), necrotic cells (upper left quadrant), and necrotic/late apoptotic cells (upper right quadrant). (B) After treatment, cells were lysed and total lysates were analyzed for cellular apoptotic markers by immunoblotting using anti-PARP-1 or antibodies specific to the activated form of caspase-3 (cleaved caspase-3). Anti-GAPDH-1 antibody was used as loading control. (C) Some cells were harvest by trypsinization, washed with PBS, and extracted. Total DNA was then analyzed on a 1.8% agarose gel. M indicates DNA size marker (base pairs). 5

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min at room temperature, and washed three times with PBS. Cells were permeabilized for 1 min in PBS containing 0.1% Triton X-100 and washed three times with PBS. Nonspecific binding sites were blocked for 30 min with 3% BSA in PBS, and the fixed cells were incubated for 60 min with anti-α-tubulin (11H10) antibody conjugated to Alexa Fluor 488 (Cell Signaling Technology, Inc., Danvers, MA, USA). Antibody dilution was carried out using PBS containing 3% BSA. After washing, the coverslips were mounted on glass slides with ProLong Antifade (Molecular Probes) according to the manufacturer’s directions. Immunofluorescence was documented with a Nikon A1 inverted confocal laser-scanning microscope (Nikon Instruments, Inc., Melville, NY, USA) equipped with an Omnichrome argon−krypton laser. Images were obtained with a 40× Plan-Neo 6 oil immersion objective (1.3 NA). Immunoblotting. Samples were resolved by 4−20% precast linear gradient SDS−Tris polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA) and then electrotransferred onto polyvinylidine fluoride membranes (Millipore, Bedford, MA, USA) in Tris−glycine buffer containing 20% methanol. Membranes were incubated in blocking buffer containing 5% nonfat dry milk (Carnation; Nestle, Glendale, CA, USA) and 0.1% Tween 20 in PBS for 1 h at room temperature. Subsequently, membranes were probed with anti-PARP-1 (D-1) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 200 ng/mL in blocking buffer overnight at 4 °C. After washing six times with PBS containing 0.1% Tween 20, membranes were incubated with HRP (horseradish peroxidase)-conjugated goat anti-mouse IgG for 1 h at room temperature and washing six more times with PBS containing 0.1% Tween 20. Antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. A similar protocol was used to detect activated caspase-3 using anticleaved caspase-3 rabbit antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) at a dilution of 1:1000.

EXPERIMENTAL SECTION

General Experimental Procedures. Physangulidine A (1) was isolated from P. angulata as previously described.15 For the sample of 1 used in these studies, HPLC and NMR spectroscopy indicated a purity of at least 95%. All experiments described herein were repeated at least two or three times. Cell Culture and Treatment. All cells were obtained from ATCC and routinely grown in a humidified incubator at 37 °C with 5% CO2. DU145 (hormone-refractory prostate cancer) cells were grown in DMEM, and PC3 (hormone-refractory prostate cancer) cells were grown in F-12K medium supplemented with 10% fetal bovine serum (Life Technologies), 62.5 μg/mL penicillin, and 100 μg/mL streptomycin (Hyclone Laboratories, Logan, UT, USA). Cells (1 × 106) in fresh complete culture medium were plated on 10 cm diameter tissue dishes for 18 h. Cells were treated by addition of physangulidine A (1) directly to the culture medium to give the final concentration indicated in the figure legends. Cells for biochemical analyses were lysed in lysis buffer (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl, 0.25% deoxycholic acid, 1% octylphenoxypolyethoxyethanol (IGEPAL CA-630), pH 7.5) containing protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA, USA) for 10 min at 4 °C and then cleared by centrifugation at 16000g for 10 min at 4 °C. All protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Clonogenic Assay. Cells in fresh complete medium were plated at very low density (3 × 102 per well) into six-well plates for 18 h. After complete adhesion, cells were treated with 1 by adding directly to the medium to give the concentration indicated in the figures. Cells were allowed to grow until visible colonies formed (10 days). Cell colonies were fixed with 4% paraformaldehyde in PBS (phosphate-buffered saline), stained with 0.25% crystal violet in 25% methanol, washed, and air-dried. Flow Cytometric Assays of Cell Cycle and Cell Death. Cells in fresh complete medium were plated (2 × 105 per well) into six-well plates for 18 h. After complete adhesion, cells were treated as indicated in the figures and harvested by trypsinization. For cell cycle distribution, cells were fixed and stained with propidium iodide using the Cycle Test Plus kit (Becton Dickinson). For cell death detection, cells were stained with annexin-V-FITC and propidium iodide using the Apoptosis Detection Kit (BD Biosciences), according to the manufacturer’s instructions. Cells were then analyzed by flow cytometry using a FACScalibur cytometer (BD Biosciences, Mountain View, CA, USA) and FlowJo program (Tree Star, Inc., Ashland, OR, USA). DNA Fragmentation Assay. After the indicated treatment, cells (2 × 106) were collected (trypsinized and floating cells in the supernatant). DNA was extracted as described previously.19 Briefly, cells were lysed in 1% NP-40, 20 mM EDTA, and 50 mM Tris-HCl, pH 7.5, for 20 min on ice and clarified by centrifugation at 16000g for 10 min at 4 °C. Lysates were incubated with 0.5% SDS (w/v), pH 8.0, containing 0.5 mg/mL RNase A (Invitrogen, Grand Island, NY, USA) for 1 h at 37 °C and subsequently with 0.25 mg/mL proteinase K (Promega, Madison, WI, USA) for 1 h at 50 °C. Afterward, the lysates were subjected to extraction using phenol−chloroform−isoamyl alcohol (25:24:1; pH 7.4, X1), and DNA was precipitated from the cell lysates by adding ammonium acetate at a final concentration of 3 M and one volume of ice cold 2-propanol followed by 1 h incubation on ice. DNA was pelleted by centrifugation at 16000g for 1 h at 4 °C before being washed in cold 80% (v/v) ethanol and air-dried. The DNA pellet was resuspended in Tris-buffer (10 mM Tris-HCl and 1 mM EDTA (pH 8.0)). The samples were subjected to electrophoresis on 1.8% agarose gel, and the DNA was stained with ethidium bromide, then visualized using ultraviolet light (302 nm). Immunofluorescence Microscopy. Cells (2.5 × 104) in fresh complete culture medium were plated on 18 mm diameter glass coverslips for 18 h. The medium was replaced with complete medium containing 1 (10 μM) or an equivalent volume of vehicle (DMSO) and incubated for 24 or 48 h. After incubation, cells were washed three times with ice-cold PBS, fixed in 4% paraformaldehyde in PBS for 30

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ASSOCIATED CONTENT

S Supporting Information *

Effect of physangulidine A (1) on the proliferation of DU145 and PC3 prostate cancer cells. Effect of 1 at 5 μM on cell cycle and apoptosis in DU145 cells. Cell cycle arrest and apoptosis induced by 1 in PC3 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 502 852 2432. Fax: 502 852 3661. E-mail: paula.bates@ louisville.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the U.S. Department of Defense, Prostate Cancer Research Program (PCRP) of the Congressionally Directed Medical Research Program (CDMRP), under grant number W81XWH-07-1-0299 (PC060331).

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