Location
Georgia
Start Date
16-5-2017 1:00 PM
Description
Chemotherapy is one of the most widely used treatments of various breast cancers; however, it has been shown that carcinoma-associated fibroblasts (CAFs) induce chemotherapy resistance of breast cancer cells (BCCs). Previous research has shown that the downregulation of caveolin-1 (cav-1), a structural component of membrane caveolae, promotes a CAF-like phenotype in stromal cells. This study was performed to evaluate the effect that downregulation of caveolin-1 in mammary fibroblasts imparts on chemotherapy resistance in BCCs. To determine the half maximal inhibitory concentration (IC50) of chemotherapy agents on BCCs, MDA-MB-231 (MDA) and MCF-7 BCCs were treated for 24 and 48 hours with doxorubicin (DOX) or tamoxifen (TAM), then cell viability was measured. The WST-1 cell viability assay showed significant cytotoxicity after 48 hours for all treatments. MDA BCCs exhibited significant cell death following treatment with 0.9 ug/mL of DOX and 7 uM of TAM. MCF-7 BCC’s had significant cell death at 0.92 ug/mL for DOX and 7.5 uM for TAM; however, these results were less consistent when multiple assays were performed on MCF-7 BCCs treated with DOX. Many attempts were made to isolate mammary gland fibroblasts from cav-1 -/- and cav-1 +/+ mice with no success. Instead, human mammary fibroblasts (HMFs) were transfected with cav-1 shRNA to induce downregulation of cav-1 protein expression. HMFs transfected with cav-1 shRNA were positively selected with puromycin, then subcultured to ensure only transfected cells remained adherent. After a 72-hour growth period, protein lysate was collected and a western blot was done to assess cav-1 expression. Results showed a significant downregulation of cav-1 in cav-1 shRNA treated samples. Overall, these results indicate that TAM is a strong cytotoxic agent against MDA and MCF-7 BCCs, while DOX is more effective against MDA BCCs. Also, the use of shRNA transfection proved to be a beneficial technique for downregulation of cav-1 in HMFs.
Embargo Period
6-26-2017
The Role of Caveolin-1 Deficient Human Mammary Fibroblasts on Chemotherapy Resistance in Breast Cancer Cells
Georgia
Chemotherapy is one of the most widely used treatments of various breast cancers; however, it has been shown that carcinoma-associated fibroblasts (CAFs) induce chemotherapy resistance of breast cancer cells (BCCs). Previous research has shown that the downregulation of caveolin-1 (cav-1), a structural component of membrane caveolae, promotes a CAF-like phenotype in stromal cells. This study was performed to evaluate the effect that downregulation of caveolin-1 in mammary fibroblasts imparts on chemotherapy resistance in BCCs. To determine the half maximal inhibitory concentration (IC50) of chemotherapy agents on BCCs, MDA-MB-231 (MDA) and MCF-7 BCCs were treated for 24 and 48 hours with doxorubicin (DOX) or tamoxifen (TAM), then cell viability was measured. The WST-1 cell viability assay showed significant cytotoxicity after 48 hours for all treatments. MDA BCCs exhibited significant cell death following treatment with 0.9 ug/mL of DOX and 7 uM of TAM. MCF-7 BCC’s had significant cell death at 0.92 ug/mL for DOX and 7.5 uM for TAM; however, these results were less consistent when multiple assays were performed on MCF-7 BCCs treated with DOX. Many attempts were made to isolate mammary gland fibroblasts from cav-1 -/- and cav-1 +/+ mice with no success. Instead, human mammary fibroblasts (HMFs) were transfected with cav-1 shRNA to induce downregulation of cav-1 protein expression. HMFs transfected with cav-1 shRNA were positively selected with puromycin, then subcultured to ensure only transfected cells remained adherent. After a 72-hour growth period, protein lysate was collected and a western blot was done to assess cav-1 expression. Results showed a significant downregulation of cav-1 in cav-1 shRNA treated samples. Overall, these results indicate that TAM is a strong cytotoxic agent against MDA and MCF-7 BCCs, while DOX is more effective against MDA BCCs. Also, the use of shRNA transfection proved to be a beneficial technique for downregulation of cav-1 in HMFs.