Makoto Usami

Accumulating evidence has demonstrated platinum-based chemotherapy followed by maintenance therapy with a poly Adenosine diphosphate (ADP)-ribose polymerase inhibitor (olaparib) show benefits in unresectable pancreatic cancer with a germline (g)BRCA1/2 mutation. Evaluation of the germline BRCA1 and BRCA2 mutation is essential for making decisions on a treatment strategy for patients with unresectable pancreatic cancer. However, the detection rates of germline BRCA1 and BRCA2 mutations and efficacy of maintenance with olaparib remain undetermined, prospectively, in Japan. In this prospective analysis, the rate of germline BRCA1 and BRCA2 mutations and efficacy of chemotherapy were analyzed in 136 patients with pancreatic cancer who underwent BRACAnalysis® (85 patients) or FoundationOne® CDx (51 patients) between January 2020 and July 2022. A total of six patients (4.4%) had a germline BRCA1 and BRCA2 mutation. Five patients were treated with modified FOLFIRINOX and one with fluorouracil and oxaliplatin. All patients continued platinum-based chemotherapy for ˃4 months and were subsequently treated with olaparib as a maintenance therapy. The response rate to platinum-based chemotherapy in the germline BRCA1 and BRCA2 mutation-positive group was significantly better than that of the germline BRCA1 and BRCA2 mutation-negative group (66% vs 23%, P = 0.04). All patients harbouring a germline BRCA1 and BRCA2 mutation were able to switch to olaparib. The median progression-free survival using olaparib was 5.7 months (range 3.0–9.2). The rate of germline BRCA1 and BRCA2 mutations found in patients with unresectable pancreatic cancer was comparable to those of previous studies. An analysis of germline BRCA1 and BRCA2 mutations has benefits for all patients with unresectable pancreatic cancer with regard to decisions on therapeutic strategies in a clinical practice setting.

MRNA expression of molecules related to the activity of nitric oxide or prostaglandin E2, the critical regulators maintaining the ductus arteriosus patency, was examined in rat ductus arteriosus at preterm (days 18.5 and 19.5 of pregnancy) and near term (days 20.5 and 21.5). The endothelial nitric oxide synthase mRNA level increased transiently at preterm and then decreased at near term. The cyclooxygenase 2 mRNA increased gradually from near-term to the term complementary to the reduced endothelial nitric oxide synthase mRNA. These results suggest that the role shift between nitric oxide and prostaglandin E2 in maintaining ductus arteriosus patency at preterm and near term may be due to complementary expression changes of endothelial nitric oxide synthase and cyclooxygenase 2 at the transcriptional level.

The metabolism of 4-methyl-2-mercaptobenzimidazole (4-MeMBI), 5-methyl-2-mercaptobenzimidazole (5-MeMBI), and 2-mercaptobenzimidazole (MBI) was examined in vitro in rat liver microsomes. The test chemicals were incubated in the presence of liver microsomes from male Sprague-Dawley rats, and their metabolism was analyzed by HPLC. The metabolism amount increased in an incubation time-dependent manner, and was similar among the test chemicals. SKF-525A, a non-selective inhibitor of cytochrome P450 (CYP) enzymes, decreased the metabolic rate of all the test chemicals, indicating the involvement of liver microsomal CYP enzymes. When liver microsomes from rats treated with CYP-inducers (β-naphthoflavone, phenobarbital, and isoniazid) were used, 4-MeMBI was more decreased than 5-MeMBI, particularly in the phenobarbital-treated group. These results, together with the reported inducibility of the drug-metabolizing activity by the test chemicals, partly explained the counteraction in the toxic effects between 4-MeMBI and 5-MeMBI in the in vivo study.

Protein expression changes were examined in day 10.5 rat embryos cultured for 24 hr in the presence of valproic acid (VPA), using two-dimensional electrophoresis and mass spectrometry. Exposure to VPA at an embryotoxic concentration of 1.2 mM resulted in quantitative changes in many embryonic protein spots (22 decreased and 29 increased). For the increased protein spots, 10 proteins were identified, including alpha-fetoprotein, phosphorylated cofilin-1, and serum albumin. These proteins are candidate protein biomarkers that may be involved in embryotoxic mechanisms.

The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13‐cis‐retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all-trans-retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.