A Cancer Specific Cell-Penetrating Peptide,BR2, for the Efficient Delivery of an scFv into Cancer Cells

Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49–57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.     

Nucleolar localization signals of LIM kinase 2 function as a cell-penetrating peptide

LIM Kinase 2 (LIMK2) is a LIM domain-containing protein kinase which regulates actin polymerization thorough phosphorylation of the actin depolymerizing factor cofilin. It is also known to function as a shuttle between the cytoplasm and nucleus in endothelial cells. A basic amino acid-rich motif in LIMK2 was previously identified to be responsible for this shuttling function, as a nucleolar localization signal (NoLS). Here it is shown that this nucleolar localization signal sequence also has the characteristic function of a cell-penetrating peptide (CPP). We synthesized LIMK2 NoLS-conjugated peptides and a protein and analyzed their cell-penetrating abilities in various types of cells. The BC-box motif of the Von Hippel-Lindau (VHL) protein was used for the peptide. This motif previously has been reported to be involved in the neural differentiation of bone marrow stromal cells and skin-derived precursor cells. Green fluorescence protein (GFP) was used as a large biologically active biomolecule for the protein. The LIMK2 NoLS-conjugated peptides and protein translocated across the cell membranes of fibroblast cells, neural stem cells, and even iPS cells. These results suggest that LIMK2 NoLS acts as a cell-penetrating peptide and its cell-penetrating ability is not restricted by cell type. Moreover, from an in vivo assay using a mouse brain, it was confirmed that NoLS has potential for transporting biomolecules across the blood-brain barrier.     

经NGR肽段遗传修饰的携带三种报告基因的腺病毒载体的构建及其实验研究

NGR(Asn-Gly-Arg)是通过噬菌体展示技术筛选出来的能够和肿瘤新生血管特异结合的三肽模体,可以将多种药物分子和病毒载体靶向运输到肿瘤或者进行血管再生的组织中。为此构建了腺病毒衣壳蛋白knob的HI环(HI-loop)经NGR肽段修饰的并同时表达三种报告基因的腺病毒载体Ad5/E1-mCherry/E3-luciferase-2A-eGFP/knob-NGR。体内、外实验研究表明,该病毒载体可成功表达三种报告基因;经NGR肽段修饰的腺病毒载体对人乳腺癌细胞系MDA-MB-231的感染效率高于未经修饰的对照腺病毒Ad5CMVeGFP。该载体的成功构建为进一步研究经NGR肽段修饰的腺病毒在肿瘤动物模型体内的靶向性及经NGR肽段修饰的并携带治疗基因的实验治疗研究奠定了基础。     

Cell biology meets biophysics to unveil the different mechanisms of penetratin internalization in cells

Although cell-penetrating peptides are widely used as molecular devices to cross membranes and transport molecules or nanoparticles inside cells, the underlying internalization mechanism for such behavior is still studied and discussed. One of the reasons for such a debate is the wide panel of chemically different cell-penetrating peptides or cargo that is used. Indeed the intrinsic physico-chemical properties of CPP and conjugates strongly affect the cell membrane recognition and therefore the internalization pathways. Altogether, the mechanisms described so far should be shared between two general pathways: endocytosis and direct translocation. As it is established now that one cell-penetrating peptide can internalize at the same time by these two different pathways, the balance between the two pathways relies on the binding of the cell-penetrating peptide or conjugate to specific cell membrane components (carbohydrates, lipids). Like endocytosis which includes clathrin- and caveolae-dependent processes and macropinocytosis, different translocation mechanisms could co-exist, an idea that emerges from recent studies. In this review, we will focus solely on penetratin membrane interactions and internalization mechanisms.     

Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.     

Design and construction of targeted AAVP vectors for mammalian cell transduction

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.     

Targeted induction of lung endothelial cell apoptosis causes emphysema-like changes in the mouse

Pulmonary gas exchange relies on a rich capillary network, which, together with alveolar epithelial type I and II cells, form alveolar septa, the functional units in the lung. Alveolar capillary endothelial cells are critical in maintaining alveolar structure, because disruption of endothelial cell integrity underlies several lung diseases. Here we show that targeted ablation of lung capillary endothelial cells recapitulates the cellular events involved in cigarette smoke-induced emphysema, one of the most prevalent nonneoplastic lung diseases. Based on phage library screening on an immortalized lung endothelial cell line, we identified a lung endothelial cell-binding peptide, which preferentially homes to lung blood vessels. This peptide fused to a proapoptotic motif specifically induced programmed cell death of lung endothelial cells in vitro as well as targeted apoptosis of the lung microcirculation in vivo. As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide. Given that these are all critical elements of the corresponding human emphysema caused by cigarette smoke, these data provide evidence for a central role for the alveolar endothelial cells in the maintenance of lung structure and of endothelial cell apoptosis in the pathogenesis of emphysema-like changes. Thus, our data enable the generation of a convenient mouse model of human emphysema. Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4(13)-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4(13)-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4(13)-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4(13)-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4(13)-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Synthesis and hypoxic-cytotoxic activity of some 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives

A series of 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives 1 have been synthesized and evaluated for their cytotoxic activity in vitro against human leukemia cell lines: Molt-4, K562, HL60, human liver cancer cell Hep-G2, human prostate cancer cell PC-3 in hypoxia. Most of the compounds showed more potent activity than TPZ. Compounds 1i and 1m displayed encouraging superior activity against Molt-4 and HL-60 cell lines. Three potential derivatives received the test of the activity in hypoxia and in normoxia against Molt-4 and HL-60 cell lines and showed obvious hypoxia selectivity. Further mechanism study revealed that the cytotoxic activities of compounds 1i and 1k in Molt-4 cells might be mediated by modulation of p53 protein expression and mitochondrial membrane potential (DeltaPsi(m)).     

A naturally processed HLA-DR-bound peptide from the IL-9 receptor alpha of HTLV-1-transformed T cells serves as a T helper epitope

Human T cell leukemia virus type 1 (HTLV-1) induced adult T cell leukemia/lymphoma (ATLL) is usually a fatal lymphoproliferative malignant disease. Thus, the enhancement of T cell immunity to ATLL through the development of therapeutic vaccines using characterized T cell peptide epitopes could be of value. We isolated and characterized HLA-DR-bound peptides from HTLV-1-transformed T cells by fractionating on reverse-phase high performance liquid chromatography and Edman NH₂-terminal sequencing and were able to identify five independent peptide sequences. One of the identified peptide sequences corresponded to a fragment of the human interleukin-9 receptor alpha (IL-9R??), which is commonly expressed by HTLV-1-infected T cell lymphoma cells. Using a synthetic peptide corresponding to the identified IL-9R?? sequence, we generated antigen-specific CD4 helper T lymphocytes in vitro, which were restricted by HLA-DR15 or HLA-DR53 molecules and could recognize and kill HTLV-1+, IL-9R??+?T cell lymphoma cells. These results indicate that IL-9R?? functions as T cell leukemia/lymphoma-associated antigen for CD4 T cells and that synthetic peptides such as the one described here could be used for T cell-based immunotherapy against IL-9R?? positive ATLL.     

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4₁₃-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4₁₃-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4₁₃-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4₁₃-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4₁₃-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4₁₃-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Design and synthesis of various quinizarin derivatives as potential anticancer agents in acute T lymphoblastic leukemia

A series of quinizarin derivatives containing quaternary ammonium salts and/or thiourea groups were synthesized and their anticancer activities against leukemia cell lines have been tested. Results showed that most of quinizarin derivatives could inhibit the proliferation of leukemia cells. Among these derivatives, compound 3 showed good inhibition activity against various leukemia cells with IC₅₀ values ranging from 0.90?±?2.55?μM to 10.90?±?3.66?μM. At the same time, compound 3 also inhibited the growth of human embryonic kidney-293 cell (HEK-293). Molt-4 and Jurkat cells, acute T lymphoblastic leukemia (T-ALL) cell lines, were selected to reveal potential anticancer mechanism of compound 3. Compound 3 inhibited the proliferation of Molt-4 and Jurkat cells in a dose- and time-dependent manner and led to a marked G0/G1 phase arrest. Analysis of Annexin V-APC and intracellular reactive oxygen species (ROS) level by flow cytometry showed that compound 3 induced significant apoptosis in Molt-4 and Jurkat cells. Western blotting assay showed that compound 3 activated the caspase-dependent apoptosis pathway and induced the degradation of Bcl-2 and c-myc protein.     

Phage matrix for isolation of glioma cell membrane proteins

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.     

The use of one-bead one-compound combinatorial library method to identify peptide ligands for α4β1 integrin receptor in non-Hodgkin's lymphoma

Summary Using a ‘one-bead one-compound’ combinatorial library method and whole cell binding assay, we have identified peptide ligands that bind preferentially to human lymphoid malignant cells but not to normal human peripheral lymphocytes. These peptides have a common motif of Leu-Asp-Ile, Leu-Asp-Phe, or Leu-Asp-Val. Anti-α4 integrin antibody blocks binding of cells to these peptides which indicates that the target receptor for these peptides is α4β1 integrin. These findings may have important therapeutic and diagnostic implications in the treatment of lymphoma, leukemia, and various immunologic disorders.     

A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells.

AP9-cd, a standardized lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by flow cytometry using specific fluorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant effect of AP9-cd. Further, caspase-3 activation correlated with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of pro-oxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-cd.     

Phospholipase C treatment of certain human target cells reduces their susceptibility to NK lysis without affecting binding or sensitivity to lytic granules.

Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme that has the capacity to release glycosyl-phosphatidyl inositol (G-PI)-anchored proteins from the cells surface. Pretreatment of the human T-cell leukemia cell line Molt-4 with PI-PLC resulted in a decrease in the susceptibility to lysis by natural killer (NK) cells. Treatment of the erythroleukemia cell line K562 with PI-PLC had no effect on its NK susceptibility. PI-PLC-treated and untreated Molt-4 bound equally well to lymphocytes in target-binding studies with effector cell preparations enriched for NK cells. Susceptibility to cytolytic granules isolated from rat LGL tumor cells remained the same after treatment of Molt-4 or K562 with PI-PLC. Combined treatment of Molt-4 with PI-PLC and rlFN-alpha or rlFN-gamma resulted in additive reductions of the NK susceptibility, suggesting that PI-PLC and interferons act on different mechanisms to protect cells from NK lysis. When expression of a number of antigens on Molt-4 and K562 was analyzed in flow cytometry, only the expression of CD58 was reduced after PI-PLC treatment. The susceptibility of Con A blasts to MLR derived cytotoxic T-cells was not altered by treatment with phospholipase. These data suggest that PI-PLC treatment reduces the capacity of some target cells to activate NK cells upon contract. The mechanism behind this phenomenon is presently unclear.     

Peptide-mediated transcytosis of phage display vectors in MDCK cells

Delivery of therapeutic macromolecules and gene vectors to certain tissues is hampered by endothelial or epithelial barriers. We show here that the transport of phage particles across epithelial cells can be facilitated by peptide ligands selected from a phage library of random peptides. Using MDCK cells, we identified a polycationic peptide sequence, RYRGDLGRR, containing a putative integrin-binding (RGD) motif that enhanced basal-to-apical transcytosis of peptide-bearing phage 1000- to 10,000-fold compared with phage with no peptide insert. Both the synthetic peptide RYRGDLGRR and the integrin-binding peptide GRGDSP inhibited phage transcytosis suggesting the involvement of integrins. Confocal immunofluorescence microscopy showed that following internalization at the basal cell surface, phage particles were delivered to the apical cytoplasm and released at the apical cell surface. These data suggest the feasibility of using short peptides for targeting transcytotic pathways and facilitating delivery of macromolecules across cellular barriers.     

流式细胞术BrdU／DNA双染法测定云芝糖肽诱导的Molt-4细胞周期阻滞

目的：探究云芝糖）Ik（PSP）对人急性淋巴母细胞白血病Molt-4细胞周期的影响。方法：采用流式细胞术BrdU／DNA双染法获得各时相细胞分布状况和细胞周期的动力学参数。结果：0．1mg／mlPSP处理12h后,G2／M期细胞百分比由对照组的11．09％减少至3．69％。DNA合成时间由12．10h延长至108．40h。24h处理组中,S期细胞百分比由对照组的43.29％增加至67．26％,而G0／G1期和G2／M期细胞百分比均减少,G0／G1期细胞百分比由对照组的37．47％减少至27．43％,G2／M期细胞百分比由对照组的19．24％降低至5.31％。DNA合成时间更是由11．95h延长至114．52h。结论：PSP对人急性淋巴母细胞白血病Molt-4细胞周期的阻滞作用在于S期．该作用与DNA合成抑制有关。