Inhibition of Rho-associated coiled-coil containing protein kinase enhances the activation of epidermal growth factor receptor in pancreatic cancer cells

<p>Abstract</p> <p>Background</p> <p>Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1.</p> <p>Results</p> <p>In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.</p> <p>Conclusions</p> <p>While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.</p

骨芽細胞系列細胞のビタミンD受容体は、in vivo において1α,25(OH)2D3の骨吸収促進活性に必須である

要旨我々は以前、活性型ビタミンD3 [1α,25(OH)2D3]製剤であるエルデカルシトールの薬用量の投与は、骨吸収を抑制することで、骨量を増加させることを報告した。この骨吸収抑制効果は、骨芽細胞系列細胞のビタミンD受容体（VDR）を介することを明らかにした。本研究において我々は、骨芽細胞系列細胞特異的VDR欠損（Ob-VDR-cKO）マウスを用いて、1α,25(OH)2D3の大量投与によって誘導される骨吸収促進が骨芽細胞系列細胞のVDRを介するか否かを調べた。4日間の野生型マウスへの1α,25(OH)2D3（5 µg/kg体重/日）投与は、骨における破骨細胞数を増加させ、骨吸収マーカーであるI型コラーゲンC末端テロペプチド（C-terminal crosslinked telopeptide of type I collagen, CTX-I）の血清濃度を上昇させた。骨吸収促進は、血清カルシウム（Ca）値、線維芽細胞増殖因子23（Fibroblast Growth Factor, FGF-23）値の上昇と、体重の減少を伴っていた。このことは、中毒量の1α,25(OH)2D3は、骨吸収促進と高Ca血症を誘導することを示している。対照的に、野生型マウスへの抗Receptor Activator of NF-κB Ligand（RANKL）中和抗体前投与は、1α,25(OH)2D3が誘導する血清CTX-I, CaおよびFGF23値の上昇を抑制した。また、抗RANKL中和抗体前投与は、1α,25(OH)2D3が誘導する体重減少を抑制した。抗RANKL中和抗体前投与マウスの所見と一致して、1α,25(OH)2D3をOb-VDR-cKOマウスに大量投与しても、破骨細胞数、血清CTX-I値、血清Ca値、血清FGF-23値は、有意に上昇せず、また体重も減少しなかった。以上、本研究において、1α,25(OH)2D3の大量投与による骨吸収促進、血清Ca値上昇および毒性作用は、骨芽細胞系列細胞のVDRを介して発揮されることを明らかにした。2020博士（歯学）松本歯科大

C57BL/KsJ-db/db-ApcMin/+ Mice Exhibit an Increased Incidence of Intestinal Neoplasms

The numbers of obese people and diabetic patients are ever increasing. Obesity and diabetes are high-risk conditions for chronic diseases, including certain types of cancer, such as colorectal cancer (CRC). The aim of this study was to develop a novel animal model in order to clarify the pathobiology of CRC development in obese and diabetic patients. We developed an animal model of obesity and colorectal cancer by breeding the C57BL/KsJ-db/db (db/db) mouse, an animal model of obesity and type II diabetes, and the C57BL/6J-ApcMin/+ (Min/+) mouse, a model of familial adenomatous polyposis. At 15 weeks of age, the N9 backcross generation of C57BL/KsJ-db/db-ApcMin/+ (db/db-Min/+) mice developed an increased incidence and multiplicity of adenomas in the intestinal tract when compared to the db/m-Min/+ and m/m-Min/+ mice. Blood biochemical profile showed significant increases in insulin (8.3-fold to 11.7-fold), cholesterol (1.2-fold to 1.7-fold), and triglyceride (1.2-fold to 1.3-fold) in the db/db-Min/+ mice, when compared to those of the db/m-Min/+ and m/m-Min/+ mice. Increases (1.4-fold to 2.6-fold) in RNA levels of insulin-like growth factor (IGF)-1, IRF-1R, and IGF-2 were also observed in the db/db- Min/+ mice. These results suggested that the IGFs, as well as hyperlipidemia and hyperinsulinemia, promoted adenoma formation in the db/db-Min/+ mice. Our results thus suggested that the db/db-Min/+ mice should be invaluable for studies on the pathogenesis of CRC in obese and diabetes patients and the therapy and prevention of CRC in these patients

Nutraceutical Approach for Preventing Obesity-Related Colorectal and Liver Carcinogenesis

Obesity and its related metabolic abnormalities, including insulin resistance, alterations in the insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) axis, and the state of chronic inflammation, increase the risk of colorectal cancer (CRC) and hepatocellular carcinoma (HCC). However, these findings also indicate that the metabolic disorders caused by obesity might be effective targets to prevent the development of CRC and HCC in obese individuals. Green tea catechins (GTCs) possess anticancer and chemopreventive properties against cancer in various organs, including the colorectum and liver. GTCs have also been known to exert anti-obesity, antidiabetic, and anti-inflammatory effects, indicating that GTCs might be useful for the prevention of obesity-associated colorectal and liver carcinogenesis. Further, branched-chain amino acids (BCAA), which improve protein malnutrition and prevent progressive hepatic failure in patients with chronic liver diseases, might be also effective for the suppression of obesity-related carcinogenesis because oral supplementation with BCAA reduces the risk of HCC in obese cirrhotic patients. BCAA shows these beneficial effects because they can improve insulin resistance. Here, we review the detailed relationship between metabolic abnormalities and the development of CRC and HCC. We also review evidence, especially that based on our basic and clinical research using GTCs and BCAA, which indicates that targeting metabolic abnormalities by either pharmaceutical or nutritional intervention may be an effective strategy to prevent the development of CRC and HCC in obese individuals

Pitavastatin suppresses diethylnitrosamine-induced liver preneoplasms in male C57BL/KsJ-db/db obese mice

<p>Abstract</p> <p>Background</p> <p>Obesity and related metabolic abnormalities, including inflammation and lipid accumulation in the liver, play a role in liver carcinogenesis. Adipocytokine imbalances, such as decreased serum adiponectin levels, are also involved in obesity-related liver tumorigenesis. In the present study, we examined the effects of pitavastatin - a drug used for the treatment of hyperlipidemia - on the development of diethylnitrosamine (DEN)-induced liver preneoplastic lesions in C57BL/KsJ-<it>db/db </it>(<it>db/db</it>) obese mice.</p> <p>Methods</p> <p>Male <it>db/db </it>mice were administered tap water containing 40 ppm DEN for 2 weeks and were subsequently fed a diet containing 1 ppm or 10 ppm pitavastatin for 14 weeks.</p> <p>Results</p> <p>At sacrifice, feeding with 10 ppm pitavastatin significantly inhibited the development of hepatic premalignant lesions, foci of cellular alteration, as compared to that in the untreated group by inducing apoptosis, but inhibiting cell proliferation. Pitavastatin improved liver steatosis and activated the AMPK-α protein in the liver. It also decreased free fatty acid and aminotransferases levels, while increasing adiponectin levels in the serum. The serum levels of tumor necrosis factor (TNF)-α and the expression of <it>TNF-α </it>and <it>interleukin-6 </it>mRNAs in the liver were decreased by pitavastatin treatment, suggesting attenuation of the chronic inflammation induced by excess fat deposition.</p> <p>Conclusions</p> <p>Pitavastatin is effective in inhibiting the early phase of obesity-related liver tumorigenesis and, therefore, may be useful in the chemoprevention of liver cancer in obese individuals.</p

Identification of mTEC precursor cells

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs