Fig 1.
ABCC3 is overexpressed in breast cancer samples and cancer cell lines.
A and B) ABCC1 and ABCC3 mRNA expression in primary normal breast tissues (n = 30) and primary grade III breast cancer tumors (n = 66) was determined by qPCR analysis. Relative quantification of ABCC1 (1A) and ABCC3 (1B) mRNA expression is standardized to β2M housekeeping gene and normalized to normal breast tissues. Error bar represent standard error of the mean (SEM); * = p<0.05, ** = p<0.01. C and D) Scatter plot shows ABCC1 (1C) and ABCC3 (1D) gene expression in immortalized breast cell lines (HBL-100 and MCF-10A) and breast cancer cell lines (BT-474, T-47D, MCF-7, MDA-MB-231 and HCC-1806) evaluated by qPCR. Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 3.
Fig 2.
ABCC3 overexpression decreases the retention of anti-cancer agents.
A and B) Bar graphs shows ABCC1 (2A) and ABCC3 (2B) gene expression in MDA-MB-231and BT-474 cells transiently transfected with pCMV (empty vector) or pCMV-ABCC1 overexpression vector (ABCC1 (OE)) or pCMV-ABCC3 overexpression vector (ABCC3 (OE)) of, evaluated by qPCR. Error bar represent standard error of the mean (SEM); *** = p<0.001, n = 3. C) Bar graphs shows the doxorubicin retention (%) in MDA-MB-231, transiently transfected with empty vector (MFI-18±1) or ABCC1 (OE), MFI-13±0.6 or ABCC3 (OE), MFI-13.5±1 and analyzed by flow cytometry. Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 3. D) Bar graphs shows the doxorubicin retention (%) in BT-474 transiently transfected with empty vector (MFI-20±1.2) or ABCC1 (OE). MFI-13.1±0.6 or ABCC3 (OE), 15±0.7 and analyzed by flow cytometry. Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 3. E) Bar graphs shows the mitoxantrone retention (%) in MDA-MB-231 transiently transfected with empty vector, MFI-19.6±0.2 or ABCC1 (OE), MFI-15.3±0.4 or ABCC3 (OE), MFI-18.1±0.67 and analyzed by flow cytometry. Error bar represent standard error of the mean (SEM); *** = p<0.001, n = 3. F) Bar graphs shows the mitoxantrone retention (%) in BT-474 transiently transfected with empty vector, MFI-21.6±0.4 or ABCC1 (OE), MFI-14.3±0.4 or ABCC3 (OE), MFI-19.1±0.97 and analyzed by flow cytometry. Error bar represent standard error of the mean (SEM); *** = p<0.001, n = 3.
Fig 3.
Chemotherapy increases ABCC3 gene expression.
A and B) qPCR analysis of ABCC1 and ABCC3 expression in tumor (chemo-naive) tissues (n = 66) and chemo-therapy treated patient tissue samples (n = 30). Relative quantification of ABCC1 (3A) and ABCC3 (3B) mRNA expression was standardized to β2M housekeeping gene and normalized to chemo-naïve tumor tissue expression. Error bar represent standard error of the mean (SEM); * = p<0.05, ** = p<0.01. C and D) FACS plots (3C) shows the doxorubicin retention in chemo-naive tumour derived cells (n = 5, MFI-9.8±0.3) and CT treated tumour derived cells (n = 5, MFI-5.95±0.4). Bar graph (3D) shows the doxorubicin retention (%). Error bar represent standard error of the mean (SEM); ** = p<0.01, n = 3. E and F) Bar graph shows ABCC1 (3E) and ABCC3 (3F) gene expression in MDA-MB-231 and BT-474 cells treated with vehicle control, doxorubicin (0.25 μM), mitoxantrone (0.25 μM) and 5-FU (3 μM) evaluated by qPCR. Error bar represent standard error of the mean (SEM); * = p<0.05, ** = p<0.01, *** = p<0.001, n = 3.
Fig 4.
Knockdown of ABCC3 enhances doxorubicin retention in breast cancer cells.
A) Bar graph shows the rhodamine-123 retention (%) in MK-571 treated MDA-MB-231 (MFI-341.1±4) and BT-474 cells (324.1±0.6) and vehicle treated MDA-MB-231(MFI-302±2) and BT-474 cells (295±5) respectively. Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 3. B) Bar graphs shows the doxorubicin retention (%) in MK-571 treated MD-MB-231 (MFI-11±0.32), BT-474 (10.5±0.6), MCF-7 (MF-13.2±0.3) and HCC-1806 (MFI-5.9±0.5) and vehicle treated MD-MB-231 (MFI-15.5±0.3), BT-474 (8.3±0.9), MCF-7 (12.1±0.2) and HCC-1806 (MFI-4.1±0.2) breast cancer cell lines respectively. C) Bar graphs shows the doxorubicin retention (%) in MK-571 treated chemo-naive tumour derived cells (MFI-14.5±0.8) compared to vehicle treated cells (MFI-9.7±0.5). Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 3. D and E) FACS plots (4D) shows the doxorubicin retention in chemo-naive tumour derived cells (n = 5, MFI-9.8±0.45), CT treated tumour derived cells (n = 5, MFI-6.1±0.3) and CT treated tumour derived cells treated with MK-571 (MFI-7.7±0.4). Scatter plot shows the doxorubicin (%) retention in tumour derived cells (4E). Error bar represent standard error of the mean (SEM); n = 3.
Fig 5.
Knockdown of ABCC3 enhances doxorubicin retention in breast cancer cells.
A and B) Bar graph shows ABCC1 (5A) and ABCC3 (5B) gene expression in MDA-MB-231 and BT-74 cells stably expressing non-targeting shRNA (NT), shABCC1 or shABCC3 shRNA evaluated by qPCR. Error bar represent standard error of the mean (SEM); *** = p<0.001, n = 3. C, D, E and F) Bar graphs shows the drug retention (%) in MDA-MB-231 and BT-474 cells stably expressing NT or shABCC1 treated with increasing concentrations of doxorubicin or mitoxantrone. Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 3. G, H, I and J) Bar graphs shows the drug retention (%) in MDA-MB-231 and BT-474 cells stably expressing NT or shABCC3 treated with increasing concentrations of doxorubicin or mitoxantrone. Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 3. K and L) Bar graphs show percentage drug retention in MDA-MB-231 and BT-474 cells stably expressing shABCC1 or shABCC3 and treated with increasing concentrations of doxorubicin. Error bar represent standard error of the mean (SEM); * = p<0.05, ** = p<0.01, *** = p<0.001, n = 3.
Fig 6.
Knockdown of ABCC3 improves sensitivity to chemotherapeutic agents.
A-F) The line graph shows the cell viability (%) in MDA-MB-231 cells stably expressing NT or shABCC1 (6A, 6B and 6C) or shABCC3 (6D, 6E and 6F) treated with increasing concentrations of doxorubicin, mitoxantrone and methotrexate for 48 hours; Error bar represent standard error of the mean (SEM); n = 3. G and H) The bar graph shows the percentage of apoptotic cells upon doxorubicin (6G) or methotrexate treatment (6H) in MDA-MB-231 cells stably expressing NT, shABCC1 and shABCC3 evaluated by Annexin V assay; Error bar represent standard error of the mean (SEM); * = p<0.05, ** = p<0.01, *** = p<0.001, n = 3.
Fig 7.
Knockdown of ABCC3 reduces stemness.
A-D) Bar graphs show gene expression of Nanog, Oct4 and Bmi1 in MDA-MB-231 and BT-474 cells stably expressing NT, shABCC1 (7A and 7B) and shABCC3 (7C and 7D) evaluated by qPCR; Error bar represent standard error of the mean (SEM); * = p<0.05, *** = p<0.001, n = 3. E and F) FACS plot shows the CD44high/CD24low expression in MDA-MB-231 cells stably expressing NT, shABCC1 and shABCC3 shRNA (7E). Bar graph shows the percentage of CD44high/24low expression (7F). Error bar represent standard error of the mean (SEM); *** = p<0.001, n = 3. G and H) The line graph shows tumor growth kinetics of MDA-MB-231 cells expressing NT or shABCC3 and injected subcutaneously (1.5x106 cells/injection) into 5 weeks NOD-SCID mice. Doxorubicin treatment was given 20 days after injection and continued for 4 weeks (7G). Tumour growth was monitored for the indicated period. At the End of the treatment tumours were isolated and tumor weight was measured (7H). Error bar represent standard error of the mean (SEM); ** = p<0.01, *** = p<0.001, n = 5.