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Background: Achieved Silica Nanoparticles (SiNPs) to encapsulate the photosensitizer [Protoporphyrin IX (PpIX)] in photodynamic therapy (PDT) application was reported in this research., Materials and Methods: Cytotoxicity for five different concentrations of encapsulated and naked PpIX was measured. Optimum concentration and optimum exposure time of encapsulated and naked PpIX that needed to destroy the cells (Osteosarcoma cells) was measured., Results: The results showed that the encapsulated PpIX has more efficacy compared to the naked PpIX and the applicability of the encapsulated PpIX-SiNPs was proved on osteosarcoma cells., Conclusion: The results established the important in-vitro photodynamic effectiveness of PpIX-SiNP, which may open a new application for PpIX in its clinical and in-vitro studies.

Angiogenesis is now well known for being involved in tumor progression, aggressiveness, emergence of metastases, and also resistance to cancer therapies. In this study, to better mimic tumor angiogenesis encountered in vivo, we used 3D culture of osteosarcoma cells (MG-63) that we deposited on 2D endothelial cells (HUVEC) grown in monolayer. We report that endothelial cells combined with tumor cells were able to form a well-organized network, and that tubule-like structures corresponding to new vessels infiltrate tumor spheroids. These vessels presented a lumen and expressed specific markers as CD31 and collagen IV. The combination of 2D endothelial cells and 3D microtissues of tumor cells also increased expression of angiogenic factors as VEGF, CXCR4 and ICAM1. The cell environment is the key point to develop tumor vascularization in vitro and to be closer to tumor encountered in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells., Competing Interests: CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Osteosarcoma is the most frequent primary bone tumor originating from adolescents and young adults. Despite improvements in the chemo- or radio- therapy of osteosarcoma patients, survival rate has not increased and drug resistance becomes a major factor that limits the effectiveness. Therefore, investigation of new treatment modalities is urgently required to optimize therapeutic options. Our previous study described an oncogenic role of miR-214 through promotion of osteosarcoma cells proliferation. In this study, we report miR-214 contributes to cisplatin resistance in osteosarcoma cells. Overexpression of miR-214 decreased the cisplatin sensitivity. By establishing an osteosarcoma cisplatin resistant cell line, we find miR-214 is significantly upregulated in cisplatin resistant cells. Moreover, we show miR-214 promotes anaerobic glycolysis rates of osteosarcoma cells but suppresses mitochondrial oxidative phosphorylation. Consistently, cisplatin resistant cells exhibit upregulated glycolysis but decreased mitochondrial oxidative phosphorylation, a phenotype called "Warburg effect". Finally, we demonstrate inhibition of glycolysis by either glycolysis inhibitor or miR-214 inhibition significantly re-sensitizes cisplatin resistant osteosarcoma cells. In summary, this study illustrates a miRNA-involved chemosensitivity of osteosarcoma and will contribute to the developments of therapeutic agents for the anti-chemoresistance treatments.

Insulin controls blood glucose while insulin-like growth factor (IGF) 1 is an important growth factor. Interestingly, both hormones have overlapping bioactivities and can activate the same intracellular signal transduction cascades. Growth control (mainly by IGF1) and metabolic function (predominantly by insulin) are believed to depend on activation of extracellular signal-regulated kinases (ERKs) 1/2 and protein kinase B (Akt/PKB), respectively. Therefore, insulin analogues that are used to normalize blood glucose are tested for their ability to preferentially activate Akt/PKB but not ERK1/2 and mitogenesis. Growth hormone, IGF1, and hyperinsulinemia are associated with increased risk of growth progression of some cancer types. To test if continuous exposure to insulin can favour tumour growth, we studied insulin/IGF1-dependent activation of ERK1/2 and Akt/PKB by Western blotting, inhibition of apoptosis by ELISA, and induction of proliferation by [ 3 H]-thymidine incorporation in Saos-2/B10 osteosarcoma cells. IGF1 and insulin both induced proliferation and prevented apoptosis effectively. Regulation of apoptosis was far more sensitive than regulation of proliferation. IGF1 and insulin activated PKB (Akt/PKB) rapidly and consistently maintained its phosphorylation. Activation of ERK1/2 was only observed in response to IGF1. Loss of p-Akt/PKB (but not of p-ERK1/2) was associated with increased apoptosis, and protection from apoptosis was lost when activation of Akt/PKB was inhibited. These findings in Saos-2/B10 cells were also replicated in the A549 cell line, originally derived from a human lung carcinoma. Therefore, IGF1 and insulin more likely (at lower concentrations) enhance tumour cell survival than proliferation, via activation and maintenance of phosphatidylinositol 3-kinase activity and p-Akt/PKB.

The physiological and pathological roles of dopamine D1 receptors (DR1) in the regulation of functions in tissues and organs have been recognized. However, whether DR1 are expressed in the osteosarcoma cells and inhibit the proliferation of these cells is unknown. In the present study, we found that DR1 were expressed in the osteosarcoma cells (OS732 cells). SKF-38393 (DR1 agonist) and the overexpression of DR1 decreased the proliferation of OS732 cells; SCH-23390 (DR1 antagonist) and the knockdown of DR1 increased the proliferation of OS732 cells, and both SCH-23390 and the knockdown of DR1 abolished the effect of SKF-38393 on the proliferation of OS732 cells. In addition, SKF-38393 down-regulated the phosphorylation of ERK1/2, PI3K, and Akt; SCH-23390 up-regulated the phosphorylation of ERK1/2, PI3K, and Akt, and SCH-23390 cancelled the effect of SKF-38393. The effect of SKF-38393 on the phosphorylation of ERK1/2, PI3K, and Akt and the proliferation of OS732 cells was similar to PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor), respectively. In conclusion, our results suggest that DR1 are expressed in the osteosarcoma cells and inhibit the proliferation of osteosarcoma cells by the down-regulation of the ERK1/2 and PI3K-Akt pathways. These findings provide a novel target for the treatment of the osteosarcoma.

Our study explored the effects of SOST gene silencing on the proliferation, apoptosis, invasion, and migration of human osteosarcoma cells through Wnt/β-catenin signaling pathway. Fresh tissues were obtained from 108 patients with osteosarcoma and 46 patients with osteochondroma. Human osteosarcoma cells (MG-63, U2-OS, HOS, and Saos-2) and normal osteoblast (hFoB1.19) were selected and cultured. Osteosarcoma cells were grouped randomly into the blank group, the scrambled control group, and the SOST-siRNA group. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis were tested by flow cytometry. Transwell and scratch test were performed to determine cell invasion and migration. The qRT-PCR and Western blotting were used to detect mRNA and protein expression level of sclerostin, Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7. The activity of caspase-3 was assessed by immunocytochemistry. Alkaline phosphatase (ALP) activity was measured using P-nitrophenylphosphate as a substrate. Low SOST mRNA and sclerostin protein expression levels were observed in osteosarcoma tissues and cells. Compared with the blank and scrambled control groups, sclerostin expression, apoptotic cells, ALP activity, and caspase-3 activity were down-regulated, while the proliferation, invasion, and migration abilities of osteosarcoma cells were evidently enhanced in the SOST-siRNA group. After SOST gene silencing, the mRNA and protein expression levels of Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7 in osteosarcoma cells and β-catenin protein expression levels in the nucleus and cytoplasm were significantly elevated. SOST gene silencing promotes the proliferation, invasion, and migration, and inhibits apoptosis of osteosarcoma cells by activating Wnt/β-catenin signaling pathway.

The use of protonated N-heterocyclic compound, i.e. 2,2'-bipyridinium cation, [bpyH + ], enabled to obtain the new nitrilotriacetate oxidovanadium(IV) salt of the stoichiometry [bpyH][VO(nta)(H 2 O)]H 2 O. The X-ray measurements have revealed that the compound comprises the discrete mononuclear [VO(nta)(H 2 O)] - coordination ion that can be rarely found among other known compounds containing nitrilotriacetate oxidovanadium(IV) moieties. The antitumor activity of [bpyH][VO(nta)(H 2 O)]H 2 O and its phenanthroline analogue, [phenH][VO(nta)(H 2 O)](H 2 O) 0.5 , towards human osteosarcoma cell lines (MG-63 and HOS) has been assessed (the LDH and BrdU tests) and referred to cis-Pt(NH 3 ) 2 Cl 2 (used as a positive control). The compounds exert a stronger cytotoxic effect on MG-63 and HOS cells than in untransformed human osteoblast cell line. Thus, the [VO(nta)(H 2 O)] - containing coordination compounds can be considered as possible antitumor agents in the osteosarcoma model of bone-related cells in culture.

G-protein-coupled receptor 120 (GPR120) and GPR40 are members of free fatty acid (FFA) receptors and mediate a variety of biological responses through binding of medium- and long-chain FFAs. Recently, it has been reported that GPR120 and GPR40 regulated cellular functions of cancer cells. In the present study, to assess whether GPR120 and GPR40 are involved in the enhancement of cell motile activity of osteosarcoma cells, we established highly migratory (MG63-R7) cells from osteosarcoma MG-63 cells. The expression level of GPR120 gene was significantly higher in MG63-R7 cells than in MG-63 cells, while no change of GPR40 expression was observed. In cell motility assay, the cell motile activity of MG63-R7 cells was approximately 200 times higher than that of MG-63 cells. The cell motile activity of MG63-R7 cells was stimulated by GW9508, which is an agonist of GPR120 and GPR40. Moreover, a GPR40 antagonist GW1100 elevated the cell motile activity of MG63-R7 cells in the presence of GW9508. To confirm the effects of GPR120 and GPR40 on the cell motile activity of MG63-R7 cells, GPR120 knockdown cells were generated from MG63-R7 cells. The cell motile activity of MG63-R7 cells was markedly suppressed by GPR120 knockdown. These results indicated that GPR120 enhanced and GPR40 inhibited the cell motile activity of highly migratory osteosarcoma cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes.

Acquisition of drug-resistant phenotypes is often associated with chemotherapy in osteosarcoma. A number of studies have demonstrated a critical role for autophagy in osteosarcoma development, therapy and drug resistance. However, the molecular mechanisms underlying the autophagy-mediated chemotherapy resistance of osteosarcoma cells remain largely unknown. In the present study, we determined the autophagy and microRNA-140 (miR-140-5p, miRBase ID: MIMAT0000431) expression induced by chemotherapeutic drugs in osteosarcoma cells. Then we determined the promotory role of miR-140-5p to the chemotherapy-induced autophagy. Our results demonstrated that miR-140-5p expression was highly induced during chemotherapy of osteosarcoma cells, and this was accompanied by up-regulated autophagy. The increased miR-140-5p expression levels up-regulated anticancer drug-induced autophagy in osteosarcoma cells and ameliorated the anticancer drug-induced cell proliferation and viability decrease. Importantly, miR-140-5p regulates this context-specific autophagy through its target, inositol 1,4,5-trisphosphate kinase 2 (IP3k2). Therefore, the results of the present study demonstrated that miR-140-5p mediated drug-resistance in osteosarcoma cells by inducing autophagy. The present study provides evidence of miRNA regulation of autophagy through modulation of IP3 signalling. The present study recognized a novel mechanism of chemoresistance in osteosarcoma cancers., (© 2016 The Author(s).)

Macroautophagy (herein termed autophagy) works as a protective mechanism in tumorigenesis and development under metabolic stress condition. Multitudes of genes have been found involved in this process during past decades. In the present study, we report that Wilm's tumor suppressor1 (WT1) is involved in autophagy in osteosarcoma (OS) cells. WT1, a transcription factor with multitude of target genes, expresses in a majority of cancer types. Though wide-ranging effect of WT1 is now well documented, the function of WT1 in tumors remains poorly defined. In this chapter, it is found that high expression of WT1 positively correlates with active autophagy in human osteosarcoma cells. And further study on cell signaling pathway illustrates that Akt/JNK pathway acts as a positive regulator of autophagy induced by WT1. Here, we present evidence that WT1 modulates Akt/JNK signaling pathway mediated autophagy by controlling the expression of growth arrest-specific 1 (Gas1). We show that WT1 is required for Gas1 transcription in osteosarcoma cells. And Gas1 is upregulated followed WT1 overexpression in a time-dependent manner. Loss of Gas1 results in a reduction of WT1-induced autophagy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling., Experimental Design: We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia., Results: It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin., Conclusion: The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy.

Background: The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells., Methods: In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown., Results: In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs., Conclusions: These findings demonstrated that the tumor microenvironment has an influence on the gene expression of healthy surrounding tissues and on the process of tumorigenicity and it is emerging as attractive targets for therapeutic strategies.

The bisphenols AF (BPAF) and S (BPS) are structural analogs of the endocrine disruptor bisphenol A (BPA), and are used in common products as a replacement for BPA. To elucidate genome-wide gene expression responses, estrogen-dependent osteosarcoma cells were cultured with 10 nM BPA, BPAF, or BPS, for 8 h and 3 months. Genome-wide gene expression was analyzed using the Illumina Expression BeadChip. Three months exposure had significant effects on gene expression, particularly for BPS, followed by BPAF and BPA, according to the number of differentially expressed genes (1980, 778, 60, respectively), the magnitude of changes in gene expression, and the number of enriched biological processes (800, 415, 33, respectively) and pathways (77, 52, 6, respectively). 'Embryonic skeletal system development' was the most enriched bone-related process, which was affected only by BPAF and BPS. Interestingly, all three bisphenols showed highest down-regulation of genes related to the cardiovascular system (e.g., NPPB, NPR3, TXNIP). BPA only and BPA/BPAF/BPS also affected genes related to the immune system and fetal development, respectively. For BPAF and BPS, the 'isoprenoid biosynthetic process' was enriched (up-regulated genes: HMGCS1, PDSS1, ACAT2, RCE1, DHDDS). Compared to BPA, BPAF and BPS had more effects on gene expression after long-term exposure. These findings stress the need for careful toxicological characterization of BPA analogs in the future., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Luteolin is a flavone in medicinal plants as well as some vegetables and spices. It is a natural anti-oxidant with less pro-oxidant potential but apparently with a better safety profile. The purpose of this study was to investigate the molecular mechanism of luteolin-mediated apoptosis of MG-63 human osteosarcoma cells. MTT assay kit was employed to evaluate the effects of luteolin on MG-63 cells proliferation. Then, we performed Annexin V-FITC/PI to analyze the apoptotic rate of the cells. Furthermore, the inhibitory effects of luteolin on the expressions of BCL-2, BAX, Caspase-3 and Survivin were detected by Western blotting. As expected, luteolin (0.5, 2.5, 12.5 µg/mL) inhibited the growth of MG-63 cells by inhibiting cell proliferation and inducing cell apoptosis. Western blotting demonstrated that luteolin (0.5, 2.5, 12.5 µg/mL) inhibited the expressions of BCL-2, Caspase-3 and Survivin, and promoted the expression of BAX in MG-63 cells with a concentration dependent way. Luteolin can inhibit osteosarcoma cell proliferation and induce apoptosis effectively in a dose dependent manner through down-regulating the expression of BCL-2, Caspase-3 and Survivin proteins levels and up-regulating the expression of BAX protein level. These findings indicated that luteolin may be used as a novel herbal medicine for the treatment of osteosarcoma.

Osteosarcoma is a primary malignancy of bone, which is characterized by the proliferation of malignant mesenchymal cells, particularly in children and adolescents. Evodiamine is extracted from a variety of traditional Chinese medicines, which has been reported to induce apoptosis in certain tumors, including cervical, prostate and breast cancer, however, its effect on oestosarcoma cells remains unclear. The aim of the present study was to investigate the effect of evodiamine on osteosarcoma cell proliferation and apoptosis, and explore the associated underlying molecular mechanism. A Cell Counting Kit 8 assay was performed to detect the effects of evodiamine on the proliferation of human osteosarcoma U2OS cells. Annexin V-fluorescein isothiocyanate/propidium iodide staining was performed to analyze the apoptotic rate of the cells. The effect of evodiamine on the protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and survivin were detected by performing western blot analysis. Evodiamine inhibited the growth of human osteosarcoma U2OS cells by inhibiting cell proliferation and inducing cell apoptosis. Western blotting demonstrated that evodiamine downregulated the expression of Bcl-2, caspase-3 and survivin, and upregulated the expression of Bax in human osteosarcoma cells. Evodiamine effectively inhibited proliferation and induced apoptosis of osteosarcoma cells in a dose-dependent manner via downregulation of Bcl-2, caspase-3 and survivin protein expression levels and upregulation of Bax protein expression levels.

Icariside II is considered one of the most important natural flavonoids with multiple bioactivities from traditional Chinese medicine Yin Yanghuo (YYH) or Horny Goat Weed (Epimedium koreanum Nakai). Previous studies show that Icariside II exhibits potent cytotoxicity against a broad spectrum of human cancer cells through various signaling transduction pathways. However, there are few reports about the effect of Icariside II on osteosarcoma cell. In this study, we found that Icariside II decreased cell proliferation in human osteosarcoma MG-63 cells and human osteosarcoma Saos-2 cells. In addition, Icariside II inactivated EGFR/mTOR signaling pathway, including EGFR, PI3K/AKT/PRAS40, Raf/MEK/ERK as well as mTOR. Furthermore, Icariside II inhibited epidermal growth factor (EGF)-induced activation of EGFR/mTOR signaling pathway. Pretreatment of EGF partially reversed cell viability decreased by Icariside II. Importantly, Icariside II inhibited the proliferation of transplantable tumors and EGFR/mTOR signaling pathway in sarcoma-180 bearing mice. In summary, these results indicate that Icariside II inhibits the proliferation of osteosarcoma cells in vitro and in vivo via EGFR/mTOR signaling pathway., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Frequent acquisition of drug resistance is often associated with the chemotherapy of malignant tumors, including osteosarcoma. A number of studies have demonstrated a critical role for autophagy in osteosarcoma development, therapy and drug resistance. However, the molecular mechanisms underlying the autophagy-mediated chemotherapy resistance of osteosarcoma cells remain largely unknown. In the present study, we determined the autophagy and microRNA-155 (miR-155) expression induced by chemotherapeutic drugs in osteosarcoma cells. Then we determined the promotory role of miR-155 to the chemotherapy-induced autophagy. Our results demonstrated that microRNA-155 (miR-155) expression was highly induced during chemotherapy of osteosarcoma cells, and this was accompanied by upregulated autophagy. The increased miR-155 expression levels upregulated anticancer drug-induced autophagy in osteosarcoma cells and ameliorated the anticancer drug-induced cell proliferation and viability decrease. Therefore, the results of the present study demonstrated that miR-155 mediated drug-resistance in osteosarcoma cells by inducing autophagy. The present study recognized a novel mechanism of chemoresistance in osteosarcoma cancers.

Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro-apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14-3-3ϵ protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14-3-3ϵ mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14-3-3ϵ using siRNA. The 14-3-3ϵ gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14-3-3ϵ is required for Bim expression and that it has an anti-cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14-3-3ϵ may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments., (© 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.

MicroRNAs (miRNAs) have emerged as potential anticancer agents, but their clinical application is limited by the lack of an effective delivery system to tumors. Exosomes are small vesicles that play important roles in intercellular communication. Here, we show that synthetic miR-143 introduced into cells is released enveloped in exosomes and that the secreted exosome-formed miR-143 is transferred to osteosarcoma cells. The delivery of exosome-formed miR-143 significantly reduced the migration of osteosarcoma cells. The delivery efficiency of exosome-formed miR-143 was less than that achieved with lipofection, but the migratory potential of osteosarcoma cells was similarly inhibited after both strategies. Our results suggest that exosomes can deliver synthetic miR-143 and are a potentially efficient and functional delivery system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Osteosarcoma is a common malignant bone tumor which threatens the life of young people worldwide. To explore alternative strategy for combating osteosarcoma, a light-emitting diode (LED) that activates methylene blue (MB) was used in the present study to investigate cell death of osteosarcoma-derived UMR106 cells., Materials and Methods: Photocytotoxicity in UMR106 cells was investigated 24h after photodynamic activation of MB using sulforhodamine B (SRB) assay and light microscopy. Apoptosis induction was observed 24h after photodynamic treatment using a confocal laser scanning microscopy (CLSM) with Hoechst 33342 staining. The change in mitochondrial membrane potential (MMP) was analyzed using a flow cytometry with rhodamine 123 staining., Results: MB under red light irradiation caused a drug-concentration (0-100μM) and light-dose (0-32J/cm(2)) dependent cytotoxicity in UMR106 cells. The SRB assay and light microscopy observed a significant decrease in the number of UMR106 cells attached to the bottom of culture well after LED light-activated MB (100μM, 32J/cm(2)). Nuclear shrinkage, chromatin condensation and fragmentation were found in the treated cells by nuclear staining. In addition, flow cytometry showed that the MMP in UMR106 cells was rapidly reduced by photo-activated MB (100μM, 32J/cm(2))., Conclusion: Photodynamic action of MB under LED irradiation could remarkably kill osteosarcoma cells and induce cell apoptosis as well as MMP collapse., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

The objective of the present study was to investigate the anticancer activity of Chinese medicine Caulis Spatholobi extract on multicentric osteosarcoma cells. Ultraviolet spectrophotometry was used to determine the total flavonoid content in each sample; vanillin sulphuric acid assay was used to determine the condensed tannin content in each sample; and the varying degrees of inhibitory activities of ethanol, ethyl acetate and n-butanol extracts of Caulis Spatholobi on human osteosarcoma Saos-2 cells were studied. The results showed that the inhibitory activity of ethyl acetate extract was the highest among the four extracts. The condensed tannin contents of 1.2 mg/mL Caulis Spatholobi water extract, ethanol extract, ethyl acetate extract and petroleum ether extract were 26.23%, 48.36%, 70.18% and 40.51% respectively; and condensed tannin content of 1.5 mg/mL Caulis Spatholobi water extract, ethanol extract, ethyl acetate extract and petroleum ether extract were 4.15%, 5.81%, 8.76% and 7.30% respectively.

Stress cytokines tumor necrosis factor α, interleukin-1 β and interleukin-6 modulate the activity of a variety of cell types including osteoblasts, and are involved in the pathogenesis of several rheumatic diseases associated with systemic bone loss. We have studied the expression of interleukin-6 induced by interleukin-1 β and tumor necrosis factor α in the osteoblast-like cell line MG-63, derived from a human osteosarcoma. We have observed marked differences in the regulation of interleukin-6 gene expression by tumor necrosis factor α or interleukin-1 β, at the level of mRNA steady state and stability and cytokine secretion. In addition, N-acetyl cysteine, a scavenger of reactive oxygen species, inhibits activation of NF-κB and induction of interleukin-6 by tumor necrosis factor α, being ineffective on interleukin-1 β activity. These data illustrate the action of stress cytokines on a cell line widely used in in vitro studies as a reliable model of osteoblast response to cytokines involved in bone resorbing diseases, an important issue for developing new strategies for treatments of bone diseases.