Cell Biochem Biophys DOI 10.1007/s12013-015-0523-x
ORIGINAL PAPER
Saurolactam Inhibits Proliferation, Migration, and Invasion of Human Osteosarcoma Cells Zhengwei Li • Hui Liu • Baizhi Li • Yanzhe Zhang Chengdong Piao
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Ó Springer Science+Business Media New York 2015
Abstract Osteosarcoma is a common type of malignant bone tumor with features of osteoid formation or osteolytic lesions of bone. New therapeutic approaches are urgently needed since it lacks response to chemotherapeutic treatments. Saurolactam, a natural compound isolated from the aerial portions of Saururus chinensis, was reported to have an anti-inflammatory activity. Here, we demonstrate that saurolactam shows anti-cancer activity against human osteosarcoma cells. Saurolactam treatment inhibited proliferation of human osteosarcoma cell lines MG-63 and HOS and decreased colony formation in soft agar in a dosedependent manner. Intraperitoneal administration of saurolactam at 25 mg/kg of body weight for 21 days dramatically inhibited the growth of MG-63 xenografts in nude mice. Flow cytometric analysis indicated that saurolactam treatment (20 lM) led to G1 cell cycle arrest and induced apoptosis in these two cell lines. Western analysis suggested that saurolactam treatment resulted in a reduction of Akt/PKB, phospho-Ser473-Akt, c-Myc, and S-phase kinase-associated protein 2 (Skp2) in MG-63 and HOS osteosarcoma cells. Akt overexpression significantly abolished saurolactam-induced decrease in protein and phosphorylation levels of Akt, c-Myc, and Skp2 protein Z. Li Y. Zhang C. Piao (&) The Second Hospital of Jilin University, No. 218 Ziqiang Street, Changchun 130041, People’s Republic of China e-mail: [email protected] H. Liu Department of Human Anatomy, College of Basic Medical Sciences, Jilin University, Changchun 130021, People’s Republic of China B. Li Institute of Frontier Medical Science of Jilin University, Changchun 130021, People’s Republic of China
levels, implying that Akt inactivation was a causal mediator of saurolactam-induced inhibition of c-Myc and Skp2. Moreover, Skp2 overexpression in MG-63 cells partly abolished the growth inhibition induced by saurolactam. Saurolactam treatment repressed migration and invasion ability, and Skp2 overexpression significantly blocked these inhibitory effects of saurolactam in MG-63 Cells. The present study indicates that saurolactam might represent a new promising agent to improve osteosarcoma treatment. Keywords Saurolactam Osteosarcoma S-phase kinaseassociated protein 2 (Skp2) Cell proliferation Tumor xenografts
Introduction Osteosarcoma is a common cancerous bone tumor primarily affecting children and young adults, and characterized by the existence of tumor cells generating an osteoid matrix [1]. Currently, the globally accepted therapeutic strategy for osteosarcoma was recommended by Rosen and his colleagues in the 1970s [2]. In the last decade, improved understanding of oncogenic pathways in osteosarcoma has contributed to the expansion of new therapeutic avenues based upon single drugs or conventional drug combinations [3]. Despite the development of polychemotherapy that has undoubtedly enhanced the survival rates of patients with osteosarcoma, the 5-year event-free survival for high-grade osteosarcoma patients still cannot break through 50 % [4]. Hence, a deep insight into the basic biology of osteosarcoma is urgently required to identify its novel therapeutic targets [5, 6]. Recently, the certain biological molecules such as Akt, C-Myc, and Skp2
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have been recognized as promising prognostic markers or therapeutic targets for osteosarcoma. Historically, natural products and their derivatives are a valuable source of therapeutic drugs. Development of novel medicinal products from natural substances becomes an important approach in the drug discovery. Various phytochemicals derived from Saururus chinensis presented a potent anti-inflammatory activity [7–10]. Saurolactam is a new compound extracted from S. chinensis. Recent studies suggest that it possesses anti-resorptive activity [11] and inhibits osteoclast differentiation, as well as induces apoptosis of mature osteoclasts [11]. However, there are no studies examining the in vitro and in vivo anti-tumor activity of saurolactam. Thus, in the present study, we investigated the anti-tumor activity of saurolactam. We assessed its effects on the pro-survival and proliferative mediators and its role in suppressing proliferation-related signaling molecules, including Akt, C-Myc, and Skp2, in two human osteosarcoma cell lines i.e., MG-63 and HOS cells.
Materials and Methods Cells and Culture Conditions The human osteosarcoma MG-63 and HOS cells acquired from ATCC were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS). Materials Saurolactam was isolated using the methods as described previously [12]. Matrigel was obtained from BD Bioscience (San Jose, CA, USA). Tissue culture dishes from Costar were from Sigma (St. Louis, MO, USA). Antibodies against Akt and phosphor-S473-Akt were from Cell Signaling Technology (Danvers, MA); anti-b-actin, c-Myc, and Skp2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Proliferation Assay Osteosarcoma cells were seeded in 96-well plates (3 9 103 cells/well). Proliferation assays were carried out under maintenance circumstances. Relative cell number was examined by assaying cellular DNA with the blue-fluorescent Hoechst (Sigma, St. Louis, MO, USA) as described before [13–20]. All values were normalized to those of the control (no saurolactam treatment). The experiment was performed three times independently. The results were presented as mean and standard deviation.
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Cell Viability Assay Osteosarcoma cells were seeded in 96-well plates (3 9 103 cells/well). After 24 h, the cells were incubated with mounting concentrations of saurolactam for 48 h. Cell viability was measured with a 3,4,5-dimethylthiazol-2-yl2-5-diphenyltetrazolium bromide (MTT) assay by determining the absorbance at 560 nm using a Benchmark Microplate Reader (Bio-Rad) [21]. All values were normalized to those of the control (no saurolactam treatment). The experiment was performed three times independently. The results were presented as mean and standard deviation. Flow Cytometric Analysis Cells were seeded into 100-mm dishes (5 9 105 cells) and cultured for 16 h prior to the addition of saurolactam. After 48 h of incubation in the presence of different concentrations of saurolactam, cells were fixed in ice-cold 70 % ethanol in phosphate buffered saline (PBS) for 12 h. Fixed cells were then rinsed with PBS, incubated with PBS containing 0.1 mg/mL RNase A for 20 min, and re-suspended in propidium iodide (PI) (50 lg/mL). Cell cycle distributions were detected using a FACScan flow cytometer (Becton–Dickinson Immunocytometry Systems) and analyzed with a ModFit software (Topsham, ME, USA) as previously described [14, 17–19, 22]. Soft Agar Colony Formation For soft agar assay, human osteosarcoma MG-63 and HOS cells (5,000 cells/plate) were suspended in 0.35 % agar containing DMEM supplemented with 10 % FBS in 35-mm cell culture plates. Cells were allowed to grow for 3 weeks at 37 °C in a CO2 incubator. The colonies were stained with crystal violet. Tumor Xenografts in Nude Mice Male Balb/c nude mice aged 6–8 weeks (NCI Frederick, MD) were subcutaneously injected into both flanks with MG-63 cells (5 9 105 cells) suspended in 0.5 ml Matrigel. Seven days after tumor inoculation, tumor growth was monitored. Mice were divided into control and saurolactam-treated groups. The control group had 8 mice bearing 16 tumors, and the saurolactam group had 8 mice bearing 11 tumors. Three weeks after inoculation, the treatment group was intraperitoneally (i.p.) administrated saurolactam (25 mg/kg of body weight) once every other day. The control groups were i.p. administrated with vehicle only. The treatment terminated on day 43 after tumor inoculation. Tumors were measured daily with calipers, and volume was calculated with the formula:
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volume = length 9 width 9 height 9 0.52 [16, 17, 23, 24]. Tumor volumes and body weight of mice were indicated as mean ± SD. Western Blotting Analysis The Western blot analyses were performed on 80 lg of protein extracts. The human osteosarcoma MG-63 and HOS cells were lysed in lysis buffer (0.5 % sodium deoxycholate, 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 % Nonidet P-40, 0.1 % SDS) containing 50 mM NaF, 5 mM EDTA, 1 mM DTT, and 10 lg/ml aprotinin. Cell lysates were resolved on SDS-PAGE according to standard protocols. After being transferred to membranes, the samples were immunoblotted with primary antibodies including phosphor-Akt-Ser473, Akt, C-Myc, Skp2, and b-actin, followed by secondary antibodies conjugated with horseradish peroxidase. Bands were revealed by using an enzyme-linked chemiluminescence detection kit (ECL, Amersham Biosciences, Piscataway, NJ) as recommended by the manufacturer, and density was quantified by use of Scion Image software (Scion Corp, Frederick, MD). Akt and Skp2 Overexpression in MG-63 Cells Ectopic expressions of Akt and Skp2 were realized by transfecting MG-63 cells with the plasmids of pcDNA3.1Skp2 (Skp2), pcDNA3.1-Akt (Akt), and pcDNA3.1 empty vector (EV) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in line with the manufacturer’s protocols [25]. MG-63 cells transfected with a pcDNA3.1 empty vector were used as controls. Transwell Migration Assay Migration assays were carried out with a transwell kit (BD Bioscience). MG-63 cells were treated with 0, 20, and 40 lM saurolactam for 48 h. Cells were then trypsinized, detached from culture plates, and washed with PBS. Saurolactam-treated cells (1 9 104) in 200 ll DMEM without serum were put in the upper chamber. The lower compartment of transwell chamber was encumbered with DMEM containing 10 % FBS. The serum-starved cells were allowed to migrate overnight at 37 °C. Cells attached to the filter were fixed and stained with Giemsa solution for 1 h at room temperature. Filters were subsequently washed with water, and the number of cells on the filter was quantified by reckoning cells in photographs.
manufacturer’s protocols. MG-63 cells were treated with various concentrations of saurolactam for 48 h. Cells were then detached from culture plates with trypsin and washed thrice with PBS. Cells were seeded (4 9 104 cells per well) in DMEM without serum in the upper chamber. As a chemoattractant, DMEM medium containing 10 % FBS was put in the lower compartment of the chamber. After overnight incubation, the non-invading cells were removed. After fixation with methanol and wash, the membranes were stained with Giemsa’s solution. Invasiveness was assessed by calculating the invading cells with a light microscope. All experiments were performed in triplicate. Statistical Analysis The data were expressed as mean ± SD. Differences were analyzed by one-way analysis of variance followed by Fisher’s protected least significant difference test. The level of significance was taken as P \ 0.05.
Results Saurolactam Repressed the Proliferation of Human Osteosarcoma Cells First, we determined the effect of saurolactam incubation on the viability and proliferation of two human osteosarcoma cell lines i.e., MG-63 and HOS osteosarcoma cells (Fig. 1). An MTT assay showed that saurolactam inhibited cell viability in a dose-dependent manner. Similarly, saurolactam dose-dependently suppressed cellular proliferation, as indicated by a Hoechst dye-based proliferation assays (Fig. 1). The EC50 for saurolactam in the MTT assay was comparable to that assayed by Hoechst dye-based assay (data not shown), implying that suppression of cell proliferation contributed to the decrease in viable cells induced by saurolactam treatment in these two human osteosarcoma cell lines. Saurolactam Treatment suppressed Colony Formation of MG-63 and HOS Cells in Soft Agar Treatment of MG-63 and HOS cells with 20 and 40 lM saurolactam strikingly suppressed the formation of their corresponding colonies in soft agar, substantiating the anticancer activity of saurolactam (Fig. 2). Saurolactam Administration Inhibited Growth of MG-63 Xenografts in Nude Mice
Transwell Invasion Assay An invasion assay was conducted with BD BioCoat Matrigel invasion chambers (BD Biosciences) in accordance with the
To determine if saurolactam could retard tumor growth in vivo, a study with MG-63 xenografts in nude mice was carried out. Intraperitoneal (i.p.) injection of saurolactam
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Fig. 1 Effect of saurolactam on viability and proliferation of human osteosarcoma cell lines. MG-63 and HOS osteosarcoma cells were incubated with increasing concentrations of saurolactam for 48 h. Relative viability and cell number of osteosarcoma cells were measured by MTT 96-well assay (a) and Hoechst dye-based
proliferation assay (b), respectively. Cell numbers were normalized to control. Results presented are mean ± SD from three independent experiments, with non-treated controls set as 100. *P \ 0.01 versus untreated cells
Fig. 2 Effect of saurolactam on colony formation. MG-63 (a) and HOS (b) cells treated with 0, 20, or 40 lM saurolactam for 12 and 15 days, respectively. Image is a representative result from three independent experiments
(25 mg/kg of body weight) once every other day for 21 days leads to a 56 % decrease in the average volume of MG-63 xenografts (Fig. 3a). Body weight of both control and saurolactam-treated mice gradually declined (Fig. 3b). These results substantiate that i.p. administration of saurolactam could inhibit the growth of osteosarcoma in vivo.
reduction in S and G2/M populations of MG-63 and HOS osteosarcoma cells (Fig. 4). Thus, treatment with 20 lM saurolactam promoted G1 cell cycle arrest in MG-63 and HOS osteosarcoma cells. In addition, we observed a significant increase in sub-G1 population, which represented apoptotic cells, in these two osteosarcoma cell lines.
Saurolactam Induced G1 Cell Cycle Arrest and Apoptosis in Osteosarcoma Cells
Saurolactam Reduced the Protein Levels of Signaling Proteins Implicated in Cell Cycle via Akt Pathway
Next, we sought to investigate if saurolactam influenced cell cycle progression of osteosarcoma cells by performing flow cytometric analysis. Treatment with 20 lM saurolactam for 48 h elicited an elevation of the G1 cell population and a
Next, we performed Western analysis to check whether saurolactam affected proteins involved in cell cycle. As indicated in Fig. 5a, saurolactam treatment resulted in a reduction of Akt/PKB, phospho-Ser473-Akt, c-Myc, and
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S-phase kinase-associated protein 2 (Skp2) in MG-63 and HOS osteosarcoma cells. To determine the causal relationship between Akt, c-Myc, and Skp2, we transfected MG-63 cells with either empty vector pcDNA3.1 (EV) or constitutively active pcDNA3.1-Akt (Akt) to overexpress Akt. As indicated in Fig. 5b, Akt overexpression significantly abolished saurolactam-induced decrease in protein and phosphorylation levels of Akt, c-Myc, and Skp2 protein levels, implying that Akt inactivation was a causal mediator of saurolactam-induced inhibition of c-Myc and Skp2. Skp2 Overexpression Partly blocked the Inhibitory Effect of Saurolactam in MG-63 Cells
Fig. 3 Effect of saurolactam on the growth of MG-63 xenografts in nude mice. MG-63 cells (5 9 105 cells) were subcutaneously injected into Male Balb/c nude mice aged 6–8 weeks. Tumors were measured daily with calipers. The formula volume = length 9 width 9 height 9 0.52 was used to calculate tumor volume. Seven days after tumor inoculation, tumor growth was monitored. Mice were divided into control and saurolactam-treated groups. The control group had 8 mice bearing 16 tumors, and the saurolactam group had 8 mice bearing 11 tumors. Three weeks after inoculation, the treatment group was intraperitoneally (i.p.) administrated saurolactam (25 mg/kg of body weight) once every other day. The control groups were i.p. administrated with vehicle only. The treatment terminated on day 43 after tumor inoculation. Tumor volumes (a) and body weight of mice (b) were indicated as mean ± SD
Fig. 4 Impact of saurolactam on cell cycle distribution of osteosarcoma cells. MG-63 (a) and HOS osteosarcoma cells (b) were treated with or without 20 lM saurolactam for 48 h. Cells were fixed with ice-cold 70 % ethanol, collected and stained with a mixture of propidium iodide and RNase. The cell cycle distribution was detected
Because saurolactam treatment contributed to a salient decrease in Skp2 expression in these two osteosarcoma cell lines (Fig. 6a), we sought to explore if overexpression of Skp2 could abolish the growth inhibition induced by saurolactam in MG-63 cells. As shown in Fig. 6b, overexpression of Skp2 in part rescued saurolactam-induced inhibition of MG-63 cell proliferation rate. Since Skp2 overexpression could not entirely inverse the growth inhibitory effects, we reasoned that other pathways or mechanisms might be elicited by saurolactam to inhibit cell growth of osteosarcoma cells. In addition, transwell migration assay (Fig. 6c) and transwell invasion assay (Fig. 6d) revealed that migration and invasion ability of MG-63 cells were inhibited dose-dependently by saurolactam treatment. Skp2 overexpression significantly blocked these inhibitory effects of saurolactam in MG-63 Cells. Discussion In the present study, we examined the anti-tumor activity of saurolactam, a compound isolated from S. chinensis,
with the FACScan flow cytometer (Becton–Dickinson Immunocytometry Systems). The representative data acquired from four independent experiments were illustrated. *P \ 0.01 versus untreated cells
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Fig. 5 Saurolactam incubation inhibits protein and phosphorylation levels of signaling proteins. a Protein expression of Akt/PKB, phospho-S473-Akt, c-Myc, Skp2, and b-actin in MG-63 and HOS osteosarcoma cells treated with 0, 20, or 40 lM saurolactam for 48 h were examined by Western analysis. b MG-63 cells were transfected
with empty vector (EV) or wild-type Akt for 24 h and then treated with or without 20 lM saurolactam for 48 h. The corresponding protein and phosphorylation levels were detected with Western analysis. Values represent relative protein expression levels
Fig. 6 Effect of Skp2 overexpression on inhibition of cell growth, migration and invasion induced by saurolactam treatment. a Protein expression of Skp2 measured by Western analysis in MG-63 EV control and MG-63 cells overexpressing Skp2. MG-63 cells overexpressing Skp2 with pcDNA3.1 vector and control MG-63 cells (empty pcDNA3.1 vector, EV) were incubated with increasing concentrations of saurolactam for 48 h and detected by the 96-well proliferation assay (b), transwell migration assay (c), and transwell invasion assay (d). *P \ 0.01 versus EV
against human osteosarcoma cells. We observed that saurolactam treatment promoted G1 cell cycle arrest and apoptosis of human osteosarcoma cells. Moreover, we demonstrate that saurolactam treatment inhibited proliferation, migration, and invasion ability of human osteosarcoma cell lines MG-63 and HOS and decreased colony formation via inhibition of c-Myc and Skp2, and that Akt inactivation was a causal mediator of saurolactam-induced suppression of c-Myc and Skp2. In addition, i.p. administration of saurolactam at 25 mg/kg of body weight for 21 days dramatically inhibited the growth of MG-63 xenografts in nude mice. Our study suggested that saurolactam may be a potential treatment for osteosarcoma. To further elucidate the related mechanisms, we performed Western blotting experiments. We demonstrate that
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the protein levels of Akt, phospho-Ser473-Akt, c-Myc, and Skp2 in human osteosarcoma cells were diminished by saurolactam treatment. The activation of PI3 K/Akt signaling plays a central role in proliferation and progression of osteosarcoma cells [26]. Akt/PKB is a serine/threonine protein kinase and implicated in cell survival pathways and hinders apoptotic processes. Akt/PKB contains a crucial phosphorylation site serine 473 that regulates its kinase activity. mTOR kinase and SIN1/MIP1 can phosphorylate serine 473 and activate Akt [27, 28]. Overactivation of the PI3 K/Akt pathway is related to poor clinical outcome of cancer [29–31]. The protein encoded by Myc (c-Myc) is a vital transcriptional regulator and a well-known multifunctional proto-oncoprotein and nuclear phosphoprotein involved in
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cell cycle progression, cell proliferation, apoptosis, and cell transformation [32, 33]. Aberrant expression of c-Myc at mRNA and protein levels induces cancer progression [34, 35] and promotes osteosarcoma cell invasion [36]. Diminution in c-Myc protein expression leads to reduced osteosarcoma cell proliferation and tumor growth [37]. In addition, dysfunction in c-Myc has been found in breast, lung, cervix, colon, and stomach cancer [38]. Therefore, Myc is regarded as a propitious target for anti-cancer drugs [39, 40]. Since saurolactam treatment repressed Akt, Akt phosphorylation at Ser473, and c-Myc, inactivation of Akt signaling and c-Myc might cause growth inhibition induced by saurolactam. S-phase kinase-associated protein 2 (Skp2) plays an important role in ubiquitination and inhibition of p27Kip1 [41, 42]. The p27Kip1 protein inhibits cell cycle progression, and amassing of p27Kip1 contributes to cell cycle arrest and decreased cell proliferation of cancer cells [17, 22, 43]. We found that saurolactam treatment caused decreased Skp2, augmented G1 cell cycle arrest and apoptosis. Overexpression of Skp2 in part rescued saurolactam-induced inhibition of MG-63 cell proliferation rate, migration, and invasion ability, suggesting that Skp2 inhibition was possibly implicated in the growth inhibition induced by saurolactam in osteosarcoma cells. Since Skp2 overexpression could not entirely inverse the growth inhibitory effects, we reasoned that other pathways or mechanisms might be elicited by saurolactam to inhibit cell growth of osteosarcoma cells. In conclusion, the present study provides systems-level insight into the mechanisms by which saurolactam treatment causes inhibition of proliferation, migration, and invasion of human osteosarcoma cells. Our results indicate that saurolactam might represent a new promising agent to improve osteosarcoma treatment.
References 1. Yang, Q. C., Zeng, B. F., Dong, Y., Shi, Z. M., Jiang, Z. M., & Huang, J. (2007). Overexpression of hypoxia-inducible factor1alpha in human osteosarcoma: Correlation with clinicopathological parameters and survival outcome. Japanese Journal of Clinical Oncology, 37, 127–134. 2. Rosen, G., Murphy, M. L., Huvos, A. G., Gutierrez, M., & Marcove, R. C. (1976). Chemotherapy, en bloc resection, and prosthetic bone replacement in the treatment of osteogenic sarcoma. Cancer, 37, 1–11. 3. Ando, K., Heymann, M. F., Stresing, V., Mori, K., Redini, F., & Heymann, D. (2013). Current therapeutic strategies and novel approaches in osteosarcoma. Cancers, 5, 591–616. 4. Picci, P., Mercuri, M., Ferrari, S., Alberghini, M., Briccoli, A., Ferrari, C., et al. (2010). Survival in high-grade osteosarcoma: Improvement over 21 years at a single institution. Annals of Oncology, 21, 1366–1373.
5. Durnali, A., Alkis, N., Cangur, S., Yukruk, F. A., Inal, A., Tokluoglu, S., et al. (2013). Prognostic factors for teenage and adult patients with high-grade osteosarcoma: An analysis of 240 patients. Medical Oncology, 30, 624. 6. Gorlick, R. (2002). Osteosarcoma: Clinical practice and the expanding role of biology. Journal of Musculoskeletal and Neuronal Interactions, 2, 549–551. 7. Lee, A. K., Sung, S. H., Kim, Y. C., & Kim, S. G. (2003). Inhibition of lipopolysaccharide-inducible nitric oxide synthase, TNF-alpha and COX-2 expression by sauchinone effects on I-kappaBalpha phosphorylation, C/EBP and AP-1 activation. British Journal of Pharmacology, 139, 11–20. 8. Hwang, B. Y., Lee, J. H., Nam, J. B., Hong, Y. S., & Lee, J. J. (2003). Lignans from Saururus chinensis inhibiting the transcription factor NF-kappaB. Phytochemistry, 64, 765–771. 9. Park, H. J., Kim, R. G., Seo, B. R., Ha, J., Ahn, B. T., Bok, S. H., et al. (2003). Saucernetin-7 and saucernetin-8 isolated from Saururus chinensis inhibit the LPS-induced production of nitric oxide and prostaglandin E2 in macrophage RAW264.7 cells. Planta Medica, 69, 947–950. 10. Moon, T. C., Seo, C. S., Haa, K., Kim, J. C., Hwang, N. K., Hong, T. G., et al. (2008). Meso-dihydroguaiaretic acid isolated from Saururus chinensis inhibits cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Archives of Pharmacal Research, 31, 606–610. 11. Kim, M. H., Ryu, S. Y., Choi, J. S., Min, Y. K., & Kim, S. H. (2009). Saurolactam inhibits osteoclast differentiation and stimulates apoptosis of mature osteoclasts. Journal of Cellular Physiology, 221, 618–628. 12. Lee, S. U., Choi, Y. H., Kim, Y. S., Min, Y. K., Rhee, M., & Kim, S. H. (2010). Anti-resorptive saurolactam exhibits in vitro antiinflammatory activity via ERK-NF-kappaB signaling pathway. International Immunopharmacology, 10, 298–303. 13. Chuu, C. P., Chen, R. Y., Hiipakka, R. A., Kokontis, J. M., Warner, K. V., Xiang, J., et al. (2007). The liver X receptor agonist T0901317 acts as androgen receptor antagonist in human prostate cancer cells. Biochemical and Biophysical Research Communications, 357, 341–346. 14. Chuu, C. P., & Lin, H. P. (2010). Antiproliferative effect of LXR agonists T0901317 and 22(R)-hydroxycholesterol on multiple human cancer cell lines. Anticancer Research, 30, 3643–3648. 15. Fukuchi, J., Kokontis, J. M., Hiipakka, R. A., Chuu, C. P., & Liao, S. (2004). Antiproliferative effect of liver X receptor agonists on LNCaP human prostate cancer cells. Cancer Research, 64, 7686–7689. 16. Chuu, C. P., Chen, R. Y., Kokontis, J. M., Hiipakka, R. A., & Liao, S. (2009). Suppression of androgen receptor signaling and prostate specific antigen expression by (-)-epigallocatechin-3gallate in different progression stages of LNCaP prostate cancer cells. Cancer Letters, 275, 86–92. 17. Chuu, C. P., Kokontis, J. M., Hiipakka, R. A., Fukuchi, J., Lin, H. P., Lin, C. Y., et al. (2011). Androgen suppresses proliferation of castration-resistant LNCaP 104-R2 prostate cancer cells through androgen receptor, Skp2, and c-Myc. Cancer Science, 102, 2022–2028. 18. Chuu, C. P., Lin, H. P., Ciaccio, M. F., Kokontis, J. M., Hause, R. J, Jr, Hiipakka, R. A., et al. (2012). Caffeic acid phenethyl ester suppresses the proliferation of human prostate cancer cells through inhibition of p70S6 K and Akt signaling networks. Cancer Prevention Research, 5, 788–797. 19. Kokontis, J. M., Hsu, S., Chuu, C. P., Dang, M., Fukuchi, J., Hiipakka, R. A., et al. (2005). Role of androgen receptor in the progression of human prostate tumor cells to androgen independence and insensitivity. The Prostate, 65, 287–298. 20. Lin, H. P., Jiang, S. S., & Chuu, C. P. (2012). Caffeic acid phenethyl ester causes p21 induction, Akt signaling reduction,
123
Cell Biochem Biophys
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
and growth inhibition in PC-3 human prostate cancer cells. PLoS ONE, 7, e31286. Lin, H. P., Kuo, L. K., & Chuu, C. P. (2012). Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells. Phytotherapy Research, 26, 122–126. Kokontis, J. M., Hay, N., & Liao, S. (1998). Progression of LNCaP prostate tumor cells during androgen deprivation: Hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Molecular Endocrinology, 12, 941–953. Chuu, C. P., Hiipakka, R. A., Kokontis, J. M., Fukuchi, J., Chen, R. Y., & Liao, S. (2006). Inhibition of tumor growth and progression of LNCaP prostate cancer cells in athymic mice by androgen and liver X receptor agonist. Cancer Research, 66, 6482–6486. Chuu, C. P., Hiipakka, R. A., Fukuchi, J., Kokontis, J. M., & Liao, S. (2005). Androgen causes growth suppression and reversion of androgen-independent prostate cancer xenografts to an androgen-stimulated phenotype in athymic mice. Cancer Research, 65, 2082–2084. Xu, H. M., Liang, Y., Chen, Q., Wu, Q. N., Guo, Y. M., Shen, G. P., et al. (2011). Correlation of Skp2 overexpression to prognosis of patients with nasopharyngeal carcinoma from South China. Chinese journal of cancer, 30, 204–212. Zhang, A., He, S., Sun, X., Ding, L., Bao, X., & Wang, N. (2014). Wnt5a promotes migration of human osteosarcoma cells by triggering a phosphatidylinositol-3 kinase/Akt signals. Cancer Cell International, 14, 15. Sarbassov, D. D., Guertin, D. A., Ali, S. M., & Sabatini, D. M. (2005). Phosphorylation and regulation of Akt/PKB by the rictormTOR complex. Science, 307, 1098–1101. Jacinto, E., Facchinetti, V., Liu, D., Soto, N., Wei, S., Jung, S. Y., et al. (2006). SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell, 127, 125–137. Lin, H. P., Lin, C. Y., Liu, C. C., Su, L. C., Huo, C., Kuo, Y. Y., et al. (2013). Caffeic Acid phenethyl ester as a potential treatment for advanced prostate cancer targeting akt signaling. International Journal of Molecular Sciences, 14, 5264–5283. Kreisberg, J. I., Malik, S. N., Prihoda, T. J., Bedolla, R. G., Troyer, D. A., Kreisberg, S., et al. (2004). Phosphorylation of Akt (Ser473) is an excellent predictor of poor clinical outcome in prostate cancer. Cancer Research, 64, 5232–5236. Liu, C. C., Hsu, J. M., Kuo, L. K., & Chuu, C. P. (2013). Caffeic acid phenethyl ester as an adjuvant therapy for advanced prostate cancer. Medical Hypotheses, 80, 617–619.
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32. Kokontis, J., Takakura, K., Hay, N., & Liao, S. (1994). Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation. Cancer Research, 54, 1566–1573. 33. Vellaichamy, A., Dezso, Z., JeBailey, L., Chinnaiyan, A. M., Sreekumar, A., Nesvizhskii, A. I., et al. (2010). ‘‘Topological significance’’ analysis of gene expression and proteomic profiles from prostate cancer cells reveals key mechanisms of androgen response. PLoS ONE, 5, e10936. 34. Edwards, J., Krishna, N. S., Witton, C. J., & Bartlett, J. M. (2003). Gene amplifications associated with the development of hormone-resistant prostate cancer. Clinical Cancer Research, 9, 5271–5281. 35. Grad, J. M., Dai, J. L., Wu, S., & Burnstein, K. L. (1999). Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA. Molecular Endocrinology, 13, 1896–1911. 36. Han, G., Wang, Y., & Bi, W. (2012). C-Myc overexpression promotes osteosarcoma cell invasion via activation of MEK-ERK pathway. Oncology Research, 20, 149–156. 37. Liu, Z., Zhang, G., Li, J., Liu, J., & Lv, P. (2014). The tumorsuppressive microRNA-135b targets c-myc in osteoscarcoma. PLoS ONE, 9, e102621. 38. Field, J. K., & Spandidos, D. A. (1990). The role of ras and myc oncogenes in human solid tumours and their relevance in diagnosis and prognosis (review). Anticancer Research, 10, 1–22. 39. Matthews, G. M., Lefebure, M., Doyle, M. A., Shortt, J., Ellul, J., Chesi, M., et al. (2013). Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma. Cell Death and Disease, 4, e798. 40. Azmi, A. S., Ali, S., Banerjee, S., Bao, B., Maitah, M. N., Padhye, S., et al. (2011). Network modeling of CDF treated pancreatic cancer cells reveals a novel c-myc-p73 dependent apoptotic mechanism. American journal of translational research, 3, 374–382. 41. Carrano, A. C., Eytan, E., Hershko, A., & Pagano, M. (1999). SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nature Cell Biology, 1, 193–199. 42. Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H., & Zhang, H. (1999). p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Current Biology, 9, 661–664. 43. Elledge, S. J., & Harper, J. W. (1994). Cdk inhibitors: On the threshold of checkpoints and development. Current Opinion in Cell Biology, 6, 847–852.
ORIGINAL PAPER
Saurolactam Inhibits Proliferation, Migration, and Invasion of Human Osteosarcoma Cells Zhengwei Li • Hui Liu • Baizhi Li • Yanzhe Zhang Chengdong Piao
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Ó Springer Science+Business Media New York 2015
Abstract Osteosarcoma is a common type of malignant bone tumor with features of osteoid formation or osteolytic lesions of bone. New therapeutic approaches are urgently needed since it lacks response to chemotherapeutic treatments. Saurolactam, a natural compound isolated from the aerial portions of Saururus chinensis, was reported to have an anti-inflammatory activity. Here, we demonstrate that saurolactam shows anti-cancer activity against human osteosarcoma cells. Saurolactam treatment inhibited proliferation of human osteosarcoma cell lines MG-63 and HOS and decreased colony formation in soft agar in a dosedependent manner. Intraperitoneal administration of saurolactam at 25 mg/kg of body weight for 21 days dramatically inhibited the growth of MG-63 xenografts in nude mice. Flow cytometric analysis indicated that saurolactam treatment (20 lM) led to G1 cell cycle arrest and induced apoptosis in these two cell lines. Western analysis suggested that saurolactam treatment resulted in a reduction of Akt/PKB, phospho-Ser473-Akt, c-Myc, and S-phase kinase-associated protein 2 (Skp2) in MG-63 and HOS osteosarcoma cells. Akt overexpression significantly abolished saurolactam-induced decrease in protein and phosphorylation levels of Akt, c-Myc, and Skp2 protein Z. Li Y. Zhang C. Piao (&) The Second Hospital of Jilin University, No. 218 Ziqiang Street, Changchun 130041, People’s Republic of China e-mail: [email protected] H. Liu Department of Human Anatomy, College of Basic Medical Sciences, Jilin University, Changchun 130021, People’s Republic of China B. Li Institute of Frontier Medical Science of Jilin University, Changchun 130021, People’s Republic of China
levels, implying that Akt inactivation was a causal mediator of saurolactam-induced inhibition of c-Myc and Skp2. Moreover, Skp2 overexpression in MG-63 cells partly abolished the growth inhibition induced by saurolactam. Saurolactam treatment repressed migration and invasion ability, and Skp2 overexpression significantly blocked these inhibitory effects of saurolactam in MG-63 Cells. The present study indicates that saurolactam might represent a new promising agent to improve osteosarcoma treatment. Keywords Saurolactam Osteosarcoma S-phase kinaseassociated protein 2 (Skp2) Cell proliferation Tumor xenografts
Introduction Osteosarcoma is a common cancerous bone tumor primarily affecting children and young adults, and characterized by the existence of tumor cells generating an osteoid matrix [1]. Currently, the globally accepted therapeutic strategy for osteosarcoma was recommended by Rosen and his colleagues in the 1970s [2]. In the last decade, improved understanding of oncogenic pathways in osteosarcoma has contributed to the expansion of new therapeutic avenues based upon single drugs or conventional drug combinations [3]. Despite the development of polychemotherapy that has undoubtedly enhanced the survival rates of patients with osteosarcoma, the 5-year event-free survival for high-grade osteosarcoma patients still cannot break through 50 % [4]. Hence, a deep insight into the basic biology of osteosarcoma is urgently required to identify its novel therapeutic targets [5, 6]. Recently, the certain biological molecules such as Akt, C-Myc, and Skp2
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have been recognized as promising prognostic markers or therapeutic targets for osteosarcoma. Historically, natural products and their derivatives are a valuable source of therapeutic drugs. Development of novel medicinal products from natural substances becomes an important approach in the drug discovery. Various phytochemicals derived from Saururus chinensis presented a potent anti-inflammatory activity [7–10]. Saurolactam is a new compound extracted from S. chinensis. Recent studies suggest that it possesses anti-resorptive activity [11] and inhibits osteoclast differentiation, as well as induces apoptosis of mature osteoclasts [11]. However, there are no studies examining the in vitro and in vivo anti-tumor activity of saurolactam. Thus, in the present study, we investigated the anti-tumor activity of saurolactam. We assessed its effects on the pro-survival and proliferative mediators and its role in suppressing proliferation-related signaling molecules, including Akt, C-Myc, and Skp2, in two human osteosarcoma cell lines i.e., MG-63 and HOS cells.
Materials and Methods Cells and Culture Conditions The human osteosarcoma MG-63 and HOS cells acquired from ATCC were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS). Materials Saurolactam was isolated using the methods as described previously [12]. Matrigel was obtained from BD Bioscience (San Jose, CA, USA). Tissue culture dishes from Costar were from Sigma (St. Louis, MO, USA). Antibodies against Akt and phosphor-S473-Akt were from Cell Signaling Technology (Danvers, MA); anti-b-actin, c-Myc, and Skp2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Proliferation Assay Osteosarcoma cells were seeded in 96-well plates (3 9 103 cells/well). Proliferation assays were carried out under maintenance circumstances. Relative cell number was examined by assaying cellular DNA with the blue-fluorescent Hoechst (Sigma, St. Louis, MO, USA) as described before [13–20]. All values were normalized to those of the control (no saurolactam treatment). The experiment was performed three times independently. The results were presented as mean and standard deviation.
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Cell Viability Assay Osteosarcoma cells were seeded in 96-well plates (3 9 103 cells/well). After 24 h, the cells were incubated with mounting concentrations of saurolactam for 48 h. Cell viability was measured with a 3,4,5-dimethylthiazol-2-yl2-5-diphenyltetrazolium bromide (MTT) assay by determining the absorbance at 560 nm using a Benchmark Microplate Reader (Bio-Rad) [21]. All values were normalized to those of the control (no saurolactam treatment). The experiment was performed three times independently. The results were presented as mean and standard deviation. Flow Cytometric Analysis Cells were seeded into 100-mm dishes (5 9 105 cells) and cultured for 16 h prior to the addition of saurolactam. After 48 h of incubation in the presence of different concentrations of saurolactam, cells were fixed in ice-cold 70 % ethanol in phosphate buffered saline (PBS) for 12 h. Fixed cells were then rinsed with PBS, incubated with PBS containing 0.1 mg/mL RNase A for 20 min, and re-suspended in propidium iodide (PI) (50 lg/mL). Cell cycle distributions were detected using a FACScan flow cytometer (Becton–Dickinson Immunocytometry Systems) and analyzed with a ModFit software (Topsham, ME, USA) as previously described [14, 17–19, 22]. Soft Agar Colony Formation For soft agar assay, human osteosarcoma MG-63 and HOS cells (5,000 cells/plate) were suspended in 0.35 % agar containing DMEM supplemented with 10 % FBS in 35-mm cell culture plates. Cells were allowed to grow for 3 weeks at 37 °C in a CO2 incubator. The colonies were stained with crystal violet. Tumor Xenografts in Nude Mice Male Balb/c nude mice aged 6–8 weeks (NCI Frederick, MD) were subcutaneously injected into both flanks with MG-63 cells (5 9 105 cells) suspended in 0.5 ml Matrigel. Seven days after tumor inoculation, tumor growth was monitored. Mice were divided into control and saurolactam-treated groups. The control group had 8 mice bearing 16 tumors, and the saurolactam group had 8 mice bearing 11 tumors. Three weeks after inoculation, the treatment group was intraperitoneally (i.p.) administrated saurolactam (25 mg/kg of body weight) once every other day. The control groups were i.p. administrated with vehicle only. The treatment terminated on day 43 after tumor inoculation. Tumors were measured daily with calipers, and volume was calculated with the formula:
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volume = length 9 width 9 height 9 0.52 [16, 17, 23, 24]. Tumor volumes and body weight of mice were indicated as mean ± SD. Western Blotting Analysis The Western blot analyses were performed on 80 lg of protein extracts. The human osteosarcoma MG-63 and HOS cells were lysed in lysis buffer (0.5 % sodium deoxycholate, 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 % Nonidet P-40, 0.1 % SDS) containing 50 mM NaF, 5 mM EDTA, 1 mM DTT, and 10 lg/ml aprotinin. Cell lysates were resolved on SDS-PAGE according to standard protocols. After being transferred to membranes, the samples were immunoblotted with primary antibodies including phosphor-Akt-Ser473, Akt, C-Myc, Skp2, and b-actin, followed by secondary antibodies conjugated with horseradish peroxidase. Bands were revealed by using an enzyme-linked chemiluminescence detection kit (ECL, Amersham Biosciences, Piscataway, NJ) as recommended by the manufacturer, and density was quantified by use of Scion Image software (Scion Corp, Frederick, MD). Akt and Skp2 Overexpression in MG-63 Cells Ectopic expressions of Akt and Skp2 were realized by transfecting MG-63 cells with the plasmids of pcDNA3.1Skp2 (Skp2), pcDNA3.1-Akt (Akt), and pcDNA3.1 empty vector (EV) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in line with the manufacturer’s protocols [25]. MG-63 cells transfected with a pcDNA3.1 empty vector were used as controls. Transwell Migration Assay Migration assays were carried out with a transwell kit (BD Bioscience). MG-63 cells were treated with 0, 20, and 40 lM saurolactam for 48 h. Cells were then trypsinized, detached from culture plates, and washed with PBS. Saurolactam-treated cells (1 9 104) in 200 ll DMEM without serum were put in the upper chamber. The lower compartment of transwell chamber was encumbered with DMEM containing 10 % FBS. The serum-starved cells were allowed to migrate overnight at 37 °C. Cells attached to the filter were fixed and stained with Giemsa solution for 1 h at room temperature. Filters were subsequently washed with water, and the number of cells on the filter was quantified by reckoning cells in photographs.
manufacturer’s protocols. MG-63 cells were treated with various concentrations of saurolactam for 48 h. Cells were then detached from culture plates with trypsin and washed thrice with PBS. Cells were seeded (4 9 104 cells per well) in DMEM without serum in the upper chamber. As a chemoattractant, DMEM medium containing 10 % FBS was put in the lower compartment of the chamber. After overnight incubation, the non-invading cells were removed. After fixation with methanol and wash, the membranes were stained with Giemsa’s solution. Invasiveness was assessed by calculating the invading cells with a light microscope. All experiments were performed in triplicate. Statistical Analysis The data were expressed as mean ± SD. Differences were analyzed by one-way analysis of variance followed by Fisher’s protected least significant difference test. The level of significance was taken as P \ 0.05.
Results Saurolactam Repressed the Proliferation of Human Osteosarcoma Cells First, we determined the effect of saurolactam incubation on the viability and proliferation of two human osteosarcoma cell lines i.e., MG-63 and HOS osteosarcoma cells (Fig. 1). An MTT assay showed that saurolactam inhibited cell viability in a dose-dependent manner. Similarly, saurolactam dose-dependently suppressed cellular proliferation, as indicated by a Hoechst dye-based proliferation assays (Fig. 1). The EC50 for saurolactam in the MTT assay was comparable to that assayed by Hoechst dye-based assay (data not shown), implying that suppression of cell proliferation contributed to the decrease in viable cells induced by saurolactam treatment in these two human osteosarcoma cell lines. Saurolactam Treatment suppressed Colony Formation of MG-63 and HOS Cells in Soft Agar Treatment of MG-63 and HOS cells with 20 and 40 lM saurolactam strikingly suppressed the formation of their corresponding colonies in soft agar, substantiating the anticancer activity of saurolactam (Fig. 2). Saurolactam Administration Inhibited Growth of MG-63 Xenografts in Nude Mice
Transwell Invasion Assay An invasion assay was conducted with BD BioCoat Matrigel invasion chambers (BD Biosciences) in accordance with the
To determine if saurolactam could retard tumor growth in vivo, a study with MG-63 xenografts in nude mice was carried out. Intraperitoneal (i.p.) injection of saurolactam
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Fig. 1 Effect of saurolactam on viability and proliferation of human osteosarcoma cell lines. MG-63 and HOS osteosarcoma cells were incubated with increasing concentrations of saurolactam for 48 h. Relative viability and cell number of osteosarcoma cells were measured by MTT 96-well assay (a) and Hoechst dye-based
proliferation assay (b), respectively. Cell numbers were normalized to control. Results presented are mean ± SD from three independent experiments, with non-treated controls set as 100. *P \ 0.01 versus untreated cells
Fig. 2 Effect of saurolactam on colony formation. MG-63 (a) and HOS (b) cells treated with 0, 20, or 40 lM saurolactam for 12 and 15 days, respectively. Image is a representative result from three independent experiments
(25 mg/kg of body weight) once every other day for 21 days leads to a 56 % decrease in the average volume of MG-63 xenografts (Fig. 3a). Body weight of both control and saurolactam-treated mice gradually declined (Fig. 3b). These results substantiate that i.p. administration of saurolactam could inhibit the growth of osteosarcoma in vivo.
reduction in S and G2/M populations of MG-63 and HOS osteosarcoma cells (Fig. 4). Thus, treatment with 20 lM saurolactam promoted G1 cell cycle arrest in MG-63 and HOS osteosarcoma cells. In addition, we observed a significant increase in sub-G1 population, which represented apoptotic cells, in these two osteosarcoma cell lines.
Saurolactam Induced G1 Cell Cycle Arrest and Apoptosis in Osteosarcoma Cells
Saurolactam Reduced the Protein Levels of Signaling Proteins Implicated in Cell Cycle via Akt Pathway
Next, we sought to investigate if saurolactam influenced cell cycle progression of osteosarcoma cells by performing flow cytometric analysis. Treatment with 20 lM saurolactam for 48 h elicited an elevation of the G1 cell population and a
Next, we performed Western analysis to check whether saurolactam affected proteins involved in cell cycle. As indicated in Fig. 5a, saurolactam treatment resulted in a reduction of Akt/PKB, phospho-Ser473-Akt, c-Myc, and
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S-phase kinase-associated protein 2 (Skp2) in MG-63 and HOS osteosarcoma cells. To determine the causal relationship between Akt, c-Myc, and Skp2, we transfected MG-63 cells with either empty vector pcDNA3.1 (EV) or constitutively active pcDNA3.1-Akt (Akt) to overexpress Akt. As indicated in Fig. 5b, Akt overexpression significantly abolished saurolactam-induced decrease in protein and phosphorylation levels of Akt, c-Myc, and Skp2 protein levels, implying that Akt inactivation was a causal mediator of saurolactam-induced inhibition of c-Myc and Skp2. Skp2 Overexpression Partly blocked the Inhibitory Effect of Saurolactam in MG-63 Cells
Fig. 3 Effect of saurolactam on the growth of MG-63 xenografts in nude mice. MG-63 cells (5 9 105 cells) were subcutaneously injected into Male Balb/c nude mice aged 6–8 weeks. Tumors were measured daily with calipers. The formula volume = length 9 width 9 height 9 0.52 was used to calculate tumor volume. Seven days after tumor inoculation, tumor growth was monitored. Mice were divided into control and saurolactam-treated groups. The control group had 8 mice bearing 16 tumors, and the saurolactam group had 8 mice bearing 11 tumors. Three weeks after inoculation, the treatment group was intraperitoneally (i.p.) administrated saurolactam (25 mg/kg of body weight) once every other day. The control groups were i.p. administrated with vehicle only. The treatment terminated on day 43 after tumor inoculation. Tumor volumes (a) and body weight of mice (b) were indicated as mean ± SD
Fig. 4 Impact of saurolactam on cell cycle distribution of osteosarcoma cells. MG-63 (a) and HOS osteosarcoma cells (b) were treated with or without 20 lM saurolactam for 48 h. Cells were fixed with ice-cold 70 % ethanol, collected and stained with a mixture of propidium iodide and RNase. The cell cycle distribution was detected
Because saurolactam treatment contributed to a salient decrease in Skp2 expression in these two osteosarcoma cell lines (Fig. 6a), we sought to explore if overexpression of Skp2 could abolish the growth inhibition induced by saurolactam in MG-63 cells. As shown in Fig. 6b, overexpression of Skp2 in part rescued saurolactam-induced inhibition of MG-63 cell proliferation rate. Since Skp2 overexpression could not entirely inverse the growth inhibitory effects, we reasoned that other pathways or mechanisms might be elicited by saurolactam to inhibit cell growth of osteosarcoma cells. In addition, transwell migration assay (Fig. 6c) and transwell invasion assay (Fig. 6d) revealed that migration and invasion ability of MG-63 cells were inhibited dose-dependently by saurolactam treatment. Skp2 overexpression significantly blocked these inhibitory effects of saurolactam in MG-63 Cells. Discussion In the present study, we examined the anti-tumor activity of saurolactam, a compound isolated from S. chinensis,
with the FACScan flow cytometer (Becton–Dickinson Immunocytometry Systems). The representative data acquired from four independent experiments were illustrated. *P \ 0.01 versus untreated cells
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Fig. 5 Saurolactam incubation inhibits protein and phosphorylation levels of signaling proteins. a Protein expression of Akt/PKB, phospho-S473-Akt, c-Myc, Skp2, and b-actin in MG-63 and HOS osteosarcoma cells treated with 0, 20, or 40 lM saurolactam for 48 h were examined by Western analysis. b MG-63 cells were transfected
with empty vector (EV) or wild-type Akt for 24 h and then treated with or without 20 lM saurolactam for 48 h. The corresponding protein and phosphorylation levels were detected with Western analysis. Values represent relative protein expression levels
Fig. 6 Effect of Skp2 overexpression on inhibition of cell growth, migration and invasion induced by saurolactam treatment. a Protein expression of Skp2 measured by Western analysis in MG-63 EV control and MG-63 cells overexpressing Skp2. MG-63 cells overexpressing Skp2 with pcDNA3.1 vector and control MG-63 cells (empty pcDNA3.1 vector, EV) were incubated with increasing concentrations of saurolactam for 48 h and detected by the 96-well proliferation assay (b), transwell migration assay (c), and transwell invasion assay (d). *P \ 0.01 versus EV
against human osteosarcoma cells. We observed that saurolactam treatment promoted G1 cell cycle arrest and apoptosis of human osteosarcoma cells. Moreover, we demonstrate that saurolactam treatment inhibited proliferation, migration, and invasion ability of human osteosarcoma cell lines MG-63 and HOS and decreased colony formation via inhibition of c-Myc and Skp2, and that Akt inactivation was a causal mediator of saurolactam-induced suppression of c-Myc and Skp2. In addition, i.p. administration of saurolactam at 25 mg/kg of body weight for 21 days dramatically inhibited the growth of MG-63 xenografts in nude mice. Our study suggested that saurolactam may be a potential treatment for osteosarcoma. To further elucidate the related mechanisms, we performed Western blotting experiments. We demonstrate that
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the protein levels of Akt, phospho-Ser473-Akt, c-Myc, and Skp2 in human osteosarcoma cells were diminished by saurolactam treatment. The activation of PI3 K/Akt signaling plays a central role in proliferation and progression of osteosarcoma cells [26]. Akt/PKB is a serine/threonine protein kinase and implicated in cell survival pathways and hinders apoptotic processes. Akt/PKB contains a crucial phosphorylation site serine 473 that regulates its kinase activity. mTOR kinase and SIN1/MIP1 can phosphorylate serine 473 and activate Akt [27, 28]. Overactivation of the PI3 K/Akt pathway is related to poor clinical outcome of cancer [29–31]. The protein encoded by Myc (c-Myc) is a vital transcriptional regulator and a well-known multifunctional proto-oncoprotein and nuclear phosphoprotein involved in
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cell cycle progression, cell proliferation, apoptosis, and cell transformation [32, 33]. Aberrant expression of c-Myc at mRNA and protein levels induces cancer progression [34, 35] and promotes osteosarcoma cell invasion [36]. Diminution in c-Myc protein expression leads to reduced osteosarcoma cell proliferation and tumor growth [37]. In addition, dysfunction in c-Myc has been found in breast, lung, cervix, colon, and stomach cancer [38]. Therefore, Myc is regarded as a propitious target for anti-cancer drugs [39, 40]. Since saurolactam treatment repressed Akt, Akt phosphorylation at Ser473, and c-Myc, inactivation of Akt signaling and c-Myc might cause growth inhibition induced by saurolactam. S-phase kinase-associated protein 2 (Skp2) plays an important role in ubiquitination and inhibition of p27Kip1 [41, 42]. The p27Kip1 protein inhibits cell cycle progression, and amassing of p27Kip1 contributes to cell cycle arrest and decreased cell proliferation of cancer cells [17, 22, 43]. We found that saurolactam treatment caused decreased Skp2, augmented G1 cell cycle arrest and apoptosis. Overexpression of Skp2 in part rescued saurolactam-induced inhibition of MG-63 cell proliferation rate, migration, and invasion ability, suggesting that Skp2 inhibition was possibly implicated in the growth inhibition induced by saurolactam in osteosarcoma cells. Since Skp2 overexpression could not entirely inverse the growth inhibitory effects, we reasoned that other pathways or mechanisms might be elicited by saurolactam to inhibit cell growth of osteosarcoma cells. In conclusion, the present study provides systems-level insight into the mechanisms by which saurolactam treatment causes inhibition of proliferation, migration, and invasion of human osteosarcoma cells. Our results indicate that saurolactam might represent a new promising agent to improve osteosarcoma treatment.
References 1. Yang, Q. C., Zeng, B. F., Dong, Y., Shi, Z. M., Jiang, Z. M., & Huang, J. (2007). Overexpression of hypoxia-inducible factor1alpha in human osteosarcoma: Correlation with clinicopathological parameters and survival outcome. Japanese Journal of Clinical Oncology, 37, 127–134. 2. Rosen, G., Murphy, M. L., Huvos, A. G., Gutierrez, M., & Marcove, R. C. (1976). Chemotherapy, en bloc resection, and prosthetic bone replacement in the treatment of osteogenic sarcoma. Cancer, 37, 1–11. 3. Ando, K., Heymann, M. F., Stresing, V., Mori, K., Redini, F., & Heymann, D. (2013). Current therapeutic strategies and novel approaches in osteosarcoma. Cancers, 5, 591–616. 4. Picci, P., Mercuri, M., Ferrari, S., Alberghini, M., Briccoli, A., Ferrari, C., et al. (2010). Survival in high-grade osteosarcoma: Improvement over 21 years at a single institution. Annals of Oncology, 21, 1366–1373.
5. Durnali, A., Alkis, N., Cangur, S., Yukruk, F. A., Inal, A., Tokluoglu, S., et al. (2013). Prognostic factors for teenage and adult patients with high-grade osteosarcoma: An analysis of 240 patients. Medical Oncology, 30, 624. 6. Gorlick, R. (2002). Osteosarcoma: Clinical practice and the expanding role of biology. Journal of Musculoskeletal and Neuronal Interactions, 2, 549–551. 7. Lee, A. K., Sung, S. H., Kim, Y. C., & Kim, S. G. (2003). Inhibition of lipopolysaccharide-inducible nitric oxide synthase, TNF-alpha and COX-2 expression by sauchinone effects on I-kappaBalpha phosphorylation, C/EBP and AP-1 activation. British Journal of Pharmacology, 139, 11–20. 8. Hwang, B. Y., Lee, J. H., Nam, J. B., Hong, Y. S., & Lee, J. J. (2003). Lignans from Saururus chinensis inhibiting the transcription factor NF-kappaB. Phytochemistry, 64, 765–771. 9. Park, H. J., Kim, R. G., Seo, B. R., Ha, J., Ahn, B. T., Bok, S. H., et al. (2003). Saucernetin-7 and saucernetin-8 isolated from Saururus chinensis inhibit the LPS-induced production of nitric oxide and prostaglandin E2 in macrophage RAW264.7 cells. Planta Medica, 69, 947–950. 10. Moon, T. C., Seo, C. S., Haa, K., Kim, J. C., Hwang, N. K., Hong, T. G., et al. (2008). Meso-dihydroguaiaretic acid isolated from Saururus chinensis inhibits cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Archives of Pharmacal Research, 31, 606–610. 11. Kim, M. H., Ryu, S. Y., Choi, J. S., Min, Y. K., & Kim, S. H. (2009). Saurolactam inhibits osteoclast differentiation and stimulates apoptosis of mature osteoclasts. Journal of Cellular Physiology, 221, 618–628. 12. Lee, S. U., Choi, Y. H., Kim, Y. S., Min, Y. K., Rhee, M., & Kim, S. H. (2010). Anti-resorptive saurolactam exhibits in vitro antiinflammatory activity via ERK-NF-kappaB signaling pathway. International Immunopharmacology, 10, 298–303. 13. Chuu, C. P., Chen, R. Y., Hiipakka, R. A., Kokontis, J. M., Warner, K. V., Xiang, J., et al. (2007). The liver X receptor agonist T0901317 acts as androgen receptor antagonist in human prostate cancer cells. Biochemical and Biophysical Research Communications, 357, 341–346. 14. Chuu, C. P., & Lin, H. P. (2010). Antiproliferative effect of LXR agonists T0901317 and 22(R)-hydroxycholesterol on multiple human cancer cell lines. Anticancer Research, 30, 3643–3648. 15. Fukuchi, J., Kokontis, J. M., Hiipakka, R. A., Chuu, C. P., & Liao, S. (2004). Antiproliferative effect of liver X receptor agonists on LNCaP human prostate cancer cells. Cancer Research, 64, 7686–7689. 16. Chuu, C. P., Chen, R. Y., Kokontis, J. M., Hiipakka, R. A., & Liao, S. (2009). Suppression of androgen receptor signaling and prostate specific antigen expression by (-)-epigallocatechin-3gallate in different progression stages of LNCaP prostate cancer cells. Cancer Letters, 275, 86–92. 17. Chuu, C. P., Kokontis, J. M., Hiipakka, R. A., Fukuchi, J., Lin, H. P., Lin, C. Y., et al. (2011). Androgen suppresses proliferation of castration-resistant LNCaP 104-R2 prostate cancer cells through androgen receptor, Skp2, and c-Myc. Cancer Science, 102, 2022–2028. 18. Chuu, C. P., Lin, H. P., Ciaccio, M. F., Kokontis, J. M., Hause, R. J, Jr, Hiipakka, R. A., et al. (2012). Caffeic acid phenethyl ester suppresses the proliferation of human prostate cancer cells through inhibition of p70S6 K and Akt signaling networks. Cancer Prevention Research, 5, 788–797. 19. Kokontis, J. M., Hsu, S., Chuu, C. P., Dang, M., Fukuchi, J., Hiipakka, R. A., et al. (2005). Role of androgen receptor in the progression of human prostate tumor cells to androgen independence and insensitivity. The Prostate, 65, 287–298. 20. Lin, H. P., Jiang, S. S., & Chuu, C. P. (2012). Caffeic acid phenethyl ester causes p21 induction, Akt signaling reduction,
123
Cell Biochem Biophys
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
and growth inhibition in PC-3 human prostate cancer cells. PLoS ONE, 7, e31286. Lin, H. P., Kuo, L. K., & Chuu, C. P. (2012). Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells. Phytotherapy Research, 26, 122–126. Kokontis, J. M., Hay, N., & Liao, S. (1998). Progression of LNCaP prostate tumor cells during androgen deprivation: Hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Molecular Endocrinology, 12, 941–953. Chuu, C. P., Hiipakka, R. A., Kokontis, J. M., Fukuchi, J., Chen, R. Y., & Liao, S. (2006). Inhibition of tumor growth and progression of LNCaP prostate cancer cells in athymic mice by androgen and liver X receptor agonist. Cancer Research, 66, 6482–6486. Chuu, C. P., Hiipakka, R. A., Fukuchi, J., Kokontis, J. M., & Liao, S. (2005). Androgen causes growth suppression and reversion of androgen-independent prostate cancer xenografts to an androgen-stimulated phenotype in athymic mice. Cancer Research, 65, 2082–2084. Xu, H. M., Liang, Y., Chen, Q., Wu, Q. N., Guo, Y. M., Shen, G. P., et al. (2011). Correlation of Skp2 overexpression to prognosis of patients with nasopharyngeal carcinoma from South China. Chinese journal of cancer, 30, 204–212. Zhang, A., He, S., Sun, X., Ding, L., Bao, X., & Wang, N. (2014). Wnt5a promotes migration of human osteosarcoma cells by triggering a phosphatidylinositol-3 kinase/Akt signals. Cancer Cell International, 14, 15. Sarbassov, D. D., Guertin, D. A., Ali, S. M., & Sabatini, D. M. (2005). Phosphorylation and regulation of Akt/PKB by the rictormTOR complex. Science, 307, 1098–1101. Jacinto, E., Facchinetti, V., Liu, D., Soto, N., Wei, S., Jung, S. Y., et al. (2006). SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell, 127, 125–137. Lin, H. P., Lin, C. Y., Liu, C. C., Su, L. C., Huo, C., Kuo, Y. Y., et al. (2013). Caffeic Acid phenethyl ester as a potential treatment for advanced prostate cancer targeting akt signaling. International Journal of Molecular Sciences, 14, 5264–5283. Kreisberg, J. I., Malik, S. N., Prihoda, T. J., Bedolla, R. G., Troyer, D. A., Kreisberg, S., et al. (2004). Phosphorylation of Akt (Ser473) is an excellent predictor of poor clinical outcome in prostate cancer. Cancer Research, 64, 5232–5236. Liu, C. C., Hsu, J. M., Kuo, L. K., & Chuu, C. P. (2013). Caffeic acid phenethyl ester as an adjuvant therapy for advanced prostate cancer. Medical Hypotheses, 80, 617–619.
123
32. Kokontis, J., Takakura, K., Hay, N., & Liao, S. (1994). Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation. Cancer Research, 54, 1566–1573. 33. Vellaichamy, A., Dezso, Z., JeBailey, L., Chinnaiyan, A. M., Sreekumar, A., Nesvizhskii, A. I., et al. (2010). ‘‘Topological significance’’ analysis of gene expression and proteomic profiles from prostate cancer cells reveals key mechanisms of androgen response. PLoS ONE, 5, e10936. 34. Edwards, J., Krishna, N. S., Witton, C. J., & Bartlett, J. M. (2003). Gene amplifications associated with the development of hormone-resistant prostate cancer. Clinical Cancer Research, 9, 5271–5281. 35. Grad, J. M., Dai, J. L., Wu, S., & Burnstein, K. L. (1999). Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA. Molecular Endocrinology, 13, 1896–1911. 36. Han, G., Wang, Y., & Bi, W. (2012). C-Myc overexpression promotes osteosarcoma cell invasion via activation of MEK-ERK pathway. Oncology Research, 20, 149–156. 37. Liu, Z., Zhang, G., Li, J., Liu, J., & Lv, P. (2014). The tumorsuppressive microRNA-135b targets c-myc in osteoscarcoma. PLoS ONE, 9, e102621. 38. Field, J. K., & Spandidos, D. A. (1990). The role of ras and myc oncogenes in human solid tumours and their relevance in diagnosis and prognosis (review). Anticancer Research, 10, 1–22. 39. Matthews, G. M., Lefebure, M., Doyle, M. A., Shortt, J., Ellul, J., Chesi, M., et al. (2013). Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma. Cell Death and Disease, 4, e798. 40. Azmi, A. S., Ali, S., Banerjee, S., Bao, B., Maitah, M. N., Padhye, S., et al. (2011). Network modeling of CDF treated pancreatic cancer cells reveals a novel c-myc-p73 dependent apoptotic mechanism. American journal of translational research, 3, 374–382. 41. Carrano, A. C., Eytan, E., Hershko, A., & Pagano, M. (1999). SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nature Cell Biology, 1, 193–199. 42. Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H., & Zhang, H. (1999). p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Current Biology, 9, 661–664. 43. Elledge, S. J., & Harper, J. W. (1994). Cdk inhibitors: On the threshold of checkpoints and development. Current Opinion in Cell Biology, 6, 847–852.
