Atsuya Habiro

Serine/Threonine Kinase AKT Is Frequently Activated in Human Bile Duct Cancer and Is Associated with Increased Radioresistance

The prognosis for patients with bile duct cancer (BDC) remains poor. Although BDC cells are essentially radioresistant, recent reports have suggested that radiation therapy, in addition to its palliative role in the management of BDC, may improve patient survival. A better understanding of the mechanisms that lead to cellular radioresistance may assist in the development of more effective BDC therapies based on radiotherapy in combination with radiosensitizing agents. The serine/threonine kinase AKT/protein kinase B, a downstream effector of phosphatidylinositol 3′-kinase, is a well-characterized kinase that is known to play a critical role in antiapoptotic signaling pathways. In this investigation, we sought to clarify the role of AKT signaling in the radioresistance in BDC cells. First, to examine whether activated AKT is expressed in BDCs, tumor specimens were obtained from 19 consecutive BDC cases. Immunohistochemical staining using an anti-phosphorylated-AKT antibody showed that phosphorylated (activated) AKT was expressed in cancer cells but not in neighboring normal mucosa in 16 cases (84.2%). Next, to evaluate the role of AKT activation in the regulation of BDC cell radiosensitivity, clonogenic assays were performed using the phosphatidylinositol 3′-kinase inhibitor LY294002 with and without irradiation. LY294002 inhibited AKT activation in BDC cells and, on irradiation, decreased clonogenic survival in a radiation dose-dependent manner. Only a small decrease in cell viability was observed in cells exposed to LY294002. Expression of constitutively active AKT in BDC cells resulted in decreased radiosensitivity, whereas a dominant-negative AKT increased radiosensitivity. Furthermore, constitutively active AKT also inhibited radiation-induced apoptosis. Collectively, these results indicate that activated AKT in BDC cells is associated with radioresistance and suggest that pharmacological or genetic modulation of AKT activity may have important therapeutic implications in BDC patients treated with radiation.

A phase I study of oral uracil-tegafur prior to weekly intravenous gemcitabine in patients with advanced pancreatic cancer

Background: Gemcitabine is widely accepted as the first-line agent for advanced pancreatic cancer. The antitumor cell activity of gemcitabine is higher when administered after 5-fluorouracil (5-FU) rather than before 5-FU in an in vitro study. The present study was conducted to define the maximum tolerated dose and dose-limiting toxicity associated with an oral fluoropyrimidine prodrug that combines uracil and tegafur (UFT), given prior to weekly intravenous gemcitabine in patients with advanced pancreatic cancer. Methods: Over a 21-day cycle, gemcitabine was given intravenously over 30 min on days 8 and 15, while UFT was given orally from days 1 to 14. The dose of UFT used was 400 mg per day, given as two doses. The dose of gemcitabine was escalated in a stepwise fashion from 800 (level 1, n = 3) to 900 mg/m2 (level 2, n = 6) and then to 1,000 mg/m2 (level 3, n = 3), such that totally 12 patients received the combination chemotherapy. Results: During the first cycle, grade 3 leukopenia was observed in 2 patients at dose level 1. Only 1 patient treated at dose level 2 experienced dose-limiting toxicity (grade 3 elevated transaminase), including additional patients treated at this dose level. No grade 3/4 toxicities occurred in patients treated at dose level 3. A significant response was observed in 1 out of 12 patients (8.3%). Seven patients (58.3%) had stable disease, while 4 patients (33.3%) showed disease progression. Conclusions: The combination chemotherapy of oral UFT and gemcitabine was convenient, well tolerated and may benefit patients with advanced pancreatic cancer. Doses recommended for further study of this schedule are gemcitabine 1,000 mg/m2 and UFT 400 mg/day.

Oleylamine, a long-chain fatty amine, induces autophagic degeneration in human pancreatic cancer cells

the cell cycle of the investigated cell lines. After 24 hours a marked accumulation of cells in the G2/M-phase and sub-Gl-phase could be observed after incubation with 0.6 ,~g/ml cbelidonine. G2/M (treated vs. control): AsPC1 69% vs. 24% (p<0.01), Pancl 33% vs. 26% (p<0.05), BxPC3 30% vs. 18% (p<0.05), MiaPaCa-2 48% vs. 20% (p<0.Ol). Sub-G1 (treated vs. control): AsPC1 13% vs. 3% (p<0.01), Pancl 20% vs. 3% (p<0.05), BxPC3 51% vs. 7% (p<0.01), MiaPaCa-2 38% vs 6% (p<0.01). Serial experiments on AsPC1 showed these effects already after 4 hour incubation. Subspecification of mitose phases by Giemsa stain revealed an accumulation of cells in prophase and metaphase following treatment by chelidonine at a concentration of 0.6/.~/ml. Analysis of cell cycle-associated proteins by Western blotting revealed a clear increase of cyclin B1 during the experiments starting after 16 hours pointing to a mitotic arrest in late G2/M-phase. A decrease of cyclin E was found at later points in time in consequence. CONCLUSION: Chelidonine leads to cell death by prophase and metaphase arrest in the investigated cell lines. This apoptotic potential in late G2/Mphase of chelidonine could mark this interesting substance as a potential new cytostatic drug for the treatment of pancreatic cancer.