Solid-state cultivation of multiple industrial strains of koji mold on different Thai unpolished rice cultivars: biotransformation of phenolic compounds and their effects on antioxidant activity.

Colored rice is abundant in polyphenols, and koji molds have potential for biotransformation. This study aimed to produce Thai-colored rice koji to study its polyphenolic biotransformation. Four industrial koji mold strains: Aspergillus oryzae 6001, A. oryzae 6020, A. sojae 7009, and A. luchuensis 8035, were cultivated on unpolished Thai-colored rice (Riceberry and Sangyod), unpolished Thai white rice (RD43), and polished Japanese white rice (Koshihikari). We discovered that koji molds grew on all the rice varieties. Methanol extracts of all rice kojis exhibited an approximately 2-fold or greater increase in total phenolic content and DPPH antioxidant activity compared to those of steamed rice. Moreover, quercetin, quercetin-3-O-glucoside, isorhamnetin-3-O-glucoside, ferulic acid, caffeic acid, protocatechuic acid, vanillic acid, (+)-catechin, and (-)-epicatechin content increased in Riceberry and Sangyod koji samples. Consequently, Aspergillus solid-state cultivation on unpolished Thai-colored rice exhibited higher functionalization than the cultivation of unpolished Thai white rice and polished Japanese white rice.

Multifunctional-separation-mode ion chromatography method for determining major metabolites during multiple parallel fermentation of rice wine.

Facile and effective analysis methods are desirable for elucidating the behaviours of metabolites during fermentation reactions. Herein, a multifunctional-separation-mode ion chromatography (MFS-IC) method was developed for the simultaneous monitoring of major metabolites during multiple parallel fermentation, including those related to central carbon metabolism (saccharification, glycolysis, alcoholic fermentation, and the tricarboxylic acid (TCA) cycle). The use of two types of sulfo-modified size-exclusion columns and phthalic acid as the eluent allowed the separation of oligosaccharides (disaccharides, trisaccharides, and tetrasaccharides), glucose, pyruvate, and major organic acids during the TCA cycle (cis-aconitate, citrate, iso-citrate, malate, fumarate, and succinate but not α-ketoglutarate) from other non-target analytes. The MFS-IC method was successfully applied to monitoring the major metabolites in the rice wine brewing process. This approach can contribute to an improved understanding of metabolite behaviour during fermentation without requiring the use of expensive advanced instrumentation methods such as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.

Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae.

Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

Comparison of taste characteristics between koji mold-ripened cheese and Camembert cheese using an electronic tongue system.

Koji mold, classified in the genus Aspergillus, is used to produce traditional Japanese fermented foods such as miso, soy sauce, and sake. In recent years, the application of koji mold to cheese ripening has attracted attention, and cheese surface-ripened with koji mold (koji cheese) has been studied. In this study, to evaluate the taste characteristics of koji cheese, an electronic tongue system was employed to measure the taste values of cheese samples ripened using 5 strains of koji mold in comparison with commercial Camembert cheese. All koji cheese samples exhibited lower sourness and greater bitterness, astringency, saltiness, and umami richness than the Camembert cheese samples. The intensity of each taste characteristic differed depending on the koji mold strain. These results indicate that koji cheese has a different taste value than conventional mold-ripened cheese. Furthermore, the results also indicate that various taste characteristics can be achieved by selecting different koji molds.

Preparation of egg-koji for developing a novel food.

While chicken eggs contain many nutrients necessary for humans and there are various cooking methods, the nutritional components are used as they are, and there are no traditional foods that utilize microorganisms. Koji-mold, containing Aspergillus oryzae, A. sojae, and A. luchuensis, which has been used in various fermented foods since ancient times, grows on raw grain materials such as rice and barley to become koji. This can give flavors not found in the raw materials that can decompose and convert the nutritional components of the raw materials. Here, we succeeded for the first time in developing egg-koji that uses only eggs and koji-mold by selecting and combining cooked egg powder (CEP) and A. oryzae AO101 as the most suitable combination. To suppress the explosive growth of harmful bacteria, we improved the sterilization method, watering method, and amount of water. In addition, it was found that egg-koji has a characteristic enzyme activity balance, in which amylase is extremely low and protease at pH 6 was high compared to grain koji, such as rice and barley. Egg-koji might produce enzymes suitable for taking in nutrients when growing into CEP and would be expected to give a flavor that could not be achieved by cooking or additives.

Volatile and soluble metabolite profiles in surface-ripened cheeses with Aspergillus oryzae and Aspergillus sojae.

To determine the impact of traditional koji molds on chemical characteristics of soft-type natural cheese, novel surface mold-ripened cheeses with Aspergillus oryzae and Aspergillus sojae were studied by non-targeted metabolite profiling. Comprehensive water-soluble and volatile metabolite profiles of koji cheese were evaluated among five Aspergillus strains and other mold-ripened cheeses. Time-course changes in the metabolite profiles and degrading enzyme activities were also compared with those of an industrial Penicillium candidum starter culture. Koji cheeses differed from Camembert, Brie, and blue cheeses in higher lactic acid, amino acid, and acetoin levels and lower methyl ketone and volatile fatty acid levels. Time-course analysis revealed the associations of rapid accumulations of glutamic, aspartic, and 3-methylbutanoic acids and 3-methylbutanal with higher proteolytic activity, and methyl ketone and fatty acid derivative suppressions with lower lipolytic activity. Ethyl butanoate, diacetyl, and malic acid also characterized koji cheeses as strain-dependent metabolites. This study highlighted the key compositional difference derived from cheese ripening with Aspergillus strains. The findings could help quality improvements of koji cheese product.

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria.

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

Koji Starter and Koji World in Japan.

Koji is made by culturing koji mold on grains. Koji has wide-ranging applications, for example, in alcoholic beverages and seasonings. The word 'mold' generally has a bad image, but in Japan, koji mold is valued for its usefulness, and over the years, efforts have been made to make safe, stable, and delicious food products from it. Koji mold spores, essential when making koji, are called koji starter in the industry. From the many available strains, those suitable for the production of each fermented food are chosen based on indicators such as growth rate and enzyme production capacity. In manufacturing using microorganisms, purity and yield are prioritized. However, the production of fermented foods using koji is more complex, with focus not only on the degree of decomposition of raw materials but also on factors influencing overall product design, including palatability, color, smell, and texture. Production can be facilitated by the variety of koji brought about by the diversity of koji mold combined with the solid culture method which increases the amount of enzyme production. In this report, we introduce the history of koji starter in Japan, the characteristics of koji mold in practice, and various fermented foods made from it. In addition, the factors affecting the quality of koji in solid culture are described.

Single assistant versus dual assistant robotic surgery for robot-assisted laparoscopic hysterectomy using da Vinci Xi or X.

BACKGROUND: To verify the usefulness of single assistant robotic surgery (SA-RS) using da Vinci Xi or X performed for total hysterectomy. METHODS: The SA-RS group (S-group) includes surgeries in which the surgeon performed all surgical operations other than that of the uterine manipulator, while the dual assistant robotic surgery group (D-group) includes surgeries performed by the surgeon with two assistants. In S-group, the forceps and camera were replaced during surgery in cases of a large uterus with limited range of motion of the instrument. A comparative study of patient background, intraoperative and postoperative results and surgery-related cost was performed between the two groups. RESULTS: No significant differences were observed between the two groups. On the contrary, S-group showed a significant reduction in preparation time and wound closure time, and a savings of $768. CONCLUSION: SA-RS for total hysterectomy was possible, which is excellent in terms of cost, esthetics, and manpower.

Rapid noninvasive monitoring of freshness variation in frozen shrimp using multidimensional fluorescence imaging coupled with chemometrics.

Shrimp is one of the most delicious and popular food commodities worldwide due to its exceptional taste and characteristics. Freshness is considered as a key factor for shrimp consumers because freshness has a significant relationship with taste and shelf-life of shrimp. However, post-mortem metabolism of shrimp differs from that of fish as they are highly susceptible to post-harvest quality loss, and it is hard to distinguish the freshness variation of shrimp at frozen state instantly. Thus, instant monitoring of frozen shrimp freshness is challenging for the seafood and aquaculture industries and a reliable, expeditious, and noninvasive technique to estimate shrimp quality is in high demand. Accordingly, this study aimed to visualize changes in post-mortem freshness of frozen shrimp using multidimensional fluorescence imaging. Live coonstripe shrimp (Pandalus hypsinotus) were harvested and instantly killed by beheading, cooled on ice for 0, 6, 24, 48, 72 and 96 h (n = 8), followed by processing into frozen peeled deveined shrimp product and stored at -60 °C. 50% of frozen shrimp were analyzed for excitation-emission matrix (EEM), ATP-related compounds, and pH using a fiber optic supported fluorescence spectrophotometer (F-7100), high performance liquid chromatography (HPLC) and pH meter, respectively at each time point (n = 4). Then, fluorescence images were obtained from the remaining 50% of frozen shrimp (n = 4) by computer vision method equipped with a charge-coupled device (CCD) camera, MAX-303 xenon light source for an excitation light (Ex. 330 nm), and an automatic filter changer for emission band-pass filters (Em. 380-610 nm at 10 nm intervals). Chemical analysis of frozen shrimp revealed that K-value and pH of shrimp increased from 1.61 to 66.56% and 6.49-7.31, respectively, during storage on ice. Repeated partial least squares regression (PLSR) models of EEM for K-value prediction suggested an efficient excitation wavelength (330 nm) and its corresponding emission wavelengths (380-610 nm) to produce fluorescence images. Spatial-temporal changes of K-value and pH were visualized successfully in frozen shrimp by fluorescence imaging. K-value visualization was then validated effectively using another group of frozen shrimp (0-72 h ice stored) with different killing method (super chilling) and the prediction accuracy was R2 = 0.80. This novel approach using a CCD camera coupled with EEM provides a state-of-the-art authentication method for practical assessment of frozen seafood freshness.

Prostate carcinoma with neuroendocrine differentiation successfully treated by early detection with imaging examination.

INTRODUCTION: Clinicopathological features of neuroendocrine differentiation of prostate carcinoma have not been totally clarified yet. It is known to be associated with poor prognosis. CASE PRESENTATION: A patient with growing prostate-specific antigen was diagnosed with prostate carcinoma and treated by laparoscopic prostatectomy. The pathological examination revealed the neuroendocrine differentiation of the tumor. Early detection of prostate carcinoma with neuroendocrine differentiation can be difficult due to its low expression of prostate-specific antigen. The imaging examination contributed to the early detection. In the follow-up period of 2 years, the patient remains cancer free. CONCLUSION: Recently, the treatment options for prostate carcinoma have been expanded. Precise assessment of immunohistochemical nature of the tumor may be helpful for individualized decision-making.

[Current Status of Prostate Cancer and Its Relation to Home Medical Care].

Because ofboth the indolent and aggressive nature ofprostate cancers, it is not easy to select the best treatment for patients receiving home medical care who already have many diseases. Since the growth ofprostate cancer is generally slow and all treatments adversely affect the quality of life to some degree, conservative treatment may well be the best option for these patients with prostate cancer. However, it is also true that we often encounter home medical care patients with a locally advanced cancer who had symptoms such as difficulty to urine, macrohematuria and/or lumbago. Such patients need to be diagnosed soon and treated with mainly hormonal therapy after consultation with the specialist. Thus, after a careful evaluation ofthe nature ofthe cancer and comorbidity, we need to provide the best option ofdiagnosis and treatment for home medical care patients to maintain their quality of life.

Gleason Pattern 5 is a Possible Pathologic Predictor for Biochemical Recurrence after Laparoscopic Radical Prostatectomy

Objective: Several prognostic factors for biochemical recurrence after radical prostatectomy have been reported, including initial prostate-specific antigen level, Gleason score, positive surgical margin, and seminal vesicle invasion. Here we investigate whether Gleason pattern 5 is a predictor for biochemical recurrence. Methods: This retrospective study included 168 patients who underwent laparoscopic radical prostatectomy from 2006 to 2015. The relationship between biochemical recurrence after laparoscopic radical prostatectomy and the presence of Gleason pattern 5, even as a tertiary pattern, was investigated. Biochemical recurrence was defined when the prostate-specific antigen level rose to >0.2 ng/ml after having decreased to <0.1 ng/ml following laparoscopic radical prostatectomy. Biochemical recurrence-free survival was estimated by the Kaplan-Meier method. Multivariate analysis was performed using a Cox proportional hazards regression model. Results: The median age was 66 years, median initial prostate-specific antigen level was 6.9 ng/ml, and median follow-up period was 47.3 months. Biochemical recurrence was recognized in 27 patients (16.1%) after laparoscopic radical prostatectomy, and 5-year biochemical recurrence-free survival was 78.6%. Gleason pattern 5 was noted in 5 patients as the primary pattern, in 10 as the secondary pattern, and in 5 as the tertiary pattern. According to multivariate analysis, presence of Gleason pattern 5 (HR = 4.75, p=0.001) and positive surgical margin (HR = 4.66, p=0.001) were independent predictive factors for biochemical recurrence-free survival. Conclusion: Gleason pattern 5 appears to be an important predictive factor for biochemical recurrence after laparoscopic radical prostatectomy.

Effectiveness of a Self-monitoring Device for Urinary Sodium-to-Potassium Ratio on Dietary Improvement in Free-Living Adults: a Randomized Controlled Trial.

BACKGROUND: Reducing the urinary sodium-to-potassium ratio is important for reducing both blood pressure and risk of cardiovascular disease. Among free-living Japanese individuals, we carried out a randomized trial to clarify the effect of lifestyle modification for lowering urinary sodium-to-potassium ratio using a self-monitoring device. METHODS: This was an open, prospective, parallel randomized, controlled trial. Ninety-two individuals were recruited from Japanese volunteers. Participants were randomly allocated into intervention and control groups. A month-long dietary intervention on self-monitoring urinary sodium-to-potassium ratio was carried out using monitors (HEU-001F, OMRON Healthcare Co., Ltd., Kyoto, Japan). All participants had brief dietary education and received a leaflet as usual care. Monitors were handed out to the intervention group, but not to the control group. The intervention group was asked to measure at least one spot urine sodium-to-potassium ratio daily, and advised to lower their sodium-to-potassium ratio toward the target of less than 1. Outcomes included changes in 24-hour urinary sodium-to-potassium ratio, sodium excretion, potassium excretion, blood pressure, and body weight in both groups. RESULTS: Mean measurement frequency of monitoring was 2.8 times/day during the intervention. Changes in urinary sodium-to-potassium ratio were -0.55 in the intervention group and -0.06 in the control group (P = 0.088); respective sodium excretion changes were -18.5 mmol/24 hours and -8.7 mmol/24 hours (P = 0.528); and corresponding potassium excretion was 2.6 mmol/24 hours and -1.5 mmol/24 hours (P = 0.300). No significant reductions were observed in either blood pressure or body weight after the intervention. CONCLUSIONS: Providing the device to self-monitor a sodium-to-potassium ratio did not achieve the targeted reduction of the ratio in "pure self-management" settings, indicating further needs to study an effective method to enhance the synergetic effect of dietary programs and self-monitoring practice to achieve the reduction. However, we cannot deny the possibility of reducing sodium-to-potassium ratio using a self-monitoring device.

[Solitary Fibrous Tumor of the Kidney].

A 59-year-old woman was referred to our hospital with the finding of an incidentaloma in her right kidney. Computed tomographyand magnetic resonance imaging showed a solid tumor, approximately8 cm in diameter, at the lower pole of the right kidney. Partial nephrectomy was performed without a renal vessel clamp. Histopathologically, fibroblast-like spindle cells were proliferating with rich collagenous fibers, and tumor cells were infiltrated into the renal parenchyma. The tumor cells were stained positive with CD34, CD99, and bcl-2. The tumor was diagnosed as solitaryfibrous tumor originating from the kidney based on these histopathological findings. The patient was followed 3 years after surgery with neither recurrence nor metastasis.

Phase Transition during Heating of Nanostructured Ultrahigh Molecular Weight Polyethylene Membranes.

Ultrahigh molecular weight polyethylene (UHMW-PE) membranes were prepared using biaxial melt-drawing and subsequent melt-shrinking. Electron microscopy observations indicate that the former membrane has more extended-chain crystals (ECCs), whereas the latter is mainly composed of folded-chain crystals (FCCs). Corresponding double-melting endotherms are recorded on differential scanning calorimetry (DSC) measurements. Detailed assignments of such double-melting components are performed using in situ X-ray measurements during heating. Wide- and small-angle X-ray diffraction and scattering (WAXD/SAXS) images were simultaneously recorded at SPring-8. Changes in WAXD images indicate that the orthorhombic reflection peak begins to decrease at 130 °C, followed by the appearance of the hexagonal reflection peak beyond 145 °C for both membranes, but the latter melt-shrunk membrane exhibits weaker hexagonal reflection intensity. Simultaneous SAXS results indicate that FCCs rapidly disappear at 135 °C for the melt-shrunk membrane, resulting in a sharper endotherm. In contrast, residual ECCs restrict the melting of FCCs for the melt-drawn membrane, resulting in a broader endotherm of FCC melting spread to a slightly higher temperature position.

Six random specimens of daytime casual urine on different days are sufficient to estimate daily sodium/potassium ratio in comparison to 7-day 24-h urine collections.

The objective of this study was to determine the optimal number and type of casual (spot) urine specimens required to estimate an individual's urinary sodium/potassium (Na/K) ratio. A total of 48 participants, 25 men and 23 women, aged between 25 and 59 years, was recruited from healthy volunteers. The Na/K ratio in each casual urine and 7-day 24-h urine sample was measured. Correlation analysis and the quality of agreement by the Bland and Altman method between casual urine and 24-h urine were analyzed. The mean Na/K ratio of 7-day 24-h urine was 4.3. The mean Na/K ratio of six random specimens of daytime (collected between 09 and 17 hours) casual urine correlated most strongly with the Na/K ratio of 7-day 24-h urine (r=0.87). The bias for the mean Na/K ratio between 7-day 24-h urine and daytime casual urine was almost negligible (0.03), and the quality of agreement for the mean of the six random, daytime casual urine specimens on different days was similar to that of the 2-day 24-h urine samples for estimating 7-day 24-h values. Our findings show that the mean Na/K ratio of six random daytime casual urine specimens on different days was a good substitute for the 2-day 24-h urine Na/K ratio.

Dasatinib is preclinically active against Src-overexpressing human transitional cell carcinoma of the urothelium with activated Src signaling.

Dasatinib is an orally administered multitargeted kinase inhibitor that targets Src family tyrosine kinases, Abl, c-Kit, and PDGFR. A preclinical study was conducted to evaluate dasatinib alone or combined with cisplatin for human transitional cell carcinoma (TCC). Expression of Src in a human TCC tissue microarray was evaluated by immunohistochemistry. The activity of dasatinib and/or cisplatin was evaluated in six human TCC cell lines. Western blot was done to assess Src and phosphorylated-Src (p-Src) expression. The activity of dasatinib alone and in combination with cisplatin was determined in murine subcutaneous xenografts. Sixty-two percent to 75% of human TCC expressed Src. Dasatinib displayed significant antiproliferative activity at nanomolar concentrations against two human TCC cell lines (RT4 and Hu456) that exhibited high Src and p-Src expression and were cisplatin-resistant. RT4 cells were the most sensitive and displayed the highest level of Src pathway activation (p-Src/Src ratio). Dasatinib downregulated p-Src in either sensitive or resistant cells. TCC cells that were sensitive to cisplatin (5637 and TCC-SUP) were highly resistant to dasatinib and exhibited low Src expression. Dasatinib showed antitumor activity in RT4 murine xenografts, and the combination of dasatinib and cisplatin was significantly more active than placebo. Combination dasatinib plus cisplatin significantly inhibited proliferation and promoted apoptosis in vivo. In conclusion, dasatinib displayed significant preclinical antitumor activity against Src-overexpressing human TCC with active Src signaling and was highly active in combination with cisplatin in vivo. Further clinical development might be warranted in selected human subjects.

Cost comparison of curative therapies for localized prostate cancer in Japan: a single-institution experience.

PURPOSE: In addition to open surgery, curative therapies for prostate cancer now include endoscopic surgery and radiation therapies. Because of the expansion and subdivision of treatment methods for prostate cancer, the medical fee point schedule in Japan was revised in fiscal year 2006. We examined changes in medical income and expenditure after this revision of the medical fee system. MATERIALS AND METHODS: We studied income and expenditure, after institution of the new medical fee schedule, for the five types of therapies for prostate cancer performed at our hospital: two surgical therapies (radical retropubic prostatectomy and laparoscopic prostatectomy) and three radiation therapies (three-dimensional conformal radiation therapy, (192)Ir high-dose-rate brachytherapy, and (125)I low-dose-rate brachytherapy). RESULTS: Low-dose-rate brachytherapy was found to be associated with a profit of yen199 per patient. Laparoscopic prostatectomy, a highly advanced medical treatment that the fee revision changed from a partially insured to an insured procedure, yielded a profit of yen75,672 per patient. However, high-dose-rate brachytherapy was associated with a loss of yen654,016 per patient. CONCLUSION: Given the loss in hospital income per patient undergoing high-dose-rate brachytherapy, the medical fee point system for this procedure should be reassessed.

Enzastaurin shows preclinical antitumor activity against human transitional cell carcinoma and enhances the activity of gemcitabine.

Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signaling through protein kinase C (PKC)-beta and the phosphatidylinositol 3-kinase/AKT pathways. We preclinically evaluated enzastaurin alone and in combination with gemcitabine for transitional cell cancer (TCC). Immunohistochemistry (IHC) was done on 105 human samples from a microarray to show the expression of PKC-beta. The preclinical antitumor activity of enzastaurin and gemcitabine as single agents and in combination against aggressive human -lines (-SUP and 5637) and murine subcutaneous xenografts bearing 5637 cells was determined. Western Blot was done on tumor cells in vitro to detect signaling through PKC-beta, GSK-3beta, and AKT. The effect on cell migration was determined in vitro. Modulation of proliferation (Ki-67), apoptosis (cleaved caspase-3), and angiogenesis (CD31) in vivo was determined by IHC. IHC done on human TCC samples from a microarray showed the expression of PKC-beta in 33% of tumors. Enzastaurin induced significant apoptosis and inhibited proliferation in vitro at low micromolar concentrations. The in vitro inhibitory activity of combination enzastaurin and gemcitabine by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay seemed synergistic. Western Blotting revealed down-regulation of Akt, PKC-beta, and GSK-3 beta phosphorylation. Enzastaurin inhibited migration at an earlier time point independent of antiproliferative activity. Combination therapy had significantly superior antitumor activity in murine xenografts compared with untreated controls, whereas single agents did not. IHC showed reduced Ki-67 and CD31 and increased cleaved caspase-3 with combination therapy compared with controls. Enzastaurin showed preclinical antitumor activity against human TCC and enhanced the activity of gemcitabine.