Transcription Of Angiogenic Factors Research Articles

The balance between proliferation and transcription of angiogenic factors of mesenchymal stem cells in hypoxia

ABSTRACTBridging large bone defects with mesenchymal stromal cells-seeded scaffolds remains a big challenge in orthopedic surgery, due to the lack of vascularization. Within such a cell–scaffold construct, cells are exposed to ischemic conditions. When human mesenchymal stem cells (hMSCs) encounter hypoxic conditions, they show higher cell proliferation than at ambient oxygen levels. However, when hMSCs are exposed to prolonged ischemia, cell proliferation ceases completely. Exposure of hMSCs to hypoxic conditions is known to result in the transcription of angiogenic factors (AGF), which can promote the development of new blood vessels. In this study, we investigated at which oxygen level hMSC proliferation and the transcription of AGF were optimal. Human bone marrow-derived hMSCs were cultured at 0.1, 1, 2, 3, 4, 5, and 21% oxygen. Cell proliferation over 14 days was assayed using a DNA quantification method. hMSC metabolic activity over 14 days was measured using a MTT test. Quantitative RT-PCR was used to assess mRNA levels of angiogenic factors at the tested oxygen percentages. hMSCs showed the highest cell proliferation rate at 1% oxygen. The highest corrected cell metabolic rate was found at 21% oxygen, followed by 2% oxygen. HIF1α transcription did not increase under hypoxic conditions compared to 21% oxygen conditions. However, transcription of VEGF and ANG-1 was significantly higher at 2% oxygen than at 21% O2. The optimum oxygen range at which hMSCs proliferated rapidly and angiogenic factors ANG-1 and VEGF simultaneously came to expression was from 1 to 2% oxygen.

Inhibition of U-87 MG glioblastoma by AN-152 (AEZS-108), a targeted cytotoxic analog of luteinizing hormone-releasing hormone

Glioblastoma multiforme is the most frequent tumor of the central nervous system in adults and has a dismal clinical outcome, which necessitates the development of new therapeutic approaches. We investigated in vivo the action of the targeted cytotoxic analog of luteinizing hormone releasing hormone, AN-152 (AEZS-108) in nude mice (Ncr nu/nu strain) bearing xenotransplanted U-87 MG glioblastoma tumors. We evaluated in vitro the expression of LHRH receptors, proliferation, apoptosis and the release of oncogenic and tumor suppressor cytokines. Clinical and U-87 MG samples of glioblastoma tumors expressed LHRH receptors. Treatment of nude mice with AN-152, once a week at an intravenous dose of 413 nmol/20 g, for six weeks resulted in 76 % reduction in tumor growth. AN-152 nearly completely abolished tumor progression and elicited remarkable apoptosis in vitro. Genomic (RT-PCR) and proteomic (ELISA, Western blot) studies revealed that AN-152 activated apoptosis, as reflected by the changes in p53 and its regulators and substrates, inhibited cell growth, and elicited changes in intermediary filament pattern. AN-152 similarly reestablished contact regulation as demonstrated by expression of adhesion molecules and inhibited vascularization, as reflected by the transcription of angiogenic factors. Our findings suggest that targeted cytotoxic analog AN-152 (AEZS-108) should be considered for a treatment of glioblastomas.

Sunitinib Exerts Only Limited Effects on the Proliferation and Differentiation of Anaplastic Thyroid Cancer Cells

Novel molecularly targeted drugs are undergoing preclinical and clinical testing to assess their efficacy against refractory thyroid carcinomas. The multikinase inhibitor Sunitinib has been shown to inhibit the kinase activity of the RET oncogene and reduce proliferation in differentiated thyroid cancer cells harboring the RET/PTC rearrangement. In this study, we evaluated its effects in human cell lines derived from differentiated (TPC-1) and anaplastic (8505C, CAL-62, and C643) thyroid cancers. The cells exposed to various concentrations of Sunitinib were examined for: (1) cell viability and presence of apoptosis, analyzed by cell counts, MTT assay, trypan blue exclusion assay, western blotting, and immunofluorescence; (2) expression of cyclin D1 and phosphorylated and nonphosphorylated extracellular signal-regulated kinase (ERK) and Akt proteins, analyzed by western blotting; and (3) transcription of genes encoding thyrocyte differentiation markers (thyroid-stimulating hormone receptor, sodium/iodide symporter, thyroglobulin, and thyroperoxidase) and proangiogenic factors (vascular endothelial growth factor A, platelet-derived growth factors A and B), measured by quantitative reverse transcriptase-polymerase chain reaction. Exposure to nanomolar concentrations of Sunitinib significantly reduced cell viability in only TPC-1 cells, and this effect was paralleled by reduction of cyclin D1 levels. Western blotting revealed reduced phosphorylation of ERK and Akt after 3 and 6 hours of drug exposure. In contrast, the growth of 8505C, CAL-62, and C-643 cells was significantly reduced only by micromolar concentrations of Sunitinib, mainly due to induced necrotic rather than apoptotic death. In these cells, Sunitinib exerted a few significant effects on the transcription of angiogenic factors or thyrocyte differentiation markers. Sunitinib has little or no effect on the growth or differentiation of anaplastic thyroid cancer cells, thus suggesting that it is unlikely to be effective in the treatment of anaplastic thyroid cancer.