Cancer Chemother Pharmacol (2014) 73:397–407 DOI 10.1007/s00280-013-2365-9

ORIGINAL ARTICLE

Induction of DNA damage and p21‑dependent senescence by Riccardin D is a novel mechanism contributing to its growth suppression in prostate cancer cells in vitro and in vivo Zhongyi Hu · Denglu Zhang · Jianrong Hao · Keli Tian · Wei Wang · Hongxiang Lou · Huiqing Yuan 

Received: 26 July 2013 / Accepted: 29 November 2013 / Published online: 10 December 2013 © Springer-Verlag Berlin Heidelberg 2013

Abstract  Purpose  Our previous studies had shown that Riccardin D (RD) exhibited cytotoxic effects by induction of apoptosis and inhibition of angiogenesis and topoisomerase II. Here, we reported that apoptosis is not the sole mechanism by which RD inhibits tumor cell growth because low concentrations of RD caused cellular senescence in prostate cancer (PCa) cells. Methods  Low concentrations of RD were used to treat PCa cells in vitro and in vivo, and senescence-associated β-galactosidase activity, DNA damage response markers, and/or colony-forming ability, cell cycle were analyzed, respectively. We then used siRNA knockdown to identify key factor in RD-triggered cellular senescence. Results  RD treatment caused growth arrest at G0/G1 phase with features of cellular senescence phenotype such as enlarged and flattened morphology, increased senescence-associated-beta-galactosidase staining cells, and decreased cell proliferation in PCa cells. Induction of cellular senescence by RD occurred through activation of DNA damage response including increases in the phosphorH2AX, inactivation of Chk1/2, and suppression of repairrelated Ku70/86 and phosphor-BRCA1 in PCa cells in vitro and in vivo. Analysis of expression levels of p53, p21CIP1, p16INK4a, p27KIP1, pRb and E2F1 and genetic knockdown

Z. Hu · D. Zhang · J. Hao · K. Tian · W. Wang · H. Yuan (*)  Department of Biochemistry and Molecular Biology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012, Shandong, People’s Republic of China e-mail: [email protected] H. Lou  Department of Natural Product Chemistry, Shandong University School of Pharmaceutical Sciences, Jinan 250012, People’s Republic of China

of p21CIP1 demonstrated an important role of p21CIP1 in RD-triggered cellular senescence. Conclusions  Involvement of the DNA damage response and p21CIP1 defines a novel mechanism of RD action and indicates that RD could be further developed as a promising anticancer agent for cancer therapy. Keywords  Riccardin D · Senescence · DNA damage · Prostate cancer

Introduction Cellular senescence is broadly defined as a signal transduction program leading to irreversible arrest of cell proliferation. The senescent state is characterized by cell cycle arrest, accompanied by distinctive changes in morphology to a flat and enlarged cell shape, along with changes in a set of genetic and proteomic biomarkers, especially by the induction of acidic senescence-associated-beta-galactosidase (SA-β-gal) activity [1]. In addition to telomeredependent onset of cellular senescence, stress-induced premature senescence is an alternative telomere-independent process that can be triggered by multiple cellular stresses including DNA damage, oxidative stress, and aberrant activation of oncogenes [2]. Upon DNA damage, cells trigger the DNA damage response (DDR), including activation of Chk1/2, key effectors of cell cycle-checkpoint protein kinases phosphorylated by ATM/ATR that initiate the checkpoint-cascade pathway, and downstream mediators of ATM/ATR signaling, leading to cell cycle arrest, DNA repair and the protection of genomic integrity. When DNA damage is hardly to be repaired, cells with damaged DNA may undergo apoptosis or initiate cellular senescence, depending on the extent of DNA lesions and cellular

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context. However, the mechanism by which DNA damage drives cellular senescence is not fully understood. It is proposed that senescence may be a consequence of failure to repair endogenous DNA damage, or activation of conserved stress response pathways promoting cellular senescence [3]. Because cellular senescence provides a tumor suppressive function by arresting mitosis, enhanced senescence in cancer cells can be considered as a prospective approach to inhibit cancer cell proliferation, making it one of the strategies for drug discovery [4]. Cellular senescence is found to be controlled by cell cycle modulators including p53, pRB, cyclin-dependent kinase (CDK) inhibitors such as p16INK4a, p21CIP1 or p27KIP1 [5]. Clinical and preclinical studies indicate that expression of these senescence-associated growth-regulatory genes in tumor cells has significant implications [6, 7]. Anticancer agents have been shown to activate p53 and p16INK4a pathways by initiating a senescent program that contributes to chemotherapeutic drug action and treatment outcome [8]. Therefore, chemotherapeutic agent-induced senescence is receiving more attention for a possible anticancer therapy via multiple mechanisms [4]. One of the most efficient chemical agents used in cancer chemotherapy are DNA damage inducers which can inflict a variety of DNA lesions. Compelling evidence indicates that cellular senescence, whether replicative senescence or premature senescence that is induced by different stressors, share a common underlying etiology, that is, DNA damage [9]. Thus, the elucidation of the biological aspects between tumor cellular senescence and DNA damage offers plausible approaches for the development of novel therapeutic strategies to arrest tumor cell proliferation including prostate cancer (PCa). PCa is one of the most common malignant tumors and a major cause of death from cancer in men [10]. Androgen deprivation therapy is initially successful in treating advanced PCa. However, the high failure rate for androgen-depletion causes progresses to hormone-refractory prostate cancer (HRPC) that is resistant to radiation, surgery, and chemotherapy [11, 12]. Currently, docetaxel is as an active agent to moderately improve quality of life and survival conditions in patients with metastatic HRPC [13, 14]. However, only about 50 % of patients treated with docetaxel have a prostate-specific antigen response [13]. Hence, many efforts have been made to investigate novel mechanisms of action of bioactive chemicals with less toxicity and high efficacy to treat HRPC [15]. Most recently, we have identified Riccardin D (RD), a macrocyclic bisbibenzyl compound from the Chinese liverwort plant Dumortiera hirsute [16], as a promising agent for its antitumor effects in vitro and in vivo, involving inhibition of angiogenesis [17], anti-inflammatory effects [18], and suppression of topoisomerase II (Topo II), leading to

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induction of rapid apoptosis [18–21]. Indeed, like other naturally occurring phytochemicals, such as well-studied docetaxel and resveratrol with a broad antitumor spectrum [22, 23], in vitro studies have demonstrated that a panel of tumor cell lines including PCa cells is responsive to RD. However, different cell lines displayed distinct modes of action (rapid apoptosis or growth arrest/senescence) in response to varied dosage of RD treatment. Here, we extended our observations in PCa cells that low concentrations of RD-induced cell cycle arrest at G0/G1 phase and generated DNA damage, subsequently leading cells to cellular senescence instead of triggering apoptotic cell death.

Materials and methods Cell culture Human LNCaP (The American Type Culture Collection (ATCC), Rockville, MD), PC3 and DU145 cells (The Cell Bank of Chinese Academy of Sciences, Shanghai) were cultured in RPMI 1,640 medium (HyClone, USA) supplemented with 10 % fetal bovine serum (HyClone). The human nonneoplastic prostate epithelial cell line RWPE1 was purchased from the ATCC and incubated in Keratinocyte medium supplemented with bovine pituitary extract and epidermal growth factor (Gibco, Grand Island, NY). Cells were maintained in 5 % CO2 at 37 °C until reaching approximately 50–70 % confluence, and then treated as indicated. For senescence studying, cells (2 × 106) were seeded in a 10-cm dish and incubated for 10 days with 0.5 μM RD in 1,640 medium with 10 % FBS. When cells reached approximately 80 % confluence, serial passage was performed (1:5 split ratio) by trypsinization with trypsin–EDTA. All experiments in which senescence was induced were performed in cells from p2 or p3. Chemicals and antibodies RD was isolated from Chinese liverwort Dumortiera hirsute, and the purity and structure determination were described previously [16]. The antibodies p53, cyclin D1, E2F1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz; Ku70 and Ku86 were obtained from Active Motif; p21CIP1 was from Anbo Biotechnology; p16INK4a, p27KIP1, Ser296-phosphorylated-Chk1, Thr68-phosphorylated-Chk2, Ser1524-phosphorylated-BRCA1, Ser139-phosphorylated histone H2AX (γH2AX), Ser807-phosphorylated-Rb, Tri-Methyl-Histone H3 (Lys9) (tri-Me-K9-H3), and horseradish peroxidase (HRP) conjugated secondary antibodies were from Cell Signaling Technology. Anti-rabbit IgG-TRITC antibody was from Abcam.

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Colony formation assay Cells were seeded into 6-well plates (300 or 500 cells/ well) and incubated with 0.5 μM RD or DMSO vehicle for 10 days. Plates were stained with Giemsa, and the number of colonies with more than 50 cells was counted. Cell cycle analysis After treatment with 0.5 μM RD for 10 days as indicated above, the cells were fixed, permeabilized, and treated with propidium iodide (PI) (Sigma) for 30 min in the dark. Cellular DNA content was analyzed by a FACS (Becton–Dickinson). Data were processed using ModFit LT Software.

HRP-conjugated secondary antibodies. Immunoblot bands were visualized by enhanced chemiluminescence detection system (Millipore) and exposed to X-ray films. Senescence‑associated β‑galactosidase staining Cells seeded in 6-well plates or tissue sections from nude mice were treated as described. After being rinsed with PBS, cells were fixed and incubated with freshly prepared senescence-associated β-Galactosidase (SA-β-Gal) staining solution at 37 °C overnight. A total of 200 cells were counted, and percentage of SA-β-Gal positive cells was calculated. Real‑time quantitative PCR analysis

BrdU assay for proliferation The proliferation of RWPE1, PC3, DU145, and LNCaP cells were examined by the BrdU Cell Proliferation Kit following the protocol (2,750, Millipore). Firstly, cells (2  × 103) in 100 μl culture medium seeded into 96-well plate were incubated with 0.5 μM RD for 10 days. BrdU was incorporated into cell culture 8 h prior to the end of the experiment incubation and detected by addition of peroxidase substrate. Spectrophotometric detection was performed at a wavelength of 450 nm by a microplate reader (BIO-RAD). The proliferation absorbance was calculated as following: the proliferation absorbance = actual absorbance  − background absorbance. Experiments were performed in triplicate and repeated three times. Immunofluorescence staining Cells were grown on cover-slips and treated as described. After being fixed with ice-cold methanol/acetone (1:1), cells were incubated with 3 % BSA in PBS with 0.1 % Triton X-100 for 20 min. Cells were then incubated with primary antibodies, immunostained with secondary antibodies and counter stained with DAPI. Fluorescence images were captured using a confocal microscopy (Carl Zeiss). Western blot analysis Whole cell lysates were prepared using RIPA buffer containing fresh protease inhibitor mixture (Fermentas). Samples containing equal amounts of proteins (60 μg) from lysates were separated by SDS–PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After being blocked with 5 % fat-free dry milk in TBS (20 mM Tris–HCl, pH 7.6, and 150 mM NaCl), the membrane was immunoblotted overnight at 4 °C with the appropriate primary antibodies and then subjected to washing and followed by incubation with

Total RNAs, extracted from cultured cells using an RNAiso plus kit (TaKaRa) according to the manufacturer’s instructions, were reverse-transcribed to cDNA using ReverTra Ace qPCR RT Kit. Quantitative PCR (qPCR) analysis was performed with SYBR Green reaction master mix (Toyobo) on a Real-time PCR System (Eppendorf International, Germany). Gene expression of mRNAs levels were normalized to the level obtained for GAPDH. Changes in the transcript levels were calculated using the ΔΔCt method. Small interference RNA treatment For optimization of siRNA transfection, different transfection procedures and siRNA concentrations were used to deliver siRNAs into cells. Transfection using Lipofectamine 2000 (Invitrogen) and 50 nM siRNA gave the best transfection efficiency and therefore was used in subsequent experiments. Cells were plated in six-well plates at 20–30 % confluency and 24 h later, transfected with 50 nM of siRNA. The next day, cells were stimulated with RD. When cells reached approximately 80 % confluence, serial passage was performed and the p2 cells were retreated with siRNA one more time to enhance the efficiencies of genetic knockdown. Chemically modified siRNAs p21CIP1i and negative control (NCi) siRNA were purchased from Invitrogen. Assessment of antitumor effect of RD on animals Tumor xenografts were established by injecting 5 × 106 PC3 cells into the right flank of 6-week-old athymic (BALB/c-nu) mice. Two weeks later, when tumors were detectable, the mice were randomized into treatment and control groups. Initial dosing was given at the time of pair matching (day 1). RD (30 mg/kg) or NS (placebo) was given every second day by i.p. injection. Mice were monitored daily for 20 days following treatment, and

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tumors were measured every second day by determining two perpendicular dimensions [length (L) and width (W)] using vernier calipers and calculated using the formula V = L × W2/2. Mice were killed then, and the tumors were collected for tumor mass measurement. Frozen tissue sections are made from the tumor tissues immediately snapfrozen in liquid nitrogen after being excised. Immunohistochemical analysis The frozen tissue sections were rewarmed at room temperature and washed with PBS. The appropriate primary antibodies were then added onto the sections and incubated overnight at 4 °C. After being stained with IgG conjugated HRP and DAB (Vector Laboratories), samples were counterstained with hematoxylin and subjected to capture images by microscopy (Nikon). Statistical analysis For the animal studies, all data are expressed as means with error bars showing SD. Comparison of end points was performed using an unpaired 2-tailed Student’s t test. For the cell culture studies, the data from three independent experiments were statistically examined by unpaired t test and are expressed as means ± SD. P 

Induction of DNA damage and p21-dependent senescence by Riccardin D is a novel mechanism contributing to its growth suppression in prostate cancer cells in vitro and in vivo.

Our previous studies had shown that Riccardin D (RD) exhibited cytotoxic effects by induction of apoptosis and inhibition of angiogenesis and topoisom...
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