Metformin alters therapeutic effects in the BALB/c tumor therapy model.

BACKGROUND: Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. METHODS: Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. RESULTS: Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. CONCLUSION: In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.

Transplantation of bone marrow derived macrophages reduces markers of neuropathology in an APP/PS1 mouse model.

BACKGROUND: We investigated early hallmarks of putative therapeutic effects following systemic transplantation of bone marrow derived macrophages (BM-M) in APP/PS1 transgenic mice. METHOD: BM-M were transplanted into the tail vein and the animals analysed 1 month later. RESULTS: BM-M transplantation promoted the reduction of the amyloid beta [37-42] plaque number and size in the cortex and hippocampus of the treated mice, but no change in the more heavily modified pyroglutamate amyloid beta E3 plaques. The number of phenotypically 'small' microglia increased in the hippocampus. Astrocyte size decreased overall, indicating a reduction of activated astrocytes. Gene expression of interleukin 6 and 10, interferon-gamma, and prostaglandin E receptor 2 was significantly lower in the hippocampus, while interleukin 10 expression was elevated in the cortex of the treated mice. CONCLUSIONS: BM-M systemically transplanted, promote a decrease in neuroinflammation and a limited reversion of amyloid pathology. This exploratory study may support the potential of BM-M or microglia-like cell therapy and further illuminates the mechanisms of action associated with such transplants.

Distribution pattern following systemic mesenchymal stem cell injection depends on the age of the recipient and neuronal health.

BACKGROUND: Mesenchymal stem cells (MSCs) show therapeutic efficacy in many different age-related degenerative diseases, including Alzheimer's disease. Very little is currently known about whether or not aging impacts the transplantation efficiency of MSCs. METHODS: In this study, we investigated the distribution of intravenously transplanted syngeneic MSCs derived from young and aged mice into young, aged, and transgenic APP/PS1 Alzheimer's disease mice. MSCs from male donors were transplanted into female mice and their distribution pattern was monitored by PCR using Y-chromosome specific probes. Biodistribution of transplanted MSCs in the brains of APP/PS1 mice was additionally confirmed by immunofluorescence and confocal microscopy. RESULTS: Four weeks after transplantation into young mice, young MSCs were found in the lung, axillary lymph nodes, blood, kidney, bone marrow, spleen, liver, heart, and brain cortex. In contrast, young MSCs that were transplanted into aged mice were only found in the brain cortex. In both young and aged mouse recipients, transplantation of aged MSCs showed biodistribution only in the blood and spleen. Although young transplanted MSCs only showed neuronal distribution in the brain cortex in young mice, they exhibited a wide neuronal distribution pattern in the brains of APP/PS1 mice and were found in the cortex, cerebellum, hippocampus, olfactory bulb, and brainstem. The immunofluorescent signal of both transplanted MSCs and resident microglia was robust in the brains of APP/PS1 mice. Monocyte chemoattractant-1 levels were lowest in the brain cortex of young mice and were significantly increased in APP/PS1 mice. Within the hippocampus, monocyte chemoattractant-1 levels were significantly higher in aged mice compared with younger and APP/PS1 mice. CONCLUSIONS: We demonstrate in vivo that MSC biodistribution post transplantation is detrimentally affected by aging and neuronal health. Aging of both the recipient and the donor MSCs used attenuates transplantation efficiency. Clinically, our data would suggest that aged MSCs should not be used for transplantation and that transplantation of MSCs into aged patients will be less efficacious.

Mesenchymal Stem Cell-Derived Microvesicles Modulate Lipopolysaccharides-Induced Inflammatory Responses to Microglia Cells.

Microglia cells are the central nervous system immune cells and have been pointed out as the main mediators of the inflammation leading to neurodegenerative disorders. Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells with very high self-renewal properties and uncomplicated in vitro culture. Research has shown that MSCs have the capacity to induce tissue regeneration and reduce inflammation. Studies demonstrated that MSCs have complex paracrine machineries involving shedding of cell-derived microvesicles (MVs), which entail part of the regulatory and regenerative activity of MSCs, as observed in animal models. We proposed MSC-derived MVs as potent regulators of microglia activation and used an in vitro model of stimulation for BV-2 cells, a microglia cell line, with lipopolysaccharides (LPS). Here we demonstrated that presence of MSCs-derived MVs (MSC-MVs) prevents Tumor necrosis factor-α, Interleukin (IL)-1ß and IL-6 upregulation by BV-2 cells and primary microglia cells toward LPS. Also, inducible isoform of nitric oxide synthases and Prostaglandin-endoperoxide synthase 2 upregulation were hampered in presence of MSC-MVs. Higher levels of the M2 microglia marker chemokine ligand-22 were detectable in BV-2 cells after coculture with MSC-MVs in presence and absence of LPS. Moreover, upregulation of the activation markers CD45 and CD11b by BV-2 cells was prevented when cocultured with MSC-MVs. Furthermore, MSC-MVs suppressed the phosphorylation of the extracellular signal kinases 1/2, c-Jun N-terminal kinases and the p38 MAP kinase (p38) molecules. Consequently, MSC-MVs might represent a modulator of microglia activation with future therapeutic impact. Stem Cells 2017;35:812-823.

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model.

The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today's research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments.

Biodistribution of in vitro-derived microglia applied intranasally and intravenously to mice: effects of aging.

BACKGROUND AIMS: The age of both the donor and the recipient has a potential influence on the efficacy of various cell therapies, but the underlying mechanisms are still being charted. We studied the effect of donor and recipient age in the context of microglia migration. METHODS: Microglia were in vitro--differentiated from bone marrow of young (3 months) and aged (12 months) mice and transplanted into young (∼ 3 months) and aged (∼ 17 months) C57BL/6 mice (n = 25) through intravenous and intranasal application routes. Recipients were not immune-suppressed or irradiated. Transplanted microglia were tracked through the use of a sex-mismatched setup or histologically with the use of cells from enhanced green fluorescent protein enhanced green fluorescent protein transgenic mice. RESULTS: No acute rejections or transplant-associated toxicity was observed. After 10 days, both intravenously and intranasally transplanted cells were detected in the brain. Transplanted cells were also found in the blood and the lymph system. The applied cells were also tracked in lungs and kidney but only after intravenous injection subjected to a "pulmonary first-pass effect." After 28 days, intravenously delivered cells were also found in the bone marrow and other organs, especially in aged recipients. Whereas in young recipients the transplanted microglia did not appear to persist, in aged brains the transplanted cells could still be identified up to 28 days after transplantation. However, when cells from aged donors were used, no signals of transplanted cells could be detected in the recipients. CONCLUSIONS: This study establishes proof of principle that in vitro--derived microglia from young but not from aged donors, intravenously or intranasally transplanted, migrate to the brain in young and aged recipients.