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FULL PAPER British Journal of Cancer (2017) 117, 1621–1630 | doi: 10.1038/bjc.2017.319 Keywords: ailanthone; non-small cell lung cancer; DNA replication; RPA1; Chinese medicine Ailanthone inhibits non-small cell lung cancer cell growth through repressing DNA replication via downregulating RPA1 Zhongya Ni1, Chao Yao1, Xiaowen Zhu1, Chenyuan Gong1, Zihang Xu2, Lixin Wang3, Suyun Li4, Chunpu Zou2 and Shiguo Zhu*,1,3 1Laboratory of Integrative Medicine, School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, 1200 Cai Lun Rd, Shanghai 201203, PR China; 2Department of Internal Classic of Medicine, School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, 1200 Cai Lun Rd, Shanghai 201203, PR China; 3Department of Immunology and Pathogenic Biology, School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, 1200 Cai Lun Rd, Shanghai 201203, PR China and 4Department of Pathology, School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, 1200 Cai Lun Rd, Shanghai 201203, PR China Background: The identification of bioactive compounds from Chinese medicine plays a crucial role in the development of novel reagents against non-small cell lung cancer (NSCLC). Methods: High throughput screening assay and analyses of cell growth, cell cycle, apoptosis, cDNA microarray, BrdU incorporation and gene expression were performed. Results: Ailanthone (Aila) suppressed NSCLC cell growth and colony formation in vitro and inhibited NSCLC tumour growth in subcutaneously xenografted and orthotopic lung tumour models, leading to prolonged survival of tumour-bearing mice. Moreover, Aila induced cell cycle arrest in a dose-independent manner but did not induce apoptosis in all NSCLC cells. Furthermore, 1222 genes were differentially expressed upon Aila administration, which were involved in 21 signal pathways, such as DNA replication. In addition, Aila dose-dependently decreased BrdU incorporation and downregulated the expression of replication protein A1 (RPA1). Conclusions: Aila inhibited the growth of NSCLC cells through the repression of DNA replication via downregulating RPA1, rather than through cell cycle arrest and apoptosis. Our findings suggested that Aila could be used as a promising therapeutic candidate for NSCLC patients. Lung cancer is the leading cause of cancer death worldwide and 5-year survival rate is still less than 5%. Therefore, it is emergently non-small cell lung cancer (NSCLC) is the most common type and necessary to develop novel treatment strategies to improve represents 80% of all lung cancers. In China, approximately efficiency and reduce mortality of lung cancer. 4 292 000 new cancer cases and 2 814 000 cancer deaths occurred in Natural products are a major resource for the development of 2015, and lung cancer is also the most common cancer and the novel drug leads (Cragg and Newman, 2013). Chinese medicine leading cause of cancer death (Chen et al, 2015). Although the has been extensively documented over the centuries, which has a 5-year survival rate has been improved owing to the advances in long history of use in cancer treatment, showing significantly the management of NSCLC over the past 30 years, the overall improved therapeutic efficacy, reduced side effects of *Correspondence: Dr S Zhu; E-mail: [email protected] Received 8 June 2017; revised 10 August 2017; accepted 18 August 2017; published online 12 October 2017 r 2017 Cancer Research UK. All rights reserved 0007 – 0920/17 www.bjcancer.com | DOI:10.1038/bjc.2017.319 1621 BRITISH JOURNAL OF CANCER Ailanthone inhibits NSCLC cell growth chemotherapy and prolonged survival (He et al, 2016a; Zheng et al, six-well plates at a density of 200 cells per well. Aila was added to 2017). Therefore, the identification of bioactive compounds from the cells at different final concentrations (0, 0.078 and 0.156 mM), Chinese medicine plays a crucial role in the development of new and then the cells were incubated for 12 days. The colonies were anticancer reagents. fixed by 4% paraformaldehyde for 15 min, stained with crystal In the present study, high throughput screening assay showed violet for 15 min and then imaged and counted under a phase- that ailanthone (Aila) possessed the potential to inhibit NSCLC cell contrast microscope. growth. Aila is a main active compound of the stem barks of Ailanthus altissima, which is traditionally used to treat ascariasis, Subcutaneous xenograft tumour models. All animal procedures, diarrhoea, spermatorrhoea, bleeding and gastrointestinal diseases, including tumour transplantation, tumour volume monitoring and and it has been recently found to have anti-inflammatory and anti- killing, were approved by the Institutional Animal Care and Use tumour activities (Kim et al, 2015; Wang et al, 2016). Analyses of Committee at Shanghai University of Traditional Chinese cell growth, cell cycle, apoptosis, cDNA microarray, BrdU Medicine. For subcutaneous xenograft model, male Balb/c nude incorporation and gene expression revealed that Aila could mice (6–8-week-old, weighing 18–20 g) were purchased from significantly inhibit NSCLC cell growth both in vitro and in vivo. Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and 6 Moreover, Aila repressed DNA replication in a dose-dependent maintained under an SPF environmental condition. Briefly, 3  10 manner and significantly downregulated replication protein A1 of H1975 cells or A549 cells were subcutaneously inoculated in the (RPA1). This finding indicated that Aila inhibited NSCLC cell back of nude mice. When tumour volume reached approximately 3 growth through targeting replication stress, suggesting that Aila 100 mm , mice were randomly assigned and administered with could be used as a promising therapeutic candidate in NSCLC different doses of Aila via intraperitoneal (IP) injection. The treatment. tumour volume and body weight were determined every 3 days. Tumour volume was calculated using the following formula: V ¼ (p/8)a  b2, where V ¼ tumour volume, a ¼ maximum tumour MATERIALS AND METHODS diameter and b ¼ minimum tumour diameter. Mice were huma- nely killed by CO2 suffocation when the tumour volume reached 3 Reagents. Aila was purchased from BioBioPha Co., Ltd (Yunnan, 2000 mm . China). Cell cycle kit was obtained from Biotime (Beijing, China). Biochemistry and histology. To evaluate the safety of Aila, mice Annexin V-FITC apoptosis kit and bromodeoxyuridine (BrdU) in the subcutaneous xenograft model were killed on day 58, and flow cytometry kit were supplied from BD Biosciences (San Jose, serum, liver and kidney were collected. Serum levels of alanine CA, USA). RNA extraction kit, PrimeScript RT MasterMix kit and aminotransferase (ALT), aspartate aminotransferase (AST), albu- SYBR Premix Ex Taq kit were provided by Takara (Dalian, China). min (ALB) and creatinine (CREA) were determined. Liver and Antibodies against caspase-3, PARP (poly ADP-ribose polymerase) kidney were fixed with 10% fresh formalin, embedded in paraffin, and actin as well as secondary antibodies were purchased from Cell cut into 4-mm sections, stained with haematoxylin–eosin and then Signaling Technology (Danvers, MA, USA). Antibodies against observed under a light microscope (  100 and  400 RPA1 and proliferating cell nuclear antigen (PCNA) were obtained magnification). from Santa Cruz (CA, USA). Mouse anti-human g-H2AX monoclonal antibody and goat anti-mouse IgG Alexa Fluro 488 Orthotopic lung tumour models and bioluminescent imaging. were purchased from Abcam (Cambridge, MA, USA). ECL western Male SCID-Bg mice (6–8-week-old) were purchased from Vital blotting substrate kit was supplied from Merck Millipore (Billerica, River Laboratory Animal Technology Co. (Beijing, China) and MA, USA). Amaxa cell line nucleofector kit T was purchased from maintained under an SPF environmental condition. Mice were Lonza (Cologne, Germany). MTT assay kit, cisplatin (DDP) and anaesthetised with 0.5% mebubarbital, and the left chest was camptothecin (CPT) were provided by Sigma-Aldrich (St Louis, shaved and exposed. Briefly, 1  106 eGFP-FFLuc þ A549 cells in MO, USA). 50 ml serum-free DMEM medium were mixed with 50 ml matrigel, and then the mixture was injected into the left lung. On day 4 after Cell culture. Human NSCLC A549, H1299 and H1975 cell lines À g were obtained from Shanghai Cell Bank of Chinese Academy of injection, mice were treated with 2 mg kg of Aila via IP Sciences. A549 cells were cultured in high glucose DMEM (Gibco, administration every 3 days, and the tumour volume was weekly Life Technologies, Grand Island, NY, USA) supplemented with monitored by imaging isoflurane-anaesthetised mice using the 10% foetal bovine serum (FBS; Gibco) and 1% penicillin and IVIS system (Xenogen, Alameda, CA, USA). streptomycin in 5% CO2. H1299 and H1975 cells were cultured in Cell cycle analysis. A total of 3  105 of A549, H1299 and H1975 RPMI 1640 (Gibco) supplemented with 10% FBS and 1% penicillin cells were respectively seeded in 6-cm plates and incubated and streptomycin in 5% CO2. overnight. Cells were then incubated with different concentrations Cell viability assay. MTT assay was performed to determine the (0, 0.625, 1.25, 2.5 and 5.0 mM) of Aila for 24 h. Subsequently, cells effect of Aila on the viability of NSCLC cells. Briefly, A549, H1299 were harvested, washed with ice-cold PBS, then fixed with 70% and H1975 cells were respectively seeded in 96-well plates at a ethanol overnight at À 20 1C and stained with propidium iodide density of 4  103 cells per well. Aila or DDP was added to the cells (PI) at 37 1C for 30 min in the dark. The DNA content was at different final concentrations (0, 0.078, 0.156, 0.3125, 0.625, 1.25, determined using a FACS Calibur cytometer (BD Biosciences) and 2.5, 5.0, 10 and 20 mM). At 24, 48 and 72 h, MTT was added to the analysed by the FCS Express v2.0 software (De Novo Software, cells at a final concentration of 0.5 mg ml À 1.

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