Targeting aberrant fatty acid synthesis and storage in endocrine resistant breast cancer cells.

Background: Lipid metabolic reprogramming is an emerging characteristic of endocrine therapy (ET) resistance in estrogen receptor-positive (ER+) breast cancer. We explored changes in lipid metabolism in ER+ breast cancer cell lines following acquired resistance to common endocrine treatments and tested efficacy of an inhibitor in current clinical trials. Methods: We derived ER+ breast cancer cell lines resistant to Tamoxifen (TamR), Fulvestrant (FulvR), and long-term estrogen withdrawal (EWD). Parental and ET resistant cells were subjected to global gene expression and unbiased lipidomic profiling. Lipid storage changes were assessed via neutral lipid staining with Oil Red O (ORO). The impact of the fatty acid synthase (FASN) inhibitor TVB-2640 on the growth and lipid storage of these cell lines was evaluated. Additionally, 13 C 2 -acetate tracing was used to examine FASN activity in parental and ET resistant cells in the absence or presence of TVB-2640. Results: Compared to parental cells, lipid metabolism and processing pathways were notably enriched in ET resistant cells, which exhibited distinct lipidomes characterized by increased triglyceride and polyunsaturated FA (PUFA) species. ET-resistant cells displayed enhanced cytoplasmic lipid droplets. Increased FASN protein levels were observed in ET-resistant cells, and TVB-2640 effectively inhibited FASN activity. FASN inhibition reduced cell growth in some but not all cell lines and ET resistance types and did not correlate to lipid storage reduction. 13 C 2 -acetate tracing confirmed reduced palmitate synthesis and enhanced PUFA synthesis in ET-resistant cells, especially when combined with FulvR. Conclusion: ET resistant breast cancer cells exhibit a shift towards enhanced triglyceride storage and complex lipids enriched with PUFA acyl chains. While targeting FASN alongside ET may not fully overcome ET resistance in our models, focusing on the unique lipid metabolic dependencies, such as PUFA pathways, may present a promising alternative strategy for treating ET resistant breast cancer.

Oncogenic Integration of Nucleotide Metabolism via Fatty Acid Synthase in Non-Hodgkin Lymphoma.

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP-NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin's lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN-PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

Transplantable delivery systems for in situ controlled release of bisphosphonate in orthopedic therapy.

IMPORTANCE OF THE FIELD: Bisphosphonates (BPs), structurally similar to pyrophosphates and functionally superior in restraining osteoclast-induced bone resorption, have been widely used as clinical drugs in the treatment of osteoporosis, bone voids and associated inflammation. However, owing to their high aqueous solubility and the consequently high rate of loss during oral administration, the loading and targeting of BPs pose major challenges in practice. Alternative delivery routes such as nasal, subcutaneous/intramuscular injection have contributed little to improving the bioavailiability and efficacy of BPs. To improve and optimize the delivery efficiency and efficacy of BPs, numerous strategies have been developed and adopted. Studies on controlled release of BPs provide important information on the fabrication of BP delivery systems for in situ treatment. As BPs play an important therapeutic role in osteoporosis and similar diseases, it has become essential and vital to survey various reported fabrication methodologies of these systems and the consequential orthopedic treatments so as to keep abreast with advances in their clinical use. AREAS COVERED IN THIS REVIEW: Transplantable delivery systems for controlled release of BP are reviewed from literature published since 2000. The fabrication pathways and the release of BPs from various material systems are discussed in case studies. Recent progress in CaP models based on the strong and specific chelation between BPs and calcium phosphate crystals is highlighted. WHAT THE READER WILL GAIN: This review offers an outline of the advances in BP controlled release and delivery systems for orthopedic therapy. TAKE HOME MESSAGE: Understanding the cutting-edge BP controlled release and delivery systems for in situ treatment is key to the successful design of a more promising and perfect delivery system for orthopedic therapy. Moreover, developing such delivery systems incorporating the numerous advantages of BPs and controlled release environment requires substantially more flexible models to control better the fate of BP drugs.

Therapeutic cell delivery and fate control in hydrogels and hydrogel hybrids.

Hydrogels are synthetic or natural polymer networks that closely mimic native extracellular matrices. As hydrogel-based vehicles are being increasingly employed in therapeutic cell delivery, two inherent traits of most common hydrogels, namely low cell affinity and high cell constraint, have significantly drawn the attention of biomedical community. These two properties lead to the unfavourable settlement of anchorage-dependent cells (ADCs) and unsatisfactory cell delivery or tissue formation in hydrogel matrices. Tissue engineers have correspondingly made many efforts involving chemical modification or physical hybridisation to facilitate ADC settlement and promote tissue formation. On the other hand, these two 'bio-inert' characteristics have particularly favoured oncological cell therapists, who expect to utilize hydrogels to provide sufficiently high confinement of the delivered cells for anti-cancer purposes. In general, control of cell fate and behaviours in these three-dimensional (3D) microenvironments has become the central aim for hydrogel-mediated cell delivery, towards which various models based on hydrogels and their hybrids have emerged. In this paper, we will first review the development of strategies aiming to overcome the aforementioned two 'shortcomings' by (i) establishing ADC survival and (ii) creating space for tissue formation respectively, and then introduce how people take advantage of these 'disadvantages' of hydrogel encapsulation for (iii) an enhanced confinement of cell motion.

Microsphere-based drug releasing scaffolds for inducing osteogenesis of human mesenchymal stem cells in vitro.

In this study, in vitro osteogenesis was successfully achieved in human mesenchymal stem cells (hMSCs) by controlled release of the osteogenesis-inducing drugs dexamethasone, ascorbic acid (AA) and beta-glycerophosphate (GP) from poly(lactic-co-glycolic acid) (PLGA) sintered microsphere scaffolds (SMS). We investigated the osteogenesis of human MSCs (hMSCs) on dexamethasone laden PLGA-SMS (PLGA-Dex-SMS), and dexamethasone, AA and GP laden PLGA-SMS (PLGA-Com-SMS). hMSCs cultured on the microsphere systems, which act as drug release vehicles and also promote cell growth/tissue formation-displayed a strong osteogenic commitment locally. The osteogenic commitment of hMSCs on the scaffolds were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real-time PCR and immunohistochemistry analysis. The results indicated hMSCs cultured on PLGA-Com-SMS exhibited superior osteogenic differentiation owing to significantly high phenotypic expression of typical osteogenic genes-osteocalcin (OC), type I collagen, alkaline phosphatase (ALP), and Runx-2/Cbfa-1, and protein secretion of bone-relevant markers such as osteoclast and type I collagen when compared with PLGA-Dex-SMS. In conclusion, by promoting osteogenic development of hMSCs in vitro, this newly designed controlled release system opens a new door to bone reparation and regeneration.

Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system.

A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-beta3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-beta3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-beta3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.

In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels.

Synovium-derived mesenchymal stem cells (SMSC), a novel line of stem cells, are regarded as a promising cell source for cartilage tissue engineering. The goal of this study was to investigate rabbit SMSC coupled with injectable gellan hydrogels for in vitro engineered cartilage. SMSC were isolated from rabbit synovial tissue, amplified to passage 4 in monolayer, and encapsulated in injectable gellan hydrogels, constructs of which were cultured in chondrogenic medium supplemented with TGF-beta1, TGF-beta3 or BMP-2 for up to 42 days. The quality of the constructs was assessed in terms of cell proliferation and chondrocytic gene/protein expression using WST-1 assay, real-time RT-PCR, biochemical analysis, histology and immunohistochemical analysis. Results indicate that the viability of SMSC in hydrogels treated with TGF-beta1, TGF-beta3 and BMP-2 remained high at culture time. The constructs formed cartilaginous tissue with the expression of chondrocytic genes (collagen type II, aggrecan, biglycan, SOX 9) and cartilaginous matrix (sulphated glycosaminoglycan and collagen) as early as 21 days in culture. Both TGF-beta1 and TGF-beta3 treated SMSC-laden hydrogels showed more chondrogenesis compared with BMP-2 treated SMSC-laden hydrogels. It demonstrates that injectable SMSC-laden gels, when treated with TGF-beta1, TGF-beta3 or BMP-2, are highly competent for in vitro engineered cartilage formation, which lays a foundation for their potential application in clinical cartilage repair.

Engineering osteogenesis and chondrogenesis with gene-enhanced therapeutic cells.

For bone and cartilage regeneration, a direct dose of growth factors or the viral or non-viral vector-mediated delivery of growth factor genes to the site of osteal or chondral wounds has disadvantages. These limitations include the short half-life and instability of the proteins, resulting in low efficacy with the repeated administration of the therapy, and the nonspecific targeting of the therapy that elevates general toxicity and systemic immunogenicity. To address these challenges, the focus of gene therapy for bone and cartilage repair has shifted in recent years to the use of autologous cells, typically osteocytes or chondrocytes, or their progenitors, transfected with therapeutic genes; the cells are cultivated in vitro before in vivo transplantation. These gene-enhanced therapeutic cells provide sustained autocrine/paracrine stimulation and localized gene expression. An important advantage of the cell-based approach is that factors contributing to off-target toxicity and immunogenicity are metabolically cleared during the in vitro incubation of the transfected cells prior to being administered to the transplant recipients. This review focuses on gene therapy approaches for treating bone and joint disorders, and specifically discusses the development of cell-based delivery approaches.

Antisense makes sense in engineered regenerative medicine.

The use of antisense strategies such as ribozymes, oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) in gene therapy, in conjunction with the use of stem cells and tissue engineering, has opened up possibilities in curing degenerative diseases and injuries to non-regenerating organs and tissues. With their unique ability to down-regulate or silence gene expression, antisense oligonucleotides are uniquely suited in turning down the production of pathogenic or undesirable proteins and cytokines. Here, we review the antisense strategies and their applications in regenerative medicine with a focus on their efficacies in promoting cell viability, regulating cell functionalities as well as shaping an optimal microenvironment for therapeutic purposes.

Synovium-derived mesenchymal stem cells: a new cell source for musculoskeletal regeneration.

Ever since synovium-derived mesenchymal stem cells (SMSCs) were first identified and successfully isolated in 2001, as a brand new member in MSC families, they have been increasingly regarded as a promising therapeutic cell species for musculoskeletal regeneration, particularly for reconstructions of cartilage, bones, tendons, and muscles. Besides the general multipotency in common among the MSC community, SMSCs excel other sourced MSCs in higher ability of proliferation and superiority in chondrogenesis. This review summarizes the latest advances in SMSC-related studies covering their specific isolation methodologies, biological insights, and practical applications in musculoskeletal therapeutics of which an emphasis is cast on engineered chondrogenesis.

In-vitro osteogenesis of synovium stem cells induced by controlled release of bisphosphate additives from microspherical mesoporous silica composite.

In this study, in-vitro osteogenesis was successfully induced in the highly chondrogenic synovium mesenchymal stem cells (SMSCs) by controlled release of a nitrogenous bisphosphonate additive--alendronate (AL) from a mesoporous silica (MS)-hydroxyapatite (HA) composite that was mediated in poly(lactic-co-glycolic acid) (PLGA) microspheres. This microspherical based controlled release system is constructed with three levels of degradable structures: (1) the AL drug was first hybridized with HA nanoparticles; (2) the HA-AL complexes were filled into the mesopores of MS particles by self-assembly in situ; and (3) the HA-AL-laden MS constructs (MSH-AL) were built in the bulk of PLGA microspheres. In comparison with any mono-component construct, the superiority of this multi-component system comes from two aspects of functionalities: (1) significantly greater loading capacity of the extremely hydrophilic drug-AL; and (2) better controlled profile of AL release. Based on this newly developed PLGA/MSH-AL releasing system, as recipients the SMSCs, which usually exhibit exclusively high chondrogenesis, demonstrated a strong osteogenic commitment. The results were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real time PCR and immunohistochemistry analysis. Considering the renewable source and high proliferative profile of SMSCs, the achievement of engineered SMSC osteogenesis with this PLGA/MSH-AL controlled release system would open a new door to major bony reparation and regeneration.

TGF-beta3: A promising growth factor in engineered organogenesis.

BACKGROUND: Engineered organogenesis is one of the most challenging areas on the cutting edge of regenerative medicine. Growth factors can affect cell proliferation, migration and differentiation profoundly, and thus play a critical role in tissue regeneration. TGF-betas produce a wide range of effects in different cells and tissues. TGF-beta3 is relatively recently discovered and studied. OBJECTIVE: To provide a broader understanding of the current state of TGF-beta3 in engineered osteogenesis, chondrogenesis, palate development, scar-free wound healing, odontogenesis and neurogenesis. METHODS: This review summarizes studies that explore or apply TGF-beta3 for organogenesis with engineering methodology and a regenerative medical perspective. RESULTS/CONCLUSION: TGF-beta3 has proven to be a competent growth factor in engineered organogenesis in vitro. In recent years, using TGF-beta3, more and more in vivo studies have yielded significant therapeutic achievements in animal models, which bear much promise for future medical application.

Gene transfer and living release of transforming growth factor-beta3 for cartilage tissue engineering applications.

Cartilage restoration continues to present a tremendous clinical challenge due to its nonvascular nature. Many studies have demonstrated that chondrogenesis of progenitor cells can be achieved in vitro by manual dose of growth factors; however, it remains a vital difficulty in feeding growth factors to implanted therapeutic cells in vivo. Herein, we constructed recombinant adenovirus encoding human transforming growth factor-beta3 (hTGF-beta3) and practiced it in rat bone marrow-derived mesenchymal stromal cells and articular chondrocytes for cartilage regeneration. Optimal viable transduction and transgenic hTGF-beta3 production were achieved; consequently, positive expression of cartilage marker-collagen type II was enabled in the infected progenitors. We thus conclude that recombinant adenovirus encoding TGF-beta3 gene has been successfully established and validated for cartilage tissue engineering applications.