Strategy For Cancer Gene Therapy Research Articles

Antitumor effects of a dual-specific lentiviral vector carrying the Escherichia coli purine nucleoside phosphorylase gene

The Escherichia coli purine nucleoside phosphorylase/Fludarabine phosphate (ePNP/Fludara) suicide system has several drawbacks, such as side-effects and the low efficiency of ePNP expression. In this study, we evaluated the antitumor effects of the dual-specific 8HSEs-hTERTp-ePNP/Fludara suicide system under hyperthermia in vitro and in vivo. Luciferase activities from the 8HSEs-hTERT and CMV promoters were compared using the dual luciferase assay in SW480 (high hTERT expression) and MKN74 cells (hTERT-negative) in the presence and absence of hyperthermia. Then, we investigated the effects of overexpressing the suicide gene ePNP using 8HSEs-hTERT-driven lentiviral vectors with Fludara on in vitro cell viability, side-effects, apoptosis, cycle distribution, colony formation and in vivo xenograft tumor growth. At 43°C, luciferase activity from the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN74 cells. Importantly, luciferase activities from the 8HSEs-hTERT promoter were much higher than from the CMV promoter in hTERT-expressing SW480 cells under heated conditions. The in vitro quantitative analysis showed a 4-fold higher ePNP protein expression from the 8HSEs-hTERT promoter at 43°C than at 37°C in SW480 cells and the ePNP mRNA expression in SW480 cells at 43°C was also higher than at 37°C. Conversely, ePNP mRNA and protein expression were low, almost absent, in hTERT-negative MKN74 cells with or without hyperthermia. After Fludara addition, cell cytotoxicity assays showed that the significant inhibitory effect of the 8HSEs-hTERTp-ePNP on SW480 cells was dose- and time-dependent with hyperthermia. The 8HSEs-hTERTp-ePNP/Fludara suicide system significantly inhibited SW480 cell viability, colony formation, cell cycle progression and induced apoptosis in vitro, but also induced significant bystander effects, especially under the heated conditions. At the protein level, the suicide system significantly promoted Bax, caspase-3 and p53 expression and suppressed Bcl-2 expression. In sections from mouse xenografts, TUNEL assays showed that the suicide system reduced xenograft growth and induced SW480 apoptosis. These results indicated that the combinatorial cancer- and heat-specific promoter system has great potential for improving the efficacy of cancer treatment with hyperthermia. The 8HSEs-hTERTp-ePNP/Fludara system may serve as a powerful strategy for cancer gene therapy combined with hyperthermia.

ICT1 knockdown inhibits breast cancer cell growth via induction of cell cycle arrest and apoptosis.

The protein encoded by immature colon carcinoma transcript1 (ICT1) is a component of the human mitochondrial ribosome, and is reported to be implicated in cell proliferation, viability and apoptosis of HeLa cells. This study was conducted to investigate the role of ICT1 in human breast cancer. Oncomine database was used to investigate ICT1 expression in human breast cancer tissues compared to normal tissues. The results showed that ICT1 was highly overexpressed in various human breast cancer subtypes. Then short hairpin RNA (shRNA)-mediated knockdown of ICT1 was performed in human breast cancer ZR-75-30 and T-47D cells. A series of functional analysis, including MTT, colony formation and flow cytometry assays were conducted after ICT1 knockdown. Our results demonstrated that knockdown of ICT1 significantly suppressed cell viability and proliferation through cell cycle arrest at the G2/Mphase and induced apoptosis in breast cancer cells. Furthermore, knockdown of ICT1 altered signaling pathways associated with cell growth and apoptosis, including phospho‑BAD (Ser112), phospho-PRAS40 (Thr246) and induction of phospho‑AMPKα (Thr172). Additionally, it was further confirmed by western blot analysis that ICT1 knockdown altered the expression of apoptosis- or cell cycle‑related proteins such as Bcl-2, caspase-3, CDK1, CDK2 and cyclinB. In conclusion, targeting ICT1 in breast cancer cells may provide a new strategy for breast cancer gene therapy.

IRES-Mediated Protein Translation Overcomes Suppression by the p14ARF Tumor Suppressor Protein.

Internal ribosome entry sites (IRES elements) have attracted interest in cancer gene therapy because they can be used in the design of gene transfer vectors that provide bicistronic co-expression of two transgene products under the control of a single promoter. Unlike cellular translation of most mRNAs, a process that requires a post-translational 5' modification of the mRNA known as the cap structure, IRES-mediated translation is independent of the cap structure. The cellular conditions that may intervene to modulate IRES-mediated, cap-independent versus cap-dependent translation, however, remain poorly understood, although they could be critical to the choice of gene transfer vectors. Here we have compared the effects of the p14ARF (Alternate Reading Frame) tumor suppressor, a translational suppressor frequently overexpressed in cancer, on cap-dependent translation versus cap-independent translation from the EMCV viral IRES often used in bicistronic gene transfer vectors. We find that ectopic overexpression of p14ARF suppresses endogenous and ectopic cap-dependent protein translation, consistent with other studies. However, p14ARF has little or no effect on transgene translation initiated within an IRES element. This suggests that transgenes placed downstream of an IRES element will retain efficient translation of their gene products in the presence of high levels of ectopic or endogenous p14ARF, a finding that could be particularly relevant to therapeutic gene therapy strategies for cancer.

Virus-stimulated neutrophils in the tumor microenvironment enhance T cell-mediated anti-tumor immunity.

The tumor microenvironment (TME) fosters tumors by attenuating anti-tumor immunity, reinforcing tumor cell survival and increasing angiogenesis. Among the constituents of the TME, here, we focused on tumor-associated neutrophils (TANs). First, we found that the combination of poly I:C and inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) synergistically suppressed tumor growth in the B16-F10 melanoma mouse model. In this model, poly I:C contributed to the recruitment of CD11b+Ly6G+ neutrophils to the TME, and co-injection of poly I:C and HVJ-E increased CD11b+Ly6G+FAS+ TAN in the TME. Depletion of neutrophils abolished the synergistic anti-tumor effect of HVJ-E and poly I:C in B16-F10 tumors. We revealed that C-X-C motif chemokine ligand 2 (CXCL2) is produced in the TME by poly I:C, but HVJ-E enhanced neutrophil infiltration of the TME does not occur. An anti-CXCL2 antibody inhibited the tumor suppression by HVJ-E+poly I:C. HVJ-E in combination with recombinant CXCL2 protein or CXCL2 pDNA suppressed mouse melanoma by increasing cytotoxic T lymphocyte activity against B16-F10 melanoma, which was abolished by an anti-Ly6G antibody. HVJ-E directly and indirectly increased FAS and ICAM-1 expression in cultured bone marrow-derived naïve neutrophils. Thus, HVJ-E activates anti-tumor immunity via anti-tumorigenic neutrophils in the TME. An HVJ-E vector containing the CXCL2 gene may be applicable as a novel cancer gene therapy strategy.

851 Designing of a Novel Strategy for Cancer Gene Therapy by Selective Delivery of Adenovirus-Based Toxin

851 Designing of a Novel Strategy for Cancer Gene Therapy by Selective Delivery of Adenovirus-Based Toxin

Polyelectrolyte complexes of hTERT siRNA and polyethyleneimine: Effect of degree of PEG grafting on biological and cellular activity

Gene silencing by siRNA (short interfering RNA)-targeted human telomerase reverse transcriptase (hTERT) is considered a successful strategy for cancer gene therapy. Polyelectrolyte complexes (PEC) of siRNA and cationic polymers such as polyethyleneimine (PEI) have been widely used for cellular transfection; however, they demonstrate some disadvantages such as cytotoxicity and extracellular matrix restrictions. PEG grafting technology was used in an attempt to improve the biocompatibility of PECs. Considering that this technology may compromise the cellular uptake of PECs, we aimed to study the effect of degree of PEI PEGylation on the carrier cytotoxicity, cellular association, and transfection efficiency of hTERT siRNA in the lung cancer cell line A549. Activated NHS ester of methoxy PEG–COOH 5 KDa was grafted to hyperbranched PEI 25 KDa in the molar ratios of 0.2 and 1. The copolymers were characterized by 1H-NMR spectroscopy. PECs of PEI or PEG-g-PEI with siRNA, alone or co-incubated with heparin sulfate, were studied by the ethidium bromide exclusion assay. Cytotoxicity of the polymers (PEG-g-PEI vs PEI), alone and upon formation of PEC nanoparticles with hTERT siRNA, was determined by a validated MTT assay, in comparison to a scrambled control sequence, in A549 human lung carcinoma cells. The cellular uptake of the PECs of FITC-labeled siRNA was investigated by flow cytometry at different N/P ratios, and the silencing effect of the transfected siRNA was compared to that of the control sequence for different PECs by real time RT-PCR. The cytotoxicity of PEI decreased significantly by PEG grafting, even at a low degree of PEGylation. Moreover, the nonspecific cytotoxicity of PECs decreased by PEG grafting. PECs of PEG-g-PEI showed more biologic stability on incubation with heparin sulfate. Average particle size and zeta potential of PEC nanoparticles were diminished for those of PEG-g-PEI. The cellular association was more pronounced at an N/P ratio of 2.5 for PECs of PEI and PEG-g-PEI alike. The level of silencing of hTERT mRNA by PEC of PEG-g-PEI was sequence-dependent, and determined non-inferior when compared to the native PEI. Conclusively, the biocompatibility of PEI was improved by a low degree of PEGylation, with no adverse effect on the cellular uptake and the transfection activity. PEC nanoparticles of hTERT siRNA and PEG-g-PEI could act as a promising weapon against A549 cells, which has to be considered for an in vivo evaluation.

24. Targeted Anticancer Approach Through microRNA-Regulated Tumor-Specific RNA Replacement

We have previously presented that trans-splicing ribozymes based on Tetrahymena group I intron could be potent anti-cancer devices through diminishing target RNA level and simultaneously triggering anti-cancer gene activity only in cancer cells expressing the target RNA. Particularly, hTERT-targeting ribozyme was shown to specifically and efficiently retard tumors in various xenograft carcinomatosis nude mouse models. However, concerns for the hTERT-targeting approach include potential side effects to proliferating hTERT+ normal cells. In this study, to improve cancer-specificity of the ribozyme, we developed cancer-specific ribozyme expression construct via microRNA regulation. To circumvent hepatocellular carcinoma (HCC) as target models, we created tissue-specific microRNA-regulated ribozyme system by incorporating perfect antisense sequence against normal hepatocyte-selective miRNA-122a that is down-regulated in HCC to the 3’-UTR region of ribozyme. Adenovirus encoding the modified hTERT-targeting ribozyme specifically and efficiently induced transgene and cytotoxicity in liver cancers through cancer-specific RNA replacement. Moreover, specificity of cancer regression was highly increased without compromising its anti-cancer efficacy in xenograft orthotopic HCC model. Furthermore, to assess the effect of this system in syngeneic model, we constructed mTERT-targeting ribozyme harboring miRNA-122a target sites. Adenoviral vector encoding the modified ribozyme selectively and efficiently regressed mTERT+ HCC with minimal liver toxicity due to least ribozyme expression in the normal livers in syngeneic orthotopic mouse model. Noticeably, cytotoxicity caused by the adenovirus induced specific immunogenic cancer cell death in the syngeneic model, thereby completely suppressing secondary tumor challenge in the mouse. Therefore, specifically targeted transgene induction through microRNA-regulated RNA replacement provides novel strategies for efficient and safe cancer gene therapy.

Nanostructured lipid carriers as novel drug delivery system for lung cancer gene therapy

Context: Nanostructured lipid carriers (NLC) are potentially good colloidal drug carriers for gene delivery. They are advised to be the second lifetime of lipid nanocarriers.Objective: The aim of this study is to develop novel modified NLC as nanomedicine for delivery of plasmid-containing enhanced green fluorescence protein (pEGFP). This system could target the lung cancer cells through receptor-mediated pathways to increase the nuclear uptake of genetic materials.Methods: In the present study, pEGFP-loaded NLC (NLC/pEGFP) were prepared. Transferrin (Tf) containing ligands were used for the surface coating of the vectors. In vitro transfection efficiency of the modified vectors was evaluated in human alveolar adenocarcinoma cell line (A549 cells) and in vivo transfection efficiency of the modified vectors was evaluated on mice bearing A549 cells model.Results: Tf-modified NLC/pEGFP (Tf-NLC/pEGFP) has a particle size of 157 nm, and ∼82% of gene loading quantity. Tf-NLC/pEGFP displayed remarkably higher transfection efficiency than non-modified NLC/pEGFP both in vitro and in vivo.Conclusion: The results demonstrate that the novel NLC gene delivery system offers an effective strategy for lung cancer gene therapy.

Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells.

The conventional strategy for cancer gene therapy offers limited control of specificity and efficacy. A possible way to overcome these limitations is to construct logic circuits. Here we present modular AND gate circuits based on CRISPR-Cas9 system. The circuits integrate cellular information from two promoters as inputs and activate the output gene only when both inputs are active in the tested cell lines. Using the luciferase reporter as the output gene, we show that the circuit specifically detects bladder cancer cells and significantly enhances luciferase expression in comparison to the human telomerase reverse transcriptase-renilla luciferase construct. We also test the modularity of the design by replacing the output with other cellular functional genes including hBAX, p21 and E-cadherin. The circuits effectively inhibit bladder cancer cell growth, induce apoptosis and decrease cell motility by regulating the corresponding gene. This approach provides a synthetic biology platform for targeting and controlling bladder cancer cells in vitro.

AB113. BAX apoptosis gene selectively affects bladder cancer cells by artificial hTERT promoter

BackgroundBecause of the lack of biological components that selectively identifies cancer cells and robustly drives therapeutic gene expression, the conventional strategy for cancer gene therapy offers limited control of specificity and efficacy. Consequently, looking for a specific and effective promoter component can help us overcome these limitations.MethodsHere, we transfected the artificially hTERT promoter sequence into cancer cells and kept the specificity of the promoter. Then we used the Dual luciferase reporter gene system to prove the artificial hTERT promoter’s specificity and efficacy. Last, we demonstrated its modularity by replacing the luciferase reporter gene with other cellular functional genes like hBAX.ResultsUsing the Dual luciferase reporter gene system, we have shown that the aritificial hTERT promoter specifically detected bladder cancer cells and significantly enhanced luciferase expression in comparison to the WT-hTERT-renilla luciferase (Rluc) construct. The functional module of artificial hTERT promoter effectively inhibited bladder urothelial carcinoma cell growth and induced apoptosis by regulating the down-stream gene.ConclusionsThis functional module provides a synthetic biology platform for targeting and controlling bladder cancer.

AB111. Synthesizing an AND gate genetic circuit for identification of bladder cancer cells based on CRISPR-Cas9

BackgroundBecause of the lack of an ideal promoter that selectively identifies cancer cells and robustly drives therapeutic gene expression, the conventional strategy for cancer gene therapy offers limited control of specificity and efficacy. A possible way to overcome these limitations is to construct logic circuits capable of integrating cellular information from multiple promoters as inputs.MethodsHere, we designed and constructed a modular AND gate circuit based on CRISPR-cas9 system through combining the cancer-specific promoter of human telomerase reverse transcriptase (hTERT) gene with the bladder-specific promoter of human uroplakin II gene. The AND gate circuit integrates cellular information from the two promoters as inputs and activates the CMV promoter of output gene repressed by LacI only when both inputs are active in the tested cell lines. The integration occurs via an interaction between the Cas9 protein and sgRNA targeting LacI.ResultsUsing the luciferase reporter as the output gene, we have shown that the circuit specifically detected bladder cancer cells and significantly enhanced luciferase expression in comparison to the hTERT-renilla luciferase (Rluc) construct. We also demonstrated the modularity of this circuit by replacing the output with other cellular functional genes including hBAX, p21, and E-cadherin. The circuit effectively inhibited bladder urothelial carcinoma cell growth, induced apoptosis, and decreased cell motility by regulating the corresponding gene.ConclusionsThis approach provides a synthetic biology platform for targeting and controlling bladder cancer.

Co-delivery of plasmid DNA and doxorubicin by solid lipid nanoparticles for lung cancer therapy.

The co-delivery of DNA and antitumor drugs has the potential to treat cancer. In this study, we aimed to develop surface-modified, co-encapsulated solid lipid nanoparticles (SLN) containing enhanced green fluorescence protein plasmid (pEGFP) and doxorubicin (DOX) in order to create a multifunctional delivery system that targets lung cancer cells, in an effort to improve the efficacy of cancer therapy. DOX- and pEGFP-loaded SLN were prepared separately and then mixed to form co-encapsulated SLN (SLN/DE). Transferrin (Tf)-containing ligands were used for the surface coating of the vectors. The in vitro transfection efficiency of the modified vectors was evaluated using a human alveolar adenocarcinoma cell line (A549 cells) and the in vivo transfection efficiency of the modified vectors was evaluated using mice bearing A549 tumors. The Tf-modified DOX and pEGFP co-encapsulated SLN (T-SLN/DE) had a particle size of 267 nm with a 42 mV surface charge. The in vitro cytotoxicity of T-SLN/DE was low (cell viability was between 80 and 100% compared with the controls). T-SLN/DE displayed a remarkable therapeutic effect both in drug delivery and gene therapy. In conclusion, our results demonstrate that the multifunctional delivery system can improve the efficacy of cancer therapy through the combination of gene therapy and chemotherapy. In addition, the coating of active targeting ligands can improve the efficacy of the carriers at targeting lung cancer cells. Thus, the novel gene and drug delivery system offers an effective strategy for lung cancer gene therapy.

Antitumor activities of an oncolytic adenovirus equipped with a double siRNA targeting Ki67 and hTERT in renal cancer cells

RNA interference has been proven to be a powerful tool for gene knockdown. Our previous study demonstrated that a Ki67 shRNA carried by an adenovirus reduced Ki67 expression. In this study, we constructed novel oncolytic adenoviruses in which the Ki67 core promoter drove expression of the E1A gene. These adenoviruses were equipped with either a Ki67 small interfering RNA (siRNA), a human telomerase reverse transcriptase (hTERT) siRNA or a double siRNA targeting Ki67 and hTERT. We identified the antitumor activities of oncolytic adenoviruses in 3 renal cancer cell lines, human normal renal tube cell HK-2 and also in nude mice bearing KETR-3-xenografted tumors. Our results showed that these oncolytic adenoviruses, especially Ki67-ZXC2-double siRNA, could effectively induce silencing of the Ki67 and hTERT genes, allow efficient viral replication and induce significant apoptosis of renal cancer cells in vitro and in nude mice. We concluded that a dual siRNA mediated by oncolytic virotherapy could be an effective strategy for cancer gene therapy.

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

Study of the UTMD-Based Delivery System to Induce Cervical Cancer Cell Apoptosis and Inhibit Proliferation with shRNA targeting Survivin

Apoptosis induction by short hairpin RNA (shRNA) expression vectors could be an efficient and promising strategy for cancer gene therapy. Ultrasound-targeted microbubble destruction (UTMD) is an appealing technique. In this study, we investigated the apoptosis induction and suppression of cell proliferation in vivo transfected by the UTMD-based shRNA delivery system. Nude mice with transplanted tumors of cervical cancer were randomly arranged into three groups: control group, plasmid injection and ultrasound (P + US), P + UTMD group. Expressions of Survivin and proliferating cell nuclear antigen (PCNA), Bcl-2, Bax, Caspase-3, Ki-67, nucleostemin (NS) were investigated by immunohistochemistry. Furthermore, microvessel density (MVD) was detected by CD34 protein expressions and apoptotic index (AI) was measured by TUNEL. As compared with those in the control and P + US groups, protein expressions of PCNA, Ki-67, Bcl-2, Survivin and NS in P + UTMD groups were down-regulated markedly, while those of Bax, Caspase-3 were up-regulated significantly (p < 0.05). MVD decreased significantly, whereas AI increased remarkably (p < 0.05). We suggested that UTMD-based shRNA delivery system could induce apoptosis and inhibit proliferation significantly, without causing any apparently adverse effect, representing a new, promising technology that would be used in the future gene therapy and research.

Combination of oncolytic adenovirus and endostatin inhibits human retinoblastoma in an in vivo mouse model

There is a critical need for new paradigms in retinoblastoma (RB) treatment that would more efficiently inhibit tumor growth while sparing the vision of patients. Oncolytic adenoviruses with the ability to selectively replicate and kill tumor cells are a promising strategy for cancer gene therapy. Exploration of a novel targeting strategy for RB utilizing combined oncolytic adenovirus and anti-angiogenesis therapy was applied over the course of the current study with positive results. The oncolytic adenoviruses Ad-E2F1 p-E1A and Ad-TERT p-E1 were constructed. The E1 region was regulated by the E2F-1 promoter or the human telomerase reverse transcriptase (hTERT) promoter, respectively. Effects on both replication and promotion of enhanced green fluorescent protein (EGFP) expression were observed in the replication-defective adenovirus Ad-EGFP in diverse cancer cell lines, HXO-RB44, Y79, Hep3B, NCIH460, MCF-7 and HLF. The cancer cell death induced by these agents was also explored. The in situ RB model demonstrated that mice with tumors treated with the oncolytic adenovirus and replication-defective adenovirus Ad-endostatin exhibited notable cancer cell death. This anticancer effect was further examined by stereo microscope, and the survival rate of experimental mice was determined. Both Ad-E2F1 p-E1A and Ad-TERT p-E1 replicated specifically in cancer cells in vitro and promoted EGFP expression in Ad-EGFP, although Ad-E2F1 p-E1A demonstrated superior EGFP promotion activity than Ad-TERT p-E1. In Hep3B, NCIH460 and MCF-7 cells, the number of Ad-TERT p-E1 copies was observed to exceed of the number of Ad-E2F1 p-E1A copies by a minimum of 10-fold. Furthermore, Ad-TERT p-E1 demonstrated significantly superior oncolytic effects in the RB mouse model, and Ad-endostatin effectively suppressed tumor growth and extended the overall lifespan of subjects; however, the Ad-E2F1 p-E1A was clearly less effective in attaining these goals. Most notably, the antitumor effect and survival rate of subjects in the combined Ad-TERT p-E1 + Ad-endostatin group were higher than those treated with either single Ad-TERT p-E1 (p=0.097, p=0.022, respectively) or Ad-endostatin (p=0.037, p=0.006, respectively). In conclusion, application of transcription factor E2F-1 and human telomerase reverse transcriptase (hTERT) promoters to control E1 offer some guarantee that not only is RB gene therapy effective, but it is also safe. Combination therapy using the oncolytic adenovirus Ad-TERT p-E1 and replication-defective adenovirus Ad-endostatin demonstrates desirable oncolysis in the in situ RB mouse model. Additionally, E1B19K is important in the RB tumor suppression effect of oncolytic adenoviruses.

Development of a gene therapy strategy to target hepatocellular carcinoma based inhibition of protein phosphatase 2A using the α-fetoprotein promoter enhancer and pgk promoter: an in vitro and in vivo study

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Current therapies are insufficient, making HCC an intractable disease. Our previous studies confirmed that inhibition of protein phosphatase 2A (PP2A) may provide a promising therapeutic strategy for cancer. Unfortunately, constitutive expression of PP2A in normal tissues limits the application of PP2A inhibition. Thus, a HCC-specific gene delivery system should be developed. The α-fetoprotein (AFP) promoter is commonly used in HCC-specific gene therapy strategies; however, the utility of this approach is limited due to the weak activity of the AFP promoter. It has been shown that linking the AFP enhancer with the promoter of the non-tissue-specific, human housekeeping phosphoglycerate kinase (pgk) gene can generate a strong and HCC-selective promoter.MethodsWe constructed a HCC-specific gene therapy system to target PP2A using the AFP enhancer/pgk promoter, and evaluated the efficiency and specificity of this system both in vitro and in vivo.ResultsAFP enhancer/pgk promoter-driven expression of the dominant negative form of the PP2A catalytic subunit α (DN-PP2Acα) exerted cytotoxic effects against an AFP-positive human hepatoma cell lines (HepG2 and Hep3B), but did not affect AFP-negative human hepatoma cells (SK-HEP-1) or normal human liver cells (L-02). Moreover, AFP enhancer/pgk promoter driven expression of DN-PP2Acα inhibited the growth of AFP-positive HepG2 tumors in nude mice bearing solid tumor xenografts, but did not affect AFP-negative SK-HEP-1 tumors.ConclusionsThe novel approach of AFP enhancer/pgk promoter-driven expression of DN-PP2Acα may provide a useful cancer gene therapy strategy to selectively target HCC.

Efficient inhibition of intraperitoneal ovarian cancer growth in nude mice by liposomal delivery of short hairpin RNA against STAT3

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the tumor formation, prognosis and chemoresistance of ovarian cancer. Our goal was to investigate the effect of silencing STAT3 on ovarian cancer cell apoptosis, proliferation, angiogenesis and expression of key targets in vitro and in vivo. The ovarian cancer cell lines A2780CP and A2780s were used. STAT3 was knocked down by the plasmid-based short hairpin RNA (shRNA) expression system. In vitro, a colony formation assay and Hoechst staining were used to examine cell proliferation and apoptosis. The expression level of STAT3 and apoptosis-related proteins were determined by Western blot. The A2780CP intraperitoneal model was used to evaluate the effect of shSTAT3 on tumor growth in mice. Proliferation, apoptosis, and angiogenesis in tumor tissues were measured by proliferating cell nuclear antigen, TUNEL and CD31 immunostaining, respectively. Treatment with shSTAT3 resulted in apoptosis and inhibition of cell proliferation in vitro. Western blot analysis demonstrated that shSTAT3 induced the expression of cleaved caspase-3 and reduced the expression of survivin, Bcl-2 and vascular endothelial growth factor. In vivo, the tumor weight was reduced to 13.46% of 5% glucose by shSTAT3/lipoplexes (P < 0.01), accompanied with apoptosis induction (P < 0.01), proliferation inhibition (P < 0.01) and angiogenesis inhibition (P < 0.01). We find that treatment with shSTAT3 inhibits tumor growth in vitro and in vivo by inducing apoptosis and inhibiting cell proliferation. This work should provide the scientific foundation for future investigation of shSTAT3 as a strategy for ovarian cancer gene therapy and the combination of gene therapy with chemotherapy.

Erratum: The radiation dose-regulated AND gate genetic circuit, a novel targeted and real-time monitoring strategy for cancer gene therapy

Erratum: The radiation dose-regulated AND gate genetic circuit, a novel targeted and real-time monitoring strategy for cancer gene therapy

Oncolytic adenovirus armed with IL-24 Inhibits the growth of breast cancer in vitro and in vivo

BackgroundInterleukin-24 (IL-24) is a cytokine that belongs to the IL-10 family. It can selectively induce cancer cell apoptosis which has been utilized as a cancer gene therapy strategy.MethodsA recombinant type five adenovirus containing IL-24 gene (designated CNHK600-IL24) was constructed, whose replication is activated only in tumor cells. The replication of CNHK600-IL24 in breast tumor cells and fibroblasts were assessed by TCID50 and MTT assay; the secretion of IL-24 was measured by ELISA and western blotting. The in vivo anti-tumor effect of CNHK600-IL24 was investigated in nude mice carrying orthotopic or metastatic breast tumor.ResultsWe observed that CNHK600-IL24 could replicate efficiently and resulted in high level IL-24 expression and massive cell death in human breast cancer cell MDA-MB-231 but not in normal fibroblast cell MRC-5. In addition, orthotopic breast tumor growth in the nude mice model was significantly suppressed when CNHK600-IL24 was administered. In the metastatic model generated by tail vein injection, CNHK600-IL24 virotherapy significantly improved survival compared with the same virus expressing EGFP (median survival CNHK600-IL24, 55 days vs. CNHK600-EGFP, 41 day, p < 0.05 Mantal-Cox test). A similar phenomenon was observed in the metastatic model achieved by left ventricular injection as suggested by in vivo luminescence imaging of tumor growth.ConclusionThe oncolytic adenovirus armed with IL-24, which exhibited enhanced anti-tumor activity and improved survival, is a promising candidate for virotherapy of breast cancer.