Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models

Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 micromol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.     

B7.1 expression by the weakly immunogenic F98 rat glioma does not enhance immunogenicity

Enhanced immunogenicity has been reported following transfection of a variety of immunogenic tumors with the B7.1 co-stimulatory molecule. The purpose of the present study was to determine if transfection of a weakly immunogenic rat brain tumor, the F98 glioma, with the gene encoding B7.1 could enhance its immunogenicity. F98 cells were transfected with a plasmid containing the B7.1 gene, and stable transfectants (F98/B7.1) were obtained. Flow cytometric analysis confirmed the expression of B7.1 and MHC class I antigens on the cell surface. To investigate the effects of B7.1 expression on the tumorigenicity of the F98 glioma, Fischer rats were implanted intracerebrally with either F98 (wild-type) or F98/B7.1 transfected cells. No significant differences in survival times were noted. Mean survival times of 21.8 and 24.0 days were observed for the respective groups at a challenge dose of 103 cells. These differences in survival time were not significant. To determine if expression of B7.1 enhanced the immunogenicity of the F98 glioma, rats were vaccinated weekly for 3 weeks with 107 mitomycin C-treated F98 or F98/B7.1 cells injected subcutaneously and then challenged intracerebrally with F98 cells 1 week later. Unvaccinated animals or those that received wild-type F98 cells as a vaccine had a survival time (mean +/- s.d.) of 22.3 +/- 1.5 days following tumor challenge versus 20.0 +/- 1.7 days for rats that had been vaccinated with F98/B7.1. Although we recognize that it might be possible to design more effective vaccination regimes, nevertheless, our data indicate that transfection of the B7.1 gene into the F98 rat glioma did not enhance its immunogenicity, and that other approaches will be required.     

Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model

The prognosis of patients with malignant glioma is extremely poor, despite the extensive surgical treatment that they receive and recent improvements in adjuvant radio- and chemotherapy. In the present study, we propose the use of gene-modified mesenchymal stem cells (MSCs) as a new tool for gene therapy of malignant brain neoplasms. Primary MSCs isolated from Fischer 344 rats possessed excellent migratory ability and exerted inhibitory effects on the proliferation of 9L glioma cell in vitro. We also confirmed the migratory capacity of MSCs in vivo and showed that when they were inoculated into the contralateral hemisphere, they migrated towards 9L glioma cells through the corpus callosum. MSCs implanted directly into the tumor localized mainly at the border between the 9L tumor cells and normal brain parenchyma, and also infiltrated into the tumor bed. Intratumoral injection of MSCs caused significant inhibition of 9L tumor growth and increased the survival of 9L glioma-bearing rats. Gene-modification of MSCs by infection with an adenoviral vector encoding human interleukin-2 (IL-2) clearly augmented the antitumor effect and further prolonged the survival of tumor-bearing rats. Thus, gene therapy employing MSCs as a targeting vehicle would be promising as a new therapeutic approach for refractory brain tumor.     

Treatment of experimental glioma by administration of adenoviral vectors expressing Fas ligand.

Fas ligand (FasL) is a cytokine, produced by activated T cells and NK cells, that triggers apoptosis of Fas-positive target cells including human glioma cells. As shown here, in vitro infection of rat F98 and human LN18 glioma cell lines with recombinant adenovirus (rAd) expressing FasL cDNA under control of the cytomegalovirus promoter (rAd-CMV-FasL) induced striking cytotoxicity in Fas-positive glioma cell lines but not in the Fas-negative F98 glioma subline F98/ZH. The extent of FasL-mediated cytotoxic effects outranged the expectations based on expression of beta-galactosidase (beta-Gal) by F98 cells infected with a control virus expressing the lacZ gene (rAd-CMV-lacZ). The detection of FasL bioactivity in supernatants of infected cells provides evidence of a bystander mechanism involving the cytotoxic action of FasL on uninfected cells. In F98 tumor-bearing rats, infection with rAd-CMV-FasL increased the mean survival time by 50% compared with infection with rAd-CMV-lacZ or untreated controls. These data suggest that viral vector transduction of the FasL gene could be part of a successful glioma gene therapy.     

The role of thrombin in gliomas

BACKGROUND: In a previous study we found that intracerebral infusion of argatroban, a specific thrombin inhibitor, reduces brain edema and neurologic deficits in a C6 glioma model. OBJECTIVES: To examine the role of thrombin in gliomas and whether systemic argatroban administration can reduce glioma mass and neurologic deficits and extend survival time in C6 and F98 gliomas. METHODS: The presence of thrombin in human glioblastoma samples and rat C6 glioma cells (in vitro and in vivo) was assessed using immunohistochemistry. The effect of thrombin on C6 cell proliferation in vitro was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The role of thrombin in vivo was assessed in rat C6 and F98 glioma cell models using argatroban, a thrombin inhibitor. The effects of argatroban on tumor mass, neurologic deficits and survival time were investigated. RESULTS: Thrombin immunoreactivity was found in cultured rat C6 glioma cells and human glioblastomas. Thrombin induced C6 cell proliferation in vitro. In C6 glioma, argatroban reduced glioma mass (P < 0.05) and neurologic deficits (P < 0.05) at day 9. In F98 glioma, argatroban prolonged survival time (P < 0.05). CONCLUSION: These results suggest that thrombin plays an important role in glioma growth. Thrombin may be a new therapeutic target for gliomas.     

Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines.

Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma.     

New transition state-based inhibitor for human ornithine decarboxylase inhibits growth of tumor cells

Pyridoxal 5'-phosphate (PLP)-dependent ornithine decarboxylase (ODC) is the key enzyme in polyamine synthesis. ODC is overexpressed in many tumor cells and thus a potential drug target. Here we show the design and synthesis of a coenzyme-substrate analogue as a novel precursor inhibitor of ODC. Structural analysis of the crystal structure of human ODC disclosed an additional hydrophobic pocket surrounding the epsilon-amino group of its substrate ornithine. Molecular modeling methods showed favorable interactions of the BOC-protected pyridoxyl-ornithine conjugate, termed POB, in the active site of human ODC. The synthesized and purified POB completely inhibited the activity of newly induced ODC activity at 100 micromol/L in glioma LN229 and COS7 cells. In correlation with the inhibition of ODC activity, a time-dependent inhibition of cell growth was observed in myeloma, glioma LN18 and LN229, Jurkat, COS7, and SW2 small-cell lung cancer cells if DNA synthesis and cell number were measured, but not in the nontumorigenic human aortic smooth muscle cells. POB strongly inhibited cell proliferation not only of low-grade glioma LN229 cells in a dose-dependent manner (IC(50) approximately 50 micromol/L) but also of high-grade glioblastoma multiforme cells. POB is much more efficient in inhibiting proliferation of several types of tumor cells than alpha-DL-difluoromethylornithine, the best known irreversible inhibitor of ODC.     

Etoposide nanocarriers suppress glioma cell growth by intracellular drug delivery and simultaneous P-glycoprotein inhibition.

In cancer treatment, efficient therapeutic strategies could be impeded by cellular mechanisms such as the multidrug resistance. Recently, drug-loaded nanoparticles have been reported to be useful, since they allow entering the cancer cell and act as an intracellular anti-cancer drug reservoir. A new approach is proposed here by the use of lipid nanocapsules (LNC) which were hypothesized to reverse multidrug resistance additionally by their P-glycoprotein (P-gp) inhibiting surfactant. LNC (mean diameter 25 to 100 nm) were loaded with etoposide, tested for the drug release and their efficiency to reduce cell growth in cell culture for C6, F98, and 9L glioma cell lines. Sustained etoposide release can be provided over a period of 1 week (t10%: 1.4+/-0.1h; t50%: 15.9+/-2.8h). The P-gp inhibiting activity in-vitro was found to be independent from the LNC size. In cell culture, an internalization of LNC was observed in all glioma cell types. Etoposide LNC showed a generally higher efficiency than the drug solution while blank LNC were found to be less inhibitory than the pure drug at equivalent concentrations (IC50: C6: etoposide: 25.2 microM; LNC: 2.6-8.9 microM, F98: etoposide: 46.5 microM; LNC: 1.4-14.7 microM, 9L: etoposide: 58.2 microM; LNC: 4.4-12.7 microM). This effect was found to be particle size dependent within a range of an 8- (C6) to 33-fold (F98) increased cytotoxicity for smallest particles. When cells were incubated with etoposide solution in the presence of blank LNC, a slight growth inhibition was observed, however, distinctly lower than the drug-trapping particles. Moreover, cell toxicity on astrocytes was similar for etoposide LNC and etoposide solution. The mechanism of action of etoposide LNC was proposed to be a cell uptake followed by a sustained drug release from the LNC in combination with an intracellular P-gp inhibition ensuring a higher anticancer drug concentration inside the cancer cells.     

Butyric acid prodrugs are histone deacetylase inhibitors that show antineoplastic activity and radiosensitizing capacity in the treatment of malignant gliomas

Histone modification has emerged as a promising approach to cancer therapy. We explored the efficacy of a novel class of histone deacetylase inhibitors in the treatment of malignant gliomas. Treatment of glioma cell lines with two butyric acid derivatives, pivaloylomethyl butyrate (AN-9) and butyroyloxymethyl butyrate (AN-1), induced hyperacetylation, increased p21(Cip1) expression, inhibited proliferation, and enhanced apoptosis. Histone deacetylase inhibitor-induced apoptosis was mediated primarily by caspase-8. Treatment of cells with AN-1 or AN-9 for 24 hours before exposure to gamma-irradiation potentiated further caspase-8 activity and resultant apoptosis. Clonogenic survival curves revealed marked reductions in cell renewal capacity of U251 MG cells exposed to combinations of AN-1 and radiation. Preliminary in vivo experiments using human glioma cell lines grown as xenografts in mouse flanks suggest in vivo efficacy of AN-9. The data suggest that novel butyric acid prodrugs provide a promising treatment strategy for malignant gliomas as single agents and in combination with radiation therapy.     

Targeted delivery of methotrexate to epidermal growth factor receptor-positive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates

We have constructed a drug delivery vehicle that targets the epidermal growth factor receptor (EGFR) and its mutant isoform EGFRvIII. The monoclonal antibody, cetuximab, previously known as C225, which binds to both EGFR and EGFRvIII, was covalently linked via its Fc region to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. As measured by mass spectrometry and UV/vis spectroscopy, the resulting bioconjugate, designated C225-G5-MTX, contained 12.6 molecules of methotrexate per unit of dendrimer. Specific binding and cytotoxicity of the bioconjugate was evaluated against the EGFR-expressing rat glioma cell line F98(EGFR). Using a competitive binding assay, it was shown that the bioconjugate retained its affinity for F98(EGFR) cells, with a 0.8 log unit reduction in its EC(50). Only cetuximab completely inhibited binding of the bioconjugate, which was unaffected by methotrexate or dendrimer. Cetuximab alone was not cytotoxic to F98(EGFR) cells at the concentration tested, whereas the IC(50) of the bioconjugate was 220 nmol/L, which was a 2.7 log unit decrease in toxicity over that of free methotrexate. The biodistribution of C225-G5-MTX in rats bearing i.c. implants of either F98(EGFR) or F98(WT) gliomas was determined 24 hours following convection enhanced delivery of (125)I-labeled bioconjugate. At this time, 62.9 +/- 14.7% ID/g tumor was localized in rats bearing F98(EGFR) gliomas versus 11.3 +/- 3.6% ID/g tumor in animals bearing F98(WT) gliomas, thereby showing specific molecular targeting of the tumor. The corresponding radioactivity of normal brain from the F98(EGFR) tumor-bearing right and non-tumor-bearing left cerebral hemisphere were 5.8 +/- 3.4% and 0.8 +/- 0.6% ID/g, respectively. Based on these results, therapy studies were initiated in F98(EGFR) glioma-bearing rats. Animals that received C225-G5-MTX, cetuximab, or free methotrexate had median survival times of 15, 17, and 19.5 days, respectively, which were not statistically different from each other or untreated control animals. Our results, which are both positive and negative, show that specific molecular targeting is but one of several requirements that must be fulfilled if an antibody-drug bioconjugate will be therapeutically useful.     

HDAC Inhibition Suppresses Primary Immune Responses,Enhances Secondary Immune Responses,and Abrogates Autoimmunity During Tumor Immunotherapy

Histone deacetylase inhibitors (HDACi) can modulate innate antiviral responses and render tumors more susceptible to oncolytic viruses (OVs); however, their effects on adaptive immunity in this context are largely unknown. Our present study reveals an unexpected property of the HDACi MS-275 that enhances viral vector-induced lymphopenia leading to selective depletion of bystander lymphocytes and regulatory T cells while allowing expansion of antigen-specific secondary responses. Coadministration of vaccine plus drug during the boosting phase focuses the immune response on the tumor by suppressing the primary immune response against the vaccine vector and enhancing the secondary response against the tumor antigen. Furthermore, improvement of T cell functionality was evident suggesting that MS-275 can orchestrate a complex array of effects that synergize immunotherapy and viral oncolysis. Surprisingly, while MS-275 dramatically enhanced efficacy, it suppressed autoimmune pathology, profoundly improving the therapeutic index.     

Phage probes for malignant glial cells

Early diagnosis and effective treatment of malignant gliomas, which are heterogeneous brain tumors with variable expression of cell surface markers, are inhibited by the lack of means to characterize and target tumor-selective molecules. To create molecular profiles for RG2 rat glioma cells, we used phage display technology, an approach capable of producing valuable ligands to unknown cell surface targets. The ligands were selected from libraries of peptides displayed as fusion molecules on phage particles. Modifications of the selection conditions resulted in identification of three distinctive families of peptide ligands for malignant glioma cells. The first family with V (D)/(G) L P (E)/(T) H(3) binding motif appeared to target a marker that is common for glioma cells, normal brain cells, and cells of non-brain origin. The second group of peptide-presented phage displayed D (T)/S/(L) T K consensus sequence and contained peptides with pronounced glioma-selective properties. Phage clones expressing peptides with E (L)/V/(S) R G D S motif were found in cell lysates and represented the third family of glioma-specific ligands. All peptides within this family contain the RGD amino acid sequence, which is known to bind to a number of integrins. Phage clones that belong to this family were internalized by RG2 glioma cells about 63-fold more efficiently than by astrocytes. The approach described could be applicable for accurate detection of glioma expression patterns in individual tumors. Such patterns could be beneficial in the design of effective combinations of drugs for anti-glioma treatments.     

Sonodynamic Therapy for Malignant Glioma Using 220-kHz Transcranial Magnetic Resonance Imaging-Guided Focused Ultrasound and 5-Aminolevulinic acid

Sonodynamic therapy (SDT) is used to treat various malignancies and can be applied to brain tumors using a transcranial magnetic resonance imaging-guided focused ultrasound (TcMRgFUS) device. This study investigated the efficacy of 220-kHz TcMRgFUS combined with 5-aminolevulinic acid (5-ALA) on malignant glioma in vitro and in vivo. F98 cells were irradiated with focused ultrasound (FUS) (4000 J, 20 W, 240 s, 100% duty cycle, target medium temperature <40°C) after treatment with 200 µg/mL 5-ALA, and cell viability and apoptosis were evaluated with the water-soluble tetrazolium-1 assay, triple fluorescent staining and Western blot analysis 20 h later. The anti-tumor effects of 5-ALA combined with FUS (500 J, 18 W, 30 s, 100% duty cycle, 10 repeats, target tissue temperature ≤42°C) were assessed on the basis of changes in tumor volume determined by MRI and histopathological analysis before and after treatment. The FUS/5-ALA combination reduced cell viability by inducing apoptosis and suppressed tumor proliferation and invasion as well as angiogenesis in vivo, while causing minimal damage to normal brain tissue. SDT with 220-kHz TcMRgFUS and 5-ALA can be safely used for the treatment of malignant glioma.     

Detection of histone deacetylase inhibition by noninvasive magnetic resonance spectroscopy

Histone deacetylase (HDAC) inhibitors are new and promising antineoplastic agents. Current methods for monitoring early response rely on invasive biopsies or indirect blood-derived markers. Our goal was to develop a magnetic resonance spectroscopy (MRS)-based method to detect HDAC inhibition. The fluorinated lysine derivative Boc-Lys-(Tfa)-OH (BLT) was investigated as a (19)F MRS molecular marker of HDAC activity together with (31)P MRS of endogenous metabolites. In silico modeling of the BLT-HDAC interaction and in vitro MRS studies of BLT cleavage by HDAC confirmed BLT as a HDAC substrate. BLT did not affect cell viability or HDAC activity in PC3 prostate cancer cells. PC3 cells were treated, in the presence of BLT, with the HDAC inhibitor p-fluoro-suberoylanilide hydroxamic acid (FSAHA) over the range of 0 to 10 micromol/L, and HDAC activity and MRS spectra were monitored. Following FSAHA treatment, HDAC activity dropped, reaching 53% of control at 10 micromol/L FSAHA. In parallel, a steady increase in intracellular BLT from 14 to 32 fmol/cell was observed. BLT levels negatively correlated with HDAC activity consistent with higher levels of uncleaved BLT in cells with inhibited HDAC. Phosphocholine, detected by (31)P MRS, increased from 7 to 16 fmol/cell following treatment with FSAHA and also negatively correlated with HDAC activity. Increased phosphocholine is probably due to heat shock protein 90 inhibition as indicated by depletion of client proteins. In summary, (19)F MRS of BLT, combined with (31)P MRS, can be used to monitor HDAC activity in cells. In principle, this could be applied in vivo to noninvasively monitor HDAC activity.     

组蛋白去乙酰化酶抑制剂对乳腺癌细胞株MCF-7增殖周期的影响

目的 研究组蛋白去乙酰化酶抑制剂对乳腺癌细胞株增殖周期的影响.方法 培养乳腺癌细胞株MCF-7,用TSA、SAHA、CS055、MS-275 4种不同类型的组蛋白去乙酰化酶抑制剂分别作用于细胞,于24、48、72、96 h用MTT比色法观察细胞生长情况,初筛出高效的抑制剂.用所选出的抑制剂的不同浓度处理细胞,流式细胞术检测S期细胞的比例和周期素Cyclin D1、Cyclin A2,并进行统计分析.结果 在TSA、SAHA、CS055和MS-275 4种不同类型的组蛋白去乙酰化酶抑制剂中,SAHA显示出较强的抑制能力(P＜0.05);在一定时间和浓度范围内,其抑制能力有明显的时效关系;在抑制剂作用下,S期细胞比例和周期素Cyclin A2表达下降,Cyclin D1表达上升(P均＜0.05).结论 组蛋白去乙酰化酶抑制剂SAHA是乳腺癌细胞增殖的高效抑制剂,其抑制作用有一定的时效关系;周期素Cyclin D1、Cyclin A2参与了抑制剂对细胞增殖周期的调控.     

Sonodynamic therapy induces oxidative stress,DNA damage and apoptosis in glioma cells

Malignant glioma remains one of the most challenging diseases to treat because of the invasive growth of glioma cells and the existence of the blood–brain barrier (BBB), which blocks drug delivery to the brain. New strategies are urgently needed to overcome these shortcomings and improve the outcomes. Ultrasound represents a promising noninvasive and reversible BBB opening approach and the related sonodynamic therapy (SDT) is rapidly emerging. This study aims to explore the ultrasound parameters for BBB opening and the cell killing effect of SDT in human glioma U373 cells by using a recently reported sonosensitizer, sinoporphyrin sodium (DVDMS). The in vitro BBB model indicated that SDT caused a time-dependent permeability increase, which peaked at 2 h post treatment and then recovered gradually. The results of toxicology tests showed significant U373 cell viability loss and apoptosis increase after DVDMS-SDT, accompanied by enhanced cleaved-caspase-3 level and DNA fragmentation, in which reactive oxygen species (ROS) were a major triggering intermediate during DVDMS-SDT. Furthermore, DVDMS-SDT produced DNA damage and the underlying mechanisms were evaluated, in order to provide a fundamental basis for DVDMS-SDT application in glioma therapy. The findings indicated that the DNA molecules could be temporarily regulated by SDT and DNA double-strand breaks (DSBs), which increased the difficulty of cellular self-repair, thus aggravating cell apoptosis and inhibiting glioma cell invasive growth. Therefore, this study supports the use of SDT as an alternative approach for glioma therapy.

Malignant glioma remains one of the most challenging diseases to treat because of the invasive growth of glioma cells and the existence of the blood–brain barrier (BBB), which blocks drug delivery to the brain.     

FK228 augmented temozolomide sensitivity in human glioma cells by blocking PI3K/AKT/mTOR signal pathways

Temozolomide is a novel cytotoxic agent currently used as first-line chemotherapy for glioblastoma multiforme (GBM). Romidepsin (FK228), a histone deacetylase inhibitor, is a promising new class of antineoplastic agent with the capacity to induce growth arrest and/or apoptosis of cancer cells. However, combination of the two drugs in glioma remains largely unknown. In the present study, we evaluated the combinatory effects of FK228 with TMZ in glioma, and its molecular mechanisms responsible for these effects. Glioma cell lines were treated with TMZ, FK228 or the combination of drugs. The resistance effect including cytotoxicity and apoptosis was determined in glioma cells, respectively. We further evaluated the effects of FK228 in the PI3K/Akt-signaling pathway in vitro. Mice engrafted with 5 × 10⁶ LN382 cells were treated with TMZ, FK228 or the combination of two drugs, and tumor weights and volumes were measured, respectively. FK228 enhanced the cytotoxic effects of TMZ in glioma cells compared to vehicle-treated controls or each drug alone. The combination of FK228 and TMZ-induced apoptosis was demonstrated by increased expression of cleaved-Caspase 3, Bax, cleaved-PARP, and decreased Bcl-2 expression. Furthermore, the expression of key components of the PI3K/Akt-signaling pathway showed that combination of FK228 and TMZ block PI3K/Akt pathways in vitro. This block effect was also confirmed in vivo in mice models. Mice treated with both FK228 and TMZ drugs showed significantly reduced tumor weights and volumes, compared to each drug alone. Our results suggested that FK228 augmented temozolomide sensitivity in human glioma cells partially by blocking PI3K/AKT/mTOR signal pathways. It thus may provide a promising target for improving the therapeutic outcome of TMZ-resistant gliomas, although further studies will be needed.     

姜黄素调节NB4细胞P53和组蛋白H3乙酰化抑制细胞增殖的研究

目的 研究姜黄素对NB4细胞组蛋白H3和非组蛋白P53的乙酰化和细胞增殖的作用，探讨姜黄素抗白血病机制。方法 应用小同浓度（50，25，12．5，6．25，3．125μmol／L）的姜黄素作用NB4细胞不同时间（0，4，8，12，24h），MTT法测定姜黄素对NB4细胞增殖的影响，Western blot法检测乙酰化组蛋白H3和乙酰化P53的水平。结果 姜黄素以时间和剂量依赖方式抑制NB4细胞增殖，在24h和36h的IC值分别为40μmol／L和25μmol／L；能明显上调组蛋白H3的乙酰化水平，促进P53的表达和P53的乙酰化。结论 姜黄素具有去乙酰化酶抑制剂作用，能上调组蛋白H3乙酰化水平，促进肿瘤抑制凶子P53表达和活化，抑制白血病细胞增殖。