Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 22
Filter
1.
J Interferon Cytokine Res ; 38(9): 378-387, 2018 09.
Article in English | MEDLINE | ID: mdl-30230984

ABSTRACT

Melanoma is one of the most lethal skin malignancies in the world. Interferons (IFNs) have been also demonstrated in response to tumor cell and IFNs such as IFN-α have been used for melanoma treatment. The long chain double-stranded RNA (dsRNA) (from a variety of nonviral sources) is a potent activator of the IFN system and an inducer of cell apoptosis. Panaxadiolsaponins (PDS) is a major Panax ginseng-derived active component with known antitumor activity and immune modulation. Here, we investigated a hypothesis that the combination of PDS and total natural dsRNA (as opposed to the synthetic dsRNA) will suppress tumor growth better than the individual agents. We have evaluated the antitumor and immunostimulatory effects of the combination of natural long chain dsRNA (derived from yeast) and PDS on melanoma cell line B16 and mice xenograft model. The underlying mechanisms of growth suppression were investigated by analyzing dsRNA-activated pathways, apoptosis, and cell cycle. Natural dsRNA and PDS exert superior anticancer effects than either agent alone. Natural dsRNA and PDS combination might be a promising strategy for treating malignancies, including melanoma.


Subject(s)
Antineoplastic Agents/pharmacology , Biological Products/pharmacology , Melanoma, Experimental/drug therapy , RNA, Double-Stranded/pharmacology , Sapogenins/pharmacology , Animals , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Melanoma, Experimental/pathology , Mice , Panax/chemistry , Tumor Cells, Cultured
2.
Sheng Wu Gong Cheng Xue Bao ; 33(4): 653-663, 2017 Apr 25.
Article in Chinese | MEDLINE | ID: mdl-28920398

ABSTRACT

To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration. The purity reached 99% with a yield of 33%. Electron microscopy analysis revealed that both the purified recombinant HEV VLPs from HP/HEV2.3 and natural hepatitis E virus particles appear identical of being 32 nm. The resulting DNA sequence obtained from VLPs is identical to the published HEV sequence. The SDS-PAGE analysis has revealed that the protein molecular weight of the HEV VLPs is 56 kDa, and the expression product HEV VLPs were accumulated up to 26% of total cellular protein. The expression level is 1.0 g/L. Western blotting, enzyme-linked immunosorbent assay (ELISA) results of the protein and ED50 of the vaccine showed that the HEV VLPs have good antigenicity and immunogenicity. In summary, the recombinant HEV VLPs from Hansenula polymorpha can be used in the manufacture of a new genetically engineered vaccine against hepatitis E.


Subject(s)
Fermentation , Hepatitis E virus/immunology , Immunogenicity, Vaccine , Pichia/virology , Vaccines, Virus-Like Particle/immunology , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Hepatitis E , Hepatitis E virus/growth & development , Humans , Microorganisms, Genetically-Modified
3.
Cytokine ; 89: 160-172, 2017 01.
Article in English | MEDLINE | ID: mdl-26778055

ABSTRACT

Bacteria act as pro- or anti- tumorigenic agents. Whole bacteria or cytotoxic or immunogenic peptides carried by them exert potent anti-tumor effects in the experimental models of cancer. The use of attenuated microorganism(s) e.g., BCG to treat human urinary bladder cancer was found to be superior compared to standard chemotherapy. Although the phase-I clinical trials with Salmonella enterica serovar Typhimurium, has shown limited benefits in human subjects, a recent pre-clinical trial in pet dogs with tumors reported some subjects benefited from this treatment strain. In addition to the attenuated host strains derived by conventional mutagenesis, recombinant DNA technology has been applied to a few microorganisms that have been evaluated in the context of tumor colonization and eradication using mouse models. There is an enormous surge in publications describing bacterial anti-cancer therapies in the past 15years. Vectors for delivering shRNAs that target oncogenic products, express tumor suppressor genes and immunogenic proteins have been developed. These approaches have showed promising anti-tumor activity in mouse models against various tumors. These can be potential therapeutics for humans in the future. In this review, some conceptual and practical issues on how to improve these agents for human applications are discussed.


Subject(s)
Microorganisms, Genetically-Modified/genetics , Neoplasms/therapy , Salmonella typhimurium/genetics , Animals , Dogs , Humans , Microorganisms, Genetically-Modified/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Salmonella typhimurium/metabolism
4.
Int J Clin Exp Pathol ; 7(11): 7497-507, 2014.
Article in English | MEDLINE | ID: mdl-25550785

ABSTRACT

GRIM-19 has been demonstrated as an important regulator for the normal tissue development. Recently, more evidences regarded GRIM-19 as the new tumor suppressor. However, the possible mechanisms underlying GRIM-19 suppressing cancer growth are unclear. In the present study, Paired hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues were obtained from 54 patients who underwent primary surgical HCC tissue resection. GRIM-19 protein expression in HCC tissues was performed by immunohistochemistry. Cells were transfected by lentiviruses plasmid expressing GRIM-19. RT-PCR and Western blot analyses were performed to confirm the expression of GRIM-19 mRNA or protein. Cell proliferation was assessed by MTT and FCM analyses. Mitochondrial membrane potential and apoptosis were respectively determined by using fluorescence microscopy and FCM analyses. AKT1, pAKT1, cyclinD1, CDK4, PCNA, Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3, and cytochrome C were detected by Western blot and immunofluorescence. GRIM-19 protein expression was markedly lower in HCC than in paired adjacent non-tumor liver tissues. GRIM-19 overexpression in HCC cells significantly induced cell cycle arrest and enhanced apoptosis. We also found that AKT1 expression and phosphorylation were regulated by the expression of GRIM-19. Collectively, our study demonstrated that GRIM-19 overexpression suppressed HCC growth and downregulated AKT1 expression, suggesting that GRIM-19 might play a crucial role in hepatocarcinogenesis through negatively regulating the PI3K/AKT signaling pathway.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Carcinoma, Hepatocellular/metabolism , Gene Expression Regulation, Neoplastic , Liver Neoplasms/metabolism , NADH, NADPH Oxidoreductases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Apoptosis , Apoptosis Regulatory Proteins/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/surgery , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Electron Transport , Gene Expression , Humans , Liver Neoplasms/pathology , Liver Neoplasms/surgery , Membrane Potential, Mitochondrial , Mitochondria/metabolism , NADH, NADPH Oxidoreductases/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
5.
Genome Announc ; 1(4)2013 Aug 22.
Article in English | MEDLINE | ID: mdl-23969054

ABSTRACT

Attenuated Salmonella enterica serovar Typhi strain Ty21a is an important vaccine for controlling typhoid fever and serves as an oral vector for delivering heterologous antigens. The key attenuating features of this randomly mutated strain remain in question. Genome sequencing has revealed 679 single nucleotide polymorphisms (SNPs), and will help define alterations contributing to Ty21a safety and immunogenicity.

6.
J Cancer Res Clin Oncol ; 139(6): 971-80, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23463096

ABSTRACT

OBJECTIVES: To investigate the therapeutic utility of an attenuated bacterium carrying a plasmid that co-expresses Endostatin, an inhibitor of tumor neovasculogenesis, and a shRNA that targets Stat3 to suppress prostate cancer growth. METHODS: Plasmid pEndo-Si-Stat3 was constructed and introduced into an attenuated strain of Salmonella enterica serovar typhimurium. The resultant recombinant bacterium was used as a vector to deliver the plasmid to tumor cells growing in vivo. Tumor-associated gene and protein expression changes were measured by using RT-PCR and Western blot analyses. Expression of Endostatin in tumor tissue was detected by ELISA. The presence of vector bacteria in tissues was monitored and tumor destruction was assessed by using TUNEL and H&E staining assays. RESULTS: Bacterially delivered pEndo-Si-Stat3 decreased Stat3 levels and increased Endostatin expression in mouse tumors, resulting in a significant suppression of tumor growth (P < 0.01). Expression of Bcl-2 and PCNA was down-regulated and Caspase3 expression was up-regulated to promote apoptosis of tumor cells. CONCLUSIONS: Successful delivery by attenuated Salmonella of the combination therapeutic plasmid simultaneously knocked down the expression of Stat3 and resulted in over-expression of Endostatin, which synergistically inhibited prostate cancer growth.


Subject(s)
Endostatins/genetics , Gene Transfer Techniques , Prostatic Neoplasms/therapy , RNA, Small Interfering/genetics , STAT3 Transcription Factor/genetics , Animals , Antigens, CD34/metabolism , Apoptosis/genetics , Blotting, Western , Cell Line, Tumor , Combined Modality Therapy , Endostatins/metabolism , Gene Expression , Genetic Therapy , Genetic Vectors/genetics , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Plasmids/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/metabolism , Salmonella typhimurium/genetics , Time Factors , Tumor Burden/genetics , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
7.
Cancer Lett ; 335(1): 242-50, 2013 Jul 10.
Article in English | MEDLINE | ID: mdl-23435374

ABSTRACT

The E6 protein of the oncogenic HPV-16 functions by interfering with the normal cell cycle control mechanisms, particularly those controlled by p53. In this study, we developed a dual expression plasmid that coexpressed-E6-specific siRNA and wild type p53, and to evaluate its effects on cervical cancer growth. We found that simultaneous expression of pSi-E6-P53 caused a robust suppression of tumor growth when compared to the controls either E6-specific siRNA or p53 alone. In conclusion, our findings demonstrate that a combined strategy of co-expressed E6-specific siRNA and p53 synergistically and more effectively suppressed cervical tumor growth when compared with single treatment.


Subject(s)
Cell Proliferation , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/therapy , RNA, Small Interfering/genetics , Repressor Proteins/genetics , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/therapy , Animals , Apoptosis , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Cell Movement , Cell Survival , Female , Gene Expression , Gene Knockdown Techniques , Genetic Therapy/methods , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/metabolism , Papillomavirus Infections/pathology , Plasmids/genetics , Repressor Proteins/metabolism , Salmonella typhimurium/genetics , Transformation, Bacterial , Tumor Burden , Tumor Suppressor Protein p53/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology
8.
Asian J Androl ; 14(4): 536-45, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22580637

ABSTRACT

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 (GRIM-19). Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium (S. typhimurium) to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitro and in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Carcinoma/therapy , Genetic Therapy , Inhibitor of Apoptosis Proteins/metabolism , NADH, NADPH Oxidoreductases/metabolism , Prostatic Neoplasms/therapy , RNA, Small Interfering/therapeutic use , Animals , Apoptosis , Apoptosis Regulatory Proteins/genetics , Carcinoma/metabolism , Caspase 3/metabolism , Cell Line, Tumor , Cyclin D1/metabolism , Gene Expression , Humans , Inhibitor of Apoptosis Proteins/genetics , Ki-67 Antigen/metabolism , Male , Mice , NADH, NADPH Oxidoreductases/genetics , Plasmids , Prostate/metabolism , Prostatic Hyperplasia/metabolism , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-myc/metabolism , RNA, Small Interfering/metabolism , STAT3 Transcription Factor/metabolism , Salmonella typhimurium , Survivin , Vascular Endothelial Growth Factor A/metabolism , bcl-X Protein/metabolism
9.
Biochem Biophys Res Commun ; 416(3-4): 270-6, 2011 Dec 16.
Article in English | MEDLINE | ID: mdl-22093819

ABSTRACT

Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed decreased expressions of MMP-2 and MMP-9 at the mRNA and protein levels. Taken together, the present findings suggest that Neu3 is a promising molecular target for the prevention of prostate cancer metastasis.


Subject(s)
Genetic Therapy/methods , Neuraminidase/antagonists & inhibitors , Prostatic Neoplasms/therapy , RNA, Small Interfering/genetics , Salmonella typhimurium , Animals , Cell Line, Tumor , Cell Movement , Humans , Male , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness , Neuraminidase/genetics , Prostatic Neoplasms/pathology , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , Transfection/methods
10.
Virus Genes ; 43(3): 319-26, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21701857

ABSTRACT

The incidence of measles in China has increased over the last decade. To evaluate the genetic variation of measles strains, 16 measles wild-type virus strains were isolated from 14 vaccinated cases and 2 nonvaccinated cases in Jilin Province during 2005-2006, and their nucleoprotein (N) and hemagglutinin (H) genes were amplified by RT-PCR. The amplified products were sequenced and compared with the Edmonston virus and the existing vaccine strains (Changchun-47 and Shanghai-191). The results showed that the variation rate between the vaccine and wild-type strains was 9.8-12.0% in the N gene and 5.9-6.9% in the H gene, respectively. In addition, cross-neutralization assays revealed that although sera obtained from infants following primary vaccination effectively neutralized vaccine strains, the capacity in neutralizing H1 wild-type measles virus isolates was decreased fourfold. Antigenic ratios testing revealed that the antigenic relatedness between wild-type measles viruses and existing vaccine strains was notably low. These data suggest that the increased incidence of measles in Jilin Province may be attributed to the antigenic drift between wild-type and vaccine strains. Our findings strengthen the recommendation of supplemental immunization with existing vaccines and also strongly suggest a need for developing new vaccines to better control measles virus outbreaks.


Subject(s)
Measles virus/classification , Measles virus/genetics , Measles/epidemiology , Phylogeny , Adolescent , Adult , Child , Child, Preschool , China/epidemiology , Female , Genotype , Humans , Incidence , Infant , Male , Measles/prevention & control , Measles/virology , Measles Vaccine/genetics , Measles Vaccine/immunology , Measles virus/immunology , Measles virus/isolation & purification , Molecular Sequence Data , Young Adult
11.
J Pharmacol Exp Ther ; 338(1): 173-83, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21444629

ABSTRACT

Our objective was to evaluate cell growth and death effects by inhibiting Murine Double Minute 2 (MDM2) expression in human prostate cancer cells overexpressing the wild-type (WT) p53 gene. Prostate PC-3 tumor cells were transfected with a plasmid containing either mdm2 small interfering (Si-mdm2) or the WT p53 gene (Pp53) alone, or both (Pmp53), using Lipofectamine in vitro and attenuated Salmonella enterica serovar Typhi vaccine strain Ty21a (Salmonella Typhi Ty21a) in vivo. Cell growth, apoptosis, and the expression of related genes and proteins were examined in vitro and in vivo by flow cytometry and Western blot assays. We demonstrated that human prostate tumors had increased expression of MDM2 and mutant p53 proteins. Transfection of the PC-3 cells with the Pmp53 plasmid in vitro offered significant inhibition of cell growth and an increase in apoptotic cell death compared with that of the Si-mdm2 or Pp53 group. These effects were associated with up-regulation of p21 and down-regulation of hypoxia-inducible factor 1α expression in Pmp53-transfected cells. To validate the in vitro findings, the nude mice implanted with PC-3 cells were treated with attenuated Salmonella Typhi Ty21a carrying the plasmids, which showed that the Pmp53 plasmid significantly inhibited the tumor growth rate in vivo compared with that of the Si-mdm2 or Pp53 plasmid alone. Tumor tissues from mice treated with the Pmp53 plasmid showed increased expression of p21 and decreased expression of hypoxia-inducible factor 1α proteins, with an increased apoptotic effect. These results suggest that knockdown of mdm2 expression by its specific small interfering RNA with overexpression of the WT p53 gene offers synergistic inhibition of prostate cancer cell growth in vitro and in vivo.


Subject(s)
Gene Expression Regulation, Neoplastic , Genes, p53/physiology , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-mdm2/biosynthesis , Proto-Oncogene Proteins c-mdm2/genetics , RNA, Small Interfering/biosynthesis , RNA, Small Interfering/genetics , Animals , Cell Line, Tumor , Humans , Male , Mice , Mice, Nude , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Random Allocation , Xenograft Model Antitumor Assays/methods
12.
Vaccine ; 29(15): 2761-71, 2011 Mar 24.
Article in English | MEDLINE | ID: mdl-21300096

ABSTRACT

Foam drying, a modified freeze drying process, was utilized to produce a heat-stable, live attenuated Salmonella Typhi 'Ty21a' bacterial vaccine. Ty21a vaccine was formulated with pharmaceutically approved stabilizers, including sugars, plasticizers, amino acids, and proteins. Growth media and harvesting conditions of the bacteria were also studied to enhance resistance to desiccation stress encountered during processing as well as subsequent storage at elevated temperatures. The optimized Ty21a vaccine, formulated with trehalose, methionine, and gelatin, demonstrated stability for approximately 12 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1log(10)CFU) and no loss in titer at 4 and 25°C following storage for the same duration. Furthermore, the foam dried Ty21a elicited a similar immunogenic response in mice as well as protection in challenge studies compared to Vivotif™, the commercial Ty21a vaccine. The enhanced heat stability of the Ty21a oral vaccine, or Ty21a derivatives expressing foreign antigens (e.g. anthrax), could mitigate risks of vaccine potency loss during long-term storage, shipping, delivery to geographical areas with warmer climates or during emergency distribution following a bioterrorist attack. Because the foam drying process is conducted using conventional freeze dryers and can be readily implemented at any freeze drying manufacturing facility, this technology appears ready and appropriate for large scale processing of foam dried vaccines.


Subject(s)
Bacterial Vaccines/immunology , Drug Carriers , Genetic Vectors , Microbial Viability/radiation effects , Salmonella typhi/genetics , Administration, Oral , Animals , Drug Stability , Humans , Mice , Salmonella typhi/physiology , Salmonella typhi/radiation effects , Temperature , Vaccines, Attenuated/immunology , Vaccines, Synthetic/immunology
13.
Methods Mol Biol ; 487: 161-87, 2009.
Article in English | MEDLINE | ID: mdl-19301647

ABSTRACT

RNAi is a powerful research tool for specific gene silencing and may also lead to promising novel therapeutic strategies. However, the development of RNAi-based therapies has been slow due to the lack of targeted delivery methods. The biggest challenge in the use of siRNA-based therapies is the delivery to target cells. There are many additional obstacles to in vivo delivery of siRNAs, such as degradation by endogenous enzymes and interaction with blood components leading to nonspecific uptake into cells, which govern biodistribution and availability of siRNA in the body. Naked unmodified synthetic siRNA including plasmid-carried-shRNA-expression constructs cannot penetrate cellular membranes, and therefore, systemic application is unlikely to be successful. The success of gene therapy by siRNAs relies on the development of safe, economical, and efficacious in vivo delivery systems into the target cells. Attenuated Salmonella have been employed recently as vectors to deliver silencing hairpin RNA (shRNA) expression plasmids into mammalian cells. This approach has achieved gene silencing in vitro and in vivo. The facultative anaerobic, invasive Salmonella have a natural tropism for solid tumors including metastatic tumors. Genetically modified, attenuated Salmonella have been used recently both as potential antitumor agents by themselves, and to deliver specific tumoricidal therapies. This chapter describes the use of attenuated bacteria as tumor-targeting delivery systems for cancer therapy.


Subject(s)
Drug Delivery Systems/methods , Genetic Therapy/methods , Liver Neoplasms/therapy , Prostatic Neoplasms/therapy , RNA, Small Interfering/administration & dosage , Salmonella typhi/genetics , Animals , Blotting, Northern , Blotting, Western , Gene Silencing , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Liver Neoplasms/microbiology , Liver Neoplasms/secondary , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Plasmids , Prostatic Neoplasms/microbiology , Prostatic Neoplasms/pathology , RNA, Messenger/analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
14.
Int J Med Microbiol ; 299(4): 233-46, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19121604

ABSTRACT

The attenuated live bacterial vaccine strain Salmonella enterica Serovar Typhi Ty21a is the main constituent of Vivotif, the only licensed oral vaccine against typhoid fever. The strain was developed in the 1970s by chemical mutagenesis. In the course of this mutagenesis, a number of mutations were introduced into the vaccine strain. Characterisation of the vaccine strain during development as well as release of master- and working seed lots (MSL and WSL) and commercial batches is based on phenotypic assays assessing microbiological and biochemical characteristics of Ty21a. In the current study, we have analysed by DNA sequencing the specific mutations originally correlated with the attenuation of strain Ty21a. These data demonstrate the stability of these mutations for MSLs and WSLs of Ty21a produced between 1980 and 2005. Finally, we have confirmed the correlation of these genetic mutations with the expected phenotypic attenuations for the seed lots used in vaccine manufacture over 25 years.


Subject(s)
Genomic Instability , Salmonella Vaccines/genetics , Salmonella typhi/genetics , DNA, Bacterial/genetics , Humans , Mutation, Missense , Point Mutation , Sequence Analysis, DNA , Vaccines, Attenuated/genetics , Virulence Factors/genetics
15.
Clin Cancer Res ; 14(2): 559-68, 2008 Jan 15.
Article in English | MEDLINE | ID: mdl-18223232

ABSTRACT

PURPOSE: Persistent activation of signal transducers and activators of transcription 3 (Stat3) and its overexpression contribute to the progression and metastasis of several different tumor types. For this reason, Stat3 is a reasonable target for RNA interference-mediated growth inhibition. Blockade of Stat3 using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth in mice. However, RNA interference does not fully ablate target gene expression in vivo, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Stat3-specific shRNA, we applied a combination treatment involving gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), another inhibitor of STAT3, along with shRNA. EXPERIMENTAL DESIGN: The coding sequences for GRIM-19, a cellular STAT3-specific inhibitor, and Stat3-specific shRNAs were used to create a dual expression plasmid vector and used for prostate cancer therapy in vitro and in mouse xenograft models in vivo. RESULTS: The coexpressed Stat3-specific shRNA and GRIM-19 synergistically and more effectively suppressed prostate tumor growth and metastases when compared with treatment with either single agent alone. CONCLUSION: The simultaneous use of two specific, but mechanistically different, inhibitors of STAT3 activity exerts enhanced antitumor effects.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , NADH, NADPH Oxidoreductases/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Small Interfering/metabolism , STAT3 Transcription Factor/metabolism , Animals , Apoptosis , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , NADH, NADPH Oxidoreductases/genetics , Plasmids , Prostate/metabolism , Prostatic Neoplasms/genetics , STAT3 Transcription Factor/genetics , Transcription, Genetic , Xenograft Model Antitumor Assays
16.
Cancer Res ; 67(12): 5859-64, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17575154

ABSTRACT

The facultative anaerobic, invasive Salmonella enterica serovar typhimurium (S. typhimurium) has been shown to retard the growth of established tumors. We wondered if a more effective antitumor response could be achieved in vivo if these bacteria were used as tools for delivering specific molecular antitumor therapeutics. Constitutively activated transcription factor signal transducer and activator of transcription 3 (STAT3) promotes the survival of a number of human tumors. In this study, we investigated the relative efficacies of attenuated S. typhimurium alone or combined with Stat3-specific small interfering RNA (siRNA) in terms of tumor growth and metastasis. The bacteria preferentially homed into tumors over normal liver and spleen tissues in vivo. S. typhimurium expressing plasmid-based Stat3-specific siRNAs significantly inhibited tumor growth, reduced the number of metastastic organs, and extended the life time for C57BL6 mice bearing an implanted prostate tumor, versus bacterial treatment alone. These results suggest that attenuated S. typhimurium combined with an RNA interference approach might be more effective for the treatment of primary as well as metastatic cancer.


Subject(s)
Prostatic Neoplasms/therapy , RNA, Small Interfering , STAT3 Transcription Factor/genetics , Salmonella typhimurium , Animals , Apoptosis , Blotting, Northern , Blotting, Western , Cell Proliferation , Genetic Therapy/methods , Genetic Vectors , Humans , Male , Mice , Mice, Inbred C57BL , Plasmids , Prostatic Neoplasms/microbiology , RNA Interference
17.
Acta Pharmacol Sin ; 27(3): 347-52, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16490172

ABSTRACT

AIM: To study the effect of pSilencer1.0-U6-siRNA-stat3 on the growth of human laryngeal tumors in nude mice. METHODS: Hep2 cells were transplanted into nude mice, then at the time of tumor formation, growth rates were observed. After the tumor formed, pSilencer1.0-U6-siRNA-stat3 was injected. Tumor volumes were calculated, and growth curves were plotted. Representative histological sections were taken from mice bearing transplantation tumors in both treated and control groups, and stat3, pTyr-stat3, Bcl-2, cyclin D1, and survivin expression were detected by Western blotting. survivin mRNA levels were detected by Northern blotting, hematoxylin and eosin staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay to confirm the apoptosis of tumors. RESULTS: In nude mice, pSilencer1.0-U6-siRNA-stat3 significantly suppressed the growth of tumors compared with controls (P<0.01). It suppressed stat3 expression, and downregulated BcL2, cyclin D1, and survivin expression within the tumor. This significantly induced apoptosis of the tumors. CONCLUSION: pSilencer1.0-U6-siRNA-stat3 was able to inhibit the growth of transplanted human laryngeal tumors in nude mice and induce apoptosis.


Subject(s)
Laryngeal Neoplasms/pathology , Microtubule-Associated Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , RNA Interference , RNA, Small Interfering/biosynthesis , STAT3 Transcription Factor/biosynthesis , Animals , Apoptosis , Cell Line, Tumor , Cyclin D1/metabolism , Humans , Inhibitor of Apoptosis Proteins , Laryngeal Neoplasms/metabolism , Mice , Mice, Nude , Microtubule-Associated Proteins/genetics , Neoplasm Proteins/genetics , Neoplasm Transplantation , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , STAT3 Transcription Factor/genetics , Survivin
18.
Clin Cancer Res ; 11(17): 6333-41, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-16144938

ABSTRACT

PURPOSE: Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector-based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model. EXPERIMENTAL DESIGN: To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector-based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo. RESULTS: Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil domain, respectively, strongly suppressed the expression of Stat3 in PC3 and LNCaP cells. The Stat3-1 siRNA, which targeted the DNA-binding domain, exerted no effect on Stat3 expression, indicating that the gene silencing efficiency of siRNA may be dependent on the local structure of Stat3 mRNA. The Stat3 siRNAs down-regulated the expression of Bcl-2 (an anti-apoptotic protein), and cyclin D1 and c-Myc (cell growth activators) in prostate cancer cells. Inhibition of Stat3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro and in tumors implanted in nude mice. CONCLUSIONS: These data indicate that Stat3 signaling is a promising molecular target for prostate cancer therapy and that vector-based Stat3 siRNA may be useful as a therapeutic agent for treatment of prostate cancer.


Subject(s)
DNA-Binding Proteins/metabolism , Genetic Vectors , Prostatic Neoplasms/prevention & control , RNA, Small Interfering/pharmacology , Trans-Activators/metabolism , Animals , Apoptosis , Base Pairing , Base Sequence , Cell Cycle , Cell Proliferation , Cyclin D1/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Down-Regulation , Humans , Male , Mice , Mice, Nude , Molecular Sequence Data , Prostatic Neoplasms/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-myc/metabolism , RNA, Small Interfering/genetics , STAT3 Transcription Factor , Trans-Activators/antagonists & inhibitors , Trans-Activators/genetics , Tumor Cells, Cultured
19.
Zhonghua Nan Ke Xue ; 11(1): 29-33, 37, 2005 Jan.
Article in Chinese | MEDLINE | ID: mdl-15704678

ABSTRACT

OBJECTIVE: To study the effects of pSilencer 1.0-U6-siRNA-STAT3 on the growth of PC3 and LNCaP cells. METHODS: Three pairs of DNA template coding siRNA were synthesized against STAT3 to reconstruct pSilencer 1.0-U6-STAT3-siRNA, which was transfected into PC3 and LNCaP cells. The STAT3 expression in PC3 cells and LNCaP were transfected with pSilencer 1.0-U6-siRNA-STAT3, and it was detected by Western blot and Northern blot. MTT and FCM were used to observe the growth-inhibiting ratio and apoptosis in PC3 cells. RESULTS: Western blot and Northern blot analyses demonstrated that pSilencer 1.0-U6-siRNA-STAT3 could significantly inhibit the expression of STAT3 in PC3 and LNCaP cells; MIT and FCM results showed that it could suppress the growth of PC3 cells and LNCaP and induce apoptosis of PC3 cells in vitro. CONCLUSION: PSilencer 1.0-U6-siRNA-STAT3 could significantly inhibit STAT3 expression, suppress the growth of PC3 and LNCaP cells and induce the apoptosis of PC3 cells.


Subject(s)
Apoptosis , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Small Interfering , STAT3 Transcription Factor/biosynthesis , Cell Line, Tumor , Humans , Male , Plasmids , RNA, Messenger/genetics , STAT3 Transcription Factor/genetics , Transcription, Genetic
20.
Acta Pharmacol Sin ; 26(3): 377-83, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15715937

ABSTRACT

AIM: To determine the inhibitory effect of the synthetic STAT3 siRNA on the expression of STAT3 gene in human laryngeal cancer cell lines Hep2 and to investigate the effect of STAT3 siRNA on growth and apoptosis in Hep2 cells. METHODS: A pair of DNA templates coding siRNA against STAT3-mRNA was synthesized to reconstruct plasmid of pSilencer1.0-U6 siRNA-STAT3. Hep2 cells were transfected with RPMI-1640 media (untreated), plasmid (empty), and STAT3 siRNA, respectively. Northern blot and Western blot analysis of STAT3 and pTyr-STAT3 expression in Hep2 cells and Western blot analysis of Bcl-2 expression in the Hep2 cell was performed 72 h after transfection. MTT, flow cytometry, and AO/EB assay were used for determination of cells proliferation and apoptosis in Hep2 cells. RESULTS: pTyr-STAT3 was markedly expressed in untreated Hep2 cells and the vector-treated Hep2 cells, whereas pTyr-STAT3 expression was significantly reduced in STAT3 siRNA-transfected Hep2 cells, indicating that STAT3 siRNA inhibited the activity of STAT3. Transfection of Hep2 cells with STAT3 siRNA significantly inhibited STAT3 expression at both mRNA and protein level in Hep2 cells and the inhibition was characterized by time-dependent transfection. Treatment of Hep2 cells with STAT3 siRNA resulted in dose-dependent growth inhibition of Hep2, this significantly increased apoptotic cell rate, and decreased Bcl-2 expression level in Hep2 cells. STAT3 siRNA had an effect on induction of either early or late stage apoptosis. CONCLUSION: This study demonstrates that STAT3 siRNA effectively inhibits STAT3 gene expression in Hep2 cells leading to growth suppression and induction of apoptosis in Hep2 cells. The use of siRNA technique may provide a novel therapeutic approach to treat laryngeal cancer and other malignant tumors expressing constitutively activated STAT3.


Subject(s)
Apoptosis , DNA-Binding Proteins/biosynthesis , Laryngeal Neoplasms/pathology , RNA Interference , RNA, Small Interfering/biosynthesis , Trans-Activators/biosynthesis , Cell Line, Tumor , Cell Proliferation , DNA-Binding Proteins/genetics , Humans , Laryngeal Neoplasms/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , STAT3 Transcription Factor , Trans-Activators/genetics , Transfection
SELECTION OF CITATIONS
SEARCH DETAIL
...