Loperamide potentiates doxorubicin sensitivity in triple‐negative breast cancer cells by targeting MDR1 and JNK and suppressing mTOR and Bcl‐2: In vitro and molecular docking study
34
Citation
62
Reference
10
Related Paper
Citation Trend
Abstract:
Multidrug resistance (MDR) is the leading cause of treatment failure in triple-negative breast cancer (TNBC) patients treated with doxorubicin (DXR). We aimed to investigate the potential of the antidiarrheal drug Loperamide (LPR) in sensitizing TNBC cells to DXR and elucidate the underlying molecular mechanisms. Therefore, we examined the effects of DXR alone or in combination with LPR on MDA-MD-231 cells viability using MTT assay, cell cycle, and apoptosis by flow cytometry, and the expression of the MDR-related genes (MDR1 and JNK1) and cell cycle/survival genes (p21, mTOR, and Bcl-2) by quantitative reverse transcription polymerase chain reaction. Results showed that adding LPR to DXR potentiated its antiproliferation effect and reduced its IC50 by twofolds compared with DXR alone. The value of the combination index of LPR/DXR was <1 indicating a synergistic effect. Combined DXR/LPR treatment also caused G1 arrest and potentiated apoptosis more than DXR-single treatment. At the molecular levels, LPR/DXR treatment downregulated the mRNA of MDR1 (1.35-folds), JNK1 (2.5-folds), mTOR (6.6-folds), Bcl-2 (9.5-folds); while upregulated p21 gene (8-folds) compared with DXR alone. Molecular docking analyses found LPR antagonizes MDR1 and JNK1 proteins, and hence supports the in vitro studies. In conclusion, the results confirmed the potential of LPR in sensitizing TNBCs to DXR by targeting MDR1 and JNK1 and suppressing Bcl-2 and mTOR genes, while upregulating the cell cycle inhibitor gene p21. Additionally, LPR could be repurposed to reduce the therapeutic doses of DXR as indicated by the dose reduction index (DRI) and subsequently decrease its side effects.Keywords:
Triple-negative breast cancer
MTT assay
Viability assay
Background : Cell viability is an important factor in radiaon therapy and thus is a method to quanfy the effect of the therapy. Materials and Methods : The viability of human hepatoma (HepG2) cells exposed to radiaon was evaluated by both the MTT and Trypan blue assays. The cells were seeded on 96 well-plates at a density of 1 x 10 4 cells/well, incubated overnight, and irradiated with 1-100 Gy. Results: The cell viability was decreased in a dose- andme- dependent manner when using the Trypan blue assay, but no significant changes in the response to dose could be detected using the MTT assay. It indicated that the MTT assay was not efficient at a cell density of 1 x 10 4 cells/well on 96 well-plates to determine cell viability. Subsequently, the relaonship between cell viability and lower cell density (1 x 10 3 , 3 x 10 3 , and 5 x 10 3 cells/well) was invesgated. A cell density of 1 x 10 3 was found to be the most effecve when using the MTT assay. Results show that the cell density is most important when using the MTT assay in 96 well-plates to follow in radiaon effects. Furthermore, the radiao n-induced cell viability dependent on cell density was confirmed by using the tradional Clonogenic assay. Conclusion: Our results suggest that the MTT and Trypan blue assays are rapid methods to detect radiaon-induced cell viability of HepG2 cells in about 3 days as compared with 14 days of assayme in the Clonogenic assay. To obtain accurate cell viability measures using both rapid assays, an incubaonme of at least 3 days is needed a6er irr adiaon.
Viability assay
MTT assay
Trypan blue
Clonogenic assay
Cite
Citations (18)
Viability assay
Clonogenic assay
MTT assay
Propidium iodide
Cite
Citations (46)
Viability assay
MTT assay
Cite
Citations (0)
To evaluate the expression of uncoupling protein 2 (UCP2) in a retinal pigment epithelium cell line (ARPE-19), under oxidative stress (OS).ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2O2; 0, 150, 300, 500, 700, and 900 µmol/L) for 24h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2O2 was investigated by reverse transcription-polymerase chain reaction (RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry (FCM). Further, UCP2-siRNA treated cultures were exposed to H2O2 (0, 75, 150, and 300 µmol/L) for 2h and cell viability determined by MTT assay.Cells treated with higher concentrations of H2O2 appeared shrunken; their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 µmol/L H2O2 (P<0.05). Cell metabolic activity was decreased with increased concentrations of H2O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2O2 alone (P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2O2-treated groups compared with controls (P<0.05). FCM analysis showed that cell reactive oxygen species (ROS) levels were increased in H2O2-treated groups and further upregulated by UCP2-siRNA treatment (P<0.05).Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.
Viability assay
MTT assay
Cite
Citations (2)
Chlorogenic Acid
Viability assay
MTT assay
IC50
Repeatability
Cite
Citations (0)
Objective To assess the role of MTT colorimetric assay in measuring mitochondrial succinate-dehydrogenase(SDH)and establish a new way to determine pretransplantational cell viability.Methods Human or rat testicular Leydig cell suspensions were incubated with MTT medium,isopropanol was used to dissolve MTT fornlazan,then the absorbance of supertants was measured at 563 nm wavelength.Cell culture and testosterone production were used to aaseas the reliability of MTT colorimetric assay with conlparimn with Trypan bluedye.Results 1 g/L MTT concentration,3 h incubation,and impropanol as a solvent were the best optimum for the cell culture in vitro and testosterone determination showed that MTT colorimetric assay was more sensitive and accurate than Trypan bluedye.MTT colorimetric assay was employed to successfully measure cell viability for 10 cases of human testicular Leydig cell transplantation.Conelusion MTT colorimetric assay provided a reliable,objective,accurate method for determining the pretransplantational cell viability.
Key words:
MTT colorimetric assay; Cell/viability; Cell/transplantation; Trypan btuedye
MTT assay
Viability assay
Formazan
Trypan blue
Colorimetry
Cite
Citations (0)
MTT assay
Viability assay
Cite
Citations (265)
Triple-negative breast cancer
Cite
Citations (1)
The purpose of this experiment is to investigate the effects of forskolin-mediated cAMP activation on the viability of LPS-treated Schwann cells. The immortalized rat RT4-D6P2T (ATCC #CRL-2768) and S16 (ATCC #CRL-2941) cell lines were cultured and received one of the following treatments: 0.1, 1, or 10 μg/mL of LPS, in N2 media (control) or N2 media supplemented with 2 µM of forskolin, for 1, 3, 12, or 24 hours, and the CyQUANT MTT Cell Viability Assay Kit (Thermo Fisher) was used to perform the viability assay.
Viability assay
MTT assay
Cite
Citations (0)
OBJECTIVE: To study the killing effect of TRAIL combined with doxorubicin on osteosarcoma cells so as to explore the new method of clinical chemotherapy.METHODS:TRAIL and doxorubicin were used on the osteosarcoma cells respectively or jointly. The inhibition rate was measured by MTT assay. The apoptotic rate were analyzed with the flow cytometry. Changes of celluar ultrastructure were observed with a microscope. RESULTS:Twenty-four hours after the application of 50 ng/mL TRAIL and 5 μg/mL doxorubicin jointly on OS-732 cells, the inhibition rate was 85.47% which was significantly higher than that after the application of 50 ng/mL TRAIL (9.68%) and 5 μg/mL doxorubicin (18.41%) respectively,P0.01. Changes of celluar ultrastructure and apoptotic rate indicated apoptosis-inducing effect of joint application was much stronger than those of respective application.CONCLUSION: Combination of TRAIL and doxorubicin is obviously conducive to apoptosis of tumor cells.
MTT assay
Cite
Citations (0)