Katsuhiko Sakaguchi

Activation of NF-κB by the RANKL/RANK system up-regulates snail and twist expressions and induces epithelial-to-mesenchymal transition in mammary tumor cell lines

BackgroundIncreased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT), which occurs during cancer progression and metastasis. Recent studies have indicated the expression of receptor activator of nuclear factor-κB (RANK) in various solid tumors, including breast cancer. Although activation of the RANK ligand (RANKL)/RANK system promotes cell migration, metastasis, and anchorage-independent growth of tumor-initiating cells, it remains to be investigated if RANKL induces EMT in breast cancer cells. In this study, we investigated whether RANKL induces EMT in normal breast mammary epithelial cells and breast cancer cells, and the mechanism underlying such induction.MethodsExpression levels of vimentin, N-cadherin, E-cadherin, Snail, Slug, and Twist were examined by real-time polymerase chain reaction. Cell migration and invasion were assessed using Boyden chamber and invasion assays, respectively. The effects of RANKL on signal transduction molecules were determined by western blot analyses.ResultsWe found that stimulation by RANKL altered the cell morphology to the mesenchymal phenotype in normal breast epithelial and breast cancer cells. In addition, RANKL increased the expression levels of vimentin, N-cadherin, Snail, and Twist and decreased the expression of E-cadherin. We also found that RANKL activated nuclear factor-κB (NF-κB), but not extracellular signal-regulated kinase 1/2, Akt, mammalian target of rapamycin, c-Jun N-terminal kinase, and signal transducer and activator of transcription 3. Moreover, dimethyl fumarate, a NF-κB inhibitor, inhibited RANKL-induced EMT, cell migration, and invasion, and upregulated the expressions of Snail, Twist, vimentin, and N-cadherin.ConclusionsThe results indicate that RANKL induces EMT by activating the NF-κB pathway and enhancing Snail and Twist expression. These findings suggest that the RANKL/RANK system promotes tumor cell migration, invasion, and metastasis via the induction of EMT.

Treatment of Severe Hypothyroidism Reduced Serum Creatinine Levels in Two Chronic Renal Failure Patients

Short-term hypothyroidism has been associated with a reversible rise in serum creatinine levels in patients with normal renal function. A remarkable decline in serum creatinine levels associated with a treatment of severe and prolonged hypothyroidism has rarely been reported so far. We present here 2 patients with chronic renal failure in whom treatment for hypothyroidism resulted in a significant and sustained reduction of their serum creatinine levels. These cases indicate that because hypothyroidism may aggravate the serum creatinine levels, TSH should be considered in screening procedures of patients with chronic renal failure presenting with recent accelerated aggravation of renal function. Hypothyroidism per se, one of its complications or one of its associated autoimmune diseases might play a role in modifying the underlying renal problem.

Association of adiponectin with blood pressure in healthy people

With the increasing prevalence of diseases related to obesity, metabolic syndrome and its key player adiponectin are now attracting considerable attention. Hypoadiponectinaemia is reported to be a risk factor for hypertension and associated with endothelial dysfunction, which is closely related to complications of obesity such as hypertension. As there is limited information regarding serum adiponectin levels in normotensive people, we undertook the large‐scale study to determine the association of adiponectin with blood pressure (BP) in mainly normotensive people.

By inhibiting Src, verapamil and dasatinib overcome multidrug resistance via increased expression of Bim and decreased expressions of MDR1 and survivin in human multidrug-resistant myeloma cells

The calcium channel blocker verapamil inhibits the transport function of multidrug resistance protein 1 (MDR1). Although verapamil acts to reverse MDR in cancer cells, the underlying mechanism remains unclear. In the present study, we investigated the mechanism of reversing MDR by verapamil in anti-cancer drug-resistant multiple myeloma (MM) cell lines. We found that verapamil suppresses MDR1 and survivin expressions and increases Bim expression via suppression of Src activation. Furthermore, dasatinib reversed the drug-resistance of the drug-resistant cell lines. These findings suggest that Src inhibitors are potentially useful as an anti-MDR agent for the treatment of malignant tumor cells.

Inhibition of the tumour necrosis factor-alpha autocrine loop enhances the sensitivity of multiple myeloma cells to anticancer drugs

Several autocrine soluble factors, including macrophage inflammatory protein-1α and tumour necrosis factor-alpha (TNF-α), promote the survival and growth of multiple myeloma (MM) cells. We hypothesised that inhibition of the TNF-α autocrine loop may enhance the cytotoxic effect of anticancer drugs in MM cell lines. In the present study, a TNF-α-neutralizing antibody suppressed cell proliferation and enhanced the cytotoxic effect of anticancer drugs on MM cells. In addition, combination treatment with the TNF-α-neutralizing antibody and the chemotherapy agent melphalan inhibited nuclear factor κB (NF-κB) p65 nuclear translocation and mammalian target of rapamycin (mTOR) activation and upregulated the expression of Bax and Bim. Treatment of ARH-77 cells with the NF-κB inhibitor dimethyl fumarate or the mTOR inhibitor rapamycin suppressed NF-κB p65 nuclear translocation and enhanced the cytotoxic effect of melphalan. Furthermore, infliximab, a monoclonal antibody against TNF-α, also enhanced the cytotoxic effect of anticancer drugs in ARH-77 cells. These results indicated that TNF-α-neutralizing antibodies or infliximab enhanced the cytotoxic effect of anticancer drugs by suppressing the TNF receptor/mTOR/NF-κB pathways. The inhibition of TNF-α may thus provide a new therapeutic approach to control tumour progression and bone destruction in MM patients.

Nitrogen-containing bisphosphonates inhibit RANKL- and M-CSF-induced osteoclast formation through the inhibition of ERK1/2 and Akt activation

BackgroundBisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7.ResultsIt was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF-κB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-κB ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation.ConclusionsThis indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.

下垂体への小転移を伴う胃癌原発の髄膜癌腫症に cerebral salt wasting syndrome が続発した1例

A 68-year-old man with disturbed consciousness had repeatedly developed light-headedness and dizziness since the summer of 1996 and was admitted to a hospital for detailed examinations on October 8, 1996. On admission, he weighed 49 kg and showed subclinical hypothyroidism with low T3 syndrome. The adrenal function and serum electrolytes were normal. Since the stool samples were positive for occult blood, gastroscopy was performed. Examination of the biopsy specimens demonstrated gastric cancer. On October 21, blood examination showed hyponatremia (127 mEq/l). On October 22, marked disturbance of consciousness developed. On October 24, the serum Na level further decreased to 116 mEq/l. On November 8, he was referred to our hospital. On admission, his skin and tongue showed marked dehydration, and severe disturbance of consciousness and neck stiffness were observed. The central venous pressure was 4 cmH2O. In the cerebrospinal fluid, atypical cells were observed, and a diagnosis of meningeal carcinomatosis was made. Syndrome of inappropriate secretion of antidiuretic hormone (SIADH) was excluded because of marked dehydration, a normal blood ADH level, and because plasma osmotic pressure was greater than urinary osmotic pressure. Considering the possibility of cerebral salt wasting syndrome (CSWS) or hypoadrenocorticism, Na supplementation and drip infusion of prednisolone (20 mg/day) were performed. The serum Na has normalized (140.1 mEq/l), and his consciousness improved. He died of aggravation of the general condition on December 16. Pathological examination demonstrated a small metastatic lesion in the infundibular part of the pituitary gland and a small metastatic lesion in the parenchyma of the bilateral adrenal glands. However, since neither hypotension nor hypoglycemia was observed before treatment, and the blood cortisol level and the serum K level were normal, hypoadrenocorticism was excluded. Hypoaldosteronism was also excluded because of a normal serum K level. CSWS has been reported to be caused by head trauma, subarachnoid hemorrhage, or trans-sphenoidal pituitary operation. This patient is a rare case of CSWS developed in the presence of meningeal carcinomatosis accompanied by a small pituitary metastatic lesion from gastric cancer. The aged with decreased ability to retain water and sodium in the body are more susceptible to CSWS than the young. In the aged with central hyponatremia, the possibility of CSWS should be considered, and early diagnosis and treatment are necessary.

Oncogenic osteomalacia secondary to nasal tumor with decreased urinary excretion of cAMP

A 53-year-old woman visited our hospital on June 8, 1999, for examination of a nasal tumor. She had suffered from pentalgia since 1984, and was revealed to have multiple bone fractures, including the ischial bone, pubic bone, right 9th and 10th, and left 10th ribs, on Xray examinations by her family doctor in 1985. She was referred to Sumitomo Hospital in 1987, where laboratory examinations revealed severe hypophosphatemia (0.61 mmol/L) and decreased serum levels of 1,25(OH)2D (36 pmol/L), although serum levels of calcium (2.10 mmol/L), PTH (3.21pmol/L), 25-hydroxyvitamin D (25-OHD; 32 nmol/L), and 24,25-dihydroxyvitamin D (24,25-(OH)2D; 4.8nmol/L) were within the normal ranges. Oral administration of 1α-hydroxyvitamin D3 (1α-OHD3) at a dose of 8–14 μg/day was required to normalize serum levels of inorganic phosphate. With normalization of serum levels of phosphate, pentalgia was ameliorated, and she required the active form of vitamin D for the next several years until admission to our hospital. General survey for a possible tumor was performed by computed tomography (CT) under the suspected diagnosis of oncogenic osteomalacia in 1994, but CT failed to detect any tumor at that time. Nasal bleeding developed in 1997 and recurred, but she did not report it to her doctor. After an episode of massive nasal bleeding, magnetic resonance imaging examination in May 1999 revealed a mass extending from the right superior nasal concha to the nasal cavity (Fig. 1). She did not have any past history of malabsorption. There was no family history of rickets or hypophosphatemia, either. On admission, the patient was 1.56 m in height and 58 kg in weight. Blood pressure and pulse rate were 164/82mmHg and 78/min, respectively. Dual

Endogenous platelet-activating factor and anti-platelet-activating factor in patients with renovascular hypertension

Renovascular hypertension is relieved by percutaneous transluminal renal angioplasty. In four patients with renovascular hypertension, platelet-activating factor (PAF) was found to be released into the ipsilateral renal venous blood after percutaneous transluminal renal angioplasty, but was not found in the contralateral renal venous blood following this procedure. Anti-platelet-activating factor with a lipid-like property was also found, and its polarity was slightly lower than that of PAF judging by its behavior on thin layer chromatography. Anti-platelet-activating factor completely blocked the aggregation of rabbit platelets induced by PAF, ADP or arachidonic acid. These results indicate that PAF and anti-platelet-activating factor are released into renal venous blood following percutaneous transluminal renal angioplasty in patients with renovascular hypertension.

Partial purification and properties of a plasma ouabain-like inhibitor of Na+, K+-ATPase in patients with essential hypertension.

Plasma levels of an ouabain-like inhibitor of Na+,K+-ATPase were higher in patients with essential hypertension compared with normal levels. The ouabain-like inhibitor was correlated significantly with blood pressure and was increased by a high-salt diet. The substance was partially purified by high performance liquid chromatography which revealed lipid-like properties, but the elution time was different from that of free unsaturated fatty acid on silica-gel high performance liquid chromatography. Its molecular weight was 600 or less, as estimated by high performance liquid chromatography with an HSG-15H column. The ouabain-like substance inhibited Na+,K+-ATPase in competition with KCI and showed positive ouabain-like immunoreactivity, whereas lysophosphatidylcholine was a non-competitive inhibitor. The ouabain-like substance was unstable at room temperature and decomposed to smaller molecular compounds which did not inhibit Na+,K+-ATPase. The inhibitory fraction gave a positive thiobarbituric acid reaction test. The mobility of the ouabain-like inhibitor on silica-gel thin-layer chromatography was different from that of prostaglandins and arachidonic acid. These results indicate that the plasma ouabain-like inhibitor of patients with essential hypertension is a lipid which is different from free fatty acid or lysophosphatidylcholine, and may be an unstable peroxide.