Investigating the Anti-tumor and Apoptosis-inducing Effects of Coumarin Derivatives as Potent 15-Lipoxygenase Inhibitors on PC-3 Prostate Cancer Cells.

INTRODUCTION: Prostate cancer is the second most prevalent cancer among men. Despite different treatments, including surgery, chemotherapy, radiation therapy, hormone therapy and immunotherapy for this disease, patients ultimately progress to advanced states. Thus, there is a need for new treatment options targeting cell growth and apoptosis to better control the proliferation and metastasis of these cells. There are many reports indicating overexpression of the 15-lipoxygenase-1 (15-LOX-1) enzyme in prostate tumors. Studies have also shown that inhibition of this enzyme prevents the progression of prostate cancer. OBJECTIVE: This study was conducted to assess the anti-cancer properties of some coumarin derivatives as possible 15- LOX-1 inhibitors, on PC-3 prostate cancer cells. METHODS: In this study, the activity of 15-LOX-1 was evaluated in PC-3 cells by a spectrophotometric assay. In addition, due to high similarity between the 15-LOX-1 and soybean 15-lipoxygenase (SLO) (L1; EC 1, 13, 11, 12) active sites, the soybean SLO was used to investigate inhibitory effects of synthetic coumarin compounds 8- isopentenyloxycoumarin (8-IC), 8-isopentenyloxy-3-carboxycoumarin (8-ICC), 8-geranyloxycoumarin (8-GC), 8- geranyloxy-3-carboxycoumarin (8-GCC), and 8-farnesyloxy-3-carboxycoumarin (8-FCC) on this enzyme. Moreover, the cytotoxic and anticancer effects of the coumarin compounds were examined on PC-3 (Prostate Cancer) and HDF-1 (Human Dermal Fibroblast) cells by alamarBlue assay. Finally, apoptosis-inducing effects of all synthetic compounds were determined by flow cytometry. RESULTS: The IC50 values obtained by the alamarBlue test revealed that 8-IC, 8-GC and 8-GCC had cytotoxic effects on PC-3 cells. Treating both PC-3 and HDF-1 cells with 8-ICC and 8-FCC did not significantly reduce cell number. Furthermore, the IC50 values of 8-IC on HDF-1 cells showed cytotoxic effects, while treating these cells with 8-GC and 8- GCC did not show any significant cytotoxicity on these normal human fibroblasts. Assessing the ability of 4-MMPB (as a specific inhibitor of 15-LOX-1), 8-GC, and 8-GCC compounds to inhibit SLO revealed that these compounds exerted strong 15-LOX-1 inhibitory activity, while 8-IC and 8-FCC had a weak inhibitory effect and also 8-ICC showed no inhibitory effect on SLO enzyme. In addition, flow cytometric analysis by FITC (fluorescein isothiocyanate)- annexin V and propidium iodide showed that treatment with IC50 values of 8-GC and 8-GCC induced apoptosis in 35.2% and 30.8% of PC-3 cells, respectively. CONCLUSION: Thus, 8-GC and 8-GCC can be introduced as effective anticancer agents with apoptosis-inducing properties. Furthermore, our results suggest that the cytotoxic effects of these compounds might be related to the inhibition of 15-LOX-1 enzyme in PC-3 cells. On the other hand, the cytotoxic effects of 8-IC might be due to the inhibition of other signaling pathways in PC-3 cells. However, further in vivo experiments are required to determine the exact mechanisms involved in the anticancer effects of these coumarin compounds.

Investigating the expression of pluripotency-related genes in human amniotic fluid cells: A semi-quantitative comparison between different subpopulations, from primary to cultured amniocytes.

Various classifications have been proposed for human amniotic subpopulations, including classification of spindle-shaped (SS) and round-shaped (RS) cells, as well as the more referred triple-category of epithelioid (E-type) cells, amniotic fluid-specific (AF-type) cells and fibroblastoid (F-type) cells. The present study aims to investigate these amniotic subpopulations regarding the expression of some stem cell markers, including OCT4, NANOG, SOX2, C-KIT (CD117), C-MYC, KLF4, and THY1 (CD90). Flow cytometry was performed to characterize the isolated clonal subpopulations for a hematopoietic and a mesenchymal marker using PE-CD31 and FITC-CD90, respectively. A semi-quantitative RT-PCR analysis was carried out on the isolates in the second half of their lifespan when the cells were at the stationary phase of the growth curve. Characterization of isolated cells demonstrated that all clones including both epithelioid and fibroblastoid cells, had mesenchymal, not hematopoietic, lineage. RT-PCR analysis also revealed a higher expression of the target genes in epithelioid cells. Furthermore, the expression pattern of the genes and their correlations were remarkably different between primary- and long term-cultured amniocytes. Taken together, our results showed that the primary-cultured cells express the stemness genes equally, whereas the long term-cultured amniocytes exhibited a highly variable manner in the expression pattern of the genes. Diverse derivation site of amniocytes and individual genetic background can potentially explain the observed variation in the expression level of the target genes. These can also explain why amniocytes differ in many respects observed in our study, including survival rate, plastic adhesion, and growth characteristics.

The importance of 15-lipoxygenase inhibitors in cancer treatment.

Cancer-targeted therapy is an expanding and successful approach in treatment of many types of cancers. One of the main categories of targeted therapy is use of small molecule inhibitors. 15-Lipoxygenase (15-LOX) is an enzyme which reacts with polyunsaturated fatty acids and produces metabolites that are implicated in many important human diseases, such as cancer. Considering the role of 15-LOX (mainly 15-LOX-1) in the progression of some cancers, the discovery of 15-LOX inhibitors could potentially lead to development of novel cancer therapeutics and it can be claimed that 15-LOX inhibitors might be suitable as chemotherapy agents in the near future. This article reviews relevant publications on 15-LOX inhibitors with focus on their anticancer activities in vitro and in vivo. Many 15-LOX inhibitors have been reported for which separate studies have shown their anticancer activities. This review paves the way to further explore the mechanism of their antiproliferative effects via 15-LOX inhibition.

The effect of platelet-rich plasma on human mesenchymal stem cell-induced bone regeneration of canine alveolar defects with calcium phosphate-based scaffolds.

OBJECTIVES: Autologous bone transplantation known as the "gold standard" to reconstruction of osseous defects has known disadvantages. This study was designed to explore the effects of hydroxy-apatite/tricalcium-phosphate (HA/TCP) and platelet-rich plasma (PRP) on the osteogenesis ability of human adipose-derived mesenchymal stem cells (hAdMSCs) in vitro and in vivo. MATERIALS AND METHODS: hAdMSCs were incubated with HA/TCP granules and/or PRP in vitro and then, cell proliferation and differentiation was assessed by MTT assay, AZR S staining and SEM examination. In vivo, four cylindrical defects were drilled in the mandibular bones of 5 mongrel dogs and divided randomly into the following groups: I-autologous crushed bone, II- no filling material, III- HA/TCP and PRP, IV- PRP-enriched hAdMSCs seeded on HA/TCP granules. Inserted hAdMSCs were labeled to trace their contribution to bone tissue regeneration. Finally, cell tracing and tissue regeneration were evaluated by immunohistochemistry and histomorphometry methods, respectively. RESULTS: In vitro, co-incubation with HA/TCP granules significantly reduced proliferation and osteogenic differentiation ability of hAdMSCs; while PRP application promoted these capacities (P<0.05). In vivo, PRP-enriched hAdMSCs seeded on HA/TCP granules induced considerable bone formation in osseous defects (P<0.05). It was obviously shown that hAdMSCs were incorporated into the newly-formed bone. CONCLUSION: Based on this study, application of stem cells could offer a helpful therapeutic tool in bone tissue regeneration. Although inserted hAdMSCs were identifiable throughout the newly-formed bone tissue, their few number could be an indicator of indirect role of hAdMSCs in tissue regeneration.

Effects of Human Adipose-derived Stem Cells and Platelet-Rich Plasma on Healing Response of Canine Alveolar Surgical Bone Defects.

BACKGROUND: Due to the known disadvantages of autologous bone grafting, tissue engineering approaches have become an attractive method for ridge augmentation in dentistry. To the best of our knowledge, this is the first study conducted to evaluate the potential therapeutic capacity of PRP-assisted hADSCs seeded on HA/TCP granules on regenerative healing response of canine alveolar surgical bone defects. This could offer a great advantage to alternative approaches of bone tissue healing-induced therapies at clinically chair-side procedures. METHODS: Cylindrical through-and-through defects were drilled in the mandibular plate of 5 mongrel dogs and filled randomly as following: I- autologous crushed mandibular bone, II- no filling material, III- HA/TCP granules in combination with PRP, and IV- PRP-enriched hADSCs seeded on HA/TCP granules. After the completion of an 8-week period of healing, radiographic, histological and histomorphometrical analysis of osteocyte number, newly-formed vessels and marrow spaces were used for evaluation and comparison of the mentioned groups. Furthermore, the buccal side of mandibular alveolar bone of every individual animal was drilled as normal control samples (n=5). RESULTS: Our results revealed that hADSCs subcultured on HA/TCP granules in combination with PRP significantly promoted bone tissue regeneration as compared with those defects treated only with PRP and HA/TCP granules (P<0.05). CONCLUSION: In conclusion, our results indicated that application of PRP-assisted hADSCs could induce bone tissue regeneration in canine alveolar bone defects and thus, present a helpful alternative in bone tissue regeneration.

Pluripotency induction in HEK293T cells by concurrent expression of STELLA, OCT4 and NANOS2.

Germline stem cells (GSCs) are attractive biological models because of their strict control on pluripotency gene expression, and their potential for huge epigenetic changes in a short period of time. Few data exists on the cooperative impact of GSC-specific genes on differentiated cells. In this study, we over-expressed 3 GSC-specific markers, STELLA, OCT4 and NANOS2, collectively designated as (SON), using the novel polycistronic lentiviral gene construct FUM-FD, in HEK293T cells and evaluated promoter activity of the Stra8 GSC marker gene We could show that HEK293T cells expressed pluripotency and GSC markers following ectopic expression of the SON genes. We also found induction of pluripotency markers after serum starvation in non-transduced HEK293T cells. Expression profiling of SON-expressing and serum-starved cells at mRNA and protein level showed the potential of SON factors and serum starvation in the induction of ESRRB, NANOG, OCT4 and REX1 expression. Additionally, the data indicated that the mouse Stra8 promoter could only be activated in a subpopulation of HEK293T cells, regardless of SON gene expression. We conclude that heterogeneous population of the HEK293T cells might be easily shifted towards expression of the pluripotency markers by ectopic expression of the SON factors or by growth in serum depleted media.

Chemically primed bone-marrow derived mesenchymal stem cells show enhanced expression of chemokine receptors contributed to their migration capability.

OBJECTIVES: The limited homing potential of bone-marrow-derived mesenchymal stem cells (BM-MSC) is the key obstacle in MSC-based therapy. It is believed that chemokines and chemokine receptor interactions play key roles in cellular processes associated with migration. Meanwhile, MSCs express a low level of distinct chemokine receptors and they even lose these receptors on their surface after a few passages which influence their therapeutic applications negatively. This study investigated whether treatment of BM-MSCs with hypoxia-mimicking agents would increase expression of some chemokine receptors and cell migration. MATERIALS AND METHODS: BM-MSCs were treated at passage 2 for our gene expression profiling. All qPCR experiments were performed by SYBR Green method in CFX-96 Bio-Rad Real-Time PCR. The Boyden chamber assay was utilized to investigate BM-MSC homing. RESULTS: Possible approaches to increasing the expression level of chemokine receptors by different hypoxia-mimicking agents such as valproic acid (VPA), CoCl2, and desferrioxamine (DFX) are described. Results show DFX efficiently up-regulate the CXCR7 and CXCR4 gene expression while VPA increase only the CXCR7 gene expression and no significant change in expression level of CXCR4 and the CXCR7 gene was detectable by CoCl2 treatment. Chemotaxis assay results show that pre-treatment with DFX, VPA, and Cocl2 enhances significantly the migration ability of BM-MSCs compared with the untreated control group and DFX treatment accelerates MSCs homing significantly with a higher rate than VPA and Cocl2 treatments. CONCLUSION: Our data supports the notion that pretreatment of MSC with VPA and DFX improves the efficiency of MSC therapy by triggering homing regulatory signaling pathways.

Mesenchymal stem cells can survive on the extracellular matrix-derived decellularized bovine articular cartilage scaffold.

OBJECTIVE S: The scarcity of articular cartilage defect to repair due to absence of blood vessels and tissue engineering is one of the promising approaches for cartilage regeneration. The objective of this study was to prepare an extracellular matrix derived decellularized bovine articular cartilage scaffold and investigate its interactions with seeded rat bone marrow mesenchymal stem cells (BM-MSCs). MATERIALS AND METHODS: Bovine articular cartilage that was cut into pieces with 2 mm thickness, were decellularized by combination of physical and chemical methods including snap freeze-thaw and treatment with sodium dodecyl sulfate (SDS). The scaffolds were then seeded with 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) labeled BM-MSCs and cultured for up to two weeks. RESULTS: Histological studies of decellularized bovine articular cartilage showed that using 5 cycles of snap freeze-thaw in liquid nitrogen and treatment with 2.5% SDS for 4 hr led to the best decellularization, while preserving the articular cartilage structure. Adherence and penetration of seeded BM-MSCs on to the scaffold were displayed by histological and florescence examinations and also confirmed by electron microscopy. CONCLUSION: ECM-derived decellularized articular cartilage scaffold provides a suitable environment to support adhesion and maintenance of cultured BM-MSCs and could be applied to investigate cellular behaviors in this system and may also be useful for studies of cartilage tissue engineering.

Ferutinin, an apoptosis inducing terpenoid from Ferula ovina.

A current hurdle in cancer management is the intrinsic or acquired resistance of cancer cells to chemical agents that restricts the efficacy of therapeutic strategies. Accordingly, there is an increasing desire to discover new natural compounds with selective toxicity to combat malignancies. In present study, the cytotoxic and apoptosis- inducing activities of ferutinin, a terpenoid derivative from Ferula ovina, were investigated on human breast (MCF7) and bladder (TCC) cancer cells as well as normal fibroblasts (HFF3).The toxicity and DNA damage inducing effects of ferutinin were studied by MTT and comet assays, DAPI and PI staining and DNA laddering. The IC50 values of ferutinin were identified and compared with routine prescribed drugs, doxorubicin and vincristine, by MTT test. Alkaline comet assay and DAPI staining revealed DNA damage due to ferutinin, which was significantly (p<0.001) higher in MCF7 and TCC than HFF3 cells. Apoptosis induction was evidenced by PI staining and DNA laddering. Our results suggest that ferutinin could be considered as an effective anticancer agent for future in vivo and clinical experiments.