Article pubs.acs.org/jnp
Withaferin A, a Steroidal Lactone from Withania somnifera, Induces Mitotic Catastrophe and Growth Arrest in Prostate Cancer Cells Ram V. Roy, Suman Suman, Trinath P. Das, Joe E. Luevano, and Chendil Damodaran* Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas 79905, United States
ABSTRACT: Cell cycle deregulation is strongly associated with the pathogenesis of prostate cancer. Clinical trials of cell cycle regulators that target either the G0/G1 or G2/M phase to inhibit the growth of cancers including prostate cancer are increasing. The present study focused on the cell cycle regulatory potential of the withanolide withaferin A (1) on prostate cancer cells. Compound 1 induced G2/M arrest in both prostate cancer cell lines (PC-3 and DU-145) when treated for 48 h. The G2/M arrest was accompanied by upregulation of phosphorylated Wee-1, phosphorylated histone H3, p21, and Aurora B. On the other hand, downregulation of cyclins (A2, B1, and E2) and a reduction in phosphorylated Cdc2 (Tyr15) were observed in 1-treated prostate cancer cells. In addition, decreased levels of phosphorylated Chk1 (Ser345) and Chk2 (Thr68) were evident in prostate cancer cells on treatment with 1. These results suggest that activation of Cdc2 leads to arrest in the M phase, with abnormal duplication, and initiation of mitotic catastrophe that results in cell death. In conclusion, these results show clearly the potential of 1 as a regulator of the G2/M phase of the cell cycle and as a therapeutic agent for prostate cancer.
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translocation of Cdc25C from the cytoplasm, while unphosphorylated Cdc25C will migrate to the nucleus.11,12 In recent years, natural compounds have drawn a great deal of attention because of their ability to suppress cancers as well as their potential to reduce the risk of cancer development.13 Withaferin A (1), a major constituent of the medicinal plant Withania somnifera (L.) Dunal (Solanaceae), is a well-studied bioactive natural product. Extracts of W. somnifera have been used historically for the treatment of cancer, inflammation, and neurological disorders.14,15 Withaferin A (1) exhibits an inhibitory effect against several different types of cancer cells. Thus, in human leukemia cells, 1 induced apoptosis in association with JNK and AKT signaling along with inhibition of NF- κB activity.16 In addition, 1 also inhibited the growth of breast cancer cells in vitro and in vivo by inhibiting molecularly the levels of both transmembrane and cleaved Notch-1, but inducing Notch-2 and -4 signaling cascades in MCF-7 and MDA-231 cells.17 Previously, it was reported that 1 induces apoptosis in castration-resistant prostate cancer cells but not in androgen-responsive and
rostate cancer is the third most common cause of cancerrelated deaths for men in Western countries.1 In recent years, the development of cutting-edge technologies for early detection of prostate cancer and more effective therapeutic strategies has prevented prostate cancer-related deaths. The pathogenesis of prostate tumors is initially androgen-dependent. Hence, hormone ablation therapy is the primary treatment for prostate cancer;2 however, many patients relapse to castration-resistant prostate cancer.3 In the mammalian cell cycle, the transition to G2/M is tightly regulated by the kinase Cdc2/cyclin B.4 Inhibition of Cdc2 activity occurs by specific phosphorylation at Tyr15 and Thr14 by two major regulators, Wee-1 and myelin transcription factor 1 (Myt-1).5 On the other hand, Cdc25C dephosphorylates Tyr15 on Cdc2 and converts this kinase to the active form, which is required for mitosis entry.6 The activity of Cdc2, Cdc25C, and Wee-1 proteins is regulated stringently at every phase of the cell cycle.7−9 The cyclin B−Cdc2 complex localizes to the cytosol in the G2 phase, and during prophase the complex is activated and shuttles to the nucleus.10 Cdc25 is regulated by the checkpoint kinase (Chk1) and 14-3-3 proteins. Chk1 phosphorylates Cdc25C at Ser216, creating a consensus binding site for 14-3-3. The binding of 14-3-3 inhibits nuclear © 2013 American Chemical Society and American Society of Pharmacognosy
Received: June 18, 2013 Published: September 30, 2013 1909
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Prostate cancer cell lines (PC-3 and DU-145) were treated with different concentrations (0.5, 1, 2, 3, and 4 μM) of 1, and cell viability was measured with a trypan blue exclusion assay. A dose-dependent loss of cell viability was observed in both PC-3 and DU-145 cells after 24 h of treatment with 1 (Figure 1A, B). To elucidate the mechanism underlying 1-mediated growth arrest in these prostate cancer cell lines, they were treated with the IC50 doses of 1 for 12, 24, and 48 h. Treatment with 1 significantly induced G2/M arrest in both cell lines as early as 8 h (data not shown). As seen in Figure 1C, the G2/M phase accounted for 62.7% of the total population at 24 h and 60.7% at 48 h, whereas 28.3% and 25.3% of vehicle-treated cells were in G2/M arrest at both time points, respectively. Similarly, DU145 cells in the G2/M phase accounted for 36.75% of the total population at 24 h and 24.75% at 48 h, whereas 22% of vehicletreated cells were in G2/M arrest at both time points (Figure 1D). These results suggest that 1 arrests prostate cancer cells in the G2/M phase. Mechanism Underlying 1-Induced G2/M Cell Cycle Arrest. Having ascertained that 1 induces G2/M cell cycle arrest in prostate cancer cell lines, the molecular mechanism by which this withanolide causes this arrest was probed. The expression patterns of cyclins were investigated by Western blot analysis. The expression levels of cyclins E2, A2, and B1 were all downregulated by 1 (Figure 2A). As the G2/M checkpoint for mitosis entry requires cyclin B and A expression,19,20 the decreased levels of these proteins in this experiment suggest that prostate cancer cells were arrested at the G2 phase of the cell cycle. Interestingly, increased expression of p21 at both the mRNA (Figure 2C) and protein (Figure 2B) levels was found following treatment with 1. Western blot analysis revealed the appearance of two closely migrating bands, suggesting that p21 exists in a hyper-phosphorylated form upon treatment with 1
normal prostate epithelial cells, by inducing the pro-apoptotic protein prostate apoptosis response-4 (Par-4).18 Withaferin A (1) interferes with the ubiquitin-mediated proteasome pathway, leading to increased levels of poly-ubiquitinated proteins and subsequent inhibition of proliferation in human umbilical vein endothelial cells (HUVECs). This molecule also inhibits sprouting of HUVECs via inhibition of NF-κB activation.15 In the present study, the cell cycle regulatory potential of 1 was determined on prostate cancer cell lines. Hence, activation of Cdc2 by 1 leads to the accumulation of cells in the M phase and results in abnormal duplication and mitotic catastrophe that initiates prostate cancer cell death.
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RESULTS AND DISCUSSION Inhibition of Cell Viability and Induction of the G2/M Phase of Cell Cycle Arrest by 1 in Prostate Cancer Cells.
Figure 1. Inhibition of cell growth and induction of G2/M cell cycle arrest by 1 in prostate cancer cell lines. (A) PC-3 and (B) DU-145 cells were treated with either DMSO or the indicated dose of 1 for 24 h, and the percentage of viable cells was determined using the trypan blue exclusion method. (C) PC-3 and (D) DU-145 cells were treated with 1 (1 μM) for 12, 24, or 48 h and subjected to cell cycle analysis by flow cytometry. Values represent mean ± standard error mean of the three independent experimental samples (*p < 0.01 and **p < 0.005 versus vehicle). 1910
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Figure 3. Regulatory effects of 1 on Wee-1/Cdc2. PC-3 cells were treated with 1 (1 μM) for the indicated time periods, and cell lysates were subjected to Western blot analysis for (A) Wee-1, phospho-Wee1, phospho-Cdc2Tyr15, and total Myt-1 expression. Actin was used as a loading control. (B) PC-3 cells were treated with either 1 (1 μM) or DMSO for 24 h and analyzed for Cdc2 (Tyr15) using immunofluorescent staining. Representative photographs were taken under a fluorescence microscope. A decrease in the punctate expression pattern was observed in the G2/M-arrested cells (yellow arrow).
Figure 2. Decreased cyclin A2, E2, and B1 protein levels in PC-3 cells treated with 1. (A) PC-3 cells were treated with either DMSO (untreated, UT) or 1, and cell lysates were subjected to Western blot analysis for cyclin A2, cyclin E2, and cyclin B1 expression. (B) PC-3 cells were treated with either DMSO (untreated, UT) or 1, and cell lysates were subjected to Western blot analysis for p21 expression. (C) RT-PCR confirming the expression of p21 protein upon 1 treatment in PC-3 cells.
activation of Cdc2 through Cdc25C, allowing cells to enter the M phase but to arrest in the pro-metaphase. Published results suggest that Wee-1 is an important factor that acts as a mitotic inhibitor; however, Wee-1 expression in cancer is controversial. For example, overexpression of Wee-1 has been reported for hepatocellular carcinoma,27 breast,28 and glioblastoma tumor tissues,23 while colon29 and prostate tumor30 tissues express very low levels of Wee-1. Thus, Wee-1 may function as a tumor suppressor, and activating Wee-1 could be a useful therapeutic target for prostate cancer. Importantly, the results demonstrated that 1 activates Wee-1 and induces growth arrest. Role of Chk1 and Chk2 Activation in 1-Induced G2/M Arrest. To determine the role of Chk1 and Chk2 levels in 1treated PC-3 cells, Western blot analysis was performed. A reduction in phosphorylated Chk1Ser345 and Chk2Thr68 was evident in 1-treated PC-3 cells (Figure 4A). No significant changes occurred in the kinase activity of Chk1 or the Cdc2− cyclin B complex in 1-treated PC-3 cells compared with control cells (Figure 4B and C). These results suggest that 1 caused G2/M arrest without altering the kinase activity of Chk1 or the Cdc2−cyclin B complex in PC-3 cells. Activation of Chk1 and Chk2 results in phosphorylation of Cdc25C during the G1/S phase transition and phosphorylation of Cdc25C during G2/ M.31 In addition, activation of Chk1 induces Wee-1 and also arrests the cells in G2/M. Another G2/M regulator is 14-3-3,32 which is activated by p53. However, in these studies, no significant differences were noted in the expression of 14-3-3 in untreated and 1-treated PC-3 cells (Figure 4D). G2/M Arrest Involves Accumulation of Aurora B and Defects in α-Tubulin Depolymerization in 1-Treated Prostate Cancer Cells. During cell division, chromatin
(Figure 2B). This may facilitate p21 binding to cyclin B1 to form a complex with Cdc2, thereby promoting kinase activation and G2/M progression.21 p53 is the major activator of p21; however, PC-3 cells do not express p53. Thus, 1 may induce p53-independent p21 activation in PC-3 cells. Induction of p21 has been reported for both G1 and G2/M cell cycle arrest that eventually led to growth arrest.22 These results clearly demonstrate that the 1-induced G2/M cycle arrest in prostate cancer cell lines is accompanied by decreased levels of cyclins A2, E2, and B1 and increased activation of p21. Accumulation of Active Wee-1 by 1 in the G2/M Phase of Prostate Cancer Cells. Transition to the G2/M phase of the cell cycle is regulated by Cdc2 activity, and Wee-1 and Myt1 negatively regulate Cdc2 function in mammalian cells.10,12,21,23−25 Hence, the expression pattern of these regulatory proteins was examined in 1-treated PC-3 cells. An upregulation of phospho-Wee-1 (Ser642) was seen at 12 and 24 h after 1 treatment in PC-3 cells (Figure 3A). On the other hand, there was reduced phosphorylation of Cdc2 at Tyr15 (Figure 3A) and of Cdc25c at Ser216 following treatment with 1 (data not shown). In addition, Myt-1 expression was downregulated in PC-3 cells (Figure 3A), indicating that Myt-1 may not play a role in the regulation of the Cdc2/Cdc25c complex. Previous reports have documented that during the M phase the Cdc2−cyclin B complex is exported to the nucleus.9,26 In accordance, a pattern of punctate staining in the cell membrane with a decrease in cytoplasmic levels of phospho-Cdc2 (Tyr15) was found (Figure 3B). These results demonstrate that 1 induces a delay in phospho-Wee-1 degradation followed by 1911
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phosphorylation of H3 (Figure 5A) after treatment with 1 suggests that Aurora B activation may initiate H3 activation in PC-3 cells. Expression of Aurora B reaches a maximum at the G2/M phase, and activated Aurora B mediates spindle positioning. When cells are preparing for division, Aurora B phosphorylates histone H3Ser10, dissociates the heterochromatin protein 1 from the chromosome, and affects the heterochromatin formation.33 Therefore, confocal microscopy was utilized to analyze the localization of Aurora B in control and 1treated PC-3 cells. Aurora B distributed uniformly in the cytoplasm of nondividing cells (Figure 5B). In 1-treated PC-3 cells, Aurora B was increased in the centromeric regions of chromosomes, as determined by its punctate distribution. These findings suggest that the cells are arrested at the G2/M phase of the cell cycle (Figure 5B). As more 1-treated cells became arrested in the M phase, the proportion of cells with punctate staining was higher than that of the control cells. Similarly, localization and accumulation of phospho-H3 in the nucleus of 1-treated PC-3 cells were observed as compared to control cells (Figure 5C). Thus, phospho-H3 staining levels were higher in the treated cells and remained uniformly associated with the chromosomes inside the nucleus. These results demonstrate that more cells are in the process of active recruitment of condensation factors, and 1 did not inhibit early chromatin remodeling factors. One of the characteristic features of active cell division is redistribution of tubulin. PC-3 cells treated with 1 were examined for distribution of α-tubulin. Cells treated with 1 exhibited bipolar spindles (Figure 6A), suggesting defects in depolymerization of α-tubulin. As a result, cells failed to progress to the next phase. In control cells, microtubule bundles converged to well-defined single poles, and their corresponding chromosomes were situated in the equatorial plane, whereas in 1-treated cells, the spindles were highly distorted and consist of a curving microtubule array (Figure 6A). To confirm whether 1 directly interferes with tubulin polymerization, tubulin dynamics were studied in a cell-free system. As seen in Figure 6B, 1 inhibited the tubulin polymerization in a time-dependent manner. These results suggested that 1 can inhibit tubulin polymerization in a cell-free system as well as cell culture models, to induce cell cycle arrest and growth arrest in prostate cancer cells. In conclusion, the results reported herein demonstrate that withaferin A (1) induces cell cycle arrest at the G2/M phase and that this arrest may inhibit the growth of prostate cancer cells. At the molecular level, cell cycle arrest is associated with inhibition of Wee-1 expression, which results in mitotic arrest and eventually leads to cell death. Further studies are required to elucidate the potential therapeutic role of 1 in the treatment of prostate cancer.
Figure 4. Inhibition of Chk1 and Chk2 expression by 1 in PC-3 cells. (A) PC-3 cells were treated with vehicle or 1, and cell lysates were subjected to Western blot analysis for Chk1, phospho-Chk1Ser345, Chk2, and phospho-Chk2Thr68. Actin was used as a loading control. (B) Checkpoint kinase activation in 1-treated PC-3 cells. No significant changes in Chk1 kinase activity were observed between vehicle- and 1-treated PC-3 cells. (C) Cdc2−cyclin B kinase activation in 1-treated PC-3 cells. No significant change was observed in Cdc2− cyclin B kinase activity between vehicle and 1-treated PC-3 cells. (D) PC-3 cells were treated with vehicle or 1, and cell lysates were subjected to Western blot analysis for total 14-3-3. Actin was used as a loading control.
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EXPERIMENTAL SECTION
Chemicals and Antibodies. Cyclin A2, cyclin E2, cyclin B1, p21, phospho-Wee-1, phospho-Cdc2, Myt-1, phospho-Chk1/2, 14-3-3, phospho-H3, H3, and Aurora B antibodies were obtained from Cell Signaling (Danvers, MA, USA) and horseradish peroxidase-conjugated anti-mouse, anti-goat, and anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Propidium iodide was purchased from Sigma (St. Louis, MO, USA). Alexa Fluor 568 Phalloidin, and ProLong Gold antifade mounting medium with DAPI were purchased from Invitrogen (Grand Island, NY, USA). Withaferin A (1) was purchased from Chromadex (Irvine, CA, USA). For the samples of withaferin A (1) used in this study,
rearrangement is coordinated by a group of proteins called the chromosomal passage proteins, of which Aurora B is a member. To understand the distribution of Aurora B during 1-induced G2/M arrest, Western blot and confocal analyses were performed. Withaferin A induced Aurora B expression at 12 and 24 h in PC-3 cells (Figure 5A). Activation of Aurora B resulted in phosphorylation of histone H3 at serine 10, leading to initiation of chromosomal condensation.33 Also, site-specific 1912
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Figure 5. Mitotic arrest, alteration of the mitotic spindles, and accumulation of passage proteins altered by 1. (A) Western blot analysis of H3, phospho-H3Ser10, and Aurora B expression in 1-treated PC-3 cells. Actin was used as a loading control. PC-3 cells were immunostained with antibodies to (B) Aurora B and (C) phospho-H3Ser10 after being treated with 1. HPLC, NMR, and IR analysis by the manufacturer indicated a purity of not less than 98%. Cell Culture. The PC-3 and DU-145 human prostate cancer cell lines were grown in Dulbecco’s modified Eagle’s medium and RPMI1640, respectively, and supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotics in multiwell plates at 37 °C in a humidified atmosphere containing 5% CO2. After overnight culture to allow cells to adhere, the medium was replaced with or without 1 at the indicated concentrations. Withaferin A (1) was dissolved in dimethyl sulfoxide (DMSO), and the cells were treated with DMSO at a final concentration of 0.002%. Cell Viability. PC-3 and DU-145 cells were plated at a density of 3 × 105 cells/well in six-well plates in medium containing 10% fetal bovine serum. Cells were cultured for 24 h and treated with the addition of different concentrations of 1. Control cells were treated with the same volumes of DMSO, which never exceeded 0.002% of the
total volume of the medium. After each treatment, cells were incubated at 37 °C for 24 h in an atmosphere of 5% CO2. Viable cells were counted by trypan blue exclusion using a hemocytometer. Results are expressed as a percentage of the number of cells in DMSO-treated control cultures, and the half maximal inhibitory concentration (IC50) values were calculated. Western Blot Analysis. Cells were treated with 1 at IC50 concentrations that had been optimized for each cell line based on preliminary studies for various time periods, and cell lysates were prepared using Pierce M-PER lysis buffer containing 1× halt protease inhibitor cocktail (Pierce, Rockford, IL, USA). Proteins (25 μg) were subjected to Western blot analysis. Cdc2, Chk1, Chk2, cyclin A2, cyclin E2, cyclin B1, H3, phospho-H3, p21, Wee-1, phospho-Wee-1, phospho-Cdc2, Myt-1, phospho-Chk1/2, 14-3-3, Aurora B proteins, and loading control β-actin were detected according to the method described.34 1913
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reaction). A similar approach was used to detect Cdc2/cyclinB1 kinase activation in 1-treated PC-3 cells. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR). Total RNA was isolated from cells using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized using the Applied Biosystems cDNA synthesis kit. Quantitative RT-PCR was performed using the 2−ΔΔCT method and SYBR Green supermix (Qiagen, Hilden, Germany) on an Applied Biosystems multicolor real-time PCR detection system. Cycle threshold values were normalized to amplification measured for βactin. Primers used for human p21 were (forward) 5′-CGATGCCAACCTCCTCAACGA-3′ and (reverse) 5′-TCGCAGACCTCCAGCATCCA-3′. Amplification was performed under the following conditions: 95 °C for 5 min, followed by 45 cycles of 94 °C for 30 s and 55 °C for 40 s, with an extension at 72 °C for 45 s. Tubulin Polymerization Assay. To determine the role of 1 on tubulin polymerization, an absorbance-based tubulin polymerization assay kit (Cytoskeleton, Denver, CO, USA) was used as per the manufacturer’s instructions. Briefly, tubulin protein (3 mg/mL) was incubated with 1 in prewarmed 96-well plates along with a negative (vehicle) and a positive control (paclitaxel). The polymerization of tubulin was measured every 10 min for 1 h at 37 °C using a Fluostar Optima reader (BMG Labtech, Inc., Cary, NC, USA) set at 405 nm. Statistical Analysis. Data are presented as the means ± standard error of the mean (SEM). Significant differences between the groups were determined using the unpaired Student’s t-test. Significant differences were established at p < 0.05. All statistical analyses were performed with GraphPad Prism 6 software.
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AUTHOR INFORMATION
Corresponding Author
Figure 6. Inhibition of microtubule polymerization in PC-3 cells and a cell-free system. (A) α-Tubulin was stained with Alexa Fluor 488, and the nucleus was stained with DAPI. (B) Determination of the effect of 1 on microtubule polymerization using a cell-free tubulin polymerization assay.
*Tel: 915-215-4228. Fax: 915-783-5221. E-mail: C.damoaran@ ttuhsc.edu. Notes
The authors declare no competing financial interest.
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Cell Cycle Analysis. Cells (PC-3 and DU-145) were plated at a density of 3 × 105 cells/well in six-well plates. After overnight attachment, cells were treated with 1 μM 1 for the indicated times. The cells were stained with 0.5 g/L propidium iodide and analyzed using FACScan (Accuri Flow Cytometers, Inc., MI, USA), as described previously.35 Immunofluorescence Staining. PC-3 cells were plated on glass coverslips and allowed to attach and grow to 60% confluence overnight. Following treatment with 1 for 24 h, cells were washed three times with PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, and permeabilized with 0.2% Triton X-100 for 20 min. Cells were incubated with antibodies to detect the localization and expression of phosphor-Cdc-2Tyr15, α-tubulin, phospho-H3Ser10, and Aurora B. Cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 (green)/Alexa Fluor 568 Phalloidin (Red). Finally, the cells were incubated with mounting medium with DAPI. Cells were analyzed using a Nikon laser scanning confocal microscope. Kinase Assay. To determine the role of Chk1 and Chk2 levels in 1-treated PC-3 cells, a CycLex checkpoint kinase assay kit-1 (CY-1162; MBL International, MA, USA) was used. Briefly, PC-3 proteins were precoated with a substrate corresponding to recombinant Cdc25C, which contains serine residues that can be phosphorylated by checkpoint kinases, including Chk1 and Chk2. The detector antibody is specific to the phosphorylated serine216 residue on Cdc25C. To perform the test, the sample was diluted in kinase buffer, which was pipetted into the wells and allowed to phosphorylate the bound substrate after the addition of Mg2+ and ATP. The amount of phosphorylated substrate was measured via a binding reaction with a horseradish peroxidase-conjugated anti-phospho-Cdc25C Ser216 specific antibody, and this binds to and catalyzes the conversion of the chromogenic substrate tetramethylbenzidine from a colorless solution to a blue solution (or yellow after the addition of reagent to stop the
ACKNOWLEDGMENTS This study was supported by the ACS-RSG-10-186-01-TBE, R01CA140605 and R01CA138797.
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