Bangladesh Journal of Pharmacology Research Article Enhanced antitumor activity of cu- curbitacin B combined with cerulenin in osteosarcoma BJP Introduction Osteosarcoma one of the commonly detected bone tumors has a five-year survival rate of ~70% in children and adolescents. The poor prognosis of the osteosarcoma patients along with less than 20% overall survival rate demands for the development more efficient and novel therapeutic strategies (Jaffe, 2009). Osteo- sarcoma has a characteristic radiographic appearance and has been the target for clinicians throughout the globe (Janeway et al., 2009; Marina et al., 2004). Cucurbitacin B (Figure 1), an oxygenated triterpene is isolated from Trichosanthes kirilowii Maximowicz (Cucurbitaceae family). The plant has a long traditional medicinal importance and is used for its anti- inflammatory, antidiabetic and abortifacient effects. Cucurbitacins are reported to exhibit in vivo anti- inflammatory activities, and preventive and curative effects against CCl4-induced hepatotoxicity (Chen et al., 2005). They also exhibit cytotoxicity and anti-cancer activity (Jayaprakasam et al., 2003). Cucurbitacin B exhibits antiproliferative effects and acts a dual inhibitor of the activation of both JAK2 and STAT3 in some malignancies (Sun et al., 2995). Recent, reports demonstrate that cucurbitacin B has antiproliferative activity against human breast cancer, glioblastoma multiforme, and myeloid leukemia cells (Wakimoto et al., 2008; Yin et al., 2008). It inhibits growth by cell cycle arrest in G2/M phase and increases apoptosis by inhibi- tion of the JAK/STAT pathway (Toyonaga et al., 2003). Cerulenin has been isolated from Cephalosporium caerulens. It acts an inhibitor of fatty acid synthase (FASN) by reacting with the keto-acyl synthase domain of FASN. Various studies have been performed to investigate the anti-tumor activity of cerulenin against the malignant neoplasms (Elbaz et al., 2010; Wang et al., 2008). The present study demonstrates the effects of the combination of cucurbitacin B and cerulenin on the rate of osteosarcoma cell proliferation, suppression of SENP5 expression and induction of apoptosis. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 956-961 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index; ISSN: 1991-0088 Abstract In the present study, the effect of cucurbitacin B and cerulenin combination on osteosarcoma was investigated to develop an effective treatment regimen. The IC50 values of cerulenin and cucurbitacin B combination in the proportion of 1:1, 1:2 and 2:1 were 3.5, 7.2 and 8.6 μg/mL, respectively. The combination index (CI) values of <0.95 for inhibition of growth and <0.93 for inhibition of SENP5 expression clearly indicated synergism between the two. The Q-value for combination of 1 μg/mL cerulenin and 1 μg/mL cucurbitacin B was 1.2. AI calculated for tumor tissues treated with a combination of cucurbitacin B and cerulenin (26.2 ± 8.4%) was higher than that of the tissues treated separately with cucurbitacin B (14.5 ± 6.8%) or cerulenin (12.6 ± 7.5%). The AI for untreated tumor tissues was 4.2 ± 1.5%. Thus, the combination of cucurbitacin B and cerulenin can be a promising regimen in the treatment of osteosarcoma. Article Info Received: 19 August 015 Accepted: 15 September 2015 Available Online: 20 November 2015 DOI: 10.3329/bjp.v10i4.24601 Cite this article: Zhang Z, Shen Y, Wu J, Cai L. En- hanced antitumor activity of cucur- bitacin B combined with cerulenin in o s t eo sa r co ma . B a n gl a d e sh J Pharmacol. 2015; 10: 956-61. Enhanced antitumor activity of cucurbitacin B combined with cerulenin in osteosarcoma Zhe Zhang1,2 , Ying Shen2 , Jian Wu2 and Lin Cai1 1Zhongnan Hospital of Wuhan University, Wuhan 430071, China; 2Xianning Central Hospital, Xianning 437100, China. Materials and Methods Reagents and drugs Cerulenin, cucurbitacin B and dimethyl sulphoxide were purchased from the Sigma (USA). Cell growth assay Human osteosarcoma cell line, Saos-2 was obtained from the American Type Culture Collection (Manassas, USA). The cells were grown in 10% fetal bovine serum- Dulbecco's modified Eagle's medium (FBS-DMEM) (HyClone Laboratories, USA) in an incubator with 5% CO2 atmosphere. MTT assay Saos-2 cells were distributed at a density of 2.5 x 105 cells per well on to the 96-well tissue culture plates containing FBS-DMEM supplemented with 2 mM L- glutamine and cultured for 12 hours. To each of the well different concentrations of cerulenin, cucurbitacin B or combination of cerulenin and cucurbitacin B were added and incubated. Following incubation for 36 hours 3- (4,5-dimeth-ylthiazol‑2-yl)‑2,5-diphenyltetrazolium bromide (MTT) was put into each the of well and incu- bated again for 2 hours. Dimethyl sulfoxide was added to each well for dissolution of farmazan crystals formed. The microplate reader (SpectraMax Plus; Molecular Devices) was employed to measure the absorbance at 455 nm for each of the well three times. The IC50 values were determined by using Originpro 7.5 program from the plot of concentration verses viability curves. For all the combinations of cerulenin and cucurbitacin B, combina- tion index (CI) was calculated which indicated the nature of interaction. The values of less than 0.95 indicated synergism, between 0.95 and 1.05 indicated antagonism and above 1.05 indicated additive interactions. Analysis of apoptosis and cell cycle using fluorescence- activated cell sorting (FACS) Saos-2 cells after incubation for 24 hours with various concentrations of cerulenin, cucurbitacin B and their combinations were analyzed using EPICS XL flow cytometer. The proportion of apoptotic cells and population of cells in different phases of cell cycle were determined using System II software. The equation 1 was used for the determination of Jin's Q value. Q = E(A+B)/[EA + (1 - EA) x EB] -------------------- 1 Where, EA represents percentage of apoptotic cells at different doses of cerulenin, EB represents percentage of apoptotic cells at different doses of cucurbitacin B and E (A+B) stands for apoptotic cell percentage using different combinations of cerulenin and cucurbitacin B. Western blot analysis Saos-2 cells (2 x 105) after incubation with cerulenin, cucurbitacin B and combination of cerulenin and cucurbitacin B were rinsed twice with PBS. The cells were then treated with 2 mL lysis buffer (50 mM Tris- HCl pH 7.4, 137 mM NaCl, 10% glycerol, 100 mM sodium vanadate, 1 mM PMSF, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1% NP-40, and 5 mM cocktail). The concentration of proteins in the cell lysates was determined by BCA method was. For the isolation of proteins electrophoresis on 10% polyacrylamide gel was performed. The proteins were then transferred on to the PVDF membrane Gelman Science (Ann Arbor, MI). The non-specific binding sites on the membranes were blocked by incubation with 5% non-fat dry milk overnight. The membrane was washed with TBST followed by incubation primary antibodies for 12 hours. After incubation the membrane was washed again and then incubated with secondary antibodies for 2 hours. Then X-ray autoradiography was performed and the gray scale images were analysed. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays The tumour tissues stored at -78˚C under liquid nitro- gen were embedded in paraffin and sliced into thin 2 µM sections. TUNEL assays was performed using Situ Cell Death Detection kit (Roche Diagnostics Corp., USA). The tissue sections after deparaffinization in xylene were rehydrated using gradient ethanol and then boiled with sodium chloride-sodium citrate buffer (pH 7.0) for 30 min at 80˚C. The sections were washed followed by 1 hour treatment with proteinase K. The sections were incubated with fluorescein-labeled deoxy- uridine triphosphate (dUTP) and TUNEL reagents according to the manufacturer’s instructions. The sec- tions after washing were incubated with horseradish peroxidase (HRP)‑conjugated fluorescein antibody followed by counterstaining with 0.5% methyl green. The apoptotic index (AI) was calculated by dividing the number of brown-stained cells with total number of tumor cells. Statistical analysis All the data expressed are the mean of ± SD. Student’s t- test was used to analyse the significance. p<0.05 was Bangladesh J Pharmacol 2015; 10: 956-961 957 Figure 1: Structure of cucurbitacin B HO O O H OH O H HO O H O considered to indicate a statistically significant result. Results Synergistic effect of cerulenin and cucurbitacin B on antitumor activity in Saos-2 cells The results from MTT assay indicated that Saos-2 cells were sensitive to both cerulenin and cucurbitacin B. Both the agents caused an inhibition in growth of Saos- 2 cells in a concentration-dependent manner (Figure 2A). The IC50 values of cerulenin and cucurbitacin B for antitumor activity against Saos-2 cells were 5.1 and 21.3 μg/mL, respectively. However, when Saos-2 cells were treated with a combination of cerulenin and cucurbi- tacin B in the proportion of 1:1, 1:2 and 2:1, the values for IC50 were 3.5, 7.2 and 8.6 μg/mL, respectively (Figure 2B). The calculated CI values for all the three combi- nations were <0.95, indicating that cerulenin and cucur- bitacin B exhibited synergistic effect on inhibition of growth in Saos-2 cells. Synergistic effect of cerulenin and cucurbitacin B on inhibition of SENP5 expression in osteosarcoma cell lines The results from quantitative PCR and western blotting analysis are shown Figure 3A and B. It is clear from the figure that SENP5 is significantly overexpressed in Saos -2 osteosarcoma cell line compared with HOB cells (human osteoblasts isolated from normal human bone). Figure 2: Inhibitory effect of cucurbitacin B, cerulenin or combination treatment on the growth of Saos-2 cells. (A) Cucurbitacin B and cerulenin both inhibited Saos-2cell growth in a dose-dependent manner. (B) Cucurbitacin B was applied to Saos-2 cells ad- junctively with cerulenin in a proportion of 1:1, 1:2 and 2:1. The adjunctive treatment also inhibited Saos-2 cell growth in a dose- dependent manner Cucurbitacin B: Cerulenin in = 1:2 Cucurbitacin B: Cerulenin in = 1:1 Cucurbitacin B: Cerulenin in = 2:1 % a ge in hi bi tio n 80 70 60 50 40 30 20 10 0 Concentration (µg/mL) 1 5 10 15 20 25 30 A % a ge in hi bi tio n 30 25 20 15 10 5 0 Concentration (µg/mL) 1 5 10 15 20 25 Cucurbitacin B Cerulenin in Control B 958 Bangladesh J Pharmacol 2015; 10: 956-961 Treatment of Saos-2 osteosarcoma cell line with cucurbitacin B or cerulenin resulted in inhibition of SENP5 expression in a dose and time-dependent manner (Figure 3C, D). We treated osteosarcoma cells with 1:1, 1:2 and 2:1 proportion of cucurbitacin B and cerulenin. The results from RT-PCR analysis clearly demonstrated a significant inhibition of SENP5 expre- ssion on treatment 1:1, 1:2 and 2:1 proportion of cucurbitacin B and cerulenin. The values for IC50 were 4.7, 8.5 and 9.4 μg/mL, respectively. The calculated CI values for all the three combinations were <0.93, indicating that cerulenin and cucurbitacin B exhibited synergistic effect on inhibition of SENP5 expression in Saos-2 cells. Synergistic effect of cerulenin and cucurbitacin B on induction of apoptosis in Saos-2 cells The Saos-2 cell cultures were treated with a range of cucurbitacin B, cerulenin and their combination concentrations for 24 hours. The results from FACS analysis indicated a concentration dependent induction of apoptosis in Saos-2 cell (Figure 4). There was apoptosis in 17.3, 26.1 and 31.5% cells treated with 1, 5 and 10 μg/mL of cucurbitacin B, respectively. The Q- value for combination of 1 μg/mL cerulenin and 1 μg/ mL cucurbitacin B was 1.2 which indicated synergistic effect between the two. Synergistic effect of cerulenin and cucurbitacin B on induction of apoptosis in tumor cells Treatment of tumor tissues with cucurbitacin B, ceru- lenin or their combination induced apoptosis in tumor cells (Figure 5). However the AI calculated for tumor tissues treated with a combination of cucurbitacin B and cerulenin (26.2 ± 8.4%) was higher than that of the tissues treated separately with cucurbitacin B (14.5 ± 6.8%) or cerulenin (12.6 ± 7.5 %). The AI for untreated tumor tissues was 4.2 ± 1.5%. Discussion The results revealed that the use of the two agents in combination exhibited synergistic effect on antitumor activity, inhibition of SENP5 expression and induction of apoptosis in osteosarcoma cell lines. The IC50 values for cerulenin and cucurbitacin B combination in the proportion of 1:1, 1:2 and 2:1 were 3.5, 7.2 and 8.6 μg/ mL, respectively. The calculated CI values for all the three combinations were <0.95, indicating that cerule- nin and cucurbitacin B exhibited synergistic effect on inhibition of growth in Saos-2 cells. The combination of cucurbitacin B and cerulenin also inhibited SENP5 expression in Saos-2 cells through synergistic effect. Among three proportions tested the calculated CI values for all the three were <0.93, indicating that cerulenin and cucurbitacin B exhibited synergistic effect on inhibition of SENP5 expression in Saos-2 cells. There was apoptosis in 17.3, 26.1 and 31.5% cells treated with 1, 5 and 10 μg/mL of cucurbitacin B, respectively. The Q-value for combination of 1 μg/mL cerulenin and 1 μg/mL cucurbitacin B was 1.2 which Figure 3: SENP5 is overexpressed in osteosarcoma cell lines. (A) mRNA expression and (B) protein levels of SENP5 in Saos-2 oste- osarcoma cell lines. Inhibition of SENP5 expression by cucurbitacin B and cerulenin treatment. (C) Concentration-dependent inhi- bition of SENP5 expression in Saos-2 cells. (D). Time-dependent inhibition of SENP5 expression in Saos-2 cells 8 7 6 5 4 3 2 1 0 SE N P5 m R N A le ve l HOB Saos -2 A R el at iv e SE N P5 m R N A le ve l Saos –2 cells Untreated Cucurbitacin Cerulenin Cucurbitacin + Cerulenin 8 7 6 5 4 3 2 1 0 SENP5 Β-actin Control Cucurbitacin B Cerulenin Cucurbitacin + Cerulenin Β-actin SENP5 Saos -2 HOB B C D Bangladesh J Pharmacol 2015; 10: 956-961 959 indicated synergistic effect between the two. AI calculated for tumor tissues treated with a combination of cucurbitacin B and cerulenin (26.2 ± 8.4%) was higher than that of the tissues treated separately with cucurbitacin B (14.5 ± 6.8%) or cerulenin (12.6 ± 7.5%). The AI for untreated tumor tissues was 4.2 ± 1.5%. Conclusion A synergistic effect exits between cerulenin and cucurbitacin B for anti-OS in vitro and a synergistic effect in vivo. It has been suggested that cerulenin combined with cucurbitacin B may be a potential Figure 4: FACS analysis of U2-OS cells treated with epirubicin, cerulenin and the combination treatment for 24 hours. (A) Few apoptotic cells were noted in the control group. Yet, the percentage of apoptotic cells was (B) 31.5% when treated with 10 μg/mL epirubicin and (C) 38.8% when treated with 20 μg/mL cerulenin. (D) The percentage of apoptotic cells rose to 66.2% when treated with 10 μg/mL epirubicin in conjunction with 20 μg/mL cerulenin. FACS, fluorescence-activated cell sorting; PI, propidium io- dide Figure 5: TUNEL assays of tumor tissue slides. The apoptotic index was significantly higher than that in the control, cerulenin and epirubicin alone groups 1 (µg/mL) 2 (µg/mL) 5 (µg/mL) Cucurbitacin + Cerulenin Cucurbitacin 1 2 3 4 5 % A po pt os is 60 50 40 30 20 10 0 Ap op tti c in de x 30 25 20 15 10 5 0 1 2 3 4 960 Bangladesh J Pharmacol 2015; 10: 956-961 Author Info Lin Cai (Principal contact) e-mail: cailin09123@gmail.com First two authors contributed equally treatment regimen for OS. Conflict of Interest The authors declare that there is no conflict of interests in publishing this paper. References Cesari M, Alberghini M, Vanel D, et al., Periosteal osteo- sarcoma: A single-institution experience. Cancer 2011; 117: 1731-35. Chen JC, Chiu MH, Nie RL, Cordell GA, Qiu SX. Cucurbitacins and cucurbitane glycosides: Structures and biological activi- ties. Nat Prod Rep. 2005; 22: 386-99. Elbaz A, Wu X, Rivas D, et al., Inhibition of fatty acid biosyn- thesis prevents adipocyte lipotoxicity on human osteoblasts in vitro. J Cell Mol Med. 2010; 14: 982-91. Jaffe N. Osteosarcoma: Review of the past, impact on the future. The American experience. Cancer Treat Res. 2009; 152: 239-62. Janeway K, Gorlick R, Bernstein M. Osteosarcoma. In: Onco- logy of infancy and childhood. Orkin S, Fisher D, Look A, Lux S, Ginsburg D, Nathan D (eds). Philadelphia, Saunders Elsevier, 2009, pp 871-910. Jayaprakasam B, Seeram NP, Nair MG. Anticancer anti-infla- mmatory activities of cucurbitacins from Cucurbita andreana. Cancer Lett. 2003; 189: 11-16. Marina N, Gebhardt M, Teot L, Gorlick R. Biology and thera- peutic advances for pediatric osteosarcoma. Oncologist 2004; 9: 422-41. Menendez JA, Mehmi I, Atlas E, et al., Novel signaling mole- cules implicated in tumor-associated fatty acid synthase- dependent breast cancer cell proliferation and survival: Role of exogenous dietary fatty acids, p53-p21 WAF1/CIP1, ERK1/2 MAPK, p27KIP1, BRCA1, and NF-κB. Int J Oncol 2004; 24: 591-608. Sun J, Blaskovich MA, Jove R, Livingston SK, Coppola D, Sebti SM. Cucurbitacin Q: A selective STAT3 activation inhibitor with potent antitumor activity. Oncogene 2005; 24: 3236-45. Toyonaga T, Nakano K, Nagano M, Zhao G, Yamaguchi K, Kuroki S et al., Blockade of constitutively activated Janus kinase/signal transducer and activator of transcription-3 pathway inhibits growth of human pancreatic cancer. Cancer Lett. 2003; 201: 107-16. Wang WQ, Zhao XY, Wang HY, et al., Increased fatty acid syn- thase as a potential therapeutic target in multiple myeloma. J Zhejiang Univ Sci B. 2008; 9: 441-47. Wakimoto N, Yin D, O’Kelly J, et al. Cucurbitacin B has a potent antiproliferative effect on breast cancer cells in vitro and in vivo . Cancer Sci. 2008; 99: 1793-97. Yin D, Wakimoto N, Xing H, et al. Cucurbitacin B markedly inhibits growth and rapidly affects the cytoskeleton in glioblastoma multiforme. Int J Cancer. 2008; 123: 1364-75. Bangladesh J Pharmacol 2015; 10: 956-961 961