Event Title

The role of caveolin-1 deficient murine mammary fibroblast on chemotherapy resistance in breast cancer cells

Location

Suwanee, GA

Start Date

14-5-2019 1:00 PM

End Date

14-5-2019 4:00 PM

Description

Chemotherapy resistance is a hurdle to achieving successful outcomes for the treatment of breast cancer. One mechanism which may contribute to chemotherapy resistance is the decreased expression of fibroblast caveolin-1 (cav-1), membrane invaginations which play a role in signal transduction. The decreased expression of cav-1 has been reported to contribute to a more aggressive breast tumor signature. As a result, it’s possible that decreased expression of cav-1 may promote breast cancer cell resistance to chemotherapy. To address this question, we sought to downregulate cav-1 in human mammary fibroblasts (HMFs), then co-culture these with breast cancer cells to determine whether the down-regulated expression of cav-1 imparted chemotherapy resistance. First, metastatic MDA-231-GFP breast cancer cells (BCC’s) were treated with various concentrations of doxorubicin for 24 and 48 hours. Analyzing GFP fluorescence intensity, the results showed that 3.0ug/ml of doxorubicin resulted in viability of 65.31% at 24 hours and thus was selected for use in subsequent co-culture experiments. Non-metastatic MCF7 BCC’s were treated with various concentrations of tamoxifen for 24 and 48 hours. Analyzing results from the WST-1 viability assay showed that 7.5uM of tamoxifen resulted in viability at 86% at 24 hours and 68% at 48 hours. Next, HMFs were transfected with either a GFP plasmid, a control scrambled plasmid or the shRNA cav-1 plasmid. Following transfection, the HMFs were treated with puromycin to eliminate non-transfected cells. Control and cav-1 shRNA transfected HMFs were cultured and passaged a total of 5 times. It was determined that passages 2 and 3 of cav-1 shRNA treated HMFs exhibited the most down-regulation of cav-1. Next, co-cultures of cav-1 downregulated HMFs with MDA-231 GFP BCCs were set up in a 1:1 ratio. Preliminary results for 1:1 ratio show that MDA-231 GFP BCCs co-cultured with HMF-siCav1 demonstrated chemotherapy resistance by having a higher viability (fluorescent) reading after treatment with doxorubicin for 24 hours compared to the control. These results are expected to elucidate whether the downregulation of cav-1 in fibroblasts leads to chemotherapy resistance in breast cancer cells.

Embargo Period

1-28-2020

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COinS
 
May 14th, 1:00 PM May 14th, 4:00 PM

The role of caveolin-1 deficient murine mammary fibroblast on chemotherapy resistance in breast cancer cells

Suwanee, GA

Chemotherapy resistance is a hurdle to achieving successful outcomes for the treatment of breast cancer. One mechanism which may contribute to chemotherapy resistance is the decreased expression of fibroblast caveolin-1 (cav-1), membrane invaginations which play a role in signal transduction. The decreased expression of cav-1 has been reported to contribute to a more aggressive breast tumor signature. As a result, it’s possible that decreased expression of cav-1 may promote breast cancer cell resistance to chemotherapy. To address this question, we sought to downregulate cav-1 in human mammary fibroblasts (HMFs), then co-culture these with breast cancer cells to determine whether the down-regulated expression of cav-1 imparted chemotherapy resistance. First, metastatic MDA-231-GFP breast cancer cells (BCC’s) were treated with various concentrations of doxorubicin for 24 and 48 hours. Analyzing GFP fluorescence intensity, the results showed that 3.0ug/ml of doxorubicin resulted in viability of 65.31% at 24 hours and thus was selected for use in subsequent co-culture experiments. Non-metastatic MCF7 BCC’s were treated with various concentrations of tamoxifen for 24 and 48 hours. Analyzing results from the WST-1 viability assay showed that 7.5uM of tamoxifen resulted in viability at 86% at 24 hours and 68% at 48 hours. Next, HMFs were transfected with either a GFP plasmid, a control scrambled plasmid or the shRNA cav-1 plasmid. Following transfection, the HMFs were treated with puromycin to eliminate non-transfected cells. Control and cav-1 shRNA transfected HMFs were cultured and passaged a total of 5 times. It was determined that passages 2 and 3 of cav-1 shRNA treated HMFs exhibited the most down-regulation of cav-1. Next, co-cultures of cav-1 downregulated HMFs with MDA-231 GFP BCCs were set up in a 1:1 ratio. Preliminary results for 1:1 ratio show that MDA-231 GFP BCCs co-cultured with HMF-siCav1 demonstrated chemotherapy resistance by having a higher viability (fluorescent) reading after treatment with doxorubicin for 24 hours compared to the control. These results are expected to elucidate whether the downregulation of cav-1 in fibroblasts leads to chemotherapy resistance in breast cancer cells.