Enhancement of cisplatin cytotoxicity in combination with herniarin in vitro.

Combinatorial chemotherapy is a valuable route, which can be conducted by different approaches. Use of cisplatin has been approved by the U.S. Food and Drug Administration for different kinds of cancers including bladder cancer. Herniarin is a member of simple coumarins, which are a group of common secondary metabolites in plants. In this study, the enhancing effects of herniarin on cisplatin cytotoxicity were investigated. Cytotoxicity of herniarin on transitional cell carcinoma (TCC) cells was first investigated in comparison with umbelliferone, the parent compound for a large number of coumarins including herniarin, by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. In order to test the effects of herniarin on cisplatin cytotoxicity, TCC cells were also treated with various combining concentrations of herniarin and cisplatin. In these experiments same amounts of dimethyl sulfoxide were used as controls. After 24, 48 and 72 h of treatments, the effects of herniarin on cisplatin cytotoxicity were evaluated by MTT assay. The level of chromatin condensation which represents the apoptotic morphology was also investigated by 4',6-diamidino-2-phenylindole (DAPI) staining. Results indicated that unlike umbelliferone, its methoxy analog, herniarin, had no significant cytotoxicity on TCC cells. On the other hand, the combination of 80 µg/mL herniarin with 5 µg/mL cisplatin, significantly enhanced the cytotoxicity of cisplatin. Furtheremore, DAPI staining revealed that combining concentrations of herniarin and cisplatin resulted in increased chromatin condensation in comparison with controls. This study is another confirmation for bioactivity of herniarin and shows that it might be a good candidate for further experiments investigating its mechanism of action.

In vitro investigation of anticancer, cell-cycle-inhibitory, and apoptosis-inducing effects of diversin, a natural prenylated coumarin, on bladder carcinoma cells.

Chemotherapy is one of the main strategies for reducing the rate of cancer progression or, in some cases, curing the tumour. Since a great number of chemotherapeutic agents are cytotoxic compounds, i. e. similarly affect normal and neoplastic cells, application of antitumour drugs is preferred in cancer management and therapy. In this study, the cytotoxicity of diversin was evaluated in 5637 cells, a transitional cell carcinoma (TCC) subline (bladder carcinoma), and normal human fibroblast cells using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Chromatin condensation and DNA damage induced by diversin were also determined by means of 4',6-diamidino-2-phenylindole (DAPI) staining and the comet assay, respectively. In addition, the mechanism of action of diversin was studied in more detail by the caspase 3 colourimetric assay and flow cytometry-based cell-cycle analyses (PI staining). Our results revealed that diversin has considerable cytotoxic effects in 5637 cells, but not on HFF3 (human foreskin fibroblast) and HDF1 (human dermal fibroblast) cells. Further studies showed that diversin exerts its cytotoxicity via induction of chromatin condensation, DNA damage, and activation of caspase 3 in 5637 cells. In addition, flow cytometric analyses revealed that 5637 cells are mostly arrested at the G2 phase of the cell cycle in the presence of diversin.

The cytotoxic activities of 7-isopentenyloxycoumarin on 5637 cells via induction of apoptosis and cell cycle arrest in G2/M stage.

BACKGROUND: Bladder cancer is the second common malignancy of genitourinary tract, and transitional cell carcinomas (TCCs) account for 90% of all bladder cancers. Due to acquired resistance of TCC cells to a wide range of chemotherapeutic agents, there is always a need for search on new compounds for treatment of these cancers. Coumarins represent a group of natural compounds, which some of them have exerted valuable anti-tumor activities. The current study was designed to evaluate anti-tumor properties and mechanism of action of 7-isopentenyloxycoumarin, a prenyloxycoumarin, on 5637 cells (a TCC cell line). RESULTS: MTT results revealed that the cytotoxic effects of 7-isopentenyloxycoumarin on 5637 cancerous cells were more prominent in comparison to HDF-1 normal cells. This coumarin increased the amount of chromatin condensation and DNA damage in 5637 cells by 58 and 33%, respectively. The results also indicated that it can induce apoptosis most probably via activation of caspase-3 in these cells. Moreover, propidium iodide staining revealed that 7-isopentenyloxycoumarin induced cell cycle arrest at G2/M stage, after 24 h of treatment. CONCLUSION: Our results indicated that 7-isopentenyloxycoumarin had selective toxic effects on this bladder cancer cell line and promoted its effects by apoptosis induction and cell cycle arrest. This coumarin can be considered for further studies to reveal its exact mechanism of action and also its anti-cancer effects in vivo.

Immediate administration of hTERT-MSCs-IDO1-EVs reduces hypoalbuminemia after spinal cord injury.

Spinal cord injury (SCI) presents challenging and unpredictable neurological recovery. During inflammatory conditions, the amount of serum albumin and nutrition consumption decreases. Currently, it is proposed to measure serum albumin and glucose content in human or animal subjects to predict the recovery rate and the efficiency of treatments following SCI. In this study, the effect of extra-cellular vesicles (EVs) from immortalized human adipose tissue-derived mesenchymal stem cells (hTERT-MSCs) equipped with the ectopic expression of the human indoleamine 2,3-dioxygenase-1 (IDO1) gene on serum albumin and glucose levels was investigated. After pre-clearing steps of 72-hr conditioned media, small EVs (sEVs) were isolated based on the ultra-filtration method. They were encapsulated with a chitosan-based hydrogel. Five experimental groups (female rats, N = 30, ~ 230 g) were considered, including SCI, sham, hydrogel, control green fluorescent protein (GFP)-EVs and IDO1-EVs. The 60.00 µL of hydrogel or hydrogels containing 100 µg sEVs from GFP or IDO1-EVs were locally injected immediately after SCI (laminectomy of the T10 vertebra and clip compression). After 8 weeks, non-fasting serum glucose and albumin levels were measured. The results indicated that the level of serum albumin in the animals received IDO1-EVs (3.52 ± 0.04) was increased in comparison with the SCI group (3.00 ± 0.94). Also, these animals indicated higher glucose levels in their serum (250.17 ± 69.61) in comparison with SCI ones (214 ± 45.34). Although these changes were not statistically significant, they could be considered as evidence for the beneficial effects of IDO1-EVs administration in the context of SCI to reduce hypoalbuminemia and improve energy consumption. More detailed experiments are required to confirm these results.

Extracellular vesicles and their cells of origin: Open issues in autoimmune diseases.

The conventional therapeutic approaches to treat autoimmune diseases through suppressing the immune system, such as steroidal and non-steroidal anti-inflammatory drugs, are not adequately practical. Moreover, these regimens are associated with considerable complications. Designing tolerogenic therapeutic strategies based on stem cells, immune cells, and their extracellular vesicles (EVs) seems to open a promising path to managing autoimmune diseases' vast burden. Mesenchymal stem/stromal cells (MSCs), dendritic cells, and regulatory T cells (Tregs) are the main cell types applied to restore a tolerogenic immune status; MSCs play a more beneficial role due to their amenable properties and extensive cross-talks with different immune cells. With existing concerns about the employment of cells, new cell-free therapeutic paradigms, such as EV-based therapies, are gaining attention in this field. Additionally, EVs' unique properties have made them to be known as smart immunomodulators and are considered as a potential substitute for cell therapy. This review provides an overview of the advantages and disadvantages of cell-based and EV-based methods for treating autoimmune diseases. The study also presents an outlook on the future of EVs to be implemented in clinics for autoimmune patients.

Investigating the effects of IDO1, PTGS2, and TGF-ß1 overexpression on immunomodulatory properties of hTERT-MSCs and their extracellular vesicles.

The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-ß1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases.

Dedifferentiation Effects of Rabbit Regenerating Tissue on Partially Differentiated Cells.

Cell Stemness can be achieved by various reprogramming techniques namely, somatic cell nuclear transfer, cell fusion, cell extracts, and introduction of transcription factors from which induced pluripotent stem cells (iPSCs) are obtained. iPSCs are valuable cell sources for drug screening and human disease modeling. Alternatives to virus-based introduction of transcription factors include application of DNA-free methods and introduction of chemically defined culturing conditions. However, the possibility of tumor development is still a hurdle. By taking advantage of NTERA-2 cells, a human embryonal carcinoma cell line, we obtained partially differentiated cells and examined the dedifferentiation capacity of regenerative tissue from rabbit ears. Results indicated that treatment of partially differentiated NTERA-2 cells with the regenerating tissue-conditioned medium (CM) induced expression of key pluripotency markers as examined by real-time polymerase chain reaction, flow cytometry, and immunocytochemistry techniques. In this study, it is reported for the first time that the CM obtained from rabbit regenerating tissue contains dedifferentiation factors, taking cells back to the pluripotency. This system could be a simple and efficient way to reprogram the differentiated cells and generate iPSCs for clinical applications as this system is not accompanied by any viral vector, and reprograms the cells within 10 days of treatment. The results may convince the genomic experts to study the unknown signaling pathways involved in the dedifferentiation by regenerating tissue-CM to authenticate the reprogramming model.