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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.
Proc Natl Acad Sci U S A ; 110(36): E3425-34, 2013 Sep 03.
Article in English | MEDLINE | ID: mdl-23959876

ABSTRACT

Vision loss from ischemic retinopathies commonly results from the accumulation of fluid in the inner retina [macular edema (ME)]. Although the precise events that lead to the development of ME remain under debate, growing evidence supports a role for an ischemia-induced hyperpermeability state regulated, in part, by VEGF. Monthly treatment with anti-VEGF therapies is effective for the treatment of ME but results in a major improvement in vision in a minority of patients, underscoring the need to identify additional therapeutic targets. Using the oxygen-induced retinopathy mouse model for ischemic retinopathy, we provide evidence showing that hypoxic Müller cells promote vascular permeability by stabilizing hypoxia-inducible factor-1α (HIF-1α) and secreting angiogenic cytokines. Blocking HIF-1α translation with digoxin inhibits the promotion of endothelial cell permeability in vitro and retinal edema in vivo. Interestingly, Müller cells require HIF--but not VEGF--to promote vascular permeability, suggesting that other HIF-dependent factors may contribute to the development of ME. Using gene expression analysis, we identify angiopoietin-like 4 (ANGPTL4) as a cytokine up-regulated by HIF-1 in hypoxic Müller cells in vitro and the ischemic inner retina in vivo. ANGPTL4 is critical and sufficient to promote vessel permeability by hypoxic Müller cells. Immunohistochemical analysis of retinal tissue from patients with diabetic eye disease shows that HIF-1α and ANGPTL4 localize to ischemic Müller cells. Our results suggest that ANGPTL4 may play an important role in promoting vessel permeability in ischemic retinopathies and could be an important target for the treatment of ME.


Subject(s)
Angiopoietins/metabolism , Capillary Permeability , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Retinal Neurons/metabolism , Angiopoietin-Like Protein 4 , Angiopoietins/genetics , Animals , Blotting, Western , Cell Hypoxia , Cells, Cultured , Diabetic Retinopathy/metabolism , Female , Gene Expression Profiling , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immunohistochemistry , Ischemia/metabolism , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Retinal Neurons/cytology , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
3.
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
4.
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
5.
Cancer Immunol Immunother ; 61(11): 1977-87, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22527247

ABSTRACT

Hepatocellular carcinoma (HCC) is one of the most aggressive carcinomas. Limited therapeutic options, mainly due to a fragmented genetic understanding of HCC, and major HCC resistance to conventional chemotherapy are the key reasons for a poor prognosis. Thus, new effective treatments are urgent and gene therapy may be a novel option. Signal transducer and activator of transcription 3 (Stat3) is a highly studied member of the STAT family. Inhibition of Stat3 signaling has been found to suppress tumor growth and improve survival, providing a molecular target for cancer therapy. Furthermore, HCC is a hypervascular tumor and angiogenesis plays a crucial role in tumor growth and metastasis. Thus, anti-angiogenic therapy, combined with inhibition of Stat3, may be an effective approach to combat HCC. We tested the effect that the combination therapy consisting of endostatin (a powerful angiogenesis inhibitor) and Stat3-specific small interfering RNA, using a DNA vector delivered by attenuated S. typhimurium, on an orthotopic HCC model in C57BL/6 mice. Although antitumor effects were observed with either single therapeutic treatment, the combination therapy provided superior antitumor effects. Correlated with this finding, the combination treatment resulted in significant alteration of Stat3 and endostatin levels and that of the downstream gene VEGF, decreased cell proliferation, induced cell apoptosis and inhibited angiogenesis. Importantly, combined treatment also elicited immune system regulation of various immune cells and cytokines. This study has provided a novel cancer gene therapeutic approach.


Subject(s)
Carcinoma, Hepatocellular/therapy , Endostatins/genetics , Genetic Therapy/methods , Liver Neoplasms, Experimental/therapy , Liver Neoplasms/therapy , RNA, Small Interfering/genetics , STAT3 Transcription Factor/antagonists & inhibitors , Salmonella typhimurium , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Silencing , Genetic Vectors , Male , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/therapy , STAT3 Transcription Factor/genetics , Vascular Endothelial Growth Factor A/analysis
6.
PLoS One ; 6(4): e19103, 2011 Apr 29.
Article in English | MEDLINE | ID: mdl-21559457

ABSTRACT

BACKGROUND: Kaposi's sarcoma (KS) is a vascular neoplasm characterized by the dysregulated expression of angiogenic and inflammatory cytokines. The driving force of the KS lesion, the KSHV-infected spindle cell, secretes elevated levels of vascular endothelial growth factor (VEGF), essential for KS development. However, the origin of VEGF in this tumor remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that the KSHV G protein-coupled receptor (vGPCR) upregulates VEGF in KS through an intricate paracrine mechanism. The cytokines secreted by the few vGPCR-expressing tumor cells activate in neighboring cells multiple pathways (including AKT, ERK, p38 and IKKß) that, in turn, converge on TSC1/2, promoting mTOR activation, HIF upregulation, and VEGF secretion. Conditioned media from vGPCR-expressing cells lead to an mTOR-dependent increase in HIF-1α and HIF-2α protein levels and VEGF upregulation. In a mouse allograft model for KS, specific inhibition of the paracrine activation of mTOR in non-vGPCR-expressing cells was sufficient to inhibit HIF upregulation in these cells, and abolished the ability of the vGPCR-expressing cells to promote tumor formation in vivo. Similarly, pharmacologic inhibition of HIF in this model blocked VEGF secretion and also lead to tumor regression. CONCLUSIONS/SIGNIFICANCE: Our findings provide a compelling explanation for how the few tumor cells expressing vGPCR can contribute to the dramatic amplification of VEGF secretion in KS, and further provide a molecular mechanism for how cytokine dysregulation in KS fuels angiogenesis and tumor development. These data further suggest that activation of HIF by vGPCR may be a vulnerable target for the treatment of patients with KS.


Subject(s)
Calcium-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Viral , Herpesvirus 8, Human/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Pathologic , Receptors, G-Protein-Coupled/metabolism , Sarcoma, Kaposi/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cytokines/metabolism , Endothelial Cells/cytology , Female , Humans , Inflammation , Mice , Mice, Nude , Mice, Transgenic , Neoplasm Transplantation , Vascular Endothelial Growth Factor A/metabolism
7.
Oral Oncol ; 47(5): 371-5, 2011 May.
Article in English | MEDLINE | ID: mdl-21421336

ABSTRACT

Kaposi's sarcoma (KS) remains among the most common causes of oral cancer in HIV-infected individuals. Infection with the KS-associated herpesvirus (KSHV/HHV8) is a necessary event for disease development. Emerging evidence suggests that KSHV infects vascular endothelial (or endothelial progenitor) cells promoting the formation of the KS tumor (or spindle) cell. These cells elaborate angiogenic growth factors and cytokines that promote the dysregulated angiogenesis and profuse edema that characterizes this unusual vascular tumor. Central among these secreted factors is the potent endothelial cell mitogen, vascular endothelial growth factor (VEGF). Indeed, VEGF has proven to be a key player in KSHV pathogenesis and is a molecular hallmark of KS lesions. We have recently shown that a second angiogenic factor, Angiopoietin-like 4 (ANGPTL4), may also play a critical role in KS development. Here we demonstrate that ANGPTL4 is upregulated both directly and indirectly by the KSHV oncogene, vGPCR. We further show that ANGPTL4 is a molecular hallmark of oral KS lesions. Indeed, expression of this protein was observed in more tumor cells and in more biopsies specimens than expression of VEGF (23/25 or 92% vs. 19/25 or 76%, respectively) in oral KS. These surprising results support a key role for ANGPTL4 in Kaposi's sarcomagenesis and further suggest that this angiogenic factor may provide a novel diagnostic and therapeutic marker for oral KS patients.


Subject(s)
Angiopoietins/metabolism , Herpesvirus 8, Human/metabolism , Mouth Neoplasms/metabolism , Sarcoma, Kaposi/metabolism , Vascular Endothelial Growth Factor A/metabolism , Adult , Angiogenesis Inducing Agents , Angiopoietin-Like Protein 4 , Angiopoietins/genetics , Cell Line, Tumor , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Endothelium, Vascular/virology , Female , Herpesvirus 8, Human/genetics , Humans , Male , Middle Aged , Mouth Neoplasms/pathology , Mouth Neoplasms/virology , Neoplasm Proteins/metabolism , Sarcoma, Kaposi/pathology , Sarcoma, Kaposi/virology , Up-Regulation , Vascular Endothelial Growth Factor A/genetics
8.
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
9.
Proc Natl Acad Sci U S A ; 107(32): 14363-8, 2010 Aug 10.
Article in English | MEDLINE | ID: mdl-20660728

ABSTRACT

Kaposi's sarcoma (KS) is an enigmatic vascular tumor thought to be a consequence of dysregulated expression of the human herpesvirus-8 (HHV-8 or KSHV)-encoded G protein-coupled receptor (vGPCR). Indeed, transgenic animals expressing vGPCR manifest vascular tumors histologically identical to human KS, with expression of the viral receptor limited to a few cells, suggestive of a paracrine mechanism for vGPCR tumorigenesis. Both human and vGPCR experimental KS lesions are characterized by prominent angiogenesis and vascular permeability attributed to the release of angiogenic molecules, most notably vascular endothelial growth factor. However, the relative contribution of these paracrine mediators to the angiogenic and exudative phenotype of KS lesions remains unclear. Here we show that vGPCR up-regulation of Angiopoietin-like 4 (ANGPTL4) plays a prominent role in promoting the angiogenesis and vessel permeability observed in KS. Indeed, ANGPTL4 expression is a hallmark of vGPCR experimental and human KS lesions. Inhibition of ANGPTL4 effectively blocks vGPCR promotion of the angiogenic switch and vascular leakage in vitro and tumorigenesis in vivo. These observations suggest that ANGPTL4 is a previously unrecognized target for the treatment of patients with KS. As angiogenesis and increased vessel permeability are common themes in all solid tumors, these findings may have a broad impact on our understanding and treatment of cancer.


Subject(s)
Angiopoietins/biosynthesis , Capillary Permeability , Neovascularization, Pathologic , Receptors, Chemokine/physiology , Sarcoma, Kaposi/physiopathology , Angiopoietin-Like Protein 4 , Angiopoietins/genetics , Cell Line , Host-Pathogen Interactions , Humans , Paracrine Communication , Vascular Endothelial Growth Factor A
10.
Cancer Res ; 68(20): 8361-8, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18922908

ABSTRACT

Rapamycin (or sirolimus), the prototypical inhibitor of the mammalian target of rapamycin (mTOR) and an immunosuppressant used for the prevention of renal transplant rejection, has recently emerged as an effective treatment for Kaposi's sarcoma (KS), an enigmatic vascular tumor and a model for pathologic angiogenesis. Indeed, recent work supports a role for mTOR as a central player in the transformation of endothelial cells by the KS-associated herpesvirus-encoded G protein-coupled receptor (vGPCR), the viral oncogene believed to be responsible for causing KS. However, emerging evidence that rapamycin may transiently promote the activation of Akt may limit its use as an anti-KS therapy. Here, we show that activation of Akt in endothelial cells expressing vGPCR is augmented by treatment with rapamycin, resulting in the up-regulation of several Akt proliferative and survival pathways. However, use of a novel dual phosphatidylinositol 3-kinase alpha (PI3Kalpha)/mTOR inhibitor, PI-103, effectively and independently blocked activation of both PI3K and mTOR in vGPCR-expressing endothelial cells. This resulted in more effective inhibition of endothelial cell proliferation and survival in vitro and tumor growth in vivo. Our results suggest that PI-103 may be an effective therapeutic option for the treatment of patients with KS. Moreover, as KS may serve as a model for pathologic angiogenesis, our results further provide the basis for the early assessment of PI-103 as an antiangiogenic chemotherapeutic.


Subject(s)
Antineoplastic Agents/pharmacology , Furans/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinases/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Sarcoma, Kaposi/drug therapy , Sirolimus/pharmacology , Angiogenesis Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Cell Line , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Furans/therapeutic use , Immunosuppressive Agents/pharmacology , Mice , Neoplasm Transplantation , Protein Kinases/physiology , Proto-Oncogene Proteins c-akt/physiology , Pyridines/therapeutic use , Pyrimidines/therapeutic use , Receptors, G-Protein-Coupled/analysis , Receptors, G-Protein-Coupled/physiology , Signal Transduction/drug effects , TOR Serine-Threonine Kinases , Transplantation, Homologous
11.
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
12.
Cancer Res ; 67(13): 6212-20, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17616678

ABSTRACT

Signal transducers and activators of transcription 3 (STAT3) was originally identified as a transcription factor that mediates cytokine-induced responses. In these pathways, Janus-activated kinase (JAK)-induced transient tyrosine phosphorylation of STAT3 promotes gene expression in response to a number of cytokines, which is inhibited by feedback mechanisms. A number of studies have shown that STAT3 is constitutively activated in human cancer cells, leading to cell proliferation. It is unclear, apart from a chronic tyrosyl phosphorylation of STAT3, what mechanisms contribute to the STAT3 deregulation in tumors. Earlier, we have isolated a novel growth inhibitory gene product, gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), using a genetic approach. GRIM-19 is an IFN/retinoic acid-regulated growth suppressor. Subsequent analyses have shown that GRIM-19 binds to STAT3 and prevents interleukin-6-induced transcription of cellular genes. However, its effects on a constitutively active STAT3 and cellular transformation are unknown. In this study, we show that GRIM-19 suppresses constitutive STAT3-induced cellular transformation in vitro and in vivo by down-regulating the expression of a number of cellular genes involved in cell proliferation and apoptosis.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/physiology , Gene Expression Regulation, Neoplastic , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/physiology , STAT3 Transcription Factor/metabolism , Animals , Apoptosis , Cell Death , Cell Proliferation , Cell Transformation, Neoplastic , Humans , Interleukin-6/metabolism , Mice , Phosphorylation , Rats , Transcription, Genetic , Tretinoin/metabolism
13.
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
14.
Cancer Cell ; 10(2): 133-43, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16904612

ABSTRACT

The Kaposi's sarcoma-associated herpesvirus (KSHV), the infectious causative agent of Kaposi's sarcoma (KS), encodes a G protein-coupled receptor (vGPCR) implicated in the initiation of KS. Here we demonstrate that Kaposi's sarcomagenesis involves stimulation of tuberin (TSC2) phosphorylation by vGPCR, promoting the activation of mTOR through both direct and paracrine mechanisms. Pharmacologic inhibition of mTOR with rapamycin prevented vGPCR sarcomagenesis, while overactivation of this pathway was sufficient to render endothelial cells oncogenic. Moreover, mice haploinsufficient for TSC2 are predisposed to vascular sarcomas remarkably similar to KS. Collectively, these results implicate mTOR in KS initiation and suggest that the sarcomagenic potential of KSHV may be a direct consequence of the profound sensitivity of endothelial cells to vGPCR dysregulation of the TSC2/mTOR pathway.


Subject(s)
Cell Transformation, Neoplastic , Endothelial Cells/pathology , Herpesvirus 8, Human/genetics , Protein Kinases/metabolism , Receptors, Chemokine/physiology , Tumor Suppressor Proteins/metabolism , Viral Proteins/physiology , Animals , Cell Line , Cell Proliferation , Humans , Mice , Mice, Nude , Mice, Transgenic , Oncogene Protein v-akt/antagonists & inhibitors , Oncogene Protein v-akt/metabolism , Paracrine Communication , Phosphorylation , Protein Kinases/genetics , Receptors, Chemokine/genetics , Sarcoma, Kaposi/pathology , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/genetics , Viral Proteins/genetics
15.
Cancer Res ; 66(1): 168-74, 2006 Jan 01.
Article in English | MEDLINE | ID: mdl-16397229

ABSTRACT

The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a G protein-coupled receptor (vGPCR) that has been implicated in the initiation of Kaposi's sarcoma, identifying vGPCR as an attractive target for preventing Kaposi's sarcoma. However, as only a fraction of cells in advanced Kaposi's sarcoma lesions express vGPCR, it is unclear whether this unique viral oncogene contributes to Kaposi's sarcoma progression. We therefore set out to determine whether the few cells that express vGPCR in established tumors represent an appropriate therapeutic target for the treatment of patients with preexisting Kaposi's sarcoma. To this end, we generated endothelial cell lines stably expressing vGPCR or key KSHV latently expressed proteins (vCyclin, vFlip, and LANA1). The endothelial cell line expressing vGPCR was rendered sensitive to treatment with the nucleoside analogue ganciclovir by using a bicistronic construct coexpressing the herpes simplex virus 1 thymidine kinase. S.c. injection into nude mice with mixed-cell populations formed tumors that approximate the ratio of vGPCR-expressing and KSHV latent gene-expressing cells. These mice were then treated with ganciclovir to specifically target only the vGPCR-expressing cells. Surprisingly, despite the expression of KSHV latent genes in the vast majority of tumor cells, specifically targeting only the few vGPCR-expressing cells in established tumors resulted in tumor regression. Moreover, we observed an increase in apoptosis of latent gene-expressing cells after the pharmacologic deletion of the vGPCR-expressing cells. These findings indicate that vGPCR may play a key role in Kaposi's sarcoma progression and provide experimental justification for developing molecular-based therapies specifically targeting vGPCR and its effectors for the treatment of Kaposi's sarcoma patients.


Subject(s)
Herpesvirus 8, Human/physiology , Receptors, G-Protein-Coupled/physiology , Sarcoma, Kaposi/metabolism , Sarcoma, Kaposi/therapy , Animals , Cell Transformation, Viral , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelial Cells/virology , Female , Ganciclovir/pharmacology , Herpesvirus 1, Human/enzymology , Herpesvirus 1, Human/genetics , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/metabolism , Mice , Mice, Nude , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/genetics , Sarcoma, Kaposi/genetics , Sarcoma, Kaposi/virology , Thymidine Kinase/biosynthesis , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Transfection
16.
J Interferon Cytokine Res ; 25(11): 666-73, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16318580

ABSTRACT

Interferon-gamma (IFN-gamma) regulates a number of cellular genes using a variety of cellular signaling pathways. Previously, we identified a novel IFN-regulated element, IFN-gamma-activated transcriptional element (GATE), in the promoter of the murine IFN regulatory factor-9 (IRF-9) gene. This element binds to novel factors. We have recently characterized a novel regulatory factor, GATE binding factor 1 (GBF1), which promoted IFN-gamma-induced transcription. Although GBF1 was a potent inducer of transcription, it did not bind to DNA well in vitro. To understand its role in IFN-gamma-induced actions, we raised monoclonal antibodies (mAb) against GBF1. These antibodies are highly useful in Western, immunoprecipitation, and immunocytochemical analyses. Employing these antibodies, we show that GBF1 is recruited to the endogenous IRF-9 promoter. We also show GBF1 interacts with CAAAT/enhancer binding protein-beta (C/EBP-beta), the other GATE binding factor. Furthermore, other cytokines, such as interleukin-1 (IL-1) and IL-6, induced the expression of GBF1. These antibodies may be useful tools for investigating the role of GBF1 in cytokine-induced responses.


Subject(s)
Antibodies, Monoclonal/chemistry , CCAAT-Enhancer-Binding Protein-beta/metabolism , Guanine Nucleotide Exchange Factors/chemistry , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , Interferon-gamma/metabolism , Interleukin-1/metabolism , Animals , Blotting, Western , Cell Line , Chromatin Immunoprecipitation , Cytokines/metabolism , DNA/chemistry , Enzyme-Linked Immunosorbent Assay , Guanine Nucleotide Exchange Factors/immunology , HeLa Cells , Humans , Immunohistochemistry , Immunoprecipitation , Interferons/metabolism , Mice , Promoter Regions, Genetic , Protein Binding , Recombinant Proteins/chemistry , Signal Transduction , Time Factors , Transcription, Genetic
17.
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
18.
Proc Natl Acad Sci U S A ; 100(16): 9342-7, 2003 Aug 05.
Article in English | MEDLINE | ID: mdl-12867595

ABSTRACT

GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-beta- and retinoic acid-induced cell death, codes for a approximately 16-kDa protein that induces apoptosis in a number of cell lines. Antisense ablation of GRIM-19 caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells. To understand the molecular bases for its cell death regulatory activity, we used a yeast two-hybrid screen and identified that the transcription factor STAT3 (signal transducer and activator of transcription 3) binds to GRIM-19. GRIM-19 inhibits transcription driven by activation of STAT3, but not STAT1. It neither inhibits the ligand-induced activation of STAT3 nor blocks its ability to bind to DNA. Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19 binding. Because GRIM-19 does not bind significantly to other STATs, our studies identify a specific inhibitor of STAT3. Because constitutively active STAT3 up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also demonstrates an antioncogenic effect exerted by biological therapeutics.


Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , NADH, NADPH Oxidoreductases/physiology , Trans-Activators/antagonists & inhibitors , Animals , Apoptosis , Apoptosis Regulatory Proteins , Cell Death , DNA/metabolism , DNA Mutational Analysis , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Humans , Interferon-beta/pharmacology , Ligands , Mice , Oligonucleotide Array Sequence Analysis , Oligonucleotides, Antisense/pharmacology , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , STAT1 Transcription Factor , STAT3 Transcription Factor , Serine/chemistry , Trans-Activators/metabolism , Trans-Activators/physiology , Transfection , Tretinoin/pharmacology , Two-Hybrid System Techniques , Up-Regulation
19.
J Interferon Cytokine Res ; 22(10): 1017-26, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12433281

ABSTRACT

A combination of interferon-beta (IFN-beta) and all-trans retinoic acid (IFN/RA) induces tumor cell apoptosis via some unknown mechanisms. Apoptosis is a gene-directed process that limits the proliferation of undesired cells. Several genes are required to regulate cell death in the higher-order animals. Earlier, we employed a gene expression knockout technique to isolate cell death-related genes. A novel gene, the gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), was found to be essential for tumor cell death induced by IFN/RA. Here, we describe the development and characterization of three monoclonal antibodies (mAbs) against GRIM-19. GRIM-19 is present in the nucleus and cytoplasm. Its expression is induced by the IFN/RA combination. We also show that GRIM-19 inhibits the cell-transforming property of viral oncogenic protein viral IFN regulatory factor-1 (vIRF-1) via a physical interaction. mAbs developed in this study should be useful for studying the other physiologic roles of GRIM-19 and serve as a potent tool for studying tumor responses to IFN/RA therapy.


Subject(s)
Antibodies, Monoclonal/immunology , Apoptosis/drug effects , Gene Expression Regulation/drug effects , Genes, Tumor Suppressor , Interferon-beta/pharmacology , NADH, NADPH Oxidoreductases/immunology , 3T3 Cells , Animals , Apoptosis Regulatory Proteins , Humans , Immunologic Factors/pharmacology , Mice , Mice, Mutant Strains , NADH, NADPH Oxidoreductases/metabolism , Nuclear Proteins/immunology , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Tretinoin/pharmacology , Tumor Cells, Cultured
20.
Clin Cancer Res ; 8(10): 3210-8, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12374691

ABSTRACT

The IFN and retinoic acid (RA) combination suppresses cell growth by inducing apoptosis in the cultured tumor cells. Using a genetic technique, we have isolated several "genes associated with retinoid-IFN-induced mortality" (GRIM) that participate in this death pathway. One such gene, GRIM-12, encodes the redox enzyme thioredoxin reductase (TR). Antisense-mediated inhibition of TR abrogates cell death. To test the in vivo relevance of TR for growth suppression, we have conducted the following study. A wild-type TR or a catalytically defective mutant were expressed in MCF-7 breast carcinoma cells and transplanted into athymic nude mice. These mice were treated with IFN-beta and all-trans RA combination. Tumors expressing the vector or wild-type TR were readily suppressed by the IFN/RA combination. In contrast, the tumors bearing a mutant TR were resistant to regression. We further show that markers of apoptosis are stimulated in the regressing tumors. These studies show a prominent role for TR in tumor-growth suppression.


Subject(s)
Antineoplastic Agents/pharmacology , Interferon-beta/pharmacology , Mammary Neoplasms, Experimental/enzymology , Thioredoxin-Disulfide Reductase/physiology , Tretinoin/pharmacology , Animals , Annexin A5/metabolism , Apoptosis , Blotting, Western , Caspase 8 , Caspase 9 , Caspases/metabolism , Down-Regulation , Enzyme Activation , Female , Gene Expression , Humans , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Mutagenesis, Site-Directed , Neoplasm Transplantation , Transfection , Tumor Cells, Cultured/drug effects
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