The Protein Deacetylase SIRT3 Prevents Oxidative Stress-induced Keratinocyte Differentiation

Keratinocyte differentiation is a key process in the formation and maintenance of the protective skin barrier. Dysregulation in the balance of reactive oxygen species homeostasis may play a role in keratinocyte differentiation. We have identified the mitochondrial deacetylase SIRT3 as a key regulator of mitochondrial reactive oxygen species in keratinocytes. Our studies demonstrate that SIRT3 expression is down-regulated during keratinocyte differentiation, consistent with an increase in mitochondrial superoxide levels. Importantly, loss of SIRT3 expression in keratinocytes increased superoxide levels and promoted the expression of differentiation markers, whereas overexpression decreased superoxide levels and reduced the expression of differentiation markers. These findings identify a new role for SIRT3 in the suppression of epidermal differentiation via lowering oxidative stress.     

Histone Deacetylase Inhibition Induces Apoptosis in Neuroblastoma

Histone deacetylase inhibitors constitute a promising new treatment for cancer due to their novel site of action and low toxicity. Almost all histone deacetylase inhibitors currently in clinical development have anti-proliferate activities against cells in cultures, and specially cause cell cycle arrest, differentiation and apoptosis. Interestingly, despite their rapid advance into clinical use, the cellular responses leading to these effects remain unclear. We recently reported that histone deacetylase inhibitor treatment induces apoptosis of neuroblastoma cells by increasing the acetylation of Ku70 in the cytoplasm, resulting in the release of Bax from Ku70. Subsequently, Bax releases cytochrome c from mitochondria causing apoptosis. Here we will discuss these findings and the implications of our model for the further clinical development of histone deacetylase inhibitors in the treatment of cancer.     

The Antiparasitic Clioquinol Induces Apoptosis in Leukemia and Myeloma Cells by Inhibiting Histone Deacetylase Activity

The antiparasitic clioquinol (CQ) represents a class of novel anticancer drugs by interfering with proteasome activity. In the present study, we found that CQ induced blood cancer cell apoptosis by inhibiting histone deacetylases (HDACs). CQ accumulated the acetylation levels of several key proteins including histone H3 (H3), p53, HSP90, and α-tubulin. In the mechanistic study, CQ was found to down-regulate HDAC1, -3, -4, and -5 in both myeloma and leukemia cells. Computer modeling analysis revealed that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp-267. Moreover, co-treatment with CQ and zinc/copper chloride led to decreased Ac-H3. Furthermore, CQ inhibited the activity of Class I and IIa HDACs in the cell-free assays, demonstrating that CQ interfered with HDAC activity. By inhibiting HDAC activity, CQ induced expression of p21, p27, and p53, cell cycle arrest at G₁ phase, and cell apoptosis. This study suggested that the HDAC enzymes are targets of CQ, which provided a novel insight into the molecular mechanism of CQ in the treatment of hematological malignancies.     

Sensitization of Chemo-Resistant Human Chronic Myeloid Leukemia Stem-Like Cells to Hsp90 Inhibitor by SIRT1 Inhibition

Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44^high K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44^high K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44^high cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.     

Fludarabine Induces Differential Expression of Proteins in Human Leukemia and Lymphoma Cells

The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt's lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 μ M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.     

VentX促进人类造血干细胞向红系分化

GATA-2作为GATA家族成员,其通过与靶基因的GATA结合位点结合,在造血系统发育中起关键性的调节作用。VentX是非洲爪蟾蜍xvent基因同源的哺乳动物基因,最近研究发现,其参与了中胚层的分化定型及造血干细胞的维持,并且在细胞衰老、增殖、分化及炎症反应等的调节中发挥功能。为探索VentX基因与GATA-2的关系及其对造血干细胞红系分化的调节功能,首先在K562细胞株中进行了VentX启动子分析,发现GATA-2可以通过结合到VentX启动子区两个GATA结合位点来顺式调控Vent X;继而在人骨髓(造血)干细胞中的实验显示,过表达GATA-2或过表达VentX,均可抑制CD34~+细胞的增殖,促进CD34~+细胞向红系分化。以上实验结果为临床红细胞来源提供了有价值的研究线索。     

Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.     

Measles Virus Infection Induces Terminal Differentiation of Human Thymic Epithelial Cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G₀/G₁ phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

一种用作阴性对照的siRNA可诱导人K562细胞向红系分化

我们发现,一种在RNA干扰实验中用作阴性对照的商业化siRNA具有明显的诱 导人慢性髓性白血病K562细胞系向红系方向分化的作用.它表现为K562细胞瞬时转 染该siRNA后,红系分化的特异表面标志CD235及ε 、γ 和β 珠蛋白的表达升高,GATA-2的表达降低,细胞增殖速度减慢,软琼脂克隆形成率降低,并且此 过程不伴随细胞凋亡. 而生物信息学分析显示,该siRNA序列与目前所有已知人类 基因均无明显同源性.研究结果提示,该siRNA不适于用作红系分化实验中的阴性 对照. siRNA的作用远比人们目前所知的要复杂得多,siRNA的脱靶效应应当引起 研究者的足够重视,在RNA干扰实验中阴性对照siRNA的选择会极大地影响对实验 结果的判读.     

Inhibition of Breast Cancer Resistance Protein (ABCG2) in Human Myeloid Dendritic Cells Induces Potent Tolerogenic Functions during LPS Stimulation

Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c⁺ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c⁺ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4⁺ T cells and promoted expansion of CD25⁺FOXP3⁺ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.     

Inhibition of Histone Deacetylase Impacts Cancer Stem Cells and Induces Epithelial-Mesenchyme Transition of Head and Neck Cancer

The genome is organized and packed into the nucleus through interactions with core histone proteins. Emerging evidence suggests that tumors are highly responsive to epigenetic alterations that induce chromatin-based events and dynamically influence tumor behavior. We examined chromatin organization in head and neck squamous cell carcinoma (HNSCC) using acetylation levels of histone 3 as a marker of chromatin compaction. Compared to control oral keratinocytes, we found that HNSCC cells are hypoacetylated and that microenvironmental cues (e.g., microvasculature endothelial cells) induce tumor acetylation. Furthermore, we found that chemical inhibition of histone deacetylases (HDAC) reduces the number of cancer stem cells (CSC) and inhibits clonogenic sphere formation. Paradoxically, inhibition of HDAC also induced epithelial-mesenchymal transition (EMT) in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. Importantly, we observed co-expression of vimentin and acetylated histone 3 at the invasion front of human HNSCC tumor tissues. Collectively, these findings suggest that environmental cues, such as endothelial cell-secreted factors, modulate tumor plasticity by limiting the population of CSC and inducing EMT. Therefore, inhibition of HDAC may constitute a novel strategy to disrupt the population of CSC in head and neck tumors to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior.     

Induction of Growth Inhibition and Differentiation of Human Leukemia HL-60 Cells by a Tunisian Gerboui Olive Leaf Extract

Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.     

Histone Deacetylase Inhibitor SAHA Induces Expression of Fatty Acid-Binding Protein 4 and Inhibits Replication of Human Cytomegalovirus

Virologica Sinica - Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic...     

HL－60细胞诱导分化过程中蛋白质酪氨酸磷酸化水平的改变

对ＲＡ、ＨＨＴ和ＷＢ_（６５２）诱导ＨＬ－６０细胞过程中,细胞浆和膜溶脱部分的蛋白质酪氨酸磷酸化水平变化进行了对比研究,结果发现,在胞浆部分主要有四种含有Ｐ－Ｔｙｒ的蛋白,而且８０ｋＤ蛋白酪氨酸磷酸化水平随着诱导发生变化。诱导前后内源性蛋白上Ｐ－Ｔｙｒ百分含量也发生了改变。     

过表达Runx2促进C2C12细胞成骨分化

利用Tet-on(Tetracycline-on)基因表达系统,通过强力霉素(doxycycline,DOX)诱导Runx2基因在C2C12细胞中的表达,探究Runx2促成骨分化功能,为其分子机制的研究提供一个理想的实验平台.先后将调控质粒pTet-on和反应质粒pTRE-Flag-Runx2转染入C2C12细胞,并用G418和潮霉素分别进行2轮筛选,运用实时荧光定量PCR选择对强力霉素诱导敏感的细胞克隆.用不同浓度DOX诱导C2C12/Tet/pTRE-Flag-Runx2细胞,蛋白免疫印迹检测Runx2的表达,确定DOX的最佳诱导浓度与时间,并检测C2C12细胞的成骨分化能力.结果表明,诱导细胞最佳DOX浓度为10μg/ml;最佳诱导时间为12h;诱导后Runx2基因高表达,C2C12细胞向成骨方向分化(P0.05).成功建立Tet调控Runx2基因表达C2C12细胞系,为进一步研究Runx2基因功能分子机制提供理想的细胞模型.     

Inhibition of the Differentiation of Monocyte-Derived Dendritic Cells by Human Gingival Fibroblasts

We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.     

Matrine Induces Apoptosis in Human Acute Myeloid Leukemia Cells via the Mitochondrial Pathway and Akt Inactivation

Acute myeloid leukemia (AML) is a hematological malignancy characterized by a rapid increase in the number of immature myeloid cells in bone marrow. Despite recent advances in the treatment, AML remains an incurable disease. Matrine, a major component extracted from Sophora flavescens Ait, has been demonstrated to exert anticancer effects on various cancer cell lines. However, the effects of matrine on AML remain largely unknown. Here we investigated its anticancer effects and underlying mechanisms on human AML cells in vitro and in vivo. The results showed that matrine inhibited cell viability and induced cell apoptosis in AML cell lines as well as primary AML cells from patients with AML in a dose- and time-dependent manner. Matrine induced apoptosis by collapsing the mitochondrial membrane potential, inducing cytochrome c release from mitochondria, reducing the ratio of Bcl-2/Bax, increasing activation of caspase-3, and decreasing the levels of p-Akt and p-ERK1/2. The apoptotic effects of matrine on AML cells were partially blocked by a caspase-3 inhibitor Z-DEVD-FMK and a PI3K/Akt activator IGF-1, respectively. Matrine potently inhibited in vivo tumor growth following subcutaneous inoculation of HL-60 cells in SCID mice. These findings indicate that matrine can inhibit cell proliferation and induce apoptosis of AML cells and may be a novel effective candidate as chemotherapeutic agent against AML.