p-Coumaric Acid Inhibits Osteosarcoma Growth by Inhibiting PI3K/Akt Signaling Pathway.

P-coumaric acid (p-CA) is a kind of phenylpropionic acid derived from aromatic amino acids, which is widely distributed in many plants and human diets. It has strong pharmacological and inhibitory effects on a variety of tumors. However, the role of p-CA in osteosarcoma, a tumor with a poor prognosis, is still unknown. Therefore, we aimed to evaluate the effect of p-CA on osteosarcoma and explore its potential mechanism. This study aimed to investigate whether p-CA has an inhibitory effect on the growth of osteosarcoma cells and explore its potential mechanism. MTT assay and clonogenic assay were used to detect the effect of p-CA on the proliferation of osteosarcoma cells. The effect of p-CA on apoptosis of osteosarcoma cells was detected by the Hoechst staining and flow cytometry. The effects of p-CA on the migration and invasion of osteosarcoma cells were detected by scratch healing assay and Transwell invasion assay. Western blot and PI3K/Akt pathway activator 740Y-P were used to detect the anti-tumor mechanism of p-CA on osteosarcoma cells. The effect of p-CA on osteosarcoma cells in vivo was verified by an orthotopic osteosarcoma tumor animal model in nude mice. MTT assay and clonogenic assay showed that p-CA inhibited the proliferation of osteosarcoma cells. Hoechst stain and flow cytometry showed that p-CA could induce apoptosis of osteosarcoma cells and lead to G2 phase arrest of osteosarcoma cells. Transwell assay and scratch healing assay found that p-CA could inhibit the migration and invasion of osteosarcoma cells. Western blot showed that p-CA could inhibit the activity of the PI3K/Akt signaling pathway in osteosarcoma cells, and 740Y-P could reverse its inhibitory effect. In vivo mouse models, p-CA has an antitumor effect on osteosarcoma cells in vivo, and at the same time, it has less toxic side effects on mice. This study demonstrated that p-CA could effectively inhibit the proliferation, migration and invasion of osteosarcoma cells and promote apoptosis. p-CA may play an anti-osteosarcoma role by inhibiting PI3K/Akt signaling pathway.

Yang B ，Wang B ，Wang G ，Cao W ，Wang Q ，Pu H ，An W ... -  《-》

Cinnamaldehyde Inhibits the Function of Osteosarcoma by Suppressing the Wnt/β-Catenin and PI3K/Akt Signaling Pathways.

Osteosarcoma (OS) is a primary bone tumor associated with locally aggressive growth and early metastatic potential that typically occurs in children and adolescents. Chinese traditional medicine Cinnamomum cassia Presl has been shown to have significant tumor-killing effect, in which cinnamaldehyde (CA) is the main active ingredient. To explore the anticancer effect of CA on the osteosarcoma cells and the possible molecular mechanism. Crystal violet assay, MTT assay and colony-forming assay were used to confirm the inhibitory role of CA in the proliferation of 143B and MG63 osteosarcoma cells. Hoechst 33258 staining and flow cytometry were used to observe apoptosis. The migration and invasion role of OS cells were evaluated using transwell assays and wound healing assays. Western blotting was used to analyse the protein expression levels. Nude mice were inoculated with 143B cells to establish an orthotopic OS tumor animal model and to investigate the effects of CA on OS tumors. According to crystal violet assay, MTT assay and colony-forming assay, CA significantly inhibited cell proliferation. Hoechst 33258 staining and flow cytometry analysis showed that CA-induced apoptosis in a concentration-dependent manner. In addition, transwell assays and wound healing assays showed that CA inhibited the migration and invasion of osteosarcoma cells. In vivo mouse models, CA inhibited the growth of osteosarcoma. The potential mechanisms could be that CA inhibited the transcriptional activity of Wnt/β-catenin and PI3K/Akt of the osteosarcoma. CA may inhibit the proliferation, migration, invasion and promote apoptosis of OS cells by inhibiting Wnt/β-catenin and PI3K/Akt signaling pathways. CA may be a potentially effective anti-tumor drug.

Huang Y ，Chen J ，Yang S ，Tan T ，Wang N ，Wang Y ，Zhang L ，Yang C ，Huang H ，Luo J ，Luo X ... -  《Drug Design Development and Therapy》

Genipin Inhibits the Development of Osteosarcoma through PI3K/AKT Signaling Pathway.

Osteosarcoma is a highly invasive and early metastatic tumor. At present, the toxic and side effects of chemotherapy affect the quality of life of cancer patients to varying degrees. Genipin is an extract of the natural medicine gardenia with various pharmacological activities. The purpose of this study was to investigate the effect of Genipin on osteosarcoma and its potential mechanism of action. Crystal violet staining, MTT assay and colony formation assay were used to detect the effect of genipin on the proliferation of osteosarcoma. The effects of vitexin on migration and invasion of osteosarcoma were detected by scratch healing assay and transwell assay. Hoechst staining and flow cytometry were used to detect the effect of genipin on apoptosis of osteosarcoma cells. The expression of related proteins was detected by Western blot. An orthotopic tumorigenic animal model was used to verify the effect of genipin on osteosarcoma in vivo. The results of crystal violet staining, MTT method and colony formation method proved that genipin significantly inhibited the proliferation of osteosarcoma cells. The results of the scratch healing assay and transwell assay showed that gen significantly inhibited the migration and invasion of osteosarcoma cells. The results of Hoechst staining and flow cytometry showed that genipin significantly promoted the apoptosis of osteosarcoma cells. The results of animal experiments show that genipin has the same anti-tumor effect in vivo. Genipin may inhibit the growth of osteosarcoma through PI3K/AKT signaling. Genipin can inhibit the growth of human osteosarcoma cells, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway.

Huang X ，Jiwa H ，Xu J ，Zhang J ，Huang Y ，Luo X ... -  《-》

Knockdown of TBRG4 suppresses proliferation, invasion and promotes apoptosis of osteosarcoma cells by downregulating TGF-β1 expression and PI3K/AKT signaling pathway.

Transforming growth factor beta regulator 4 (TBRG4) is a novel regulator in tumorigenic progression of several tumors. However, so far, the expression and functions of TBRG4 in osteosarcoma are unknown. The aim of this study was to investigate the potential biological functions of TBRG4 in osteosarcoma. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of TBRG4 in osteosarcoma tissues and cell lines. The levels of TBRG4 protein in osteosarcoma tissues were assessed by immunohistochemistry. Lentivirus-mediated short hairpin (sh) RNA was employed to knock down TBRG4 in osteosarcoma cells, and the expressions of TBRG4 mRNA and protein were determined by qRT-PCR and Western blot assay, respectively. Subsequently, the proliferation, clonogenic ability, apoptosis and invasion of osteosarcoma cells were measured using high content screening analysis and CCK8 assay, tumor sphere formation assay, flow cytometry and Transwell invasion assays, respectively. Furthermore, the osteosarcoma cells growth and metastasis in vivo were detected, and the effect of TBRG4 on the transforming growth factor β1 (TGF-β1) and PI3K/AKT signaling pathway was explored by qRT-PCR and Western blot assay, respectively. The results showed the levels of TBRG4 were overexpressed in osteosarcoma tissues and cell lines, confirming that the high TBRG4 expression was related to advanced tumor stages, large tumor size, and lymph node metastasis. Functional assays showed knockdown of TBRG4 could inhibit proliferation, invasion and induce apoptosis of osteosarcoma cells in vitro, and could also suppress osteosarcoma growth and metastasis in vivo. By examining the expression levels of TGF-β1, p-PI3K, PI3K, p-AKT and AKT, it showed that the suppression of TBRG4 would reduce TGF-β1 expression and inactivate the PI3K/AKT signaling pathway. These results showed for the first time that TBRG4 knockdown could suppress osteosarcoma progression, suggesting TBRG4 might be a promising therapeutic target for osteosarcoma treatment.

Huang F ，Zhou P ，Wang Z ，Zhang XL ，Liao FX ，Hu Y ，Chang J ... -  《-》

RILP inhibits tumor progression in osteosarcoma via Grb10-mediated inhibition of the PI3K/AKT/mTOR pathway.

Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism. Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed. Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial-mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway. Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment.

Wei Z ，Xia K ，Zheng D ，Gong C ，Guo W ... -  《-》