MICA-G129R: A bifunctional fusion protein increases PRLR-positive breast cancer cell death in co-culture with natural killer cells.

Breast cancer cells were reported to up-regulate human prolactin receptor (PRLR) to assist their growth through the utilization of prolactin (PRL) as the growth factor, which makes PRLR a potential therapeutic target for breast cancer. On the other hand, advanced cancer cells tend to down-regulate or shed off stress signal proteins to evade immune surveillance and elimination. In this report, we created a fusion protein consisting of the extracellular domain of MHC class I chain-related protein (MICA), a stress signal protein and ligand of the activating receptor NKG2D of natural killer (NK) cells, and G129R, an antagonistic variant of PRL. We hypothesize that the MICA portion of the fusion protein binds to NKG2D to activate NK cells and the G129R portion binds to PRLR on breast cancer cells, so that the activated NK cells will kill the PRLR-positive breast cancer cells. We demonstrated that the MICA-G129R fusion protein not only binds to human natural killer NK-92 cells and PRLR-positive human breast cancer T-47D cells, but also promotes NK cells to release granzyme B and IFN-γ and enhances the cytotoxicity of NK cells specifically on PRLR-positive cells. The fusion protein, therefore, represents a new approach for the development of breast cancer specific immunotherapy.

Rapid isolation of lentivirus particles from cell culture media via a hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber stationary phase.

Lentiviruses are increasingly used as gene delivery vehicles for vaccines and immunotherapies. However, the purification of clinical-grade lentivirus vectors for therapeutic use is still troublesome and limits preclinical and clinical experiments. Current purification methods such as ultracentrifugation and ultrafiltration are time consuming and do not remove all of the impurities such as cellular debris, membrane fragments, and denatured proteins from the lentiviruses. The same challenges exist in terms of their analytical characterization. Presented here is the novel demonstration of the chromatographic isolation of virus particles from culture media based on the hydrophobicity characteristics of the vesicles. A method was developed to isolate lentivirus from media using a hydrophobic interaction chromatography (HIC) method performed on a polyester, capillary-channeled polymer (PET C-CP) stationary phase and a standard liquid chromatography apparatus. The method is an extension of the approach developed in this laboratory for the isolation of extracellular vesicles (EVs). Quantitative polymerase chain reaction (qPCR) was used to verify and quantify lentiviruses in elution fractions. Load and elution mobile phase compositions were optimized to affect high efficiency and throughput. The process has been visualized via scanning electron microscopy (SEM) of the fiber surfaces following media injection, the elution of proteinaceous material, and the elution of lentiviruses. This effort has yielded a rapid (<10 min), low-cost (< $15 per column, providing multiple separations), and efficient method for the isolation/purification of lentivirus particles from cell culture media at the analytical scale.

Expression-based prediction of human essential genes and candidate lncRNAs in cancer cells.

MOTIVATION: Essential genes are required for the reproductive success at either cellular or organismal level. The identification of essential genes is important for understanding the core biological processes and identifying effective therapeutic drug targets. However, experimental identification of essential genes is costly, time consuming and labor intensive. Although several machine learning models have been developed to predict essential genes, these models are not readily applicable to lncRNAs. Moreover, the currently available models cannot be used to predict essential genes in a specific cancer type. RESULTS: In this study, we have developed a new machine learning approach, XGEP (eXpression-based Gene Essentiality Prediction), to predict essential genes and candidate lncRNAs in cancer cells. The novelty of XGEP lies in the utilization of relevant features derived from the TCGA transcriptome dataset through collaborative embedding. When evaluated on the pan-cancer dataset, XGEP was able to accurately predict human essential genes and achieve significantly higher performance than previous models. Notably, several candidate lncRNAs selected by XGEP are reported to promote cell proliferation and inhibit cell apoptosis. Moreover, XGEP also demonstrated superior performance on cancer-type-specific datasets to identify essential genes. The comprehensive lists of candidate essential genes in specific cancer types may be used to guide experimental characterization and facilitate the discovery of drug targets for cancer therapy. AVAILABILITY AND IMPLEMENTATION: The source code and datasets used in this study are freely available at https://github.com/BioDataLearning/XGEP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Bioactive Sesquiterpene Lactones Isolated from the Whole Plants of Vernonia cinerea.

Twelve sesquiterpene lactones were isolated from the whole plants of Vernonia cinerea. These included eight new compounds, vercinolides A-H (1-8), along with four known substances (9-12). The structures of the new compounds were determined by 1D and 2D NMR experiments and mass spectrometric methods. The absolute configurations of compounds 1-8 were determined by Mosher experiments and ECD data. Compounds 1-8 are the first examples of a new class of sesquiterpene lactones possessing a rare 4α,10α-ether ring and a 2,14-ether ring. Compounds 1-4, 6, 8, 10, and 12 were evaluated for their cytotoxic and anti-inflammatory activities. Compounds 10 and 12 exhibited inhibitory effects against nitric oxide production in lipopolysaccharide-activated RAW 264.7 mouse macrophage cells with IC50 values of 21 and 23 µM, respectively. Both compounds were inactive for HeLa cells (IC50 > 10 µM).

Lentiviral delivery of novel fusion protein IL12/FasTI for cancer immune/gene therapy.

Many of the cytokine-based cancer immunotherapies are hindered by the devastating side effects of systemic delivery of the cytokines. To address this problem, we previously described a novel approach to locally achieve high doses of interleukin-12 (IL-12) in tumors and demonstrated that bi-functional fusion protein mIL-12/FasTI expressed by stable clones of TC-1 cells efficiently suppressed tumor proliferation by activating natural killer (NK) cells and other cytolytic killer cells and sending apoptotic signals into tumor cells. In the present study, we employed a lentiviral vector-based gene delivery system to deliver this fusion construct directly into tumor cells. We show that lentiviral vector efficiently delivers the fusion constructs into Hela cells in vitro as assayed by RT-PCR and immunohistochemistry (IHC). We also confirm that fusion protein mIL-12/FasTI delivered by the viral vector significantly enhanced killer cell activation, increased caspase-3 activity and decreased tumor growth in vitro. This study offers a further step for fusion protein cancer therapy for cancer patients.

Investigation of composition, structure and bioactivity of extracellular polymeric substances from original and stress-induced strains of Thraustochytrium striatum.

This paper was the first to study extracellular polymeric substances (EPSs) of Thraustochytrium striatum on composition, structure and bioactivities. Two strains of T. striatum including original (ori) and high-biomass (mut) strains (induced by high-nitrogen stress) were compared. The EPSs from both strains mainly contained polysaccharide (41-64%, w/w, dry basis) and protein (25-40%, w/w, dry basis), which was shown by the morphology study with an AFM. The monosaccharide profile of the EPS polysaccharide was consisted of glucose, galactose, arabinose, and trace amount of xylose. Glucose and arabinose took up to 82-90% (w/w, dry basis) of the total polysaccharide. The structure and functional groups of EPSs were determined by FTIR and NMR. The NMR results revealed that the major structural linkages of the polysaccharides of both ori and mut EPSs were 1 → 6-ß-glucan and 1 → 4-α-galactan branched with l-α-arabinose. The EPSs were found to have anti-tumor activities against mouse melanoma B16-F0, human prostate carcinoma DU145, human cervical carcinoma HeLa, and human lung carcinoma A549. The EPSs also showed antioxidant and anti-inflammatory activities and antibacterial activity against Pseudomonas aeruginosa.

MHC class I chain-related A: Polymorphism, regulation and therapeutic value in cancer.

MICA and MICB are stress-induced molecules recognized by NKG2D, one of major activation receptors of natural killer (NK) cells. Upon binding to NKG2D, NKG2D-mediated cytolytic immune response of immune effector cells will be activated against virally infected and tumor cells expressing MICA. In the early oncogenic development, membrane-bound MICA serves as a key signal to recruit anti-tumor immune effectors. Nevertheless, both MICA polymorphic features and its dysregulated expression in evolving tumors have resulted in tumor evasion in various cancer types. Therefore, in order to reconstitute tumor immunosurveilance, it is of great significance that we understand MICA genetics, polymorphisms, mechanisms of MICA-associated tumor escape and molecular/cellular modulation of MICA. In this review, the MICA-associated co-expression networks involving microRNAs (miRNAs) and novel candidate long non-coding RNAs (lncRNAs) were also discussed. Given the current importance in the study of MICA gene, this review paper focuses on the role of MICA in different cancer types, and strategies that we manipulate MICA regulation against tumor proliferation.

Fusion Proteins of NKG2D/NKG2DL in Cancer Immunotherapy.

NKG2D (natural killer group 2, member D) is an important activating receptor in natural killer (NK) cells and some T cells. NKG2D ligands (NKG2DLs) are specifically expressed on most tumor cells. The engagement of these ligands on tumor cells to NKG2D on NK cells will induce cell-mediated cytotoxicity and have target cells destroyed. This gives NKG2D/NKG2DLs great potential in cancer therapeutic application. The creation of NKG2D/NKG2DL-based multi-functional fusion proteins is becoming one of the most promising strategies in immunotherapy for cancer. Antibodies, cytokines, and death receptors have been fused with NKG2D or its ligands to produce many powerful fusion proteins, including NKG2D-based chimeric antigen receptors (CARs). In this article, we review the recent developments of the fusion proteins with NKG2D/NKG2DL ligands in cancer immunotherapy.

MICA/IL-12: A novel bifunctional protein for killer cell activation.

Natural killer (NK) cells have the potential to be effective killers of tumor cells. They are governed by inhibitory and activating receptors such as NKG2D, whose ligands are normally upregulated in cells that are stressed, like cancer cells. Advanced cancer cells, however, have ways to reduce the expression of these ligands, leaving them less detectable by NK cells. Along with these receptors, NK cells also require activating cytokines, such as IL-12. A previous study in our laboratory showed that a fusion protein of the extracellular domain of mouse UL-16 binding protein-like transcript 1 (MULT1E) and mouse interleukin 12 (IL-12) can effectively activate mouse NK cells by in vitro assays and in vivo in animal tumor models. The aim of the present study was to expand the concept of developing a novel bifunctional fusion protein for enhanced NK cell activation to human killer cells. The proposed protein combines the extracellular domain of a human NKG2D ligand, MHC class I polypeptide-related sequence A (MICA) and IL-12. It is hypothesized that when expressed by tumor cells, the protein will activate human NK and other killer cells using the NKG2D receptor, and deliver IL-12 to the NK cells where it can interact with the IL-12R and enhance cytotoxicity. The fusion protein, when expressed by engineered tumor cells, indeed activated NK92 cells as measured by an increase in interferon-γ (IFN-γ) production and an increase in cytotoxicity of tumor cells. The fusion protein was also able to increase the proliferation of human peripheral blood mononuclear cells (PBMCs) and augment their production of IFN-γ. This study along with the data from the previous mouse studies suggest that the MICA/IL-12 bifunctional fusion protein represents an effective activator of killer cells for cancer treatment.

Bovine serum albumin coated nanoparticles for in vitro activated fluorescence.

A fluorophore modified nanoparticle was developed that can only fluoresce when a specific environmental parameter interacts with the system. The model system consisted of an azide modified bovine serum albumin (azBSA) that had been covalently attached to an alkyne modified silicon phthalocyanine (alSiPc) derivative through a copper catalyzed azide/alkyne Huisgen cycloaddition (click reaction). The azBSA/alSiPc assembly was then clicked to a ca. 67 nm poly(propargyl acrylate) (PA) nanoparticle (PA/azBSA/alSiPc). The resulting particles did not exhibit any florescence when the alSiPc was excited. Incubating the particles at 37 °C for 30 min with a proteolytic enzyme (trypsin) degraded the linking BSA and resulted in the appearance of florescence that was attributed to a "free" silicon phthalocyanine. The PA/azBSA/alSiPc particles were incubated with human non-small cell lung cancer cells (A549) and the florescence of the initially quenched particles was achieved with cellular uptake.

Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

A new fatty acid ester disaccharide, 2-O-(ß-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-ß-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

Mouse interleukin-12/FasTI: A novel bi-functional fusion protein for cancer immuno/gene therapy.

Whereas cancer immunotherapy with cytokines in recent research was demonstrated effective in activating immune response against tumor cells, one major obstacle with the use of these cytokines is their severe side effects when delivered systemically at high doses. Another challenge is that advanced tumor cells often evade immunosurveillance of the immune system as well as of the Fas-mediated apoptosis by various mechanisms. We report the design and preliminary evaluation of the antitumor activity of a novel fusion protein-mIL-12/FasTI, consisting of mouse interleukin-12 and the transmembrane and intracellular domains of mouse Fas. The fusion construct (pmIL-12/FasTI) was transfected into mouse lung carcinoma cell line TC-1. Stable cell clones expressing the fusion protein were established as assayed by RT-PCR and immunohistochemistry. ELISA and cell proliferation analyses demonstrated that NK cells were effectively activated by the fusion protein with increased IFN-γ production and cytotoxicity. Enhanced caspase-3 activity of the clones when co-cultured with NK cells indicated that apoptosis was induced through Fas/FasL signaling pathway. The preliminary results suggest a synergized anticancer activity of the fusion protein. It may represent a promising therapeutic agent for cancer treatment.

Sequestering survivin to functionalized nanoparticles: a strategy to enhance apoptosis in cancer cells.

Survivin belongs to the family of inhibitor of apoptosis proteins (IAP) and is present in most cancers while being below detection limits in most terminally differentiated adult tissues, making it an attractive protein to target for diagnostic and, potentially, therapeutic roles. Sub-100 nm poly(propargyl acrylate) (PA) particles were surface modified through the copper-catalyzed azide/alkyne cycloaddition of an azide-terminated survivin ligand derivative (azTM) originally proposed by Abbott Laboratories and speculated to bind directly to survivin (protein) at its dimer interface. Using affinity pull-down studies, it was determined that the PA/azTM nanoparticles selectively bind survivin and the particles can enhance apoptotic cell death in glioblastoma cell lines and other survivin over-expressing cell lines such as A549 and MCF7 relative to cells incubated with the original Abbott-derived small molecule inhibitor.

Cerebrospinal fluid extracellular vesicles undergo age dependent declines and contain known and novel non-coding RNAs.

Brain development requires precise orchestration of cellular events through the coordinate exchange of information between distally located cells. One mechanism by which intercellular communication is achieved is through the transfer of extracellular vesicles (EVs). Exosomes are EVs that carry lipids, nucleic acids, and proteins and are detectable in most biological fluids including cerebrospinal fluid (CSF). Here we report that CSF EV concentrations undergo age dependent fluctuations. We characterized EV RNA content by next generation small RNA sequencing and miRNA microarray analysis and identified a temporal shift in CSF EV content. CSF EVs encapsulated miRNAs that contain a conserved hnRNPA2/B1 recognition sequence. We found that hnRNPA2/B1-containing EVs were produced by choroid plexus epithelial cells and that hnRNPA2/B1 containing EVs decreased with age. These results provide insight into EV exchange of miRNAs within the central nervous system and a framework to understand how changes in EVs may have an important impact on brain development.

A biotin-streptavidin-biotin bridge dramatically enhances cell fusion.

Although the generation of hybrid cells by cell fusion plays a significant role in biotechnology and biomedicine, the low cell-fusion rates and the limitation of large-scale cell fusion for clinical applications of the two widely used approaches, polyethylene-glycol (PEG)-mediated cell fusion and electrofusion, hinder the application of this critical technology in certain key areas, including cancer immunotherapy. In the present study, a simple procedure that can not only significantly increase the heterologous cell fusion but is also capable of producing fused cells on a large scale is reported. A biotin-streptavidin-biotin (BSB) bridge was created by coating one to-be-fused cell with biotin and the other with biotin-streptavidin. The BSB bridge enhances cell-fusion rates induced with PEG fusion or electrofusion by 10-30% depending on the cell types when compared with cell fusions without the bridge. The procedure described increases heterologous cell pairing and eliminates the alignment step required for the majority of electrofusions. Notably, it can be used to make large-scale cell fusions for clinical applications.

Antitumor activity of fermented noni exudates and its fractions.

Noni has been extensively used in folk medicine by Polynesians for over 2000 year. Recent studies have shown that noni has a wide spectrum of therapeutic activities including inhibition of angiogenesis, anti-inflammatory effects and anti-cancer activities. Intraperitoneal (i.p.) injection of fermented noni exudates (fNE) were previously found to induce significant tumor rejection in a S180 mouse sarcoma tumor model, while natural killer (NK) cells were demonstrated to be markedly involved in fNE-induced antitumor activity. In this study, fNE was partitioned into three fractions and their antitumor effects were examined using i.p. injection or as water supplement. The in vivo animal study results showed that when delivered by i.p. injection, n-butanol fraction of fNE (BuOH) effectively rejected (100%) tumor challenge and eradicated existing tumors (75%). When delivered as a water supplement, 62.5% of the mice receiving the n-butanol or ethyl acetate fractions resisted tumor cells. The tumor-resistant mice effectively rejected more and higher doses of tumor challenge, indicating that the immune system was activated. The findings confirm those of an earlier study showing fNE to have anti-tumor activity and demonstrating that the n-butanol fraction of fNE contains active antitumor components, to be further identified. More importantly, the antitumor effect of fNE and its fractions as water supplements renders a significant potential for identifying novel and powerful new dietary products for cancer prevention.

An IL-12/Shh-C domain fusion protein-based IL-12 autocrine loop for sustained natural killer cell activation.

The dependency of activated natural killer (NK) cells on the continuous support of exogenous interleukin (IL)-2 for their in vivo survival, tumor localization and consequently, their antitumor effect, is a major obstacle for NK cell-mediated tumor therapy. In the present study, a fusion gene between IL-12 and mouse sonic hedgehog C-terminal domain (Shh-C) was constructed. The fusion protein was autocatalytically processed to form cholesterol-modified IL-12 molecules and an autocrine loop of IL-12 was established for the sustained activation of NK cells. The transduced NK cells matured more rapidly in vitro with the enhanced expression of granule-related proteins. NKIL-12/Shh-C cells reached the same proliferation rate as NK cells transduced with enhanced green fluorescent protein (EGFP)/Shh-C (NKEGFP/Shh-C) with <10-fold IL-2 supplementation, suggesting that the fusion protein reduced the dependency of NK cells on IL-2. The amount of interferon­Î³ (IFN-γ) in the supernatants of NKIL-12/Shh-C cells 5 and 7 days after transduction was significantly higher than that in the supernatants of NKIL-12 cells. Immunofluorescent staining of lung tissues from B16-bearing mice which had received an intravenous injection of lentivirus-transduced NK cells without exogenous IL-2 confirmed that donor NK cells successfully infiltrated into the lung tissues. The survival time of the mice which had received NKIL-12/Shh-C cells was significantly prolonged compared to the mice which had received NKEGFP/Shh-C cells.

Antitumor and anti-angiogenic activities of Scutellaria barbata extracts in vitro are partially mediated by inhibition of Akt/protein kinase B.

The Akt pathway is considered a pivotal player in regulating cell survival, growth, migration and angiogenesis. Disruption of normal Akt/PKB/PTEN signaling frequently occurs in numerous types of human cancers. Therefore, this signaling pathway is regarded as an important target for effective cancer therapeutic strategies. In the present study, methanol extracts from Scutellaria barbata (S. barbata) were determined to be Akt/protein kinase B inhibitory, after screening a panel of 40 traditional Chinese herbs with the Fast Activated Cell-based ELISA (FACE) assay. S. barbata extracts were found to suppress the phosphorylation levels of Akt. This inhibition was Akt kinase-specific as it had no effect on PI3K, the upstream kinase of Akt, whereas the levels of phosphorylated Bad and FHKR, the two downstream targets of Akt, changed as the levels of Akt changed. S. barbata extracts also exhibited cytotoxicity against LoVo and human umbilical vein endothelial cells (HUVECs). Furthermore, this extract inhibited the process of in vitro angiogenesis of HUVECs on Matrigel. S. barbata may be a suitable alternative source with which to isolate small molecules for use as Akt kinase inhibitors.

Apoptosis of lung carcinoma cells induced by a flexible optical fiber-based cold microplasma.

Atmospheric pressure plasmas have been used as a therapy for cancer. However, the fairly large size and rigidity of present plasma-delivery systems obstructs the precise treatment of tumors in harder-to-reach internal organs such as the lungs, pancreas, and duodenum. In order to improve the targeted delivery of plasmas a highly flexible microplasma jet device is fabricated using a hollow-core optical fiber with an inner diameter of either 15 µm, 55 µm, or 200 µm. Described herein, based on this device, are results on lung carcinoma therapy using a microplasma cancer endoscope. Despite the small inner diameter and the low gas flow rate, the generated plasma jets are shown to be sufficiently effective to induce apoptosis, but not necrosis, in both cultured mouse lung carcinoma and fibroblast cells. Further, the lung carcinoma cells were found to be more sensitive to plasma treatment than the fibroblast cells based on the overall plasma dose conditions. This work enables directed cancer therapies using on highly flexible and precise hollow optical fiber-based plasma device and offers enhancements to microplasma cancer endoscopy using an improved method of plasma targeting and delivery.

Single-cell-level microplasma cancer therapy.

A flexible microplasma endoscope based on a 15 µm hollow-core glass optical fiber is fabricated, and tumor cell apoptotic analysis supports its potential use in targeted cancer therapies. The optical-fiber microplasma jet reveals antitumor activity at a certain plasma dose in animal studies.