WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma.

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12-Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.

TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death.

Self-aggregation of transforming growth factor ß (TGF-ß)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid ß (Aß) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aß in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aß aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aß. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aß formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.

Thyroid metastasis from intraductal papillary-mucinous carcinoma of the pancreas. A case report.

BACKGROUND: Intraductal papillary-mucinous carcinoma (IPMC) of the pancreas is a newly identified clinicopathologic entity of the exocrine pancreas. It has been considered a slowly growing and less-aggressive carcinoma with a favorable prognosis. There have been only a few documents reporting its distant metastasis and cytologic features, with no report of thyroid metastasis until the present. CASE: A case of IPMC occurred in a 45-year-old male, who was admitted with rapid growth and tenderness of the thyroid. Abdominal computed tomography showed the typical cystic dilatation of IPMC with adjacent organ metastasis. Fine needle aspiration of the thyroid yielded papillary fronds of carcinoma cells with nuclear pleomorphism, abundant cytoplasm and prominent nucleoli in a mucinous background. Immunohistochemical findings from the skin and thyroid characterized the papillary-mucinous carcinoma as having originated in the pancreas. CONCLUSION: This case suggests that papillary carcinoma fronds aspirated from the thyroid should be further differentiated from the primary site and that a pleomorphic nucleus in a mucinous background is a useful feature to exclude a thyroid origin. Before this, distant metastasis of IPMC to the skin and thyroid has not been reported. The prognosis of IPMC with wide, distant metastasis at an advanced stage is poor.

Neuronal and endothelial nitric oxide synthase expression in outer medulla of streptozotocin-induced diabetic rat kidney.

AIMS/HYPOTHESIS: Several investigations have shown that the renal medulla has a greater capacity to generate nitric oxide than the renal cortex. To further evaluate the changes of nitric oxide synthesis in the kidney, particularly in the outer medulla, in disorders involving fluid and electrolyte imbalances, we sought to determine renal nitric oxide synthase expression in the diabetic rats. METHODS: We determined renal nitric oxide synthase mRNA and urinary nitrite/nitrate excretion in 12 normal and 12 streptozotocin-induced diabetic rats by reverse transcription-polymerase chain reaction with Southern blot hybridization and with Griess reaction, respectively. Nitric oxide synthase immunoreactivity was detected by immunohistochemistry in four normal and four diabetic rats. RESULTS: Neuronal and endothelial nitric oxide synthase mRNA were 3.5-fold and 1.8-fold increased in the outer medulla of 12 diabetic rats with no difference found in the cortex and inner medulla when compared with 12 normal rats. Urinary nitrite/nitrate excretion was significantly increased from the first week after diabetic induction. In normal rats, immunohistochemical studies showed positive neuronal and endothelial nitric oxide synthase immunostaining in almost all segments of renal tubules. Diabetic rats had the greatest enhancement of immunostaining for neuronal and endothelial nitric oxide synthase in the proximal straight tubule and medullary thick ascending limb. CONCLUSION/INTERPRETATION: Our results indicate that increases in neuronal and endothelial nitric oxide synthase synthesis in the kidney, particularly in the outer medulla, possibly play an important part in the adaptation of renal function to hyperglycaemia and hyperosmolality in diabetes.

Up-regulation of adrenal cortical and medullary atrial natriuretic peptide and gene expression in rats with deoxycorticosterone acetate-salt treatment.

Our previous study demonstrated that human adrenal medulla is a site of atrial natriuretic peptide (ANP) synthesis. To further evaluate the role of adrenal ANP in body fluid homeostasis, we investigated the changes in adrenal ANP in rats receiving deoxycorticosterone acetate (DOCA)-salt treatment. In situ hybridization and immunohistochemical study showed that adrenal ANP messenger RNA (mRNA) and ANP-like immunoreactivities (ANP-LI) were mainly localized in the zona glomerulosa and medulla of vehicle-treated rats. DOCA-salt treatment activated ANP mRNA and peptide expression in all adrenal zones, especially in the zona fasciculata/reticularis from 12 h to the entire 8-day study period. Using a semiquantitative RT-PCR technique, the relative quantities of ANP mRNA in the adrenals of the DOCA-salt-treated group were significantly increased from 1 to 8 days, whereas the adrenal weights of DOCA-salt-treated rats were significantly decreased from day 2 to day 8. Our results are the first to indicate that ANP is synthesized not only in the adrenal medulla but also in the adrenal cortex and their syntheses are markedly increased in DOCA-salt-treated rats. These results imply that adrenal ANP may participate in the intraadrenal regulation of adrenal function on water-electrolyte homeostasis in an autocrine or paracrine manner.

Increased nitric oxide synthase mRNA expression in the renal medulla of water-deprived rats.

UNLABELLED: Increased nitric oxide synthase mRNA expression in the renal medulla of water-deprived rats. BACKGROUND: Experiments were performed to investigate whether renal nitric oxide synthase (NOS) mRNA and protein expression are responsive to the alteration of body volume. METHODS: Four days of water deprivation (WD) was initiated in 16 male Wistar rats, and 16 normal rats (NC) served as the control group. Neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) mRNAs and immunoreactivity were measured by reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot hybridization and immunohistochemistry, respectively. Plasma angiotensin II, vasopressin, and atrial natriuretic peptide (ANP) concentrations were measured by radioimmunoassay. RESULTS: The four-day WD increased plasma sodium and osmolality levels, but severely decreased daily urine sodium excretion and urine volume. Plasma angiotensin II and vasopressin concentrations were increased, but the plasma ANP level was significantly decreased in WD rats. nNOS, eNOS, and iNOS mRNA levels were increased by 5.2-, 3.3-, and 3. 4-fold in the outer medulla and 1.7-, 1.5-, and 1.8-fold in the inner medulla, whereas no significant difference was found in the renal cortex of WD rats as compared with NC rats. Additionally, immunohistochemistry revealed that the immunostaining intensity of nNOS, eNOS, and iNOS was clearly enhanced in the medullary thick ascending limb, proximal straight tubule, inner medullary collecting duct, and proximal convoluted tubule in WD rats. Kidney angiotensin II content as well as renin mRNA levels in renal cortex, outer medulla, and inner medulla in WD rats were apparently increased. CONCLUSIONS: Our results indicate that the increases of nNOS, eNOS, and iNOS synthesis in the kidney, particularly in the renal medulla, may have a role in the adaptation of renal function to volume depletion in the face of an increase of systemic and intrarenal vasoconstrictive substances.

Increased renal ANP synthesis, but decreased or unchanged cardiac ANP synthesis in water-deprived and salt-restricted rats.

BACKGROUND: Experiments were performed to examine the effect of water deprivation and salt restriction on ANP synthesis in the kidneys and hearts of normal rats. METHODS: A 4-day water deprivation (WD) and 7-day salt restriction (SR; 0.01% NaCl) were performed in 12 and 14 rats, respectively. Atrial natriuretic peptide (ANP) mRNA expression in the kidney was assessed with reverse transcription-polymerase chain reaction coupled with Southern blot hybridization, while the ANP mRNA in the hearts was measured by Northern blot hybridization. ANP and angiotensin II concentrations in the extracted plasma were measured by radioimmunoassay. The molecular form of renal ANP-like protein was characterized by reverse phase-high-performance liquid chromatography (RP-HPLC). RESULTS: Renal outer and inner medullary ANP mRNA showed a respective 11-fold and ninefold increase in WD rats, and an eightfold and fivefold increase in SR rats as compared to corresponding control groups. Inversely, cardiac atrial ANP mRNA and plasma ANP were decreased in WD rats, whereas they did not change in the SR group. Plasma angiotensin II concentration increased in conjunction with the decrease of urine sodium excretion in both groups. RP-HPLC analysis revealed a 45% extraction of ANP in the WD rat kidneys, whereas only 3% ANP in the control kidneys migrated in a molecular form similar to cardiac atrial proANP. CONCLUSIONS: Our results demonstrate that water deprivation and salt restriction markedly enhance renal ANP mRNA, whereas water deprivation suppresses cardiac atrial ANP mRNA and plasma ANP concentrations. The current study indicates that renal ANP and cardiac atrial ANP appear to be two distinct systems regulated by different mechanisms and possibly exhibiting different intra-renal paracrine and systemic endocrine functions.