Systematic profiling of Taxol resistance and sensitivity to tubulin missence mutations at molecular and cellular levels.

Taxol (paclitaxel) is the first approved microtubule-stabilizing agent (MSA) by binding stoichiometrically to tubulin, which is considered to be one of the most significant advances in first-line chemotherapy against diverse tumors. However, a large number of residue missence mutations harboring in the tubulin have been observed to cause acquired drug resistance, largely limiting the clinical application of Taxol and its analogs in chemotherapy. A systematic investigation of the intermolecular interactions between the Taxol and various tubulin mutants would help to establish a comprehensive picture of drug response to tubulin mutations in clinical treatment of cancer, and to design new MSA agents with high potency and selectivity to overcome drug resistance. In this study, we described an integration of in silico analysis and in vitro assay (iSiV) to profile Taxol against a panel of 149 clinically observed, cancer-associated missence mutations in ß-tubulin at molecular and cellular levels, aiming to a systematic understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity conferring from tubulin mutations. It is revealed that the Taxol-resistant mutations can be classified into three types: (I) nonbonded interaction broken due to mutation, (II) steric hindrance caused by mutation, and (III) conformational change upon mutation. In addition, we identified three new Taxol-resistant mutations (C239Y, T274I, and R320P) that can largely reduce the binding affinity of Taxol to tubulin at molecular level, in which the T274I and R320P were observed to considerably impair the antitumor activity of Taxol at cellular level. Moreover, a novel drug-susceptible mutation (M363T) was also identified, which improves Taxol affinity by 2.6-fold and decreases Taxol antitumor EC50 values from 29.4 to 18.7 µM.

Knockdown of circ­PVT1 inhibits the progression of lung adenocarcinoma and enhances the sensitivity to cisplatin via the miR­429/FOXK1 signaling axis.

Lung cancer is one of the most prevalent cancers in China, and its incidence and morbidity remain high due to various independent factors. Lung adenocarcinoma (ADC) is the most common type of non­small cell lung carcinoma. Circular RNA plasmacytoma variant translocation 1 (circ­PVT1) plays an oncogenic role in various types of cancer, but the specific role of circ­PVT1 in lung ADC has not yet been reported. In the present study, circ­PVT1 was knocked down in A549 cells and the cell viability, proliferation, migration and invasion were measured via MTT, colony formation, wound healing and Transwell assays, respectively. Then, the cell viability of A549 cells with circ­PVT1­knockdown or ­overexpression was detected after exposure to cisplatin (DDP). After confirming the associations among circ­PVT1, microRNA (miR)­429 and forkhead box k1 (FOXK1) using various tools and assays, the cellular functions of A549 cells treated with combined short hairpin (sh)RNA­circ­PVT1 and miR­429 inhibitor/pcDNA3.1­FOXK1 were tested again. The expression of circ­PVT1 was found to be increased in lung ADC cells, and shRNA­circ­PVT1 led to decreased cell viability, proliferation, migration and invasion. The expression of circ­PVT1 was higher in A549/DDP cells than that in A549 cells, and the activity of caspase­3 was also activated by DDP in A549/DDP cells transfected with shRNA­circ­PVT1, whereas it was inactivated by DDP in A549 cells transfected with circ­PVT1 overexpression plasmid. Furthermore, the decreased cell viability, proliferation, invasion and migration induced by shRNA­circ­PVT1 could be abated by transfection with miR­429 inhibitor and pcDNA3.1­FOXK1. In conclusion, interference of circ­PVT1 inhibits the progression of lung ADC and enhances its sensitivity to DDP via miR­429/FOXK1, which may provide a theoretical basis for the use of novel targets in the treatment of lung ADC.

Systematic molecular profiling of inhibitor response to the clinical missense mutations of ErbB family kinases in human gastric cancer.

The oncogenic receptor tyrosine kinase family ErbB consists of four members (ErbB1, ErbB2, ErbB3 and ErbB4); they are involved in the tumorgenesis of diverse cancers. A variety of missence mutations have been clinically observed in ErbB kinases, which would shift drug sensitivity to these kinases and cause drug resistance in targeted cancer therapy. In this study, systematic inhibitor response to ErbB missense mutations in gastric cancer (GC) is investigated by combining computational analysis and experimental assay. The response profile is created for 6 ATP-competitive, reversible inhibitors against 9, 17, 5 and 17 GC-associated missense mutations of ErbB1, ErbB2, ErbB3 and ErbB4 kinase domains, respectively. From the profile a number of potential resistant and sensitive responses are identified theoretically. It is suggested that most ErbB mutations have only a modest effect on inhibitor binding, but few that are located around the kinase active site can influence the binding significantly. Structural examination reveals that steric hindrance and allosteric effect are primarily responsible for inhibitor resistance and sensitivity, respectively. Two ErbB2 mutations, namely V777L and T862A, are predicted to cause effective resistance on inhibitors TAK285 and Lapatinib, respectively. Kinase assays consistently observe that the mutations can reduce inhibitor activity by 4.9-fold and 2.4-fold, with IC50 changing from 29 to 16 nM (wild type) to 83 and 39 nM (mutant) for TAK285 and Lapatinib, respectively.

A novel model of humanised keloid scarring in mice.

Treatments for keloid scarring are a major challenge to scientists and physicians for their unknown aetiology. Although several models, including monolayer cell culture to tissue-engineered models, were developed, further research on keloid has more or less been hindered by the lack of appropriate animal models. Because these aberrant scars are specific to humans, we obtained human normal and keloid skin tissues and isolated dermal fibroblasts from them. Cell morphology, growth and immunohistochemical staining of myofibroblastmarker α-SMA were examined, and the cell medium of 2-hour culture and 24-hour culture was implanted on the back of nude mice. The cell medium of 2-hour culture and 24-hour culture was also analysed by a protein array for the detection of distinction in inflammatory factors. We showed that keloid fibroblasts had similar morphology and growth compared to normal skin fibroblasts, but the α-SMA expression was obviously up-regulated. After 6 weeks, mice of the 2-hour keloid-derived culture medium group exhibited keloid-like hypertrophic nodules macroscopically, while mice of 24-hour keloid-derived culture medium group were similar to normal skin. Histological findings confirmed that the reconstituted skin tissues had the typical features of human keloids. The protein array data revealed that RANTES were involved in humanised fibrotic occurrence in mice, also suggesting they were important modulators of this inflammatory event. This novel model might help to understand the key events that result in the formation of these abnormal scars and provide new therapeutic options.

Mesenchymal stem cells ameliorate inflammatory cytokine-induced impairment of AT-II cells through a keratinocyte growth factor-dependent PI3K/Akt/mTOR signaling pathway.

Lung epithelium restoration subsequent to injury is of concern in association with the outcomes of diverse inflammatory lung diseases. Previous studies have demonstrated that mesenchymal stem cells (MSCs) may promote epithelial repair subsequent to inflammatory injury, however the mechanism that mediates this effect remains unclear. The current study examined the role of MSCs in alveolar type II epithelial cell (AT­II cell) restoration subsequent to an inflammatory insult. AT­II cells were firstly exposed to inflammatory cytokines including tumor necrosis factor­α, interleukin (IL)­6 and IL­1ß, then were co­cultured with MSCs in Transwell for 72 h. Cell proliferation, expression of surfactant protein A (SP­A) and expression of the α1 subunit were evaluated respectively by the Cell Counting Kit­8 assay, western blotting and semiquantitative reverse transcription-polymerase chain reaction. Keratinocyte growth factor (KGF) small interfering RNA (siRNA) was applied to knockdown the main cytoprotective factors in the MSCs. Subsequent to an inflammatory insult, AT­II cells were observed to be impaired, exhibiting the characteristics of injured cell morphology, reduced cell proliferation and reduced expression of SP­A and the α1 subunit. Co­culture with MSCs significantly ameliorated these cell impairments, while these benefits were weakened by the application of KGF siRNA. Simultaneously, expression levels of phosphorylated (p­) protein kinase B (AKT) and p­mammalian target of rapamycin (mTOR) in AT­II cells were upregulated by MSCs, suggesting activation of the phosphoinositide 3­kinase (PI3K) pathway. These data demonstrate that administration of MSCs to the inflammation-insulted AT-II cells may ameliorate the impairments through a KGF-dependent PI3K/AKT/mTOR signaling pathway.

Age-associated changes in regenerative capabilities of mesenchymal stem cell: impact on chronic wounds repair.

Mesenchymal stem cells (MSCs) represent an ideal source of autologous cell-based therapy for chronic wounds. Functional characteristics of MSCs may benefit wound healing by exerting their multi-regenerative potential. However, cell ageing resulting from chronic degenerative diseases or donor age could cause inevitable effects on the regenerative abilities of MSCs. A variety of studies have shown the relationship between MSC ageing and age-related dysfunction, but few associate these age-related impacts on MSCs with their ability of repairing chronic wounds, which are common in the elderly population. Here, we discuss the age-associated changes of MSCs and describe the potential impacts on MSC-based therapy for chronic wounds. Furthermore, critical evaluation of the current literatures is necessary for understanding the underlying mechanisms of MSC ageing and raising the corresponding concerns on considering their possible use for chronic wound repair.

High DEPTOR expression correlates with poor prognosis in patients with esophageal squamous cell carcinoma.

OBJECTIVE: The disheveled, Egl-10, and pleckstrin (DEP) domain containing mammalian target of rapamycin (mTOR)-interacting protein (DEPTOR) is a binding protein containing mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2), and an endogenous mTOR inhibitor. DEPTOR shows abnormal expressions in numerous types of solid tumors. However, how DEP-TOR is expressed in esophageal squamous cell carcinoma (ESCC) remains elusive. METHODS: The expression of DEPTOR in 220 cases of ESCC and non-cancerous adjacent tissues was detected by immunohistochemistry. DEPTOR levels in ESCC and paired normal tissue were quantified using reverse transcription-polymerase chain reaction and Western blot analysis to verify the immunohistochemical results. The relationship between DEPTOR expression and the clinicopathological features of ESCC was analyzed based on the results of immunohistochemistry. Finally, we analyzed the relationship between DEPTOR expression and the prognosis of patients with ESCC. RESULTS: Immunohistochemical staining showed that the expression rate of DEPTOR in ESCC tissues was significantly increased. DEPTOR mRNA and protein expression was significantly higher in ESCC tissues than in normal adjacent esophageal squamous tissues. High DEPTOR expression was significantly correlated with regional lymph node status in the TNM stage of patients with ESCC. Kaplan-Meier survival curves showed that the rate of overall survival was significantly lower in patients with high DEPTOR expression than in those with low DEPTOR expression. Additionally, high DEPTOR expression was an independent prognostic predictor for ESCC patients. CONCLUSION: High DEPTOR expression is an independent prognostic biomarker indicating a worse prognosis for patients with ESCC.

Cytokeratin Expression at Different Stages in Sweat Gland Development of C57BL/6J Mice.

Sweat glands exhibit a documented role in epidermal reepithelialization after wounding. However, the regenerative potential of sweat glands has remained underappreciated due to the absence of useful markers for the analysis of determination and differentiation processes in the developing eccrine sweat gland from epithelium. Although the current knowledge of keratin expression in most of the different origins has been described, it remains widely shared and not unified in eccrine sweat glands of C57BL/6J mice that are commonly used as animal models for sweat gland and wound healing studies, both at the molecular and cellular levels. Aiming to answer this question, we have investigated the changes in cytokeratin expression patterns during the embryonic, neonatal, juvenile, and young adult stages (E12.5, E17.5, P0.5, P5, and P28). In this article, we demonstrate that the morphology of murine sweat gland progenitor cells are similar to epidermal stem cells before birth (E12.5 and E17.5); at postnatal stages, the duct formed gradually and curled to glob. K8 and K19 were expressed in the eccrine sweat gland cells at all times and highly expressed after birth at both gene and protein levels. Also, histological results revealed K8 and K19 positive cells localized in the secretary portion of glands. Meanwhile, K14 strongly expressed both in vivo and in vitro at E12.5, while it weakly expressed at other stages. Moreover, K10 was rarely detected before birth, but it expressed positively in vivo and in vitro only at the protein level after birth. These data indicate the pattern of main cytokeratin expression at different stages during murine sweat gland development and might provide an efficient tool for sweat gland research and exciting potential for developing targeted therapies for wound healing.

Paracrine action of mesenchymal stromal cells delivered by microspheres contributes to cutaneous wound healing and prevents scar formation in mice.

BACKGROUND AIMS: Accumulating evidence suggests that mesenchymal stromal cells (MSCs) participate in wound healing to favor tissue regeneration and inhibit fibrotic tissue formation. However, the evidence of MSCs to suppress cutaneous scar is extremely rare, and the mechanism remains unidentified. This study aimed to demonstrate whether MSCs-as the result of their paracrine actions on damaged tissues-would accelerate wound healing and prevent cutaneous fibrosis. METHODS: For efficient delivery of MSCs to skin wounds, microspheres were used to maintain MSC potency. Whether MSCs can accelerate wound healing and alleviate cutaneous fibrosis through paracrine action was investigated with the use of a Transwell co-culture system in vitro and a murine model in vivo. RESULTS: MSCs cultured on gelatin microspheres fully retained their cell surface marker expression profile, proliferation, differentiation and paracrine potential. Co-cultures of MSCs and fibroblasts indicated that the benefits of MSCs on suppressing fibroblast proliferation and its fibrotic behavior induced by inflammatory cytokines probably were caused by paracrine actions. Importantly, microspheres successfully delivered MSCs into wound margins and significantly accelerated wound healing and concomitantly reduced the fibrotic activities of cells within the wounds and excessive accumulation of extracellular matrix as well as the transforming growth factor-ß1/transforming growth factor-ß3 ratio. CONCLUSIONS: This study provides insight into what we believe to be a previously undescribed, multifaceted role of MSC-released protein in reducing cutaneous fibrotic formation. Paracrine action of MSCs delivered by microspheres may thus qualify as a promising strategy to enhance tissue repair and to prevent excessive fibrosis during cutaneous wound healing.

Mesenchymal stem cells suppress fibroblast proliferation and reduce skin fibrosis through a TGF-ß3-dependent activation.

Recent studies showed that transplantation of mesenchymal stem cells (MSCs) significantly decreased tissue fibrosis; however, little attention has been paid to its efficacy on attenuating skin fibrosis, and the mechanism involved in its effect is poorly understood. In this work, we investigated the effects of MSCs on keloid fibroblasts and extracellular matrix deposition through paracrine actions and whether the antifibrotic properties of MSCs involved transforming growth factor-ß (TGF-ß)-dependent activation. In vitro experiments showed that conditioned media (CM) from MSCs decreased viability, a-smooth muscle actin expression, and collagen secretion of human keloid fibroblasts. In addition, TGF-ß3 secreted by MSCs was expressed at high level under inflammatory environment, and blocking the activity of TGF-ß3 apparently antagonized the suppressive activity of MSC CM, which demonstrated that TGF-ß3 played a preponderant role in preventing collagen accumulation. In vivo studies showed that MSC CM infusion in a mouse dermal fibrosis model induced a significant decrease in skin fibrosis. Histological examination of tissue sections and immunohistochemical analysis for α-smooth muscle actin revealed that TGF-ß3 of CM-mediated therapeutic effects could obviously attenuate matrix production and myofibroblast proliferation and differentiation. These findings suggest that TGF-ß3 mediates the attenuating effect of MSCs on both the proliferation and extracellular matrix production of human keloid fibroblasts and decreases skin fibrosis of mouse model, thus providing new understanding and MSC-based therapeutic strategy for cutaneous scar treatment.

Mesenchymal stromal cells enhance wound healing by ameliorating impaired metabolism in diabetic mice.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been documented to improve delayed wound healing in diabetes, but the underlying mechanism remains obscure. We aimed to investigate whether the therapeutic effects on wounds was associated with metabolic alterations by paracrine action of MSCs. METHODS: MSCs from mice with high-fat diet/streptozotocin-induced diabetes or wild-type C57BL/6 mice were evaluated for their paracrine potential in vitro using enzyme-linked immunosorbent assay and immunohistochemical staining assay. MSCs were then evaluated for their therapeutic potential in vivo using an excisional cutaneous wound model in mice with diabetes. Metabolic alterations and glucose transporter four (GLUT4) as well as PI3K/Akt signaling pathway expression after wounding were also examined. RESULTS: MSCs from normal mice expressed even more insulin-like growth factor-1 (IGF-1) than mice with diabetes, suggesting putative paracrine action. Furthermore, compared with IGF-1 knockdown MSCs, normal MSCs markedly accelerated wound healing, as revealed by higher wound closure rate and better healing quality at 21 days post-wound. By contrast, MSCs administration increased the level of insulin as well as GLUT4 and PI3K/Akt signaling pathway expression but repressed the biochemical indexes of glucose and lipid, resulting in obvious metabolic improvement. CONCLUSIONS: These findings suggest that IGF-1 is an important paracrine factor that mediates the therapeutic effects of MSCs on wound healing in diabetes, and the benefits of MSCs may be associated with metabolism improvements, which would provide a new target for treatment.

MSC attenuate diabetes-induced functional impairment in adipocytes via secretion of insulin-like growth factor-1.

The function of subcutaneous adipocytes in promoting wound healing is significantly suppressed in diabetic wounds. Recent studies have demonstrated the ability of mesenchymal stem cell (MSC) to ameliorate impaired diabetic wound healing. We hypothesized that MSC function may involve subcutaneous adipocytes. The abnormal function of subcutaneous adipocytes from STZ induced diabetic mice including glucose uptake and free fatty acid (FFA) secretion level were assessed. Then these cells were co-cultured with MSC via a transwell system to observe the changes of metabolic index and glucose transporter four (GLUT4) as well as phosphoinositide 3-kinase/protein kinase (PI3K/AKT) signaling pathway expression. The results of metabolic index suggest that MSC obviously attenuated the diabetes-induced functional impairment. Both mRNA and protein expression analyses showed that PI3K/AKT insulin signaling pathway and GLUT4 expression were up-regulated. These changes were substantially associated with a increased level of insulin-like growth factor-1 (IGF-1) secretion from MSC. These findings suggest that MSC could attenuate abnormal function of diabetic adipocytes by IGF-1secretion, which was more or less associated with the beneficial effects of MSC on improving diabetic wound healing.

Angiogenic Effect of Mesenchymal Stem Cells as a Therapeutic Target for Enhancing Diabetic Wound Healing.

Impaired wound-healing activity in diabetes could result from several factors, including severely damaged angiogenic responses, which can affect wound healing process to cause delayed wound repair. Mesenchymal stem cells (MSCs) have been shown to enhance wound healing via multiple effects, including promoting angiogenesis both in vitro and in vivo; however, the mechanisms involved in enhancing diabetic wound healing are barely understood. This article reviews the recent literatures on MSCs treatment for promoting angiogenesis or vascularization in diabetic wounds and the potential mechanisms involved, with an emphasis on the role of paracrine soluble factors. Meanwhile, the potential benefits and related risks associated with the therapeutic use of MSCs have been presented and may lead to better understanding of the influence of MSCs without increasing potential risks. Further investigation will be required to determine the molecular basis of paracrine mechanisms and regulated angiogenesis of MSCs for its rational manipulation for impaired angiogenesis repair and diabetic wound healing.

Paracrine factors from mesenchymal stem cells: a proposed therapeutic tool for acute lung injury and acute respiratory distress syndrome.

Despite extensive researches in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), current pharmacological therapies and respiratory support are still the main methods to treat patients with ALI and ARDS and the effects remain limited. Hence, innovative therapies are needed to decrease the morbidity and mortality. Because of the proven therapeutic effects in other fields, mesenchymal stem cells (MSCs) might be considered as a promising alternative to treat ALI and ARDS. Numerous documents demonstrate that MSCs can exert multiple functions, such as engraftment, differentiation and immunoregulation, but now the key researches are concentrated on paracrine factors secreted by MSCs that can mediate endothelial and epithelial permeability, increase alveolar fluid clearance and other potential mechanisms. This review aimed to review the current researches in terms of the effects of MSCs on ALI and ARDS and to analyse these paracrine factors, as well as to predict the potential directions and challenges of the application in this field.

Platelet rich plasma clot releasate preconditioning induced PI3K/AKT/NFκB signaling enhances survival and regenerative function of rat bone marrow mesenchymal stem cells in hostile microenvironments.

Mesenchymal stem cells (MSCs) have been optimal targets in the development of cell based therapies, but their limited availability and high death rate after transplantation remains a concern in clinical applications. This study describes novel effects of platelet rich clot releasate (PRCR) on rat bone marrow-derived MSCs (BM-MSCs), with the former driving a gene program, which can reduce apoptosis and promote the regenerative function of the latter in hostile microenvironments through enhancement of paracrine/autocrine factors. By using reverse transcription-polymerase chain reaction, immunofluorescence and western blot analyses, we showed that PRCR preconditioning could alleviate the apoptosis of BM-MSCs under stress conditions induced by hydrogen peroxide (H2O2) and serum deprivation by enhancing expression of vascular endothelial growth factor and platelet-derived growth factor (PDGF) via stimulation of the platelet-derived growth factor receptor (PDGFR)/PI3K/AKT/NF-κB signaling pathways. Furthermore, the effects of PRCR preconditioned GFP-BM-MSCs subcutaneously transplanted into rats 6 h after wound surgery were examined by histological and other tests from days 0-22 after transplantation. Engraftment of the PRCR preconditioned BM-MSCs not only significantly attenuated apoptosis and wound size but also improved epithelization and blood vessel regeneration of skin via regulation of the wound microenvironment. Thus, preconditioning with PRCR, which reprograms BM-MSCs to tolerate hostile microenvironments and enhance regenerative function by increasing levels of paracrine factors through PDGFR-α/PI3K/AKT/NF-κB signaling pathways would be a safe method for boosting the effectiveness of transplantation therapy in the clinic.