Plumbagin exhibits an anti‑proliferative effect in human osteosarcoma cells by downregulating FHL2 and interfering with Wnt/β‑catenin signalling

  • Authors:
    • Yuan‑Liang Xue
    • Xiang‑Qi Meng
    • Long‑Jun Ma
    • Zhen Yuan
  • View Affiliations

  • Published online on: June 15, 2016     https://doi.org/10.3892/ol.2016.4725
  • Pages: 1095-1100
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Plumbagin, a naphthoquinone constituent of Plumbago zeylanica L. (Plumbaginaceae) is widely used in traditional Chinese medicine as an antifungal, antibacterial and anti‑inflammatory agent. Plumbagin is known to exhibit proapoptotic, antiangiogenic and antimetastatic effects in cancer cells. The transcriptional co‑factor four and a half LIM domains 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of gene expression, signal transduction and cell proliferation and differentiation, and also acts as a tumor suppressor or oncoprotein depending on the tissue microenvironment. The present study investigated the effect of plumbagin on FHL2 expression, Wnt/β‑catenin signalling and its anti‑proliferative activity in various human osteosarcoma cell lines, including SaOS2, MG63, HOS and U2OS. The cells were exposed to plumbagin and the expression of FHL2 was evaluated using western blot analysis. Furthermore, the anti‑proliferative effect of plumbagin was evaluated using a 3‑(4,5 dimethyl‑thiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. In addition, since FHL2 is involved in Wnt/β‑catenin signaling, the effect of plumbagin on β‑catenin and its primary target genes, including v‑myc avian myelocytomatosis viral oncogene homolog (c‑Myc) and WNT1 inducible signaling pathway protein‑1 (WISP‑1), was evaluated using western blot analysis. It was observed that plumbagin suppressed the expression of FHL2 and exhibited significant anti‑proliferative activity in osteosarcoma cells. It also attenuated Wnt/β‑catenin signalling by downregulating β‑catenin and its target genes, including c‑Myc and WISP‑1. In conclusion, plumbagin demonstrated anti‑proliferative activity in osteosarcoma cells by downregulating FHL2 and interfering with Wnt/β‑catenin signalling.

Introduction

Osteosarcoma, a primary bone sarcoma, is generally observed in children and adolescents and comprises ~5% of pediatric tumors and ~20% of bone tumors with a predominance in males (1). The tumour commonly originates in the metaphyseal regions of the distal femur, proximal humerus and proximal tibia. Osteosarcoma presents with high metastasis that is generally characterized by local invasion to the bone and soft tissue, and it most often affects the lung in distant metastasis (2). Conventional therapeutic approaches for osteosarcoma may cause drug-resistance and yield a variety of side effects, including cardiotoxicity, hearing loss, nephrotoxicity and risk of local relapse (3). An improved understanding of the molecular mechanisms involved in oncogenesis of osteosarcoma may enable identification and development of novel strategies for therapy in this disease.

Several studies have reported the aberrant activation of Wnt signaling in a variety of bone and soft-tissue sarcomas (48). Wnt signalling cascades regulate various biological processes, including cell-cycle regulation and stem cell maintenance (911), and are also involved in bone development and homeostasis (12,13). The Wnt/β-catenin signalling cascade is the best characterized of the Wnt pathways. An accumulation of β-catenin in the nucleus or cytoplasm of a cell is key for Wnt signal activation, and has been reported in osteosarcoma (14). Upon activation of Wnt signal, β-catenin translocates to the nucleus and binds to the transcription factors T-cell factor (TCF)/ lymphoid enhancer factor, which activates genes involved in cell proliferation, including v-myc avian myelocytomatosis viral oncogene homolog (c-Myc), mettatoproteinases and cyclin D1 (15,16).

WNT1 inducible signaling pathway protein-1 (WISP-1) has been reported to be significant in the invasion and migration capabilities of osteosarcoma cells. Studies have reported that increased WISP-1 expression is associated with tumor stage. WISP-1 enhances the migration of osteosarcoma cells by upregulating the expression of matrix metalloproteinase (MMP)-2 and MMP-9 with increasing tumor stage (17). Four and a half LIM domains protein 2 (FHL-2) is a member of the LIM-only protein family. LIM-domain containing proteins are key in protein-protein interactions in vital cellular processes, including cytoskeletal organization, and have cellular roles as regulators of gene expression, cell adhesion, cell motility and signal transduction (18). Studies also suggest that FHL-2 is involved in various types of cancer, including metastatic melanoma, prostate, breast, lung, colon, gastric and bladder cancer (1921), and a mechanism by which FHL-2 induces oncogenesis is by binding to β-catenin (22). However, the effect of FHL-2 on bone cancer tumorigenesis remains unclear. Targeting the Wnt/β-catenin signaling pathway may be of important clinical value in osteosarcoma therapy. Recent studies have reported the effective modulation of the Wnt/β-catenin signaling pathway by phytochemicals (23,24).

Plumbagin is a naphthoquinone constituent of Plumbago zeylanica L. (Plumbaginaceae) (Fig. 1), and has been used as an antifungal, antibacterial, and anti-inflammatory agent in traditional medicine (25). Plumbagin is known to exhibit proapoptotic (26), antiangiogenic (27) and antimetastatic effects in cancer cells (28). In addition, plumbagin is known to inhibit NF-κB (29), c-Jun-NH(2)-terminal kinase (30), protein kinase C ε (31) and signal transducer and activator of transcription 3 (31). Plumbagin activates glycogen synthase kinase 3β by inhibiting its phosphorylation at serine 9 (32), decreases the expression of cyclin D1 (33) and modulates the acetyltransferase activity of p300 protein (34). Consequently, the present study investigated the effect of plumbagin on FHL2 expression in various osteosarcoma cell lines, and it's effect on cell proliferation and Wnt/β-catenin signaling in these cells.

Materials and methods

Cell culture

Cancer cell lines derived from various human osteosarcoma tumors (human p53-deficient SaOS2, p53 mutant MG63 and human osteosarcoma HOS and U2OS cells) (35) were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured according to the company's protocol in Dulbecco's Modified Eagle Medium (Gibco®; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% heat inactivated fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 1% L-glutamine (Sigma-Aldrich) and penicillin/streptomycin (10,000 U/ml; Sigma-Aldrich) at 37°C in a humidified atmosphere with 5% CO2.

Cell proliferation assay

The effect of plumbagin on the proliferation of human osteosarcoma SaOS2, MG63, HOS and U2OS cells was assessed using a 3-(4,5 dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the cells were seeded into 96-well plates (Thermo Fisher Scientific, Inc.) at a density of 5–8×103 cells/well. The cells were subsequently treated with various concentration of plumbagin (0, 10, 20 or 40 µg; Sigma-Aldrich) or dimethyl sulfoxide (DMSO; vehicle control; Sigma-Aldrich) for 72 h at 37°C. Following treatment, 100 µl MTT (1 mg/ml; Sigma-Aldrich) was added to each well to analyze cell growth. DMSO was added following an incubation period of 4 h at 37°C, to lyse the cells and dissolve the purple formazan crystals that had formed. Absorbance of the formed formazan product was determined at λ max of 595 nm using a fluorescence spectrophotometer (HTS 7000 Reader; MTX Lab Systems Inc., Vienna, VA, USA). By plotting the percentage survival of cells against the concentration of plumbagin, the half maximal inhibitory concentration (IC50) values were obtained from the sigmoidal curve.

Western blot analysis

The human osteosarcoma SaOS2, MG63, HOS and U2OS cells were exposed to various concentrations of plumbagin (10, 20 and 40 µg/ml) or DMSO (vehicle control) at 37°C for 24 h, and following incubation the cells were lysed. The cells were treated with ice-cold hypotonic lysis buffer (10 mM HEPES, pH 7.9; 1.5 mM MgCl2, 0.2 mM KCl; 0.5 mM dithiothreitol; 0.2 mM phenylmethylsulfonylfluoride; Sigma-Aldrich), vortexed for 2 min and were centrifuged for 10–15 min at 12,000 rpm, followed by collection of the supernatants and assaying for protein concentration using Lowry's method (3638) using a Pierce Modified Lowry Protein Assay kit (Thermo Fisher Scientific, Inc.). The proteins (40–45 µg) were then subjected to 12% sodium dodecyl sulfate polyacrylamide gel-electrophoresis. β-actin was used as a loading control. The gels were transferred onto polyvinylidene difluoride membranes by electro-blotting and membranes were treated for 1 h with blocking buffer (5% non-fat dry milk) at room temperature. Subsequently, the membranes were incubated with the following primary antibodies overnight at 4°C, at dilutions according to the manufacturer's protocol: Rabbit polyclonal anti-FHL2 (dilution, 1:1,000; catalog no., ab12327; Abcam, Cambridge, MA, USA), rabbit monoclonal anti-β-catenin (dilution, 1:1,000; catalog no., 8480; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-β-actin (dilution, 1:1,000; catalog no., 4970; Cell Signaling Technology, Inc.), rabbit monoclonal anti-c-Myc (dilution, 1:1,000; catalog no., 13987; Cell Signaling Technology, Inc.), and rabbit polyclonal anti-WISP1 (dilution, 1:1,000; catalog no., Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After incubation with primary antibodies, the membranes were wahsed with TBST 3–4 times and incubated with goat anti-rabbit polyclonal horseradish peroxidase-conjugated secondary antibody (dilution, 10,000; catalog no., ab6721; Abcam) for 1 h at room temperature. The membranes were washed three times for 5 min with phosphate-buffered saline with Tween 20 (Thermo Fisher Scientific, Inc.) and bands were visualized using hydrogen peroxidase-chemiluminescent detection kit (Frontier Laboratories, Koriyama, Japan) using Luminescent Image Analyzer LAS-3000 (Fujifilm Corp., Tokyo, Japan).

Statistical analysis

The experiments were performed three times and data are expressed as the mean ± standard deviation. SPSS version 17 (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analyses. Results were analyzed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Overexpression of FHL2 in osteosarcoma cells

The expression of FHL2 in osteosarcoma U2OS, HOS, SaOS2 and MG63 cells was determined by western blot analysis. A single band was observed at the predicted molecular weight in all osteosarcoma cells (Fig. 2A). FHL2 expressions in HOS and SaOS2 cells were decreased compared to the expressions observed in MG63 and U2OS cells. These results suggest that there is expression of FHL2 in certain human osteosarcoma cells in vitro.

Plumbagin downregulates the expression of FHL2 in human osteosarcoma cells

The osteosarcoma cells were exposed to various concentrations of plumbagin (10, 20, 40 µg/ml) for 24 h. It was observed that plumbagin inhibited the expression of FHL2 in a dose-dependent manner in MG63, U2OS and SaOS2 cells compared with the control (DMSO) (Fig. 2B). Plumbagin also effectively inhibited the expression of FHL-2 in HOS cells, which exhibited mild expressions of FHL2.

Effect of plumbagin on cell proliferation

The anti-proliferative effect of plumbagin on U2OS (Fig. 3A), SaOS2 (Fig. 3B), HOS (Fig. 3C) and MG-63 (Fig. 3D) cells was evaluated by MTT assay. A 72 h exposure of U2OS, SaOS2, HOS and MG-63 cells to various concentrations of plumbagin (0, 10, 20 and 40 µg/ml) induced a dose-dependent inhibition in cell proliferation. The effect was clearer in U2OS, SaOS2 and MG-63 cells compared with HOS cells.

Effect of plumbagin on Wnt/β-catenin signaling

Similarly to the plumbagin-induced downregulation of FHL2, the nuclear translocation of β-catenin was also attenuated dose-dependently in cells treated with plumbagin (Fig. 4A). To additionally confirm the effect of FHL2 inhibition on Wnt signaling, the expression of WISP-1 and c-Myc, which are Wnt/β-catenin-target genes, in cells treated with various concentrations of plumbagin (10, 20 and 40 µg/ml) were assessed. Plumbagin dose-dependently inhibited the expressions of WISP-1 and c-Myc (Fig. 4B and C).

Discussion

In the present study, the effect of plumbagin on the expression of FHL2 and Wnt/β-catenin signaling were assessed. An altered FHL2 expression was observed in osteosarcoma cells treated with plumbagin, similarly to the results reported in previous studies (35,39). In addition, previous studies have demonstrated that FHL2 gene expression in human soft tissue cancers is variable depending on the cell type; FHL2 was revealed to be overexpressed in breast cancer (40), glioma (41), lung cancer (42), colon carcinoma (20) and gastrointestinal cancer (43) cells compared with cells from normal tissue.

FHL2 has been reported to effectively interact with numerous transcription factors, including androgen receptor, activator protein 1, cAMP response element-binding (CREB) protein, SKI proto-oncogene and β-catenin (43,4446), and FHL2 functions as a coactivator or corepressor, depending on the cell type and the promoter that is present (22,46). Furthermore, FHL2 binds with various transcription factors concurrently and participates in multiprotein complex assemblies (47,48). As a coactivator of β-catenin it cooperates with CREB-binding protein/p300 to enhance transcription driven by the β-catenin/TCF complex (46,47).

The present study focused on the effect of plumbagin on Wnt/β-catenin signaling cascade, since FHL2 interacts with this pathway in various cells (22,49), including osteoblast progenitor cells (46). In addition, dysregulation of the pathway has been reported in osteosarcoma cells (14,50). In the present study, plumbagin dose-dependently reduced the expression of FHL2 in osteosarcoma U2OS, HOS, SaOS2 and MG63 cells and reduced the proliferation of the cells. Plumbagin-induced inhibition of FHL2 in the osteosarcoma cells also led to a reduction of β-catenin translocation into the nucleus of these cells, which was evidenced by the reduction in the expression of c-Myc, a key target protein and onco-protein. The expression of WISP-1, a direct Wnt/β-catenin target gene (51), was also effectively downregulated by plumbagin in the present study. A dose-dependent decrease in the expression of c-Myc and WIST-1 was observed. Additionally, the decrease in the expressions of c-Myc and WISP was similar to the expression levels of FHL2 in U2OS, SaOS2 and MG63 cells. By contrast, HOS cells expressed a lower amount of FHL2 and exhibited almost insignificant levels of FHL2 following plumbagin exposure and negligible expression of c-Myc and WISP-1. The underlying cause of this requires additional investigation.

Overall in the present study, plumbagin significantly reduced the proliferation of osteosarcoma cells via the inhibition of Wnt/β-catenin signalling by downregulating the Wnt co-regulator FHL2, an oncoprotein in osteosarcoma cells. Therefore, following additional preclinical and clinical evaluations, plumbagin may be effectively used in osteosarcoma therapeutics.

References

1 

Ottaviani G and Jaffe N: The epidemiology of osteosarcoma. Pediatric and Adolescent Osteosarcoma. Jaffe N, Bruland OS and Bielack S: Springer; New York, NY: pp. pp3–13. 2009, View Article : Google Scholar

2 

Guijarro MV, Ghivizzani SC and Gibbs CP: Animal models in osteosarcoma. Front Oncol. 4:1892014. View Article : Google Scholar : PubMed/NCBI

3 

Luetke A, Meyers PA, Lewis I and Juergens H: Osteosarcoma treatment-where do we stand? A state of the art review. Cancer Treat Rev. 40:523–532. 2014. View Article : Google Scholar : PubMed/NCBI

4 

Weeraratna AT, Jiang Y, Hostetter G, Rosenblatt K, Duray P, Bittner M and Trent JM: Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell. 1:279–288. 2002. View Article : Google Scholar : PubMed/NCBI

5 

Wissmann C, Wild PJ, Kaiser S, Roepcke S, Stoehr R, Woenckhaus M, Kristiansen G, Hsieh JC, Hofstaedter F, Hartmann A, et al: WIF-1, a component of the Wnt pathway, is down-regulated in prostate, breast, lung and bladder cancer. J Pathol. 201:204–212. 2003. View Article : Google Scholar : PubMed/NCBI

6 

Hoang BH, Kubo T, Healey JH, Yang R, Nathan SS, Kolb EA, Mazza B, Meyers PA and Gorlick R: Dickkopf 3 inhibits invasion and motility of SaOS-2 osteosarcoma cells by modulating the Wnt-beta-catenin pathway. Cancer Res. 64:2734–2739. 2004. View Article : Google Scholar : PubMed/NCBI

7 

Barker N and Clevers H: Mining the Wnt pathway for cancer therapeutics. Nat Rev Drug Discov. 5:997–1014. 2006. View Article : Google Scholar : PubMed/NCBI

8 

Lin YC, You L, Xu Z, He B, Mikami I, Thung E, Chou J, Kuchenbecker K, Kim J, Raz D, et al: Wnt signaling activation and WIF-1 silencing in nasopharyngeal cancer cell lines. Biochem Biophys Res Commun. 341:635–640. 2006. View Article : Google Scholar : PubMed/NCBI

9 

Huelsken J and Birchmeier W: New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev. 11:547–553. 2001. View Article : Google Scholar : PubMed/NCBI

10 

Mlodzik M: Planar cell polarization: Do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation? Trends Genet. 18:564–571. 2002. View Article : Google Scholar : PubMed/NCBI

11 

Clevers H: Wnt/beta-catenin signaling in development and disease. Cell. 127:469–480. 2006. View Article : Google Scholar : PubMed/NCBI

12 

Westendorf JJ, Kahler RA and Schroeder TM: Wnt signaling in osteoblasts and bone diseases. Gene. 341:19–39. 2004. View Article : Google Scholar : PubMed/NCBI

13 

Rawadi G and Roman-Roman S: Wnt signalling pathway: A new target for the treatment of osteoporosis. Expert Opin Ther Targets. 9:1063–1077. 2005. View Article : Google Scholar : PubMed/NCBI

14 

Haydon RC, Deyrup A, Ishikawa A, Heck R, Jiang W, Zhou L, Feng T, King D, Cheng H, Breyer B, et al: Cytoplasmic and/or nuclear accumulation of the beta-catenin protein is a frequent event in human osteosarcoma. Int J Cancer. 102:338–342. 2002. View Article : Google Scholar : PubMed/NCBI

15 

Reya T and Clevers H: Wnt signalling in stem cells and cancer. Nature. 434:843–850. 2005. View Article : Google Scholar : PubMed/NCBI

16 

Espada J, Calvo MB, Diaz-Prado S and Medina V: Wnt signaling and cancer stem cells. Clin Transl Oncol. 11:411–427. 2009. View Article : Google Scholar : PubMed/NCBI

17 

Wu CL, Tsai HC, Chen ZW, Wu CM, Li TM, Fong YC and Tang CH: Ras activation mediates WISP-1-induced increases in cell motility and matrix metalloproteinase expression in human osteosarcoma. Cell Signal. 25:2812–2822. 2013. View Article : Google Scholar : PubMed/NCBI

18 

Kadrmas JL and Beckerle MC: The LIM domain: From the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol. 5:920–931. 2004. View Article : Google Scholar : PubMed/NCBI

19 

Gabriel B, Fischer DC, Orlowska-Volk M, zur Hausen A, Schüle R, Müller JM and Hasenburg A: Expression of the transcriptional coregulator FHL2 in human breast cancer: A clinicopathologic study. J Soc Gynecol Investig. 13:69–75. 2006. View Article : Google Scholar : PubMed/NCBI

20 

Wang J, Yang Y, Xia HH, Gu Q, Lin MC, Jiang B, Peng Y, Li G, An X, Zhang Y, et al: Suppression of FHL2 expression induces cell differentiation and inhibits gastric and colon carcinogenesis. Gastroenterology. 132:1066–1076. 2007. View Article : Google Scholar : PubMed/NCBI

21 

Amann T, Egle Y, Bosserhoff AK and Hellerbrand C: FHL2 suppresses growth and differentiation of the colon cancer cell line HT-29. Oncol Rep. 23:1669–1674. 2010.PubMed/NCBI

22 

Martin B, Schneider R, Janetzky S, Waibler Z, Pandur P, Kühl M, Behrens J, von der Mark K, Starzinski-Powitz A and Wixler V: The LIM only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts. J Cell Biol. 159:113–122. 2002. View Article : Google Scholar : PubMed/NCBI

23 

Zhang J, Cao H, Zhang B, Cao H, Xu X, Ruan H, Yi T, Tan L, Qu R, Song G, et al: Berberine potently attenuates intestinal polyps growth in ApcMin mice and familial adenomatous polyposis patients through inhibition of Wnt signalling. J Cell Mol Med. 17:1484–1493. 2013. View Article : Google Scholar : PubMed/NCBI

24 

Kim JH, Kim YH, Song GY, Kim DE, Jeong YJ, Liu KH, Chung YH and Oh S: Ursolic acid and its natural derivative corosolic acid suppress the proliferation of APC-mutated colon cancer cells through promotion of β-catenin degradation. Food Chem Toxicol. 67:87–95. 2014. View Article : Google Scholar : PubMed/NCBI

25 

Padhye S, Dandawate P, Yusufi M, Ahmad A and Sarkar FH: Perspectives on medicinal properties of plumbagin and its analogs. Med Res Rev. 32:1131–1158. 2012. View Article : Google Scholar : PubMed/NCBI

26 

Xu TP, Shen H, Liu LX and Shu YQ: Plumbagin from Plumbago zeylanica L induces apoptosis in human non-small cell lung cancer cell lines through NF-kB Inactivation. Asian Pac J Cancer Prev. 14:2325–2331. 2013. View Article : Google Scholar : PubMed/NCBI

27 

Sinha S, Pal K, Elkhanany A, Dutta S, Cao Y, Mondal G, Iyer S, Somasundaram V, Couch FJ, Shridhar V, et al: Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo. Int J Cancer. 132:1201–1212. 2013. View Article : Google Scholar : PubMed/NCBI

28 

Hafeez BB, Zhong W, Fischer JW, Mustafa A, Shi X, Meske L, Hong H, Cai W, Havighurst T, Kim K and Verma AK: Plumbagin, a medicinal plant (Plumbago zeylanica)-derived 1,4-naphthoquinone, inhibits growth and metastasis of human prostate cancer PC-3M-luciferase cells in an orthotopic xenograft mouse model. Mol Oncol. 7:428–439. 2013. View Article : Google Scholar : PubMed/NCBI

29 

Ahmad A, Banerjee S, Wang Z, Kong D and Sarkar FH: Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2. J Cell Biochem. 105:1461–1471. 2008. View Article : Google Scholar : PubMed/NCBI

30 

Hsu YL, Cho CY, Kuo PL, Huang YT and Lin CC: Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo. J Pharmacol Exp Ther. 318:484–494. 2006. View Article : Google Scholar : PubMed/NCBI

31 

Hafeez BB, Zhong W, Mustafa A, Fischer JW, Witkowsky O and Verma AK: Plumbagin inhibits prostate cancer development in TRAMP mice via targeting PKCε, Stat3 and neuroendocrine markers. Carcinogenesis. 33:2586–2592. 2012. View Article : Google Scholar : PubMed/NCBI

32 

Kuo PL, Hsu YL and Cho CY: Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther. 5:3209–3221. 2006. View Article : Google Scholar : PubMed/NCBI

33 

Gomathinayagam R, Sowmyalakshmi S, Mardhatillah F, Kumar R, Akbarsha MA and Damodaran C: Anticancer mechanism of plumbagin, a natural compound, on non-small cell lung cancer cells. Anticancer Res. 28:785–792. 2008.PubMed/NCBI

34 

Ravindra KC, Selvi BR, Arif M, Reddy BA, Thanuja GR, Agrawal S, Pradhan SK, Nagashayana N, Dasgupta D and Kundu TK: Inhibition of lysine acetyltransferase KAT3B/p300 activity by a naturally occurring hydroxynaphthoquinone, plumbagin. J Biol Chem. 284:24453–24464. 2009. View Article : Google Scholar : PubMed/NCBI

35 

Khanna C, Prehn J, Yeung C, Caylor J, Tsokos M and Helman L: An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential. Clin Exp Metastasis. 18:261–271. 2000. View Article : Google Scholar : PubMed/NCBI

36 

Davies EM: Protein assays: A review of common techniques. Am Biotech Lab. 6:28–37. 1988.

37 

Legler G, Müller-Platz CM, Mentges-Hettkamp M, Pflieger G and Julich E: On the chemical basis of the Lowry protein determination. Anal Biochem. 150:278–287. 1985. View Article : Google Scholar : PubMed/NCBI

38 

Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein measurement with the Folin Phenol Reagent. J Biol Chem. 193:267–275. 1951.

39 

Jaffe N: Osteosarcoma: Review of the past, impact on the future. The American experience. Cancer Treat Res. 152:239–262. 2009. View Article : Google Scholar : PubMed/NCBI

40 

Martin BT, Kleiber K, Wixler V, Raab M, Zimmer B, Kaufmann M and Strebhardt K: FHL2 regulates cell cycle-dependent and doxorubicin-induced p21Cip1/Waf1 expression in breast cancer cells. Cell Cycle. 6:1779–1788. 2007. View Article : Google Scholar : PubMed/NCBI

41 

Li M, Wang J, Ng SS, Chan CY, Chen AC, Xia HP, Yew DT, Wong BC, Chen Z, Kung HF and Lin MC: The four-and-a-half-LIM protein 2 (FHL2) is overexpressed in gliomas and associated with oncogenic activities. Glia. 56:1328–1338. 2008. View Article : Google Scholar : PubMed/NCBI

42 

Tanahashi H and Tabira T: Alzheimer's disease-associated presenilin 2 interacts with DRAL, an LIM-domain protein. Hum Mol Genet. 9:2281–2289. 2000. View Article : Google Scholar : PubMed/NCBI

43 

Muller JM, Isele U, Metzger E, Rempel A, Moser M, Pscherer A, Breyer T, Holubarsch C, Buettner R and Schüle R: FHL2, a novel tissue-specific coactivator of the androgen receptor. EMBO J. 19:359–369. 2000. View Article : Google Scholar : PubMed/NCBI

44 

Fimia GM, De Cesare D and Sassone-Corsi P: A family of LIM-only transcriptional coactivators: Tissue-specific expression and selective activation of CREB and CREM. Mol Cell Biol. 20:8613–8622. 2000. View Article : Google Scholar : PubMed/NCBI

45 

Morlon A and Sassone-Corsi P: The LIM-only protein FHL2 is a serum-inducible transcriptional coactivator of AP-1. Proc Natl Acad Sci USA. 100:3977–3982. 2003. View Article : Google Scholar : PubMed/NCBI

46 

Wei Y, Renard CA, Labalette C, Wu Y, Lévy L, Neuveut C, Prieur X, Flajolet M, Prigent S and Buendia MA: Identification of the LIM protein FHL2 as a coactivator of beta-catenin. J Biol Chem. 278:5188–5194. 2003. View Article : Google Scholar : PubMed/NCBI

47 

Labalette C, Renard CA, Neuveut C, Buendia MA and Wei Y: Interaction and functional cooperation between the LIM protein FHL2, CBP/p300 and beta-catenin. Mol Cell Biol. 24:10689–10702. 2004. View Article : Google Scholar : PubMed/NCBI

48 

Yang Y, Hou H, Haller EM, Nicosia SV and Bai W: Suppression of FOXO1 activity by FHL2 through SIRT1-mediated deacetylation. EMBO J. 24:1021–1032. 2005. View Article : Google Scholar : PubMed/NCBI

49 

Hamidouche Z, Haÿ E, Vaudin P, Charbord P, Schüle R, Marie PJ and Fromigué O: FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/beta-catenin signaling-dependent Runx2 expression. FASEB J. 22:3813–3822. 2008. View Article : Google Scholar : PubMed/NCBI

50 

Entz-Werle N, Lavaux T, Metzger N, Stoetzel C, Lasthaus C, Marec P, Kalifa C, Brugieres L, Pacquement H, Schmitt C, et al: Involvement of MET/TWIST/APC combination or the potential role of ossification factors in pediatric high-grade osteosarcoma oncogenesis. Neoplasia. 9:678–688. 2007. View Article : Google Scholar : PubMed/NCBI

51 

Nusse R: Wnt signaling in disease and in development. Cell Res. 15:28–32. 2005. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

August-2016
Volume 12 Issue 2

Print ISSN: 1792-1074
Online ISSN:1792-1082

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Xue YL, Meng XQ, Ma LJ and Yuan Z: Plumbagin exhibits an anti‑proliferative effect in human osteosarcoma cells by downregulating FHL2 and interfering with Wnt/β‑catenin signalling. Oncol Lett 12: 1095-1100, 2016
APA
Xue, Y., Meng, X., Ma, L., & Yuan, Z. (2016). Plumbagin exhibits an anti‑proliferative effect in human osteosarcoma cells by downregulating FHL2 and interfering with Wnt/β‑catenin signalling. Oncology Letters, 12, 1095-1100. https://doi.org/10.3892/ol.2016.4725
MLA
Xue, Y., Meng, X., Ma, L., Yuan, Z."Plumbagin exhibits an anti‑proliferative effect in human osteosarcoma cells by downregulating FHL2 and interfering with Wnt/β‑catenin signalling". Oncology Letters 12.2 (2016): 1095-1100.
Chicago
Xue, Y., Meng, X., Ma, L., Yuan, Z."Plumbagin exhibits an anti‑proliferative effect in human osteosarcoma cells by downregulating FHL2 and interfering with Wnt/β‑catenin signalling". Oncology Letters 12, no. 2 (2016): 1095-1100. https://doi.org/10.3892/ol.2016.4725