Additive Polyplexes to Undertake siRNA Therapy against CDC20 and Survivin in Breast Cancer Cells.

Small interfering RNA (siRNA) delivered to silence overexpressed genes associated with malignancies is a promising targeted therapy to decrease the uncontrolled growth of malignant cells. To create potent delivery agents for siRNA, here we formulated additive polyplexes of siRNA using linoleic acid-substituted polyethylenimine and additive polymers (hyaluronic acid, poly(acrylic acid), dextran sulfate, and methyl cellulose) and characterized their physicochemical properties and effectiveness. Incorporating polyanionic polymer along with anionic siRNA in polyplexes was found to decrease the ζ-potential of polyplexes but enhance the cellular delivery of siRNA. The CDC20 and survivin siRNAs delivered by additive polyplexes showed promising efficacy in breast cancer MDA-MB-231, SUM149PT, MDA-MB-436, and MCF7 cells. However, the side effects of the siRNA delivery were observed in nonmalignant cells, and a careful formulation of siRNA/polymer polyplexes was needed to minimize side effects on normal cells. Because the efficacy of siRNA delivery by additive polyplexes was independent of breast cancer phenotypes used in this study, these polyplexes could be further developed to treat a wide range of breast cancers.

Evolution of the duplicated intracellular lipid-binding protein genes of teleost fishes.

Increasing organismal complexity during the evolution of life has been attributed to the duplication of genes and entire genomes. More recently, theoretical models have been proposed that postulate the fate of duplicated genes, among them the duplication-degeneration-complementation (DDC) model. In the DDC model, the common fate of a duplicated gene is lost from the genome owing to nonfunctionalization. Duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicate genes acquires a new function. Both processes occur either by loss or gain of regulatory elements in the promoters of duplicated genes. Here, we review the genomic organization, evolution, and transcriptional regulation of the multigene family of intracellular lipid-binding protein (iLBP) genes from teleost fishes. Teleost fishes possess many copies of iLBP genes owing to a whole genome duplication (WGD) early in the teleost fish radiation. Moreover, the retention of duplicated iLBP genes is substantially higher than the retention of all other genes duplicated in the teleost genome. The fatty acid-binding protein genes, a subfamily of the iLBP multigene family in zebrafish, are differentially regulated by peroxisome proliferator-activated receptor (PPAR) isoforms, which may account for the retention of iLBP genes in the zebrafish genome by the process of subfunctionalization of cis-acting regulatory elements in iLBP gene promoters.

Comparative evolutionary genomics of medaka and three-spined stickleback fabp2a and fabp2b genes with fabp2 of zebrafish.

Here we describe the evolutionary relationship of the duplicated intestinal fatty acid binding protein genes fabp2a and fabp2b from medaka and three-spined stickleback by comparing them to the well-studied fabp2 gene from zebrafish. The duplicated fabp2 genes from medaka and three-spined stickleback consist of four exons separated by three introns, which code for a polypeptide of 132 amino acids. Fabp2a and Fabp2b of medaka and three-spined stickleback share highest sequence identity with zebrafish Fabp2. All Fabp2/FABP2 sequences from vertebrates form a distinct clade in a neighbor-joining phylogenetic tree with a robust 100% bootstrap value, which indicates that the medaka and three-spined stickleback fabp2a and fabp2b are orthologs of zebrafish fabp2. The syntenic genes of fabp2a and fabp2b from medaka and three-spined stickleback were shown to be conserved with the syntenic genes of fabp2/FABP2 from zebrafish and human, evidence that the duplicated fabp2 genes from medaka and three-spined stickleback most likely arose from the teleost-specific whole-genome duplication. The tissue-specific distribution of medaka and three-spined stickleback fabp2a and fabp2b transcripts, and zebrafish fabp2 transcripts, assayed by RT-qPCR suggests the acquisition of new function(s) by the medaka fabp2a, and the distinct evolution of fabp2b compared with fabp2a in the medaka and three-spined stickleback genomes.

Comparative genomic organization and tissue-specific transcription of the duplicated fabp7 and fabp10 genes in teleost fishes.

A whole-genome duplication (WGD) early in the teleost fish lineage makes fish ideal organisms to study the fate of duplicated genes and underlying evolutionary trajectories that have led to the retention of ohnologous gene duplicates in fish genomes. Here, we compare the genomic organization and tissue-specific transcription of the ohnologous fabp7 and fabp10 genes in medaka, three-spined stickleback, and spotted green pufferfish to the well-studied duplicated fabp7 and fabp10 genes of zebrafish. Teleost fabp7 and fabp10 genes contain four exons interrupted by three introns. Polypeptide sequences of Fabp7 and Fabp10 show the highest sequence identity and similarity with their orthologs from vertebrates. Orthology was evident as the ohnologous Fabp7 and Fabp10 polypeptides of teleost fishes each formed distinct clades and clustered together with their orthologs from other vertebrates in a phylogenetic tree. Furthermore, ohnologous teleost fabp7 and fabp10 genes exhibit conserved gene synteny with human FABP7 and chicken FABP10, respectively, which provides compelling evidence that the duplicated fabp7 and fabp10 genes of teleost fishes most likely arose from the well-documented WGD. The tissue-specific distribution of fabp7a, fabp7b, fabp10a, and fabp10b transcripts provides evidence of diverged spatial transcriptional regulation between ohnologous gene duplicates of fabp7 and fabp10 in teleost fishes.

Self-Assembling Peptide Nanoscaffold That Activates Human Mast Cells.

Engineering biomaterials to manipulate the immune response to elicit specific therapeutic outcomes is a burgeoning field of research. Mast cells play a distinct and central role in the innate immune response, and are characterized by their rapid release of a myriad of proinflammatory mediators in response to stimulation. These mediators are central to protective actions such as wound healing, angiogenesis, and host defense against pathogens and animal venoms. Considering that mast cells are widely distributed in tissues that interface with the external environment, and are loaded with large amounts of preformed protective compounds, they are ideal targets for novel immunotherapies. Here we report that, by using an engineered nanoscaffold, human mast cells can be contact activated in cell and primary human skin tissue culture using a specific receptor-ligand mechanism. The IgE independent PAMP-12 peptide activates human mast cells through the recently identified Mas-related G-protein coupled receptor member X2 (MRGPRX2) receptor. The PAMP-12 motif was conjugated, via a glycine spacer, with the self-assembling peptide (RADA)4 and mixed with unmodified (RADA)4 to form a nanofiber matrix; mast cell activation was influenced directly by this ratio. Moreover, conjugating the PAMP-12 motif within the matrix was shown to only activate local, tissue-resident mast cells. The result of ex vivo human skin tissue tests confirmed that the engineered nanoscaffold successfully activated skin-resident mast cells by contact. Thus, this nanoscaffold design may provide a new platform to modulate localized mast cell functions thereby facilitating their protective role in the skin.

Combinational siRNA delivery using hyaluronic acid modified amphiphilic polyplexes against cell cycle and phosphatase proteins to inhibit growth and migration of triple-negative breast cancer cells.

Triple-negative breast cancer is an aggressive form of breast cancer with few therapeutic options if it recurs after adjuvant chemotherapy. RNA interference could be an alternative therapy for metastatic breast cancer, where small interfering RNA (siRNA) can silence the expression of aberrant genes critical for growth and migration of malignant cells. Here, we formulated a siRNA delivery system using lipid-substituted polyethylenimine (PEI) and hyaluronic acid (HA), and characterized the size, ζ-potential and cellular uptake of the nanoparticulate delivery system. Higher cellular uptake of siRNA by the tailored PEI/HA formulation suggested better interaction of complexes with breast cancer cells due to improved physicochemical characteristics of carrier and HA-binding CD44 receptors. The siRNAs against specific phosphatases that inhibited migration of MDA-MB-231 cells were then identified using library screen against 267 protein-tyrosine phosphatases, and siRNAs to inhibit cell migration were further validated. We then assessed the combinational delivery of a siRNA against CDC20 to decrease cell growth and a siRNA against several phosphatases shown to decrease migration of breast cancer cells. Combinational siRNA therapy against CDC20 and identified phosphatases PPP1R7, PTPN1, PTPN22, LHPP, PPP1R12A and DUPD1 successfully inhibited cell growth and migration, respectively, without interfering the functional effect of the co-delivered siRNA. The identified phosphatases could serve as potential targets to inhibit migration of highly aggressive metastatic breast cancer cells. Combinational siRNA delivery against cell cycle and phosphatases could be a promising strategy to inhibit both growth and migration of metastatic breast cancer cells, and potentially other types of metastatic cancer. STATEMENT OF SIGNIFICANCE: The manuscript investigated the efficacy of a tailored polymeric siRNA delivery system formulation as well as combinational siRNA therapy in metastatic breast cancer cells to inhibit malignant cell growth and migration. The siRNA delivery was undertaken by non-viral means with PEI/HA. We identified six phosphatases that could be critical targets to inhibit migration of highly aggressive metastatic breast cancer cells. We further report on specifically targeting cell cycle and phosphatase proteins to decrease both malignant cell growth and migration simultaneously. Clinical gene therapy against metastatic breast cancer with effective and safe delivery systems is urgently needed to realize the potential of molecular medicine in this deadly disease and our studies in this manuscript is intended to facilitate this endeavor.

Polymeric Delivery of siRNA against Integrin-ß1 (CD29) to Reduce Attachment and Migration of Breast Cancer Cells.

Cell surface integrins, which play important roles in the survival, proliferation, migration, and invasion of cancer cells, are a viable target for treatment of metastatic breast cancer. This line of therapy still remains challenging due to the lack of proper identification and validation of effective targets as well as the lack of suitable therapeutic agents for treatment. The focus is on one such molecular target for this purpose, namely integrin-ß1, and effective lowering of integrin-ß1 levels on a breast cancer model (MDA-MB-231 cells) is achieved by delivering a dicer-substrate short interfering RNA (siRNA) targeting integrin-ß1 with lipid-modified low molecular weight polyethylenimine polymers. Reduction of integrin-ß1 levels leads to reduced adhesion of MDA-MB-231 cells to extracellular matrix component fibronectin as well as to human bone marrow cells. A reduced migration of the breast cancer cells is also observed after integrin-ß1 silencing in "scratch" and "transwell" migration assays. These results highlight the importance of integrin-ß1 for the migration of metastatic breast cancer cells by effectively silencing this target with a practical dose of siRNA.

Targeting CXCR4/SDF-1 axis by lipopolymer complexes of siRNA in acute myeloid leukemia.

In spite of high complete remission rates in Acute Myeloid Leukemia (AML), little progress has been made in the long-term survival of relapsing AML patients, urging for the development of novel therapies. The CXCR4/SDF-1 axis is a potential therapeutic target in AML to reduce the enhanced survival and proliferation of leukemic cells, with current drug development efforts focusing on antagonists and blocking antibodies. The RNAi technology mediated by siRNA is a promising alternative; however, further development of clinically relevant siRNA carriers is needed since siRNA on its own is an incompetent silencing agent. Here, we report on lipid-substituted polymeric carriers for siRNA delivery to AML cells, specifically targeting CXCR4. Our results demonstrate an effective suppression of CXCR4 protein with the polymeric siRNA delivery in AML THP-1 cells. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), decreased THP-1 cell numbers due to reduced cell proliferation. The reduced proliferation was also observed in the presence of human bone marrow stromal cells (hBMSC), suggesting that our approach would be effective in the protective bone marrow microenvironment. The combination of CXCR4 silencing and cytarabine treatment resulted in more effective cytotoxicity when the cells were co-incubated with hBMSC. We observed a decrease in the toxicity of the lipopolymer/siRNA complexes when THP-1 cells were treated in the presence of hBMSC but this effect did not negatively affect CXCR4 silencing. In addition, siRNA delivery to mononuclear cells derived from AML patients led to significant CXCR4 silencing in 2 out of 5 samples, providing a proof-of-concept for clinical translation. We conclude that decreasing CXCR4 expression via lipopolymer/siRNA complexes is a promising option for AML therapy and could provide an effective alternative to current CXCR4 inhibition strategies.

Multiple siRNA delivery against cell cycle and anti-apoptosis proteins using lipid-substituted polyethylenimine in triple-negative breast cancer and nonmalignant cells.

Conventional breast cancer therapies have significant limitations that warrant a search for alternative therapies. Short-interfering RNA (siRNA), delivered by polymeric biomaterials and capable of silencing specific genes critical for growth of cancer cells, holds great promise as an effective, and more specific therapy. Here, we employed amphiphilic polymers and silenced the expression of two cell cycle proteins, TTK and CDC20, and the anti-apoptosis protein survivin to determine the efficacy of polymer-mediated siRNA treatment in breast cancer cells as well as side effects in nonmalignant cells in vitro. We first identified effective siRNA carriers by screening a library of lipid-substituted polyethylenimines (PEI), and PEI substituted with linoleic acid (LA) emerged as the most effective carrier for selected siRNAs. Combinations of TTK/CDC20 and CDC20/Survivin siRNAs decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth. The effects of combinational siRNA therapy was higher when complexes were formulated at lower siRNA:polymer ratio (1:2) compared to higher ratio (1:8) in nonmalignant cells. The lead polymer (1.2PEI-LA6) showed differential transfection efficiency based on the cell-type transfected. We conclude that the lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets in breast cancer therapy. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3031-3044, 2016.

Targeting Cell Cycle Proteins in Breast Cancer Cells with siRNA by Using Lipid-Substituted Polyethylenimines.

The cell cycle proteins are key regulators of cell cycle progression whose deregulation is one of the causes of breast cancer. RNA interference (RNAi) is an endogenous mechanism to regulate gene expression and it could serve as the basis of regulating aberrant proteins including cell cycle proteins. Since the delivery of small interfering RNA (siRNA) is a main barrier for implementation of RNAi therapy, we explored the potential of a non-viral delivery system, 2.0 kDa polyethylenimines substituted with linoleic acid and caprylic acid, for this purpose. Using a library of siRNAs against cell cycle proteins, we identified cell division cycle protein 20 (CDC20), a recombinase RAD51, and serine-threonine protein kinase CHEK1 as effective targets for breast cancer therapy, and demonstrated their therapeutic potential in breast cancer MDA-MB-435, MDA-MB-231, and MCF7 cells with respect to another well-studied cell cycle protein, kinesin spindle protein. We also explored the efficacy of dicer-substrate siRNA (DsiRNA) against CDC20, RAD51, and CHEK1, where a particular DsiRNA against CDC20 showed an exceptionally high inhibition of cell growth in vitro. There was no apparent effect of silencing selected cell cycle proteins on the potency of the chemotherapy drug doxorubicin. The efficacy of DsiRNA against CDC20 was subsequently assessed in a xenograft model, which indicated a reduced tumor growth as a result of CDC20 DsiRNA therapy. The presented study highlighted specific cell cycle protein targets critical for breast cancer therapy, and provided a polymeric delivery system for their effective down-regulation.

siRNA therapy in cutaneous T-cell lymphoma cells using polymeric carriers.

Cutaneous T-cell lymphomas (CTCLs) arise from specific molecular aberrations that lead to uncontrolled cell proliferation. RNA interference (RNAi) with short interfering RNAs (siRNAs) is a feasible approach to interrupt aberrant signal processing in CTCL cells, but functional biomaterial carriers are needed to effectively deliver siRNAs intracellularly. Towards this goal, we explored the utility of lipid-substituted polyethylenimines (PEI) carriers in a cell model of CTCL. Using caprylic and linoleic acid substituted 2 kDa PEI (PEI-CA and PEI-LA, respectively), we showed effective delivery of siRNA to T-lymphocyte Hut78 and Jurkat cells, but silencing of a model protein (Green Fluorescent Protein, GFP) was possible only in the Hut78 cells. To enhance siRNA delivery to Hut78 cells, a high siRNA: carrier ratio used to assemble the complexes and centrifugation of cells in the presence of complexes were found effective. The toxicities of PEI-CA and PEI-LA were significantly lower than other commercial carriers, 25 kDa PEI and Lipofectamine(®) RNAiMAX. This might have contributed to reduced siRNA delivery efficiency of the latter carriers. Screening several endogenous targets led us to identify phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and cyclin-dependent kinase 18 (CDK18) as viable targets to induce siRNA-mediated cell growth inhibition. The results of this study identified promising polymeric carriers and molecular targets that could control proliferation of CTCL cells based on RNAi therapy.

Duplicated crabp1 and crabp2 genes in medaka (Oryzias latipes): gene structure, phylogenetic relationship and tissue-specific distribution of transcripts.

Here we report the genomic organization of duplicated cellular retinoic acid-binding protein genes, crabp1 and crabp2, in medaka (Japanese ricefish; Oryzias latipes), the phylogenetic relationship of medaka Crabp1a, Crabp1b, Crabp2a and Crabp2b with other Crabp/CRABP sequences from teleosts/tetrapods, and the tissue-specific distribution of crabp1a, crabp1b, crabp2a, and crabp2b transcripts in adult medaka. The duplicated medaka crabp1 and crabp2 genes contain four exons separated by three introns, which encode polypeptides of 137 and 142 amino acids, respectively. Sequence alignment revealed that medaka Crabp sequences share highest sequence identity and similarity with their orthologs from vertebrates. Phylogenetic analysis confirmed the orthology of the medaka Crabps as they form a distinct clade with their orthologous polypeptides from vertebrates. Conserved gene synteny was evident between the duplicated crabp1 and crabp2 genes from medaka, and CRABP1 and CRABP2 genes from human, which provides compelling evidence that the identified duplicated crabp1 and crabp2 genes from medaka most likely arose owing to teleost-specific whole-genome duplication. The tissue-specific distribution of zebrafish (Danio rerio) and medaka crabp1a, crabp1b, crabp2a, and crabp2b gene transcripts suggests acquisition of new function by these genes in medaka, which may explain potential evolutionary processes that led to the retention of sister duplicates of crabp1 and crabp2 genes in the medaka genome.

Genomic organization and transcription of the medaka and zebrafish cellular retinol-binding protein (rbp) genes.

In this study, we examined the evolutionary trajectories and the common ancestor of medaka rbp genes by comparing them to the well-studied rbp/RBP genes from zebrafish and other vertebrates. We describe here gene structure, sequence identity, phylogenetic analysis and conserved gene synteny of medaka rbp genes and their putative proteins as well as the tissue-specific distribution of rbp transcripts in adult medaka and zebrafish. Medaka rbp genes consist of four exons separated by three introns that encode putative polypeptides of 134-138 amino acids, a genomic organization characteristic of rbp genes. Medaka Rbp sequences share highest sequence identity and similarity with their orthologs in vertebrates, and form a distinct clade with them in phylogenetic analysis. Conserved gene synteny was evident among medaka, zebrafish and human rbp/RBP genes, which provides compelling evidence that the medaka rbp1, rbp2a, rbp2b, rbp5, rbp7a and rbp7b genes arose from a common ancestor of vertebrates. Moreover, the duplicated rbp2 and rbp7 genes most likely exist owing to a whole-genome duplication (WGD) event specific to the teleost fish lineage. Selection pressure and the nonparametric relative rate test of the medaka and zebrafish duplicated rbp2 and rbp7 genes suggest that these duplicated genes are subjected to purifying selection and one paralog might have evolved at an accelerated rate compared to its sister duplicate since the WGD. The steady-state levels of medaka and zebrafish rbp1, rbp2a, rbp2b and rbp5 transcripts in various tissues suggest that medaka rbp1, rbp2a and rbp2b genes have retained the regulatory elements of an ancestral RBP1 and RBP2 genes, and the medaka rbp5 gene has acquired new function. Furthermore, the tissue-specific regulations of rbp7a and rbp7b genes have diverged markedly in medaka and zebrafish since the teleost-specific WGD.

Comparative genomics and evolutionary diversification of the duplicated fabp6a and fabp6b genes in medaka and three-spined stickleback.

We describe the evolutionary diversification of the duplicated ileal fatty acid-binding protein genes (fabp6a and fabp6b) from Japanese ricefish (Oryzias latipes; medaka) and three-spined stickleback (Gasterosteus aculeatus). The fabp6a and fabp6b genes from medaka and three-spined stickleback encode polypeptides of 125-127 amino acids, which share highest sequence identity with their orthologs in teleost fishes and tetrapods. All Fabp6a and Fabp6b from different species cluster together in a distinct clade in phylogenetic analysis and the topology of the tree suggests that fabp6a and fabp6b from medaka and three-spined stickleback are most likely duplicated genes of an ancestral FABP6 owing to teleost-specific whole-genome duplication. However, the topology of an alternate phylogenetic tree revealed that the duplication of the ancestral FABP6 that gave rise to the extant fabp6a and fabp6b possibly occurred before the divergence of tetrapods and fishes. Conserved gene synteny was evident between the teleost fabp6a and fabp6b genes and the human FABP6 gene. The tissue-specific distribution of fabp6a transcripts suggests the retention of ancestral function(s) of the fabp6a gene in medaka and three-spined stickleback with acquisition of new function(s) in different tissues. However, the tissue-specific regulation of the fabp6b gene has diverged markedly in medaka and three-spined stickleback since the duplication of the fabp6 gene.

The evolutionary relationship of the transcriptionally active fabp11a (intronless) and fabp11b genes of medaka with fabp11 genes of other teleost fishes.

Here we describe the structure of the fatty acid-binding protein 11a and 11b genes (fabp11a and fabp11b) in medaka, and their evolutionary relationship to fabp11 genes from other teleost fishes. Initial studies indicated that the medaka fabp11a gene is intronless, but the fabp11b gene consists of four exons separated by three introns, a genomic organization that is characteristic of most members of the intracellular lipid-binding protein family. Based on genomic sequence, we conclude that the intronless fabp11a gene most likely arose as a result of reverse transcription of its mRNA transcript into cDNA followed by integration into chromosomal DNA. The ancestral intron-containing fabp11a gene was presumably lost from the medaka genome. The duplicated fabp11 genes extant in medaka encode polypeptides of 134 amino acids, which share highest sequence identity and similarity, and cluster in a distinct phylogenetic clade, with their orthologs in other teleost fishes. The fabp11a and fabp11b genes in medaka are therefore orthologs of the fabp11a and fabp11b genes, respectively, of other teleost fishes. No conserved gene synteny was found between medaka fabp11a and fabp11a genes from other teleost fishes, supporting our suggestion as to how this intronless gene arose. However, conserved gene synteny was evident between medaka fabp11b and fabp11b genes from other teleost fishes. The tissue-specific distribution of transcripts for medaka and zebrafish fabp11a and fabp11b genes revealed acquisition of a new function(s) in various tissues by the medaka fabp11b gene, which may explain the retention of sister duplicates of fabp11 in the medaka genome.