Other Oxygenases/Oxidases

When tumors reached an average of 50mm3, mice were randomized into 6 treatment groups based on caliper measurements and total IVIS signal (Supplementary figure 1)

When tumors reached an average of 50mm3, mice were randomized into 6 treatment groups based on caliper measurements and total IVIS signal (Supplementary figure 1). blot, and reverse phase protein array. In vivo, HER2+ BC xenografts were treated with enzalutamide, everolimus, trastuzumab, and combinations of these drugs. AR antagonists inhibited proliferation of both HER2+ and TNBC cell lines. Combining AR antagonist and either everolimus or trastuzumab resulted in synergistic inhibition of proliferation. Dihydrotestosterone caused increased phosphorylation of HER2 and/or HER3 that was attenuated by AR inhibition. Everolimus caused an increase in total AR, phosphorylation of HER2 and/or HER3, and these effects were abrogated by enzalutamide. Growth of trastuzumab-resistant HER2+ xenograft tumors was inhibited by enzalutamide, and combining enzalutamide with everolimus decreased tumor Rodatristat viability more than either single agent. AR antagonists synergize with FDA-approved BC therapies such as everolimus and trastuzumab through distinct mechanisms. Treatment combinations are effective in trastuzumab-resistant HER2+ BC cells in vivo. treatments Xenograft studies were approved by the University of Colorado Institutional Animal Care and Use Committee (IACUC protocol 83614(01)1E). All experiments were conducted in accordance with the NIH Guidelines of Care and Use of Laboratory Animals. Tras-resistant BT474-HR20 BC cells were stably transfected with the NES-TGL vector, which contains GFP-luciferase. A total of 2106 BT474-HR20 cells were mixed in 100uL growth factor reduced Matrigel (BD Biosciences) and injected bilaterally into the mammary fat pads of female NOD/SCID mice (Taconic). Mice also received estradiol pellets made in-house using silastic tubing and 1.5mg pharmaceutical-grade estradiol; Rodatristat these pellets allow for extended release of estradiol and minimal toxicity in NOD/SCID mice. Tumor growth was measured weekly by caliper and by luciferase signal using an in vivo preclinical imaging system (IVIS). When tumors reached an average of 50mm3, mice were randomized into 6 treatment groups based on caliper measurements and total IVIS signal (Supplementary figure 1). Mice received Enza via their chow (equivalent to 50mg/kg dose). Enza was mixed with ground mouse chow at a concentration of 0.43 mg/g chow (Research Diets, Inc.). Everol was administered intraperitoneally twice weekly at a dose of 2mg/kg. Tras was administered intraperitoneally twice weekly at a dose of 5mg/kg. Mice were euthanized by CO2 asphyxiation and cervical dislocation. Tumors, mammary glands, and colons were harvested for immunohistochemical and gene expression analyses. Statistical Analyses Data were analyzed with GraphPad Prism 6, using Student’s t tests for comparisons of 2 conditions, or 1-way ANOVA with Bonferroni correction when making multiple comparisons. P-values 0.05 were considered statistically significant. Standard deviations are indicated by error bars, except for studies, where standard error of the mean is indicated by error bars. Synergy was calculated using CalcuSyn Software (Biosoft Inc), which uses the Median Effect method(30), where a combination index (CI) 0.9 indicates synergy, CI = 0.9-1.1 indicates additivity, and CI 1.1 indicates antagonism. Experiments were performed in biological triplicate, and mean values were imported to CalcuSyn for synergy calculations. To compare the effect of treatment on tumor growth over time, a repeated measures design was used. Assumptions for different types of repeated measures analyses were tested (i.e., normal distribution, equal variances, balanced data, no missing data or unequal time measurements). Normal distributions were determined by graphing the data to check for a symmetrical data distribution without outliers and by the Shapiro-Wilk test. Data failing this assumption were transformed. A repeated measures ANOVA was used if there were no missing data, there were equal numbers in each treatment group, the measurement time points were equal, and there were no missing data points. If this model failed the assumptions of sphericity (Mauchly’s Test), either a p-value correction (Huynh-Feldt) was reported or a multivariate ANOVA was used to determine differences in treatment groups over time. If there were missing data, or unbalanced Rodatristat data, or unequal time points, a repeated measures mixed models approach was used. The appropriate covariance structure for the mixed model was tested and the covariance structure leading to the best model fit (lowest Akaike Information Criterion and Bayesian Information Criterion values) was used. The data were transformed by taking the square root to meet the assumption of normality. Adjusted p-values using Tukeys’ method were used to determine differences between individual treatment groups over time. The repeated measures analyses were performed using SAS ver 9.4 (SAS Institute, Cary, NC). Significance was set at p 0.05. Results Enzalutamide inhibits phosphorylation of HER2 and HER3, and enhances trastuzumab efficacy in vitro Treatment of mutations treated with Everol for 48 hours demonstrated an increase in AR protein (Figure 2A and ?and2B)2B) and gene expression (Figure Rabbit Polyclonal to Trk C (phospho-Tyr516) 2C and ?and2D).2D). Additionally, Everol treatment resulted in increased total- and phospho-HER3 protein in the BT474-HR20 cells (Figure 2A), and phospho-HER2 and phospho-HER3 in.