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Background: Pancreatic cancer often goes undiagnosed until late stage disease due in part to suboptimal early detection. Our goal was to develop a Syndecan-1 tagged liposome containing fluorescent dye as an improved contrast agent for detection of pancreatic adenocarcinoma in vivo using multispectral optoacoustic tomography. Results: The diagnostic capabilities and specificity to pancreatic adenocarcinoma of Syndecan-1 targeted liposomes were evaluated both in vitro and in vivo. Immunocytochemistry showed that liposomes preferentially bound to and released their contents into cells expressing high levels of insulin-like growth factor 1 receptor. We determined that the contents of the liposome were released into the cell as noted by the change in propidium iodide fluorescence from green to red based upon nucleic acid binding. In an orthotopic mouse model, the liposomes preferentially targeted the pancreatic tumor with little off-target binding in the liver and spleen. Peak accumulation of the liposomes in the tumor occurred at 8 h post-injection. Multispectral optoacoustic tomographic imaging was able to provide high-resolution 3D images of the tumor and liposome location. Ex vivo analysis showed that non-targeted liposomes accumulated in the liver, suggesting that specificity of the liposomes for pancreatic adenocarcinoma was due to the presence of the Syndecan-1 ligand. Conclusions: This study demonstrated that Syndecan-1 liposomes were able to release cargo into IGF1-R expressing tumor cells. The Syndecan-1 liposomes demonstrated tumor specificity in orthotopic pancreatic cancer as observed using multispectral optoacoustic tomography with reduced kidney and liver uptake. By targeting the liposome with Syndecan-1, this nanovehicle has potential as a targeted theranostic nanoparticle for both drug and contrast agent delivery to pancreatic tumors. [ABSTRACT FROM AUTHOR]

Background: Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10. Results: The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes. Conclusion: Expression and secretion of mature IP-10 was efficiently achieved by L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant L. lactis represents a potentially useful tool to be used as a live vaccine in vivo. [ABSTRACT FROM AUTHOR]

Background: PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. Methods: PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Results: Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Conclusion: Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling pathways provided here will further enhance insights on the mechanisms of E2F-1-induced cancer cell apoptosis as a strategy for cancer therapy. [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer often goes undiagnosed until late stage disease due in part to suboptimal early detection. Our goal was to develop a Syndecan-1 tagged liposome containing fluorescent dye as an improved contrast agent for detection of pancreatic adenocarcinoma in vivo using multispectral optoacoustic tomography., Results: The diagnostic capabilities and specificity to pancreatic adenocarcinoma of Syndecan-1 targeted liposomes were evaluated both in vitro and in vivo. Immunocytochemistry showed that liposomes preferentially bound to and released their contents into cells expressing high levels of insulin-like growth factor 1 receptor. We determined that the contents of the liposome were released into the cell as noted by the change in propidium iodide fluorescence from green to red based upon nucleic acid binding. In an orthotopic mouse model, the liposomes preferentially targeted the pancreatic tumor with little off-target binding in the liver and spleen. Peak accumulation of the liposomes in the tumor occurred at 8 h post-injection. Multispectral optoacoustic tomographic imaging was able to provide high-resolution 3D images of the tumor and liposome location. Ex vivo analysis showed that non-targeted liposomes accumulated in the liver, suggesting that specificity of the liposomes for pancreatic adenocarcinoma was due to the presence of the Syndecan-1 ligand., Conclusions: This study demonstrated that Syndecan-1 liposomes were able to release cargo into IGF1-R expressing tumor cells. The Syndecan-1 liposomes demonstrated tumor specificity in orthotopic pancreatic cancer as observed using multispectral optoacoustic tomography with reduced kidney and liver uptake. By targeting the liposome with Syndecan-1, this nanovehicle has potential as a targeted theranostic nanoparticle for both drug and contrast agent delivery to pancreatic tumors.

Background: PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells., Methods: PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit., Results: Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway., Conclusion: Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling pathways provided here will further enhance insights on the mechanisms of E2F-1-induced cancer cell apoptosis as a strategy for cancer therapy.