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Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2014; 18: 3877-3888 Synergistic effects of arsenic trioxide combined with ascorbic acid in human osteosarcoma MG-63 cells: a systems biology analysis X.C. HUANG, X.Y.M. MAIMAITI 1, C.W. HUANG 2, L. ZHANG 3, Z.B. LI, Z.G. CHEN 3, X. GAO 4, T.Y. CHEN 3 Rehabilitation Department, Zhongshan Hospital of Fudan University, Shanghai, China 1Microscope Repairing Surgery Section, the First Affiliated Hospital of Xinjiang Medical University, Xinjiang Uygur Autonomous Region, China 2Medical Department, Zhongshan Hospital of Fudan University, Shanghai, China 3Department of Orthopedic Surgery, Zhongshan Hospital of Fudan University, Shanghai, China 4Genergy Biotechnology (Shanghai) Co., Ltd, Shangai, China Xiaochun Huang and Xiayimaierdan Maimaiti are regarded as co-first author Abstract. – OBJECTIVE : To further under - Key Words: stand the synergistic mechanism of As 2O3 and Arsenic trioxide, Osteosarcoma, Synergistic effect, asscorbic acid (AA) in human osteosarcoma Systems biology analysis. MG-63 cells by systems biology analysis. MATERIALS AND METHODS: Human os - teosarcoma MG-63 cells were treated by As 2O3 (1 µmol/L), AA (62.5 µmol/L) and combined drugs (1 µmol/L As 2O3 plus 62.5 µmol/L AA). Dynamic mor - Introduction phological characteristics were recorded by Cell- IQ system, and growth rate was calculated. Illumi - Osteosarcoma is the most common and ag - na beadchip assay was used to analyze the differ - gressive type of bone cancer 1, which may not on - ential expression genes in different groups. Syn - ergic effects on differential expression genes ly cause extremity disability, but also threaten (DEGs) were analyzed by mixture linear model life by metastasis. Surgery was the only form of and singular value decomposition model. KEGG treatment in the pre-chemotherapy era, when the pathway annotations and GO enrichment analysis survival rate was less than 20% 2. Recently limb were performed to figure out the pathways in - salvage surgery with large dose neoadjuvant volved in the synergic effects. chemotherapy saves many patients’ limbs and RESULTS: We captured 1987 differential expres - lives. However, most of chemotherapeutics, such sion genes in combined therapy MG-63 cells. FAT1 gene was significantly upregulated in all as ifosfamide, cis-platinum, adriamycin and three groups, which is a promising drug target as methotrexate, have a major problem that large an important tumor suppressor analogue; mean - dose brings about effectiveness on tumor as well while, HIST1H2BD gene was markedly downregu - as severe side effects, among which tiredness, lated in the As 2O3 monotherapy group and the nausea, headache, edema, diarrhea and vomit are combined therapy group, which was found to be common ones, and leukocytosis and retinoic acid upregulated in prostatic cancer. These two genes 3 might play critical roles in synergetic effects of syndrome are the most severe ones . Thus, novel therapeutic strategies with less systemic toxicity AA and As 2O3, although the exact mechanism needs further investigation. KEGG pathway analy - and higher effectiveness are demanded urgently. sis showed many DEGs were related with tight Arsenicals have been used to treat diseases, junction, and GO analysis also indicated that such as ulcer, haemorrhoids and dysentery, for DEGs in the combined therapy cells gathered in thousands of years in Chinese traditional medi - occluding junction, apical junction complex, cell cine 4. In the recent years arsenic trioxide junction, and tight junction. CONCLUSIONS: AA potentiates the efficacy of (As 2O3) was found to be effective on recurrent acute promyelocytic leukemia (APL, M3) 5 and As 2O3 in MG-63 cells. Systems biology analysis 6 showed the synergic effect on the DEGs. solid tumor . More importantly As 2O3 did not Corresponding Author: Zenggan Chen, MD, Ph.D; e-mail: [email protected]; Zebing Li, BS; e-mail: [email protected] 3877 X.C. Huang, X.Y.M. Maimaiti, C.W. Huang, L. Zhang, Z.B. Li, Z.G. Chen, X. Gao, T.Y. Chen accumulate in blood plasma or induce myelo - Cell Culture and Drug Administration suppression in most of the patients. In addition, Human osteosarcoma MG-63 cells were cul - As 2O3 inhibits proliferation of osteosarcoma tured in the DMEM medium containing 10% cells in a time-dependent and dose-dependent FBS, 100 IU/mL penicillin, 100 µg/mL strepto - manner, the inhibition rate can reach 70% when mycin at 37°C in 5% CO 2. Cells in logarithmic the concentration of As 2O3 was higher than 1 growth phase were planted as 30 000 cells/well 7 µmol/L . Whereas, arsenic compounds have in 24-well plates. As 2O3 (1 µ mol/L ), AA (62.5 double-edged effects (anticancer and carcino - µmol/L) and combined drugs (1 µmol/L As 2O3 genic effects) dependent on dosage 8. Drug com - plus 62.5 µmol/L AA ) were administrated to the bination could be a valuable way to lower sys - cells respectively for 72 h. Dynamic morphologi - temic toxicity and increase effectiveness of anti - cal characteristics were recorded every 2 h and cancer drugs such as As 2O3. analyzed by Cell-IQ continuous live cell imaging Ascorbic acid (AA) has antioxidative effects, and analysis platform (Chip-man Technologies which can decrease reduced glutathione with no Ltd., Tampere, Finland). toxicity itself. Previous researches 9 have found AA enhanced anticancer activity of As 2O3; AA Illumina Beadchip Assay had sensitization effect on apoptosis-inducing ac - After the treatment, total RNA was extracted tivity of As 2O3 in human multiple myeloma RP - from cells using Trizol (Invitrogen, Carlsbad, MI8226 cells, which depended on overexpression CA, USA) reagent following the manufacturer’s of death receptor 4 (DR4) 10 ; AA enhanced cyto - instruction. Reverse transcription, purification, toxicity of As 2O3 in human leukemia HL-60 hybridization, image reading and data analysis cells, as malondialdehyde (MDA) level was in - were performed using commercial kits and sys - creased by increasing dosage of AA, and AA co- tems from Illumina (San Diego, CA, USA). treated with As 2O3 increased reactive oxygen species (ROS) generation 11 ; AA enhanced an - Identification of Synergic 12 timyeloma effects of As 2O3 . Furthermore, it was Drug Interaction Effects demonstrated AA has preferable tolerance on re - To facilitate the identification of heterotypic current/intractable myeloma 13 and recurrent ma - interaction effects on global gene expression in 14 lignant tumor in lymphatic system . mixed co-treated (1 µmol/L As 2O3 plus 62.5 In this study, systems biology analysis was µmol/L AA) cells, we performed background ad - performed to investigate synergistic effects of justment and normalized the gene expression us - As 2O3 and AA in human osteosarcoma MG-63 ing Quantile normalization method (Supplement cells, in order to further understand the syner - 1). After preprocessing, we averaged the biologi - gistic mechanism of the two drugs, and figure cal replicates and then used mixture linear model out better drug combinative strategy for os - to fit the normalization. To determine the contri - teosarcoma. bution of each drug to the combined synergic ef - fects of gene expression pattern in the linear re - gression model (Supplement 2), the expression levels of the mono-treated (1 µmol/L As 2O3 or Materials and Methods 62.5 µmol/L AA) cells are the predictors and the expression levels of the co-treated cells, the re - Cell Line and Reagents sponse. Interaction effects represented as gene Human osteosarcoma MG-63 cells were from expression changes. The ratio larger than 2 or Institute of biochemistry and Cell Biology, Chi - less than 0.5 represents the relevant positive or nese Academy of Sciences (Shanghai, China). negative interaction effect on the MG-63 cells Fetal bovine serum (FBS) and Dulbecco’s Modi - from As 2O3 or AA. fied Eagle Medium (DMEM ) (high glucose) were purchased from Gibco (Carlsbad, CA, Gene Expression Decomposition Analysis USA). AA and As 2O3 powder were purchased Distinct influences of varying magnitude with - from Beijing Dingguo Changsheng Biotechnolo - in genes can be analyzed using SVD (singular gy Co., Ltd (Beijing, China) and Beijing Double- value decomposition, Supplement 3) approach. crane Pharmaceutical Co., Ltd (Beijing, China) SVD is an unsupervised algebric method that respectively, and dissolved in phosphate buffered mathematically separates a gene expression ma - saline (PBS). trix into a set of modes determined by the quanti - 3878 Osteosarcoma: a systems biology analysis tative composition of the data. Each mode is was found to be upregulated in prostatic cancer 18 . manifested in the data as a global expression Thus, we speculate these two genes might play component that influences the expression of each critical roles in synergetic effects of AA and gene to a varying degree. SVD has also proved As 2O3, although the exact mechanism needs fur - useful in linear modeling of gene expression, and ther investigation. gene classification, dimensional reduction and network modeling. Here, we used SVD method Genes Responsible for Synergistic Effects to deconvolute gene expression into component Through drug combination data, we used lin - modes, which can reveal the significant gene sets ear mixture regression models to fit the normal - those function in genetic or transcriptional regu - ized co-treated (As 2O3 and AA) MG-63 expres - lation in co-treated (1 µmol/L As 2O3 plus 62.5 sion profile. Using threshold of interaction effect µmol/L AA) cells or mono-type (1 µmol/L As 2O3 computed from the fitted models, we captured or 62.5 µmol/L AA ) cells. 1987 genes, in which 730 positive-effected genes and 1257 negative-effected genes for MG-63 Bioinformatic Analysis cells, respectively (Table IV). Based on previous The DAVID Bioinformatics Resources 6.7 knowledge, As 2O3 and AA synergic interaction (http://david.abcc.ncifcrf.gov/home.jsp) was used can double the cell therapy effects than As 2O3 or to make KEGG pathway annotations 15 .