Xin Hua Long

Inhibition of fatty acid synthase suppresses osteosarcoma cell invasion and migration via downregulation of the PI3K/Akt signaling pathway in vitro

In the present study, the effect of fatty acid synthase (FASN) inhibition on cell invasion and migration in vitro was investigated. A recombinant plasmid containing a microRNA targeting the FASN gene was used to inhibit FASN expression in U2‑OS cells. Cell migration and invasion were investigated using wound healing and Transwell invasion assays. We found that cell invasion and migration were suppressed by inhibiting FASN. In addition, the effect of inhibition of FASN on phosphorylation of Akt was investigated by detecting the expression levels of pAkt using western blot analysis. Furthermore, protein expression levels of nuclear factor‑κB (NF‑κB; p65) and matrix metalloproteinase (MMP)‑2 and ‑9 were also measured by western blot analysis. Results demonstrated that expression levels of pAkt, NF‑κB (p65) and MMP‑2 and ‑9 proteins were reduced significantly by inhibiting FASN. Therefore, we confirmed that inhibition of FASN by RNA interference suppresses osteosarcoma cell metastasis via downregulation of the phosphoinositide 3‑kinase/Akt/NF‑κB signaling pathway in vitro.

Demethylation-mediated miR-129-5p up-regulation inhibits malignant phenotype of osteogenic osteosarcoma by targeting Homo sapiens valosin-containing protein (VCP)

Previous studies demonstrated that increased Homo sapiens valosin-containing protein (VCP) may be involved in osteosarcoma (OS) metastasis. However, the underlying mechanism of VCP over-expression in OS remains unknown. In the present study, we found a significantly negative correlation between miR-129-5p and VCP protein expression in OS tissues with pulmonary metastasis (Spearmans rho, rs = −0.948). Bioinformatical prediction, Luciferase reporter assay, Western blot, and RT-PCR assays performed on OS cells indicated that VCP is a target of miR-129-5p. In addition, three CPG islands in the region of miR-129-5p promoter were detected by bioinformatical prediction, and significantly higher expression of miR-129-5p and lower methylation level of miR-129-2 gene in OS cells treated with 5-Aza-2′-deoxycytidine (a potent DNA demethylating agent) than in those untreated cells were observed. Furthermore, lower migratory and invasive ability was found in cells with elevated miR-129-5p than in those with decreased miR-129-5p. These findings indicated that increased miR-129-5p may be mediated by demethylation and inhibit OS cell migration and invasion by targeting VCP in OS, and targeting miR-129-5p/VCP signaling pathway may serve as a therapeutic strategy for OS management, although further studies will be necessary.

LY294002 inhibits the malignant phenotype of osteosarcoma cells by modulating the phosphatidylinositol 3‑kinase/Akt/fatty acid synthase signaling pathway in vitro

Increasing evidence suggests that fatty acid synthase (FASN) is crucial in the carcinogenesis of various types of tumor. In addition, the phosphatidylinositol 3‑kinase (PI3K)/Akt signaling pathway, which is closely associated with cellular metabolism, affects cancer biology. However, whether the malignant phenotype of osteosarcoma (OS) cells is regulated by the PI3K/Akt/FASN signaling pathway and how the PI3K family specific inhibitor, 2‑(4‑morpholinyl)‑8‑phenyl‑chromone (LY294002) affects the malignant phenotype of OS cells remains to be elucidated. In the present study, U2‑OS and MG‑63 cells were treated with LY294002 and subsequently western blot analysis was used to examine Akt, p‑Akt and FASN protein expression. Additionally, FASN mRNA was detected by reverse transcription quantitative polymerase chain reaction. MTT and fluorescence‑activated cell sorting assays were used to assess proliferation and apoptosis. Migration and invasion were investigated using wound healing and transwell invasion assays. The results demonstrated that LY294002 suppressed the PI3K/Akt/FASN signaling pathway. However, the malignant phenotypes of OS cells mentioned above were significantly inhibited. The present results indicated that LY294002 inhibits the malignant phenotype of OS cells via modulation of the PI3K/Akt/FASN signaling pathway in vitro and may be a new therapeutic strategy for the management of OS.

Inhibiting valosin-containing protein suppresses osteosarcoma cell metastasis via AKT/nuclear factor of kappa B signaling pathway in vitro

BACKGROUND AND AIM The strategies of targeting valosin-containing protein (VCP) may have therapeutic potential for treating cancer metastasis. In this study, we aim to investigate the correlation of VCP protein expression in osteosarcoma (OS) tissues with pulmonary metastasis and its possible molecular mechanism. MATERIALS AND METHODS Expression of VCP in 60 OS specimens was detected by immunohistochemistry (IHC) and the relationship with metastasis was analyzed. An artificial micro ribonucleic acid, targeting VCP, was performed to silence the expression of VCP in U2-OS cells. Cell mobility was detected by wound healing and Transwell assays. Western blot and real-time polymerase chain reaction were performed to investigate the expression of VCP in U2-OS cells. Furthermore, the protein of pAKT (phosphorylated serine/threonine protein kinase) and nuclear factor of kappa B protein 65 were measured by western blot to evaluate the effect of silencing VCP on AKT/nuclear factor of kappa B (NF-kB) signaling pathway. RESULTS The results showed that the expression level of VCP protein in cases with pulmonary metastases was significantly higher than that in those without metastasis (P = 0.004). The invasion and migration of U2-OS cells were suppressed by silencing VCP. Furthermore, silencing VCP could down-regulate the phosphorylation of AKT and nuclear transfer of NF-kB. CONCLUSIONS Our findings suggested that inhibition of VCP could suppress OS cells invasion and migration through down-regulating AKT/NF-kB signaling pathway.

Positive feedback regulation between Akt phosphorylation and fatty acid synthase expression in osteosarcoma

The activation of PI3K/Akt and the overexpression of fatty acid synthase (FASN) are frequently observed in human osteosarcoma (OS). In the present study, in order to investigate the possible association between the phosphorylation of Akt and FASN expression, immunohistochemical staining was conducted on 24 OS specimens from patients with pulmonary metastasis, which revealed a significant positive correlation between phosphorylated Akt (p-Akt) and the expression of FASN (R=0.469, P=0.04). To investigate the association between p-Akt and FASN in vitro, human U2-OS OS cells were treated with FASN-specific RNAi plasmid or LY294002 (an inhibitor of PI3k/Akt). The mRNA levels of Akt and FASN were measured by real-time PCR. Western blot analysis was also performed to detect the protein experession of PI3K, Akt, p-Akt and FASN. The results demonstrated that the PI3K/Akt signaling pathway modulates FASN expression; the inhibition of FASN resulted in the downregulation of p-Akt in the U2-OS cells. Furthermore, the effects induced by the inhibition of the activity of p-Akt or FASN on the malignant phenotype of U2-OS cells were investigated, demonstrating that the malignant phenotype was inhibited by suppressing the activity of PI3K/Akt or FASN in the U2-OS cells. The findings from our study suggest the existence of a positive feedback regulation between Akt phosphorylation and FASN expression and that this loop may play an important role in the malignant phenotype of OS cells.

Inhibition of Aurora-B suppresses osteosarcoma cell migration and invasion.

Previous studies have suggested that Aurora-B may be involved in cancer metastasis. However, its role has been poorly evaluated in osteosarcoma (OS). The aim of this study was to investigate the correlation between Aurora-B expression and metastasis in human OS. The human OS cell line, U2-OS, and OS biopsy specimens were used in the study. The expression of Aurora-B protein was examined using immunohistochemistry and western blotting in OS tissues and U2-OS cells, respectively. AZD1152-hydroxyquinazoline-pyrazol-anilide, an inhibitor of Aurora-B, was used to inhibit Aurora-B expression in U2-OS cells. The effect of Aurora-B inhibition on U2-OS cell proliferation, invasion and migration was assessed using MTT, colony formation, wound healing and Transwell assays. The results showed that positive expression of the Aurora-B protein was observed in the nucleus, and that Aurora-B expression levels in the cases with pulmonary metastases were significantly higher than in those without metastasis. In vitro, the proliferation, invasion and migration of U2-OS cells were suppressed by the inhibition of Aurora-B. These results suggest that Aurora-B may be involved in OS metastasis, and may be a promising target in the treatment of OS metastasis.

RNA interference-mediated knockdown of Aurora-B alters the metastatic behavior of A549 cells via modulation of the phosphoinositide 3-kinase/Akt signaling pathway

Accumulating evidence has revealed that an elevated expression level of Aurora-B is associated with metastasis in various types of malignant tumor. However, it is currently unclear whether this molecule is involved in non-small lung cancer (NSCLC) metastasis, and the molecular mechanisms associated with Aurora-B and metastasis remain unknown. In the present study, in order to investigate whether Aurora-B is involved in the development and metastasis of NSCLC, the Aurora-B protein expression in NSCLC tissues was detected by immunohistochemistry and its association with metastasis was analyzed. The results revealed that the expression levels of the Aurora-B protein in tissues obtained from NSCLC patients with lymph node metastasis were significantly higher than those without metastatic disease. Furthermore, the effect of Aurora-B inhibition on A549 cell migration and invasion, as well as the activity of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was evaluated. Aurora-B was inhibited in the A549 cells using short hairpin RNA, and the cell migration and invasion rates were investigated using wound healing and Transwell invasion assays. In addition, the expression of the main proteins in the PI3K/Akt/nuclear factor-κB (NF-κB) signaling pathway, and matrix metalloproteinase (MMP)-2 and -9 were measured by western blot analysis. The results demonstrated that cell migration and invasion were decreased as a result of silencing Aurora-B. Furthermore, the activity of the PI3K/Akt/NF-κB signaling pathway and the expression of MMP-2 and -9 protein were suppressed by silencing Aurora-B. The results of the present study indicate that the knockdown of Aurora-B suppresses A549 cell invasion and migration via the inhibition of the PI3K/Akt signaling pathway in vitro and thus, targeting Aurora-B may present a potential treatment strategy for NSCLC.

HELQ reverses the malignant phenotype of osteosarcoma cells via CHK1-RAD51 signaling pathway

HELQ is a DNA helicase important for repair of DNA lesions and has been linked to several types of cancer. However, little is known about its relationship with osteosarcoma (OS) and its mechanism. In the present study, the expression of HELQ and its downstream mediators in OS cells was assayed by quantitative PCR and western blot analysis. The function of HELQ in OS cells was investigated by Transwell invasion, wound healing, CCK8 assays and Comet assay. The results demonstrated that HELQ gene and protein were expressed in OS cells. OS cell invasion, migration, proliferation and DNA damage repair were enhanced by HELQ knock-down with shRNA-lentivirus and inhibited by HELQ overexpression with lentivirus transfection. Furthermore, the antitumor activities of HELQ may be associated with upregulated expression of the DNA damage-related proteins CHK1 and RAD51. Our findings indicated that HELQ confers an anti-invasive phenotype on OS cells by activating the CHK1-RAD51 signaling pathway and suggested that HELQ could be recognized as a promising therapeutic target for OS and other types of malignant tumors.

LY294002 suppresses the malignant phenotype and sensitizes osteosarcoma cells to pirarubicin chemotherapy.

Pirarubicin is frequently used in chemotherapy against tumors. However, clinical use is severely limited by the development of progressive dose-dependent cardiomyopathy and acquired drug resistance. LY294002 is a commonly used pharmacologic inhibitor, which selectively inhibits the phosphoinositide 3-kinase-AKT nexus. The aim of this study was to investigate the combined inhibitory effect of LY294002 and pirarubicin on human osteosarcoma (OS) cells in vitro. The inhibitory effect of LY294002 plus pirarubicin on U2-OS and MG-63 OS cell proliferation, apoptosis, migration and invasion was investigated by cell proliferation, wound healing and Transwell invasion assays. The results revealed that LY294002 and pirarubicin synergistically induced apoptosis, and inhibited the growth, migration and invasion of OS cells. This indicates that LY294002 enhanced the effects of pirarubicin on OS in vitro. LY294002 combined with pirarubicin may thus be a future therapeutic strategy in OS.

Transcription factor NF‑YA promotes a malignant phenotype by upregulating fatty acid synthase expression

Recent studies have revealed that increased expression of the alpha subunit of nuclear transcription factor Y (NF-YA) is associated with the malignant phenotype of various tumors. However, whether elevated expression of NF-YA promotes a malignant phenotype in osteosarcoma (OS), and the molecular mechanisms underlying this predicted effect is currently unknown. In the present study, small hairpin RNA (shRNA)-mediated knockdown of endogenous NF-YA significantly inhibited the migration and invasion capabilities of OS cells in vitro, whereas ectopic expression of NF-YA increased the migration and invasion capabilities of these cells. In addition, the induction of upregulated NF-YA expression on the malignant phenotype of OS cells was attenuated by silencing fatty acid synthase (FASN) expression. Furthermore, the expression level of FASN was increased by upregulating NF-YA, while decreased FASN expression was observed following NF-YA silencing in OS cells. The results of the present study suggest that NF-YA may promote a malignant phenotype in OS cells, in part, by activating the FASN signaling pathway, which may represent a promising target for the management of OS.