Studies on molecular mechanism of tumor suppression by the drs gene

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2001～2002課題番号: 13670211研究代表者: 井上 寛一（滋賀医科大学・医学部・助教授）研究協力者: 原口 清輝（滋賀医科大学・医学部・助手）研究協力者: 旦部 幸博（滋賀医科大学・医学部・助手

Cryptotanshinone, a novel PDK 4 inhibitor, suppresses bladder cancer cell invasiveness via the mTOR/β‑catenin/N‑cadherin axis.

The phosphorylation of pyruvate dehydrogenase (PDH) by pyruvate dehydrogenase kinase (PDK) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is upregulated in various types of human cancer. Because PDK4 regulation is critical for metabolic changes in cancer cells, it is an attractive target for cancer therapy given its ability to shift glucose metabolism. It was previously shown that a novel PDK4 inhibitor, cryptotanshinone (CPT), suppressed the three‑dimensional (3D)‑spheroid formation of pancreatic and colorectal cancer cells. In the present study, the effects of CPT on the invasiveness of bladder cancer cells were investigated. CPT significantly suppressed the invasiveness and 3D‑spheroid formation of T24 and J82 bladder cancer cells. CPT also suppressed the phosphorylation of PDH and β‑catenin, as well as the expression of N‑cadherin, which are all critical for inducing epithelial‑mesenchymal transition (EMT). The knockdown of β‑catenin or PDK4 using specific small interfering RNAs suppressed N‑cadherin expression and invasiveness in T24 cells. An mTOR inhibitor also suppressed the phosphorylation of β‑catenin and N‑cadherin expression. Furthermore, CPT injection significantly suppressed pancreatic tumor growth and peritoneal dissemination of highly metastatic SUIT‑2 pancreatic cancer cells in a mouse orthotopic pancreatic cancer model, without evident toxicity. Moreover, immunohistochemistry analyses demonstrated decreased β‑catenin expression in CPT‑treated pancreatic tumors compared with control tumors. Taken together, these results indicate that CPT reduced the invasiveness and metastasis of bladder cancer cells by suppressing EMT via the mTOR/β‑catenin/N‑cadherin pathway

Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS‑activated pancreatic cancer cells

Pyruvate dehydrogenase kinase 4 (PDK4) is an important regulator of energy metabolism. Previously, knockdown of PDK4 by specific small interfering RNAs (siRNAs) have been shown to suppress the expression of Κirsten rat sarcoma viral oncogene homolog (KRAS) and the growth of lung and colorectal cancer cells, indicating that PDK4 is an attractive target of cancer therapy by altering energy metabolism. The authors previously reported that a novel small molecule, cryptotanshinone (CPT), which inhibits PDK4 activity, suppresses the in vitro three‑dimensional (3D)‑spheroid formation and in vivo tumorigenesis of KRAS‑activated human pancreatic and colorectal cancer cells. The present study investigated the molecular mechanism of CPT‑induced tumor suppression via alteration of glutamine and lipid metabolism in human pancreatic and colon cancer cell lines with mutant and wild‑type KRAS. The antitumor effect of CPT was more pronounced in the cancer cells containing mutant KRAS compared with those containing wild‑type KRAS. CPT treatment decreased glutamine and lipid metabolism, affected redox regulation and increased reactive oxygen species (ROS) production in the pancreatic cancer cell line MIAPaCa‑2 containing mutant KRAS. Suppression of activated KRAS by specific siRNAs decreased 3D‑spheroid formation, the expression of acetyl‑CoA carboxylase 1 and fatty acid synthase (FASN) and lipid synthesis. The suppression also reduced glutathione‑SH/glutathione disulfide and increased the production of ROS. Knockdown of FASN suppressed lipid synthesis in MIAPaCa‑2 cells, partially promoted ROS production and mildly suppressed 3D‑spheroid formation. These results indicated that CPT reduced tumorigenesis by inhibiting lipid metabolism and promoting ROS production in a mutant KRAS‑dependent manner. This PDK4 inhibitor could serve as a novel therapeutic drug for KRAS‑driven intractable cancers via alteration of cell metabolism

Role of drs tumor suppressor gene on host defense mechanism against infection

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2008～2010課題番号: 20590383研究代表者: 旦部 幸博（滋賀医科大学・医学部・助教

Regulation of metabolic shift by drs tumor suppressor gene

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2011～2013課題番号: 23590457研究代表者: 旦部 幸博（滋賀医科大学・医学部・助教

Suppression of Viral Replication by Stress-Inducible GADD34 Protein via the Mammalian Serine/Threonine Protein Kinase mTOR Pathway▿

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2α nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense