Viability Of Osteosarcoma Cells Research Articles

Deoxyelephantopin Induces Reactive Oxygen Species-Mediated Apoptosis and Autophagy in Human Osteosarcoma Cells

Background/Aims: Osteosarcoma is the predominant form of primary bone malignancy. Although the combinational application of neoadjuvant chemotherapy and surgical resection significantly increases the survival rate, the therapeutic outcome remains unsatisfactory. Deoxyelephantopin (DET), an active ingredient of Elephantopus scaber, has been reported to have an anti-tumor effect in recent publications. This study aimed to investigate whether DET has antineoplastic effects on osteosarcoma cells and its underlying mechanism. Methods: Cell viability and morphological changes were assessed by MTT and Live/dead assays. Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were detected utilizing Annexin V-FITC/PI double staining, DCFH-DA and JC-1 probes, respectively. Autophagy was detected by mRFP-GFP-LC3 adenovirus transfection and western blot. Results: DET dose-dependently reduced the viability of osteosarcoma cells following the increase in intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) reversed this effect. Furthermore, DET induced mitochondrial apoptosis. Depolarized cells were increased, and apoptosis-related proteins, such as Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3 and cleaved ploy ADP-ribose polymerase, were activated. Additionally, we found that DET could induce autophagy in osteosarcoma cells, but autophagy inhibition did not affect the decrease in cell viability. Conclusion: DET induced apoptosis in osteosarcoma cells through ROS generation, mitochondrial dysfunction and caspase activation; in addition, autophagy was involved in the effects of DET on osteosarcoma cells.

Ferulic acid promoting apoptosis in human osteosarcoma cell lines.

Objective:To explore the promoting apoptosis and antitumor activities of ferulic acid (FA) in human osteosarcoma and its potential mechanism.Methods:The SaOS-2 and MG63 osteosarcoma cell lines were opted to experiment and these cells were, respectively, cultured with various concentrations of FA (0 μM, 10 μM, 20 μM, 40 μM) for 72 hours at 37°C. The viabilities of the FA treated cells were monitored by MTT. Apoptosis cells were evaluated using annexin V/PI by flow cytometry. Apoptosis proteins caspase-3, procaspase-3, Bcl-2 and Bax were detected by western blot. Expressions of apoptotic genes Bcl-2 and Bax were quantified by qPCR.Results:The cell viabilities were critically declined in the concentration-dependent manner in FA groups (P < 0.01). The apoptosis cells were increased proportionately with the concentration of FA (P < 0.05). The procaspase-3 protein contents, and Bcl-2 mRNA and protein contents were significantly decreased while caspase-3 protein contents, and Bax mRNA and protein contents were concomitantly increased in the concentration-dependent manner in FA groups (P < 0.05). The response to FA by the SaOS-2 osteosarcoma cell was similar with the MG63 osteosarcoma cell (P > 0.05).Conclusion:Ferulic acid could significantly descend osteosarcoma cell viability through the promoting apoptosis pathway in which FA activates both caspase-3 and Bax and inactivates Bcl-2.

Silencing LPAATβ inhibits tumor growth of cisplatin-resistant human osteosarcoma in vivo and in vitro.

Cisplatin-resistance has become a major impediment in the medical treatment of cancers such as osteosarcoma, the most common primary malignancy of bone. Since lysophosphatidic acid acyltransferase β (LPAATβ) was reported to be critically involved in osteosarcoma, our study investigated the role of LPAATβ in human osteosarcoma with cisplatin-resistance. Expression of LPAATβ or other relevant proteins were analyzed in 40 osteosarcoma patients by immunohistochemistry analysis (IHC), and in cisplatin‑resistant sublines by real-time PCR and western blotting. Next, the synthesized siRNA was inserted into the lentivirus vector and silencing of LPAATβ expression was employed to determine the effect of LPAATβ on cisplatin-resistant osteosarcoma cell viability invitro and osteosarcoma tumor growth in vivo with cisplatin treatment. Exogenous LPAATβ mediated by heritable RNAi decreased cisplatin‑resistant sensitivity through activating the PI3K/Akt/mTOR signaling pathway. We further demonstrate that silencing LPAATβ effectively inhibited tumor growth in nude mice with xenografts of cisplatin‑resistant osteosarcoma cells. IHC assay results showed that PI3K/Akt/mTOR signaling pathway was also involved in this process. Our results suggested that LPAATβ may play an important role in osteosarcoma and silencing LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of cisplatin-resistance.

Zebularine enhances apoptosis of human osteosarcoma cells by suppressing methylation of ARHI.

ARHI is an imprinted tumor suppressor gene and its methylation suppresses ARHI transcription levels to cause the development and progression of malignant tumors. Zebularine exerts a demethylation function for tumor suppressor genes. Our study aims to investigate the effect and mechanism of action of zebularine on the epigenetic modification of the ARHI gene, and whether this effect may modulate the viability and apoptosis of human osteosarcoma cells. We found that zebularine inhibited the viability and promoted apoptosis in osteosarcoma cells. Zebularine potentiated the expression of ARHI at both the protein and mRNA level. This was related to the downregulation of methylation of ARHI caused by zebularine. Zebularine suppressed the interaction of DNA methyltransferase 1 (DNMT1) with histone methyltransferase G9a, but had no effect on G9a alone. Knockdown of DNMT1 or G9a can induce a reduction of ARHI methylation. Therefore, we inferred that zebularine was likely to directly repress DNMT1 alone, but G9a was necessary to regulate the function of DNMT1 on ARHI methylation. Moreover, knockdown of ARHI rescued cell viability and apoptosis under the zebularine‐treated condition. We showed that zebularine inhibited viability and promoted apoptosis by disturbing the interaction between DNMT1 and G9a, thereby resulting in lower ARHI methylation and elevated ARHI expression in osteosarcoma cells.

MicroRNA-212 suppresses the proliferation and migration of osteosarcoma cells by targeting forkhead box protein A1.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as critical gene regulators by targeting the 3' untranslated region (UTR) of mRNA, causing translational repression or mRNA degradation. Deregulation of specific miRNAs, including miR-212, has been identified in patients with osteosarcoma. However, the underlying mechanism is yet to be fully elucidated. The present study aimed to reveal the regulatory mechanism of miR-212 in osteosarcoma cell viability and migration. Quantitative polymerase chain reaction data revealed that miR-212 was significantly downregulated in osteosarcoma tissues compared with normal bone tissues. miR-212 was also downregulated in osteosarcoma cell lines compared with normal osteoblast cell lines. Overexpression of miR-212 significantly suppressed the viability and migration of human osteosarcoma MG-63 and Saos-2 cell lines. In addition, forkhead box protein A1 (FOXA1), an oncogene in osteosarcoma, was predicted to be a putative target of miR-212 by bioinformatical analysis. Furthermore, luciferase reporter assay data confirmed that miR-212 could directly bind to the seed sequences within the 3'UTR of FOXA1 mRNA, and miR-212 negatively mediated the protein levels of FOXA1 in osteosarcoma MG-63 and Saos-2 cells. Moreover, knockdown of FOXA1 also led to a significant decrease in the viability and migration of osteosarcoma MG-63 and Saos-2 cells and the expression levels of FOXA1 were significantly upregulated in osteosarcoma tissues and cell lines. These data suggest that miR-212 inhibits the viability and migration of osteosarcoma cells by targeting FOXA1. Accordingly, miR-212 may become a potential candidate for osteosarcoma therapy.

APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH–proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21Waf1, and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

Paris saponin VII suppresses osteosarcoma cell migration and invasion by inhibiting MMP‑2/9 production via the p38 MAPK signaling pathway.

Metastasis is the primary cause of mortality in osteosarcoma. Targeting metastasis is a major strategy in osteosarcoma treatment. As a traditional Chinese medicine, Trillium tschonoskii Maxim has been widely used in the therapy of various diseases, including cancer. However, currently there is no evidence regarding the anti-metastasic effect of Paris saponin VII (PS VII), which is extracted from Trillium tschonoskii Maxim, on osteosarcoma cells and its underling mechanisms. The present study aimed to examine the effect of PS VII on the migration and invasion of osteosarcoma cells. Viability and proliferation of osteosarcoma cells were examined by MTT assay. Migration and invasion of osteosarcoma cells was then detected using scratch wound healing assays and Transwell assays, respectively. Additionally, the expression of matrix metalloproteinase (MMP)-2 and -9 was determined at the mRNA and protein level following treatment with PS VII. Mitogen-activated protein kinase (MAPK) expression was also detected by western blot analysis. Finally, an inhibitor of p38 MAPK was used to verify the effect of PS VII on the expression of MMP-2 and -9, as well as the migration and invasion osteosarcoma cells. This demonstrated that the proliferation, migration and invasion of the osteosarcoma cells were suppressed following treatment with PS VII. PS VII downregulated the expression of MMP-2 and -9 in a dose- and time-dependent manner. PS VII also exerted its ability to downregulate the phosphorylation of p38 MAPKs. Furthermore, by using a p38 inhibitor, SB203580, the role of PS VII in MMP-2 and -9 expression and osteosarcoma cell invasion was revealed. Taken together, these results demonstrated that PS VII suppresses the migration and invasion of osteosarcoma cells via the p38 MAPK signaling pathway.

Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase

Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1-2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability.

A single nucleotide polymorphism in the 3'-untranslated region of the KRAS gene disrupts the interaction with let-7a and enhances the metastatic potential of osteosarcoma cells.

The objective of the present study was to explore the molecular mechanism with which a single nucleotide polymorphism (rs61764370) interferes with the interaction between the 3'-untranslated region(3'-UTR) of Kirsten rat sarcoma viral oncogene homolog(KRAS) and let-7a, and its association with the metastasis of osteosarcoma(OS). In this study, we confirmed that KRAS is a target of let-7a in OS cells, and the introduction of rs61764370 minor allele into KRAS 3'-UTR significantly compromised the microRNA(miRNA)/mRNA interaction using a luciferase reporter system. Additionally, a total of 36OS tissue samples of three different genotypes (TT,22; TG,10; GG,4) were obtained, and the expression of let-7a and KRAS was determined. We showed that let-7a mRNA expression was similar between each group whereas the mRNA and protein expression of KRAS in the TT genotype group was significantly lower than that in the GT or GG genotype groups. Moreover, we identified a negative regulatory relationship between let-7a and KRAS. Furthermore, we demonstrated that let-7a and KRAS interfered with the viability, invasiveness and migration of OS cells genotyped as TT. In the OScells genotyped as TG, let-7a exerted minimal effects, and the effect of KRAS siRNA remained. Taken together, the findings of the present study demonstrated that the KRAS3'-UTRrs61764370 polymorphism interfered with miRNA/mRNA interaction, and showed that the minor allele was associated with an elevated risk of developing metastatic disease in OS.

Ablation of MCL1 expression by virally induced microRNA-29 reverses chemoresistance in human osteosarcomas.

Osteosarcoma is a rare disease diagnosed as malignant bone tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The reversal of chemoresistance is a major clinical challenge to improve the prognostic outcome of osteosarcoma patients. We developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301 (telomelysin) and assessed its synergistic effects with chemotherapeutic agents (cisplatin and doxorubicin) using human osteosarcoma cell lines and a xenograft tumor model. The molecular mechanism underlying the chemosensitizing effect of OBP-301 was evaluated in aspects of apoptosis induction. OBP-301 inhibits anti-apoptotic myeloid cell leukemia 1 (MCL1) expression, which in turn leads to chemosensitization in human osteosarcoma cells. The siRNA-mediated knockdown of MCL1 expression sensitized human osteosarcoma cells to common chemotherapeutic agents. We also found that upregulation of microRNA-29 targeting MCL1 via virally induced transcriptional factor E2F-1 activation was critical for the enhancement of chemotherapy-induced apoptosis in osteosarcoma cells. Telomerase-specific oncolytic adenovirus synergistically suppressed the viability of human osteosarcoma cells in combination with chemotherapeutic agents. The combination treatment also significantly inhibited tumor growth, as compared to monotherapy, in an osteosarcoma xenograft tumor model. Our data suggest that replicative virus-mediated tumor-specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients.

In Situ Spectroscopic Screening of Osteosarcoma Living Cells on Stoichiometry-Modulated Silicon Nitride Bioceramic Surfaces

Osteosarcoma cell viability, proliferation, and differentiation into osteoblasts on a silicon nitride bioceramic were examined as a function of chemical modifications of its as-fired surface. Biological and spectroscopic analyses showed that (i) postsintering annealing in N2 gas significantly improved apatite formation from human osteosarcoma (SaOS-2) cells; (ii) in situ Raman spectroscopic monitoring revealed new metabolic details of the SaOS-2 cells, including fine differences in intracellular RNA and membrane phospholipids; and (iii) the enhanced apatite formation originated from a high density of positively charged surface groups, including both nitrogen vacancies (VN3+) and nitrogen N-N bonds (N4+) formed during annealing in N2 gas. At homeostatic pH, these positive surface charges promoted binding of proteins onto an otherwise negatively charged surface of deprotonated silanols (SiO-). A dipole-like electric-charge, which includes VN3+/N4+ and SiO- defective sites, is proposed as a mechanism to explain the attractive forces between transmembrane proteins and the COO- and NH2+ termini, respectively. This is analogous to the mechanism occurring in mineral hydroxyapatite where protein groups are specifically displaced by the presence of positively charged calcium loci (Ca+) and off-stoichiometry phosphorus sites (PO42-).

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53.

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.

Nitidine chloride suppresses epithelial-to-mesenchymal transition in osteosarcoma cell migration and invasion through Akt/GSK-3β/Snail signaling pathway.

Metastasis is the main cause of death in osteosarcoma. Targeting the process of metastasis is a main strategy for osteosarcoma therapy. As a traditional Chinese medicine, Zanthoxylum nitidum (Roxb) has been applied to treat various diseases, including cancer. However, no evidence has been shown on the anti-metastasis effect of nitidine chloride (NC) that was extracted from Zanthoxylum nitidum (Roxb) on osteosarcoma cells, or its underling mechanisms. In the present study, we aimed to demonstrate the role of NC on the migration and invasion of osteosarcoma cells. Viability and proliferation of osteosarcoma cells were examined by MTT assay. Then, by appling scratch wound healing assay and Transwell assays, we evaluated migratory and invasive ability of the cells, respectively. Moreover, the expression of epithelial-to-mesenchymal transition (EMT) markers were determined after treatment with NC. Furthermore, the expression of Akt, GSK-3β and Snail were detected by western blot analysis. In addition, the GSK-3β activity was examined by GSK-3β kinase assay. Finally, an inhibitor of GSK-3β, lithium chloride (LiCl) was applied to testify the effect of NC on the expression of EMT markers and Snail. We found that the proliferative, migratory and invasive ability of the U2OS osteosarcoma cells were all suppressed when treated with NC. NC increased the expression of E-cadherin and decreased the expression of N-cadherin, vimentin and fibronectin in a dose-dependent manner. NC also exerted its ability to suppress the phosphorylation of Akt and GSK-3β so as to activate GSK-3β. Then, by using an GSK-3β inhibitor, LiCl, we revealed the effect of GSK-3β in the expression of EMT markers. The expression of Snail was inhibited when treated with NC and LiCl also reversed the NC-inhibited Snail expression. Taken together, these results revealed that NC suppressed EMT and decreased the invasive ability of osteosarcoma cells via the Akt/GSK-3β/snail signaling pathway.

Fuse-binding protein 1 is a target of the EZH2 inhibitor GSK343, in osteosarcoma cells.

Osteosarcoma is the primary cancer of leaf tissue and is regarded as a differentiation disease caused by genetic and epigenetic changes which interrupt the osteoblast differentiation from mesenchymal stem cells. Because of its high malignancy degree and rapid development, the morbidity and mortality are high. The enhancer of zeste homolog 2 (EZH2) is a catalytic subunit of polycomb repressive complex2(PRC2) and has been demonstrated to be involved in a variety of biological processes, such as cell proliferation and program cell death. EZH2 impairs gene expression by catalyzing the tri-methylation of histone H3 lysine 27 (H3K27me3) which controls gene transcription epigenetically. It is reported that EZH2 expression is higher in osteosarcoma than in osteoblastoma and the highest expression of EZH2 is found in osteosarcoma with metastasis. In the past few years, several potent inhibitors of EZH2 have been discovered, and GSK343 is one of them. In this study, we found that GSK343 inhibited osteosarcoma cell viability, restrained cell cycle transition and promoted programmed cell death. GSK343 not only inhibited the expression of EZH2 and its target, c-Myc and H3K27me3, but it also inhibited fuse binding protein 1 (FBP1) expression, another c-Myc regulator. Furthermore, we found that FBP1 physically interacts with EZH2. Based on these results, we believe that GSK343 is a potential molecule for osteosarcoma clinical treatment. Other than the inhibition on EZH2-c-Myc signal pathway, we postulate that the inhibition on FBP1-c-Myc signal pathway is another potential underlying mechanism with which GSK343 inhibits osteosarcoma cell viability.

Actein Inhibits Cell Proliferation and Migration in Human Osteosarcoma.

BackgroundOsteosarcoma is one of the most common malignant bone cancers worldwide. Although the traditional chemotherapies have made some progression in the past decades, the mortality of osteosarcoma in children and adolescent is very high. Herein, the role of actein in osteosarcoma was explored.Material/MethodsCell viability assay was performed in osteosarcoma cell lines 143B and U2OS. Colony formation analysis was included when cells were treated with different doses of actin. Cell cycle assay was conducted to further examine the role of actein. Cell apoptotic rate and the relative activities of caspase-3, caspase-8, and caspase-9 were detected in 143B and U2OS osteosarcoma cells. Moreover, transwell assays were used to explore the effects of actein on cell metastasis.ResultsActein significantly inhibited osteosarcoma cell viability in a time- and dose-dependent manner. Actein also dramatically suppressed the colony formation ability in osteosarcoma143B and U2OS cells. It was revealed that osteosarcoma cells were arrested in G0/G1 phase in the cell cycle progression and induced to apoptosis by administration of actein. The activities of pro-apoptotic factors such as caspase-3 and caspase-9 were significantly increased by actein. Furthermore, administration of actein decreased cell migrated and invasive abilities in both 143B and U2OS cell lines.ConclusionsActein inhibits tumor growth by inducing cell apoptosis in osteosarcoma. The inhibitive roles of actein in cell proliferation, migration and invasion suggest that actein may serve as a potential therapeutic agent in the treatment of osteosarcoma.

Triptolide reduces the viability of osteosarcoma cells by reducing MKP-1 and Hsp70 expression.

Osteosarcoma is the most common type of malignant bone tumor found in adolescents and young adults. The aim of the present study was to determine whether triptolide, a diterpene epoxide extracted from the Tripterygium plant, was able effectively decrease the viability of osteosarcoma cells. The underlying molecular mechanisms are also investigated. The human osteosarcoma cell lines U-2 OS and MG-63 were used in this study. The U-2 OS and MG-63 cells were treated with 0, 5, 10, 25 or 50 nM triptolide. Cells treated with dimethyl sulfoxide only were used as the no drug treatment control. A commercial MTT kit was used to determine the effects of triptolide on cells. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is frequently overexpressed in tumor tissues, possibly related to the failure of a number of chemotherapeutics. Heat shock protein 70 (Hsp70) is a chaperone molecule that is able to increase drug resistance. The protein expression levels of MKP-1 and Hsp70 were determined using western blot analysis. The results indicate that triptolide effectively reduced the viability of the osteosarcoma cells. Furthermore, triptolide was found to effectively reduce MKP-1 expression and Hsp70 levels. Further analysis showed that triptolide reduced MKP-1 mRNA expression in the U-2 OS and MG-63 cells. Triptolide reduced Hsp70 mRNA expression levels in U-2 OS and MG-63 cells. These results suggest that triptolide effectively decreases the viability of osteosarcoma cells. These effects may be associated with the decreased expression of MKP-1 and Hsp70 levels. These results suggest that triptolide may be used in the treatments of osteosarcoma.

Abstract B11: Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling

Abstract Purpose: The objective of this study is to characterize the function of porcupine inhibitors in osteosarcoma and to determine the contribution of the porcupine enzyme to osteosarcoma cell growth. Each year, approximately 1000 children and adults in the United States are diagnosed with osteosarcoma (OS), the most common primary bone malignancy among children. Although overall survival is relatively good in OS patients, the need for aggressive surgery (often involving limb amputation) to prevent relapse significantly impacts the quality of life of survivors. OS has been well characterized to display aberrantly activated Wnt signaling. The necessity for active Wnt signaling pathways in OS patient tissue and cultured cell lines has been well established. Recently, a group of Wnt pathway inhibitors targeting the porcupine enzyme have been developed and used in treatment of pancreatic adenocarcinoma, BRAF mutant colorectal cancer and other tumors reported to exhibit aberrant Wnt signaling. Porcupine is a membrane bound O-acyl transferase which lipid-modifies the Wnt proteins prior to their secretion. This processing step regulates their release and paracrine or autocrine activity in stimulating both canonical and non-canonical Wnt signaling. These drugs have proven effective in initial studies which have applied them in vitro, in mouse models of Wnt- dependent cancers, and a recent phase 1 clinical trial. Our data show the first report of increased porcupine enzyme expression in human OS in vitro cell lines, compared to primary human osteoblasts. These findings have been confirmed by quantitative real-time PCR (qRT-PCR) analysis of PPN mRNA expression. Surprisingly, we found that inhibition of the porcupine enzyme with newly developed porcupine inhibitors significantly increased OS cell viability. While we observed only slight decreases in canonical Wnt pathway signaling with PPN inhibitor treatment (as measured by TOPflash reporter assay), we found that both NFATc1 and Cyclin D1 mRNA expression was upregulated. These data indicate that treatment with PPN inhibitors may stimulate a pro-tumorigenic response that is mediated primarily through non-canonical Wnt signaling. Continuing work is being done to establish the mechanism of action of these drugs, as well as unique roles that PPN-mediated non-canonical Wnt signaling may play in promoting OS cell growth. In conclusion, this study is aimed to provide insight into the function of a group of novel Wnt-targeting therapies in osteosarcoma, a disease which is well established to exhibit aberrant Wnt signaling. We aim to utilize these insights to rationally exploit Wnt signaling as a therapeutic measure for osteosarcoma. Methods: Western blotting was performed according to accepted methods using BioRad Criterion™ 10% gel and an antibody polyclonal to known porcupine splice variants (anti-rabbit) obtained from GeneTex. Cell viability was determined by CellTiter 96 ® (Promega) proliferation assay. C59 (Novartis) was purchased from Cayman Chemical and reconstituted in DMSO. Quantitative PCR performed using an Applied Biosystems ABI 7900HT Fast Real Time PCR system. FuGENE® HD utilized for transfection of TOPflash components. Citation Format: Carl T. Gustafson, Avudai Maran, Michael J. Yaszemski. Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B11.

(Z)-3,4,3',5'-tetramethoxystilbene, a natural product, induces apoptosis and reduces viability of paclitaxel-and cisplatin-resistant osteosarcoma cells.

Osteosarcoma is a common bone tumor and the development of drug resistance in therapy of osteosarcoma is a general rule. The natural compounds isolated from medicinal plants represent a valuable resource for anticancer therapeutics. (Z)-3,4,3', '-tetramethoxystilbene is one of them. In this work, we investigated the potential anti-cancer activities of (Z)-3,4,3' ,'5-tetramethoxystilbene in paclitaxel- and cisplatin-resistant osteosarcoma cells. ATP assay was used to examine cell viability. Cell nuclei staining assays with Hoechst or propidium iodide (PI) were used to evaluate cell apoptosis. Xenograft tumor model was used to evaluate the in vivo anti-cancer activities of (Z )-3,4,3',5'-tetramethoxystilbene. TUNEL staining assay was used to evaluate the apoptosis of tumor cells. We found that (Z)-3,4,3',5'-tetramethoxystilbene could effectively reduce viability of both paclitaxel-and cisplatin-resistant osteosarcoma cells. Moreover, (Z)-3,4,3',5'-tetramethoxystilbene induced dramatic apoptosis in resistant cells. Importantly, (Z)-3,4,3',5'-tetramethoxystilbene significantly suppressed in vivo tumor growth of cisplatin-resistant osteosarcoma. This is the first report on anti-cancer activity of (Z)-3,4,3','5- tetramethoxystilbene in resistant osteosarcoma cells. Our studies suggest that (Z)-3,4,3',5'-tetramethoxystilbene is a promising therapeutic drug for overcoming drug resistance in osteosarcoma.

Correlation between TRAIL and caspase-8 expression and their relationship with cell proliferation and apoptosis in human osteosarcoma.

Osteosarcoma is a common malignant bone tumor that mainly affects children and adolescents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily. Caspase-8 appears in the upstream of apoptosis signaling pathway among caspases. We investigated TRAIL and caspase-8 levels in osteosarcoma patients to determine their correlation with cell proliferation and apoptosis. Osteosarcoma and osteochondroma patients receiving surgery in our hospital were selected. TRAIL and caspase-8 expression levels in tissue were determined by immunohistochemistry, and protein levels in cells were evaluated by western blotting. Human osteosarcoma cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The osteosarcoma and osteochondroma cell cycles and apoptosis were investigated by flow cytometry. Correlation analysis was applied to TRAIL and caspase-8 levels during cell apoptosis. Positive TRAIL and caspase-8 expression rates in osteosarcoma tissue were significantly lower than in the controls (P < 0.05). TRAIL (0.114 ± 0.002) and caspase-8 (0.352 ± 0.124) levels in experimental cells were obviously lower than in the controls (P < 0.05). Osteosarcoma cells in the experimental group demonstrated higher proliferation and lower apoptosis at 24, 48, and 72 h (P < 0.05). The experimental cell number increased in the G1 stage and decreased in the S stage (P < 0.05). TRAIL and caspase-8 proteins showed positive correlation with apoptosis in osteosarcoma (P < 0.05). Human osteosarcoma presented reduced TRAIL and caspase-8 levels with enhanced cell proliferation and reduced apoptosis. TRAIL and caspase-8 expression levels were positively correlated with apoptosis in osteosarcoma.

Aspirin Suppresses the Growth and Metastasis of Osteosarcoma through the NF-κB Pathway.

Aspirin has recently been reported to reduce both the incidence and the risk of metastasis in colon cancer. However, there is no evidence at the cellular levels or in the animal models for such an effect of aspirin on cancer metastasis. MTT assay, colony formation assay, and apoptosis assay were employed to analyze the effects of aspirin on the osteosarcoma cell viability in vitro. The NF-κB activity was measured by the NF-κB p65 luciferase reporter. Western blotting was used to analyze the proteins in cells. The migration and invasion abilities of osteosarcoma cells in vitro were measured by the Transwell assay. Xenograft-bearing mice were used to assess the roles of aspirin in both tumor growth and metastasis of osteosarcoma in vivo (n = 5-8 mice/group). An unpaired Student t test or ANOVA with the Bonferroni post hoc test were used for the statistical comparisons. Aspirin reduced cell viability in a dose- and time-dependent manner in osteosarcoma cell lines, and aspirin synergistically sensitized osteosarcoma cells to cisplatin (DDP) in vitro and in vivo (P < 0.001). Moreover, aspirin markedly repressed the migration and invasion of osteosarcoma cells in vitro (P < 0.001), and dramatically diminished the occurrence of osteosarcoma xenograft metastases to the lungs in vivo (P < 0.001). Mechanistically, aspirin diminishes osteosarcoma migration, invasion, and metastasis through the NF-κB pathway. Aspirin suppresses both the growth and metastasis of osteosarcoma through the NF-κB pathway at the cellular level and in the animal models.