技术资料

The epithelial-mesenchymal transition (EMT) is activated in cancer cells by ZEB1,a member of the zinc finger/homeodomain family of transcriptional repressors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in human carcinoma cells. The present studies in breast cancer cells demonstrate that the oncogenic MUC1-C subunit induces expression of ZEB1 by a NF-$$B (nuclear factor kappa B) p65-dependent mechanism. MUC1-C occupies the ZEB1 promoter with NF-$$B p65 and thereby promotes ZEB1 transcription. In turn,ZEB1 associates with MUC1-C and the ZEB1/MUC1-C complex contributes to the transcriptional suppression of miR-200c,an inducer of epithelial differentiation. The co-ordinate upregulation of ZEB1 and suppression of miR-200c has been linked to the induction of EMT. In concert with the effects of MUC1-C on ZEB1 and miR-200c,we show that MUC1-C induces EMT and cellular invasion by a ZEB1-mediated mechanism. These findings indicate that (i) MUC1-C activates ZEB1 and suppresses miR-200c with the induction of EMT and (ii) targeting MUC1-C could be an effective approach for the treatment of breast and possibly other types of cancers that develop EMT properties. View Publication

Zoledronic acid,a third-generation bisphosphonate,has been shown to reduce cell migration,invasion,and metastasis. However,the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT),a cellular process essential to the metastatic cascade,remain unclear. Therefore,the effects of zoledronic acid on EMT,using triple-negative breast cancer (TNBC) cells as a model system,were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers,N-cadherin,Twist,and Snail,and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability,induced cell-cycle arrest,and decreased the proliferative capacity of TNBC,suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype,the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins,BMI-1 and Oct-4,and both prevented and eliminated mammosphere formation. To understand the mechanism of these results,the effect of zoledronic acid on established EMT regulator NF-$$B was investigated. Zoledronic acid inhibited phosphorylation of RelA,the active subunit of NF-$$B,at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter,providing a direct link between inactivation of NF-$$B signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased,correlating modulation of EMT to decreased self-renewal. On the basis of these results,it is proposed that through inactivation of NF-$$B,zoledronic acid reverses EMT,which leads to a decrease in self-renewal. View Publication

Recently,the use of herbal medicines has been increased all over the world due to their therapeutic effects and fewer adverse effects as compared to the modern medicines. However,many herbal drugs and herbal extracts despite of their impressive in-vitro findings demonstrates less or negligible in-vivo activity due to their poor lipid solubility or improper molecular size,resulting in poor absorption and hence poor bioavailability. Nowadays with the advancement in the technology,novel drug delivery systems open the door towards the development of enhancing bioavailability of herbal drug delivery systems. For last one decade many novel carriers such as liposomes,microspheres,nanoparticles,transferosomes,ethosomes,lipid based systems etc. have been reported for successful modified delivery of various herbal drugs. Many herbal compounds including quercetin,genistein,naringin,sinomenine,piperine,glycyrrhizin and nitrile glycoside have demonstrated capability to enhance the bioavailability. The objective of this review is to summarize various available novel drug delivery technologies which have been developed for delivery of drugs (herbal),and to achieve better therapeutic response. An attempt has also been made to compile a profile on bioavailability enhancers of herbal origin with the mechanism of action (wherever reported) and studies on improvement in drug bioavailability,exhibited particularly by natural compounds. View Publication

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched,full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell,HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units,CD56+CD3- placenta-derived NK cells,termed pNK cells,were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were textgreater80% CD56+CD3-,comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D,NKp46,and NKp44 (p textless 0.001,p textless 0.001,and p textless 0.05,respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile,immunophenotype,and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development,pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options. View Publication

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues,first identified in zebrafish,remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development. View Publication

Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here,we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells,human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h,the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell–cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels,inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity,highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary,we have developed microengineered three-dimensional vascular structures that can be used as a model to test the effects of drugs and study the interaction between different human vascular cell types. In the future,the model may be integrated into larger tissue constructs to advance the development of organs-on-chips. View Publication

Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome,slow,and variable. Alternatively,human fibroblasts can be directly converted into induced neuronal (iN) cells. However,with present techniques conversion is inefficient,synapse formation is limited,and only small amounts of neurons can be generated. Here,we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin,form mature pre- and postsynaptic specializations,and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples,our approach enables large-scale studies of human neurons for questions such as analyses of human diseases,examination of human-specific genes,and drug screening View Publication

Overexpression of the adverse prognostic marker ERBB2 occurs in 30% of breast cancers and is associated with aggressive disease and poor outcomes. Our recent findings have shown that NR1D1 and the peroxisome proliferator-activated receptor-γ (PPARγ)-binding protein (PBP) act through a common pathway in upregulating several genes in the de novo fatty acid synthesis network,which is highly active in ERBB2-positive breast cancer cells. NR1D1 and PBP are functionally related to PPARγ,a well-established positive regulator of adipogenesis and lipid storage. Here,we report that inhibition of the PPARγ pathway reduces the aldehyde dehydrogenase (ALDH)-positive population in ERBB2-positive breast cancer cells. Results from in vitro tumorsphere formation assays demonstrate that the PPARγ antagonists GW9662 and T0070907 decrease tumorsphere formation in ERBB2-positive cells,but not other breast cells. We show that the mechanism by which GW9662 treatment causes a reduction in ALDH-positive population cells is partially due to ROS,as it can be rescued by treatment with N-acetyl-cysteine. Furthermore,global gene expression analyses show that GW9662 treatment suppresses the expression of several lipogenic genes,including ACLY,MIG12,FASN and NR1D1,and the stem-cell related genes KLF4 and ALDH in BT474 cells. Antagonist treatment also decreases the level of acetylation in histone 3 and histone 4 in BT474 cells,compared with MCF7 cells. In vivo,GW9662 pre-treatment inhibits the tumor-seeding ability of BT474 cells. Together,these results show that the PPARγ pathway is critical for the cancer stem cell properties of ERBB2-positive breast cancer cells. View Publication

Cancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor 1 (ANTXR1) has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f(+)/EpCAM(-)/Lin(-)) expressed higher levels of ANTXR1 compared with mature luminal cells. CD49f(+)/EpCAM(-),CD44(+)/EpCAM(-),CD44(+)/CD24(-),or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44(+)/CD24(-)/ANTXR1(+) cells displayed enhanced self-renewal as measured by mammosphere assay compared with CD44(+)/CD24(-)/ANTXR1(-) cells. Activation of ANTXR1 by its natural ligand C5A,a fragment of collagen VI $$3,increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor-lipoprotein receptor-related protein 6 (LRP6),phosphorylation of GSK3$$/$$,and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression,self-renewal,invasion,tumorigenicity,and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A,which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Furthermore,ANTXR1 is among the 10% most overexpressed genes in breast cancer and is coexpressed with collagen VI. Thus,ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment,linking it to a stemness signaling network that drives metastatic progression. View Publication

We previously demonstrated that RARα2 expression is increased in CD138 selected plasma cells of relapsed multiple myelomas (MMs),and increased expression was linked to poor prognosis in newly diagnosed MM patients. In the present study,we demonstrate that increased RARα2 confers myeloma stem cell features. Higher expression of RARα2 was identified in the multiple myeloma stem cell (MMSC) fraction. Overexpression of RARα2 in bulk MM cell lines resulted in: 1) increased drug resistance; 2) increased clonogenic potential; 3) activation of both Wnt and Hedgehog (Hh) pathways; 4) increased side population and aldehyde dehydrogenase levels; and 5) increased expression of embryonic stem cell genes. The opposite effects were seen with RARα2 knockdown. We demonstrate that RARα2 induces drug resistance by activating the drug efflux pump gene ABCC3 and anti-apoptotic Bcl-2 family members. Inhibition of Wnt signaling or ABCC3 function could overcome drug resistance in RARα2 overexpressing MM cells. We also showed that in the 5TGM1 mouse model,targeting of the Wnt and Hh pathways using CAY10404,cyclopamine,or itraconazole significantly reduced the myeloma tumor burden and increased survival. Targeting RARα2 or its downstream signaling pathways provides a potential strategy to eliminate MMSC. View Publication

While inhibitory Siglec receptors are known to regulate myeloid cells,less is known about their expression and function in lymphocytes subsets. Here we identified Siglec-7 as a glyco-immune checkpoint expressed on non-exhausted effector memory CD8+ T cells that exhibit high functional and metabolic capacities. Seahorse analysis revealed higher basal respiration and glycolysis levels of Siglec-7+ CD8+ T cells in steady state,and particularly upon activation. Siglec-7 polarization into the T cell immune synapse was dependent on sialoglycan interactions in trans and prevented actin polarization and effective T cell responses. Siglec-7 ligands were found to be expressed on both leukemic stem cells and acute myeloid leukemia (AML) cells suggesting the occurrence of glyco-immune checkpoints for Siglec-7+ CD8+ T cells,which were found in patients' peripheral blood and bone marrow. Our findings project Siglec-7 as a glyco-immune checkpoint and therapeutic target for T cell-driven disorders and cancer. View Publication

BACKGROUND Ageing is a complex phenomenon that leads to decreased proliferative activity,loss of function of the cells,and cellular senescence. Senescence of the immune system exacerbates individual's immune response,both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets - CD4+,CD8+,CD19+,CD56+. RESULTS Median telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively,p??=??0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (na{\{i}}ve and effector memory) CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22 3%) than high-risk patients (9 3%) p??=??0 04. CONCLUSION Our data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features characteristic for physiological ageing (telomeric shortening immunophenotype) when compared to their respective donors. However a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore it is safe to conclude that the observed subtle differences between recipients' and donors' cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts' and recipients' microenvironments." View Publication