Minoru Fujimori

Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors.

A fundamental obstacle in gene therapy for cancer is the specific delivery of an anticancer gene product to a solid tumor, and yet no systemic delivery system that specifically targets solid tumors currently exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized and proliferated in several types of mouse solid tumors after systemic application. In this report, we further describe a novel approach to cancer gene therapy in which genetically engineered Bifidobacterium is used as a tumor-specific vector. Similarly to wild-type B. longum, genetically engineered B. longum could be detected in tumor tissue only and was not found in a large survey of normal mouse tissues after intravenous injection. This finding strongly suggests that obligate anaerobic bacteria such as Bifidobacterium can be used as highly specific gene delivery vectors for cancer gene therapy.

Suppression of acute and chronic rejection by hepatocyte growth factor in a murine model of cardiac transplantation: Induction of tolerance and prevention of cardiac allograft vasculopathy

Background—Although treatment with immunosuppressive agents has contributed to overcoming acute rejection and improving the midterm survival of transplanted hearts, cardiac allograft vasculopathy (CAV) has remained the main cause of primary graft failure. Recent approaches have shown that hepatocyte growth factor (HGF) exhibits cardiotrophic functions. We therefore addressed whether HGF would regulate acute and chronic rejection in cardiac transplantation. Methods and Results—We used a murine heterotopic cardiac transplantation model between fully incompatible strains and administered 500 &mgr;g · kg−1 · d−1 HGF during the initial 14 days after transplantation. The HGF-treated allografts showed significantly prolonged survival (42.3±4.1 days, P<0.001) compared with the controls (11.1±0.6 days), with tolerance induction in 47.4%. Histopathologically, the number of infiltrating cells was significantly decreased and myocardial necrosis was less prominent with a reduction of apoptosis in the allografts by HGF treatment during acute rejection. In the long-term surviving allografts, HGF significantly inhibited the development of CAV and interstitial fibrosis. With respect to intragraft cytokine mRNA expression, HGF treatment reduced the early expression of interferon-&ggr; and enhanced the expression of transforming growth factor-&bgr;1 during the acute phase and of interleukin-10 continuously through the acute phase to the chronic phase. Conclusions—Our findings demonstrate that HGF can prolong the survival of allografts by its cardioprotective and immunomodulative potencies. Thus, HGF administration may constitute a new therapeutic approach to preventing cardiac graft failure that has not been overcome by conventional immunosuppressive agents.

Increased nuclear localization of transcription factor Y-box binding protein 1 accompanied by up-regulation of P-glycoprotein in breast cancer pretreated with paclitaxel.

Purpose: The Y-box binding protein 1 (YB-1) regulates expression of P-glycoprotein encoded by the MDR1 gene. There have been no previous studies regarding the involvement of YB-1 in the development of resistance to paclitaxel. The present study was done to examine how paclitaxel affects the localization and expression of YB-1 in breast cancer. Experimental Design: We evaluated the expression and localization of YB-1 and P-glycoprotein in breast cancer tissues obtained from 27 patients before and after treatment with paclitaxel. The effect of paclitaxel on localization of cellular YB-1 was examined by using GFP-YB-1. Interaction of YB-1 with the Y-box motif of the MDR1 promoters was studied by electrophoretic mobility shift assay. The effects of paclitaxel on MDR1 promoter activity were examined by luciferase assay. Results: Of 27 breast cancer tissues treated with paclitaxel, nine (33%) showed translocation of YB-1 from the cytoplasm to the nucleus together with increased expression of P-glycoprotein during the course of treatment. Twelve breast cancer tissues (44%) showed neither translocation of YB-1 nor increased expression of P-glycoprotein. Nuclear translocation of YB-1 was correlated significantly with increased expression of P-glycoprotein (P = 0.0037). Confocal analysis indicated that paclitaxel induced nuclear translocation of green fluorescent fused YB-1 in MCF7 cells. Furthermore, binding of YB-1 to the Y-box of MDR1 promoter was increased in response to treatment with paclitaxel. In addition, MDR1 promoter activity was significantly up-regulated by paclitaxel in MCF7 cells (P < 0.001). Conclusions: The results of the present study suggested that YB-1 may be involved in the development of resistance to paclitaxel in breast cancer.

Increased expression of proapoptotic BMCC1 , a novel gene with the BNIP2 and Cdc42GAP homology (BCH) domain, is associated with favorable prognosis in human neuroblastomas

Differential screening of the genes obtained from cDNA libraries of primary neuroblastomas (NBLs) between the favorable and unfavorable subsets has identified a novel gene BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1). Its 350u2009kDa protein product possessed a Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP2) and Cdc42GAP homology domain in the COOH-terminus in addition to P-loop and a coiled-coil region near the NH2-terminus. High levels of BMCC1 expression were detected in the human nervous system as well as spinal cord, brain and dorsal root ganglion in mouse embryo. The immunohistochemical study revealed that BMCC1 was positively stained in the cytoplasm of favorable NBL cells but not in unfavorable ones with MYCN amplification. The quantitative real-time reverse transcription–PCR using 98 primary NBLs showed that high expression of BMCC1 was a significant indicator of favorable NBL. In primary culture of newborn mice superior cervical ganglion (SCG) neurons, mBMCC1 expression was downregulated after nerve growth factor (NGF)-induced differentiation, and upregulated during the NGF-depletion-induced apoptosis. Furthermore, the proapoptotic function of BMCC1 was also suggested by increased expression in CHP134 NBL cells undergoing apoptosis after treatment with retinoic acid, and by an enhanced apoptosis after depletion of NGF in the SCG neurons obtained from newborn mice transgenic with BMCC1 in primary culture. Thus, BMCC1 is a new member of prognostic factors for NBL and may play an important role in regulating differentiation, survival and aggressiveness of the tumor cells.

Targeting solid tumors with non‐pathogenic obligate anaerobic bacteria

Molecular‐targeting drugs with fewer severe adverse effects are attracting great attention as the next wave of cancer treatment. There exist, however, populations of cancer cells resistant to these drugs that stem from the instability of tumor cells and/or the existence of cancer stem cells, and thus specific toxicity is required to destroy them. If such selectivity is not available, these targets may be sought out not by the cancer cell types themselves, but rather in their adjacent cancer microenvironments by means of hypoxia, low pH, and so on. The anaerobic conditions present in malignant tumor tissues have previously been regarded as a source of resistance in cancer cells against conventional therapy. However, there now appears to be a way to make use of these limiting factors as a selective target. In this review, we will refer to several trials, including our own, to direct attention to the utilizable anaerobic conditions present in malignant tumor tissues and the use of bacteria as carriers to target them. Specifically, we have been developing a method to attack solid cancers using the non‐pathogenic obligate anaerobic bacterium Bifidobacterium longum as a vehicle to selectively recognize and target the anaerobic conditions in solid cancer tissues. We will also discuss the existence of low oxygen pressure in tumor masses in spite of generally enhanced angiogenesis, overview current cancer therapies, especially the history and present situation of bacterial utility to treat solid tumors, and discuss the rationality and future possibilities of this novel mode of cancer treatment. (Cancer Sci 2010)

Ret/PTC3 is the most frequent form of gene rearrangement in papillary thyroid carcinomas in Japan.

AbstractRearrangements of the RET and TRK proto-oncogenes, which generate fusion oncogenes, are frequent in papillary thyroid carcinomas in Caucasian populations. To determine the spectrum of gene rearrangements in Japanese patients, we systematically examined 40 papillary thyroid carcinomas for all possible types of gene fusion events involving RET or TRK genes. RET rearrangements were found in ten tumors (25%): ret/PTC1 had occurred in two tumors, ret/PTC2 in one, ret/PTC3 in six, and a novel RET rearrangement in the remaining patient. In this last patient, the 5′ novel sequence was fused in-frame to the RET amino acid sequence; thus, the fusion gene may encode a protein with a RET kinase domain at the carboxy terminus. The RET gene was fused to 5′ donor sequences at the beginning of exon 12 in all ten tumors. No rearrangements involving the TRK gene were found in this panel of carcinomas. Our results indicated that constitutive activation of the RET by gene rearrangement is a frequent mechanism of papillary thyroid carcinogenesis in Japanese adults.

Prognostic Significance of Magnetic Resonance Findings in Advanced Papillary Thyroid Cancer

We assessed the prognostic importance of magnetic resonance (MR) findings in locally advanced papillary thyroid cancer. MR findings, clinical data, and pathologic (and surgical) data for 66 patients, including 51 women and 15 men with a mean age of 57 years, who had primary surgery for papillary thyroid cancers were correlated with prognosis. Mean follow-up was 27.5 months (range, 5-117 months). Recurrence was seen in 18 patients (27%). In univariate analyses, age of 60 years or more (p = 0.0066), male gender (p = 0.0373), six MR findings (tumor size of > or = 4 cm ([p = 0.0002], ill-defined margins ([p < 0.0001], tumor extension of the trachea [p = 0.0337], carotoid artery [p = 0.0028]), esophagus [p < 0.0001], and lymph nodes [p = 0.0005]), and three pathologic findings (tumor extension of soft tissues [p = 0.0288], carotid artery [p = 0.0013], and esophagus [p < 0.0001]) had a significant adverse effect on disease-free survival. In multivariate analyses, tumor size (p = 0.0169) and nodal metastasis (p = 0.0393) determined on MR imaging and pathologic esophageal invasion (p = 0.0016) were the only significant independent variables. Esophageal invasion was accurately diagnosed with MR imaging (94% accuracy). MR findings may contain prognostic importance of locally advanced papillary thyroid cancer.

High levels of DJ‐1 protein and isoelectric point 6.3 isoform in sera of breast cancer patients

In patients with cancer and Parkinsons disease, the DJ‐1 protein may be secreted into the serum during the impaired response of the underlying cell‐protective mechanisms. In order to determine the clinical significance of DJ‐1 protein in the sera of breast cancer patients, we examined blood samples from a breast cancer group (n = 180) and a non‐cancerous control group (n = 300). Higher levels of DJ‐1 were detected in the breast cancer group (mean level, 42.7 ng/mL) than the control group (28.3 ng/mL) by ELISA (P = 0.019). Higher DJ‐1 levels were significantly associated with advanced clinical grade, according to the TNM classification, negative hormone receptor status, and high Ki‐67 labeling index, of biopsied materials; samples showed low DJ‐1 protein expression despite upregulated DJ‐1 mRNA. DJ‐1 isoforms could be detected clearly in 17 blood samples (from 11 breast cancer patients, and 6 non‐cancerous controls) by 2‐D gel electrophoresis and immunoblot analysis. The isoform at the pI of 6.3 showed the highest intensity in all 11 cancer cases. Conversely, in the 6 non‐cancerous cases, isoforms other than the pI 6.3 isoform were highly expressed, and there was a significant difference in the isoform pattern between breast cancer cases and controls (P = 0.00025). These data indicate that high levels of DJ‐1, probably of isoform at pI 6.3, is a candidate serum marker of breast cancer.

CSLEX (Sialyl Lewis X) is a Useful Tumor Marker for Monitoring of Breast Cancer Patients

BACKGROUNDnCSLEX is a type II carbohydrate antigen that interacts with the CSLEX-1 monoclonal antibody. CSLEX in combination with carbohydrate antigen 15-3 may be more useful than Carcinoembryonic Antigen with carbohydrate antigen 15-3 as tumor markers for monitoring of breast cancer.nnnMETHODSnThe serum levels of tumor markers, including CSLEX, were measured in 480 consecutive breast cancer patients with or without metastasis who visited the outpatient clinic of the Division of Breast and Endocrine Surgery, Shinshu University Hospital, between April 2007 and September 2007.nnnRESULTSnSerum levels of each of the tumor markers correlated significantly with the status of metastasis (P < 0.01). Combinations of Carcinoembryonic Antigen and carbohydrate antigen 15-3, Carcinoembryonic Antigen and Nation Cancer Center-Stomach-439, Carcinoembryonic Antigen and CSLEX, carbohydrate antigen 15-3 and Nation Cancer Center-Stomach-439, and carbohydrate antigen 15-3 and CSLEX levels also correlated significantly with the status of metastasis (P < 0.01). The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were almost the same for CSLEX and Nation Cancer Center-Stomach-439, which are both type II carbohydrate antigens. The cutoff indexes of serum CSLEX and Nation Cancer Center-Stomach-439 for detection of breast cancer metastasis were 38.8 ± 52.7-fold and 22.1 ± 27.8-fold, respectively (P = 0.16).nnnCONCLUSIONSnThese data suggest that the diagnostic values of CSLEX and Nation Cancer Center-Stomach-439 are similar in single or combined use. However, the cutoff index of serum CSLEX tended to be higher than that of Nation Cancer Center-Stomach-439, which may make CSLEX more useful for detection of breast cancer metastasis.

Changes in thyroid function and immunological parameters long after subtotal thyroidectomy for Graves' disease.

OBJECTIVEnTo find out whether subtotal thyroidectomy results in long term stable functional and immunological remission in patients with Graves disease.nnnDESIGNnRetrospective study.nnnSETTINGnTeaching hospital, Japan.nnnSUBJECTSn176 patients who underwent subtotal thyroidectomy for Graves disease, 1970-79.nnnINTERVENTIONnFollow up surveys in 1984 and 1992.nnnMAIN OUTCOME MEASURESnChanges in thyroid function, antibody titres, and lymphocyte subsets.nnnRESULTSn29/79 patients (39%) who were euthyroid in 1984 had evidence of thyroid dysfunction in 1992. Of the 8 patients with latent hypothyroidism in 1984, 3 (38%) had become euthyroid by 1992, and none required treatment. Of the 29 patients who were hypothyroid in 1984, 5 had latent hypothyroidism and 1 was euthyroid in 1992, and of the 18 patients with recurrent hyperthyroidism in 1984, 1 had become euthyroid by 1992. The number of positive titres to TSH-binding inhibitory immunoglobulin was significantly higher in the recurrence group (31/36, 86%) compared with the hypothyroid (7/26, 27%), latent hypothyroidism (8/37, 22%), and euthyroid (22/77, 29%) groups (p < 0.01). There were also significant differences in the mean (SD) number of Leu HLA DR subsets between the control (17 (3), n = 18) and recurrence (21 (6), n = 38), hypothyroid (22 (6), n = 35), latent hypothyroidism (22 (6), n = 22), and euthyroid (22 (9), n = 64) groups (p < 0.002). There were no differences in the number of T cell subsets among the groups.nnnCONCLUSIONnTreatment of Graves disease by subtotal thyroidectomy does not necessarily result in stable late functional or immunological remission. Long term follow up of such patients may be necessary.