Maria P. Morgan

Microcalcifications Associated with Breast Cancer: An Epiphenomenon or Biologically Significant Feature of Selected Tumors?

Radiographic mammary calcifications occur in 30–50% of breast cancers and constitute one of the most important diagnostic markers of both benign and malignant lesions of the breast. The presence of oxalate-type microcalcification appears to be a reliable criterion in favor of the benign nature of the lesion or, at most, of a lobular carcinoma in situ. In contrast, calcium hydroxyapatite (HA) crystals are associated with both benign and malignant breast tumors. Although the diagnostic value of microcalcifications in breast cancer is of great importance, the genesis of these calcifications is unclear. Despite numerous histological ultrastructure studies of HA deposits in breast carcinomas, to date there have been limited investigations of the potential role of these crystals in breast cancer. We review the literature examining the biological effects of HA crystals in breast cancer cell lines, specifically the mechanism of HA-induced mitogenesis and upregulation of gene expression.

A novel anti-cancer bifunctional platinum drug candidate with dual DNA binding and histone deacetylase inhibitory activity

The successful design and synthesis of a novel multifunctional platinum drug candidate with DNA binding, histone deacetylase inhibitory activity and enhanced selectivity for cancer cells are described.

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.

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 E2 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.

Novel trans-platinum complexes of the histone deacetylase inhibitor valproic acid; synthesis, in vitro cytotoxicity and mutagenicity.

The first examples of Pt complexes of the well known anti-epilepsy drug and histone deacetylase inhibitor, valproic acid (VPA), are reported. Reaction of the Pt(II) am(m)ine precursors trans-[PtCl(2)(NH(3))(py)] and trans-[PtCl(2)(py)(2)] with silver nitrate and subsequently sodium valproate gave trans-[Pt(VPA(-1H))(2)(NH(3))(py)] and trans-[Pt(VPA(-1H))(2)(py)(2)], respectively. The valproato ligands in both complexes are bound to the Pt(II) centres via the carboxylato functionality and in a monodentate manner. The X-ray crystal structure of trans-[Pt(VPA(-1H))(2)(NH(3))(py)] is described. Replacement of the dichlorido ligands in trans-[PtCl(2)(py)(2)] and trans-[PtCl(2)(NH(3))(py)] by valproato ligands (VPA(-1H)) to yield trans-[Pt(VPA(-1H))(2)(py)(2)] and trans-[Pt(VPA(-1H))(2)(NH(3))(py)] respectively, significantly enhanced cytotoxicity against A2780 (parental) and A2780 cisR (cisplatin resistant) ovarian cancer cells. The mutagenicity of trans-[Pt(VPA(-1H))(2)(NH(3))(py)] and trans-[Pt(VPA(-1H))(2)(py)(2)] was determined using the Ames test and is also reported.

Microcalcifications in breast cancer: Lessons from physiological mineralization

Mammographic mammary microcalcifications are routinely used for the early detection of breast cancer, however the mechanisms by which they form remain unclear. Two species of mammary microcalcifications have been identified; calcium oxalate and hydroxyapatite. Calcium oxalate is mostly associated with benign lesions of the breast, whereas hydroxyapatite is associated with both benign and malignant tumors. The way in which hydroxyapatite forms within mammary tissue remains largely unexplored, however lessons can be learned from the process of physiological mineralization. Normal physiological mineralization by osteoblasts results in hydroxyapatite deposition in bone. This review brings together existing knowledge from the field of physiological mineralization and juxtaposes it with our current understanding of the genesis of mammary microcalcifications. As an increasing number of breast cancers are being detected in their non-palpable stage through mammographic microcalcifications, it is important that future studies investigate the underlying mechanisms of their formation in order to fully understand the significance of this unique early marker of breast cancer.

Phosphocitrate Inhibits Calcium Hydroxyapatite Induced Mitogenesis and Upregulation of Matrix Metalloproteinase-1, Interleukin-1β and Cyclooxygenase-2 mRNA in Human Breast Cancer Cell Lines

Microcalcifications containing calcium hydroxyapatite (HA) are often associated with malignant human breast lesions. Frequently, they are the only mammographic features that indicate the presence of a tumoural lesion. We previously reported the induction of both mitogenesis and prostaglandin E2 (PGE2) production and the increased activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in normal human mammary epithelial cells and breast cancer cell lines, treated with HA. In the present study we attempted to elucidate the mechanism of these biological effects. Firstly, we found that direct cell–crystal contact was required for induction of mitogenesis as the effect was not merely a result of isotopic exchange of calcium into the culture medium. Treatment with bafilomycin A1, a proton pump inhibitor, abrogated HA-induced mitogenesis to control cell levels. These results suggest that phagocytosis and intracellular crystal dissolution is required for HA-induced mitogenesis. We also demonstrated that the increase in prostaglandin E2, previously reported, is due, at least in part, to HA-induced upregulation of cyclooxygenase-2 (COX-2) in Hs578T cells. An accumulation of MMP-1 mRNA was also shown in response to HA stimulation in Hs578T cells. Furthermore, a HA-induced increase in interleukin-1β (IL-1β), a potent inducer of MMP-1 gene expression, was demonstrated in Hs578T cells at 2 and 4 h. Treatment with phosphocitrate (PC) (a naturally occurring inhibitor of calcium phosphate crystallisation, which is known to block a number of HA-induced biological effects in other cell types) blocked HA-mediated mitogenesis, as well as, COX-2, MMP-1 and IL-1β induction, at the transcriptional level. These results show that calcium HA crystals are capable of exerting significant biological effects on surrounding cells which can be abrogated by PC and emphasise the role of calcium HA in amplifying the pathological process involved in breast cancer.

Signaling mechanisms involved in crystal-induced tissue damage.

The association of crystal deposition with osteoarthritis and joint destruction is well established. Recent advances in understanding the mechanisms whereby calcium crystals contribute to cartilage damage are highlighted in this review. In vitro studies have shown that when calcium-containing crystals come in contact with cells they cause an influx in Ca2+ concentration and activation of p42/44 mitogen-activated protein kinases. This is followed by induction of proto-oncogenes (c-fos, c-jun) and induction of the nuclear transcription factors activator protein-1 and nuclear factor-&kgr;B, which in turn lead to crystal-induced modulation of normal gene expression. Some of the downstream effects known to date include increased mitogenesis, up-regulation of members of the matrix metalloproteinase family, down-regulation of tissue inhibitor of metalloproteinase-1 and -2 in fibroblasts, induction of neutrophil chemotactic chemokines such as interleukin-8, activation and degranulation of neutrophils, and inhibition of neutrophil apoptosis. Because no known drug prevents or treats the consequences of basic calcium phosphate crystal deposition, an improved understanding of the molecular mechanisms leading to crystal-induced joint degeneration is essential to the development of a rational approach to target the consequences of crystal deposition.

Osteomimicry of Mammary Adenocarcinoma Cells In Vitro; Increased Expression of Bone Matrix Proteins and Proliferation within a 3D Collagen Environment

Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.

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.