Response to trastuzumab and investigation of expression profiles of matrix metalloproteinase-related proteins in primary breast cancer stem cells.

Breast cancer (BC) is the leading cause of cancer deaths in women. One of the reasons for the failure of BC treatment is reportedly the ineffectiveness of chemotherapeutic drugs against breast cancer stem-like cells (BCSCs). HER2 receptors have an important role in the self-renewal of BCSCs. Matrix metalloproteinase (MMP) and cytokine levels were found to be higher in BCSCs, which demonstrates their potential metastatic capacity. Therefore, the aim of this study was to evaluate the response of BCSCs to trastuzumab and to investigate the MMP levels in primary breast cancer cells and HER2+ BCSCs. Tumour tissue samples were obtained during surgical intervention from ten breast cancer patients, and primary culture cells were established from these tissues. Four major molecular subgroups were sorted from the primary culture: HER2+ BCSCs (CD44+CD24-HER2+), HER2- BCSCs (CD44+CD24-HER2-), HER2- primary culture cells (CD44+CD24+HER2-) and triple positive primary culture cells (CD44+CD24+HER2+). These cells were cultured and treated with trastuzumab, paclitaxel, carboplatin, and the combination of those three drugs for 96 h. Cellular responses to these drugs were determined by XTT cytotoxicity test. MMPs and cytokine array analysis showed that MMPs and TIMP-1, TIMP-2 proteins were expressed more in HER2+ BCSCs than in primary culture. HER2- BCSCs were more resistant to drugs than HER2+ BCSCs. Our findings suggest that the presence of HER2- BCSCs may be responsible for primary trastuzumab resistance in HER2+ BC cell population. Further studies investigating the function of MMPs are needed for drug targeting of BCSCs.

Fomes fomentarius and Tricholoma anatolicum (Agaricomycetes) Extracts Exhibit Significant Multiple Drug-Resistant Modulation Activity in Drug-Resistant Breast Cancer Cells.

Multiple drug resistance is one of the main problems that hinder successful cancer chemotherapy. Investigations on the development of effective chemotherapeutic agents and drug resistance inhibitors motivate studies on the effects of natural compounds on drug-resistant cancer cells. For this purpose, aqueous, methanol, and ethanol extracts of Fomes fomentarius and Tricholoma anatolicum were prepared. The extracts were evaluated to assess their anticancer and multiple drug resistance modulation activities. Cytotoxic effects of F. fomentarius and T. anatolicum extracts on paclitaxel and vincristine resistant P-glycoprotein over-expressing MCF-7 cell lines were investigated by cytotoxicity test (XTT). P-glycoprotein reversing ability and MDR modulation effects of the extracts were determined by flow cytometry through Rhodamine 123 exclusion assay. Furthermore, 11 phenolic compounds in the extracts were characterized by HPLC. As a result of the cytotoxicity assay, IC50 values of the extracts for MCF-7/Vinc were between 1.08 and 1.80 mg/mL, and IC50 values for MCF-7/Pac were found between 1.11 and 2.83 mg/mL. Strikingly, methanol extract of F. fomentarius and ethanol extract of T. anatolicum have potential value to become MDR reversing agents for drug-resistant breast cancer cells.

Cytotoxic and Genotoxic Assessment of Silicon Dioxide Nanoparticles by Allium and Comet Tests.

Silicon nanoparticles gained a great interest due to its use in biomedical research. It is considered as safe and has been used in nanomedicine. But literature still states its toxicity depending upon the size and dose of silicon nanoparticles. So, current study was aimed to evaluate the cytotoxicity and genotoxicity of silicon dioxide nanoparticles (SiO2NPs) by Allium anaphase-telophase and Comet tests. Characterization of SiO2NPs showed the particle size as 16.12 ± 3.07 nm. The mean diameter of SiO2NPs was having range of 404.66 ± 93.39 nm in solution. Highest total anomalies (18.80 ± 0.45) were observed at 100 µg/mL, whereas least (11.2 ± 0.84) were observed by the 12.5 µg/mL concentration. There was concentration-response association in increased CAs and DNA damage. The highest concentration (100 µg/mL) of SiO2NPs induced the significant DNA damage (149.67 ± 1.15), whereas the least was observed by the negative control (2.67 ± 0.58). The current study revealed the cytotoxic and genotoxic effects of SiO2NPs on the root meristem cells of A. cepa.

Determination of the target proteins in chemotherapy resistant breast cancer stem cell-like cells by protein array.

Breast cancer comes second among the causes of cancer deaths of women. Although new generation hormone therapy is a promising strategy, re-occurrence or emergence of drug resistance limits the success. According to the theory of cancer stem cells (CSCs); CSCs are immortal, tumor inducing and self renewing pluripotent cells and multiply as chemotherapy proceeds, making the chemotherapy inefficient. Emerging scientific reports indicate that the mechanisms of drug resistance are the main features that CSCs gain actually. Due to this fact, cancer stem cell markers should be clarified to target CSCs and this will play important role to reverse drug resistance. In this study, MCF-7/Pac, a cell line resistant to microtubule inhibitor paclitaxel and multiple drugs permanently, was used as a reference cell line for drug resistant mammary cancer. It has some properties that breast cancer stem cells possess so it is considerable to isolate breast cancer stem cell-like cells from MCF-7/Pac population. The chemotherapy resistant breast cancer stem-like (BCSC-like) cells were sorted from MCF-7/Pac population by using markers CD44, CD24 and ALDH. At the next step the proteins that are up-regulated in BCSC-like cells were determined by protein array analysis. Additionally the effect of paclitaxel on BCSC-like cell proliferation was determined. The MCF-7/Pac population contains 12.4% BCSC-like cells. The cells bearing BCSC-like cell phenotype exhibited resistance to paclitaxel. The over-expressed growth factors, MMP proteins, Frizzled proteins and IL-23 were found to be related to the BCSC-like cell proliferation. These results will guide both basic science and medical science.

Drug resistance restricts the efficacy of short term low dose Mitomycin-C treatment in UMUC-3 bladder cancer cells.

OBJECTIVE: Mitomycin-c (MMC) is the most used intravesical adjuvant agent in non-muscle invasive bladder cancer to prevent recurrence. However, a consensus on about appropriate dosage and treatment schedule of MMC is lacking. We, therefore, aimed to evaluate the most appropriate MMC dosage using an in vitro model of high-grade human bladder cancer. METHODS: UMUC-3 cells, a model for high-grade bladder cancer, were exposed to MMC in different time courses to assess its toxicological effects. XTT cell proliferation kit was used to evaluate the effect of MMC on the proliferation of UMUC-3 cell line. Gene expression analysis for the MDR1, BCL2 and ANXA5 genes was performed by Real-time PCR and flow cytometry analysis were conducted to evaluate the cell death mechanism and acquired resistance after MMC exposure. An ANXA5 kit was used to detect apoptotic cells, and 7-AAD was used to detect necrotic cells. RESULTS: Cell proliferation was prevented to a large extent (IC50, 0.17-0.081 mg/mL) and cytotoxic effects were observed after 5 µg/mL and 10 µg/mL MMC administrations for 1 and 2-h, after the 4th and 2nd dose cycles, respectively. Moreover, cell death was observed at 5 µg/mL and 10 µg/mL MMC applications for 1-h and 2-h by the sixth and second week, respectively. Flow cytometry exhibits increased subpopulation of drugextruding UMUC-3 cells after a single dose of MMC for 1-h. MMC did not increase the number of apoptotic or necrotic cells; yet, MDR1 (multiple drug resistance) and ANXA5 (apoptotic) expression levels were increased and BCL2 (anti-apoptotic) expression was decreased. LIMITATIONS: In-vitro nature of the study and working with only one cell culture are inherit limitations of this project. CONCLUSION: A single dose of MMC administration for 1 or 2-h results in drug-resistance. If maintenance treatment is administered for one hour, it should be continued throughout a 6-week period.

Drug resistance restricts the efficacy of short term low dose Mitomycin-C treatment in UMUC-3 bladder cancer cells

OBJECTIVE: Mitomycin-c (MMC) is the most used intravesical adjuvant agent in non-muscle invasive bladder cancer to prevent recurrence. However, a consensus on about appropriate dosage and treatment schedule of MMC is lacking. We, therefore, aimed to evaluate the most appropriate MMC dosage using an in vitro model of high-grade human bladder cancer. METHODS: UMUC-3 cells, a model for high-grade bladder cancer, were exposed to MMC in different time courses to assess its toxicological effects. XTT cell proliferation kit was used to evaluate the effect of MMC on the proliferation of UMUC-3 cell line. Gene expression analysis for the MDR1, BCL2 and ANXA5 genes was performed by Real-time PCR and flow cytometry analysis were conducted to evaluate the cell death mechanism and acquired resistance after MMC exposure. An ANXA5 kit was used to detect apoptotic cells, and 7-AAD was used to detect necrotic cells. RESULTS: Cell proliferation was prevented to a large extent (IC50, 0.175-0.081 mg/mL) and cytotoxic effects were observed after 5 μg/mL and 10 μg/mL MMC administrations for 1 and 2-h, after the 4th and 2nd dose cycles, respectively. Moreover, cell death was observed at 5 μg/mL and 10 μg/mL MMC applications for 1-h and 2-h by the sixth and second week, respectively. Flow cytometry exhibits increased subpopulation of drugextruding UMUC-3 cells after a single dose of MMC for 1-h. MMC did not increase the number of apoptotic or necrotic cells; yet, MDR1 (multiple drug resistance) and ANXA5 (apoptotic) expression levels were increased and BCL2 (anti-apoptotic) expression was decreased. Limitations: In-vitro nature of the study and working with only one cell culture are inherit limitations of this project. CONCLUSION: A single dose of MMC administration for 1 or 2-h results in drug-resistance. If maintenance treatment is administered for one hour, it should be continued throughout a 6-week period

OBJETIVO: La Mitomicina C (MMC) es el agente intravesical adyuvante más utilizado para prevenir recurrencias en cáncer vesical no musculo invasivo.Sin embargo, no hay un consenso sobre la dosis y el régimen terapeutico apropiado. Buscamos evaluar la dosis más apropiada de MMC utilizando un modelo in vitro de cáncer vesical humano de alto grado. MÉTODOS: Se realizó una exposición de las células UMUC 3, un modelo de cáncer vesical de alto grado, a MMC en diferentes regímenes temporales para evaluar sus efectos toxicológicos. Se utilizó el kit de proliferación celular XTT para evaluar el efecto de la MMC sobre la proliferación de la línea celular UMUC-3. Se analizó la expresión génica de los genes MDR1, BCL2 y ANXA5 mediante PCR a tiempo real y análisis de citometría de flujo para evaluar el mecanismo de muerte celular y resistencia adquirida después de la exposición a MMC. Se utilizó un Kit ANXA5 para detectar células apoptóticas, y AAD-7 para detectar células necróticas. RESULTADOS: La proliferación celular fue impedida en gran medida (IC50, 0,175-0,081 mg/mL) y se observaron efectos citotóxicos con la administración de 5 μg/mL y 10 mig/mL de MMC durante 1 y 2 horas después del 4º y 2º ciclo de dosis, respectivamente. Además, se observó muerte celular con la aplicación de MMC en dosis de 5 mig/mL y 10 μg/mL durante 1 y 2 horas en la sexta y segunda semana, respectivamente. La citometría de flujo mostró aumento de la subpoblación de células UMUC-3 que extruían el fármaco después de una dosis única durante 1 hora. La MMC no aumentó el número de células apoptóticas o necróticas; pero, los niveles de expresión de MDR1 (Múltiple drug resistance- resistencia múltiple a fármacos) y ANXA5 (apoptóticas) aumentaron y los de BCL2 (anti-apoptóticas) disminuyeron. Limitaciones: La naturaleza in-vitro del estudio y trabajar sólo con un cultivo celular son limitaciones esenciales de este proyecto. CONCLUSIONES: La administración de una dosis única de MMC durante 1-2 horas da como resultado resistencia a fármacos. El tratamiento debe continuarse durante un periodo de 6 semanas

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.

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.

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.

Two different docetaxel resistant MCF-7 sublines exhibited different gene expression pattern.

The objective of the present study was to investigate gene expression pattern of two docetaxel resistant MCF-7 breast carcinoma sublines step wisely selected in 30 and 120 nM docetaxel. Cell proliferation assay was performed in order to demonstrate development of docetaxel resistance. cDNA microarray analysis was performed using Affymetrix(®) Human Genome U133 Plus 2.0 Arrays in duplicate experiments. Quantitative and semi-quantitative gene expression analysis was also performed to confirm gene expression analysis for selected genes. XTT results demonstrated that 30 (MCF-7/30nM DOC) and 120 nM (MCF-7/120nM DOC) docetaxel selected cells were 13- and 47-fold resistant, respectively. cDNA microarray analysis demonstrated that expression profiles of MCF-7 and MCF-7/30nM DOC were more similar to each other where expression profile of MCF-7/120nM DOC was different as examined by line graphs and scatter plots. 2,837 and 4,036 genes were significantly altered in 30 and 120 nM docetaxel resistant sublines, respectively. Among these, 849 genes were altered in common in two docetaxel resistant sublines. Antiapoptotic gene expression (e.g., Bcl-2 and APRIL) were noticeably altered in MCF-7/30nM DOC. However, docetaxel resistance in MCF-7/120nM DOC were more complicated with the involvement of ECM related gene expression, cytokine and growth factor signaling, ROS metabolism and EMT related gene expression together with higher level of MDR1 expression. Expression profiles in 30 and 120 nM docetaxel resistant sublines changed gradually with increasing resistance index. Drug resistance development seems to be step wise event in MCF-7 cells.

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

PURPOSE: One 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. METHODS: Doxorubicin-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. RESULTS: Results 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. CONCLUSIONS: The present study gives an idea about the efficiency of verapamil and promethazine on MDR reversal both in gene expression and in transport activity levels.

Drug resistant MCF-7 cells exhibit epithelial-mesenchymal transition gene expression pattern.

PURPOSE: Multidrug resistance is resistance to structurally unrelated anticancer agents. Large-scale expression analysis by using high-density oligonucleotide microarrays may provide information about new candidate genes contributing to MDR. This study demonstrates alterations in expression levels of several genes related to epithelial-mesenchymal transition (EMT) in paclitaxel, docetaxel, and doxorubicin resistant MCF-7 cells. METHODS: Resistant sublines were developed from sensitive cells by selective paclitaxel, docetaxel, and doxorubicin applications in dose increments. cDNA microarray analysis was performed for sensitive and resistant cells. Genes having statistically significantly altered expression levels more than two-folds compared to the sensitive MCF-7 cells were considered. Genes encoding the determinants of the EMT were evaluated. Immunostaining was performed for relevant protein expressions. RESULTS: Key elements of EMT were transcriptionally activated in paclitaxel, docetaxel and doxorubicin resistant sublines. One of the upregulated genes was Slug, a transcription factor of E-cadherin, occludin repression, and N-cadherin, vimentin activation. Decreased estrogen receptor-α (ER) levels in cells might have stimulated Slug expression. Increased expression levels of TGF-beta receptor2 (TGFBR2) together with SMAD3 might have stimulated EMT in resistant cells. Immunocytochemistry results confirmed loss of ER and E-cadherin, together with high vimentin levels. CONCLUSIONS: EMT was induced in multidrug resistant MCF-7 cells indicating a relationship of this process and drug resistance. However, the relationship of each specific component of EMT with drug resistance requires further analysis.

Drug resistant breast cancer cells overexpress ETS1 gene.

PURPOSE: Multidrug resistance (MDR) is resistance to wide range of structurally unrelated anticancer agents. MDR is a serious limitation to the effective chemotherapy. Involvement of ETS1 overexpression in upregulation of MDR1 gene expression is implicated. In the present study the aim was to assess the involvement of ETS1 and the genes, which encode the proteins interacting with ETS1 in drug resistant MCF-7 breast cancer cells. METHODS: Drug resistant sublines to paclitaxel (MCF-7/Pac), docetaxel (MCF-7Doc), vincristine (MCF-7/Vinc) and doxorubicin (MCF-7/Dox) 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. Microarray data was supported by immunocytochemistry and western blot for drug resistance protein, P-gp, encoded by MDR1 gene. RESULTS: According to microarray data MDR1 and ETS1 genes were highly overexpressed in all of the resistant sublines. Matrix metalloproteinase-1 gene (MMP-1) was also tremendously upregulated only in vincristine resistant cells. Immunocytochemistry and western blot results confirmed that P-gp was highly overexpressed in resistant sublines compared to original MCF-7 cells. CONCLUSION: High ETS1 expression levels in all resistant MCF-7 sublines may lead to the upregulation of the transcription of MDR1 gene. Overexpression of ETS1 gene in resistant cells may have contributed to the development of resistance in the cells. Furthermore, the significant upregulation of MMP1 and MMP9 in MCF-7/Vinc may also be related to an acquired invasive behavior of MCF-7 cell line due to vincristine treatment.

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

PURPOSE: Since 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. METHODS: Resistant 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. RESULTS: Genes 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. CONCLUSIONS: Based 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.

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.

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.

Effect of doxorubicin on telomerase activity and apoptotic gene expression in doxorubicin-resistant and -sensitive MCF-7 cells: an experimental study.

BACKGROUND: Dose- and time-dependent effects of doxorubicin on telomerase activity (TA) and expression levels of hTERT, Bcl-2, Bcl-x(L) 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-x(L)level 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.

In vitro evaluation of zoledronic acid resistance developed in MCF-7 cells.

BACKGROUND: Zoledronic acid is an important osteotropic compound used in combination with anticancer agents to reduce the incidence of hypercalcemia and skeletal morbidity in patients with advanced breast cancer and bone metastases. Ineffectiveness of anticancer drugs during chemotherapy is a frequently observed situation in cancer chemotherapy. The resistance of tumor cells to more than one cytotoxic drugs is defined as multidrug resistance. Drug resistance may be caused by altered gene expression levels and altered activities of proteins related to drug transport or cell death. MATERIALS AND METHODS: To investigate the potential development of zoledronic acid resistance in breast cancer, parental MCF-7 cells were selected by increasing doses of zoledronic acid. MTT cytotoxicity assays, RT-PCR and Western blot were performed. The anticancer drugs paclitaxel, docetaxel, vincristine and doxorubicin were tested in combination to assess their combined antiproliferative effects and cross-resistance profiles. RESULTS: Results demonstrated that the drug-adapted cells are resistant to zoledronic acid compared to parental MCF-7 and de novo expression of resistance genes, such as BCRP and LRP, were found. Up-regulation of Bcl-2 gene expression in resistant cells was also found. Synergistic cytotoxic effects of the combination of zoledronic acid with paclitaxel, docetaxel and vincristine were confirmed by fractional inhibitory indices, and zoledronic acid resistant cells were also found to be cross-resistant to these agents. CONCLUSION: Zoledronic acid may cause resistance in MCF-7 cells. Overexpression of BCRP and LRP genes and an increase in Bcl-2 gene expression may have roles in the development of zoledronic acid resistance in the MCF-7 cell line. On the other hand, MDR1 and MRP1 genes do not seem to contribute to the zoledronic acid resistance significantly.

Development of rational in vitro models for drug resistance in breast cancer and modulation of MDR by selected compounds.

BACKGROUND: [corrected] The effectiveness of chemotherapy is limited by the emergence of multidrug resistance (MDR). MDR is caused by the activity of various ATP binding cassette (ABC) transporters that pump anticancer drugs out of the cells in an ATP-dependent manner. Additionally some other cellular mechanisms of MDR have been reported. The purpose of this study was to investigate mechanisms of MDR in drug resistant MCF-7 cell lines and to modulate P-glycoprotein (P-gp) and MRP1-based MDR. MATERIALS AND METHODS: Paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), doxorubicin (MCF-7/Dox) and vincristine (MCF-7/Vinc) resistant sublines were developed from the parent MCF-7 cell line (MCF-7/S) by stepwise selection in dose increments over two years. Flow cytometry, MTT cytotoxicity assay, RT-PCR, caspase-3 activity assay and checkerboard combination assay were performed to investigate the degree of resistance developed in sublines and to reverse drug resistance phenotype. RESULTS: The flow cytometry histograms of drug accumulation assays demonstrated that the drug-resistant cell lines are P-gp and MRPI positive. RT-PCR results showed that the resistant sublines express both MDR1 and MRP1 genes. Resistance indices of each subline to each anticancer drug were determined using the MTT cytotoxicity assay and it was found that all the sublines were resistant to their respective agents. Caspase-3 activities of the cell lines were also determined. Caspase-3 activity is an important indicator of apoptosis in the cell. The reversal of MDR was attempted by two cinnamylidene ketone and two organosilicon compounds. The results indicated that these compounds modulated P-gp effectively, but they were not very effective at reversing MRP1 activity in the MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and four synthetic MDR modulators [2-(2-methoxycinnamylidene) indan-1-one (cinnamylidene-1), 2-(2- methoxycinnamylidene)-3, 4-dihydro-2H-naphthelen-1-one) (cinnamylidene-2), ALIS 409 and ALIS 421] were applied to the sublines in combination. The fractional inhibitory indices illustrated that combined applications of cinnamylidene ketones and organosilicon compounds with paclitaxel, docetaxel or vincristine exerted significant antiproliferative effects on the resistant sublines. CONCLUSION: This report demonstrates the development of rational models for drug resistance MCF-7 cell lines and reversal of acquired drug resistance.