Shuji Terao

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 12C or 13C. 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.

Zoledronate Stimulates γδ 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 patients immunity. The purpose of this study was to examine the effect of zoledronate (ZOL) on γδ T cells, serum prostate-specific antigen (PSA) levels, and velocities. The effect of ZOL, with and without IL-2, on γδ T cell activation was examined in vitro. Furthermore, the activated state and the number of γδ 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 γδ T cells, and the number of γδ T cell was increased when IL-2 was administered with ZOL in vitro. Comparisons before and after the first ZOL infusion revealed that γδ 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 γδ T cells in vivo and in vitro. This study provides further insight into the ability of γδ T cells to induce an antitumor immune response.

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.

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

PurposeHeterozygous 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.MethodsNineteen 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.ResultsmRNA 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.ConclusionResults 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.

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.

Abstract  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.

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

Abstract  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.

Gamma-delta T Cells may Function as Carrier Vehicles in Adenovirus Vector-based Gene Therapy

Developing new treatments that reduce prostate cancer progression is important. Therapeutic efficacy of conventional gene therapy for metastatic prostate cancer is still low. Lower induction rate of naked genes into target cell is due to reduced expression of adenovirus receptor on cancer cell and also due to high seroprevalence of anti-Ad antibodies in adults. Therefore, efficient Ad carrier systems that circumvent these problems should be developed. Gamma-delta T cells have demonstrated high affinity to cancer cells. CD46, which leads to broad tropism in Ad35 vectors, is expressed in hematopoietic cells, including γδ T cells. In this study, we demonstrate the potential of γδ T cells as “vehicles” for transporting Ad5/F35 vectors and genes into cancer cells.

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&mgr;M ADP and change of aggregation was monitored by an aggregometer. NM adjusted to the final concentrations of 0.1 (1.9 × 10–7), 1.0 (1.9 × 10–6), 10, (1.9 × 10–5), and 100 (1.9 × 10-4) &mgr;g/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 &mgr;g/mln or higher. Moreover, NM almost completely disaggregated at 100 &mgr;g/ml. This NM concentration of 1.0 &mgr;g/ml was lower than the therapeutic concentration in ECC of HD (i.e., 10–5M). 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.

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 × 107 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.

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.