Triptolide enhances the sensitivity of pancreatic cancer PANC-1 cells to gemcitabine by inhibiting TLR4/NF-κB signaling.

BACKGROUND: This study aimed to investigate roles of Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling in triptolide (TPL)-induced sensitivity of pancreatic cancer cells to gemcitabine (GEM). METHODS: In vitro, pancreatic cancer PANC-1 cells were treated with lipopolysaccharide (LPS) to activate TLR4, TLR4-siRNA, GEM alone, or GEM plus TPL. In vivo, nude mice bearing PANC-1 cell xenografts were treated with GEM, TPL, or both. Cell proliferation was detected by MTT assay and Ki-67 staining. Apoptosis was assessed by flow cytometry and TUNEL assay. A double luciferase reporter gene was used to detect NF-κB activity. RESULTS: The sensitivity of PANC-1 cells to GEM was reduced by LPS but enhanced by TLR4-siRNA. TPL inhibited expression of TLR4/NF-κB signaling components, which was reversed by LPS. The TPL+GEM group showed more apoptosis than the LPS+TPL+GEM group. Moreover, the activity of NF-κB and the expression of TLR4, p-p65 Survivin, CyclinD1 and Bcl-2 in the TPL+GEM group were lower than in the LPS+TPL+GEM group, whereas Bax expression was higher. The volume of transplanted tumors in the TPL+GEM group was lower than that in the TPL or GEM group. Phospho-p65, Survivin, CyclinD1 and Bcl-2 expression in transplanted tumors was lower in TPL+GEM group than in either single drug group. The Ki-67 staining score of the TPL+GEM group was lower and tumor cells apoptosis rate was increased when compared with TPL or GEM alone. CONCLUSIONS: TPL enhances the sensitivity of pancreatic cancer PANC-1 cells to GEM by inhibiting TLR4/NF-κB signaling.

Triptolide induces apoptosis and inhibits the growth and angiogenesis of human pancreatic cancer cells by downregulating COX-2 and VEGF.

Triptolide (TPL) inhibits the growth and proliferation of a wide range of human cancer cells, but the underlying mechanism is largely unknown. Here, we report that TPL induces apoptosis and inhibits proliferation of PANC-1 pancreatic cancer cells by downregulating cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF). Cell viability and apoptosis were measured by MTT assay and flow cytometry. Real-time PCR and Western blot were used to examine the expression of COX-2 and VEGF. The Matrigel angiogenesis and Transwell migration were employed to assess tube formation and cell migration. Pancreatic cancer mouse xenografts were established to investigate the in vivo antitumor effects of TPL. TUNEL staining and immunohistochemistry were used to detect the apoptosis rate and protein expression in tumor tissues. TPL inhibited the proliferation of pancreatic cancer cells in a time and concentration-dependent manner and decreased the expression of COX-2 and VEGF in vitro. Furthermore, medium from TPL-treated PANC-1 cells inhibited the proliferation, migration, and tube formation of HUVECs. TPL significantly reduced the growth of pancreatic cancer mouse xenografts, accompanied by an induction of apoptosis, inhibition of angiogenesis, and reduction of COX-2 and VEGF. Our data indicate that suppressing the expression of COX-2 and VEGF may be one of the molecular mechanisms by which TPL induces apoptosis and inhibits the growth and angiogenesis of human pancreatic cancer cells.

Expression of cyclooxygenase-2 and matrix metalloproteinase-9 in gastric carcinoma and its correlation with angiogenesis.

OBJECTIVE: To investigate the expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 in gastric carcinomas, and to correlate this expression with clinicopathological parameters and angiogenesis. METHODS: Ninety-six resected tumor specimens from patients with gastric carcinoma were obtained, and 30 corresponding paracancerous normal tissues were randomly selected as a control. Immunohistochemical staining was used for detecting the expression of COX-2 and MMP-9. Monoclonal antibody against CD34 was used for displaying vascular endothelial cells, and microvascular density (MVD) was calculated by counting of CD34-positive vascular endothelial cells. RESULTS: The positive expression rates of COX-2, MMP-9 and MVD in the cancerous tissue were 80.2%, 74.0%, and 32.5 +/- 8.3, respectively, which were significantly higher than those in the normal tissue (P < 0.01). COX-2, MMP-9 expression rates and MVD in the patients with stages III and IV were 91.4%, 84.5% and 34.9 +/- 8.7, respectively, which were significantly higher than those in the patients with stages I and II (P < 0.01). In addition, the Spearman rank correlation test showed that tumor MVD was closely associated with COX-2 (r = 0.311, P < 0.01) and MMP-9 (r = 0.349, P < 0.01) expressions. CONCLUSIONS: Overexpression of COX-2 and MMP-9 is related to tumor invasion and lymph node metastasis in the gastric carcinoma. These results provide evidence that COX-2 contribute to gastric cancer development by promoting MMP-9 expression and angiogenesis.

[Expression of glutathione S-transferase-pi and DNA topoisomerase II alpha and their implications in colorectal carcinoma].

OBJECTIVE: To investigate the expression of GST-pi and Topo II-alpha, and their relationships with clinicopathological parameters in colorectal carcinoma. METHODS: The expression of GST-pi and Topo II-alpha were detected by avidin-biotin-peroxide complex (ABC) method in tumor specimens, matched paratumor tissues from 60 cases with colorectal carcinoma and normal colonic tissues from 15 cases. RESULTS: The expression rates of GST-pi and Topo II-alpha were 90.0% and 86.7% respectively in tumor tissues, significantly higher than those in matched paratumor tissues and normal tissues (P< 0.01). The expressions of GST-pi and Topo II-alpha were associated with cellular differentiation, Dukes stage and lymph node metastasis (all P< 0.01), but not with tumor size and histological type (all P > 0.05). The expression level of GST-pi was significantly higher in poorly differentiated tumors than that in well differentiated tumors. The expression level of Topo II-alpha in well-differentiated tumors were stronger than that in poorly differentiated tumors. CONCLUSIONS: The detection of GST-pi and Topo II-alpha expressions may be helpful to judge the malignant behavior, metastasis and prognosis in human colorectal carcinoma.

[Relationship between cyclooxygenase- 2 expression and angiogenesis in patients with gastric cancer].

OBJECTIVE: To investigate COX-2 expression in patients with gastric cancer and its relationship with angiogenesis and clinicopathologic features of gastric cancer. METHODS: COX-2 expression and CD34-stained microvessel density (MVD) were detected by immunohistochemical methods in specimens from 96 patients with gastric cancer. The correlations among COX-2 expression, MVD and clinicopathologic features were analyzed. RESULTS: The COX-2 positive rate and MVD in gastric cancer were significantly higher than those in the normal gastric mucosa (80.2% vs. 13.3%; 32.5+/- 8.3 vs. 13.1+/- 2.4, all P< 0.01). The COX-2 positive rate and MVD in the patients with stage III and IV were significantly higher (91.4% and 34.9+/- 8.7 respectively, P< 0.01), than that in the patients with stage I and II. The COX-2 positive rate and MVD in the cases with lymph node metastasis were 87.9% and (35.0+/- 8.5) respectively, higher than those in the cases without lymph node metastasis (P< 0.05). The Spearman rank correlation test showed a significant correlation between COX-2 expression and tumor MVD (r=0.311, P< 0.01). CONCLUSIONS: COX-2 plays an important role in gastric cancer angiogenesis. COX-2 and angiogenesis induced by COX-2 contribute to tumor invasion and lymph node metastasis.

Efficacy of omeprazole and amoxicillin with either clarithromycin or metronidazole on eradication of Helicobacter pylori in Chinese peptic ulcer patients.

AIM: One-week triple therapy with proton pump inhibitors, clarithromycin and amoxicillin has recently been proposed as the first-line treatment for Helicobacter pylori (H. pylori) infection; however, data regarding the effects of this regimen in China are scarce. The aim of this prospective and randomized study was to compare the efficacy of clarithromycin and metronidazole when they were combined with omeprazole and amoxicillin on eradication of H. pylori and ulcer healing in Chinese peptic ulcer patients. METHODS: A total of 103 subjects with H. pylori-positive peptic ulcer were randomly divided into two groups, and accepted triple therapy with omeprazole 20 mg, amoxicillin 1000 mg and either clarithromycin 500 mg (OAC group, n = 58) or metronidazole 400 mg (OAM group, n = 45). All drugs were given twice daily for 7 d. Patients with active peptic ulcer were treated with omeprazole 20 mg daily for 2-4 wk after anti-H. pylori therapy. Six to eight weeks after omeprazole therapy, all patients underwent endoscopies and four biopsies (two from the antrum and two others from the corpus of stomach) were taken for rapid urease test and histological analysis (with modified Giemsa staining) to examine H. pylori. Successful eradication was defined as negative results from both examination methods. RESULTS: One hundred patients completed the entire course of therapy and returned for follow-up. The eradication rate of H. pylori for the per-protocol analysis was 89.3% (50/56) in OAC group and 84.1% (37/44) in OAM group. Based on the intention-to-treat analysis, the eradication rate of H. pylori was 86.2% (50/58) in OAC group and 82.2% (37/45) in OAM group. There were no significant differences in eradication rates between the two groups on either analysis. The active ulcer-healing rate was 96.7% (29/30) in OAC group and 100% (21/21) in OAM group (per-protocol analysis, P>0.05). Six patients in OAC group (10.3%) and five in OAM group (11.1%) reported adverse events (P>0.05). CONCLUSION: One-week triple therapy with omeprazole and amoxicillin in combination with either clarithromycin or metronidazole is effective for the eradication of H. pylori. The therapeutic regimen comprising metronidazole with low cost, good compliance and mild adverse events may offer a good choice for the treatment of peptic ulcers associated with H. pylori infection in China.

Roles of Helicobacter pylori infection and cyclooxygenase-2 expression in gastric carcinogenesis.

AIM: Cyclooxygenase (COX)-2 is over expressed in gastrointestinal neoplasm. Helicobacter pylori (H pylori) infection is causally linked to gastric cancer. However, the expression of COX-2 in various stages of H pylori-associated gastric carcinogenesis pathway has not been elucidated. Therefore, the aim of this study was to clarify the role of H pylori induced COX-2 expression during carcinogenesis in the stomach. METHODS: Gastric biopsies from 138 subjects (30 cases of chronic superficial gastritis (CSG), 28 cases of gastric glandular atrophy (GA), 45 cases of gastric mucosal intestinal metaplasia (IM), 12 cases of moderate gastric epithelial dysplasia and 23 cases of gastric cancer) were enrolled. H pylori infection was assessed by a rapid urease test and histological examination (modified Giemsa staining). The expression of COX-1 and COX-2 in human gastric mucosa was detected by immunohistochemical staining. RESULTS: H pylori infection rate was 64.3% in GA and 69.5% in gastric cancer, which was significantly higher than that (36.7%) in CSG (P<0.05). The positive expression rates of COX-2 were 10.0%, 35.7%, 37.8%, 41.7% and 69.5% in CSG, GA, IM, dysplasia and gastric cancer, respectively. From CSG to GA, IM, dysplasia and finally to gastric cancer, expression of COX-2 showed an ascending tendency, whereas COX-1 expression did not change significantly in the gastric mucosa. The level of COX-2 expression in IM and dysplasia was significantly higher in H pylori-positive than in H pylori-negative subjects (P<0.01). CONCLUSION: COX-2 expression induced by H pylori infection is a relatively early event during carcinogenesis in the stomach.