scholarly journals Dose-Dependent Effect of Cordycepin on Viability, Proliferation, Cell Cycle, and Migration in Dental Pulp Stem Cells

2021 ◽  
Vol 11 (8) ◽  
pp. 718
Author(s):  
Nezar Boreak ◽  
Ahmed Alkahtani ◽  
Khalid Alzahrani ◽  
Amani Hassan Kenani ◽  
Wafa Hussain Faqehi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Growth Curve ◽  
Dental Pulp ◽  
Phase Cell ◽  
Ros Generation ◽  
Dependent Manner ◽  
Significant Difference ◽  
Dose Dependent

Objective: To examine the effect of Cordycepin on the viability, proliferation, and migratory properties of dental pulp-derived mesenchymal stem cells. Materials and methods: The pulp was derived from human premolar teeth extracted for orthodontic purposes after obtaining informed consent. The samples were transferred to the laboratory for processing. DPSCs were expanded and characterized using flow cytometry and differentiation to the bone, adipose, and cartilage cells was examined. MTT Assay was performed using various concentrations of Cordycepin. The growth curve was plotted for 13 days. Cell cycle analysis was performed by flow cytometry. Migratory ability was assessed by wound healing assay. ROS generation was detected by flow cytometry. Gene expression was quantified by RT-qPCR. Statistical analysis was performed. p < 0.05 was considered as significant and p < 0.01 was considered as highly significant (* p < 0.05, and ** p < 0.01). Results: DPSCs expressed characteristic MSC-specific markers and trilineage differentiation. Cordycepin at lower concentrations did not affect the viability of DPSCs. The growth curve of cells showed a dose-dependent increase in cell numbers till the maximum dose. DPSCs treated with 2.5 µM Cordycepin was found to have a reduced G1 phase cell percentage. DPSCs treated with 2.5 µM and 5 µM Cordycepin showed a significant decrease in G2 phase cells. No significant difference was observed for S phase cells. Cordycepin treatment affected the migratory ability in DPSCs in a concentration-dependent manner. Conclusion: Cordycepin can be used at therapeutic doses to maintain stem cells.

scholarly journals METTL3-mediated M6a Modification Regulates Cell Cycle Progression of Dental Pulp Stem Cells

2021 ◽  
Author(s):  
Haiyun Luo ◽  
Wenjing Liu ◽  
Yanli Zhang ◽  
Xiao Jiang ◽  
Shiqing Wu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Differentially Expressed Genes ◽  
Dental Pulp ◽  
Cell Cycle Control ◽  
Dental Pulp Stem Cells ◽  
Differentially Expressed ◽  
Expression Level

Abstract Background: Dental pulp stem cells (DPSCs) exhibited self-renewal, pluripotency capacity and served as promising cells source in endodontic regeneration and tissue engineering. Meanwhile, the regenerative capacity of DPSCs is limited and reduced in long lifespan. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs. The methyltransferases complex and demethylases mediated m6A methylation and cooperated to impact various biological processes associated with stem cell fate determination. However, the biological effect of m6A methylation in DPSCs remained unclear. Methods: Cell surface markers and differentiation potential of primary DPSCs were identified and m6A immunoprecipitation with deep sequencing (m6A RIP-seq) was used to uncover characteristics of m6A modifications in DPSCs transcriptome. Expression level of m6A-related genes were evaluated in immature/mature pulp tissues and cells. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence and apoptosis were further analyzed by β-galactosidase, TUNEL staining and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq was used to functionally enrich overlapped genes and screen target of METTL3. Cell cycle distributions were assayed by flow cytometry and m6A RIP-qPCR was used to confirm METTL3 mediated m6A methylation in DPSCs. Results: Here, m6A peaks distribution, binding area and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relative high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. Furthermore, Conjoint analysis of m6A RIP and RNA-sequencing showed differentially expressed genes affected by METTL3 depletion was mainly enriched in cell cycle, mitosis and alteration of METTL3 expression resulted in cell cycle arrest which indicated METTL3 make essential effect in cell cycle control. To further investigate underlying mechanisms, we explored proteins interaction network of differentially expressed genes and Polo-like Kinase 1 (PLK1), a critical cycle modulator was identified as target of METTL3-mediated m6A methylation in DPSCs. Conclusions: These results revealed m6A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and serve new insight in stem cells based tissue engineering.

scholarly journals Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21Cip1/Waf1Pathway

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Mingyu Zhang ◽  
Yue Du ◽  
Renzhong Lu ◽  
You Shu ◽  
Wei Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cellular Senescence ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Marrow Mesenchymal Stem Cells ◽  
Dose Dependent ◽  
Increased Activity

In the present study, we demonstrated that bone marrow mesenchymal stem cells (BMSCs) of the 3rd passage displayed the senescence-associated phenotypes characterized with increased activity of SA-β-gal, altered autophagy, and increased G1 cell cycle arrest, ROS production, and expression of p53 andp21Cip1/Waf1compared with BMSCs of the 1st passage. Cholesterol (CH) reduced the number of SA-β-gal positive cells in a dose-dependent manner in aging BMSCs induced by H2O2and the 3rd passage BMSCs. Moreover, CH inhibited the production of ROS and expression of p53 andp21Cip1/Waf1in both cellular senescence models and decreased the percentage of BMSCs in G1 cell cycle in the 3rd passage BMSCs. CH prevented the increase in SA-β-gal positive cells induced by RITA (reactivation of p53 and induction of tumor cell apoptosis, a p53 activator) or 3-MA (3-methyladenine, an autophagy inhibitor). Our results indicate that CH not only is a structural component of cell membrane but also functionally contributes to regulating cellular senescence by modulating cell cycle, autophagy, and the ROS/p53/p21Cip1/Waf1signaling pathway.

scholarly journals Tri-ortho-cresyl phosphate induces autophagy of rat spermatogonial stem cells

Reproduction ◽  
2015 ◽  
Vol 149 (2) ◽  
pp. 163-170 ◽  
Author(s):  
Meng-Ling Liu ◽  
Jing-Lei Wang ◽  
Jie Wei ◽  
Lin-Lin Xu ◽  
Mei Yu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flame Retardants ◽  
Spermatogonial Stem Cells ◽  
Ultrastructural Observation ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Transmission Electron ◽  
Dose Dependent

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame retardants in industry and reported to have a deleterious effect on the male reproductive system in animals besides delayed neurotoxicity. Our preliminary results found that TOCP could disrupt the seminiferous epithelium in the testis and inhibit spermatogenesis, but the precise mechanism is yet to be elucidated. This study shows that TOCP inhibited viability of rat spermatogonial stem cells in a dose-dependent manner. TOCP could not lead to cell cycle arrest in the cells; the mRNA levels of p21, p27, p53, and cyclin D1 in the cells were also not affected by TOCP. Meanwhile, TOCP did not induce apoptosis of rat spermatogonial stem cells. After treatment with TOCP, however, both LC3-II and the ratio of LC3-II/LC3-I were markedly increased; autophagy proteins ATG5 and beclin 1 were also increased after treatment with TOCP, indicating that TOCP could induce autophagy in the cells. Ultrastructural observation under the transmission electron microscopy indicated that autophagic vesicles in the cytoplasm containing extensively degraded organelles such as mitochondria and endoplasmic reticulum increased significantly after the cells were treated with TOCP. In summary, we have shown that TOCP can inhibit viability of rat spermatogonial stem cells and induce autophagy of the cells, without affecting cell cycle and apoptosis.

scholarly journals Inducible expression of Oct-3/4 reveals synergy with Klf4 in targeting Cyclin A2 to enhance proliferation during early reprogramming

2021 ◽  
Author(s):  
Lamuk Zaveri ◽  
Jyotsna Dhawan
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Mesenchymal To Epithelial Transition ◽  
Cyclin A2 ◽  
Slow Cycling ◽  
Induced Pluripotent ◽  
Dose Dependent

AbstractDuring reprogramming of somatic cells, heightened proliferation is one of the earliest changes observed. While other early events such as mesenchymal-to-epithelial transition have been well studied, the mechanisms by which the cell cycle switches from a slow cycling state to a faster cycling state are still incompletely understood. To investigate the role of Oct-3/4 in this early feature of reprogramming, we created a 4-Hydroxytamoxifen dependent Oct-3/4 Estrogen Receptor fusion (OctER). We show that OctER can substitute for Oct-3/4 to reprogram mouse embryonic fibroblasts to induced pluripotent stem cells. While over-expression of OctER or Klf4 individually did not affect cell proliferation, in combination, these factors hasten the cell cycle, in a tamoxifen dose-dependent manner, supporting a key role for OctER. Oct-3/4 + Klf4 increased proliferation by enhancing expression of Cyclin A2. We verified occupancy of endogenous Oct-3/4 and Klf4 at bioinformatically identified binding sites in the Cyclin A2 promoter in mouse embryonic stem cells (mESC). Using inducible OctER along with Klf4, we show dose-dependent induction of Cyclin A2 promoter-reporter activity and mRNA levels. Taken together, our results provide further evidence of the interdependence of pluripotency and the rapid cell cycle seen in mESC, and identify CyclinA2 as a key early target.

Myricanol-9-acetate, a novel naturally occurring derivative of myricanol, induces ROS-dependent mitochondrial-mediated Apoptosis in MCF-7 cancer cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Gazanfar Ahmad ◽  
Sameer Ahmad Mir ◽  
Loveleena Kour Anand ◽  
Faheem Hyder Pottoo ◽  
Neerupma Dhiman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Ros Generation ◽  
Cycle Arrest ◽  
Naturally Occurring ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Mcf 7

Background: Low therapeutic efficacy and drug-induced systemic toxicity of currently used anti-cancerous chemotherapeutic agents are major compelling factors for finding out clinically efficient molecules with high efficiency and less toxicity. Objective: The current research work was undertaken to evaluate the anticancer potential of Myricanol-9-acetate (MA), a novel naturally occurring derivative of myricanol. Methods: MCF-7, MiaPaCa-2, and HCT 116 were used for cytotoxicity determination of the MA and ML (Myricanol) by MTT assay. The mechanistic study involved the determination of cell cycle arrest, ΔΨm loss, ROS generation, western blot assay, and flow cytometry by reported methods on MCF-7 cells. Results: MA exhibited anticancer activity against all three cell lines; however, the molecule was found most active against the MCF-7 cell line. We observed IC50 20μM with MA treatment as compared to the IC50 of 42 μM for myricanol treatment. Detailed mechanistic studies revealed that MA induced apoptosis on MCF-7 cell line through ROS generation; a dose-dependent drop in mitochondrial membrane potential was found to be associated with cell cycle arrest at G0/G1 phase. Our results further demonstrated down-regulation of Bcl2 and activation of the caspase cascade as the events involved in the MA-induced apoptosis. Flow cytometry results indicated an increase in early and late apoptotic population in a dose-dependent manner with an apoptotic population of about 20% at 30 μM of MA, thus supporting our results. Conclusion: Present findings thus suggest that MA might serve as a promising novel drug candidate having high scope for further evaluation in preclinical and clinical studies.

scholarly journals METTL3-mediated m6A modification regulates cell cycle progression of dental pulp stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Haiyun Luo ◽  
Wenjing Liu ◽  
Yanli Zhang ◽  
Yeqing Yang ◽  
Xiao Jiang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Fate ◽  
Dental Pulp ◽  
Interaction Network ◽  
Dental Pulp Stem Cells ◽  
Tunel Staining

Abstract Background Dental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs associated with stem cell fate determination. In this study, we aim to explore the biological effect of m6A methylation in DPSCs. Methods m6A immunoprecipitation with deep sequencing (m6A RIP-seq) demonstrated the features of m6A modifications in DPSC transcriptome. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence, and apoptosis were analyzed by β-galactosidase, TUNEL staining, and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq functionally enriched overlapped genes and screened target of METTL3. Cell cycle distributions were assayed by flow cytometry, and m6A RIP-qPCR was used to confirm METTL3-mediated m6A methylation. Results Here, m6A peak distribution, binding area, and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relatively high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. A conjoint analysis of m6A RIP and RNA sequencing showed METTL3 depletion associated with cell cycle, mitosis, and alteration of METTL3 resulted in cell cycle arrest. Furthermore, the protein interaction network of differentially expressed genes identified Polo-like kinase 1 (PLK1), a critical cycle modulator, as the target of METTL3-mediated m6A methylation in DPSCs. Conclusions These results revealed m6A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and served new insight into stem cell-based tissue engineering.

scholarly journals Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Dental Pulp Stem Cells (DPSCs) Display a Similar Profile with Pericytes

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Shao Yue Zhu ◽  
Chang Yong Yuan ◽  
Yi Fan Lin ◽  
Hao Liu ◽  
Yan Qi Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mrna Expression ◽  
Dental Pulp ◽  
Dental Pulp Stem Cells ◽  
Deciduous Teeth ◽  
Potential Candidate ◽  
Migration Ability ◽  
Significant Difference ◽  
Human Exfoliated Deciduous Teeth

Background. Pericytes play an important role in forming functional blood vessels and establishing stable and effective microcirculation, which is crucial for vascular tissue engineering. The slow ex vivo expansion rate, limited proliferative capacity, and variability of tissue-specific phenotypes would hinder experimental studies and clinical translation of primary pericytes. In this study, the angiogenic and pericyte functions of stem cells from human exfoliated deciduous teeth (SHEDs) and postnatal human dental pulp stem cells (DPSCs) were investigated. Methods. Osteogenic and adipogenic induction assays were performed to evaluate the mesenchymal potential of SHEDs, DPSCs, and pericytes. An in vitro Matrigel angiogenesis assay was conducted to reveal the ability of SHEDs, DPSCs, and pericytes to stabilize vascular-like structures. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed to evaluate mRNA expression. Flow cytometry, western blotting, and immunostaining were used to assess the protein expression. Wound healing and transwell assays were performed to evaluate the migration ability of SHEDs, DPSCs, and pericytes. Results. The osteogenic and adipogenic induction assays showed that SHEDs, DPSCs, and pericytes exhibited similar stem cell characteristics. The mRNA expression levels of PDGFR-β, α-SMA, NG2, and DEMSIN in SHEDs and DPSCs cultured in EC medium were significantly higher than those in the control groups on day 7 ( P < 0.05 ), but significantly higher than those in the pericytes group on day 14 ( P < 0.05 ). Flow cytometry showed that high proportions of SHEDs and DPSCs were positive for various pericyte markers on day 7. The DPSCs, SHEDs, and pericytes displayed strong migration ability; however, there was no significant difference among the groups ( P > 0.05 ). Conclusion. The SHEDs and DPSCs display a profile similar to that of pericytes. Our study lays a solid theoretical foundation for the clinical use of dental pulp stem cells as a potential candidate to replace pericytes.

scholarly journals Sulforaphane induces S-phase arrest and apoptosis via p53-dependent manner in gastric cancer cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuan Wang ◽  
Huazhang Wu ◽  
Nannan Dong ◽  
Xu Su ◽  
Mingxiu Duan ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Apoptosis ◽  
S Phase ◽  
Phase Cell ◽  
Dependent Manner ◽  
Gastric Cancer Cells ◽  
Dose Dependent

AbstractSulforaphane (SFN) extracted from broccoli sprout has previously been investigated for its potential properties in cancers, however, the underlying mechanisms of the anticancer activity of SFN remain not fully understood. In the present study, we investigate the effects of SFN on cell proliferation, cell cycle, cell apoptosis, and also the expression of several cell cycle and apoptosis-related genes by MTT assay, flow cytometry and western blot analysis in gastric cancer (GC) cells. The results showed that SFN could impair the colony-forming ability in BGC-823 and MGC-803 cell lines compared with the control. In addition, SFN significantly suppressed cell proliferation by arresting the cell cycle at the S phase and enhancing cell apoptosis in GC cells in a dose-dependent manner. Western blot results showed that SFN treatment significantly increased the expression levels of p53, p21 and decreased CDK2 expression, which directly regulated the S phase transition. The Bax and cleaved-caspase-3 genes involved in apoptosis executive functions were significantly increased in a dose-dependent manner in BGC-823 and MGC-803 cells. These results suggested that SFN-induced S phase cell cycle arrest and apoptosis through p53-dependent manner in GC cells, which suggested that SFN has a potential therapeutic application in the treatment and prevention of GC.

Ternary Copper (II) Complex Induced Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Sathiavani Arikrishnan ◽  
Jian Sheng Loh ◽  
Xian Wei Teo ◽  
Faris bin Norizan ◽  
May Lee Low ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Annexin V ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Colorectal Cancer Cells ◽  
Dose Dependent ◽  
Parp 1 ◽  
Ht 29

Background: The lack of specificity, severe side effects, and development of drug resistance have largely limited the use of platinum-based compounds in cancer treatment. Therefore, copper complexes have emerged as potential alternatives to platinum-based compounds. Objective: Ternary copper (II) complex incorporated with 1-10-phenanthroline and L-tyrosine was investigated for its anti-cancer effects in HT-29 colorectal cancer cells. Methods: Cytotoxic effects of ternary copper (II) complex in HT-29 cells were evaluated using MTT assay, Real-Time Cell Analysis (RTCA), and lactate dehydrogenase (LDH) assay. Cell cycle analysis was performed using flow cytometry. Apoptosis induction was studied by Annexin V-FITC/propidium iodide (PI) staining and mitochondrial membrane potential analysis (JC-10 staining) using flow cytometry. Intracellular reactive oxygen species (ROS) were detected by DCFH-DA assay. The expression of proteins involved in the apoptotic signalling pathway (p53, caspases, and PARP-1) was evaluated by western blot analysis. Results: Ternary copper (II) complex reduced the cell viability of HT-29 cells in a time- and dose-dependent manner, with IC50 of 2.4 ± 0.4 and 0.8 ± 0.04 µM at 24 and 48 hours, respectively. Cell cycle analysis demonstrated induction of S-phase cell cycle arrest. Morphological evaluation and Annexin V-FITC/PI flow cytometry analysis confirmed induction of apoptosis that was further supported by cleavage and activation of caspase-8, caspase-9, caspase-3, and PARP-1. Mutant p53 was also downregulated in a dose-dependent manner. No LDH release, mitochondrial membrane potential disruption, and ROS production were observed. Conclusion: Ternary copper (II) complex holds great potential to be developed for colorectal cancer treatment.

scholarly journals The Study on Biocompatibility of Porous nHA/PLGA Composite Scaffolds for Tissue Engineering with Rabbit ChondrocytesIn Vitro

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Lei Chen ◽  
Wei-Min Zhu ◽  
Zhi-Qiang Fei ◽  
Jie-Lin Chen ◽  
Jian-Yi Xiong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Flow Cytometry ◽  
Mtt Assay ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Control Group ◽  
Dependent Manner ◽  
Plga Scaffold ◽  
Significant Difference

Objective. To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA) composite and evaluate its feasibility as a scaffold for cartilage tissue engineering.Methods. Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffoldin vitroand the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM). The cell cycle profile was analyzed by flow cytometry.Results. The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P>0.05).Conclusion. The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

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