Tumor Biol. DOI 10.1007/s13277-015-3477-0

RESEARCH ARTICLE

Induction of cell cycle arrest, DNA damage, and apoptosis by nimbolide in human renal cell carcinoma cells Yi-Hsien Hsieh 1,2 & Chien-Hsing Lee 3,4 & Hsiao-Yun Chen 6 & Shu-Ching Hsieh 5 & Chia-Liang Lin 6 & Jen-Pi Tsai 7,8

Received: 20 February 2015 / Accepted: 20 April 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract Nimbolide is a tetranortriterpenoid isolated from the leaves and flowers of Azadirachta indica which has been shown to exhibit anticancer, antioxidant, anti-inflammatory, and anti-invasive properties in a variety of cancer cells. However, the anti-tumor effect on human renal cell carcinoma (RCC) cells is unknown. In this study, we found that nimbolide treatment had a cytotoxic effect on 786-O and A-498 RCC cells in a dose-dependent manner. According to flow cytometric analysis, nimbolide treatment resulted in G2/ M arrest in 786-O and A-498 cells accompanied with an increase in the phosphorylation status of p53, cdc2, cdc25c, and decreased expressions of cyclin A, cyclin B, cdc2, and cdc25c. Nimbolide also caused DNA damage in a dosedependent manner as determined by comet assay and Yi-Hsien Hsieh and Chien-Hsing Lee contributed equally to this work. * Jen-Pi Tsai [email protected]

measurement of γ-H2AX. In addition, apoptotic cells were observed in an Annexin V-FITC/propidium iodide doublestained assay. The activities of caspase-3, -9, and poly ADPribose polymerase (PARP) were increased, and the expression of pro-caspase-8 was decreased in nimbolide-treated 786-O and A-498 cells. Western blot analysis revealed that the levels of intrinsic-related apoptotic proteins Bax and extrinsicrelated proteins (DR5, CHOP) were significantly increased in nimbolide-treated 786-O and A-498 cells. In addition, the expressions of Bcl-2 and Mcl-1 were decreased in 786-O and A-498 cells after nimbolide treatment. We conclude that nimbolide can inhibit the growth of human RCC cells by inducing G2/M phase arrest by modulating cell cycle-related proteins and cell apoptosis by regulating intrinsic and extrinsic caspase signaling pathways. Nimbolide may be a promising therapeutic strategy for the treatment of RCC. Keywords Apoptosis . Cell cycle . Nimbolide . DNA damage . Renal cell carcinoma

1

Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan

2

Clinical laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan

Introduction

3

Division of Pediatric Surgery, Changhua Christian Hospital, Changhua, Taiwan

4

Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan, Taiwan

5

Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

6

Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan

7

School of Medicine, Tzu Chi University, Hualien, Taiwan

8

Department of Nephrology, Buddhist Dalin Tzu Chi General Hospital, No 2, Minsheng Road, Dalin Township, Chiayi, Taiwan

Kidney cancer is the ninth most common cancer in men and the 14th most common in women worldwide, with renal cell carcinoma (RCC) accounting for more than 90 % of new cases in 2012 [1]. Kidney cancer is also the 16th most common cause of death from all cancers worldwide. Until the mid2000s, the standard treatment for RCC was surgical excision and immunotherapy, which mostly consisted of interferon or interleukin regimens [2]. Since then, the introduction of targeted therapy with tyrosine kinases and mTOR inhibitors has reduced the mortality rate [3]; however, RCC is generally resistant to chemotherapy, radiotherapy, and hormone therapy,

Tumor Biol.

and some patients will develop metastasis, recurrence, and eventually die [4]. It has been reported that natural and synthetic external substances such as resveratrol and chrysin, which possess antioxidant and anti-inflammatory properties, can suppress renal carcinogenesis [5, 6]. Nimbolide (5, 7, 4′-trihydroxy-3′, 5′diprenylflavanone), a tetranortriterpenoid with an α, βunsaturated ketone system, and a γ-lactone ring, was first derived from the leaves and flowers of neem, which is a traditional medicinal plant of the Meliaceae family that has attracted much attention as a promising candidate for the chemoprevention of malignancies [7, 8]. It has been reported that nimbolide has exhibited anticancer activity in multiple cancer cell lines including human hepatocarcinoma cells, colon cancer cells, and breast cancer cells [9–11]. In an in vivo study, nimbolide was found to have anti-proliferative and apoptotic effects in a hamster buccal pouch carcinogenesis model [12]. The possible mechanisms of nimbolide against cancer cells reported in the literature include the induction of apoptosis, inhibition of tumor cell proliferation, and suppression of NF-κB activation [13]. Cell cycle progression is a highly ordered and tightly regulated process that involves multiple checkpoints that assess extracellular growth signals, cell size, and DNA integrity. The cell cycle has been widely investigated, and the cyclindependent kinase 1 (CDK1; Cdc2)/cyclin B complex has been found to play a significant role in the regulation of the G2/M phase [14]. Cyclin A binds with CDK2, and this complex plays an important role in the S phase. In late G2 and early M phases, cyclin A forms a complex with CDK1 to promote entry into the M phase [15, 16]. Cdc25, which is the CDKactivating kinase, is necessary for the activation of CDK1 and further progression through the cell cycle. The activity of Cdc25 is inhibited by phosphorylation and dysregulation of Cdc25C, which catalyzes cyclin B/CDK1, thereby allowing for the unscheduled activation of CDK-cyclins, which are associated with tumor formation [15]. The p53 tumor suppressor gene has been shown to regulate the expression of p21, which is a CDK inhibitor that can inactivate the CDK-cyclin complex [16]. Several studies have shown that nimbolide can inhibit a variety of cancer cells through causing cell arrest by modulating these cell cycle-related proteins [17–19]. Apoptosis is a form of cell death that can be activated through two major signaling pathways: the intrinsic mitochondrial-mediated pathway and the extrinsic death receptor-induced pathway. The intrinsic pathway is triggered by anticancer medicines, growth factor withdrawal, or hypoxia with a resulting increase in the permeability of the outer mitochondrial membrane and release of apoptogenic factors such as cytochrome c from the mitochondrial inter-membrane space into the cytosol [20, 21], where they bind with Apaf1 and pro-caspase-9 to activate caspase-9, which in turn proteolytically activates caspase-3. Activated caspase-3 cleaves

many substrates including poly ADP-ribose polymerase (PARP) which triggers cell death and subsequently activates the caspase cascade [22, 23]. The extrinsic pathway is activated by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binding to its receptors, death receptor (DR4, also called TRAIL-R1) and death receptor 5 (DR5, also called TRAIL-R2), which transduce an apoptotic signal downward to recruit the Fas-associated death domain and pro-caspase-8 in a death-inducing complex [24]. Recently, the induction of apoptosis by nimbolide has received much attention because it has been shown to induce apoptosis of multiple cancer cell lines through individual or both pathways [25], demonstrating its potential as a tumor-inhibiting agent for adjuvant cancer treatment. Previous reports have demonstrated the apoptosisinducing effects of nimbolide in human choriocarcinoma (BeWo) and cervical cancer (HeLa) cells [19, 26]. However, the effect of nimbolide on the intrinsic and extrinsic pathways and DNA damage signaling in human renal cancer cells is unknown. Therefore, the aim of this study was to investigate the anticancer effect of nimbolide in RCC cells.

Materials and methods Reagents and antibodies Antibodies against p-p53, p-cdc2, p-cdc25c, cyclin A, cyclin B, cdc2, cdc25c, γ-H2AX, DR4, DR5, CHOP, caspase-8, and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against cleaved-caspase3, cleaved-caspase-9, and cleaved-PARP were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Enhanced chemiluminescence Western blot detection reagents were purchased from Amersham Life Sciences Inc. (Arlington Heights, IL, USA). Nimbolide, Z-DEVD-fmk (caspase-3 inhibitor), Z-LEHD-fmk (caspase-9 inhibitor), and Z-IETD-fmk (caspase-8 inhibitor) were purchased from BioVision Inc. (Milpitas, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and all other chemicals used were of analytical grade and were purchased from SigmaAldrich (St. Louis, MO, USA). Cell culture Human renal tubule HK-2 cells (BCRC 60097), human renal carcinoma 786-O cells (BCRC 60243), and A-498 cells (BCRC 60241) were obtained from the Bioresources Collection and Research Center, Food Industry Research and Development Institute (Hsinchu, Taiwan). The 786-O cells were grown in RPMI medium, and A-498 cells were grown in MEM medium; all cells were supplemented with 10 % FBS and 1 % antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). The human renal tubule HK-2 cells were grown in

Tumor Biol.

keratinocyte serum-free medium supplemented with bovine pituitary extract, human recombinant epidermal growth factor, and 1 % antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). The cells were maintained at 37 °C in a humidified atmosphere containing 5 % CO2. Upon reaching confluency, the cells were trypsinized and passaged. Determination of cell viability (MTT assay) Cells were seeded in 24-well culture plates at a density of 2× 104 cells/well before being treated with a series of concentrations of nimbolide. After treatment for 24 h, 100 μl of MTT (5 mg/ml) was added to each well and incubated with the cells for another 4 h and subsequently solubilized in isopropanol. Absorbance at 570 nm was measured using an automated microplate spectrophotometer (Sunrise, Tecan, Austria). The half maximal inhibitory concentration value was taken to be the concentration that would cause 50 % inhibition of cell viability and was calculated by the logit method.

(H2O2) for 24 h. The treated cells and controls were then trypsinized, and comet slides were prepared immediately after treatment. Half of the slides were then treated with 50 μM H2O2 for 5 min and washed twice with PBS. Fifty images were randomly captured per slide, and fluorescence microscopy images were quantified using Comet Assay III software (Perceptive Instruments Ltd., Haverhill, UK). The migration of DNA from the nucleus of each cell was measured with computer software using the parameter of comet moment. Statistical analysis The results are expressed as mean±SEM for three independent experiments. Statistical differences between groups were determined with the Student’s t test by using Instate software (GraphPad Prism 4, San Diego, CA, USA). Differences were considered to be statistically significant if the p value was