hymecromone and Breast-Neoplasms

The malignant phenotype of various cancers is linked to enhanced expression of hyaluronan, a pro-angiogenic glycosaminoglycan whose expression is suppressed by 4-methylumbelliferone (4-MU), a non-toxic oral agent used as a dietary supplement to improve health and combat prostate cancer. In this study, we investigated the role of 4-MU in mammary carcinoma cells with distinct malignant phenotypes and estrogen receptor (ER) status, a major prognostic factor in the clinical management of breast cancers. We focused on two breast cancer cell lines, the low metastatic and ERα+ MCF-7 cells, and the highly-aggressive and ERα- MDA-MB-231 cells. Treatment with 4-MU caused a dose-dependent decrease of hyaluronan accumulation in the extracellular matrix as well as within the breast cancer cells, most prevalent in cells lacking ERα. This decrease in hyaluronan was accompanied by suppression of Hyaluronan Synthase 2 (HAS2), the major enzyme responsible for the synthesis of hyaluronan, and by induction of hyaluronidases (HYALs) -1 and -2. Moreover, 4-MU induced intense phenotypic changes and substantial loss of CD44, a major hyaluronan receptor, from cell protrusions. Importantly, 4-MU evoked differential effects depending on the absence or presence of ERα. Only the ERα+ cells showed signs of apoptosis, as determined by cleaved PARP-1, and anoikis as shown by concurrent loss of E-cadherin and β-catenin. Interestingly, 4-MU significantly reduced migration, adhesion and invasion of ERα- breast cancer cells, and concurrently reduced the expression and activity of several matrix degrading enzymes and pro-inflammatory molecules with tumor-promoting functions. Collectively, our findings suggest that 4-MU could represent a novel therapeutic for specific breast cancer subtypes with regard to their ER status via suppression of hyaluronan synthesis and regulation of HAS2, CD44, matrix-degrading enzymes and inflammatory mediators.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; MCF-7 Cells; Receptors, Estrogen

Numerous studies have demonstrated the importance of altered hyaluronan metabolism to malignant progression of multiple tumor types, including breast carcinomas. Increased hyaluronan (HA) metabolism in the stroma of primary tumors promotes activation of oncogenic signaling pathways that impact tumor initiation, growth, and invasion. Carcinoma cell synthesis and assembly of HA-rich pericellular matrices induces a stromal-independent phenotype, which is associated with cancer progression. Although the pro-tumorigenic role of stromal HA is well established, a novel but unexplored hypothesis is that carcinoma cell-associated HA pericellular matrices promote metastasis of circulating tumor cells. Here, we report the development of an in vitro assay that employs microfluidic techniques to directly measure the importance of an HA-rich pericellular matrix in the entry of carcinoma cells into ectopic sites. This model provides the capability to visualize specific steps in metastasis, which is difficult using animal models. The results show that the presence of a HA-rich pericellular matrix correlates to the invasive and metastatic potential of breast carcinoma cells. Furthermore, enzymatic removal or pharmacologic inhibition of HA synthesis significantly inhibits carcinoma cell extravasation and invasion in this model system. These results implicate pericellular HA-rich carcinoma cell associated pericellular matrices in colonization of ectopic sites by circulating tumor cells and support specific targeting of this matrix to limit metastasis in patients.

Topics: Animals; Breast Neoplasms; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cell Movement; Extracellular Matrix; Female; Green Fluorescent Proteins; Human Umbilical Vein Endothelial Cells; Humans; Hyaluronic Acid; Hymecromone; Mice; Mice, Nude; Microfluidics; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype

Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Enzyme Activation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Glucuronosyltransferase; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; Lung Neoplasms; MCF-7 Cells; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta1

4-methylumbelliferone (4-MU) has received considerable attention due to its potential for cancer treatment since it inhibits cell proliferation, migration and invasion. An increasing body of evidence suggests that extracellular matrix (e.g. hyaluronan) and nutrients (e.g. glucose) in the tumor microenvironment may affect cellular responses to extracellular signals. This study investigates the role of hyaluronan and glucose on 4-MU-inhibited cell proliferation in breast carcinoma cells.. 4-MU-inhibited cell proliferation was determined using the soluble formazan dye, whose absorbance is directly proportional to the number of living cells, under conditions of competent vs. deficient in producing hyaluronan, or low vs. high glucose in culture media of breast carcinoma cells.. Cellular sensitivity to 4-MU-inhibited cell proliferation was altered by changes in the amount of hyaluronan or glucose in the tumor microenvironment.. Increased amounts of hyaluronan or glucose in the tumor microenvironment reduced cellular sensitivity of breast carcinoma cells to 4-MU-inhibited cell proliferation.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Knockdown Techniques; Glucose; Humans; Hyaluronic Acid; Hymecromone; Mice

Recently, photoresponsive nanoparticles have received significant attention because of their ability to provide spatial and temporal control over the drug release. In the present work, we report for the first time photoresponsive multifunctional magnetic nanoparticles (MNPs) fabricated using coumarin-based phototrigger and Fe/Si MNPs for controlled delivery of anticancer drug chlorambucil. Further, newly fabricated photoresponsive multifunctional MNPs were also explored for cell luminescence imaging. In vitro biological studies revealed that coumarin tethered Fe/Si MNPs of ~9 nm size efficiently delivered the anticancer drug chlorambucil into cancer cells and thereby improving the drug action to kill the cancer cells upon irradiation. Such multifunctional MNPs with strong fluorescence, good biocompatibility and efficient photocontrolled drug release ability will be of great benefit in the construction of light-activated multifunctional nano drug delivery systems.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; Chlorambucil; Drug Delivery Systems; Female; Humans; Hydrogen-Ion Concentration; Hymecromone; Light; Magnetic Fields; Metal Nanoparticles; Microscopy, Electron, Transmission; Photochemical Processes; Ultraviolet Rays

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Previous studies demonstrated that inhibition of HA suppressed the tumorigenicity of various malignant tumors including breast cancer. 4-methylumbelliferone (MU) has been reported to inhibit HA synthesis in several cell types. However, few studies have focused on the effects of HA inhibition in breast cancer cells by MU, nor the effects on bone metastasis. We hypothesized that MU would suppress the progression of bone metastasis via inhibition of HA synthesis. Here, we investigated the effects of MU on HA expression in MDA-MB-231 breast cancer cell line in addition to their tumorigenicity in vitro and in vivo. HAS2 mRNA expression was downregulated after 6 and 24 hr treatment with MU. Quantitative analysis of HA revealed that MU significantly inhibited the intracellular and cell surface HA. MU significantly inhibited cell growth and induced apoptosis as determined by cell proliferation and TUNEL assays, respectively. Phosphorylation of Akt was suppressed after 12 and 24 hr treatment with MU. MU treatment also inhibited cell motility as well as cell invasiveness. MU also inhibited cell growth and motility in murine fibroblast cell line NIH3T3. In vivo, administration of MU inhibited the expansion of osteolytic lesions on soft X-rays in mouse breast cancer xenograft models. HA accumulation in bone metastatic lesions was perturbed peripherally. These data suggest that MU might be a therapeutic candidate for bone metastasis of breast cancer via suppression of HA synthesis and accumulation.

Topics: Animals; Apoptosis; Bone Neoplasms; Breast Neoplasms; Cartilage, Articular; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Extracellular Matrix; Female; Glucuronosyltransferase; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; Mice; NIH 3T3 Cells; Oncogene Protein v-akt; Phosphorylation; RNA, Messenger

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem-like cells (CSC) defined by CD44(+)/CD24(-)/ESA(+) phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Adhesion; Cell Movement; Disease Progression; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glucuronosyltransferase; Humans; Hyaluronan Synthases; Hymecromone; Macrophages; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplastic Stem Cells; Stromal Cells; Up-Regulation

Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA Primers; Down-Regulation; Female; Glucuronosyltransferase; Humans; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; Melanoma; Neoplasm Invasiveness; Neoplasms; RNA, Messenger; RNA, Neoplasm; Uridine Diphosphate Glucuronic Acid