Biosafety studies of carrier cells infected with a replication-competent adenovirus introduced by IAI.3B promoter.

The use of carrier cells infected with oncolytic viruses in cancer gene therapy is an attractive method because it can overcome viral immunogenicity and induce tumor immunity and significant antitumor activity. To enable human clinical trials of this treatment, acute and chronic toxicity tests must first be performed to ensure safety. IAI.3B promoter, oncolytic adenovirus AdE3-IAI.3B introduced by IAI.3B promoter, and A549 carrier cells infected with AdE3-IAI.3B were highly active in cancer cells but not in normal cells. Freeze-thawing increased the antitumor effect of A549 carrier cells by promoting the translocation of oncolytic adenovirus particles from the nucleus to the cytoplasm following the rupture of the nuclear membranes. No deaths or abnormal blood test data resulted from acute toxicity tests conducted in nude mice after a single dose. In chronic toxicity tests in rabbits, there were no serious side effects after eight doses of 1.25 × 10(7) cells/kg or less for 4 weeks; a significant immune response is known to elicit increased numbers of antiadenovirus antibodies and enlarge the spleen. From these results, it could be concluded that cancer gene therapy of recurrent solid tumors using carrier cells can be safely trialed in humans.

Potential tumor markers of renal cell carcinoma: α-enolase for postoperative follow up, and galectin-1 and galectin-3 for primary detection.

The diagnosis of renal cell carcinoma is currently based on imaging techniques, mainly because there is no blood marker available for its detection. Thus, there is still the need for the development of novel tumor markers. We examined plasma levels of eight proteins in 15 renal cell carcinoma patients before and after surgery, and in 51 healthy controls using enzyme-linked immunosorbent assay. Plasma levels of α-enolase, calnexin, galectin-1, galectin-3 and lectin mannose-binding 2 were significantly higher in renal cell carcinoma patients than in controls (P < 0.05). Among these proteins, the sensitivities for galectin-1 and galectin-3 were higher than those for calnexin and lectin mannose-binding 2 in the specificity range from 80% to 100%. A combined use of galectin-1 and galectin-3 showed 98% specificity and 47% sensitivity. In addition, the assays showed that plasma α-enolase levels decreased significantly 4 weeks after nephrectomy (P = 0.0034), and this tendency continued until 12 weeks after nephrectomy (P = 0.0156). These findings suggest that α-enolase could be used in the postoperative follow up of renal cell carcinoma patients, whereas the combined use of galectin-1 and galectin-3 might represent a useful tool for primary detection.

Improved gene transfer into bladder cancer cells using adenovirus vector containing RGD motif.

BACKGROUND: The transduction efficacy of adenovirus serotype 5 (Ad5) vector in high-grade human bladder cancer cells is generally extremely low due to the non-expression of coxsackie and adenoviral receptor (CAR). We investigated whether fiber-modified adenovirus vector containing an RGD motif in the HI loop of the adenovirus fiber knob could increase the transduction efficiency of Ad5 into human bladder cancer cells in vitro. MATERIALS AND METHODS: We examined the expressions of CAR, and of α(v), ß(3) and ß(5) integrin, and the transduction efficacy of fiber-modified adenovirus vector in four human bladder cancer cell lines (TCC-SUP, 253J, T24 and KK47). RESULTS: The expression of CAR was lower and those of α(v) and ß(3) integrin were higher in four human cancer cell lines compared with the control cell line, KK47. The transduction efficacy of fiber-modified adenovirus vector increased by 20- to 470-fold compared with Ad5. CONCLUSION: Fiber-modified adenovirus vector may be useful in order to establish new effective gene therapy strategies for the treatment of high-grade human bladder cancer.

Fiber-substituted conditionally replicating adenovirus for oncolysis of human renal carcinoma cells.

BACKGROUND: Adenovirus vectors have lately been highlighted in gene therapies. We investigated the oncolytic effects of a chimeric adenovirus type 5 (Ad5) with replacement of Ad5 fiber knob with adenovirus type 35 (Ad35) fiber knob (Ad5F35) on human renal cell carcinoma (RCC). MATERIALS AND METHODS: The conditionally replicating Ad5F35 vector was constructed and infected into RCC cell lines 786-O, ACHN, and RCC4-VHL. For these cells, reverse transcription-polymerase chain reaction and western blotting were carried out and the cell viability was assayed. RESULTS: In all RCC cell lines, it was found that CD46, a cell surface target of Ad35, was well-expressed, while coxsackie and adenovirus receptor (CAR), a cell surface target of Ad5, was considerably less expressed. The Ad5F35 vector induced oncolysis of RCC cells, with significantly higher efficacy as compared with that for the Ad5 vector. CONCLUSION: Ad5F35 vector could be a candidate for promising gene therapy of human RCC.

Enhancement of IL-2-induced cytotoxicity by interferon-alpha in renal cell carcinoma.

Metastatic renal cell carcinoma (mRCC) treatment consists of molecular targeted agents and cytokines that have fundamentally different mechanisms of action. Clinical responses also differ; complete response is rare with molecular targeted agents but is sometimes achieved with cytokine therapies. Because of the relatively high efficacy of combination therapy with low-dose interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) against mRCC, it is important to reevaluate cytokine therapies in vitro. Here, we show that when IL-2 is administered in combination with IFN-alpha, a stronger cytotoxic effect of PBMCs on RCC cell lines is observed than when IL-2 is administered alone. The upregulation of TNF-related apoptosis-inducing ligand on NK cell by IL-2 and suppression of regulatory T cells (Tregs) by IFN-alpha were recognized at the same time when cytotoxicity of peripheral blood mononuclear cells (PBMCs) was enhanced. IL-2 is known to activate natural killer cell cytotoxicity; however, IL-2 also stimulates Treg expansion, which enhances immunosuppression. On the other hand, IFN-alpha negatively regulates Treg cells, thereby increasing the function of immune effector cells. Our in vitro results may explain, at least in part, the clinical efficacy of combination low-dose IL-2 and IFN-alpha therapy against mRCC.

The antitumour effect of {gamma}{delta} T-cells is enhanced by valproic acid-induced up-regulation of NKG2D ligands.

UNLABELLED: The aim of this study wsa to evaluate the additive effect of valproic acid (VPA) to γδ T-cell cytotoxicity against bladder cancer cells. MATERIALS AND METHODS: Human bladder cancer cell lines TCCSUP and 253J were treated with VPA and mRNA expression of natural killer group 2D (NKG2D) ligands was determined. The antitumour effect of expanded γδ T-cells against zoledronic acid (ZOL) and VPA pre-treated cancer cells was subsequently determined. RESULTS: VPA increased mRNA expression of NKG2D ligands on both cancer cell types. A blocking study revealed that 253J cells were recognised through NKG2D, while TCCSUP cells were mainly recognised through γδ T-cell receptor. VPA pre-treatment increased sensitivity to cytolysis by γδ T-cells for both cancer cell types, whereas ZOL pre-treatment was only effective against TCCSUP. CONCLUSION: Induction of NKG2D ligands by VPA increased the susceptibility of cancer cells that are recognised by NKG2D to cytolysis by γδ T-cells.

Zoledronate stimulates gamma delta T cells in prostate cancer patients.

Androgen deprivation therapy is the mainstay of treatment for prostate cancer. Given its frequent failure, new therapy that reduces prostate cancer progression would be a breakthrough in treating this disease. Bisphosphonates are well-established agents for treating skeletal-related events (SREs) in prostate cancer patients with bone metastases. Exposure to bisphosphonates may not only reduce the incidence of SREs, but also have anticancer effects by modulating a patient's immunity. The purpose of this study was to examine the effect of zoledronate (ZOL) on gamma delta T cells, serum prostate-specific antigen (PSA) levels, and velocities. The effect of ZOL, with and without IL-2, on gamma delta T cell activation was examined in vitro. Furthermore, the activated state and the number of gamma delta T cells and changes in serum PSA levels were examined for patients who received ZOL infusion for the prevention of SREs. We found that ZOL activated gamma delta T cells, and the number of gamma delta T cell was increased when IL-2 was administered with ZOL in vitro. Comparisons before and after the first ZOL infusion revealed that gamma delta T cells in peripheral blood were activated by ZOL. Moreover, after the first ZOL treatment, reduction in serum PSA was observed in 3 of 11 patients, and reduction in PSA velocity was observed in 5 of 10 patients. Our findings indicate that ZOL stimulates gamma delta T cells in vivo and in vitro. This study provides further insight into the ability of gamma delta T cells to induce an antitumor immune response.

Improving gene transfer in human renal carcinoma cells: Utilization of adenovirus vectors containing chimeric type 5 and type 35 fiber proteins.

The transduction efficacy of adenovirus serotype 5 (Ad5) vector in human renal carcinoma cells is generally low due to the down-regulated expression of Coxsackie and adenovirus receptor (CAR) in target cells. By contrast, the infectivity of adenovirus serotype 35 vectors depends on the binding rate to CD46 receptor, independent of CAR. In this study, we examined whether an adenovirus vector containing chimeric type 5 and type 35 fiber proteins (Ad5/F35) increases transduction efficiency compared to Ad5 vector in human renal carcinoma cells in vitro. The expression of CAR was much lower in the human renal carcinoma cells than in control HEK293 cells. By contrast, the expression of CD46 was similar and perhaps at a higher level in the human renal carcinoma cells than in the HEK293 cells. The transduction efficacy of Ad5/F35 vector was dramatically higher compared to that of Ad5 in human renal carcinoma cells, and was correlated to the expression of CD46. Thus, Ad5/35 vector may be useful for the development of novel gene therapy approaches to renal cell carcinoma.

Improved gene transfer into renal carcinoma cells using adenovirus vector containing RGD motif.

UNLABELLED: The transduction efficacy of adenovirus serotype 5 (Ad5) vector in human renal carcinoma cells is generally extremely low due to the non-expression of Coxsackie and adenoviral receptor (CAR). We investigated whether fiber-modified Ad vector containing an RGD motif in the HI loop of the Ad fiber knob could increase the transduction efficiency of Ad5 in human renal carcinoma cells in vitro. MATERIALS AND METHODS: We examined both expressions of CAR, and alpha(v), beta(3) and beta(5) integrins, and the transduction efficacy of fiber-modified adenovirus vector in all cell lines. RESULTS: The expression of CAR was lower and those of alpha(v) and beta(3) integrins were higher in all cell lines compared with control cell line, KK47. The transduction efficacy of fiber-modified Ad vector increased by 125- to 1,800-fold compared with Ad5. CONCLUSION: The fiber-modified Ad vector may be useful to establish effective new gene therapy strategies for the treatment of renal cell carcinoma.

A pilot study of quality of life of patients with hormone-refractory prostate cancer after gene therapy.

BACKGROUND: The effects on quality of life (QOL) after a Phase I/II clinical trial of a combination of osteocalcin promoter-driven herpes simplex virus thymidine kinase (Ad-OC-TK) gene therapy and valacyclovir (VAL) were investigated for patients with hormone-refractory prostate cancer (HRPC). PATIENTS AND METHODS: The QOL of six patients was prospectively assessed after gene therapy on days 0, 14, and 28. A modified questionnaire was created based on the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire's prostate cancer-specific module (EORTC QLQ-PR25). RESULTS: The scores of all items significantly improved along with the total score. Further, bodily pain was significantly reduced on day 28. Moreover, the rate of change in the serum prostate-specific antigen levels from day 0 to day 28 was significantly correlated with the rate of change in bodily pain. CONCLUSION: In this clinical trial, Ad-OC-TK plus VAL treatment significantly improved the short-term QOL and bodily pain of patients with localized recurrence or bone metastases of HRPC.

Expression of the fluoropyrimidine-metabolizing enzymes in bladder cancers as measured by the Danenberg tumor profile.

5-Fluorouracil (5-FU) and its prodrugs are used to treat various cancers. Response to 5-FU-based chemotherapy and expression of 5-FU-related enzymes differ among cancers. The objective of the present study was to investigate the relationship between the expression of 5-FU-related enzymes and clinicopathologic factors in bladder cancer. Formalin-fixed, paraffin-embedded sections of 44 bladder cancers and 27 normal bladders were included in this study. After laser capture microdissection, "Danenberg tumor profile," was performed for the measurement of 5-FU-related enzymes. There was no significant difference between dihydropyrimidine hydrogenase (DPD) mRNA expression in bladder cancer and in normal bladder. On the contrary, mRNA expressions of orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS), and thymidine phosphorylase (TP) in bladder cancer were higher than those in normal bladder. Compared with previously reported DPD mRNA expressions in other types of cancer, DPD mRNA expression in bladder cancer was relatively low. The 5-FU-related enzymatic condition of bladder cancer is favorable for 5-FU.

Quantitative proteomic analysis to discover potential diagnostic markers and therapeutic targets in human renal cell carcinoma.

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2-nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six (12)C or (13)C. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI-TOF MS. NBS-labeled peptides with a 6 Da difference were identified by MS/MS. Thirty-four proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT-PCR. The results suggest that NBS-based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC.

Long-term outcome of phase I/II clinical trial of Ad-OC-TK/VAL gene therapy for hormone-refractory metastatic prostate cancer.

We evaluated the long-term safety and efficacy of Ad-OC-TK (recombinant adenoviral vector carrying an osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) gene therapy for hormone-refractory prostate cancer. Ad-OC-TK/VAL therapy is the first in vivo adenovirus-mediated gene therapy to be used to treat metastatic prostate cancer, including bone metastasis. Six patients were enrolled in this trial, and two doses of Ad-OC-TK (2.5 x 10(9) or 2.5 x 10(10) plaque-forming units) were injected into locally recurrent tumor or bone metastasis on day 1 and day 8. Patients were also given VAL (3 g/day) for 21 days. Safety and efficacy were evaluated for at least 8 months in each patient. All patients tolerated this therapy with no serious adverse events. One prostate-specific antigen (PSA) response (from 318.3 to 4.9 ng/ml) was observed with a time to PSA progression (TTP) of 12 months. Docetaxel (30 mg/m2 per week) and estramustine (560 mg/day) combination chemotherapy (DE) was given to three docetaxel-naive patients on PSA failure after gene therapy. All three patients had a PSA response to DE therapy with 21, 7, and 4 months of TTP. These results suggest that additional trials are warranted.

Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model.

OBJECTIVES: To develop a novel therapeutic strategy against human bladder cancer using Ad-MK-E1a-a midkine (MK) promoter-regulated, conditionally replicating, adenovirus. METHODS: We tested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative polymerase chain reaction, MK promoter activity, after plasmid transfection, using a luciferase assay, and the transduction efficiency by co-transfection with the cytomegalovirus-beta-gal plasmid. In these cells, we assessed the cell type-specific replication of Ad-MK-E1a virus by measuring the E1a DNA copy number by real-time polymerase chain reaction and the cell growth inhibition due to this virus using the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with phosphate-buffered saline or Ad5-CMV-LacZ or Ad-MK-E1a. RESULTS: The MK mRNA expression level and MK promoter-driven luciferase activity were relatively greater and markedly increased, respectively, in the 5637, A549, and KK47 cells than in the T24 and H891 cells. After Ad-MK-E1a infection, the E1a DNA copy number increased more significantly in the KK47, 5637, and A549 cells than in the T24 and H891 cells. At a multiplicity of infection of 0.01, Ad-MK-E1a significantly inhibited KK47 and 5637 cell growth. In vivo, Ad-MK-E1a injection markedly inhibited KK47 tumor growth. CONCLUSIONS: We have demonstrated the antitumor effect of Ad-MK-E1a in a human bladder cancer model overexpressing MK mRNA.

Progress report on phase I/II clinical trial of Ad-OC-TK plus VAL therapy for metastatic or locally recurrent prostate cancer: Initial experience at Kobe University.

There is no effective therapy for hormone-refractory prostate cancer and a novel therapeutic modality, such as a gene therapy, should be actively pursued. Previously, Gardner and Chung conducted a phase I clinical trial of Ad-OC-TK (recombinant adenoviral vector containing osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) for the treatment of hormone-refractory prostate cancer at the University of Virginia. We report on our ongoing phase I/II clinical trial of Ad-OC-TK plus VAL for the treatment of advanced prostate cancer at the Kobe University Hospital, Japan.

Effects of nafamostat mesilate on ADP-induced platelet aggregation and disaggregation in hemodialysis patients.

Nafamostat mesilate (NM), a synthetic protease inhibitor, is the most commonly used anticoagulant in the setting of extracorporeal circulation (ECC) in patients with bleeding tendency. It inhibits both platelet aggregation and activation of coagulation factors. Although it has been reported that NM disaggregates aggregated platelets, little is known about such an effect in the setting of hemodialysis therapy (HD). We examined the effects of NM on adenosine 5'-diphosphate (ADP)-induced platelet aggregation and disaggregation using platelet-rich plasma obtained from 6 HD patients. The platelet aggregation was stimulated by 3 microM ADP and change of aggregation was monitored by an aggregometer. NM adjusted to the final concentrations of 0.1 (1.9 x 10(-7)), 1.0 (1.9 x 10(-6)), 10, (1.9 x 10(-5)), and 100 (1.9 x 10(-4)) microg/ml (M) or veronal-buffered saline (VBS) as control was added before or after to the stimulation of ADP. NM not only inhibited platelet aggregation, but also disaggregated already aggregated platelets at concentrations of 1.0 microg/mln or higher. Moreover, NM almost completely disaggregated at 100 microg/ml. This NM concentration of 1.0 microg/ml was lower than the therapeutic concentration in ECC of HD (i.e., 10 M(-5)). Both inhibitory and disaggregatory effects of NM expressed a dose-related dependency. Our results suggest that NM can exert both aggregation inhibitory and disaggregatory effects on platelets of HD patients within the therapeutic concentration.

EGFR mRNA is upregulated, but somatic mutations of the gene are hardly found in renal cell carcinoma in Japanese patients.

PURPOSE: Heterozygous somatic mutations of epidermal growth factor receptor (EGFR) in exons 18, 19, and 21 were recently reported to be associated with response to gefitinib in patients having nonsmall cell lung cancer. Such mutations are more frequently found among Japanese than Europeans. In this work, the frequency of mutations was investigated in renal cell carcinoma (RCC) samples obtained from Japanese subjects to examine the potential of gefitinib as a therapeutic agent for RCC. METHODS: Nineteen patients with RCC, who gave written informed consent, were enrolled in this study. mRNA expression levels of EGFR were measured in RCC and its adjacent noncancerous renal tissue via the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Somatic mutations were determined using genomic DNA extracted from RCC by direct sequencing method. RESULTS: mRNA expression was confirmed to be about 19 times higher in RCC than in adjacent noncancerous renal tissues, but no such mutations were detected in both. CONCLUSION: Results from this study do not support the validity of further clinical trials on gefitinib for RCC with genotyping even in Japanese patients, although EGFR plays a key role in tumor progression.

Recombinant interleukin-2 enhanced the antitumor effect of ADV/RSV-HSV-tk/ACV therapy in a murine bladder cancer model.

BACKGROUND: Previous studies demonstrated the antitumor effects of IL-2 and ADV/RSV-HSV-tk in bladder tumor models. In our study, we employed the intramuscular injection of recombinant IL-2 combined with ADV/RSV-HSV-tk gene therapy in the MBT-2 murine bladder tumor model. MATERIALS AND METHODS: In the in vitro study, after adenoviral gene transduction efficiency had been assessed, the cytotoxicity of ADV/RSV-HSV-tk/ACV was examined. In the in vivo study, ADV/RSV-HSV-tk was injected into MBT-2 subcutaneous tumors, ACV was injected intraperitoneally daily for 13 days and recombinant IL-2 was injected intramuscularly daily for 10 days. RESULTS: The X-gal staining of MBT-2 cells infected with 125 multiplicity of injection (MOI) indicated > 20% adenoviral gene transduction efficiency. The cell growth of MBT-2 infected with 125 MOI was significantly inhibited by 40 microM of ACV. In the in vivo study, the combination therapy significantly inhibited tumor growth in the MBT-2 tumor model. CONCLUSION: The systemic administration of recombinant IL-2 in combination with HSV-tk gene therapy exhibited an enhanced antitumor effect.

Granulocyte-colony stimulating factor producing urothelial carcinoma of renal pelvis.

We report a case of granulocyte-colony stimulating factor (G-CSF) producing urothelial carcinoma of the renal pelvis in a 39-year old man. The patient was admitted to Kobe University Hospital, Kobe, Japan, complaining of macrohematuria and a 6-month history of left abdominal swelling. Abdominal computed tomography showed a large mass in the left kidney and para-aortic lymph node enlargement. A remarkable degree of leukocytosis was detected without any acute infectious disease. Enzyme immunoassay of the serum demonstrated a remarkable high concentration of G-CSF. The patient underwent left nephroureterectomy and para-aortic lymphadenectomy. Histochemical examination revealed urothelial carcinoma. Immunohistochemical staining with an anti-G-CSF antibody demonstrated G-CSF secreting cells. The patient died 8 weeks after the surgical operation. To our knowledge, this is the second case of G-CSF producing urothelial carcinoma of renal pelvis reported in the English literature.

[Gene therapy].

We selected bone-metastatic prostate cancer as the target form of recurrent prostate cancer and developed a suicide-gene therapy based on an adenovirus vector with an organ-specific osteocalcin promoter. Related clinical studies have already been conducted in the United States at the University of Virginia, where results so far have established the safety of this therapy. In the present paper, in addition to presenting the results of these gene-therapy studies from the basic research to the clinical stage, we discuss the clinical studies begun by our group in August 2003. In the 21st century, therapeutic systems in use are undergoing major changes. Gene therapy is likely to become an important therapeutic option in recurrent prostate cancer. In terms of theory and technology however, this form of treatment is still at a very immature stage of development. We look forward to evolution in this field to provide an established treatment for recurrent prostate cancer and are committed to actively continuing with the development of gene therapy through translational research.