Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression and chemoresistance by enhancing the secretion of chemotherapy-induced senescence-associated secretory phenotype via activation of DNA damage response pathway.

Senescence frequently occurs in cancer cells in response to chemotherapy (called therapy-induced senescence). Senescent cells can exert paracrine effects through the senescence-associated secretory phenotype (SASP) promoting cancer recurrence and chemoresistance. The altered gut microbiota has been closely associated with cancer progression through the direct interaction with cancer cells. However, little is known about the relationship between the gut microbiota and therapy-induced senescent cells. This study aimed to explore the impact of the gut microbiota on therapy-induced senescent cells and the SASP. We found that esophageal squamous cell carcinoma (ESCC) cells were induced into senescence following platinum-based chemotherapy, accompanied by the secretion of a robust SASP. Furthermore, senescent ESCC cells exerted a tumor-promoting effect through the SASP both in vitro and in vivo. Through 16S rRNA gene sequencing and fluorescence in situ hybridization, we identified that Fusobacterium nucleatum (F. nucleatum) was abundant in human ESCC cancerous tissues and correlated with poor prognosis in ESCC patients. Notably, F. nucleatum further promoted the secretion of the SASP by senescent ESCC cells. Compared with the conditioned medium from senescent ESCC cells, the conditioned medium from F. nucleatum-treated senescent ESCC cells accelerated tumor growth in xenograft models, enhanced migration and invasion abilities, and potentiated chemoresistance both in vitro and in vivo. Mechanistically, F. nucleatum invaded and survived in senescent ESCC cells and induced an increase in DNA damage to further activate the DNA damage response pathway, thus enhancing the SASP. Altogether, these findings reveal for the first time that F. nucleatum promotes the secretion of chemotherapy-induced SASP to drive ESCC progression and chemoresistance, which supports F. nucleatum as a potential target for ESCC therapy.

Targeting mitochondrial transcription factor A sensitizes pancreatic cancer cell to gemcitabine.

BACKGROUND: The survival of pancreatic cancer cells, particularly cancer stem cells which are responsible for tumor relapse, depends on mitochondrial function. Mitochondrial transcription factor A (TFAM) is critical for the regulation of mitochondrial DNA and thus mitochondrial function. However, the possible involvement of TFAM in pancreatic cancer is unknown. METHODS: Human samples were obtained from pancreatic cancers and their adjacent tissues; human pancreatic cell lines were cultured in RPMI1640 medium. TFAM expressions in pancreatic tissues and cultured cells were determined using immunohistochemistry, ELISA, and reverse transcription polymerase chain reaction (RT-PCR). The effect of TFAM on cell growth, migration, colony formation and apoptosis were evaluated. Mitochondrial biogenesis in pancreatic cancer and normal cells were examined. RESULTS: The majority of pancreatic cancer tissues exhibited higher TFAM expression compared to the adjacent counterparts. Consistently, TFAM mRNA and protein levels were higher in pancreatic cancer cell lines than in immortalized normal pancreatic epithelial cells. There was no difference on TFAM level between gemcitabine-sensitive and resistant pancreatic cancer cells. Functional analysis demonstrated that TFAM overexpression activated pancreatic normal and tumor cells whereas TFAM inhibition effectively inhibited the growth of pancreatic cancer cells. TFAM inhibition enhanced gemcitabine's cytotoxicity and suppressed growth, anchorage-independent colony formation and survival of gemcitabine-resistant pancreatic cancer cells. Mechanistic studies showed that TFAM inhibition resulted in remarkable mitochondrial dysfunction and energy crisis followed by oxidative stress. The basal mitochondrial biogenesis level correlated well with TFAM level in pancreatic cancer cells. CONCLUSIONS: TFAM played essential roles in pancreatic cancer via regulating mitochondrial functions which highlighted the therapeutic value of inhibiting TFAM to overcome gemcitabine resistance.

Senescent cells in cancer therapy: why and how to remove them.

Cellular senescence is a stress response that imposes a growth arrest on cancer and nonmalignant cells during cancer therapy. By secreting a plethora of proinflammatory factors collectively termed the senescence-associated secretory phenotype (SASP), therapy-induced senescent cells can promote tumorigenesis. Moreover, the SASP from senescent cells is also able to drive therapy resistance and mediate many adverse effects of cancer therapy. Because senescent cell production often occurs during cancer therapy, it is important to carefully consider these potential detrimental effects. Senotherapy, which refers to selective removal of senescent cells, has been proposed as a promising adjuvant approach to eliminate the adverse effects of senescent cells. Thus, in this review we summarize in detail the mechanisms by which senescent cells contribute to tumorigenesis and therapeutic resistance. Also, we thoroughly discuss the potential strategies regarding how to effectively circumvent the undesirable effects of therapy-induced senescent cells.

Neutrophils injure gallbladder interstitial Cajal-like cells in a guinea pig model of acute cholecystitis.

Acute cholecystitis is a common disease with gallbladder dysmotility. Disease pathogenesis involves immune cell infiltration as well as changes in gallbladder interstitial Cajal-like cells (ICLCs). However, it remains unclear if or how the immune cells affect ICLC morphology, density, distribution, and function in gallbladder tissue during acute cholecystitis. In this study, we explored the acute cholecystitis-related alterations in gallbladder ICLCs in a guinea pig model, focusing on the effects of neighboring neutrophils. Adult guinea pigs were randomly divided into four groups (control, 24 hr common bile duct ligation [CBDL], 48-hr CBDL, and antipolymorphonuclear neutrophil [PMN] treated) and analyzed using methylene blue staining and immunofluorescence. Gallbladder contractility was also monitored. To culture gallbladder ICLCs, collagenase digestion was performed on tissue from 10- to 15-day-old guinea pigs. Neutrophils isolated from the peripheral blood of experimental animals 48-hr postsurgery were also cocultured with the gallbladder ICLCs. Intracellular calcium was detected with Fluo-4 AM dye. Our results showed that gallbladder ICLC density significantly declined during acute cholecystitis and was accompanied by shortening of the cellular processes and damage to their network-like structure. However, pretreatment with anti-PMN partially prevented these changes. Gallbladder contraction was also significantly decreased during acute cholecystitis, and this appeared to be mediated by the neutrophils. Moreover, ICLCs cocultured with neutrophils also had shortened and reduced processes and impaired network-like structure formation. Intracellular calcium transient was less sensitive to contraction agonists and inhibitors when cocultured with neutrophils. Taken together, neutrophils greatly affect gallbladder ICLCs and dysmotility during acute cholecystitis.

Acute Cholecystitis Reduces Interstitial Cells of Cajal in Porcine Gallbladder Through Decreased mRNA Synthesis.

BACKGROUND/AIMS: Acute cholecystitis is a common gastrointestinal disorder, often characterized by acute cholecystitis with gallbladder motility disorder. Interstitial cells of Cajal (ICCs) are the pacemaker cells of gut motility in the gastrointestinal tract. Disruption of ICC function is related to motility disorders. The aim of this study was to explore the cellular and molecular mechanisms of ICCs in acute cholecystitis and after the resolution of acute inflammation. MATERIALS AND METHODS: Fifty adult guinea pigs were randomly divided into five groups: a sham-administered group (control group); two groups that were intraperitoneally administered an anti-polyclonal neutrophil (PMN) antibody 24 h before common bile duct ligation (CBDL); and two groups of guinea pigs that were subjected to CBDL without receiving the PMN antibody. Guinea pigs that underwent CBDL were held for 24 h or 48 h after surgery before being subjected to laparotomy and cholecystectomy. Immunohistochemistry, TUNEL assays, western blotting, and real-time PCR were performed to determine ICC morphology and density, to detect ICC apoptosis, and to examine stem cell factor (SCF) and c-kit protein expression and SCF and c-kit mRNA levels, respectively. RESULTS: Both hematoxylin-eosin staining and histological inflammation scores in the PMN groups were lower than those in the control groups (P < 0.01). No differences were observed in ICC morphology between groups. During acute cholecystitis, ICCs numbers were reduced. Conversely, the density of ICCs increased after inflammation was relieved (P < 0.01). In addition, SCF and c-kit protein and mRNA expression levels decreased during acute cholecystitis (P < 0.05) and increased after inflammation was relieved (P < 0.05). Furthermore, ICC apoptosis increased during acute cholecystitis and decreased after resolution of acute cholecystitis (P < 0.01). CONCLUSIONS: In acute cholecystitis, ICC injury may be related to gallbladder motility disorder.

Effect of Neutrophils on Gallbladder Interstitial Cajal-Like Cells in Guinea Pig Model of Acute Cholecystitis.

BACKGROUND: Acute cholecystitis is a common condition in gallbladder motility disorder. Interstitial Cajal-like cells (ICLCs) in the gallbladder are known as one of the players in the complex motility mechanisms affecting gallbladder motility. AIM: This study explored morphological symptoms and molecular mechanisms underlying gallbladder ICLC changes induced by acute cholecystitis. MATERIALS AND METHODS: Fifteen adult guinea pigs were randomly divided into 3 groups: sham-operated group (healthy controls) and 2 experimental groups wherein these guinea pigs were subjected to common bile duct ligation to induce acute cholecystitis. Neutrophils were isolated from the peripheral blood of sham-operated animals and from the experimental animals at 24 and 48 h after surgery, and co-cultured with gallbladder ICLCs. The morphology of gallbladder ICLCs was examined by laser confocal immunofluorescence microscopy, TUNEL assay was used to detect apoptosis, and western blot and real-time PCR were performed to detect stem cell factor (SCF) and c-kit protein and mRNA expression, respectively. RESULTS: No morphological differences in the gallbladder ICLCs were observed between single-culture and co-culture with healthy control neutrophil groups. However, the ICLCs in all co-culture groups with acute inflammation were impaired. In the co-culture groups, the rate of ICLC apoptosis was significantly higher than that in the single-culture group. SCF and c-kit protein and mRNA expression levels decreased in all co-culture groups as well. CONCLUSION: We demonstrated that the neutrophils are involved in gallbladder ICLC injury in acute cholecystitis cases and associated with gallbladder motility disorder.

The Role of Interstitial Cells of Cajal in Acute Cholecystitis in Guinea Pig Gallbladder.

BACKGROUND/AIMS: Acute cholecystitis is common in gallbladder motility disorder. Interstitial cells of Cajal (ICCs) in the gallbladder are involved in the regulation of gallbladder motility. The aim of this study was to explore the change of gallbladder ICCs in acute cholecystitis. METHODS: Thirty adult guinea pigs were randomly divided into 3 groups: a sham-operated group (healthy controls) and 2 study groups. The animals in the study group were subjected to bile duct ligation and then to laparotomy and cholecystectomy at 24 and 48 hours after surgery. Immunohistochemistry, immunohistofluorescence, and laser confocal microscopy were performed to observe the shape, size, morphology, and density of gallbladder ICCs. Western blot and real-time PCR were performed to detect stem cell factor and c-kit protein and mRNA expression, respectively. RESULTS: There were no differences in the shape, size, and morphology of the gallbladder ICCs in the control and the two acute cholecystitis groups. Density of gallbladder ICCs, SCF level, and c-kit protein and mRNA expression all decreased in the acute cholecystitis groups. Further, SCF level and c-kit protein and mRNA expression decreased with progress of acute cholecystitis (all P < 0.05). CONCLUSION: Acute cholecystitis can decrease ICCs through repression of SCF and c-kit expression and that ICCs loss play a role in acute cholecystitis.

Risk factors for proton pump inhibitor refractoriness in Chinese patients with non-erosive reflux disease.

AIM: To analyze risk factors for refractoriness to proton pump inhibitors (PPIs) in patients with non-erosive reflux disease (NERD). METHODS: A total of 256 NERD patients treated with the PPI esomeprazole were enrolled. They were classified into symptom-free and residual symptoms groups according to Quality of Life in Reflux and Dyspepsia (QolRad) scale. All subjects completed questionnaires on psychological status (self-rating anxiety scale; self-rating depression scale) and quality of life scale (Short Form 36). Multivariate analysis was used to determine the predictive factors for PPI responses. RESULTS: According to QolRad, 97 patients were confirmed to have residual reflux symptoms, and the remaining 159 patients were considered symptom free. There were no significant differences between the two groups in lifestyle factors (smoking and alcohol consumption), age, Helicobacter pylori infection, and hiatal hernia. There were significant differences between the two groups in relation to sex, psychological distress including anxiety and depression, body mass index (BMI), and irritable bowel syndrome (IBS) (P < 0.05). Logistic regression analysis found that BMI < 23, comorbid IBS, anxiety, and depression were major risk factors for PPI resistance. Symptomatic patients had a lower quality of life compared with symptom-free patients. CONCLUSION: Some NERD patients are refractory to PPIs and have lower quality of life. Residual symptoms are associated with psychological distress, intestinal disorders, and low BMI.

Deoxycholic acid inhibits smooth muscle contraction via protein kinase C-dependent modulation of L-type Ca2+ channels in rat proximal colon.

The aim of this study was to investigate the effects of deoxycholic acid (DCA) on the contractions of rat proximal colonic smooth muscle (PCSM) in vitro. The contractile response of rat PCSM strips was tested using a polyphysio-graph. The whole cell patch-clamp technique was also used in rat colonic smooth muscle cells (SMCs) isolated by an enzymatic procedure to record the L-type calcium current (I(Ca-L)) prior to and following the application of various concentrations of DCA. The application of DCA (10(-6)-10(-4) M) decreased the amplitude of spontaneous contractions of the PCSM strips in a dose-dependent manner. The administration of DCA (10(-5) M) caused the relaxation of isolated smooth muscle strips pre-contracted by acetylcholine (Ach) or KCl (by 12.2±1.5 and 16.3±6.9%, respectively). The concentration-response curve of CaCl2 was shifted to the right. Pre-treatment of the strips with the protein kinase C (PKC) inhibitor chelerythrine (1 µM) significantly attenuated the effects of DCA on the strips pre-contracted by Ach. DCA reduced the peak I(Ca-L) by 6.02±0.87% at 10(-6) M, 15.02±1.73% at 10(-5) M and 47.14±3.79% at 10(-4) M. DCA shifted the current-voltage (I-V) curve of ICa-L upward, but the contour of the I-V curve was unchanged, and the peak current-induced voltage remained at 0 mV. Pre-treatment with chelerythrine (1 µM) blocked the actions of DCA on the I(Ca-L). Taken together, the actions of DCA on I(Ca-L) in rat colonic SMCs contributed to a negative inotropic effect. These actions appear to be mediated through protein kinase C. Furthermore, this study suggests another possible mechanism for the DCA-related modulation of gastrointestinal motility.

[Significance of caveolin-1 protein detected by quantum dots technique in human lung cancer invasiveness and metastasis.].

OBJECTIVE: Fluorescent semiconductor nanocrystals [also known as quantum dots (QDs)] are nanometer-sized light-emitting particles and are emerging as a new class of fluorescent probes for cancer detection, due to their unique optical and electronic properties. The aim of this study was to investigate the expression of caveolin-1 (Cav-1), extracellular matrix metalloproteinase inducer (CD(147)/EMMPRIN), matrix metalloproteinase-2 (MMP-2) proteins in the human lung cancer tissue microarray (TMA) by QDs immunofluorescence histochemistry (QDs-IHC) and therefore to evaluate the relationship between Cav-1 protein and lung cancer invasiveness and metastasis. METHODS: QDs-IHC combined with TMA were used to detect the expression of Cav-1, CD(147) and MMP-2 proteins in 70 cases of human lung cancers and 5 cases of noncancerous lung tissues. RESULTS: The average immunofluorescence intensity of Cav-1 protein in the lung cancer group was 55 +/- 23, significantly lower than that in the control group (80 +/- 4, t = 2.461, P = 0.016). The expression of Cav-1 was not associated with the age and the gender of the patients, nor with the histology type of lung cancer (P > 0.05). The average immunofluorescence intensity of Cav-1 protein was associated significantly with TNM staging (t = 2.466, P = 0.016) and lymph node metastasis (t = 2.972, P = 0.004). A negative correlation was observed between Cav-1 and CD(147) protein expression (r = -0.331, P = 0.005), but no correlation was observed between Cav-1 and MMP-2 protein expression (P = 0.193). CONCLUSIONS: QDs-IHC could accurately and quantitatively detect different protein location in lung cancer TMA. A close relationship was detected between Cav-1 protein and the development of lung cancer. High expression of Cav-1 may be involved in invasiveness and metastasis of lung cancer, possibly through the regulation of CD(147) rather than MMP-2 activition.

Increase of mast cells may be associated with infiltration of eosinophils and proliferation of microvessels in gastric eosinophilic granuloma.

BACKGROUND: Gastric eosinophilic granuloma (GEG) is a rare disease. Recently this disease has begun to increase in China. In the present study, the function and the role of mast cells (MC) in the pathogenesis of GEG were investigated. METHODS: Paraffin-embedded tissue sections from 23 GEG patients and 15 gastric ulcer (GU) patients, were stained with antihuman mast cell tryptase for counting the MC and degranulated MC. Antihuman CD34 antibody was used for detecting the microvessel density (MVD) with immunohistochemical technique. Mast cell degranulation was also studied using electron microscopy. RESULTS: The quantity of both MC and degranulated MC were higher in both GEG and GU than in normal gastric mucosa. The proportion of degranulated MC was higher in the GEG but in GU it was similar to normal mucosa. The MVD was higher in both GU and GEG than that in the normal gastric mucosa and it was higher in the high-MC group than in the low-MC group in GEG. The positive correlation between eosinophil and MC was present only in GEG, not in GU. CONCLUSIONS: The infiltration of eosinophils and MVD may be associated with the increase of MC in GEG. This suggests that in addition to eosinophils, MC might be the important cells in the pathogenesis of GEG.

Ovarian hormone modulates 5-hydroxytryptamine 3 receptors mRNA expression in rat colon with restraint stress-induced bowel dysfunction.

AIM: To examine the effects of ovarian hormone on the expression of 5-hydroxytryptamine 3 receptors (5-HT3R) in rat colon of restraint stress-induced bowel dysfunction. METHODS: Twenty-four female Sprague-Dawley rats were randomly divided into three groups of 8 each: sham operation, ovariectomy (OVX) and ovariectomy with estrogen (E2) and progesterone (P) replacement therapy (OVX+E2+P). The rats were subjected to 1-h restraint stress 4 wk after operation. The changes of defecation were monitored by collection of fecal pellets. The gonadal steroids were measured in duplicate by radioimmunoassay (RIA). The expression of 5-HT3R mRNA in the colon was studied by RT-PCR. RESULTS: Compared with sham group and OVX+E2+P group, OVX group showed increase in fecal pellets and decrease in the time of vitreous pellets excretion (P<0.01). Serum levels of E2 and P were suppressed in OVX group and restored following treatment with ovarian steroids (P<0.01), and the levels of 5-HT3R mRNA in the colon of ovariectomized rats were significantly increased, the expression of 5-HT3R mRNA was significantly decreased in hormone replacement therapy group (P<0.01). CONCLUSION: Ovarian hormone plays a role in the regulation of 5-HT3R expressions in restraint stress-induced bowel dysfunction of rats. The interactions between ovarian steroids and gastrointestinal tract may have major pathophysiological implications in 5-HT-related disorders, such as irritable bowel syndrome (IBS).

[Nimesulide, a selective cyclooxygenase-2 inhibitor inhibits telomerase activity by blocking activation of PKB in gastric cancer cell line].

OBJECTIVE: To study the effects of nimesulide, a selective COX-2 inhibitor, on cell viability, telomerase and PKB activities in human gastric cancer cell line SGC7901 and to explore its molecular mechanism of selective growth inhibition. METHODS: MTT assay was used to determine cell viability after incubation for 0, 12, 24, and 48 h in different concentrations (0, 25, 50, 100, 200 micro mol/L) of nimesulide and/or okadaic acid (300 nmol/L). Telomerase and protein kinase B (PKB) activities were detected using TRAP PCR-ELISA and nonradioactive IP-kinase assay. RESULTS: Nimsulide caused a time and dose-dependent reduction of cell numbers of SGC7901. The telomerase and PKB activities were significantly inhibited, and the inhibition of telomerase activity was partly associated with decrease in PKB activity. CONCLUSION: Selective COX-2 inhibitor nimesulide inhibits telomerase activity of gastric cancer cells by partly blocking the activation of protein kinase B. The results suggest an additional signaling pathway underlying the anti-cancer effect of COX-2 inhibitor.

Expression of NF-kappaB and human telomerase reverse transcriptase in gastric cancer and precancerous lesions.

AIM: To investigate the expression of NF-kappaBp65 protein and human telomerase reverse transcriptase (hTERT) and their correlation in gastric cancer and precancerous lesions. METHODS: Forty-one patients with primary gastric cancer, 15 with dysplasia, 23 intestinal metaplasia and 10 with normal gastric mucosa were included in this study. Expression of NF-kappaBp65 protein, hTERT mRNA and protein were determined by immunohistochemistry and in situ hybridization. RESULTS: The rate of p65 expression in normal gastric mucosa, intestinal metaplasia, dysplasia and carcinoma was 0%, 34.78%, 53.33% and 60.98%, respectively, while the rate of hTERT mRNA expression was 10.00%, 39.13%, 66.67% and 85.37% and the rate of hTERT protein expression was 0%, 30.43%, 60.00% and 78.05%, respectively. All the three parameters were significantly increased in dysplasia and carcinoma compared to normal mucosa, while the expression levels were also significantly higher in carcinoma than in intestinal metaplasia (P<0.05). In gastric cancer tissues, nuclear staining rates of p65 and hTERT protein were both significantly associated with the degree of differentiation, lymph node metastasis, clinical stage and invasion depth (P<0.05). However, hTERT mRNA expression was only significantly associated with clinical stage. There was a positive correlation between p65 and hTERT mRNA (rs=0.661-0.752, P<0.01), and between hTERT protein and hTERT mRNA (rs=0.609-0.750, P<0.01). CONCLUSION: NF-kappaBp65 and hTERT expressions are upregulated at the early stage of gastric carcinogenesis. NF-kappaB activation may contribute to hTERT expression and thereby enhance telomerase activity, which represents an important step in carcinogenesis progress.

[Clinical significance of detecting VEGF, CD44v6, MMP-2, and MMP-9 in malignant ascites].

BACKGROUND & OBJECTIVE: Cytological examination, despite of its high specificity, has been found to have a low sensitivity in the diagnosis of malignant ascites due to the high percentage of false negative results. So it is indispensable to seek appropriate cancerous markers in ascites. The aim of this study was to determine vascular endothelial growth factor (VEGF), CD44v6 levels and matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) activities in ascites so as to provide scientific basis for clinical diagnosis and treatment of malignant ascites. METHODS: A total of 67 samples were collected, including cirrhotic ascites (36), tuberculosis ascites (8), and malignant ascites (23). VEGF and CD44v6 levels were determined using enzyme-linked immunosorbent assay (ELASA). MMP-2 and MMP-9 activities were determined by gelatin zymography. RESULTS: VEGF and CD44v6 levels in malignant ascites were 640.74+/-264.81 ng/L and 89.22+/-38.20 microg/L, respectively, which were significantly higher than those in cirrhotic ascites (67.05+/-51.91 ng/L, 44.79+/-18.02 microg/L) and tuberculosis ascites (88.25+/-24.12 ng/L, 50.25+/-12.57 microg/L) (P< 0.01, respectively). The activities of MMP-2 and MMP-9 could not be detected in cirrhotic ascites and tuberculosis ascites but could be detected in 20 and 18 out of 23 malignant ascites respectively. CONCLUSION: VEGF, CD44v6 levels increase significantly and MMP-2, MMP-9 activities are obvious in cancerous ascites. The detection of VEGF, CD44v6 levels and MMP-2, MMP-9 activities are useful in differential diagnosis of benign and malignant ascites.

Ameliorative effects of sodium ferulate on experimental colitis and their mechanisms in rats.

AIM: To investigate the ameliorative effects of sodium ferulate (SF) on acetic acid-induced colitis and their mechanisms in rats. METHODS: The colitis model of Sprague-Dawley rats was induced by intracolon enema with 8% (V/V) of acetic acid. The experimental animals were randomly divided into model control, 5-aminosalicylic acid therapy group and three dose of SF therapy groups. The 5 groups were treated intracolonically with normal saline, 5-aminosalicylic acid (100 mg x kg(-1)), and SF at the doses of 200, 400 and 800 mg x kg(-1) respectively and daily (8:00 am) for 7 days 24 h following the induction of colitis. A normal control group of rats clystered with normal saline instead of acetic acid was also included in the study. Pathological changes of the colonic mucosa were evaluated by the colon mucosa damage index (CMDI) and the histopathological score (HS). The insulted colonic mucosa was sampled for a variety of determinations at the end of experiment when the animals were sacrificed by decapitation. Colonic activities of myeloperoxidase (MPO) and superoxide dismutase (SOD), and levels of malondialdehyde (MDA) and nitric oxide (NO) were assayed with ultraviolet spectrophotometry. Colonic contents of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) were determined by radioimmunoassay. The expressions of inducible nitric oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2) and nuclear factor kappa B (NF-kappaB) p65 proteins in the colonic tissue were detected with immunohistochemistry. RESULTS: Enhanced colonic mucosal injury, inflammatory response and oxidative stress were observed in the animals clystered with acetic acid, which manifested as the significant increase of CMDI, HS, MPO activities, MDA and NO levels, PGE2 and TXB2 contents, as well as the expressions of iNOS, COX-2 and NF-kappaB p65 proteins in the colonic mucosa, although the colonic SOD activity was significantly decreased compared with the normal control (CMDI: 2.9+/-0.6 vs 0.0+/-0.0; HS: 4.3+/-0.9 vs 0.7+/-1.1; MPO: 98.1+/-26.9 vs 24.8+/-11.5; MDA: 57.53+/-12.36 vs 9.21+/-3.85; NO: 0.331+/-0.092 vs 0.176+/-0.045; PGE2: 186.2+/-96.2 vs 42.8+/-32.8; TXB2: 34.26+/-13.51 vs 8.83+/-3.75; iNOS: 0.365+/-0.026 vs 0.053+/-0.015; COX-2: 0.296+/-0.028 vs 0.034+/-0.013; NF-kappaB p65: 0.314+/-0.026 vs 0.039+/-0.012; SOD: 28.33+/-1.17 vs 36.14+/-1.91; P<0.01). However, these parameters were found to be significantly ameliorated in rats treated locally with SF at the given dose protocols, especially at 400 mg x kg(-1) and 800 mg x kg(-1) doses (CMDI: 1.8+/-0.8, 1.6+/-0.9; HS: 3.3+/-0.9, 3.1+/-1.0; MPO: 63.8+/-30.5, 36.2+/-14.2; MDA: 41.84+/-10.62, 37.34+/-8.58; NO: 0.247+/-0.042; 0.216+/-0.033; PGE2: 77.2+/-26.9, 58.4+/-23.9; TXB2: 18.07+/-14.83; 15.52+/-8.62; iNOS:0.175+/-0.018, 0.106+/-0.019; COX-2: 0.064+/-0.018, 0.056+/-0.014; NF-kappaBp65: 0.215+/-0.019,0.189+/-0.016; SOD: 32.15+/-4.26, 33.24+/-3.69; P<0.05-0.01). Moreover, a therapeutic dose protocol of 800 mg x kg(-1) SF was observed as effective as 100 mg x kg(-1) of 5-ASA in the amelioration of colonic mucosal injury as evaluated by CMDI and HS. CONCLUSION: Administration of SF intracolonically may have significant therapeutic effects on the rat model of colitis induced by acetic acid enema, which was probably due to the mechanism of antioxidation, inhibition of arachidonic acid metabolism and NF-kappaB expression.

Detection of type IV collagenase activity in malignant ascites.

AIM: Type IV collagenase participates in invasion and metastasis of cancer cells. Malignant ascites is a manifestation of advanced malignant disease that is associated with invasion and metastasis of the peritoneal cavity. Thus, it is reasonable to hypothesize that type IV collagenase is linked to malignant ascites. The purpose of our study was to detect type IV collagenase activity in malignant ascites so as to provide the scientific basis for clinic diagnosis and treatment of malignant ascites. METHODS: Cirrhotic ascites (n=36), tuberculous ascites (n=8) and malignant ascites (n=23) from patients with gastric cancer (n=6), colon cancer (n=5), ovarian cancer (n=8) and other cancers (n=4), including 2 hepatocellular cancers, 1 pancreatic cancer, 1 primary peritoneal carcinoma were collected by paracentesis. The ascites were made cell-free by centrifugation and stored frozen at -70 degrees C before determination. Type IV collagenase activity was determined by gelatin zymography. RESULTS: The activity of matrix metalloproteinases-2 and -9 could not be detected in ascites of hepatic cirrhosis and tuberculous peritonitis but could be detected in 20 and 18 out of 23 malignant ascites respectively. The positive rate of type IV collagenase (MMP-2, 87.0% and MMP-9, 78.3%) was higher than that by routine ascites tests (P<0.01) in malignant ascites. Furthermore, the activity of MMP-2 was higher than that of MMP-9 (P=0.022<0.05). CONCLUSION: Type IV collagenase is positive in malignant ascites. Detection of type IV collagenase activity is useful in qualitative diagnosis of ascites. Type IV collagenase may play an important role in malignant ascites formation.

Role of VEGF and CD44v6 in differentiating benign from malignant ascites.

AIM: To detect the vascular endothelial growth factor (VEGF) and soluble splice variant 6 of CD44 (sCD44v6) levels in ascites and to explore their role in differentiating benign from malignant ascites. METHODS: Cirrhotic ascites (n=36), tuberculosis ascites (n=8) and malignant ascites (n=23) were collected and studied. Concentrations of soluble VEGF and sCD44v6 in various kinds of ascites (n=67) were measured using a sandwich enzyme-linked immunoadsorbent assay. RESULTS: VEGF and sCD44v6 levels in malignant ascites were 640.74+/-264.81 pg/ml and 89.22+/-38.20 ng/ml, respectively, both of which were significantly higher than those in cirrhotic ascites and tuberculous ascites (q=18.98, 11.89 and q=8.92, 5.09; P<0.01). However, the levels of VEGF and sCD44v6 in cirrhotic and tuberculous ascites had no significant difference (q=0.48, 0.75; P>0.05). Furthermore, VEGF levels in malignant ascites in patients with ovarian cancer were higher than those with gastric and colon cancer (q=5.03, 6.79; P<0.01, respectively). But differences of VEGF levels between gastric and colon cancer were not significant (q=1.90, P>0.05). Whereas, sCD44v6 levels in malignant ascites from patients with ovarian, gastric and colon cancer had no significant difference (q=0.06, 0.91, 0.35; P>0.05, respectively). In comparison with cirrhotic and tuberculous ascites, when the upper limit of its VEGF mean levels 119.44 pg/ml (70.90+/-48.54) and sCD44v6 mean levels 63.59 ng/ml (44.42+/-19.17) was taken as the minimum cutoff limit, the sensitivity and specificity of VEGF and sCD44v6 of this assay to the diagnosis of malignant ascites were 91.3%, 90.9% and 73.9%, 88.7% respectively. CONCLUSION: Elevated levels of VEGF and sCD44v6 may be useful in differential diagnosis of benign and malignant ascites.

Antiproliferative effect of octreotide on gastric cancer cells mediated by inhibition of Akt/PKB and telomerase.

AIM: To investigate the antiproliferative effect of octreotide, a long-acting analogue of somatostatin, on gastric cancer cell line SGC7901 and its possible molecular mechanisms. METHODS: Gastric cancer cell line SGC7901 employed in the study was treated with 0.008, 0.04, 0.2, 1, 5 and 25 microg.ml(-1) of octreotide respectively for 24 h to evaluate the antiproliferative effect of somatostatin analog on the tumor cells by MTT assay method. To elucidate the underlying mechanism, the cells were exposed to 1 microg.ml(-1) of octreotide for 0, 12, 24 and 48 h, when their Akt/PKB and telomerase activities were respectively determined using PCR-ELSIA and nonradioactive protein kinase assay protocols. The same experimental procedures were also performed in the control cells that were treated with corresponding vehicles instead of somatostatin analog. RESULTS: After exposed to octreotide for 24 h at the concentrations of more than 1 microg.ml(-1), SGC7901 cells exhibited a dose-dependent inhibition of growth with the inhibiting rate to be as high as 34.66% when 25 microg.ml(-1) of octreotide was applied. The Akt/PKB and telomerase activity of SGC7901 cells was significantly inhibited when the cells were exposed to 1 microg.ml(-1) of octreotide for 12, 24 and 48 h compared with that of their control counterparts (P<0.01), both of which exhibited in a time-dependent manner. CONCLUSION: The antiproliferative effect of octreotide on SGC7901 cells might be mediated by the inhibition of Akt/PKB and telomerase.