Treatment Of Human Osteosarcoma Research Articles

Long non‑coding RNA GHET1 promotes osteosarcoma development and progression via Wnt/β‑catenin signaling pathway.

Osteosarcoma (OS) is known as a malignant tumor with a high mortality rate of children and adults worldwide. Long non‑coding RNAs (lncRNAs) have been revealed as oncogenes or tumor suppressors that are involved in the tumorigenesis and metastasis of some types of cancer. However, the biological role of long non‑coding RNA gastric carcinoma proliferation enhancing transcript1 (lncGHET1) and its regulatory mechanism in OS progression have not been elucidated. The aim of the present study was to investigate the role of lncGHET1 in OS. The present study explored lncGHET1 expression using a reverse transcription‑quantitative (RT‑q)PCR assay. Furthermore, the Cell Counting Kit‑8 assay, flow cytometry detection, wound healing and transwell invasion assays were performed to evaluate its biological role and the underlying mechanisms invitro. Additionally, the effect of lncGHET1 was evaluated invivo in a xenograft model. lncGHET1 expression was significantly upregulated in OS cell lines compared with in an osteoblastic cell line according to the RT‑qPCR assay. The results of a knockdown functional experiment suggested that inhibition of lncGHET1 attenuated cell proliferation, migration, invasion and epithelial‑to‑mesenchymal transition, and promoted apoptosis, partly through regulating the Wnt/β‑catenin signaling pathway in OS. These findings indicated that lncGHET1 may serve an essential regulatory role in the biological processes of OS. The present study identified a novel therapeutic target for diagnosis and treatment of human OS.

Effect of cisplatin on NOD1 and NOD2 expression in SaOS-2 cell line

Objective To investigate the effects of cisplatin on the expression of nucleotide-binding oligomerization domain-like receptor(NOD) 1 and 2 in human osteosarcoma SaOS-2 cell line, and to explore the mechanism of cisplatin in the treatment of human osteosarcoma. Methods CCK-8 assay, real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) and immumofluorescence methods were used to determine the growth survival rate and expression levels of NOD1 and NOD2 in osteosarcoma SaOS-2 cell line treated with cisplatin (0, 5, 10, 20 μmol/L, named group S0, group S5, group S10, group S20, respectively) for 24, 48, 72 hours. Results After treatment with cisplatin for 48 h or 72 h, the growth survival rates of SaOS-2 cells were significantly decreased in group S5 than those in group S0(65.53% vs.100.00%; 46.43% vs.100.00%, χ2=8.64, 73.97, all P<0.01). Moreover, after treatment with cisplatin for 24 h, 48 h or 72 h, the growth survival rates of SaOS-2 cells were significantly decreased in group S10 or group S20 than those in group S0(80.60% vs.100.00%, 42.94 vs.100.00%, 27.90% vs.100.00%; 62.54% vs.100.00%, 33.09% vs.100.00%, 22.95% vs.100.00%, χ2=20.99, 79.72, 112.50; 45.40, 67.56, 125.20, all P<0.01), and the growth survival rates were significantly lower in group S20 than those in group S5(62.54% vs.93.78%, 33.09% vs.65.53%, 22.95% vs.46.43%, χ2=28.47, 21.78, 11.71, all P<0.01). The expression levels of NOD1 mRNA and NOD2 mRNA in group S5 were significantly increased at 48 h or 72 h than those at 24 h, and were higher than group S0 when treated with 5μmol/L cisplatin[(3.64±0.44) vs.(4.47±1.22) vs.(1.79±0.44) vs (1.00±0.00); (6.88±2.79) vs.(6.86±2.40) vs (2.29±0.70) vs.(1.00±0.00), F=29.12, 24.11, all P<0.01]. And the expression levels of NOD1 protein had an increased tendency after 48 h or 72 h treatment with 5μmol/L cisplatin.Furthermore, the expression level of NOD1 mRNA was positively correlated with NOD2 mRNA(n=36, r=0.92, P<0.01). Conclusion Cisplatin can elevate the function of osteosarcoma cell in a dose- and time-dependent manners, cisplatin may act as a efficient drug to cure osteosarcoma desease, which may be related to NOD1 and NOD2 signal pathway. Key words: Osteosarcoma; Tumor cells, cultured; Cisplatin; Polymerase chain reaction; Nucleotides; Adaptor proteins, signal transducing; Drug fvaluation

Targeting the Wnt/β-catenin pathway in human osteosarcoma cells.

Aberrant activation of Wnt signaling has been implicated in human osteosarcoma, which may provide a genetic vulnerability that can be targeted in osteosarcoma treatment. To test whether Wnt activation is necessary for osteosarcoma growth, colony formation, invasion, and metastasis, we treated human osteosarcoma cells with a small molecule inhibitor of Wnt/β-catenin, PRI-724, which suppresses Wnt/β-catenin-mediated transcription. We found increased protein levels of endogenous active-β-catenin in five human osteosarcoma cell lines. Treatment with PRI-724 was sufficient to inhibit human osteosarcoma 143B and SJSA-1 cell proliferation. Suppressed Wnt signaling was confirmed by decreased protein levels of the Wnt target Cyclin D1. Furthermore, we revealed significant inhibitory effects on cell migration, invasion, and colony formation in the human osteosarcoma cells. Using deposited data from next generation sequencing studies, we analyzed somatic mutations and gene expression of components in the Wnt/β-catenin pathway. We found somatic mutations and upregulated gene expression of many components in the Wnt/ β-catenin pathway, indicating activated Wnt signaling. Taken together, our results illustrate the critical role of Wnt/β-catenin signaling in human osteosarcoma pathogenesis and growth, as well as the therapeutic potential of Wnt inhibitors in the treatment of human osteosarcoma.

HDAC2 depletion promotes osteosarcoma\u2019s stemness both in vitro and in vivo: a study on a putative new target for CSCs directed therapy

BackgroundCancer stem cells (CSCs) play a key role in cancer initiation, progression and chemoresistance. Epigenetic alterations have been identified as prominent factors that contribute to the CSCs phenotype. Here, we investigated the effects of the HDAC inhibitor valproic acid (VPA) and the demethylating agent, 5’azacytidine (DAC) on the stem phenotype of MG63 and Saos2 osteosarcoma cell lines.MethodsSaos2 and MG63 cells were treated with DAC and VPA, alone and in combination. Untreated and treated cells were examined for stemness phenotype by cytometry and real-time PCR. Sarcospheres and colonies formation were also evaluated. Moreover, histone modification and methylation were tested by flow cytomery and western blotting. HDAC2 depleted cells were examined for stemness phenotype and their ability to generate tumors in NOD/SCID IL2R-gamma-0 (NSG) mice. HDAC2 expression on human osteosarcoma tissues was evaluated.ResultsWe found that DAC and VPA induce an increased expression of stem markers including CD133, OCT4, SOX2 and NANOG, and an increased ability in sarcospheres and colonies formation efficiency. Interestingly, we showed that DAC and VPA treatment decreased repressive histone markers, while increased the active ones. These histone modifications were also associated with an increase of acetylation of histones H3, a decrease of DNA global methylation, HDAC2 and DNMT3a. Furthermore, HDAC2 silenced-MG63 and Saos2 cells acquired a stem phenotype, and promoted in vivo tumorigenesis. In human osteosarcoma tissues, HDAC2 was strongly expressed in nucleus.ConclusionsCollectively, our results suggest that VPA and DAC induce an expansion of osteosarcoma CSCs, and we report for the first time that HDAC2 is a key factor regulating both CSCs phenotype and in vivo cancer growth. In conclusion, we have identified HDAC2 as a potential therapeutic target in human osteosarcoma treatment.

MicroRNA-96 acts as a tumor suppressor gene in human osteosarcoma via target regulation of EZRIN

AimsmicroRNA-96 (miR-96) is considered as a tumor suppressor in multiple malignancies. Some studies have indicated that EZRIN could be regulated by the miR-96 in several kinds of cancer cells. However, the function of miR-96 in human osteosarcoma has not been investigated intensively so far. In this study, we mainly explored the relationship between miR-96 and the expression of EZRIN in human osteosarcoma cells. Main methodsThe levels of miR-96 and EZRIN in osteosarcoma tissues and paracancerous tissues of patients were evaluated with qPCR. The targeted regulation between miR-96 and EZRIN was further confirmed with Dual luciferase assay. Overexpression of miR-96 and EZRIN was induced in human osteosarcoma cell line, and the changing of cell viability was analyzed to determine the role of miR-96 in human osteosarcoma in vitro and in vivo. Key findingsOur results showed that the expression level of miR-96 was much lower in human osteosarcoma tissues compared with human paracancerous tissues. Dual luciferase assay indicated that EZRIN as the direct target of miR-96 in osteosarcoma cells. Besides, the up-regulation of miR-96 could inhibit cell proliferation, cell migration and invasion, tumor formation ability, and increase the percentage of cell apoptosis in osteosarcoma cells through inhibiting EZRIN. SignificanceTherefore, our study indicated that miR-96 hold an important role for the development and progress of human osteosarcoma, and this miRNA might become a novel target in the diagnosis and treatment of human osteosarcoma and even other cancers.

Selenocysteine inhibits human osteosarcoma cells growth through triggering mitochondrial dysfunction and ROS-mediated p53 phosphorylation.

Osteosarcoma represents the most common primary malignant bone tumor in children and adolescents, which shows severe resistance toward standard chemotherapy because of high invasive capacity and growing incidence. Selenocysteine (SeC) is a naturally available Se-containing amino acid that displays splendid anticancer activities against several human tumors. However, little information about SeC-induced growth inhibition against human osteosarcoma is available. Herein, the anticancer efficiency and underlying mechanism of SeC against human osteosarcoma were evaluated in vitro and in vivo. The results revealed that SeC significantly inhibited MG-63 human osteosarcoma cells growth in vitro through induction of S-phase arrest and apoptosis, as reflected by the decrease of cyclin A and CDK-2, PARP cleavage, and caspases activation. SeC treatment also resulted in mitochondrial dysfunction through affecting Bcl-2 family expression. Moreover, SeC triggered p53 phosphorylation by inducing reactive oxygen species (ROS) overproduction. ROS inhibition effectively blocked SeC-induced cytotoxicity and p53 phosphorylation. Importantly, MG-63 human osteosarcoma xenograft growth in nude mice was significantly suppressed in vivo through triggering apoptosis and p53 phosphorylation. These results indicated that SeC had the potential to inhibit human osteosarcoma cells growth in vitro and in vivo through triggering mitochondrial dysfunction and ROS-mediated p53 phosphorylation, which validated the potential application of Se-containing compounds in treatment of human osteosarcoma.

Expression and therapeutic implications of cyclin-dependent kinase 4 (CDK4) in osteosarcoma

Overexpression and/or hyperactivation of cyclin-dependent kinase 4 (CDK4) has been found in many types of human cancers, and a CDK4 specific inhibitor, palbociclib, has been recently approved by the FDA for the treatment of breast cancer. However, the expression and the therapeutic potential of CDK4 in osteosarcoma remain unclear. In the present study, CDK4 was found to be highly expressed in human osteosarcoma tissues and cell lines as compared with normal human osteoblasts. Elevated CDK4 expression correlated with metastasis potential and poor prognosis in osteosarcoma patients as determined by immunohistochemical analysis in a human osteosarcoma tissue microarray (TMA). CDK4 inhibition by either palbociclib or specific small interference RNA (siRNA) exhibited dose-dependent inhibition of osteosarcoma cell proliferation and growth, accompanied by suppression of the CDK4/6-cyclinD-Rb signaling pathway. Flow cytometry analysis showed that CDK4 knockdown arrested osteosarcoma cells in the G1 phase of the cell cycle and induced cell apoptosis. Furthermore, inhibition of CDK4 significantly decreased osteosarcoma cell migration in vitro determined by the wound healing assay. These data highlight that CDK4 may be a potential promising therapeutic target in the treatment of human osteosarcoma.

SphK2 over-expression promotes osteosarcoma cell growth.

It is needed to explore novel biological markers for early diagnosis and treatment of human osteosarcoma. Sphingosine kinase 2 (SphK2) expression and potential functions in osteosarcoma were studied. We demonstrate that SphK2 is over-expressed in multiple human osteosarcoma tissues and established human osteosarcoma cell lines. Silence of SphK2 by targeted-shRNAs inhibited osteosarcoma cell growth, and induced cell apoptosis. On the other hand, exogenous over-expression of SphK2 could further promote osteosarcoma cell growth. Notably, microRNA-19a-3p (“miR-19a-3p”) targets the 3′ UTR (untranslated region) of SphK2 mRNA. Remarkably, forced-expression of miR-19a-3p silenced SphK2 and inhibited osteosarcoma cell growth. In vivo, SphK2 silence, by targeted-shRNA or miR-19a-3p, inhibited U2OS tumor growth in nude mice. These results suggest that SphK2 could be a novel and key oncotarget protein for OS cell progression.

Cold Atmospheric Plasma in the Treatment of Osteosarcoma.

Human osteosarcoma (OS) is the most common primary malignant bone tumor occurring most commonly in adolescents and young adults. Major improvements in disease-free survival have been achieved by implementing a combination therapy consisting of radical surgical resection of the tumor and systemic multi-agent chemotherapy. However, long-term survival remains poor, so novel targeted therapies to improve outcomes for patients with osteosarcoma remains an area of active research. This includes immunotherapy, photodynamic therapy, or treatment with nanoparticles. Cold atmospheric plasma (CAP), a highly reactive (partially) ionized physical state, has been shown to inherit a significant anticancer capacity, leading to a new field in medicine called “plasma oncology.” The current article summarizes the potential of CAP in the treatment of human OS and reviews the underlying molecular mode of action.

The anti-tumor effect of pachymic acid on osteosarcoma cells by inducing PTEN and Caspase 3/7-dependent apoptosis.

Pachymic acid (PA) is a lanostane type triterpenoid isolated from Poria cocos, which possesses an anti-tumor effect in breast cancer, prostate cancer, lung cancer, and bladder cancer cells. In this study, we investigated the effect of PA on the growth and apoptosis of human immortalized cell line (HOS) and primary osteosarcoma cells by a Cell Counting Kit-8 (CCK-8) and Annexin V and propidium iodide (PI) staining, respectively. Western blot was used to measure the expression of cleaved Caspase 3, PTEN, and AKT, as well as the AKT phosphorylation. The Caspase 3 activity was determined using the Caspase-3 Colorimetric Assay Kit. From the results, PA significantly reduced cell proliferation in a concentration- and time-dependent manner. PA also induced cell apoptosis in a dose-dependent fashion. PA treatment led to increased Caspase 3 activation and PTEN expression, as well as reduced AKT phosphorylation. Moreover, Ac-DEVD-CHO (a Caspase 3/7 inhibitor) pre-treatment or PTEN knockdown partially blocked the effects of PA on cell proliferation and apoptosis. Caspase 3/7 inhibitor had an additive effect with PTEN knockdown. Collectively, our results suggested that induction of apoptosis by PA was mediated in part by PTEN/AKT signaling and Caspase 3/7 activity. This study provides evidence that PA might be useful in the treatment of human osteosarcoma.

Abstract 3124: Expression and therapeutic implications of cyclin dependent kinase 4 (CDK4) in osteosarcoma

Abstract Overexpression and/or hyperactivation of cyclin-dependent kinase 4 (CDK4) has been found in many types of human cancers, and a CDK4 specific inhibitor, palbociclib, has been recently approved by the FDA for the treatment of breast cancer. However, the expression and the therapeutic potential of CDK4 in osteosarcoma remain unclear. In the present study, CDK4 was found to be highly expressed in human osteosarcoma tissues and cell lines as compared with normal human osteoblasts. Elevated CDK4 expression correlated with metastasis potential and poor prognosis in osteosarcoma patients as determined by immunohistochemical analysis in a human osteosarcoma tissue microarray (TMA). CDK4 inhibition by either palbociclib or specific small interference RNA (siRNA) exhibited dose-dependent inhibition of osteosarcoma cell proliferation and growth, accompanied by suppression of the CDK4/6-cyclinD-Rb signaling pathway. Flow cytometry analysis showed that CDK4 knockdown arrested osteosarcoma cells in the G1 phase of the cell cycle and induced cell apoptosis. Furthermore, inhibition of CDK4 significantly decreased osteosarcoma cell migration in vitro determined by the wound healing assay. These data highlight that CDK4 may be a potential promising therapeutic target in the treatment of human osteosarcoma. Citation Format: Yubing Zhou, Francis Hornicke, Zhenfeng Duan. Expression and therapeutic implications of cyclin dependent kinase 4 (CDK4) in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3124. doi:10.1158/1538-7445.AM2017-3124

MicroRNA-203 inhibits proliferation and invasion, and promotes apoptosis of osteosarcoma cells by targeting Runt-related transcription factor 2

Accumulating evidence indicates that microRNA-203 (miR-203) is abnormally expressed in many human tumor tissues and significantly associated with the occurrence, development and clinical outcomes of human tumors. The aim of this study was to determine the target genes and functional significance of miR-203 in osteosarcoma cells. We found reduced expression of miR-203 in osteosarcoma tissues and cells (MG63 and U2-OS) compared with the adjacent normal tissues and normal osteoblastic cells (hFOB1.19), respectively. In vitro studies further demonstrated that exogenous miR-203 overexpression inhibited osteosarcoma cell proliferation and invasion, and promoted apoptosis. At the molecular level, our results confirmed that apoptosis, cell cycle and invasion-related proteins were regulated by miR-203. Our findings also revealed that Runt-related transcription factor 2 (RUNX2) was directly negatively regulated by miR-203. These results suggested that miR-203 may function as a tumor suppressor and may therefore have therapeutic potential in the treatment of human osteosarcoma.

Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells.

Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF) antibody and ligand cluster of differentiation 80 (CD80) for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA). The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTMNPO CRL-2836) at four different concentrations (0.1, 1.0, 10.0, and 100.0 μg/mL) of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF). Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 μg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 μg/mL of CD80+VEGF and 0.1 μg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 μg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 μg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test) induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum species having spontaneous OSA, such as the dog, and on a human OSA cell line model.

Apigenin inhibits the proliferation and invasion of osteosarcoma cells by suppressing the Wnt/β-catenin signaling pathway.

Osteosarcoma (OS) is the most common type of bone cancer. Even with early diagnosis and aggressive treatment, the prognosis for OS is poor. In the present study, we investigated the proliferation and invasion inhibitory effect of apigenin on human OS cells and the possible molecular mechanisms involved. The cell viability of U2OS and MG63 human OS cell lines was detected by MTT assay. Cell cycle progression and invasion were assessed by flow cytometry and the Matrigel Boyden chamber assay, respectively, and the involvement of molecular mechanisms was examined by western blot analysis. We demonstrated that apigenin inhibited proliferation and reduced invasion in human OS cells, and downregulated the expression of β-catenin in OS cells. Furthermore, the inhibitory effect of apigenin on OS cells was reversed by overexpression of β-catenin, but enhanced by knockdown of β-catenin. Collectively, our results showed that apigenin inhibits the tumor growth of OS cells by inactivating Wnt/β-catenin signaling. Therefore, apigenin is a promising chemotherapeutic agent that may be used in the treatment of human OS.

Abstract 1689: Synergistic effect of JQ1 and rapamycin for treatment of human osteosarcoma

Abstract Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facillitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity againts many hematological and solid cancers, mainly through inhibition of c-myc and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment, due to the frequent copy number gain and overexpression of c-myc in OS cells. JQ1 significantly inhibited the proliferation and survival of seven OS cells (U2OS, G292, MG-63, HT-161, MNNG/HOS, SAOS-2, SJSA), at a relatively higher dose than other cancers (median IC50 = 7.35 μM). JQ1 induced G1 cell cycle arrest and premature senescence of OS cells, but produced little apoptosis. Interestingly, c-myc protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable, as noted by Western blots. Although effective in vitro, JQ1 failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice in vivo. To overcome the resistance of OS cells to JQ1 treatment, we tested JQ1 in combination with rapamycin. Rapamycin inhibits the assembly of mTOR complexes which is one of the important growth signaling pathways in many cancers including OS. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro. The drug combination nearly abrogated expression of c-myc protein, enhanced levels of p21, and induced apoptosis in OS cells in vitro. After 24 days of treatment in vivo, JQ1 (50mg/kg body weight) or rapamycin (1.5 mg/kg body weight) alone showed little effect on the size of human OS xenografts in nude mice. However, combination of both drugs significantly inhibited tumor growth by 85% and 73% (p&amp;lt;0.05 for both) compared to either JQ1 or rapamycin alone, respectively. In conclusion, JQ1 inhibited proliferation and survival of OS cells in vitro but failed to reduce growth of OS xenografts in immunodeficient mice. When combined with rapamycin, JQ1 synergistically inhibited proliferation of OS cells both in vitro and in vivo. Citation Format: Dhong Hyun Tony Lee, Jun Qi, James Bradner, Jonathan Said, Ngan Doan, Charles Forscher, H Phillip Koeffler. Synergistic effect of JQ1 and rapamycin for treatment of human osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1689. doi:10.1158/1538-7445.AM2014-1689

A New Synthetic Ursolic Acid Derivative IUA with Anti-Tumor Efficacy Against Osteosarcoma Cells via Inhibition of JNK Signaling Pathway

Background: Osteosarcoma is the most common primary malignant bone tumor in children and adolescents and is characterized by frequent metastasis and resistance to chemotherapy. Because osteosarcoma cells are not highly susceptible to current chemotherapy drugs, new alternative strategies for the treatment of osteosarcoma are needed. This study was undertaken to investigate the inhibitory effects of a new synthetic ursolic acid derivative IUA on osteosarcoma cells and to explore its molecular mechanism. We also intended to identify new therapeutic candidates. Methods: We used MTT assay to assess the effect of IUA on the proliferation of osteosarcoma cells. Western-blot analysis was performed to examine downstream molecular events. The Annexin V method was used to evaluate the effect of IUA on apoptosis of osteosarcoma cells. The cell cycle of IUA-treated cells was examined by flow cytometry, and the in vivo effects of this new ursolic acid derivative were evaluated in a mouse osteosarcoma model. Results: The results showed that the new synthetic ursolic acid derivative IUA significantly decreased viability of osteosarcoma cells in vitro and in vivo. It could also induce apoptosis and G1 phase arrest of osteosarcoma cells. The JNK signaling pathway was significantly inhibited, and cleaved caspase-3 protein was increased. Conclusion: We concluded that the new synthetic ursolic acid derivative IUA induces proliferation inhibition and apoptosis of osteosarcoma cells in vitro and in vivo via the down-regulation of the JNK signaling pathway, making it a promising agent for the prevention and treatment of human osteosarcoma.

SGP-2, an acidic polysaccharide from Sarcandra glabra, inhibits proliferation and migration of human osteosarcoma cells

An acidic polysaccharide (SGP-2), with a molecular weight of 1880 kDa, was purified from the defatted whole-plant of Sarcandra glabra (Thunb.) Nakai. SGP-2 is mainly composed of glucose, galactose, mannose, arabinose and galacturonic acid in a molar ratio of 12.19 : 8.68 : 6.03 : 1.00 : 15.24. The primary structure analysis reveals that SGP-2 consists of 1,4-linked α-D-galacturonic acid, methyl-esterified 1,4-linked α-D-galacturonic acid, 1,5-linked α-L-arabinose, 1,4-linked α-D-mannose, 1,6-linked β-D-glucose and 1,3-linked β-D-galactose with branch chains of 1,4,6-linked β-D-glucose, 1,3,6-linked α-D-mannose and 1,4,6-linked α-D-galactose. The results of a cell viability assay and colony formation assay indicate that SGP-2 has a potent anti-proliferation activity on human osteosarcoma MG-63 cells. SGP-2 increases the proportion of apoptotic cells and activates caspase-3. In addition, the anti-proliferation effect induced by SGP-2 is blocked by the pan-caspase inhibitor. Moreover, SGP-2 inhibits the migratory capacity of MG-63 cells accompanied with the inhibition of receptor for advanced glycation end-products (RAGE) and nuclear factor-kappa B (NF-κB). Taken together, these results suggest that SGP-2 has anti-cancer potential in the treatment of human osteosarcoma.

Dihydroartemisinin inhibits tumor growth of human osteosarcoma cells by suppressing Wnt/β-catenin signaling

Osteosarcoma (OS) is the most common type of bone cancer. Even with early diagnosis and aggressive treatment, the prognosis for OS is poor. In the present study, we investigated the proliferation and invasion inhibitory effect of dihydroartemisinin (DHA) on human OS cells and the possible molecular mechanisms involved. We demonstrated that DHA can inhibit proliferation, decrease migration, reduce invasion and induce apoptosis in human OS cells. Using an in vivo tumor animal model, we confirmed that DHA can prevent OS formation and maintain intact bone structure in athymic mice. In addition, we examined the possible molecular mechanisms mediating the function of DHA. We found that the total protein levels and transcriptional activity of β-catenin in OS cells are reduced by DHA treatment, and this may result from the increased catalytic activity of glycogen synthase kinase 3β (GSK3β). Moreover, the inhibitory effect of DHA on OS cells is reversed by overexpression of β-catenin, but is further enhanced by knockdown of β-catenin, respectively. Collectively, our results reveal that DHA can inhibit tumor growth of OS cells by inactivating Wnt/β-catenin signaling. Therefore, DHA is a promising chemotherapy agent in the treatment of human OS.

Effect of bevacizumab on angiogenesis and growth of canine osteosarcoma cells xenografted in athymic mice

Objective-To investigate the effects of bevacizumab, a human monoclonal antibody against vascular endothelial growth factor, on the angiogenesis and growth of canine osteosarcoma cells xenografted in mice. Animals-27 athymic nude mice. Procedures-To each mouse, highly metastasizing parent osteosarcoma cells of canine origin were injected into the left gastrocnemius muscle. Each mouse was then randomly allocated to 1 of 3 treatment groups: high-dose bevacizumab (4 mg/kg, IP), low-dose bevacizumab (2 mg/kg, IP), or control (no treatment). Tumor growth (the number of days required for the tumor to grow from 8 to 13 mm), vasculature, histomorphology, necrosis, and pulmonary metastasis were evaluated. Results-Mice in the high-dose bevacizumab group had significantly delayed tumor growth (mean ± SD, 13.4 ± 3.8 days; range, 9 to 21 days), compared with that for mice in the low-dose bevacizumab group (mean ± SD, 9.4 ± 1.5 days; range, 7 to 11 days) or control group (mean ± SD, 7. 2 ± 1.5 days; range, 4 to 9 days). Mice in the low-dose bevacizumab group also had significantly delayed tumor growth, compared with that for mice in the control group. Conclusions and Clinical Relevance-Results indicated that bevacizumab inhibited growth of canine osteosarcoma cells xenografted in mice, which suggested that vascular endothelial growth factor inhibitors may be clinically useful for the treatment of osteosarcoma in dogs. Impact for Human Medicine-Canine osteosarcoma is used as a research model for human osteosarcoma; therefore, bevacizumab may be clinically beneficial for the treatment of osteosarcoma in humans.

Abstract 3546: Cucurbitacin B as a direct mTOR inhibitor synergizes with methotrexate for treatment of human osteosarcoma

Abstract The mammalian target of rapamycin (mTOR) pathway is one of the most important oncogenic pathways in cancer, and the upregulation of the mTOR pathway is frequently observed in human osteosarcoma (OS). In this study, we demonstrated for the first time that cucurbitacin B, a plant-derived tetracyclic triterpenoid, is a direct mTOR inhibitor in OS using drug affinity responsive target stability (DARTS) analysis. Binding of cucurbitacin B to mTOR resulted in the inhibition of mTOR phosphorylation in MG-63 and SAOS-2 human OS cells in vitro, which was further confirmed by the inhibition of ribosomal S6 protein kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). This finding led us to hypothesize the possible use of cucurbitacin B as a single agent as well as in combination with methotrexate (MTX) for OS treatment. Cucurbitacin B in combination with MTX synergistically inhibited OS cell growth in vitro such that combination index (CI) &amp;lt; 0.9. Murine xenografts treated with either low-dose cucurbitacin B (LD-CuB, 0.5 mg/kg body weight) or low-dose MTX (LD-MTX, 150 mg/kg) alone failed to decrease tumor size. However, combined therapy at identical concentrations inhibited tumor growth by 62% vs LD-CuB and 81% vs LD-MTX (p&amp;lt;0.001). The effect persisted even at a two-thirds decrease (50 mg/kg) in the MTX dose. In conclusion, cucurbitacin B is a direct inhibitor of mTOR, and the combined use of cucurbitacin B with MTX showed promising antiproliferative activity against human OS. The use of this combination should be explored further in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3546. doi:10.1158/1538-7445.AM2011-3546