Woong-Shick Ahn

A Therapy Modality Using Recombinant IL-12 Adenovirus plus E7 Protein in a Human Papillomavirus 16 E6/E7-Associated Cervical Cancer Animal Model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.

Pharmacodynamics of insulin in polyethylene glycol-coated liposomes

To reduce the injection frequency and toxicity of intravenously administered protein drugs, it is necessary to develop safe and sustained injectable delivery systems. In this study, to evaluate liposomes as safe and sustained injectable delivery systems of proteins, we chose insulin as a model protein drug and tested its incorporation efficiency and pharmacodynamics in various liposomes with and without polyethylene glycol (PEG)-derivatized phospholipid. The liposomes coated with PEG showed 3-fold higher efficiency of insulin incorporation than did the liposomes without PEG. Moreover, among the liposomes coated with PEG, dipalmitoylphosphocholine (DPPC) liposomes showed higher incorporation efficiency than did dimyristoylphosphocholine (DMPC) liposomes. For pharmacodynamic study, insulin (2 IU/kg) was administered in various formulations, such as insulin alone in phosphate-buffered saline and insulin in the DPPC liposomes with and without PEG, to streptozotocin-treated diabetic rats. The pharmacodynamics of insulin alone, however, could not be measured due to the immediate death of rats caused by hypoglycemic shock. In contrast, all the rats treated with liposomal insulin survived, probably by the sustained release of insulin from liposomes. Pharmacodynamics of liposomal insulin showed that PEG-coated liposomes induced the lowest level of blood glucose-the nadir-1 h later than did the liposomes without PEG. These results indicate that PEG-coated liposomes could be developed as a relatively safe and sustained injectable delivery system for insulin with improved incorporation efficiency. Moreover, it is suggested that the liposomes coated with PEG might have a potential as safe injectable delivery systems for other protein and peptide drugs.

Enhancement of polyethylene glycol (PEG)‐modified cationic liposome‐mediated gene deliveries: effects on serum stability and transfection efficiency

In this study, we modified cationic liposomes either by polyethylene glycol (PEG)‐grafting or PEG‐adding methods, and compared the physical properties of transfection complexes and transfection efficiency in‐vitro and prolonged circulation in‐vivo. The PEG‐grafted transfection complexes were prepared by mixing plasmid DNA with PEG‐grafted cationic liposomes, which were composed of DSPE‐PEG 2000 and cationic lipids. The PEG‐added transfection complexes were prepared by adding DSPE‐PEG 2000 to the mixture of cationic liposomes and plasmid DNA. The particle sizes of the PEG‐modified transfection complexes (˜200 nm) changed a little over 4 weeks compared with the conventional transfection complexes. In the presence of serum, the transfection efficiency of the conventional transfection complexes was lowered whereas the transfection efficiency of the PEG‐modified transfection complexes was maintained. Moreover, the transfection efficiency of the conventional transfection complexes was significantly reduced when they were stored. However, the transfection efficiency was stable for the PEG‐modified transfection complexes, even after two weeks of storage. Of the in‐vitro transfection efficiencies, there was no difference between PEG‐grafted and PEG‐added transfection complexes. When the conventional, PEG‐grafted, and PEG‐added transfection complexes were administered into mice by the tail vein, the PEG‐added transfection complexes showed a prolonged circulation of plasmid DNA compared with other transfection complexes. These results suggest that the PEG‐added transfection complexes could be a useful non‐viral vector because of their simplicity in preparation, enhanced stability and prolonged circulation compared with the conventional transfection complexes.

CpG-ODN-stimulated dendritic cells act as a potent adjuvant for E7 protein delivery to induce antigen-specific antitumour immunity in a HPV 16 E7-associated animal tumour model.

We previously reported that both E7 and CpG‐oligodeoxynucleotide (ODN) are required for protecting animals from human papillomavirus (HPV) 16 E7‐associated tumour challenge. Here we investigate dendritic cells (DC)‐based approach in this protection. In the study, we isolated bone marrow‐derived DC and stimulated DC with E7 and ODN. In vitro stimulation of DC with E7 plus ODN resulted in more production of interleukin‐12, as compared to that with E7 or ODN alone. Further injection with E7+ODN‐stimulated DC resulted in more significant tumour protection, as compared to stimulation with E7 or ODN alone. We further evaluated the levels of immune responses induced by DC stimulated with E7+ODN. We observed little enhancement of E7‐specific antibody and T helper cell proliferative responses by E7+ODN stimulation, as compared to E7 stimulation. However, there was some enhancement of interferon‐γ (IFN‐γ) production from CD4+ T cells and a more significant production of IFN‐γ from CD8+ T cells by E7+ODN stimulation, as compared to E7 stimulation alone. This was consistent with intracellular IFN‐γ staining levels of CD8+ T cells. Tumour protection further appeared to be mediated by CD8+ T cells, as determined by in vivo T‐cell depletion. Thus, these data suggest that upon ODN stimulation DC might function as a potent adjuvant for E7 protein delivery for induction of protective cellular immunity against HPV E7‐associated tumour challenge.

Lys-63-specific Deubiquitination of SDS3 by USP17 Regulates HDAC Activity

SDS3 is a key component of the histone deacetylase (HDAC)-dependent Sin3A co-repressor complex, serving to maintain its HDAC activity. Here, we report both exogenous and endogenous functional interaction between deubiquitinating enzyme USP17 and human SDS3 by MALDI-TOF-MS, co-immunoprecipitation assay, and GST pull-down assay. In this study, we demonstrated that SDS3 readily undergoes endogenous polyubiquitination, which is associated specifically with Lys-63-branched polyubiquitin chains and not with Lys-48-branched polyubiquitin chains. Further, we also demonstrated that USP17 specifically deubiquitinates Lys-63-linked ubiquitin chains from SDS3 and regulates its biological functions. The deubiquitinating activity of USP17 on SDS3 negatively regulates SDS3-associated HDAC activity. The constitutive expression of USP17 and its substrate SDS3 was involved in the inhibition of anchorage-independent tumor growth and blocks cell proliferation, leading to apoptosis in cervical carcinoma cells. Furthermore, we showed that USP17 and SDS3 mutually interact with each other to regulate cancer cell viability. These data support the possibility that SDS3, being a substrate of USP17, may play an important role in developing a novel therapeutic means to inhibit specific HDAC activities in cancer.

Photodynamic therapy‐generated tumor cell lysates with CpG‐oligodeoxynucleotide enhance immunotherapy efficacy in human papillomavirus 16 (E6/E7) immortalized tumor cells

Immunotherapy with photodynamic therapy (PDT) offers great promise as a new alternative for cancer treatment; however, its use remains experimental. In this study, we examined the immunotherapeutic significance of human papillomavirus (HPV)‐immortalized tumor cell lysates induced by PDT with CpG‐oligodeoxynucleotide (ODN). PDT‐cell lysates were generated by irradiating Radachlorin (5 µg/mL) preloaded TC‐1 cells carrying HPV 16 E7. PDT‐cell lysates plus ODN coinjection for protection against E7‐expressing tumors as well as specific immune responses were evaluated with the following tests: heat shock protein 70 (HSP70) enzyme‐linked immunosorbent assay, in vitro and in vivo tumor growth inhibition, interferon‐γ (IFN‐γ) and tumor necrosis factor‐α (TNF‐α) assay, cytotoxic T‐lymphocyte assay, and fluorescence activated cell sorting (FACS) analysis. PDT‐cell lysates plus ODN coinjection showed a significant suppression of tumor growth at both prophylactic and therapeutic levels, compared to PDT (or F/T)‐cell lysates or ODN alone. In addition, we evaluated the level of the immune response with the coinjection. HSP70, an important regulator of inflammatory and immune response, was observed in abundance in the PDT‐cell lysates. IFN‐γ production and cytotoxic T lymphocytes (CTL) responses were induced by PDT‐cell lysates plus ODN injection. The coinjection resulted in PDT‐cell lysate‐specific antibodies (IgG1, IgG2a, IgG2b, and IgG3) and T‐helper cell responses significantly higher than PDT‐cell lysates alone. Moreover, IFN‐γ production and CTL responses were significantly induced in the PDT‐cell lysate plus ODN immunized groups. These enhanced immune responses appeared to be mediated by CD8+ T cells only. These data suggest that PDT‐cell lysates plus ODN injection may be an effective approach to induce CTL immune responses as a possible immunotherapeutic strategy for cancer therapy. (Cancer Sci 2007; 98: 747–752)

Hypoxic activation of unoccupied estrogen-receptor-alpha is mediated by hypoxia-inducible factor-1 alpha.

The estrogen receptor (ER) plays an important role in breast cancer development and progression. Hypoxia has been shown to modulate the level of ERalpha expression, which is intimately associated with the biology of breast carcinomas. However, the effect of hypoxia on ERalpha-mediated transactivation is largely unknown. In this report, we have examined ligand-independent transcriptional activation of ERalpha by hypoxia. The hypoxia-induced ERalpha-mediated transcriptional response was inhibited by the ER antagonist ICI 182,780 as determined by transient expression of ERalpha and ER-responsive reporter plasmids in the HEK 293 cells. Hypoxic activation of ERalpha was dependent on the increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha), as examined in HEK 293 cells under conditions of normoxia. These results indicate that hypoxia activates ERalpha in a ligand-independent manner, possibly through the interaction between HIF-1alpha and ERalpha.

Photodynamic Effects of Radachlorin® on Cervical Cancer Cells

PURPOSEnPhotodynamic therapy (PDT) is a novel treatment modality, which produces local tissue necrosis with laser light following the prior administration of a photosensitizing agent. Radachlorin has recently been shown to be a promising PDT sensitizer. In order to elucidate the antitumor effects of PDT using Radachlorin on cervical cancer, growth inhibition studies on a HPV-associated tumor cell line, TC-1 cells in vitro and animals with an established TC-1 tumor in vivo were determined.nnnMATERIALS AND METHODSnTC-1 tumor cells were exposed to various concentrations of Radachlorin and PDT, with irradiation of 12.5 or 25 J/cm(2) at an irradiance of 20 mW/cm(2) using a Won-PDT D662 laser at 662 nm in vitro. C57BL/6 mice with TC-1 tumor were injected with Radachlorin via different routes and treated with PDT in vivo. A growth suppression study was then used to evaluate the effects at various time points after PDT.nnnRESULTSnThe results showed that irradiation of TC-1 tumor cells in the presence of Radachlorin induced significant cell growth inhibition. Animals with established TC-1 tumors exhibited significantly smaller tumor sizes over time when treated with Radachlorin and irradiation.nnnCONCLUSIONnPDT after the application of Radachlorin appears to be effective against TC-1 tumors both in vitro and in vivo.

Comparative evaluation of ELISA and Luminex panel reactive antibody assays for HLA alloantibody screening

BACKGROUNDnFor the detection of HLA antibodies, solid-phase tests using purified HLA antigens are increasingly used. In this study, we analyzed the panel reactive antibody (PRA) test results using ELISA and Luminex methods, and the results were compared with those of crossmatch test.nnnMETHODSnA total of 111 sera including 90 sera from kidney transplanted patients were tested. ELISA-PRA was performed using Lambda Antigen Tray Class I and II Mixed kits (One Lambda Inc., USA) and additional test was performed to identify HLA specificities. Luminex-PRA tests were performed using LABScreen Mixed kits (One Lambda Inc., USA) and LIFECODES LifeScreen Deluxe kits (Tepnel Co., USA).nnnRESULTSnThe positive rates of PRA were higher in Tepnel (P=0.006) and One Lambda Luminex (P<0.001) methods than ELISA, without significant difference between two Luminex methods (P=0.087). The overall concordance rate among the three PRA tests was 62.2% (69/111). The positive and negative predictive values of PRA tests for the flow cytometric crossmatch were 33.3-45.7% and 85.7-89.5%, respectively. Of the two Luminex methods, One Lambda showed higher positive rate than Tepnel for the detection of class I antibodies. The sensitivity of pretransplant PRA for the detection of posttransplant acute rejection episodes was higher in Luminex (P=0.007 for Tepnel, P=0.003 for One lambda) than ELISA method.nnnCONCLUSIONSnDifferent methods used to detect HLA antibodies showed discrepant results. As the Luminex method was more sensitive than ELISA for the detection of HLA antibodies, it can be used as a routine test in the transplantation laboratory.

Estrogen activities and the cellular effects of natural progesterone from wild yam extract in mcf-7 human breast cancer cells.

We studied the estrogenic activity and cellular effect of wild yam extract in MCF-7 human breast cancer cells. The extract increased the activity of the progesterone receptor and pS2 genes at the mRNA levels in human breast cancer MCF-7 cells, although the effects were not as prominent as those of 17beta-estradiol (E(2)). Western blot analysis showed that the level of estrogen receptor alpha protein was down-regulated after treatment with E(2) or wild yam extract. Wild yam extract also inhibited proliferation of MCF-7 cells. These data indicate that wild yam extract acts as a weak phytoestrogen and protects against proliferation in human breast carcinoma MCF-7 cells.