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

2016 ◽  
Vol 39 (6) ◽  
pp. 2297-2307 ◽  
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.

miR-184 Inhibits Hypertrophic Scar Through Regulating Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (AKT) Signaling Pathway

2020 ◽  
Vol 10 (1) ◽  
pp. 133-138
Author(s):  
Peng Zhao ◽  
Junxia Qin ◽  
Lili Liang ◽  
Xinzhong Zhang
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Hypertrophic Scar ◽  
Scar Tissue ◽  
Akt Signaling ◽  
Scar Formation ◽  
Akt Signaling Pathway

Hypertrophic scar (HS) is a process of tissue repair and healing, and excessive fibrosis of local tissue leads to scar formation. During HS formation, fibroblasts (Fb) proliferate, synthesize and secrete and promote HS development. miR-184 regulates skin formation and tissue development. However, miR-184’s role in HS remains unclear. miR-184 expression in HS patients and normal healthy (Control) tissues was measured by real-time PCR. pAKT expression was analyzed by Western blot. Fb cells from human HS were cultured and divided into 2 groups, siRNA NC group and miR-184 siRNA group followed by analysis of miR-184 expression by real time PCR, cell proliferation by MTT assay, secretion of inflammatory factors IL-1β and IL-6 by ELISA, as well as expression of pAKT and AKT by western blot. Compared with control group, miR-184 and pAKT expression was significantly increased in the HS group. Transfection of miR-184 siRNA into Fb significantly downregulated miR-184 expression, inhibited cell proliferation, promoted Caspase 3 activity, decreased IL-1β and IL-6 secretion, and reduced pAKT level (P < 0.05). miR-184 expression is increased in hypertrophic scar tissue. Down-regulation of miR-184 expression in proliferative scar tissue fibroblasts can down-regulate PI3K/AKT signaling pathway, inhibit inflammation, promote apoptosis, inhibit fibroblast proliferation, and regulate hypertrophic scar formation.

scholarly journals Diosmetin inhibits cell proliferation and promotes apoptosis through STAT3/c-Myc signaling pathway in human osteosarcoma cells

2021 ◽  
Vol 54 (1) ◽  
Author(s):  
Rende Ning ◽  
Guang Chen ◽  
Run Fang ◽  
Yanhui Zhang ◽  
Wenjuan Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Osteosarcoma Cells ◽  
Antitumor Property ◽  
Human Osteosarcoma ◽  
Cycle Arrest ◽  
Stat3 Phosphorylation ◽  
Human Osteosarcoma Cells ◽  
M Phase

Abstract Background Diosmetin is a bioflavonoid compound naturally abundant in citrus fruits. It is found to perform a variety of activities, while its antitumor property in osteosarcoma, a malignant tumor with unmet clinical treatment, remained unknown. Methods Colony formation assay, cell cycle analysis and apoptosis analysis were conducted respectively to observe the effect of diosmetin on cell proliferation and apoptosis in human osteosarcoma cells. Western blot and immunoprecipitation were used to detect the expression of apoptotic molecules and activation of STAT3/c-Myc pathway in Saos-2 and U2SO cells. Results Diosmetin significantly inhibited cell proliferation, induced cell cycle arrest at G2/M phase and promoted cell apoptosis in both Saos-2 and U2SO cells. Moreover, Diosmetin downregulated the expression of anti-apoptotic protein Bcl-xL while upregulated the levels of pro-apoptotic proteins including cleaved Caspase-3, cleaved-PARP and Bax. Furthermore, diosmetin dose-dependently inhibited STAT3 phosphorylation, reduced the expression of its downstream protein c-Myc and impeded the interaction between STAT3 molecules. Conclusions These results suggest that diosmetin exerts anti-osteosarcoma effects by suppressing cell proliferation and inducing apoptosis via inhibiting the activation of STAT3/c-Myc signaling pathway, which provide the possibility for diosmetin to be a chemotherapeutic candidate for osteosarcoma.

scholarly journals Effect of Quercetin on Cell Cycle and Cyclin Expression in Ovarian Carcinoma and Osteosarcoma Cell Lines

2015 ◽  
Vol 10 (8) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Daniela Catanzaro ◽  
Eugenio Ragazzi ◽  
Caterina Vianello ◽  
Laura Caparrotta ◽  
Monica Montopoli
Keyword(s):  
Cell Cycle ◽  
Ovarian Carcinoma ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Cyclin B1 ◽  
Osteosarcoma Cell ◽  
Chemotherapy Drugs ◽  
Cell Cycle Modulation ◽  
M Phase ◽  
Flavonoid Quercetin

Resistance to chemotherapeutic drugs is a major problem in cancer treatment. The search for new interventions able to overcome this resistance may involve compounds of natural origin, such as flavonoids, ubiquitously present in many foods. In the present study, the cytotoxic effects and cell cycle modulation of the flavonoid quercetin were investigated in ovarian carcinoma (SKOV3) and osteosarcoma (U2OS) human cell lines and in their cisplatin (CDDP)-resistant counterparts (SKOV3/CDDP and U2OSPt cells, respectively). Quercetin (10–50 μM) caused evident changes in the distribution of cell cycle phases in the CDDP-resistant SKOV3/CDDP ovarian cell line. The levels of cyclin D1 and cyclin B1 were determined by means of Western blot in all cell lines incubated with quercetin (50 μM) for 48 hours. The cyclin D1 expression was significantly decreased following the treatment with quercetin in SKOV3 and U2OSPt cells, but not in SKOV3/CDDP and U2OS cells. The reduction of cyclin D1 level could be linked to the G1/S phase alteration found in quercetin-treated cells. Although cyclin B1 is required for G2/M phase, and despite our observation that quercetin influenced the G2/M phase of cell cycle, the flavonoid did not affect cyclin B1 levels in all cell lines, indicating the involvement of other possible mechanisms. These results suggest that quercetin, exceeding the resistance to CDDP, might become an interesting tool to evaluate cytotoxic activity in combination with chemotherapy drugs.

Activation of G Protein-coupled Receptor 30 Promotes Proliferation of Goat Mammary Epithelial Cells via MEK/Erk&PI3K/Akt Signaling Pathway

2020 ◽  
Author(s):  
Ying Zhao ◽  
Haokun Liu ◽  
Mingzhen Fan ◽  
Yuyang Miao ◽  
Xiaoe Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Cyclin D1 ◽  
Mammary Epithelial Cells ◽  
Cyclin B1 ◽  
Akt Signaling ◽  
Mammary Epithelial ◽  
M Phase ◽  
G Protein Coupled ◽  
Goat Mammary Epithelial Cells

Abstract BackgroundGoat is an important dairy animal. During lactation, maintaining a high proliferative activity in goat mammary epithelial cells (GMECs) is significant to improve the yield and composition of goat milk. Estrogen is an essential hormone in epithelial cell proliferation and ductal morphogenesis of mammary gland. G protein-coupled receptor 30 (GPR30) is a novel membrane receptor of estrogen. However, the relationship between estrogen/GPR30 signaling and proliferation of goat mammary epithelial cells has not been reported. And the molecular mechanisms underlying the proliferative effect of estrogen via GPR30 on GMECs remain unclear.ResultsTo investigate the effect of estrogen/GPR30 signaling on GMECs proliferation, goat mammary epithelial cells, which expressed cytokeratin 18 and β-casein, were isolated and identified, defining their mammary alveolar epithelium origination. Estrogen and GPR30 agonist G1 obviously promoted the proliferation of GEMCs, in contrast, GPR30 antagonist G15 partly abolished estrogen-induced cell proliferation. Remarkably, the stimulatory effect of estrogen and G1 on GMECs growth was suppressed by GPR30 knockdown detected by cell counting assay, CCK-8 assay, and BrdU assay, suggesting that estrogen/GPR30 signaling was involved in GMECs proliferation. Additionally, G15 decreased cyclin D1, cyclin B1, CDK1, and p-CDK1 expression, resulting in cell cycle arrest in the G2/M phase via a down-regulated phosphorylation of Erk1/2 and Akt compared with estrogen alone. What’s more, knock-down GPR30 led to an accumulation in the G2/M phase and inhibition of cyclin D1, cyclin B1, CDK1, and p-CDK1 expression via a down-regulation of phosphorylated Erk1/2 and Akt despite the presence of estrogen and G1. Furthermore, MEK inhibitor and PI3K inhibitor decreased the expression of cyclin D1, cyclin B1, CDK1, and p-CDK1, and repressed estrogen-induced and G1-driven promotion of cell growth. It indicated that estrogen/GPR30 signaling played an important role in GMECs proliferation by affecting cell cycle progression via MEK/Erk&PI3K/Akt signaling pathway.ConclusionThis study may provide a new insight into the effect of estrogen/GPR30 signaling on the regulatory action of goat mammary gland development.

scholarly journals The Suppression of CCL2 Expression Decrease the Proliferation of HL-60 Cells through Downregulation of Cyclin d1 Via Arresting Cells at the G1 Phase

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4809-4809
Author(s):  
Jin Yang ◽  
Dan Hong ◽  
Qi Zhou ◽  
Jun-jie Fan ◽  
Le Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cyclin D1 ◽  
Real Time ◽  
Signaling Pathway ◽  
G1 Phase ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Ccl2 Expression

Abstract Background and purpose: Chemokine (C-C motif) ligand 2(CCL2) is a member of the CC subfamily which displays chemotactic activity for monocytes and basophils. It has played a very important role in many solid tumors and changes in bone marrow microenvironment. However, its role in acute myeloid leukemia (AML) has not yet been clear. In this point, we established a cell line with CCL2 down-expression to explore the effect of CCL2 gene on leukemogenesis. Methods: Lentivirus with CCL2-knockdown was successfully constructed after screening effective CCL2 shRNA sequence and tranfected into HL-60 cells which was validated on the level of mRNA and protein by real-time PCR and Western blot. The cells coming from parental, sh-Vectors and shCCL2 were detected for cell growth viability by CCK-8 assay, cell cycles and apoptosis by Flow cytometry. We applied exon sequencing technology to identify the gene profiling between the CCL2 knockdown and the control, of which, Cyclin d1 was selected for further experiments as its expression level was significantly downregulated. Then we successfully down regulated cyclin d1 expression in HL-60 by means of RNA interference to detect the cell proliferation through CCK-8 assay, cell cycles and apoptosis through Flow cytometry. Results: HL-60 cell line expressed the highest level of CCL2 among acute leukemia cell lines (Figure 1). Among 4 pairs of CCL2 interference sequences, only pair 2 had the most efficient potential in knockdown CCL2 expression which was constructed into sh-Vector, GV248, and validated by real-time RT-PCR and Western blot(Figure 2). Low expression of CCL2 significantly decreased HL-60 cell growth. Meanwhile, the CCL2-shRNA-mediated HL-60 cells showed about 12% more cells arrested in G1 phase compared with controls (Table 1, Figure 3). The results of expression profiling showed that there were total 159 genes differentially expressed (Figure 4), of which, ten top pathways were illustrated in Table 2. Cyclin D1 was related to cell cycle, NOD-like receptor signaling pathway, TNF signaling pathway and NF-kappa B signaling pathway which was the lowest expression among cell cycle gene related in HL-60 cells transfect with shCCL2(Table 2, highlighted raw) and further validated by real-time RT-PCR and Western blot (Figure 5). After Cyclin D1 was decreased on the level of mRNA and protein of HL-60, the cell proliferation was evidently slow and cell cycle analysis also indicated a similar pattern of CCL2 (Figure 6). Conclusion: CCL2 involved in cell proliferation which was mediated by cyclin D1 via blocking more cells at G1 phase. Figure 3. Knockdown of CCL2 inhibits cell proliferation via G1 phase arrested. A: Down regulation of CCL2 influenced cell proliferation. From day 2 to 5, the proliferation rate of HL-60 cells transfected by shCCL2 grew significantly slower than controls. B: CCL2 played a role in cell cycle process. More cells transfected shCCL2 were arrested in G1 phase compared with controls. *Indicate significant differences with P-values <0.05 Figure 5. Cylin D1 was the most influenced gene among cell proliferation related genes profile. A: quantitative RT-PCR analysis showed that mRNA levels of preliferation related genes: PCNA, cyclin D1, c-jun, surviving, erk1, and erk2. Among them, Cyclin D1 was expressed lowest. B: Western blot analysis confirmed that the protein expression of total cyclin D1 was much lower compared with controls. Figure 6. The effect of Cyclin D1 on cell proliferation. A: Cyclin D1 was successfully knockdowned in HL-60. mRNA levels were determined by quantitative RT-PCR and revealed that Cyclin D1 expression was knockdowned by the vector of shccnd. B: Knockdown of cyclin D1 inhibits cell proliferation. Compared with control, HL-60 cells with low level of Cyclin D grew significant slowly. C: Down regulation of Cyclin D1 influences more cells arrested in G1 phase compared with control. *P-values <0.05 Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Knockdown of UBE2T Inhibits Osteosarcoma Cell Proliferation, Migration, and Invasion by Suppressing the PI3K/Akt Signaling Pathway

2016 ◽  
Vol 24 (5) ◽  
pp. 361-369 ◽  
Author(s):  
Yu Wang ◽  
Hui Leng ◽  
Hui Chen ◽  
Lei Wang ◽  
Nan Jiang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Migration And Invasion ◽  
Osteosarcoma Cell ◽  
Akt Signaling Pathway ◽  
Inhibitory Effects ◽  
Study Results ◽  
Ubiquitin Conjugating Enzyme

Ubiquitin-conjugating enzyme E2T (UBE2T), a member of the E2 family, was found to be overexpressed in a great many cancers such as bladder cancer, lung cancer, and prostate cancer. However, there have been no reports on the role of UBE2T in osteosarcoma. In this study, we tried to make the effects of UBE2T on osteosarcoma clear. The study results showed that UBE2T was overexpressed in osteosarcoma tissues and cell lines. Moreover, UBE2T knockdown inhibited osteosarcoma cell proliferation, migration, and invasion. We also observed that UBE2T downregulation could suppress the activity of the PI3K/Akt signaling pathway. Therefore, we concluded that UBE2T exerted its inhibitory effects on osteosarcoma cells via suppressing the PI3K/Akt signaling pathway. These findings indicated that UBE2T may be a potential therapeutic target for osteosarcoma treatment.

PI3K/Akt Signaling Interacts With Wnt/β-Catenin Signaling But Does Not Induce An Accumulation Of β-Catenin In The Nucleus Of Acute Lymphoblastic Leukemia Cell Lines

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4886-4886 ◽  
Author(s):  
Tina-Susann Langhammer ◽  
Catrin Roolf ◽  
Saskia Krohn ◽  
Christin Kretzschmar ◽  
Rayk Huebner ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Signaling Pathways ◽  
Cell Line ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Akt Signaling ◽  
Stimulation Of

Abstract Signaling pathways play essential roles in biological processes as development, cell proliferation and homeostasis. The accurate modulation of signaling pathways, their adapted interaction and their time- and tissue-specific adjusted regulation are required for normal cell development. PI3K/Akt and Wnt/β-Catenin signaling pathways act as key regulators in cell proliferation, differentiation and growth. Both signaling pathways include GSK3β as a common protein, which may mediate an interaction and cross-talk between the pathways. Aberrant activation of PI3K/Akt signaling has been linked to different types of leukemia while Wnt/β-Catenin signaling is known to be deregulated in some solid tumors. However, a potential role of Wnt/β-Catenin signaling for pathogenesis of acute lymphoblastic leukemia (ALL) has not yet been analyzed. In our study we analyzed both signaling pathways in different B- and T-ALL cell lines (RS4;11, SEM, REH, CEM, Jurkat, MOLT-4), thereby focusing mainly on their potential interaction via the protein GSK3β. Western Blot experiments were performed to evaluate the expression of specific PI3K/Akt and Wnt/β-Catenin key proteins. To evaluate the activation status of Wnt signaling immunofluorescence and protein fractionation experiments were performed, analyzing the activation linked nucleic localization of β-Catenin. The effect of pathway activation and inhibition on cell proliferation via chemical compounds was analyzed by WST-1 test. High pAkt levels were detected in B-ALL cell line SEM and T-ALL cell line CEM, indicating a hyperactive PI3K/Akt signaling, whereas other analyzed cell lines diplayed lower pAkt status. Among all cell lines analyzed SEM and CEM also showed the highest cytoplasmic β-Catenin levels, indicating a direct interaction of both signaling pathways. However, immunofluorescence and fractionation experiments revealed that a translocation of β-Catenin into the nucleus did not occur. To further investigate the role and interaction of PI3K/Akt and Wnt/β-Catenin signaling, pathway inhibiting and stimulating experiments were performed. Treatment of cells with Wnt3a led to activation of the Wnt/β-Catenin signaling cascade, characterized by nuclear β-Catenin accumulation. Inhibition of cell proliferation was detected after treatment with high concentrations Wnt3a (≥ 500 ng/ml). PI3K inhibition by LY294002 led to decreased phosphorylation of GSK3β at Ser9 and an increased decay of β-Catenin. Stimulation of PI3K/Akt signaling using activating ligand FLT3L induced GSK3β phosphorylation at Ser9 and accumulation of cytoplasmic β-Catenin. However a translocation of β-Catenin into the nucleus seems not to occur. In summary our results indicate that PI3K/Akt and Wnt/β-Catenin signaling can interact through their common protein GSK3β, but stimulation of the PI3K/Akt signaling pathway by addition of PI3K/Akt specific activators does not fully activate Wnt/β-Catenin signaling in ALL cells. Complete activation of the Wnt cascade characterized by translocation of β-Catenin into the nucleus can only be induced by use of specific Wnt effectors. Disclosures: No relevant conflicts of interest to declare.

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