The Inhibitory Effect of Ginger Extract on Ovarian Cancer Cell Line; Application of Systems Biology

This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers.Purpose: Ginger is a natural compound with anti-cancer properties. The effects of ginger and its mechanism on ovarian cancer and its cell line model, SKOV-3, are unclear. In this study, we have evaluated the effect of ginger extract on SKOV-3. Methods: SKOV-3 cells were incubated with ginger extract for 24, 48 and 72 hours. Cell toxicity assay was performed. Different data mining algorithms were applied to highlight the most important features contributing to ginger inhibition on the SKOV-3 cell proliferation. Moreover, Real-Time PCR was performed to assay p53, p21 and bcl-2 genes expression. For co-expression meta-analysis of p53, mutual ranking (MR) index and transformation to Z-values (Z distribution) were applied on available transcriptome data in NCBI GEO data repository. Results: The ginger extract significantly inhibited cancer growth in ovarian cancer cell line. The most important attribute was 60 μg/ml concentration which received weights higher than 0.50, 0.75 and 0.95 by 90%, 80% and 50% of feature selection models, respectively. The expression level of p53 was increased sharply in response to ginger treatment. Systems biology analysis and meta-analysis of deposited expression value in NCBI based on rank of correlation and Z-transformation approach unraveled the key co-expressed genes and coexpressed network of P53, as the key transcription factor induced by ginger extract. High co-expression between P53 and the other apoptosis-inducing proteins such as CASP2 and DEDD was noticeable, suggesting the molecular mechanism underpinning of ginger action. Conclusion: We found that the ginger extract has anticancer properties through p53 pathway to induce apoptosis

Human Mesenchymal Stem Cell Transplantation Improved Functional Outcomes Following Spinal Cord Injury Concomitantly with Neuroblast Regeneration

Purpose: Spinal cord injury (SCI) is damage to the spinal cord that resulted in irreversible neuronal loss, glial scar formation and axonal injury. Herein, we used the human amniotic fluid mesenchymal stem cells (hAF-MSCs) and their conditioned medium (CM), to investigate their ability in neuroblast and astrocyte production as well as functional recovery following SCI. Methods: Fifty-four adult rats were randomly divided into nine groups (n=6), included: Control, SCI, (SCI+DMEM), (SCI+CM), (SCI+MSCs), (SCI+Astrocyte), (SCI+Astrocyte+DMEM), (SCI+Astrocyte+CM) and (SCI+Astrocyte+MSCs). Following laminectomy and SCI induction, DMEM, CM, MSCs, and astrocytes were injected. Western blot was performed to explore the levels of the Sox2 protein in the MSCs-CM. The immunofluorescence staining against doublecortin (DCX) and glial fibrillary acidic protein (GFAP) was done. Finally, Basso-Beattie-Brenham (BBB) locomotor test was conducted to assess the neurological outcomes. Results: Our results showed that the MSCs increased the number of endogenous DCX-positive cells and decreased the number of GFAP-positive cells by mediating juxtacrine and paracrine mechanisms (P<0.001). Transplanted human astrocytes were converted to neuroblasts rather than astrocytes under influence of MSCs and CM in the SCI. Moreover, functional recovery indexes were promoted in those groups that received MSCs and CM. Conclusion: Taken together, our data indicate the MSCs via juxtacrine and paracrine pathways could direct the spinal cord endogenous neural stem cells (NSCs) to the neuroblasts lineage which indicates the capability of the MSCs in the increasing of the number of DCX-positive cells and astrocytes decline

Generation of embryonic stem cell lines from IVF embryos and parthenotes

Efficient isolation and maintenance of pluripotent cell lines in livestock species have proven to be a major hurdle despite tremendous efforts over 3 decades. Little is known about the signalling pathways controlling pluripotency in mammals other than rodents and primates. To understand these mechanisms in the bovine due to a biomedical model for human diseases, biotechnology applications and commercial use, embryonic stem cells (ESCs) were isolated from IVF embryos in a conventional medium and in a medium with 3 small-molecule inhibitors (3i) which permit efficient self-renewal of ESCs in mice and rats. SU5402 inhibits the FGF receptor tyrosine kinases and PD184352 inhibits the ERK cascade. CHIR99021 inhibits GSK3 to increase ES-cell proliferation. The effect of three inhibitors “3i” on 1) the efficiency of ESC isolation and 2) maintenance of established bovine ESC lines (bESCs) was examined in the different media. Colony formation and attachment to the feeder layer differed among media and media containing the 3i components resulted in better growth and maintenance of putative ESCs compared with the others. Expression of 6 pluripotency related genes (Oct4, rex1, sox2, ssea1, alp and nanog) and four proteins (Oct4, SSEA1, SSEA4, Alkaline Phosphatase) were assessed. The results indicate that 3i can improve bovine ESC isolation and increase the pluripotent component of putative bovine ES cells. The second series of experiments investigated the generation of autologous cell lines. ES cells from parthenogenetically activated oocytes can provide autologous transplantable cells, which are immuno-compatible for the oocyte donors as well as an invaluable tool for genetic engineering for transplantation and epigenetic studies. We report the efficient isolation of 8 putative bovine parthenogenetic ESC lines in 3i medium from 15 in vitro produced parthenotes. The cells displayed pluripotency similar to IVF ESC lines and differentiated in suspension culture to form embryoid bodies (EBs) expressing markers of the three embryonic germ layers. The third series of experiments investigated the long-term maintenance of pluripotent bESCs during in vitro culture further and developed a robust method of cryopreservation which is essential requirements to maximize the use of ESCs in research and for biotechnology applications. 1) Eight putative bESC lines established in N2B27-3i medium and 1 established in conventional medium were cultured over 30 passages (> 240 days) and characterized at P23-30. All cell lines expressed markers of pluripotency. The cells formed EBs containing cells of the three embryonic germ layers. For cryopreservation, vitrification was compared with conventional slow-freezing. Survival rate and colony morphology of vitrified cell lines after thawing was significantly higher than after slow-freezing, (97% v/s 56% respectively; p<0.01). Cell lines survived and proliferated, with good morphology, following vitrification in both N2B27-3i, 95% (104/110) and conventional medium, 97% (93/96). Post-thawed vitrified cell lines were cultured for an additional 12 passages (P¬18+12, P19+12 and P23+12) and shown to retain pluripotency. The cells formed EBs in suspension culture and expressed genes representative of the three embryonic germ layers. Vitrification did not change the karyotype of cells. Finally, three cell lines isolated in conventional medium were maintained for 80-95 passages and vitrified. These cell lines also retained expression of pluripotent markers. In summary, the results in this thesis present 1) efficient derivation and characterization of pluripotent putative bESCs from IVF and parthenotes in 3i medium and 2) maintenance of cell lines in both conventional and 3i media which express pluripotent markers and retain the ability to differentiation in vitro after long-term culture, and 3) successful cryopreservation of cell lines, especially by vitrification. This thesis adds significantly to the knowledge on isolation, characterization and cryopreservation of pluripotent cell lines in livestock, especially the bovine

The Toxic Effect of Silibinin and Paclitaxel Combination on Endometrial Cancer Cell Line

Background & objectives: Among gynecologic malignancies, endometrial cancer is the fourth most frequent cause of cancer death all over the world. Paclitaxel is one of the chemotherapy regimens that is used against this cancer. Treatment of tumor with Paclitaxel induces apoptosis, but it is also associated with serious side effects. Thus, it is imperative to search for more effective and safer chemotherapeutic regimens. Silibinin is a milk thistle plant extract that its antioxidant effects against some cancers have been studied. The aim of this study was to examine the effect of Paclitaxel and Silibinin combination on endometrial cancer cell line (Hela). Methods: Hela cell line was cultured in 25cm2 flask in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Then the numbers of live cells were calculated with trypan blue staining method and then the cells were seeded in to 96-well flat-bottomed culture plates and treated with Silibilin, Paclitaxel and Paclitaxel plus Silibilin together with the control without treatment. MTT assay was used to evaluate cytotoxicity of different treatments. Results: After 48 hours of treatment, Paclitaxel and Silibilin combination inhibited cell growth significantly compared with the other groups (p<0.05). Conclusions: It is indicated that combination of Paclitaxel and Silibilin can affect the growth arrest of Hela cancer cell line more&nbsp; effective than other treatments and is needed to be examined in vitro

Anti-Cancer Effect of Silibinin on Epithelial Ovarian Cancer Cell Line and P21 Gene Expression

Background & objectives: Epithelial ovarian carcinoma seems to be one of the most lethal cancer types among all gynecological malignancies. The conventional course of therapy includes chemotherapy. Actually most cancers respond to chemotherapy but in the long run drug resistance and side effects cause treatment failure. In addition, milk thistle (silibinin), a plant that has been used from ancient time because of its good effects on different organs, determined to have powerful antioxidant activity. &nbsp;The aim of this study was to examine the effect of silibinin on SKOV-3 cancer cell line after 48 hours of treatment and P21 gene expression which involves in cell cycle progression. Methods: The human epithelial ovarian cancer cell line SKOV-3 was cultured as monolayer in 25 cm2 flask in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Then the numbers of live cells were calculated using hemocytometer method and the cells were seeded in 96-well flat-bottomed culture plates and treated with different concentration of Silibinin. MTT assay was carried out to determine cell viability. To study P21 gene expression, RNA extraction and cDNA synthesis were carried out and real-time PCR was done. Results: Cell growth was inhibited considerably by Silibinin treated groups compared with control after 48 hours. P21 gene expression was increased as well. Conclusions: According to the results, Silibinin can be used as an effective drug in cancer treatment. More studies on animal models are also suggested

The efficient generation of cell lines from bovine parthenotes

The generation of embryonic stem cell (ESC) lines from parthenogenetically activated oocytes can provide transplantable cells, which are immunocompatible for the oocyte donors as well as an invaluable tool for genetic engineering and epigenetic studies. We report the efficient isolation of eight putative bovine parthenogenetic embryonic stem cell (bpESC) lines from 15 in vitro produced parthenotes. Five of these cell lines were maintained for more than 15 passages (>140 days) and analyzed. The cells displayed typical ESC morphology, stained positive for alkaline phosphate by histochemical staining, expressed Oct4, Nanog, and either stage-specific embryonic antigens, SSEA1, or SSEA4, detected by immunofluorescence staining. RT-PCR analysis of the cells demonstrated expression of Oct4, Rex1, SSEA1, and ALP. All the cell lines except one had a normal karyotype of 60, XX. The cells differentiated in suspension culture to form embryoid bodies (EBs) expressing markers of the three embryonic germ layers as assessed by RT-PCR. In conclusion, we report efficient derivation of putative ESCs from bovine parthenogenetic embryos. The cells express pluripotent markers, have the ability to form EBs, and differentiate into cells of the three embryonic germ layers. This is the first report of characterized putative parthenogenetic bovine ESC lines

Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC

Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression of OCT4, SOX2 NANOG, and STAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers both in vitro by embryoid bodies (EBs) formation and in vivo by teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors

Unravelling evolution of Nanog, the key transcription factor involved in self-renewal of undifferentiated embryonic stem cells, by pattern recognition in nucleotide and tandem repeats characteristics

Nanog, an important transcription factor in embryonic stem cells (ESC), is the key factor in maintaining pluripotency to establish ESC identity and has the ability to induce embryonic germ layers. Nanog is responsible for self-renewal and pluripotency of stem cells as well as cancer invasiveness, tumor cell proliferation, motility and drug-resistance. Understanding the underlying mechanisms of Nanog evolution and regulation can lead to future advances in treatment of cancers. Recent integration of machine learning models with genetics has provided a powerful tool for knowledge discovery and uncovering evolutionary pathways. Herein, sequences of 47 Nanog genes from various species were extracted and two datasets of features were computationally extracted from these sequences. At the first dataset, 76 nucleotide acid attributes were calculated for each Nanog sequence. The second dataset was prepared based on the 10,480 repeated nucleotide sequences (from 5 to 50. bp lengths). Then, various data mining algorithms such as decision tree models were applied on these datasets to find the evolutionary pathways of Nanog diversion. Attribute weighting models were highlighted features such as the frequencies of AA and GC as the most important genomic features in Nanog gene classification and differentiation. Similar findings were obtained by tree induction algorithms. Results from the second database showed that some short sequence strings, such as ACTACT, TCCTGA, CCTGA, GAAGAC, and TATCCC can be effectively used to identify Nanog genes in various species. The outcomes of this study, for the first time, unravels the importance of particular genomic features in Nanog gene evolution paving roads toward better understanding of stem cell development and human targeted disorder therapy. © 2015 Elsevier B.V

Bidirectional and Opposite Effects of Naive Mesenchymal Stem Cells on Tumor Growth and Progression

Cancer has long been considered as a heterogeneous population of uncontrolled proliferation of different transformed cell types. The recent findings concerning tumorigeneses have highlighted the fact that tumors can progress through tight relationships among tumor cells, cellular, and non-cellular components which are present within tumor tissues. In recent years, studies have shown that mesenchymal stem cells (MSCs) are essential components of non-tumor cells within the tumor tissues that can strongly affect tumor development. Several forms of MSCs have been identified within tumor stroma. Naïve (innate) mesenchymal stem cells (N-MSCs) derived from different sources are mostly recruited into the tumor stroma. N-MSCs exert dual and divergent effects on tumor growth through different conditions and factors such as toll-like receptor priming (TLR-priming), which is the primary underlying causes of opposite effects. Moreover, MSCs also have the contrary effects by various molecular mechanisms relying on direct cellto- cell connections and indirect communications through the autocrine, paracrine routes, and tumor microenvironment (TME). Overall, cell-based therapies will hold great promise to provide novel anticancer treatments. However, the application of intact MSCs in cancer treatment can theoretically cause adverse clinical outcomes. It is essential that to extensively analysis the effective factors and conditions in which underlying mechanisms are adopted by MSCs when encounter with cancer. The aim is to review the cellular and molecular mechanisms underlying the dual effects of MSCs followed by the importance of polarization of MSCs through priming of TLRs

Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells - Fig 4

<p>A and D) total population of cells are presented and highlighted population are transferred and tested for different markers, B) CD90-EP positive cells, C) CD44-FITC positive cells, and E) CD45 negative cells and F) C31 negative cells population.</p