Angiogenic responses are enhanced by recombinant human erythropoietin in a model of periventricular white matter damage of neonatal rats through EPOR-ERK1 signaling.

Recombinant human erythropoietin (rh-EPO) has been shown to stimulate neurogenesis and angiogenesis, both of which play crucial roles in the repair of brain injuries. Previously, we observed that rh-EPO treatment effectively reduced brain damage and enhanced angiogenesis in a neonatal rat model of periventricular white matter damage (PWMD). The objective of this research is to investigate the specific mechanism through which rh-EPO regulates angiogenesis following PWMD in premature neonates. We conducted experiments utilizing a neonatal PWMD model. Following rh-EPO treatment, the levels of erythropoietin receptor (EPOR) were found to be increased in the damaged brain of rats. Although the total amount of extracellular signal-regulated kinase (ERK), a downstream protein in the EPO signaling pathway, remained unchanged, there was clear upregulation of phosphorylated ERK1 (p-ERK1) levels. The increase in levels of p-ERK1 was inhibited by an ERK kinase inhibitor, while the total amount of ERK remained unchanged. Conversely, the levels of EPOR were not affected by the inhibitor. Notably, the introduction of rh-EPO led to a significant increase in the frequency of angiogenesis-related cells and the expression levels of angiogenic factors. However, these effects were nullified when the ERK pathway was blocked. These findings indicate that rh-EPO enhances angiogenic responses through the EPOR-ERK1 pathway in a neonatal PWMD model.

Changes in mesenchymal stem cells following long-term culture in vitro.

Mesenchymal stem cells (MSCs), which can be isolated from umbilical cords and induced to differentiate into multiple cell types in vitro, represent an ideal source for cell and gene therapy. MSCs are typically expanded in culture prior to their therapeutic application. However, similar to other types of stem cell, MSCs undergo senescence following a certain number of cell expansion passages in vitro, and eventually stop proliferating. The objective of the present study was to measure the changes that occur over successive passages of MSCs during long­term in vitro culture, and to detect the effect of aging on MSC morphology, phenotype, proliferation, cell cycle, differentiation, intracellular reactive oxygen species (ROS) levels and gene expression. To understand the importance of oxidative stress in the aging of adult stem cells, the current study established a cell model of H2O2­induced MSC premature senescence. Analysis of the biological characteristics of human umbilical cord MSCs during replicative and premature senescence revealed the importance of extrinsic factors in the aging of stem cells, particularly ROS. The findings of the present study suggest that cellular senescence, a state of irreversible growth arrest, can be triggered by ROS. Thus, it is important to improve the extrinsic culture environment of MSCs to retain the phenotype of expanded cells and delay the process of senescence prior to their clinical application.

miR-21: A gene of dual regulation in breast cancer.

Breast cancer is characterized by an elevated capacity for tumor invasion and lymph node metastasis, but the cause remains to be determined. Recent studies suggest that microRNAs (miRNAs) can regulate the evolution of malignant behavior by regulating multiple target genes. A key oncomir in carcinogenesis is miR-21, which is consistently upregulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and searched for miR-21 targets.Total RNA from breast cancer tissue and corresponding adjacent normal tissue was extracted and used to detect miR-21 expression by quantificational real-time polymerase chain reaction (qRT-PCR), followed by analysis of the correlation between gonad hormone indices in peripheral blood and miR-21 expression in cancerous tissues from the same patients. Cell proliferation, colony formation, migration and invasion were then examined to determine the role of miR-21 in regulating breast cancer cells. Finally, western blotting was performed to determine if miR-21 regulated expression of signal transducers and activators of transcription 3 (STAT3), and assays of cell proliferation, colony formation, migration and invasion were performed to examine the role of STAT3 in regulation of breast cancer cells. We found that expression of miR-21 increased from normal through benign to cancerous breast tissues. Enhanced miR-21 expression was associated with serum levels of follicle-stimulating hormone, estradiol, ß-human chorionic gonadotropin, testosterone and prolactin in patients with breast cancer. Furthermore, cell proliferation, colony formation, migration and invasion were increased after overexpression of miR-21 in breast cancer cells and reduced by miR-21 suppression. In addition, we identified a putative miR-21 binding site in the 3'-untranslated region of the STAT3 gene using an online bioinformatical tool. We found that protein expression of STAT3 was significantly downregulated when breast cancer cells were transfected with miR-21 mimics, and was significantly upregulated in breast cancer cells transfected with a miR-21 inhibitor. Finally, we found that cell proliferation, colony formation, migration and invasion were decreased by treatment with 2.5 nM of Stattic, an inhibitor of STAT3 activation. Our data suggest that miR-21 expression is increased in breast cancer and plays an important role as a tumor gene by targeting STAT3, which may act as a double-response controller in breast cancer.

Cytotoxic effects of 4-methylimidazole on bone marrow mesenchymal stem cells in vitro.

4-Methylimidazole (4-MI) is found in a great number of food products. The National Toxicology Program (NTP) revealed that 4-MI is carcinogenic and can also cause anemia and weight loss. Mesenchymal stem cells (MSCs) are able to support hematopoiesis and migrate to the site of tumors. To investigate whether 4-MI has an impact on MSCs, we have measured the ability of cell (osteoblast, adipocyte) proliferation, apoptosis, cell cycle, gene expression, migration and differentiation between control group and the 4-MI group. The results showed that higher concentrations of 4-MI (≥150 µg/ml) had significant effects on BMSCs viability while lower concentrations (≤100 µg/ml) had no significant effects on cell proliferation, apoptosis, migration, differentiation, and expression of relevant marker genes of hematopoietic cytokines, including TPO, SCF, VEGF and FLt3. The results also indicated that 4-MI (≤100 µg/ml) may have no significant effect on the biological characteristics of MSCs. Low concentration of 4-MI in foods and beverages have no toxic effect on BMSCs. The anemia and weight loss of animals caused by 4-MI may not be due to its effect on BMSCs.

Umbilical cord-derived mesenchymal stem cells promote proliferation and migration in MCF-7 and MDA-MB-231 breast cancer cells through activation of the ERK pathway.

Mesenchymal stem cells (MSCs) are known to migrate to tumor tissues and to play an important role in cancer progression. However, the effects of MSCs on tumor progression remain controversial. The purpose of the present study was to detect the effects of human umbilical cord-derived MSCs (hUC­MSCs) on the human breast cancer cell lines MDA-MB­231 and MCF-7 in vitro and the underlying mechanisms. MSCs were isolated and identified from umbilical cord tissues. MDA-MB­231 and MCF-7 cells were treated with conditioned medium (CM) from 10 and 20% umbilical cord MSCs (UC-MSCs), and the resulting changes in proliferation and migration were investigated. The 3-(4,5-dimethyl-2-thiazolyl)­2,5-diphenyl­2-H-tetrazolium bromide (MTT) and plate clone formation assays were used to assess the effect on proliferation, and the effects of CM on MDA-MB-231 and MCF-7 migration were assessed through scratch wound and Transwell migration assays. The expression of cell proliferation- and metastasis-related genes and proteins and activation of the ERK signaling pathway were analyzed by RT-PCR and western blot assays. UC-MSCs are characteristically similar to bone marrow MSCs (BM-MSCs) and exhibit multipotential differentiation capability (i.e., osteoblasts and adipocytes). The MTT, plate clone formation, scratch wound and Transwell migration assay results revealed that 10 and 20% CM promoted the proliferation and migration to higher levels than those observed in the control group. Our findings showed that UC-MSC-CM inhibited E-cadherin expression, increased the expression of N-cadherin and proliferating cell nuclear antigen (PCNA) and enhanced the expression of ZEB1, a transcription factor involved in epithelial­to­mesenchymal transition (EMT), through activation of the ERK pathway. U0126, an inhibitor of ERK, reversed the effects of UC-MSC-CM on breast cancer cell proliferation and migration. We conclude that UC-MSCs promote the proliferation and migration of breast cancer cell lines via activation of the ERK pathway.