All-trans-retinoic Acid-dependent Inhibition of E-Cadherin-based Cell Adhesion with Concomitant Dephosphorylation of β-Catenin in Metastatic Human Renal Carcinoma Cells

We previously described an in vitro invasion assay model, using a monolayer of vascular endothelial cells grown on collagen gel, that mimics the metastatic abilities of the highly metastatic human renal carcinoma cell lines, MM-1,3 and 8 and their poorly metastatic counterparts, SN12C and Q-8. MM-1, 3 and 8 cells were observed to penetrate the monolayer of vascular endothelial cells and grew in a spreading or scattering manner with loose cell-cell contact on collagen gel or on vascular endothelial cells. SN12C and Cl-8 cells failed to penetrate and grew in a clustering manner with tight cell-cell contact. Treatment with all- trans -retinoic acid (ATRA) at non-toxic concentrations induced clustering or growth of MM-1, 3 and 8 cells on collagen gel or on vascular endothelial cells with tight cell-cell contact, and inhibited penetration. The clustering induced by ATRA was virtually blocked in the presence of anti-E cadherin antibody. E-Cadherin and β -catenin were each localized mainly at the cell-cell adherent junctions of colonizing cell populations that had been treated with ATRA. While the cellular levels of E-cadherin and β -catenin did not change significantly following ATRA treatment, the tyrosine residue of β -catenin was rapidly dephosphorylated. The concomitant administration of Na vanadate, an inhibitor of tyrosine dephosphorylase, inhibited both the ATRA-induced clustering and the dephosphorylation of β -catenin tyrosine. ATRA-induced clustering of MM-3 cells may be linked to the state of tyrosine phosphorylation of β -catenin.     

The inhibition of RANKL/RANK signaling by osteoprotegerin suppresses bone invasion by oral squamous cell carcinoma cells

Oral squamous cell carcinomas (OSCCs) are malignant tumors that frequently invade the maxilla and mandibular bone. However, the molecular mechanisms underlying bone invasion by OSCC are unclear. Recent studies showed that receptor activator of nuclear factor κB (RANK) was expressed not only in osteoclast precursors but also in tumor cells. Therefore, we examined whether RANK ligand (RANKL)/RANK signaling regulates bone invasion by OSCC cells in vivo and in vitro. We first injected human OSCC B88 cells into the masseter region of nude mice. Mice were treated for 3 weeks with osteoprotegerin (OPG), the decoy receptor for RANKL. Treatment with OPG decreased bone invasion by B88 cells, reduced the number of osteoclasts and increased B88 cell apoptosis. However, OPG did not affect apoptosis and proliferation in B88 cells in vitro, suggesting that the effects of OPG on apoptosis in B88 cells are restricted in a bone environment. RANK was expressed in the B88 cells and in OSCC cells from patients. RANKL induced NF-κB activation and extracellular signal-regulated kinase phosphorylation in B88 cells and enhanced B88 cell migration in a modified chemotaxis chamber equipped with a gelatin-coated filter. OPG inhibited RANKL-induced NF-κB activation, extracellular signal-regulated kinase phosphorylation and cell migration. Our data clearly indicate that RANKL/RANK inhibition suppresses bone invasion by inhibiting osteoclastogenesis and cancer cell migration and by inducing apoptosis of cancer cells via indirect anticancer action in vivo.     

Selective inhibition of NF-κB suppresses bone invasion by oral squamous cell carcinoma in vivo

Nuclear factor-κB (NF-κB) is constitutively activated in many cancers, including oral squamous cell carcinoma (OSCC), and is involved in the invasive characteristics of OSCC, such as growth, antiapoptotic activity and protease production. However, the cellular mechanism underlying NF-κB's promotion of bone invasion by OSCC is unclear. Therefore, we investigated the role of NF-κB in bone invasion by OSCC in vivo. Immunohistochemical staining of OSCC invading bone in 10 patients indicated that the expression and nuclear translocation of p65, a main subunit of NF-κB, was increased in OSCC compared with normal squamous epithelial cells. An active form of p65 phosphorylated at serine 536 was present mainly in the nucleus in not only differentiated tumor cells but also tumor-associated stromal cells and bone-resorbing osteoclasts. We next injected mouse OSCC SCCVII cells into the masseter region of C(3) H/HeN mice. Mice were treated for 3 weeks with a selective NF-κB inhibitor, NBD peptide, which disrupts the association of NF-κB essential modulator (NEMO) with IκB kinases. NBD peptide treatment inhibited TNFα-induced and constitutive NF-κB activation in SCCVII cells in vitro and in vivo, respectively. Treatment with NBD peptide decreased zygoma and mandible destruction by SCCVII cells, reduced number of osteoclasts by inhibiting RANKL expression in osteoblastic cells and SCCVII cells, increased apoptosis and suppressed the proliferation of SCCVII cells. Taken together, our data clearly indicate that inhibition of NF-κB is useful for inhibiting bone invasion by OSCC.     

Immunogold and freeze etch electron microscopic studies of merosin localization in basal lamina of human skeletal muscle fibers

Merosin is a basement-membrane-associated protein found in striated muscle, peripheral nerve and placenta, the deficiency of which causes the muscle wasting condition in C57BL/6J-dy/dy, so-called dy/dy mouse. Moreover, merosin is the binding protein of 156 kDa α-dystroglycan which binds dystrophin by way of 43 kDa β-dystroglycan. Therefore, merosin is an important component of the basal lamina of normal skeletal myofibers. We investigated the ultrastructural localization of merosin antibody in normal human skeletal myofibers by using immunogold electron microscopy and freeze etch electron microscopy. The ultrastructure of the basal lamina showed the presence of the lamina lucida, lamina densa and lamina reticularis. The lamina lucida appeared electron translucent with the exception of fuzzy fibrils. The immunogold electron microscopy disclosed that the merosin was present at the innermost layer (lamina lucida) of the basal lamina of normal human skeletal myofibers. With freeze etch replica electron microscopy, short cross-bridge fine fibrils were noted in the lamina lucida, connecting the basal lamina to the outer leaflet of the muscle plasma membrane. They measured 3–13 nm in diameter, 20–90 nm in length and were distributed with a spacing of 30–40 nm. The immunogold particles showing the presence of the merosin epitope were associated with these connecting structures. Received: 3 April 1996 / Revised, accepted: 15 July 1996     

Preoperative systemic chemotherapy (FAC) in 3 cases of advanced breast cancer

Three cases of advanced breast cancer treated with preoperative systemic chemotherapy (FAC) were reported. Radical mastectomy was performed in all three cases after partial response to the systemic chemotherapy. Systemic chemotherapy itself is easy to manage and its resulting response rates and side effects are comparable to those of intra-arterial infusion chemotherapy.     

Freeze–fracture analysis of muscle plasma membrane in Becker's muscular dystrophy

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities in skeletal muscle plasma membranes from eight patients with Becker*s muscular dystrophy (BMD) were analysed by the freeze–fracture technique. The results showed almost normal IMP density with the significant decrease of OA and OASP densities in BMD. The group mean densities ± SE of IMPs on the protoplasmic faces with and without OASPs, and on extracellular faces/μm² were 2137 ± 207, 1839 ± 68 and 895 ± 108, respectively in controls; whereas those of BMD were 1989 ± 259, 1837 ± 203 and 900 ± 239, respectively ( P > 0.1 by two–tailed t –test). The group median density of OAs and their pits/nm² was 4.89 with mid–ranges (25˜75% values of the counts) of 2.66 ˜ 10.18 in controls; whereas that in BMD was 2.15 with mid–ranges of 1.14 ˜ 4.31 ( P < 0.01 by Wilcoxon rank–sum test). The group mean density ± SE of OASPs in controls was 15.99 ± 1.83; whereas that in BMD was 13.47 ± 1.07 ( P < 0.01 by two–tailed t –test). However, the diminution of OA and OASP densities in BMD muscle plasma membranes was not as severe as in Duchenne's muscular dystrophy. There was a relationship between OA density and clinical severity in BMD patients; the decrease of OA density in a severe BMD patient was more marked than that in mildly affected BMD patients. Therefore, it seems that marked depletion of OA density may lead to the severe disability in muscular dystrophies.     

Diagnostic value of signs and symptoms of mammary candidosis among lactating women.

Mammary candidosis in lactating women is not well defined and is most often presumptively diagnosed by signs and symptoms. This study evaluates the sensitivity, specificity, positive predictive value, and likelihood ratios of signs and symptoms of mammary candidosis based on the presence of Candida species on the nipple/areola or in the milk. In this prospective cohort study, the nipple/areola skin and milk of 100 healthy breastfeeding mothers were cultured from each breast at 2 weeks postpartum, and mothers were interviewed regarding signs and symptoms associated with mammary candidosis between 2 and 9 weeks postpartum. Positive predictive value for Candida colonization was highest when there were 3 or more signs or symptoms simultaneously or when flaky or shiny skin of the nipple/areola was reported together or in combination with breast pain.     

High-density lipoprotein and apolipoprotein A-I deficiency induced by combination therapy with probucol and bezafibrate

The effects of the administration of slow-release bezafibrate to hypercholesterolaemic patients who were already receiving long-term probucol treatment (mean 865 days, 500–1000 mg·day^–1) were investigated. Bezafibrate was administered at either 200 mg·day^–1 (13 males, 13 females, mean age 55.2 years) or 400 mg·day^–1 (11 males, 14 females, mean age 57.2 years), and blood was taken at 0, 3, 6 and 12 months after the beginning of combination therapy. Overall, serum total cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL)-TC, high-density lipoprotein (HDL)-TG, VLDL-TG, VLDL-phospholipid (PL), lipoprotein (a) [Lp(a)], apolipoprotein (apo) C-III, apo E levels and LCAT activity decreased significantly with this combination therapy, while HDL cholesterol (C), HDL3-C, HDL-PL, apo A-I and apo A-II levels significantly increased, as assessed by analysis of variance (ANOVA). Five patients (one receiving 200 mg·day^–1, four receiving 400 mg·day^–1 bezafibrate) showed drastic reductions in HDL-C (HDL-C levels were reduced by a mean of 46.2%, 59.3% and 61.6% at 3, 6 and 12 months, respectively) after beginning combination therapy. These HDL-C reductions were maintained for the 1 year of combination therapy, but then returned to pre-combination treatment levels 1 month after discontinuation of bezafibrate. Serum probucol concentrations and cholesteryl ester transfer protein (CETP) mass were assayed at 6 months, and the probucol concentration was higher in the HDL-deficient group (56.2 vs 26.5 g/ml). In contrast, CETP mass was significantly lower in HDL-deficient patients than in non-HDL-deficient patients (2.08 vs 2.87 mg·l^–1). When the patients in the non-HDL-deficient group were divided into two groups, receiving low (200 mg·day^–1, n–25) and high (400 mg·day^–1, n–21) doses of bezafibrate, the former group showed a significant increase in probucol-lowered HDL-C and apo A-I, although these levels did not return to pre-probucol treatment levels, while the latter group showed no changes in HDL. These data suggest that the addition of a low dose of bezafibrate to probucol tended to reverse probucol-induced HDL lowering, while 9.8% (5 of 51 patients) of the patients exhibited a severe HDL deficiency. Since it is unclear whether or not such an extreme HDL reduction is harmful, HDL deficiency should be carefully monitored with this combination therapy.Part of this work was presented at the XIth International Symposium on Drugs Affecting Lipid Metabolism (DALM), 15 May 1992, Florence, Italy.