Synergistic effects of nimesulide and 5-fluorouracil on tumor growth and apoptosis in the implanted hepatoma in mice.

AIM: To compare the effect of nimesulide or/and 5-fluorouracil (5-FU) on tumor growth inhibition and apoptosis in mice with the implanted hepatoma and to observe their possible interactions. METHODS: The inhibitory effects on tumor growth was evaluated by inhibition rate. Apoptosis was assessed by the ultrastructural, flow cytometry features and the DNA ladder demonstrated by agarose gel electrophoresis. PGE(2) level was determined by radioimmunoassay. Expression levels of c-jun, c-fos and p53 were evaluated by western blotting. RESULTS: Nimesulide or 5-FU alone inhibited the growth of hepatoma, while a synergistic effect was observed for a combined use of both. More pronounced morphologic changes for tumor cell apoptosis and the DNA ladder were found for the latter treatment. Expression levels of c-jun and p53 were found to be elevated for the tumors from mice treated with nimesulide and 5-FU comparing to those with either of them, but a reduced PGE(2) level was observed only for the treatment with nimesulide. No change was detected on c-fos expression. CONCLUSION: Nimesulide and 5-FU appear to have synergistic effects for the growth inhibition and apoptosis induction. Both were found to be overexpressed in p53 and c-jun proteins, rather than that of c-fos, associations with the resulted apoptosis.

Changes of NF-kB, p53, Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells: role of reactive oxygen species.

AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. CONCLUSION: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.

Serum from rabbit orally administered cobra venom inhibits growth of implanted hepatocellular carcinoma cells in mice.

AIM: To investigate the inhibitory effect of serum preparation from rabbits orally administered cobra venom (SRCV) on implanted hepatocellular carcinoma (HCC) cells in mice. METHODS: An HCC cell line, HepA, was injected into mice to prepare implanted tumors. The animals (n=30) were divided randomly into SRCV, 5-fluorouracil (5-FU), and distilled water (control) groups. From the second day after transplantation, 20 mg/kg 5-FU was administered intraperitoneally once a day for 9 days. SRCV (1,000 mg/kg) or distilled water (0.2 mL) was given by gastrogavage. Tumor growth inhibition was described by the inhibitory rate (IR). Apoptosis was detected by transmission electron microscopy (TEM), flow cytometry (FCM), and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). Student's t-test was performed for statistical analysis. RESULTS: The tumor growth was inhibited markedly by SRCV treatment compared to that in the control group (P<0.01). The treatment resulted in a significant increase in the apoptotic rate of cancer cells by the factors of 10.5+/-2.4 % and 20.65+/-3.2 % as demonstrated through TUNEL and FCM assays, respectively (P<0.01). The apoptotic cells were also identified by characteristic ultrastructural features. CONCLUSION: SRCV can inhibit the growth of implanted HepA cells in mice, and the apoptosis rate appears to elevate during the process.

JTE-522-induced apoptosis in human gastric adenocarcinoma [correction of adenocarcinoma] cell line AGS cells by caspase activation accompanying cytochrome C release, membrane translocation of Bax and loss of mitochondrial membrane potential.

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Loss of C-terminal alpha-helix decreased SDF-1alpha-mediated signaling and chemotaxis without influencing CXCR4 internalization.

AIM: To investigate the possibility that a novel alpha-helix-defective mutant of stromal cell-derived factor-1alpha (SDF-1alpha) (SDF-1/54R) acts as an antagonist of CXC chemokine receptor 4 (CXCR4). METHODS: According to the genetic sequence of natural SDF-1alpha, a recombinant alpha-helix-defective mutant of SDF-1alpha was designed and some biologic characteristics of this mutant were demonstrated. The migration of Jurkat cells was assessed with chemotactic assay. ERK phosphorylation was analyzed by Western blot with a specific anti-phospho-ERK1/2 antibody. Intracellular calcium influx was examined by flow cytometer with a calcium indicator dye Fluo-3AM. The CXCR4 on the cell surface was detected by flow cytometer with a PE conjoined anti-human CXCR4 antibody. RESULTS: Compared with native SDF-1alpha, SDF-1/54R displayed apparent decrease in chemotactic ability, ERK1/2 activation, and intracellular calcium influx in Jurkat cells. However, the binding to CXCR4 and inducing CXCR4 internalization of SDF-1/54R did not change outstandingly. Moreover, a competitive inhibitory effect of SDF-1/54R on the migration of Jurkat cells induced by native SDF-1alpha was confirmed. CONCLUSION: Alpha-helix-defective mutant of SDF-1alpha, SDF-1/54R that remained both the N-terminus and the central beta-sheet region, decreased SDF-1alpha-mediated signaling and chemotaxis but did not influence CXCR4 internalization, which suggested that SDF-1/54R might be developed as an anti-CHIV inhibitor with high biological potency and low side-effect.

Total saponins of Panax notoginseng protected rabbit iliac artery against balloon endothelial denudation injury.

AIM: To investigate whether total Panax notoginseng saponins (PNS) could protect endothelium of rabbit iliac artery against balloon endothelial denudation (BED) injury. METHODS: The morphology changes of the endothelium were observed with scanning electron microscope (SEM) and hematoxylin and eosin stain after BED of rabbit iliac artery at 0, 4, 6, and 8 week respectively. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) was also determined by immunohistochemistry. PNS 10, 30, and 50 mg/kg were administered iv per day from 2 d before to 4 weeks after operation. RESULTS: The endothelium was denudated completely after BED. At the 4th week the endothelium was repaired in some degree, then recovered gradually at 6 and 8 week. The degree of intimal thickening at 4 week was significantly greater than that at 0, 6, or 8 week. The sequence of VEGF or MMP-2 staining from strong to weak was 4, 6, 0, 8 week, and normal control. However at 4 week, endothelial regeneration in PNS 30 and 50 mg/kg groups was significantly faster than that in saline group. The intimal thickness was significantly decreased and expressions of VEGF and MMP-2 were both down-regulated in PNS 30 or 50 mg/kg groups compared with saline control group. CONCLUSION: PNS promoted the endothelial regeneration and reduced ECM thickening, which was related to regulation of the expression of VEGF and MMP-2. PNS may have sustained antirestenotic effect after BED.

Synergism between heparin and adriamycin on cell proliferation and apoptosis in human nasopharyngeal carcinoma CNE2 cells.

AIM: To measure the effect of addition of heparin to adriamycin (ADM) on cell proliferation and apoptosis in CNE2 cells and investigate the possible molecular mechanisms of heparin and ADM interactions. METHODS: Cell viability and cell cycle were determined by MTT assay and flow cytometry. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and agarose gel electrophoresis. The expression of Bax, Bcl-2, p53, and p21 was examined by Western blot. RESULTS: ADM (5 mg/L) alone inhibited the growth of CNE2 cells, which was magnified when heparin was added. ADM elicited typical apoptotic morphologic changes. Compared with ADM or heparin alone, ADM plus heparin obviously enhanced the number of TUNEL positive cells from 12.6 % 1.1 % to 65.7 % 1.3 %, and the DNA ladder was more clearly observed. After exposure to different concentrations of heparin (with or without ADM) for 24 h, CNE2 cells were accumulated in G0/G1 phase. There was a decrease in the number of cells in S phase by the combined heparin and ADM treatment compared to heparin or ADM alone. The ratio of Bax/Bcl-2 was elevated, and p53 and p21 mRNA were over-expression. CONCLUSION: Heparin and ADM appear to interact in a synergistic manner, which may be related to the over-expression of p53 and p21 mRNA and the elevated ratio of Bax/Bcl-2 mRNA.

JTE-522, a selective COX-2 inhibitor, inhibits cell proliferation and induces apoptosis in RL95-2 cells.

AIM: To investigate whether JTE-522 [4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide], a selective COX-2 inhibitor, can induce apoptosis and inhibit cell proliferation in human endometrial cancer cell line RL95-2 cells and to explore the molecular mechanisms. METHODS: [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), DNA ladder, enzyme-linked immunosorbent assay (ELISA), flow cytometry, RT-PCR, and Western blot analysis were employed to investigate effect of JTE-522 on human endometrial cancer cell line RL95-2 cells and the related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of RL95-2 cells and induced the apoptosis. Furthermore, it arrested G0/G1 phase and inhibited S phase in RL95-2 cells. JTE-522 inhibited the expressions of COX-2 mRNA, phosphorylated Rb, and CDK4 proteins, while increased the levels of p53, p21, cyclin D1 proteins, and the activity of caspase-3 in RL95-2 cells. CONCLUSION: JTE-522 inhibits cell proliferation and induces apoptosis in RL95-2 cells, which may be associated with the activation of caspase-3-like proteases, down-regulation of the expression of COX-2 mRNA, phosphorylated Rb, and CDK4 proteins, and up-regulation of the expressions of p53, p21, and cyclin D1 proteins.

Nimesulide inhibits tumor growth in mice implanted hepatoma: overexpression of Bax over Bcl-2.

AIM: To investigate whether nimesulide could suppress tumor growth and induce apoptosis in implanted hepatoma mice and to explore the molecular mechanisms. METHODS: Male mice received nimesulide 10 mg/kg, 20 mg/kg, and 40 mg/kg ig daily for 21 d. Electron microscopy (EM), flow cytometry (FCM), DNA ladder, radioimmunoassay (RIA), and Western blot analysis were employed to investigate effect of nimesulide on mice hepatoma and the related molecular mechanisms. RESULTS: Nimesulide inhibited the growth of hepatoma (from 14 % to 62 %) and elicited typical apoptotic morphologic changes. The DNA ladder of high dose nimesulide was more clearly observed and apoptotic rate was 51.3 %+/-1.5 %. Nimesulide also decreased cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and Bcl-2 expression, while increased the level of Bax protein. CONCLUSION: Nimesulide suppresses tumor growth and induces apoptosis by inhibiting COX-2 and PGE2 expression, which may be related to overexpression of Bax over Bcl-2.

An increase in opening of BK(Ca) channels in smooth muscle cells in streptozotocin-induced diabetic mice.

AIM: To investigate the changes of function of large conductance of calcium-activated potassium channels (BK(Ca) channels) in thoracic aortic smooth muscle cells in early stage of streptozotocin (STZ)-induced diabetic C57BL/6J mice. METHODS: Vascular muscle tension in the isolated thoracic aortic rings of mice was compared, and the role of BK(Ca) channels in relaxation of isolated mice thoracic aortic rings induced by acetylcholine (ACh) was determined. Meanwhile, single vascular smooth muscle cells (VSMCs) were isolated by collagenase, and BK(Ca) currents were recorded by patch-clamp single channel recording technique in symmetric high potassium solution. RESULTS: Tetraethylammonium (TEA) 1 mmol/L, a selective calcium-activated potassium channel blocker, caused significant rightward shift in the concentration-response curves of ACh in the isolated thoracic aortic rings of diabetic mice and pD2 value of ACh-induced relaxation was decreased notably after TEA treatment [(6.3+/-0.4) vs (6.9+/-0.5), n=10 rings from 7 mice, P<0.01]. But pD2 value of ACh-induced relaxation in age-matched control mice did not change in presence and absence of TEA 1 mmol/L [(6.4+/-0.15) vs (6.5+/-0.5), n=7 rings from 6 mice, P>0.05]. Furthermore, conductance of BK(Ca) channels in single thoracic aortic smooth muscle cells was decreased [(199+/-15) pS, n=10 cells from 7 mice vs (266+/-11) pS, n=12 cells from 6 mice, P<0.01], but probability of open of BKCa channels was increased [(0.51+/-0.28) vs (0.11+/-0.06), n=6 cells from 6 mice, P<0.01], and the mean closed time in diabetic mice was reduced [(15+/-15) vs (132+/-98), n=6 cells from 6 mice, P<0.05]. CONCLUSION: The opening of BK(Ca) channels was increased in thoracic aortic smooth muscle cells in the early stage of STZ-induced diabetic C57BL/6J mice by reducing mean closed time, but the conductance of BK(Ca) channels was decreased.