Effects of the Co-Overexpression of the BCL and BDNF Genes on the Gamma-Aminobutyric Acid-Ergic Differentiation of Wharton's-Jelly-Derived Mesenchymal Stem Cells.

One of the problems with using MSCs (mesenchymal stem cells) to treat different neurodegenerative diseases of the central nervous system is their low ability to spontaneously differentiate into functional neurons. The aim of this study was to investigate how the co-overexpression of the BCL and BDNF genes affects the ability of genetically modified MSCs to differentiate into GABA-ergic neurons. A co-overexpression of two genes was performed, one of which, BCL, was supposed to increase the resistance of the cells to the toxic agents in the brain environment. The second one, BDNF, was supposed to direct the cells onto the neuronal differentiation pathway. As a result, the co-overexpression of both BCL2 + BDNF and BCLXL + BDNF caused an increase in the MAP2 gene expression level (a marker of the neuronal pathway) and the SYP gene that is associated with synaptogenesis. In both cases, approximately 18% of the genetically modified and then differentiated cells exhibited the presence of the GAD protein, which is characteristic of GABA-ergic neurons. Despite the presence of GAD, after both modifications, only the BCL2 and BDNF co-overexpression correlated with the ability of the modified cells to release gamma-aminobutyric acid (GABA) after depolarization. Our study identified a novel model of genetically engineered MSCs that can be used as a tool to deliver the antiapoptotic proteins (BCL) and neurotrophic factor (BDNF) directly into the brain microenvironment. Additionally, in the investigated model, the genetically modified MSCs could easily differentiate into functional GABA-ergic neurons and, moreover, due to the secreted BCL and BDNF, promote endogenous neuronal growth and encourage synaptic connections between neurons.

NO-induced apoptosis in human melanocytes from lightly and darkly pigmented skin.

Introduction: Nitric oxide (NO) is a potent modulator of programmed cell death, with the ability to both induce and prevent apoptosis. Some of the factors that are capable of triggering apoptosis of skin cells also cause NO overproduction in the epidermis. Unlike keratinocytes, melanin-producing melanocytes are highly resistant to apoptotic death. Aim: To investigate whether NO can induce apoptosis in normal human epidermal melanocytes and whether the pigmentation phenotype of the cells can affect their response to NO. Material and methods: Human epidermal melanocytes, derived from lightly and darkly pigmented neonatal foreskins, were cultured in the presence of various concentrations of SPER/NO. The effect of NO released from its donor on the cell morphology, viability, and proliferation was assessed. The ability of NO to induce cell apoptosis was evaluated by Hoechst 33342 staining, DNA fragmentation assay, flow cytometry with annexin V and propidium iodide staining, determination of caspase 3/7, 8, and 9 activities, and assessment of changes in the cell expression levels of BAX and BCL-2. Results: We have shown that NO is capable of inducing apoptosis in normal human epidermal melanocytes in vitro, with preferential activation of the intrinsic (mitochondrial) pathway. Melanocytes from darkly pigmented skin showed a strong increase in BCL-2 expression in response to NO and were significantly more resistant to apoptosis than those from lightly pigmented skin. Conclusions: The pigmentation phenotype may be an important factor modulating the response of human epidermal melanocytes to proapoptotic activity of extracellular NO.

The Combination of Baicalin with Knockdown of mir148a Gene Suppresses Cell Viability and Proliferation and Induces the Apoptosis and Autophagy of Human Glioblastoma Multiforme T98G and U87MG Cells.

BACKGROUND: Glioblastoma multiforme (GBM) is a heterogeneous and highly vascularized brain tumor that avoids apoptosis due to P-glycoprotein (P-gp) mediated multidrug resistance. Therefore, the development of new therapeutic strategies that induce apoptosis and inhibit proliferation is urgently warranted. OBJECTIVES: We examined the efficacy of the combination of baicalin (BAI) and knockdown of miR-148a gene in human glioblastoma T98G and U87MG cell lines. METHODS: T98G and U87MG cells were transfected with miR148a siRNA. The influence of miR- 148a siRNA in combination with BAI on T98G and U87MG cell viability, proliferation, apoptosis, and autophagy was evaluated as well. Alterations in the mRNA expression of autophagy-related genes were analyzed using RT-qPCR. RESULTS: The transfection of T98G and U87MG cells with miR148a specific siRNA and exposition on baicalin led to a significant reduction in cell viability and proliferation, the accumulation of sub G1-phase cells and a reduced population of cells in the S and G2/M phases (only in U87MG cell line), increased population of cells in the S phase in T98G cell line and apoptosis or necrosis induction and induction of autophagy for both cell lines. CONCLUSION: The siRNA-induced miR-148a mRNA knockdown in combination with baicalin may offer a novel therapeutic strategy to more effectively control the growth of human GBM cells. Thus, knockdown of this gene in combination with baicalin inhibits proliferation (cell cycle arrest in the S phase in T98G but not in U87MG cells), induces apoptosis, and regulates autophagy in T98G and U87MG cells. However, further studies are urgently needed to confirm a positive phenomenon for the treatment of GBM.

Survival and Neurogenesis-Promoting Effects of the Co-Overexpression of BCLXL and BDNF Genes on Wharton's Jelly-Derived Mesenchymal Stem Cells.

The main problem with using MSC (mesenchymal stem cells) to treat the deficient diseases of the central nervous system is the low cell survival rate after the transplant procedure and their low ability to spontaneously differentiate into functional neurons. The aim of this study was to investigate the effects of genetically modifying MSC. A co-overexpression of two genes was performed: BCLXL was supposed to increase the resistance of the cells to the toxic agents and BDNF was supposed to direct cells into the neuronal differentiation pathway. As a result, it was possible to obtain the functional overexpression of the BCLXL and BDNF genes. These cells had an increased resistance to apoptosis-inducing toxicants (staurosporine, doxorubicin and H2O2). At the same time, the genes of the neuronal pathway (CHAT, TPH1) were overexpressed. The genetically modified MSC increased the survival rate under toxic conditions, which increased the chance of surviving a transplant procedure. The obtained cells can be treated as neural cell progenitors, which makes them a universal material that can be used in various disease models. The production of neurotransmitters suggests that cells transplanted into the brain and subjected to the additional influence of the brain's microenvironment, will be able to form synapses and become functional neurons.

Study of the influence of NGF-ß gene overexpression in human mesenchymal stem cells on the expression level of SOX1 and neural pathway genes.

BACKGROUND: Nerve growth factor (NGF) is a protein exhibiting an influence on the neural development and also, its' impact on the stem cells remains a great potential treatment strategy. The influence of its overexpression on the neural pathway differentiation on Wharton's Jelly derived MSC (WJ-MSC) has not been studied so far, but considering the fact that these cells are relatively easy to obtain, using them may indicate an innovative change in stem cell therapies. The aim of this study was to evaluate the effect of NGF overexpression in human mesenchymal stem cells (MSC) on SOX1 and genes related to the neural pathway. METHODS AND RESULTS: The lentiviral transduction was performed in order to obtain the NGF overexpression, as well as RT-PCR to evaluate the expression level SOX1, SOX2, NES, NGF under influence of overexpressed NGF protein in WJ-MSC. During the study we have observed a decrease in SOX1 expression as the marker of neural stem cells. Other than that an increase of SOX2, NES and NGF was noticed, as they all are markers of early-neural as well as already differentiated neural cells. The results show a great potential of using those examined genes' expression as a form of a new stem cell therapy. CONCLUSIONS: The achieved overexpression of NGF in this study, led the modified MSC onto the neural pathway as well as caused a decrease of SOX1 expression and an increase of expression of genes related to neural differentiated cells.

Synergistic Effect of the Long-Term Overexpression of Bcl-2 and BDNF Lentiviral in Cell Protecting against Death and Generating TH Positive and CHAT Positive Cells from MSC.

Mesenchymal stem cells (MSC) are potentially a good material for transplantation in many diseases, including neurodegenerative diseases. The main problem with using them is the low percentage of surviving cells after the transplant procedure and the naturally poor ability of MSC to spontaneously differentiate into certain types of cells, which results in their poor integration with the host cells. The aim and the novelty of this work consists in the synergistic overexpression of two genes, BCL2 and BDNF, using lentiviral vectors. According to our hypothesis, the overexpression of the BCL2 gene is aimed at increasing the resistance of cells to stressors and toxic factors. In turn, the overexpression of the BDNF gene is suspected to direct the MSC into the neural differentiation pathway. As a result, it was shown that the overexpression of both genes and the overproduction of proteins is permanent and persists for at least 60 days. The synergistically transduced MSC were significantly more resistant to the action of staurosporine; 12 days after transduction, the synergistically transduced MSC had a six-times greater survival rate. The overexpression of the Bcl-2 and BDNF proteins was sufficient to stimulate a significant overexpression of the CHAT gene, and under specific conditions, the TH, TPH1, and SYP genes were also overexpressed. Modified MSC are able to differentiate into cholinergic and dopaminergic neurons, and the release of acetylcholine and dopamine may indicate their functionality.

Transient Excursions to Membrane Core as Determinants of Influenza Virus Fusion Peptide Activity.

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid-water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.

Anticancer Activity of the Acetylenic Derivative of Betulin Phosphate Involves Induction of Necrotic-Like Death in Breast Cancer Cells In Vitro.

Betulin (BT) is a natural pentacyclic lupane-type triterpene exhibiting anticancer activity. Betulin derivatives bearing propynoyloxy and phosphate groups were prepared in an effort to improve the availability and efficacy of the drug. In this study, a comparative assessment of the in vitro anticancer activity of betulin and its four derivatives was carried out using two human breast cancer cell lines: SK-BR-3 and MCF-7. In both studied cell lines, 30-diethoxyphosphoryl-28-propynoylbetulin (compound 4) turned out to be the most powerful inhibitor of growth and inducer of cellular death. Detailed examination of that derivative pertained to the mechanisms underlying its anticancer action. Treatment with compound 4 decreased DNA synthesis and up-regulated p21WAF1/Cip1 mRNA and protein levels in both cell lines. On the other hand, that derivative caused a significant increase in cell death, as evidenced by increased lactate dehydrogenase (LDH) release and ethidium homodimer uptake. Shortly after the compound addition, an increased generation of reactive oxygen species and loss of mitochondrial membrane potential were detected. The activation of caspase-3 and fragmentation of genomic DNA suggested an apoptotic type of cell death. However, analysis of cellular morphology did not reveal any nuclear features typical of apoptosis. Despite necrosis-like morphology, dead cells exhibited activation of the cascade of caspases. These observations have led to the conclusion that compound 4 pushed cells to undergo a form of necrotic-like regulated cell demise.

Effect of apigenin, kaempferol and resveratrol on the gene expression and protein secretion of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) in RAW-264.7 macrophages.

Polyphenols such as apigenin, kaempferol or resveratrol are typically found in plants, including fruits, vegetables, herbs and spices, which have a wide range of biological functions such as antioxidative, anti-inflammatory, vasodilative, anticoagulative and proapoptotic. Discovering such multifunctional compounds in widely consumed plant-based products - ones that both inhibit the release of TNF-α from tissue macrophages and at the same time enhance the secretion of IL-10 - would be an important signpost in the quest for effective pharmacological treatment of numerous diseases that have an inflammatory etiology. The aim of the study is to investigate the impact of biologically active polyphenols such as apigenin, resveratrol and kaempferol on gene expression and protein secretion of IL-10 and TNF-α in line RAW-264.7. Cells were cultured under standard conditions. IL-10 and TNF-α genes expression were examined using QRT-PCR and to assess cytokines concentration ELISA have been used. Apigenin, kaempferol and resveratrol at a dose 30µM significantly decrease the TNF-α expression and secretion. Apigenin decrease the IL-10 expression and secretion. Furthermore, increase in IL-10 secretion after administration of kaempferol and resveratrol were observed. In the process of administration of tested compounds before LPS, which activate macrophages, decrease of TNF-α secretion after apigenin and kaempferol and increase of IL-10 secretion after resveratrol were observed. The results of present work indicate that 1) apigenin, resveratrol and kaempferol may reduce the intensity of inflammatory processes by inhibiting the secretion of proinflammatory cytokine TNF-α, and resveratrol and kaempferol additionally by increasing the secretion of anti-inflammatory cytokine IL-10 2) the studies indicate the potentially beneficial - anti-inflammatory - impact of diet rich in products including apigenin, resveratrol and kaempferol.

The influence of TSA and VPA on the in vitro differentiation of bone marrow mesenchymal stem cells into neuronal lineage cells: Gene expression studies.

INTRODUCTION: Epigenetic mechanisms regulate the transcription of genes, which can affect the differentiation of MSCs. The aim of the current work is to determine how the histone deacetylase inhibitors TSA and VPA affect the expression of neuronal lineage genes in a culture of rat MSCs (rMSCs). MATERIALS AND METHODS: We analyzed the expression of early neuron marker gene (Tubb3), mature neuron markers genes (Vacht, Th, Htr2a) and the oligodendrocyte progenitor marker gene (GalC). Moreover, changes in the gene expression after three different periods of exposure to TSA and VPA were investigated for the first time. RESULTS: After six days of exposition to TSA and VPA, the expression of Tubb3 and GalC decreased, while the expression of Th increased. The highest increase of VAChT expression was observed after three days of TSA and VPA treatment. A decrease in Htr2a gene expression was observed after TSA treatment and an increase was observed after VPA treatment. We also observed that TSA and VPA inhibited cell proliferation and the formation of neurospheres in the rMSCs culture. DISCUSSION: The central findings of our study are that TSA and VPA affect the expression of neuronal lineage genes in an rMSCs culture. After exposure to TSA or VPA, the expression of early neuronal gene decreases but equally the expression of mature neuron genes increases. After TSA and VPA treatment ER of the oligodendrocyte progenitor marker decreased. TSA and VPA inhibit cell proliferation and the formation of neurospheres in rMSCs culture.

Methylglyoxal (MGO) inhibits proliferation and induces cell death of human glioblastoma multiforme T98G and U87MG cells.

Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor and it is characterized by a poor prognosis and short survival time. Current treatment strategies for GBM using surgery, chemotherapy and/or radiotherapy are ineffective. Thus new therapeutic strategies to target GBM are urgently needed. The effect of methylglyoxal (MGO) on the cell cycle, cell death and proliferation of human GBM cells was investigated. The T98G and U87MG cell lines were cultured in modified EMEM supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were exposed to methylglyoxal (0.025mM) per 72h. The influence of MGO on T98G and U87MG cell cycle, proliferation and apoptosis was evaluated as well. Cell cycle phase distribution, proliferation, apoptosis were analyzed by flow cytometry. MGO causes changes in cell cycle and induces accumulation of G1/G0-phase cells and reduced fraction of cells in S and G2/M phases. We have also observed inhibition of cell proliferation and induction of apoptosis in cancer cells. We have also revealed that MGO induces senescence of U87MG but not T98G cells, but further studies are necessary in order to clarify and check mechanism of action of methylglyoxal and it Is a positive phenomenon for the treatment of GBM.

Combination Therapy with AKT3 and PI3KCA siRNA Enhances the Antitumor Effect of Temozolomide and Carmustine in T98G Glioblastoma Multiforme Cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor, and it is characterized by a poor prognosis and short survival time. Current treatment strategies for GBM, using surgery, chemotherapy and/or radiotherapy, are ineffective. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in this cancer, and it is connected with regulation of the cell cycle, apoptosis, and autophagy. OBJECTIVES: The current study was undertaken to examine the effect of small interfering RNA (siRNA) targeting the AKT3 and PIK3CA genes on the susceptibility of T98G cells to temozolomide (TMZ) and carmustine (BCNU). METHODS: T98G cells were transfected with AKT3 or PI3KCA siRNA. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy. The influence of AKT3 and PI3KCA siRNA in combination with TMZ and BCNU on T98G cell viability, proliferation, apoptosis, and autophagy was evaluated as well. Alterations in messenger RNA (mRNA) expression of apoptosis-related and autophagy-related genes were analyzed using quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Transfection of T98G cells with AKT3 or PI3KCA siRNA and exposure to TMZ and BCNU led to a significant reduction in cell viability, accumulation of subG1-phase cells, and reduction of cells in the S and G2/M phases, as well as induction of apoptosis or necrosis, and regulation of autophagy. CONCLUSION: The siRNA-induced AKT3 and PI3KCA mRNA knockdown in combination with TMZ and BCNU inhibited proliferation and induced apoptosis and autophagy in T98G cells. Thus, knockdown of these genes in combination with TMZ and BCNU may offer a novel therapeutic strategy to more effectively control the growth of human GBM cells, but further studies are necessary to confirm a positive phenomenon for the treatment of GBM.

Knockdown of AKT3 and PI3KCA by RNA interference changes the expression of the genes that are related to apoptosis and autophagy in T98G glioblastoma multiforme cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor and it is characterized by a poor prognosis and short survival time. The PI3K/AKT/PTEN signaling pathway plays a crucial role in GBM development and it is connected with the regulation of apoptosis and autophagy. Akt is involved in various aspects of cancer cell biology such as cell survival, in addition to both apoptosis and autophagy. The current study was undertaken to examine the effect of the siRNAs that target AKT3 and PI3KCA genes on the apoptosis and autophagy of T98G cells. METHODS: T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. Alterations in the mRNA expression of apoptosis- and autophagy-related genes were analyzed using QRT-PCR. LC3IIA protein-positive cells were identified using flow cytometry with specific antibodies. RESULTS: Our findings demonstrate for the first time that the siRNAs that target AKT3 and PI3KCA change the expression of the genes that are related to apoptosis and autophagy and change the expression of the LC3IIA protein in T98G cells. CONCLUSIONS: Thus, there is a high probability that the knockdown of these genes induces apoptosis and autophagy in T98G cells, but further studies are necessary in order to clarify and check whether autophagy induction is a positive phenomenon for the treatment of GBM.

Affect of antidepressants on the in vitro differentiation of rat bone marrow mesenchymal stem cells into neuronal cells.

BACKGROUND: Bone marrow is a valuable source of mesenchymal stem cells (MSCs) that can be used in regenerative medicine. MSCs are able to differentiate into cells from all three germ layers under specific conditions. The aim of the current work was to study the differentiation of rat MSCs (rMSCs) into neuron-like cells. NEW METHOD: We investigated how the antidepressants imipramine, desipramine, fluoxetine and tianeptine affect the differentiation of rMSCs. Furthermore, we present differentiation cocktails using a cortex astrocyte-conditioned medium (CACM) separately or in conjunction with each of the antidepressants and investigated their additive effect on the efficiency of differentiation. We also observed how various differentiation conditions affect the number of primary dendrites and branching dendrites per cell. RESULTS: Gene expression for an early neuronal marker (ß-III-tubulin) and markers that are typical for adult neurons such as Th, Htr2A and Slc6a4 were observed. The Tubb3 and Htr2A gene expression were up-regulated, Th decreased slightly and Slc6a4 was down-regulated after differentiation We observed a two-fold higher percentage of ß-III-tubulin positive cells after treatment with antidepressants and two-fold increase of neuron-like cells after using CACM with imipramine or fluoxetine simultaneously. Differentiation using imipramine or in conjunction with CACM and desipramine or fluoxetine simultaneously increased the number of cell dendrites. COMPARISON WITH EXISTING METHODS: The results that were obtained are completely new and need further investigations in the nearest future. CONCLUSIONS: These results suggest that antidepressants improve differentiation efficiency of rMSCs and may be useful in the preparation of rMSCs for transplantation. Differentiation efficiency is higher after long-term exposure to antidepressants, than after a 24-h exposure. Nearly additive effect of CACM and imipramine or fluoxetine suggests a beneficial role of antidepressants after transplantation.

Differentiation of adult rat mesenchymal stem cells to GABAergic, dopaminergic and cholinergic neurons.

BACKGROUND: Mesenchymal stem cells (MSCs) are able to differentiate into cells from all three germ layers. The aim of the current work was the differentiation rat MSCs into GABAergic, cholinergic and dopaminergic cells. NEW METHOD: In this paper, we present differentiation cocktails with a hippocampal astrocyte conditioned medium and with a glioblastoma conditioned medium. We wanted to maximize the role of endogenous secreted substances by cells from the central nervous system in both combinations. These modifications create a microenvironment of differentiation that is similar to natural conditions. Moreover, the presence of the Cxcr4 receptor on neuron-like cells was investigated first time. RESULTS: Our results show that a differentiation cocktail with a hippocampal astrocyte conditioned medium is the most effective and that 17% Gad67(+) and 7% Acht(+) cells were observed using this protocol. After differentiation using the glioblastoma conditioned medium, 12% Gad67 (+) was observed. The presence of the Cxcr4 migration receptor on Gad67(+) and Th(+) cells were observed, which might suggest the transplantation potential of differentiated cells. COMPARISON WITH EXISTING METHODS: Our results are slightly lower than those of previous studies but when differences in counting cells is taken into account, a comparison of results is really difficult. CONCLUSIONS: These new differentiation cocktails should be further investigated and in the next experiment only a part of MSCs that expressed the Cxcr4 receptor will be differentiated. We suppose that the Cxcr4(+) cells may differentiate more easily and as a result, we may achieve a homogenous population of one phenotype of neurons.

Knockdown of the AKT3 (PKBγ), PI3KCA, and VEGFR2 genes by RNA interference suppresses glioblastoma multiforme T98G cells invasiveness in vitro.

Glioblastoma multiforme (GBM) is the most common primary brain malignancy, having a very poor prognosis and is characterized by extensive brain invasion as well as resistance to the therapy. The phosphoinositide 3-kinase (PI3K)/Akt/PTEN signaling pathway is deregulated in GBM. Besides, florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF) occur very often. The present study was designed to examine the inhibitory effect of AKT3, PI3KCA, and VEGFR2 small interfering RNAs (siRNAs) on GBM cell invasiveness. T98G cells were transfected with AKT3, PI3KCA, and/or VEGFR2 siRNAs. VEGFR2 protein-positive cells were identified by flow cytometry using specific monoclonal anti-VEGFR2 antibodies. Alterations in messenger RNA (mRNA) expression of VEGF, VEGFR2, matrix metalloproteinases (MMPs) (MMP-2, MMP-9, MMP-13, MMP-14), tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, TIMP-3), c-Fos, c-Jun, hypoxia-inducible factor-1α (HIF-1α), ObRa, and cathepsin D genes were analyzed by qRT-PCR. Cells treated with specific siRNA were also analyzed for invasion using the Matrigel invasion assay. We have found significantly lower mRNA levels of MMPs, cathepsin D, VEGF, VEGFR2, HIF-1α, and c-Fos/c-Jun ratio, as well as significantly higher mRNA level of TIMPs in AKT3 and PI3KCA siRNA transfected cells compared to untransfected cells, while significantly lower mRNA levels of MMPs (MMP-2, MMP-9, MMP-14) and TIMP-1, as well as significantly higher mRNA level of TIMP-3, were shown only in cells transfected with VEGFR2 siRNA. The positive correlation between MMP-13 and ObRa mRNA copy number has been found. Summarizing, transfection of T98G cells with AKT3, PI3KCA, or VEGFR2 siRNAs leads to a significant reduction in cell invasiveness. The siRNA-induced AKT3, PI3KCA, and VEGFR2 mRNA knockdown may offer a novel therapeutic strategy to reduce the invasiveness of GBM cells.

Knockdown of AKT3 (PKBγ) and PI3KCA suppresses cell viability and proliferation and induces the apoptosis of glioblastoma multiforme T98G cells.

Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor that is difficult to treat and has a very poor prognosis. Thus, new therapeutic strategies that target GBM are urgently needed. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in a wide range of cancers. The present study was designed to examine the inhibitory effect of AKT3 or PI3KCA siRNAs on GBM cell growth, viability, and proliferation.T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. AKT3 and PI3KCA protein-positive cells were identified using FC and Western blotting. The influence of specific siRNAs on T98G cell viability, proliferation, cell cycle, and apoptosis was evaluated as well using FC. Alterations in the mRNA expression of AKT3, PI3KCA, and apoptosis-related genes were analyzed using QRT-PCR. Knockdown of AKT3 and/or PI3KCA genes in T98G cells led to a significant reduction in cell viability, the accumulation of subG1-phase cells and, a reduced fraction of cells in the S and G2/M phases. Additionally, statistically significant differences in the BAX/BCL-2 ratio and an increased percentage of apoptotic cells were found. The siRNA-induced AKT3 and PI3KCA mRNA knockdown may offer a novel therapeutic strategy to control the growth of human GBM cells.

Influence of betulin and 28-O-propynoylbetulin on proliferation and apoptosis of human melanoma cells (G-361).

INTRODUCTION: Pentacyclic triterpenes are a group of compounds known to have anticancer activity. One of the best characterized triterpenes is betulin, which can be isolated from bark of birch trees and modified into new compounds with various interesting medical properties. Betulin is involved in activation of the caspase cascade and promotes cell death. The aim of the study was to investigate the effect of betulin and its acetylenic derivative, 28-O-propynoylbetulin, on proliferation and apoptosis in a human melanoma cell line. MATERIALS AND METHODS: The G-361 melanoma cell line was used. To evaluate growth arrest and caspase-3 activity, cells were treated with betulin and its derivative at a wide range of concentrations from 0.1 to 10 µg/mL. RESULTS: Betulin and 28-O-propynoylbetulin inhibited cell proliferation in a concentration-dependent manner. The cell cycle analysis revealed an increase of the sub-G1 cell fraction (representing dead cells) after incubation of cells with betulin and 28-O-propynoylbetulin. The observed cytotoxic effects were more pronounced for 28-O-propynoylbetulin. Activity of caspase-3 in 28-O-propynoylbetulin treated cells was nearly 2-fold greater compared to cells incubated with betulin. DISCUSSION: Our results show that betulin and 28-O-propynoylbetulin were effective in inhibition of cell growth and induction of apoptosis in a human melanoma cell line. The addition of the propynoyl group at the C-28 hydroxyl group of betulin led to a greater proapoptotic and antiproliferative effect in comparison to unmodified betulin. These observations suggest that the obtained derivative is a potent anti-melanoma agent.

Heat shock protein 70 gene polymorphisms are associated with paranoid schizophrenia in the Polish population.

HSP70 genes have been considered as promising schizophrenia candidate genes based on their protective role in the central nervous system under stress conditions. In this study, we analyzed the potential implication of HSPA1A +190G/C, HSPA1B +1267A/G, and HSPA1L +2437T/C polymorphisms in the susceptibility to paranoid schizophrenia in a homogenous Caucasian Polish population. In addition, we investigated the association of the polymorphisms with the clinical variables of the disease. Two hundred and three patients with paranoid schizophrenia and 243 healthy controls were enrolled in the study. Polymorphisms of HSPA1A, -1B, and -1L genes were genotyped using the PCR-RFLP technique. Analyses were conducted in entire groups and in subgroups that were stratified according to gender. There were significant differences in the genotype and allele frequencies of HSPA1A polymorphism between the patients and controls. The +190CC genotype and +190C allele were over-represented in the patients and significantly increased the risk for developing schizophrenia (OR = 3.45 and OR = 1.61, respectively). Interestingly, such a risk was higher for females with the +190CC genotype than for males with the +190CC genotype (OR = 5.78 vs. OR = 2.76). We also identified the CGT haplotype as a risk haplotype for schizophrenia and demonstrated the effects of HSPA1A and HSPA1B genotypes on the psychopathology and age of onset. Our study provided the first evidence that the HSPA1A polymorphism may potentially increase the risk of developing paranoid schizophrenia. Further independent analyses in different populations to evaluate the role of gender are needed to replicate these results.

Association of interleukin 2 (IL-2), interleukin 6 (IL-6), and TNF-alpha (TNFα) gene polymorphisms with paranoid schizophrenia in a Polish population.

Numerous reports have brought attention to the potential role of cytokines in schizophrenia. The aim of the study was to determine whether polymorphisms of IL-2, IL-6, and TNFα genes are risk factors for development of paranoid schizophrenia in a Polish population. Promoter polymorphisms of IL-6 (rs1800795), TNFα (rs1800629), and IL-2 (rs2069762) genes in patients (N=115) and controls (N=135) were genotyped by PCR-RFLP and AS-PCR methods, respectively. Genotype TT and allele T for IL-2 polymorphism, and genotype AA and allele A for TNFα polymorphism were found to be significantly associated with paranoid schizophrenia. Similarly, haplotypes CTA and GTA increased the risk (4.4 times and 5.9 times, respectively) of schizophrenia. To reveal associations between Positive and Negative Symptom Scale subscales and age at onset of schizophrenia, the authors used a novel method called Grade Correspondence Analysis. This analysis revealed that patients with early age at onset have higher scores on the Negative and General subscales of PANSS, and, in that group of patients, haplotype CTA was the most represented. As far as is known, this analysis was used for the first time with reference to genetic data.