Sinensetin suppresses breast cancer cell progression via Wnt/ß-catenin pathway inhibition.

Background: Although there are many treatments for breast cancer, such as surgery, radiotherapy, chemotherapy, estrogen receptor antagonists, immune checkpoint inhibitors and so on. However, safer and more effective therapeutic drugs for breast cancer are needed. Sinensetin, a safer therapeutic drugs, come from citrus species and medicinal plants used in traditional medicine, while its role and underlying mechanism in breast cancer remain unclear. Our study aimed to investigate the role and mechanism of sinensetin in breast cancer. Methods: Cell Counting Kit-8 (CCK-8) was used to determine the safe concentration of sinensetin in MCF-10A, MCF7 and MDA-MB-231 cells; 120 µM sinensetin was used in subsequent experiments. Real time polymerase chain reaction (RT-PCR), Western blotting, Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay, Transwell invasion assay and Clone formation assay were used in this study to determine cell viability, mRNA expression, protein levels, apoptosis, proliferation, invasion and so on. Results: Herein, our results showed that 120 µM sinensetin suppressed the cell viability and promoted apoptosis of MCF7 and MDA-MB-231 cells. Treatment with 120 µM sinensetin for 24 h showed no significant toxicity to normal mammary cells; 120 µM sinensetin decreased cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and downregulated ß-catenin, lymphatic enhancing factor 1 (LEF1), T-cell factor (TCF) 1/TCF7, and TCF3/TCF7L1 expression in MCF7 and MDA-MB-231 cells. The Wnt agonist SKL2001 reversed the inhibitory effect of sinensetin on cell survival, metastasis, and EMT. Sinensetin-induced downregulation of ß-catenin, LEF1, and TCF1/TCF7 expression were upregulated by SKL2001 in MCF7 and MDA-MB-231 cells. Conclusions: In summary, sinensetin suppressed the metastasis of breast cancer cell via inhibition of Wnt/ß-catenin pathway and there were no adverse effects on normal breast cells. Our study confirmed the role of sinensetin in breast cancer cells and provided a better understanding of the underlying mechanism.

Evaluation of Facial Trauma Scars After Treating by Refining Plastic Surgery Techniques: A Follow-Up Study.

BACKGROUND: Although early debridement and refining plastic surgery techniques have been shown to be effective in the treatment of facial scars after trauma, their postoperative outcomes have not been quantitatively evaluated by the relevant Scar Cosmesis Assessment and Rating (SCAR) Scale. This study was designed to provide a fair assessment of the appearance and local symptoms of scars after treatment by refining plastic surgery techniques and to share the operational skills of surgical repairs. PATIENTS AND METHODS: Patients who received refining plastic surgery techniques were followed up, and facial scars were taken as high-definition photos, which were presented to 6 professional observers, 6 lay observers, and patients themselves to score the facial scars, including: scar spread, erythema, dyspigmentation, track marks or suture marks, hypertrophy/atrophy, itch and pain according to the SCAR. RESULTS: There were 56 patients who met the inclusion criteria and 25 agreed to participate in the study. No hypertrophic scar was found, and all patients were satisfied with the scar control effect. The scores showed that the treatment was achieved good results in scar spread (pro group: 0.85±0.55, lay group: 0.96±0.68, patients: 0.92±0.64), erythema (pro group: 0.34±0.26, lay group: 0.45±0.37, patients: 0.32±0.48), hypertrophy/atrophy (pro group: 0.21±0.27, lay group: 0.21±0.31, patients: 0.32±0.48), and there was no significant difference in the scores of the 3 observation groups ( P >0.05). However, it is difficult to eliminate dyspigmentation (pro group: 0.29±0.26, lay group: 0.30±0.30, patients: 0.40±0.50), track marks or suture marks (pro group: 0.45±0.33, lay group: 0.59±0.30, patients: 0.36±0.49). Two (8%) patients complained of itch and 1 (4%) patient complained of both itch and pain in the past 24 hours. CONCLUSIONS: The appearance of facial scars is satisfactory, the local symptoms are mild, and the evaluation among different aesthetics is affirmative after receiving refining plastic surgery techniques, which is just in line with the purpose of seeking beauty for the patients, and meanwhile can provide a good foundation for the comprehensive treatment of late scars, so that the treatment plan should be promoted.

Protective Effect of Fat-Tissue-Derived Products against Ultraviolet Irradiation-Induced Photoaging in Mouse Skin.

BACKGROUND: Exposure to ultraviolet radiation causes erythema, inflammation, and photoaging. Mechanical micronization of adipose tissue can concentrate functional cells and has great potential as an alternative for regenerative medicine. Stromal vascular fraction gel is produced by means of a series of mechanical processes of lipoaspirates and can be injected intradermally. This study aimed to assess the therapeutic effect of stromal vascular fraction gel on photoaging skin. METHODS: A photoaging model was established in nude mice. Photoaging mice received treatments of stromal vascular fraction gel, fat, tretinoin, or phosphate-buffered saline. Photoaging skin was characterized by histologic and immunohistochemical analyses. Expression of collagen synthesis-related or photoaging-related genes was assessed. RESULTS: Stromal vascular fraction gel, fat, and tretinoin reversed photoaging, whereas stromal vascular fraction gel demonstrated the greatest therapeutic effect. Treatment with stromal vascular fraction gel restored intradermal fat tissue content and increased dermal collagen density. Injection of stromal vascular fraction gel had the strongest effect on stimulating fibroblasts and increasing the expression of transforming growth factor ß1 (TGF-ß1), propeptide of type-I procollagen, and Smad 2, decreasing the expression of Smad 3, compared with fat and tretinoin. Expression of photoaging-related genes was significantly reduced, whereas expression of fibulin-5 was significantly increased after stromal vascular fraction gel treatment. CONCLUSIONS: Stromal vascular fraction gel demonstrated remarkable therapeutic effects in reversing photoaging skin. Stromal vascular fraction gel can be injected intradermally and survive within dermal layer after grafting. This product increased TGF-ß1expression and activated fibroblasts to produce propeptide of type I procollagen, thus increasing the amount of collagen I, leading to thickening of the dermis of photoaging skin.

Induced Beige Adipocytes Improved Fat Graft Retention by Promoting Adipogenesis and Angiogenesis.

BACKGROUND: Fat grafting is commonly used in treating soft-tissue defects. However, the basic biology behind fat grafting is still not fully understood. Evidence of adipose browning into beige adipose tissue after fat grafting was revealed, but its role in fat grafting remains unclear. METHODS: Induced beige adipocytes and adipose-derived stem cells were obtained from human lipoaspirates and labeled with green fluorescent protein. Nude mice were each injected with 300 mg of human lipoaspirate containing green fluorescent protein-labeled adipose-derived stem cells, green fluorescent protein-labeled induced beige adipocytes, or phosphate-buffered saline. Grafted fat was harvested after 1, 4, 8, and 12 weeks for immunohistochemistry and histologic examination. Graft retention, vascularization, and adipogenic gene expression were compared. RESULTS: After 7 days' induction, adipocytes achieved browning with multilocular lipid droplets, increased mitochondria, and up-regulated browning gene expression. Fat graft retention rates at week 12 were significantly higher after injection of induced beige adipocytes than after injection of phosphate-buffered saline (46.0 ± 4.9 percent versus 31.0 ± 3.6 percent; p = 0.01), but were similar after injection of induced beige adipocytes and adipose-derived stem cells (p > 0.05). Induced beige adipocytes underwent rewhitening into white adipocytes and showed up-regulation of peroxisome proliferator-activated receptor-γ expression. Induced beige adipocytes enhanced angiogenesis, but were not active in forming vessel structures. CONCLUSIONS: Induced beige adipocytes and adipose-derived stem cells were comparable in improving fat graft retention rates. Induced beige adipocytes promote angiogenesis in a paracrine manner and are prone to rewhitening after fat grafting.

Novel pre-vascularized tissue-engineered dermis based on stem cell sheet technique used for dermis-defect healing.

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis (TED). To solve these problems, we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet (PBMCS) and pre-vascularized fibroblasts cell sheet (PFCS) by cell sheet technology, and then superimposed or folded them together to construct a pre-vascularized TED (PTED), aiming to mimic the real dermis structure. The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1, 7 and 14 via the methods of histochemistry and immunohistochemistry. The results showed that PTED could rapidly promote the wound closure, especially at Day 14, and the wound-healing rate of three-layer PTED could reach 97.2% (P < 0.01), which was faster than the blank control group (89.1%), PBMCS (92.4%), PFCS (93.8%) and six-layer PTED (92.3%). In addition, the vessel density in the PTED group was higher than the other groups on the 14th day. Taken together, it is proved that the PTED, especially three-layer PTED, is more conducive to the full-thickness dermis-defect repair and the construction of the three-dimensional vascular networks, indicating its potential application in dermis-defect repair.

3D co-culture model of endothelial colony-forming cells (ECFCs) reverses late passage adipose-derived stem cell senescence for wound healing.

BACKGROUND: Extensive passage of adipose-derived stem cells (ASCs) in vitro leads to loss of function. Endothelial colony-forming cells (ECFCs) can be isolated from adult peripheral blood. A 3D co-culture system may rescue in vitro ASC senescence. METHODS: A 3D co-culture model was successfully established using hyaluronic acid (HA) gel and a 10:1 ratio of late-passage ASCs and ECFCs. Cell density and culture conditions were optimized. Stem cell phenotype was characterized by flow cytometry. ELISA was used to measure the trophic effect of angiogenic growth factors and compare the effects of these factors between the 3-D co-culture and single-cell culture. Therapeutic potential of ASC/ECFC 3-D co-cultures was evaluated in a mouse chronic injury model. RESULTS: Following incubation in a HA substrate 3D co-culture system, ASC morphology, phenotype, secretory profile, and differentiation capacity were restored. The ASC/ECFC co-culture increased the secretion of cytokines, such as hepatocyte growth factor, compared with single-cell 3D culture or monolayer culture. Mice radiation-ulcer wounds treated with ASC/ECFC 3-D co-cultures (spheroids) showed epithelialization and improved healing compared with wounds treated with ASCs or ECFCs alone. Further, transplanted ASC/ECFC spheroids exhibited superior angiogenic potential due to the ability of the ASCs to transdifferentiate into pericytes. CONCLUSION: 3D co-culture of ECFCs and ASCs in vitro restored native ASC properties by reversing cellular senescence and loss of trophic function. Transplant of ASC/ECFC 3D spheroids in vivo demonstrated pro-angiogenic capacity with improved therapeutic potential.

The effects of macrophage-mediated inflammatory response to the donor site on long-term retention of a fat graft in the recipient site in a mice model.

Inflammatory responses mediated by macrophages play a role in tissue repair. However, it is unclear whether the repair in the donor site after liposuction would have any effects on fat graft retention in the recipient site. This study is designed to evaluate the effects of a macrophage-mediated inflammatory response in donor sites on long-term retention of fat grafting. In this study, mice were randomly divided into two groups. One underwent simulated liposuction, called the fat procurement plus grafting (Pro-Grafting) group, and the other underwent sham surgery, called the fat grafting only (Grafting Only) group. The prepared fat (0.3 ml each) was engrafted and cellular events over a 90-day period were assessed. We found macrophages were infiltrated into adipose tissue at the recipient site in the Grafting Only group within 7 days and the repair essentially completed within 30 days. By contrast, few macrophages infiltrated the recipient site in the Pro-Grafting group within 7 days and the entire remodeling process took 30 days longer in the Pro-Grafting than the Grafting Only group. Moreover, C-reactive protein levels were immediately upregulated after surgery, and the inflammatory factors' expression was higher at the donor rather than the recipient site. However, the repair processes and the long-term retention rate became normal when the adipose tissue was grafted after the donor site did not require macrophages for repair. Therefore, we suggest higher inflammatory factors promote macrophage infiltration and the adipose tissue regeneration process at the donor site. This process is delayed at the recipient site, which may affect long-term retention of fat grafts.

Mechanical process prior to cryopreservation of lipoaspirates maintains extracellular matrix integrity and cell viability: evaluation of the retention and regenerative potential of cryopreserved fat-derived product after fat grafting.

BACKGROUND: Cryopreservation of fat grafts facilitates reinjection for later use. However, low temperature and thawing can disrupt tissues and cause lipid leakage, which raises safety concerns. Here, we compared the cryopreservation potential of stromal vascular fraction (SVF) gel processed from lipoaspirate with that of fat. METHODS: Human SVF gel and fat were cryopreserved at - 20 °C without cryoprotectant for 1 month. Fresh SVF gel and fat were used as controls. Tissue viability, adipose-derived stem cell (ASC) function, and the extracellular content were evaluated. At 3 months after transplanting the specimens to immunocompromised mice subcutaneously, the grafts were examined for retention, tissue engraftment, and inflammatory levels. The regenerative effect of cryopreserved SVF gel was evaluated in a murine ischemic wound healing model. RESULTS: At 1 month, the cell death rate in the SVF gel group was 36 ± 2%. The survived ASCs not only could be isolated via explant culture but also preserved colony-forming and differentiation. However, prolonged cryopreservation exacerbated apoptosis. Assessment of recovered tissues showed that the morphology, cell viability, and extracellular protein enrichment were better in SVF gel-preserved tissues than in frozen fat. At 3 months after lipotransfer, the retention ability of 1-month cryopreserved fat was 41.1 ± 9% compared to that of 1-month cryopreserved SVF gel. Immunostaining results showed that adipose tissue regeneration and integrity in the 1-month cryopreserved SVF gel group were superior to those of the cryopreserved fat group. The cryopreserved SVF gel also accelerated healing of the ischemic wound, compared with cryopreserved fat. CONCLUSION: Cryopreserved SVF gel maintained tissue integrity and cell viability and resulted in a better long-term retention rate than that of cryopreserved fat. Cryopreserved SVF gel also showed superior regenerative potential and improved ischemic wound healing.

Reduced expression of the long non-coding RNA AI364715 in gastric cancer and its clinical significance.

Long non-coding RNA (lncRNA), which is greater than 200 nucleotides, is a class of RNA molecules without protein coding function. In recent years, studies have shown that lncRNAs are associated with cancers. They are affecting the occurrence and development of cancers. However, the diagnostic significances of lncRNAs in gastric cancer are largely unknown. In this study, we focused on AI364715, one typical lncRNA. A total of 186 samples were collected from two cancer centers. To find the potential association between its level and gastric cancer, we first collected 75 paired gastric cancer tissues and normal tissues, which are 5 cm away from the edge of carcinoma. Besides, 18 human healthy gastric mucosa and 18 gastric precancerous lesions (dysplasia) were also collected. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was first used to detect the expression level of AI364715 at multiple stages of gastric tumorigenesis. Then, the relationships between AI364715 level and the clinicopathological factors of patients with gastric cancer were analyzed. The results showed that the expression level of AI364715 in gastric cancer tissues was downregulated. Meanwhile, its expression level was closely associated with tumor size and differentiation. More importantly, AI364715 expression level was significantly changed in dysplasia, the typical precancerous lesions. Taken together, AI364715 may be a potential biomarker for the diagnosis of gastric cancer.