Effect of Paclitaxel upon the differentiation ability of hMSCs.

<p>(A) and (B) After 21 days of induced adipogenic differentiation in the presence of either 0, 10, or 10,000 nM Paclitaxel, cells were fixed and stained with the lipophilic dye Nile Red and Hoechst 33342. Image samples were taken of each culture and area of staining was standardized to nuclei counts for that area. A decrease in lipid accumulation of approximately 40% when compared to control was observed in hMSCs treated with either 10 or 10,000 nM Paclitaxel. (C) Microtubule staining via immunocytochemistry reveled an apparent dose-dependence upon cytoskeletal organization in the differentiated hMSCs. At 10 nM characteristic bundling was observed. At 10,000 nM over-accumulation of microtubules was observed. Results are displayed as mean ± standard error. *** P < 0.01. Scale bar (B) 150 μm, (C) 15 μm.</p

Western blot analysis of P-glycoprotein in hMSCs treated with 10nM Paclitaxel over time.

<p>Results show that the hMSCs do not natively express P-glycoprotein (P-gp), nor is expression induced by exposure to Paclitaxel. Positive control is cell lysate from the human colorectal adenocarcinoma cell line HCT-15, which constitutively expresses high levels of P-gp. β-actin was used as a loading control.</p

Cell cycle regulation may provide Paclitaxel resistance in hMSCs.

<p>By inducing cells to enter quiescence before the G2/M transition needed for Paclitaxel action, it is possible that hMSCs utilize cell cycle regulation to protect themselves from the cytotoxic effects of Paclitaxel.</p

Primer list for real-time PCR.

<p>Primer list for real-time PCR.</p

Proliferation and viability of hMSCs in the presence of Paclitaxel.

<p>(A) Human MSCs were incubated with 30–250,000 nM Paclitaxel for 72 hrs, then treated with dimethyl thiazolyl diphenyl tetrazolium salt (MTT) and processed. Absorbance was measured at 600 nM and compared with controls to produce relative values. (B) Human MSCs were incubated for 72 hrs with Paclitaxel at various concentrations. After incubation, cells were counted via hemocytometer and compared with controls to determine relative viability. After 72 hrs viability was determined to be above 90% in hMSCs treated with up to 100,000 nM Paclitaxel. (C) Growth curves were generated by counting stained nuclei of hMSCs treated with either 0, 10, or 10,000 nM Paclitaxel for various lengths of time. It was revealed that Paclitaxel at both 10 and 10,000 nM completely inhibited proliferation of hMSCs. Results for each experiment are displayed as mean ± standard deviation.</p

Characterization of the fibroblast-like state hMSCs adopt when treated with Paclitaxel.

<p>(A) After 72hrs of exposure to either 10 or 10,000 nM Paclitaxel hMSCs transition from the typical spindle-shaped morphology, seen in the untreated hMSCs, to one that is more broad and flat. Quantitative Real-Time PCR was used to determine expression levels of (B) CD9 (C) CD106 (D) CD146 (E) CD166 (F) integrin alpha 11 (ITGA11) (G) matrix metalloproteinase 1 (MMP-1) and (H) MMP-3 in hMSCs treated with 10 nM Paclitaxel over time. Results are displayed as mean ± standard deviation. ** P < 0.05, *** P < 0.01. Scale bar (A) 150 μm.</p

Quantitative real-time PCR of growth arrest specific factor 1 (GAS1) in hMSCs treated with Paclitaxel over time.

<p>Human MSCs were treated with 10 nM Paclitaxel for up to 12 days, with samples being taken at various time points. Relative expression for each gene was calculated via the 2^^(-ΔΔCt) method with Day 0 expression acting as the basis for comparison. Results are displayed as mean ± standard deviation. *** P < 0.01</p

Differentially Expressed Transcripts and Dysregulated Signaling Pathways and Networks in African American Breast Cancer

<div><p>African Americans (AAs) have higher mortality rate from breast cancer than that of Caucasian Americans (CAs) even when socioeconomic factors are accounted for. To better understand the driving biological factors of this health disparity, we performed a comprehensive differential gene expression analysis, including subtype- and stage-specific analysis, using the breast cancer data in the Cancer Genome Atlas (TCGA). In total, 674 unique genes and other transcripts were found differentially expressed between these two populations. The numbers of differentially expressed genes between AA and CA patients increased in each stage of tumor progression: there were 26 in stage I, 161 in stage II, and 223 in stage III. Resistin, a gene that is linked to obesity, insulin resistance, and breast cancer, was expressed more than four times higher in AA tumors. An uncharacterized, long, non-coding RNA, LOC90784, was down-regulated in AA tumors, and its expression was inversely related to cancer stage and was the lowest in triple negative AA breast tumors. Network analysis showed increased expression of a majority of components in p53 and BRCA1 subnetworks in AA breast tumor samples, and members of the aurora B and polo-like kinase signaling pathways were also highly expressed. Higher gene expression diversity was observed in more advanced stage breast tumors suggesting increased genomic instability during tumor progression. Amplified resistin expression may indicate insulin-resistant type II diabetes and obesity are associated with AA breast cancer. Expression of LOC90784 may have a protective effect on breast cancer patients, and its loss, particularly in triple negative breast cancer, could be having detrimental effects. This work helps elucidate molecular mechanisms of breast cancer health disparity and identifies putative biomarkers and therapeutic targets such as resistin, and the aurora B and polo-like kinase signaling pathways for treating AA breast cancer patients. </p> </div

Functional Classification of Differentially Expressed Genes in Human Prostate Cancer According to PANTHER Protein Class (A) and Biological Process Gene Ontology Terms (B).

<p>(A) “Nucleic Acid Binding” includes RNA and DNA binding, nucleases, and helicases. “Transcription Factor” includes zinc finger, helix-turn-helix, high mobility group box, basic helix-loop-helix, and basic leucine zipper transcription factors; cofactors; and nuclear hormone receptors. “Hydrolase” refers to proteases, phosphatases, esterases, lipases, deaminases, phosphodiesterases, glycosidases, deacetylases, pyrophosphatases, glucosidases, galactosidases, and amylases. “Receptor” includes protein kinase receptors, nuclear hormone receptors, cytokine receptors, ligand-gated ion channels, and G-protein coupled receptors. “Enzyme Modulator” includes G protein, kinase, phosphatase, and protease modulators. (B) “Metabolic Process” features carbohydrate, cellular amino acid, lipid, protein, and nucleobase-containing compound metabolism; and the tricarboxylic acid cycle. “Cellular Process” categories are cell-cell signaling, cell cycle, growth and proliferation, cell component movement, and cytokinesis. “Biological Regulation” includes the regulation of apoptosis, metabolism, cell cycle, translation, catalytic activity, and homeostasis. “Developmental Process” categories are system, ectoderm, mesoderm, and endoderm development; cell differentiation; death; anatomical structure morphogenesis; embryo development; sex determination; and pattern specification processes. “Localization” includes transport proteins, protein and RNA localization processes.</p

Magnitude of gene expression differences between tumor and non-malignant human prostate cancer samples.

<p>In this one-dimensional scatter plot the magnitude of gene expression changes represented by log₂ fold ratios are shown. Each point represents a gene or transcript. Significantly differentially expressed genes and transcripts are shown as solid red diamonds.</p