Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition

Abstract Background: Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism. Methods: We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1). Results: Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells. Conclusion: These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS. Disclosures No relevant conflicts of interest to declare.

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Src Kinases Act Downstream Akt and Are Required for the Transforming Properties of Constitutively Activated Forms of STAT5A and STAT5B

Blood ◽

10.1182/blood.v118.21.2378.2378 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2378-2378

Author(s):

Remy Nyga ◽

Lea Savay ◽

Ingrid Marcq ◽

Aline Regnier ◽

Gandhi Damaj ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Cell Survival ◽

Conflicts Of Interest ◽

Cell Counting ◽

Parp Cleavage ◽

Blue Dye ◽

Mouse Bone Marrow ◽

Src Kinases ◽

Dependent Manner

Abstract Abstract 2378 Background: Stat5A and Stat5B transcription factors play an important role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia. Moreover, expression of constitutively active Stat5 (caStat5) mutants in primary hematopoietic cells can lead to leukaemia. The transforming properties of CaStat5 have been previously shown to require Akt activation. However, the mechanisms by which CaStat5 mutants and Akt are activated remain enigmatic. In this study we aimed to determine the roles of src kinases in these processes, Methods: For this purpose, we used murine Ba/F3 cells transfected or not with CaSta5A (Stat5A1*6) or CaStat5B (Stat51*B6) or with the oncogenic fusion proteins Tel-JAK2 and Tel-Abl as controls. Primary hematopoietic progenitors obtained from mouse bone marrow were also used in this sutdy. Cells were treated or not with the pan-src inhibitors PP1, PP2, and SU 6656 (and with PP3 an inactive analogue of PP1 as a control), imatinib (Abl inhibitor) and AG490 (JAK inhibitor). In some experiments cells were treated with U0126 (MEK inhibitor) or with the highly transducible TaT-dominant negative (dn) Akt fusion protein. Cell proliferation was assessed by cell counting. Cell death was evaluated using the trypan blue dye exclusion test. Cell apoptosis was assessed by analysing annexin V staining using flow cytometry and PARP cleavage using western blot. Expression of kinases and their phosphotylated forms were examined by western blot. Results: CaStat5A and CaStat5B cells were shown to express constitutively phosphorylated Lyn, Hck, Src kinases as well as Akt, IKK and ERK1/2. Stat1, Stat3, and JAK family members (TYK2 and JAK1-3) were not phosphorylated in these cells. Use of pan-Src inhibitors inhibited proliferation of CaStat5 cells and induced apoptosis of a fraction of these cells, in a dose-dependent manner. By contrast, imatinib and AG490 had no effect on the growth and survival of CaStat5 cells. Moreover, pan-src inhibitors prevented Erk but not Akt, IKK or STAT5 phosphorylation. By contrast, inhibition of Akt by means of TaT-dnAkt abolished Lyn, Src and ERK1/2 phosphorylation. Importantly, inhibition of MAPK did not interfere with Akt phosphorylation and did not interfere with CaStat5 cell survival. Conclusion: Altogether our data indicate that Akt acts upstream in the CaStat5-associated signalling cascade, then activates src kinases and thereby promotes MAPK-dependent cell proliferation and MAPK-independent cell survival. These results shed light onto a hitherto undescribed role of Src kinases acting downstream Akt in the mechanisms of action of CaStat5. Disclosures: No relevant conflicts of interest to declare.

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Expression of YAP and Inhibitory Effect By IGF-1R Suppressor in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.892.892 ◽

2016 ◽

Vol 128 (22) ◽

pp. 892-892 ◽

Cited By ~ 1

Author(s):

Xiangxiang Zhou ◽

Ying Li ◽

Ya Zhang ◽

Yangyang Xu ◽

Dai Yuan ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Conflicts Of Interest ◽

B Cell Lymphoma ◽

Inhibitory Effect ◽

Cell Counting ◽

Hippo Signaling ◽

Dependent Manner ◽

Proliferation And Apoptosis ◽

Reactive Hyperplasia

Abstract Introduction: Yes-associated protein (YAP), a direct downstream effector of the tumor suppressive Hippo signaling, functions as a transcriptional coactivator in the modulation of cell growth, proliferation and apoptosis in a number of cancers. Given that Hippo signaling does not have an extracellular ligand nor a specific membrane receptor, its activation depends on the crosstalk mechanisms. Receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF-1R) is implicated in various tumor entities by modulating the proliferation, survival and metastasis of cancer cells. Strategies to block IGF-1R pathway in solid malignancies are being tested in clinical trials. However, the potential significance of IGF-1R and YAP in DLBCL remains ill defined. We assume that IGF-1R modulates the proliferation and apoptosis of DLBCL cells partly by regulating YAP in DLBCL. We aimed to investigate the functional significance of IGF-1R and its regulatory effect on YAP in DLBCL. Methods: Lymph node biopsies from 30 de novo DLBCL patients and 20 reactive hyperplasia cases were collected with informed consents. Immunohistochemistry (IHC) was performed to assess the expression of YAP and IGF-1R in lymphoma tissues. Protein expression levels of YAP and IGF-1R in DLBCL cell lines were detected by western blotting. Peripheral blood mononuclear cells were isolated with informed consents from healthy volunteers. Effects of AG1024 on cell viabilities and apoptosis were assessed by cell counting kit-8 and Annexin-V/7-AAD staining. Propidium iodide staining was conducted to assess cell cycle of DLBCL cells. Results: Compared to the reactive hyperplasia group, DLBCL patients revealed significantly higher protein levels of both YAP and IGF-1R (Fig. 1A). Overexpression of YAP and IGF-1R protein in DLBCL cell lines (LY1 and LY8) were further confirmed by immunoblotting (Fig. 1B). AG1024, a selective inhibitor of IGF-1R, significantly restrained the viabilities of LY1 and LY8 cells in a dose dependent manner (Fig. 1C). Apoptotic rates in LY1 and LY8 cells treated with IGF-1R inhibitor were markedly increased (Fig. 1D). Moreover, addition of IGF-1Rsuppressor induced remarkable cell cycle arrest in G2/M phase in DLBCL cells (Fig. 1E). In LY1 and LY8 cells treated with AG1024, we observed that the reduced phosphorylation of IGF-1R was accompanied by significantly declined level of YAP protein level (Fig. 1F). Results of the immunofluorescense verified the inhibitory effect of AG1024 on expression of YAP protein in LY1 and LY8 cell (Fig. 1G). Conclusions: We identified the aberrant overexpression of YAP and IGF-1R in DLBCL patients and cell lines, which suggesting the dysregulation of Hippo and IGF-1 signaling in DLBCL. IGF-1R inhibitor exhibits proliferation-inhibitory and pro-apoptotic effect via inhibiting the activation YAP. We provided a novel mechanism involved in the function of IGF-1R in DLBCL. Further interrogation on the biological function of YAP in DLBCL will highlight the crosstalk between these two pathways and presents a promising therapeutic strategy in DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Ser68 phosphoregulation is essential for CENP-A deposition, centromere function and viability in mice

10.1101/2021.11.23.469796 ◽

2021 ◽

Author(s):

Yuting Liu ◽

Kehui Wang ◽

Li Huang ◽

Jicheng Zhao ◽

Xinpeng Chen ◽

...

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Histone H3 ◽

Dependent Manner ◽

Centromere Function ◽

Histone H3 Variant ◽

A Cell ◽

Cycle Dependent ◽

Spatiotemporal Control

Centromere identity is defined by nucleosomes containing CENP-A, a histone H3 variant. The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner. We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68. However, a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading. Here, we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability. We also find that mice harboring the corresponding mutations are lethal. Together, these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.

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Abstract 286: The Role of Gut Microbiota in Neointimal Hyperplasia After Vascular Injury

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.286 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Karen J Ho ◽

Liqun Xiong ◽

Nathaniel Hubert ◽

Anuradha Nadimpalli ◽

Eugene B Chang ◽

...

Keyword(s):

Cell Cycle ◽

Vascular Injury ◽

Sodium Butyrate ◽

Gut Microbiome ◽

Neointimal Hyperplasia ◽

Cell Counting ◽

Rrna Gene ◽

Control Analysis ◽

Dependent Manner ◽

Microbial Composition

Introduction: There is increasing evidence that the gut microbiome regulates susceptibility to certain diseases through systemic effects of microbe-derived metabolites. Sodium butyrate is a short chain fatty acid that is produced by microbial fermentation of dietary fiber and has known anti-proliferative and anti-migratory effects on vascular smooth muscle cells (VSMC). We hypothesized that perturbation of the gut microbiome with antibiotics would alter systemic serum butyrate concentration and impact neointimal hyperplasia after vascular injury. Methods: 10-wk-old male Lewis Inbred rats were treated with vancomycin (“vanco”) in the drinking water (0.5mg/mL) ± sodium butyrate (“buty”, 0.5mg/mL) for 4 wks prior to undergoing left carotid angioplasty. Serum butyrate concentration was assessed by gas chromatography. Gut microbial composition was assessed by 16S rRNA gene surveys of fecal samples. VSMC were treated with butyrate (0-5mM) and assessed for cell proliferation using cell counting, cell migration using a transwell assay, and cell cycle progression using FACS. Results: Post-angioplasty carotid arteries from vanco-treated rats developed 38% more neointima than controls (0.032±0.004mm2 vs. 0.044±0.003 mm2, P=0.02), but vanco+buty treatment prevented this increase in intimal area (0.035±0.004 mm2, P=.62 vs. control). Analysis of gut microbial communities revealed unique shifts in bacterial clustering by treatment group, which correlated with changes in serum butyrate levels, with the lowest butyrate level detected in vanco-treated rats (0.54±0.1 μmol/mL control, 0.017±0.1 μmol/mL vanco, 0.45±0.1 μmol/mL vanco+buty, P=.008). In vitro, butyrate treatment inhibited VSMC proliferation at 24-48 hrs in a dose-dependent manner, which correlated with induction of G0/G1 cell cycle arrest (P=.001) and a reduction in chemotaxis (P=.03). Conclusions: Oral vancomycin treatment induced a shift in the gut microbial community that was associated with decreased serum butyrate levels and increased neointimal hyperplasia, both of which were reversed by oral butyrate supplementation. These data demonstrate proof-of-concept that there is a correlation between gut microbial dysbiosis and susceptibility to neointimal hyperplasia.

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Carvedilol Inhibits Angiotensin II-Induced Proliferation and Contraction in Hepatic Stellate Cells through the RhoA/Rho-Kinase Pathway

BioMed Research International ◽

10.1155/2019/7932046 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Ying Wu ◽

Zhen Li ◽

Sining Wang ◽

Aiyuan Xiu ◽

Chunqing Zhang

Keyword(s):

Cell Cycle ◽

Angiotensin Ii ◽

Liver Fibrosis ◽

Cell Cycle Progression ◽

Rho Kinase ◽

Cell Counting ◽

Type I ◽

Dependent Manner ◽

Ang Ii ◽

Cycle Progression

Aim. Carvedilol is a nonselective beta-blocker used to reduce portal hypertension. This study investigated the effects and potential mechanisms of carvedilol in angiotensin II- (Ang II-) induced hepatic stellate cell (HSC) proliferation and contraction. Methods. The effect of carvedilol on HSC proliferation was measured by Cell Counting Kit-8 (CCK-8). Cell cycle progression and apoptosis in HSCs were determined by flow cytometry. A collagen gel assay was used to confirm HSC contraction. The extent of liver fibrosis in mice was evaluated by hematoxylin-eosin (H&E) and Sirius Red staining. Western blot analyses were performed to detect the expression of collagen I, collagen III, α-smooth muscle actin (α-SMA), Ang II type I receptor (AT1R), RhoA, Rho-kinase 2 (ROCK2), and others. Results. The results showed that carvedilol inhibited HSC proliferation and arrested the cell cycle at the G0/G1 phase in a dose-dependent manner. Carvedilol also modulated Bcl-2 family proteins and increased apoptosis in Ang II-treated HSCs. Furthermore, carvedilol inhibited HSC contraction induced by Ang II, an effect that was associated with AT1R-mediated RhoA/ROCK2 pathway interference. In addition, carvedilol reduced α-SMA expression and collagen deposition and attenuated liver fibrosis in carbon tetrachloride (CCl4)-treated mice. The in vivo data further confirmed that carvedilol inhibited the expression of angiotensin-converting enzyme (ACE), AT1R, RhoA, and ROCK2. Conclusions. The results indicated that carvedilol dose-dependently inhibited Ang II-induced HSC proliferation by impeding cell cycle progression, thus alleviating hepatic fibrosis. Furthermore, carvedilol could inhibit Ang II-induced HSC contraction by interfering with the AT1R-mediated RhoA/ROCK2 pathway.

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Gcn5-mediated acetylation at MBF-regulated promoters induces the G1/S transcriptional wave

Nucleic Acids Research ◽

10.1093/nar/gkz561 ◽

2019 ◽

Vol 47 (16) ◽

pp. 8439-8451 ◽

Cited By ~ 1

Author(s):

Alberto González-Medina ◽

Elena Hidalgo ◽

José Ayté

Keyword(s):

Cell Cycle ◽

Molecular Mechanisms ◽

Histone H3 ◽

Dependent Manner ◽

Saga Complex ◽

Lysine Residues ◽

Physical Barriers ◽

A Cell ◽

Cycle Dependent ◽

Regulated Promoters

Abstract In fission yeast, MBF-dependent transcription is inactivated at the end of S phase through a negative feedback loop that involves the co-repressors, Yox1 and Nrm1. Although this repression system is well known, the molecular mechanisms involved in MBF activation remain largely unknown. Compacted chromatin constitutes a barrier to activators accessing promoters. Here, we show that chromatin regulation plays a key role in activating MBF-dependent transcription. Gcn5, a part of the SAGA complex, binds to MBF-regulated promoters through the MBF co-activator Rep2 in a cell cycle-dependent manner and in a reverse correlation to the binding of the MBF co-repressors, Nrm1 or Yox1. We propose that the co-repressors function as physical barriers to SAGA recruitment onto MBF promoters. We also show that Gcn5 acetylates specific lysine residues on histone H3 in a cell cycle-regulated manner. Furthermore, either in a gcn5 mutant or in a strain in which histone H3 is kept in an unacetylated form, MBF-dependent transcription is downregulated. In summary, Gcn5 is required for the full activation and correct timing of MBF-regulated gene transcription.

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Cell Cycle Association of the Retinoblastoma Protein Rb and the Histone Demethylase LSD1 with the Epstein-Barr Virus Latency Promoter Cp

Journal of Virology ◽

10.1128/jvi.01412-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3428-3437 ◽

Cited By ~ 23

Author(s):

Charles M. Chau ◽

Zhong Deng ◽

Hyojueng Kang ◽

Paul M. Lieberman

Keyword(s):

Cell Cycle ◽

Epstein Barr Virus ◽

Affinity Purification ◽

Histone H3 ◽

Histone Demethylase ◽

Stable Complex ◽

Dependent Manner ◽

Barr Virus ◽

A Cell ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus C promoter (Cp) regulates the major multicistronic transcript encoding the EBNA-LP, 1, 2, and 3 genes required for B-cell proliferation during latency. The growth-transforming potential of these viral genes suggests that they must be tightly regulated with the host cell cycle and differentiation process. To better understand Cp regulation, we used DNA affinity purification to identify cellular and viral proteins that bind to Cp in latently infected cells. Several previously unknown factors were identified, including the cell cycle regulatory proteins E2F1 and Rb. E2F1 bound to a specific site in Cp located in the core Cp region 3′ of the known EBNA2-responsive RBP-Jk (CSL, CBF1) binding site. The histone H3 K4 demethylase LSD1 (BCC110) was also identified by DNA affinity and was shown to form a stable complex with Rb. Coimmunoprecipitation assays demonstrated that E2F1, Rb, and LSD1 bind to Cp in a cell cycle-dependent manner. Rb and LSD1 binding to Cp increased after the S phase, corresponding to a decrease in histone H3 K4 methylation and Cp transcription. Coimmunoprecipitation and immunofluorescence assays reveal that LSD1 interacts with Rb. Surprisingly, LSD1 did not coimmunoprecipitate with E2F1, suggesting that it associates with Rb independently of E2F1. Depletion of LSD1 by small interfering RNAs inhibited Cp basal transcription levels, and overexpression of LSD1 altered the cell cycle profile in p53-positive (p53+), but not p53-negative (p53−), HCT cells. These findings indicate that Cp is a cell cycle-regulated promoter that is under the control of Rb and the histone demethylase LSD1 in multiple latency types.

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Interactions Between BAFF and BAFF Receptors Are Involved in Macrophage-Induced Bortezomib Resistance in Myeloma

Blood ◽

10.1182/blood.v128.22.4429.4429 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4429-4429

Author(s):

Jing Chen ◽

Donghua He ◽

Xing Guo ◽

Qingxiao Chen ◽

Xuanru Lin ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Resistance ◽

Western Blot ◽

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Counting ◽

Therapeutic Modality ◽

Second Primary ◽

Induced Apoptosis

Abstract Background:B-cell-activating factor (BAFF) is a member of the TNF family that critical for maintenance of B-cell development and homeostasis. BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) are three BAFF receptors. It has been reported that BAFF is expressed by neutrophils, monocytes, dentritic cells and macrophages and modulates the proliferation, survival and drug resistance of multiple myeloma (MM) cells. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by direct interaction with MM cells. We hypothesized that BAFF/BAFF receptors play a role in macrophage-induced bortezomib resistance in myeloma. Methods: First, the expression levels of BAFF and its three receptors in primary MM cells, MM cell lines and peripheral blood monocyte(PBMC)-induced macrophages were detected by semiquantitative real time-polymerase chain reaction (qPCR),Western blot and flow-cytometry. Also the concentration of BAFF in the supernatants of MM patients' bone marrow, MM cell lines and macrophages were determined by ELISA. Second, Primary MM cells and MM cell lines were cocultured with macrophages for the indicated time (usually 4-6h and 24h), for some experiments, we added bortezomib to the coculture system. Cell viability and apoptosis of MM cells were verified by Cell Counting Kit-8(CCK8) after treated with recombinant human (rh) BAFF, BAFF neutralizing antibody and BAFF siRNA. The interactions between BAFF and its receptors are unveiled by flow-cytometry. Then, cell survival signaling activations that may confer MM drug resistance were examined by Western blot. Results: Two receptors of BAFF, TACI and BCMA were highly expressed in various MM cell lines. The expressions of BAFF in PBMC-induced macrophages were heterogeneous. Functional studies showed that rhBAFF promoted RPMI8226 and ARP1 myeloma cells growth (P<0.05) and protected them from bortezomib-induced apoptosis (P<0.05). Then we verified macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, RPMI8226) with PBMC-derived macrophages from healthy donors. The macrophage-induced bortezomib resistance was attenuated by neutralizing antibodies(P<0.05) and siRNA of BAFF(P<0.01) . Next we found that in MM cells cocultured with macrophages, bortezomib-induced PARP and caspase-3 cleavages were highly repressed and phosphorylated Src ,AKT and Erk1/2 were upregulated which indicated that BAFF-mediated MM drug resistance may be through ERK1/2 and Src pathway .In addition, BAFF induced activation of NF-κB2,a pathway critical for the growth and survival of these cells. Conclusions: Our data show that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors, leading to a reduction in caspase proteins and subsequent activation of Src and Erk1/2 kinases and NF-κB2 pathways .These studies reveal a promising unknown role for BAFF/BAFF receptors, suggesting that targeting macrophage-MM interactions may represent a promising therapeutic modality. Disclosures No relevant conflicts of interest to declare.

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Qilan Preparation Inhibits Proliferation and Induces Apoptosis by down-regulating microRNA-21 in Human Tca8113 Tongue Squamous Cell Carcinoma Cells

10.21203/rs.3.rs-344688/v1 ◽

2021 ◽

Author(s):

Jiamin Ding ◽

Zuoliang Chen ◽

Wanlu Chen ◽

Zhongxiong Ma ◽

Yunde Xie ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Squamous Cell Carcinoma ◽

Cell Growth ◽

Cell Carcinoma ◽

Squamous Cell ◽

Tongue Squamous Cell Carcinoma ◽

Cell Counting ◽

Dependent Manner ◽

Pdcd4 Expression

Abstract Background: Qilan preparation, a complex Chinese herbal medicine consisting of ingredients extracted from Radix Astragali, Gynostemma Pentaphyllum, Rhizoma Chuanxiong and selenium- rich green tea and known for ‘fortifying the spleen and boosting qi, quickening the blood and transforming stasis, and resolving toxins and relieving pain, is used for the prevention and management of oral diseases. The aim of this study was to examine the antitumor effects of Qilan preparation on oral squamous cell carcinoma (OSCC) in vitro and to explore its underlying mechanisms of action. Methods: Human Tca8113 tongue squamous cell carcinoma (TSCC) cells were tested. Cell proliferation, cell cycle distribution and apoptosis were examined using cell counting kit-8 (CCK8) and flow cytometry (FCM). The expression of PTEN and PDCD4 were determined by western blot. Changes in miR-21 levels were quantified using TaqMan stem-loop real-time PCR. After miR-21 was transiently transfected into Tca8113 cells using Lipofectamine®3000, cell proliferation, apoptosis and miR-21 and PDCD4 expression levels were measured.Results: Qilan preparation inhibited Tca8113 cell growth in a dose- and time-dependent manner by inducing apoptosis and cell cycle arrest in S-phase, decreasing miR-21 levels and increasing PTEN and PDCD4 expression. MiR-21 overexpression reversed the Qilan preparation-induced suppression of cell proliferation and induction of apoptosis while also blocking the increase in PDCD4.Conclusions: Our study revealed, for the first time, the ability of Qilan preparation to suppress TSCC cell growth and elucidated that Qilan preparation elicits its anti-cancer actions via either the miR-21/PDCD4 or PTEN pathway.

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Oxymatrine Induces Apoptosis in Human Myeloma Cells By Cell Cycle Arrest and Activation of Caspase-Dependent Apoptosis

Blood ◽

10.1182/blood.v124.21.5727.5727 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5727-5727

Author(s):

Wenjun Wu ◽

Cai Wu ◽

Fuming Zi ◽

Yi Li ◽

Li Yang ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Growth Inhibition ◽

Signaling Pathway ◽

Cell Lines ◽

Western Blotting ◽

Cell Apoptosis ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Rt Pcr

Abstract Background : Multiple myeloma (MM) is a B cell malignant hematologic cancer. Despite the introduction of new drugs and improvement of chemotherapy, MM is still an incurable disease. Oxymatrine (OMT), the active ingredients of traditional Chinese herbal medicine sophora, has been reported to have antitumor activity. This study was to estimate the therapeutic efficacy of OMT in MM. Methods: The growth inhibition of myeloma cell lines (RPMI8226, U266, ARP-1) or primary cells by OMT was assessed by MTT assay. Apoptosis of MM cells was examined by annexin V-FITC using flow cytometry analysis. DNA content was analyzed by flow cytometry. RT-PCR and western-blot analysis were used to assess the expression of Bcl-2 family proteins and the IAP family proteins. Western blotting was also used to elucidate the signaling pathway that may mediate OMT-induced apoptosis of MM cells. Results: OMT treatment resulted in cell growth inhibition and apoptosis in primary MM cells and all tested MM cell lines in a dose-dependent manner (P <0.05). To elucidate OMT -induced MM cell apoptosis, MM cell lines were treated with or without OMT for 24h and assessed for caspase activation and signaling pathway by Western blotting. The results showed the cleavage of PARP, caspase-3, and caspase-9, and p-AKT were down-regulated after OMT treatment. The mRNA expression of survivin and HIAP by RT-PCR was down-regulated. OMT treatment at 5mM for 48h resulted in increased G-phase cells and decreased S-phase cells in MM cell lines (P <0.05). Cell cycle repressor P21 protein was up-regulated while CDK4, CDK6 and CyclinD1 expression was down-regulated. Our finding also showed a synergistic anti-MM activity of OMT and dexamethasone or adriamycin at a low does (CI<1). In addition, LC3-II expression was significantly increased both in RPMI8226 and U266 cells after treatment with OMT. However, treatment with different doses of OMT and 5 mM autophagy inhibitor 3-MA, significant increased cell apoptosis (P <0.05). Conclusion: Our findings demonstrate the anti-MM activity of OMT and indicate that OMT alone or together with other MM chemotherapeutics may be a prospective treatment for MM. Disclosures No relevant conflicts of interest to declare.

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