scholarly journals The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-20 ◽  
Author(s):  
Xiao Liang ◽  
Chang Xu ◽  
Wanchun Wang ◽  
Xiang Li
Keyword(s):  
Dna Methyltransferase ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Mouse Xenograft Model ◽  
Human Osteosarcoma Cells ◽  
Oct4 Protein ◽  
U2os Cells ◽  
Dose Dependent

The DNA methyltransferase 1 (DNMT1)/miR-34a axis promoted carcinogenesis of various types of cancers. However, no literature reported its contribution to the stemness of osteosarcoma cancer stem-like cells (OSLCs). We sought to determine whether the DNMT1/miR-34a axis facilitates the stemness of OSLCs. We here revealed the higher DNMT1 activity and expression, lower miR-34a expression with high methylation of its promoter, and stronger stemness of OSLCs, as manifested by elevated sphere and colony formation capacities; CD133, CD44, ABCG2, Bmi1, Sox2, and Oct4 protein amounts in vitro; and carcinogenicity in a nude mouse xenograft model, when compared to the parental U2OS cells. 5-Azacytidine (Aza-dC) repressed DNMT1 activation and upregulated miR-34a expression by promoter demethylation and suppressed the stemness of OSLCs in a dose-dependent manner. DNMT1 knockdown increased miR-34a and reduced the stemness of OSLCs. Transfection with a miR-34a mimic repressed the stemness of OSLCs but did not alter DNMT1 activity and expression. Conversely, DNMT1 overexpression declined miR-34a levels, promoting the stemness of U2OS cells. Transfection with a miR-34a inhibitor enhanced the stemness of U2OS cells, without affecting the DNMT1 activity and expression. Importantly, reexpression of miR-34a could rescue the effects of DNMT1 overexpression on miR-34a inhibition as well as the stemness promotion without affecting the activity and expression of DNMT1. Our results revealed that aberrant activation of DNMT1 caused promoter methylation of miR-34a, leading to miR-34a underexpression, and the role of the DNMT1/miR-34a axis in promoting and sustaining the stemness of OSLCs.

scholarly journals Novel Repositioning Therapy for Drug-Resistant Glioblastoma: In vivo Validation Study of Clindamycin Treatment Targeting the mTOR Pathway and Combination Therapy with Temozolomide

2021 ◽  
Author(s):  
Takeyoshi Eda ◽  
Masayasu Okada ◽  
Ryosuke Ogura ◽  
Yoshihiro Tsukamoto ◽  
Yu Kanamaru ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Dna Methyltransferase ◽  
Cultured Cells ◽  
Radiation Treatment ◽  
Xenograft Model ◽  
Mtor Pathway ◽  
Dependent Manner ◽  
Ki 67 ◽  
Mouse Xenograft Model

Multimodal therapy including surgery, radiation treatment and temozolomide (TMZ) is performed on glioblastoma (GBM). However, the prognosis is still poor and there is an urgent need to develop effective treatments to improve survival. Molecular biological analysis was conducted to exam-ine the signal activation patterns at GBM specimens and remains an open problem. Advanced macrolides, such as azithromycin, reduce the phosphorylation of p70 ribosomal protein S6 kinase (p70S6K), a downstream mammalian target of rapamycin (mTOR) effector, and suppress the proliferation of T-cells. We focused on its unique profile and screened for the antitumor activity of approved macrolide antibiotics. Clindamycin (CLD) reduced the viability of GBM cells in vitro. We assessed the effects of the candidate macrolide on the mTOR pathway through Western blotting. CLD attenuated p70S6K phosphorylation in a dose dependent manner. These effects of on GBM cells were enhanced by co-treatment with TMZ. Furthermore, CLD inhibited the expression of O6-methylguanine-DNA methyltransferase (MGMT) protein in cultured cells. In the mouse xenograft model, CLD and TMZ co-administration significantly suppressed the tumor growth and markedly decreased the number of Ki-67 (clone MIB-1) positive cells within the tumor. These results suggest that CLD suppresses GBM cell growth by the inhibiting mTOR signaling. Moreover, CLD and TMZ showed promising synergistic antitumor activity.

scholarly journals Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zizhen Si ◽  
Lei Yu ◽  
Haoyu Jing ◽  
Lun Wu ◽  
Xidi Wang
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Cancer Proliferation ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

scholarly journals Lumican Inhibits Osteoclastogenesis and Bone Resorption by Suppressing Akt Activity

2021 ◽  
Vol 22 (9) ◽  
pp. 4717
Author(s):  
Jin-Young Lee ◽  
Da-Ae Kim ◽  
Eun-Young Kim ◽  
Eun-Ju Chang ◽  
So-Jeong Park ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Formation ◽  
Map Kinases ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Dual Action ◽  
Dose Dependent ◽  
Resorptive Activity

Lumican, a ubiquitously expressed small leucine-rich proteoglycan, has been utilized in diverse biological functions. Recent experiments demonstrated that lumican stimulates preosteoblast viability and differentiation, leading to bone formation. To further understand the role of lumican in bone metabolism, we investigated its effects on osteoclast biology. Lumican inhibited both osteoclast differentiation and in vitro bone resorption in a dose-dependent manner. Consistent with this, lumican markedly decreased the expression of osteoclastogenesis markers. Moreover, the migration and fusion of preosteoclasts and the resorptive activity per osteoclast were significantly reduced in the presence of lumican, indicating that this protein affects most stages of osteoclastogenesis. Among RANKL-dependent pathways, lumican inhibited Akt but not MAP kinases such as JNK, p38, and ERK. Importantly, co-treatment with an Akt activator almost completely reversed the effect of lumican on osteoclast differentiation. Taken together, our findings revealed that lumican inhibits osteoclastogenesis by suppressing Akt activity. Thus, lumican plays an osteoprotective role by simultaneously increasing bone formation and decreasing bone resorption, suggesting that it represents a dual-action therapeutic target for osteoporosis.

scholarly journals Metformin reduces glycometabolism of papillary thyroid carcinoma in vitro and in vivo

2017 ◽  
Vol 58 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Chen-Tian Shen ◽  
Wei-Jun Wei ◽  
Zhong-Ling Qiu ◽  
Hong-Jun Song ◽  
Xin-Yun Zhang ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Papillary Thyroid Carcinoma ◽  
Thyroid Carcinoma ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Papillary Thyroid ◽  
Fdg Uptake ◽  
Dose Dependent

More aggressive thyroid cancer cells show a higher activity of glycometabolism. Targeting cancer cell metabolism has emerged as a novel approach to prevent or treat malignant tumors. Glucose metabolism regulation effect of metformin in papillary thyroid cancer was investigated in the current study. Human papillary thyroid carcinoma (PTC) cell lines BCPAP and KTC1 were used. Cell viability was detected by CCK8 assay. Glucose uptake and relative gene expression were measured in metformin (0–10 mM for 48 h)-treated cells by 18F-FDG uptake assay and western blotting analysis, respectively. MicroPET/CT imaging was performed to detect 18F-FDG uptake in vivo. After treatment with metformin at 0, 2.5, 5 and 10 mM for 48 h, the ratio of p-AMPK to total AMPK showed significant rising in a dose-dependent manner in both BCPAP and KTC1, whereas p-AKT and p-mTOR expression level were downregulated. 18F-FDG uptake reduced after metformin treatment in a dose-dependent manner, corresponding to the reduced expression level of HK2 and GLUT1 in vitro. Xenograft model of PTC using BCPAP cells was achieved successfully. MicroPET/CT imaging showed that in vivo 18F-FDG uptake decreased after treatment with metformin. Immunohistochemistry staining further confirmed the reduction of HK2 and GLUT1 expression in the tumor tissue of metformin-treated PTC xenograft model. In conclusion, metformin could reduce glucose metabolism of PTC in vitro and in vivo. Metformin, by targeting glycometabolism of cancer cells, could be a promising adjuvant therapy alternative in the treatment modality of advanced thyroid carcinoma.

scholarly journals Leptin Upregulates the Expression of β3-Integrin, MMP9, HB-EGF, and IL-1β in Primary Porcine Endometrium Epithelial Cells In Vitro

2020 ◽  
Vol 17 (18) ◽  
pp. 6508
Author(s):  
Hongfang Wang ◽  
Jinlian Fu ◽  
Aiguo Wang
Keyword(s):  
Signaling Pathways ◽  
Embryo Implantation ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Reproductive Disorders ◽  
Β3 Integrin ◽  
Epithelium Cells ◽  
Dose Dependent

Obesity has become a global health problem. Research suggests that leptin, a hormone that responds to fat deposition, may be involved in mammalian reproduction; however, its precise role in embryo implantation is poorly understood. Here, primary porcine endometrium epithelium cells (PEECs) were cultured in vitro and used to evaluate the regulatory role of different leptin levels on β3-integrin, MMP9, HB-EGF, and IL-1β, which are, respectively, involved in four critical steps of embryo implantation. Results showed that only 0.01 nM leptin significantly improved β3-integrin mRNA expression (p < 0.05). MMP9 and HB-EGF mRNA expressions were upregulated by 0.10–10.00 nM leptin (p < 0.05). The IL-1β expression level was only increased by 10.00 nM leptin (p < 0.05). β3-integrin, MMP9, HB-EGF, and IL-1β mRNA and protein have a similar fluctuant response to increased leptin. Leptin’s influence on β3-integrin, MMP9, HB-EGF, and IL-1β disappeared when the JAK2, PI(3)K, or MAPK signaling pathways were blocked, respectively. In conclusion, leptin affected porcine implantation by regulating the expression of β3-integrin, MMP9, HB-EGF, and IL-1β in a dose-dependent manner. The signaling pathways of JAK2, PI(3)K, and MAPK may participate in this regulatory process. These findings will contribute to further understanding the mechanisms of reproductive disorders in obesity.

scholarly journals Opposing Roles of Leptin and Ghrelin in the Equine Corpus Luteum Regulation: AnIn VitroStudy

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
António Galvão ◽  
Angela Tramontano ◽  
Maria Rosa Rebordão ◽  
Ana Amaral ◽  
Pedro Pinto Bravo ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Reproductive Function ◽  
Secretory Activity ◽  
Dependent Manner ◽  
Angiogenic Activity ◽  
Metabolic Hormones ◽  
Protein Levels ◽  
Dose Dependent

Metabolic hormones have been associated with reproductive function modulation. Thus, the aim of this study was: (i) to characterize the immunolocalization, mRNA and protein levels of leptin (LEP), Ghrelin (GHR) and respective receptors LEPR and Ghr-R1A, throughout luteal phase; and (ii) to evaluate the role of LEP and GHR on progesterone (P4), prostaglandin (PG) E2and PGF2α, nitric oxide (nitrite), tumor necrosis factor-α(TNF); macrophage migration inhibitory factor (MIF) secretion, and on angiogenic activity (BAEC proliferation), in equine corpus luteum (CL) from early and mid-luteal stages. LEPR expression was decreased in late CL, while GHR/Ghr-R1A system was increased in the same stage. Regarding secretory activity, GHR decreased P4in early CL, but increased PGF2α, nitrite and TNF in mid CL. Conversely, LEP increased P4, PGE2, angiogenic activity, MIF, TNF and nitrite during early CL, in a dose-dependent manner. Thein vitroeffect of LEP on secretory activity was reverted by GHR, when both factors acted together. The present results evidence the presence of LEP and GHR systems in the equine CL. Moreover, we suggest that LEP and GHR play opposing roles in equine CL regulation, with LEP supporting luteal establishment and GHR promoting luteal regression. Finally, a dose-dependent luteotrophic effect of LEP was demonstrated.

scholarly journals Discovery of LAMP-2A as Potential Biomarkers for Glioblastoma Development by Modulating Apoptosis through N-CoR Degradation

2020 ◽  
Author(s):  
Yongjie Wang ◽  
Buyi Zhang ◽  
Jianli Wang ◽  
Haijian Wu ◽  
Shenbin Xu ◽  
...  
Keyword(s):  
Xenograft Model ◽  
Therapeutic Modality ◽  
Clinical Samples ◽  
Low Grade ◽  
Valuable Insight ◽  
Potential Biomarker ◽  
Mouse Xenograft Model ◽  
Novel Therapeutic

Abstract Background: Lysosome-associated membrane protein type 2A (LAMP-2A) is the key component of chaperone-mediated autophagy (CMA), a cargo-selective lysosomal degradation pathway. Aberrant LAMP-2A expression and CMA activation have been demonstrated in various human malignancies. The study focusing on the intrinsic role of LAMP-2A and CMA in glioblastoma (GBM), and downstream mechanism could provide valuable insight into the pathogenesis and novel therapeutic modality of GBM. Methods: The levels of LAMP-2A, nuclear receptor co-repressor (N-CoR), unfolded protein reaction (UPR) and apoptosis were examined in clinical samples. LAMP-2A siRNA and shRNA were constructed to manipulate CMA activation. The role of CMA and downstream mechanism through degradation of N-CoR and arresting UPR mediated apoptosis were explored in GBM cells and nude mouse xenograft model. Results: Elevated LAMP-2A and associated decreased N-CoR expression were observed in GBM as compared with peritumoral region and low-grade glioma. Inhibited UPR and apoptosis were observed in GBM with high LAMP-2A expression. In vitro study demonstrated co-localization and interaction between LAMP-2A and N-CoR. LAMP-2A silencing up-regulated N-CoR and aroused UPR pathway, leading to apoptosis, while N-CoR silencing led to an opposite result. In vivo study further confirmed that LAMP-2A inhibition arrested tumor growth by promoting apoptosis.Conclusions: Our results demonstrated the central role of CMA in mediating N-CoR degradation and protecting GBM cells against UPR and apoptosis, and provided evidence of LAMP-2A as potential biomarker. Further research focusing on CMA with other tumorigenic process is needed and selective modulators of LAMP-2A remain to be investigated to provide a novel therapeutic strategy for GBM.

scholarly journals Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

2020 ◽  
Author(s):  
Sophie H. L. Austin ◽  
Lachlan Harris ◽  
Oana Paun ◽  
Piero Rigo ◽  
François Guillemot ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Direct Role ◽  
Adult Nscs ◽  
Hippocampal Networks ◽  
Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

Drug Design Structural Alert

2011 ◽  
Vol 30 (5) ◽  
pp. 546-550 ◽  
Author(s):  
Martin E. Dowty ◽  
George Hu ◽  
Fengmei Hua ◽  
F. Barclay Shilliday ◽  
Heather V. Dowty
Keyword(s):  
Drug Design ◽  
Dependent Manner ◽  
Safety Testing ◽  
Cellular Debris ◽  
Safety Studies ◽  
Structural Alerts ◽  
Testicular Injury ◽  
Dose Dependent

In the process of drug design, it is important to consider potential structural alerts that may lead to toxicosis. This work illustrates how using trifluoroethane as a part of a novel chemical entity led to cytochrome P450 – mediated N-dealkylation and the formation of trifluoroacetaldehyde, a known testicular toxicant, in exploratory safety studies in rats. Testicular toxicosis was noted microscopically in a dose-dependent manner as measured by testicular spermatocytic degeneration and necrosis and excessive intratubular cellular debris in the epididymis. This apparent toxic effect correlated well with the dose-dependent formation of trifluoroacetaldehyde, identified from in vitro rat liver microsome metabolism studies. A similar safety study performed with an N-tetrazole substitution in place of the N-trifluoroethane showed no evidence of testicular injury, implicating further the role of trifluoroacetaldehyde in the testicular lesion observed. These results highlight the relevance of early metabolic and safety testing in assessing potential structural alerts in drug design.

scholarly journals Proteolytic Cleavage of Cyclin E Leads to Inactivation of Associated Kinase Activity and Amplification of Apoptosis in Hematopoietic Cells

2002 ◽  
Vol 22 (7) ◽  
pp. 2398-2409 ◽  
Author(s):  
Suparna Mazumder ◽  
Bendi Gong ◽  
Quan Chen ◽  
Judith A. Drazba ◽  
Jeffrey C. Buchsbaum ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Cyclin E ◽  
Genotoxic Stress ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Dose Dependent

ABSTRACT Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G1 to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E276-395 (where cyclin E276-395 is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E1-275, cyclin E276-395 deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E276-395, but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.

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