Antitumor Effect of TRAIL on Oral Squamous Cell Carcinoma using Magnetic Nanoparticle-Mediated Gene Expression

We developed a new magnetic nanovector to improve the efficiency and targeting of transgene therapy for oral squamous cell carcinoma (OSCC). Positively charged polymer PEI-modified Fe₃O₄ magnetic nanoparticles were tested as gene transfer vectors in the presence of a magnetic field. The Fe₃O₄ nanoparticles were prepared by a co-precipitation method and had good dispersibility in water. These nanoparticles modified by PEI were combined with negatively charged pACTERT-EGFP via electrostatic interaction. The transfection efficiency of the magnetic nano-gene vector with the magnetic field was determined by a fluorescence-inverted microscope and flow cytometry. The results showed significant improvement compared with the control group (p < 0.05). The magnetic complexes also exhibited up to 6-times higher transfection efficiency compared with commonly used PEI or lipofectin. On the basis of these results, the antitumor effect with suicide gene therapy using pACTERT-TRAIL in vitro and vivo was evaluated. In vitro apoptosis was determined with the Annexin V-FITC Apoptosis Detection Kit. The results suggested that PEI-modified Fe₃O₄ nanoparticles could mediate the killing of Tca83 cells. Furthermore, treatment with pACTERT-TRAIL delivered by magnetic nanoparticles showed a significant cytostatic effect through the induction of apoptosis in a xenograft model. This indicates that magnetic nano-gene vectors could improve the transgene efficiency for Tca83 cells and could exhibit antitumor functions with the plasmid pACTERT-TRAIL. This may be a new way to treat OSCC.     

新型聚乙烯亚胺衍生物在Hep G2 细胞中转染和毒性的研究

目的:研究以对苯二甲醛( Terephthalaldehyde)为连接剂的聚乙烯亚胺(Polyethyleneimine,PEI)衍生物PEI-Tp对肝癌细胞Hep G2的转染活性和细胞毒性的影响.方法:以荧光素酶质粒作为报告基因,研究高分子和DNA的复合物在Hep G2细胞中的转染活性,用MTT的方法研究高分子对Hep G2细胞的毒性.结果:Hep G2细胞转染结果显示构建的聚乙烯亚胺衍生物PEI-Tp具有高效输送质粒的能力;细胞毒性结果显示PEI-Tp随着浓度的增加,其毒性显著低于PEI25 kDa.结论:Hep G2细胞实验数据显示PEI-Tp是一种高效、低毒,在基因治疗领域有相当前景的非病毒载体.     

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.     

Inactivation of PTEN is responsible for the survival of Hep G2 cells in response to etoposide-induced damage

The chemo-resistance character of human hepatocellular carcinoma cells is well known but the anomalies associated with such resistance character are not completely understood. In this study, etoposide-induced signaling events in human hepatocellular carcinoma cell line, Hep G2 has been compared with Chang Liver cells, a normal human liver cell line. Hep G2 cells are resistant to etoposide when compared with Chang Liver cells. Etoposide-induced γH2AX foci in Hep G2 cells are persisted for a longer time without affecting cell cycle, indicating that Hep G2 cells are able to maintain its growth with damaged DNA. Further, Akt signaling pathway is deregulated in Hep G2 cells. The upstream negative regulator of Akt, PTEN remains inactive, as it is hyperphosphorylated in Hep G2 cells. Inhibition of PI-3K pathway by wortmannin partially reverses the etoposide-resistance character of Hep G2 cells. Either Hep G2 or Chang Liver cells when transfected with plasmid carrying active Akt (myr-Akt) become resistance towards etoposide compared to the cells transfected with empty vectors or kinase defective Akt. Transient transfection of wild type PTEN in Hep G2 cells does not change its response towards etoposide whereas Chang Liver cells become sensitive after transfection with same plasmid. These results suggest that inactivation of PTEN, which renders activation of Akt, may contribute largely for the etoposide-resistance character of Hep G2 cells.     

Comparison of Transfection Efficiency of Nonviral Gene Transfer Reagents

This study compared six commercially available reagents (Arrest-In, ExpressFect, FuGENE HD, jetPEI, Lipofectamine 2000, and SuperFect) for gene transfection. We examined the efficiency and cytotoxicity using nine different cell lines (MC3T3-E1 mouse preosteoblasts, PT-30 human epithelial precancer cells, C3H10T1/2 mouse stem cells, MCF-7 human breast cancer cells, HeLa human cervical cancer, C2C12 mouse myoblasts, Hep G2 human hepatocellular carcinoma, 4T1 mouse mammary carcinoma, and HCT116 human colorectal carcinoma), and primary cells (HEKn human epidermal keratinocytes) with two different plasmid DNAs encoding luciferase or β-galactosidase in the presence or absence of serum. Maximal transfection efficiency in MC3T3-E1, C3H10T1/2, HeLa, C2C12, Hep G2, and HCT116 was seen using FuGENE HD, in PT-30, 4T1, and HEKn was seen using Arrest-In, and in MCF-7 was seen using jetPEI. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetPEI and ExpressFect. These results suggest that FuGENE HD is the most preferred transfection reagent for many cell lines, followed by Arrest-In and jetPEI. These results may be useful for improving nonviral gene and cell therapy applications.     

Cationic lipid polymerization as a novel approach for constructing new DNA delivery agents

In vivo gene delivery mediated by cationic lipids is often compromised by aggregation due to complexation with proteins in the blood. To improve the stability of cationic lipid-DNA complexes, the present study aimed to develop a novel approach in which a poly(cationic lipid) (PCL) is utilized to form stable cationic polyplexes for gene transfection. Hydrogenation of the acrylamide analogue of betaAE-DMRI, the polymerizable precursor of PCL, provided a monomeric lipid derivative (MHL) which was used for direct comparison of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol (Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G2 cells as indicated by an LDH leakage assay. Furthermore, PCL was significantly less toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were resistant to aggregation even after 24 h of exposure to solutions containing 50 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectamine extensively aggregated after 24 h of exposure to 50% FBS. To examine the influence of lipid polymerization on gene transfer activity, liposome-mediated transfections of a luciferase vector (pGL3) were performed in Hep G2 and Alexander cell lines. The luciferase activity of the PCL formulations in Hep G2 cells were similar to those of the MHL, DOTAP-Chol and lipofectamine formulations, demonstrating that lipid polymerization does not compromise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the novel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most resistant to serum-induced aggregation and had comparable transfection activity when coformulated with cholesterol. This novel polymerization approach for the development of stable and active polyplexes may prove a valuable alternative for in vivo gene delivery.     

Plasmid DNA-entrapped nanoparticles engineered from microemulsion precursors: in vitro and in vivo evaluation

Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provide protection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle delivery system was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing with cationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 degrees C with emulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply cooling the O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperature while stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM. Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, the nanoparticles were coincubated with simulated biological media to assess their stability. In vitro hepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coated with pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containing I-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on the hydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm in diameter) were engineered within minutes from warm O/W microemulsion precursors. The nanoparticles were negatively charged (-6 to -15 mV) and spherical. An anionic exchange column was used to separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was approximately 30%. Free 'naked' pDNA was completely digested by serum nucleases while the entrapped pDNA remained intact. Moreover, in vitro transfection studies in Hep G2 cells showed that pullulan-coated nanoparticles resulted in enhanced luciferase expression, compared to both pDNA alone and uncoated nanoparticles. Preincubation of the cells with free pullulan inhibited the transfection. Finally, 30 min after tail vein injection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over 40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles in the blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.     

Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.     

葡聚糖磁性纳米颗粒外加磁场对人树突状细胞基因转染效率的研究

目的研究葡聚糖磁性纳米颗粒（the dextran coated magnetic iron oxide nanoparticles,DMN）在外加钕一铁一硼稀土固定磁场的作用下对人树突状细胞转染效率以及安全性的影响。方法先通过磁力计对DMN进行分析;再将修饰有多聚赖氨酸（Poly-L—Lysine,PLL）的DMN携带绿色荧光蛋白pEGFP—Cl质粒报告基因,在钕-铁-硼稀土周定强磁场的作用下,体外转染人树突状细胞,用荧光显微镜直接观察和流式细胞仪检测来评价外加磁场对DMN作为人树突状细胞转染载体效率的影响;在转染后采用MTT比色法测定在磁场干预下的DMN对人树突状细胞增殖和功能的影响以了解其细胞毒性。结果DMN的核心直径〈30nm,具有明硅的超顺磁性,比饱和磁化强度也明显高于相同Fe3O4含量的普通磁块;DMN作为基因载体在外加磁场作用下,转染12h即可将报告基因转染至人树突状细胞内并成功表达,在荧光显微镜下可观察到绿色荧光细胞,24h转染率可达到最高（约为27％）,转染效率较未加磁场组提高了2～4倍。而且转染后的人树突状细胞增殖活性及功能未因DMN外加磁场及其作用时间的长短而受到影响。结论超顺磁性的DMN在外加磁场作用下可以明显、安全、有效地提高对人树突状细胞的转染效率。     

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells

Summary. Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.     

Dual-responsive magnetic core-shell nanoparticles for nonviral gene delivery and cell separation

We present the synthesis of dual-responsive (pH and temperature) magnetic core-shell nanoparticles utilizing the grafting-from approach. First, oleic acid stabilized superparamagnetic maghemite (γ-Fe(2)O(3)) nanoparticles (NPs), prepared by thermal decomposition of iron pentacarbonyl, were surface-functionalized with ATRP initiating sites bearing a dopamine anchor group via ligand exchange. Subsequently, 2-(dimethylamino)ethyl methacrylate (DMAEMA) was polymerized from the surface by ATRP, yielding dual-responsive magnetic core-shell NPs (γ-Fe(2)O(3)@PDMAEMA). The attachment of the dopamine anchor group on the nanoparticle's surface is shown to be reversible to a certain extent, resulting in a grafting density of 0.15 chains per nm(2) after purification. Nevertheless, the grafted NPs show excellent long-term stability in water over a wide pH range and exhibit a pH- and temperature-dependent reversible agglomeration, as revealed by turbidimetry. The efficiency of γ-Fe(2)O(3)@PDMAEMA hybrid nanoparticles as a potential transfection agent was explored under standard conditions in CHO-K1 cells. Remarkably, γ-Fe(2)O(3)@PDMAEMA led to a 2-fold increase in the transfection efficiency without increasing the cytotoxicity, as compared to polyethyleneimine (PEI), and yielded on average more than 50% transfected cells. Moreover, after transfection with the hybrid nanoparticles, the cells acquired magnetic properties that could be used for selective isolation of transfected cells.     

Apoptosis of Hepatocellular Carcinoma Cells Induced by Nanoencapsulated Polysaccharides Extracted from Antrodia Camphorata

Antrodia camphorata is a well-known medicinal mushroom in Taiwan and has been studied for decades, especially with focus on anti-cancer activity. Polysaccharides are the major bioactive compounds reported with anti-cancer activity, but the debates on how they target cells still remain. Research addressing the encapsulation of polysaccharides from A. camphorata extract (ACE) to enhance anti-cancer activity is rare. In this study, ACE polysaccharides were nano-encapsulated in chitosan-silica and silica (expressed as ACE/CS and ACE/S, respectively) to evaluate the apoptosis effect on a hepatoma cell line (Hep G2). The results showed that ACE polysaccharides, ACE/CS and ACE/S all could damage the Hep G2 cell membrane and cause cell death, especially in the ACE/CS group. In apoptosis assays, DNA fragmentation and sub-G₁ phase populations were increased, and the mitochondrial membrane potential decreased significantly after treatments. ACE/CS and ACE/S could also increase reactive oxygen species (ROS) generation, induce Fas/APO-1 (apoptosis antigen 1) expression and elevate the proteolytic activities of caspase-3, caspase-8 and caspase-9 in Hep G2 cells. Unsurprisingly, ACE/CS induced a similar apoptosis mechanism at a lower dosage (ACE polysaccharides = 13.2 μg/mL) than those of ACE/S (ACE polysaccharides = 21.2 μg/mL) and ACE polysaccharides (25 μg/mL). Therefore, the encapsulation of ACE polysaccharides by chitosan-silica nanoparticles may provide a viable approach for enhancing anti-tumor efficacy in liver cancer cells.     

Poly(DMAEMA-NVP)-b-PEG-galactose as gene delivery vector for hepatocytes

A block copolymer composed of cationic polymer and poly(ethylene glycol) (PEG) was used as a DNA carrier. Poly(2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-N-vinyl-2-pyrrolidone (NVP)) having a terminal carboxylic group was synthesized by free radical polymerization using an initiator, 4,4'-azobis(4-cyanovaleric acid). The terminal carboxylic acid was activated by N-hydroxysuccinimide (NHS) with dicyclohexylcarbodiimide (DCC) and then conjugated with PEG-bis(amine). For specific gene targeting to asialoglycoprotein receptor of hepatocytes, a galactose moiety was incorporated into the PEG terminal end of poly(DMAEMA-NVP)-b-PEG by reductive coupling using lactose and sodium cyanoborohydride. RSV luciferase plasmid was used as a reporter gene, and in vitro gene transfection efficiency was measured in HepG2 human hepatocarcinoma cells. Poly(DMAEMA-NVP)-b-PEG-galactose/DNA complexes formed at 0.5-2 polymer/plasmid weight ratio had compacted structures around 200 nm particle size and exhibited slightly negative surface charge. These complexes were coated with a cationic, pH sensitive, endosomolytic peptide, KALA, to generate positively charged poly(DMAEMA-NVP)-b-PEG-galactose/DNA/KALA complex particles. In the presence of serum proteins, both the PEG block and the galactose moiety of poly(DMAEMA-NVP)-b-PEG-galactose greatly enhanced the gene transfection efficiency, which was very close to that of Lipofectamine plus. Irrespective of the presence of serum proteins, as the KALA/DNA weight ratio increased, the transfection efficiency of poly(DMAEMA-NVP)-b-PEG-galactose was enhanced due to the pH dependent endosomal disruptive property of KALA. This study demonstrates that sufficient transfection efficiency as high as that of commercial agent could be attained by judicious formulation of molecular engineered poly(DMAEMA-NVP)-b-PEG-galactose in combination with an endosomolytic peptide, KALA.     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.     

Suppression of E-cadherin Mediates Gallotannin Induced Apoptosis in Hep G2 Hepatocelluar Carcinoma Cells

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.     

Immobilization of HIV-1 TAT peptide on gold nanoparticles: A feasible approach for siRNA delivery

RNA interference is one of the prosperous approaches for cancer treatment. However, small interfering RNA (siRNA) delivery to cancer cells has been faced with various challenges restricting their clinical application over the decades. Since ROR1 is an onco-embryonic gene overexpressed in many malignancies, suppression of ROR1 by siRNA can potentially fight cancer. Herein, a delivery system for ROR1 siRNA based on HIV-1 TAT peptide-capped gold nanoparticles (GNPs) was developed to treat breast cancer. Besides, we introduced a new feasible method for conjugating the peptide to the nanoparticles. Since the GNPs have high affinity to the sulfur, the findings demonstrated the peptide successfully conjugated to the nanoparticles via Au–S bonds. As positively charged nanoparticles showed high cellular uptake, we could use a low concentration of nanoparticles led to high efficient gene transfection with negligible cytotoxicity that was confirmed by flow cytometry, confocal microscopy, gel retardation, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Following transfection, downregulation of ROR1 and its targeted gene, CCND1, induced apoptosis in cancer cells. In conclusion, the reported capped GNPs could be potentially utilized for delivering negatively charged therapeutic agents in particular genes.     

Formation and characterization of three dimensional human hepatocyte cell line spheroids on chitosan matrix for in vitro tissue engineering applications

Chitosan was used as a matrix to induce three-dimensional spheroids of HepG2 cells. Chitosan films were prepared and used for culturing Hep G2 cells. Attachment kinetics of the cells was studied on the chitosan films. The optimum seeding density of the Hep G2 cells, required for three-dimensional spheroid formation was determined and was found to be 5 × 10⁴/ml. The growth kinetics of Hep G2 cells was studied using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, and morphology of the cells was studied through optical photographs taken at various days of culture. The liver cell functions of the spheroids were determined by measuring albumin and urea secretions. The results obtained from these studies have shown that the culture of Hep G2 cells on chitosan matrix taking appropriate seeding density resulted in the formation of three-dimensional spheroids and exhibited higher amount of albumin and urea synthesis compared to monolayer culture. These miniature “liver tissue like” models can be used for in vitro tissue engineering applications like preliminary evaluation of the toxicity of drugs and chemicals.     

Senescence marker protein-30 (SMP30) induces formation of microvilli and bile canaliculi in Hep G2 cells

Senescence marker protein-30 (SMP30) is an androgen-independent factor that decreases with aging. To elucidate the physiological functions of SMP30, we transfected human SMP30 cDNA into the human hepatoma cell line, Hep G2. These Hep G2/SMP30 transfectants, which stably expressed large amounts of SMP30, proliferated at a slower rate and synthesized less DNA than mock transfectants (Hep G2/pcDNA3 controls). Thus, enhanced expression of SMP30 retarded the growth of Hep G2/SMP30 cells. Ultrastructural studies by scanning electron microscopy revealed numerous microvilli covering the surfaces of Hep G2/SMP30 cells, whereas few microvilli appeared on control cells. Subsequently, transmission electron microscopy revealed that groups of Hep G2/SMP30 cells exhibited bile canaliculi and possessed specialized adhesion contacts, such as tight junctions and desmosomes, at interplasmic membranes. However, in controls, units of only two cells were seen, and these lacked specialized adhesion junctions. Moesin and ZO-1 are known to be concentrated in microvilli and at tight junctions, respectively. Double-immunostaining was performed to examine whether moesin and ZO-1 were expressed in bile canaliculi with microvilli at the apical regions of Hep G2/SMP30 cells. The intensity of moesin and ZO-1 staining in the contact regions of each cell was markedly higher in Hep G2/SMP30 than in control cells. Moreover, moesin stained more interior areas, which corresponded to the microvilli of bile canaliculi. Clearly, bile canaliculi with microvilli formed at the apical ends of Hep G2/SMP30 cells. These results indicate that SMP30 has an important physiological function as a participant in cell-to-cell interactions and imply that the down-regulation of SMP30 during the aging process contributes to the deterioration of cellular interactivity.Akihito Ishigami and Toshiko Fujita contributed equally to this work.This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (to S.H., A.I., and N.M.), and a grant from the Health and Labour Sciences Research Grants for Comprehensive Research on Aging and Health and Research on Dementia and Fracture supported by the Ministry of Health, Labour, and Welfare, Japan (to A.I and N.M.), and a Grant-in-Aid for Smoking Research Foundation, Japan (to N.M.).