Makoto Fujime

Ki26894, a novel transforming growth factor‐β type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line

Transforming growth factor (TGF)‐β signaling has been shown to promote tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF‐β signaling may thus be a novel strategy for treatment of patients with such cancers. In this study, we investigated the effects of a novel TGF‐β type I receptor (TβR‐I) kinase inhibitor, Ki26894, on bone metastasis of a highly bone‐metastatic variant of human breast cancer MDA‐MB‐231 cells, termed MDA‐MB‐231–5a‐D (MDA‐231‐D). Ki26894 blocked TGF‐β signaling in MDA‐231‐D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF‐β‐responsive reporter activity. Moreover, Ki26894 decreased the motility and the invasion of MDA‐231‐D cells induced by TGF‐βin vitro. Ki26894 also suppressed transcription of plasminogen activator inhibitor‐1 (PAI‐1), parathyroid hormone‐related protein (PTHrP), and interleukin‐11 (IL‐11) mRNA of MDA‐231‐D cells, which were stimulated by TGF‐β. X‐ray radiography revealed that systemic Ki26894 treatment initiated 1 day before the inoculation of MDA‐231‐D cells into the left ventricle of BALB/c nu/nu female mice resulted in decreased bone metastasis of breast cancer cells. Moreover, Ki26894 prolonged the survival of mice inoculated with MDA‐231‐D cells compared to vehicle‐treated mice. These findings suggest that TβR‐I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers. (Cancer Sci 2007; 98: 127–133)

Transforming Growth Factor-β Promotes Survival of Mammary Carcinoma Cells through Induction of Antiapoptotic Transcription Factor DEC1

Transforming growth factor-beta (TGF-beta) signaling facilitates tumor growth and metastasis in advanced cancer. In the present study, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stra13) as a downstream target of TGF-beta signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC(A) and 4T1, the TGF-beta type I receptor kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-beta induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TGF-beta type I receptor kinase inhibitors as well as by expression of dominant-negative TGF-beta type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC(A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-beta-promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC(A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-beta-mediated cell survival and metastasis of cancer.

Promoter CpG hypomethylation and transcription factor EGR1 hyperactivate heparanase expression in bladder cancer

Heparanase plays a critical role in the degradation of extracellular matrix and cell membrane and is frequently upregulated in malignant tumors. Transcription factor, early growth response 1 (EGR1), is closely associated with inducible transcription of the heparanase gene. We hypothesized that promoter CpG hypomethylation with increased EGR1 expression could determine heparanase expression during the pathogenesis of bladder cancer. Bladder cancer cell lines (J82, T24 and transitional cell carcinoma) significantly restored heparanase expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24 bladder cancer cell line significantly downregulated heparanase expression compared to the control siRNA transfection. In 54 bladder cancer and paired normal bladder samples, heparanase expression was significantly higher in bladder cancer than in normal bladder (P<0.01). We performed methylation-specific PCR targeting the CpG sites within the core-binding consensus motifs of EGR1 (GGCG) and Sp1 (GGGCGG). Methylation prevalence was significantly higher in normal bladder than in bladder cancer (P<0.05) and inversely correlated with heparanase expression (P=0.055). In the total series of bladder cancer and normal bladder samples, the combination of promoter CpG methylation and EGR1 expression regulated heparanase expression in a stepwise manner, where heparanase expression was the lowest in methylation-positive and EGR1-negative samples and the highest in methylation-negative and EGR1-positive samples. To our knowledge, this is the first study demonstrating that increased heparanase expression during the pathogenesis of bladder cancer is due to promoter hypomethylation and transcription factor EGR1.

Expression of MUC1 mucins inversely correlated with post-surgical survival of renal cell carcinoma patients

Surgical specimens of the normal kidney and of renal cell carcinoma (RCC) tissues at different stages of progression and of various histological grades were examined for the expression of MUC1 mucins with sialylated carbohydrates (sialylated MUC1 mucins) using a monoclonal antibody MY.1E12. Immunohistochemical studies revealed that the binding sites for this antibody were localized to the apical side of the epithelial cells of the distal convoluted tubules, Henle’s loops and collecting ducts. However, proximal convoluted tubules, where RCC is considered to originate, were not stained. This antibody also bound strongly to RCC at advanced stages of progression and at metastatic sites, and to RCC of histologically high grades (undifferentiated). The epitope, presumably sialylated MUC1 mucin, was detected not only along the surface of the cell membranes but also in the cytoplasm. The level of expression of sialylated MUC1 mucins was inversely correlated with the survival of the patients with RCC and the disease-free survival period after curative surgery. Western blot analysis demonstrated that the electrophoretic mobility of sialylated MUC1 mucins of RCC was greater than that from the normal kidney. It is suggested that high levels of expression of sialylated MUC1 mucins in certain human RCC populations correlate with the aggressiveness of the disease, such as the tendency to form metastasis.

Loss of anti-apoptotic genes in aging rat crura

PURPOSE Erectile dysfunction is a common problem in aging men. The molecular mechanisms associated with aging erectile dysfunction are not completely understood. We hypothesized that apoptosis is a downstream event in erectile dysfunction, and pro-apoptotic (Bak and Bax) and anti-apoptotic (Bcl-2 and Bcl-x) factors are involved in the etiology of aging erectile dysfunction. To test this hypothesis intracavernous pressure in aging rats was measured to assess erectile function. Gene and protein expression of pro-apoptotic and anti-apoptotic factors was then analyzed in aging rat crura to assess its role in aging erectile dysfunction. MATERIALS AND METHODS A total of 25 male Sprague-Dawley rats divided into 5 groups of 5 each based on age (6, 12, 18, 24 and 28 months old, respectively) were used in functional and apoptotic studies. In addition, 5, 3-month-old male Sprague-Dawley rats were used in the apoptotic study. The rats were anesthetized with pentobarbital. Erectile function was assessed by measuring intracavernous pressure after electrostimulation of the cavernous nerves. After completion of the functional study the penile crura were immediately harvested for histochemical and molecular studies. Gene expression of pro-apoptotic (Bak and Bax) and anti-apoptotic (Bcl-2 and Bcl-x) factors were then analyzed by reverse transcription-polymerase chain reaction. Protein expressions of these apoptotic factors were also analyzed by immunohistochemistry using specific antibodies. RESULTS Mean intracavernous pressure plus or minus SD in 18, 24 and 28-month-old rats was significantly lower than in 6-month-old rats (101.6 +/- 24.8, 77.7 +/- 24.5 and 45.7 +/- 7.3 versus 136.7 +/- 11.4 cm. water, respectively; p <0.05). The reduction in intracavernous pressure was an age dependent phenomenon. Gene and protein expression of the pro-apoptotic genes Bak and Bax was detected in the crura of all age groups but there was no significant difference in young and old rat crura. The anti-apoptotic Bcl-2 and Bcl-x genes were expressed in 3, 6 and 12-month-old crura, whereas this expression was lost at 18, 24 and 28 months. Anti-apoptotic Bcl-2 and Bcl-x proteins were also expressed in young rat crura, whereas this expression was lost in old rat crura. CONCLUSIONS The decline in intracavernous pressure correlated significantly with a loss of anti-apoptotic genes in aging rat crura. To our knowledge this is the first report to show the loss of anti-apoptotic factors in aging rat crura and suggest their role in the pathogenesis of aging erectile dysfunction.

Primary carcinoid tumor in a horseshoe kidney

A case of primary carcinoid tumor arising within a horseshoe kidney in a 51‐year‐old woman is reported. The tumor was found incidentally by computed tomography (CT) during a check‐up for a suspected gall bladder polyp. Histologic, immunohistochemical and electron microscopic analyses of this tumor revealed features typical of carcinoid tumor. Primary carcinoid tumor of the kidney is extremely rare and only 32 cases were previously reported, including five cases in horseshoe kidneys. None of these five cases in horseshoe kidneys demonstrated any evidence of local or distant metastases and all were alive at the time of reporting without evidence of disease after up to 3 years of follow up. The present case, even with accompanying lymph nodal metastasis, also has had no evidence of local recurrence or distant metastasis for 3 years post operation. Primary carcinoid tumor arising within horseshoe kidneys appear to be more benign than those within non‐horseshoe kidneys.

Expression of sialylated MUC1 in prostate cancer: Relationship to clinical stage and prognosis

Aim: MUC1 is distributed among a variety of normal epithelial tissues, and overexpression of MUC1 is detected in several human cancers. This study aimed to elucidate whether sialylated MUC1 expression correlated with: (i) clinical stage of prostate cancer; (ii) pathological grade of prostate cancer; (iii) pretreatment serum level of prostate‐specific antigen (PSA); or (iv) the disease prognosis in patients with prostate cancer who received endocrine therapy.

Case of combined adrenal cortical adenoma and myelolipoma

We report a case of myelolipoma 10 mm in size within a functional cortical adenoma that was 33 × 22 × 17 mm in size. A 29‐year‐old woman was referred to hospital for transient hypertension. A right adrenal tumor was detected by computed tomography (CT) scan and magnetic resonance imaging (MRI). Her cortisol levels indicated a loss of the normal diurnal pattern, and urinary 17‐hydroxycorticosteroids was elevated. She underwent a right adrenalectomy under the diagnosis of adrenal adenoma with Cushings syndrome. The tumor was fairly well encapsulated by a thin layer of connective tissue. The major tumor portion was composed of two distinct cell types, clear cells and eosinophilic cells, arranged in an alveolar structure. These findings were representative of cortical adenoma. The adrenal cortical adenoma centrally included well‐demarcated adipose tissue admixed with scattered islands of myelopoietic elements: erythroblasts, myeloid cell series and lymphocytic cells, which was eventually recognized as myelolipoma. Recently, adrenal myelolipoma has commonly been found because of the ease of detecting it as an incidentaloma by CT scan or MRI. However, the present adrenal myelolipoma case is uncommon because it is combined with a functioning cortical adenoma. Only six similar cases have previously been reported in English and Japanese publications. Furthermore, in the present case, the myelolipoma formed a tumor nodule, and to our knowledge, this is the first reported case of a radiographically recognizable tumor nodule. We discuss the etiology of myelolipoma and suggest that myelolipoma can develop in the course of endocrine hyperfunction.

Impact of Dry Ejaculation Caused by Highly Selective α1A-blocker: Randomized, Double-blind, Placebo-controlled Crossover Pilot Study in Healthy Volunteer Men

INTRODUCTION Dry ejaculation with loss of seminal emission is reported in patients who have been administered silodosin, an alpha1A-adrenoceptor antagonist. AIM We investigated the impact of dry ejaculation caused by orally administered silodosin on orgasmic function. METHODS In a double-blind crossover study, 50 healthy volunteer men were randomly assigned to receive either a single dose of 4-mg silodosin or placebo with 3 days of washout before crossover. Subjects masturbated 4 hours after administering agents. MAIN OUTCOME MEASURES Numerical rating scale (NRS) score from 0 (highest) to 10 (lowest) for subjective quality of orgasm, the subjective number of contractions of the bulbocavernosus/pelvic floor muscles, and the amount of semen were examined. Results. After the administration of silodosin, the NRS score worsened by 1.3 points (P = 0.003), the number of contractions of the bulbocavernosus/pelvic floor muscles decreased by about 1 (P = 0.003), and there was a decrease of 1.8 mL in the amount of semen produced (P < 0.0001). Eleven men overall (22%) on silodosin administration had less than a 50% decrease from baseline in the amount of semen. CONCLUSIONS Silodosin may adversely affect the subjective orgasmic function by causing an abnormal ejaculation with decreased (or no) semen discharge and a decrease in the number of bulbocavernosus/pelvic floor muscle contractions. Semen passing through the urethra and sufficient rhythmic contraction of the muscle of the pelvic floor may contribute to the subjective pleasure of orgasm.

Stone attenuation value and cross-sectional area on computed tomography predict the success of shock wave lithotripsy.

Purpose To identify the parameters on noncontrast computed tomography (NCCT) that best predict the success of shock wave lithotripsy (SWL). Materials and Methods We reviewed the records of 75 patients who underwent SWL for urinary calculi measuring 5 to 20 mm. Using NCCT images, we estimated the largest stone cross-sectional area and contoured the inner edge of the stone. Clinical outcome was classified as successful (stone-free or <4 mm in diameter) or failed (stone fragments, ≥4 mm). The impact of preoperative parameters was evaluated by univariate and multivariate analysis. Results The overall success rate was 73.3%. Average stone attenuation value, stone length, and stone cross-sectional area in the success and failure groups were 627.4±166.5 HU (Hounsfield unit) vs. 788.1±233.9 HU (p=0.002), 11.7±3.8 mm vs. 14.2±3.6 mm (p=0.015), and 0.31±0.17 cm2 vs. 0.57±0.41 cm2 (p<0.001), respectively. In the multivariate analysis, stone attenuation value was the only independent predictor of SWL success (p=0.023), although stone cross-sectional area had a tendency to be associated with SWL success (p=0.053). Patients were then classified into four groups by using cutoff values of 780 HU for stone attenuation value and 0.4 cm2 for cross-sectional area. By use of these cutoff values, the group with a low stone attenuation value and a low cross-sectional area was more than 11.6 times as likely to have a successful result on SWL as were all other groups (odds ratio, 11.6; 95% confidence interval, 3.9 to 54.7; p<0.001). Conclusions Stone attenuation value and stone cross-sectional area are good predictors of extracorporeal SWL outcome.