An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model : Role of High Level Matrix Metalloproteinase 9

The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.Glioblastoma (GBM) is the most aggressive glioma and is associated with extensive invasion into surrounding brain tissue. The highly invasive nature of GBMs precludes total surgical resection and contributes to the recurrence of GBM. Effective treatments depend on experimental models that closely resemble human GBM characteristics for testing new therapy and providing an accurate understanding of the molecular basis of widespread glioma invasion. Unfortunately, mechanistic investigations into GBM invasion and drug development have been hampered by a paucity of representative experimental models that recapitulate the invasive GBM tumor growth.A number of animal models have been developed by intracranial implantation of human GBM cell lines in rodents to test novel therapeutic agents targeting different features of human GBM, ie, angiogenesis and invasion,^1,2,3,4,5 and to study the mechanisms of tumor invasiveness.^6,7 Human U251 and U87 GBM xenograft animal models are the most common GBM animal models and have been widely used to test therapeutic approaches to human GBM cells in vivo^8,9,10 since they recapitulate some features of the human GBM, ie, tumor formation, necrosis, or angiogenesis. Moreover, the U87 model has also been applied and proved useful when assessing GBM angiogenesis and anti-angiogenic therapeutic approaches.^11,12,13 In addition, the rat C6 glioma cell animal model shows diffuse brain invasion.^14,15 However, tumor invasion in these models is not as extensive as in spontaneous GBM in humans, although peritumoral infiltration of the brain parenchyma¹⁶ and individual cells infiltration^14,15 were detected around the tumor. In addition, no any individual GBM model assembles extensive infiltration, necrosis, and vascular proliferation.These nonextensive infiltrative GBM models have made it difficult to predict the outcome of novel therapies. Even though novel therapies have been proven successful to reduce the tumor mass in these GBM models, they may fail to inhibit the tumor invasion when they are translated to human patients. There is therefore a compelling need for more accurate and reproducible intracranial tumor models, which should recapitulate key features of the human GBM, especially extensive invasive nature of these tumors.Tumor invasiveness involves proteolytic degradation of the extracellular matrix (ECM) as well as depends on the adhesion, mobility, proliferation, apoptosis, and senescence of tumor cells. Increased protease expression has been correlated with the invasive properties of invading cells.^17,18,19,20 The two classes of proteases involved in GBM tumor invasion are matrix metalloproteinases (MMPs) and serine proteases. Increased expression/activity of MMP-9 has been found in anaplastic astrocytomas and GBM^19,21,22 and correlated with the highly malignant progression of human gliomas in vivo.¹⁹ The activity level of MMP-9 is increased during the growth of glial tumors in nude mice following intracerebral injection of glioblastoma cells.²³ Even though in vitro and in vivo experimental models demonstrated that inhibiting MMP-9 function significantly decreased SNB19 glioma cell invasion in vitro and tumor formation in the xenograft model,²⁴ the SNB19 glioma cells-formed tumors displayed well-circumscribed tumor mass without extensive infiltration. Therefore, the mechanism underlying the association of MMP-9 expression and extensive invasion of GBM remains to be elucidated.Epidermal growth factor receptor (EGFR) activation increases MMP-9 expression and activity in other cancer cell types through the JAK3/ERK pathway and PI3K pathway,^25,26,27 and epidermal growth factor (EGF) is expressed in normal brain, benign, and malignant glia tumors.²⁸ EGFR overexpression/amplification occurs in 40% to 60% of all primary GBMs and has been associated with poor prognosis.^29,30,31,32 Both short interfering (si)RNA-targeted EGFR and transfection of EGFR cDNA antisense in the human malignant glioma cell line TJ905 decreased expression and enzymatic activities of MMP-9, as well as cell invasion.³³ However, the role of EGF-stimulated MMP-9 and the signal pathway in GBM cell invasion have not been reported.In this study, we characterized an extensive infiltrative human glioma xenograft mouse model. High levels of MMP-9 contributed to single, clustered, perivascular, and subpial cellular infiltration as well as infiltrating tumor-induced vascular proliferation. Our U1242 MG xenograft model would be a useful experimental and preclinical model for studying the mechanisms underlying the extensive invasion of GBM and testing novel therapeutic agents. Finally, MMP-9 was essential for the EGFR/Ras/MEK and PI3K/AKT-dependent increase in invasion and colony formation of U1242 MG cells.     

IL-1beta-induced increase in intestinal epithelial tight junction permeability is mediated by MEKK-1 activation of canonical NF-kappaB pathway

IL-1β is a proinflammatory cytokine that plays a central role in the inflammatory process of the gut. IL-1β causes an increase in intestinal epithelial tight junction (TJ) permeability, but the intracellular pathways that mediate intestinal TJ permeability remain unclear. The major aims of this study were to delineate the protein kinases that regulate the IL-1β modulation of intestinal TJ barrier function and to determine the intracellular mechanisms involved, using filter-grown Caco-2 monolayers as the in vitro model system. Our results showed that IL-1β caused a rapid activation of MEKK-1 and NIK. The knockdown of MEKK-1, but not NIK, inhibited the IL-1β increase in Caco-2 TJ permeability. IL-1β caused an activation of both canonical and noncanonical NF-κB pathways; MEKK-1 regulated the activation of the canonical pathway, while NIK regulated the activation of the noncanonical pathway. Inhibition of MEKK-1 activation of the canonical pathway prevented the IL-1β increase in TJ permeability. Our data also indicated that inhibitory κB kinase was the catalytic subunit primarily involved in canonical pathway activation and TJ barrier opening. MEKK-1 also played an essential role in myosin light chain kinase gene activation. In conclusion, our data show for the first time that MEKK-1 plays an integral role in IL-1β modulation of Caco-2 TJ barrier function by regulating the activation of the canonical NF-κB pathway and the MLCK gene.     

Routine vaginal Pap test is not useful in women status-post hysterectomy for benign disease

The US Preventive Services Task Force (USPTF) has recommended that routine vaginal Pap (V-Pap) screening is unnecessary for women status-post (S/P) total hysterectomy (T-Hyst) for benign disease (Guide to Clinical Preventive Services. 2nd ed. Baltimore, MD: Williams & Wilkins; 1996. p 105-118). However, many US women continue to have V-Pap despite no risk for cervical cancer and minimal risk for primary vaginal cancer (JAMA 2004;291:2990). Herein, we report our experience with such patients.Computerized data of patients S/P T-Hyst for benign conditions over a 6-yr-period were retrospectively evaluated. Pap diagnoses of epithelial abnormalities (Ep Abnl), negative for intraepithelial lesion or malignancy with/without nonneoplastic findings (NILM-NN and NILM), were reviewed based on three age groups: group A, 18-44 yr; group B, 45-64 yr; and group C, > or =65 yr (JAMA 2004;291:2990). A control group was used.Of 1,860 T-Hyst 1,303 (70%) were for benign disease. Of these 581/1303 (44.5%) patients had 819 current V-Paps (range, 1-5; mean, 1.4). The 581 patients were group A, 288 (49.5%); group B, 272 (46.8%); and group C, 21 (3.6%). Overall, the 819 V-Paps showed Ep Abnl, 28 (3.4%); NILM-NN, 252 (30.7%); and NILM, 539 (65.8%). Of the 28 Ep Abnl, 19 (67.8%) were atypical squamous cells of undetermined significance (ASCUS), and 9 (32%) were low-grade vaginal intraepithelial lesions (LG-VAIN). The NILM-NN findings included organisms, atrophy, and endometriosis. On the basis of individual age groups, Ep Abnl were only seen in V-Paps of 7/288 (2%) of group A and 21/272 (8%) of group B patients. In 23 control patients, 7/71 (9.8%) current V-Paps showed Ep Abnl (ASCUS, 4 (57%); LG-VAIN, 3 (43%)).Continued V-Pap in women S/P T-Hyst for benign disease does not appear to be useful, particularly in those aged > or =65 yr.     

Systematic review of prospective studies investigating “remission” from amphetamine,cannabis, cocaine or opioid dependence

Aims

To review and summarize existing prospective studies reporting on remission from dependence upon amphetamines, cannabis, cocaine or opioids.

Methods

Systematic searches of the peer-reviewed literature were conducted to identify prospective studies reporting on remission from amphetamines, cannabis, cocaine or opioid dependence. Searches were limited to publication between 1990 and 2009. Reference lists of review articles and important studies were searched to identify additional studies. Remission was defined as no longer meeting diagnostic criteria for drug dependence or abstinence from drug use; follow-up periods of at least three years were investigated. The remission rate was estimated for each drug type, allowing pooling across studies with varying follow-up times.

Results

There were few studies examining the course of psychostimulant dependence that met inclusion criteria (one for amphetamines and four for cocaine). There were ten studies of opioid and three for cannabis dependence. Definitions of remission varied and most did not clearly assess remission from dependence. Amphetamine dependence had the highest remission rate (0.4477; 95%CI 0.3991, 0.4945), followed by opioid (0.2235; 95%CI 0.2091, 0.2408) and cocaine dependence (0.1366; 95%CI 0.1244, 0.1498). Conservative estimates of remission rates followed the same pattern with cannabis dependence (0.1734; 95%CI 0.1430, 0.2078) followed by amphetamine (0.1637; 95%CI 0.1475, 0.1797), opioid (0.0917; 95%CI 0.0842, 0.0979) and cocaine dependence (0.0532; 95%CI 0.0502, 0.0597).

Conclusions

The limited prospective evidence suggests that “remission” from dependence may occur relatively frequently but rates may differ across drugs. There is very little research on remission from drug dependence; definitions used are often imprecise and inconsistent across studies and there remains considerable uncertainty about the longitudinal course of dependence upon these most commonly used illicit drugs.     

Cancer mortality-to-incidence ratios in Georgia: describing racial cancer disparities and potential geographic determinants

BACKGROUND:

The objective of this study was to evaluate racial cancer disparities in Georgia by calculating and comparing mortality‐to‐incidence ratios (MIRs) by health district and in relation to geographic factors.

METHODS:

Data sources included cancer incidence (Georgia Comprehensive Cancer Registry), cancer mortality (Georgia Vital Records), and health factor (County Health Rankings) data. Age‐adjusted incidence and mortality rates were calculated by cancer site (all sites combined, lung, colorectal, prostate, breast, oral, and cervical) for 2003‐2007. MIRs and 95% confidence intervals were calculated overall and by district for each cancer site, race, and sex. MIRs were mapped by district and compared with geographic health factors.

RESULTS:

In total, 186,419 incident cases and 71,533 deaths were identified. Blacks had higher MIRs than whites for every cancer site evaluated, and especially large differentials were observed for prostate, cervical, and oral cancer in men. Large geographic disparities were detected, with larger MIRs, chiefly among blacks, in Georgia compared with national data. The highest MIRs were detected in west and east central Georgia, and the lowest MIRs were detected in and around Atlanta. Districts with better health behavior, clinical care, and social/economic factors had lower MIRs, especially among whites.

CONCLUSIONS:

More fatal cancers, particularly prostate, cervical, and oral cancer in men were detected among blacks, especially in central Georgia, where health behavior and social/economic factors were worse. MIRs are an efficient indicator of survival and provide insight into racial cancer disparities. Additional examination of geographic determinants of cancer fatality in Georgia as indicated by MIRs is warranted. Cancer 2012. © 2012 American Cancer Society     

Molecular imaging of breast tumors using a near-infrared fluorescently labeled clusterin binding peptide

Several reports have shown that secreted clusterin (sCLU) plays multiple roles in tumor development and metastasis. Here, we report on a 12-mer sCLU binding peptide (designated P3378) that was identified by screening a phage-display peptide library against purified human sCLU. Differential resonance perturbation nuclear magnetic resonance using P3378 and a scrambled control peptide (designated P3378R) confirmed the P3378-sCLU interaction and demonstrated that it was sequence specific. P3378 and P3378R peptides were conjugated to an Alexa680 near infrared fluorophore (NIRF) and assessed for their tumor homing abilities in in vivo time-domain fluorescence optical imaging experiments using living 4T1 tumor bearing BALB/c mice. When injected in separate animals, both peptides accumulated at the tumor site, however the NIRF-labeled P3378 peptide was retained for a significant longer period of time than the P3378R peptide. Similar observations were made after simultaneously injecting the same tumor-bearing animal with a peptide mixture of P3378 DyLight (DL)680 and the P3378R-DL800. Coinjection of P3378-DL680 with excess unlabeled P3378 blocked tumor accumulation of fluorescent signal while excess P3378R control peptide did not confirming the sequence specificity of the tumor accumulation. Finally, ex vivo fluorescence microscopy of these tumors confirmed the presence of P3378-DL680 in the tumor and its colocalization with CLU. These results confirm the tumor targeting specificity of the P3378 CLU-binding peptide and suggest its usefulness for the in vivo monitoring of solid tumors secreting detectable levels of CLU.     

Anti-leukemic activity of Wattakaka volubilis leaf extract against human myeloid leukemia cell lines

Ethnopharmacological relevance

Wattakaka volubilis has been traditionally used in Ayurvedic medicine in India for treatment of several ailments such as bronchial asthma, inflammations, tumors, piles, leucoderma, application to boils, rat bite etc.

Aim of the study

The present study was designed to investigate anti-leukemic activity of the crude aqueous methanolic extract and to identify active compounds from the leaves of Wattakaka volubilis.

Materials and methods

The leaves of Wattakaka volubilis were extracted with aqueous methanol. Liquid–liquid fractionation of the crude methanolic extract with different organic solvents was done and the fractions were screened for in vitro anti-leukemic activity using different leukemic cell lines. The active fractions were then subjected to chromatographic separation for isolation of bioactive compounds. Structure of isolated compound was elucidated by spectroscopic methods. The in vitro anti-leukemic activities of different extracts of the leaves and isolated compound WVP were studied in U-937, HL-60 and K-562 cell-lines by using cell count, MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] and DNA laddering assays, flow-cytometric and confocal microscopic techniques.

Results

Kaempferol-3-O-[α-l-rhamnopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→6)-O]-β-d-glucopyranoside (WVP) was isolated from crude leaves extract of Wattakaka volubilis. Both the n-butanolic extract (WVB) of Wattakaka volubilis and its isolate WVP were found to be responsible for in vitro anti-leukemic activity. The IC₅₀ values of WVB were found be 120, 100 and 50 (μg/ml) in U937, K562, and HL-60 cell lines, respectively. Whereas, the pure isolate WVP exhibited anti-leukemic activity with IC₅₀ values of 13.5, 10.8, and 13.2 (μg/ml) in U937, K562, and HL-60 cell lines, respectively. The flow-cytometric analysis confirms that the cell cycle arrest occurs at G1 phase in case of U937 and K562 cell lines and G2/M phase in case of HL60 cell lines. Similarly both confocal microsocopic analysis and DNA laddering assay confirm the apoptosis and cell cycle arrests of leukemic cells.

Conclusion

The overall results provide evidence for the ethnopharmacological relevance for use of the plant Wattakaka volubilis in developing novel agents for the treatment of leukemia.     

Effect of thymoquinone on the lung pathology and cytokine levels of ovalbumin-sensitized guinea pigs

Different pharmacological effects from Nigella sativa have been demonstrated in guinea pig tracheal chains in previous studies. In the present study, the prophylactic effects of thymoquinone on lung pathology as well as blood IL-4 and IFN-γ levels in sensitized guinea pigs were examined. Three groups of guinea pigs sensitized to ovalbumin were given drinking water alone (group S) or drinking water containing low (LTQ) or high (HTQ) concentrations of thymoquinone (groups S + LTQ and S + HTQ). The lung pathology as well as blood IL-4 and IFN-γ levels of the sensitized and the control guinea pigs were evaluated in three sensitized and one control group (n = 8, for all groups). The lungs of the S group showed significant pathological changes (p < 0.001). Blood IL-4 and IFN-γ levels were increased in the sensitized animals compared to those of controls (p < 0.01 and p < 0.001, respectively). Treatment of the S animals with thymoquinone significantly improved their pathological changes to the lung and decreased their IL-4 levels (p < 0.05 to p < 0.001) but increased their IFN-γ levels (p < 0.001). These results showed a preventive effect of thymoquinone on lung inflammation in sensitized guinea pigs.     

Evaluation of CA 242 as a Tumor Marker in Gallbladder Cancer

Purpose

Gallbladder and pancreas share common embryological origin, and malignancies of these organs may share common tumor antigens. CA 242 is a tumor marker for pancreatic cancer, but has not been studied in gallbladder cancer (GBC). We measured serum CA 242 levels in patients with GBC and compared it with those in patients with gallstones (GS) and healthy volunteers.

Methods

We enrolled consecutive patients with GBC (cases), GS (disease controls), and healthy volunteers (healthy controls). Serum CA 242, CEA, and CA 19?C9 levels were measured using ELISA. Receiver operator curve was plotted for all the three markers.

Results

We studied 117 patients with GBC, 58 with GS, and 10 healthy volunteers. Among patients with GBC, 81 (69%) also had GB calculi. Patients with GBC more often had elevated CA 242 levels (64%) compared to those with GS (17%; p?<?0.001) and healthy controls (0%; p?<?0.001). The median levels of CA 242 was higher in the GBC group (59 [199] U/ml) compared to the GS group (10 [13] U/ml; p?<?0.001) and the control group (3 [14.5] U/ml; p?<?0.001). The sensitivity, specificity, positive predictive value (PPV), and negative predictive values of CA 242 for diagnosis of GBC were 64%, 83%, 88%, and 53%, respectively. At a cutoff of 45?U/ml, the specificity and PPV increased to 100%. CA 242 had higher AOC (0.759) compared to CEA (0.528) and CA 19?C9 (0.430).

Conclusions

CA 242 is a promising tumor marker for GBC and performs better than CEA and CA 19-9.