Increased Cell Differentiation Research Articles

Targeting HDAC9 Contributes to Degradation of MLL Fusion Oncoproteins in KMT2A-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia with MLL rearrangement (MLLr-AML) is a highly aggressive subtype of AML. Existing treatment strategies are nonspecific and ineffective. The MLL fusion gene is one of the driver oncogenes in leukemia and is central to the pathogenesis of MLLr-AML, and more research has recently begun to focus on targeting MLL fusion proteins. Targeting MLL fusion proteins has been shown to have a tumor suppressor effect, demonstrating efficacy in preclinical models of MLLr-AML, and in 2022 Scott A Armstrong ‘s team suggested that degradation of MLL-AF9 can cause an increase in cell differentiation and apoptosis, confirming the efficacy of targeting MLL fusions. However, specific targets that degrade MLL fusion proteins as well as clinically translatable targeted therapies are still lacking and need to be further explored. Study confirmed that MLL-rearranged ALL cells are highly sensitive to the broad-spectrum HDAC inhibitor panobinostat. A recent clinical study by Mireia Camós’ team proposed that HDAC9 has a significantly high expression in childhood MLL-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and AML, but that article did not address any mechanistic studies. The potential value of HDAC9 in adult lymphoid hematologic diseases has been gradually revealed, however, the expression profile and precise molecular mechanisms of HDAC9 in adult myeloid hematologic tumors, have not been reported, which deserves to be further explored. In order to investigate the expression and role of HDAC9 in MLLr-AML, we analyzed the second-generation sequencing data from the Beat AML dataset and found that HDAC9 has a significantly high expression in MLLr-AML, while only HDAC9 is characterized by this feature in HDAC family molecules. Further we analyzed the data of 300 adult AML specimens in our hospital (including 20 MLLr-AML patients), and the results further confirmed that only HDAC9 among the HDAC family had a significantly higher expression level in the MLLr-AML patient group than that in the Non-MLLr-AML patient group (P<0.001), and the high expression of HDAC9 in the MLLr-AML patient group was a common phenomenon and not limited to a particular class of MLL chaperone molecules. Further studies revealed that overexpression of MLL fusion proteins in HEK293T could cause up-regulation of HDAC9 protein levels, and this experiment clarified the direct correlation between the MLL fusion genes and the high expression of HDAC9. The HDAC IIa inhibitor (TMP-269), which can target HDAC9, was significantly more sensitive in MLLr-AML cell lines than in Non-MLLr-AML cell lines, and also that MLLr-AML cell survival was dependent on HDAC9, and that knockdown of HDAC9 induced an increase in apoptosis and a slowdown in proliferation in MLLr-AML cells. We subsequently demonstrated that knockdown of HDAC9 downregulates the expression of MLL fusion proteins through degradation of MLL proteins. Furthermore, when exploring therapeutic options for targeting MLLr-AML, we found that combining the BCL-2 inhibitor Venetoclax (VEN) with an MLL-Menin specific inhibitor (MEN1i) specifically downregulated the expression of HDAC9 and had a favorable synergistic killing effect on MLLr-AML in both in vitro and in vivo models, further suggesting an important role of HDAC9 in the treatment of MLLr-AML. To sum up, through this study, we can shed light on the role of specific HDAC molecules in MLLr-AML and provide a new potential target for the degradation of MLL fusion protein to eradicate MLLr-AML.

Rhamnolipid 89 Biosurfactant Is Effective against Streptococcus oralis Biofilm and Preserves Osteoblast Behavior: Perspectives in Dental Implantology.

Biofilm-related peri-implant diseases represent the major complication for osteointegrated dental implants, requiring complex treatments or implant removal. Microbial biosurfactants emerged as new antibiofilm coating agents for implantable devices thanks to their high biocompatibility. This study aimed to assess the efficacy of the rhamnolipid 89 biosurfactant (R89BS) in limiting Streptococcus oralis biofilm formation and dislodging sessile cells from medical grade titanium, but preserving adhesion and proliferation of human osteoblasts. The inhibitory activity of a R89BS coating on S. oralis biofilm formation was assayed by quantifying biofilm biomass and microbial cells on titanium discs incubated up to 72 h. R89BS dispersal activity was addressed by measuring residual biomass of pre-formed biofilms after rhamnolipid treatment up to 24 h. Adhesion and proliferation of human primary osteoblasts on R89BS-coated titanium were evaluated by cell count and adenosine-triphosphate quantification, while cell differentiation was studied by measuring alkaline phosphatase activity and observing mineral deposition. Results showed that R89BS coating inhibited S. oralis biofilm formation by 80% at 72 h and dislodged 63-86% of pre-formed biofilms in 24 h according to concentration. No change in the adhesion of human osteoblasts was observed, whereas proliferation was reduced accompanied by an increase in cell differentiation. R89BS effectively counteracts S. oralis biofilm formation on titanium and preserves overall osteoblasts behavior representing a promising preventive strategy against biofilm-related peri-implant diseases.

OsARF4 regulates leaf inclination via auxin and brassinosteroid pathways in rice.

Leaf inclination is a vital agronomic trait and is important for plant architecture that affects photosynthetic efficiency and grain yield. To understand the molecular mechanisms underlying regulation of leaf inclination, we constructed an auxin response factor (arf) rice mutant—osarf4—showing increased leaf inclination using CRISPR/Cas9 gene editing technology. OsARF4 encodes a nuclear protein that is expressed in the lamina joint (LJ) at different developmental stages in rice. Histological analysis indicated that an increase in cell differentiation on the adaxial side resulted in increased leaf inclination in the osarf4 mutants; however, OsARF4-overexpressing lines showed a decrease in leaf inclination, resulting in erect leaves. Additionally, a decrease in the content and distribution of indole-3-acetic acid (IAA) in osarf4 mutant led to a greater leaf inclination, whereas the OsARF4-overexpressing lines showed the opposite phenotype with increased IAA content. RNA-sequencing analysis revealed that the expression of genes related to brassinosteroid (BR) biosynthesis and response was different in the mutants and overexpressing lines, suggesting that OsARF4 participates in the BR signaling pathway. Moreover, BR sensitivity assay revealed that OsARF4-overexpressing lines were more sensitive to exogenous BR treatment than the mutants. In conclusion, OsARF4, a transcription factor in auxin signaling, participates in leaf inclination regulation and links auxin and BR signaling pathways. Our results provide a novel insight into l leaf inclination regulation, and have significant implications for improving rice architecture and grain yield.

Ubiquitin E3 ligase FBXO21 regulates cytokine-mediated signaling pathways, but is dispensable for steady-state hematopoiesis

Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways. One pathway, the ubiquitin proteasome system (UPS), controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. Ubiquitin E3 ligases serve as the substrate-recognition component of the UPS. By investigating the FBOX family of E3 ligases, we discovered that Fbxo21 was highly expressed in the hematopoietic stem and progenitor cell (HSPC) population, and exhibited low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a hematopoiesis-specific Fbxo21 conditional knockout (cKO) mouse model. We found that silencing FBXO21 in HSPCs led to a loss in colony formation and an increase in cell differentiation in vitro. Additionally, stressing the HSPC populations in our Fbxo21 cKO mouse with 5-fluorouracil injections resulted in a decrease in survival, despite these populations exhibiting minimal alterations during steady-state hematopoiesis. Although FBXO21 has previously been proposed to regulate cytokine signaling via ASK and p38, our results indicate that depletion of FBXO21 led to altered ERK signaling in vitro. Together, these findings suggest ubiquitin E3 ligase FBXO21 regulates HSPCs through cytokine-mediated pathways.

3226 – THE ROLE OF UBIQUITIN E3 LIGASE FBXO21 IN REGULATING HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPC) THROUGH CYTOKINE MEDIATED PATHWAYS

Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways, one being the ubiquitin proteasome system (UPS) which controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The substrate recognition component of the UPS is the ubiquitin E3 ligase. Through investigating a specific family of ubiquitin E3 ligases, the Fbox family of proteins, we discovered that Fbxo21 was highly expressed in the HSPC population and was more highly expressed when compared to other Fbox genes. Western blot and qRT-PCR analysis confirmed high expression of FBXO21 in HSPCs, and revealed low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a conditional Fbxo21 knockout (KO) mouse model. We found that silencing FBXO21 in human CD34+ and mouse HSPC cells led to a loss in colony formation and an increase in cell differentiation. In our conditional Fbxo21 KO, we found a decrease in the number of colonies formed after replating and minimal differences in the production of specific hematopoietic cell populations in steady-state hematopoiesis. However upon 5-FU injections, we see a decrease in survival when Fbxo21 is knocked down. When further investigating targets of FBXO21, we found an increase in the activation of proteins involved in cytokine signaling when Fbxo21 is knocked down. FBXO21 has been previously linked to MAPK signaling which leads us to hypothesize that the ubiquitin E3 ligase FBXO21 regulates hematopoietic stem and progenitor cells through cytokine mediated pathways. Deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.

Characterization of a new small-molecule inhibitor of HDAC6 in glioblastoma

Histone deacetylase 6 (HDAC6) is an epigenetic modifier that is an attractive pharmacological target in cancer. In this work, we show that HDAC6 is elevated in glioblastoma, the most malignant and common brain tumor in adults, in which its high levels correlate with poor patient survival and is more abundant in glioma stem cell subpopulation. Moreover, we identified a new small-molecule inhibitor of HDAC6, which presents strong sensitivity for HDAC6 inhibition and exerts high cytotoxic activity, alone or in combination with temozolomide. It is also able to significantly reduce tumor growth in vivo. Transcriptomic analysis of patient-derived glioma stem cells revealed an increase in cell differentiation and cell death pathways, as well as a decrease in cell-cycle activity and cell division by the treatment with the compound. Finally, the comparison with a pan-HDAC inhibitor, Vorinostat (SAHA), or HDAC6-specific inhibitor, Tubastatin A, showed higher target specificity and antitumor activity of the new HDAC6 inhibitor. In conclusion, our data reveal the efficacy of a novel HDAC6 inhibitor in glioblastoma preclinical setting.

MicroRNA-495 inhibits the high glucose-induced inflammation, differentiation and extracellular matrix accumulation of cardiac fibroblasts through downregulation of NOD1

BackgroundMicroRNAs (miRNAs) have physiological and pathophysiological functions that are involved in the regulation of cardiac fibrosis. This study aimed to investigate the effects of miR-495 on high glucose-induced cardiac fibrosis in human cardiac fibroblasts (CFs) and to establish the mechanism underlying these effects.MethodsHuman CFs were transfected with an miR-495 inhibitor or mimic and incubated with high glucose. The levels of NOD1 and miR-495 were then determined via quantitative RT-PCR. Pro-inflammatory cytokine levels, cell differentiation and extracellular matrix accumulation were respectively detected using ELISA, quantitative RT-PCR and western blot assays. The luciferase reporter assay, quantitative RT-PCR and western blot were used to explore whether NOD1 was a target of miR-495. The effects of miR-495 on the NF-κB and TGF-β1/Smad signaling pathways were also detected via western blot.ResultsOur results show that high glucose can significantly increase the expression of NOD1 in a time-dependent manner. Upregulation of miR-495 significantly alleviated the high glucose-induced increases in cell differentiation and collagen accumulation of CFs. Moreover, the bioinformatics analysis predicted that NOD1 was a potential target gene for miR-495. The luciferase reporter assay showed that miR-495 can directly target NOD1. The introduction of miR-495 could significantly inhibit the high glucose-activated NF-κB and TGF-β1/Smad signaling pathways.ConclusionUpregulation of miR-495 ameliorates the high glucose-induced inflammatory, cell differentiation and extracellular matrix accumulation of human CFs by modulating both the NF-κB and TGF-β1/Smad signaling pathways through downregulation of NOD1 expression. These results provide further evidence for the protective effect of miR-495 overexpression in cases of high glucose-induced cardiac fibrosis.

The application of a mechanistic model to analyze the factors that affect the lactation curve parameters of dairy sheep in Mexico

Pollott́s mechanistic model has been designed to describe lactation curve parameters based on the known biology of milk production and can be useful for analyzing the factors that affect this process. A total of 553 lactations (9956 weekly test-day records) of crossbred dairy sheep from four commercial farms located in Mexico, were analyzed to investigate environmental factors that influenced lactation curve parameters, using Pollott’s 5-parameter additive model. This model was fitted to each lactation using an iterative nonlinear procedure. The estimated parameters were maximum milk secretion potential (MSmax), relative rate of increase in cell differentiation (GR), maximum secretion loss (MSLmax), relative rate of decline in cell numbers (DR) and the proportion of parenchyma cells dead at parturition. A general linear model procedure was used to determine the effect of type of lambing, lambing number, flock and lambing season on total lactation milk yield (TMY), lactation length and estimated parameters of the Pollott model. Ewes had an average milk yield of 74.4 L with an average lactation length of 140 days. Flock had a significant (P < 0.05) effect on most of the analyzed traits, which can be explained by the different farmś management practices. The TMY were significantly (P = 0.005) higher for twin-lambing than single-lambing lactations. Sheep in their first lambing had lower TMY than those in their fourth lambing (P = 0.01), possibly explained by the lower values of MSmax (2.85 vs. 5.3 L) and the decrease in DR throughout life (P = 0.03). However, the relative GR was greatest (P = 0.04) during first lambing and then decreased as lambing number increased. Both lambing number and type of lambing also affected milk yield. The parameters of the Pollott model can be useful to explain, with a biological approximation, the dynamics of differentiation, secretion and death of mammary cells in dairy sheep.

Protective Effects of Human Chorionic Gonadotropin Against Breast Cancer: How Can We Use This Information to Prevent/Treat the Disease?

Breast cancers (BCs) are the most common malignancies among women worldwide. Giving birth to a first child before 24 years of age decreases the BC risk by about half, when women reach menopausal years. The scientific evidence suggests that the actions of human chorionic gonadotropin (hCG) are responsible for this decrease. Human BC cells and tissues contain hCG/luteinizing hormone receptors. The activation of the receptors results in an increase in cell differentiation and apoptosis. Conversely, it decreases the cell proliferation, invasion, and survival. The hCG actions are primarily cyclic adenosine monophosphate/protein kinase A mediated, require the presence of receptors, and involve blocking the activation and nuclear translocation of the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The women with a higher hCG levels during pregnancy tend to have a lower BC incidence and those with the receptor-positive tumors have a longer metastasis-free survival. The long-term benefits of pregnancy/hCG seem to come from permanent signature genomic imprinting and expression changes, which are characterized by low cell proliferation, increased efficiency of DNA repair mechanisms, cell differentiation, and cells resistance to carcinogenesis. These findings could provide clinical opportunities to use hCG for the prevention of BC in this modern era of increasing number of young women in our societies waiting longer than ever to have their first child. In addition, hCG may be useful to reduce and/or eliminate cellular targets of carcinogenic changes during an active ongoing disease.

MicroRNA-98 inhibits TGF-β1-induced differentiation and collagen production of cardiac fibroblasts by targeting TGFBR1.

To investigate the effects of miR-98 on TGF-β1-induced cardiac fibrosis in human cardiac fibroblasts (HCFs), and to establish the mechanism underlying these effects, HCFs were transfected with miR-98 inhibitor or mimic, and then treated with or without TGF-β1. The level of miR-98 was determined by qRT-PCR in TGF-β1-induced HCFs. Cell differentiation and collagen accumulation of HCFs were detected by qRT-PCR and Western blot assays, respectively. The mRNA and protein expressions of TGFBR1 were determined by qRT-PCR and Western blotting. In this study, the outcomes showed that TGF-β1 could dramatically decrease the level of miR-98 in a time- and concentration-dependent manner. Upregulation of miR-98 dramatically improved TGF-β1-induced increases in cell differentiation and collagen accumulation of HCFs. Moreover, bioinformatics analysis predicted that the TGFBR1 was a potential target gene of miR-98. Luciferase reporter assay demonstrated that miR-98 could directly target TGFBR1. Inhibition of TGFBR1 had the similar effect as miR-98 overexpression. Downregulation of TGFBR1 in HCFs transfected with miR-98 inhibitor partially reversed the protective effect of miR-98 overexpression on TGF-β1-induced cardiac fibrosis in HCFs. Upregulation of miR-98 ameliorates TGF-β1-induced differentiation and collagen accumulation of HCFs by downregulation of TGFBR1. These results provide further evidence for protective effect of miR-98 overexpression on TGF-β1-induced cardiac fibrosis.

Neuroprotective effects of erythropoietin on neurodegenerative and ischemic brain diseases: the role of erythropoietin receptor.

Erythropoietin (Epo) is a fundamental hormone in the regulation of hematopoiesis, and other secondary roles mediated by the binding of the hormone to its specific receptor (EpoR), which leads to an activation of key signaling pathways that induce an increase in cell differentiation, apoptosis control and neuroprotection. It has been suggested that their function depends on final conformation of glycosylations, related with affinity to the receptor and its half-life. The presence of EpoR has been reported in different tissues including central nervous system, where it has been demonstrated to exert a neuroprotective function against oxidative stress conditions, such as ischemic injury and neurodegenerative diseases. There is also evidence of an increase in EpoR expression in brain cell lysates of Alzheimer's patients with respect to healthy patients. These results are related with extensive in vitro experimental data of neuroprotection obtained from cell lines, primary cell cultures and hippocampal slices. Additionally, this data is correlated with in vivo experiments (water maze test) in mouse models of Alzheimer's disease where Epo treatment improved cognitive function. These studies support the idea that receptor activation induces a neuroprotective effect in neurodegenerative disorders including dementias, and especially Alzheimer's disease. Taken together, available evidence suggests that Epo appears to be a central element for EpoR activation and neuroprotective properties in the central nervous system. In this review, we will describe the mechanisms associated with neuroprotection and its relation with the activation of EpoR in order with identify new targets to develop pharmacological strategies.

Osteogenic/Odontogenic Bioengineering with co-Administration of Simvastatin and Hydroxyapatite on Poly Caprolactone Based Nanofibrous Scaffold.

Purpose: Statin is an effective factor for promoting osteogenesis. The aim of the present study was to evaluate the effect of simvastatin (SIM) and/or HA addition on changes in osteogenesis levels by human DPSCs transferred onto three-dimensional (3D) nanofibrous Poly (ε-caprolactone) (PCL)/Poly lactic acide (PLLA) polymeric scaffolds. Methods: For this purpose, a 3D nanofibrous composite scaffold of PCL/PLLA/HA was prepared by electrospinning method. SIM was added to scaffolds during DPSCs culturing step. Cell proliferation and osteogenic activity levels were assessed by using MTT assay and Alizarin Red assay methods. In addition, the expression of genes responsible for osteogenesis, including BMP2, Osteocalcin, DSPP and RUNX2, were determined before and 2 weeks after incorporation of SIM. Results: The MTT assay showed that PCL/PLLA/HA scaffolds seeded with DPSCs has significant (p<0.05) more proliferative effect than PCL/PLLA or DMEM cultured cells, additionally SIM administration improved this result over the PCL/PLLA/HA scaffolds without SIM treatment. SEM imaging revealed improved adhesion and probably osteogenic differentiation of DPSCs on PCL/PLLA/HA nanofibers treated with SIM, moreover the alizarin red assay ensured significant (p<0.05) higher mineralization of this group. Finally, real time PCR confirmed the positive regulation (P<0.05) of the expression of osteo/odontogenesis markers BMP2, Osteocalcin, DSPP and RUNX2 genes in PLLA-PCL-HA (0.1)-SIM group. Conclusion: As a result, addition of simvastatin with incorporation of hydroxyapatite in PCL-PLLA scaffolds might increase the expression of osteogenesis markers in the DPSCs, with a possible increase in cell differentiation and bone formation.

Preclinical results of gemcitabinebased regimens for preoperative chemotherapy of spontaneous feline breast cancer

Comparative preclinical study of chemotherapy regimen doxorubicin (DOX) + gemcitabine (GEM) for preoperative chemotherapy of spontaneous feline breast cancer and standard monochemotherapy with DOX alone was performed on transplantable murine mammary adenocarcinoma Ca55 in BDF1 hybrid mice model. The total GEM doses of 100 and 200 mg/kg for the triple course in combination with the single injection of 7.5 mg/kg of DOX were chosen to treat the established Ca755 tumor of 595.5 mm 3 average volume (preoperative treatment modeling). The combined chemotherapy showed an advantage of DOX + GEM regimen with GEM course dose of 200 mg/kg (70, 70 and 60 mg/kg, accordingly) as compared to other groups in terms of Tumor Growth Inhibition (TGI max=100% versus 96%; number of complete remissions (CR) 30% versus 0%, and enhanced Life Expectancy (LE) 163% versus 84-85%). The most effective regimen demonstrated dose-dependent therapeutic pathomorphism of grade II-III with 1.5-fold decrease of cell polymorphism and increase of cell differentiation. At the same time, the number of cells in mitosis decreased in 3-4 times and the number of apoptotic cells increased in 5-10 times. The results suggest that DOX + GEM combination with the unchanged components doses ratio has good potential for preoperative chemotherapy of locally advanced feline breast cancer and may be recommended for clinical study of this disease.

A Serum Component Mediates Food Restriction–Induced Growth Attenuation

Proper nutrition in terms of calories and essential food components is required to maximize longitudinal growth in children. Our previous study showed that prepubertal male rats subjected to 10 days of 40% food restriction (RES) exhibited a dramatic reduction in weight and epiphyseal growth plate height, as well as changes in gene expression and microRNAs (miRNAs) in the epiphyseal growth plate. These findings reversed rapidly after renewal of the regular food supply (catch-up [CU]). To further elucidate the mechanisms underlying the nutrition-growth association, serum collected from the RES and CU rats and control rats fed ad libitum (AL) was added to the culture medium of the chondrocyte cell line ATDC5 (instead of fetal calf serum). Serum from the RES group induced a reduction in cell viability (25%, P < .05) concomitant with an increase in cell differentiation compared with that for the AL group serum. The most interesting observation, in our opinion, was the significant reduction in the expression of specific miRNAs, including the chondro-specific miR-140. These effects were not observed for serum from refed (CU) rats. Serum levels of IGF-I, leptin, and fibroblast growth factor 21 were reduced by food restriction. The addition of IGF-I and leptin to the culture increased cell viability, whereas fibroblast growth factor 21 reduced it, suggesting the involvement of IGF-I, leptin, and possibly other still unidentified serum factors in chondrocyte cell growth. In conclusion, specific miRNAs respond to nutritional cues, and these effects are mediated by serum-borne factors. These results may promote the development of superior interventions for children with malnutrition and growth abnormalities.

Tailoring hierarchical meso-macroporous 3D scaffolds: from nano to macro.

Bone tissue regeneration requires the use of 3D scaffolds which mimic the architecture of the natural extracellular matrix, creating an adequate microenvironment for bone cell growth. Such 3D scaffolds need surface properties suitable for biological recognition in the early stage of cell adhesion, necessary to ensure complete cell colonization, retained cell functionality, and subsequently bone regeneration. Herein, hierarchical 3D scaffolds based on new hydroxyapatite/mesoporous glass nanocomposite bioceramic (MGHA) exhibiting different scales of porosity have been synthesized. These 3D scaffolds possess: (i) highly ordered mesopores with diameters of 10 nm; (ii) macropores with diameters in the 30-80 μm range with interconnections of 1-10 μm; and (iii) large macropores of ca. 500 μm. To improve their surface properties, 3D scaffolds were modified through direct functionalization with amine propyl groups, which notably improve preosteoblast adhesion, proliferation (2.3 fold), differentiation (4.8 fold) and further cell colonization of these scaffolds. The observed enhancement can be related to these amine groups which favour early adhesion, e.g., based on nonspecific protein adsorption as was demonstrated by ellipsometry. These results suggest that the combination of hierarchical structure design and amine surface modification of hydroxyapatite/mesoporous nanocomposite scaffolds yields a double increase in cell proliferation, as well as a quadruple increase in cell differentiation, demonstrating the potential of these nanocomposite materials for bone tissue regeneration purposes.

Design, Synthesis, and Cytotoxic Evaluation of Acyl Derivatives of 3-Aminonaphtho[2,3-b]thiophene-4,9-dione, a Quinone-Based System

A series of 3-acyl derivatives of the dihydronaphtho[2,3-b]thiophen-4,9-dione system were studied with respect to cytotoxicity and topoisomerase II inhibitory activity. These analogues were designed as electron-deficient anthraquinone analogues with potential intercalation ability. Derivatives 3-(diethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11m) and 3-(2-(dimethylamino)ethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11p) showed a high efficacy in cell lines that were highly resistant to treatment with doxorubicin, such as MDA-MB435 (melanoma), IGROV (ovarian), and SF-295 (glioblastoma) human cell lines. Both compounds inhibit topoisomerase II mediated relaxation of DNA, while only 11p incites arrest at the S phase in Caco-2 cells, inducing a delay of cell cycle progression and an increase of cell differentiation. The ability of these derivatives to modulate small heat shock proteins and cardiotoxicy effects was also explored. In addition, the DNA-binding properties of these compounds were investigated and discussed.

Synthesis of C 6-epimer derivatives of diacetoxy acetal derivative of santonin and their inducing effects on HL-60 leukemia cell differentiation

Induction of differentiation is a new and promising approach to leukemia therapy, well illustrated by the treatment of acute promyelocytic leukemia with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (ATRA). Using combination of either 1,25-(OH)(2)D(3) or ATRA and chemotherapy, adverse effects 1,25-(OH)(2)D(3) or ATRA such as hypercalcemic effects have decreased, and long-term survival has improved. In a previous study, we demonstrated that santonin could be chemically modified into a diacetoxy acetal derivative of santonin with strong differentiation-inducing activity. In this study, we further synthesized C(6)-epimer derivatives of diacetoxy acetal derivative of santonin and tested their effects on HL-60 cell differentiation. Some of the C(6)-epimer derivatives themselves induced increases in cell differentiation. Especially, (11S)-3,3-(ethylenedioxy) eudesmano-13-ol-6β-acetate (7) was demonstrated to induce differentiation with larger than 80% of the cells attaining a differentiated phenotype. Importantly, 7 strongly enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)(2)D(3) or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)(2)D(3) or ATRA by 7 may improve outcomes in the therapy of acute promyelocytic leukemia.

Effects and action mechanism of Diospyros kaki on the differentiation of human leukemia HL-60 cells

Diospyros kaki Thunb. (Ebenaceae) is widely distributed in North-East Asian countries. Almost all parts of this plant have been traditionally used as medicine. Human promyelocytic leukemia cells differentiate into monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or all-trans retinoic acid (ATRA). Combination of low doses of ATRA or 1,25-dihydroxyvitamin D3 that do not induce toxicity with another drug is a useful strategy for acute promyelocytic leukemia therapy. Our main aim was to investigate the effect of an acetone extract of D. kaki leaves (KV-1) on HL-60 cell differentiation in combination of ATRA or 1,25-dihydroxyvitamin D3. Treatment of HL-60 cells with zero to 100 microg/ml of KV-1 for 72 h induced a small increase in cell differentiation. Surprisingly, a synergistic induction of differentiation was observed when the HL-60 cells were treated with ATRA or 1,25-(OH)2D3 and the extract. The inhibitors of protein kinase C (PKC) (alpha and betaI) and extracellular signal-regulated kinase (ERK), but not of phosphoinositide 3-kinase (PI3-K) and c-Jun N-terminal kinase (JNK) inhibited the HL-60 differentiation induced by the extract in combination of ATRA or 1,25-(OH)2D3, suggesting that PKC and ERK were involved in the cell differentiation enhancement by the extract. The results indicate that the acetone extract of D. kaki leaves has the ability to enhance HL-60 cell differentiation and suggest that it may be useful in acute promyelocytic leukemia therapy.

Enhancement of 1,25-Dihydroxyvitamin D3- and All-transRetinoic Acid-Induced HL-60 Leukemia Cell Differentiation byPanax ginseng

Ginseng (Panax ginseng C.A. Meyer) has a wide range of therapeutic uses including cancer treatment. Human promyelocytic leukemia cells differentiate into monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (ATRA). Treatment of HL-60 cells with zero to 100 microg/ml of a methanol extract of ginseng for 72 h induced a small increase in cell differentiation. Surprisingly, a synergistic induction of differentiation was observed when HL-60 cells were treated with ATRA or 1,25-(OH)(2)D(3) and the extract. The inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), but not of phosphoinositide 3-kinase (PI3-K), inhibited the HL-60 differentiation induced by the extract in combination with ATRA or 1,25-(OH)(2)D(3), signifying that PKC and ERK were involved in the cell differentiation enhancement by the extract. These results suggest that the ability of a methanol extract of ginseng to enhance the differentiation potential of ATRA or 1,25-(OH)(2)D(3) may improve the ultimate outcome of acute promyelocytic leukemia therapy.

ENHANCEMENT OF 1,25-DIHYDROXYVITAMIN D3- AND ALL-TRANS RETINOIC ACID-INDUCED DIFFERENTIATION OF HUMAN LEUKEMIA HL-60 CELLS BY BLOOD SHELL, SCAPHARCA BROUGHTONII

Human myeloid leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) or all-trans retinoic acid (RA), respectively. In this study, the effect of ethanol fraction prepared from the blood shell, Scapharca broughtonii, on cell differentiation was investigated in an HL-60 cell culture system. Treatment of HL-60 cells with 2.5- to 20-µg/mL ethanol fraction of the blood shell for 72 h inhibited cell proliferation and induced a 20% increase in cell differentiation, as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium and nitroblue tetrazolium reduction assay. Interestingly, synergistic induction of HL-60 cell differentiation was observed when the ethanol fraction of the blood shell was combined with either 5-nM 1,25-(OH)2D3 or 50-nM all-trans RA. Flow cytometric analysis and morphologic studies confirmed that combinations of either 1,25-(OH)2D3 or all-trans RA and the ethanol fraction of the blood ark shell stimulated leukemia cell differentiation. These results suggest a possibility of blood shell in the treatment of human leukemia. PRACTICAL APPLICATIONS The ethanol fraction (SIII) of blood shell, Scapharca broughtonii, potentiates 1,25-(OH)2D3- and all-trans retinoic acid (RA)-induced differentiation in HL-60 myeloid leukemia cells. HL-60 cells might be synergistically differentiated into monocytes or granulocytes when treated with the ethanol fraction in combination with either 1,25-(OH)2D3 or all-trans RA. These results imply a possible application of blood ark shell in the treatment of leukemia patients with less toxicity.