Evaluation of pyrrolidine-based analog of jaspine B as potential SphK1 inhibitors against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitise, and its pathogenesis is complicated. Sphingosine-1-phosphate (S1P) is a lipid produced by sphingosine kinase 1 and 2 (SphK1/2), which participate in some of most-spread skeletal diseases such as rheumatoid arthritis or osteoarthritis. To explore the anti-inflammatory activity of 2-epi-jaspine B analogs as SphKs inhibitors, we used LPS-induced rheumatoid arthritis fibroblast-like synovial cells (HFLS-RA) as the research object to evaluate the anti-inflammatory activity of 16 2-epi-jaspine B analogs and the newly synthesized salt CHJ01. We found that 2-epi-jaspine B analog CHJ01 in hydrochloride salt form has excellent SphK1 inhibitory effect and better anti-RA effect. CHJ01 showed an anti-inflammatory effect similar to that of MTX in vitro, its IC50 value is 8.64 µM. Moreover, the anti-RA effect of CHJ01 was also studied by using a Complete Freund's Adjuvant (CFA)-induced arthritis (AIA) in a rat mode. Pharmacological experiments show that CHJ01 can help to significantly improve the symptoms of rheumatoid arthritis by reducing the swelling volume, arthritis score, spleen index and the level of IL-1ß, TNF-α, IL-6 of AIA rats. Therefore, CHJ01 holds high potential for the treatment of RA.

[Effect of Electroacupuncture Intervention on Gastrointestinal Motility and Expression of Insulin-like Growth Factor 1 and Its Receptor Proteins in Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention of "Zusanli"(ST 36), etc. on gastrointestinal motility and levels of insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1 R) proteins in the gastric antrum in diabetic gastroparesis (DGP) rats, so as to study its mechanism underlying improvement of DGP. METHODS: Fifty SD rats, half male and half female, were randomly allocated to control, model, EA acupoint, EA non-acupoint and medication (metoclopramide) groups (n=10 rats/group). The DGP model was induced by 2% streptozotocin (STZ, i.p.i.,55 mg/kg) and high fat-sugar forage for 8 weeks. EA (10 Hz/50 Hz, 2 mA) was applied to "Zusanli"(ST 36), "Liangmen"(ST 21), "Sanyinjiao"(SP 6) and non-acupoints (about 5 mm lateral to ST 36, ST 21 and SP 6, respectively) for 20 min, one daily for 15 days, and 1.7% metocloppramide (1 mL/100 g) was given (gavage) to rats of the medication group, once daily for 15 days. After the treatment, the rats' general conditions were scored, the blood glucose level was determined using a glucometer, and the gastrointestinal mobility was evaluated by measuring the gastric emptying rate (GER) and the intestinal propulsion rates (IPR) marked by oral-infused phenol red solution (50 mg/dL). The immunoactivity levels of IGF-1 and IGF-1 R of gastric antrum tissue were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: After modeling, the symptom integrative score, the blood glucose, IGF-1 and IGF-1 R levels were significantly increased (P<0.01), and the GER and IPR were notably decreased relevant to the control group (P<0.01). After EA stimulation of ST 36, ST 21 and SP 6, the symptom score, blood glucose, and IGF-1 and IGF-1 R levels of the gastric antrum were significantly reduced, while the GER and IPR were considerably increased (P<0.05,P<0.01). Except the GER and IPR were considerably increased in the medication group(P<0.05,P<0.01), no significant changes were found in the symptom integrative score, the blood glucose, IGF-1 and IGF-1 R levels in both EA non-acupoint and medication groups relevant to the model group (P>0.05),the GER and IPR in the EA non-acupoint group (P>0.05). Compared with the EA acupoint group, the symptom integrative score, IGF-1 and IGF-1 R levels were significantly increased in both EA non-acupoint and medication groups(P<0.05,P<0.01). CONCLUSIONS: EA stimulation can improve gastrointestinal motility and lower blood glucose in DGP rats, which may be closely associated with its effectiveness in reducing IGF-1 and IGF-1 R immunoactivity levels in gastric antrum.

[Effects of Electroacupuncture on Ultrastructure of Interstitial Cells of Cajal and Stem Cell Factor-kit Signal Pathway of Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on gastrointestinal motility and the ultrastructure of interstitial cells of Cajal (ICC) and the expressions of c-kit receptor protein and stem cell factor (SCF) mRNA in diabetic gastroparesis (DGP) rats, so as to explore its mechanism. METHODS: Fifty SD rats were randomly divided into normal, model, acupoint, non-acupoint and metoclopramide groups (n＝10 rats/group). DGP model was established by intraperitoneal injection of streptozotocin (STZ, 2%), and raised with high-sugar high-fat diet irregularly. EA (sparse-dense, 10 Hz/50 Hz, 2 mA, 20 min) was applied at "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sanyinjiao" (SP 6), and the corresponding non-acupoints of the 3 acupoints, daily for 15 days. The rats in metoclopramide group received intragastric administration of metoclopramide (1.7%, 1 mL/100 g) for 15 days, once a day. Blood sugar was determined with One Touch blood glucose test paper. The gastric emptying rate (GER) and the intestinal propulsion rate (IPR) were measured by intragastric phenol red. The ultrastructure of ICC was detected by transmission electron microscopy. The expression levels of c-kit receptor protein and SCF mRNA of gastric antrum were examined respectively by Western blot and RT-PCR. RESULTS: Compared with the normal group, the blood glucose significantly increased in the model group (P<0.01), while the GER, IRP and the expression level of SCF mRNA in the gastric antrum significantly decreased (P<0.01), and the ultrastructure of ICC appeared apoptosis-like changes. The blood glucose of the EA group was obviously decreased compared with that of the model group (P<0.05); the GER and IRP significantly increased(P<0.05, P<0.01); the expression level of SCF mRNA increased (P<0.01), the number of ICC increased and its ultrastructure was repaired. There was some relief on ICC ultrastructure in the acupoint group compared with that in the non-acupoint group; and SCF mRNA increased (P<0.05). There was no significant difference on c-kit receptor expression among all the modeling groups (P＞0.05). CONCLUSIONS: EA at ST 36, etc. can regulate the blood glucose and improve gastrointestinal emptying in DGP rats. The mechanism may be related to up-regulating SCF mRNA, repairing ICC ultrastructure, restoring the pacing function, and improving gastrointestinal motility.

Alpha 1,3-fucosyltransferase-VII regulates the signaling molecules of the insulin receptor pathway.

Two H7721 human hepatocarcinoma cell lines showing moderate and high expression of alpha1,3-fucosyltransferase (FucT)-VII cDNA were established and designated FucTVII-M and FucTVII-H, respectively. In alpha1,3-FucT-VII-transfected cells, expression of insulin receptor (InR) alpha- and beta subunits and epidermal growth factor receptor (EGFR) on the cell surface and in cells, as well as the sialyl Lewis X (SLe(x), the product of alpha1,3-FucT-VII) content of the EGFR were unchanged. However the level of SLe(x) on the InR alpha subunit (InR-alpha) was increased dramatically. Tyrosine autophosphorylation of InR-beta , but not EGFR, was elevated. Concomitantly, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Ser/Thr phosphorylation of protein kinase B (PKB; Akt), p42/44 mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), and the protein of some other signaling molecules, such as phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase (PKN), c-Raf-1 and beta-catenin were also upregulated. The activities of PKB and transcription factor TCF were concomitantly stimulated. Upregulation of InR signaling molecules and their phosphorylation was correlated with the level of SLe(x) on InR-alpha and alpha1,3-FucT-VII expression in cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different levels of alpha1,3-FucT-VII expression were attenuated significantly by the inhibitor of InR tyrosine kinase and by the mAb to SLe(x). Furthermore, insulin-induced signaling was facilitated in alpha1,3-FucT-VII-transfected cells, particularly FucTVII-H. These findings provide strong evidence that alpha1,3-FucT-VII may affect insulin signaling by upregulating the phosphorylation and expression of some signaling molecules involved in the InR-signaling pathway. These effects are likely mediated by its product, SLe(x), on the glycans of the InR. This is the first study to report that changes in the terminal structure of glycans on a surface receptor can modify cell signaling.

[Effect of epidermal growth factor signal pathway on integrin alpha5 beta1 in prostate cancer cell line DU145].

OBJECTIVE: To investigate the molecular mechanism of epidermal growth factor (EGF) signal pathway on the expression of integrin alpha5 beta1 in prostate cancer cell line DU145. METHODS: Using flow-cytometry, the effects of EGF and the mitogen-activated protein kinase (MAPK) signal pathway inhibitor PD98059 on the expression of integrin alpha5 and beta1 subunits on DU145 cell surface were analyzed. RT-PCR and Western blot methods were used to examined the expression of mRNA and cell total protein of integrin alpha5 and beta1 subunits. And the metastatic phenotypes in DU145 cell were investigated. RESULTS: The expression levels of integrin alpha5 beta1, which was the receptor for fibronectin, were changed. EGF up-regulated the protein and mRNA expression of beta1 subunit on DU145 cell surface, 231% and 248% (P < 0.01) compared to the control respectively, and it could significantly promote the ability of DU145 cell adhesion to fibronectin and migration. However PD98058, which was the inhibitor of MAPK signal pathway, down-regulated the protein and mRNA expression of beta1 subunit, 60% and 63% (P < 0.01) compared to the control respectively, and it had the contrary function on the adhesion and migration ability of DU145 cell. But both had no effect the expression of alpha5 subunit. CONCLUSIONS: EGF might promote the metastatic ability mainly by up-regulating the expression of beta1 subunit by activating MAPK signal pathway in DU145 cells. Their regulation effects are on the mRNA transcriptional level.

[Correlation between integrin subunits alpha5 and beta1 expressions in prostate cancer and its clinical implication].

OBJECTIVE: To explore the correlation between the expression of integrin subunits alpha5 and beta1 in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage. METHODS: Expressions of integrin subunits alpha5 and beta1 were examined in 30 cases of PCa and 30 cases of normal prostatic tissues by immunohistochemical assay. RESULTS: Expressions of integrin subunits alpha5 and beta1 in PCa were lower than those in normal prostatic tissues (P <0.05) respectively. CONCLUSION: Compared with normal prostatic tissues, expressions of integrin subunits alpha5 and beta1 in PCa were rather weaker or even faded. Expressions of integrin subunits alpha5 and beta1 revealed an positive correlation with tumor's Gleason grade and negative with clinical TNM stage. The results indicate that integrin subunits alpha5 and beta1 have potential values in the diagnosis and are predictable indices in the proliferation of PCa.

Down regulation of N-acetylglucosaminyltransferase V facilitates all-transretinoic acid to induce apoptosis of human hepatocarcinoma cells.

After N-acetylglucosaminyltransferase V (GnT-V) activity was down-regulated by the transfection of its antisense cDNA(GnTV-AS), apoptosis of H7721 cells was appeared and the apoptosis induced by 80 microM all-transretinoic acid (ATRA) was facilitated, while ATRA itself could not induce apparent apoptosis in mock cells transfected with the vector. In the study of the molecular mechanism of this phenomenon, it was found that GnTV-AS reduced the expressions of anti-apoptotic proteins, such as phosphorylated protein kinase B and phosphorylated Bad as well as Bcl-2 and Bcl-X (L), and elevated those of pro-apoptotic proteins, including Bax, full length caspase-3 and its activated fragments as well as anti-oncoprotein p53. In the contrast, ATRA up regulated the expressions of Bax and activated caspase-3 fragments only. After the GnTV-AS transfected cells were treated with ATRA, phosphorylated PKB and Bad were further decreased, while Bax and activated caspase-3 fragment were further increased, leading to the enhanced apoptosis in flow-cytometry analysis when compared with GnTV-AS cells not treated with ATRA. It was speculated that the decreased phospho-Bad resulted from the reduced phospho-PKB and the up regulation of p53 caused the elevated activity of Bax. The increased active caspase-3 was the consequence of the elevated Bax/ Bcl-2(Bcl-X(L)) activity ratio in the cells.

[Prostate sarcoma: a report of 14 cases].

OBJECTIVE: To discuss the diagnosis and effective treatment of prostate sarcoma. METHODS: We analysed the clinical materials of fourteen patients with prostate sarcoma treated in our hospital from Jan. 1991 to Jun. 2004. RESULTS: Prostate sarcoma accounted for 3.21% of all the prostatic malignant tumors treated in our hospital during that period. The average age was 39.5 years old (ranging from 17 to 62). Twelve cases came to our hospital because of dysuria. Large soft prostate tumors were found in all the patients in physical examination. The serum AKP, ACP and PSA levels were normal in all the 10 patients who had received the test. Pathological test revealed: 7 cases leiomyosarcoma, 1 case rhabdomyosarcoma, 4 cases fibrosarcoma, 2 cases spindle cell sarcoma. Different kinds of prostate sarcoma each his its own immuno-histochemical staining features. Two cases were at Ghavimi Stage I, 5 at Stage II, 3 at Stage III, and 4 at Stage IV. All the patients received surgery, chemotherapy and/or radiotherapy. One case failed to be followed up, 11 died 2 approximately 12 months after diagnosis. Two patients are still alive, 1 for 18 months without recurrence, and the other for through with relapse. CONCLUSION: Dysuria is always the first symptom of prostate sarcoma. DRE test may suggest prostate sarcoma but needle biopsy contributes to a definite diagnosis. The immuno-histochemical dyeing helps to differentiate the disease. The prognosis of prostate sarcoma is very poor and the main treatment is surgery followed by chemotherapy and/or radiotherapy.