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Lower oxidative capacity in skeletal muscles (SKMs) is a prevailing cause of metabolic diseases. Exercise not only enhances the fatty acid oxidation (FAO) capacity of SKMs but also increases lactate levels. Given that lactate may contribute to tricarboxylic acid cycle (TCA) flux and impact monocarboxylate transporter 1 in the SKMs, we hypothesize that lactate can influence glucose and fatty acid (FA) metabolism. To test this hypothesis, we investigated the mechanism underlying lactate-driven FAO regulation in the SKM of mice with diet-induced obesity (DIO). Lactate was administered to DIO mice immediately after exercise for over 3 wk. We found that increased lactate levels enhanced energy expenditure mediated by fat metabolism during exercise recovery and decreased triglyceride levels in DIO mice SKMs. To determine the lactate-specific effects without exercise, we administered lactate to mice on a high-fat diet (HFD) for 8 wk. Similar to our exercise conditions, lactate increased FAO, TCA cycle activity, and mitochondrial respiration in the SKMs of HFD-fed mice. In addition, under sufficient FA conditions, lactate increased uncoupling protein-3 abundance via the NADH-NAD+ shuttle. Conversely, ATP synthase abundance decreased in the SKMs of HFD mice. Taken together, our results suggest that lactate amplifies the adaptive increase in FAO capacity mediated by the TCA cycle and mitochondrial respiration in SKMs under sufficient FA abundance. NEW & NOTEWORTHY: Lactate administration post-exercise promotes triglyceride content loss in skeletal muscles (SKMs) and reduced body weight. Lactate enhances fatty acid oxidation in the SKMs of high-fat diet (HFD)-fed mice due to enhanced mitochondrial oxygen consumption. In addition, lactate restores the malate-aspartate shuttle, which is reduced by a HFD, and activates the tricarboxylic acid cycle (TCA) cycle in SKMs. Interestingly, supraphysiological lactate facilitates uncoupling protein-3 expression through NADH/NAD+, which is enhanced under high-fat levels in SKMs. [ABSTRACT FROM AUTHOR]

Regulation of proper gene expression is important for cellular and organismal survival, maintenance, and growth. Abnormal gene expression, even for a single critical gene, can thwart cellular integrity and normal physiology to cause diseases, aging, and death. Therefore, gene expression profiling serves as a powerful tool to understand the pathology of diseases and to cure them. In this study, the difference in gene expression in Flammulina velutipes was compared between the wild type (WT) mushroom and the mutant one with clogging phenomenon. Differentially expressed transcripts were screened to identify the candidate genes responsible for the mutant phenotype using the DNA microarray analysis. A total of 88 genes including 60 upregulated and 28 downregulated genes were validated using the real-time quantitative PCR analysis. In addition, proteomic differences between the WT and mutant mushroom were analyzed using two–dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Interestingly, the genes identified by these genomic and proteomic analyses were involved in stress response, translation, and energy/sugar metabolism, including HSP70, elongation factor 2, and pyruvate kinase. Together, our data suggest that the aberrant expression of these genes attributes to the mutant clogging phenotype. We propose that these genes can be targeted to foster normal growth in F. velutipes. [ABSTRACT FROM AUTHOR]

Senescent cells accumulate in various tissues over time and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular microRNAs in conditioned medium via microRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, Adriamycin-induced, and ionizing radiation-induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated β-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGF-β receptor 2, p53, and p21. mmu-miR-291a-3p in conditioned medium was enclosed in ESC-derived exosomes and exosomes purified from ESC conditioned medium inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGF-β receptor 2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases. [ABSTRACT FROM AUTHOR]

A single-pass process was developed to continuously assemble lateral nanoscaled iron (pure iron and iron oxide-hydroxide)-decorated graphitic oxide conjugated with doxorubicin and polyethylene glycol (IDGO@DOX-PEG) flakes. The resulting flakes were employed for magneto- and photo-thermal therapies as well as magnetic resonance (MR) imaging as a theranostic nanosystem. The lateral nanoscaled GO flakes were primarily biocompatible and suitable to suppress burst DOX release because of abundant oxygenated plane and edges for DOX conjugation, and the fully nanoscale system was further feasible for near-infrared induced chemo-phototherapy to kill HeLa cells. The decorated iron nanoparticles exhibited a superparamagnetic behavior. IDGO@DOX-PEG flakes, thus, further demonstrated effective chemo-magnetotherapy under alternating magnetic field as well as contrasting activity for MR imaging. This study offers a new and generalizable concept to continuously assemble composite GO nanoflakes for theranostic nanomedicine which usually requires complicated hazardous chemistries and multiple posttreatments. [ABSTRACT FROM AUTHOR]

Photoinduced assembly of Ag2S nanodots with surface thiolates was achieved in a single-pass gas stream configuration. Ag nanoagglomerates were converted into aligned Ag2S nanodots without hydrothermal reaction, separation, or purification procedures. The proposed route to fabricate hybrid nanodroplets containing Ag nanoagglomerates and 1-hexanethiol resulted in electrostatic interactions between Ag surfaces and thiol groups to assemble Ag2S nanodots. Photoirradiation (185 nm wavelength UV light) onto the nanodroplets induced electron detachments from Ag via photons (photon energy: 6.2 eV) reaching the Ag (work function: 4.3 eV) surfaces. The positively charged Ag surfaces interacted with the negatively charged surrounding thiol groups, resulting in the formation of Ag2S nanodots. Continuous photoirradiation for 6.4 s resulted in complete phase transfer; thus, inducing the isolation of the nanodots (i.e., resulting in an aligned structure of the nanodots without random aggregation) by conjugated surface thiolates. The nanodots were stably dispersible for near-infrared-radiation-induced fluorescence bioimaging without significant cytotoxic effects; furthermore, merging the nanodots with TiO2 nanoparticles significantly enhanced photocurrent generation under simulated solar irradiation. The photoinduced assembly demonstrates a new feasible safe-by-design platform to continuously produce photoresponsive nanodots without optimization concerning chemically efficient and environmentally benign concepts. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose, and its plasma levels are elevated in patients with diabetes. Studies have shown that MGO combines with the amino and sulphhydryl groups of proteins to form stable advanced glycation end products (AGEs), which are associated with vascular endothelial cell (EC) injury and may contribute to the progression of atherosclerosis. In this study, MGO induced apoptosis in a dose-dependent manner in HUVECs, which was attenuated by pre-treatment with z-VAD, a pan caspase inhibitor. Treatment with MGO increased ROS levels, followed by dose-dependent down-regulation of c-FLIPL. In addition, pre-treatment with the ROS scavenger NAC prevented the MGO-induced down-regulation of p65 and c-FLIPL, and the forced expression of c-FLIPL attenuated MGO-mediated apoptosis. Furthermore, MGO-induced apoptotic cell death in endothelium isolated from mouse aortas. Finally, MGO was found to induce apoptosis by down-regulating p65 expression at both the transcriptional and posttranslational levels, and thus, to inhibit c-FLIPL mRNA expression by suppressing NF-κB transcriptional activity. Collectively, this study showed that MGO-induced apoptosis is dependent on c-FLIPL down-regulation via ROS-mediated down-regulation of p65 expression in endothelial cells. [ABSTRACT FROM AUTHOR]

Cationic liposomes are broadly used as non-viral vectors to deliver genetic materials that can be used to treat various diseases including cancer. To circumvent problems associated with cationic liposome-mediated delivery systems such as low transfection efficiency and serum-induced inhibition, cholesterol-based cationic lipids have been synthesized that resist the effects of serum. The introduction of an ether-type linkage and extension of the aminopropyl head group on the cholesterol backbone increased the transfection efficiency and DNA binding affinity compared to a carbamoyl-type linkage and a mono aminopropyl head group, respectively. Under optimal conditions, each liposome formulation showed higher transfection efficiency in AGS and Huh-7 cells than commercially available cationic liposomes, particularly in the presence of serum. The following molecular structures were found to have a positive effect on transfection properties: (i) extended aminopropyl head groups for a strong binding affinity to plasmid DNA; (ii) an ether linkage that favors electrostatic binding to plasmid DNA; and (iii) a cholesterol backbone for serum resistance. [ABSTRACT FROM AUTHOR]

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30-40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

Doxorubicin (DOX) is one of the most effective anticancer agents used for the treatment of multiple cancers; however, its use is limited by its short half-life and adverse drug reactions, especially cardiotoxicity. In this study, we found that the conjugate of DOX with APTA12 (Gemcitabine incorporated G-quadruplex aptamer) was significantly more cancer selective and cytotoxic than DOX. The conjugate had an affinity for nucleolin, with higher uptake and retention into the cancer cells than those of DOX. Further, it was localized to the nucleus, which is the target site of DOX. Owing to its mechanism of action, DOX has the ability to intercalate into the nucleotides thus making it a suitable drug to form a conjugate with cancer selective aptamers such as APTA12. The conjugation can lead to selectively accumulate in the cancer cells thus decreasing its potential nonspecific as well as cardiotoxic side effects. The aim of this study was to prepare a conjugate of DOX with APTA12 and assess the chemotherapeutic properties of the conjugate specific to cancer cells. The DOX-APTA12 conjugate was prepared by incubation and its cytotoxicity in MCF-10A (non-cancerous mammary cells) and MDA-MB-231 (breast cancer cells) was assessed. The results indicate that DOX-APTA12 conjugate is a potential option for chemotherapy especially for nucleolin expressing breast cancer with reduced doxorubicin associated side effects.

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose, and its plasma levels are elevated in patients with diabetes. Studies have shown that MGO combines with the amino and sulphhydryl groups of proteins to form stable advanced glycation end products (AGEs), which are associated with vascular endothelial cell (EC) injury and may contribute to the progression of atherosclerosis. In this study, MGO induced apoptosis in a dose-dependent manner in HUVECs, which was attenuated by pre-treatment with z-VAD, a pan caspase inhibitor. Treatment with MGO increased ROS levels, followed by dose-dependent down-regulation of c-FLIP L . In addition, pre-treatment with the ROS scavenger NAC prevented the MGO-induced down-regulation of p65 and c-FLIP L , and the forced expression of c-FLIP L attenuated MGO-mediated apoptosis. Furthermore, MGO-induced apoptotic cell death in endothelium isolated from mouse aortas. Finally, MGO was found to induce apoptosis by down-regulating p65 expression at both the transcriptional and posttranslational levels, and thus, to inhibit c-FLIP L mRNA expression by suppressing NF-κB transcriptional activity. Collectively, this study showed that MGO-induced apoptosis is dependent on c-FLIP L down-regulation via ROS-mediated down-regulation of p65 expression in endothelial cells., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism of IF-mediated metabolic benefit and suggests that isocaloric IF can be a preventive and therapeutic approach against obesity and metabolic disorders.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is considered as a human carcinogenic or mutagenic compound that is produced from the co-condensation of creatinine and amino acids as meats cook at high temperatures. The cooking of meats at high temperatures produces fumes, and these fumes can be suspended as aerosols via the vapor-to-particle (or -droplet) process in a temperature gradient field. Size distributions of the aerosols included a significant portion of nano- and submicron-sized particles, and these can be directly deposited in the lungs and on skin by particle transport phenomena near cooking areas. In this study, for the first time, PhIP-incorporated oleic acid (OA, simulating cooking oil) (PhIP@OA) particles, including individual particulate PhIP as simulated fumes from meat cooking, were constantly produced via collison atomization and subsequent drying processes. The aerosol particles were then dispersed in phosphate-buffered saline for cytotoxicity and senescence-associated β-galactosidase assays, which were compared with dissolved PhIP in dimethyl sulfoxide. PhIP and PhIP@OA did not show significant cytotoxic effects on SHSY5Y, MRC5, and human dermal fibroblast cells compared with the dissolved PhIP but clearly induced premature senescence activities that may be caused by a limited release of PhIP molecules from the particulate PhIP.

MicroRNA (miR) can exert various biological functions by targeting oncogenes or tumor suppressor genes in numerous human malignancies. Recent evidence has shown that miR-148a increases the drug sensitivity of various cancer cells. Herein, we show that ectopic expression of miR-148a induces apoptosis, reduces clonogenicity, and increases the sensitivity to TRAIL and cisplatin in renal cancer cells. The luciferase reporter assay showed that miR-148a negatively regulated ras-related protein 14 (Rab14) expression by binding to the miR-148a binding site in the 3' untranslated region (3'UTR) of Rab14. Rab14-specific siRNA-induced downregulation of Rab14 increases the sensitivity to cisplatin, while forced expression of Rab14 lacking 3'-UTR abrogated the pro-apoptotic function of miR-148a in renal cancer cells. These findings suggest that miR-148a acts as a tumor suppressor and holds great potential for renal cancer therapy by directly targeting Rab14.

Cationic liposomes have been actively used as gene delivery vehicles despite their unsatisfactory efficiencies because of their relatively low toxicity. In this study, we designed novel heterodimeric peptides as nonviral gene delivery systems from TAT and NLS peptides using cysteine-to-cysteine disulfide bonds between the two. Mixing these heterodimeric peptides with DNA before mixing with lipofectamine resulted in higher transfection efficiencies in MCF-7 breast cancer cells than mixing unmodified TAT, NLS, and a simple mixture of TAT and NLS with DNA, but did not show an adverse effect on cell viability. In gel retardation assays, the DNA binding affinities of heterodimeric peptides were stronger than NLS but weaker than TAT. However, this enhancement was only observed when heterodimeric peptides were premixed with DNA before being mixed with lipofectamine. The described novel transfection-enhancing peptide system produced by the heterodimerization of TAT and NLS peptides followed by simple mixing with DNA, increased the gene transfer efficiency of cationic lipids without enhancing cytotoxicity.

Endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and mitochondrial biogenesis were assessed following varying intensities of exercise training. The animals were randomly assigned to receive either low- (LIT, n=7) or high intensity training (HIT, n=7), or were assigned to a control group (n=7). Over 5 weeks, the animals in the LIT were exercised on a treadmill with a 10° incline for 60 min at a speed of 20 m/min group, and in the HIT group at a speed of 34 m/min for 5 days a week. No statistically significant differences were found in the body weight, plasma triglyceride, and total cholesterol levels across the three groups, but fasting glucose and insulin levels were significantly lower in the exercise-trained groups. Additionally, no statistically significant differences were observed in the levels of PERK phosphorylation in skeletal muscles between the three groups. However, compared to the control and LIT groups, the level of BiP was lower in the HIT group. Compared to the control group, the levels of ATF4 in skeletal muscles and CHOP were significantly lower in the HIT group. The HIT group also showed increased PGC-1α mRNA expression in comparison with the control group. Furthermore, both of the trained groups showed higher levels of mitochondrial UCP3 than the control group. In summary, we found that a 5-week high-intensity exercise training routine resulted in increased mitochondrial biogenesis and decreased ER stress and apoptotic signaling in the skeletal muscle tissue of rats.

A DOTAP analog labeled by NBD on the head group (DTNBD) was designed and synthesized to label DOTAP liposome. The structure was confirmed by (1)H NMR and FAB-MS, and the fluorescence of the newly synthesized DT-NBD was observed by fluorescent microscopy. The transfection efficiency of DOTAP liposome containing DT-NBD was comparable to commonly used NBD PE in COS7 and MCF7 cells. Furthermore, the level of cellular uptake and fluorescent intensity of fluorescent liposome containing DT-NBD was higher than NBD PE. Therefore, the novel NBD-labeled DOTAP analog seems to be effectively used for investigation of the cellular interaction and transfection mechanism of DOTAP liposome.

Cholesterol-based cationic lipids have been widely used because of biocompatibility and serum resistance. However, the reason for the effectiveness of cholesterol-based cationic lipids remains unclear. We compared the transfection route of CHOL-E, a cholesterol-based cationic lipid having an amine head and an ether linker, with that of DOTAP. The luciferase assay with chemical inhibitors and microscopic observation of pathway markers revealed that clathrin mediated endocytosis is the main pathway for CHOL-E and DOTAP. However, CHOL-E showed resistance to cholesterol depletion by methyl-β-cyclodextrin. Furthermore, CHOL-E recovered the transfection efficiency of DOTAP from cholesterol depletion. These results suggested that superior transfection of CHOL-E might be partly derived from effects on the cell membrane.

A novel cholesterol-based cationic lipid containing a tri-2- hydroxyethylamine head group and ether linker (Chol- THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.

Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-alpha- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.

In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

The large circular (LC)-antisense library to the 221 unigene clone was constructed and utilized in the identification of genes functionally involved in the growth of hepatocellular carcinoma cells. We identified that 37 out of the 221 members of the antisense library exerted a marked inhibitory effect on the growth of Huh-7. The putative functional categorization of each gene was then conducted on the basis of the sequence information. The relative expression levels of target genes were measured and treated with two LC-antisense molecules by real-time PCR. LC-antisense to EIF3EIP and AFP abolished the expression of EIF3EIP and AFP to the level of approximately 7 and 39% compared to the control treatment in Huh-7 cells, respectively. LC-antisense molecules to EIF3EIP and AFP were simultaneously treated with 5-FU to Huh-7 cells. Two LC-antisense molecules showed additive effects with 5-FU compared with 5-FU alone, respectively. The combination of LC-antisense molecules and 5-FU showed a dramatic increase of sub-G1 apoptotic cell death fraction in cell cycle analysis, respectively. Therefore, these candidates may be used as target genes for drug development or adjuvant of conventional chemotherapeutic drugs.

Transforming growth factor-beta1 (TGF-beta1) is an important mediator of tissue fibrosis, including liver cirrhosis. Ribbon-type antisense oligonucleotide to TGF-beta1 (TGF-beta1 RiAS) was designed and combined with cationic peptide derived from the nuclear localization signal of human immunodeficiency virus-1 Tat protein for enhanced cellular uptake. When Hepa1c1c7 cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was reduced by >70%. TGF-beta1 RiAS, mismatched RiAS, and normal saline were each injected into mice via the tail vein, beginning the week after intraperitoneal CCl4 injection and continuing for 7 weeks, in order to determine whether TGF-beta1 RiAS prevents the fibrotic changes induced by the CCl4 injection. After 8 weeks of the experiment, all of the mice treated with TGF-beta1 RiAS survived, compared to 50% of the control group and 65% of the mismatched RiAS-treated group. Upon examining the biochemical effects on the liver, TGF-beta1 mRNA levels were reduced significantly only in the TGF-beta1 RiAS-treated group. Immunohistochemical studies showed a reduced accumulation of collagen and alpha-smooth muscle actin. Our experimental results suggest that ribbon antisense to TGF-beta1, with efficient uptake, effectively blocks the expression of TGF-beta1 and prevents fibrosis of the liver.