Biomolecular fingerprints of the effect of zoledronic acid on prostate cancer stem cells: Comparison of 2D and 3D cell culture models.

Revealing the potential of candidate drugs against different cancer types without disrupting normal cells depends on the drug mode of action. In the current study, the drug response of prostate cancer stem cells (PCSCs) to zoledronic acid (ZOL) grown in two-dimensional (2D) and three-dimensional (3D) culture systems was compared using Fourier transform-infrared (FT-IR) spectroscopy which is a vibrational spectroscopic technique, supporting by biochemical assays and imaging techniques. Based on our data, in 2D cell culture conditions, the ZOL treatment of PCSCs isolated according to both C133 and CD44 cell surface properties induced early/late apoptosis and suppressed migration ability. The CD133 gene expression and protein levels were altered, depending on culture systems. CD133 expression was significantly reduced in 2D cells upon ZOL treatment. FT-IR data revealed that the integrity, fluidity, and ordering/disordering states of the cell membrane and nucleic acid content were altered in both 2D and 3D cells after ZOL treatment. Regular protein structures decrease in 2D cells while glycogen and protein contents increase in 3D cells, indicating a more pronounced cytotoxic effect of ZOL for 2D cells. Untreated 3D PCSCs exhibited an even different spectral profile associated with IR signals of lipids, proteins, nucleic acids, and glycogen in comparison to untreated 2D cells. Our study revealed significant differences in the drug response and cellular constituents between 2D and 3D cells. Exploring molecular targets and/or drug-action mechanisms is significant in cancer treatment approaches; thus, FT-IR spectroscopy can be successfully applied as a novel drug-screening method in clinical research.

Differences and similarities in biophysical and biological characteristics between U87 MG glioblastoma and astrocyte cells.

Current cancer studies focus on molecular-targeting diagnostics and interactions with surroundings; however, there are still gaps in characterization based on topological differences and elemental composition. Glioblastoma (GBM cells; GBMCs) is an astrocytic aggressive brain tumor. At the molecular level, GBMCs and astrocytes may differ, and cell elemental/topological analysis is critical for identifying potential new cancer targets. Here, we used U87 MG cells for GBMCS. U87 MG cell lines, which are frequently used in glioblastoma research, are an important tool for studying the various features and underlying mechanisms of this aggressive brain tumor. For the first time, atomic force microscopy (AFM), scanning electron microscopy (SEM) accompanied by energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS) are used to report the topology and chemistry of cancer (U87 MG) and healthy (SVG p12) cells. In addition, F-actin staining and cytoskeleton-based gene expression analyses were performed. The degree of gene expression for genes related to the cytoskeleton was similar; however, the intensity of F-actin, anisotropy values, and invasion-related genes were different. Morphologically, GBMCs were longer and narrower while astrocytes were shorter and more disseminated based on AFM. Furthermore, the roughness values of these cells differed slightly between the two call types. In contrast to the rougher astrocyte surfaces in the lamellipodial area, SEM-EDS analysis showed that elongated GBMCs displayed filopodial protrusions. Our investigation provides considerable further insight into rapid cancer cell characterization in terms of a combinatorial spectroscopic and microscopic approach.

Doxorubicin-induced senescence promotes resistance to cell death by modulating genes associated with apoptotic and necrotic pathways in prostate cancer DU145 CD133+/CD44+ cells.

Cancer stem cells (CSCs) are the most important cause of cancer treatment failure. Traditional cancer treatments, such as chemotherapy and radiotherapy, damage healthy cells alongside malignant cells, leading to severe adverse effects. Therefore, inducing cellular senescence without triggering apoptosis, which further damages healthy cells, may be an alternative strategy. However, there is insufficient knowledge regarding senescence induction in CSCs that show resistance to treatment and stemness properties. The present study aims to elucidate the effects of senescence induction on proliferation, cell cycle, and apoptosis in prostate CSCs and non-CSCs. Prostate CSCs were isolated from DU145 cancer cells using the FACS method. Subsequently, senescence induction was performed in RWPE-1, DU145, prostate CSCs, and non-CSCs by using different concentrations of Doxorubicin (DOX). Cellular senescence was detected using the senescence markers SA-ß-gal, Ki67, and senescence-associated heterochromatin foci (SAHF). The effects of senescence on cell cycle and apoptosis were evaluated using the Muse Cell Analyzer, and genes in signaling pathways associated with the apoptotic/necrotic pathway were analyzed by real-time PCR. Prostate CSCs were isolated with 95.6 ± 1.4% purity according to CD133+/CD44+ characteristics, and spheroid formation belonging to stem cells was observed. After DOX-induced senescence, we observed morphological changes, SA-ß-gal positivity, SAHF, and the lack of Ki67 in senescent cells. Furthermore; we detected G2/M cell cycle arrest and downregulation of various apoptosis-related genes in senescent prostate CSCs. Our results showed that DOX is a potent inducer of senescence for prostate CSCs, inhibits proliferation by arresting the cell cycle, and senescent prostate CSCs develop resistance to apoptosis.

"Double hit" strategy: Removal of sialic acid from the dendritic cell surface and loading with CD44+/CD24-/low cell lysate inhibits tumor growth and metastasis by targeting breast cancer stem cells.

Cancer stem cells (CSCs), which represent the root cause of resistance to conventional treatments, recurrence, and metastasis, constitute the critical point of failure in cancer treatments. Targeting CSCs with dendritic cell (DC)-based vaccines have been an effective strategy, but sialic acids on the surface of DCs limit the interaction with loaded antigens. We hypothesized that removal of sialic acid moieties on immature DCs (iDCs) could significantly affect DC-CSC-antigen loading, thereby leading to DC maturation and improving immune recognition and activity. The lysate of CD44+/CD24-/low breast CSCs (BCSCs) was pulsed with sialidase-treated DCs to obtain mature dendritic cells (mDCs). The roles of cytoskeletal elements in antigen uptake and dendritic cell maturation were determined by immunofluorescence staining, flow cytometry, and cytokine measurement, respectively. To test the efficacy of the vaccine in vivo, CSCs tumor-bearing mice were immunized with iDC or mDC. Pulsing DCs with antigen increased the expression levels of actin, gelsolin, talin, WASp, and Arp2, especially in podosome-like regions. Compared with iDCs, mDCs expressed high levels of CD40, CD80, CD86 costimulatory molecules and increased IL-12 production. Vaccination with mDC: i) increased CD8+ and CD4 + T-cell numbers, ii) prevented tumor growth with anti-mitotic activity and apoptotic induction, iii) suppressed metastasis by decreasing Snail, Slug, and Twist expressions. This study reveals for the first time that sialic acid removal and loading with CSC antigens induces significant molecular, morphological, and functional changes in DCs and that this new DC identity may be considered for future combined immunotherapy strategies against breast tumors.

Neuroprotective Effect of Intrastriatal Caffeic Acid Phenethyl Ester Treatment in 6-OH Dopamine Model of Parkinson's Disease in Rats.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and the main cause of PD is still not known. Until now, no cure for Parkinson's disease is yet in sight. Caffeic acid phenethyl ester (CAPE) is a polyphenolic component of the propolis, which can be derived from honeybee hive propolis. We aimed to determine the effect of intrastriatal CAPE administration as a neuroprotective agent on 6-hydroxydopamine (6-OHDA)-induced PD model. Adult male Wistar rats weighing 280-320 g were used. The PD model was induced with unilateral intrastriatal 6-OHDA injection. Treatment groups received 20 µmol/5 µL/4 day and 80 µmol/5 µL/4 day CAPE 24 h after 6-OHDA injection. Eight days after 6-OHDA application, behavioral studies (adhesive tape removal test, open-field test, cylinder test, and apomorphine-induced asymmetric rotational behavior) were performed once more to compare the effects of CAPE on behavior tests. Striatal histological verifications, immunohistochemistry, and stereological quantitation were performed. Our results for the first time showed that, besides improving the motor performance, CAPE treatment also prevents 6-OHDA-induced loss of TH-positive neurons. From our results, CAPE may be a promising clinical agent in the treatment of PD.

Sonic hedgehog signaling is associated with resistance to zoledronic acid in CD133high/CD44high prostate cancer stem cells.

Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH) signal regulates stem cells in normal prostate epithelium by affecting cell behavior, survival, proliferation, and maintenance. Aberrant SHH pathway activation leads to an unsuitable expansion of stem cell lineages in the prostate epithelium and the transformation of prostate CSCs (PCSCs). Zoledronic acid (ZOL), one of the third-generation bisphosphonates, effectively prevented bone metastasis and treated advanced prostate cancer despite androgen deprivation therapy. Despite strong evidence for the involvement of the SHH in human PCSCs survival and drug resistance, the roles of SHH in the PCSCs-related resistance to ZOL remain to be fully elucidated. The present study aimed to investigate the role of the SHH pathway in ZOL resistance of PCSCs in 2D and three 3D cell culture conditions. For this purpose, we isolated CD133high/ CD44high PCSCs using a flow cytometer. Following ZOL treatment, mRNA and protein expressions of the components of the SHH signaling pathway in PCSCs and non-CSCs were analyzed using qRT-PCR and Immunofluorescence staining, respectively. Our finding suggested that SHH signaling may be activated by different mechanisms that lead to avoidance of the inhibition effect of ZOL. Thereby, SHH pathways may be associated with the resistance to ZOL developed by prostate CSCs. Inhibition of CSCs-related SHH signaling along with ZOL treatment should be considered to achieve improvement in survival or delayed treatment failure and prevention of the CSCs-related drug resistance.

The Rhythmicity of Life: A Review of the Circadian Clocks.

Physiology of the mammalian body has been adapted to diurnal cycles of around 24 h, an evolutionary situation that affects a wide spectrum of biological events including sleep-to-wake transitions, feeding/fasting, body temperature, and hormonal regulations. The patterns of the diurnal cycle occur due to rhythmic oscillations that arise from the suprachiasmatic nucleus of hypothalamus, which also can be defined as the pacemaker of the system. The clock can be defined as a molecular machinery driven by the core clock genes that encode clock proteins in a rhythmic oscillatory fashion maintained by the light/dark cycles of the environment. Although the well-established knowledge refers to the function of the circadian rhythm as maintenance of the normal physiology, growing evidence shows that disruptions in the system usually caused by genetic and/or epigenetic misregulations may have a direct effect to lead major pathological conditions, such as carcinogenesis. This review outlines the main molecular aspects of circadian physiology, and reveals the reasons for and results of the circadian disruptions at different levels. In spite of the fact that more proof is needed for a direct correlation between circadian disruptions and oncogenesis and other pathological events, data obtained from current research supports the role of circadian rhythms in malfunctioning of the normal cellular metabolism.

Immunohistochemical determination of mTOR pathway molecules in ovaries and uterus in rat estrous cycle stages.

mTOR is a member of the PI3K/Akt/mTOR signaling pathway that participates in cell growth, proliferation, protein synthesis, transcription, angiogenesis, apoptosis and autophagy. mTOR and MAPK pahways are two important key signal pathways which are related to each other. We investigated the role of mTOR and other signaling molecules in rat ovaries and uteruses in stages of the estrous cycle. Young adult female rats were divided into four groups as proestrous, estrous, metestrous and diestrous according to vaginal smears. Immunohistochemical staining of mTORC1, IGF1, PI3K, pAKT1/2/3, ERK1 and pERK1/2 was performed and pAKT1/2/3 and ERK1 were also analyzed using western blotting on ovarian and uterine tissue samples. According to our results, PI3K/Akt/mTOR and ERK/pERK showed an increase in the rat ovulation period. When all the groups were evaluated the immunoreactivities for all of the antibodies were detected in the oocytes, granulosa and theca cells, corpus luteum and stroma of ovary and lamina propria, surface and glandular epithelium of uterus with the strongest observed with anti-ERK1 antibody and then with a decreasing trend with anti-mTORC1, anti-pAkt1/2/3, anti-IGF1, anti-PI3K and anti-pERK1/2 antibodies in the proestrus and estrus stages. Differently from other parts of the ovary, highest antibody expression in the corpus luteum was observed in the metestrous stage. Moreover, the existence of pAKT1/2/3 and ERK1 proteins was confirmed with the Western blotting technique. We suggest that mTOR and mTOR-related ERK signaling molecules may participate in the rat ovulation process.

Effect of SIRT1 activators and inhibitors on CD44+/CD133+­enriched non­small cell lung cancer cells.

Lung cancer is one of the most commonly diagnosed cancers and it is associated with high rates of morbidity and mortality. Metastasis and relapse of the tumor depend on the survival and proliferation of lung cancer stem cells (LCSCs). The ability to identify CSCs may prevent recurrence and lead to more effective treatments. Sirtuins are a group of deacetylases that include seven variants (SIRT1­7), with sirtuin 1 (SIRT1) being the most intensively investigated. Evidence suggests that SIRT1 is both a tumor­suppressor gene and an oncogene. SIRT1 can deacetylate the tumor­suppressor protein p53 to decrease its activity. SIRT1 activators increase the deacetylation of p53, whereas SIRT1 inhibitors can stimulate p53 by inhibiting deacetylation. In the present study, CD44+ and CD133+­enriched A549 (non­small cell lung cancer) cells collected using the CD44 and CD133 CSC surface markers by fluorescence­activated cell sorting method were treated with SIRT1 inhibitors (tenovin­6 and sirtinol) and SIRT1 activators (resveratrol and SRT1720), and their effects on apoptosis, as well as the mRNA and protein expression of SIRT1 and p53 were investigated. Of these agents, it was found that resveratrol increased p53 expression by 4.1­fold, decreased SIRT1 expression by 0.2­fold, and it was the most potent inducer of apoptosis.

Triptolide inhibits CD133+ /CD44+ colon cancer stem cell growth and migration through triggering apoptosis and represses epithelial-mesenchymal transition via downregulating expressions of snail, slug, and twist.

High recurrence and metastatic behavior patterns are the most important reasons for the failure of treatment strategies in patients with colon cancer. Cancer stem cells (CSCs), which are considered root of cancer, are thought to be associated with therapy resistance, relapse, and metastasis, and, therefore, targeting CSCs rather than the bulk population may be an effective approach. In cancer studies, there is an increasing interest in close friendship between epithelial-mesenchymal transition (EMT) and CSCs. Triptolide (TPL) isolated from Chinese herb Tripterygium wilfordii has important effects on the prevention of migration and metastasis as well as cytotoxic effect against cancer cells. The potential lethal efficacy of TPL on CSCs that is highly resistant to the drug is an unsolved mystery. Fundamentally, the present study basically aims to find answers to two questions: (a) is it possible to target colon CSCs with TPL? and (b) what are the mechanisms underlying TPL's potential to eliminate CSCs? Cytotoxic effects of TPL on CSCs were evaluated by WST-1 and Muse count and viability assays. Apoptosis assay and cell-cycle analysis were performed to investigate the inhibitory effect of TPL. Moreover, the effects of TPL on spheroid formation capacity, migration, and EMT processes, which are associated with CSC phenotype, were also investigated. The results revealed that TPL triggered cell death and apoptosis and altered cell cycle distribution. Moreover, TPL significantly reduced the snail slug and twist expressions associated with EMT. TPL has been shown to be effective in colon CSCs by in vitro experiments, and it might be a highly effective agent against colon cancer has been implicated in need of supporting in vivo and clinical studies.

Cancer and Cancer Stem Cells: New Molecular Perspectives.

Cancer, which causes the deaths of millions of people, is an important public health problem worldwide. Despite significant advances in the diagnosis and treatment of cancer, survival rates are still insufficient in progressive cancers. Today, one of the most important reasons for not being able to reach the desired level in the fight against progressive cancer types is cancer stem cells (CSCs). The ineffectiveness of conventional therapies on CSCs has made it necessary to investigate the molecular mechanisms and signaling pathways used in the survival, drug resistance, and metastasis of CSCs. In this context, studies investigating the biology of CSCs suggest that lipid metabolism and extracellular vesicles are critical for understanding the stemness and metastasis of these cells. These studies have demonstrated that a number of molecules play a vital role in resistant to apoptosis in CSCs, including the cellular FLICE-inhibitor protein (c-FLIP), which inhibits TRAIL-induced extrinsic apoptosis. Another important output of these studies is the demonstration of the relationship of the cancer microenvironment in terms of epithelial-mesenchymal transition, which CSCs frequently use in the metastasis process. In addition, studies investigating the differences in glycosylation observed in CSCs and investigating cancer vaccines are promisng. These findings will strengthen our aresnal in the fight against cancer. In this article, we summarize current molecular studies on CSCs, an important target in novel cancer therapies.

Differential gene expression analysis of human cumulus cells.

OBJECTIVE: This study was performed to explore the possibility that each oocyte and its surrounding cumulus cells might have different genetic expression patterns that could affect human reproduction. METHODS: Differential gene expression analysis was performed for 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes from 10 patients. Same procedures related to oocyte maturation, microinjection, and microarray analyses were performed for each group of cumulus cells. Two differential gene expression analyses were performed: one for the outcome of clinical pregnancy and one for the outcome of live birth. RESULTS: Significant genes resulting from these analyses were selected and the top 20 affected pathways in each group were analyzed. Circadian entrainment is determined to be the most affected pathway for clinical pregnancy, and proteoglycans in cancer pathway is the most affected pathway for live birth. Circadian entrainment is also amongst the 12 pathways that are found to be in top 20 affected pathways for both outcomes, and has both lowest p-value and highest number of times found count. CONCLUSION: Although further confirmatory studies are necessary, findings of this study suggest that these pathways, especially circadian entrainment in cumulus cells, may be essential for embryo development and pregnancy.

Characterization of CD133+/CD44+ human prostate cancer stem cells with ATR-FTIR spectroscopy.

Current cancer treatments destroy the tumor mass but cannot prevent the recurrence of cancer. The heterogeneous structure of the tumor mass includes cancer stem cells that are responsible for tumor relapse, treatment resistance, invasion and metastasis. The biology of these cells is still not fully understood; therefore, effective treatments cannot be developed sufficiently. Herein, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, combined with unsupervised multivariate analysis, was applied to prostate cancer stem cells (CSCs), non-stem cancer cells (non-CSCs) and normal prostate epithelial cells to elucidate the molecular mechanisms and features of CSCs, which are crucial to improving the target specific therapies. This work revealed the spectral differences in the cellular mechanisms and biochemical structures among three different cell types. Particularly, prostate CSCs exhibit differences in the lipid composition and dynamics when compared to other cell types. CSCs also harbor pronounced differences in their major cellular macromolecules, including differences in the protein amount and content (mainly α-helices), the abundance of nucleic acids (DNA/RNA), altered nucleic acid conformation and carbohydrate composition. Interestingly, macromolecules containing the C[double bond, length as m-dash]O groups and negatively charged molecules having the COO- groups are abundant in prostate CSCs in comparison to prostate non-CSCs and normal prostate cells. Overall, this study demonstrates the potential use of ATR-FTIR spectroscopy as a powerful tool to obtain new insights into the understanding of the CSC features, which may provide new strategies for cancer treatment by selectively targeting the CSCs.

Glycogen synthase kinase-3 inhibition in glioblastoma multiforme cells induces apoptosis, cell cycle arrest and changing biomolecular structure.

Glioblastoma multiforme (GBM) is the most malignant and aggressive primary human brain tumors. The regulatory pathways of apoptosis are altered in GBMs, leading to a survival advantage of the tumor cells. Thus, identification of target molecules, which are effective in triggering of the cell death mechanisms in GBM, is an essential strategy for therapeutic purposes. Glycogen synthase kinase-3 (GSK-3) plays an important role in apoptosis, proliferation and cell cycle. This study focused on the effect of GSK-3 inhibitor IX in the GBM cells. Apoptosis induction was determined by Annexin-V assay, multicaspase activity and immunofluorescence analyses. Concentration-dependent effects of GSK-3 inhibitor IX on the cell cycle were also evaluated. Moreover, the effect of GSK inhibitor on the cellular biomolecules was assessed by using ATR-FTIR spectroscopy. Our assay results indicated that GSK-3 inhibitor IX induces apoptosis, resulting in a significant increase in the expression of caspase-3 and caspase-8 proteins. Cell cycle analyses revealed that GSK-3 inhibitor IX leads to dose-dependent G2/M-phase cell cycle arrest. Based on the FTIR data, treatment of GBM cells causes dysregulation in the carbohydrate metabolism and induces apoptotic cell death which was characterized by the spectral alterations in nucleic acids, an increment in the lipid amount with disordering state and compositional changes in the cellular proteins. These findings suggest that GSK-3 inhibitor IX exhibits anti-cancer effects by inducing apoptosis and changing biomolecular structure of membrane lipids, carbohydrates, nucleic acids and proteins, and thus, may be further evaluated as a potential effective candidate agent for the GBM combination therapies.

Midkine downregulation increases the efficacy of quercetin on prostate cancer stem cell survival and migration through PI3K/AKT and MAPK/ERK pathway.

AIMS: To examine the functions of growth factor midkine (MK) and a flavonoid quercetin on survival, apoptosis and migration of prostate cancer (PCa) stem cells (CSCs). MAIN METHODS: CD44+/CD133+ and CD44+ stem cells were isolated from PC3 and LNCaP cells, respectively by magnetic-activated cell sorting system. 3D cell culture was used to evaluate the ability of quercetin, MK siRNA, and the combination of both to inhibit spheroid formation, apoptosis and cell cycle arrest. Image-based cytometer, RT-qPCR, Western blotting and transwell migration assays were performed. KEY FINDINGS: Quercetin treatment for 24-72 h inhibited PC3 and CD44+/CD133+ stem cell proliferation in a time- and dose-dependent manner. Knockdown of endogenous MK expression significantly suppressed proliferation of CD44+/CD133+ and CD44+ cells as well as their parent cells. Co-administration of MK siRNA and quercetin reduced the cell survival, induced apoptosis and caused G1 phase cell cycle arrest more effectively than the individual therapy. Knockdown of MK significantly enhanced the inhibitory effect of quercetin on CD44+/CD133+ migration and spheroid formation. In addition, the combined therapy inhibited the phosphorylation of PI3K, AKT and ERK1/2, and reduced the protein expression of p38, ABCG2 and NF-κB. SIGNIFICANCE: Quercetin alone exhibited significant cytotoxic effects on CD44+/CD133+. MK plays an important role in the proliferation of CD44+/CD133+ and CD44+ cells in particular, and quercetin and MK-silencing therapy may be an important strategy in targeting CSCs that play a role in relapse, migration and drug resistance.

Effects of different drug treatments on the proliferation of human ovarian carcinoma cell line MDAH-2774

Background/aim: In this study, the effects of resveratrol as a natural polyphenol compound, gemcitabine as an antimetabolite that has nucleoside structure analogous to deoxycytidine, and para-aminophenol-derived paracetamol were investigated with single and combined applications in monolayers of the MDAH-2774 human ovarian cancer cell line. Materials and methods: Drugs were evaluated in cell culture with respect to cell proliferation, cell cytotoxicity (trypan blue dye exclusion test), synthesis phase of cell cycle, and cell structure in 24, 48, 72, and 96 h. Result: Resveratrol and gemcitabine diminished both cell proliferation and cell cycle synthesis phase indication in monolayer cell cultures (P < 0.05). All combination groups showed similar effects that were mainly more effective in respect to single usage of resveratrol and gemcitabine in monolayer cell cultures. Conclusion: The effects of gemcitabine, resveratrol, and paracetamol were investigated in monolayers of the MDAH-2774 human ovarian cancer cell line and a decrease in cell number in cell cycle synthesis phase, prevention of cell proliferation, and destruction of cell structure were observed.

Expression of the Bone Morphogenetic Protein-2 (BMP2) in the Human Cumulus Cells as a Biomarker of Oocytes and Embryo Quality.

BACKGROUND: The members of the transforming growth factor-B superfamily, as the bone morphogenetic proteins (BMPs) subfamily and anti-Müllerian hormone (AMH), play a role during follicular development, and the bone morphogenetic protein-2 (BMP2), AMH, and THY1 are expressed in ovaries. AIM: This study was designed to define whether or not the expressions of these proteins in human cumulus cells (CCs) can be used as predictors of the oocyte and embryo competence. SETTINGS AND DESIGN: The study included nine female patients who were diagnosed as idiopathic infertility, aged 25-33 years (median 30 years) and underwent Assisted Reproductive Technologies. MATERIALS AND METHODS: The CCs from 60 oocyte-cumulus complexes obtained from the nine patients were evaluated with immunofluorescence staining in respect of BMPs, AMH and THY1 markers. The CCs surrounding the same oocytes were evaluated separately according to the oocyte and embryo quality. STATISTICAL ANALYSIS: Quantitative data were statistically analyzed for differences using the two-sided Mann-Whitney U test (P < 0.05). RESULTS AND CONCLUSIONS: Significant differences in immunofluorescence staining were observed in oocyte quality and embryo quality for the BMP2 only (P < 0.05). No significant differences were observed for AMH or CD90/THY1. CONCLUSION: These results demonstrated that there is a significant difference in the expression of BMP2 in the CCs of good quality oocytes and subsequently a good embryo.

Treatment with milk thistle extract (Silybum marianum), ursodeoxycholic acid, or their combination attenuates cholestatic liver injury in rats: Role of the hepatic stem cells.

BACKGROUND/AIMS: Cholestasis, which results in hepatic cell death, fibrosis, cirrhosis, and eventually liver failure, is associated with oxidative stress. The aim of this study was to evaluate the effects of milk thistle (MT, Silybum marianum) and ursodeoxycholic acid (UDCA) or their combination on the activation of hepatic stem cells and on the severity of cholestasis liver injury in rats. MATERIALS AND METHODS: Under anesthesia, bile ducts of female Sprague Dawley rats were ligated (BDL) or had sham operation. BDL rats were administered saline, UDCA (15 mg/kg/d), MT (600 mg/kg/d), or UDCA+MT by gavage for 10 days. On the 11th day, rats were sacrificed and blood and liver samples were obtained. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic malondialdehyde (MDA) levels, and myeloperoxidase (MPO) activity were measured. Hepatic injury, a-smooth muscle actin expression, and stem cell markers c-kit, c-Myc, Oct3/4, and SSEA-1 were histologically determined. RESULTS: Histological scores, serum ALT, and hepatic MDA levels were higher in BDL group than in the sham rats, while all treatments significantly reduced these levels. The reduction in ALT was significantly greater in UCDA+MT-treated group than in other treatment groups. c-Kit, c-Myc, Oct3/4, and SSEA-1 were increased in saline-treated BDL group with respect to sham-operated control group, and these markers were significantly reduced in all treatment groups. CONCLUSION: In addition to a modulatory effect on the stem cell-induced regenerative response of the liver, UDCA, MT, and their combination demonstrated similar anti-inflammatory and antiproliferative effects on cholestasis-induced hepatic injury.

Repression of the Notch pathway prevents liver damage in streptozotocin-induced diabetic mice.

INTRODUCTION: Sunitinib is an oral inhibitor of vascular endothelial growth factor that is used to treat a variety of cancer. There are limited data regarding the effect of sunitinib on diabetes. In the liver, Notch signaling plays an important role in liver tissue development and homeostasis and its dysfunction is associated with liver pathol-ogies. The aim of the present study is to investigate the effects of sunitinib on streptozotocin (STZ)-induced diabetic liver in mice models. MATERIAL AND METHODS: An experimental diabetes mellitus (DM) model was created in 28 male CD-1 mice. Twenty-eight male CD-1 mice divided in four groups (n = 7 each) were used; control mice (C), control mice treated with sunitinib (C + S), diabetic mice (DM), and diabetic mice treated with sunitinib (DM + S) for four weeks. The histopathological changes in the liver were examined by histochemistry and immunohistochemistry. Immunoreactivity of Notch1, Jagged1, DLL-1 and VEGF were evaluated in control and diabetic mice after sunitinib treatment. RESULTS: The significant morphological changes in the liver were mostly seen in hepatocytes that were hyper-trophied in the DM mice, with an increased amount of eosinophilic granules; moreover, some hepatocytes contained empty vacuole-like structures. The livers of the DM mice revealed increased deposition of collagen fibers. After sunitinib treatment the hepatocytes and hepatic lobules had almost similar morphology to control mice. The immunoreactivities of Notch1, Jagged1, DLL-1 and VEGF in hepatocytes were significantly lower in the DM group when compared with the C, DM + S and C + S group treated with sunitinib. CONCLUSIONS: These results suggest that sunitinib effectively protects the liver from diabetes-induced damage through the inhibition of the Notch pathway.

Ribosome biogenesis mediates antitumor activity of flavopiridol in CD44+/CD24- breast cancer stem cells.

Flavopiridol is a synthetically produced flavonoid that potently inhibits the proliferation of human tumor cell lines. Flavopiridol exerts strong antitumor activity via several mechanisms, including the induction of cell cycle arrest and apoptosis, and the modulation of transcriptional regulation. The aim of the present study was to determine the effect of flavopiridol on a subpopulation of cluster of differentiation (CD)44+/CD24- human breast cancer MCF7 stem cells. The CD44+/CD24- cells were isolated from the MCF7 cell line by fluorescence-activated cell sorting and treated with 100, 300, 500, 750 and 1,000 nM flavopiridol for 24, 48 and 72 h. Cell viability and proliferation assays were performed to determine the inhibitory effect of flavopiridol. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarray. According to the results, the half maximal inhibitory concentration (IC50) value of flavopiridol was 500 nM in monolayer cells. Flavopiridol induced growth inhibition and cytotoxicity in breast cancer stem cells (BCSCs) at the IC50 dose. The present study revealed several differentially regulated genes between flavopiridol-treated and untreated cells. The result of the pathway analysis revealed that flavopiridol serves an important role in translation, the ribosome biogenesis pathway, oxidative phosphorylation, the electron transport chain pathway, carbon metabolism and cell cycle. A notable result from the present study is that ribosome-associated gene expression is significantly affected by flavopiridol treatment. The data of the present study indicate that flavopiridol exhibits antitumor activity against CD44+/CD24- MCF7 BCSCs through different mechanisms, mainly by inhibiting translation and the ribosome biogenesis pathway, and could be an effective chemotherapeutic molecule to target and kill BCSCs.