17β-Estradiol Inhibits Lysophosphatidylcholine-Induced Apoptosis in Cultured Vascular Smooth Muscle Cells

Objectives@#Coronary heart disease (CHD) risk increases in women after menopause, but menopausal hormone therapy (MHT) helps prevent CHD if started early after menopause. To explore the mechanism underlying the direct vascular actions of estrogen, the effects of 17β-estradiol (E2) on apoptosis of vascular smooth muscle cells (VSMCs) induced with lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, were investigated in the present study. @*Methods@#VSMCs were isolated from rat aortas. Apoptosis and protein expression of caspases were assessed using propidium iodide staining and Western blot analysis, respectively. Intracellular formation of reactive oxygen species (ROS) was examined using dichlorofluorescein diacetate, a cell-permeable oxidation-sensitive probe, and quantitated with flow cytometry. Nuclear factor-κB (NF-κB) activation was determined after transfection with a reporter plasmid containing the luciferase reporter gene. @*Results@#After pre-treatment for 24 hours, 17β-E2 suppressed lysoPC-induced (15 mM) apoptotic cell death in a dose-dependent manner with statistical significance at near physiological concentration. 17β-E2 (10−6 M) also increased protein levels of caspase-9 and -8 precursors and decreased the active form of caspase-3. Western blot analysis using subcellular fractions showed that 17β-E2 decreased mitochondrial Bax levels and concomitantly increased cytosolic Bax expression. Furthermore, intracellular production of ROS and NF- κB-mediated transcriptional activity were reduced with 17β-E2. In addition, estrogen effects on apoptosis were partially blocked by ICI 182,780, a specific estrogen receptor antagonist. @*Conclusions@#In cultured VSMCs treated with lysoPC, 17β-E2 reduced apoptotic cell death by down-regulating both extrinsic and intrinsic apoptosis pathways, contributing to the preventive action of MHT against CHD.

Effects of 17β-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine

Objectives@#When administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17β-estradiol (17β-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs). @*Methods@#VSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction. @*Results@#Following pre-treatment for 24 hours, 17β-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17β-E2 (10−7 M) inhibited PAI-1 gene expression, and ICI 182,780—a specific estrogen receptor antagonist—blocked the effects of 17β-E2 on the PAI-1 protein. 17β-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17β-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription. @*Conclusions@#In VSMCs stimulated with lysoPC, 17β-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17β-E2 suppressed PAI- 1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.

Cancer-Associated Fibroblasts and Desmoplastic Reactions Related to Cancer Invasiveness in Patients With Colorectal Cancer

PURPOSE: We evaluated the relationship of cancer-associated fibroblasts (CAFs) and desmoplastic reactions with cancer invasiveness and long-term outcomes in patients with colorectal cancer (CRC). METHODS: Histologic evaluation of mature CAFs and desmoplasia was performed by observing the collagen fiber structure and fibroblast cytomorphology in the intratumoral stroma and invasive front of CRC tissues. Cancer-cell invasiveness was evaluated using lymphatic invasion, vascular invasion, perineural invasion, tumor budding, and tumor growth patterns. Overall survival and systemic recurrence were analyzed. A network analysis was performed between CAF maturation, desmoplastic reaction, and cancer invasiveness. RESULTS: The proportions of mature CAFs in the intratumoral stroma and the invasive front were 57.6% and 60.3%, respectively. Epidermal growth factor receptor (EGFR) overexpression was significantly higher in the mature CAFs in the invasive front as compared to immature CAFs. Lymphatic invasion increased as the number of mature fibroblasts in the intratumoral stroma increased. Tumor budding was observed in almost half of both mature and immature stroma samples and occurred more frequently in infiltrating tumors. On network analysis, well-connected islands were identified that was associated with EGFR overexpression, CAF maturation, and infiltrating tumor growth patterns leading to tumor budding. CONCLUSION: The maturity of CAFs and desmoplastic reactions were associated with cancer invasion. However, the cytomorphologic characteristics of CAFs were insufficient as an independent prognostic factor for patients with CRC.

Increase of NKG2D ligands and sensitivity to NK cell-mediated cytotoxicity of tumor cells by heat shock and ionizing radiation

In this study, we have investigated if current cancer therapeutic modalities including hyperthermia and ionizing radiation can increase the expression of NKG2D ligands in human cancer cell lines. The expressions of NKG2D ligands were induced by both heat shock and ionizing radiation in various cell lines including KM12, NCI-H23, HeLa and A375 cells with peaks at 2 h and 9 h after treatment, respectively, although inducibility of each NKG2D ligand was various depending on cell lines. During the induction of NKG2D ligands, heat shock protein 70 was induced by heat shock but not by ionizing radiation. These results were followed by increased susceptibilities to NK cell-mediated cytolysis after treatment with heat shock and ionizing radiation. These results suggest that heat shock and ionizing radiation induce NKG2D ligands and consequently might lead to increased NK cell-mediated cytotoxicity in various cancer cells.

Identification of differentially expressed proteins in the bacterial biofilm / 대한치주과학회지

No abstract available.

Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.

Association of DNA-dependent protein kinase with hypoxia induciblefactor-1 and its implication inresistance to anticancer drugs in hypoxic tum or cells

Tumor hypoxia contributes to the progression of a malignant phenotype and resistance to ionizing radiation and anticancer drug therapy. Many of these effects in hypoxic tumor cells are mediated by expression of specific set of genes whose relation to therapy resistance is poorly understood. In this study, we revealed that DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double strand break repair, would be involved in regulation of hypoxia inducible factor-1 (HIF-1). HIF-1beta-deficient cells showed constitutively reduced expression and DNA-binding activity of Ku, the regulatory subunit of DNA-PK. Under hypoxic condition, the expression and activity of DNA- PK were markedly induced with a concurrent increase in HIF-1alpha expression. Our result also demonstrated that DNA-PK could directly interact with HIF- and especially DNA-PKcs, the catalytic subunit of DNA-PK, could be involved in phosphorylation of HIF-1alpha, suggesting the possibility that the enhanced expression of DNA- PK under hypoxic condition might attribute to modulate HIF-1alpha stabilization. Thus, the correlated regulation of DNA-PK with HIF-1 could contribute to therapy resistance in hypoxic tumor cells, and it provides new evidence for developing therapeutic strategies enhancing the efficacy of cancer therapy in hypoxic tumor cells.

Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells

The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.

Suppression of multidrug resistance via inhibition of heat shock factor by quercetin in MDR cells

MDR1 promoter has been shown to contain heat shock elements (HSE), and it has been reported that FM3A/M and P388/M MDR cells show a constitutively activated heat shock factor (HSF), suggesting that HSF might be an important target for reversing the multidrug resistance. Therefore, it was examined whether quercetin, which has been shown to interfere with the formation of the complex between HSE and HSF, and to downregulate the level of HSF1, can sensitize MDR cells against anticancer drugs by inhibition of HSF DNA-binding activity. In this study, quercetin appeared to inhibit the constitutive HSF DNA-binding activity and the sodium arsenite-induced HSF DNA-binding activity in the MDR cells. The basal and sodium arsenite-induced MDRCAT activities were remarkably suppressed by the treatment of quercetin. These results were well consistent with the finding that the treatment of quercetin decreased the expression level of P-gp, MDR1 gene product, in dose-dependent manner, and markedly increased the sensitivity of MDR cells to vincristine or vinblastine. These results suggest that quercetin can decrease the expression of P-gp via inhibition of HSF DNA-binding activity, and might be useful as a chemosensitizer in MDR cells.