Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2–3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.

Download Full-text

FAM64A Promotes Osteosarcoma Cell Growth and Metastasis and Is Mediated by miR-493

Journal of Oncology ◽

10.1155/2020/2518297 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Ying Jiang ◽

Chunlei Zhou ◽

Qiang Gao ◽

Zhi-Qi Yin ◽

Jingwen Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Poor Prognosis ◽

Migration And Invasion ◽

Tumor Proliferation ◽

Osteosarcoma Cell ◽

Aberrant Expression ◽

Upstream Gene ◽

Cancer Types

Aberrant expression of FAM64A was correlated with cell proliferation in various cancer types. We examined the expression of FAM64A and the upstream gene miR-493 in OS. The functions of miR-493 were revealed through extensive experiments. We found an increase of FAM64A gene and protein in OS tissues. Overexpression of FAM64A resulted in promoting tumor proliferation, migration, and invasion. The miR-493 targeted and negatively regulated FAM64A. Our data showed that upregulation of FAM64A in OS correlated with poor prognosis.

Download Full-text

miR-124 Regulates Caveolin-1 Expression and Affects Proliferation and Apoptosis of Osteosarcoma Cells

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2133 ◽

2019 ◽

Vol 9 (9) ◽

pp. 1245-1249

Author(s):

Huanzhi Ma ◽

Jian Wang ◽

Jun Shi ◽

Wei Zhang ◽

Dongsheng Zhou

Keyword(s):

Cell Proliferation ◽

Cell Apoptosis ◽

Luciferase Activity ◽

Cell Number ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Specific Mechanism ◽

Caveolin 1 ◽

Proliferation And Apoptosis ◽

Relative Luciferase Activity

Osteosarcoma (OS) seriously affects human health. miR-124 expression is closely related to osteosarcoma, but its specific mechanism remains unclear. Our study intends to evaluate miR-124’s effect on osteosarcoma. MG-63 cells were transfected with miR-124 mimics/NC followed by analysis of miR-124 expression by real-time PCR, cell proliferation by CCK8 assay, cell apoptosis by flow cytometry as well as the level of caveolin-1 (CAV1) by Western blot. miR-124 was significantly lower and CAV1 was increased in the four osteosarcoma cells than those in normal osteoblasts (P < 0.05). miR-124 mimics transfection significantly reduced CAV1 level and cell number (P < 0.05) and increased cell apoptosis rate (P < 0.05). Moreover, miR-124 inhibitor significantly promoted the relative luciferase activity in pmirGLO-CAV1-3′UTR-wt-transfected cells (P < 0.05). miR-124 affects osteosarcoma cell proliferation and apoptosis via targeting CAV1.

Download Full-text

Circ-FOXM1 Promotes Cell Proliferation, Migration and EMT Process in Osteosarcoma Through FOXM1-Mediated Wnt Pathway Activation

10.21203/rs.3.rs-522400/v1 ◽

2021 ◽

Author(s):

Hao Zhang ◽

Qiongqiong Zhou

Keyword(s):

Cell Proliferation ◽

Cell Migration ◽

Cell Apoptosis ◽

Biological Activities ◽

Wnt Signaling Pathway ◽

Luciferase Reporter ◽

Circular Rnas ◽

Transcriptional Level ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells

Abstract Background: As the most common primary bone tumor in adolescents and children, osteosarcoma commonly occurs with high mortality rate and metastasis. Emerging evidence has illustrated that circular RNAs (circRNAs) are important regulatory RNAs that are involved in multiple biological activities of carcinomas. Circ-FOXM1 (hsa_circ_0025033) is a recently found circRNA and promotes the cellular activities of several cancers. However, the function and molecular mechanism of circ-FOXM1 in osteosarcoma have not been interrogated yet. Methods: The qRT-PCR was utilized to test the expression of circ-FOXM1 in osteosarcoma cell lines. Loss-of-function assays including CCK-8, EdU, TUNEL, transwell and western blot assays were conducted to measure cell proliferation, cell migration, EMT process and cell apoptosis. Luciferase reporter assay and RIP assay were utilized to detect the interaction of circ-FOXM1 and RNAs. Results：We discovered the high expression of circ-FOXM1 in osteosarcoma cells. Besides, it was indicated that circ-FOXM1 knockdown inhibited cell proliferation, cell migration and EMT process, as well as induced cell apoptosis of osteosarcoma cells. Furthermore, circ-FOXM1 was discovered to upregulate the expression level of forkhead box M1 (FOXM1) at post-transcriptional level. Moreover, it was proved that circ-FOXM1 sponged miR-320a and miR-320b so as to increase FOXM1 expression. Additionally, circ-FOXM1 could activate Wnt signaling pathway through upregulating FOXM1. In the end, rescue assays certified that FOXM1 overexpression could totally rescue the circ-FOXM1 silence-repressed cellular activities of osteosarcoma cells.Conclusion: Circ-FOXM1 facilitated the progression of osteosarcoma cells via relieving FOXM1 from the inhibition by miR-320a and miR-320b.

Download Full-text

Androgen receptor is a potential novel prognostic marker and oncogenic target in osteosarcoma with dependence on CDK11

Scientific Reports ◽

10.1038/srep43941 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 4

Author(s):

Yunfei Liao ◽

Slim Sassi ◽

Stefan Halvorsen ◽

Yong Feng ◽

Jacson Shen ◽

...

Keyword(s):

Cell Growth ◽

Prognostic Marker ◽

Cell Lines ◽

Differentially Expressed Genes ◽

Transcriptional Activation ◽

Disease Free Survival ◽

Bone Cancer ◽

Differentially Expressed ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells

Abstract Osteosarcoma is the most common bone cancer in children and adolescents. Previously, we have found that cyclin-dependent kinase 11 (CDK11) signaling was essential for osteosarcoma cell growth and survival. Subsequently, CDK11 siRNA gene targeting, expression profiling, and network reconstruction of differentially expressed genes were performed between CDK11 knock down and wild type osteosarcoma cells. Reconstructed network of the differentially expressed genes pointed to the AR as key to CDK11 signaling in osteosarcoma. CDK11 increased transcriptional activation of AR gene in osteosarcoma cell lines. AR protein was highly expressed in various osteosarcoma cell lines and patient tumor tissues. Tissue microarray analysis showed that the disease-free survival rate for patients with high-expression of AR was significantly shorter than for patients with low-expression of AR. In addition, AR gene expression knockdown via siRNA greatly inhibited cell growth and viability. Similar results were found in osteosarcoma cells treated with AR inhibitor. These findings suggest that CDK11 is involved in the regulation of AR pathway and AR can be a potential novel prognostic marker and therapeutic target for osteosarcoma treatment.

Download Full-text

Cytoskeletal Actin Structure in Osteosarcoma Cells Determines Metastatic Phenotype via Regulating Cell Stiffness, Migration, and Transmigration

Current Issues in Molecular Biology ◽

10.3390/cimb43030089 ◽

2021 ◽

Vol 43 (3) ◽

pp. 1255-1266

Author(s):

Kouji Kita ◽

Kunihiro Asanuma ◽

Takayuki Okamoto ◽

Eiji Kawamoto ◽

Koichi Nakamura ◽

...

Keyword(s):

Cell Proliferation ◽

Actin Cytoskeleton ◽

Actin Polymerization ◽

Metastatic Potential ◽

New Drugs ◽

Cell Stiffness ◽

Migration And Invasion ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Metastatic Phenotype

Osteosarcoma is the most common primary malignant bone tumor. The cause of death due to osteosarcoma is typically a consequence of metastasis to the lung. Controlling metastasis leads to improved prognosis for osteosarcoma patients. The cell stiffness of several tumor types is involved in metastatic potential; however, it is unclear whether the metastatic potential of osteosarcoma depends on cell stiffness. In this study, we analyzed the cell stiffness of the low metastatic Dunn cell line and its highly metastatic LM8 subline, and compared actin organization, cell proliferation, and metastasis. Actin cytoskeleton, polymerization, stiffness, and other cellular properties were analyzed. The organization of the actin cytoskeleton was evaluated by staining F-actin with Alexa Fluor 488 phalloidin. Cell stiffness was measured using Atomic Force Microscopy (AFM). Cell proliferation, migration, invasion, and adhesion were also evaluated. All experiments were performed using mouse osteosarcoma cell lines cultured in the absence and presence of cytochalasin. In LM8 cells, actin polymerization was strongly suppressed and actin levels were significantly lower than in Dunn cells. Stiffness evaluation revealed that LM8 cells were significantly softer than Dunn. Young’s modulus images showed more rigid fibrillar structures were present in Dunn cells than in LM8 cells. LM8 cells also exhibited a significantly higher proliferation. The migration and invasion potential were also higher in LM8 cells, whereas the adhesion potential was higher in Dunn cells. The administration of cytochalasin resulted in actin filament fragmentation and decreased actin staining intensity and cell stiffness in both LM8 and Dunn cells. Cells with high metastatic potential exhibited lower actin levels and cell stiffness than cells with low metastatic potential. The metastatic phenotype is highly correlated to actin status and cell stiffness in osteosarcoma cells. These results suggest that evaluation of actin dynamics and cell stiffness is an important quantitative diagnostic parameter for predicting metastatic potential. We believe that these parameters represent new reliable quantitative indicators that can facilitate the development of new drugs against metastasis.

Download Full-text

RNF38 inhibits osteosarcoma cell proliferation by binding to CRY1

Biochemistry and Cell Biology ◽

10.1139/bcb-2021-0093 ◽

2021 ◽

pp. 1-7

Author(s):

Jian Zhou ◽

Zhen-yu Tang ◽

Xiao-liang Sun

Keyword(s):

Cell Proliferation ◽

Western Blotting ◽

Colony Formation ◽

Akt Pathway ◽

Osteosarcoma Cell ◽

Mrna Levels ◽

Osteosarcoma Cells ◽

Protein Levels ◽

Qrt Pcr

The PI3K/AKT pathway plays an important role in the development of osteosarcoma. RNF38 interferes with activation of the AKT pathway. Cryptochrome1 (CRY1) inhibits osteosarcoma proliferation through the AKT pathway. We aimed to clarify whether RNF38 affects the proliferation of osteosarcoma cells by regulating the PI3K/AKT pathway through its interaction with CRY1. The mRNA levels of RNF38 were determined using qRT-PCR. Protein levels of RNF38, p-p70S6, p70S6, +p-AKT, AKT, p-mTOR, mTOR, and CRY1 were detected by western blotting. The proliferation of osteosarcoma cells was detected using CCK-8 and colony formation assays. The interaction between CRY1 and RNF38 was detected by co-immunoprecipitation and GST pull-down assays. RNF38 expression was higher in Saos-2 and U20S cells than in hFOB cells. Overexpression of RNF38 promoted the proliferation of osteosarcoma cells, the number of colonies, and p-AKT and p-mTOR levels, suggesting that overexpression of RNF38 activated the PI3K/AKT pathway. In addition, RNF38 directly binds to the N-terminal of CRY1. The simultaneous knockdown of RNF38 and CRY1 restored the level of p-AKT, which was reduced by RNF38 knockdown alone. RNF38 affects the proliferation of osteosarcoma cells by regulating the PI3K/AKT pathway through its interaction with CRY1.

Download Full-text

Using Bioinformatics Analysis to Detect the Effect of Zoledronic Acid on the Biological Behavior of Osteosarcoma Cells

Journal of Medical Imaging and Health Informatics ◽

10.1166/jmihi.2019.2773 ◽

2019 ◽

Vol 9 (8) ◽

pp. 1568-1574

Author(s):

Sheng Li ◽

Jianjun Li

Keyword(s):

Cell Proliferation ◽

Zoledronic Acid ◽

Side Effects ◽

Bioinformatics Analysis ◽

Biological Behavior ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Migration Ability ◽

And Migration ◽

Proliferation And Migration

Objective: Osteosarcoma is a malignant bone tumor commonly seen in adolescents. Drug treatment for osteosarcoma is often accompanied by systemic toxicity and side effects, while zoledronic acid has few side effects but has anti-tumor effects. Methods: The bioinformatics analysis and scratch test were used to detect changes in cell proliferation, migration, and apoptosis in two osteosarcoma cell lines 24, 48, and 72 hours after adding zoledronic acid (0, 25, 50, 100, and 200 μM). Flow cytometry and transmission electron microscopy were used to observe the changes in cell apoptosis in the control and experimental groups after a 50% inhibitory dose of zoledronic acid was given. Results: The inhibition of cell proliferation and migration ability, as well as apoptosis increased with the increase in zoledronic acid exposure time and concentration. The 50% inhibitory rate occurred 48 hours after treatment with 100 M zoledronic acid. Conclusion: Zoledronic acid inhibited proliferation and migration and promoted apoptosis of osteosarcoma cells in vitro.

Download Full-text

The proliferation and invasion of osteosarcoma are inhibited by miR-101 via targetting ZEB2

Bioscience Reports ◽

10.1042/bsr20181283 ◽

2019 ◽

Vol 39 (2) ◽

Cited By ~ 5

Author(s):

Haopeng Lin ◽

Xiaodong Zheng ◽

Ting Lu ◽

Yang Gu ◽

Canhao Zheng ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Molecular Mechanisms ◽

Bone Cell ◽

Luciferase Reporter ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Human Osteosarcoma ◽

Direct Target ◽

Proliferation And Invasion

AbstractHaving a better grasp of the molecular mechanisms underlying carcinogenesis and progression in osteosarcoma would be helpful to find novel therapeutic targets. Different types of cancers have presented abnormal expression of miRNA-101 (miR-101). Nevertheless, we still could not figure out what expression of miR-101 in human osteosarcoma is and its biological function. Thus, we conducted the present study to identify its expression, function, and molecular mechanism in osteosarcoma. We detected the expression of miR-101 in osteosarcoma samples and cell lines. The effects of miR-101 on osteosarcoma cells’ proliferation and invasion were evaluated. Luciferase reporter assay was applied to identify the direct target of miR-101. Compared with adjacent normal specimens and normal bone cell line by using qPCR, the expression levels of miR-101 in osteosarcoma specimens and human osteosarcoma cell lines distinctly decreased. According to function assays, we found that overexpression of miR-101 significantly inhibited the cell proliferation and invasion in osteosarcoma cells. Moreover, we confirmed that zinc finger E-box binding homeobox 2 (ZEB2) was a direct target of miR-101. In addition, overexpression of ZEB2 could rescue the inhibition effect of proliferation and invasion induced by miR-101 in osteosarcoma cells. MiR-101 has been proved to be down-regulated in osteosarcoma and has the ability to suppress osteosarcoma cell proliferation and invasion by directly targetting ZEB2.

Download Full-text

Cisplatin inhibits the proliferation of Saos-2 osteosarcoma cells via the miR-376c/TGFA pathway

Bosnian Journal of Basic Medical Sciences ◽

10.17305/bjbms.2020.4485 ◽

2020 ◽

Author(s):

Yuan Wang ◽

Yichao Wu ◽

Awei Cai ◽

Chengxiao Ma ◽

Shang Cai ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Protein Expression ◽

Mrna Expression ◽

Transforming Growth Factor ◽

Combined Treatment ◽

Luciferase Reporter ◽

Osteosarcoma Cells ◽

Inhibitory Effects ◽

Reverse Transcription Pcr

Transforming growth factor alpha (TGFA) gene is involved in the proliferation and metastasis of various tumors, but its role in cell sensitivity to cisplatin chemotherapy is unclear. In this study, we investigated the mechanism underlying inhibitory effects of cisplatin on growth and proliferation of osteosarcoma cells. Osteosarcoma and normal skeletal muscle tissues were collected from 26 patients by biopsy. TGFA was silenced or overexpressed in Saos-2 osteosarcoma cells by transfection with TGFA-shRNA or TGFA ORF clone, respectively. MiR-376c was inhibited or overexpressed by transfection of Saos-2 cells with miR-376c sponge or miR-376c mimics, respectively. Cell growth was analyzed by MTT assay and cell proliferation by BrdU assay. MiR-376c and TGFA mRNA expression was detected by quantitative reverse transcription PCR and TGFA protein expression by Western blot. The target relationship between miR-376c and TGFA was assessed by luciferase reporter assay. Both in osteosarcoma tissues and Saos-2 cells, miR-376c expression was significantly decreased and TGFA mRNA expression was significantly increased compared with control. Transfection of Saos-2 cells with TGFA-shRNA silenced TGFA expression and significantly inhibited cell growth and proliferation. TGFA mRNA and protein expression in Saos-2 cells significantly decreased with increasing cisplatin concentrations (2.5–10 mg/L). Transfection with TGFA ORF clone reversed the inhibitory effects of cisplatin on Saos-2 cell proliferation. Compared with cisplatin (10 mg/L) treatment alone, the combined treatment with cisplatin and miR-376c mimics inhibited the proliferation of Saos-2 cells more significantly. MiR-376c suppressed TGFA expression by directly interacting with its 3'-UTR region. Overall, cisplatin inhibited the proliferation of Saos-2 cells by upregulating miR-376c and downregulating TGFA expression.

Download Full-text

Inhibition of ERK1/2 Signaling Pathway is Involved in Melatonin's Antiproliferative Effect on Human MG-63 Osteosarcoma Cells

Cellular Physiology and Biochemistry ◽

10.1159/000447922 ◽

2016 ◽

Vol 39 (6) ◽

pp. 2297-2307 ◽

Cited By ~ 23

Author(s):

Lifeng Liu ◽

Ying Xu ◽

Russel J. Reiter ◽

Yutao Pan ◽

Di Chen ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Cyclin D1 ◽

Signaling Pathways ◽

Signaling Pathway ◽

Cyclin B1 ◽

Akt Signaling ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

M Phase

Background: In a previous study, we found that melatonin inhibits MG-63 osteosarcoma cell proliferation; however, the underlying mechanisms remain elusive. Mitogen-activated protein kinase (MAPK) and Akt signaling pathways play key roles in the anticancer effects of melatonin. Aims: The present study investigated whether MAPK and Akt signaling pathways are involved in melatonin's antiproliferative actions on the human MG-63 osteosarcoma cells. Methods/Results: Western blot analysis confirmed that melatonin significantly inhibited phosphorylation of ERK1/2 but not p38, JNK, or Akt. The expression of ERK1/2, p38, JNK, and Akt was not altered by melatonin. PD98059 and melatonin alone, and especially in combination, significantly inhibited cell proliferation. The changes included G1 and G2/M phase arrest of the cell cycle, and a downregulation of the expression at both the protein and mRNA levels of cyclin D1 and CDK4 (related to the G1 phase) and of cyclin B1 and CDK1 (related to the G2/M phase) as measured by flow cytometry after propidium iodide staining, and both western blot and real-time PCR, respectively. Furthermore, the combination of PD98059 and melatonin synergistically and markedly augmented the action of either agent alone. Co-immunoprecipitation further confirmed that there was an interaction between p-ERK1/2 and cyclin D1, CDK4, cyclin B1, or CDK1, which was blunted in the presence of melatonin or PD98059. Conclusion: These findings suggest that melatonin's antiproliferative action is mediated by inhibition of the ERK1/2 signaling pathway rather than the p38, JNK, or Akt pathways.

Download Full-text