Uptake and distribution of carboxylated quantum dots in human mesenchymal stem cells: cell growing density matters.

BACKGROUND: Human mesenchymal stem cells (MSCs) have drawn much attention in the field of regenerative medicine for their immunomodulatory and anti-inflammatory effects. MSCs possess specific tumor-oriented migration and incorporation highlighting the potential for MSCs to be used as an ideal carrier for anticancer agents. Bone marrow is the main source of MSCs for clinical applications. MSCs tracking in vivo is a critical component of the safety and efficacy evaluation of therapeutic cell products; therefore, cells must be labeled with contrast agents to enable visualization of the MSCs migration in vivo. Due to their unique properties, quantum dots (QDs) are emerging as optimal tools in long-term MSC optical imaging applications. The aim of this study was to investigate the uptake dynamics, cytotoxity, subcellular and extracellular distribution of non-targeted carboxylated quantum dots in human bone marrow MSCs at different cell growing densities. RESULTS: QDs had no negative impact on MSC viability throughout the experiment and accumulated in all observed cells efficiently; however, in some MSCs QDs induced formation of lipid droplets. At low cell growing densities QDs distribute within MSCs cytoplasm already after 1 h of incubation reaching saturation after 6 h. After 24 h QDs localize mainly in the perinuclear region of the cells in endosomes. Interestingly, in more confluent culture QDs localize mostly outside MSCs. QDs abundantly mark MSC long filopodia-like structures attaching neighboring cells. At high cell density cultivation, we for the first time demonstrated that carboxylated QDs localize in human bone marrow MSC extracellular matrix. Moreover, we observed that average photoluminescence lifetime of QDs distributed in extracellular matrix are longer than lifetimes of QDs entrapped in endocytic vesicles; thus, for the first time showing the possibility to identify and distinguish localization of QDs in various extracellular and intracellular structures using fluorescence-lifetime imaging microscopy without additional staining assays. CONCLUSION: Carboxylated QDs can be used as nonspecific and effective dye for staining of human bone marrow MSCs and their specific extracellular structures. These results are promising in fundamental stem cell biology as well as in cellular therapy, anticancer drug delivery and tissue engineering.

MX2 mediates establishment of interferon response profile, regulates XAF1, and can sensitize melanoma cells to targeted therapy.

MX2 is an interferon inducible gene that is mostly known for its antiviral activity. We have previously demonstrated that MX2 is also associated with the tumorigenesis process in melanoma. However, it remains unknown which molecular mechanisms are regulated by MX2 in response to interferon signaling in this disease. Here, we report that MX2 is necessary for the establishment of an interferon-induced transcriptional profile partially through regulation of STAT1 phosphorylation and other interferon-related downstream factors, including proapoptotic tumor suppressor XAF1. MX2 and XAF1 expression tightly correlate in both cultured melanoma cell lines and in patient-derived primary and metastatic tumors, where they also are significantly related with survival. MX2 mediates IFN growth-inhibitory signals in both XAF1 dependent and independent ways and in a cell type and context-dependent manner. Higher MX2 expression renders melanoma cells more sensitive to targeted therapy drugs such as vemurafenib and trametinib; however, this effect is XAF1 independent. In summary, we uncovered a new mechanism in the complex regulation of interferon signaling in melanoma that can influence both survival and response to therapy.

MX 2 is a novel regulator of cell cycle in melanoma cells.

MX2 protein is a dynamin-like GTPase2 that has recently been identified as an interferon-induced restriction factor of HIV-1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome-wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context-dependent.

The influence of photodynamic therapy with 5-aminolevulinic acid on senescent skin cancer cells.

BACKGROUND: Senescent cells, which are resistant to apoptosis, accumulate with age and after ultraviolet (UV) radiation, chemotherapy and radiation therapy. Preventing or eliminating senescent cells may be crucial for protection against skin cancer development and improving tumour treatment. The aim of the present study was to investigate the potential of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) to induce senescence in skin cancer cells and to eliminate senescent cells induced by chemotherapy (bleomycin) or UVA (315-400nm) exposure. METHODS: WM115 and A431 cells were incubated with 1mM ALA for 2 and 4h, respectively, before exposure to blue light (10mW/cm2, 0-80s, 0-0.8J/cm2). WM115 cells were treated once with 106J/cm2 (58.4mW/cm2, 30.25min) UVA 6days before ALA-PDT or with 0.24IU/ml bleomycin for 7days to induce senescence before ALA-PDT. Cell viability was monitored by the MTT colorimetric assay. Senescent cells were detected using senescence-associated-beta-galactosidase (SA-ß-Gal) staining and morphological changes (enlarged, flat cells). RESULTS: ALA-PDT caused a light dose dependent increase in senescence. ALA-PDT induced senescence very effectively only in WM115 cells but not in A431 cells, while similar cytotoxic effects were observed in both cell lines. After ALA-PDT with 0.4J/cm2 around 70% of survived WM115 cells were senescent, while only around 5% of A431 cells were senescent after ALA-PDT with 0.8J/cm2. CONCLUSION: ALA-PDT can induce premature senescence and kill senescent cells induced by ALA-PDT itself, UVA and chemotherapy (bleomycin). Light doses must be properly chosen to photoinactivate ALA-PDT-induced senescent cells.

Folic acid and its photoproducts, 6-formylpterin and pterin-6-carboxylic acid, as generators of reactive oxygen species in skin cells during UVA exposure.

Folic acid (FA) is the synthetic form of folate (vitamin B9), present in supplements and fortified foods. During ultraviolet (UV) radiation FA is degraded to 6-formylpterin (FPT) and pterin-6-carboxylic acid (PCA) which generate reactive oxygen species (ROS) and may be phototoxic. The aim of the present study was to investigate the production of ROS and phototoxicity of FA, FPT and PCA in skin cells during UVA exposure. The production of ROS and phototoxicity of FA, FPT and PCA were studied in the immortal human keratinocytes (HaCaT) and malignant skin cells (A431 and WM115) during UVA exposure. Increased ROS production and the photoinactivation of cells in vitro were observed during UVA exposure in the presence of FA, FPT and PCA. HPLC analysis revealed that 10 µM FA photodegradation was around 2.1 and 5.8-fold faster than that of 5 µM and 1 µM FA. Photodegradation of FA is concentration dependent, and even non-phototoxic doses of FA and its photoproducts, FPT and PCA, generate high levels of ROS in vitro. FA, FPT and PCA are phototoxic in vitro. The photodegradation of topical or unmetabolized FA during UV exposure via sunlight, sunbeds or phototherapy may lead to ROS production, to the cutaneous folate deficiency, skin photocarcinogenesis and other deleterious skin effects. Further studies are needed to confirm whether UV exposure can decrease cutaneous and serum folate levels in humans taking FA supplements or using cosmetic creams with FA.

Phototherapy and vitamin D.

The skin is the site for the photosynthesis of vitamin D and is a target tissue for the active metabolite of vitamin D. An increasing body of evidence indicates that vitamin D produced during phototherapy may be responsible for the positive effects observed during treatment of some skin diseases. Topical or oral application of vitamin D derivatives are used alone or with phototherapy. This paper reviews what is known about the use of phototherapy to enhance vitamin D levels, the use of vitamin D analogues with phototherapy, the efficacy of combination therapies, and controversies regarding some of the outcomes. Vitamin D can play a beneficial role in treating psoriasis, even though the exact role of vitamin D in the pathogenesis and severity of psoriasis remains unclear. The role of vitamin D in vitiligo, atopic dermatitis, polymorphic light eruption, and mycosis fungoides must be further investigated.