Assessment of the efficacy of anticancer drugs by amino acid metabolomics using fluorescence derivatization-HPLC.

Metabolomic studies conducted for evaluating cancer pathogenesis and progression by monitoring the amino acids metabolic balance hold great promise for assessing current and future anticancer treatments. We performed a comprehensive quantification of 21 amino acids concentrations in cultured human colorectal adenocarcinoma cells treated with the anticancer drugs 5-fluorouracil, irinotecan, and cisplatin. A precolumn fluorescence derivatization-HPLC method involving 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate was used. Amino acid concentration data were analyzed by principal-component analysis and partial least-squares multivariate statistical methods to represent samples on two-dimensional graphs. The hierarchical cluster analysis and linear discriminant analysis were used to classify the samples on the score plots. Unlike the cluster analysis approach, the linear discrimination analysis classification successfully distinguished anticancer drug-treated samples from the untreated controls. Moreover, three candidate amino acids (serine, aspartic acid, and methionine) were identified from the loading plots as potential biomarkers. Our proposed method might be able to evaluate the effectiveness of anticancer therapy even in small laboratories or medical institutions.

Fully automated reagent peak-free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column-switching system and fluorous scavenging derivatization.

In this study, we combined a column-switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak-free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess of the unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column-switching system placed in front of an analytical reversed-phase column. The signal of the fluorous-tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type-II model mice and their control.

Accuracy of differential diagnosis for pancreatic cancer is improved in the combination of RCAS1 and CEA measurements and cytology in pancreatic juice.

Improvement of diagnostic accuracy for pancreatic cancer in pancreatic disease patients was investigated by examining the combination of three diagnostic methods, i.e., measurements of RCAS1 and CEA levels in pancreatic juice and pancreatic juice cytology. Pancreatic juice was collected from 12 pancreatic cancer (PC) and 26 non-PC patients. RCAS1 and CEA levels were measured by using ELISA. RCAS1 expression on surgically resected tissue was immunohistochemically examined for 2 PC patients. By setting the cutoff level of RCAS1 at 10 U/ml and that of CEA at 18.5 µg/ml, sensitivity of RCAS1 was 42% and that of CEA was 50%. On the other hand, sensitivity and specificity increased from 42% and 85% of RCAS1 alone to 75% and 85% in the examination of RCAS1 + CEA + cytology, and the false-negative rate was also reduced to 25% in this combination. Immunohistochemically, a patient with a high RCAS1 level in pancreatic juice had numerous RCAS1-positive tumor cells in the pancreatic juice. We concluded that RCAS1 and CEA measurements together with cytology in pancreatic juice would be a useful combination method for making a differential diagnosis of PC from non-PC.

Association between lipid accumulation and the cannabinoid system in Huh7 cells expressing HCV genes.

Evidence from clinical and laboratory studies has accumulated indicating that the activation of the cannabinoid system is crucial for steatosis, especially in non-alcoholic fatty liver disease. However, the association between hepatitis C virus (HCV) infection and the cannabinoid system has not been well investigated and it is unclear whether steatosis in chronic hepatitis C develops via activation of the endocannabinoid/cannabinoid receptor signaling pathway. In this study, we examined the expression of a cannabinoid receptor (CB1) and the lipid accumulation in the hepatic Huh7 cell line, expressing HCV genes. We utilized Huh7/Rep-Feo-1b cells stably expressing HCV non-structural proteins (NS) 3, NS4, NS5A, and NS5B, as well as Tet-On Core-2 cells, in which the HCV core protein expression is inducible. Significantly higher levels of stored triglycerides were found in Huh7/Rep-Feo-1b cells compared to Huh7 cells. Also, triglyceride accumulation and CB1 receptor expression were down-regulated in Huh7/Rep-Feo-1b cells after HCV reduction by IFNα. Moreover, lipid accumulation appeared to increase after CB1 agonist treatment, while it decreased after CB1 antagonist treatment, although significant differences were not found compared to untreated cells. In Tet-On Core-2 cells, induction of HCV core protein expression did not affect CB1 expression or triglyceride accumulation. The results of this study in cultured cells suggest that HCV infection may activate the cannabinoid system and precede steatosis, but the core protein by itself may not have any effect on the cannabinoid system.

Analysis of cell cycle arrest and apoptosis induced by RCAS1.

A tumor-associated antigen RCAS1 (receptor binding cancer antigen expressed on SiSo cells) induces cell cycle arrest and apoptosis to a putative RCAS1 receptor (RCAS1-R) expressing cells such as T, B, and natural killer cells. Its expression is related with clinical poor prognosis of some malignant tumors. It is suggested that the expression of RCAS1 in tumor cells plays an important role in evasion from host immune system resulting tumor progression, invasion and metastasis. However, the mechanism of RCAS1 induced cell cycle arrest and apoptosis has not been clarified. In this study, we established a mouse L cell line transformed with tetracycline-induced rcas1 gene expression system and analyzed the RCAS1 functions. We showed that RCAS1 induced cytochrome c release and activation of caspase-3 for apoptosis. Moreover, we investigated cell cycle associated proteins and revealed that cyclin D3 decreased significantly and no change was seen in the expression levels of the other proteins. These results suggest that cyclin D3 is one of the key target molecules in the RCAS1-RCAS1-R signaling pathway.

NPC1L1 inhibitor ezetimibe is a reliable therapeutic agent for non-obese patients with nonalcoholic fatty liver disease.

BACKGROUND: We recently examined the distribution of abdominal fat, dietary intake and biochemical data in patients with nonalcoholic fatty liver disease (NAFLD) and found that non-obese NAFLD patients did not necessarily exhibit insulin resistance and/or dysregulated secretion of adipocytokines. However, dietary cholesterol intake was superabundant in non-obese patients compared with obese patients, although total energy and carbohydrate intake was not excessive. Therefore, excess cholesterol intake appears to be one of the main factors associated with NAFLD development and liver injury. METHODS: We reviewed a year of follow-up data of non-obese NAFLD patients treated with Niemann-Pick C1 like 1 inhibitor ezetimibe to evaluate its therapeutic effect on clinical parameters related to NAFLD. Without any dietary or exercise modification, 10 mg/day of ezetimibe was given to 8 patients. In 4 of 8 patients, ezetimibe was administered initially. In the remaining 4 patients, medication was switched from ursodeoxycholic acid to ezetimibe. RESULTS: In each patient, body mass index was maintained under 25 kg/m2 during the observation period. Serum ALT levels significantly decreased within 6 months and in 4 patients levels reached the normal range (<30 U/L), which was accompanied with at least a 10% decrease in serum total cholesterol and LDL-cholesterol. However, ultrasonographic findings of fatty liver did not show obvious improvement for a year. CONCLUSION: We conclude that the cholesterol absorption inhibitor ezetimibe can suppress hepatic injury in non-obese patients with NAFLD and that ezetimibe may offer a novel treatment for NAFLD.

Detection of amyloid beta protein in the urine of Alzheimer's disease patients and healthy individuals.

To seek for a new valid biomarker using non-invasive specimens for the diagnosis of Alzheimer's disease (AD) and mild cognitive impairment (MCI), we carried out the detection of amyloid beta (Abeta) protein in urine. Ten-millilitre urine samples were first sedimented with trichloroacetic acid, and the pellets were resuspended for further analysis by Western blotting with anti-Abeta antibody. The detection sensitivity of the method was 40pg/ml. Rates of subjects positive for monomeric Abeta according to their clinical dementia rating (CDR) were 11.1% for CDR 0, 62.5% for CDR 0.5, 83.3% for CDR 1, 54.5% for CDR 2 and 0% for CDR 3. A single Abeta band relative to the CDR score reflects an alteration in the production, solubility and clearance of Abeta in the brain. Thus, the method could be used as both a diagnostic and monitoring tool in assessing AD and MCI patients during disease-modifying therapies.