epidermal-growth-factor and fasudil

Both RhoA and Ezrin are confirmed to play an important role in the development and metastasis of tumors. However, the mechanism remains unclear. This study rudimentally investigates the regulatory effect of RhoA on the expression of Ezrin.. After MDA-MB-231 cells were treated with epidermal growth factor (EGF) or following pretreatment of fasudil (the special inhibitor of RhoA), the expression of RhoA, p-RhoA and Ezrin in MDA-MB-231 cells was detected by western blot.. Stimulation of EGF triggered RhoA phosphorylation in MDA-MB-231 cells which reached the maximum at 30 min; RhoA expression did not change; Ezrin expression was enhanced and reached the maximum at 24 h. However, pretreatment of fasudil before EGF stimulation decreased RhoA phosphorylation and Ezrin expression in MDA-MB-231 cells by 72.73% and 51.28%, respectively.. RhoA may regulate the invasion and metastasis of breast cancer cells as an upstream signaling of Ezrin.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cytoskeletal Proteins; Epidermal Growth Factor; Humans; Phosphorylation; rhoA GTP-Binding Protein; Signal Transduction; Time Factors

To investigate the role of the signal routes P38, ERK, and Rho in the differentiation of bone marrow mesenchymal stem cells (MSCs) into epidermoid cells.. (1) MSCs were separated from the bone marrow of Wistar rats by Ficoll-Pague lymphocyte separating medium and proliferated in culture medium. Then the MSCs were immunocytochemically stained to detect the expression of surface antigens. (2) The MSCs were randomly divided into 3 groups: control group; pure induction induced group, cultured with epithelial growth factor (EGF) added into the culture fluid, and Rho inhibition group, cultured with EGF and HA1077, a ROK inhibitor, added into the culture fluid. One, 3, 5, and 7 days later FC was used to detect the levels of phosphorylated P38 and ERK. (3) MSCs were randomly divided into 4 groups: control group, cultured with low-sugar DMEM complete culture fluid; pure induction group, cultured with supernatant of rat fibroblasts and EGF added into the culture fluid, p38 blocking group, with SB203580, inhibitor of P38 added into the culture fluid; and ERK blocking group, with PD98059, inhibitor of ERK added into the culture fluid. Seven days later, SP method was used to detect the expression of CK5/8 and CK19 induced by MSCs. (4) MSCs were randomly divided into 4 groups: control group; pure induction group, with supernatant of rat fibroblasts and EGF added into the culture fluid; and RHO blocking group, with HA1007 added into the culture fluid. Seven days later, FC was used to detect the expression of CK5/8 and CK19.. (1) Both FC and immunocytochemistry showed that the MSCs were uniformly positive in CD29 and CD44, but did not express CD34 and CD45. (2) The phosphorylated P38 rate remained 0.01% in the control group. The phosphorylated P38 rate was 0.04%, significantly higher than that of the control group (0.01%, P < 0.05) at day 5, and then lowered to 0.01% at day 5 in the pure induction group; and became 6.17%, 4.13%, 3.97%, and 0.41% respectively at day 1, 3, 5, and 7, all significantly higher than those of the control group (all P < 0.05), in the Rho inhibition group. The phosphorylated ERK level was 4.23% in the control group; became 0.39% and 0.40% at day 3 and day 5 (both P < 0.05), and then returned to 5.10% at day 7 in the pure induction group; and was not significantly changed at days 1, 3, and 5, and then became 0.41%, significantly lower than that of the control group (P < 0.05), in the Rho blocking group, (3) The control group was CK5/8 and CK19 negative. The CK5/8 and CK19 rates at day 7 of the pure induction group were 3.01% and 6.47% respectively, both significantly higher than those of the p38 inhibition group (1.43% and 5.41% respectively, both P < 0.05). The CK5/8 and CK19 expression rates of the ERK inhibition group were 5.54% and 7.56% respectively, both significantly higher than those of the pure induction group (both P < 0.05), (4) The CK5/8 and CK19 expression rates of the HA1077 group were 21.65% and 39.41% pure, both significantly higher than those of the pure induction group (1.81% and 10.19% respectively, both P < 0.05).. p38 route may play an active role in the differentiation of MSCs into epidermoid cells. Blocking of the upstream signal Rho may enhance the activation of p38 route and then promote the differentiation of MSCs into epidermoid cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epidermis; Female; Flavonoids; Flow Cytometry; Hyaluronan Receptors; Imidazoles; Immunohistochemistry; Integrin beta1; Keratin-19; Keratins; Male; Mechanotransduction, Cellular; Mesenchymal Stem Cells; Pyridines; Rats; Rats, Wistar; Time Factors

The role of actin organization in occupancy-induced receptor internalization remains poorly defined. Here we report that treatment of mouse Swiss 3T3 cells with latrunculin A, a potent inhibitor of actin polymerization (including cortical actin), inhibited the internalization of the endogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judged by uptake of (125)I-labeled GRP or fluorescent Cy3-labeled bombesin. In contrast, cells pretreated with cytochalasin D showed minimal inhibition of bombesin/GRP receptor internalization. Similarly, pretreatment of Swiss 3T3 cells with the potent Rho-kinase inhibitor HA-1077, at concentrations (10-20 microM) that abrogated bombesin-mediated stress fiber formation, did not significantly alter receptor-mediated internalization of (125)I-GRP. These results indicate that bombesin/GRP receptor internalization depends on latrunculin A-sensitive cortical actin rather than on rapidly turning over actin stress fibers that are disrupted by either cytochalasin D or HA-1077. The rates and total levels of internalization of the endogenously expressed endothelin A receptor and epidermal growth factor receptor were also markedly reduced by latrunculin A in Swiss 3T3 cells. The potency of latrunculin A for inhibiting G protein-coupled receptor endocytosis was comparable to that for reducing internalization of the epidermal growth factor tyrosine kinase receptor. We conclude that cortical actin structures, disrupted by latrunculin A, are necessary for occupancy-induced receptor internalization in animal cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Actins; Animals; Bombesin; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gastrin-Releasing Peptide; GTP-Binding Proteins; Iodine Radioisotopes; Kinetics; Mice; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Receptors, Bombesin; Receptors, Endothelin; Signal Transduction; Thiazoles; Thiazolidines

Insulin and growth factors increase glycogen synthesis via complex pathways including protein phosphorylation/dephosphorylation processes. We investigated the involvement of 90-kDa S6 kinase in the control of insulin- or epidermal growth factor (EGF)-stimulated glycogen synthase activation using newly synthesized compounds which selectively inhibit 90-kDa S6 kinase. HH-5709 (1-(5-hydroxynaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine) inhibited 90-kDa S6 kinase at lower concentrations than observed for protein kinases A or C. The inhibition by HH-5709 was competitive with respect to ATP with a Ki value of 1.3 microM. H-7, an inhibitor of protein kinases A and C, and HA-1077 (1-(5-isoquinolinesulfonyl)-homopiperazine), where the naphthalene ring of HH-5709 was replaced with isoquinoline, also inhibited 90-kDa S6 kinase to a similar extent as HH-5709. In 3Y1 fibroblasts, H-7 and HA-1077 attenuated the activation of glycogen synthase. HH-5709, however, failed to affect the glycogen synthase activation by either insulin or EGF. These findings suggest that 90-kDa S6 kinase is unrelated or insufficient to mediate activation of glycogen synthase and that unidentified pathway(s) sensitive to H-7 or HA-1077 would be involved in the activation of glycogen synthase by insulin or EGF in 3Y1 fibroblasts.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cattle; Dose-Response Relationship, Drug; Epidermal Growth Factor; Glycogen Synthase; Insulin; Protein Serine-Threonine Kinases; Rabbits; Ribosomal Protein S6 Kinases