The Effects of Rhodiola Crenulata Extract on Proliferation and Differentiation in Glioblastoma Multiforme

Purpose: Purpose of the study was to evaluate the effects of rhodiola crenulata plant extract on glioblastoma in vitro. Methods: U-87MG glioblastoma multiforme cell line was utilized for evaluation in this study. Cells were treated with 100ug/ml or 200ug/ml of rhodiola crenulata and compared to ethanol vehicle control. Proliferation was measured at 24, 48, 72, and 96 hours after treatment utilizing an MTS proliferation assay. To further assess proliferation a clonogenicity assay was conducted. These cells were treated with ethanol vehicle control, 100ug/ml of rhodiola, radiation, or combined rhodiola/radiation treatment. To evaluate differentiation the expression of glial fibrillary acidic protein (GFAP), a protein marker of differentiation, was assessed with immunocytochemistry. Results: Effects on proliferation were initially noted at 48hours after treatment and observed through the 96-hour period. The effects on proliferation were noted in both treatment groups. At 96-hours after treatment significant difference was noted between the 100ug/ml of rhodiola and control group (p=0.0065) and significant difference noted between the 200ug/ml of rhodiola and control group (p=0.0006). Cell clonogenicity was reduced in the cells treated with 100ug/ml of rhodiola. The decreased number of colonies was significant when comparing the radiation treated cells with 100ug/ml rhodiola treated cells (p=0.0030). GFAP was overexpressed in the rhodiola treatment group when compared to expression in the control group (Figure 1). Conclusion: Rhodiola crenulata extract effectively decreases proliferation and increases differentiation of glioblastoma cells in vitro. Further work is required to fully understand the extent and full effects rhodiola crenulata has glioblastoma cells

A Novel Approach to Targeted Oncologic Therapy - Co-culture Viability of Polymer Prodrug Conjugation to Mesenchymal Stem Cells

Background/Purpose: Conjugation of polymer prodrugs to tumor homing cells, such as Mesenchymal Stem Cells (MSCs), could provide a vehicle for actively targeted delivery of polymer prodrugs. Methods: Human Bone Marrow MSCs were conjugated to either a doxorubicin polymer prodrug or free doxorubicin and were co-cultured with T-cells. Viability was assessed through the use of a Vi-cell counter. In Vivo Migration Analysis was performed NOD SCID mice implanted with subcutaneous MDA MB-231 breast cancer xenografts. Following tumor establishment, mice were injected via lateral tail vein injection with either saline or polymer loaded MSCs. Five days following stem cell injection, mice were euthanized, tumors were harvested and sections were analyzed using fluorescent microscopy and immuno-histochemical staining for cd105. Results: T-cell viability was reduced when co-cultured with MSCs conjugated to free doxorubicin although cells co-cultured with MSCs conjugated to doxorubicin polymer did not exhibit reduced viability. Polymer loaded MSCs displayed intact tumor homing migratory ability in vivo (Figure 1). Conclusion: MSCs conjugated to doxorubicin released the drug, resulting in reduced neighboring T-cells viability. MSCs loaded with polymer maintained their migratory capacity were able to migrate to tumors in vivo. MSCs therefore represent a potential vehicle for targeted drug delivery. Future work will focus on developing methods for releasing the drug upon successful delivery to the target in vivo

Mice Deficient in SFRP1 Exhibit Increased Adiposity, Dysregulated Glucose Metabolism

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Wnt signaling plays an important role in preadipocyte differentiation and adipogenesis. The expression of a Wnt antagonist, secreted frizzled related protein 1 (SFRP1), is increased in response to initial weight gain, then levels are reduced under conditions of extreme obesity in both humans and animals. Here we report that loss of Sfrp1 exacerbates weight gain and glucose homeostasis in mice in response to diet induced obesity (DIO). Sfrp1-/- mice fed a high fat diet (HFD) exhibited an increase in body mass accompanied by increases in body fat percentage, visceral WAT mass, and adipocyte size. Fasting glucose levels are elevated, glucose clearance is impaired, hepatic gluconeogenesis regulators are aberrantly upregulated, and glucose transporters are repressed in Sfrp1-/- mice fed a HFD. Additionally, we observed increased steatosis in the livers of Sfrp1-/- mice. Our findings demonstrate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular signaling in response to the onset of obesity

Antineoplastic Effects of Rhodiola crenulata on B16F10 Melanomas

Hypothesis: Rhodiola crenulata extract is derived from Tibetan plant’s roots and has been shown to have anti-cancer properties. Previously, we have shown that Rhodiola extract has toxic effects on B16 F10 mouse melanoma cells in vitro. The purpose of this project was to determine if a daily topical application of Rhodiola extract on melanoma tumors in mice leads to a reduction in tumor size and improved survival. Methods: 1x106 B16F10 melanoma cells were subcutaneously injected above the scapular fat pad in C57/BL6 mice. Rhodiola extract was dissolved in a 10% DMSO Eucerine based cream. Twenty-four hours following tumor implantation, daily topical Rhodiola treatment began. Tumor volume measurements began on the fifth day of therapy and were measured daily thereafter. Results: Tumors treated with the topical Rhodiola cream tended to grow more radially, rather than vertically when compared to vehicle control. Mice treated with the vehicle control reached a tumor volume of 500mm3 in 10 days, whereas mice treated with the 5% and 10% Rhodiola reached a tumor volume of 500mm3 in 11 and 15 days respectively. All mice treated with vehicle control met requirements for euthanasia by day 14. In contrast, mice treated with the 5% Rhodiola met requirements for euthanasia by day 15 except for one mouse who exhibited tumor regression and survived for over 30 days. All mice treated with the 10% Rhodiola cream met requirements for euthanasia by day 20. Conclusion: Although analysis from our experiment is still in progress, we have observed a gross difference in tumors of mice treated with topical application of Rhodiola cream in comparison with mice treated with a topical application of a vehicle control cream. Future work will focus on histological evaluation of harvested tumors to determine microscopic differences in tumor characteristics

In Vivo Evaluation of a Biomimetic Polymer-Doxorubicin Conjugate for Cancer Therapy

This poster will describe a novel polymer pro-drug platform designed for conjugation and delivery of chemotherapeutics. Specifically, polymer pro-drugs were prepared from functional polymer zwitterions and doxorubicin (DOX), and evaluated in vivo to assess toxicological, pharmacokinetic and therapeutic properties. The biocompatible polymer scaffold (PolyMPC) consists of zwitterionic phosphorylcholine pendent groups, which mimic the natural hydrophilic moieties of phospholipids in cell membranes, and hydrazone linkages that allow for pH-triggered release of DOX. PolyMPC-DOX pro-drugs were isolated as dry solids using a facile strategy that allows for precise control of molecular weight and DOX incorporation. In vivo toxicity of PolyMPC and PolyMPC-DOX was assessed in a murine model. The maximum tolerated dose of the pro-drug was five times greater than that of free DOX, while PolyMPC alone exhibited no toxicity even at a dose of 800 mg/kg. A pharmacokinetic study in tumor-bearing mice demonstrated a significant increase in circulation half-life of conjugated DOX (t1/2=2 hours) compared to free DOX (t1/2=15 minutes), with conjugated DOX detectable in blood serum for longer than 24 hours. This pronounced enhancement in circulation time was attributed to the macromolecular scaffold, which precludes rapid renal clearance compared to native DOX. Examination of mice given PolyMPC-DOX five days after injection in the PK study showed a three-fold increase of drug accumulated in tumor tissue compared to that of mice treated with free DOX and drug accumulation in off-target organs was reduced for mice given DOX conjugate. The therapeutic efficacy of the PolyMPC-DOX conjugates was then assessed in an orthotopic murine breast cancer model. The treatment group given PolyMPC-DOX exhibited a two-fold increase in overall survival and a significant reduction in average tumor volume compared to the free DOX and saline control groups. A study evaluating the therapeutic efficacy of PolyMPC-DOX in a human ovarian xenograft tumor model is ongoing

Pregnancy Induces Persistent Changes that Potentiate Apoptotic Signaling and Responses to DNA Damage

A full-term pregnancy reduces the lifetime risk of breast cancer by up to 50%. This effect is mediated, in part, by p53-dependent pathways. Gene expression profiling was used to investigate the mechanisms that alter apoptotic responses to DNA damage in the mammary gland. Radiation-induced responses in BALB/c-Trp53+/+ and BALB/c-Trp53-/- mice identified 121 genes that were altered by radiation and p53 status (p53-IR). To determine the effect of parity, mice were mated, force-weaned and mammary glands were allowed to involute for 21 days (parous) and compared with age-matched nulliparous mice. Gene expression profiles were determined in mammary tissues from nulliparous (N), parous (P), irradiated nulliparous (N-IR) and irradiated parous (P-IR) mice. The p53-IR gene signature did not differ among the N-IR and P-IR groups indicating that transcriptional activity of p53 was not altered by parity. However, expression profiles of apoptosis-related genes differed significantly in the parous group. The alterations in parous mammary tissues was accompanied by over-representation of biological processes that included “signal transduction” (e=1.69E-05). Within this set, Wnt signaling was especially pronounced (e Parity-regulated genes collaborate with p53-dependent targets, which act as a “switch”, to elicit apoptosis following ionizing radiation. The epigenetic states of the parity-regulated genes Tgfb2 and Wnt5a provide a mechanism for the persistent alterations in gene expression and apoptosis in parous mammary epithelial cells

Curcumin Ingestion Inhibits Mastocytosis and Suppresses Intestinal Anaphylaxis in a Murine Model of Food Allergy

IgE antibodies and mast cells play critical roles in the establishment of allergic responses to food antigens. Curcumin, the active ingredient of the curry spice turmeric, has anti-inflammatory properties, and thus may have the capacity to regulate Th2 cells and mucosal mast cell function during allergic responses. We assessed whether curcumin ingestion during oral allergen exposure can modulate the development of food allergy using a murine model of ovalbumin (OVA)-induced intestinal anaphylaxis. Herein, we demonstrate that frequent ingestion of curcumin during oral OVA exposure inhibits the development of mastocytosis and intestinal anaphylaxis in OVA-challenged allergic mice. Intragastric (i.g.) exposure to OVA in sensitized BALB/c mice induced a robust IgE-mediated response accompanied by enhanced OVA-IgE levels, intestinal mastocytosis, elevated serum mMCP-1, and acute diarrhea. In contrast, mice exposed to oral curcumin throughout the experimental regimen appeared to be normal and did not exhibit intense allergic diarrhea or a significant enhancement of OVA-IgE and intestinal mast cell expansion and activation. Furthermore, allergic diarrhea, mast cell activation and expansion, and Th2 responses were also suppressed in mice exposed to curcumin during the OVA-challenge phase alone, despite the presence of elevated levels of OVA-IgE, suggesting that curcumin may have a direct suppressive effect on intestinal mast cell activation and reverse food allergy symptoms in allergen-sensitized individuals. This was confirmed by observations that curcumin attenuated the expansion of both adoptively transferred bone marrow-derived mast cells (BMMCs), and inhibited their survival and activation during cell culture. Finally, the suppression of intestinal anaphylaxis by curcumin was directly linked with the inhibition of NF-kappaB activation in curcumin-treated allergic mice, and curcumin inhibited the phosphorylation of the p65 subunit of NF-kappaB in BMMCs. In summary, our data demonstrates a protective role for curcumin during allergic responses to food antigens, suggesting that frequent ingestion of this spice may modulate the outcome of disease in susceptible individuals

Gene Expression Profiles Identify Features Common to Lobular and Ductal Premalignant Breast Lesions

Premalignant lesions have been identified in both the ductal and lobular units of the breast epithelium. These lesions have a 4-fold increase in risk of progression to invasive breast cancer, but 80% will remain indolent. This may be due, in part, to the uncertainty of diagnoses as inter-observer reproducibility is poor. When treated with prophylactic hormone therapies blocking the estrogen receptor, up to 40% of women still develop tumors. Therefore the challenge is to develop diagnostic tests that identify the subset of high-risk lesions and provide appropriate prophylactic therapies. We undertook genome-wide expression studies to define sets of genes that show reproducible alterations in atypical hyperplastic lesions. Patients with sporadic atypical hyperplasias and no evidence of breast cancer for at least 2 years following the initial biopsy were selected. RNA quality from formalin-fixed, paraffin-embedded (FFPE) was assessed using the ratio of 150 and 500 bp amplicons of B-actin determined by RT-qPCR. A total of 23 patients were included with diagnoses of pure flattened epithelial atypia (FEA, n=2), atypical lobular lesions (n=9), and atypical ductal lesions (n=12). The atypical lesions and histologically normal breast epithelium were microdissected separately from 6 um thick tissue sections from each patient. RNA was amplified linearly, labeled and hybridized to Affymetrix ST1.0 arrays. Genes differentially expressed by \u3e2-fold between the lesion and normal epithlium within each patient were used to identify gene expression signatures of atypical hyperplasias. Hierarchical clustering of a 512 gene signature yielded 3 major groupings: Benign, Intermediate, Atypia. These results reveal that atypia of the lobular and ductal structures share common underlying transcriptional features. The gene profile provides markers that can improve the reproducibility of diagnoses of atypia. Expression profiling of individuals who subsequently progress to invasive carcinoma will provide biomarkers of high-risk premalignancies and assist selection of therapeutic choices

Genetic Mutations Associated with Hormone-Positive Breast Cancer in a Small Cohort of Ethiopian Women

In Ethiopia, a breast cancer diagnosis is associated with a prognosis significantly worse than that of Europe and the US. Further, patients presenting with breast cancer in Ethiopia are far younger, on average, and patients are typically diagnosed at very late stages, relative to breast cancer patients of European descent. Emerging data suggest that a large proportion of Ethiopian patients have hormone-positive (ER+) breast cancer. This is surprising given 1) that patients have late-stage breast cancer at the time of diagnosis, 2) that African Americans with breast cancer frequently have triple negative breast cancer (TNBC), and 3) these patients typically receive chemotherapy, not hormone-targeting drugs.To further examine the similarity of Ethiopian breast tumors to those of African Americans or of those of European descent, we sequenced matched tumor and normal adjacent tissue from Ethiopian patients from a small pilot collection. We identified mutations in 615 genes across all three patients, unique to the tumor tissue. Across this analysis, we found far more mutations shared between Ethiopian patient tissue and White patients (103) than we did comparing to African Americans (3). Several mutations were found in extracellular matrix encoding genes with known roles in tumor cell growth and metastasis. We suggest future mechanistic studies on this disease focus on these genes first, toward finding new treatment strategies for breast cancer patients in Ethiopia

Gene expression signature of atypical breast hyperplasia and regulation by SFRP1

BACKGROUND: Atypical breast hyperplasias (AH) have a 10-year risk of progression to invasive cancer estimated at 4-7%, with the overall risk of developing breast cancer increased by ~ 4-fold. AH lesions are estrogen receptor alpha positive (ERalpha+) and represent risk indicators and/or precursor lesions to low grade ERalpha+ tumors. Therefore, molecular profiles of AH lesions offer insights into the earliest changes in the breast epithelium, rendering it susceptible to oncogenic transformation. METHODS: In this study, women were selected who were diagnosed with ductal or lobular AH, but no breast cancer prior to or within the 2-year follow-up. Paired AH and histologically normal benign (HNB) tissues from patients were microdissected. RNA was isolated, amplified linearly, labeled, and hybridized to whole transcriptome microarrays to determine gene expression profiles. Genes that were differentially expressed between AH and HNB were identified using a paired analysis. Gene expression signatures distinguishing AH and HNB were defined using AGNES and PAM methods. Regulation of gene networks was investigated using breast epithelial cell lines, explant cultures of normal breast tissue and mouse tissues. RESULTS: A 99-gene signature discriminated the histologically normal and AH tissues in 81% of the cases. Network analysis identified coordinated alterations in signaling through ERalpha, epidermal growth factor receptors, and androgen receptor which were associated with the development of both lobular and ductal AH. Decreased expression of SFRP1 was also consistently lower in AH. Knockdown of SFRP1 in 76N-Tert cells resulted altered expression of 13 genes similarly to that observed in AH. An SFRP1-regulated network was also observed in tissues from mice lacking Sfrp1. Re-expression of SFRP1 in MCF7 cells provided further support for the SFRP1-regulated network. Treatment of breast explant cultures with rSFRP1 dampened estrogen-induced progesterone receptor levels. CONCLUSIONS: The alterations in gene expression were observed in both ductal and lobular AH suggesting shared underlying mechanisms predisposing to AH. Loss of SFRP1 expression is a significant regulator of AH transcriptional profiles driving previously unidentified changes affecting responses to estrogen and possibly other pathways. The gene signature and pathways provide insights into alterations contributing to AH breast lesions