CRISPR-Cas9-Mediated Silencing of CD44 in Human Highly Metastatic Osteosarcoma Cells

Background/Aims: Metastasis is the major cause of death in patients with osteosarcoma. There is an urgent need to identify molecular markers that promote metastasis. Cluster of differentiation 44 is a receptor for hyaluronic acid (HA) and HA-binding has been proven to participate in various biological tumor activities, including tumor progression and metastasis. Methods: We performed a meta-analysis to investigate the relationship between CD44 expression, survival, and metastasis in patients with osteosarcoma. We then utilized the CRISPR-Cas9 system to specifically silence CD44 in highly metastatic human osteosarcoma cells (MNNG/HOS and 143B) and further determined the functional effects of CD44 knockout in these cells. Results: The meta-analysis demonstrated that a high level of CD44 may predict poor survival and higher potential of metastasis in patients with osteosarcoma. The expression of CD44 in highly metastatic human osteosarcoma cell lines was efficiently blocked by CRISPR-Cas9. When CD44 was silenced, the proliferation and spheroid formation of these osteosarcoma cells was inhibited under 3-D culture conditions. Furthermore, the migratory and invasive functions were also impaired in these highly metastatic osteosarcoma cells. Conclusion: These results suggest that developing new strategies to target CD44 in osteosarcoma may prevent metastasis and improve the clinical outcome of osteosarcoma patients.

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Antiproliferative Properties of Oleuropein in Human Osteosarcoma Cells

Natural Product Communications ◽

10.1177/1934578x1601100418 ◽

2016 ◽

Vol 11 (4) ◽

pp. 1934578X1601100 ◽

Cited By ~ 1

Author(s):

Jose M. Moran ◽

Olga Leal-Hernandez ◽

Maria L. Canal-Macías ◽

Raul Roncero-Martin ◽

Rafael Guerrero-Bonmatty ◽

...

Keyword(s):

Cell Lines ◽

Osteosarcoma Cell ◽

Dependent Manner ◽

Osteosarcoma Cells ◽

Probit Regression ◽

Cytotoxic Effects ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Mg63 Cells ◽

Human Osteosarcoma Cell

In this study, we evaluated the antiproliferative activity on two human osteosarcoma cell lines (MG-63 and Saos2) of oleuropein, an olive oil compound traditionally found in the Mediterranean diet. Oleuropein exhibited obvious cytotoxic effects on human osteosarcoma cells in a concentration- and time-dependent manner. Statistical analysis of IC50 by the Probit regression method suggested that oleuropein had similar toxic effects on both cell lines tested (IC50 range from 247.4–475.0 μM for MG63 cells and from 798.7–359.9 μM for Saos2 cells).

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β-Phenethyl Isothiocyanate Induces Cell Death in Human Osteosarcoma through Altering Iron Metabolism, Disturbing the Redox Balance, and Activating the MAPK Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/5021983 ◽

2020 ◽

Vol 2020 ◽

pp. 1-23

Author(s):

Huanhuan Lv ◽

Chenxiao Zhen ◽

Junyu Liu ◽

Peng Shang

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Iron Metabolism ◽

Ros Generation ◽

Osteosarcoma Cell ◽

Phenethyl Isothiocyanate ◽

Osteosarcoma Cells ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell

Osteosarcoma is the most common primary malignancy of the skeleton in children and adults. The outcomes of people with osteosarcomas are unsatisfied. β-Phenethyl isothiocyanate (PEITC) exhibits chemoprevention and chemotherapeutic activities against many human cancers. The molecular mechanism underlying its action on osteosarcoma is still unknown. This study was aimed at investigating the effect of PEITC on human osteosarcoma both in vitro and in vivo. The results showed that PEITC reduced cell viability, inhibited proliferation, and caused G2/M cell cycle arrest in four human osteosarcoma cell lines (MNNG/HOS, U-2 OS, MG-63, and 143B). Then, we found that PEITC altered iron metabolism related to the processes of iron import, storage, and export, which resulted in increased labile iron. Expectedly, PEITC caused oxidative stress as a consequence of GSH depletion-inducing ROS generation and lipid peroxidation. Multiple cell death modalities, including ferroptosis, apoptosis, and autophagy, were triggered in human osteosarcoma cells. Three MAPKs (ERK, p38, and JNK) were all activated after PEITC treatment; however, they presented different responses among the four human osteosarcoma cell lines. ROS generation was proved to be the major cause of PEITC-induced decreased proliferative potential, altered iron metabolism, cell death, and activated MAPKs in human osteosarcoma cells. In addition, PEITC also significantly delayed tumor growth in a xenograft osteosarcoma mouse model with a 30 mg/kg administration dose. In conclusion, this study reveals that PEITC simultaneously triggers ferroptosis, apoptosis, and autophagy in human osteosarcoma cells by inducing oxidative stress.

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Bioinspired-Metalloporphyrin Magnetic Nanocomposite as a Reusable Catalyst for Synthesis of Diastereomeric (−)-Isopulegol Epoxide: Anticancer Activity Against Human Osteosarcoma Cells (MG-63)

Molecules ◽

10.3390/molecules24010052 ◽

2018 ◽

Vol 24 (1) ◽

pp. 52 ◽

Cited By ~ 4

Author(s):

Lucas Dias ◽

Ana Batista de Carvalho ◽

Sara Pinto ◽

Gilberto Aquino ◽

Mário Calvete ◽

...

Keyword(s):

Reusable Catalyst ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Human Osteosarcoma Cell Line ◽

Anticancer Effect ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell ◽

Ic50 Values

In the present study, we developed a green epoxidation approach for the synthesis of the diastereomers of (−)-isopulegol benzyl ether epoxide using molecular oxygen as the oxidant and a hybrid manganese(III)-porphyrin magnetic reusable nanocomposite as the catalyst. High activity, selectivity, and stability were obtained, with up to four recycling cycles without the loss of activity and selectivity for epoxide. The anticancer effect of the newly synthesized isopulegol epoxide diastereomers was evaluated on a human osteosarcoma cell line (MG-63); both diastereomers showed similar in vitro potency. The measured IC50 values were significantly lower than those reported for other monoterpene analogues, rendering these epoxide isomers as promising anti-tumor agents against low prognosis osteosarcoma.

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Ginsenoside Rg5 Inhibits Human Osteosarcoma Cell Proliferation and Induces Cell Apoptosis through PI3K/Akt/mTORC1-Related LC3 Autophagy Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/5040326 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Ming-Yang Liu ◽

Fei Liu ◽

Yan-Jiao Li ◽

Jia-Ning Yin ◽

Yan-Li Gao ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Apoptosis ◽

Caspase 3 ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell ◽

Ginsenoside Rg5 ◽

Autophagy Pathway

The function and mechanism underlying the suppression of human osteosarcoma cells by ginsenoside-Rg5 (Rg5) was investigated in the present study. MG-63, HOS, and U2OS cell proliferation was determined by MTT assay after Rg5 treatment for 24 h. Rg5 inhibited human osteosarcoma cell proliferation effectively in a dose-dependent manner. The range of effective inhibitory concentrations was 160-1280 nM. Annexin V-FITC and PI double-staining assay revealed that Rg5 induced human osteosarcoma cell apoptosis. Western blotting, qRT-PCR, and FACS experiments revealed that Rg5 inhibited human osteosarcoma cells via caspase-3 activity which was related to the LC3-mediated autophagy pathway. Rg5 decreased the phosphorylation of PI3K, Akt, and mTORC1 activation. In contrast, LC3-mediated autophagy and caspase-3 activity increased significantly. A PI3K/AKT stimulator, IGF-1, reversed Rg5-induced cell autophagy and apoptosis in MG-63 cells. Collectively, the current study demonstrated that Rg5 induced human osteosarcoma cell apoptosis through the LC3-mediated autophagy pathway. Under physiological conditions, activation of PI3K/AKT/mTORC1 inhibits LC3 activity and caspase-3-related cell apoptosis. However, Rg5 activated LC3 activity by inhibiting the activation of PI3K/AKT/mTORC1. The present study indicated that Rg5 could be a promising candidate as a chemotherapeutic agent against human osteosarcoma.

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Anti-Metastatic and Anti-Angiogenic Effects of Curcumin Analog DK1 on Human Osteosarcoma Cells In Vitro

Pharmaceuticals ◽

10.3390/ph14060532 ◽

2021 ◽

Vol 14 (6) ◽

pp. 532

Author(s):

Muhammad Nazirul Mubin Aziz ◽

Nurul Fattin Che Rahim ◽

Yazmin Hussin ◽

Swee Keong Yeap ◽

Mas Jaffri Masarudin ◽

...

Keyword(s):

Cell Lines ◽

Safety Profile ◽

Quantitative Polymerase Chain Reaction ◽

Tube Formation ◽

Osteosarcoma Cell ◽

Curcumin Analog ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell

Osteosarcoma (OS) is a life-threatening malignant bone tumor associated with poor prognosis among children. The survival rate of the patient is still arguably low even with intensive treatment provided, plus with the inherent side effects from the chemotherapy, which gives more unfavorable outcomes. Hence, the search for potent anti-osteosarcoma agent with promising safety profile is still on going. Natural occurring substance like curcumin has gained a lot of attention due to its splendid safety profile as well as it pharmacological advantages such as anti-metastasis and anti-angiogenesis. However, natural curcumin was widely known for its poor cellular uptake, which undermines all potential that it possesses. This prompted the development of synthetically synthesized curcuminoid analog, known as (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2- en-1-one (DK1). In this present study, in vitro scratch assay, transwell migration/invasion assay, HUVEC tube formation assay, and ex vivo rat aortic ring assays were performed in order to investigate the anti-metastatic and anti-angiogenic potential of DK1. For further comprehension of DK1 mechanism on human osteosarcoma cell lines, microarray gene expression analysis, quantitative polymerase chain reaction (qPCR), and proteome profiler were adopted, providing valuable forecast from the expression of important genes and proteins related to metastasis and angiogenesis. Based on the data gathered from the bioassays, DK1 was able to inhibit the metastasis and angiogenesis of human osteosarcoma cell lines by significantly reducing the cell motility, number of migrated and invaded cells as well as the tube formation and micro-vessels sprouting. Additionally, DK1 also has significantly regulated several cancer pathways involved in OS proliferation, metastasis, and angiogenesis such as PI3K/Akt and NF-κB in both U-2 OS and MG-63. Regulation of PI3K/Akt caused up-regulation of genes related to metastasis inhibition, namely, PTEN, FOXO, PLK3, and GADD45A. Meanwhile, NF-κB pathway was regulated by mitigating the expression of NF-κB activator such as IKBKB and IKBKE in MG-63, whilst up-regulating the expression of NF-κB inhibitors such as NFKBIA and NFKBIE in U-2 OS. Finally, DK1 also has successfully hindered the metastatic and angiogenic capability of OS cell lines by down-regulating the expression of pro-metastatic genes and proteins like MMP3, COL11A1, FGF1, Endoglin, uPA, and IGFBP2 in U-2 OS. Whilst for MG-63, the significantly down-regulated oncogenes were Serpin E1, AKT2, VEGF, uPA, PD-ECGF, and Endoglin. These results suggest that curcumin analog DK1 may serve as a potential new anti-osteosarcoma agent due to its anti-metastatic and anti-angiogenic attributes.

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Rhotekin 2 silencing inhibits proliferation and induces apoptosis in human osteosarcoma cells

Bioscience Reports ◽

10.1042/bsr20181384 ◽

2018 ◽

Vol 38 (6) ◽

Author(s):

Xiong Wang ◽

Lei Zhang ◽

Wenji Wang ◽

Yuchen Wang ◽

Ye Chen ◽

...

Keyword(s):

Cell Lines ◽

Phase Cell ◽

Osteosarcoma Cell ◽

Cyclin Dependent Kinase ◽

Potential Therapeutic Target ◽

Human Osteosarcoma ◽

Cycle Arrest ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell ◽

Induced Apoptosis

Human osteosarcoma is the most frequent primary malignant of bone, and often occurs in adolescents. However, molecular mechanism of this disease remains unclear. In the present study, we found that the level of Rhotekin 2 (RTKN2) was up-regulated in osteosarcoma tissues and cell lines. In addition, silencing of RTKN2 of human osteosarcoma cell lines U2OS, inhibited proliferation, and induced G1 phase cell cycle arrest via reducing the level of the cyclin-dependent kinase 2 (CDK2). Furthermore, RTKN2 knockdown in the U2OS cells induced apoptosis by increasing the level of Bax and decreasing the level of Bcl2. These results suggested that RTKN2 is involved in the progression of human osteosarcoma, and may be a potential therapeutic target.

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Micro-RNA 122 and micro-RNA 96 affected human osteosarcoma biological behavior and associated with prognosis of patients with osteosarcoma

Bioscience Reports ◽

10.1042/bsr20201529 ◽

2020 ◽

Vol 40 (12) ◽

Author(s):

Bin Liu ◽

Shuqiang Yao ◽

Jiping Zhou

Keyword(s):

Independent Predictor ◽

Micro Rna ◽

Biological Behavior ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Rt Pcr ◽

Normal Tissues ◽

Human Osteosarcoma ◽

Tumor Tissues ◽

Human Osteosarcoma Cell

Abstract Osteosarcoma (OS) is the most common bone malignancy in both children and adolescents. In the present study, we aimed to explore the association of miRNA-122 and miRNA-96 expression with the clinical characteristics and prognosis of patients with osteosarcoma. The expression of miRNA-122 and miRNA-96 in human osteosarcoma cell lines and tissues were detected in the present study. Reverse transcriptase-PCR (RT-PCR) was used to determine the expression levels of miRNA-122 and miRNA-96 in 68 human OS samples. We found that MiRNA-122 and miRNA-96 were widely up-regulated in osteosarcoma, gastric cancer and pancreatic cancer. In HOS, Saos-2 and U2OS osteosarcoma cells, miRNA-122 and miRNA-96 were up-regulated significantly, while down-regulated in MG-63 cells. After further investigation, we found that miRNA-122 and miRNA-96 concentrations were significantly higher in the tumor tissues than those in the normal tissues (P<0.01). Moreover, the cell proliferation of LV-miRNA-122-RNAi and LV-miRNA-96-RNAi transfected SaOS2 was significantly decreased compared with the LV- miRNA-122-RNAi-CN and LV- miRNA-96-RNAi group. After adjusting for competing risk factors, we found combined high miRNA-122 and miRNA-96 expression was identified as independent predictor of overall survival.

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Neohesperidin Induces Cell Cycle Arrest, Apoptosis, and Autophagy via the ROS/JNK Signaling Pathway in Human Osteosarcoma Cells

The American Journal of Chinese Medicine ◽

10.1142/s0192415x21500609 ◽

2021 ◽

pp. 1-24

Author(s):

Shubin Wang ◽

Zongguang Li ◽

Wei Liu ◽

Guojun Wei ◽

Naichun Yu ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Signaling Pathway ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Jnk Signaling ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Jnk Signaling Pathway ◽

Induced Apoptosis

Neohesperidin has anti-oxidative and anti-inflammatory properties and exerts extensive therapeutic effects on various cancers. In this study, the osteosarcoma cell lines were exposed to different concentrations of neohesperidin. Cell proliferation and viability were assessed by CCK-8 and colony-formation assays. The role of neohesperidin in cell cycle progression and apoptosis were analyzed by flow cytometry and western blotting. To identify autophagosomes and autolysosomes, we used a tandem GFP-mRFP-LC3B lentiviral construct. In addition, autophagy was evaluated by examining autophagosome formation using transmission electron microscopy. Intracellular reactive oxygen species (ROS) production was detected by fluorescence microscopy and flow cytometry. Subsequently, the activation of the ROS/JNK signaling pathway was investigated. Neohesperidin could inhibit proliferation and induce apoptosis in SJSA and HOS cells. The formation of autophagosomes indicated that autophagy occurred in neohesperidin-treated cells and the apoptotic effect of neohesperidin was significantly increased after the use of autophagy inhibitors. Subsequently, we found that neohesperidin-induced apoptosis and autophagy were related to the increase in ROS generation and were significantly inhibited by GSH. Moreover, neohesperidin induced activation of the c-Jun N-terminal kinase (JNK) signaling pathway and inhibition of JNK with SP600125 attenuated neohesperidin-induced apoptosis and autophagy simultaneously. Our data indicated that neohesperidin caused G2/M phase arrest and induced apoptosis and autophagy by activating the ROS/JNK pathway in human osteosarcoma cells, suggesting that neohesperidin is a potential drug candidate for the treatment of osteosarcomas.

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MicroRNA-133a Inhibits Osteosarcoma Cells Proliferation and Invasion via Targeting IGF-1R

Cellular Physiology and Biochemistry ◽

10.1159/000438653 ◽

2016 ◽

Vol 38 (2) ◽

pp. 598-608 ◽

Cited By ~ 32

Author(s):

Guangnan Chen ◽

Tingting Fang ◽

Zhongming Huang ◽

Yiying Qi ◽

Shaohua Du ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Luciferase Reporter ◽

Osteoblastic Cell ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Invasion And Metastasis ◽

Osteoblastic Cell Line ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

Background/Aims: MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. Downregulated microRNAs and their roles in cancer development have attracted much attention. A growing body of evidence showed that microRNA-133a (miR-133a) has inhibitory effects on cell proliferation, migration, invasion, and metastasis of osteosarcoma. Methods: MiR-133a expression in human osteosarcoma cell lines and human normal osteoblastic cell line hFOB was investigated by real-time PCR (RT-PCR). The role of miR-133a in human osteosarcoma growth and invasion was assessed in cell lines in vitro and in vivo. Then, luciferase reporter assay validated IGF-1R as a downstream and functional target of miR-133a, and functional studies revealed that the anti-tumor effect of miR-133a was probably due to targeting and repressing of IGF-1R expression. Results: MiR-133a was lower expressed in human osteosarcoma cell lines than human normal osteoblastic cell line hFOB and its effect on inhibiting proliferation, invasion and metastasis is mediated by its direct interaction with the IGF-1R. Furthermore, the tumour-suppressive function of miR-133a probably contributed to inhibiting the activation AKT and ERK signaling pathway. Conclusion: MiR-133a suppresses osteosarcoma progression and metastasis by targeting IGF-1R in human osteosarcoma cells, providing a novel candidate prognostic factor and a potential anti-metastasis therapeutic target in osteosarcoma.

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Elevated expression of serine/threonine phosphatase type 5 correlates with malignant proliferation in human osteosarcoma.

Acta Biochimica Polonica ◽

10.18388/abp.2014_951 ◽

1970 ◽

Vol 64 (1) ◽

Cited By ~ 2

Author(s):

Kun Han ◽

Zhihua Gan ◽

Shuchen Lin ◽

Haiyan Hu ◽

Zan Shen ◽

...

Keyword(s):

Cell Lines ◽

Functional Role ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Facs Analysis ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell ◽

Elevated Expression ◽

Common Primary Malignant Bone

Osteosarcoma is the most common primary malignant bone tumor in adolescents and young adults. However, the involvement of serine/threonine phosphatase type 5 (PP5) in osteosarcoma remains unclear. The aim of this study was to evaluate the functional role of PP5 in osteosarcoma cells. Firstly, we found that PP5 is widely expressed in several human osteosarcoma cell lines. Then we used lentivirus-delivered siRNA to silence PP5 expression in Saos-2 and U2OS cell lines. Knockdown of endogenous PP5 expression by shRNA-expressing lentivirus significantly decreased the viability and proliferation of the osteosarcoma cells. Moreover, FACS analysis showed that knockdown of PP5 expression induced a significant arrest in the G0/G1 phase of the cell cycle, which was associated with the inhibition of cell proliferation. Therefore, knockdown of PP5 is likely to provide a novel alternative to targeted therapy of osteosarcoma and deserves further investigation.

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