Meltem Demirel Kars

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.

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.

Interaction of tomato lectin with ABC transporter in cancer cells: Glycosylation confers functional conformation of P-gp

Phospho-glycoprotein (P-gp) is a polytopic plasma membrane protein whose overexpression causes multidrug resistance (MDR) responsible for the failure of cancer chemotherapy. P-gp 170 is a member of the ATP-binding cassette (ABC) transporter superfamily and has two potentially interesting regions for drugs interfering with its efflux function, namely the oligosaccharides on the first extracellular loop with unknown function and the two intracellular ATP-binding regions providing the energy for drug efflux function. The polylactoseamine oligosaccharides on the first loop can specifically bind the tomato lectin (TL). The P-gp efflux activities of TL-pre-treated MDR resistant cells were measured in the presence of structurally unrelated resistance modifiers such as phenothiazines, terpenoids and carotenoids. The inhibition of efflux activity was measured via the increased rhodamine uptake by mouse lymphoma cells transfected in human MDR1 gene and in human brain capillary endothelial cells. The tested resistance modifiers inhibit the function of ABC transporter resulting in increased R123 accumulation in MDR1 expressing cells. TL prevented the inhibitory action of phenothiazine and verapamil on brain capillary endothelial and MDR1-lymphoma cells, presumably due to the stabilization of the functional active conformation of P-gp. Our results indicate that the polylactosamine chains of P-gp are part of the functionally active protein conformation.

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.

Femtosecond laser induced photodynamic therapy on 5-ALA treated SKMEL-30 cells: An efficient theranostic strategy to combat melanoma

PURPOSE Photodynamic therapy (PDT) is a type of photo-chemotherapy that is based on the application of photosensitizer and irradiation of the region by laser sources. Photosensitizer and light interaction will develop reactive oxygen radicals ((1)O2) in the cells and elimination of cells by apoptosis or necrosis. METHODS Metastatic skin cancer cells SKMEL-30 were treated by 5-ALA in dark and then they were irradiated by 90-femtosecond (fs) laser with different pulse powers for different durations. The effects of 5-ALA mediated photodynamic therapy on the cells were determined by XTT proliferation kit and by flow cytometry measurements of Annexin V, 7-AAD and mitochondrial membrane potential alterations. Fluorescent accumulation of protoporphyrin IX was investigated by fluorometry and confocal laser microscope. RESULTS The viability tests for SKMEL-30 cells treated with different 5-ALA doses and femtosecond laser power and durations demonstrated that 635 nm, 45 mW pulse energy at 90 fs laser pulse applications for 60 sec to 1mM 5-ALA exposed cells decreased the cell proliferation by 30%. Flow cytometric measurements exhibit that PDT caused 63% of mitochondria membrane potential alteration, 30% of cell death in the population by apoptosis and 39% of cells by necrosis. There was 1mM 5-ALA exposure that also exhibited about 32% accumulation of fluorescence in the cells. CONCLUSION The pretreatment of the cells with the precursor 5-ALA lets the imaging due to increased protoporphyrin IX fluorescence. This treatment method may be proposed as an effective theranostic strategy for melanoma because of its rapid and effective anticancer consequences.

Anticancer Effects of the Organosilicon Multidrug Resistance Modulator SILA 421

1,3-dimethyl-1,3-bis(4-fluorophenyl)-1,3-bis{3-[1(4-butylpiperazinyl)]-propyl}-disiloxan-tetrahydrochlorid (SILA 421) is a compound that was developed as modulator of the ABC cassette transporter P-glycoprotein. Furthermore, it exerted antimicrobial toxicity, vascular effects, downregulation of chaperone induction and plasmid curing in bacterial cells. Here, this drug was found to possess cytotoxic activity against a panel of human cancer cell lines that do not overexpress P-gp, with 50% inhibitory concentrations ranging between 1.75±0.38 μM for GLC14 small cell lung cancer and 34.00±4.75 μM for PC-3 prostate cancer cells. HL-60 leukemia and MDA-MB-435 breast cancer cells exhibited cell cycle arrest and apoptotic cell death in response to SILA 421. Assessment of global gene expression of SILA 421-treated HL-60 cells was employed to identify cellular pathways affected by the compound and revealed disturbance of DNA replication, transcription and production of apparently misfolded proteins. Endoplasmatic reticulum stress and downregulation of cell cycle, cellular repair mechanisms and growth factor-related signaling cascades eventually resulted in induction of apoptosis in this cell line. In addition to the well established P-gp inhibitory effect of SILA compounds, reversal of resistance to taxanes, which had been reported for SILA 421 and the related molecule SILA 409, may be linked to downregulation of gene expression of kinesins. Interference with DNA replication and transcription seems to be the common denominator of antimicrobial activity and plasmid curing, as well as anticancer toxicity in human cell lines. Thus, in consideration of the full range of putative cellular targets found in the present work, the application of these SILA compounds for treatment of tumors should be further evaluated.

Exploring a natural MDR reversal agent: potential of medicinal food supplement Nerium oleander leaf distillate

OBJECTIVE To investigate the molecular effects of Nerium oleander leaf distillate on paclitaxel and vincristine resistant (MCF-7/Pac and MCF-7/Vinc) cells and sensitive (MCF-7/S) cell lines. METHODS Nerium oleander (N. oleander) leaf extract was obtained by hydrodistillation method. The toxicological effects of N. oleander distillate, previously suggested as medicinal food supplement, on drug resistant cells were evaluated by XTT tests. MDR modulation potential of the plant material was evaluated by flow cytometry and fluorescent microscopy. Paclitaxel and vincristine were applied to the sublines in combination with N. oleander distillate. RESULTS Fractional inhibitory indices show that N. oleander distillate did not increase the antiproliferative effects of anticancer drugs. N. oleander treatment in to MCF-7/Pac and MCF-7/Vinc did not inhibit P-gp activity and MDR1 gene expression level. CONCLUSIONS As a result it may be suggested that although N. oleander distillate has some medicinal effects as food supplement it may not be suitable as an MDR modulator for drug resistant breast cancer cells.

Production and characterization of titanium (Ti), platinum (Pt) and tantalum (Ta) thin films for native DNA biosensors

The use of the femtosecond (fs) laser pulses for ablation applications have several advantageous and Laser-Induced Forward Transfer (LIFT) is an ablation-driven transfer process. The use of fs laser pulses for LIFT is gaining a great attraction nowadays. The most of the Direct Writing (DW) methods are laser based techniques and the LIFT technique is the one of them. This spectacular technique allows high resolution without lithographic processes. In this study, we have grown Ti, Pt and Ta thin films on the microscope slides by Pulse Laser Deposition (PLD) technique using Nd:YAG laser in the high vacuum condition. As a result, thin films produced in this work is a good candidate to produce native DNA biosensors based on LIFT technique.

Implications from a pharmacogenomic analysis: Nerium oleander leaf distillate supplemented diet regulates cholesterol metabolism in rats

Abstract Context: Despite the usage of Nerium oleander L. (Apocynaceae) for anticancer studies and traditional remediation, the regulatory effect of N. oleander leaf distillate on cholesterol metabolism is not disclosed sufficiently. Objective: Cholesterol is an important biological molecule and the synthesis rate is regulated by the amount of cholesterol uptake from the diet. The aim of this study was to investigate the regulation of cholesterol metabolism in response to a high-fat diet (HFD) and the effects of N. oleander leaf distillate-supplemented diet (NOHFD) in rats. Materials and methods: Microarray technology was used to clarify the regulation of cholesterol mechanism in HFD and NOHFD-fed rats (375 μg/0.5 mL distilled water applied by gavage). The treatment period was 90 days. Rat liver tissues were used for microarray analysis using the Affymetrix GeneChip Rat Genome platform. Results of groups were statistically analyzed with the Partek 6.6 bioinformatic program. Results: The HFD group exhibited alterations in the expression levels of about 1945 genes with respect to the normal diet (ND) group. The results showed that expression levels of 47 genes were altered related to cholesterol metabolism in HFD and NOHFD groups. The expression levels of seven genes in the NOHFD group were significantly closer to those in the ND group than those of the HFD group. Discussion and conclusion: To conclude, findings suggest that N. oleander leaf distillate-supplemented food has considerable beneficial effects on cholesterol metabolism-related gene expression levels.