Microcalcifications in breast cancer: From pathophysiology to diagnosis and prognosis.

The implementation of mammographic screening programmes in many countries has been linked to a marked increase in early detection and improved prognosis for breast cancer patients. Breast tumours can be detected by assessing several features in mammographic images but one of the most common are the presence of small deposits of calcium known as microcalcifications, which in many cases may be the only detectable sign of a breast tumour. In addition to their efficacy in the detection of breast cancer, the presence of microcalcifications within a breast tumour may also convey useful prognostic information. Breast tumours with associated calcifications display an increased rate of HER2 overexpression as well as decreased survival, increased risk of recurrence, high tumour grade and increased likelihood of spread to the lymph nodes. Clearly, the presence of microcalcifications in a tumour is a clinically significant finding, suggesting that a detailed understanding of their formation may improve our knowledge of the early stages of breast tumourigenesis, yet there are no reports which attempt to bring together recent basic science research findings and current knowledge of the clinical significance of microcalcifications. This review will summarise the most current understanding of the formation of calcifications within breast tissue and explore their associated clinical features and prognostic value.

Associations between single-nucleotide polymorphisms of ADIPOQ, serum adiponectin and increased type 2 diabetes mellitus risk in Bahraini individuals.

This study aimed to estimate the frequency of the SNPs (+45T>G and +276G>T) genotypes and investigate the association between the two SNPs and adiponectin concentration, metabolic parameters and risk of T2DM in the Bahraini population. We genotyped the two ADIPOQ SNPs in 140 unrelated T2DM patients and 66 nondiabetic controls using the polymerase chain reaction-restriction fragment length polymorphism assay. Lipid profile was measured by enzymatic methods. Total serum adiponectin levels were measured by immunoassay. T2DM patients had reduced adiponectin levels compared with controls. +45T>G was more prevalent in patients than controls. The rare G allele of +45T>G occurred more frequently than the common T allele in T2DM patients compared with controls, and was associated with lower serum adiponectin levels. There was no significant difference in allele and genotype frequencies of +276G>T between type T2DM patients and controls. There was no association between both SNPs and metabolic parameters.

Polymeric prodrug combination to exploit the therapeutic potential of antimicrobial peptides against cancer cells.

Antimicrobial Peptides (AMPs) have unique anticancer properties, but their clinical application is currently limited by an inadequate margin of safety. A prodrug strategy associated with a combination therapy approach could address this limitation by increasing their therapeutic index and their efficacy. Accordingly, the first targeted anticancer polymeric prodrug candidates of AMPs, intended for combination therapy with another polymeric prodrug of an approved antineoplastic agent (doxorubicin), were synthesized as either a PEG-based dual-release prodrug or two individual pegylated prodrugs. The latter are based on a cathepsin B-labile peptide linker and an acid-sensitive acyl hydrazone bond for the AMP and doxorubicin prodrugs, respectively. Anticancer activities and toxicity differentials achieved with the free peptide and its polymer conjugates against ovarian, cancer and non-malignant, cells, indicate that protease-dependent reversible pegylation could be implemented to increase the therapeutic indices of AMPs in cancer therapy. The results obtained also show that this approach can be developed if the releasable PEG linker can be optimised to conciliate the attributes and restrictions of pegylation against proteases. In addition, combination of the polymeric prodrugs of the AMP and of doxorubicin provides additive antitumor effects which could be exploited to enhance the efficacy of the AMP candidate.

Associations between single-nucleotide polymorphisms of ADIPOQ, serum adiponectin and increased type 2 diabetes mellitus risk in Bahraini individuals

This study aimed to estimate the frequency of the SNPs [+45T>G and +276G>T] genotypes and investigate the association between the two SNPs and adiponectin concentration, metabolic parameters and risk of T2DM in the Bahraini population. We genotyped the two ADIPOQ SNPs in 140 unrelated T2DM patients and 66 nondiabetic controls using the polymerase chain reaction-restriction fragment length polymorphism assay. Lipid profile was measured by enzymatic methods. Total serum adiponectin levels were measured by immunoassay. T2DM patients had reduced adiponectin levels compared with controls. +45T>G was more prevalent in patients than controls. The rare G allele of +45T>G occurred more frequently than the common T allele in T2DM patients compared with controls, and was associated with lower serum adiponectin levels. There was no significant difference in allele and genotype frequencies of +276G>T between type T2DM patients and controls. There was no association between both SNPs and metabolic parameters

La présente étude avait pour objectif de mesurer la fréquence des polymorphismes mononucléotidiques [+45T>G et +276G>T] des génotypes et d'évaluer l'association entre ces deux polymorphismes et la concentration d'adiponectine, les paramètres métaboliques et le risque de diabète non insulino-dépendant [DNID] dans la population bahreinienne. Nous avons génotypé les deux polymorphismes mononucléotidiques du gène ADIPOQ chez 140 patients atteints de DNID sans lien de parenté et 66 témoins non diabétiques en recourant à l'analyse du polymorphisme de longueur des fragments de restriction par réaction en chaîne de polymérase. Le profil lipidique a été mesuré au moyen de méthodes enzymatiques. Les concentrations d'adiponectine totale sérique ont été mesurées par immunodosage. Les patients atteints de DNDI affichaient des concentrations d'adiponectine réduites par rapport aux témoins. Le polymorphisme +45T>G avait une prévalence plus élevée chez les patients que chez les témoins. L'allèle rare G du polymorphisme +45T>G apparaissait plus fréquemment que l'allèle commun T chez les patients atteints de DNID que chez les témoins, et était associé à des concentrations d'adiponectine sérique plus faibles. Il n'existait pas de différence significative entre les fréquences des allèles et des génotypes du polymorphisme +276G>T entre les patients atteints de DNID et les témoins. Aucune association entre les deux polymorphismes et les paramètres métaboliques n'a été notée

Derivatisation of buforin IIb, a cationic henicosapeptide, to afford its complexation to platinum(ii) resulting in a novel platinum(ii)-buforin IIb conjugate with anti-cancer activity.

Herein we report the synthesis of buforin IIb, its novel malonate derivative malBuf and its Pt(ii) complex cis-[Pt(NH3)2(malBuf-2H)]. We decided to harness the cell targeting, cell-penetrating and anti-proliferative effects of buforin IIb to help target a cytotoxic dose of a Pt DNA binding species, {Pt(NH3)2} to cancer cells whilst also delivering a peptide with potent anti-cancer properties. Preliminary in vitro data shows cis-[Pt(NH3)2(malBuf-2H)] to be more cytotoxic against the cisplatin resistant ovarian cancer cell line (A2780cisR) relative to buforin IIb, cisplatin and cis-[Pt(NH3)2(malonate)].

Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation.

BACKGROUND: Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood. METHODS: We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice. RESULTS: Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice. CONCLUSION: This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development.

Microsomal prostaglandin E2 synthase 1 expression in basic calcium phosphate crystal-stimulated fibroblasts: role of prostaglandin E2 and the EP4 receptor.

OBJECTIVE: Basic calcium phosphate (BCP) crystals have been implicated in the pathogenesis of osteoarthritis (OA), in part because of their ability to upregulate cyclooxygenase and prostaglandin E(2) (PGE(2)) production. The aim of this work was to investigate the expression of terminal PGE(2) synthases and PGE(2) receptors (EP) in BCP crystal-stimulated fibroblasts. METHODS: Cultured fibroblasts were stimulated with BCP crystals in vitro. mRNA expression was measured by real-time polymerase chain reaction, and protein production by western blotting. RESULTS: Basal expression of microsomal prostaglandin E(2) synthase 1 (mPGES1) in osteoarthritic synovial fibroblasts (OASF) was found to be 30-fold higher than in human foreskin fibroblasts (HFF). BCP crystals increased mPGES1 expression fourfold in HFF, but not in OASF. EP4 expression was downregulated twofold by BCP crystals in OASF, but not in HFF. Exogenous PGE(2) also downregulated EP4 expression; this effect was blocked by co-administration of L-161,982, a selective EP4 antagonist. While administration of exogenous PGE(2) significantly upregulated mPGES1 expression in OASF, mPGES1 expression was threefold higher in the OASF treated with BCP crystals and PGE(2) as compared with OASF treated with PGE(2) alone. CONCLUSIONS: The differing effects of BCP crystals on mPGES1 expression in HFF and OASF may be explained by BCP crystal-induced EP4 downregulation in OASF, likely mediated via PGE(2). These data underline the complexity of the pathways regulating PGE(2) synthesis and suggest the existence of a compensatory mechanism whereby mPGES1 expression can be diminished, potentially reducing the stimulus for further PGE(2) production.

Mechanism of basic calcium phosphate crystal-stimulated cyclo-oxygenase-1 up-regulation in osteoarthritic synovial fibroblasts.

OBJECTIVES: Basic calcium phosphate (BCP) crystals have been implicated in the pathogenesis of OA and stimulate cyclo-oxygenase (COX) expression and PGE(2) production. This study aimed to elucidate the mechanism of COX-1 up-regulation by BCP crystals and to characterize the PGs produced in OA synovial fibroblasts (OASFs) in response to BCP crystals. METHODS: OASFs were stimulated with BCP crystals in vitro. mRNA expression was measured by real-time PCR, PG production by EIA and protein production by western blot. RESULTS: Maximal (19-fold) up-regulation of COX-1 mRNA occurred 32 h after stimulation with BCP crystals; increased COX-1 protein production was also seen. At 32 h post-stimulation with BCP crystals, PGE(2) (and prostacyclin) production was COX-1 dependent. In contrast, maximal (17-fold) up-regulation of COX-2, with corresponding COX-2-dependent PG production, occurred 4 h after BCP crystal stimulation. There was no appreciable increased production of other PGs such as PGF(2alpha), thromboxane A(2) or cyclopentanone PGs including 15d-PGJ(2). Inhibition of protein kinase C (PKC) and extracellular regulated kinase 1/2 (ERK1/2) signal transduction pathways blocked BCP crystal-induced COX-1 mRNA expression. Bafilomycin A1, an inhibitor of intra-lysosomal BCP crystal dissolution, diminished BCP crystal-induced COX-1 mRNA expression. CONCLUSIONS: These findings indicate that BCP crystals can augment PG production in OASF through induction of COX-1 and COX-2. Intra-lysosomal BCP crystal dissolution and activity of the PKC and ERK1/2 signal transduction pathways are required for BCP crystal-induced COX-1 up-regulation. These data add to the evidence suggesting that the constitutive COX-1/inducible COX-2 concept is an over-simplification and suggest that non-selective COX inhibition may be preferable to COX-2 selective inhibition in BCP crystal-associated OA.

Mechanism of basic calcium phosphate crystal-stimulated matrix metalloproteinase-13 expression by osteoarthritic synovial fibroblasts: inhibition by prostaglandin E2.

OBJECTIVE: To determine the mechanism of matrix metalloproteinase (MMP)-13 upregulation in osteoarthritic synovial fibroblasts (OASF) in response to stimulation with basic calcium phosphate (BCP) crystals and to investigate the effect of prostaglandin (PG)E2 on BCP crystal-stimulated MMP expression. METHODS: Primary OASF were stimulated with BCP crystals; mRNA expression was measured by real-time reverse transcription-polymerase chain reaction and protein levels were assessed by Western blotting. RESULTS: BCP crystals upregulated MMP-13 mRNA expression over 20-fold and increased MMP-13 protein production in OASF. BCP crystal-stimulated MMP-13 mRNA expression was blocked by inhibition of the extracellular regulated kinase (ERK1/2) and p38 mitogen activated protein kinase (MAPK) pathways and inhibition of the activation of nuclear factor kappaB. Addition of exogenous PGE2 downregulated BCP crystal-stimulated MMP-13 expression. In contrast, PGE2 upregulated, and had no effect, on BCP crystal stimulated MMP-3 and MMP-1 mRNA expression, respectively. These effects of PGE2 were diminished by L-161,982, a selective EP4 receptor antagonist, and mimicked by CAY10399, a selective EP2 receptor agonist, and forskolin, an adenylate cyclase activator. CONCLUSIONS: These data suggest that BCP crystal induction of MMP-13 expression may involve the ERK1/2 and p38 MAPK pathways and activation of nuclear factor kappaB; this upregulation of MMP-13 may contribute to the accelerated cartilage breakdown in BCP crystal-associated osteoarthritis. PGE2 had contrasting effects on BCP crystal-stimulated MMP-3 and MMP-13 mRNA expression, mediated in an EP2/EP4/cAMP-dependent manner, suggesting that PGE2 may have beneficial as well as deleterious effects in BCP crystal-associated osteoarthritis.

BCP crystals increase prostacyclin production and upregulate the prostacyclin receptor in OA synovial fibroblasts: potential effects on mPGES1 and MMP-13.

OBJECTIVE: To investigate the potential involvement of prostacyclin in basic calcium phosphate (BCP) crystal-induced responses in osteoarthritic synovial fibroblasts (OASF). METHODS: OASF grown in culture were stimulated with BCP crystals. Prostacyclin production was measured by enzyme immunoassay. Expression of messenger RNA (mRNA) transcripts was assessed by real-time polymerase chain reaction (PCR). Expression of prostacyclin synthase (PGIS) and the prostacyclin (IP) receptor was measured. The effects of iloprost, a prostacyclin analogue, on expression of genes implicated in osteoarthritis such as microsomal prostaglandin E2 synthase 1 (mPGES1) and matrix metalloproteinases (MMPs) were also studied. FPT inhibitor II, a farnesyl transferase inhibitor, was used to antagonize iloprost-induced responses. RESULTS: BCP crystal stimulation led to a five-fold increase in prostacyclin production in OASF compared to untreated cells. This induction was attenuated by cyclooxygenase (COX)-2 and COX-1 inhibition at 4 and 32h, respectively. PGIS and IP receptor transcripts were constitutively expressed in OASF. BCP crystals upregulated IP receptor expression two-fold. While iloprost diminished BCP crystal-stimulated IP receptor upregulation, the inhibitory effect of iloprost was blocked by the farnesyl transferase inhibitor. In addition, iloprost upregulated mPGES1 and downregulated MMP-13 expression in BCP crystal-stimulated OASF, effects that were not influenced by the farnesyl transferase inhibitor. CONCLUSIONS: These data showed for the first time that BCP crystals can increase prostacyclin production and upregulate expression of the IP receptor in OASF. The potential of prostacyclin to influence BCP crystal-stimulated responses was supported by the effects of iloprost on the expression of the IP receptor, mPGES1 and MMP-13. These data demonstrate the potential involvement of prostacyclin in BCP crystal-associated osteoarthritis (OA) and suggest that inhibition of PG synthesis with non-steroidal anti-inflammatory drugs may have both deleterious and beneficial effects in BCP crystal-associated OA.

Calcium hydroxyapatite promotes mitogenesis and matrix metalloproteinase expression in human breast cancer cell lines.

Radiographic mammary microcalcifications are one of the most pertinent diagnostic markers of breast cancer. Breast tissue calcification in the form of calcium hydroxyapatite (HA) is strongly associated with malignant disease. We tested the hypothesis that calcium HA may exert biological effects on surrounding cells, thereby facilitating breast cancer progression. Our findings showed that HA crystals enhanced mitogenesis in breast cancer cell lines MCF-7 and Hs578T and also in normal human mammary epithelial cells. HA crystals were also found to upregulate the production of a variety of matrix metalloproteinases (MMPs), including MMP-2, -9, and -13 in MCF-7 and MMP-9 in human mammary epithelial cell lines. HA crystals were found to greatly augment prostaglandin E(2) levels in Hs578T cells, and treatment with a cyclooxygenase inhibitor, aspirin, abrogated the HA-induced mitogenesis. These results suggest that calcium HA crystals may play an active role in amplifying the pathological process involved in breast cancer.

Development of a shortened version of the Breathing Problems Questionnaire suitable for use in a pulmonary rehabilitation clinic: a purpose-specific, disease-specific questionnaire.

One hundred and thirty-eight chronic obstructive pulmonary disease (COPD) patients completed the Breathing Problems Questionnaire (BPQ) before and after a comprehensive programme of rehabilitation. Examination of the changes on individual items showed improvement on 22 items, of which four items were significant at p < 0.05 and deterioration on nine items, of which two were significant at p < 0.01. All deteriorating items were consistent with lifestyle adaptations encouraged as part of the rehabilitation programme. We examined the psychometric properties of a reduced ten item version of the BPQ limited to the items most sensitive to change. We recommend the purpose-specific, disease-specific COPD scale for measuring change in pulmonary rehabilitation assessment in contrast to the longer 33 item questionnaire, which, however, may be more useful for cross-sectional assessment.

Treatment of posttraumatic stress disorder with eye movement desensitization.

This case report describes the successful treatment of a posttraumatic stress disorder (PTSD) using eye movement desensitization (EMD). The client, a 40-year-old male, presented with an 8-year history of PTSD following an incident in which he was shot with a hand gun and left dying. Using EMD treatment, this trauma was quickly desensitized. Two earlier traumas with similar themes then emerged and they too were desensitized. Test results, taken pre-treatment and posttreatment, along with the client's verbatim account of cognitive and behavioral changes 8 months later, converged to document the successful treatment outcome.

Human ciliary body epithelium in culture.

Human ciliary body epithelial cells have been maintained in vitro and have been partially characterized by the determination of growth rate, morphology, and ultrastructural parameters. The dissection technique employed allows the separation of pure ciliary body epithelium with a predominance of cells being from the nonpigmented layer. Growth curves indicate this cell population follows a prolonged rate of growth compared to other primary cell cultures. Loss of pigment granules noted by light microscopy were documented by morphometric analysis of electron micrographs. Thirty-two percent of the cultures attempted were successful. Maintenance of these cells in vitro may provide a means for studying their enzyme systems, growth factors, reactions to various stimuli, and the effects of this cell population on other intraocular tissues.