Lack of hippocalcin causes impairment in Ras/extracellular signal-regulated kinase cascade via a Raf-mediated activation process.

Hippocalcin (Hpca) is a member of the neuronal calcium sensor protein family and is highly expressed in hippocampal neurons. Hpca-deficient (Hpca(-/-)) mice display a defect in cAMP response element-binding protein (CREB) activation associated with impaired spatial and associative memory. Here we examine the involvement of Hpca in the extracellular signal-regulated kinase (ERK) cascade leading to CREB activation, because application of PD98059, a broad ERK cascade inhibitor, has resulted in similar levels of CREB activation in Hpca(-/-) hippocampus. N-methyl-D-aspartate (NMDA)- and KCl-induced phosphorylation of ERK was significantly attenuated in Hpca(-/-) hippocampal slices, as was ionomycin-induced phosphorylation of ERK, whereas forskolin and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation yielded indistinguishable levels of ERK phosphorylation in both wild-type and Hpca(-/-) slices. In an in vitro reconstitution assay system, recombinant Hpca affected neither Raf-1 protein kinase activity with recombinant MEK-1 as a substrate nor MEK-1 kinase activity with ERK2 as a substrate. Activation of Ras by NMDA and KCl stimulation of hippocampal slices showed no obvious changes between the two genotypes; however, phosphorylation of Raf-1 was significantly lower in Hpca(-/-) slices. These results suggest that Hpca plays an important role in the activation of Raf conducted by Ras.

Evaluation of 5-fluorouracil applicability by the collagen gel droplet embedded drug sensitivity test with area under the curve analysis.

We have evaluated the 5-fluorouracil sensitivity of cancer cells from colorectal cancer patients using the collagen gel droplet embedded drug sensitivity test under multiple drug concentrations and contact durations. After converting drug concentration and contact time to the area under the curve (AUC) and plotting against the growth inhibition rate, the correlation between AUC and the growth inhibition rates was approximated to the logarithmic regression curve. In this study, to further validate the reliability of the regression curve, the growth inhibition rate was calculated from the regression curve and the actual growth inhibition rate was compared at AUC of 48 mug h/ml. No significant difference was observed in the growth inhibition rates between the two groups by paired t-test (P=0.590). A strong positive correlation was found between the two groups by regression analysis (y=0.7555x+10.514, R=0.8236). This result strongly suggests that in-vitro antitumor effect of 5-fluorouracil depends on the AUC in colorectal cancer and the AUC-inhibition rate curve is reliable. We can obtain the inhibition rate from AUC and vice versa using the AUC-inhibition rate curve. We can also calculate the individualized AUCIR50, AUC value that gives 50% growth inhibition, using the AUC-inhibition rate curve. This could be useful to establish individualized chemotherapy using the collagen gel droplet embedded drug sensitivity test.

Prognostic impact of orotate phosphoribosyl transferase activity in resectable colorectal cancers treated by 5-fluorouracil-based adjuvant chemotherapy.

BACKGROUND AND OBJECTIVES: Phosphoribosylation of 5-fluorouracil (5-FU) is an essential step which leads to tumor growth inhibition and orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in this conversion of 5-FU to 5-fluorouridine monophosphate. This retrospective study was aimed to evaluate the correlation between tumor OPRT activity and the clinical outcome in colorectal cancer (CRC) patients treated by oral 5-FU-based adjuvant chemotherapy. METHODS: Surgical specimen was obtained from resectable 124 CRC patients who were subsequently treated by oral 5-FU-based adjuvant chemotherapy. OPRT activity in the extract of tumor tissue was enzymatically determined. The cut-off value of intratumor OPRT activity against disease free survival was determined by maximal chi2 method. The disease free survival and overall survival in each group were calculated using the Kaplan-Meier method. RESULTS: Patients were divided into two groups by determined cut-off value of intratumor OPRT (0.147 nmol/min/mg protein) (high group: n = 102, low group: n = 22). Five-year DFS (P = 0.035) and OS (P = 0.020) were significantly better for high OPRT group. CONCLUSIONS: This study demonstrated that an assay of tumor OPRT contributes to the determination of 5-FU-based adjuvant chemotherapy outcome and application in clinical practice should be included in tumor analysis prior to 5-FU-based adjuvant chemotherapy.

Prognostic impact of orotate phosphoribosyl transferase among 5-fluorouracil metabolic enzymes in resectable colorectal cancers treated by oral 5-fluorouracil-based adjuvant chemotherapy.

Orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in phosphoribosylation of 5-fluorouracil (5-FU), an essential step that leads to tumor growth inhibition. In our study, the prognostic relevance of OPRT, thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in resectable colorectal cancer (CRC) patients treated by oral 5-FU were compared to further clarify the prognostic value of OPRT. Tumor tissue was collected from 90 CRC patients and the patients were followed for 5.2 years (Median). TS, DPD and OPRT activities in the extract of tumor tissue were determined enzymatically. The cut-off value of OPRT (0.147 nmol/(min mg), TS (0.044 pmol/mg) and DPD (72.10 pmol/(min mg) were determined by maximal chi(2) method. Among these 5-FU metabolic enzymes, only high OPRT group demonstrated significantly better disease-free survival (DFS) (p = 0.0152) and better overall survival (p = 0.0078). In Cox regression analysis, node status (p < 0.0005) and OPRT (p = 0.044) were significant factors for DFS. OPRT activity in tumor tissue was a predictor of prognosis in resectable CRC patients treated by oral 5-FU-based adjuvant chemotherapy, and was useful to pick-up high risk patients independent from known prognosis factors.

Impact of orotate phosphoribosyl transferase activity as a predictor of lymph node metastasis in gastric cancer.

Orotate phosphoribosyl transferase (OPRT) is an essential nucleotide metabolic enzyme for cell proliferation and also a key enzyme for conversion of 5-FU to its active form in tumor tissue. The association between tumor OPRT activity and pathophysiological status, including lymph node metastasis [pN+], and the impact of OPRT for predicting pN+ were investigated in gastric cancer. The lymph node status of 73 resectable gastric cancer patients was analyzed preoperatively by computed tomography (CT), ultrasonography and magnetic resonance, and the OPRT activity of collected tumor tissue was measured. Then these data were compared with pathological observation of a surgical lymph node specimen. OPRT activity in the tumor tissue decreased as the depth of invasion increased. An OPRT test demonstrated superior sensitivity and comparable accuracy and sensitivity for predicting pN+, against current imaging diagnoses. Furthermore, the analysis of node negative patients by CT revealed that 80% of false negative patients were retrieved by this OPRT test. Thus, OPRT activity in tumor tissue was a powerful predictor of pN+ in resectable gastric cancer, and the preoperative OPRT test, when it becomes possible, would provide a basis for accurate evaluation of disease status, which is indispensable for the planning of personalized therapy.

Evaluation of 5-fluorouracil applicability by multi-point collagen gel droplet embedded drug sensitivity test.

The drug sensitivity of tumor cells is one of key issues to explore individualized therapy for cancer patients. One of such methods is in vitro anticancer drug sensitivity test which is generally based on one drug concentration and contact time. In this study, 5-fluorouracil (5-FU) sensitivity of cancer cells from colorectal cancer patients was evaluated by collagen gel droplet embedded drug sensitivity test (CD-DST) under multiple drug concentrations and contact durations. Cancer cells from 19 patients were measured for 9 drug concentration/contact time conditions (cohort 1) and from 34 patients were measured for 2 drug concentration/contact time conditions (cohort 2) using CD-DST. There was not significant difference in growth inhibition rate for 1.0 microg/ml for 24 h and 0.2 microg/ml for 120 h, which gives the same area under the curve (AUC) (p=0.832) in all 53 patients (cohort 1 and 2). In cohort 1, 9 conditions were successfully measured in 18 of 19 cohort 1 patients (94.7%). The drug concentrations and growth inhibition rate approximated to logarithmic curve for all 3 contact times and 50% inhibitory concentration (IC50) values at 3 contact times could be calculated in these 18 patients. Growth inhibition rate and AUC also approximated to logarithmic curve. These values varied several orders of magnitude among patients. In vitro antitumor effect of 5-FU depended on AUC in colorectal tumor and it might support the use of continuous infusion or oral therapy which generates significant AUC with manageable toxicity. Some patients demonstrating low 5-FU sensitivity could not be indicated for 5-FU based therapy, and non-5-FU therapy should be explored for them.

Usefulness of the perineum pusher in performing rectal procedures.

We invented the Perineum Pusher to prevent excessive extension of the rectum by elevating the bottom of the pelvis. In the treatment of cancers of the middle and lower thirds of the rectum, a clear operative view can be maintained for a long time by using the Perineum Pusher. Consequently, a sphincter-saving rectal resection with coloanal anastomosis can be performed easily and safely. In addition, intraoperative rectal irrigation can be performed using the Perineum Pusher. No complications resulting from the Perineum Pusher have been experienced in 27 rectal cancer patients. As the Perineum Pusher can be used very simply and effectively in sphincter-saving rectal resections with coloanal anastomosis, this new surgical instrument is therefore highly recommended for use when performing various rectal procedures.

[Correlation between clinical pathophysiologic factors and expression of orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) in colorectal cancer].

We measured the activity of orotate phosphoribosyl transferase (OPRT), the amount of thymidylate synthase (TS) enzyme, and the activity of dihydropyrimidine dehydrogenase (DPD) for individual tissue types in order to study the contribution of these substances to the effects of the pyrimidine fluoride anticancer drug 5-fluorouracil (5-FU). We also studied the correlation between these 3 enzymes and clinical pathophysiologic characteristics (age, sex, extent of tumor invasion, extent of metastasis to the lymph nodes, lymphatic invasion and the venous invasion of the colorectal wall). Sixty-eight patients with colorectal carcinoma who had undergone surgical resection in our department were studied. There was a significant (p < 0.01) elevation of OPRT activity in the tumor tissue compared with regions of normal tissue. OPRT activity levels in the tumor tissue were lowest in patients with mucinous carcinoma while TS enzyme levels showed the highest activity in tumor tissue in poorly differentiated adenocarcinoma. DPD also showed high activity levels in tumor tissue in poorly differentiated adenocarcinoma and mucinous carcinoma. It is possible that the expression of enzymes with respect to the antitumor effects of 5-FU is a factor contributing to the poor prognosis for patients with poorly differentiated adenocarcinoma and mucinous carcinoma. In the present study of clinical pathophysiologic characteristics, we found that metastasis to the lymph nodes was associated with a significant reduction in the OPRT tumor/normal (T/N) ratio. Our results indicate that it may be possible to predict lymphatic metastasis by determining the T/N ratio for OPRT before surgery.