Enhanced efficacy of photodynamic therapy by inhibiting ABCG2 in colon cancers.

BACKGROUND: Photodynamic therapy (PDT) contains a photosensitizing process, which includes cellular uptake of photosensitizer and delivery of light to the target. ATP-binding cassette subfamily G2 (ABCG2) regulates endogenous protoporphyrin levels. In human colon cancers, it is not fully examined the role of ABCG2 in porphyrin-based photodynamic therapy. METHODS: SW480 and HT29 cells were selected because they showed low and high ABCG2 expression levels, respectively. Pyropheophorbid-a (PPa) was used as a photosensitizer. Cells were exposed to a 670 nm diod laser. Cell viability and necrosi apoptosis was examined. Production level of singlet oxygen was detected with the photomultiplier-tube s/ -based singlet oxygen detection system. RESULTS: SW480 cells, which expressed lower level of ABCG2, showed the higher uptake of PPa than HT-29 cells. The uptake level of PPa was significantly correlated with the decreased cell viability after PDT. Pretreatment with a ABCG2 inhibitor, Ko-143, significantly enhanced the PDT efficacy in HT29 cells compared to vehicle-pretreated cells. To confirm the ABCG2 effect on PDT, we established ABCG2 over-expressing stable cells in SW480 cells (SW480/ABCG2). Furthermore, SW480/ABCG2 cells showed significantly decreased PDT effect compared to the control cells. The increased or decreased cell survival was significantly correlated with the production level of singlet oxygen after PDT. CONCLUSION: ABCG2 plays an important role in determining the PDT efficacy by controlling the photosensitizer efflux rate. This implies the control of ABCG2 expression may be a potential solution to enhance photosensitivity.

Synergistic antitumor immune response mediated by paclitaxel-conjugated nanohybrid oncolytic adenovirus with dendritic cell therapy.

Dendritic cell (DC)-based vaccines have emerged as a promising strategy in cancer immunotherapy due to low toxicity. However, the therapeutic efficacy of DC as a monotherapy is insufficient due to highly immunosuppressive tumor environment. To address these limitations of DC as immunotherapeutic agent, we have developed a polymeric nanocomplex incorporating (1) oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and (2) arginine-grafted bioreducible polymer with PEGylated paclitaxel (APP) to restore antitumor immune surveillance function in tumor milieu and potentiate immunostimulatory attributes of DC vaccine. Nanohybrid complex (oAd/APP) in combination with DC (oAd/APP+DC) induced superior expression level of antitumor cytokines (IL-12, GM-CSF, and interferon gamma) than either oAd/APP or DC monotherapy in tumor tissues, thus resulting in superior intratumoral infiltration of both endogenous and exogenous DCs. Furthermore, oAd/APP+DC treatment led superior migration of DC to secondary lymphoid organs, such as draining lymph nodes and spleen, in comparison with either monotherapy. Superior migration profile of DCs in oAd/APP+DC treatment group resulted in more prolific activation of tumor-specific T cells in these lymphoid organs and greater intratumoral infiltration of T cells. Additionally, oAd/APP+DC treatment led to lower subset of tumor infiltrating lymphocytes and splenocytes being immunosuppressive regulatory T cells than any other treatment groups. Collectively, oAd/APP+DC led to superior induction of antitumor immune response and amelioration of immunosuppressive tumor microenvironment to elicit potent tumor growth inhibition than either monotherapy.

Proproliferative functions of Drosophila small mitochondrial heat shock protein 22 in human cells.

Aging is a complex process accompanied by a decreased capacity of cells to cope with random damages induced by reactive oxygen species, the natural by-products of energy metabolism, leading to protein aggregation in various components of the cell. Chaperones are important players in the aging process as they prevent protein misfolding and aggregation. Small chaperones, such as small heat shock proteins, are involved in the refolding and/or disposal of protein aggregates, a feature of many age-associated diseases. In Drosophila melanogaster, mitochondrial Hsp22 (DmHsp22), is localized in the mitochondrial matrix and is preferentially up-regulated during aging. Its overexpression results in an extension of life span (>30%) (Morrow, G., Samson, M., Michaud, S., and Tanguay, R. M. (2004) FASEB J. 18, 598-599 and Morrow, G., Battistini, S., Zhang, P., and Tanguay, R. M. (2004) J. Biol. Chem. 279, 43382-43385). Long lived flies expressing Hsp22 also have an increased resistance to oxidative stress and maintain locomotor activity longer. In the present study, the cross-species effects of Hsp22 expression were tested. DmHsp22 was found to be functionally active in human cells. It extended the life span of normal fibroblasts, slowing the aging process as evidenced by a lower level of the senescence associated beta-galactosidase. DmHsp22 expression in human cancer cells increased their malignant properties including anchorage-independent growth, tumor formation in nude mice, and resistance to a variety of anticancer drugs. We report that the DmHsp22 interacts and inactivates wild type tumor suppressor protein p53, which may be one possible way of its functioning in human cells.

Efficacy of horse oil on lipopolysaccharide-induced inflammation in human keratinocyte.

OBJECTIVE: To investigate the efficacy of horse oil on lipopolysaccharide (LPS)-induced inflammation in human keratinocytes. METHODS: Western blot analysis was performed to measure the expression of cyclooxygenase-2 (COX-2) and IκBα. ELISA was used to analyze prostaglandin E2 (PGE2) levels. RESULTS: Horse oil decreased LPS-induced COX-2 and PGE2 levels in a dose-dependent manner. Nuclear factor-kappa B (NF-κB) plays a key role in the expression of inflammatory cytokines and mediators. Therefore, we investigated the influence of horse oil on the NF-κB signaling pathways. Horse oil inhibited translocation of NF-κB from the cytosol to the nucleus. Furthermore, LPS-induced degradation of IκBα was recovered by horse oil. The activation of p38 mitogen-activated protein kinase (MAPK) reportedly induces degradation of IκBα In agreement with this, LPS activated p38 MAPK and caused IκBα degradation. Conversely, horse oil inhibited LPS-induced p38 MAPK activation and IκBα degradation. In addition, a specific p38 MAPK inhibitor, SB203580, blocked IκBα degradation. CONCLUSION: Horse oil decreased COX-2 and PGE2 by inhibiting p38 MAPK activation, IκBα degradation, and the translocation of NF-κB.

p62 manipulation affects chlorin e6-mediated photodynamic therapy efficacy in colorectal cancer cell lines.

p62 is a multifunctional protein that mediates cell signaling pathways, autophagy and tumorigenesis, and participates in important regulation processes at the intersection between autophagy and cancer. Photodynamic therapy (PDT) is a treatment that involves photosensitizing agents and light to kill cancer cells. However, whether the efficacy of PDT depends on the expression level of p62 in colorectal cancer cell lines is not known. The present study aimed to examine the role of p62 expression levels in chlorin e6-based PDT in colorectal cancer cells. To study the effect of p62 on cancer cell death, we used PDT to treat a stable cell line overexpressing p62. Cells overexpressing p62 showed a higher cell death rate than cells not expressing this protein. Overexpression of p62 may contribute to colorectal cancer cell death. These results provide preliminary evidence for use of p62 as a therapy target to treat colorectal cancer.

Photodynamic Therapy Using Photosensitizer-Encapsulated Polymeric Nanoparticle to Overcome ATP-Binding Cassette Transporter Subfamily G2 Function in Pancreatic Cancer.

Chlorin-based photosensitizers are commonly used in photodynamic therapy (PDT). These drugs are effluxed by cell membrane transporters, such as the ATP-binding cassette subfamily G member 2 (ABCG2). PDT efficacy is limited in tumor cells expressing high levels of these proteins. Pancreatic cancer cell lines AsPC-1 and MIA PaCa-2, which have high and low ABCG2 expression, respectively, were used, and ABCG2-overexpressing MIA PaCa-2 cells were generated. We compared PDT efficacy between chlorin e6 (Ce6) and cationic photosensitizer-encapsulated polymeric nanoparticle (PS-pNP), which is comprised with Ce6, polyethylene glycol, and polyethylenimine. The intracellular concentration of Ce6 was significantly higher in MIA PaCa-2 cells than in AsPC-1 or ABCG2-overexpressing MIA PaCa-2 cells. PS-pNP increased intracellular levels of the photosensitizer in all cell lines. The cell viability experiments indicated increased Ce6 resistance in ABCG2-overexpressing cells. In contrast, PS-pNP produced similar levels of cytotoxicity in each of the cancer cell lines tested. Singlet oxygen production was higher in cells treated with PS-pNP than in those treated with Ce6. Furthermore, in heterotopic and orthotopic AsPC-1 xenograft mouse models, PDT using PS-pNP significantly reduced tumor volume in comparison with that of Ce6 treatment. PS-pNP could increase intracellular Ce6 concentration, which was related with reduced ABCG2-mediated efflux of Ce6, thereby enhancing the effects of PDT in pancreatic cancer cells. Mol Cancer Ther; 16(8); 1487-96. ©2017 AACR.

Fluorescence lifetime measurement with confocal endomicroscopy for direct analysis of tissue biochemistry in vivo.

Confocal endomicroscopy is a powerful tool for in vivo real-time imaging at cellular resolution inside a living body without tissue resection. Microscopic fluorescence lifetime measurement can provide information about localized biochemical conditions such as pH and the concentrations of oxygen and calcium. We hypothesized that combining these techniques could assist accurate cancer discrimination by providing both biochemical and morphological information. We designed a dual-mode experimental setup for confocal endomicroscopic imaging and fluorescence lifetime measurement and applied it to a mouse xenograft model of activated human pancreatic cancer generated by subcutaneous injection of AsPC-1 tumor cells. Using this method with pH-sensitive sodium fluorescein injection, we demonstrated discrimination between normal and cancerous tissues in a living mouse. With further development, this method may be useful for clinical cancer detection.

Direct measurement of singlet oxygen by using a photomultiplier tube-based detection system.

The effective dosimetry for photodynamic therapy (PDT) can be specified by direct measurement of singlet oxygen ((1)O2) production. The purpose of this study was to investigate the feasibility of a newly developed photomultiplier tube (PMT)-based singlet oxygen detection (SOD) system. The lowest and highest (1)O2 concentrations detectable by the PMT-SOD system were 15nM and 10µM, respectively. Dose-dependent quenching, by NaN3, of the fluorogenic reaction was observed, which was negatively correlated with the (1)O2 level measured by the PMT-SOD system. The lifetime of (1)O2, as measured by the PMT-SOD system, was found to be lengthened when H2O was replaced with deuterium oxide. (1)O2 photon counts were significantly and dose-dependently correlated with intracellular fluorescence intensity after photosensitizer treatments. In vitro cell viability test and in vivo xenografted-tumor mass shrinkage showed a positive association between PDT-induced cytotoxicity and (1)O2 production concomitantly measured by the PMT-SOD system. It was concluded that the PMT-SOD system is capable of measuring (1)O2 production directly and accurately, demonstrating that this system can be useful in the determination of dosimetry for PDT.

Development of bead-based immunoassay to quantify neutralizing antibody for human papillomavirus 16 and 18.

Human papillomavirus (HPV) has drawn great attention globally because of its association with virtually all (99 %) cases of cervical cancer. HPV virus-like particles (VLPs) have been implicated as an effective HPV vaccine candidate. In this study, we optimized the relevant parameters for bacterial production of high-risk HPV16 and HPV18 VLP L1 proteins. The combination of glutathione S-transferase fusion and late log phase culture induction enhanced the solubility and yield of HPV L1 proteins. For detection and quantification of HPV-16 and -18 antibodies, a Luminex-based competitive immunoassay was developed for use in vaccine clinical trials. The characteristics of the assay that were optimized included monoclonal antibody specificity, conjugation of VLP to microspheres, VLP concentration, antibody concentration, dilution of samples, and incubation time. No cross-reactivity occurred. This immunoassay was proven to be sensitive and accurate, and is potentially valuable for vaccine candidate evaluation and clinical use.

Comparison of single-, double- and triple-combined testing, including Pap test, HPV DNA test and cervicography, as screening methods for the detection of uterine cervical cancer.

Cervical cancer is a serious disease that threatens the health of women worldwide. This study compared the sensitivities and false-positive rates of cervical cytology (Pap smear), human papilloma virus (HPV) DNA test, cervicography, first double-combined testing (cervical cytology and HPV DNA test), second double-combined testing (cervical cytology and cervicography) and triple-combined testing (cervical cytology, HPV DNA test and cervicography). The study included 261 patients screened for uterine cervical cancer. All women simultaneously underwent cervical cytology, HPV DNA test and cervicography for uterine cervical cancer screening and colposcopically directed biopsy for diagnostic evaluation. The triple-combined testing was consistently the most sensitive among the cervical screening tests. The second double-combined testing, with a sensitivity rate of 98.1% was more sensitive than the first double-combined test (92.3%). However, cervical cytology was most specific (93.5%) and showed the highest positive predictive value (77.8%). The sensitivity of cervical cytology was markedly improved in combination with HPV DNA test and cervicography. Thus, the triple-combined testing, which improves the high false negativity of cervical cytology, may be an effective tool in uterine cervical cancer screening, pending confirmation of the effectiveness in a mass screening study.

Identification of candidate biomarkers using the Experion™ automated electrophoresis system in serum samples from ovarian cancer patients.

Ovarian cancer is the most common cause of disease-related death in women globally. Detection of ovarian cancer using new biomarkers is necessary for early diagnosis. To date, there have been no obvious biomarkers for ovarian cancer detection in the incipient stage. In this study, we discovered potential diagnostic serological biomarkers for ovarian cancer using the Experion™ automated electrophoresis system. Sera from 14 healthy women and 84 ovarian cancer patients at stages I- IV were applied to the Experion to compare the protein expression levels. To examine the protein expression pattern of Experion data, proteins in the samples were resolved using 10 and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. The candidate biomarkers elevated in ovarian cancer were purified and determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. α-2-macroglobulin (173.7 kDa), ceruloplasmin (147 kDa), inter-α-trypsin inhibitor family heavy chain-related protein (126 kDa), C-1 inhibitor (115.2 kDa) and hemoglobin α/ß (14.4 kDa were overexpressed in the ovarian cancer sera. This study documents a novel way to measure ovarian cancer or cancer-related proteins for biomarkers using the Experion assay system, which should be easily adaptable for high-throughput diagnosis to establish databases of ovarian cancer for clinical applications.

Target-based molecular signature characteristics of cervical adenocarcinoma and squamous cell carcinoma.

There is an urgent need for molecular marker studies of adenocarcinoma (AC) and squamous cell carcinoma (SCC) of the uterine cervix. This study utilized oligomicroarray and pathway analyses to characterize a transcriptomic signature with molecular networks associated with AC and SCC. A 10K oligomicroarray was used to identify potential transcripts that were differentially expressed in cervical cancers from 28 patients and common reference RNAs from 17 different normal cervixes. Molecular networks were correlated using genomics tools to globally explore cellular pathways. Gene expression levels of 46 transcripts separated cancer samples into AC and SCC groups. Genes including: KRT17, IGFBP2, CALCA and VIPR1 were differentially expressed in AC and SCC. In addition, we identified a transcriptomic signature that predicted tumor classification and progression based upon its cellular processes. The downregulated signatures for SCC were cell death of pheochromocytoma cells (P=0.0037), apoptosis of neurons (P=0.009) and damage to DNA (P=0.0038). By contrast, the upregulated molecular signatures in AC were immunological disorder (P=0.006), splenomegaly (P=0.0053) and hepatic system disorder (P=0.006). The G2/M DNA damage checkpoint regulation pathway (P=0.05) was found to be significantly linked to IGF1R as a new regulatory component of a putative cytoplasmic signaling cascade in SCC. By contrast, the antigen presenting canonical pathway (P=0.038) appeared to be linked to PPARγ in AC. Taken together, these experiments provide important new information regarding the role of molecular networks in mediating SCC and AC, possibly through two independent pathways, and contribute to provide new targets for the prevention and treatment of cervical cancer.

Synthesis of chlorin-based unsaturated fatty acid conjugates: their in vitro phototoxicity on TC-1 cancer cell line.

Chlorin-based photosensitizers in photodynamic therapy are the promising anticancer agents, but some of their properties such as specific-targeting to tumor need to be improved. The aim of this study was to synthesize chlorin-based unsaturated fatty acid conjugates to obtain an optimal photosensitizers. Thus four chlorin-based fatty acid conjugates were successfully synthesized through an esterification reaction using carbodiimide coupling reagents in enough yields. Then, structures of these conjugates were confirmed by (1)H NMR, MALDI-MS, and UV-vis spectroscopy. Furthermore, their in vitro phototoxicity and cellular uptake were evaluated on TC-1 lung cancer cell line and HeLa cell line.

Selenium enhances the efficacy of Radachlorin mediated-photodynamic therapy in TC-1 tumor development.

Selenium, an essential trace element possessing anti-carcinogenic properties, can induce apoptosis in cancer cells. Our goal was to investigate the enhanced antitumor effects of photodynamic therapy (PDT) plus selenium in TC-1 tumor cells and implanted mice. Cell viability was evaluated at various time intervals after PDT treatment and/or selenium by methyl thiazolyl tetrazolium (MTT) assay. When only PDT treatment was administered to TC-1 tumor cells, TC-1 cell growth recovered over time. On the other hand, co-treatment of PDT and selenium extended the inhibition time of tumor cell growth. Co-treatment of PDT and selenium showed serious morphological changes in TC-1 cells and induced a more apoptotic population by FACS analysis. By signal transduction pathway SuperArray analysis, genes closely involved in the NFκB, p53 and phopholipase C pathways, such as VCAM1, MDM2 and FOS, were significantly downregulated at least 10-fold in TC-1 cells following PDT and selenium cotreatment. In an in vivo study, tumor-bearing mice were intravenously injected with Radachlorin 3 h before irradiation with 300 J/cm2 of light. Selenium was administered daily for 20 days. Combination therapy against the mouse tumors generated by TC-1 cells was more effective than PDT or selenium alone. These data suggest that selenium plus PDT can induce a significant tumor suppression response compared with PDT alone. Additionally, it can be an effective anticancer therapy strategy.

Serum selenium levels in Korean hepatoma patients.

The aim of this study was to determine serum selenium (Se) levels during the development of liver disease as well as the possible Se supplementation benefits in liver disease patients. Serum was collected from 187 patients with liver diseases and 120 normal healthy people living in Seoul. The samples were collected at the Kangnam St. Mary's Hospital College of Medicines, The Catholic University of Korea, in accordance with procedures approved by the Institutional Review Board of the Catholic University of Korea. Serum Se levels were quantified by inductively coupled plasma mass spectrometry and were compared between healthy and liver diseases patients. Se levels were 92.65 ± 32.50 µg/l in hepatitis infection, 92.33 ± 30.66 µg/l in hepatitis B virus infection and 96.41 ± 51.50 µg/l in hepatitis C virus infection, 96.42 ± 32.80 µg/l in cirrhosis, and 67.47 ± 14.30 µg/l in hepatoma patients. Findings were significantly lower in hepatitis and hepatoma as compared with the healthy participants (P < 0.001). The Se level of the healthy population was 108.38 ± 29.50, 119.37 ± 28.31 for males and 97.87 ± 26.99 µg/l for females. Our data shows the same parallelism between liver disease progression and decrease of Se levels except in the case of liver cirrhosis. And also, our study confirms the previous findings of significantly lower Se levels in Korean hepatoma patients. Se levels that decrease parallel to liver disease progression should be further integrated and analyzed with liver function blood biomarkers.

Multiplexed bead-based immunoassay of four serum biomarkers for diagnosis of ovarian cancer.

The purpose of the present study was to evaluate multiplex liquid assay-based measurement of multiple ovarian cancer-associated biomarkers such as hemoglobin, haptoglobin and apolipoprotein E, together with CA125, which has been widely used in the diagnosis of ovarian cancer, in order to provide a higher diagnostic power. We measured the serum levels of CA125, hemoglobin, haptoglobin and apolipoprotein E from the serum of 76 healthy individuals and 69 ovarian cancer patients using a multiplex liquid assay system, Luminex 100. The results were analyzed according to normal versus ovarian cancer, tumor stages and tumor histology. In addition, to validate the use of these biomarkers for the diagnosis of ovarian cancer, the sensitivity and specificity of each biomarker was analyzed by its receiver operating characteristics (ROC) curve. The serum levels of all four biomarkers in ovarian cancer patients were significantly higher than those of healthy individuals. When CA125 was combined with the biomarkers, the overall sensitivity and specificity were significantly improved in the ROC curve, which showed 95 and 75% sensitivity and specificity, respectively. At 95% specificity for all stages the sensitivity increased to 75% compared to 41% for CA125 alone. For stage I+II increased the sensitivity to 68% from 36% for CA125 alone. For stage III+IV the corresponding values were 100 and 95%, respectively. Taken together, the new combination of hemoglobin, haptoglobin and apolipoprotein E with CA125 significantly improved both the sensitivity and the specificity of ovarian cancer diagnosis compared with those of individual biomarkers. These findings suggest the benefit of the combination of these markers for the diagnosis of ovarian cancer.

Differential DNA copy number aberrations in the progression of cervical lesions to invasive cervical carcinoma.

Host genomic alterations in addition to human papillomavirus (HPV) are needed for cervical precursor lesions to progress to invasive cancer because only a small percentage of women infected by the virus develop disease. However, the genomic alterations during the progression of cervical lesions have not been systematically examined. The aim of this study was to identify differential genomic alterations among cervical intraepithelial neoplasia CIN1, CIN2, CIN3 and cervical squamous cell carcinoma (SCC). Genomic alterations were examined for 15 cases each of CIN1, CIN2, CIN3 and SCC by array-based comparative genomic hybridization (array CGH). The chromosomal regions showing significant differential in DNA copy number aberrations (DCNAs) among CIN1, CIN2, CIN3 and SCC were successfully identified by resampling-based t-test. The chromosomal regions of 5q35.3 and 2q14.3 showed significant DCNAs between CIN1 and CIN2, and between CIN2 and CIN3, respectively, while a significant difference in DCNAs between CIN3 and SCC was observed at 1q24.3, 3p14.1, 3p14.2, 5q13.2, 7p15.3, 7q22.1 and 13q32.3. In addition, the status of DCNAs in 1q43, 2p11.2, 6p11.2, 7p21.1, 7p14.3, 10q24.1, 13q22.3, 13q34 and 16p13.3 was conserved throughout the progression of CIN to SCC. The presence of differential and common DCNAs among CIN1, CIN2, CIN3 and SCC supports that the CIN progression may include continual clonal selection and evolution. This approach also identified 34 probe sets consistently overexpressed when amplified, suggesting an unbiased identification of candidate genes in SCC during cervical cancer progression.

Colocalization of interferon regulatory factor 7 (IRF7) with latent membrane protein 1 (LMP1) of Epstein-Barr virus.

Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.

Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C.

In this study we constructed an artificial flavohemoprotein by fusing Vitreoscilla hemoglobin (VHb) with D-amino acid oxidase (DAO) of Rhodotorula gracilis to determine whether bacterial hemoglobin can be used as an oxygen donor to immobilized flavoenzyme. This chimeric enzyme significantly enhanced DAO activity and stability in the bioconversion process of cephalosporin C. In a 200-mL bioreactor, the catalytic efficiency of immobilized VHb-DAO against cephalosporin C was 12.5-fold higher than that of immobilized DAO, and the operational stability of the immobilized VHb-DAO was approximately threefold better than that of the immobilized DAO. In the scaled-up bioprocess with a 5-L bioreactor, immobilized VHb-DAO (2500 U/L) resulted in 99% bioconversion of 120 mM cephalosporin C within 60 min at an oxygen flow rate of 0.2 (v/v) x min. Ninety percent of the initial activity of immobilized VHb-DAO could be maintained at up to 50 cycles of the enzymatic reaction without exogenous addition of H(2)O(2) and flavin adenine dinucleotide (FAD). The purity of the final product, glutaryl-7-aminocephalosporanic acid, was confirmed to be 99.77% by high-performance liquid chromatography (HPLC) analysis. Relative specificity of immobilized VHb-DAO on D-alpha-aminoadipic acid, a precursor in cephalosporin C biosynthesis, increased twofold, compared with that of immobilized DAO, suggesting that conformational modification of the VHb-DAO fusion protein may be altered in favor of cephalosporin C.