Published OnlineFirst July 22, 2015; DOI: 10.1158/1078-0432.CCR-15-0198 Cancer Therapy: Preclinical Clinical Cancer Research Aspirin Suppresses the Growth and Metastasis of Osteosarcoma through the NF-kB Pathway Dan Liao, Li Zhong, Tingmei Duan, Ru-Hua Zhang, Xin Wang, Gang Wang, Kaishun Hu, Xiaobin Lv, and Tiebang Kang Abstract Purpose: Aspirin has recently been reported to reduce both t test or ANOVA with the Bonferroni post hoc test were used for the the incidence and the risk of metastasis in colon cancer. statistical comparisons. However, there is no evidence at the cellular levels or in the Results: Aspirin reduced cell viability in a dose- and time- animal models for such an effect of aspirin on cancer dependent manner in osteosarcoma cell lines, and aspirin syn- metastasis. ergistically sensitized osteosarcoma cells to cisplatin (DDP) Experimental Design: MTT assay, colony formation assay, and in vitro and in vivo (P < 0.001). Moreover, aspirin markedly apoptosis assay were employed to analyze the effects of aspirin on repressed the migration and invasion of osteosarcoma cells the osteosarcoma cell viability in vitro. The NF-kB activity was in vitro (P < 0.001), and dramatically diminished the occurrence measured by the NF-kB p65 luciferase reporter. Western blotting of osteosarcoma xenograft metastases to the lungs in vivo was used to analyze the proteins in cells. The migration and (P < 0.001). Mechanistically, aspirin diminishes osteosarcoma invasion abilities of osteosarcoma cells in vitro were measured migration, invasion, and metastasis through the NF-kB pathway. by the Transwell assay. Xenograft-bearing mice were used to assess Conclusion: Aspirin suppresses both the growth and metasta- the roles of aspirin in both tumor growth and metastasis of sis of osteosarcoma through the NF-kB pathway at the cellular osteosarcoma in vivo (n ¼ 5–8 mice/group). An unpaired Student level and in the animal models. Clin Cancer Res; 1–11. Ó2015 AACR. Introduction level and in the animal models to argue that aspirin may be an excellent auxiliary drug for treating patients with osteosarcoma. Osteosarcoma, which has a peak incidence in adolescence, is an extremely aggressive primary malignant bone tumor of childhood Materials and Methods (1). Currently, the 5-year overall survival is 75% to 77% for the primary nonmetastatic disease, however, no more than 20% in Reagents and cell culture metastatic osteosarcoma (2, 3). Patients with metastasis or recur- U2OS and MG63 cells were cultured according to the instruc- rence present a formidable challenge despite modern multimodal tions from the ATCC. ZOS and U2OS/MTX300 cells were therapy (4, 5). Therefore, finding new strategies for the patients described previously (15). HEK-293T cells were purchased from with osteosarcoma are necessary and urgent. the ATCC, cultured in DMEM (Invitrogen) with 10% FBS Aspirin, an NSAID, was used primarily for anti-inflammation (GIBCO), 1 mmol/L glutamine, and 100 U/mL each of penicillin and analgesic (6). Interestingly, a recent population study and streptomycin. All cell lines used in this study were authen- fi reported that acetylsalicylic acid (aspirin) reduces both the inci- ticated using short-tandem repeat pro ling less than 6 months dence and the risk of metastasis in colon cancer (7). However, ago when this project was initiated, and the cells have not been in there is no evidence at the cellular level or in animal models for culture for more than 2 months. such an effect of aspirin on cancer metastasis, even though aspirin Aspirin was purchased from Sigma-Aldrich. Antibodies against is widely used for both the prevention and treatment of many p65(C22B4), XIAP(3B6), CIAP1(D5G9), CIAP2(58C7), BCL2 a diseases, including cancers in clinical settings (7–14). In this (50E3), Survivin(6E4), Livin(D61D1), and -Tublin(2148S) report, we tried to provide comprehensive evidence at the cellular were purchased from Cell Signaling Technology. An antibody against GAPDH(SC-166574) was purchased from Santa Cruz Biotechnology. State Key Laboratory of Oncology in South China, Collaborative Plasmids Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer The full-length p65 cDNA was cloned into the pBABE vector Center, Guangzhou, China. from human 293T cells. P65 was generated by PCR amplification, Note: Supplementary data for this article are available at Clinical Cancer subcloned into the pBABE and pCDNA3.1 vectors, and verified by Research Online (http://clincancerres.aacrjournals.org/). DNA sequencing. The p65 shRNA was purchased from Sigma- Corresponding Author: Tiebang Kang, State Key Laboratory of Oncology in Aldrich. The CMV-luciferase plasmid was a gift from Prof. Xiaom- South China, Sun Yat-Sen University Cancer Center, 651 Dongfeng East Road, ing Xie (Sun Yat-Sen University, Guangzhou, China; ref. 16). Guangzhou 510060, China. Phone: 86-20-8734-3183; Fax: 86-20-8734-3170; E-mail: [email protected] Cell viability assay doi: 10.1158/1078-0432.CCR-15-0198 U2OS, MG63, or ZOS cells were seeded in 96-well plates at a Ó2015 American Association for Cancer Research. density of 3,000 cells per well. They were treated with different www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst July 22, 2015; DOI: 10.1158/1078-0432.CCR-15-0198 Liao et al. 50 nmol/L siRNA. The transfections were performed as described Translational Relevance previously (15). Briefly, asynchronously growing cells seeded at 2.5 Osteosarcoma, an extremely aggressive form of primary  105 cells per well in a 6-well plate or at 1  106 cells in a 10-cm malignant bone tumor in childhood, has a high rate of lung plate were transfected with 2 or 12 mg plasmid DNA, respectively, metastasis. A recent population study reported that aspirin using Lipofectamine 2000 (Invitrogen). Cell lines that stably reduces both the incidence and the risk of metastasis in colon expressed the NC or p65 shRNAs were established by the Sigma cancer. Our results showed that aspirin inhibits osteosarcoma shRNA system according to the manufacturer's instructions. metastasis and enhances the chemotherapy of osteosarcoma in vitro and in vivo, which is the first evidence at the cellular level Dual-luciferase reporter assay and in an animal model for the inhibition of aspirin on cancer Transfection was performed as described previously (15). The metastasis. This strongly argues that aspirin may be an excel- transfected p65-siRNA cells incubated for 24 hours or U2OS cells lent auxiliary drug for treating patients with osteosarcoma, stably overexpressing p65 were transfected with NF-kB p65 lucif- suggesting that it is worthy to design a clinical trial using the erase reporter and pRL-TK Renilla luciferase construct (Promega) combination of aspirin with the current standard therapies for per well using Lipofectamine 2000 (Invitrogen). After 24 hours, patients with osteosarcoma. the cells were treated with aspirin (5 mmol/L). The cells were then analyzed after an additional 24 hours according to the Dual- Luciferase Assay System protocol (Promega). concentrations of Aspirin (0, 1, 2.5, 5, 10, or 20 mmol/L) and/or Boyden chamber assays chemotherapy drugs (DDP, 10mmol/L) for the indicated times, The migration and invasion of osteosarcoma cells were exam- m and the cell viability was measured by MTT assays as described ined using 24-well Boyden chambers with 8- m inserts coated previously (15, 17). without (migration) or with Matrigel (invasion) as previously described (21). A total of 5  104 cells per well were plated on the o Clone formation assay inserts and cultured at 37 C in the upper chambers without Colony formation assays were performed as described previ- serum and supplemented with Aspirin (5 mmol/L). After 24 ously (15, 18). ZOS or U2OS cells plated in triplicate at 500 hours, the cells that crossed the inserts were stained with crystal cells per well in 6-well plates were treated with vehicle (DMSO), violet (0.005%, sigma) and counted as the number of cells per fi 10 mmol/L DDP, 5 mmol/L aspirin, or 10mmol/L DDP plus 5 eld of view under phase-contrast microscopy. mmol/L aspirin for 48 hours, and then cultured for 10 days. The Western blotting cell clones were washed with PBS, and then fixed in methanol and Western blotting was performed as previously described (18). dyed with 0.1% crystal violet, and the colonies that contained Briefly, the cells were lysed in RIPA buffer containing protease more than 50 cells were counted. inhibitor and phosphatase inhibitor cocktails (Thermo Scientific). The nuclear protein was isolated according to the protocol pro- fl Apoptosis ow-cytometry assay vided by the Nuclear Protein Extraction Kit (Thermo Scientific). Drug-induced apoptosis was detected using an Annexin V- The protein extracts were resolved using SDS-PAGE and trans- EGFP apoptosis detection kit (KeyGEN). Treated ZOS, MG63, or ferred to polyvinylidene difluoride membranes for Western blot- m U2OS cells (negative control, 10 mol/L DDP, 5 mmol/L aspirin, ting using ECL detection reagents (Beyotime Co. Haimen). or 10 mmol/L DDP plus 5 mmol/L aspirin for 48 hours) were harvested, the assay were performed according to the manufac- Mouse xenograft turer's instruction. The stained cells were analyzed using a Animal experiments were approved by the Animal Research Cytomics FC500 flow cytometer (Beckman Coulter). Committee of Sun Yat-sen University Cancer Center and were performed in accordance with established guidelines. For osteo- The effect of combination of DDP and aspirin sarcoma xenograft growth of orthotopic animal model, U2OS/ This effect of the combination of DDP and aspirin was assessed MTX300 cells were used as previously described (15, 22). Two fl by the Chou-Talalay method (19, 20). Brie y, the cells were weeks after the cells were injected, the mice were randomly treated with serial dilutions of DDP and aspirin separately or separated into four groups (n ¼ 6).