Co‐encapsulation of anti‐BCR‐ABL siRNA and imatinib mesylate in transferrin receptor‐targeted sterically stabilized liposomes for chronic myeloid leukemia treatment

Chronic myeloid leukemia (CML) is triggered by the BCR‐ABL oncogene. Imatinib is the first‐line treatment of CML; however imatinib resistance and intolerance have been detected in many patients. Therefore, new therapeutic approaches are required. The present work aimed at the development and application of transferrin receptor (TrfR) targeted liposomes co‐encapsulating anti‐BCR‐ABL siRNA and imatinib at different molar ratios. The encapsulation yields and drug loading of each molecule was evaluated. Anti‐leukemia activity of the developed formulations co‐encapsulating siRNA and imatinib and of the combination of Trf‐liposomes carrying siRNA and free imatinib under two different treatment schedules of pre‐sensitization was assessed. The results obtained demonstrate that the presence of imatinib significantly decreases the encapsulation yields of siRNA, whereas imatinib encapsulation yields are increased by the presence of siRNA. Cytotoxicity assays demonstrate that the formulations co‐encapsulating siRNA and imatinib promote a 3.84‐fold reduction on the imatinib IC₅₀ (from 3.49 to 0.91 µM), whereas a 8.71‐fold reduction was observed for the pre‐sensitization protocols (from 42.7 to 4.9 nM). It was also observed that the formulations with higher siRNA to imatinib molar ratios promote higher cell toxicity. Thus, the present work describes a novel triple targeting strategy with one single system: cellular targeting (through the targeting ligand, transferrin) and molecular targeting at the BCR‐ABL mRNA and Bcr‐Abl protein level. Biotechnol. Bioeng. 2010;107: 884–893. © 2010 Wiley Periodicals, Inc.     

CD155 knockdown promotes apoptosis via AKT/Bcl‐2/Bax in colon cancer cells

CD155, one of the nectin‐like molecule family members, is involved in cell adhesion and motility. CD155 is overexpressed in several human cancers, but its role in proliferation and apoptosis of colorectal cancer remains unclear. We found that CD155 was up‐regulated in colorectal cancer tissues. CD155 knockdown via shRNA lentiviruses inhibited colon cancers cell migration and invasion, with a reduction in the expression of FAK, Src and MMP‐2. CD155 down‐regulation also suppressed colon cancer cell proliferation, accompanied by changing expressions of some molecules related to cell cycle. Finally, CD155 knockdown increased the expression ratio between Bax and Bcl‐2, resulting in a significant increase in colon cancer cell apoptosis. Taken together, these results demonstrate that CD155 is involved in not only migration and invasion but also proliferation and survival abilities of colon cancer cells, suggesting that CD155 is one of key molecules promoting the growth and metastasis of colorectal cancer.     

AFM‐ and NSOM‐based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4⁺ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4⁺ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4⁺ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4⁺ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.     

Epiregulin promotes osteogenic differentiation and inhibits neurogenic trans‐differentiation of adipose‐derived mesenchymal stem cells via MAPKs pathway

Mesenchymal stem cells (MSCs) exists low efficiency to trans‐differentiate into other germinal layer cell types. One key issue is to discover the effect of important factor on MSCs differentiation abiltiy. In this study, we investigated the role and mechanism of epiregulin (EREG) on the osteogenic differentiation and neurogenic trans‐differentiation in adipose‐derived stem cells (ADSCs). We discovered that the depletion of EREG inhibited the osteogenic differentiation in vitro. And 25 ng/mL recombinant human epiregulin protein (rhEREG) effectively improved the osteogenic differentiation of EREG‐depleted‐ADSCs. Depletion of EREG promoted the formation of neural spheres, and increased the expressions of nestin, βIII‐tubulin, NeuroD, NCAM, TH, and NEF in ADSCs. Then, 25 ng/mL rhEREG significantly inhibited these neurogenic differentiation indicators. Inhibition of p38 MAPK, JNK, or Erk1/2 signaling pathway separately, blocked the rhEREG‐enhanced osteogenic differentiation ability and the rhEREG‐inhibited neurogenic trans‐differentiation ability of ADSCs. In conclusions, EREG promoted the osteogenic differentiation and inhibited the neurogenic trans‐differentiation potentials of ADSCs via MAPK signaling pathways.     

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX‐1 and markers of pluripotency

UCB (human umbilical cord blood) contains cells able to differentiate into non‐haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic‐like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM‐CSF (granulocyte–macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT‐PCR and immunocytochemistry. The results show a dramatic expansion (>150‐fold) of haematopoietic progenitors (CD45⁺/CD133⁺) which also expressed embryo markers of pluripotency (nanog, kfl‐4, sox‐2, oct‐3/4 andc‐myc), nestin, and pancreatic markers such as pax‐4, ngn‐3, pdx‐1 and syt‐1 (that is regulated by pdx‐1 and provides the cells with a Ca⁺⁺ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.     

Changes in cell adhesivity and cytoskeleton‐related proteins during imatinib‐induced apoptosis of leukemic JURL‐MK1 cells

The fusion protein Bcr–Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr–Abl protein used in front‐line CML therapy, on the adhesivity of JURL‐MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL‐MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr–Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL‐MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr–Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F‐actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors. J. Cell. Biochem. 111: 1413–1425, 2010. © 2010 Wiley‐Liss, Inc.     

SR‐BI,CD36, and caveolin‐1 contribute positively to cholesterol efflux in hepatic cells

In non‐hepatic cells, scavenger receptor class B type I (SR‐BI), cluster of differentiation 36 (CD36), and caveolin‐1 were described as mediators of cholesterol efflux, the first step of reverse cholesterol transport (RCT). Stable transformants of HepG2 cells overexpressing SR‐BI, CD36, or caveolin‐1 were generated, as well as cells overexpressing both caveolin‐1 and SR‐BI or caveolin‐1 and CD36 in order to address the effect of caveolin‐1 on both receptor activities. These cells were analyzed for their ability to efflux cholesterol to HDL₃. Our results show that overexpressing SR‐BI, CD36, or caveolin‐1 increases cholesterol efflux by 106, 92, and 48%, respectively. Moreover, the dual overexpressions of caveolin‐1 and SR‐BI or caveolin‐1 and CD36 lead to a more prominent increase in cholesterol efflux. Studies were also conducted with primary cultures of SR‐BI knockout (KO), CD36 KO, and SR‐BI/CD36 double‐KO (dKO) mice. SR‐BI KO and SR‐BI/CD36 dKO hepatic cells show 41 and 56% less cholesterol efflux, respectively, than normal hepatic cells. No significant difference was observed between the efflux of normal and CD36 KO cells. The difference between the role of human and murine CD36 correlated with the absence of CD36 dimers in mouse caveolae/rafts. Overall, our results show that SR‐BI is clearly involved in cholesterol efflux in mouse and human hepatic cells, while CD36 plays a significant role in human cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Unusual sterolic mixture,and 24‐isopropylcholesterol,from the sponge Ciocalypta sp. reduce cholesterol uptake and basolateral secretion in Caco‐2 cells

An unusual sterolic mixture (82.3% of 24‐isopropylated sterols) and its major component, 24‐isopropylcholesterol, isolated from a marine sponge, Ciocalypta sp. (Halichondriidae), reduce cholesterol uptake, basolateral secretion and ACAT‐2 mRNA expression and increase the expression of ABCA1 mRNA in Caco‐2 cells. The decreases of cholesterol uptake and secretion induced by 24‐isopropylcholesterol alone were more than that of both the sterolic mixture and β‐sitosterol. These data add a new sterol, 24‐isopropylcholesterol, to sterols that may reduce intestinal cholesterol absorption. J. Cell. Biochem. 106: 659–665, 2009. © 2009 Wiley‐Liss, Inc.     

Differential effects of 24‐hydroxycholesterol and 27‐hydroxycholesterol on tyrosine hydroxylase and α‐synuclein in human neuroblastoma SH‐SY5Y cells

Evidence suggests that environmental and dietary factors may contribute to the pathogenesis of Parkinson’s disease (PD). High dietary intake of cholesterol is such a factor that has been shown to increase or decrease the risk of PD. However, because circulating cholesterol does not cross the blood–brain barrier, the mechanisms linking dietary cholesterol to the pathogenesis of PD remain to be understood. In contrast to cholesterol, the oxidized cholesterol metabolites (oxysterols), 24S‐hydroxycholesterol (24‐OHC) and 27‐hydroxycholesterol (27‐OHC), can cross the blood–brain barrier and may place the brain at risk of degeneration. In this study, we incubated the human neuroblastoma SH‐SY5Y cells for 24 h with 24‐OHC, 27‐OHC, or a mixture of 24‐OHC plus 27‐OHC, and have determined effects on tyrosine hydroxylase (the rate‐limiting enzyme in dopamine synthesis) levels, α‐synuclein levels, and apoptosis. We demonstrate that while 24‐OHC increases the levels of tyrosine hydroxylase, 27‐OHC increases levels of α‐synuclein, and induces apoptosis. Our findings show for the first time that oxysterols trigger changes in levels of proteins that are associated with the pathogenesis of PD. As steady state levels of 24‐OHC and 27‐OHC are tightly regulated in the brain, disturbances in these levels may contribute to the pathogenesis of PD.     

Up‐regulation of β‐amyloidogenesis in neuron‐like human cells by both 24‐ and 27‐hydroxycholesterol: protective effect of N‐acetyl‐cysteine

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27‐hydroxycholesterol (27‐OH) and 24‐hydroxycholesterol (24‐OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK‐N‐BE human neuroblastoma cells with patho‐physiologically relevant amounts of 27‐OH and 24‐OH showed that both oxysterols induce a net synthesis of Aβ_1‐42 by up‐regulating expression levels of amyloid precursor protein and β‐secretase, as well as the β‐secretase activity. Interestingly, cell pretreatment with N‐acetyl‐cysteine (NAC) fully prevented the enhancement of β‐amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β‐amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols‐induced Aβ toxic peptide accumulation in the brain.     

Role of T‐bet,the master regulator of Th1 cells,in the cytotoxicity of murine CD4+ T cells

Although CD4⁺ T cells are generally regarded as helper T cells, some activated CD4⁺ T cells have cytotoxic properties. Given that CD4⁺ cytotoxic T lymphocytes (CTLs) often secrete IFN‐γ, CTL activity among CD4⁺ T cells may be attributable to Th1 cells, where a T‐box family molecule, T‐bet serves as the “master regulator”. However, although the essential contribution of T‐bet to expression of IFN‐γ has been well‐documented, it remains unclear whether T‐bet is involved in CD4⁺ T cell‐mediated cytotoxicity. In this study, to investigate the ability of T‐bet to confer cytolytic activity on CD4⁺ T cells, the T‐bet gene (Tbx21) was introduced into non‐cytocidal CD4⁺ T cell lines and their cytolytic function analyzed. Up‐regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4⁺ T cells but not in untransfected parental cells. In one cell line, T‐bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas^? target cells. Although T‐bet was found to repress up‐regulation of CD40L (CD154), which controls FasL‐mediated cytolysis, the extent of CD40L up‐regulation on in vitro‐differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T‐bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.

     

MicroRNA‐300 promotes apoptosis and inhibits proliferation,migration, invasion and epithelial‐mesenchymal transition via the Wnt/β‐catenin signaling pathway by targeting CUL4B in pancreatic cancer cells

The study aims to verify the hypothesis that up‐regulation of microRNA‐300 (miR‐300) targeting CUL4B promotes apoptosis and suppresses proliferation, migration, invasion, and epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells by regulating the Wnt/β‐catenin signaling pathway. Pancreatic cancer tissues and adjacent tissues were collected from 110 pancreatic cancer patients. Expression of miR‐300, CUL4B, Wnt, β‐catenin, E‐cadherin, N‐cadherin, Snail, GSK‐3β, and CyclinD1 were detected using qRT‐PCR and Western blot. CFPAC‐1, Capan‐1, and PANC‐1 were classified into blank, negative control (NC), miR‐300 mimics, miR‐300 inhibitors, siRNA‐CUL4B, and miR‐300 inhibitors + siRNA‐CUL4B groups. The proliferation, migration, invasion abilities, the cell cycle distribution, and apoptosis rates were measured in CCK‐8 and Transwell assays. Pancreatic cancer tissues showed increased CUL4B expression but decreased miR‐300 expression. When miR‐300 was lowly expressed, CUL4B was upregulated which in‐turn activated the Wnt/β‐catenin pathway to protect the β‐catenin expression and thus induce EMT. When miR‐300 was highly expressed, CUL4B was downregulated which in‐turn inhibited the Wnt/β‐catenin pathway to prevent EMT. Weakened cell migration and invasion abilities and enhanced apoptosis were observed in the CUL4B group. The miR‐300 inhibitors group exhibited an evident increase in growth rate accompanied the largest tumor volume. Smaller tumor volume and slower growth rate were observed in the miR‐300 mimics and siRNA‐CUL4B group. Our study concludes that lowly expressed miR‐300 may contribute to highly expressed CUL4B activating the Wnt/β‐catenin signaling pathway and further stimulating EMT, thus promoting proliferation and migration but suppressing apoptosis of pancreatic cancer cells.