NBM-BMX,an HDAC8 Inhibitor,Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the β-Catenin/c-Myc/SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of β-catenin was reversed by proteasome inhibitor via the β-catenin/ GSK3β signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the β-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.     

Enhanced Anti-Tumor Activity in Mice with Temozolomide-Resistant Human Glioblastoma Cell Line-Derived Xenograft Using SN-38-Incorporated Polymeric Microparticle

Glioblastoma multiforme (GBM) has remained one of the most lethal and challenging cancers to treat. Previous studies have shown encouraging results when irinotecan was used in combination with temozolomide (TMZ) for treating GBM. However, irinotecan has a narrow therapeutic index: a slight dose increase in irinotecan can induce toxicities that outweigh its therapeutic benefits. SN-38 is the active metabolite of irinotecan that accounts for both its anti-tumor efficacy and toxicity. In our previous paper, we showed that SN-38 embedded into 50:50 biodegradable poly[(d,l)-lactide-co-glycolide] (PLGA) microparticles (SMPs) provides an efficient delivery and sustained release of SN-38 from SMPs in the brain tissues of rats. These properties of SMPs give them potential for therapeutic application due to their high efficacy and low toxicity. In this study, we tested the anti-tumor activity of SMP-based interstitial chemotherapy combined with TMZ using TMZ-resistant human glioblastoma cell line-derived xenograft models. Our data suggest that treatment in which SMPs are combined with TMZ reduces tumor growth and extends survival in mice bearing xenograft tumors derived from both TMZ-resistant and TMZ-sensitive human glioblastoma cell lines. Our findings demonstrate that combining SMPs with TMZ may have potential as a promising strategy for the treatment of GBM.     

Preparation of magnetic poly(lactic‐co‐glycolic acid) microspheres with a controllable particle size based on a composite emulsion and their release properties for curcumin loading

Because of their unique magnetic features and good biocompatibility, magnetic poly(lactic‐co‐glycolic) acid (PLGA) microspheres have great application potential in magnetic targeted drug‐delivery systems. In this research, magnetic PLGA microspheres with controllable particle sizes were successfully prepared from a composite emulsion with a T‐shaped microchannel reactor. A water‐in‐oil‐in‐water composite emulsion was generated by the injection of a dichloromethane/gelatin water‐in‐oil initial emulsion into the microchannel together with a coating aqueous phase, that is, the aqueous solution of glucose and poly(vinyl alcohol). The mean particle size of the microspheres could be controlled by the manipulation of the osmotic pressure difference between the internal and external aqueous phases via changes in the glucose concentration. Curcumin, a drug with an inhibitory effect on tumor cells, was used to exemplify the release properties of the magnetic PLGA microspheres. We found that the mean particle size of the microspheres ranged from 16 to 207 μm with glucose concentrations from 0 to 20 wt %. The resulting microspheres showed a rapid magnetic response, good superparamagnetism, and a considerable magnetocaloric effect, with a maximum magnetic entropy of 0.061 J·kg^?1·K^?1 at 325 K. An encapsulation efficiency of up to 77.9% was achieved at a loading ratio of 3.2% curcumin. A release ratio of 72.4% curcumin from the magnetic PLGA microspheres was achieved within 120 h in a phosphate‐buffered solution. The magnetic PLGA microspheres showed potential to be used as drug carriers for magnetic targeted tumor therapy. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43317.     

Preparation and characterization of new nano-composite scaffolds loaded with vascular stents

In this study, vascular stents were fabricated from poly (lactide-ɛ-caprolactone)/collagen/nano-hydroxyapatite (PLCL/Col/nHA) by electrospinning, and the surface morphology and breaking strength were observed or measured through scanning electron microscopy and tensile tests. The anti-clotting properties of stents were evaluated for anticoagulation surfaces modified by the electrostatic layer-by-layer self-assembly technique. In addition, nano-composite scaffolds of poly (lactic-co-glycolic acid)/polycaprolactone/nano-hydroxyapatite (PLGA/PCL/nHA) loaded with the vascular stents were prepared by thermoforming-particle leaching and their basic performance and osteogenesis were tested in vitro and in vivo. The results show that the PLCL/Col/nHA stents and PLGA/PCL/nHA nano-composite scaffolds had good surface structures, mechanical properties, biocompatibility and could guide bone regeneration. These may provide a new way to build vascularized-tissue engineered bone to repair large bone defects in bone tissue engineering.     

Morphological and Functional Expression of Fibroblast on Poly(lactide-co-glycolide)/β-Tricalcium Phosphate/Nature Bone

The in-vitro biocompatibility of degradable and bioactive composites consisting of poly(lactide-co-glycolide) (PLGA), β-tricalcium phosphate (β-TCP), and nature bone (NB) was investigated by culturing fibroblasts on the PLGA/β-TCP/NB substrates, and the cell morphology, proliferation, alkaline phosphatase (ALP) activity, and von Kossa mineralization were evaluated. The results showed that the incorporation of β-TCP and NB benefited fibroblasts attachment and the fibroblasts cultured on the PLGA/β-TCP/NB composite substrates spread better as compared to those on the pure PLGA after culturing for different times. MTT assay showed that the fibroblasts cultured on the PLGA/β-TCP/NB extraction revealed a higher proliferation rate than those on the pure PLGA extraction. ALP and von Kossa assay indicated that β-TCP and NB could promote biomineralization in vitro. All of the results showed that the addition of β-TCP and NB into PLGA could stimulate fibroblasts to proliferate and differentiate.     

Synergistic inhibition of human glioma cell line by temozolomide and PAMAM‐mediated miR‐21i

Temozolomide (TMZ) is a promising chemotherapeutic agent for treating glioblastomas. However, resistance develops quickly and with a high frequency. Efforts to overcome chemoresistance are, therefore, critically needed. In present study, a poly(amidoamine; PAMAM) dendrimer was used as a vector to deliver microRNA‐21 inhibitor (miR‐21i) into U87 cells and the chemosensitivity of the combination effect of miR‐21i and TMZ for glioma therapy was investigated. Flow cytometry analysis showed the uptake efficiency of microRNA‐21 inhibitor after complexation with PAMAM. Real‐time PCR and in situ hybridization indicated that, compared with TMZ or miR‐21i treated cells, cells simultaneously treated with miR‐21i and TMZ showed a remarkable decrease in the microRNA‐21 (miR‐21) level. The transfection of miR‐21i enhanced the chemosensitivity by significantly decreasing the IC50 value of TMZ to glioma cells. Knockdown of miR‐21 promoted the cells' apoptosis, and at the same time, inhibited cell invasion. In conclusion, the combination treatment of glioma cells with TMZ and miR‐21i could yield a synergistic effect in inhibition of human glioma cell line. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

TRPML2 Mucolipin Channels Drive the Response of Glioma Stem Cells to Temozolomide and Affect the Overall Survival in Glioblastoma Patients

The survival of patients with glioblastoma (GBM) is poor. The main cause is the presence of glioma stem cells (GSCs), exceptionally resistant to temozolomide (TMZ) treatment. This last may be related to the heterogeneous expression of ion channels, among them TRPML2. Its mRNA expression was evaluated in two different neural stem cell (NS/PC) lines and sixteen GBM stem-like cells by qRT-PCR. The response to TMZ was evaluated in undifferentiated or differentiated GSCs, and in TRPML2-induced or silenced GSCs. The relationship between TRPML2 expression and responsiveness to TMZ treatment was evaluated by MTT assay showing that increased TRPML2 mRNA levels are associated with resistance to TMZ. This research was deepened by qRT-PCR and western blot analysis. PI3K/AKT and JAK/STAT pathways as well as ABC and SLC drug transporters were involved. Finally, the relationship between TRPML2 expression and overall survival (OS) and progression-free survival (PFS) in patient-derived GSCs was evaluated by Kaplan–Meier analysis. The expression of TRPML2 mRNA correlates with worse OS and PFS in GBM patients. Thus, the expression of TRPML2 in GSCs influences the responsiveness to TMZ in vitro and affects OS and PFS in GBM patients.     

Intrinsic factor and vitamin B12 complex‐loaded poly[lactic‐co‐(glycolic acid)] microspheres: preparation,characterization and drug release

BACKGROUND: Vitamin B12 is an essential vitamin required by all mammals. Absorption of vitamin B12 is facilitated by binding of intrinsic factor–vitamin B₁₂ complex to specific receptors in the ileum. In humans a deficiency of this vitamin or a lack of intrinsic factor leads to pernicious anaemia. The major objective of the present study was to prepare intrinsic factor–vitamin B12 complex‐loaded poly[lactic‐co‐(glycolic acid)] (PLGA)‐based microparticles and to investigate their release kinetics. RESULTS: PLGA copolymer was synthesized by the ring‐opening polymerization method and characterized using gel permeation chromatography, Fourier transform infrared spectroscopy and ¹H NMR. The glass transition temperature measurement showed a single T_g at 40 °C. The intrinsic factor–vitamin B12 complex‐loaded PLGA microspheres were prepared by a water‐in‐oil‐in‐water double emulsion solvent extraction/evaporation technique. An environmental scanning electron microscopy investigation demonstrated that the PLGA particles had a mean particle diameter of 38 µm. Interestingly, different drug release patterns (bi‐ and triphasic ones) were observed for vitamin B12‐loaded and intrinsic factor–vitamin B12 complex‐loaded microspheres. In contrast to the rapid release of vitamin B12 by itself, in vitro release tests showed that intrinsic factor and vitamin B12 in the complex were released from PLGA microspheres in a sustained manner over 15 days. CONCLUSION: PLGA microspheres can be an effective carrier for the intrinsic factor–vitamin B12 complex. Copyright © 2007 Society of Chemical Industry     

Prolonged drug release using PCL–TMZ nanofibers induce the apoptotic behavior of U87 glioma cells

In situ prolonged delivery of drugs at the site of tumor can be satisfactorily accelerated patient recovery. We compared the effect of temozolomide while incorporated by polycaprolactone nanofibers on the apoptotic behavior of U87 glioma cells. After biocompatibility evaluation of nanofibers by scanning electron microscope and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide analysis, the apoptosis of U87 cells was evaluated using p53, Bcl2 and Bax genes expression. It was found that nanofiber-temozolomide group showed a greater ability to induce apoptosis as well as have a significantly diminished initial burst release of drug compared with other groups and have promising potential in treating cancer.     

Dual Delivery of BMP-2 and bFGF from a New Nano-Composite Scaffold,Loaded with Vascular Stents for Large-Size Mandibular Defect Regeneration

The aim of this study was to investigate the feasibility and advantages of the dual delivery of bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) from nano-composite scaffolds (PLGA/PCL/nHA) loaded with vascular stents (PLCL/Col/nHA) for large bone defect regeneration in rabbit mandibles. Thirty-six large bone defects were repaired in rabbits using engineering bone composed of allogeneic bone marrow mesenchymal stem cells (BMSCs), bFGF, BMP-2 and scaffolds composed of PLGA/PCL/nHA loaded with PLCL/Col/nHA. The experiments were divided into six groups: BMSCs/bFGF/BMP-2/scaffold, BMSCs/BMP-2/scaffold, BMSCs/bFGF/scaffold, BMSCs/scaffold, scaffold alone and no treatment. Sodium alginate hydrogel was used as the carrier for BMP-2 and bFGF and its features, including gelling, degradation and controlled release properties, was detected by the determination of gelation and degradation time coupled with a controlled release study of bovine serum albumin (BSA). AlamarBlue assay and alkaline phosphatase (ALP) activity were used to evaluate the proliferation and osteogenic differentiation of BMSCs in different groups. X-ray and histological examinations of the samples were performed after 4 and 12 weeks post-implantation to clarify new bone formation in the mandible defects. The results verified that the use of sodium alginate hydrogel as a controlled release carrier has good sustained release ability, and the combined application of bFGF and BMP-2 could significantly promote the proliferation and osteogenic differentiation of BMSCs (p < 0.05 or p < 0.01). In addition, X-ray and histological examinations of the samples exhibited that the dual release group had significantly higher bone formation than the other groups. The above results indicate that the delivery of both growth factors could enhance new bone formation and vascularization compared with delivery of BMP-2 or bFGF alone, and may supply a promising way of repairing large bone defects in bone tissue engineering.     

CuZn-SOD乳酸-羟乙酸共聚物缓释微球的制备及体外、体内释放研究

目的 探讨CuZn-SOD包裹入乙酸-羟乙酸共聚物制备的药物微球的缓释效果。方法 采用复乳法制备CuZn-SOD乙酸-羟乙酸共聚物(PLGA)药物微球,分别用恒温摇瓶法和ELISA法检测体外和体内的释放效果。结果 有机相/分散相体积比、内水相体积、蛋白质浓度等因素影响微球的性质,由于聚合物的吸附作用,CuZn-SOD的体外释放呈明显的两段释放,释放不完全;体内释放显示微球制剂有一定的缓释效果。结论 该微球有比较好的缓释作用。     

Synthesis and characterization of star-shaped poly (lactide-co-glycolide) and its drug-loaded microspheres

The star-shaped poly (lactide-co-glycolide) (PLGA) was synthesized via the ring-opening polymerization of d,l-lactide and glycolide, with pentaerythritol as a multifunctional initiator and stannous 2-ethyl hexanoate as a catalyst. The structures of these polymers were characterized by ¹³C-NMR spectroscopy, while the molecular weight and polydispersity index (PDI) were determined by gel permeation chromatography (GPC). The glass transition temperature (T _g) of copolymer was determined by differential scanning calorimetry (DSC). Bovine serum albumin (BSA) loaded microspheres were fabricated using star-shaped PLGA by a W/O/W double emulsion solvent evaporation method. The results of characterization demonstrated that the particle size of the PLGA microspheres were about 80–150 μm, the maximum loading capacity and encapsulation efficiency of BSA-loaded microspheres were 67.51 μg/(mg microspheres) and 78.39%, respectively, which were better than linear PLGA. The in vitro release profiles of BSA in phosphate buffer saline (PBS) lasted for 37 h. Drug release profiles can be affected by polymer molecular weight and the ratio of polymer to drug. The maximum release percentage was 80%.     

Functional Cell Adhesion Receptors (Integrins) in Polymeric Architectures

Integrins, as transmembrane heterodimeric receptors, have important functions in cell adhesion, migration, proliferation, survival apoptosis and signal transduction, in many physio‐ as well as pathophysiological settings. Characterisation of integrins and their ligand/antagonist binding is notoriously difficult, due to high integrin redundancy and ubiquity. Bypassing the intrinsic difficulties of cell‐based integrin expression, purification and reconstitution, we present for the first time the synthesis of a heterodimeric integrin receptor and its assembly into a block‐copolymeric membrane mimic. We present comprehensive data to demonstrate the synthesis of functionally active integrin α_vβ₃, generated by in vitro membrane‐assisted protein synthesis (iMAPS). This work represents the first step towards a robust and adaptable polymer‐based platform for characterisation of integrin–ligand interactions.     

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte^® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.     

快速膜乳化法制备载醋酸曲普瑞林PLGA微球

以生物可降解聚合物聚(乳酸?羟基乙酸)(PLGA)为载体,以160 g/L明胶水溶液为内水相、含500 g/L PLGA的二氯甲烷为油相,采用快速膜乳化和溶剂蒸发法制备了粒径均一的载醋酸曲普瑞林PLGA微球,微球粒径约30 mm,粒径分布系数Span<0.8,醋酸曲普瑞林包埋率达80.12%,药物在磷酸盐缓冲液中释放36 d的释放率为72.60%,体外释放行为良好.     

The Integrin Ligand c(RGDf(NMe)Nal) Reduces Neointimal Hyperplasia in a Polymer‐Free Drug‐Eluting Stent System

The use of highly active and selective integrin ligands in combination with stent implantation is emerging as a promising alternative to the release of classical immunosuppressive drugs by current drug‐eluting stents (DES), which has been associated with delayed vascular healing and late stent thrombosis. Herein we present the development and biological evaluation of the integrin ligand c(RGDf(NMe)Nal) as a potent anti‐proliferative molecule that targets coronary artery smooth muscle cells (CASMCs). This peptide showed an antagonistic activity for αvβ3 and αvβ5 in the low‐nanomolar range, and selectivity against the platelet receptor αIIbβ3. In vitro, it efficiently inhibited the proliferation of CASMCs, displaying higher potency than the anti‐tumor drug candidate cilengitide. This peptide was then loaded into a polymer‐free bare metal stent (BMS), and its release studied at different time points. Up to seven days of elution, the peptide‐coated stents retained high anti‐proliferative activity toward CASMCs. Finally, the peptide was examined in vivo in a polymer‐free DES system in a rabbit iliac artery model. After 28 days of implantation, histopathological analysis revealed that the peptide clearly decreased neointimal growth and improved vessel healing and re‐endothelialization compared with the FDA‐approved Cypher DES. Our study shows that this type of lipophilic integrin ligand, when eluted from a polymer‐free stent system, has the potential to successfully decrease in‐stent restenosis in the absence of delayed vascular healing.     

快速膜乳化法制备载生长激素释放肽-6的PLGA微球

采用快速膜乳化法制备了聚(乳酸-羟基乙酸)(PLGA)微球,得到制备PLGA微球的优化条件为：过膜压力5 kPa,水相中PVA浓度19 g/L,油/水相体积比1:10,该条件下所制空白微球的平均粒径约为24 mm,粒径分布系数Span<0.7. 在此基础上制备载生长激素释放肽-6(GHRP-6)微球,油相乳化剂浓度2.5 g/L、外水相中NaCl浓度10 g/L条件下所制载GHRP-6微球包埋率最高可达85%,初乳制备方式对药物包埋率及体外释放行为均有较大影响,超声法制备的初乳所得微球内部结构紧密,药物包埋率较高(85%),但释药缓慢;而均质法制备的初乳所得微球内部结构疏松,药物包埋率较低(76.8%),但在体外释放更完全.     

Tamoxifen‐loaded microspheres based on mixtures of poly(D,L‐lactide‐co‐glycolide) and poly(D,L‐lactide) polymers: Effect of polymeric composition on drug release and in vitro antitumoral activity

Mixtures of different bioerosionable polyesters were used to prepare microparticulated tamoxifen delivery systems to achieve anticancer effects in breast malignant cancer cells. Tamoxifen (TMX) was included into microspheres (MS) formulated via spray‐drying. Mixtures of poly(D ,L ‐lactide‐co‐glycolide) (PLGA) of different lactide/glycolide proportions (50 : 50 and 75 : 25) and poly(D ,L ‐lactic acid) (PLA) were used. The average diameter of the resultant TMX‐loaded microparticles was in the range 1.04 ± 0.51–1.55 ± 0.95 μm. The encapsulation efficiency of TMX was between 97.8% [48.9 ± 0.1 TMX (μg)/MS (mg)] and 69.6% [36.6 ± 0.1 TMX (μg)/MS (mg)] depending on the polymeric composition of the formulation. Drug burst effect was not observed. TMX was released from the polymeric matrices in a sustained release manner between 11 and 58 days depending on polymeric composition of microspheres. TMX‐loaded microspheres showed high efficacy in causing cell death in MCF7 breast malignant cancer cells. Thus, these TMX‐loaded PLGA‐based microspheres hold potential to treat breast malignant cancer cells. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

O6-Methylguanine-Methyltransferase (MGMT) Promoter Methylation Status in Glioma Stem-Like Cells is Correlated to Temozolomide Sensitivity Under Differentiation-Promoting Conditions

Glioblastoma (GBM) is the most malignant type of primary brain tumor with a very poor prognosis. The actual standard protocol of treatment for GBM patients consists of radiotherapy and concomitant temozolomide (TMZ). However, the therapeutic efficacy of this treatment is limited due to tumor recurrence and TMZ resistance. Recently isolated, glioma stem-like cells (GSCs) are thought to represent the population of tumorigenic cells responsible for GBM resistance and recurrence following surgery and chemotherapy. In addition, MGMT (O6-methylguanine-methyltransferase) methylation is considered as one of the principal mechanisms contributing to TMZ sensitivity of GBM. In this study we have isolated GSCs from 10 adult GBM patients and investigated the relationship between MGMT methylation status and Temozolomide (TMZ) sensitivity of these lines grown either in stem-like or differentiation promoting conditions. Sensitivity to TMZ was significantly associated with MGMT methylation status in cells committed to differentiation but not in stem-like cells. In addition, patients harboring highly methylated MGMT promoters had a longer overall survival. These results reveal the importance of the differentiation process when considering the predictive value of MGMT status in GSCs for clinical response to TMZ.