The Role of Caveolin-1 Deficient Human Mammary Fibroblasts on Chemotherapy Resistance in Breast Cancer Cells

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

On a Generalization of the Frobenius Number

We consider a generalization of the Frobenius Problem where the object of interest is the greatest integer which has exactly $j$ representations by a collection of positive relatively prime integers. We prove an analogue of a theorem of Brauer and Shockley and show how it can be used for computation.Comment: 5 page

The Role of Caveolin-1 Deficient Human Mammary Fibroblasts in Chemotherapy Resistance in Breast Cancer Cells

Chemotherapy is one of the most widely used treatments of many types of breast cancer, however, it has been shown that carcinoma-associated fibroblasts (CAFs) induce chemotherapy resistance in breast cancer cells. 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 cav-1 in mammary fibroblasts imparts on chemotherapy resistance in breast cancer cells (BCCs). To determine the half maximal inhibitory concentration (IC50) of chemotherapy agents on breast cancer cells, MDA-MB-231 and MCF-7 BCCs were treated for 24 and 48 hours with doxorubicin or tamoxifen, then cell viability was measured. The WST-1 cell viability assay revealed that there was significant cytotoxicity after 48 hours for both cell lines and treatment types. Specifically, the MDA-MB-231 BCCs showed significant cell death following treatment with 0.9 ug/mL of doxorubicin and 7 uM of tamoxifen. For the MCF-7 BCC’s results, significant cell death was observed at 0.92 ug/mL for doxorubicin and 7.5 uM for tamoxifen. Multiple attempts were made to isolate mammary gland fibroblasts from cav-1 -/- and cav1 +/+ mice with no success. As an alternative, human mammary fibroblasts (HMFs) were i transfected with cav-1 shRNA to induce downregulation of cav-1 gene expression. HMFs transfected with cav-1 shRNA were positively selected with puromycin, then trypsinized and subcultured to ensure only transfected cells remained adherent. Following a growth period of 72 hours, protein lysate from whole transfected HMF cells was collected and a western blot was performed to assess cav-1 expression. Results showed a significant downregulation of cav-1 in cav-1 shRNA treated samples compared to HMFs plated in transfection medium only. Transfected HMFs were then co-cultured with MCF-7 and MDA-MB-231 BCCs for 24 hours then treated with tamoxifen for 24 and 48 hours. WST-1 assays showed there was no significant increase in cell proliferation of 24 and 48 hour tamoxifen treated MCF-7 BCCs co-cultured with cav-1 shRNA transfected HMFs or 24 hour tamoxifen treated MDA-MB-231 BCCs co-cultured with cav-1 shRNA transfected HMFs. However, significant increase in cell proliferation was revealed in the 48 hour tamoxifen treated MDA-MB-231 BCCs co-cultured with cav-1 shRNA transfected HMFs through a WST-1 assay. Overall, these results indicate that tamoxifen is a strong cytotoxic agent against MDA-MB-231 and MCF-7 BCCs, while doxorubicin is more effective against MDA-MB-231 BCCs. Additionally, the use of shRNA transfection proved to be a beneficial technique for downregulation of cav-1 in HMFs. Lastly, cav-1 shRNA transfected HMFs co-cultured with MDA-MB-231 BCCs was associated with a decrease in the cytotoxic effects of tamoxifen against the MDA-MB231 BCCs after 48 hours tamoxifen treatment

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer

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Androgen Receptor mRNA Expression in Urothelial Carcinoma of the Bladder: A Retrospective Analysis of Two Independent Cohorts

INTRODUCTION: Gender-specific differences have led to the androgen receptor (AR) being considered a possible factor in the pathophysiology of urothelial carcinoma of the bladder (UCB), but the exact role remains unclear. MATERIALS AND METHODS: The association of AR mRNA expression with clinicopathological features was retrospectively analyzed in two previously described cohorts. The first cohort consisted of 41 patients with all stages of UCB treated at Aarhus University Hospital, Denmark. The second cohort consisted of 323 patients with muscle-invasive bladder cancer (MIBC) accumulated by the Cancer Genome Atlas (TCGA) Research Network. RESULTS: AR mRNA expression is significantly higher in non-muscle-invasive bladder cancer (NMIBC) when compared to MIBC (P = .0004), with no relevant changes within the different stages of MIBC. AR mRNA expression was significantly associated with TCGA molecular subtypes (P < .0001). In the total cohort, there was no association between AR expression and gender (P = .23). When analyzed separately, females showed a significantly worse disease-free (P = .03) and overall survival (P = .02) when expressing AR mRNA above median level, while the same was not observed for men. Multivariable Cox's regression analyses revealed AR mRNA expression to be an independent prognostic marker for disease-free survival in women (P = .007). CONCLUSIONS: AR mRNA expression is significantly higher in NMIBC than in MIBC, while high AR mRNA expression is associated with worse survival in females with MIBC. Further studies need to investigate the gender-specific role of AR in UCB