CoresRI, Core Research Facilities in Rhode Island. Facility Instrument/Services Manufacturer/Description Functions; 3D Facility for Biomedical Sciences: Animation. Imaris is Bitplane’s core scientific software module that delivers all the necessary functionality for data visualization, analysis, segmentation and interpretation. Drug Efflux Transporters Are Overexpressed in Short-Term Tamoxifen-Induced MCF7 Breast Cancer Cells. Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single. Grants to subsidize Core facility use, funded through the University of Chicago Institute for Translational Medicine; The Chicago Biomedical Consortium's Core-Based. Olympus FV3000 Description The FLUOVIEW FV3000 Series is designed to meet some of the most difficult challenges in modern science. With the high sensitivity and speed. Drug Efflux Transporters Are Overexpressed in Short- Term Tamoxifen- Induced MCF7 Breast Cancer Cells. Department of Medical Pharmacy, Faculty of Medicine, University of Indonesia, Jakarta 1. Indonesia. 2Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Indonesia, Jakarta 1. Indonesia. 3Master Program in Biomedicine, Faculty of Medicine, University of Indonesia, Jakarta 1. Indonesia. 4Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Indonesia, Jakarta 1. Indonesia. Copyright . This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tamoxifen is the first line drug used in the treatment of estrogen receptor- positive (ER+) breast cancer. Research System Microscoope BX51/BX61 BX2 Series Everything for digital imaging Photos courtesy of: Junko Kyozuka, Associate Professor, Graduate School of.The development of multidrug resistance (MDR) to tamoxifen remains a major challenge in the treatment of cancer. One of the mechanisms related to MDR is decrease of drug influx via overexpression of drug efflux transporters such as P- glycoprotein (P- gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein). We aimed to investigate whether the sensitivity of tamoxifen to the cells is maintained through the short period and whether the expressions of several drug efflux transporters have been upregulated. We exposed MCF7 breast cancer cells with tamoxifen 1. The result showed that MCF7 began to lose their sensitivity to tamoxifen from the second passage. MCF7 (T) also showed a significant increase in all transporters examined compared with MCF7 parent cells. The result also showed a significant increase of CC5. MCF7 (T) compared to that in MCF7 (9. In conclusion, we suggest that the expression of several drug efflux transporters such as P- glycoprotein, MRP2, and BCRP might be used and further studied as a marker in the development of tamoxifen resistance. Introduction. Tamoxifen has been used as first line treatment for estrogen receptor alpha- (ER. However, resistance to tamoxifen occurs in many patients, although ER. Several mechanisms have been proposed to explain MDR, one of which is decreased intracellular drug accumulation which resulted from a decrease of drug influx via overexpression of drug efflux transporters such as P- glycoprotein (P- gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein) . Studies have shown that the overexpression of multidrug resistance (MDR) played an important role in the development of cancer resistance to tamoxifen . Although these cell- based studies of MDR have been an important source of understanding about the mechanism of resistance, no data showed whether overexpression of drug efflux transporters, which leads to resistance to tamoxifen, occurs in a much shorter regimen. In this study, we used a short- term tamoxifen treatment to MCF7 cells and determined whether the sensitivity of drugs to the cells is maintained through the short period and whether the expression of several drug efflux transporters has been upregulated. Materials and Methods. Materials. MCF7 cell line for breast cancer was a kind gift from the Laboratory of the Agency for the Assessment and Application Technology (BPPT), Serpong, Indonesia. Tamoxifen and DMSO were purchased from Sigma- Aldrich (Singapore). Dulbecco Minimal Essential Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin/Streptomycin, Gentamicin, Fungizone, Dulbecco Phosphate Buffer Solution (D- PBS), and Triple Express were obtained from Gibco, Ltd. Tripure Isolation Reagents were from Roche Diagnostics (Singapore), primers were from 1st BASE Ltd., Singapore, and q. RT- PCR kit used was KAPA SYBR FAST One- Step q. RT- PCR Kit Universal from KAPA Biosystem, USA. MTS assay kit was obtained from Promega, USA. Cell Culture. MCF7 cells were cultured in DMEM supplemented with 1. Medium was routinely changed every day. The cells were subcultured when reaching 8. Tamoxifen- Induced MCF7 Cells. MCF7 cells were grown in a medium containing tamoxifen 1. When reaching confluence, cells were subcultured and counted using trypan blue exclusion method. For the purpose of cell viability counting, we normalized the data to DMSO, as control, and show the data as % viability over control. Cells from passage 1 (day 5) and passage 1. RNA isolation and q. RT- PCR for drug efflux transporters (P- glycoprotein, MRP2, and BCRP). Cell Morphology. MCF7 and MCF7 (T) cells morphology were photographed under confocal microscope (Olympus Fluoview FV1. Confocal Laser Scanning Microscope, Olympus, Japan). Photo observation is done using gray scale, Pseudo 3. D DIC by Transmitted Nomarski System. RNA Isolation. Total RNA was isolated using Tripure Isolation Reagents (Roche) according to the manufacturer. Quantity and purity of RNA were determined by measuring 2. Nano. Drop spectrophotometer. RNA obtained then was subjected to quantitative real- time reverse transcription polymerase chain reactions (q. RT- PCR). 2. 6. Quantitative real- time reverse transcription polymerase chain reaction (q. RT- PCR) was performed using KAPA SYBR FAST One- Step q. RT- PCR Kit on Universal Biorad Chromo 4 Real- Time PCR Detection System. The sequences of the primers were . Primers used for the determination of drug efflux transporters were described previously . Primers used to determine the m. RNA expressions of Caspase- 3 and Caspase- 9 were described previously by Iwao et al. Primers used to determine the m. RNA expression of progesterone receptor were used previously by Shanker et al. Relative changes in m. RNA transporter expression levels were calculated using Livak method . MTS Assay (Cell Proliferation Assay)Cytotoxicity concentration of tamoxifen to MCF7 cells before and after 4. MTS assay (Promega). Cells were plated at a density of 2. CC5. 0 values were calculated from linear regression equation of dose- response curves. Statistical Analysis. Data were presented in the form of means . Graphs were created using Graph. Pad Prism software 6 (Graph. Pad, USA). Statistical significance was calculated using - test or ANOVA One- Way followed by post hoc test, with considered as significant. Results. Cell morphology of MCF7 cells treated with tamoxifen continuously is shown on Figure 1. Figure 1: (a) Cell morphology of MCF7 cells (parent). Photographed under confocal microscope. Our result showed that cancer cells maintained their sensitivity towards tamoxifen only in the first passage. Cells began to lose their sensitivity to drug from the second passage (or about 9 days of tamoxifen treatment). Afterwards, MCF7 treated with tamoxifen had stable overgrowth compared with MCF7 cells treated with DMSO only (Figure 2). Figure 2: Percentage of viable cells over control (DMSO) after treatment with tamoxifen 1. We found a significant increase of CC5. MCF7 (T) compared to that in MCF7 (9. We found that Caspase- 3 and Caspase- 9 expressions were significantly increased compared with parent cells, which proved that apoptosis process, which were still in place. Figure 4: Level of Caspase- 3 and Caspase- 9 m. RNA expressions after treatment with tamoxifen 1. Results were shown as mean . Results were shown as mean . The result showed that the expressions of P- gp, MRP2, and BCRP had been elevated from the first passage. MCF7 (T) showed a significant increase in all transporters examined compared with MCF7 parent cells (Figure 6). Figure 6: Level of m. RNA expressions of P- glycoprotein, MRP2, and BCRP after 5 days of treatment (MCF7- P1) or 4. MCF7- T) with tamoxifen 1. Results were shown as mean . Discussion. Tamoxifen currently is still the mainstay of endocrine therapies for ER. Unfortunately, majority of patients treated with tamoxifen eventually develop resistance, leading to disease progression and death . Tamoxifen- resistant breast cancer cells often overexpress drug efflux transporter, which lower the effective drug concentration in a cell by pumping out tamoxifen out of the cells . To our knowledge, this is the first to describe the expressions of several drug transporters after short period of tamoxifen treatment. Previous studies had described the development of resistance of breast cancer cells to tamoxifen by exposing the drug for years . In this study, we use 1. Fewer studies had used lower dose of tamoxifen compared to this study . Our own preliminary result (data not shown) using tamoxifen 0. Therefore, we thought tamoxifen in lower dosages had little effect on cell viability and thus would result differently in selection of cells to induce resistance. After 1. 0 passages of tamoxifen treatment, photographs using confocal microscope indicate that there might be slight changes in cell morphology. We found more mesenchymal- like cells in MCF7 (T) compared to MCF7 parents cells which showed more cobblestone- like cells. Other studies had shown that epithelial- mesenchymal transition process played significant roles in the development of tamoxifen resistance . In this study, we did not confirm the markers of EMT, as we mainly aimed to determine drug efflux expressions in tamoxifen- resistant cells. Our result showed that downstream regulation of ER had occurred, as confirmed with downregulation of progesterone receptor. This is in accordance with previous results that tamoxifen resistance is accompanied with the reduced expressions of both ER and PR. In his study, Johnston et al. Tamoxifen had failed to suppress cancer cell growth as early as second passage. We found that apoptosis process was still ongoing in passage 4 as shown by increased expressions of Caspase- 3 and Caspase- 9. Previous studies using MCF7 and MDA- MB- 2. ER, is able to induce apoptosis process trough cleavage of retinoblastoma (Rb) protein and activation of Caspase- 3 . Our result suggests that tamoxifen dramatically increased m. RNA expressions of P- glycoprotein, MRP2, and BCRP. The expressions of the three drug transporters m. RNA had even began to increase from first passage.
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