Anti-osteosarcoma Biological Activity Evaluation and Complete Chloroplast Genome Sequencing of Populus yunnanensis.

Recently, the Populus yunnanensis extract has drawn the attention of most researchers, because of their anti-cancer activity. In this present research, the anti-cancer activity of the Populus yunnanensis extract was measured with Cell Counting Kit-8 (CCK-8) detection kit on the cancer cells. Then, the inhibitory activity of the Populus yunnanensis extract on the migration and invasion ability of the cancer cells was also determined in this present research with trans-well assay. Subsequently, to reveal the evolutionary genome evolution evaluation of the Populus yunnanensis and other Populus species, the high-throughput Illumina pair-end sequencing was performed and the chloroplast (cp) genome of Populus yunnanensis was determined, and the phylogenetic analysis was finished as wells. The results of the CCK-8 assay indicated that the Populus yunnanensis extract showed inhibitory effect on the cancer cell viability. Besides, the migration and invasion ability of the cancer cell was also reduced by the Populus yunnanensis extract. The complete chloroplast genome sequence results revealed that the Populus yunnanensis has a 156,505 bp circular cp genome. The phylogenetic analysis further revealed that the Populus yunnanensis has closely relationship with Populus simonii.

Neohesperidin promotes the osteogenic differentiation of bone mesenchymal stem cells by activating the Wnt/ß-catenin signaling pathway.

BACKGROUND: Osteoporosis is a common disease in aging populations. However, osteoporosis treatment is still challenging. Here, we aimed to investigate the role of neohesperidin (NEO) in osteoporosis progression and the potential mechanism. METHODS: Bone mesenchymal stem cells (BMSCs) were isolated and treated with different concentrations of NEO (0, 10, 30, 100 µM). Cell proliferation was analyzed by cell count kit-8 (CCK-8) assay. RNA-sequencing was performed on the isolated BMSCs with control and NEO treatment. Differentially expressed genes were obtained by R software. Alkaline phosphatase (ALP) staining and Alizarin red staining (ARS) were performed to assess the osteogenic capacity of the NEO. qRT-PCR was used to detect the expression of osteoblast markers. Western blot was used to evaluate the protein levels in BMSCs. RESULTS: NEO treatment significantly improved hBMSC proliferation at different time points, particularly when cells were incubated with 30 µM NEO (P < 0.05). NEO dose-dependently increased the ALP activity and calcium deposition than the control group (P < 0.05). A total of 855 differentially expressed genes were identified according to the significance criteria of log2 (fold change) > 1 and adj P < 0.05. DKK1 partially reversed the promotion effects of NEO on osteogenic differentiation of BMSCs. NEO increased levels of the ß-catenin protein in BMSCs. CONCLUSION: NEO plays a positive role in promoting osteogenic differentiation of BMSCs, which was related with activation of Wnt/ß-catenin pathway.

microRNA-539 suppresses tumor growth and tumorigenesis and overcomes arsenic trioxide resistance in hepatocellular carcinoma.

AIMS: Dysregulation of microRNAs (miRNAs) plays a critical role in tumor growth and progression. In this study, we sought to explore the expression and biological roles of miR-539 in hepatocellular carcinoma (HCC). MAIN METHODS: The expression of miR-539 in human HCC tissues and cell lines was examined. The effects of miR-539 overexpression on cell growth, tumorigenicity, arsenic trioxide resistance of HCC cells were determined. The signaling pathways involved in the action of miR-539 in HCC were also investigated. KEY FINDINGS: miR-539 was downregulated in HCC tissues and cells, relative to corresponding controls. Overexpression of miR-539 inhibited HCC cell viability and colony formation in vitro and impaired tumorigenesis of HCC cells in vivo. Transfection with miR-539 mimic significantly induced apoptosis in HepG2 cells, which was coupled with reduced expression of anti-apoptotic proteins Bcl-2 and Bcl-xL and decreased phosphorylation of Stat3. Overexpression of a constitutively active form of Stat3 partially blocked miR-539-mediated apoptosis. Enforced expression of miR-539 resensitized arsenic trioxide-resistant HCC cells to arsenic trioxide. Intratumoral delivery of miR-539 mimic significantly retarded the growth of xenograft tumors from arsenic trioxide-resistant HCC cells by about 35%, compared to delivery of control miRNA (P<0.05). In combination with arsenic trioxide, miR-539 mimic yielded about 80% decrease in tumor burden. SIGNIFICANCE: miR-539 functions as a tumor suppressor in HCC and reexpression of this miRNA offers a potential therapeutic strategy for this disease.

Bufalin Inhibits the Differentiation and Proliferation of Cancer Stem Cells Derived from Primary Osteosarcoma Cells through Mir-148a.

BACKGROUND/AIMS: Osteosarcoma (OS) is the second leading cause of cancer-related death in children and young adults. Chemoresistance is the most important cause of treatment failure in OS, largely resulting from presence of cancer stem cells (CSCs). However, CSCs isolated from cancer cell lines do not necessarily represent those from primary human tumors due to accumulation of genetic aberrations that increase with passage number. Therefore, studies on CSCs from primary OS may be more important for understanding the mechanisms driving the chemoresistance of CSCs in OS. METHODS: We established a primary culture of OS cells, known as C1OS, from freshly resected tumor tissue. We further isolated CSCs from C1OS cells (C1OS-CSCs). We analyzed the effects of bufalin, a traditional Chinese medicine, on the stemness of C1OS-CSCs. We also analyzed the microRNA (miR) targets of bufalin on the stemness of C1OS-CSCs. Moreover, we examined these findings in the OS specimen. RESULTS: Bufalin inhibited the stemness of C1OS-CSCs. Moreover, we found that miR-148a appeared to be a target of bufalin, and miR-148a further regulated DNMT1 and p27 to control the stemness of OS cells. This mechanism was further confirmed in OS specimen. CONCLUSION: Our data suggest that bufalin may be a promising treatment for OS, and its function may be conducted through regulation of miR-148a.

Bufalin exerts inhibitory effects on IL-1ß-mediated proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) proliferate abnormally and resist apoptosis. Bufalin inhibits cell proliferation and induces apoptosis in human cancer cells. In this study, we explored the effects of bufalin on interleukin-1beta (IL-1ß)-induced proliferation and apoptosis of RAFLSs. The cell proliferation and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and annexin V/propidium iodide staining, respectively. Bufalin dose-dependently inhibited IL-1ß-induced RAFLS proliferation. Mechanistically, bufalin decreased the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB), both of which are involved in IL-1ß-mediated RAFLS proliferation. Moreover, bufalin induced apoptosis and mitochondrial damage of RAFLSs, which was associated with Bcl-2 downregulation, Bax upregulation, mitochondrial cytochrome c release, and enhanced cleavages of caspase-3 and poly-(ADP-ribose) polymerase. Collectively, our results reveal that bufalin suppresses IL-1ß-induced proliferation of RAFLSs through MAPK and NF-κB signaling pathways and induces RAFLS apoptosis via the mitochondria-dependent pathway.

Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

Liver X receptor α inhibits osteosarcoma cell proliferation through up-regulation of FoxO1.

BACKGROUND: Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. METHODS: We analyzed liver X receptor α (LXRα) mRNA expression in 16 pairs of human osteosarcoma tissues and adjacent noncancerous tissues. Moreover, we investigated LXRα's potential role in regulating cell proliferation in Saos-2 and U2OS cells. RESULTS: We found that activation of LXRα, a member of nuclear receptor, was able to inhibit cell proliferation in Saos-2 and U2OS cells. At the molecular level, our results further revealed that expression of tumor suppressor gene, FoxO1, was up-regulated by LXRα activation. LXRα activates FoxO1 transcription through a direct binding on its promoter region. CONCLUSION: LXRα acts as a tumor suppressor for osteosarcoma, which may offer a new way in molecular targeting cancer treatment.