Özlem Darcansoy İşeri

Effect of MDR modulators verapamil and promethazine on gene expression levels of MDR1 and MRP1 in doxorubicin-resistant MCF-7 cells

PurposeOne of the major problems of cancer chemotherapy is the development of multidrug resistance (MDR) phenotype. Among the numerous mechanisms of MDR, a prominent one is the increased expression of membrane transporter proteins, the action of which leads to decreased intracellular drug concentration and cytotoxicity of drugs. Among them, P-gp and MRP1, encoded by MDR1 and MRP1 genes, respectively, have been associated with MDR phenotype. Chemical modulators can be used to reverse MDR. These chemicals can either modulate MDR due to their substrate analogy (such as calcium channel blocker verapamil) or interact with phospholipid membranes (such as antihistaminic drug promethazine). This study focuses on the effect of verapamil and promethazine on the expression levels of MDR1 and MRP1 genes and the drug transport activity in doxorubicin-resistant MCF-7 breast carcinoma cell line.MethodsDoxorubicin-resistant MCF-7 (MCF-7/Dox) cells were incubated with either verapamil or promethazine, and total RNA was isolated. Real-time PCR (qPCR) was carried out by using specific primers for MDR1, MRP1, and ß-actin genes. Intracellular doxorubicin accumulation was also examined by confocal laser scanning microscopy in treated cells.ResultsResults demonstrated a significant decrease in both MDR1 and MRP1 expression levels after promethazine applications. It has also been shown that treatment of the cells with verapamil results in significant decrease in MDR1 mRNA levels. Confocal laser scanning microscopy images demonstrated that the intracellular accumulation of doxorubicin was increased after verapamil treatment in MCF-7/Dox cells.ConclusionsThe present study gives an idea about the efficiency of verapamil and promethazine on MDR reversal both in gene expression and in transport activity levels.

Reversal of Multidrug Resistance by Synthetic and Natural Compounds in Drug-Resistant MCF-7 Cell Lines

Background: Ineffectiveness of anticancer drugs is frequently observed in cancer chemotherapy. The resistance of tumor cells to various cytotoxic drugs is defined as multidrug resistance (MDR). The purpose of this study is to investigate the potential reversal effect of some synthetic and natural chemicals on drug-resistant MCF-7 cell lines. The effects of potential MDR modulators combined with some anticancer drugs were also studied. Methods: Flow cytometry, MTT cytotoxicity assays and checkerboard combination assays were performed to study the reversal of drug resistance and to investigate the antiproliferative effects of the combination of anticancer drugs and the potential modulators. The results indicated that verapamil, capsanthin, zeaxanthin and promethazine inhibited P-gp effectively, but chrysin was not effective at reversing the resistance in MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and 4 effective MDR modulators (verapamil, capsanthin, zeaxanthin and promethazine) were applied to the sublines in combination. Results and Conclusion: Fractional inhibitory indices show that verapamil and zeaxanthin seem to be the most effective MDR reversal agents that may be used together with paclitaxel, docetaxel, vincristine and doxorubicin in drug-resistant mammary carcinoma sublines. In conclusion, this report represents the importance to find out active and efficient drug resistance modulators for improving the efficacy of chemotherapy.

Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins.

PurposeSince multidrug resistance is a multifactorial phenomenon, a large-scale expression analysis of drug-resistant cells by using high-density oligonucleotide microarrays may provide information about new candidate genes contributing to resistance. Extracellular matrix (ECM) is responsible for many aspects of proliferation and invasive/metastatic behavior of tumor cells. This study demonstrates alterations in gene expression levels of several ECM components, matrix metalloproteinases (MMPs), adamalysins (ADAMs and ADAMTSs) and tissue inhibitors of metalloproteinases (TIMPs) in paclitaxel, docetaxel, vincristine and doxorubicin-resistant MCF-7 cells.MethodsResistant MCF-7 cells were developed by stepwise selection of cells in increasing concentrations of drugs. Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array was used for hybridizations. Statistical significance was determined by independent sample t test. The genes having altered expression levels in drug-resistant sublines were selected and filtered by volcano plots.ResultsGenes up/downregulated more than twofolds were selected and listed. Expression of 25 genes encoding ECM proteins (including collagen, finronectin and syndecan) and integrin receptor subunits were found to be upregulated in drug-resistant cells. In addition, expression levels of, 13 genes encoding MMPs, ADAMs, ADAMTSs and TIMPs (including MMP1, MMP9, ADAM9 and TIMP3) were found to be altered in drug-resistant sublines when compared with sensitive MCF-7.ConclusionsBased on the expression analysis profiles, this report provides a preliminary insight into the relationship between drug resistance and ECM components, which are related to invasion and metastasis. Correlation of each specific ECM component with drug resistance requires further analysis.

A microarray based expression profiling of paclitaxel and vincristine resistant MCF-7 cells

Resistance to the broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. Drug resistant sublines to paclitaxel (MCF-7/Pac) and vincristine (MCF-7/Vinc) that were developed from sensitive MCF-7 cells (MCF-7/S) were used. cDNA microarray analysis was performed for the RNA samples of sensitive and resistant cells in duplicate experiments. GeneSpring GX 7.3.1 Software was used in data analysis. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of the paclitaxel and vincristine drug resistance. Additionally the upregulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant downregulation of apoptotic genes (i.e. PDCD2/4/6/8) and upregulation of some cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Drug resistant cancer cells exhibit different gene expression patterns depending on drug treatment, and each drug resistance phenotype is probably genetically different. Further functional studies are needed to demonstrate the complete set of genes contributing to the drug resistance phenotype in breast cancer cells.

Effect of Doxorubicin on Telomerase Activity and Apoptotic Gene Expression in Doxorubicin-Resistant and -Sensitive MCF-7 Cells

Background: Dose- and time-dependent effects of doxorubicin on telomerase activity (TA) and expression levels of hTERT, Bcl-2, Bcl-xL and Bax were investigated in doxorubicin-resistant and -sensitive MCF-7 cells. Methods: Doxorubicin-resistant MCF-7/R was developed from sensitive MCF-7 breast carcinoma cell line and acquired resistance was demonstrated by XTT and mRNA analysis of MDR1 and MRP1 genes. Expression levels were determined by RT-PCR. Newly developed rapid and simple TRAP-silver staining assay was used to assess TA levels. Results: Doxorubicin-selected MCF-7 cells were 107-fold resistant to the drug and overexpress MDR1 and MRP1 genes. 72 h doxorubicin incubation caused a decrease in TA in parallel with a small decrease in hTERT level in both sensitive and resistant cells. Bcl-2 expression level decreased upon doxorubicin application in sensitive cells. However, the Bcl-xLlevel increased in sensitive cells after 72 h of doxorubicin incubation. Conclusion: This report demonstrates the inhibitory effects of doxorubicin on TA in both resistant and sensitive MCF-7 cells possibly through modulation of the apoptotic pathway genes.

beta-Adrenoreceptor antagonists reduce cancer cell proliferation, invasion, and migration

Abstract Context: Propranolol, atenolol, and ICI118,551 are non-selective β-adrenergic receptor (AR), β1-AR, and β2-AR antagonists, respectively. Objective: We investigated the efficacy of propranolol, atenolol, and ICI118,551 on proliferation, migration, and invasion of non-stimulated breast (MCF7), colon (HT-29), and hepatocellular (HepG2) cancer cells. Materials and methods: β-AR expression profiling of cells was performed by real time PCR. Cell proliferation was determined by MTT. Boyden chamber and scratch assays were performed to evaluate invasion and migration. Results and discussion: All cell lines expressed β-ARs. ICI118,551 was the most cytotoxic, whereas atenolol was the least effective β-AR antagonist for 24, 48, and 72 h. Cell invasion was inhibited by ICI118,551 (45, 46, and 50% for MCF7, HT29, and HepG2, respectively) and propranolol (72, 65, and 90% for MCF7, HT29, and HepG2, respectively). Propranolol, atenolol, and ICI118,551 reduced migration of MCF7, HT-29, and HepG2 cells to varying extents depending on the application concentration and duration. Propranolol and atenolol reduced migration of MCF7 and HT-29 in a concentration-dependent manner, whereas migration of these cells decreased after 48 and 72 h of ICI118,551 applications. Conclusion: Beta2-AR antagonist seemed to be the most cytotoxic β-blocker on non-stimulated cancer cells. Propranolol and ICI118,551 were more effective than atenolol in inhibiting invasion and migration of non-stimulated MCF7 and HT-29 cells; ICI118,551 being the most potent. Concordantly, β2-selective blockage seemed to be more effective for non-stimulated cells. Effect of the selective β-AR antagonists showed variation depending on the concentration, incubation time, and histological origin of cells.

Development of radioresistance in drug resistant human MCF-7 breast cancer cells

Abstract Background and purpose: Radiotherapy is used for the treatment of malignant tumours, and may be usedas the primary therapy. It is also common to combine radiotherapy with surgery, chemotherapy, hormonetherapy or some combination of them. Even if the tumour is treated intensively, women diagnosed withbreast cancer may develop a recurrence. Most recurrences may be in the form of distant metastases,development of multi-drug resistance phenotype or both together. This study demonstrated that some ofthe multi-drug resistant cancer cells may also become radioresistant.Materials and Methods: Chemoresistance in paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), vincristine(MCF-7/Vinc), doxorubicin (MCF-7/Dox) and zoledronic acid (MCF-7/Zol) resistant MCF-7 cells weredemonstrated by XTT assay. MDR1 gene expression was detected by real-time PCR in human MCF-7 breastcancer cells. Drug resistant and sensitive cells were exposed to g-radiation and development of radio-resistance was investigated.Results: Results have indicated that paclitaxel, docetaxel, vincristine, doxorubicin and zoledronic acidselected cells gained varying degrees of resistance to their selective drugs when compared with originalMCF-7/S. MCF-7/Pac, MCF-7/Doc, MCF-7/Vinc and MCF-7/Dox cells have all acquired MDR1 expression.Among the resistant sub-lines, MCF-7/Pac and MCF-7/Doc cells were significantly cross-resistant toirradiation compared to the sensitive cells.Conclusion: MCF-7/Pac and MCF-7/Doc cell lines were found radioresistant to g-radiation. On the contrary,doxorubicin, vincristine and zoledronic acid resistant cancer cells were still sensitive to radiation.

High-antibacterial activity of Urtica spp. seed extracts on food and plant pathogenic bacteria.

The aim of this study was to comparatively evaluate antibacterial activities of methanol (MetOH) and aqueous (dw) leaf (L), root (R) and seed (S) extracts of Urtica dioica L. (Ud; stinging nettle) and Urtica pilulifera L. (Up; Roman nettle) on both food- and plant-borne pathogens, with total phenolic contents and DPPH radical scavenging activities (DRSA). MetOH extracts of leaves and roots of U. dioica had the highest DRSA. Extracts with high antibacterial activity were in the order Up-LMetOH (13/16) > Ud-SMetOH (11/16) > Up-SMetOH (9/16). Results obtained with Up-SMetOH against food spoiling Bacillus pumilus, Shigella spp. and Enterococcus gallinarum with minimum inhibitory concentrations (MICs) in 128–1024 μg/ml range seem to be promising. Up-SMetOH also exerted strong inhibition against Clavibacter michiganensis with a considerably low MIC (32 μg/ml). Ud-SMetOH and Up-LMetOH were also effective against C. michiganensis (MIC = 256 and 1024 μg/ml, respectively). Ud-SMetOH and Ud-RMetOH had also antimicrobial activity against Xanthomonas vesicatoria (MIC = 512 and 1024 μg/ml, respectively). Results presented here demonstrate high-antibacterial activity of U. pilulifera extracts and U. dioica seed extract against phytopathogens for the first time, and provide the most comprehensive data on the antibacterial activity screening of U. pilulifera against food-borne pathogens. Considering limitations in plant disease control, antibacterial activities of these extracts would be of agricultural importance.

Genotoxic and cytotoxic effects of doxorubicin and silymarin on human hepatocellular carcinoma cells

The aim of this study was to investigate genotoxic and cytotoxic effects of doxorubicin, silymarin, or in combination on HepG2 cells for 24 and 48 h. Both doxorubicin and silymarin caused dose-dependent inhibition of cell proliferation. After 48 h of treatment, doxorubicin application caused dramatically increased ratio of apoptotic cells. Both 24 and 48 h of silymarin and doxorubicin–silymarin combination caused significant increases in the rate of apoptotic cells. Applications of doxorubicin and silymarin separately for 24 h led to deoxyribonucleic acid (DNA) damages. After 48 h of incubation, doxorubicin-induced genotoxic damage was 2-fold higher than the silymarin-induced damage. After 24 and 48 h, DNA damage in response to combined applications of doxorubicin and silymarin was indifferent from silymarin- and doxorubicin-induced damage respectively. There was not any difference in genotoxicity levels between incubation periods in combined applications of doxorubicin and silymarin. Lipid peroxidation levels increased in all applications. Biopharmacotherapy with chemotherapeutic agents are of interest in the issue of adjuvant therapy. Here, we demonstrate in vitro potential genotoxic and cytotoxic antitumor effect of silymarin on HepG2 cells at achievable plasma level concentrations.

Screening of Nasturtium officinale Extracts for Biological Activities: Implications for Plant Pathogens

Abstract Nasturtium officinale (Brassicaceae) is a succulent, rhizomatous perennial aquatic weed, and naturally found in Turkey. The aim of the present study was to screen biological activity of leaf (L), root (R), and seed extracts (S). Extracts were prepared using methanol (MetOH) and water (dw). Total phenol contents were; Ldw>LMetOH>SMetOH>RMetOH>Sdw>Rdw. DPPH· scavenging activities were; Ldw>LMetOH>SMetOH>Rdw>RMetOH>Sdw. Extracts were tested on 11 food-borne and 5 plant-borne strains for antibacterial activity which were; LMetOH(15/16)>RMetOH(3/16)>L-Rdw (2/16)>SMetOH-dw (1/16). All strains tested were susceptible to LMetOH (7.6<IZ<12.3 mm) except for the Pseudomonas aeruginosa. Highest activity was against Klebsiella pneumoniae and Shigella spp. (MIC: 1024 µg.mL-1). Listeria monocytogenes was also susceptible to RMetOH and Rdw (MIC: 2048 µg.mL-1). LMetOH, RMetOH, and Rdw had high antibacterial activity against Xanthomonas vesicatoria, with MIC of 256 and 512 µg.mL-1, respectively. LMetOH was also effective against Pseudomonas tomato and Erwinia caratovora (MIC: 1024 µg.mL-1). Considering eco-safety and effectiveness, antibacterial activities of plant extracts would be important in phytopathogen control.