Evaluation of Boric Acid Treatment on microRNA-127-5p and Metastasis Genes Orchestration of Breast Cancer Stem Cells.

Coregulation of microRNAs (miRNAs) and cancer stem cells (CSCs) is very important in carcinogenesis. miR-127-5p is known to be downregulated in breast cancer. In this study, we aimed to investigate how boric acid (BA), known for its previously unstudied anti-cancer properties, would affect the expression of miR127-5p and genes responsible for breast cancer stem cells (BC-SCs) metastasis. BC-SCs were isolated from human breast cancer cells (MCF-7) by immunomagnetic cell separation and characterized with flow cytometry and sphere formation. The viability of BC-SCs and the determination of its IC50 value in response to boric acid (BA) were assessed via the MTT assay. Boric acid exhibited dose- and time-dependent inhibition of cell viability in cells. The IC50 doses of boric acid in MCF-7 cells and BC-SCs were 45.69 mM and 41.27 mM, respectively. The impact of BA on the expression of metastatic genes and miR127-5p was elucidated through RT-qPCR analysis. While the expression of the COL1A1 (p < 0.05) and VIM (p < 0.01) was downregulated, the expression of the miR-127-5p, ZEB1 (p < 0.01), CDH1 (p < 0.05), ITGB1 (p < 0.05), ITGA5 (p < 0.05), LAMA5 (p < 0.01), and SNAIL (p < 0.05), was up-regulated in dose-treated BC-SCs (p < 0.001) to the RT-qPCR results. Our findings suggest that boric acid could induce miR-127-5p expression. However, it cannot be said that it improves the metastasis properties of breast cancer stem cells.

Crosstalk among miR-29, α-SMA, and TGFß1/ß3 in melatonin-induced exosome (Mel-prExo) treated human limbal mesenchymal stem cells (hLMSCs): An insight into scarless healing of the cornea.

Inflammatory mediators that infiltrate the corneal stroma after corneal infections, trauma or refractive surgery can trigger the transformation of corneal keratocytes into myofibroblasts, resulting in highly irregular collagen deposition and subsequently corneal scarring. Mesenchymal stem cells (MSCs) can be used as therapeutic agents to regenerate corneal and conjunctival tissue damage, regulate inflammation, and reduce the development of limbal stem cell failure. The use of MSC-derived exosomes as a cell-free therapeutic vector is a novel therapeutic approach. This study aimed to assess the effect of exosomes obtained from melatonin (Mel)-treated human limbal mesenchymal stem cells (hLMSCs) on naïve hLMSCs and to determine their influence on the antifibrotic and pro-regenerative pathways involved in corneal scarring. hLMSCs were treated with varying concentrations of Mel, followed by isolation and characterization of the procured exosomes (Mel-prExos). These exosomes were added to the cell culture media of naïve hLMSCs to examine their antifibrotic and pro-regenerative effects. The expression of miR-155, miR-29, TGFß1, TGFß3, PPARγ, and α-SMA miRNAs and genes were compared between Mel-treated hLMSCs and Mel-prExo-treated hLMSCs by using real-time PCR. We found that at 1 µM Mel and in the presence of Mel-prExos, TGFß1 was expressed 0.001-fold, while TGFß3 was expressed 0.6-fold. miR-29 expression was increased 38-fold in the control-Exo group compared to that in the control group. Changes in TGFß1/ß3 and α-SMA expression are associated with miR-29 and miR-155. This approach could prove beneficial for ocular surface tissue engineering applications.

The analysis of boric acid effect on epithelial-mesenchymal transition of CD133 + CD117 + lung cancer stem cells.

Targeting lung cancer stem cells (LC-SCs) for metastasis may be an effective strategy against lung cancer. This study is the first on epithelial-mesenchymal transition (EMT) properties of boric acid (BA) in LC-SCs. LC-SCs were isolated using the magnetic cell sorting (MACS) method. Tumor-sphere formation and flow cytometry confirmed CSC phenotype. The cytotoxic effect of BA was measured by MTT analysis, and the effect of BA on EMT was examined by migration analysis. The expression levels of ZEB1, SNAIL1, ITGA5, CDH1, ITGB1, VIM, COL1A1, and LAMA5 genes were analyzed by RT-qPCR. E-cadherin, Collagen-1, MMP-3, and Vimentin expressions were analyzed immunohistochemically. Boric acid slightly reduced the migration of cancer cells. Increased expression of transcription factor SNAIL (p < 0.001), but not ZEB1, was observed in LC-SCs. mRNA expression levels of ITGB1 (p < 0.01), ITGA5 (p < 0.001), COL1A1 (p < 0.001), and LAMA5 (p < 0.001) increased; CDH1 and VIM decreased in LC-SCs. Moreover, while E-cadherin (p < 0.001) and Collagen-1 (p < 0.01) immunoreactivities significantly increased, MMP-3 (p < 0.001) and Vimentin (p < 0.01) immunoreactivities decreased in BA-treated LC-SCs. To conclude, the current study provided insights into the efficacy and effects of BA against LC-SCs regarding proliferation, EMT, and cell death for future studies.

Boric Acid Affects the Expression of DNA Double-Strand Break Repair Factors in A549 Cells and A549 Cancer Stem Cells: An In Vitro Study.

DNA double-strand break (DSB) repair genes interact with tumor stemness- and resistance-associated processes in cancer stem cells (CSCs). Therefore, targeting DNA DSB genes in cancer treatment is important for the CSC phenotype. Although the anti-cancer effect of boric acid (BA) has been studied, its effect on DNA DSB is unclear. Moreover, no studies investigate BA's effects on DNA DSB of lung cancer stem cells (LC-SCs). To fill the gap, we aimed to assess the effects of BA on A549 cancer stem cells. CSCs were isolated from human non-small cell lung cancer cells (A549) and characterized by flow cytometry. Different concentrations of BA (at doses ranging from 1 to 100 mM) were applied to cancer stem cells. Cytotoxic activities were determined using the cell viability assay (MTT assay) at 24 and 48 h. Expression levels of DNA DSB genes that BRCA1, BRCA2, RAD51, KU70/80, ATM, and XRCC4 were evaluated by RT-qPCR. Additionally, immunofluorescence staining analysis was exploited for caspase-3 and E-cadherin. ATM expression increased significantly (p < 0.001). No significant change was observed in the expression of other genes. Moreover, BA up-regulated caspase-3 and E-cadherin expression. Consequently, we can say that BA affects DNA DSB and the apoptotic abilities of LC-SCs.

Animal Models for Limbal Stem Cell Deficiency: A Critical Narrative Literature Review.

This literature review will provide a critical narrative overview of the highlights and potential pitfalls of the reported animal models for limbal stem cell deficiency (LSCD) and will identify the neglected aspects of this research area. There exists significant heterogeneity in the literature regarding the methodology used to create the model and the predefined duration after the insult when the model is supposedly fully fit for evaluations and/or for testing various therapeutic interventions. The literature is also replete with examples wherein the implementation of a specific model varies significantly across different studies. For example, the concentration of the chemical, as well as its duration and technique of exposure in a chemically induced LSCD model, has a great impact not only on the validity of the model but also on the severity of the complications. Furthermore, while some models induce a full-blown clinical picture of total LSCD, some are hindered by their ability to yield only partial LSCD. Another aspect to consider is the nature of the damage induced by a specific method. As thermal methods cause more stromal scarring, they may be better suited for assessing the anti-fibrotic properties of a particular treatment. On the other hand, since chemical burns cause more neovascularisation, they provide the opportunity to tap into the potential treatments for anti-neovascularisation. The animal species (i.e., rats, mice, rabbits, etc.) is also a crucial factor in the validity of the model and its potential for clinical translation, with each animal having its unique set of advantages and disadvantages. This review will also elaborate on other overlooked aspects, such as the anaesthetic(s) used during experiments, the gender of the animals, care after LSCD induction, and model validation. The review will conclude by providing future perspectives and suggestions for further developments in this rather important area of research.

Developing a Novel Platelet-Rich Plasma-Laden Bioadhesive Hydrogel Contact Lens for the Treatment of Ocular Surface Chemical Injuries.

Permanent injury to corneal limbal stem cells after ocular surface chemical and thermal injuries is a major cause of corneal blindness. In this study, a PRP-laden GelMA hydrogel contact lens is manufactured which is aimed to support the limbal niche after ocular surface insults thereby preventing limbal stem cell failure. GelMA with varying platelet-rich plasma (PRP) concentrations (5%, 10%, and 20%) is photopolymerized using a visible light crosslinking system followed by characterizations of mechanical properties, growth factor release, enzymatic degradation, and in vitro cytotoxicity. The addition of 10% PRP into 10% GelMA hydrogel precursor solution results in the highest tensile and compressive modulus (38 and 110 kPa, respectively) and burst pressure (251±37.66 mmHg). Degradation time varies according to the concentration of the collagenase enzyme tested (0, 2.5, 5, and 40 µg/mL) and is most prolonged with 20% PRP. EGF and TGF-ß release profiles suggest an initial burst release followed by sustained release, most consistent in the 10% PRP sample. Although cell viability decreases on day 1, rapid recovery is observed and is approximately 120% after day 21. PRP-laden GelMA in the form of a contact lens may be a promising biomaterial-based treatment approach for the maintenance of limbal epithelial stem cells after ocular surface insults.

Parathyroid Allotransplant for Persistent Hypocalcaemia: A New Technique Involving Short-Term Culture.

OBJECTIVES: To develop a new parathyroid allotransplant method for the treatment of permanent hypoparathyroidism. MATERIALS AND METHODS: Parathyroid cells 50 × 10(6) derived from a parathyroid hyperplasia patient were transferred to a 61-year-old patient who had thyroidectomy 17 years earlier, allowing to papillary thyroid cancer; he was admitted to our outpatient clinic with symptomatic chronic hypocalcemia. Cell isolation, cryopreservation, and culturing were conducted according to a new protocol. RESULTS: During a follow-up of 5 months, the patient had no complications that could indicate rejection, and clinical symptoms completely resolved without requiring any drug supplementation. CONCLUSIONS: Here, we report a new method, enabling fast and cost-effective parathyroid allotransplant with maintained tissue viability sufficient to treat persistent hypocalcemia.

Parathyroid Allotransplant With a New Technique: A Prospective Clinical Trial.

OBJECTIVES: Parathyroid allotransplant is a valuable alternative in treating permanent hypoparathyroidism. However, it is a difficult process that requires several trained staff and advanced laboratory equipment, which makes the costs high. Here, we identify a new parathyroid allotransplant technique. MATERIALS AND METHODS: After obtaining informed consent from patients, parathyroid cell suspensions obtained from 4 donors who had undergone a subtotal parathyroidectomy owing to chronic renal failure were transplanted in 10 patients with permanent hypoparathyroidism after short-term cell cultivation. Prednisolone were used as immunosuppressant for the first 10 days and discontinued thereafter. RESULTS: Allograft function was observed in 7 patients (70%) at a mean follow-up of 12 months. Daily oral calcium and vitamin D supplementations discontinued totally in 7 patients. No major or minor complication was observed. CONCLUSIONS: Our technique is simple, fast, and has a low cost, with a 70% success rate at a mean follow-up of 12 months. It requires few staff, minimal equipment, and short-term immunosuppressant use for maintenance. The technical developments of parathyroid allotransplant, as mentioned in this study, may be important in treating permanent hypoparathyroidism.

A novel non-surgical, minimally invasive technique for parathyroid autotransplantation: a case report.

We present a case report of intramuscular autotransplantation of the parathyroid cell suspension acquired after total parathyroidectomy. A 15-yr-old female patient who had been undergoing hemodialysis due to chronic renal failure for eight yr was diagnosed with secondary hyperthyroidism and subsequently underwent total parathyroidectomy. The parathyroid cells were acquired from the resected tissues, processed through isolation and cultivation phases, and counted using a cell counter. A total of two million cells were injected into the left deltoid muscle using a 22-gauge needle. After surgery, five and 10 million cells were injected in the fifth and 12 week, respectively. The desired serum levels of parathyroid hormones and calcium were not achieved after the first two transplantations. In addition, there was no regression in the patient's symptoms. However, at four wk after the third transplantation, serum parathyroid hormone level did not decrease to <3 pg/mL, the patient was asymptomatic, and the oral treatment was stopped. Our findings indicate that this new technique is applicable because it is minimally invasive, and it can be easily repeated.