Evaluation of anti-inflammatory activity of compounds isolated from the rhizome of Ophiopogon japonicas.

BACKGROUND: Ophiopogon japonicas (L.f) Ker-Gawl has been used as a traditional Chinese medicine to cure acute and chronic inflammation and cardiovascular diseases including thrombotic diseases for thousands of years. Previous phytochemical studies showed that O. japonicus contained compounds with anti-inflammatory activity. The aim of this study was to identify and isolate compounds with anti-inflammatory activity from the rhizome of O. japonicas. METHODS: Compounds were isolated by various column chromatography and their structures were identified in terms of nuclear magnetic resonance spectrum (NMR) and mass spectrum (MS). To measure the anti-inflammatory effects of thirteen compounds in LPS-induced RAW 264.7 macrophage cells, we used the following methods: cell viability assay, nitric oxide assay, enzyme-linked immunosorbent assay, quantitative real-time PCR analysis and western blotting analysis. RESULTS: One new and twelve known compounds (mainly homoisoflavonoids) were extracted from O. japonicas, in which 4'-O-Demethylophiopogonanone E (10) was considered as a new compound, additionally, compounds 4-O-(2-Hydroxy-1- hydroxymethylethyl)-dihydroconiferyl alcohol (2) and 5,7-dihydroxy-6-methyl-3-(2', 4'-dihydroxybenzyl) chroman-4-one (12) were isolated from the rhizome of O. japonicas for the first time. The isolated compounds Oleic acid (3), Palmitic acid (4), desmethylisoophiopogonone B [5,7-dihydroxy-3-(4'-hydroxybenzyl)-8- methyl- chromone] (5), 5,7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chromone (7) and 10 significantly suppressed the production of NO in LPS-induced RAW 264.7 cells. Especially compound 10 showed the strongest effect against the production of the pro-inflammatory cytokine IL-1ß and IL-6 with the IC50 value of 32.5 ± 3.5 µg/mL and 13.4 ± 2.3 µg/mL, respectively. Further analysis elucidated that the anti-inflammatory activity of compound 10 might be exerted through inhibiting the phosphorylation of ERK1/2 and JNK in MAPK signaling pathways to decrease NO and pro-inflammatory cytokines production. CONCLUSIONS: Our results indicated that 4'-O-Demethylophiopogonanone E can be considered as a potential source of therapeutic medicine for inflammatory diseases.

China-Africa cooperation initiatives in malaria control and elimination.

Malaria has affected human health globally with a significant burden of disease, and also has impeded social and economic development in the areas where it is present. In Africa, many countries have faced serious challenges in controlling malaria, in part due to major limitations in public health systems and primary health care infrastructure. Although China is a developing country, a set of control strategies and measures in different local settings have been implemented successfully by the National Malaria Control Programme over the last 60 years, with a low cost of investment. It is expected that Chinese experience may benefit malaria control in Africa. This review will address the importance and possibility of China-Africa collaboration in control of malaria in targeted African countries, as well as how to proceed toward the goal of elimination where this is technically feasible.

[The clinical, endoscopic and pathologic features of Crohn's disease in the differentiation from intestinal tuberculosis].

OBJECTIVE: To investigate the clinical, endoscopic and pathologic features in the differential diagnosis between Crohn's disease(CD) and intestinal tuberculosis (ITB). METHODS: The complete clinical data of 107 patients with CD and 69 patients with ITB in our hospital from January 2011 to January 2012 were retrospectively analyzed. The diagnostic value of the clinical and endoscopic scoring system was evaluated. RESULTS: CD occurred mainly in male. The salient features of CD included long duration of disease high incidence of colectomy. Comparing with patients with ITB, patients with CD have more cases of diarrhea, hematochezia, abdominal mass, intestinal obstruction, intestinal hemorrhage, perianal lesions, and extraintestinal manifestations (all P < 0.05).It's more frequent to have positive results of anti-Saccharomyces cerevisiae antibody (ASCA), perinuclear antineutrophil cytoplasmic antibody (pANCA) and fecal occult blood in CD patients, as well as low albumin, high C-reactive protein ( CRP), elevated platelet count and hematocrit (P < 0.05 or P < 0.01). The salient features of ITB included low fever, night sweats, active parenteral tuberculosis, increased erythrocyte sedimentation rate (ESR), chest X-ray abnormalities, the positive PPD (purified protein derivatives tuberculin) and T-SPOT (P < 0.05 or P < 0.01). Based on the imaging, CD often involved the small intestine, such as the intestinal stricture and abdominal abscess (P < 0.05), while mesenteric lymphadenopathy was more common in ITB (P < 0.05). The endoscopic examination showed that some patterns of disease involvement such as fissure-shape ulcer [41.12% (44/107) vs 5.80% (4/69)], cobblestone sign[15.89% (17/107) vs 4.35% (3/69)], lesions over four segment [24.30% (26/107) vs 7.25% (5/69)], rectum involvement [17.76% (19/107) vs 5.80% (4/69)], ileocecal valve stenosis [21.50% (23/107) vs 8.70% (6/69)] and mucosal bridge[5.61% (6/107) vs 0(0/69)] were more frequent in CD patients than those in ITB patients(P < 0.01 or P < 0.05). However circular ulcers[37.68% (26/69) vs 9.35% (10/107)], rat-bite-like ulcers[24.64% (17/69) vs 12.15% (13/107)], persistent open ileocecal valves [39.13% (27/69) vs 19.63% (21/107)], tuberous and polypoid lesions[36.23% (25/69) vs 20.56% (22/107), 37.68% (26/69) vs 22.43% (24/107)] were more common in ITB (P < 0.01 or P < 0.05). In terms of pathological findings, certain characteristic features such as transmural inflammation [5.61% (6/107) vs 0(0/69)], fissure-liked ulcers [14.02% (15/107) vs 4.35% (3/69)], non-caseous granulomas [5.61% (6/107) vs 0(0/69)], lymphoid hyperplasia [16.82% (18/107) vs 5.80% (4/69)] and crypt abscess [9.35% (10/107) vs 1.45% (1/69)] were more common in CD than those in ITB(P < 0.05). According to the clinical and endoscopic scoring system, the positive diagnostic rate of CD was 50.47% (54/107) and of ITB was 66.67% (46/69) (P < 0.05) . CONCLUSIONS: The differential diagnosis between CD and ITB should be considered carefully based on clinical, endoscopic, pathological characteristics. The clinical and endoscopic scoring system may contribute to distinguish CD and ITB.

Interleukin-18 and -12 synergistically enhance cytotoxic functions of tumor-infiltrating lymphocytes.

BACKGROUND: The role of tumor-infiltrating lymphocytes (TILs) in the immunopathogenesis of individual cancer is not clear and is a challenge for anti-tumor immunotherapy. This study aimed to investigate the effects of interleukin (IL)-18 and -12 on cytotoxic functions of TILs. METHODS: TILs from postoperative gastric cancer patients were costimulated with IL-18 and IL-12. SGC-7901 tumor cells were pre-incubated with TILs and subcutaneously injected into BALB/C SCID mice. The function of TILs was evaluated by measuring tumor sizes in tumor-bearing mice, T helper (Th)1 (tumor necrosis factor (TNF)-α, interferon (IFN)-γ) and Th2 cytokine levels (IL-10 and IL-4) in serum and cytotoxicity of mouse natural killer (NK) and CD8(+) T cells. RESULTS: IL-18 and IL-12 synergistically inhibited the growth of SGC-7901 cells in vivo and significantly extended the survival rate of SGC-7901-bearing mice (66.7% vs. 13.7%, P < 0.01). Moreover, TILs could promote the secretion of TNF-α and IFN-γ ((130.34 ± 7.65) vs. (210.63 ± 12.31) pg/ml, P < 0.01; (14.23 ± 1.97) vs. (30.52 ± 2.12) pg/ml, P < 0.01), and downregulate IL-10 and IL-4 secretion ((103.72 ± 11.21) vs. (61.36 ± 5.41) pg/ml, P = 0.021; (49.36 ± 4.67) vs. (28.48 ± 3.86) pg/ml, P = 0.024). CONCLUSION: IL-18 and IL-12 can synergistically enhance cytotoxic functions of TILs from human gastric cancer.

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo.

AIM: To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS: We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad-p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS: The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was >or= 50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G(0)/G(1), while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION: p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G(0)/G(1). Therefore, p27mt can induce apoptosis in colorectal cells.

Flow-dependent regulation of vascular function and gene expression in rat superior mesenteric artery.

BACKGROUND: Mesenteric artery thrombosis is prone to occur at specific arterial regions with different fluid flow patterns, yet mechanistic links between blood flow and vascular function remain unclear. This study aimd to investigate the role of blood flow in regulation of vascular function and gene expression in rats. METHODS: Isometric tension was recorded in wire myograph to examine vascular function of specific regions (trunk parts and proximal parts from the origin) with different blood flow in superior mesenteric artery (SMA). Endothelial nitric oxide syntheses (eNOS), phosphorylated-eNOS (p-eNOS), serine-threonine kinase Akt and phosphorylated-Akt (p-Akt) protein expressions in SMA were examined by Western blotting. Significance was analyzed using a Student's t test or analysis of variance (ANOVA) followed by a Dunnett's multiple-comparison post hoc test. RESULTS: Compared with trunks, proximal parts exhibited severely impaired relaxant responses to acetylcholine (Ach) (1 nmol/L to 10 micromol/L) (P < 0.01). p-eNOS and p-Akt protein levels were significantly reduced in proximal parts of SMA (0.37 +/- 0.03, 0.42 +/- 0.03 respectively) versus trunk parts (0.82 +/- 0.03, 0.72 +/- 0.03 respectively, both P < 0.05) while total eNOS and Akt expressions remain comparable in both regions by Western blotting analysis (0.70 +/- 0.03 vs 0.82 +/- 0.03; 0.70 +/- 0.03 vs 0.77 +/- 0.03 respectively, both P > 0.05). CONCLUSION: Critical components that drive the vascular function and influence the localization of mesenteric artery thrombosis are flow-responsive elements within the vascular endothelium.

Antitumor bioactivity of adenovirus-mediated p27mt in colorectal cancer cell line SW480.

AIM: To explore the antitumor bioactivity of adenovirus-mediated mutant type p27(kip1) gene in a colorectal cancer cell line SW480. METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27(kip1) gene (ad-p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber and SW480 tumor cell growth inhibition in vitro and in vivo. RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ad-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G(1)/S arrest in SW480 cells and inhibited cell migration. CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.

Expression of angiopoietin-2 is correlated with vascularization and tumor size in human colorectal adenocarcinoma.

Angiopoietins are endothelial growth factors, which play crucial roles in normal vascular development and tumor angiogenesis. We examined the expression profiles of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), and Tie-2, a receptor for Ang-1 and Ang-2, in both colorectal adenocarcinoma and adjacent normal tissues, as judged by histology, in order to elucidate their relationships with microvascular density (MVD) and clinicopathologic properties. Higher MVD was associated with a lower degree of differentiation of colorectal adenocarcinoma. Immunohistochemical analysis revealed that the expression of Ang-2 and VEGF was significantly increased in colorectal adenocarcinoma compared to adjacent normal tissues (p < 0.01), and the expression of Ang-2 positively correlated with that of VEGF (r = 0.997, p < 0.01). In contrast, the expression of Ang-1 was lower in adenocarcinoma tissues than in adjacent normal tissues (p < 0.01), while there was no significant difference in Tie-2 expression in both tissues. Moreover, MVD was increased in Ang-2- and VEGF-expressing adenocarcinoma tissues compared to the Ang-2- and VEGF-negative tissues, respectively (p < 0.01). Importantly, MVD was lower in Ang-1-expressing adenocarcinoma tissues relative to Ang-1-negative tissues (p < 0.01). Furthermore, expression of Ang-2 as well as VEGF was significantly up-regulated in colorectal adenocarcinoma with diameters > or = 5 cm or with lymph-node metastases (p < 0.01). In conclusion, the increased expression of Ang-2 and the decreased expression of Ang-1 may be responsible for blood vessel formation and rapid growth of the colorectal adenocarcinoma.

[Effects of anti-angiopoietin-2 antibody on vascularization of an implanted model of human colonic carcinoma on chick embryo].

OBJECTIVE: To establish an implanted model of human colonic carcinoma on chick embryo, and to study the effects of anti-angiopoietin-2 antibody on its vascularization. METHODS: The human colonic adenocarcinoma cell line HT-29 was transplanted on the chick embryo's chorioallantoic membrane(CAM), and the angiogenesis characteristics were observed by stero-microscope, light microscope and immunohistochemistry. Furthermore, the effects of anti-angiopoietin-2 antibody on angiogenesis and tumor growth were also investigated. RESULTS: Three to seven days after HT-29 cell line was implanted into CAM, tumors grew rapidly and new blood vessels grew toward tumors. Five days after anti-angiopoietin-2 antibody was given, the number of blood vessels in anti-angiopoietin-2 antibody group was significantly down-regulated than that in tumor control group observed by stero-microscope (37.2+/-4.6 vs 56.8+/-7.4, P<0.01), but was up-regulated than that in normal control group (37.2+/-4.6 vs 9.6+/-2.4, P<0.01). Microvessel density(MVD) in anti-angiopoietin-2 antibody group was much lower than that in tumor control group by histological examination (9.6+/-2.4 vs 20.2+/-5.8, P<0.01). CONCLUSION: Angiopoietin-2 antibody is able to inhibit the angiogenesis induced by colorectal cancer cell line HT-29 obviously. The anti-angiopoietin-2 antibody may be potentially useful for clinical treatment of colonic carcinoma.

Curcumin regulated shift from Th1 to Th2 in trinitrobenzene sulphonic acid-induced chronic colitis.

AIM: To investigate the therapeutic effects of curcumin (Cur) on trinitrobenzene sulphonic acid (TNBS)-induced colitis and the effects of Cur on the balance of Th1/Th2 cytokines. METHODS: Colitis was induced by TNBS and treated with Cur (30 mg/kg/d, ip), dexamethasone (Dex, 2 mg/kg/d), or Cur plus dexamethasone (Cur+Dex, 30 mg/kg/d Cur ip+2 mg/kg/d Dex,ip). mRNA in colon mucosa were detected by real-time quantitative polymerase chain reaction. Intracellular cytokines were detected by flow cytometry and concentrations of cytokines in sera were detected by enzyme-linked immunosorbent analysis. RESULTS: Both Cur and Dex improved body weight loss, ameliorated histological images and decreased macroscopic score and myeloperoxidase activity. Cur decreased the expression of Th1 cytokines (IL-12, IFN-gamma, TNF-alpha, IL-1) and increased the expression of Th2 cytokines (IL-4 and IL-10) in colon mucosa. Cur also increased the proportion of IFN-gamma/IL-4 in splenocytes and circulation. Dex and Cur+Dex decreased the expression of Th1 cytokines but could not increase the expression of Th2 cytokines and the proportion of IFN-gamma/IL-4. CONCLUSION: Cur exerted therapeutic effects on colitis by regulating the shift from Th1 to Th2.

Curcumin-attenuated trinitrobenzene sulphonic acid induces chronic colitis by inhibiting expression of cyclooxygenase-2.

AIM: To explore the possible mechanisms of curcumin in rat colitis induced by trinitrobenzene sulfonic (TNBS) acid. METHODS: Rats with TNBS acid-induced colitis were treated with curcumin (30 mg/kg or 60 mg/kg per day ip). Changes of body weight and histological scores as well as survival rate were evaluated. Leukocyte infiltration was detected by myeloperoxidase (MPO) activity assay. The expression of cyclooxygenase-2 (COX-2) was detected by RT-PCR and Western blot. Inflammation cytokines were determined by RT-PCR. Local concentration of prostaglandin E(2) (PGE(2)) in colon mucosa was determined by ELISA. RESULTS: Curcumin improved survival rate and histological image, decreased the macroscopic scores and MPO activity. Also curcumin reduced the expression of COX-2 and inflammation cytokines. In addition, treatment with curcumin increased the PGE(2) level. CONCLUSION: Curcumin has therapeutic effects on TNBS acid-induced colitis, the mechanisms seem to be related to COX-2 inhibition and PGE(2) improvement.

[Correlations of expressions of Glut1 and HIF-1alpha to cellular proliferation of colorectal adenocarcinoma].

BACKGROUND & OBJECTIVE: Malignant cells exhibit increased glucose metabolism and microenvironment hypoxia. Glucose transporter-1 (Glut1) and hypoxia-inducible factor 1 alpha (HIF-1alpha) enhance glucose metabolism under hypoxia. This study was to investigate the expressions of Glut1 and HIF-1alpha in colorectal adenocarcinoma, and explore their correlations to cell proliferation. METHODS: Immunohistochemistry was used to detect expressions of Glut1, HIF-1alpha, and proliferating cell nuclear antigen (PCNA) in 60 specimens of colorectal adenocarcinoma and 20 specimens of normal colorectal tissues. Their interrelations were analyzed. RESULTS: Positive rates of Glut1 and HIF-1alpha were significantly higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (58.3% vs. 0, P < 0.01; 73.3% vs. 0, P < 0.01). PCNA level was significantly higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (65.25+/-16.35 vs. 15.20+/-3.47, P < 0.01). Glut1 and HIF-1alpha levels were positively correlated with PCNA level (r(1)=0.409, P < 0.01; r(2)=0.323, P < 0.05), and were associated with Dukes' stage and lymph node metastasis. CONCLUSIONS: The overexpressions of Glut1 and HIF-1alpha play important roles in carcinogenesis and progression of colorectal adenocarcinoma, and closely correlate with cell proliferation of colorectal adenocarcinoma.

[Expression of NFkappaB p65 and its target genes in gastric cancer and precancerous lesions].

OBJECTIVE: To study the expression of NFkappaB p65 and its target genes in intestinal metaplasia (IM), dysplasia (Dys), gastric cancer (GC) infected with Helicobacter pylori (Hp) and explore the mechanism of infection by cytotoxin-associated antigen A expressing Hp (CagA(+)Hp) in the development of gastric cancer. METHODS: CagA antibody in blood sample of 289 patients was determined by ELISA. Hp was detected by rapid urease test and Warthin starry staining. Expression of NFkappaB p65 and its target genes in IM, Dys and GC was examined by immunohistochemistry. RESULTS: In IMI approximately II, IMIII, DysI, DysII approximately III and GC, the expression of NFkappaB p65 was significantly higher in patients with CagA(+)Hp infection than those without CagA Hp infection. In IMIII and DysII approximately III, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in patients with CagA Hp infection than those without CagA Hp infection. In gastric cancer infected with CagA(+)Hp, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in intestinal type than in diffuse type. CONCLUSION: There are different mechanisms in intestinal type and diffuse type in the development of gastric cancer. The occurrence of intestinal type gastric cancer is associated with CagA(+)Hp infection which by NFkappaB p65 upregulating the expression of c-myc, CyclinD(1),bcl-xl in patients with IMIII, DysII approximately III. It may be an effective method to prevent gastric cancer by inhibiting NFkappaB p65.

Association of tumor necrosis factor genetic polymorphism with chronic atrophic gastritis and gastric adenocarcinoma in Chinese Han population.

AIM: To investigate the association of TNF polymorphisms with chronic atrophic gastritis (CAG) and gastric adenocarcinoma in Chinese Han patients. METHODS: The TNFa-e 5 microsatellites and 3 RFLP sites were typed using PCR technique, followed by high-voltage denaturing PAGE with silver staining and restriction enzyme digestion respectively in specimens from 53 patients with CAG and 56 patients with gastric adenocarcinoma and 164 healthy controls. The PCR products were cloned and sequenced. RESULTS: The frequency of TNF-beta Ncol*1/2 genotype was higher in patients with chronic atrophic gastritis than in healthy controls, but no significant difference was observed (60.38% vs 46.34%, P=0.076). The frequency of TNa10 allele was significantly higher in patients with chronic atrophic gastritis than in healthy controls (19.81% vs 11.89%, P=0.04). However, it did not relate to age, gender, atrophic degree or intestinal metaplasia in patients with chronic atrophic gastritis. The frequency of TNF-beta Ncol*1/2 and d2/d6 genotypes were significantly higher in patients with gastric adenocarcinoma than in healthy individuals (P>0.05). However, TNF-beta Ncol*1/2 and d2/d6 genotypes did not relate to age, gender, grade of differentiation and clinicopathologic stage in patients with gastric adenocarcinoma. The frequency of TNFa6b5c1 haplotype homozygote was significantly lower in patients with gastric adenocarcinoma than in healthy controls (1.79% vs 15.85%, P=0.006). CONCLUSION: TNFa10 allele may be a risk factor for chronic atrophic gastritis. TNF-beta Ncol*1/2 and d2/d6 genotypes are associated with the susceptibility to gastric adenocarcinoma, whereas TNFa6b5c1 haplotype homozygote may contribute to the resistance against gastric adenocarcinoma.

Expression of nuclear factor-kappa B and target genes in gastric precancerous lesions and adenocarcinoma: association with Helicobactor pylori cagA (+) infection.

AIM: To examine the expression of nuclear factor kappaB (NF-kappaB) and its target genes in intestinal metaplasia (IM), dysplasia (DYS) and gastric carcinoma (GC) infected with Helicobacter pylori (H pylori) and to investigate the mechanism underlying H pylori cytotoxin associated gene A (cag A) infection leading to gastric adenocarcinoma. METHODS: Expressions of NF-kappaB/p65 and its target genes: c-myc, cyclinD1 and bcl-xl were immunohistochemically examined in 289 cases of gastric biopsy and resection specimens from patients with IM, DYS and GC infected with H pylori. H pylori in the above mentioned tissues was detected by Warthin-Starry stain and rapid urease tests. IgG antibody to cagA in sera of the patients was measured by ELISA. RESULTS: The positive rates of NF-kappaB/p65 were significantly higher in groups with cagA of IMI-II(28/33), IM III(48/52), DYSI(27/31), DYS II-III(28/32), GC(35/40) than in groups without cagA of IMI-II(4/17), IMIII(3/20), DYSI(3/20), DYSII-III(6/21), GC(10/23). The expressions of c-myc, cyclinD1, and bcl-xl were significantly higher in groups with cagA of IM III(47/52, 49/52, 46/52), DYSII-III(29/32, 26/32, 25/32) than in groups without cagA of IM III(8/20, 7/20, 5/20), DYSII-III(10/21, 8/21, 3/21), which were in conformity with the expression of NF-kappaB in IM III, and DYSII-III. A significantly higher expression level of NF-kappaB/p65, c-myc, cyclinD1 and bcl-xl was detected in intestinal type GC(27/28, 18/28, 22/28, 24/28) than in diffuse type GC(8/12, 3/12, 3/12, 6/12), respectively. CONCLUSION: There may be two different molecular mechanisms in the occurrence of intestinal and diffuse type gastric carcinomas. Intestinal type gastric carcinoma is strongly associated with high expression of c-myc, cyclinD1 and bcl-xl through NF-kappaB/p65 activated by H pylori cagA. Inhibiting the activity of NF-kappaB is an effective and promising way to prevent intestinal type gastric carcinoma.

[Synergistic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and doxorubicin on human colon cancer SW480 cells].

BACKGROUND & OBJECTIVE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) could selectively kill tumor cells. This effect could be improved using some therapeutic agents. The aim of this study was to determine the sensitivity of SW480 cells to TRAIL, the interaction of TRAIL and doxorubicin against colon cancer cells and its possible mechanism. METHODS: SW480 cells were cultured with RPMI1640 medium in regular condition. Cytotoxicity was examined by MTT assay. Cell apoptosis was detected by flow cytometry. The subcellular morphology was observed by electron microscopy. The changes of p53 and Bcl-2 in protein level were quantified by Western blot analysis. RESULTS: (1)SW480 cells were not sensitive to TRAIL,and the IC(50) was more than 1,000 ng/ml. 100 ng/ml of TRAIL could only kill 7.8% of the cells. (2)Concentration-dependent cytotoxicity of doxorubicin was exhibited in SW480 cells, with IC(50) of 65 micromol/L. 0.86 micromol/L of doxorubicin did not affect cell growth. (3)The combination of TRAIL and doxorubicin exhibited synergistic effect on SW480 cells. Subtoxic TRAIL(100 ng/ml)and subtoxic doxorubicin(0.86 micromol/L) killed 80% of the cells. The synergistic cytotoxicity was large partly attributed to cell apoptosis, which was proved by simultaneous flow cytometry assay and electron microscopy. However, TRAIL and doxorubicin did not affect p53 and Bcl-2 protein expression. CONCLUSION: TRAIL in combination with subtoxic doxorubicin could effectively kill SW480 cells, which did not respond to TRAIL alone. Apoptosis was the main manner of this killing effect, and the apoptotic pathway induced by TRAIL and doxorubicin did not involved in the change of p53 and Bcl-2 protein expression.

Role of inducible nitric oxide synthase expression in aberrant crypt foci-adenoma-carcinoma sequence.

AIM: To investigate the expression of inducible nitric oxide synthase (iNOS) in aberrant crypt foci (ACF) -adenoma-carcinoma sequence and its relation with tumor cell apoptosis, proliferation and angiogenesis. METHODS: The expression of iNOS, proliferating cell nuclear antigen (PCNA) and microvessel density (MVD) in different stages of colorectal cancer were studied by immunohistochemical method from 30 normal tissues, 30 nonhyperplastic ACF, 30 hyperplastic ACF, 30 dysplastic ACF, 30 adenomas and 60 carcinomas. The apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method using an Apop Tag in situ detection kit. RESULTS: The immunoreactivity of iNOS significantly increased in the transition from hyperplastic ACF to dysplastic ACF. This transition was associated with a significant decrease in the apoptotic index (AI) (0.73+/-0.37 vs 0.61+/-0.35, P<0.05) and significant increases in the PCNA labeling index (LI) (27.3+/-2.80 vs 40.3+/-3.11, P<0.01) and microvessel density (MVD) (55+/-11.5 vs 70+/-13.2, P<0.01). The expression of iNOS was in low levels and positively correlated with PCNA-LI (r=0.812, P<0.01) and MVD (r=0.863, P<0.01) during transition from normal mucosa to nonhyperplastic ACF and hyperplastic ACF. The expression of iNOS was in high levels and positively correlated with AI (r=0.901, P<0.01) after transition from hyperplastic ACF to dysplastic ACF, adenoma and carcinoma. CONCLUSION: The results suggest that the transition from hyperplastic ACF to dysplastic ACF may be a crucial step in the ACF-adenoma-carcinoma sequence, in which iNOS plays an important role by regulating tumor cell apoptosis, proliferation and angiogenesis.

Synergistic antitumor effect of TRAIL and doxorubicin on colon cancer cell line SW480.

AIM: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent. Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms. METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents. Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT) assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detect apoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay. RESULTS: (1) SW480 cells were not sensitive to TRAIL, with IC(50)>1 mg/L(-1) and dose-independent cytotoxicity. (2) SW480 cells were sensitive to doxorubicin at a certain degree, with dose-dependent cytotoxicity and IC(50)=65.25+/-3.48 micromol/L(-1). (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC(50) of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 microg/L(-1)), combined with subtoxic doxorubicin (0.86 micromol/L(-1)), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12+/-2.67 %, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82+/-1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (P>0.05). CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentration of doxorubicin to induce apoptosis effectively. The status of p53 protein is not involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubicin against colon cancers.