Ferroptosis-dependent breast cancer cell-derived exosomes inhibit migration and invasion of breast cancer cells by suppressing M2 macrophage polarization.

Aim: Ferroptosis, a novel type of iron-dependent cell death, plays a vital role in breast cancer progression. However, the function of ferroptosis-induced cancer cell-derived exosomes in breast cancer remains unclear. In this study, we attempted to investigate the impact of breast cancer cells-derived exosomes induced by ferroptosis on the polarization of macrophages and the progression of breast cancer. Methods: Erastin was used to induce ferroptosis and breast cancer cell-derived exosomes were identified by transmission electron microscopy. Western blot, quantitative reverse transcription PCR, immunofluorescence, flow cytometry, and ELISA were used to determine the role of exosomes in macrophage polarization. Transwell assays were used to detect breast cancer cell migration, and invasion. Results: Our results showed that erastin promoted ferroptosis in breast cancer cells with increased Fe2+ level and ROS production. Breast cancer cell-derived exosomes induced by ferroptosis were successfully isolated and verified to be internalized by macrophages. In addition, ferroptosis-induced breast cancer cell-derived exosomes (Fe-exo) remarkably diminished M2 marker, Arg-1 expression. The ratio of CD206+ macrophages was significantly decreased after Fe-exo treatment. CD206 protein expression and Arg-1 level were dramatically reduced in M2 macrophages incubated by Fe-exo. Moreover, autophagy PCR array showed that the expression of 84 autophagy-related genes were altered after macrophages were incubated by Fe-exo. Furthermore, macrophages incubated by Fe-exo repressed the migration and invasion of breast cancer cells. Conclusion: Ferroptosis-dependent cancer cell-derived exosomes inhibited M2 polarization of macrophages, which in turn inhibited migration and invasion of breast cancer cells. This study provides novel therapeutic strategies for patients with breast cancer.

Low-numerical aperture confined-doped long-tapered Yb-doped silica fiber for a single-mode high-power fiber amplifier.

A low-numerical aperture (NA) confined-doped long-tapered (LCT) Yb-doped fiber is proposed and fabricated by modified chemical vapor deposition combined with solution doping technique. The LCT fiber owns the core NA of ∼0.05 and the gain dopant doping diameter ratio of ∼77%, with a core/cladding diameter of 25/400 µm at both ends and 37.5/600 µm in the middle. The laser performance is demonstrated by a bidirectional pumping all-fiber amplifier, of which a 4.18-kW single-mode (M2 factor ∼1.3) laser output is achieved with a slope efficiency of ∼82.8%. Compared with the conventional fiber, the co-pumped and counter-pumped transverse mode instability thresholds and beam quality of the LCT fiber are remarkably enhanced. Throughout the continuous operation, the LCT fiber amplifier presents high power stability with fluctuation of < 1%. These results indicate that LCT fiber has great potential in power scaling remaining excellent beam quality.

Helicobacter pylori promotes gastric cancer progression by upregulating semaphorin 5A expression via ERK/MMP9 signaling.

Helicobacter pylori (H. pylori) infection is the strongest risk factor for the occurrence and development of gastric carcinoma. However, the molecular mechanism underlying H. pylori-induced pathogenesis has not yet been fully characterized. Here, we explored whether H. pylori upregulates semaphorin 5A to promote gastric cancer progression via the extracellular regulated protein kinases/matrix metalloproteinase (ERK/MMP9) signaling pathway. In this study, H. pylori upregulated semaphorin 5A expression in vitro and in vivo. Using the human gastric carcinoma cell lines SGC7901, SGC7901-siScrambled, and SGC7901-siSema 5A, our studies showed that H. pylori increased the proliferation, growth, migration, and invasiveness of gastric cancer cells via its effects on semaphorin 5A and that H. pylori increased the expression of MMP9 in gastric cancer cells via the semaphorin 5A-mediated ERK signaling pathway. Further analysis revealed that the ERK inhibitor PD98059 and MMP9 antibody (Ab) attenuated H. pylori-induced gastric cancer cell invasion and metastasis in vitro through a semaphorin 5A-dependent mechanism. In conclusion, H. pylori could promote gastric cancer progression in a semaphorin 5A-dependent manner via the ERK/MMP9 signaling pathway. Semaphorin 5A and its related signaling molecules potentially represent latent targets for H. pylori-related gastric cancer therapy.

Clinical characteristics and management of primary granulocytic sarcoma of the breast: A case report.

RATIONALE: Primary granulocytic sarcoma of the breast is a rare and poor-prognosis malignancy. Clinicians do not have sufficient knowledge of this disease and often misdirect it as other soft tissue sarcomas or inflammation. PATIENT CONCERNS: A 42-year-old female presented with a self-discovered asymptomatic growing and palpable right breast mass that had been present for 4 months. DIAGNOSES: The patient was diagnosed as primary myeloid sarcoma. INTERVENTIONS: The patient received modified radical mastectomy in the right breast and sentinel lymph node biopsy. Pathological diagnosis is primary granulocytic sarcoma. Then the patient accepted acute myeloid leukemia-induction chemotherapy. OUTCOMES: The follow-up of this patient has no evidence of disease progression or spread during 1 year. LESSONS: Granulocytic sarcoma in the breast tissue is rare. But it still should be considered in the differential diagnosis of any tumor in the breast. The present study discusses comprehensively the clinical and pathological characteristics to improve the understanding of myeloid sarcoma.

Trends of incidence and survival in patients with gastrointestinal mucinous adenocarcinoma.

The epidemiology and clinical outcome of gastrointestinal mucinous adenocarcinoma (MA) are not well illustrated. The present study aimed to explore the evolving epidemiology and prognostic factors that affect the survival of patients with MA in the gastrointestinal tract. A retrospective and population-based study was conducted to determine the annual age-adjusted incidence, overall survival (OS) and survival trend of gastrointestinal mucinous MA using nationally representative data from the Surveillance, Epidemiology, and End Results (SEER) program between 2000 and 2014. A Kaplan-Meier curve and a Cox proportional regression model were used to evaluate prognostic factors for this disease. Of the 51632 cases, females accounted for 50.5% (26058). The annual incidence of MA steadily decreased from 2000 to 2014. This trend occurred across all stages, grades and sites, apart from the appendix. In the SEER 18 registry grouping (2000-2014), the highest incidence was 3.333 per 100,000 persons for the colon. The median OS varied significantly between different primary sites, stages, grades, and age of clinical diagnosis, and the time period of diagnosis, according to a multivariable analysis. The five-year OS of gastrointestinal MA improved gradually between 2000 and 2014. The improvement in survival over the same interval was more pronounced in the subgroup of distant gastrointestinal MA. All sites along the alimentary tract, with the exception of the appendix, showed a decrease in the incidence of MA. Improved survival rates were observed for most of the gastrointestinal tract, especially for patients with advanced stage disease. MA in the upper gastrointestinal tract was less frequent but had poorer survival than colorectal MA. Clinicians should consider the primary tumour site when making therapeutic guidelines and treatment decisions for gastrointestinal MA.

Marital Status and Survival of Patients with Hormone Receptor-Positive Male Breast Cancer: A Surveillance, Epidemiology, and End Results (SEER) Population-Based Study.

BACKGROUND Although marital status has been reported as a prognostic factor in different cancer types, its prognostic effect on hormone receptor (HR) positive male breast cancer (MBC) is unclear. The objective of the present analysis was to assess the effects of marital status on survival in patients with HR positive MBC. MATERIAL AND METHODS Patients diagnosed with HR positive MBC from 1990 to 2014 in the Surveillance, Epidemiology, and End Results (SEER) database were included. Kaplan-Meier survival analysis and Cox proportional hazard regression were used to identify the effects of marital status on cancer-specific survival (CSS) and overall survival (OS). RESULTS A total of 3612 cases were identified in this study. Married patients had better 5-year CSS and 5-year OS than unmarried men. In multivariate Cox regression models, unmarried patients also showed higher mortality risk for both CSS and OS, independent of age, race, grade, stage, PR status, HER2 status, and surgery. Subgroup survival analysis according to different ER/PR status showed that married patients had beneficial CSS results only in ER+/PR+ subtype, and CSS in the married and unmarried groups did not significantly differ by TNM stage. The results were further confirmed in the 1: 1 matched group. CONCLUSIONS Marital status was an important prognostic factor for survival in patients with HR positive MBC. Unmarried patients are at greater risk of death compared with married groups. The survival benefit for married patients remained even after adjustment, which indicates the importance of spousal support in MBC.

Differentiation of rat adipose-derived mesenchymal stem cells into corneal-like epithelial cells driven by PAX6.

Corneal integrity, transparency and vision acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by corneal limbal stem cells (LSCs). Deficiency of CECs and/or LSCs is associated with numerous ocular diseases. Paired box (PAX)6 is an eye development-associated transcription factor that is necessary for cell fate determination and differentiation of LSCs and CECs. In the present study, the PAX6 gene was introduced into adipose-derived rat mesenchymal stem cells (ADMSCs) to investigate whether PAX6-transfected cells were able to transdifferentiate into corneal-like epithelial cells and to further verify whether the cells were suitable as a cell source for corneal transplantation. The ADMSCs were isolated from the bilateral inguinal region of healthy Sprague Dawley rats. The characteristics of ADMSCs were identified using flow cytometric analysis. After subculture, ADMSCs underwent transfection with recombinant plasmid containing either PAX6-enhanced green fluorescent protein (EGFP) complementary (c)DNA or EGFP cDNA (blank plasmid group), followed by selection with G418 and determination of the transfection efficiency. Subsequently, the morphology of the ADMSCs and the expression profiles of corneal-specific markers CK3/12 and epithelial-specific adhesion protein were determined. E-cadherin was detected using immunofluorescence staining and western blot analysis at 21 days following transfection. An MTT cell proliferation and a colony formation assay were performed to assess the proliferative activity and clonogenicity of PAX6-transfected ADMSCs. Finally, the PAX6-expressing ADMSCs were transplanted onto the cornea of a rabbits with limbal stem cell deficiency (LSCD). At 21 days after transfection, the ADMSCs with PAX6 transfection exhibited a characteristic flagstone-like appearance with assembled corneal-like epithelial cells, and concomitant prominent expression of the corneal-specific markers cytokeratin 3/12 and E-cadherin. Furthermore, the proliferation and colony formation ability of PAX6-overexpressing ADMSCs was significantly retarded. The transplantation experiment indicated that PAX6-reprogramed ADMSCs attached to and replenished the damaged cornea via formation of stratified corneal epithelium. Taken together, these results suggested that conversion of ADMSCs into corneal-like epithelium may be driven by PAX6 transfection, which makes ADMSCs a promising cell candidate for the treatment of LSCD.

PLD4 promotes M1 macrophages to perform antitumor effects in colon cancer cells.

Phospholipase D4 (PLD4) is a newly identified protein expressed in microglia. However, the function of PLD4 in tumor-associated macrophages (TAMs) is unknown. In the present study, we revealed that the expression of PLD4 was located in macrophages in the colon cancer mesenchymal and lymph nodes as shown by immunohistochemical analysis. furthermore, its expression was associated with clinical staging of colon cancer. Then, THP-1 as a cell model induced into TAMs. Western blot and RT-PCR analysis showed that PLD4 was mainly presented in M1 phenotype TAMs. The secretion of pro-inflammatory cytokines in M1 macrophages was significantly reduced after the expression of PLD4 inhibited by PLD4-siRNA. Furthermore, co-cultured with condition-medium from control or PLD4-siRNA M1 macrophages for 24 h, cell apoptosis, cycle and proliferation of cancer cells improved compared to control. These results indicated that PLD4 could be involved in the activation process of M1 phenotype macrophages.

Mesenchymal stem cell-mediated suppression of hypertrophic scarring is p53 dependent in a rabbit ear model.

INTRODUCTION: Mesenchymal stem cells (MSCs) are considered to play important roles in wound repair and tissue remodeling. Hypertrophic scar (HTS) is a cutaneous condition characterized by deposits of excessive amount of collagen after an acute skin injury. However, currently there is little knowledge about the direct relationship between MSCs and HTS. METHODS: The hypertrophic scar model was established on rabbit ears. MSCs were isolated from rabbit femur bone marrow and transplanted through ear artery injection. Hypertrophic scar formation was examined using frozen-section analysis, hematoxylin and eosin (HE) staining, Masson's trichrome staining, and scar elevation index. The role of p53 in the MSCs-mediated anti-scarring effect was examined by gene knockdown using p53 shRNA. RESULTS: In this study, MSCs engraftment through ear artery injection significantly inhibited the hypertrophic scarring in a rabbit ear hypertrophic scar model, while this anti-scarring function could be abrogated by p53 gene knockdown in MSCs. Additionally, we found that MSCs down-regulated the expression of TGF-ß receptor I (TßRI) and alpha-smooth muscle actin (α-SMA) at both mRNA and protein levels in a paracrine manner, and this down-regulation was rescued by p53 gene knockdown. Moreover, our results showed that MSCs with p53 gene knockdown promoted the proliferation of fibroblasts through increasing nitric oxide (NO) production. CONCLUSIONS: These results suggest that MSCs inhibit the formation of HTS in a p53 dependent manner through at least two mechanisms: inhibition of the transformation of HTS fibroblast to myofibroblast; and inhibition of the proliferation of fibroblasts through inhibition of NO production.

[Differentiation of adipose-derived mesenchymal stem cells after transfection with Pax6 gene].

OBJECTIVE: To investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene. METHODS: The adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs. The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A), pcDNA3.1 empty vector transfection (group B), and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C), respectively. At 48 hours after transfection, the cells in groups B and C were selected with G418. The cell morphology changes were observed under the inverted microscope. Pax6 protein and level of corneal epithelial cells specific molecular--cytokeratin 12 (CK-12) were measured by Western blot. Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12. RESULTS: No morphology change was observed in groups A and B. Two different cell clones were found in group C. No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells; No.2 selected clone showed a net-like appearance, with 3-7 cell processes. The Western blot results showed the Pax6 protein expression in 2 clones of group C, but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C, and no expression in the others. The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64 ± 0.73, which was significantly higher than that of No.2 selected clone of group C (0.55 ± 0.42), group B (1.36 ± 0.40), and group A (1.00 ± 0.00) (P < 0.05), and there was no significant difference among groups A, Band No.2 selected clone of group C (P > 0.05). CONCLUSION: Pax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels. This result provides a promising strategy of generating corneal epithelium-like cells for construction of tissue engineered cornea.

Expression, purification of recombinant human mitochondrial transcription termination factor 3 (hMTERF3) and preparation of polyclonal antibody against hMTERF3.

In mammalian cells, a family of mitochondrial transcription termination factors (MTERFs) regulates mitochondrial gene expression. Mitochondrial transcription termination factor 3 (MTERF3) is the most conserved member of the MTERF family and a negative regulator of mammalian mitochondrial DNA transcription. To create a specific polyclonal antibody against human MTERF3 (hMTERF3), we first cloned hMTERF3 into prokaryotic expression vector pGEX-4T-1, and GST-hMTERF3 was efficiently expressed in Escherichia coli after induction by IPTG. The expressed GST-tagged hMTERF3 fusion protein was purified by passive electro-elution process and then used to immunize BALB/c mice, we obtained anti-GST-hMTERF3 polyclonal antibody purified by protein A column and determined the sensitivity and specificity of the antibody against human MTERF3 by enzyme-linked immunosorbent assay and Western blot assay. Furthermore, the full-length hMTERF3 protein expressed in human embryonic kidney 293T cells was detected by anti-GST-hMTERF3 in western blot analysis and immunofluorescence staining. Taken together, our results demonstrate the functionality of the mouse anti-GST-hMTERF3 polyclonal antibody which will provide a useful tool for further characterization of hMTERF3.

Clinical features and molecular phenotypes of breast cancer in patients with type-2 diabetes mellitus.

OBJECTIVE: To investigate the clinical features, molecular phenotypes and clinical prognosis of breast cancer patients with type-2 diabetes mellitus, thereby providing a basis for individualized therapy of breast cancer. METHODS: 105 breast cancer patients with type-2 diabetes mellitus (DM) presenting from January 2005 to December 2010 were enrolled in this study. 200 breast cancer non-diabetic patients in the same period were randomly selected as the control group. The clinical data of DM group and control group were retrospectively analyzed. The SPSS12.0 software was used for statistics and survival analysis. RESULTS: The mean age of the patients in DM group were of 57.2∓11.8 years, which was older compared with the control group. The percentage of postmenopausal patients was 71.4% and the ratio of grade II+III was 98.8%, which was higher than the control group. The neoadjuvant chemotherapy response rate of DM group was 67.5%, which was lower than control group. The patients in DM group had later clinical stage and more lymph metastasis. The proportion of advanced breast cancer was 68.57% and the ratio of lymph node metastasis was 66.01%. All the difference was significant (P<0.05). But there was no significant difference in tumor size and molecular phenotype between the diabetic group with breast cancer and the control group. Disease-free survival and overall survival rates of DM group were 80.2% and 84.2%, which were worse than those in the control group. All the differences were significant (P<0.05). After excluding the patients with other causes of death, results of overall survival still showed worse in DM group, but the difference was not statistically significant (P>0.05). Serum insulin at fasting and two hours postprandial were higher than normal value in DM group, but serum insulin levels in the control group changed in the normal range. CONCLUSION: There were older patients, higher proportion of high pathological grade, more lymph node metastasis, later clinical stages in the diabetic group with breast cancer. Breast cancer patients with type-2 diabetes mellitus were at risk of a poor prognosis. Hyperinsulinemia may be the real cause of poor prognosis in breast cancer patients with type-2 diabetes.

Vaccination of goats with 31 kDa and 32 kDa Schistosoma japonicum antigens by DNA priming and protein boosting.

Two Schistosoma japonicum vaccine candidate antigens Sj 31 and Sj 32, which have shown particular promise to induce protective immunity in mice, were used to immunize goats by using a DNA priming-protein boosting strategy in present work. DNA vaccine formulations of the two antigens (VRSj31 and VRSj32) were produced and injected intramuscularly twice at a 2-week interval and then recombinant proteins (rSj31 and rSj32) together with Freund Complete Adjuvant (FCA) were used to boost the goats. The experiment was repeated in different batche cercariae. A strong anamnestic antibody response was induced after boost. A significant reduction of liver egg counts and miracidial hatching was showed in both experiments. Significant protections against challenge infection were elicited with 31.6% of percentage reduction for worm recovery in the second experiment and 20.9% in the first experiment, respectively.