Glioblastoma stem cells: a new target for metformin and arsenic trioxide.

The high malignancy of glioblastoma has been recently attributed to the presence, within the tumor, of glioblastoma stem cells (GSC) poorly responsive to chemo- and radiotherapy. Here, the potential employment of metformin and arsenic trioxide (ATO) in glioblastoma therapy is discussed focusing on their effects on GSC. Metformin exerts anticancer effects by primarily blocking the pivotal LKB1/AMPK/mTOR/S6K1 pathway-dependent cell growth, induces selective lethal effects on GSC by impairing the GSC-initiating spherogenesis and inhibits the proliferation of CD133+ cells, while having a low or null effect on differentiated glioblastoma cells and normal human stem cells. Metformin and ATO induce autophagy and apoptosis in glioma cells by inhibiting and stimulating the PI3K/Akt and the mitogen-activated protein kinase pathways, respectively. Both drugs promote differentiation of GSC into non-tumorigenic cells. In this regard, metformin acts via activation of the AMPK-FOXO3 axis, whereas ATO blocks the interleukin 6-induced promotion of STAT3 phosphorylation. Blood-brain barrier, easily crossed by metformin but not by ATO, undergoes important glioblastoma-induced alterations that increase its permeability, thus allowing ATO to distribute more into the glioblastoma bulk than in the normal brain parenchyma. A prompt clinical assessment of metformin and ATO in glioblastoma patients would represent a valid attempt to improve their survival.

Paraneoplastic syndromes with connective tissue involvement. "It's not always lupus!".

Paraneoplastic syndromes (PNS) are remote effects of cancer that are, by definition, caused neither by invasion of the tumor or its metastases nor by infection, ischemia, metabolic and nutritional deficits, surgery or other forms of tumor treatment. The purpose of the current review was to present the challenging elements of differential diagnosis in oncology, as they may represent the main clinical problem in a patient diagnosed with cancer, even though the complete knowledge of both their clinical aspects and pathogenesis remain quite poor. This review focuses on the paraneoplastic syndromes related to dermatology and rheumatology, as the most frequent manifestations come from connective tissues that might determine a patient to ask for consultation by a general practitioner.

The colorectal cancer stem-like cell hypothesis: a pathologist's point of view.

Colorectal cancer is the fourth most common cancer in men and the third most common cancer in women worldwide. The partial failure of classic therapeutic options makes scientists to doubt the efficacy of systemic treatments in targeting the essential cell populations and achieving cure as a final goal. Overgrowing data suggest that cancer is a disease closely linked to stem cells (SCs). It is well known that the first identification of cancer stem-like cells in acute myeloid leukaemia was soon followed by similar results in solid malignancies, including colorectal cancer, and the classic model for colon carcinogenesis supports the development of sudden mutations that will lead to the activation or inactivation of certain oncogenes or tumor suppressors. Thus, this process may go on for years before the first symptoms and the only cells able to withstand for many years, avoid apoptosis and have a high regenerative capacity are the progenitor cells found at the lower part of colon crypts. A more profound study of the mechanisms and molecular signalling pathways that control the basic characteristics of SCs, such as asymmetrical division or self-renewal, may help comprehend the basic mechanisms of cancer genesis and progression. This will result in the development of new therapeutic agents that may target chemoresistant cell populations and improve the therapeutic results. In the current review we point out the importance of cancer stem-like cells in colorectal oncology from a pathologist's point of view, stating the obvious correlation between histology, embryology and surgical pathology.

Cancer stem cells and malignant gliomas. From pathophysiology to targeted molecular therapy.

High grade gliomas, the most frequent and most malignant brain cancers, grow rapidly and infiltrate the cerebrospinal axis causing deficits in cognition, mobility, balance or speech and are typically resistant to radiation and chemotherapy. Despite recent progress, WHO grade III and IV gliomas still represent a great challenge in oncology, with overall poor outcomes and inevitable lethality. While radiotherapy and temozolomide are considered the standard first-line approach for therapy of newly diagnosed malignant gliomas, the treatment protocols for recurrent tumors remain ill-defined. Increasing evidence suggests that tumors of the central nervous system are derived from proliferatively active neural stem cells residing in defined neuropoietic niches of the adult brain. These cancer stem cells, also identified in other tumors, provide a reservoir of cells with self-renewal capabilities, can maintain the tumor by generating differentiated non-stem tumor cells and are responsible for recurrences after ablative neurosurgical therapy and chemoradiotherapy. The only way to successfully control recurrent malignant gliomas and even hope for a cure in the future is by combining standard chemotherapy with immunotherapy. Despite the apparent improvements of current treatments, it should be realized that the characteristic brain tumor niche may provide recurrent gliomas an "escape mechanism" from anticancer treatments. Thus, the use of targeted molecular therapy drugs may effectively inhibit or at least slow down cancer stem cell proliferation and stop the brain microenvironment from allowing furtive invasion and proliferation of highly aggressive malignant gliomas.

Antidiabetic pharmacology: a link between metabolic syndrome and neuro-oncology?

One of the main topics of the annual meeting of the American Society for Clinical Oncology in 2011 were the results presented on breast cancer chemotherapy and concomitant administration of the oral antidiabetic metformin. The overall agreement was that current evidence is just enough to dramatically change the clinical practice of oncology, and in our case, brain cancer treatment, and that further research is needed to address the relationship between diabetes, metabolism, insulin analogues and neoplasia. Still, it is very interesting to explore the potentially beneficial effects of metformin in glioma chemo/immunotherapy and wait for results in the clinic. In the current paper we present the cell and molecular aspects of the metabolic syndrome, metformin administration and cancer chemotherapy, with a special emphasis in neuro-oncology, since brain tumors are usually devastating diseases with an extremely high mortality within two years of diagnosis even when surgical, radiotherapeutic and chemotherapeutic interventions are applied.

Metformin plus temozolomide-based chemotherapy as adjuvant treatment for WHO grade III and IV malignant gliomas.

PURPOSE: Glioblastoma multiforme (GBM) remains one of the most devastating diseases known to man and affects more than 17,000 patients in the United States alone every year. This malignancy infiltrates the brain early in its course and makes complete neurosurgical resection almost impossible. Recent years have brought significant advances in tumor biology, including the discovery that many cancers, including gliomas, appear to be supported by cells with stem-like properties. In the current study we have investigated the effects of combining metformin with the standard treatment-of-care, as this drug, already used in the treatment of diabetes mellitus, has shown surprising results in the treatment of breast cancer, being also associated with lower mortality in several other malignancies. METHODS: The subjects of the current study were 8 patients with newly diagnosed high-grade gliomas, operated at the Department of Neurosurgery - Clinical University Emergency Hospital, Cluj Napoca. Tumor tissue cultures were established and characterized using immunofluorescence microscopy and PCR analysis and the sensitivity to metformin, epidermal growth factor (EGF) and temozolomide (TMZ) was tested. Microvascular density (MVD) assay was performed on the tumor samples. RESULTS: Seven of the 8 cases had a positive correlation between the number of endothelial cells, the phenotype of isolated tumor cells and the response to adjuvant chemoradiotherapy. The isolated tumor cells had a stem-like behavior, being resistant to conventional drugs. In most cases there was no statistical significant difference between TMZ alone and TMZ plus EGF arms, but there was a important difference between TMZ alone and TMZ plus metformin arms in 6 of the cases. CONCLUSION: New drugs and targeted molecular therapies are important for future therapeutics, but sometimes we must not exclude drugs already used in the clinic that might have remarkable results. Such is the case of metformin, a drug used for decades in the treatment of type 2 diabetes mellitus that has proven to enhance the effect of TMZ in the treatment of breast cancer and, starting with this paper, of brain cancer.

A systematic approach on the frequency of cleft lip/palate in pediatric patients with leukemia.

PURPOSE: The purpose of this study was to reveal the frequency between cleft lip/palate and leukemia in pediatric patients by a systematic analysis of the current literature. MATERIALS AND METHODS: Electronic search on three database (PubMed, Web of Science, Cochrane) was carried out using the following keywords: cleft lip, cleft palate, facial cleft, oral cleft, orofacial cleft, leukemia, acute myeloid leukemia, acute lymphocytic leukemia, lymphoma. Studies published until March 2020 reporting an association between leukemia and cleft lip/palate (CL/P) were included in our research. RESULTS: Five articles (2 case-controls, 3 cohorts), met the inclusion criteria. Case-control studies involved 268 patients with acute lymphocytic leukemia (ALL) and 177 patients with acute myeloid leukemia (AML), of which 9 patients had CL/P. The cohorts studies involved 10 patients with ALL, of which 6 patients with CL/P, 2 patients with cleft palate and 1 patient with cleft lip and palate. CONCLUSION: This research was able to indicate a limited evidence of the association between CL/P and leukemia. In order to draw a clear conclusion, studies with larger cohorts are needed to establish this correlation.

Mesenchymal stem cell irradiation in culture engages differential effect of hyper-fractionated radiotherapy for head and neck cancers.

PURPOSE: The purpose of this study was to challenge current knowledge on the potential therapeutic advantages of stem cells in radiotherapy by developing an in vitro model of the healthy tissue surrounding or replacing the widely resected tumor. After radical surgery, the start of radiotherapy is often delayed due to wound healing process, with potential loss of the opportunity for treating microscopic disease instead of macroscopic early recurrence. Hyperfractionated radiotherapy, contrary to the standard one, can extend the limits of radical surgery and shorten the gap before the onset of postoperative radiotherapy, with potential improvement in local control. METHODS: By using both mesenchymal stem cells and pre-differentiated osteoblasts, cultured in proper pro-osteogenic media after cell irradiation, we investigated both the differences in the response to DNA damage between lineages undergoing differentiation in culture and the intensity of the mineralization process. RESULTS: Ionizing radiation stimulated stem cell proliferation and differentiation at 0.5 Gy and 1 Gy, thus confirming in vitro the clinical results of hyperfractionated irradiation randomized trials in head and neck cancers -HNCs-. CONCLUSION: To our knowledge, this study is the first to investigate the biophysics of low dose gamma irradiation on stem cell culture, focusing on the potential applications in radiation oncology. For advanced oral cavity and oropharyngeal cancers, as radical surgery often implies major bone resection, the use of mesenchymal stem cells as bone reconstruction vectors might shorten the onset of adjuvant hyperfractionated radiotherapy which enhances the mineralization process. As postoperative radiotherapy has recently being revisited for osteosarcoma, this scenario could impact also on bone reconstruction process in this pathology.

Arsenic trioxide sensitizes cancer stem cells to chemoradiotherapy. A new approach in the treatment of inoperable glioblastoma multiforme.

PURPOSE: glioblastoma multiforme (GBM) still bears a very dismal prognosis even with complete resection followed by adjuvant chemoradiation. The aim of the current study was to evaluate in vitro the antitumor efficacy of arsenic trioxide (ATO) in combination with ionizing radiation plus temozolomide and bevacizumab against cultured glioblastoma stem-like cells, as possible way to increase the therapeutic index in patients diagnosed with recurrent, therapy-refractory GBM. METHODS: stem-like tumor cells isolated from a GBM biopsy were established by cell proliferation assays and upregulation of stem cell markers, as proven by reverse transcription - polymerase chain reaction (RT-PCR). Low concentrations of ATO were added prior to temozolomide, bevacizumab and ionizing irradiation. RESULTS: molecular analysis showed that cells expressed CXCR4, Oct-3/4 and GAPDH when compared to placental mesenchymal stem cells, as well as nestin, GFAP and neurofilament protein. Low concentrations of ATO led to morphologic differentiation, with fewer stem cells in Go state and differentiation-associated cytochemical features, like increased sensitivity to cytostatic drugs and radiotherapy. CONCLUSION: ATO exposure before conventional postoperative chemoradiotherapy for GBM might increase treatment efficacy. Further in vivo experiments on laboratory animals and analysis of absorption rate and side effects are required.

Functional and molecular characterization of glioblastoma multiforme-derived cancer stem cells.

PURPOSE: Brain tumors are the leading cause of cancer mortality in children and remain incurable despite advances in surgery and adjuvant therapies. The failure of malignant gliomas to respond to conventional treatment reflects the unique biology of these tumors, linked to a small population of stem-like precursors. This study describes the characteristics of stem cells isolated from glioblastoma multiforme (GM) and gives insight into the mechanism of brain tumorigenesis. METHODS: Tumor stem-like precursors were identified from primary human GM-derived cell culture using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). Cells were cultured in vitro in stem cell medium supplemented with growth factors and then the capacity of the surviving stem-like precursors to form tumor spheres and to continue to proliferate after chemoradiotherapy were tested. RESULTS: The tumor cells expressed the cellular markers CD133, CD105, CD90, Nanog, Oct 3/4, CXCR4, nestin, glial fibrillary acidic protein (GFAP), neurofilament protein (NF) and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells also displayed a high proliferative potential despite chemotherapy and irradiation and also had the ability to form spheroids in suspension. CONCLUSION: High grade gliomas contain stem-like precursors, which exhibit neural stem cell properties with tumorigenicity, establishing a novel developmental paradigm in the study of brain carcinogenesis and providing a powerful tool to develop patient-tailored therapy for this devastating disease.

Placental stem cell differentiation into islets of Langerhans-like glucagon-secreting cells.

BACKGROUND AND PURPOSE: For the past few years, in an attempt to find new sources of cells that may be used in cell therapy, numerous researchers have highlighted the particular properties of mesenchymal stem cells. Mesenchymal stem cells can be isolated from adult tissues such as the bone marrow or adipose tissue, but also from other organs such as the human placenta. Our study focuses adult stem cells isolated from the chorionic villi in an attempt to differentiate them into islets of Langerhans in order to study their differentiation potential, as a future background for cell therapy. EXPERIMENTAL DESIGN: Full-term placentas were prelevated from volunteer women that have just delivered a normal pregnancy. After a mechanical fragmentation of the placenta, the chorion fragments are transferred in a dish with dispase before the enzyme is inactivated using fetal calf serum. The cell suspension is filtered in order to obtain a single-cell suspension. After the adherence of the first cells, the proliferation rate increased progressively and cell morphology is kept the same for several passages. In order to correctly differentiate placental stem cells into glucagon-secreting cells, we used a culture method on a scaffold with sequential exposure to different growth factors. The underlying substrate used contained type IV collagen, chytosan, Matrigel and laminin. Molecular biology techniques were carried out to investigate the gene expression of the stem cells. RESULTS: Our results show that exendin-4 is able to induce the differentiation of placental stem cells into glucagon-secreting cells. We also notice the absence of the insulin gene, a conclusion that may be explained by the fact that our phenotype is a partial one, incomplete, closer to islet cell progenitors than to insulin-producing progenitors. CONCLUSIONS: The identification of the placenta as a valid source for stem cells has important practical advantages because it is easily accessible, it raises no ethical issues and cells are easily to isolate in a large enough number to use. The future knowledge and manipulation of the signaling pathways that determines the dramatic phenotype shift may provide the basis for efficient cell differentiation, with great impact on regenerative medicine and tissue engineering.

Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.

BACKGROUND AND PURPOSE: Ovarian cancers are composed of heterogeneous cell populations, including highly proliferative immature precursors and differentiated cells that may belong to different lineages. The main reason why epithelial ovarian cancer is difficult to treat is the unusual mechanism of dissemination that involves local invasion of pelvic and abdominal organs. But, unlike many other carcinomas, initial dissemination rarely requires blood or lymph vessels. Because it has been proven that aggregates of malignant cells within the ascites of patients diagnosed with ovarian cancer represent an impediment to cure such cancers, in the present study we adopted suspension culture combined with anti-cancer regimens as a laboratory strategy for research of the initial process of peritoneal micrometastasis. EXPERIMENTAL DESIGN: MLS human ovarian cancer cells were cultured in serum-free medium. Cells of passage eight were treated in combination with the anticancer agent doxorubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The acquired increased aggressiveness properties was confirmed by multidrug resistance assays and by their ability to grow in an anchorage-independent manner in vitro as tumor spheroids. RESULTS: Cells selected after chemotherapy had a increased proliferative potential, eliminated Rhodamine 123 in culture and also formed spheroids in suspension. CONCLUSIONS: Here we present direct evidence that the metastasis of human ovarian cancer may be a result of transformation and dysfunction of immature precursor cells in the ovary. Also, spheroid formation may represent a key component of chemotherapy recurrence and a better understanding of these 3D structures can contribute to the development of new treatments for metastatic carcinoma.

Effects of 60Co gamma-rays on human osteoprogenitor cells.

BACKGROUND: Radiation therapy is one of the most efficient treatments of neoplastic diseases used worldwide. However, patients who undergo radiotherapy may develop side effects that can be life threatening because tissue complications caused by radiation-induced stem cell depletion may result in structural and functional alterations of the surrounding matrix. This treatment also damages the osteogenic activity of human bone marrow by suppressing osteoblasts, leading to post-irradiation sequelae. Even if widely used in oncology, there is still little information on the fate and potential therapeutic efficacy of electromagnetic rays. MATERIAL AND METHODS: We addressed this question using both human mesenchymal stem cells and osteoblasts. Monoclonal antibody characterization identified specific surface markers for stem cells (SSEA-4, CD29, CD105, Oct 3, Nanog and SOX2) and osteoblasts (Osteopontin and Osteonectin). The technique of anti-alkaline phosphatase FITC-staining demonstrated the presence of this specific ectoenzyme. Cells were cultured in complex osteogenic medium (DMEM, 15% fetal calf serum, non-essential amino acids, L-glutamine, dexametazone, ascorbic acid, insulin, TGF-beta, BMP-2 and beta-glycero-phosphate) after being irradiated at 0.5 Gy, 1 Gy, 2 Gy and 4 Gy using a Theratron 1000 60Co source. The viability of irradiated cells was assessed using Trypan Blue staining. The comparison between cell lineages after culture in osteogenic media regarding phenotypical characterization and the intensity of the mineralization process included histology stainings (Alizarin Red S, Alcian Blue and von Kossa), and the MTT-based proliferation assay. RESULTS: After irradiation, the proliferation and differentiation of osteoprogenitor cells is dose-dependent. CONCLUSIONS: This study is one among the first papers investigating the biophysics of low-dose gamma-irradiation on stem cell culture, focusing on the potential applications in radiation oncology and various palliative treatments.

Seroprevalence of Bartonella species, Coxiella burnetii and Toxoplasma gondii among patients with hematological malignancies: A pilot study in Romania.

Patients receiving immunosuppressive cancer treatments in settings where there is a high degree of human-animal interaction may be at increased risk for opportunistic zoonotic infections or reactivation of latent infections. We sought to determine the seroprevalence of selected zoonotic pathogens among patients diagnosed with haematologic malignancies and undergoing chemotherapeutic treatments in Romania, where much of the general population lives and/or works in contact with livestock. A convenience sample of 51 patients with haematologic cancer undergoing chemotherapy at a referral clinic in Cluj-Napoca, Romania, was surveyed regarding animal exposures. Blood samples were obtained and tested for evidence of infection with Bartonella species, Coxiella burnetii and Toxoplasma gondii, which are important opportunistic zoonotic agents in immunocompromised individuals. 58.8% of participants reported living or working on a farm, and living or working on a farm was associated with contact with livestock and other animals. 37.5% of participants were IgG seroreactive against one or more of five Bartonella antigens, and seroreactivity was statistically associated with living on farms. Farm dwellers were 3.6 times more likely to test IgG seroreactive to Bartonella antibodies than non-farm dwellers. 47.1% of the participants tested T. gondii IgG positive and 13.7% tested C. burnetii IgG positive, indicating past or latent infection. C. burnetii IgM antibodies were detected in four participants (7.8%), indicating possible recent infection. These results indicate that a large proportion of patients with haematologic cancer in Romania may be at risk for zoonotic infections or for reactivation of latent zoonotic infections, particularly with respect to Bartonella species. Special attention should be paid to cancer patients' exposure to livestock and companion animals in areas where much of the population lives in rural settings.

First Successful Haploidentical Stem Cell Transplantation in Romania.

Hematopoietic stem cell transplantation is an established treatment for many malignant and non-malignant haematological disorders. In the current case report, we describe the first haploidentical stem cell transplantation, used for the first time in Romania, the case of a 33 year-old young woman diagnosed with Hodgkin's lymphoma that has underwent a haploSCT after she relapsed from several chemotherapy regimens, as well as after an autologous stem cell transplantation. This success represents a prèmiere in Romanian clinical hematology, being the first case of a haploSCT in Romania, as well as in South-Eastern Europe.

HLA Genotyping using Next Generation Sequencing.

From an oncological perspective, the second most common malignancies in children are brain tumors. Despite the recent therapeutic breakthroughs in this field, concerning surgery, radiotherapy and chemotherapy alike, some cases still have poor outcomes in curability. This is especially the case in patients with high-risk histological types of tumors, and those suffering from residual, remitting and disseminated diseases. Due to the unique neuroanatomical emplacement of brain tumors and their aggressive infiltrative behavior, their total removal remains a demanding task. This can be perceived in the high rates of failure treatment and disease recurrence. Furthermore, the adjacent healthy brain tissue is inevitably damaged in the surgical process of effectively removing these tumors. Thus, stem cell transplantation may be a viable solution for the clinical management of these malignancies, as proven by various recent breakthroughs. In the current concise review, we present the role of next generation sequencing in HLA typing for stem cell transplantation in primary CNS pediatric malignancies.

Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by 'trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics.

Redistribution of iron towards deposits in erythroblastopenic anemia as a consequence of decreased erythroferrone production.

Most of the body iron is found within hemoglobin in red cells (the erythron), a smaller amount being distributed in other tissues such as muscles and in deposits. Iron homeostasis is a finely tuned process in which the most important regulators are probably the liver-derived hepcidin which blocks iron absorption and directs iron towards deposits and the recently discovered erythroblast-derived erythroferrone which inhibits hepcidin synthesis and therefore increases availability of iron for hemoglobin synthesis. Hepcidin secretion is increased by inflammatory cytokines and erythroferrone production increases when there is active, expanding erythropoiesis, for example after acute blood loss. We hypothesize that in pathological situations associated with erythroid precursor suppression (erythroblastopenia), anemia is the result of two major mechanisms: (1) direct erythroblast suppression leading to decreased production of red cells and (2) low iron availability due to high hepcidin levels arising as a result of low erythroferrone production. Additionally, infectious episodes and other inflammatory conditions that often complicate the course of these diseases may further promote hepcidin synthesis through increased cytokine production leading to even lower iron availability and a vicious circle of worsening anemia.

Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma.

OBJECTIVES: Metformin, the most used oral antidiabetic drug for the treatment of type 2 diabetus mellitus, has proved encouraging results when used in the treatment of various types of cancer such as triple-negative breast cancer. Despite compelling evidence of a role of metformin as an anticancer drug, the mechanisms by which metformin exerts its oncostatic actions are not fully understood yet. Therefore, we tried to bring new insights by analyzing the anti-neoplastic effect of metformin for hepatocellular carcinoma-derived stem-like cells treated with conventional combination chemotherapy. METHODS: Cancer stem-like cells previusly isolated from a hepatocellular carcinoma biopsy were treated with metformin, PIAF chemotherapy regimen and the combination of these two protocols. Measurements of lipid peroxidation, reduced glutathione, fluorescein diacetate and proliferation rates were determined, apart from the autophagy assay and apoptosis determination by chip flow cytometry. RESULTS: Metformin alone and especially metformin in association with PIAF increases oxidative stress within the cells by increasing the levels of lipid peroxids as well as decreasing the levels of reduced glutathione. The MTT cell proliferation assay showed decreased prolife-ration rates for the arm treated with metformin and with the combination of drugs in comparison with the control arm, proving high correlation with the oxidative stress results. The autophagy assay and determination of apoptosis by chip flow cytometry confirmed the results obtained in the previous assays. CONCLUSION: Metformin could be used in chemotherapy treatments to induce reactive oxygen species and increase the cytostatics effects within the tumor cell. Still, further experiments must be carried out on murine models before we can move on and use this drugs in the adjuvant setting for unresectable primary liver cancer.

Establishment of an embryonic stem cell line from blastocyst stage mouse embryos.

Embryonic stem cells have the ability to remain undifferentiated and proliferate in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. The aim of the present study was to establish mouse ES lines from blastocyst stage embryos obtained after CD1/EGFP mice superovulation. We isolated, cultured and determined the characteristics of mouse embryonic stem cells in early passages, which were first described by Evans M and Kaufman M. Therefore, we evaluated the morphological criteria for the approval of ES cells in early expansion stage. Two cell lines were isolated (CDE1 and CDE2) and analyzed. They showed similar characteristics to those reported earlier for blastocyst-derived ES cell lines.