Dynamics of activities of matrix metalloproteinases-9 and -2, and the tissue inhibitors of MMPs in fetal fluid compartments during gestation and at parturition in the mare

During late gestation in the mare, rapid fetal growth is accompanied by considerable placental growth and further invasion of the endometrium by microvilli. This growth requires extensive remodeling of the extracellular matrix (ECM). In early pregnancy, we know that matrix metalloproteinase (MMP)-9 and -2 are involved in the endometrial invasion during endometrial cup formation. The present study investigated whether MMPs are found in fetal fluids later in gestation and during parturition, and if there was a difference in their activities between normal and preterm delivery. Amniotic fluids were collected from pony mares during the latter half of gestation, and amniotic and allantoic fluids from pony and thoroughbred mares at foaling. The fluids were analysed for the activity of MMP-9 and -2, and TIMPs using zymography techniques. There was an increase (P = 0.002) in activity of latent MMP-9 when approaching normal foaling, and a decrease (P < 0.001) during foaling. MMP-2 activity did not change through gestation, or during foaling. When comparing samples from pregnancies resulting in preterm deliveries with samples from foaling mares, the activity of MMP-9 was lower (P < 0.001) and MMP-2 activity was higher (P = 0.004) during foaling than preceding preterm delivery. The activity of MMP-9 was lower (P = 0.002) prior to preterm delivery than before delivery of a live foal at term, whereas no difference (P = 0.07) was demonstrated for latent MMP-2 activity when comparing the same groups. The activity of TIMP-2 was higher (P < 0.001) in the pre-parturient period before normal foaling than preceding preterm delivery. These results suggest that MMPs may have a role as markers for high risk pregnancy in the mare.     

Potent and selective 2-naphthylsulfonamide substituted hydroxamic acid inhibitors of matrix metalloproteinase-13

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would provide a disease modifying therapy for the treatment of arthritis, although this goal still continues to elude the pharmaceutical industry due to issues with safety. Our efforts have resulted in the discovery of a series of hydroxamic acid inhibitors of MMP-13 that do not significantly inhibit MMP-2 (gelatinase-1). MMP-2 has been implicated in the musculoskeletal side effects resulting from pan-MMP inhibition due to findings from spontaneously occurring human MMP-2 deletions. Analysis of the SAR of hundreds of previously prepared hydroxamate based MMP inhibitors lead us to 2-naphthylsulfonamide substituted hydroxamates which exhibited modest selectivity for MMP-13 versus MMP-2. This Letter describes the lead optimization of 1 and identification of inhibitors exhibiting >100-fold selectivity for MMP-13 over MMP-2.     

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease

Background

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.

Methods

Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.

Results

ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.

Conclusions

ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.

General significance

The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.     

Soft drug-resistant ovarian cancer cells migrate via two distinct mechanisms utilizing myosin II-based contractility

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the migration mechanisms of drug-resistant cancer cells remain incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are more contractile and softer than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR cells, indicative of a protease-dependent mode of migration in CisR cells and a protease-independent mode of migration in PacR. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin II signaling pathway, regulates both modes of migration. Confined migration experiments establish the role of myosin IIA and IIB in mediating nuclear translocation and regulation of proteolytic activity. Collectively, our results highlight the importance of myosin II as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.     

Label-free FTIR spectroscopy detects and visualizes the early stage of pulmonary micrometastasis seeded from breast carcinoma

An application of FTIR spectroscopic imaging for the identification and visualization of early micrometastasis from breast cancer to lungs in a murine model is shown. Spectroscopic and histological examination is focused on lung cross-sections derived from animals at the early phase of metastasis (early micrometastasis, EM) as compared to healthy control (HC) and late phase of metastasis (advanced macrometastasis, AM) using murine model of metastatic breast cancer with 4T1 cells orthotopically inoculated. FTIR imaging allows for a detailed, objective and label-free differentiation and visualization of EM foci including large and small micrometastases as well as single cancer cells grouped in clusters. An effect of the EM phase on the entire lung tissue matrix as well as characteristic biochemical profiles for HC and advanced macrometastasis were determined from morphological and spectroscopic points of view. The extraordinary sensitivity of FTIR imaging toward EM detection and discrimination of AM borders confirms its applicability as a complementary tool for the histopathological assessment of the metastatic cancer progression.     

Arylpropionic acid-derived NSAIDs: New insights on derivatization,anticancer activity and potential mechanism of action

NSAIDs displayed chemopreventive and anticancer effects against several types of cancers. Moreover, combination of NSAIDs with anticancer agents resulted in enhanced anticancer activity. These findings have attracted much attention of researchers working in this field. The 2-arylpropionic acid-derived NSAIDs represent one of the most widely used anti-inflammatory agents. Additionally, they displayed antiproliferative activities against different types of cancer cells. Large volume of research was performed to identify molecular targets responsible for this activity. However, the exact mechanism underlying the anticancer activity of profens is still unclear. In this review article, the anticancer potential, structure activity relationship and synthesis of selected profen derivatives were summarized. This review is focused also on non-COX targets which can mediate the anticancer activity of this derivatives. The data in this review highlighted profens as promising lead compounds in future research to develop potent and safe anticancer agents.     

血管生长抑素抑制小鼠胚胎植入的最低有效量及其作用机理

血管生长抑素是一种新的抑制血管生成的因子，它能够特异性地抑制血管生成。本文通过宫角注射法对血管生长抑素抑制小鼠胚胎植入的最低有效量进行了探讨，并通过明胶酶谱分析和免疫印迹等方法研究了不同剂量血管生长抑素对MMP-2和MMP-9活性及蛋白表达的影响。实验表明，宫角注射4μg血管生长抑素是抑制小鼠胚胎植入的最低有效剂量，这一最低有效剂量显著抑制了小鼠子宫MMP-2和MMP-9的活性及蛋白的表达[动物学报49(3)：332—338，2003]。     

Importance of Balance between Extracellular Matrix Synthesis and Degradation in Basement Membrane Formation

The epidermal basement membrane (BM) plays important roles in adhesion between epidermis and dermis and in controlling epidermal differentiation. In a skin-equivalent (SE), components of the epidermal BM such as laminin 5 and type IV and VII collagens were detected in conditioned media and in basal keratinocytes. Despite production of these BM components, however, BM was rarely observed at the dermal-epidermal junction. One possible explanation for the absence of BM in SEs is that matrix metalloproteinases (MMPs) degrade newly synthesized extracellular matrices. In fact, several MMPs, such as MMPs-1, 2, 3, and 9, were observed to be present in conditioned media and some of them were in active forms. Tissue inhibitor of metalloproteinase (TIMP)-2 was not detected, although TIMP-1 was present. BM degradation activity presumably exceeds BM formation activity in the SE, resulting in the absence of lamina densa at the dermal-epidermal junction. Synthetic MMP inhibitors CGS27023A and MMP inhibitor I, which inhibit MMPs 1, 2, 3, and 9, markedly augmented deposition of laminin 5 and type IV and VII collagens at the dermal-epidermal junction, resulting in formation of continuous epidermal BM. These results suggest that the balance between synthesis and degradation of BM components is important for BM formation.