Eda Acikgoz

Induced growth inhibition, cell cycle arrest and apoptosis in CD133+/CD44+ prostate cancer stem cells by flavopiridol

Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity, apoptosis and G1-phase arrest in a number of human tumor cell lines. Flavopiridol is currently undergoing investigation in human clinical trials. The present study focused on the effect of flavopiridol in cell proliferation, cell cycle progression and apoptosis in prostate cancer stem cells (CSCs). Therefore, cluster of differentiation 133 (CD133)+high/CD44+high prostate CSCs were isolated from the DU145 human prostate cancer cell line. The cells were treated with flavopiridol in a dose- and time-dependent manner to determine the inhibitory effect. Cell viability and proliferation were analyzed and the efficiency of flavopiridol was assessed using the sphere-forming assay. Flavopiridol was applied to monolayer cultures of CD133high/CD44high human prostate CSCs at the following final concentrations: 100, 300, 500 and 1000 nM. The cultures were incubated for 24, 48 and 72 h. The half maximal inhibitory concentration (IC50) value of the drug was determined as 500 nM for monolayer cells. Dead cells were analyzed prior and subsequent to exposure to increasing flavopiridol doses. Annexin-V and immunofluorescence analyses were performed for the evaluation of apoptotic pathways. According to the results, flavopiridol treatment caused significant growth inhibition at 500 and 1000 nM when compared to the control at 24 h. G0/G1 analysis showed a statistically significant difference between 100 and 500 nM (P<0.005), 100 and 1000 nM (P<0.001), 300 and 1000 nM (P<0.001), and 500 and 1000 nM (P<0.001). Flavopiridol also significantly influenced the cells in the G2/M phase, particularly at high-dose treatments. Flavopiridol induced growth inhibition and apoptosis at the IC50 dose (500 nM), resulting in a significant increase in immunofluorescence staining of caspase-3, caspase-8 and p53. In conclusion, the present results indicated that flavopiridol could be a useful therapeutic agent for prostate CSCs by inhibiting tumor growth and malignant progression, and inducing apoptosis.

miR-15a enhances the anticancer effects of cisplatin in the resistant non-small cell lung cancer cells

Platinum-based chemotherapies have long been used as a standard treatment in non-small cell lung cancer. However, cisplatin resistance is a major problem that restricts the use of cisplatin. Deregulated cell death mechanisms including apoptosis and autophagy could be responsible for the development of cisplatin resistance and miRNAs are the key regulators of these mechanisms. We aimed to analyse the effects of selected miRNAs in the development of cisplatin resistance and found that hsa-miR-15a-3p was one of the most significantly downregulated miRNAs conferring resistance to cisplatin in Calu1 epidermoid lung carcinoma cells. Only hsa-miR-15a-3p mimic transfection did not affect cell proliferation or cell death, though decreased cell viability was found when combined with cisplatin. We found that induced expression of hsa-miR-15a-3p via mimic transfection sensitised cisplatin-resistant cells to apoptosis and autophagy. Our results demonstrated that the apoptosis- and autophagy-inducing effects of hsa-miR-15a-3p might be due to suppression of BCL2, which exhibits a major connection with cell death mechanisms. This study provides new insights into the mechanism of cisplatin resistance due to silencing of the tumour suppressor hsa-miR-15a-3p and its possible contribution to apoptosis, autophagy and cisplatin resistance, which are the devil’s triangle in determining cancer cell fate.

Enhanced G2/M Arrest, Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells.

Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133+high/CD44+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in prostate CSCs; and may be a potential effective therapeutic agent against prostate cancer.

Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells.

Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle–cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and carcinogenesis may prove crucial in developing novel therapeutics that specifically target cancer cells.

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.

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.

Effects of flavopiridol on critical regulation pathways of CD133high/CD44high lung cancer stem cells

Background: Flavopiridol a semisynthetic flavone that inhibits cyclin-dependent kinases (CDKs) and has growth-inhibitory activity and induces a blockade of cell-cycle progression at G1-phase and apoptosis in numerous human tumor cell lines and is currently under investigation in phase II clinical trials. Cancer stem cells (CSCs) are comprised of subpopulation of cells in tumors that have been proposed to be responsible for recurrence and resistance to chemotherapy. The aim of the present study was to investigate the effects of flavopiridol in cancer stem cell cytoskeleton, cell adhesion, and epithelial to mesenchymal transition in CSCs. Methods: The cells were treated with flavopiridol to determine the inhibitory effect. Cell viability and proliferation were determined by using the WST-1 assay. Caspase activity and immunofluorescence analyses were performed for the evaluation of apoptosis, cell cytoskeleton, and epithelial-mesenchymal transition (EMT) markers. The effects of flavopiridol on the cell cycle were also evaluated. Flow cytometric analysis was used to detect the percentages of CSCs subpopulation. We analyzed the gene expression patterns to predict cell cycle and cell cytoskeleton in CSCs by RT-PCR. Results: Flavopiridol-induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspases activity. Cell cycle analyses revealed that flavopiridol induces G1 phase cell cycle arrest. Flavopiridol significantly decreased the mRNA expressions of the genes that regulate the cell cytoskeleton and cell cycle components and cell motility in CSCs. Conclusion: Our results suggest that Flavopiridol has activity against lung CSCs and may be effective chemotherapeutic molecule for lung cancer treatment.

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.

Effects of sunitinib on immunoreactivity of vimentin, E-cadherin and S100 in kidneys of streptozotocin induced diabetic mice

Abstract Diabetes mellitus (DM) affects many organs including kidney. Tyrosine kinase can cause hypoglycemia and sunitinib is an inhibitor of tyrosine kinase. We investigated the possible effects of sunitinib on the kidney of streptozotocin (STZ) induced type 1 diabetic mice. We used 28 CD 1 type male mice divided into four groups of seven. Type 1 diabetes was induced by injection of STZ. Group 1 was the untreated control. Group 2 comprised non-diabetic mice + sunitinib. Both groups 1 and 2 exhibited normal blood glucose levels. Group 3 comprised STZ treated diabetic mice + saline. Group 4 were diabetic mice + sunitinib treatment. Kidneys were removed after 8 weeks. The immunoreactivities of vimentin, E-cadherin and S100 were assessed. Immunostaining of vimentin, E-cadherin and S100 was located in both the glomeruli and tubules of the kidney. We found that the number of vimentin and E-cadherin positive glomeruli and tubules were increased after sunitinib treatment compared to saline treated diabetic mice. The number of vimentin labeled tubules was decreased in the sunitinib treated group compared to diabetic + saline groups. Differences in the number of S100 positive tubules and glomeruli between groups 3 and 4 were not statistically significant. The effect of sunitinib on experimental diabetic mice appears to be related to levels of vimentin, E-cadherin and S100 in the glomeruli and tubules of the kidney, and sunitinib may protect against renal damage from DM.

Deciphering the biochemical similarities and differences among mouse embryonic stem cells, somatic and cancer cells using ATR-FTIR spectroscopy

Cellular macromolecules play important roles in cellular behaviors and biological processes. In the current work, cancer (KLN205), normal (MSFs) and mouse embryonic stem cells (mESCs) are compared using ATR-FTIR spectroscopy. Modifications in the composition, concentration, structure and function-related changes in the cellular components were deciphered using the infrared spectra. Our results revealed that cancer and embryonic stem cells are very similar but highly different from the normal cells based on the spectral variations in the protein, lipid, carbohydrate and nucleic acid components. The longest lipid acyl chains exist in mESCs, while cancer cells harbor the lowest lipid amount, short lipid acyl chains, a high content of branched fatty acids and thin cell membranes. The highest cellular growth rate and accelerated cell divisions were observed in the cancer cells. However, the normal cells harbor low nucleic acid and glycogen amounts but have a higher lipid composition. Any defect in the signaling pathways and/or biosynthesis of these cellular parameters during the embryonic-to-somatic cell transition may lead to physiological and molecular events that promote cancer initiation, progression and drug resistance. We conclude that an improved understanding of both similarities and differences in the cellular mechanisms among the cancer, normal and mESCs is crucial to develop a potential clinical relevance, and ATR-FITR can be successfully used as a novel approach to gain new insights into the stem cell and cancer research. We suggest that targeting the cellular metabolisms (glycogen and lipid) can provide new strategies for cancer treatment.