IL-33/ST2 Axis Regulates Vasculogenic Mimicry via ERK1/2-MMP-2/9 Pathway in Melanoma.

BACKGROUND: Melanoma, an extremely malignant form of cancer, poses a significant health risk. Vasculogenic mimicry (VM), blood vessels formed by tumor cells instead of endothelial cells, is an important factor for the rapid progression of melanoma. Interleukin (IL)-33 is an inflammatory factor commonly found in the tumor microenvironment and plays an important role in the progression of many tumors. IL-33 acts on immune cells and tumor cells through its receptor ST2. This study hypothesized that IL-33 directly affects the progression of melanoma. OBJECTIVES: This study was designed to investigate the effect of IL-33 on VM of melanoma and its potential mechanism of action. METHODS: The expression of ST2 was evaluated in 66 cases of melanoma collected from human patients, and the differences were analyzed. In vitro experiments were conducted to study the effects of the IL-33/ST2 axis on cell migration and invasion and to elucidate possible mechanisms. RESULTS: ST2 expression is associated with that of matrix metalloproteinase (MMP)-2 and VM in melanoma of patients. IL-33 increases the abilities of proliferation, migration and invasion of melanoma cells and VM tube formation through ST2. IL-33 induces the production of MMP-2/9 via ERK1/2 phosphorylation. CONCLUSION: IL-33 can directly act on melanoma cells and promote its development.

Twist1 expression induced by sunitinib accelerates tumor cell vasculogenic mimicry by increasing the population of CD133+ cells in triple-negative breast cancer.

BACKGROUND: Hypoxia induced by antiangiogenic agents is linked to the generation of cancer stem cells (CSCs) and treatment failure through unknown mechanisms. The generation of endothelial cell-independent microcirculation in malignant tumors is defined as tumor cell vasculogenic mimicry (VM). In the present study, we analyzed the effects of an antiangiogenic agent on VM in triple-negative breast cancer (TNBC). METHODS: Microcirculation patterns were detected in patients with TNBC and non-TNBC. Tientsin Albino 2 (TA2) mice engrafted with mouse TNBC cells and nude mice engrafted with human breast cancer cell lines with TNBC or non-TNBC phenotypes were administered sunitinib and analyzed to determine tumor progression, survival, microcirculation, and oxygen concentration. Further, we evaluated the effects of hypoxia induced with CoCl2 and the expression levels of the transcription factor Twist1, in the presence or absence of a Twist siRNA, on the population of CD133(+) cells and VM in TNBC and non-TNBC cells. RESULTS: VM was detected in 35.8 and 17.8% of patients with TNBC or with non-TNBC, respectively. The growth of tumors in TNBC and non-TNBC-bearing mice was inhibited by sunitinib. The tumors in TA2 mice engrafted with mouse TNBCs and in mice engrafted a human TNBC cell line (MDA-MB-231) regrew after terminating sunitinib administration. However, this effect was not observed in mice engrafted with a non-TNBC tumor cell line. Tumor metastases in sunitinib-treated TA2 mice was accelerated, and the survival of these mice decreased when sunitinib was withdrawn. VM was the major component of the microcirculation in sunitinib-treated mice with TNBC tumors, and the population of CD133+ cells increased in hypoxic areas. Hypoxia also induced MDA-MB-231 cells to express Twist1, and CD133(+) cells present in the MDA-MB-231 cell population induced VM after reoxygenation. Moreover, hypoxia did not induce MDA-MB-231 cells transfected with an sh-Twist1 siRNA cell to form VM and generate CD133(+) cells. Conversely, hypoxia induced MCF-7 cells transfected with Twist to form VM and generate CD133+ cells. CONCLUSIONS: Sunitinib induced hypoxia in TNBCs, and Twist1 expression induced by hypoxia accelerated VM by increasing population of CD133(+) cells. VM was responsible for the regrowth of TNBCs sunitinib administration was terminated.

HER2/neu expression correlates with vasculogenic mimicry in invasive breast carcinoma.

Vasculogenic mimicry (VM) refers to the condition in which tumour cells mimic endothelial cells to form extracellular matrix-rich tubular channels. VM is more extensive in more aggressive tumours. The human epidermal growth factor receptor 2 (HER2) gene is amplified in 20-30% of human breast cancers and has been implicated in mediating aggressive tumour growth and metastasis. However, thus far, there have been no data on the role of HER2 in VM formation. Immunohistochemical and histochemical double-staining methods were performed to display VM in breast cancer specimens. Transfection in MCF7 cells was performed and clones were selected by G418. The three-dimensional Matrigel culture was used to evaluate VM formation in the breast cancer cell line. According to statistical analysis, VM was related to the presence of a positive nodal status and advanced clinical stage. The positive rate of VM increased with increased HER2 expression. In addition, cases with HER2 3+ expression showed significantly greater VM channel count than those in other cases. The exogenous HER2 overexpression in MCF-7 cells induced vessel-like VM structures on the Matrigel and increased the VM mediator vascular endothelial (VE) cadherin. Our data provide evidence for a clinically relevant association between HER2 and VM in human invasive breast cancer. HER2 overexpression possibly induces VM through the up-regulation of VE cadherin. Understanding the key molecular events may provide therapeutic intervention strategies for HER2+ breast cancer.

Clinical characteristics, allergic response to autologous semen, and desensitization in patients with postorgasmic illness syndrome.

Introduction: Postorgasmic illness syndrome (POIS) is rare and includes a cluster of physical and cognitive symptoms that occur after ejaculation. The pathogenesis and effective treatments remain unclear. Aim: This study aimed to characterize the symptomatology of POIS, study the allergic response of autologous semen in patients and controls, and evaluate the effects of desensitization therapy. Methods: The clinical characteristics of 24 Chinese patients with POIS were analyzed. Skin prick tests, intracutaneous tests, and specific IgE detection were performed with autologous semen. Five patients were desensitized via subcutaneous injections of autologous semen. Outcomes: Evaluated outcomes included the clinical features of POIS; scores of the Self-rating Anxiety Scale (SAS), Self-rating Depression Scale (SDS), and visual analog scale (VAS) of symptoms; skin reactions; desensitization with diluted autologous seminal fluid; and the IgE reactivity patterns of immunoblotting and enzyme-linked immunosorbent assay in vitro. Results: The most common symptom cluster was the general cluster, and the most prevalent symptoms were extreme fatigue and inattention. A total of 66.67% (14/21) of the patients had no symptoms or milder symptoms after nocturnal emission than after intercourse or masturbation. Of the patients, 87.5% (21/24) had psychiatric symptoms and 53.85% (7/13) had abnormal sex hormone levels. The SAS and SDS scores of the high and low VAS groups were significantly higher than those of the control group. Pearson analysis showed that the correlation coefficient between the SAS and VAS was 0.607 (P < .01) and that between the SDS and VAS was 0.490 (P < .05). The patients and healthy donors all had positive intracutaneous test results with their own semen, negative skin prick test results, and no IgE specific to autologous semen. Most patients (4/5) did not achieve ideal therapeutic effects with desensitization. Clinical Implications: Allergy is not the main pathogenesis of POIS, and desensitization with autologous semen is not effective for most patients. Strengths and Limitations: This project included the largest number of patients with POIS in China and assessed the allergic response to autologous semen and the effect of desensitization therapy. There is no objective method for evaluating the efficacy of desensitization with autologous semen. Conclusions: IgE-mediated semen allergy is not the main pathogenesis of POIS, and there is a positive chance that POIS is related to psychological factors. Most patients do not respond to desensitization with autologous semen, and POIS treatment should be individualized, especially in cases with uncertain causes.

Promotion of tumor cell metastasis and vasculogenic mimicry by way of transcription coactivation by Bcl-2 and Twist1: a study of hepatocellular carcinoma.

UNLABELLED: The antiapoptotic protein Bcl-2 plays multiple roles in apoptosis, immunity, and autophagy. Its expression in tumors correlates with tumor grade and malignancy. The recapitulation of the normal developmental process of epithelial-mesenchymal transition (EMT) contributes to tumor cell plasticity. This process is also a characteristic of metastatic cells and vasculogenic mimicry. In the present study we report functional and structural interactions between Bcl-2 and the EMT-regulating transcription factor Twist1 and the relationship with metastasis and vascular mimicry. Bcl-2 and Twist1 are coexpressed under hypoxia conditions. The Bcl-2 can bind to Twist1 in vivo and in vitro. This interaction involves basic helix-loop-helix DNA binding domain within Twist1 and through two separate domains within Bcl-2 protein. Formation of the Bcl-2/Twist1 complex facilitates the nuclear transport of Twist1 and leads to transcriptional activation of wide ranges of genes that can increase the tumor cell plasticity, metastasis, and vasculogenic mimicry. Finally, nuclear expression of Bcl-2 and Twist1 is correlated with poor survival of these patients in a cohort of 97 cases of human hepatocellular carcinoma. CONCLUSION: The results describe a novel function of Bcl-2 in EMT induction, provide insight into tumor progression, and implicate the Bcl-2/Twist1 complex as a potential target for developing chemotherapeutics.

Bioinformatic Analysis and Cellular Assays Identify Substance P Influencing Th17/Treg Differentiation via the MyD88 Pathway as a Potential Contributor to the Progression of Asthma and Allergic Rhinitis.

OBJECTIVE: This study is aimed at investigating the role of substance P (SP) in the development of asthma. METHODS: The Gene Expression Omnibus (GEO) database was used to characterize SP expression in allergic rhinitis (AR) and asthma. Peripheral blood was collected from patients with asthma or AR. The expression of relevant cytokines and neuropeptides was measured. Enzyme-linked immunosorbent assay (ELISA) was also performed. The mast cell line LAD2 and the lung bronchial epithelial cell line BEAS-2B were treated with different concentrations of SP concentration. Then, the qRT-PCR method was used to determine the mRNA expression. Furthermore, p38 and p65 and their associated phosphorylated proteins (p-p38 and p-p65) were further validated by western blotting. RESULT: Clinical and GSE75011 data analysis suggested that MyD88 expression was upregulated in AR and asthma. Through the gene set variation analysis (GSVA), MyD88-related pathways were noticed and further investigated. ELISA results suggested that the SP expression was significantly increased in AR and asthma and IL-10 expression was decreased, whereas the expression of IL-6, IL-17A, IL-23, and TGF-ß expressions increased. The mast cell line LAD2 was treated with different SP concentrations, and ELISA results showed that the expression of IL-6, IL-17A, IL-23, and TGF-ß in the cell supernatant gradually increased with increasing SP concentrations, whereas that of IL-10 decreased. The lung bronchial epithelial cell line BEAS-2B was treated with different SP concentrations, and the expression of myeloid differentiation factor 88 (MyD88) and its related proteins was elevated. The expression of p38 and p-p38 proteins was elevated after SP treatment, and their expression levels elevated as SP concentrations increased. Finally, MyD88 expression at the single-cell level was also demonstrated. CONCLUSION: SP may affect the cytokine expression through the MyD88 pathway, thereby influencing Th17/Treg differentiation and eventually participating in the pathological process of asthma and AR. There are many pathological similarities between allergic rhinitis (AR) and bronchial asthma. In the present study, SP was found to possibly activate downstream inflammatory signaling pathways via MyD88, thereby affecting Th17/Treg differentiation and ultimately participating in the pathological process of asthma and AR.

MiRNA-221-5p suppressed the Th17/Treg ratio in asthma via RORγt/Foxp3 by targeting SOCS1.

BACKGROUND: This study was designed to investigate the mechanism and effects of miRNA-221-5p on the T-helper 17 (Th17)/T-regulatory (Treg) ratio in asthma. METHODS: BALB/c mice were intranasally challenged with 100 µg OVA on 14 and 21 day. Mice were rechallenged with 2.5% OVA-PBS on 22 and 28 day. Mice were sacrificed using on day 30 under 35 mg/kg pentobarbital sodium. PBMCs were induced vitro model of asthma using 500 ng of lipopolysaccharides (LPS) for 4 h. RESULTS: The expression of miRNA-221-5p was reduced in in vivo model, compared sham group. The vitro model of asthma treated with miRNA-221-5p mimic resulted in the reduction of IL-6, IL-17, IL-21 and IL-22 levels, and induction of IL-10, IL-35 and TGF-ß levels. In addition, down-regulation of miRNA-221-5p induced the protein expression of suppressor of cytokine signaling 1 (SOCS1) and receptor-related orphan receptor-gamma-t (RORγt) and suppressed that of FOXP3 in in vitro model of asthma. Over-expression of miRNA-221-5p induced the protein expression of FOXP3, and suppressed that of SOCS1 and RORγt in in vitro model of asthma. The inhibition of SOCS1 or RORγt attenuated the effects of anti-miRNA-221-5p on Th17/Treg ratio in asthma. CONCLUSION: miRNA-221-5p may play critical roles in driving the differentiation of Th17/Treg ratio via RORγt/Foxp3 by Targeting SOCS1, reduced the function of Th17 cells by directly inhibiting RORγt/SOCS1 and promoted the function of Treg cells via Foxp3/ SOCS1 in asthma.

Genetic diagnosis in a Chinese Hailey-Hailey disease pedigree with novel ATP2C1 gene mutation.

Hailey-Hailey disease (HHD) is an autosomal dominant skin disorder characterized by recurrent eruption of vesicles and bullae at the sites of friction and in the intertriginous areas. Mutations in the ATP2C1 gene encoding the human secretory pathway calcium ATPase 1 (hSPCA1) have been identified as the causative mutations in HHD. In this study, we used direct sequencing and restriction endonuclease digestion to analyze mutations of the ATP2C1 gene in a Chinese three-generation pedigree. A heterozygous T-to-C transition at nucleotide 1004 in exon 12 of ATP2C1 gene was detected. After summarizing the reported cases with ATP2C1 mutation, we concluded that the T1004C transition resulted in a novel missense mutation of leucine condon (CTG) to proline (CCG) at amino acid residue 335(L335P) in hSPCA1. Here, a genetic diagnosis was made for the proband's daughter before the clinical presentation. The study realized the molecular diagnosis in the HHD pedigree. Our findings should be useful for genetic counseling and prenatal diagnosis for the affected family and in demonstrating the critical role of the ATP2C1 gene in the pathogenesis of HHD further.

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed.

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.

The in-vitro spheroid culture induces a more highly differentiated but tumorigenic population from melanoma cell lines.

Cancer stem cells (CSCs) have been identified in various malignancies, and different properties have been examined to characterize CSCs: tumorigenicity in immunocompromised mice, stem cell surface markers, label-retaining properties, and proliferation as nonadherent spheres. This study explored the consistency and efficiency among these methods. Among the melanoma cell lines examined (A375, A875, MUM-2b, and MUM-2c), only A375 and MUM-2c grew as nonadherent spheres and continuously propagated in a defined serum-free medium in vitro. Flow cytometry and immunofluorescence analysis indicated that sphere-derived cells contained a smaller proportion of cells expressing the candidate surface markers of melanoma stem cells such as ABCB5, CD133, CD20 and CD271, and a larger proportion of cells expressing melanocytic differentiation markers such as HMB45 and S100 protein, compared with adherent cells. Surprisingly, the more highly differentiated sphere-derived melanoma cells exhibited increased tumorigenic potential in vivo, as indicated by shorter tumor incubation (A375) and smaller number of cells required to initiate tumor formation (A375 and MUM-2c) compared with those of parental cells. Despite the similarity in histopathological characteristics, the expression profile indicated that xenografts derived from sphere-derived melanoma cells exhibited a more tumorigenic phenotype with respect to the stem or the differentiation markers detected by immunohistochemical analysis. Therefore, sphere formation in nonadherent cultures may not be a preferred surrogate in-vitro method for enriching melanoma stem cells according to candidate markers but may be a favorable condition for activating potential CSCs.

Hypoxia-induced senescence contributes to the regulation of microenvironment in melanomas.

Senescence, an irreversible state of cell cycle arrest, maintains metabolic activity. Although being a barrier against tumor development, senescence could also promote tumor progression by influencing the microenvironment. Necrosis is a common feature of various malignant tumors, which also has two opposing effects: pro-tumor by chronic inflammation and anti-tumor by effective cell clearance. However, the role of senescence in melanoma and whether it is associated with necrosis remain unclear. By detecting senescence-associated ß-galactosidase activity and pimonidazole (hypoxia probe), we found that senescent cells (SA-ß-gal positive) are mainly located around the necrotic/hypoxic areas of melanoma from C57BL/6J mice. Moreover, treatment of hypoxia induced irreversibly cellular senescence in vitro. In addition, the senescent cells may facilitate microenvironment modulation and promote the invasion of melanoma cells by secreting matrix metalloproteinase-2(MMP-2). Moreover, Kaplan-Meier analysis showed that the presence of necrosis in melanomas had an inverse correlation with patient survival and may serve as an independent prognostic marker. Therefore, hypoxic stress imposed on melanomas may lead to cellular senescence surrounding necrotic areas, and the adverse effects of necrosis in tumor may be attributed to the adjacent senescent cells with senescence-associated secretion phenotype (SASP), including secretion of MMP-2.

Hypoxia-induced vasculogenic mimicry formation via VE-cadherin regulation by Bcl-2.

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Hypoxia plays a pivotal role in the formation of VM. Hypoxia-induced Bcl-2 overexpression is observed in many types of tumors including melanoma, in which it is associated with tumorigenicity and angiogenesis. VE-cadherin, the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation, is also overexpressed in melanoma. Despite these connections, whether hypoxia induces VM formation via VE-cadherin regulation by Bcl-2 is not confirmed. We used human melanoma cells to upregulate or knockdown the expression of Bcl-2 to investigate the possible molecular mechanism of VM formation under hypoxia. Bcl-2 overexpression increased VE-cadherin expression and VM formation under normoxia, whereas Bcl-2 siRNA significantly decreased VE-cadherin expression and VM formation under hypoxia. We then demonstrated that Bcl-2 regulated VE-cadherin transcription activity by Western blot, three-dimensional cultures, reporter gene assay, and clinical analysis. Therefore, Bcl-2-dependent VE-cadherin overexpression may be an important mechanism by which hypoxia induces VM.

A pilot study on acute inflammation and cancer: a new balance between IFN-gamma and TGF-beta in melanoma.

Recent data have redefined the concept of inflammation as a critical component of tumor progression. However, there has been little development on cases where inflammation on or near a wound and a tumor exist simultaneously. Therefore, this pilot study aims to observe the impact of a wound on a tumor, to build a new mouse tumor model with a manufactured surgical wound representing acute inflammation, and to evaluate the relationship between acute inflammation or wound healing and the process of tumor growth. We focus on the two phases that are present when acute inflammation influences tumor. In the early phase, inhibitory effects are present. The process that produces these effects is the functional reaction of IFN-gamma secretions from a wound inflammation. In the latter phase, the inhibited tumor is made resistant to IFN-gamma through the release of TGF-beta to balance the inflammatory factor effect on the tumor cells. A pair of cytokines IFN-gamma/TGF-beta established a new balance to protect the tumor from the interference effect of the inflammation. The tumor was made resistant to IFN-gamma through the release of TGF-beta to balance the inflammatory effect on the tumor cells. This balance mechanism that occurred in the tumor cells increased proliferation and invasion. In vitro and in vivo experiments have confirmed a new view of clinical surgery that will provide more detailed information on the evaluation of tumors after surgery. This study also provides a better understanding of the relationship between tumor and inflammation, as well as tumor cell attacks on inflammatory factors.