Gene Therapy Of Liver Cancer Research Articles

Effects of Raf kinase inhibitor protein on biological characteristics of hepatocellular carcinoma cells and its potential therapeutic effects

Raf kinase inhibitor protein (RKIP) is an important member of the phosphatidylethanolamine-binding protein family. This study investigated the inhibitory effect of RKIP on the malignant biological behavior of liver cancer cells in vivo and in vitro. An RKIP gene expression vector was constructed using the pcDNA3.1 vector and transfected into human hepatoma cell line HepG2 to overexpress the RKIP gene. Growth, proliferation, the cell cycle, apoptosis, migration, and invasion of the cell line were analyzed. For in vivo experiments, the vector was transfected into tumor tissue of hepatoma-bearing animals and then tumor growth was analyzed and compared with the control to assess its anti-tumor effect. Compared with the control group, cell growth in the RKIP gene transfection group (HEP-RKIP) was significantly slower. The average colony formation rate of the HEP-RKIP group was significantly lower than that of the other two groups (p < 0.05). The proportion of HEP-RKIP cells in G0/G1 phase was significantly higher than that of the control group (p < 0.05), while the proportion of cells in G2/M phase was significantly lower than that in the control group (p < 0.05). The apoptosis rate of HEP-RKIP cells was approximately 6.4%, which was significantly higher than that of the control group (p < 0.05). Migration of HEP-RKIP cells was significantly slower than that of the other two groups (p < 0.05). In an inhibition experiment of the liver tumor animal model, the tumor inhibition rate of the RKIP in vivo transfection group was significantly higher than that of each control group (p < 0.05). RKIP inhibits malignant biological behavior of liver cancer cells in vitro and has a tumor inhibitory effect in vivo, which may be a potential target for gene therapy of liver cancer.

Enhanced fluorescence/magnetic resonance dual imaging and gene therapy of liver cancer using cationized amylose nanoprobe.

Recently, various technologies for targeted gene release in cancer treatment have emerged. However, most of these strategies are facing the challenge of untraceable distribution and poor antitumour treatment effects. In this study, we constructed a gene delivery system that integrated a series of components to assemble multifunctional NPs, providing a promising theranostic nanoplatform for hepatocellular carcinoma (HCC) therapy. Cationized amylose (CA), superparamagnetic iron oxide (SPIO) nanoparticles (NPs), and tetraphenylethylene (TPE) were self-assembled to form nanospheres (CSP/TPE). The prepared NPs was modified with SP94 pepide through amidation reaction, and then survivin small interfering RNA (siRNA) were loaded into the NPs to form CSP/TPE@siRNA-SP94 NPs. Our results showed that the prepared NPs had good size distribution, high RNA condensation and transfection ability. CSP/TPE@siRNA-SP94 NPs exhibited excellent fluorescence and magnetic resonance (MR) imaging properties in vitro and in vivo. The prepared targeted NPs improved Huh-7 cellular uptake in vitro, and the biodistribution of CSP/TPE@siRNA-SP94 in vivo was observed through in/ex vivo fluorescence imaging system and MRI. As survivin siRNA effectively retained in tumour cells, CSP/TPE@siRNA-SP94 NPs considerably inhibited tumour growth in vivo. In addition, H&E staining results showed that all the prepared CSP-based NPs had good biocompatibilities, as few histological changes or tumour metastasis were observed in major organs of the mice in the treatment group. Therefore, we envisage that the prepared CSP/TPE@siRNA-SP94 NPs can represent a promising strategy for HCC diagnosis and treatment.

Cell membrane breakage and triggering T cell infiltration are involved in human telomerase reverse transcriptase (hTERT) promoter-driven novel peptide KK-64 for liver cancer gene therapy

ABSTRACT Liver cancer is an aggressive malignancy with exhibits both high mortality and morbidity. The current treatment options are associated with several limitations, novel specific anti-cancer drugs are urgently needed to improve liver cancer treatment. In this study, a new peptide KK-64 was designed, and it showed strong cytotoxicity against liver cancer cells. To obtain the tumor targeting property, a plasmid that contains KK-64 DNA fragment and driven by human telomerase reverse transcriptase (hTERT) promoter was constructed. pcTERT-kk-64 plasmid was found to specifically inhibit the viability of liver cancer cells HepG2, induce substantial apoptosis as well as damage to the cell membranes, but had minimal effects toward normal liver HL-7702 cells. Furthermore, pcTERT-kk-64 plasmids was also noted to significantly attenuate migration and invasion of HepG2 cells. The anti-tumor effect of pcTERT-kk-64 plasmid was also observed in H22 cell-bearing mice, and it appeared to cause significant tumor regression, trigger tumor cell apoptosis, and infiltrate cytotoxicity T cells to the tumor tissues after plasmids injection. Thus, pcTERT-kk-64 plasmids showed both strong cytotoxicity and tumor selectivity in vitro and in tumor-bearing mice in liver cancer models.

Targeted suicide gene therapy for liver cancer based on ribozyme-mediated RNA replacement through post-transcriptional regulation

Hepatocellular carcinoma (HCC) has high fatality rate and limited therapeutic options. Here, we propose a new anti-HCC approach with high cancer-selectivity and efficient anticancer effects, based on adenovirus-mediated Tetrahymena group I trans-splicing ribozymes specifically inducing targeted suicide gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To confer potent anti-HCC effects and minimize hepatotoxicity, we constructed post-transcriptionally enhanced ribozyme constructs coupled with splicing donor and acceptor site and woodchuck hepatitis virus post-transcriptional regulatory element under the control of microRNA-122a (miR-122a). Adenovirus encoding post-transcriptionally enhanced ribozyme improved trans-splicing reaction and decreased human TERT (hTERT) RNA level, efficiently and selectively retarding hTERT-positive liver cancers. Adenovirus encoding miR-122a-regulated ribozyme caused selective liver cancer cytotoxicity, the efficiency of which depended on ribozyme expression level relative to miR-122a level. Systemic administration of adenovirus encoding the post-transcriptionally enhanced and miR-regulated ribozyme caused efficient anti-cancer effects at a single dose of low titers and least hepatotoxicity in intrahepatic multifocal HCC mouse xenografts. Minimal liver toxicity, tissue distribution, and clearance pattern of the recombinant adenovirus were observed in normal animals administered either systemically or via the hepatic artery. Post-transcriptionally regulated RNA replacement strategy mediated by a cancer-specific ribozyme provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Visualization of gene therapy with a liver cancer-targeted adeno-associated virus 3 vector.

Background: To evaluate the feasibility of a self-complementing recombinant adeno-associated virus 3 (scrAAV3) vector targeting liver cancer and non-invasively monitor gene therapy of liver cancer.Materials and methods: An scrAAV3-HSV1-TK-kallistatin (ATK) gene drug was constructed, which contained the herpes virus thymidine kinase (HSV1-TK) reporter gene and human endogenous angiogenesis inhibitor (kallistatin) gene for non-invasive imaging of gene expression. Subcutaneous xenografted tumors of hepatoma in nude mice were generated for positron emission tomography/computed tomography (PET/CT) imaging. The ATK group was injected with the ATK gene through the tail vein, and an imaging agent was injected 2 weeks later. PET/CT imaging was performed at 1 hour after injection of the imaging agent. The control group was injected with phosphate-buffered saline at the same volume as the ATK gene drug. HE staining is used for pathological observation of tumor sections. HSV1-TK and kallistatin expression was identified by immunofluorescence, real-time quantitative PCR, and western blotting.Results: Radioactivity on PET/CT images was significantly higher in the ATK group compared with the control group. 18F-FHBG uptake values of left forelegs in ATK and control groups were 0.591±0.151% and 0.017 ± 0.011% ID/g (n=5), respectively (P<0.05). After injection of the ATK gene drug, mRNA and protein expression of HSV1-TK and kallistatin in subcutaneous xenograft tumors was detected successfully. In vitro analysis demonstrated significant differences in the expression of HSV1-TK and kallistatin between ATK and control groups (P<0.05).Conclusions: The scrAAV3 vector has a strong liver cancer-targeting ability, and the ATK gene drug can be used for targeted and non-invasive monitoring of liver cancer gene therapy.

Effects of melanoma differentiation associated gene-7 (MDA-7/IL-24) on apoptosis of liver cancer cells via regulating the expression of B-cell lymphoma-2.

The present study investigated the mechanism of selective killing of liver cancer cells of melanoma differentiation associated gene-7 (MDA-7, also called IL-24α) in order to provide a theoretical basis for gene therapy of liver cancer. A recombinant eukaryotic expression vector (pcDNA3-MDA-7) containing human MDA-7 gene was constructed, which was then delivered to liver cancer cell line HepG2 and normal liver cell line L02. The positive cell clone was screened by G418. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to confirm the occurrence of MDA-7 transcription in the transfected cells. The protein expression of MDA-7 was determined by western blot analysis. The effects of MDA-7 on liver cancer cell proliferation and apoptosis were investigated through MTT assay and flow cytometry by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining. The mitochondrial protein was extracted from the normal liver cell line L02 and liver cancer cell line HepG2 at 3 day post-culture, in which the alterations of anti-apoptotic B-cell lymphoma-2 (Bcl-2), pro-apoptotic Bcl-2 associated X protein (Bax), mitochondria-released cytochrome c and caspase 9 were determined by western blot analysis. pcDNA3-MDA-7 mediated the expression of foreign gene MDA-7 in HepG2 and L02 cells. MDA-7 promoted liver cancer cell apoptosis and inhibited cell proliferation; while no effect was exerted on normal liver cells, as determined by the MTT assay and flow cytometry. Relative to the L02 cells, the protein expression of Bcl-2 was downregulated in the HepG2 cells, while that of Bax, cytochrome c and caspase 9 were upregulated. In the study, the eukaryotic expression vector pcDNA3-MDA-7 was successfully constructed, it can mediate the expression of MDA-7 in human liver cancer cells and normal liver cells and inhibits the proliferation of human liver cancer cells through the restored expression of mitochondrial pro-apoptotic Bcl-2.

The function of Trim72 gene in hepatocellular carcinoma metastasis

Objective To investigate the role of non-small cell lung cancer metastasis-related Trim72 gene in hepatocellular carcinoma (HCC) using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 system. Methods Hepa1-6 (Cas9) HCC cells were established with stable expression of Cas9 protein, and then specific gene knockout was performed using sgRNA targeting Trim72 gene. After obtaining the Hepa1-6 (Trim72-KO) cells, the metastasis and invasion abilities of the cells were evaluated by in vitro Transwell assay and in vivo subcutaneous lung metastasis examination. Results Hepa1-6 (Trim72-KO) cell line was successfully established by the CRISPR-Cas9 system. Transwell assay indicated that the mobility of Hepa1-6 (Trim72-KO) cells was increased compared to the control cells. Transwell assay indicated that the metastasis and invasion of Hepa1-6 (Cas9) HCC cells were enhanced after the knockout of Trim72 gene. The pathological examination of lung metastasis of subcutaneous tumor in vivo showed that the subcutaneously metastatic ability of Hepa1-6 (Trim72-KO) cells (the experimental group) was significantly stronger than Hepa1-6 (Cas9) cells (the control troup) that were not transferred to the corresponding sgRNA. Conclusions The trim72 gene knocked-out HCC cells were obtained by CRISPR-Cas9 system, which showed stronger metastasis and invasion abilities than the control cells. It is suggested that Trim72 gene may play an important role in the invasion and metastasis of HCC, and Trim 72 gene is expected to be a potential target for gene therapy of liver cancer. Key words: CRISPR-Cas9 system; Trim72; Gene knock out; Hepatocellular carcinoma

Advances in gene therapy of liver cancer

Advances in gene therapy of liver cancer

Microencapsulation of recombinant adenovirus within poly-DL-lactide-poly(ethylene glycol) microspheres for enhanced gene transfection efficiency and inhibitory effects on hepatocellular carcinoma cells in vitro.

When gene therapy is performed for the treatment of malignant tumors, gene transfer efficiency and selectivity are highly important. Polymer vehicle microspheres are a novel type of therapy, which have been developed rapidly in recent years and are able to control drug release, prolong the biological half-life of drugs, decrease side effects and achieve targeted delivery. The present study was designed to construct a polymer microsphere-encapsulated recombinant adenovirus with human tissue inhibitors of the matrix metalloproteinase-1 (TIMP-1) gene, and to discuss its characterization for the purpose of liver cancer gene therapy. The microsphere was prepared from biodegradable poly-DL-lactide-poly(ethylene glycol) (PELA) encapsulating rAdTIMP-1, the recombinant adenovirus carrying TIMP-1, by a modified double-emulsion method. The particle morphology, diameter, virus encapsulation, loading rate and release kinetics of the rAd-microspheres were determined in vitro. Hepatocellular carcinoma (HCC) HepG2 cells were transfected with the rAd-microsphere and the efficiency of transfection was assessed by fluorescent microscopy. The production and expression of TIMP-1 was identified by gelatin zymography and western blot analysis, and the invasiveness was detected by a matrigel matrix invasion assay. The microsphere encapsulating rAdTIMP-1 was successfully constructed with a diameter of 1.965 μm, encapsulation efficiency of 60.0%, a viral load of 10.5 x 10(8)/mg, a virus release of ~60% within 120 h and a total release time of >240 h. The resultant rAd-microspheres were able to efficiently transfect HepG2 cells with the transfection efficiency enhanced by ~90%. As a result, the transfected HepG2 cells had significantly increased TIMP-1 enzyme activity and the expression of TIMP-1 was detected by western blot analysis. In addition, the proliferation and invasion ability of the HCC cells was markedly inhibited by the rAd-microspheres. The resultant rAd-microspheres, PELA-encapsulated recombinant TIMP-1 adenovirus, had enhanced transfection efficiency and were able to markedly inhibit the in vitro biological behavior of HepG2 cells. This provides an experimental basis for this polymer application and may pave the way for prospective in vivo clinical trials and further comprehensive therapy for liver cancer.

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

ObjectivesTo build GPC3 gene short hairpin interference RNA (shRNA) slow virus vector, observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer. MethodsHepatocellular carcinoma cell line Huh-7 was transfected by a RNA interference technique. GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR. Targeted GPC3 gene sequences of small interfering RNA (siRNA) PGC-shRNA-GPC3 were restructured. Stable expression cell lines of siRNA were screened and established with the help of liposomes (lipofectamineTM2000) as carrier transfection of human liver cell lines. In order to validate siRNA interference efficiency, GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot. The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects of GPC3 gene on Huh-7 cell proliferation and apoptosis were observed. ResultsIn the liver cancer cell lines Huh-7, GPC3 gene showed high expression. PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation. Stable recombinant plasmid transfected into liver cancer cell lines Huh-7 can obviously inhibit GPC3 mRNA expression level. ConclusionsThe targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

Potential Usefulness of Baculovirus-Mediated Sodium-Iodide Symporter Reporter Gene as Non-Invasively Gene Therapy Monitoring in Liver Cancer Cells: An In Vitro Evaluation

Primary liver cancer has one of the highest mortality rates of all cancers, and the main current treatments have a poor prognosis. This study aims to examine the efficiency of baculovirus vectors for transducing target gene into liver cancer cells and to evaluate the feasibility of using baculovirus vectors to deliver the sodium-iodide symporter (NIS) gene as a reporter gene through co-vector administration approach to monitor the expression of the target therapeutic gene in liver cancer gene therapy. We constructed (green fluorescent protein) GFP- and NIS-expressing baculovirus vectors (Bac-GFP and Bac-NIS), and measured the baculovirus transduction efficiency in HepG2 cells and other tumor cells (A549, SW1116 and 8505C), and it showed that the transduction efficiency and target gene expression level rose with increasing viral multiplicity of infection (MOI) in HepG2 cells, and HepG2 cells had a significantly higher transduction efficiency (60.8% at MOI = 200) than other tumor cells. Moreover, the baculovirus transduction was not cytotoxic to HepG2 cells at a higher MOI (MOI 5 400). We also performed dynamic iodide uptake trials, and found that Bac-NIS-transduced HepG2 cells exhibited efficient iodide uptake which could be inhibited by sodium perchlorate (NaClO₄). And we measured the correlation of fluorescent intensities and 125I uptake amount in HepG2 cells after co-vector administration with Bac-NIS and Bac-GFP at different MOIs, and found a high correlation coefficient (r(2) = 0.8447), which provides a good basis for successfully evaluating the feasibility of baculovirus-mediated NIS reporter gene monitoring target gene expression in liver cancer therapy. Therefore, this study indicates that baculovirus vector is a potential vehicle for delivering therapeutic genes in studying liver cancer cells. And it is feasible to use a baculovirus vector to deliver NIS gene as a reporter gene to monitor the expression of target genes. It therefore provides an effective approach and a good basis for future baculovirus-mediated therapeutic gene delivering or therapeutic gene expression monitoring in liver cancer cells studies.

Therapeutic efficiency of folated poly(ethylene glycol)-chitosan-graft-polyethylenimine-Pdcd4 complexes in H-ras12V mice with liver cancer

BackgroundChitosan and chitosan derivatives have been proposed as alternative and biocompatible cationic polymers for nonviral gene delivery. However, the low transfection efficiency and low specificity of chitosan is an aspect of this approach that must be addressed prior to any clinical application. In the present study, folated poly(ethylene glycol)-chitosan-graft-polyethylenimine (FPCP) was investigated as a potential folate receptor-overexpressed cancer cell targeting gene carrier.MethodsThe FPCP copolymer was synthesized in two steps. In the first step, folate-PEG was synthesized by an amide formation reaction between the activated carboxyl groups of folic acid and the amine groups of bifunctional poly(ethylene glycol) (PEG). In the second step, FPCP was synthesized by an amide formation reaction between the activated carboxyl groups of folate-PEG and amine groups of CHI-g-polyethyleneimine (PEI). The composition of FPCP was characterized by 1H nuclear magnetic resonance.Results:FPCP showed low cytotoxicity in various cell lines, and FPCP-DNA complexes showed good cancer cell specificity as well as good transfection efficiency in the presence of serum. Further, FPCP-Pdcd4 complexes reduced tumor numbers and progression more effectively than PEI 25 kDa in H-ras12V liver cancer mice after intravenous administration.ConclusionOur data suggest that FPCP, which has improved transfection efficiency and cancer cell specificity, may be useful in gene therapy for liver cancer.

Bioreducible PEI‐siRNA Nanocomplex for Liver Cancer Therapy: Transfection, Biodistribution, and Tumor Growth Inhibition In Vivo

A bioreducible polyethylenimine (SS‐PEI) was successfully applied as a nonviral carrier for the delivery of plasmid DNA and VEGF‐siRNA in vitro and in vivo. The SS‐PEI could strongly condense DNA or siRNA into nanosized complexes (below 200 nm) with positive surface charges. In vitro transfection experiments using GFP plasmid as gene reporter showed that the complexes of SS‐PEI/DNA were able to efficiently transfect HepG2 cells, with efficiency comparable to that of polyethylenimine, a gold standard for nonviral gene delivery. Moreover, the complexes of SS‐PEI/VEGF‐siRNA could lead to reduced levels of VEGF protein in HepG2 cells in vitro. Treatment with the complexes of SS‐PEI/VEGF‐siRNA efficiently inhibited HepG2 tumor growth in an xenograft mouse model. The data of this study imply that the SS‐PEI is a potent nucleic acid carrier applicable for liver cancer gene therapy.

HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a cancer targeting gene-viro-therapy strategy

BackgroundIn previously published studies, oncolytic adenovirus-mediated gene therapy has produced good results in targeting cancer cells. However, safety and efficacy, the two most important aspects in cancer therapy, remain serious challenges. The specific expression or deletion of replication related genes in an adenovirus has been frequently utilized to regulate the cancer cell specificity of a virus. Accordingly, in this study, we deleted 24 bp in E1A (bp924-bp947) and the entirety of E1B, including those genes encoding E1B 55kDa and E1B19kDa. We used the survivin promoter (SP) to control E1A in order to construct a new adenovirus vector named Ad.SP.E1A(Δ24).ΔE1B (briefly Ad.SPDD). HCCS1 (hepatocellular carcinoma suppressor 1) is a novel tumor suppressor gene that is able to specifically induce apoptosis in cancer cells. The expression cassette AFP-HCCS1-WPRE-SV40 was inserted into Ad.SPDD to form Ad.SPDD-HCCS1, enabling us to improve the safety and efficacy of oncolytic-mediated gene therapy for liver cancer.ResultsAd.SPDD showed a decreased viral yield and less toxicity in normal cells but enhanced toxicity in liver cancer cells, compared with the cancer-specific adenovirus ZD55 (E1B55K deletion). Ad.SPDD-HCCS1 exhibited a potent anti-liver-cancer ability and decreased toxicity in vitro. Ad.SPDD-HCCS1 also showed a measurable capacity to inhibit Huh-7 xenograft tumor growth on nude mice. The underlying mechanism of Ad.SPDD-HCCS1-induced liver cancer cell death was found to be via the mitochondrial apoptosis pathway.ConclusionsThese results demonstrate that Ad.SPDD-HCCS1 was able to elicit reduced toxicity and enhanced efficacy both in vitro and in vivo compared to a previously constructed oncolytic adenovirus. Ad.SPDD-HCCS1 could be a promising candidate for liver cancer therapy.

Gene-viro-therapy targeting liver cancer by a dual-regulated oncolytic adenoviral vector harboring IL-24 and TRAIL

Cancer-targeting gene-viro-therapy is a promising cancer therapeutic strategy that strengthens the antitumor effect of oncolytic viruses by expressing an inserted foreign antitumor gene. To achieve liver cancer targeting and to improve the safety of the ZD55 vector (a widely-used E1B55KD gene-deleted oncolytic adenoviral vector (OV), we previously constructed), we designed a novel OV named Ad·AFP·D55 that selectively replicates in hepatocellular carcinoma (HCC) cells by replacing the E1A promoter with the liver-cancer specific α-Fetoprotein (AFP) promoter based on the ZD55 vector. We found that the oncolytic adenoviruses Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL express tumor-suppressor gene interleukin-24 (IL-24) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), respectively, significantly suppressed the HCC cell growth in vitro by inducing apoptosis by the caspase-8 and mitochondria-dependent caspase-9 signaling pathways. Furthermore, the combined treatment of Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL showed strong antitumor effects in vivo by significantly inhibiting the tumor growth in HCC HuH-7 cell xenograft mice, and markedly increasing animal survival rate. Therefore, this novel HCC cell-targeting OV carrying tumor-suppressor genes may provide a promising approach for liver cancer gene therapy.

Effect of Recombinant Plasmid pEGFP-AFP-hTNF on Liver Cancer Cells (HepG2 Cells) in vitro when Delivered by PEG-PEI/Fe 3O 4 Nanomagnetic Fluid

Gene delivery into liver cancer cells has been a problem. This study aimed to understand the effect of using PEGI/Fe₃O₄ nanomagnetic fluid as a gene vector for liver cancer gene therapy. An AFP enhancer aids in the expression of tumor-specific foreign genes in AFP-producing cancer cells like HepG2 cells, and was utilized in the delivery method in this study. We constructed recombinant plasmid PEGFP-AFP-hTNFα, which was transfected into AFP positive HepG2 cells and AFP negative Hela cells by PEG-PEI/Fe₃O₄ nanomagnetic fluid. Fluorescence microscopy was used to evaluate the transfection rate of the hTNFα gene in the HepG2 cells 12 hours after transfection. Reverse transcription polymerase chain reaction (RT-PCR) and western blot were used to detect expression of hTNFα gene in the HepG2 cells 48 hours after transfection. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the inhibitory effect of hTNFα on the proliferation of HepG2 cells. Flow cytometry was used to analyze the apoptosis of HepG2 cells. Plasmid PEGFP-AFP-hTNFα delivered by PEG-PEI/Fe₃O₄ nanomagnetic fluid was successfully transfected into HepG2 cells and expressed in the HepG2 cells. The transfection efficacy of hTNFα gene delivered by PEG-PEI/Fe₃O₄ nanomagnetic fluid was superior to that of hTNFα gene delivered by lipofectamine in HepG2 cells. RT-PCR and western blot demonstrated that hTNFα gene was expressed in HepG2 cells that were transfected with complexes of nanomagnetic fluid/PEGFP-AFP-hTNFα. MTT and flow cytometry showed that the hTNFα gene markedly exerted a cell killing effect. PEG-PEI/Fe₃O₄ nanomagnetic fluid successfully transfected PEGFP-AFP-hTNFα into HepG2 cells and induced expression of hTNFα gene in the HepG2 cells, thus showing promise as a gene vector for liver cancer gene therapy. Furthermore, an AFP enhancer can specifically increase the expression of target genes in cells positive for AFP.

Gene therapy of liver cancer: an update

Le cancer du foie est l’une des maladies les plus importantes au monde, en raison de sa haute incidence et de sa resistance notable aux traitements classiques. Le carcinome hepatocellulaire (CHC) represente plus de 80 % des cancers primitifs du foie. Le poids du CHC en Afrique est remarquable, car certains des facteurs etiologiques majeurs, tels que l’hepatite B chronique et l’exposition aux mycotoxines, sont particulierement frequents dans ce continent. Outre le besoin urgent de campagnes de prevention, il faut de nouvelles armes therapeutiques pour ameliorer la prise en charge des patients atteints de CHC. La therapie genique (TG) est une discipline experimentale en evolution rapide, qui vise a surmonter les obstacles importants identifies dans les premiers essais cliniques. Malgre les difficultes particulieres inherentes au transfert de genes dans des tumeurs, le cancer reste l’un des principaux domaines d’application de la TG. Les recents progres scientifiques et techniques en TG et en therapie mixte geneticocellulaire, associes aux nouvelles techniques de radiotherapie et d’immunotherapie chez les patients atteints de CHC, ouvrent de nouvelles perspectives dans ce domaine. Nous resumons ici les tendances actuelles de la TG pour le traitement du CHC.

Killing effects of PNP-CD chimeric suicide gene vector on HCC cells

Objective To investigate the cytotoxic effects and mechanism of PNP-CD chimeric gene vector originated from PNP/MeP-dR system on HCC cells. Methods The fusion suicide gene PNP-CD obtained by site directed mutagenesis technique was subcloned into pcDNA3.0 to construct a eukaryotic expression vector containing a chimeric gene, pcDNA3.0/ PNP-CD. After being identified by recombinant enzyme, PCR and subsequent sequencing, it was transfected into HepG2 cells by liposome-mediation method. The G418-resistant cellular clone with stable transfection of pcDNA3.0/PNP-CD, HepG2/PNP-CD was established by selection. The expression of PNP-CD gene was also verified by RT-PCR and Western blotting. The curve of cellular growth was assayed by Trypan blue exclusion. The cellular sensitivity of HepG2/PNP-CD to its specific prodrugs and its bystander effects were also assayed by MTT method. Results The chimeric gene, PNP-CD, was inserted into pcDNA3.0 correctly, and the stable expression of pcDNA3.0/PNP-CD in HepG2 cells was confirmed.This cellular clone was highly sensitive to its corresponding prodrugs. It was indicated that its bystander effects with the synergetic treatment of its specific prodrugs were substantially higher than those caused by the same vector with the administration of only a single prodrug, MeP-dR. Conclusion The bi-functional fusion suicide gene vector, pcDNA3.0/PNP-CD, yields powerful cytotoxic effects on HCC cells in the presence of the synergetic treatment of its specific prodrugs, which would be a high-performance therapeutic vector in gene therapy for liver cancer. Key words: Carcinoma hepatocellular; PNP/MeP-dR suicide gene system; CD/5-FC suicide gene system; Chimeric gene; Gene therapy

Selenium-Bifidobacterium longum as a delivery system of endostatin for inhibition of pathogenic bacteria and selective regression of solid tumor

Bifidobacterium longum (B. longum) as a delivery system for endostatin was shown to have definite antitumor effects. Moreover, it was found that the enrichment of selenium was able to enhance the immunity of mice. In order to further evaluate the safety and efficacy of B. longum carrying pBV22210-endostatin (B. longum-En) enriched with selenium (Se-B. longum-En), we determined the biochemical characteristics of Se-B. longum-En. We then investigated its effect on macrophage activity, as well as its inhibitory effect on the multiplication of pathogenic bacteria in vitro and the antitumor effects on murine hepatic (H22) tumor-bearing mice. The results showed that Se-B. longum-En exhibited similar biochemical characteristics to that of wild-type B. longum, i.e., Se-B. longum-En strongly enhanced macrophage phagocytosis in rats and inhibited the growth of pathogenic bacteria. In addition, Se-B. longum-En showed a definite inhibitory effect of tumor growth when H22 tumor-bearing mice were fed through oral or tail vein delivery. These results suggested that Se-B. longum is able to retain the advantages of wild-type B. longum and be used as a novel gene delivery system for liver cancer gene therapy.

Selenium-Bifidobacterium longum as a delivery system of endostatin for inhibition of pathogenic bacteria and selective regression of solid tumor

Bifidobacterium longum (B. longum) as a delivery system for endostatin was shown to have definite antitumor effects. Moreover, it was found that the enrichment of selenium was able to enhance the immunity of mice. In order to further evaluate the safety and efficacy of B. longum carrying pBV22210-endostatin (B. longum-En) enriched with selenium (Se-B. longum-En), we determined the biochemical characteristics of Se-B. longum-En. We then investigated its effect on macrophage activity, as well as its inhibitory effect on the multiplication of pathogenic bacteria in vitro and the antitumor effects on murine hepatic (H22) tumor-bearing mice. The results showed that Se-B. longum-En exhibited similar biochemical characteristics to that of wild-type B. longum, i.e., Se-B. longum-En strongly enhanced macrophage phagocytosis in rats and inhibited the growth of pathogenic bacteria. In addition, Se-B. longum-En showed a definite inhibitory effect of tumor growth when H22 tumor-bearing mice were fed through oral or tail vein delivery. These results suggested that Se-B. longum is able to retain the advantages of wild-type B. longum and be used as a novel gene delivery system for liver cancer gene therapy.