ω-3 polyunsaturated fatty acids inhibits RHO-ROCK1 signaling pathway in SGC7901 gastric cancer cells / 中国医师杂志

Objective This study was aimed to investigate the effects of ω-3 polyunstaurated fatty acids (ω-3 PUFAs) on the growth of gastric cancer cells in nude mice,and to find whether the Ros homolog gene Rho-associated coiled-coil containing protein kinase 1 (RHO-ROCK1) signaling pathway is involved.Methods 16 BALB/C nude mice were injected subcutaneously with SGC7901 gastric cancer cells to establish the tumor-bearing mouse model.The mice were randomized:control group (normal saline) and intervention group (ω-3 PUFAs).The mRNA expression of Ros homolog gene family,member A (RHOA),RHOC,and ROCK1 in tumor tissue were detected by quantitative polymerase chain reaction (qPCR).Immunofluorescence and Western blot were used to detect RHOA,RHOC,and ROCK1 protein expression.Results The volume and weight of the tumors in the ω-3 PUFAs group were slightly smaller than that in the control group (P ＞ 0.05).Compared to the control group,hematoxylin and eosin staining showed multifocal tumor necrosis in the ω-3 PUFAs group,while the tumors of the control group showed abundant blood supply.qPCR and Western blot showed that the mRNA and proteins expression of RHOA and ROCK1 in the ω-3 PUFAs group was significantly lower than those in the control group (P ＜ 0.05).The immunofluorescence redults also showed that the expression of these proteins in the ω-3 PUFAs group was slightly lower than that in the control group.Conclusions These results suggested that ω-3 PUFAs may affect the growth of gastric cancer in nude mice by affecting the expression of RHOA,RHOC and ROCK1,thus inhibiting the excessive proliferation of gastric cancer cells and leading to tumor necrosis.

Effects of butylphthalide on brain edema, blood-brain barrier permeability and RhoA expression in cortex in focal cerebral infarction in rats / 国际脑血管病杂志

Objective To investigate the effects of butylphthalide on brain edema,blood-brain barrier permeability and RhoA expression in cortex in focal cerebral infarction in rats.Methods A total of 220 male Sprague Dawley rats were divided into a control,a model and a butylphthalile group (40 mg/kg,once a day,gavage) according to the random number table method.A model of rat focal cerebral infarction was induced by photochemical method.At 3,12,24,72,and 144 h after modeling,wet-dry weight method and Evans blue extravasation method were used to detect the brain water content and blood-brain barrier permeability.At 24 h after modeling,immunohistochemical staining and Western blot were used to detect the expression of RhoA protein in the periinfarction cortex.Results Compared to the control group,the brain water content (except at 6 h) and blood-brain barrier permeability in the model group and the butylphthalide group were increased significantly (all P ＜ 0.05).The immunohistochemical staining and Western blot suggested that the RhoA expression in the periinfarction cortex was upregulated significantly (all P ＜ 0.05).Compared to the model group,the brain water content and blood-brain barrier permeability at different time points in the butylphthalide group were decreased significantly (all P ＜ 0.05).The expression levels of RhoA were also decreased significantly (P ＜ 0.05).Conclusions Butylphthalide may reduce the brain edema of focal cerebral infarction in rats,inhibit disruption of the blood-brain barrier,and down-regulate the expression of RhoA.

Prevention of Vasculogenic Erectile Dysfunction in Rat Model by the Chronic Administration of Oral Rho Kinase Inhibitor / 대한비뇨기과학회지

PURPOSE: Since Rho-Rho kinase calcium sensitizing pathway is being regarded as a potential target not only for the treatment of erectile dysfunction but also atherosclerosis, we designed a study to prevent vasculogenic erectile dysfunction in an atherosclerotic rat model by chronic administration of fasudil, oral Rho kinase inhibitor. MATERIALS AND METHODS: Rats (3 months old) were divided into 3 groups (n=10 in each group): control (group 1), atherosclerosis (group 2), and fasudil-treated (group 3). The group 2, 3 received atherosclerosis-prone treatment but group 3 was concurrently treated by fasudil (30mg/kg/day) for 6 weeks. Following the treatment, the erectile function and the amount of pelvic atherosclerosis amount were determined. Cavernosal tissues were prepared for Western blot and malondialdehyde (MDA) assay. RESULTS: Compared to group 2, the progression of atherosclerosis in iliac and pudendal arteries was significantly suppressed by the chronic administration of fasudil. Also the treatment lowered the level of malondialdehyde in the cavernosal tissue. The results of Western blot revealed that systemically administered fasudil could ameliorate cavernosal molecular environment characterized by the decreased expression of Rho A, transforming growth factor-beta 1 and overexpression of endothelial nitric oxide synthase. As a result, erectile function was maintained by the treatment. CONCLUSIONS: These results indicate that Rho/Rho kinase pathway is substantially involved in the development of penile erection and pelvic atherosclerosis, both of which could be prevented by chronic treatment of fasudil. Thus, Rho-kinase might be considered as a novel target for prevention of vasculogenic erectile dysfunction.

Phosphorylation of 46-kappa Da protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kappa Da protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca2+/ calmodulin (CaM), which causes the small GTP- binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM- dependnet protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca2+/CaM directly or indirectly through GTP-binding protein(s) and Ca2+/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca2+/CaM may be important for the regulation of transporters and neurosecretion.

The cytotoxicity of histone deacetylase inhibitor plus paclitaxel to lung cancer cells / 解放军医学杂志

Objective To evaluate the cytotoxicity of trichostatin A (TSA),a histone deacetylase inhibitor,plus paclitaxel to H1299 strain lung cancer cells. Methods H1299 cells were exposed to TSA and/or paclitaxel in different concentrations in different ways. The proliferation rates were determined by MTT assay,the 50% inhibition concentration (IC50) of paclitaxel was calculated and the growth curve was plotted. The H1299 cells were then divided into 4 groups:control group (cells were normally cultivated for 36h),TAX group (cells were treated with 10nmol/L of paclitaxel for 24h),TSA group (cells were treated with 300nmol/L of TSA for 24h) and TF group (cells were exposed to TAX for 24h after being treated with TSA). Cell cycle and apoptosis were determined by flow cytometry. The morphological changes in nuclei as stained with Hoechst 33342 were observed by fluorescence microscopy. The protein expression levels of p21 and cleaved poly-ADP ribose polymerase (PARP) were determined by Western blotting. Results TSA significantly enhanced the inhibition of paclitaxel in lung cancer cell lines H1299. When combined with TSA,the IC50 of paclitaxel decreased significantly from 110.6?38.7nmol/L to 63.7?11.8nmol/L in H1299 cells (P0.05). Conclusion The HDAC inhibitor TSA,combined with TAX,may enhance the cytotoxicity of paclitaxel to,and promote the death of,lung cancer cell line H1299,which may not be related to apoptosis.