Pancreatic adenocarcinoma cell lines were treated with Agaricus blazei Murrill hot water extract (AbE).

Pancreatic cancer is one of the most aggressive human malignancies with 7.1% five-year survival rate due to late-stage diagnosis and lack of effective treatment. A novel active compound to be useful for treating pancreatic cancer is absolutely demanded. Agaricus blazei (A. blazei) Murrill has been shown to have an anti-cancer activity, however, its specific efficacy and underlying mechanism is poorly understood. Here, we demonstrated that A. blazei hot water extract (AbE) showed significant growth inhibition of cultured pancreatic cancer cells with less suppressive effect on the normal human pancreatic duct epithelial cells. The pancreatic cancer cells committed G0/G1 cell cycle arrest and caspase-dependent apoptosis. Moreover, we uncovered significant alterations of global gene expression in the pancreatic cancer cells by AbE treatment. Signature of alteration of gene expression indicated that AbE induced repression of genes associated with kinetochore function, spindle formation, centromere maintenance, and cyclins and cyclin dependent kinases, which were essential for cell cycle progression. The gene expression analysis also showed upregulation of proapoptotic genes. These results indicate that AbE could be useful for treatment of pancreatic cancer and a good candidate to isolate potent active compound for development of novel drug for pancreatic cancer. Cells of a human pancreatic adenocarcinoma cell line were seeded at 250,000 cells/10 cm dish with an appropriate growth medium. After 24 hours of the seeding, the growth medium was replaced with the growth medium with 0.045 % Agaricus blazei Murrill hot water extract (AbE) as treated or the growth medium with water in the same volume of AbE as control (mock). Adherent cells were collected after 48 hours of the treatment. Total RNAs were extracted from both control and treated cells using RNeasy midi kit (QIAGEN, Hilden, Germany) according to the supplier’s instructions. cRNAs labelled with Cyanin 3 were synthesized by means of the T7-linear amplification method from 500 ng of the total RNAs employing Low RNA Input Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, U.S.A.) and purified using RNeasy mini kit (QIAGEN). For each hybridization, 1.65 µg cRNAs were fragmented at 60 ºC for 30 minutes using Gene Expression Hybridization Kit (Agilent Technologies) and hybridized at 65 ºC for 17 hours to an 4×44K Whole Human Genome Microarray slide (Agilent Technologies) in accordance with the manufacture’s instructions. Miroarrays were scanned, and signals in the scanned image were converted to intensity values that were subsequently normalized by Hokkaido System Science Co., Ltd. (Sapporo, Japan). Cell lines employed for this study were three human pancreatic cancer cell lines, MIA PaCa-2, PCI-35, and PK-8. The experiment was performed using the three cell lines and independently triplicated in each cell line. Correlation coefficients were referred for quality control, and one of AbE treated PK-8 was excluded because its correlation coefficient to the other replicates was below 0.01.