Reversine suppresses osteosarcoma cell growth through targeting BMP-Smad1/5/8-mediated angiogenesis.

Reversine, or 2-(4-morpholinoanilino)-6cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule exhibits tumor-suppressive activities through different molecular mechanisms. In this study, in vitro and in vivo angiogenic models were used to elucidate the effect of Reversine on angiogenesis in the tumor suppression. Firstly, we grafted osteosarcoma-derived MNNG/HOS cell aggregates onto chick embryonic chorioallantoic membrane (CAM) to examine the vascularization of these grafts following Reversine treatment. Following culture, it was determined that Reversine inhibited MNNG/HOS grafts growth, and decreased the density of blood vessels in the chick CAM. We then used CAM and chick embryonic yolk-sac membrane (YSM) to investigate the effects of Reversine on angiogenesis. The results revealed Reversine inhibited the proliferation of endothelial cells, where cells were mainly arrested at G1/S phase of the cell cycle. Scratch-wound assay with HUVECs revealed that Reversine suppressed cell migration in vitro. Furthermore, endothelial cells tube formation assay and chick aortic arch sprouting assay demonstrated Reversine inhibited the sprouting, migration of endothelial cells. Lastly, qPCR and western blot analyses showed BMP-associated Smad1/5/8 signaling expressions were up-regulated by Reversine treatment. Our results showed that Reversine could suppress tumor growth by inhibiting angiogenesis through BMP signaling, and suggests a potential use of Reversine as an anti-tumor therapy.

Autophagy inhibition by reversine and its suppressive effects on human hepatocellular carcinoma cells.

INTRODUCTION: Therapy for human hepatocellular carcinoma (HCC) remains a great challenge for physicians and patients worldwide. The anti-tumor effects of reversine have attracted much more concerns. MATERIALS AND METHODS: This study evaluated the growth regulatory effects of reversine on HCC cells lines. Meanwhile, the underlying mechanism including autophagy modulation was also identified. RESULTS: reversine markedly inhibited the proliferation of both HCC cells and induced cell apoptosis and multinuclear in a dose-dependent manner. In addition, the decreased ratio of LC3II/LC3I as well as elevated p62 expression were observed under reversine treatment, indicating the autophagy inhibition by reversine in HepG2 cell line. Moreover, modulation of autophagy with rapamycin and chloroquine significantly attenuated and enhanced the cytostatic effects of reversine, respectively. CONCLUSIONS: reversine could reduce the cell viability of HCC cells via inducing cell apoptosis and polyploidy. In addition, cell autophagy was involved and might play a protective role in HCC cells, the joint use of autophagy inhibitor enhanced reversine-mediating antitumor effects. Our data offered novel ideas for comprehensive therapeutic regimes on human hepatocellular carcinoma.

Effects of Different Concentrations of Reversine on Plasticity of Mesenchymal Stem Cells.

Dedifferentiation can be induced by small molecules. One of these small molecules used in this study in order to increase the plasticity of differentiation of stem cells was reversine. The objective of present study was to investigate the effect of different concentrations of reversine on the plasticity of ovine fetal bone-marrow mesenchymal stem cells (BM-MSCs). BM-MSCs were extracted from ovine fetal and cultured. Passaged-3 cells were evaluated for their differentiation potential into osteocytes and adipocytes cells. In the present study, BM-MSCs were culture plated in the presence of 0, 300, 600, 900 and 1200 nM of reversine. The number of viable cells was determined by MTT test after addition of different concentrations of reversine. Furthermore, expression of the nanog gene was evaluated. The culture without reversine was taken as the control group. Expression of nanog was analysed by immunocytochemistry. Multi-lineage differentiation showed that the BM-MSCs could be differentiated into adipose cells and osteocytes. Our results indicated that the addition of 1200 nM of reversine to medium significantly decreased overall proliferation compared to the other treatment groups (p > 0.05). Real-time PCR analysis showed that after addition of 600 nM of reversine significantly increased nanog expression compared to other treatments. All treatments received reversine were seen to be relative expression of nanog. Our findings confirm that low concentrations reversine increases the plasticity of ovine BM-MSCs.

Reversine promotes browning of white adipocytes by suppressing miR-133a.

Brown adipocytes are characterized by a high number of uncoupling protein 1 (UCP1)-positive mitochondrial content and increased thermogenic capacity. As UCP1-enriched cells can consume lipids by generating heat, browning of white adipocytes is now highlighted as a promising approach for the prevention of obesity and obesity-associated metabolic diseases. Upon cold exposure or ß-adrenergic stimuli, downregulation of microRNA-133 (miR-133) elevates the expression levels of PR domain containing 16 (Prdm16), which has been shown to be a brown adipose determination factor, in brown adipose tissue and subcutaneous white adipose tissues (WAT). Here, we show that treatment of reversine to white adipocytes induces browning via suppression of miR-133a. Reversine treatment promoted the expression of brown adipocyte marker genes, such as Prdm16 and UCP1, increasing the mitochondrial content, while decreasing the levels of miR-133a and white adipocyte marker genes. Ectopic expression of miR-133a mimic reversed the browning effects of the reversine treatment. Moreover, intraperitoneal administration of reversine in mice upregulated thermogenesis and resulted in resistance to high-fat diet-mediated weight gain as well as browning of subcutaneous and epididymal WAT. Taken together, we found a novel way to promote browning of white adipocytes through downregulation of miR-133a followed by activation of Prdm16, with a synthetic chemical, reversine.

Relation of AURKB over-expression to low survival rate in BCRA and reversine-modulated aurora B kinase in breast cancer cell lines.

BACKGROUND: New therapeutic drug for breast cancer (BRCA), especially triple negative BRCA (TNBC), is urgently needed. Even though 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (reversine) is an aurora kinase inhibitor, it also inhibits some cancer cells and human BRCA cells. However, the potential roles of reversine as a novel therapeutic agent for the treatment of BRCA remains unknown and must be further investigation. Thus, the relationship of reversine to aurora kinase in BCRA has not been reported. The relationship between AURKB and survival rate in BRCA has never been reported. Herein, we tested the roles of reversine on different BRCA cell line subtypes. We also investigated the relationship between AURKB and survival rate in BRCA as well as reversine to Aurora kinase expression in BCRA cell lines, including TNBC subtype, 4T1, MDA-MB-231, and luminal subtype MCF-7. METHODS: Cell viability and apoptosis were detected using Cell Counting Kit-8 and flow cytometry analysis, respectively. Apoptotic and tumor-related proteins were tested using Western blot analysis. Important microRNAs that regulate BRCA were analyzed using RT-PCR. UALCAN public databases were used to analyze the targeted gene profiles, and the PROGgeneV2 database was used to study the prognostic implications of genes. RESULTS: Reversine inhibits cell proliferation and induces cell apoptosis by modulating caspase-3 and bax/bcl-2 among the three cell lines. Data from the UALCAN public database show that BRCA tissues expressed high gene levels of AURKB, TIMP1, MMP9, and TGFB1 compared with the normal tissue. Among the over-expressed genes in BRCA, AURKB ranks 9th in TNBC, 49th in luminal subtype, and 48th in HER2 subtype. High AURKB level in BRCA is highly related to the low survival rate in patients displayed in 18 databases searched via PROGgeneV2. The protein levels of aurora B kinase (Aurora B), which is encoded by AURKB gene, are highly suppressed by reversine in the three cell lines. The tumor-related proteins TGF-ß1, TIMP1, and MMP9 are partially suppressed by reversine but with different sensitivity in the three cell lines. The reversine-affected microRNAs, such as miR129-5p, miR-199a-3p, and miR-3960, in MDA-MB-231 cell line might be the research targets in TNBC regulation. CONCLUSIONS: In BRCA, the level of AURKB are over-expressed and is related to low survival rate. Reversine contributes to anti-growth effect in BRCA cell lines, especially for TNBC, by modulating the aurora B. However, the invasiveness, metastasis, and anti-tumor effects of reversine in vivo and in vitro must be further investigated.

Reversine inhibits MMP-1 and MMP-3 expressions by suppressing of ROS/MAPK/AP-1 activation in UV-stimulated human keratinocytes and dermal fibroblasts.

UVB has been shown to stimulate the generation of reactive oxygen species (ROS), which subsequently results in the activation of various intracellular signalling pathways and transcription factors (AP-1, NF-κB). These transcription factors are regulated by MAPKs, which increase cytokine and MMP expression. We examined the preventive effects of reversine on MMP-1 and MMP-3 expressions in NHEKs and NHDFs exposed to UVB irradiation. Also, we confirmed that reversine decreased pro-inflammatory cytokine expression in NHEKs. The mechanism underlying the MMP inhibitory effects of reversine occurred via the suppression of UVB-induced ROS generation and MAPK/AP-1 activation. Therefore, reversine is an effective therapeutic candidate for preventing skin photoageing.

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment.

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C-MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.

Synthesis of 2,6-Diamino-Substituted Purine Derivatives and Evaluation of Cell Cycle Arrest in Breast and Colorectal Cancer Cells.

Reversine is a potent antitumor 2,6-diamino-substituted purine acting as an Aurora kinases inhibitor and interfering with cancer cell cycle progression. In this study we describe three reversine-related molecules, designed by docking calculation, that present structural modifications in the diamino units at positions 2 and 6. We investigated the conformations of the most stable prototropic tautomers of one of these molecules, the N6-cyclohexyl-N6-methyl-N2-phenyl-7H-purine-2,6-diamine (3), by Density Functional Theory (DFT) calculation in the gas phase, water and chloroform, the last solvent considered to give insights into the detection of broad signals in NMR analysis. In all cases the HN(9) tautomer resulted more stable than the HN(7) form, but the most stable conformations changed in different solvents. Molecules 1⁻3 were evaluated on MCF-7 breast and HCT116 colorectal cancer cell lines showing that, while being less cytotoxic than reversine, they still caused cell cycle arrest in G2/M phase and polyploidy. Unlike reversine, which produced a pronounced cell cycle arrest in G2/M phase in all the cell lines used, similar concentrations of 1⁻3 were effective only in cells where p53 was deleted or down-regulated. Therefore, our findings support a potential selective role of these structurally simplified, reversine-related molecules in p53-defective cancer cells.

Inhibition of Suicidal Erythrocyte Death by Reversine.

BACKGROUND/AIMS: The A3 adenosine receptor antagonist reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine) influences cellular differentiation, inhibits cell proliferation, induces cell-cycle arrest, triggers apoptosis, causes cell swelling with polyploidy and stimulates autophagy. The effect on apoptosis involves mitochondria and caspases. Erythrocytes are lacking mitochondria but express caspases and are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), energy depletion and oxidative stress. The present study explored, whether reversine influences eryptosis. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), Ca2+ loading (30 minutes treatment with 1 µM Ca2+ ionophore ionomycin), or oxidative stress (15 min exposure to 0.3 mM tert-butylhydroperoxide). RESULTS: A 48 hours exposure of human erythrocytes to reversine (1-10 µM) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, Ca2+ loading, and oxidative stress were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of each, Ca2+ loading, energy depletion and oxidative stress on annexin-V-binding were significantly blunted in the presence of reversine (1-10 µM). The effect of ionomycin, but not the effects of energy depletion and oxidative stress on forward scatter were again significantly blunted in the presence of reversine (≥1 µM]. CONCLUSIONS: Reversine is a powerful inhibitor of cell membrane scrambling following energy depletion, Ca2+ loading and oxidative stress.

Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase.

Monopolar spindle 1 (Mps1, also known as TTK) is a protein kinase crucial for ensuring that cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules. Incomplete chromosomal attachment leads to abnormal chromosome counts in the daughter cells (aneuploidy), a condition common in many solid cancers. Therefore Mps1 is an established target in cancer therapy. Mps1 kinase inhibitors include reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine), a promiscuous compound first recognized as an inhibitor of the Aurora B mitotic kinase. Here, we present the 3.0-Å resolution crystal structure of the Mps1 kinase domain bound to reversine. Structural comparison of reversine bound to Mps1 and Aurora B, indicates a similar binding pose for the purine moiety of reversine making three conserved hydrogen bonds to the protein main chain, explaining the observed promiscuity of this inhibitor. The cyclohexyl and morpholinoaniline moieties of reversine however, have more extensive contacts with the protein in Mps1 than in Aurora B. This is reflected both in structure-based docking energy calculations, and in new experimental data we present here, that both confirm that the affinity of reversine towards Mps1 is about two orders of magnitude higher than towards Aurora B. Thus, our data provides detailed structural understanding of the existing literature that argues reversine inhibits Mps1 more efficiently than Aurora B based on biochemical and in-cell assays. Proteins 2016; 84:1761-1766. © 2016 Wiley Periodicals, Inc.

Effect of reversine on cell cycle, apoptosis, and activation of hepatic stellate cells.

Experimental and clinical evidence show that liver fibrosis is potentially reversible. Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis. Some studies have shown that reversine could induce cell apoptosis. We attempted to elucidate the effect of reversine on cell cycle, apoptosis, and activation of HSCs. Data showed that reversine induced morphological changes in HSCs, inhibited cell proliferation, and induced cell-cycle arrest at the G2/M phase. Reversine induced cell apoptosis through caspase-dependent and mitochondria-dependent pathways. Reversine inhibited the activation of HSCs through TGF-ß signaling pathway and degraded extracellular matrix protein collagen-I. The decreased TIMP1 and TGF-ß1 proteins promoted fibrosis reversion. Reversine might be a promising drug for liver fibrosis reversion because it induces HSCs apoptosis, restrains cell proliferation, reduces HSCs activation, and degrades extracellular matrix in vitro.

Ovine fetal mesenchymal stem cell differentiation to cardiomyocytes, effects of co-culture, role of small molecules; reversine and 5-azacytidine.

The aim of the present study was to investigate the effect of small molecules: Reversine and 5-azacytidine (5-AC), in an indirect co-culture condition with the cardiac fibroblasts as well as non co-culture condition, in order to explore the effect of such molecules in the process of differentiation of the ovine bone-marrow mesenchymal stem cells (BM-MSCs) towards cardiomyocytes. Surface antigens of the isolated cells were analysed using flow-cytometry. In addition, following to three passages cells were examined for their differentiation capacity into osteocytes and adipose cells, in order to ensure the mesenchymal origin of the stem cells. Six types of treatments were carried out in the present investigation, such that, in the first treatment BM-MSCs were cultured for 28 days as control group; the second treatment was composed of culturing ovine fetal cardiac fibroblasts on inserts, aiming to use these inserts for culturing plates which were seeded with BM-MSCs (Chamber group). As the third treatment, BM-MSCs were supplemented with 10-µM 5-AC and incubated for 48 h. The fourth treatment was composed of supplementing BM-MSCs with the 600-nM reversine, incubated for 48 h, and subsequently the incubation was further extended for another 48 h in the presence of 5-AC. The fifth treatment was composed of supplementing the chamber group with 10-µM 5-AC and incubation for 48 h, and the last or the sixth treatment was such that chamber group was supplemented with 600-nM reversine and an incubation period of 48 h. Following to the incubation, medium was replaced with 10-µM 5-AC and further incubated for another round of 48 h. In all treatments, following to addition of the small molecules incubations were carried out for 28 days; same as controls. Expression of cardiac alpha-actinin was analysed by immunocytochemistry. BM-MSCs have shown to express CD44 and CD166 along with a weak expression of the CD90, CD34, in addition to CD45. Multilineage differentiation has indicated that BM-MSCs could differentiate into adipose and osteocytes cells as well. In the treatment 4 it was observed that FGF signalling involved genes and all cardiac-related genes (ANP, MYH6 and Troponin I) were significantly expressed, except connexin 43 compared to other treatments. All treatments received small molecules, either alone or as a co-culture were seen to express sarcomeric alpha-actinin. This finding was partially supported by immunocytochemistry. These results validate that reversine and 5-AC have an effect on ovine BM-MSC differentiation into cardiomyocytes. Copyright © 2016 John Wiley & Sons, Ltd.

A chemical approach to myocardial protection and regeneration.

The possibility of generating induced pluripotent stem cells from mouse embryonic fibroblasts and human adult fibroblasts has introduced new perspectives for possible therapeutic strategies to repair damaged hearts. However, obtaining large numbers of adult stem cells is still an ongoing challenge, and the safety of genetic reprogramming with lenti- or retro-viruses has several drawbacks not easy to be addressed. Furthermore, the majority of adult stem cell-based clinical trials for heart regeneration have had generally poor and controversial results. Nonetheless, it is now clear that the injected cells activate the growth and differentiation of progenitor cells that are already present in the heart. This is achieved by the release of signalling factors and/or exosomes carrying them. Along this line, chemistry may play a major role in developing new strategies for activating resident stem cells to regenerate the heart. In particular, this review focuses on small molecule approaches for cell reprogramming, cell differentiation, and activation of cell protection.

Reversine induces multipotency of lineage-committed cells through epigenetic silencing of miR-133a.

Reversine has been shown to induce dedifferentiation of C2C12 murine myoblasts into multipotent progenitor cells. However, little is known about the key regulators mediating the dedifferentiation induced by reversine. Here, we show that large scale miRNA gene expression profiling of reversine-treated C2C12 myoblasts identifies a down-regulated miRNA, miR-133a, involved in dedifferentiation of myoblasts. Reversine treatment results in up- and down-regulated miRNA profiles. Among miRNAs affected by reversine, the level of muscle-specific miR-133a, which has been shown to be up-regulated during muscle development and to suppress differentiation into other lineages, is markedly reduced by treatment of C2C12 myoblasts with reversine. In parallel, reversine decreases the expression and recruitment of myogenic factor, SRF, to the enhancer regions of miR-133a. Sequentially, down-regulation of miR-133a by reversine is accompanied by a decrease in active histone modifications including trimethylation of histone H3K4 and H3K36, phosphorylation of H3S10, and acetylation of H3K14 on the miR-133a promoter, leading to dissociation of RNA polymerase II from the promoter. Furthermore, inhibition of miR-133a by transfection of C2C12 myoblasts with miR-133a inhibitor increases the expression of osteogenic lineage marker, Ogn, and adipotenic lineage marker, ApoE, similar to that in response to reversine. In contrast, the co-overexpression of miR-133a mimic reversed the effect of reversine on C2C12 myoblast dedifferentiation. Taken together, the results indicate that reversine induces a multipotency of C2C12 myoblasts by suppression of miR-133a expression through depletion of active histone modifications, and suggest that miR-133a is a potential miRNA regulating the reversine-induced dedifferentiation. Collectively, our findings provide a mechanistic rationale for the application of reversine to dedifferentiation of somatic cells.

Reversine increases the plasticity of lineage-committed preadipocytes to osteogenesis by inhibiting adipogenesis through induction of TGF-ß pathway in vitro.

Reversine has been reported to reverse differentiation of lineage-committed cells to mesenchymal stem cells (MSCs), which then enables them to be differentiated into other various lineages. Both adipocytes and osteoblasts are known to originate from common MSCs, and the balance between adipogenesis and osteogenesis in MSCs is reported to modulate the progression of various human diseases, such as obesity and osteoporosis. However, the role of reversine in modulating the adipogenic potential of lineage-committed preadipocytes and their plasticity to osteogenesis is unclear. Here we report that reversine has an anti-adipogenic function in 3T3-L1 preadipocytes in vitro and alters cell morphology and viability. The transforming growth factor-ß (TGF-ß) pathway appears to be required for the anti-adipogenic effect of reversine, due to reversine-induced expression of genes involved in TGF-ß pathway and reversal of reversine-inhibited adipogenesis by inhibition of TGF-ß pathway. We show that treatment with reversine transformed 3T3-L1 preadipocytes into MSC-like cells, as evidenced by the expression of MSCs marker genes. This, in turn, allowed differentiation of lineage-committed 3T3-L1 preadipocytes to osteoblasts under the osteogenic condition in vitro. Collectively, these findings reveal a new function of reversine in reversing lineage-committed preadipocytes to osteogenesis in vitro, and provide new insights into adipose tissue-based regeneration of osteoblasts.

Reversine inhibits proliferation and induces apoptosis of human osteosarcoma cells through targeting MEK1.

Reversine, or 2-(4-morpholinoanilino)-6-cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule shows anti-tumor potential by playing a central role in the inhibition of several kinases related to cell cycle regulation and cytokinesis. In this study, systematic review demonstrated the feasibility and pharmacological mechanism of anti-tumor effect of reversine. Firstly, we grafted MNNG/HOS, U-2 OS, MG-63 osteosarcoma cell aggregates onto chicken embryonic chorioallantoic membrane (CAM) to examine the tumor volume of these grafts after reversine treatment. Following culture, reversine inhibited the growth of osteosarcoma cell aggregates on CAM significantly. In vitro experiment, reversine suppressed osteosarcoma cell viability, colony formation, proliferation, and induced apoptosis and cell cycle arrest at G0-G1 phase. Scratch wound assay demonstrated that reversine restrained cell migration. Reversine increased the protein expression of E-cadherin. The mRNA expression of Rac1, RhoA, CDC42, PTK2, PXN, N-cadherin, Vimentin in MNNG/HOS, U-2 OS and MG-63 cells were suppressed and PTEN increased after reversine treatment. Network pharmacology prediction, molecular docking and systematic review revealed MEK1 can be used as an effective target for reversine to inhibit osteosarcoma. Western blot results show the regulation of MEK1 and ERK1/2 by reversine was not consistent in different osteosarcoma cell lines, but we found that reversine significantly inhibited the protein expression of MEK1 in MNNG/HOS, U-2 OS and MG-63. All these suggested that reversine can exert its anti-tumor effect by targeting the expression of MEK1.

In Vitro Characterization of Reversine-Treated Gingival Fibroblasts and Their Safety Evaluation after In Vivo Transplantation.

Reversine is a purine derivative that has been investigated with regard to its biological effects, such as its anticancer properties and, mostly, its ability to induce the dedifferentiation of adult cells, increasing their plasticity. The obtained dedifferentiated cells have a high potential for use in regenerative procedures, such as regenerative dentistry (RD). Instead of replacing the lost or damaged oral tissues with synthetic materials, RD uses stem cells combined with matrices and an appropriate microenvironment to achieve tissue regeneration. However, the currently available stem cell sources present limitations, thus restricting the potential of RD. Based on this problem, new sources of stem cells are fundamental. This work aims to characterize mouse gingival fibroblasts (GFs) after dedifferentiation with reversine. Different administration protocols were tested, and the cells obtained were evaluated regarding their cell metabolism, protein and DNA contents, cell cycle changes, morphology, cell death, genotoxicity, and acquisition of stem cell characteristics. Additionally, their teratoma potential was evaluated after in vivo transplantation. Reversine caused toxicity at higher concentrations, with decreased cell metabolic activity and protein content. The cells obtained displayed polyploidy, a cycle arrest in the G2/M phase, and showed an enlarged size. Additionally, apoptosis and genotoxicity were found at higher reversine concentrations. A subpopulation of the GFs possessed stem properties, as supported by the increased expression of CD90, CD105, and TERT, the existence of a CD106+ population, and their trilineage differentiation capacity. The dedifferentiated cells did not induce teratoma formation. The extensive characterization performed shows that significant functional, morphological, and genetic changes occur during the dedifferentiation process. The dedifferentiated cells have some stem-like characteristics, which are of interest for RD.

Reversine attenuates cholestatic ductular reaction in rats.

Ductular reaction (DR) is usually observed in biliary disorders or various liver disorders, including nonalcoholic fatty liver disease. Few studies have focused on interrupting the DR process in the cholestatic environment. Here, we investigated the impact of reversine on DR in rats that had undergone bile duct ligation (BDL). Cholestatic injury was induced in rats 2 weeks following BDL. DR was assessed with biliary markers by immunohistochemistry. Biliary epithelial cells (BECs) were isolated for the analysis of proliferation and biliary factor gene expression. The effects of reversine on DR and fibrosis were analyzed in vivo via intraperitoneal injection in rats for 2 weeks. Chemically-induced BEC formation was used to investigate the biliary markers affected by reversine in vitro. DR with increased BEC expansion was identified in cholestatic liver injury, as indicated by CK7, CK19, and EpCAM expression around the portal vein in BDL rats. BDL-induced DR cells showed the increased expression of genes regulating cell proliferation (Ki67, Foxm1, and Pcna) and biliary markers (Krt7, Krt19, Epcam, Sox9, Cftr, and Asbt). Reversine attenuated cholestatic fibrosis and DR in rats. Reversine affected chemically-induced BEC formation, with the decreased expression of biliary Krt7, Cftr, and Ggt1 genes in vitro. BDL-induced Notch activation was attenuated upon reversine treatment in vivo, in part via the Notch/Sox9 pathway. In conclusion, reversine attenuated cholestatic ductular reaction and fibrosis in rats and reduced the bile duct formation associated with Dlk1/Notch/Sox9 signaling. Reversine may be regarded as a potential drug for cholangiopathies for preventing a ductular reaction.

Treatment of CHO cells with Taxol and reversine improves micronucleation and microcell-mediated chromosome transfer efficiency.

Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research.

Reversine enhances the conversion of dedifferentiated fat cells into mature cardiomyocytes.

Background: There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn't fully understood. Methods: We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. Results: ANOVA with Tukey's post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p < 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p =0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p= 0 .001) increased significantly from day 7 until day 21. Conclusions: Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to cell stemness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.