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The use of messenger RNA (mRNA) as a cancer vaccine and gene therapy requires targeted vehicle delivery to the site of disease. Here, we designed a mRNA-encapsulating lipid nanoparticle (LNP) conjugated with anti-programmed death-ligand 1 (PD-L1) DNA aptamer that delivers mRNA encoding a tumor suppressor gene, namely phosphatase and tensin homolog (PTEN), to castration-resistant prostate cancer (CRPC) cells expressing PD-L1 on the cell surface. The DNA aptamer-conjugated LNP-based mRNA delivery system (Apt-LNP[PTEN mRNA]) mediated efficient mRNA delivery and transfection in CRPC cells than LNPs without targeting ligands. Cancer-targeted PTEN mRNA delivery using Apt-LNPs achieved significantly higher PTEN expression via aptamer-mediated endocytosis in target cancer cells compared with non-targeted LNP delivery, resulting in significant downregulation of AKT phosphorylation. This enhanced PI3K/AKT pathway regulation, and in turn reduced cell migration after two days along with a 70 % decrease in cell viability, leading to effective apoptotic cell death. In a CRPC xenograft model, Apt-LNP[PTEN mRNA] led to an approximate 60 % reduction in tumor growth, which was attributable to the effective PTEN restoration and PI3K/AKT signaling pathway regulation. PTEN expression was significantly enhanced in CRPC tumor tissues, which abolished cancer cell tumorigenicity. These findings demonstrated the potential of Apt-LNPs for targeted mRNA delivery to cancer cells, thus providing a promising tool for targeted mRNA delivery to a range of cancers and tissues using a conventional LNP systems.
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Aptámeros de Nucleótidos , Nanopartículas , Fosfohidrolasa PTEN , ARN Mensajero , Masculino , Fosfohidrolasa PTEN/genética , Humanos , Animales , Nanopartículas/química , ARN Mensajero/administración & dosificación , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/química , Línea Celular Tumoral , Lípidos/química , Ratones Desnudos , Neoplasias de la Próstata Resistentes a la Castración , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Próstata , Ratones Endogámicos BALB C , Supervivencia Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , LiposomasRESUMEN
Many different types of nanoparticles have been suggested for tumor-targeted theranosis. However, most systems were prepared through a series of complicated processes and could not even overcome the blood-immune barriers. For the accurate diagnosis and effective treatment of cancers, herein we suggested the lipid micellar structure capturing quantum dot (QD) for cancer theranosis. The QD/lipid micelles (QDMs) were prepared using a simple self-assembly procedure and then conjugated with anti-epidermal growth factor receptor (EGFR) antibodies for tumor targeting. As a therapeutic agent, Bcl2 siRNA-cholesterol conjugates were loaded on the surface of QDMs. The EGFR-directed QDMs containing Bcl2 siRNA, so-called immuno-QDM/siBcl2 (iQDM/siBcl2), exhibited the more effective delivery of QDs and siBcl2 to target human colorectal cancer cells in cultures as well as in mouse xenografts. The effective in vivo targeting of iQDM/siBcl2 resulted in a more enhanced therapeutic efficacy of siBcl2 to the target cancer in mice. Based on the results, anti-EGFR QDM capturing therapeutic siRNA could be suggested as an alternative modality for tumor-targeted theranosis.
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Receptores ErbB , Proteínas Proto-Oncogénicas c-bcl-2 , Puntos Cuánticos , ARN Interferente Pequeño , Puntos Cuánticos/química , Animales , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inhibidores , Humanos , ARN Interferente Pequeño/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Ratones , Línea Celular Tumoral , Nanopartículas/química , Lípidos/química , Nanomedicina Teranóstica/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , MicelasRESUMEN
We investigated methods for cryopreserving sperm from the endangered gudgeon, Microphysogobio rapidus, by examining the effects of cryoprotective agent (CPA) concentration, diluent, and dilution ratio on post-thaw sperm quality. The quality of frozen sperm was evaluated in terms of motility and kinematic parameters, viability, DNA damage, and fertilization rate. We evaluated methanol, glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol as CPAs. Sperm motility, velocity, and viability were significantly higher when methanol was used as the CPA (p < 0.05). The diluents tested were Ringer's solution, Kurokura's Extender, Common Carp Sperm Extender (CCSE), and buffered sperm motility-inhibiting saline solution (BSMIS); post-thaw motility was highest when Ringer's solution was used as the diluent. Next, various quantities of methanol were combined with Ringer's solution to identify the optimal dose of methanol. The dilution ratios tested ranged from 1:1 to 1:7. Cryopreserved sperm was thawed at 20 °C for 15 s. The use of 10% methanol with Ringer's solution at a dilution ratio of 1:5 resulted in the highest post-thaw sperm motility, viability, and velocity including VAP, VCL, and VSL. Post-thaw sperm showed significantly greater DNA damage than the control (fresh sperm) (p < 0.05). The fertilization rate was highest with fresh sperm (p < 0.05), followed by sperm frozen with 10% methanol + Ringer's solution. We recommend that the best way to preserve sperm in the studied species is to use a combination of Ringer's solution and 10% methanol at a 1:5 dilution ratio. Our findings will facilitate the artificial fertilization of M. rapidus.
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Criopreservación , Crioprotectores , Cyprinidae , Dimetilsulfóxido , Metanol , Preservación de Semen , Motilidad Espermática , Espermatozoides , Animales , Masculino , Criopreservación/métodos , Criopreservación/veterinaria , Crioprotectores/farmacología , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Cyprinidae/fisiología , Metanol/farmacología , Dimetilsulfóxido/farmacología , Glicerol/farmacología , Glicol de Etileno/farmacología , Daño del ADN/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , FemeninoRESUMEN
Background: As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, Dryophytes suweonensis, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, D. japonicus. In particular, D. suweonensis, D. japonicus, and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence. Methods: We collected 57 individuals from six localities where D. suweonensis is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (SIAH, TYR, POMC, RAG1, and C-MYC) were analyzed to determine their parental species and hybrid status. Results: The HRM analysis showed that the melting temperature of D. suweonensis was in the range of 79.0-79.3 °C, and that of D. japonicus was 77.7-78.0 °C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be D. suweonensis, eight were found to be D. japonicus, and the remaining 38 individuals were found to be hybrids. Conclusion: Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for D. suweonensis conservation and restoration research.
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Anuros , Genes myc , Humanos , Animales , Anuros/genética , Animales Salvajes , Biodiversidad , Especies en Peligro de ExtinciónRESUMEN
Microphysogobio rapidus, an endemic cyprinid fish species found exclusively in Korea, has been identified in only two tributaries of the Nakdong River. The species predominantly occupies the near-gravel bottom waters within shallow sections of the middle and lower reaches of the river, characterized by swift currents. M. rapidus is currently recognized as a critically endangered species due to its distinct habitat preference, as well as the negative impacts of stream dam development and water environment pollution. In this study, we used 10 microsatellite markers to examine the genetic diversity of M. rapidus in the upper Nam (UN), lower Nam (LN), and Deokcheon Rivers (DC) in Korea, with a specific focus on assessment of the impact of dam development. Fish sampled from the UN and LN showed a greater average number of alleles and allelic richness (A = 18.3-18.4, AR = 13.8) compared to those from DC (A = 11.8, AR = 11.5). The observed heterozygosity among the fish examined ranged from HO = 0.748 (LN) to 0.766 (DC). All three fish groups exhibited a significant departure from Hardy-Weinberg equilibrium (HWE) (p < 0.05). Despite having the largest effective population size (Ne = 175 and 157, respectively), the fish sampled from UN and LN showed the highest inbreeding coefficients (FIS = 0.056-0.053, respectively), which were highly significant (p < 0.01). In contrast, the fish sampled from DC exhibited the smallest effective population size (Ne = 61) and showed an inbreeding coefficient close to zero (p > 0.05). BOTTLENECK analysis and estimated M-ratio values (0.341-0.372) revealed indications of past population size reduction in all fish groups examined. No significant genetic differentiation (FST < 0.05) was detected using the DAPC, STRUCTURE, and AMOVA among the fish studied. However, pairwise comparisons of FST between fish sampled from the Nam and Deokcheon Rivers revealed significant values (p < 0.001) ranging from 0.013 to 0.014. In addition, the closest genetic distance (0.026) was observed between UN and LN, while the greatest distance (0.087) was found between UN and DC. Analysis of gene flow rates among the fish examined indicated asymmetrical gene exchange within the Nam River, which was 31.51% in the downstream direction (from UN to LN), with a minimal gene flow rate (0.41%) in the upstream (from LN to UN) direction. The opposite trend was recorded between DC and LN, with a higher gene flow rate (29.74%) in the upstream direction compared to the downstream direction (0.12%). Our study highlighted the importance of implementing long-term conservation efforts focused on maintaining river integrity by removing water barriers such as weirs that impede fish migration and implementing active protection measures, such as aquaculture breeding and reasonable stocking practices, to preserve M. rapidus in the study area.
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Cipriniformes , Especies en Peligro de Extinción , Animales , Genética de Población , Endogamia , República de CoreaRESUMEN
BACKGROUND: The freshwater fish Gobiobotia naktongensis (Teleostei, Cypriniformes, and Gobionidae) is an endangered class I species whose population size has been greatly reduced. OBJECTIVE: To successfully protect and restore the highly endangered freshwater fish G. naktongensis from the Geum River in South Korea. METHODS: The mitogenome was characterized using the primer walking method with phylogenetic relationships. RESULTS: The complete mitogenome of G. naktongensis Geum River was 16,607 bp, comprising 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA (tRNA) genes. Seventeen substitutions were found by comparing the tRNA regions between G. naktongensis Geum and Nakdong Rivers and G. pappenheimi; most were specific to G. naktongensis Nakdong River, with changes in their secondary structures. The comparison between G. naktongensis Geum River and G. pappenheimi revealed differences in the lengths of the D-loop and two tRNAs (tRNAArg and tRNATrp) and the secondary structures in the TΨC-arm of tRNAHis. In the phylogenetic tree, G. naktongensis Geum River did not cluster with its conspecific specimen from the Nakdong River in South Korea, but showed the closest relationship to G. pappenheimi in mainland China. CONCLUSIONS: Our results support the existence of the Paleo-Huanghe River connecting the Korean peninsula and mainland China, suggesting that G. naktongensis in the Geum River should be treated as a different evolutionarily significant unit separated from that in the Nakdong River. The complete mitogenome of G. naktongensis Geum River provides essential baseline data to establish strategies for its conservation and restoration.
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Cipriniformes , Genoma Mitocondrial , Geum , Animales , Cipriniformes/genética , Especies en Peligro de Extinción , Agua Dulce , Genoma Mitocondrial/genética , Geum/genética , Filogenia , ARN de Transferencia/genética , RíosRESUMEN
Cancer cells have various immune evasion mechanisms that resist the immune cells by reprogramming the tumor microenvironment (TME), such as programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase-1 (IDO1) overexpression. One of the approaches to restore antitumor immune response by T-cells is through induction of immunogenic cell death (ICD). Thus, drug carrier containing IDO1 siRNA and ICD inducer would be effective anticancer regimen to modulate the immunosuppressive TME by reversing the IDO1-mediated immunosuppression in a synergistic combination with ICD induction. However, numerous nanocarrier platforms for co-delivery of multiple drugs mostly depend on the enhanced permeation and retention (EPR), which is insufficient to achieve selectivity in tumor sites harboring various types of cells. We designed a targeted drug delivery system using nano-sized liposomes functionalized with anti-CD44 and anti-PD-L1 DNA aptamers, which target breast cancer cells and inhibit PD-1/PD-L1 interaction between cancer cells and T-cells. To reverse immunosuppressive TME and reactivate immune response, cancer-targeting nano-liposomes were prepared to contain immunogenic cell death inducer (Doxorubicin, DOX) and IDO1 siRNA, namely Aptm[DOX/IDO1]. The Aptm[DOX/IDO1] specifically delivered the loaded DOX and IDO1 siRNA into target breast cancer cells through aptamer-mediated endocytosis. Cancer-targeted DOX/IDO1 siRNA delivery enhanced ICD and suppressed IDO1 expression with significantly high toxicity in cancer cells. We demonstrated that Aptm[DOX/IDO1] could achieve synergistic antitumor effects by facilitating ICD response and simultaneous reversal of the immunosuppressive TME with IDO1 knockdown in the subcutaneous breast cancer model mice, thus reducing tumor size. These antitumor effects were exerted with intratumoral infiltration of CD8+ cytotoxic T lymphocyte as well as attenuation of regulatory T-cell recruitment in the tumor sites. We further proved that our Aptm[DOX/IDO1] strategy significantly reduced tumor metastasis in tumor-xenograft mice through a synergistic combination of cancer cell-targeted ICD induction and reversal of the IDO1-mediated immunosuppressive TME. Our nanocarrier platform based on cationic liposomes containing DOX and IDO1 siRNA, which are conjugated with two DNA aptamers targeting the cancer cell surface, accomplished synergistic chemoimmunotherapy through tumor-specific immune modulation into immune-favorable TME in vivo.
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Antineoplásicos , Aptámeros de Nucleótidos , Animales , Línea Celular Tumoral , Doxorrubicina , Humanos , Terapia de Inmunosupresión , Liposomas , Ratones , Ratones Endogámicos BALB C , ARN Interferente Pequeño/genéticaRESUMEN
Breast cancer in women is one of the most common life-threatening malignancies. Despite of the development for the improved treatment, there are still many limitations to overcome. Among them, cancer stem cells (CSCs) are well known for tumor formation, development, cellular heterogeneity, and cancer recurrence. Therefore, to completely cure breast cancer, treatment of both cancer and CSC is required. To selectively target CSCs, we generated a liposome-based smart nano complex using CEACAM 6 (CD66c) antibody (Ab), a novel cell-surface biomarker of breast-derived CSCs (BCSCs) discovered in our previous research. Selective and increased cellular uptake was observed in BCSCs treated with CD66c Ab-conjugated rhodamine-labeled liposomes (CDRHOL) depending on the expression level of CD66c. CD66c Ab-conjugated doxorubicin (DOX)-loaded liposomes (CDDOXL) selectively showed increased cell killing effects in BCSCs with high CD66c expression levels. In an in vivo animal study, CDRHOL showed enhanced accumulation in xenografted BCSC tumors with low delivery into non-target organs. Moreover, mice treated with CDDOXL have assessed the decreased induction ability of immune response by low expression levels of pro-inflammatory cytokines and reduced liver toxicity by histopathological analysis. Finally, the improved antitumor effect of CDDOXL was evaluated in a metastatic BCSC mouse model via systemic administration. Collectively, our study is the first to demonstrate that a multi-functional nano complex using a novel surface biomarker of BCSC may be a more effective therapeutic agent for the treatment of cancer and CSCs.
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Liposomas , Recurrencia Local de Neoplasia , Femenino , Ratones , Animales , Liposomas/metabolismo , Recurrencia Local de Neoplasia/patología , Biomarcadores/metabolismo , Células Madre Neoplásicas/metabolismo , Línea Celular TumoralRESUMEN
The full-length mitochondrial genome of the yellow catfish, Tachysurus fulvidraco was analyzed by the primer walking method. Its assembled mitochondrial genome was found to be 16,527 bp, consisting of 37 genes (13 protein-coding genes, 22 tRNA gens, and 2 rRNA gens). The gene content and order of T. fulvidraco were congruent with those of typical vertebrate fishes. In the phylogenetic tree, it showed the closet relationship to the another conspecific specimen from China and Pseudobagrus koreanus and well separated from the other species in the family Bagridae.
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In this study, we developed a drug-loading liposome linked with two types of DNA aptamers targeting the surface marker transmembrane glycoprotein mucin 1 antigen (MUC1) on breast cancer cells and cell surface glycoprotein CD44 antigen (CD44) on their cancer stem cells (CSCs). Dual-aptamer-conjugated liposomes, called dual-aptamosomes, were prepared to encapsulate doxorubicin (Dox) and tested for Dox delivery to the 3D-cultured breast cancer cells as well as CSCs. Dox was readily delivered into the cancer cells via ligand-mediated cellular uptake of dual-aptamosomes. Dual-aptamosomes harboring Dox (dual-Apt-Dox) showed a significantly higher cytotoxicity to both CSCs and cancer cells than to liposomes lacking the aptamers. Furthermore, we demonstrated inhibitory activity of dual-Apt-Dox against metastasis of breast cancer stems cells in athymic nude mice. Thus, the dual-aptamer-conjugated liposomal system can be a useful drug delivery carrier for treatment of breast cancer, given its efficiency in targeting both breast cancer cells and their CSCs.
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BACKGROUND: Efficient target-specific siRNA delivery has always been a primary concern in the field of siRNA clinical application. PURPOSE: In this study, four different types of anti-epidermal growth factor receptor (EGFR) antibody-conjugated immunonanoparticles were prepared and tested for cancer cell-targeted therapeutic siRNA delivery. MATERIALS AND METHODS: The prepared nanoparticles encapsulating siRNAs were character-ized by gel retardation and particle analysis using a Zetasizer. In vitro transfection and reduction of target genes, vimentin and JAK3, were determined using quantitative reverse transcription polymerase chain reaction. In vivo tumor targeting and antitumoral efficacies of the nanoparticles were evaluated in mice carrying tumors. RESULTS: Among these immunonanoparticles, anti-EGFR immunolipoplexes and immunoviroplexes exhibited remarkable cell binding and siRNA delivery to EGFR-expressing tumor cells compared to immunoliposomes and immunovirosomes. Especially, the anti-EGFR immunoviroplexes exhibited the most efficient siRNA transfection to target tumor cells. Therefore, antitumoral vimentin and Janus kinase-3 siRNAs were loaded in the anti-EGFR immunolipoplexes and immunoviroplexes, which were tested in mice carrying SK-OV-3 tumor xenografts. In fact, the therapeutic siRNAs were efficiently delivered to the tumor tissues by both delivery vehicles, resulting in significant inhibition of tumor growth. Moreover, administration of doxorubicin in combination with anti-EGFR immunoviroplexes resulted in remarkable and synergistic tumor growth inhibition. CONCLUSION: This study provides experimental proof that cancer cell-targeted immunoviroplexes are an efficient siRNA delivery system for cancer therapy. Moreover, this study also suggests that a combination of conventional chemotherapy and tumor-directed anticancer siRNA therapy would be a better modality for cancer treatment.
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Receptores ErbB/inmunología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Administración Intravenosa , Animales , Antineoplásicos/metabolismo , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Receptores ErbB/metabolismo , Femenino , Humanos , Janus Quinasa 3/metabolismo , Liposomas/administración & dosificación , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Nanopartículas/química , Proteínas de Neoplasias/metabolismo , ARN Interferente Pequeño/genética , Transfección , Vimentina/metabolismoRESUMEN
Aptamers are single-stranded oligonucleotides that specifically bind and interact with their corresponding targets, including proteins and cells, through unique three-dimensional structures. Numerous aptamers have been developed to target cancer biomarkers with high specificity and affinity, and some are employed as versatile guiding ligands for cancer-specific drug delivery and anti-cancer therapeutics. In this review, we list the aptamers that target tumor surface biomarkers and summarize the representative applications of aptamers as agonists and antagonists that activate anti-cancer and inactivate pro-cancer biomarkers, respectively. In addition, we describe applications of aptamer-drug or aptamer-oligonucleotide conjugates that can deliver therapeutic agents, including small interfering RNAs, micro RNAs, short hairpin RNAs, and chemotherapeutic molecules, to cancer cells. Moreover, we provide examples of aptamer- conjugated nano-vehicles, in which cancer-targeting oligonucleotide aptamers are conjugated with nano-vehicles such as liposomes, micelles, polymeric nanoparticles, and quantum dots. Conjugation of aptamers with anti-cancer drugs and nano-vehicles will facilitate innovative applications of aptamer-based cancer therapeutics.
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Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/uso terapéutico , Sistemas de Liberación de Medicamentos , HumanosRESUMEN
The entire mitochondrial genome of Cobitis nalbanti (Teleostei: Cypriniformes: Cobitidae) was analyzed using the primer walking method. The mitogenome was 16,631 bp in length, comprising 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Its gene order was congruent with those of typical vertebrates. In the phylogenetic tree, C. nalbanti was clearly separated from C. lutheri, which supported the recent taxonomic revision.
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To investigate distribution, habitat characteristics, and current conservation status of the endangered endemic species, rapid small gudgeon Microphysogobio rapidus (Cyprinidae), we surveyed a total of 79 sites from the historic records (20 sites) plus additional sites (59 sites) with good habitat conditions, analyzed their sites, and compared them with historic recorded sites to reveal the factors of extinction threats and causes. We found only eight out of 79 sites in the Nam River areas. The habitats were greatly reduced and restricted compared with the historic sites, which mainly cause from habitat modification, such as various types of river renovations at the main stream and tributary streams of the Nakdong River. The present habitats are higher water temperature and more number of fish species than the absent ones, but conductivity, total nitrogen, and number of weir are lower. In addition, the present sites are lower low velocity at pool and higher mean substrate at pool. From this study, we suggest that maintaining good water quality and preventing anthropogenic impacts greatly aid conservation of the M. rapidus in South Korea.
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Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.
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Cetuximab/administración & dosificación , Venenos de Crotálidos/genética , Inmunoconjugados/administración & dosificación , Interleucina-12/genética , Neoplasias Pulmonares/terapia , Melanoma Experimental/terapia , Nanoconjugados/administración & dosificación , Administración Intravenosa , Animales , Línea Celular Tumoral , Cetuximab/uso terapéutico , Terapia Combinada , Terapia Genética , Vectores Genéticos/administración & dosificación , Humanos , Liposomas/administración & dosificación , Neoplasias Pulmonares/secundario , Células MCF-7 , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor-directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off-target effects of gene transfection. METHODS: The system consists of a well-verified cationic O,O'-dimyristyl-N-lysyl glutamate (DMKE), Sendai virus fusion (F) protein and hemagglutinin-neuraminidase (HN) protein, referred to as cationic Sendai F/HN virosomes. To achieve tumor-specific recognition, anti-epidermal growth factor (EGF) receptor antibody was coupled to the surface of the virosomes containing interleukin-12 (IL-12) and/or salmosin genes that have potent anti-angiogenetic functions. RESULTS: Among the virosomal formulations, the anti-EGF receptor (EGFR) viroplexes, prepared via complexation of plasmid DNA (pDNA) with cationic DMKE lipid, exhibited more efficient gene transfection to tumor cells over-expressing EGF receptors compared to the neutrally-charged anti-EGFR virosomes encapsulating pDNA. In addition, the anti-EGFR viroplexes with IL-12 and salmosin genes exhibited the most effective therapeutic efficacy in a mouse tumor model. Especially when combined with doxorubicin, transfection of the two genes via the anti-EGFR viroplexes exhibited an enhanced inhibitory effect on tumor growth and metastasis in lungs. CONCLUSIONS: The results of the present study suggest that anti-EGFR viroplexes can be utilized as an effective strategy for tumor-directed gene delivery. Copyright © 2016 John Wiley & Sons, Ltd.
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Venenos de Crotálidos/genética , Receptores ErbB/genética , Interleucina-12/genética , Neoplasias/genética , Virus Sendai/genética , Células A549 , Animales , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Venenos de Crotálidos/metabolismo , Doxorrubicina/farmacología , Receptores ErbB/metabolismo , Terapia Genética/métodos , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Interleucina-12/metabolismo , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/terapia , Virus Sendai/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Virosomas/genética , Virosomas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Giant grouper (Epinephelus lanceolatus) is a commercially important species, but its wild population has recently been classified as vulnerable. This species has significant potential for use in aquaculture, though a greater understanding of population genetics is necessary for selective breeding programs to minimize kinship for genetically healthy individuals. High-throughput pyrosequencing of genomic DNA was used to identify and characterize novel tetra- and trinucleotide microsatellite markers in giant grouper from Sabah, Malaysia. In total, of 62,763 sequences containing simple sequence repeats (SSRs) were obtained, and 78 SSR loci were selected to possibly contain tetra- and trinucleotide repeats. Of these loci, 16 had tetra- and 8 had trinucleotide repeats, all of which exhibited polymorphisms within easily genotyped regions. A total of 143 alleles were identified with an average of 5.94 alleles per locus, with mean observed and expected heterozygosities of 0.648 and 0.620, respectively. Among of them, 15 microsatellite markers were identified without null alleles and with Hardy-Weinberg equilibrium. These alleles showed a combined non-exclusion probability of 0.01138. The probability of individual identification (PID) value combined with in descending order 12 microsatellite markers was 0.00008, which strongly suggests that the use of the microsatellite markers developed in this study in various combinations would result in a high resolution method for parentage analysis and individual identification. These markers could be used to establish a broodstock management program for giant grouper and to provide a foundation for genetic studies such as population structure, parentage analysis, and kinship selection.
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Perciformes/genética , Alelos , Animales , Secuencia de Bases , Frecuencia de los Genes , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Desequilibrio de Ligamiento , Repeticiones de Microsatélite , Tipificación de Secuencias Multilocus , Polimorfismo GenéticoRESUMEN
The combination of therapeutic nucleic acids and chemotherapeutic drugs has shown great promise for cancer therapy. In this study, asialoglycoprotein receptors (ASGPR) targeting-ligand-based liposomes were tested to determine whether they can co-deliver vimentin siRNA and doxorubicin to hepatocellular carcinoma (HCC) selectively. To achieve this goal, we developed an ASGPR receptor targeted co-delivery system called gal-doxorubicin/vimentin siRNA liposome (Gal-DOX/siRNA-L). The Gal-DOX/siRNA-L was created via electrostatic interaction of galactose linked-cationic liposomal doxorubicin (Gal-DOX-L) on vimentin siRNA. Previous studies have shown that Gal-DOX/siRNA-L inhibited tumor growth by combined effect of DOX and vimentin siRNA than single delivery of either DOX or vimentin siRNA. These Gal-DOX/siRNA-Ls showed stronger affinity to human hepatocellular carcinoma cells (Huh7) than other cells (lung epithelial carcinoma, A549). These liposomes also have demonstrated that novel hepatic drug/gene delivery systems composed of cationic lipid (DMKE: O,O'-dimyristyl-N-lysyl glutamate), cholesterol, galactosylated ceramide, POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), and PEG2000-DSPE (distearoyl phosphatidyl ethanolamine) at 2:1:1:1:0.2 (moral ratios) can be used as an effective drug/gene carrier specifically targeting the liver in vivo. These results suggest that Gal-DOX-siRNA-L could effectively target tumor cells, enhance transfection efficacy and subsequently achieve the co-delivery of DOX and siRNA, demonstrating great potential for synergistic anti-tumor therapy.
RESUMEN
We performed a virtual screen of â¼340â¯000 small molecules against the active site of proteasomes followed by in vitro assays and subsequent optimization, yielding a proteasome inhibitor with pyrazole scaffold. The pyrazole-scaffold compound displayed excellent metabolic stability and was highly effective in suppressing solid tumor growth in vivo. Furthermore, the effectiveness of this compound was not negatively impacted by resistance to bortezomib or carfilzomib.
Asunto(s)
Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Neoplasias Experimentales/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Pirazoles/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Dominio Catalítico/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Relación Dosis-Respuesta a Droga , Humanos , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/patología , Inhibidores de Proteasoma/administración & dosificación , Inhibidores de Proteasoma/química , Pirazoles/administración & dosificación , Pirazoles/química , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
To minimize the systemic toxicity prevalent to chemotherapeutics, we designed a novel anticancer drug-encapsulating liposome conjugated with an RNA aptamer specific to the prostate specific membrane antigen (PSMA), which is expressed on the surface of prostate cancer cells. The RNA aptamer-conjugated liposome, termed an aptamosome, was prepared by the post-insertion method, in which RNA aptamer-conjugated micelles were inserted into a liposome. These nanosized (90-100 nm) aptamer-conjugated liposomes specifically bind to LNCaP prostate epithelial cells that express PSMA and thus cause the nanoparticles to have significantly enhanced in vitro cellular binding and uptake as compared with nontargeted nanoparticles that lack the PSMA aptamer. Aptamosomes encapsulated with the anticancer drug doxorubicin (Dox) were significantly more toxic to the targeted LNCaP cells than to nontargeted cancer cells. Dox-encapsulating aptamosomes administered to LNCaP xenograft nude mice were selectively retained in tumor tissue. We also demonstrated in vivo anticancer efficacy of the Dox-encapsulating PSMA-aptamosomes on tumor size regression in LNCaP xenograft mice. We suggest that the encapsulation of toxic chemicals with aptamer-conjugated liposomes will enable the use of these bioconjugates in clinical practice with fewer side effects.