A cobalt(II) coordination polymer-derived catalyst engineered via temperature-induced semi-reversible single-crystal-to-single-crystal (SCSC) dehydration for efficient liquid-phase epoxidation of olefins.

Single-crystal-to-single-crystal (SCSC) transformations provide more avenues for phase transitions, which have piqued great interest in crystal engineering. In this work, a 3D Co(II)-based coordination polymer (CP), {Co2(OH2)8(btec)}·4H2O (1), (where (btec)4- = 1,2,4,5-benzenetetracarboxylate) undergoes SCSC transition upon heating at 180 °C to afford an anhydrous phase [Co2(btec)] (1'). Room-temperature water-vapour induced semi-reversible SCSC transformation of 1' involves condensation of two water molecules coordinating to the metal cluster, yielding a new framework [Co2(OH2)2(btec)] (2). These SCSC transitions were accomplished through a sequential bond breaking and new bond formation process which was accompanied by colour changes from orange (1) → violet (1') → pink (2). All materials were structurally elucidated by single-crystal X-ray diffraction (SCXRD) and further established by various analytical techniques. According to SCXRD data, all the frameworks possess octahedral geometries around the cobalt(II) sphere. SCXRD studies further revealed that 1 is a polymeric architecture with a binodal 4-c sql topology while 1' and 2 possess (3,6)-c kgd and (4,6)-c scu 3D nets, respectively. By virtue of multitopicity exhibited by the tetracarboxylate, the coordination number of the linker around the Co(II) sphere increased from four (in 1) to eight (in 1') and then decreased to six (in 2). Most interestingly, permanent porosity could be observed for the dihydrate 2, originated from potential void space as substantiated by dinitrogen (N2) sorption isotherm. These porous frameworks were active catalysts for the aerobic epoxidation of the model substrate cyclohexene using molecular oxygen (O2) as the final oxidant in the presence of the sacrificial i-butyraldehyde (IBA) reductant. For using the dihydrous phase 2, cyclohexene and various other olefins were catalytically oxidised to their corresponding epoxides with up to 38.5% conversion and 99.0% selectivity. The catalyst 2 can be expediently recycled in four runs without significant loss of activity. This research demonstrates that a little innovation in the active-site-engineered organic-inorganic hybrid materials can significantly enhance the catalytic performance and selectivity of coordination polymer-derived heterogeneous catalysts.

Enhanced Catalytic Activity of a Copper(II) Metal-Organic Framework Constructed via Semireversible Single-Crystal-to-Single-Crystal Dehydration.

Herein, we present a copper(II) metal-organic framework, [Cu2(btec)(OH2)4]·2H2O (1) [(btec)4- = 1,2,4,5-benzenetetracarboxylate], that undergoes single-crystal-to-single-crystal transformations into two anhydrous phases 2' and 2″ with the chemical formula [Cu2(btec)], triggered by two-step dehydration at 403 and 433 K, respectively. After immersion in water for 3 days at room temperature, 2' transformed into [Cu2(btec)(OH2)] (3), while both 2' and 2″ took 1 week to revert to 1. Dynamic vapor sorption studies validated water-induced reversible structural transformations at 70% relative humidity (RH). According to single-crystal X-ray diffraction (SC-XRD), the local coordination geometry of the Cu2+ ion in 2' changed from a saturated octahedron to a coordinatively unsaturated square-based pyramid in 3, manifested by changes in color and dimensionality. From a topological point of view, all of the scaffolds show a binodal (3,6)-connected kgd topology with the point symbol {43}2{46}. In addition, the materials were thoroughly characterized using routine spectroscopic data and various analytical techniques. The catalytic activity of the microporous materials in the liquid-phase oxidation of styrene in acetonitrile, using 30% (wt) H2O2 as the oxidant, was investigated. The excellent performance of the monohydrous phase 3 was shown to be superior to the pristine framework and the anhydrous counterparts, as evidenced by a good turnover number (TON) and turnover frequency (TOF) = 82.6 and 21.0 h-1, respectively. Within 4 h, the substrates were catalytically oxidized to the desired products with up to 67% conversion and 100% benzaldehyde selectivity. It is worth noting that the accessible active metal sites and higher surface area enhanced the catalytic properties of 3. Furthermore, the maintenance of catalytic efficiency over five cycles and reusability are illustrated and discussed in terms of the structural differences of the microporous frameworks. Thus, a preliminary reaction mechanism for the selective oxidation of styrene is proposed. This study not only provides a fascinating example of MOF chromism achieved by thermal activation and rehydration but also sheds some light on the relationship between pore-surface- or metal-engineered sites in MOFs and their heterogeneous catalytic performances.

Exploring intermolecular contacts in multi-substituted benzaldehyde derivatives: X-ray, Hirshfeld surface and lattice energy analyses.

Crystal structures of six benzaldehyde derivatives (1-6) have been determined and their supramolecular networks were established by an X-ray crystallographic study. The study has shown that the compounds are linked by various intermolecular interactions such as weak C-H⋯O hydrogen bonding, and C-H⋯π, π-π and halogen bonding interactions which consolidate and strengthen the formation of these molecular assemblies. The carbonyl group generates diverse synthons in 1-6via intermolecular C-H⋯O hydrogen bonds. An interplay of C-H⋯O hydrogen bonds, and C-H⋯π and π-π stacking interactions facilitates the formation of multi-dimensional supramolecular networks. Crystal packings in 4 and 5 are further generated by type I halogen⋯halogen bonding interactions. The differences in crystal packing are represented by variation of substitution positions in the compounds. Structure 3 is isomorphous with 4 but there are subtle differences in their crystal packing. The nature of intermolecular contacts in the structures has been studied through the Hirshfeld surfaces and two-dimensional fingerprint plots which serve as a comparison in constructing different supramolecular networks. The intermolecular interaction energies are quantified utilizing theorectical calculations for the title compounds and various analogous structures retrieved from the Cambridge Structural Database (CSD). Also intermolecular interactions for the molecular pairs are exctrated from respective crystal structures. Essentially, there are some invariant and variable intermolecular contacts realized between different groups in all six structures. The ab initio DFT total lattice energy (E Tot) calculations showed a direct correlation with thermal strengths of the title compounds.

Crystal structures, spectroscopic and theoretical study of novel Schiff bases of 2-(methylthiomethyl)anilines.

New Schiff bases derived from p-methoxysalicylaldehyde and 2-(methylthiomethyl)anilines (substituted with methyl, methoxy, nitro) were synthesized and characterized by elemental analyses, FT-IR, NMR, electronic spectra and quantum chemical calculations. X-ray crystallography of two compounds showed the solid structures are stabilized by intramolecular and intermolecular H-bonds. The effect of OH⋯N interaction between the phenolic hydrogen and imine nitrogen on the proton and carbon NMR shifts, and the role of CH⋯O and CH⋯S contacts are discussed. The bond lengths and angles, (1)H and (13)C NMR data, E(LUMO-HOMO), dipole moments and polarizability of the compounds were predicted by density functional theory, DFT (B3LYP/6-31G∗∗) method. The experimental geometric parameters and the NMR shifts were compared with the calculated values, which gave good correlations. The electronic effects of aryl ring substituents (methyl, methoxy and nitro) on the properties of the resulting compounds, such as the color, NMR shifts, electronic spectra and the calculated energy band gaps, dipole moments and polarizability are discussed. Increase in electron density shifted the phenolic proton resonance to lower fields. The methoxy-substituted compound has a small dipole moment and subsequent large polarizability value. Highest polarity was indicated by the nitro compound which also showed high polarizability due to its larger size. The energy gaps obtained from E(LUMO-HOMO) calculations suggest these compounds may have applications as organic semiconducting materials.

The expression and prognostic value of ROCK I and ROCK II and their role in human breast cancer.

The role and expression of ROCKI and ROCKII in human breast cancer was investigated and the effect on clinical outcome assessed. ROCK knockdown cells (MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII were tested for their in vitro invasiveness, motility and in vivo tumour growth. Samples of fresh frozen breast tumour tissue (n=113) and normal background tissue (n=30) were processed for immunohistochemical and quantitative RT-PCR analyses. MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII cells showed significantly decreased invasiveness compared with control cells (mean +/- SEM 4.33+/-0.84 MDA-MB-231DeltaROCKI vs. 18.4+/-1.4 control, p<0.001; 6.8+/-1.2 MDA-MB-231DeltaROCKII vs. 18.4+/-1.4 control, p<0.001). Similarly, both exhibited reduced motility compared with control cells (p<0.001) and lost their response to HGF. Importantly, no significant difference existed between knockdown and control cells in in vivo tumour growth. ROCKI was significantly higher in human mammary tumours than normal background tissue (2.9+/-1.1 vs. 0.29+/-0.13, p=0.023), although expression of ROCKII was fairly consistent in both (2050+/-646 vs. 2303+/-2079). ROCKI expression was greater in patients who died from breast cancer than in patients who remained disease free (11.6+/-7.1 vs. 1.95+/-0.95). Higher levels of ROCKI were associated with increased grade (0.95+/-0.73 grade-1; 2.11+/-1.72 grade-2; and 4.06+/-1.99 grade-3). Levels of ROCKI, but not ROCKII, were significantly correlated with overall survival of patients (p=0.004, Univariate analysis, median follow-up 120 month). These results show that ROCKI and possibly ROCKII are key factors in regulation of motility/invasion of breast cancer cells. This, together with significant correlation between ROCKI and poor outcome in clinical breast cancer, indicates that it is a potential target in human breast cancer.

Is N-cadherin expression important in ductal carcinoma?

OBJECTIVES: Neural (N)-cadherin is a calcium-dependent cell adhesion molecule that is associated with invasive tumors in breast cancer, but no association with grade or nodal status has been shown in previous studies. The present study examined the expression of N-cadherin in human breast cancer and assessed its prognostic value in long-term patient follow-up (10 years). METHODS: Using real-time polymerase chain reaction, the number of N-cadherin transcripts in normal breasts (n = 32) and infiltrating ductal carcinomas (n = 90) was assessed. The results were then analyzed in relation to grade, nodal involvement, distant metastasis, TNM stage, Nottingham Prognostic Index, and survival over 10 years. RESULTS: The levels of N-cadherin transcripts (normalized to glyceraldehyde 3-phosphate dehydrogenase) in primary tumors were lower in patients with metastases (P = 0.05), with local recurrence (P = 0.037), and those who died as a result of breast cancer (P = 0.038). There was no significant association with grade, nodal status, TNM stage, or Nottingham Prognostic Index. CONCLUSION: In ductal carcinomas, decreased levels of N-cadherin (normalized to glyceraldehyde 3-phosphate dehydrogenase) in primary tumors correlate with local recurrence and death in long-term follow-up of patients.

Claudin-16 reduces the aggressive behavior of human breast cancer cells.

Claudin-16 (Paracellin-1) is a transmembrane tight junction (TJ) protein originally described as having a critical role in the re-absorption of magnesium and calcium in the kidney. This study examined expression of Claudin-16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between Claudin-16 levels and patient prognosis. Insertion of the Claudin-16 gene into MDA-MB-231 human breast cancer cells resulted in cells that were significantly less motile and invasive in behavior, with increased adhesion to matrix. These cells also exhibited significantly increased TJ functionality and "tighter" colony morphology. Moreover, growth rates were reduced in both in vitro and in vivo assays (P < 0.002). Frozen sections from breast cancer primary tumors (matched tumor 124 and background 33) were immuno-stained. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, normalized with cytokeratin-19 levels). Levels of expression of Claudin-16 were significantly decreased in node positive tumors compared to negative (P = 0.016). Expression was significantly lower in patients with node positive tumors (P = 0.016) and in those who had died from breast cancer or had general poor prognosis (P < 0.015). Immunohistochemical staining showed decreased expression of Claudin-16 in tumor sections (P < 0.00001). In conclusion, forced expression of Claudin-16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumor volume. Claudin-16 expression was reduced in human breast cancer, particularly in patients with aggressive tumors and high mortality. This suggests that Claudin-16 plays a role beyond that of an initial metastasis repressor in this cancer type.

Lymphangiogenesis and lymph node metastasis in breast cancer.

INTRODUCTION: There have been few studies on lymphangiogenesis in the past due to the lack of specific lymphatic endothelial markers, and lymphatic-specific growth factors. Recently, these limitations have been relieved by the discovery of a small number of potential lymphatic-specific markers. The relationship between lymphangiogenesis and regional or distant metastasis has not previously been investigated in humans. Using these lymphatic markers, it is possible to explore the relationship between lymphangiogenesis and tumour metastasis. This study indirectly quantified lymphangiogenesis by measuring mRNA expression of all seven lymphatic markers described above in breast cancers and correlated these markers with lymphatic involvement and survival. The cDNA from 153 frozen archived breast samples were analysed with Q-PCR for all seven lymphangiogenic markers. This was correlated with various prognostic factors as well as patient survival. RESULTS: There was significantly greater expression of all 7 markers in malignant compared to benign breast tissue. In addition, there was greater expression in lymph node positive/grade 3 tumours when compared to lymph node negative/grade 1 tumours. In 5 of the markers, there was a greater expression in poor NPI prognostic tumours when compared to favourable prognostic tumours which was not statistically significant. There was no association between recurrence risk and lymphangiogenic marker expression. CONCLUSION: In summary, the findings from this study show that lymphangiogenesis, measured by specific lymphatic marker expression, is higher in breast cancers than in normal breast tissue. Secondly, breast cancers which have metastasised to the regional lymphatics show higher expression compared to those which have not, although the individual differences for all five markers were not statistically significant.

Expression of the cell to cell adhesion molecule, ALCAM, in breast cancer patients and the potential link with skeletal metastasis.

The activated leukocyte cell adhesion molecule (ALCAM) is involved in cell migration and adhesion. Decreased levels of ALCAM expression in breast cancer tissue are known to correlate with poor prognosis. The current study specifically investigated the ALCAM expression in tumours which developed skeletal metastasis. Fresh frozen primary breast cancer tissues (n=234) and non-neoplastic mammary tissue (n=34) were used. The distribution and location of ALCAM was assessed using immunohistochemical methods and the level of ALCAM was determined using quantitative RT-PCR. The results were analysed against the clinical and pathological data. ALCAM staining was largely membranous and cytoplasmic in normal epithelial cells and is significantly stronger than in cancer cells (p=0.023) and patients who develop skeletal metastasis (p=0.048). The ALCAM transcript levels were lowest in patients with skeletal metastasis (p=0.0048) but were also significantly lower in patients who developed local recurrence (p=0.040) and in those who died from breast cancer (p=0.0075). Patients with moderate and poor prognostic indices have a lower level than those with a good index (p=0.05 and p=0.0089 respectively) and ER-positive tumours show a lower level than ER-negative (p=0.043). Ductal carcinomas, 86% of the cohort, have a similar pattern of changes with skeletal metastasis patients having significantly lower levels (p=0.015). This study has, for the first time, shown that patients who develop skeletal metastasis tend to have the lowest levels of ALCAM transcripts in their breast cancers, a finding potentially useful for clinical practice.

Deregulation of cofactor of BRCA1 expression in breast cancer cells.

Cofactor of BRCA1 (COBRA1) is an integral component of the human negative elongation factor (NELF), a four-subunit protein complex that inhibits transcription elongation. Previous in vivo work indicates that COBRA1 and the rest of the NELF complex repress estrogen-dependent transcription and the growth of breast cancer cells. In light of the COBRA1 function in breast cancer-related gene expression, we sought to examine regulation of COBRA1 expression in both established breast cancer cell lines and breast carcinoma tissues. We found that COBRA1 expression was inversely correlated with breast cancer progression, as tumor samples of patients who had distant metastasis and local recurrence expressed very low levels of COBRA1 mRNA when compared to those who were disease free for over 10 years (P = 0.0065 and 0.0081, respectively). Using both breast and prostate cancer cell lines, we also explored the possible mechanisms by which COBRA1 expression is regulated. Our results indicate that the protein abundance of COBRA1 and the other NELF subunits are mutually influenced in a tightly coordinated fashion. Small interfering RNA (siRNA) that targeted at one NELF subunit dampened the protein levels of all four subunits. Conversely, ectopic expression of COBRA1 in the knockdown cells partially rescues the co-depletion of the NELF subunits. In addition, our study suggests that a post-transcriptional, proteasome-independent mechanism is involved in the interdependent regulation of the NELF abundance. Furthermore, a lack of COBRA1 expression in breast carcinoma may serve as a useful indicator for poor prognosis.

Bone morphogenetic proteins 1 to 7 in human breast cancer, expression pattern and clinical/prognostic relevance.

BACKGROUND AND AIMS: Bone morphogenetic proteins (BMPs) have a diverse role and they act in a time, concentration and cell type specific manner. They regulate cellular motility and the cells ability to invade. Recently, BMP molecules have further been shown to have an impact on the biological behaviour of breast cancer cells. In this study, we looked, for the first time, at the expression of a panel of BMPs in breast carcinomas. METHOD: Fresh frozen primary human breast cancer tissues (n = 120) and non-neoplastic mammary tissues (n = 32) were used. The distribution and location of BMPs was assessed using immunohistochemical methods and the transcript levels of BMPs (BMP-1, -2, -3, -4, -5, -6, and -7) were determined using quantitative RT-PCR. The results were analysed against the clinical, pathological and follow-up (10 years) data. RESULTS: BMP-2 and BMP-7 exhibited contrasting patterns of expression in normal and tumour tissues, wherein BMP-2 transcript level was lower and BMP-7 was higher in breast tumours than normal tissues. BMP-2 transcript was also significantly lower in tumours from patients with a moderate and poor prognosis than from those with a good prognosis (p = 0.04). BMP-2 and BMP-7 also showed a significant difference between node positive and node negative tumours (p = 0.033 and p = 0.031 respectively). BMPs 1, 3, 4, 5 and 6 showed an inconsistent variation in transcript levels within the cancer subgroups with no statistically significant results. CONCLUSION: This study has demonstrated a differential pattern of expression of BMP molecules in breast cancer and reveals a potential prognostic value of BMP-2 and BMP-7 for patients. The findings also suggest that these BMPs may be potential therapeutic targets.

N-WASP is a putative tumour suppressor in breast cancer cells, in vitro and in vivo, and is associated with clinical outcome in patients with breast cancer.

N-WASP is a key regulator of cell migration and actin polymerisation. We examined the correlation of N-WASP, with human breast cancer, in vitro, in vivo and in clinical breast cancer tissue. Immunohistochemical study of frozen sectioned human breast mammary tissues (n=124) revealed that mammary epithelial cells stained positively for N-WASP and that cancer cells in tumour tissues stained very weakly. Quantitative RT-PCR revealed that breast cancer tissues had significantly lower levels of N-WASP compared with normal background mammary tissues (0.83+/-0.3 vs 13.6+/-13, P=0.03). Although no significantly correlation was found with tumour grade and TNM staging, lower levels of transcript were seen to correlate with clinical outcome following a ten year follow up. Thus tumours from patients with predicted poor prognosis had significantly lower levels than from those with good prognosis (0.098+/-0.14 vs 1.14+/-0.56, P=0.05). Patients with metastatic disease/died of breast cancer had significantly lower levels of N-WASP compared to those remaining disease free (0.04+/-0.02 and 0.47+/-0.3, vs 0.79+/-0.44, P=0.01 and P<0.05 respectively). During in vitro experiments, MDA-MB-231 cells stably transfected with N-WASP (MDA-MB-231(WASP+)) exhibited a significantly reduced in vitro invasiveness and motility compared with control and wild type cells (P<0.0001), had increased adhesiveness (P=0.05) and moreover MDA-MB-231(WASP+ )exhibited reduced in vivo growth (P=0.002). The motogen HGF (50 ng/ml) caused a relocation of N-WASP to the cell periphery in a temporal and spatial response. It is concluded that N-WASP, a member of the N-WASP family may act as a tumour progression suppressor in human breast cancer and may therefore have significant clinical value in this condition.

The expression of the von Hippel-Lindau gene product and its impact on invasiveness of human breast cancer cells.

The von Hippel-Lindau (VHL) gene is located on the short arm of chromosome 3, the mutations of which lead to the development of von Hippel-Lindau disease. The VHL gene is a putative tumour suppressor gene in VHL and a few other conditions, possibly by negative regulation of hypoxia- inducible factor-1 (HIF-1) and the stromal-derived factor-1 (SDF-1) receptor, CXCR4, via which the VHL protein negates angiogenesis and tumour cell migration. The current study investigated the expression of VHL at the mRNA and protein levels in clinical breast tumours and evaluated the impact of VHL on the invasion of human breast cancer cells in vitro. Primary breast cancer samples (n=124), adjacent non-cancerous breast tissues obtained from patients in cohort (n=33) and a panel of human breast cancer cells (n=12) were used. Tissue distribution of VHL protein in human breast cancer tissues was assessed using immunohistochemical analysis, and VHL transcript was determined using quantitative reverse transcription PCR. Breast cancer cell line MDA-MB-231 was transfected with a human VHL expression construct (pCR3-GFP/VHL) to allow forced overexpression of VHL in the cells. Invasiveness and migration of cancer cells were assessed using the Matrigel invasion and Cytodex-2 migration assays. Statistical analysis was performed using the Student's t-test. Our results showed that breast cancer cell lines MCF-7 and ZR-75-1 expressed very high levels of VHL transcripts, but the highly aggressive MDA-MB-231, MDA-MB-435 and MDA-MB-453 expressed either no VHL or a low level. The levels of VHL transcripts were significantly lower in grade 2 and grade 3 tumours (mean +/- SD, 1.36+/-0.55 and 0.9+/-0.37), compared with grade 1 tumours (12.3+/-7.6, p<0.002). Node-positive tumours had lower levels of VHL than node-negative tumours. Although tumours from patients with metastasis and from those who died of breast cancer had low levels of VHL, the most significant reduction in VHL was seen in tumours which developed local recurrence (p=0.03). The staining of VHL protein was most abundant in mammary epithelial cells and moderate in endothelial cells. Tumour cells in breast tissues had low to moderate VHL staining. pCR3-GFP/VHL-transfected MDA-MB-231 (MDA-MB-231VHL+) exhibited a reduced spontaneous in vitro invasiveness (14.8+/-2.7) compared with the control cells (18.4+/-1.4). MDA-MB-231VHL+ cells also lost their invasion response to HGF/SF, an invasion-inducing cytokine. The MDA-MB-231VHL+ cells had substantially reduced motility compared with that of the controls (14.8+/-0.7 for MDA-MB-231VHL+ and 20.7+/-1.2 for the control; p<0.001). Thus, VHL exerts inhibitory effects on the invasive and migratory capacity of breast cancer cells in vitro. Low levels of VHL occur in most aggressive breast tumours. Taken together, VHL is a powerful putative tumour suppressor gene in human breast cancer.

Expression of transcription factor CREB1 in human breast cancer and its correlation with prognosis.

It has been suggested that the cAMP responsive element-binding protein (CREB) may act as a transcription regulator of aromatase in breast cancer cells. However, there is little knowledge on the expression of CREB1 in human breast cancer and its clinical significance. The current study investigated the expression pattern of CREB1 in human breast cancer at the mRNA and protein level and correlated it with the clinical outcome. CREB1 staining was primarily seen in the nucleus of both normal and tumour cells. At the mRNA level, we found a significantly higher level of CREB1 in breast tumour tissues (n=120) as compared to non-neoplastic mammary tissues (n=33, p=0.0092). When compared between different histological types CREB1 expression was significantly higher in ductal carcinoma as compared to lobular and other breast carcinoma. Patients with a poor prognosis and with metastasis had a markedly raised level of CREB1 compared to patients who were disease free. In addition, node-positive tumours had higher levels of CREB1 than node-negative tumours (p=0.0018). Finally, patients with high levels of CREB1 had a significantly shorter disease-free survival [95.3 (68.4-122.3, 95% CI) months] compared with those with lower levels [133.9 (123.5-144.2) months, p=0.0193]. This study demonstrates that the level of CREB1 in breast cancer patients is elevated and is significantly raised in patients with a poor prognosis, metastatic disease and nodal involvement. We conclude that the level of CREB1 expression is aberrant in human breast cancer and is associated with disease progression in breast cancer patients.

Differential expression and prognostic implications of the CCN family members WISP-1, WISP-2, and WISP-3 in human breast cancer.

BACKGROUND: The CCN family has three Wnt-inducted secreted proteins named WISP-1, WISP-2 and WISP-3. These molecules are known to play a diverse role in cells, but their role in cancer cells remains controversial. METHODS: In this study, we analyzed the expression of the three WISP molecules at the mRNA and protein levels in a cohort of 122 human breast tumors and 32 normal breast tissues, and we correlated these findings with patients' clinical outcomes. RESULTS: WISP-1 transcripts were found in lower levels in node-positive tumors compared with node-negative tumors (P < .05); were lower in patients with a moderate (P = .01) and poor Nottingham Prognostic Index prognosis (P < .05) compared with good prognostic groups; were of significantly lower level in grade 3 differentiated tumors (P < .05) compared with grade 1; and were of lower levels in patients who developed metastasis and died from breast cancer-related causes (P < .05 in both comparisons). Almost the reverse was found to be true for WISP-2, which had greater levels of expression in node-positive tumors (P = .0043); higher levels in both moderate and poor prognostic groups compared with the good prognostic group (both P < .05); greater level in both grade 2 and 3 when compared with grade 1 (both P < .05); and higher levels in patients who went on to develop metastases (P < .01). WISP-3 transcript levels showed no statistically significant differences between groups. CONCLUSIONS: WISPs may play important but contrasting roles in breast cancer. WISP-1 seems to act as a tumor suppressor and WISP-2 as a factor that stimulates aggressiveness; WISP-3 has no definable beneficial or detrimental role.

Angiopoietins lack of prognostic significance in ductal mammary carcinoma.

Angiopoietins (Ang) have been shown to regulate the process of vasculature and angiogenesis in tumour. Different angiopoietins have different roles during the angiogenic process. The current study sought to examine the levels of the expression of Ang-1, Ang-2, Ang-3 and their receptor Tie-2 in mammary ductal carcinoma and to assess their relevance to prognosis. Fresh frozen ductal carcinoma tissues (n = 90) and adjacent non-cancerous breast tissues (n = 32) were used. The expression of Ang-1, Ang-2 and Ang-3 transcripts in cancer and normal breast tissues were examined quantitatively using quantitative RT-PCR. The protein expression of Ang-1, Ang-2 and Tie-2 was assessed by immunohistochemistry on frozen sectioned tissues. Ang-1, Ang-2 and Ang-3 were detected in mammary tissues. Ang-1 was seen in both normal epithelial cells, breast cancer cells as well as in endothelial cells. Ang-2 was seen at a higher level than Ang-1 and it is expressed in epithelial, endothelial as well as stromal cells to certain degree. Ang-1 and Ang-2 transcripts were detected almost equally in cancer and normal breast tissue, and Ang-3 was high in cancer tissue compared to normal breast but not significant (155 +/- 123 & 24.1 +/- 22.6, P > 0.05). No significant differences were seen between patients with different predicted prognosis (using the Nottingham Prognostic Index as a guide) (Ang-1 p = 0.34, Ang-2 p = 0.26 and Ang-3 p = 0.32, respectively). No significant correlation was seen between Ang-1, Ang-2 and Ang-3 with tumour grade. When the levels of the transcripts were compared against clinical outcome (disease free, developed recurrence and patients who died of breast cancer), levels of Ang-3 transcript was found to be high in breast cancer patient who had bone metastasis 33.8 +/- 28.3, although the difference was not significant (p = 0.08). No significant difference was seen with levels of Ang-1 and Ang-2 transcripts and clinical outcomes. Furthermore, no significant trend was observed between Tie-2 receptor and clinical/pathological parameters in the cohort. These data suggest that angiopoietins (Ang-1, Ang-2 and Ang-3) are expressed in mammary tissues, both in normal and tumour. These molecules have limited value in predicting the prognosis and clinical outcome in patients with mammary ductal carcinoma.

Curcuminoids, curcumin, and demethoxycurcumin reduce lead-induced memory deficits in male Wistar rats.

This study investigated the neuroprotective effects of the curcuminoids against lead-induced neurotoxicity. The results show that lead significantly increases lipid peroxidation and reduces the viability of primary hippocampal neurons in culture. This lead-induced toxicity was significantly curtailed by the co-incubation of the neurons with the curcuminoids. In a whole animal experiment, rats were trained in a water maze and thereafter dosed with lead and/or curcumin (CURC), demethoxycurcumin (DMC), or bisdemethoxycurcumin (BDMC) for 5 days. Animals treated with curcumin and demethoxycurcumin but not bisdemethoxycurcumin had more glutathione and less oxidized proteins in the hippocampus than those treated with lead alone. These animals also had faster escape latencies when compared to the Pb-treated animals indicating that CURC- and DMC-treated animals retain the spatial reference memory. The findings of this study indicate that curcumin, a well-established dietary antioxidant, is capable of playing a major role against heavy metal-induced neurotoxicity and has neuroprotective properties.

Elevated levels of tumour endothelial marker-8 in human breast cancer and its clinical significance.

Tumour endothelial marker-8 (TEM-8) belongs to a family of endothelial markers that are raised during tumour angiogenesis. We have recently reported aberrant expression of TEMs at the mRNA level in human breast cancer. This study sought to examine the level of TEM-8 expression at the protein and mRNA level in human breast cancer tissue, and in a panel of human breast cancer cell lines. We also wished to determine if TEM-8 can be used as a suitable marker for identifying tumour associated micro-vessels. At the mRNA level more tumours showed positive TEM-8 expression compared to normal background tissue. TEM-8 was detected in a variety of breast cancer cell lines, endothelial cells (HECV) and in a human fibroblast cell line (MRC5) at both the mRNA and protein level. Using immunohistochemistry the distribution of TEM-8 staining was more widespread in invasive breast cancer tissue compared to normal background tissue. Furthermore, the TEM-8 marker was found to be more discriminatory in identifying micro-vessels in tumour endothelium (2.8+/-0.83 vs. normal 1.66+/-0.52; P<0.011), compared to the vWFA marker (1.61+/-0.54 vs. normal 2.71+/-0.76; P<0.009). Raised levels of TEM-8 were associated with shorter survival outcome, but were not correlated to disease-free survival as shown by Kaplan-Meier and Cox regression analysis. We conclude that TEM-8 is a useful marker for identifying tumour associated micro-vessels and that elevated levels are associated with disease progression, which may have some bearing on the prognostic outcome in breast cancer.

Decreased pigment epithelium-derived factor expression in human breast cancer progression.

PURPOSE: The aim of this study was to correlate the expression of pigment epithelium-derived factor (PEDF), a potent endogenous antiangiogenic molecule, with severity and prognosis in breast cancer. EXPERIMENTAL DESIGN: To investigate the gene expression profile of PEDF in human breast cancer in relation to a patient's clinical variables, we examined human breast cancer tissue (n = 119), background breast tissue (n = 33), and a range of cell lines for mRNA and protein levels of PEDF by using reverse transcription PCR, real-time quantitative PCR, immunohistochemistry, and ELISA. RESULTS: By using reverse transcription PCR, real-time quantitative PCR, immunohistochemistry, and ELISA, PEDF expression was found to be dramatically decreased in breast cancer. An overall outlook for the patients inversely correlated with PEDF mRNA levels. Exogenous PEDF inhibits endothelial tubule formation induced by breast cancer cell-conditioned medium, in vitro. CONCLUSION: These observations collectively support the hypothesis that a lack of PEDF expression is a potent factor for the enhancement of tumor growth and angiogenesis in breast cancer.