Enhanced expression of human β-defensin 2 in peripheral lungs of patients with chronic obstructive pulmonary disease

Human β-defensin 2 (hBD-2) has antimicrobial activity and may play a role in airway mucosal defense, but studies have not yet examined its expression in lung tissue of patients with chronic obstructive pulmonary disease (COPD). Here we investigated hBD-2 levels in lung tissues of COPD patients and analyzed their correlations with IL-8, IL-1β, cigarette smoking and lung function in order to see whether the protein may be involved in pathogenesis of the disease. Peripheral lung tissue specimens were obtained from 51 patients who underwent lung resection for peripheral lung cancer: healthy non-smokers (n = 8), healthy current smokers (n = 7), non-smokers with COPD (n = 11), and current smokers with COPD (n = 25). RT-PCR and immunohistochemical staining were used to detect expression levels of hBD-2, IL-8 and IL-1β. Expression of hBD-2 mRNA was significantly higher in COPD patients than in healthy controls, and significantly higher in current smokers than in non-smokers (p < 0.05). Among healthy controls, hBD-2 mRNA levels were similar between current smokers and non-smokers. Immunohistochemistry showed hBD-2 protein to be expressed mainly in epithelia of distal bronchioles and its expression pattern among our patient groups mirrored that of the mRNA. IL-8 mRNA levels were significantly higher in COPD patients than in healthy controls (p < 0.05), while IL-1β mRNA levels did not differ significantly among the groups. Levels of hBD-2 mRNA positively correlated with levels of IL-8 mRNA (r = 0.545, p = 0.002), and negatively correlated with FEV1/FVC ratios and with predicted FEV1% values (r = −0.406, p = 0.011). Our results indicate that hBD-2 expression is elevated in distal airways of COPD patients and that it may be involved in pathogenesis of the disease. Our data implicate cigarette smoking as a factor that may elevate hBD-2 levels in lung tissues of COPD patients.     

Inhibition of C6 rat glioma proliferation by [Ru2Cl(Ibp)4] depends on changes in p21, p27, Bax/Bcl2 ratio and mitochondrial membrane potential

The ruthenium compound [Ru₂Cl(Ibp)₄] (or RuIbp) has been reported to cause significantly greater inhibition of C6 glioma cell proliferation than the parent HIbp. The present study determined the effects of 0-72 h exposure to RuIbp upon C6 cell cycle distribution, mitochondrial membrane potential, reactive species generation and mRNA and protein expression of E2F1, cyclin D1, c-myc, pRb, p21, p27, p53, Ku70, Ku80, Bax, Bcl2, cyclooxygenase 1 and 2 (COX1 and COX2). The most significant changes in mRNA and protein expression were seen for the cyclin-dependent kinase inhibitors p21 and p27 which were both increased (p < 0.05). The marked decrease in mitochondrial membrane potential (p < 0.01) and modest increase in apoptosis was accompanied by a decrease in anti-apoptotic Bcl2 expression and an increase in pro-apoptotic Bax expression (p < 0.05). Interestingly, COX1 expression was increased in response to a significant loss of prostaglandin E₂ production (p < 0.001), most likely due to the intracellular action of Ibp. Future studies will investigate the efficacy of this novel ruthenium-ibuprofen complex in human glioma cell lines in vitro and both rat and human glioma cells growing under orthotopic conditions in vivo.     

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI₅₀) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p < 0.0001, T-test). At 8 μg/ml (GI₇₀), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p < 0.05, T-test, n = 8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI₅₀ of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p < 0.05, T-test, n = 9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.     

Overexpression of the Interferon regulatory factor 4-binding protein in human colorectal cancer and its clinical significance

Background: IFN regulatory factor 4-binding protein (IBP) is a novel type of activator of Rho GTPases. It has been linked with differentiation and apoptosis of lymphocytes, but its function in oncogenesis remains unclear. Here we studied the expression of endogenous IBP in four human colorectal cancer cell lines, normal, adenoma and tumor colorectal tissues. Methods: Molecular (Western blot and RT-PCR), and confocal analyses were used to investigate IBP expression in human colorectal cancer cell lines. Matched normal and tumor tissue sections of 63 patients and 15 adenoma tissue sections were analyzed for IBP expression by immunohistochemistry (IHC). Results: IBP was ubiquitely expressed in human colorectal cancer cell lines. The expression of IBP can be detected at both the mRNA and protein level in SW480, SW620 and HT29 cells. Clinically, IBP were elevated in human colorectal cancer specimens in comparison to normal colorectal tissues. Substantial high expression of IBP was observed in colorectal cancer tissues (67%), whereas corresponding normal tissues and 15 adenoma tissues showed consistently absent immunoreactivity of IBP. Moreover, IBP expression is correlated with the differentiation level of colorectal cancer cells (p < 0.05) and clinical stage of patients (p < 0.01). Conclusions: Our data show, for the first time, a dysregulated expression of IBP in human colorectal cancer, offering new perspectives for its role in cancer development and progression. IBP may be a novel tumor marker and a therapeutic target for colorectal cancer.     

The clinicopathological significance of REIC expression in colorectal carcinomas

REIC is down-regulated in immortalized cell lines compared with the parental normal counterparts, and could inhibit colony formation, tumor growth and induce apoptosis. Here, its expression was examined by immunohistochemistry on tissue microarray containing colorectal non-neoplastic mucosa (NNM), adenoma and adenocarcinoma. Colorectal carcinoma tissue and cell lines were studied for REIC expression or its secretory level by Western blot, RT-PCR or enzyme-linked immunosorbent assay (ELISA). The results demonstrated that REIC was differentially expressed in Colo201, Colo205, DLD-1, HCT-15, HCT-116, HT-29, KM-12, SW480, SW620, and WiDr with its secretion concentration less than 300 pg/mL. Carcinomas showed statistically lower REIC expression than matched NNM with no difference for protein content. Immunohistochemically, REIC expression was significantly decreased from NNM, adenoma to adenocarcinoma (p<0.05). REIC expression was negatively correlated with depth of invasion, TNM staging, dedifferentiation, Capase-3 and nuclear inhibitor of growth 5 (ING5) expression (p<0.05), while not with age, sex, tumor size, lymphatic or venous invasion, or lymph node metastasis (p>0.05). Kaplan-Meier analysis indicated that REIC expression was not associated with the prognosis of colorectal carcinomas (p>0.05). Cox's analysis demonstrated that lymphatic and venous invasion, lymph node metastasis, and UICC staging were independent prognostic factors for carcinoma (p<0.05). Our study indicated that down-regulated REIC expression might play an important role in colorectal adenoma-adenocarcinoma sequence and subsequent progression. Aberrant REIC expression might be employed as a good marker of pathogenesis and development of colorectal carcinomas.     

Modulation of ephrinB2 leads to increased angiogenesis in ischemic myocardium and endothelial cell proliferation

Eph/ephrin signaling is pivotal in prenatal angiogenesis while its potential role in postnatal angiogenesis largely remains to be explored. Therefore its putative angiogenic and therapeutic effects were explored in endothelium and in myocardial ischemia. In culture of human aortic endothelial cells the fusion protein ephrinB2-Fc induced cell proliferation (p < 0.0005) and in the murine aortic ring model ephrinB2-Fc induced increased sprouting (p < 0.05). Myocardial infarction was induced by ligation of the left anterior descending artery in mouse. During the following 2 weeks mRNA of the receptor/ligand pair EphB4/ephrinB2 was expressed dichotomously (p < 0.05) and other Eph/ephrin pairs were expressed to a lesser degree. Twenty-four hours after intraperitoneal administration of ephrinB2-Fc it was detected in abundance throughout the myocardium along capillaries, showing signs of increased mitosis. After 4 weeks the capillary density was increased 28% in the periinfarcted area (p < 0.05) to a level not different from healthy regions of the heart where no change was observed. These results implicate that EphB4/ephrinB2 is an important signaling pathway in ischemic heart disease and its modulation may induce therapeutic angiogenesis.     

Ursolic acid inhibits tumor angiogenesis and induces apoptosis through mitochondrial-dependent pathway in Ehrlich ascites carcinoma tumor

Ursolic acid (UA) is a pentacyclic triterpene naturally occurring in many plant foods. In the present study, we investigated anti-cancer activity of UA in vivo in Ehrlich ascites carcinoma (EAC) tumor. 15 × 10⁶ EAC cells were implanted intraperitoneally (i.p., ascitic tumor) and subcutaneous (s.c., solid tumor) in Swiss albino mice. Mice with established tumors received UA i.p. at 25, 50 and 100 mg/kg bw for 14 d in ascitic and 100 mg/kg bw in solid tumor for 30 d. On day 15, blood samples were collected for hematological assessment of hemoglobin (Hb%), RBCs, WBCs and PCV. Tumor volume, cell viability, angiogenic, anti-angiogenic, anti-inflammatory factors and antioxidant parameters were determined. Immunohistochemistry analysis for VEGF, iNOS, CD31, caspase-3 and Bax were also performed. UA significantly inhibited tumor growth, cell viability, in both ascites and solid tumor model in vivo (p < 0·001). The anti-angiogenic effects were accompanied with decreased VEGF, iNOS, TNF-α and increased IL-12 levels. UA at 100 mg/kg bw dose significantly increased SOD and CAT activity (p < 0.01). GSH and TBARS were increased as compared to control group (p < 0.001). Furthermore, UA increased total RBCs, WBCs as well as Hb% significantly (p < 0.05) compared to cyclophosphamide (CP). Histopathological examination of tumor cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. Decreased peritoneal angiogenesis showed the anti-angiogenic potential. UA downregulated VEGF & iNOS expression whereas bax and caspase-3 expressions were upregulated suggesting drug induced tumor cell apoptosis through activating the pro-apoptotic bcl-2 family and caspase-3 and downregulation of VEGF. The present study sheds light on the potent antitumor property of the UA and can be extended further to develop therapeutic protocols for treatment of cancer.     

Decreased circulating miR-375: A potential biomarker for patients with non-small-cell lung cancer

MicroRNAs (miRNAs) are directly involved in cancer initiation, progression and metastasis. Alterations of miRNAs expression in cancer tissue may be reflected in circulation. We attempted to investigate the expression and clinical significance of plasma miR-20a, miR-31 and miR-375 in patients with non-small cell lung cancer (NSCLC). The plasma levels of miR-20a, miR-31 and miR-375 in 164 NSCLC patients and 164 healthy controls (discovery cohort) were evaluated and compared among various clinicopathological characteristics. The relationship between miRNA expression and clinical outcome of NSCLC patients was examined in an independent cohort (53 cases and 53 controls). The expression level of miR-375 in tissue was also examined. Plasma miR-375 levels in NSCLC patients were significantly decreased in both patient cohorts (P < 0.05). In addition, patients with metastatic NSCLC had lower plasma miR-375 expression than those with non-metastatic NSCLC (P < 0.05). Survival analysis showed that patients with low miR-375 expression had worse overall survival rates than those with high miR-375 expression (hazard ratios (HR) = 1.537 (1.046–2.258), P = 0.029). This association was independently validated in a separate cohort of 53 NSCLC patients (HR = 2.406, 95% CI 1.170–4.945, P = 0.017). The expression level of miR-375 was also found to be significantly down-regulated in NSCLC tissues compared with paracancerous tissues (P < 0.001). These findings indicate that miR-375 has an important role in NSCLC initiation and progression, and may be an independent poor prognostic factor in NSCLC patients.     

Prediction value of intercellular adhesion molecule-1 gene polymorphisms for epithelial ovarian cancer risk,clinical features,and prognosis

Intercellular adhesion molecule-1 (ICAM-1, encoded by ICAM-1) is implicated in tumorigenesis and tumor progression. ICAM-1 modulates the susceptibility to several types of cancer and the disease prognosis; however, its role in epithelial ovarian cancer (EOC) is unclear. Here, we evaluate single nucleotide polymorphisms (SNPs) in ICAM-1 as predictors of EOC risk and prognosis. Six ICAM-1 polymorphisms were genotyped in 408 patients with EOC and 520 controls using the MassARRAY system. The ICAM-1 mRNA levels in 89 EOC tissues and 35 normal ovarian tissues were examined using quantitative PCR. The ICAM-1 rs5498 G allele was associated with increased tumor grade (OR = 2.650) and EOC risk (OR = 1.405). This risk was more evident in females who had first-degree relatives (FDRs) with a tumor (OR = 3.475) or who experienced early menarche (OR = 2.774). The ICAM-1 expression in the cancerous tissue was elevated compared with that of normal ovarian tissues (p < 0.0001), and it was associated with an rs5498 genotype (p = 0.0002). ICAM-1 SNPs did not significantly predict the overall EOC survival (p > 0.05). However, the rs5498 G allele correlated with EOC survival time in patients whose FDRs suffered from a tumor (p = 0.001). ICAM-1 rs5498 likely confers a high risk for EOC in G allele carriers accompanied by up-regulation of ICAM-1 expression during carcinogenesis. The combination of ICAM-1 rs5498 and tumor history predicts the EOC prognosis.     

Intra-cervical application of Misoprostol at estrus alters the content of cervical hyaluronan and the mRNA expression of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and cyclooxygenase-2 in the ewe

The complex anatomy the of ovine cervix limits the success of transcervical artificial insemination in sheep, but Misoprostol (a PGE₁ analogue) relaxes the cervix and facilitates transcervical artificial insemination. However, the mechanism by which Misoprostol causes cervical relaxation is not known. This study examined if intra-cervical Misoprostol altered the hyaluronan content and the mRNA expression of COX-2, LHR, or FSHR in the cervix of the estrus ewe. Estrus was synchronized in cyclic ewes with progestagen pessaries and 48 h after sponge removal ewes were treated intra-cervically with 0 (controls), 200, or 400 μg Misoprostol. Hyaluronan content was determined by ELISA and mRNA expression of LHR, FSHR, and COX-2 was analyzed by in situ hybridization using digoxigenin-11-uridine-5′-triphosphate labeled riboprobes. The hyaluronan content of the cervix was significantly higher in sheep that received 200 (P < 0.05) or 400 (P < 0.05) μg Misoprostol compared to controls. Moreover, it was significantly (P < 0.05) higher in the vaginal region compared to mid and uterine regions. Misoprostol increased (P < 0.05) the mRNA expression of LHR and COX-2 but not FSHR. The expression for all three genes was highest in the vaginal region and lowest in uterine region. The luminal epithelium and circular smooth muscle layers had higher (P < 0.05) expression for LHR, FSHR, and COX-2 mRNAs, and the sub-epithelial stroma had the lowest (P < 0.05). We propose that the intra-cervical application of Misoprostol induces the mRNA expression of LHR, FSHR, and COX-2 through a positive feedback loop. The data suggest that softening of the cervix by Misoprostol is caused by an increase in the hyaluronan content of the cervix.     

Hyperglycemia induces elevated expression of thyroid hormone binding protein in vivo in kidney and heart and in vitro in mesangial cells

During a search for glucose-regulated abundant mRNAs in the diabetic rat kidney, we cloned thyroid hormone binding protein (THBP), also known as μ-crystallin or CRYM. The aim of this study was to investigate the effect of hyperglycemia/high glucose on the expression of THBP. THBP mRNA copy numbers were determined in kidneys and hearts of diabetic GK rats vs normoglycemic Wistar rats, and in human mesangial cells (HMCs) exposed to high glucose using real-time qPCR, and THBP protein levels were measured by Western blotting and immunofluorescence. Intracellular ROS was measured in THBP transfected cells using DCF fluorescence. Hyperglycemia significantly increased THBP mRNA in GK rat kidneys (326 ± 50 vs 147 ± 54, p < 0.05), and hearts (1583 ± 277 vs 191 ± 63, p < 0.05). Moreover, the levels of THBP mRNA increased with age and hyperglycemia in GK rat kidneys, whereas in normoglycemic Wistar rat kidneys there was a decline with age. High glucose significantly increased THBP mRNA (92 ± 37 vs 18 ± 4, p < 0.005), and protein in HMCs. The expression of THBP as a fusion protein in transfected HMCs resulted in reduction of glucose-induced intracellular ROS. We have shown that THBP mRNA is increased in diabetic kidney and heart, is regulated by high glucose in renal cells, and appears to attenuate glucose-induced intracellular ROS. These data suggest that THBP may be involved in the cellular pathways activated in response to glucose. This is the first report linking hyperglycemia with THBP and suggests that the role of THBP in diabetic complications should be further investigated.     

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H₂ (PGH₂) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC^Min/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH₂ into PGE₂, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE₂ related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE₂ levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH₂ derived prostanoids were generally enhanced, being most prominent for TxA₂ and PGD₂. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE₂ during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.