Development of Epirubicin-Loaded Biocompatible Polymer PLA-PEG-PLA Nanoparticles: Synthesis, Characterization, Stability, and In Vitro Anticancerous Assessment.

Epirubicin (EPI) is an anti-cancerous chemotherapeutic drug that is an effective epimer of doxorubicin with less cardiotoxicity. Although EPI has fewer side effects than its analog, doxorubicin, this study aims to develop EPI nanoparticles as an improved formula of the conventional treatment of EPI in its free form. METHODS: In this study, EPI-loaded polymeric nanoparticles (EPI-NPs) were prepared by the double emulsion method using a biocompatible poly (lactide) poly (ethylene glycol) poly(lactide) (PLA-PEG-PLA) polymer. The physicochemical properties of the EPI-NPs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), entrapment efficiency and stability studies. The effect of EPI-NPs on cancer cells was determined by high throughput imaging and flow cytometry. RESULTS: The synthesis process resulted in monodisperse EPI-NPs with a size of 166.93 ± 1.40 nm and an elevated encapsulation efficiency (EE) of 88.3%. In addition, TEM images revealed the spherical uniformness of EPI-NPs with no aggregation, while the cellular studies presented the effect of EPI-NPs on MCF-7 cells' viability; after 96 h of treatment, the MCF-7 cells presented considerable apoptotic activity. The stability study showed that the EPI-NPs remained stable at room temperature at physiological pH for over 30 days. CONCLUSION: EPI-NPs were successfully encapsulated within a highly stable biocompatible polymer with minimal loss of the drug. The used polymer has low cytotoxicity and EPI-NPs induced apoptosis in estrogen-positive cell line, making them a promising, safe treatment for cancer with less adverse side effects.

Proteomic analysis of soft tissue tumor implants treated with a novel polybisphosphonate.

BACKGROUND: Osteodex is a novel bi-functional macromolecular polybisphosphonate developed for treatment of bone metastases in prostate and breast cancer. High efficacy of osteodex has been demonstrated both in vitro and in vivo. The present study investigates whether osteodex is also efficacious on soft tissue tumor lesions. MATERIALS AND METHODS: Twelve female nude mice were injected with MDA-MB-231 cells orthotopically. Osteodex was administered i.v. at 2.5 mg/kg, once per week for five weeks. Tumor volumes were measured during the treatment period, the animals were sacrificed, and samples collected for proteomic analysis. RESULTS: The non-treated mice developed multiple tumors greater than 4 cm with pronounced ulceration, while the treated mice had tumors smaller than 1 cm, without ulceration. While general condition of treated mice was good, non-treated animals were in poor condition. Sixteen out of 300 identified proteins were differentially expressed, with statistically significant expression changes of more than two-fold differences between treated and non-treated groups. These proteins were identified using non-gel based nano-liquid chromatography coupled with a Synapt G2 instrument. CONCLUSION: We conclude that osteodex showed significant treatment efficacy on soft tissue tumor implants. The study provides a global view of changes in protein expression profiles following osteodex treatment. Some functions of the identified proteins might be used to explain the specific treatment efficacy of osteodex.

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers.

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

Fascin is a key regulator of breast cancer invasion that acts via the modification of metastasis-associated molecules.

The actin-bundling protein, fascin, is a member of the cytoskeletal protein family that has restricted expression in specialized normal cells. However, many studies have reported the induction of this protein in various transformed cells including breast cancer cells. While the role of fascin in the regulation of breast cancer cell migration has been previously shown, the underlying molecular mechanism remained poorly defined. We have used variety of immunological and functional assays to study whether fascin regulates breast cancer metastasis-associated molecules. In this report we found a direct relationship between fascin expression in breast cancer patients and; metastasis and shorter disease-free survival. Most importantly, in vitro interference with fascin expression by loss or gain of function demonstrates a central role for this protein in regulating the cell morphology, migration and invasion potential. Our results show that fascin regulation of invasion is mediated via modulating several metastasis-associated genes. We show for the first time that fascin down-regulates the expression and nuclear translocation of a key metastasis suppressor protein known as breast cancer metastasis suppressor-1 (BRMS1). In addition, fascin up-regulates NF-kappa B activity, which is essential for metastasis. Importantly, fascin up-regulates other proteins that are known to be critical for the execution of metastasis such as urokinase-type plasminogen activator (uPA) and the matrix metalloproteases (MMP)-2 and MMP-9. This study demonstrates that fascin expression in breast cancer cells establishes a gene expression profile consistent with metastatic tumors and offers a potential therapeutic intervention in metastatic breast cancer treatment through fascin targeting.

Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule.

INTRODUCTION: B7-H1 (PD-L1, CD274) is a T cell inhibitory molecule expressed in many types of cancer, leading to immune escape of tumor cells. Indeed, in previous reports we have shown an association of B7-H1 expression with high-risk breast cancer patients. METHODS: In the current study, we used immunohistochemistry, immunofluorescence and Western blot techniques to investigate the effect of neoadjuvant chemotherapy on the expression of B7-H1 in breast cancer cells. RESULTS: Among tested chemotherapeutic agents, doxorubicin was the most effective in downregulating cell surface expression of B7-H1 in vitro. These results were validated in vivo in a xenograft mouse model, as well as in murine heart tissue known to constitutively express B7-H1. The doxorubicin-dependent cell surface downregulation of B7-H1 was accompanied by an upregulation of B7-H1 in the nucleus. This re-distribution of B7-H1 was concurrent with a similar translocation of phosphorylated AKT to the nucleus. Inhibition of the PI3K/AKT pathway abrogated the doxorubicin-mediated nuclear up-regulation of B7-H1, suggesting an involvement of PI3K/AKT pathway in the nuclear up-regulation of B7-H1. Interestingly, siRNA knock down of B7-H1 lead to an increase in spontaneous apoptosis, as well as doxorubicin-induced apoptosis, which indicates an anti-apoptotic role for B7-H1 in breast cancer cells. The novel discovery of B7-H1 expression in the nuclei of breast cancer cells suggests that B7-H1 has functions other than inhibition of T cells. CONCLUSIONS: Our findings explain the previously reported immunomodulatory effect of anthracyclines on cancer cells, and provide a link between immunoresistance and chemoresistance. Finally these results suggest the use of dual combinatorial agents to inhibit B7-H1 beside chemotherapy, in breast cancer patients.

FOXP3+ Tregs and B7-H1+/PD-1+ T lymphocytes co-infiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy.

BACKGROUND: Recent studies have demonstrated a direct involvement of B7-H1, PD-1 and FOXP3 molecules in the immune escape of cancer. B7-H1 is an inhibitory molecule that binds to PD-1 on T lymphocytes, while FOXP3 is a marker for regulatory T cells (Tregs). We have previously demonstrated the association of B7-H1-expressing T infiltrating lymphocytes (TIL) with high-risk breast cancer patients while other studies reported the involvement of FOXP3+ Tregs as a bad prognostic factor in breast tumors. Although the co-existence between the two types of cells has been demonstrated in vitro and animal models, their relative infiltration and correlation with the clinicopathological parameters of cancer patients have not been well studied. Therefore, we investigated TIL-expressing the B7-H1, PD-1, and FOXP3 molecules, in the microenvironment of human breast tumors and their possible association with the progression of the disease. METHODS: Using immunohistochemistry, tumor sections from 62 breast cancer patients were co-stained for B7-H1, PD-1 and FOXP3 molecules and their expression was statistically correlated with factors known to be involved in the progression of the disease. RESULTS: A co-existence of B7-H1+ T lymphocytes and FOXP3+ Tregs was evidenced by the highly significant correlation of these molecules (P < .0001) and their expression by different T lymphocyte subsets was clearly demonstrated. Interestingly, concomitant presence of FOXP3+ Tregs, B7-H1+ and PD-1+ TIL synergistically correlated with high histological grade (III) (P < .001), estrogen receptor negative status (P = .017), and the presence of severe lymphocytic infiltration (P = .022). CONCLUSION: Accumulation of TIL-expressing such inhibitory molecules may deteriorate the immunity of high-risk breast cancer patients and this should encourage vigorous combinatorial immunotherapeutic approaches targeting Tregs and B7-H1/PD-1 molecules.