MiR-7-5p suppresses stemness and enhances temozolomide sensitivity of drug-resistant glioblastoma cells by targeting Yin Yang 1.

Glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system, and chemoresistance blunts the effect of temozolomide (TMZ) in the treatment of GBM. Clarifying the underlying mechanism of chemoresistance might yield novel strategies to improve the patients' response to chemotherapeutics. Mounting evidence indicates that microRNAs (miRNAs) are involved in chemoresistance and tumorigenesis. At present, miR-7-5p has been recognized as a tumor suppressor involved in multiple cancers. However, the biological effects of miR-7-5p in TMZ resistance have not been illuminated. In this study, we used RNA sequencing and high-throughput screening techniques, which revealed that miR-7-5p is significantly downregulated in TMZ resistant LN229 cells (LN229/TMZ-R) compared to control cells (LN229), and low miR-7-5p expression was correlated with recurrence in GBM patients. Ectopic overexpression of miR-7-5p sensitized LN229/TMZ-R cells to TMZ and suppressed the stemness of glioblastoma stem cells (GSCs). Further experiments demonstrated that miR-7-5p exerts its role by directly targeting the 3'-untranslated region of Yin Yang 1 (YY1). Our findings suggest that combinational use of miR-7-5p and TMZ might be a promising therapeutic strategy to increase the long-term drug response in GBM patients.

d-amino acid modification protects N-Acetyl-seryl-aspartyl-lysyl-proline from physiological hydroxylation and increases its antifibrotic effects on hepatic fibrosis.

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development in the liver. However, its extremely short half-life in vivo greatly compromises its potential applications. Here, we report an Ac-SDKP analog peptide with d-amino acid replacement (Ac-SDD KD P). The stability of Ac-SDD KD P and its prevention of liver fibrosis were investigated in vitro and in vivo. The stabilities of Ac-SDKP and Ac-SDD KD P exposed to angiotensin-1-converting enzyme (ACE) and their half-lives in rats and human sera were determined by high-performance liquid chromatography. The inhibitory effects of Ac-SDKP and Ac-SDD KD P on the proliferation and activation of hepatic stellate cells (HSC-T6) were evaluated using the Cell Counting Kit-8, Western blotting, reverse transcription quantitative polymerase chain reaction, and immunofluorescence assays. Finally, the protective effects of Ac-SDKP and Ac-SDD KD P on carbon tetrachloride (CCl4 )-induced liver fibrosis in rats were compared. d-Amino acid replacement significantly enhanced the stability of the peptide to ACE and prolonged the half-life of Ac-SDKP in rats and human sera. The Ac-SDKP-mediated inhibition of HSC-T6 cell proliferation was well preserved, and Ac-SDD KD P exerted inhibitory effects comparable to Ac-SDKP on α-smooth muscle actin (α-SMA), collagen I and III expression, and phosphorylated-Smad-2 expression. After intraperitoneal (i.p.) administration, Ac-SDD KD P exhibited significantly greater protection than Ac-SDKP against CCl4 -induced liver fibrosis in rats. The serum alanine aminotransferase, aspartate aminotransferase, albumin, and total protein levels of the Ac-SDD KD P-treated rats were significantly lower than those of the Ac-SDKP-treated rats. α-SMA, CD45, and collagen I and III expression, as well as Smad-2 phosphorylation were significantly attenuated in the livers of the Ac-SDD KD P-treated rats compared to those of the Ac-SDKP-treated rats. Furthermore, we showed that the Ac-SDD KD P concentration in the rat liver increased to a physiological level of 60 min after i.p. administration, although i.p. administration of Ac-SDKP failed to enhance the peptide concentration in the rat liver. Our findings indicate that d-amino acid replacement is a simple and effective method to enhance the stability of Ac-SDKP. Ac-SDD KD P represents potential application of Ac-SDKP in fibrosis treatment and provides a new potential treatment strategy for liver fibrosis. © 2019 IUBMB Life, 71(9):1302-1312, 2019.

Identification of Tumor Specific Peptide as EpCAM Ligand and Its Potential Diagnostic and Therapeutic Clinical Application.

Tumor targeting agents are being developed for early tumor detection and therapeutics. We previously identified the peptide SNFYMPL (SNF*) and demonstrated its specific binding to human esophageal specimens of high-grade dysplasia (HGD) and adenocarcinoma with imaging ex vivo. Here, we aim to identify the target for this peptide and investigate its potential applications in imaging and drug delivery. With SNF* conjugated affinity chromatography, mass spectrum, Western blot, enzyme-linked immunosorbent assay (ELISA), and molecular docking, we found that the epithelial cell adhesion molecule (EpCAM) was the potential target of SNF*. Next, we showed that FITC-labeled SNF* (SNF*-FITC) colocalized with EpCAM antibody on the surface of esophageal adenocarcinoma cells OE33, and SNF*-FITC binding patterns significantly changed after EpCAM knockdown or exogenous EpCAM transfection. With the data from TCGA, we demonstrated that EpCAM was overexpressed in 17 types of cancers. Using colon and gastric adenocarcinoma cells and tissues as examples, we found that SNF*-FITC bound in a pattern was colocalized with EpCAM antibody, and the SNF* binding did not upregulate the EpCAM downstream Wnt signals. Subsequently, we conjugated SNF* with our previously constructed poly(histidine)-PEG/DSPE copolymer micelles. SNF* labeling significantly improved the micelle binding with colon and gastric adenocarcinoma cells in vitro, and enhanced the antitumor effects and decreased the toxicities of the micelles in vivo. In conclusion, we identified and validated SNF* as a specific peptide for EpCAM. The future potential use of SNF* peptide in multiple tumor surveillance and tumor-targeted therapeutics was demonstrated.

Acetylation and Amination Protect Angiotensin 1-7 from Physiological Hydrolyzation and Therefore Increases Its Antitumor Effects on Lung Cancer.

The recently reported inhibitory effects of angiotensin 1-7 (Ang-(1-7)) on various cancers indicate its potential use as a therapeutic agent for primary and metastatic cancers. However, its extremely short half-life in the circulation greatly compromises its potential applications. Here, we reported an Ang-(1-7) analogue peptide with the amino and carboxy termini protected by acetylation and amination. The in vitro and in vivo degradation of the resulting analogue, Ang-AA, were determined using high-performance liquid chromatography (HPLC). At the same time, small RNA interference and competition studies were performed to evaluate the specific capacity of Ang-AA to bind to the cell surface Mas receptor. Cell Counting Kit-8 (CCK8), wound-healing, and Boyden chamber assays were performed to investigate the inhibitory effects of Ang-AA on A549 cells. Finally, the synergistic inhibitory effects of Ang-AA and paclitaxel (PTX) on A549 xenografts in mice were observed using animal imaging systems and survival observations. The toxicity of Ang-AA in mice was evaluated. Our results showed that acetylation and amination significantly inhibited the hydrolyzation of Ang-(1-7) in vitro and in vivo. The half-life of Ang-(1-7) in rats was prolonged from 2.4 ± 0.6 min to 238.7 ± 61.3 min ( p < 0.001). The specific binding of Ang-AA to the Mas receptor was well preserved, and Ang-AA exerted significantly greater inhibitory effects on the proliferation, migration, and invasion of A549 cells than Ang-(1-7). The combination of Ang-AA and PTX exhibited a significantly greater synergistic inhibitory effect on A549 xenografts than the combination of Ang-(1-7) and PTX. Ang-AA did not display obvious toxicity in mice. Our findings indicate acetylation and amination is a simple and effective method for producing Ang-(1-7) as a bioactive peptide.

Genistein From Fructus sophorae Protects Mice From Radiation-Induced Intestinal Injury.

The development of an effective pharmacological countermeasure is needed to reduce the morbidity and mortality in high-dose ionizing radiation-induced acute damage. Genistein has shown bioactivity in alleviating radiation damage and is currently synthesized by chemosynthetic methods. Due to concerns about chemical residues and high costs, the clinical application of genistein is still a major challenge. In this study, we aimed to establish an efficient method for the extraction of genistein from Fructus sophorae. The effects of extracted genistein (FSGen) on preventing intestinal injury from radiation were further investigated in this study. C57/BL mice were exposed to 7.5 Gy whole body irradiation with and without FSGen treatments. Histological analysis demonstrated significant structural and functional restitution of the intestine and bone marrow in FSGen-pretreated cohorts after irradiation. Through mRNA expression, protein expression, and small interfering RNA analyses, we demonstrated that FSGen protects IEC-6 cells against radiation damage by upregulating the Rassf1a and Ercc1 genes to effectively attenuate DNA irradiation damage. Together, our data established an effective method to extract genistein from the Fructus sophorae plant with high purity, and validated the beneficial roles of the FSGen in protecting the radiation damage. These results promise the future applications of Fructus sophorae extracted genistein in the protection of radiation related damages.

A dimeric thymosin beta 4 with novel bio-activity protects post-ischemic cardiac function by accelerating vascular endothelial cell proliferation.

BACKGROUND: Thymosin beta 4 (Tß4) is a 43-amino-acid peptide with protective properties in myocardium injury. Previously, we produced a recombinant human dimeric Tß4 (DTß4). Here, the cardioprotective effects of DTß4 and the molecular mechanisms underlying its enhanced activity were investigated. METHODS AND RESULTS: Echocardiography measurements showed that the cardioprotective effect of DTß4 in myocardial infarction mice was significantly higher than that of wild-type Tß4. Corresponding in vitro analyses demonstrated that the enhanced cardioprotection provided by DTß4 was largely due to increased stimulation of angiogenesis. HPLC analysis, western blotting and qRT-PCR indicated that the enhanced pro-angiogenesis activity of DTß4 was independent of the protein half-life and the known downstream pathways of wild-type Tß4. Transcriptome deep sequencing (RNA-seq), BrdU incorporation assays, flow cytometry analysis and RNA interference demonstrated that the enhanced angiogenic activity of DTß4 depended on MALAT1 (metastasis-associated lung adenocarcinoma transcript 1)-induced proliferation of vascular endothelial cells, which has not been reported for wild-type Tß4. Moreover, transcription factor activation screening, luciferase promoter reporter assay and immunoprecipitation assay demonstrated that DTß4 enhanced MALAT1 transcription by inhibiting the degradation of prospero-related homeobox 1 (PROX1). CONCLUSION: This study demonstrates the potential applications and the novel bioactivity of the Tß4 dimer. Moreover, to construct the dimer represents a new method for production of bioactive peptides that may have novel activities.

Cpt1c regulated by AMPK promotes papillary thyroid carcinomas cells survival under metabolic stress conditions.

Background: Cancer cells have to take metabolic transformation in tumor progression when facing need of increased energy and adequate vascularization. However, molecular mechanism is not fully known. In this study, we showed that expression of carnitine palmitoyltransferase 1C (Cpt1c), as a member of the gate-keeper enzymes , which transferring long-chain fatty acids into mitochondria to further oxidation, which is regulated by AMPK promotes papillary thyroid carcinomas cells survival under metabolic stress conditions. Methods: Firstly, we used qRT-PCR to detect expression of Cpt1c in papillary thyroid carcinomas tissues compared with paired normal tissues. Secondly, to evaluate whether Cpt1c is induced under metabolic stress, models of hypoxia (0.2% oxygen) and glucose deprivation for cultured papillary thyroid carcinomas cells were established. Lastly, KTC-1 cells were treated with AICAR (as an agonist of AMPK) and Compound C (as an inhibitor of AMPK) to investigate the correlation of AMPK activity with Cpt1c expression under metabolic stress. Results: Cpt1c is higher in papillary thyroid carcinomas tissues compared with paired normal tissues. Furthermore, Cpt1c up-regulation promotes cancer cell growth and metastasis. In addition, the results showed that Cpt1c expression is induced by metabolic stress, including hypoxia and low glucose treatment. Consistently, Cpt1c can protect cells from cancer cells death caused by hypoxia and low glucose. Lastly, Cpt1c expression is regulated by AMPK activity. Conclusion: Here we describe that induction of Cpt1c expression facing metabolic stress in papillary thyroid carcinomas is at least partly regulated by AMPK activity and ultimately contribute to development and progression of papillary thyroid carcinomas.

Identification and validation of FGFR2 peptide for detection of early Barrett's neoplasia.

The incidence of esophageal adenocarcinoma (EAC) is rising rapidly, and early detection within the precursor state of Barrett's esophagus (BE) is challenged by flat premalignant lesions that are difficult detect with conventional endoscopic surveillance. Overexpression of cell surface fibroblast growth factor receptor 2 (FGFR2) is an early event in progression of BE to EAC, and is a promising imaging target. We used phage display to identify the peptide SRRPASFRTARE that binds specifically to the extracellular domain of FGFR2. We labeled this peptide with a near-infrared fluorophore Cy5.5, and validated the specific binding to FGFR2 overexpressed in cells in vitro. We found high affinity kd = 68 nM and rapid binding k = 0.16 min-1 (6.2 min). In human esophageal specimens, we found significantly greater peptide binding to high-grade dysplasia (HGD) versus either BE or normal squamous epithelium, and good correlation with anti-FGFR2 antibody. We also observed significantly greater peptide binding to excised specimens of esophageal squamous cell carcinoma and gastric cancer compared to normal mucosa. These results demonstrate potential for this FGFR2 peptide to be used as a clinical imaging agent to guide tissue biopsy and improve methods for early detection of EAC and potentially other epithelial-derived cancers.

Local blockage of self-sustainable erythropoietin signaling suppresses tumor progression in non-small cell lung cancer.

Functional significance of co-expressed erythropoietin (EPO) and its receptor (EPOR) in non-small cell lung cancer (NSCLC) had been under debate. In this study, co-overexpression of EPO/EPOR was confirmed to be positively associated with poor survival in NSCLC. The serum EPO in 14 of 35 enrolled NSCLC patients were found elevated significantly and decreased to normal level after tumor resection. With primary tumor cell culture and patient-derived tumor xenograft (PDX) mouse model, the EPO secretion from the tumors of these 14 patients was verified. Then, we proved the patient derived serum EPO was functionally active and had growth promotion effect in EPO/EPOR overexpressed but not in EPO/EPOR under-expressed NSCLC cells. We also illustrated EPO promoted NSCLC cell proliferation through an EPOR/Jak2/Stat5a/cyclinD1 pathway. In xenograft mouse model, we proved local application of EPO neutralizing antibody and short hairpin RNA (shRNA) against EPOR effectively inhibited the growth of EPO/EPOR overexpressed NSCLC cells and prolonged survivals of the mice. Finally, EPO/EPOR/Jak2/Stat5a/cyclinD1 signaling was found to be a mediator of hypoxia induced growth in EPO/EPOR overexpressed NSCLC. Our results illustrated a subgroup of NSCLC adapt to hypoxia through self-sustainable EPO/EPOR signaling and suggest local blockage of EPO/EPOR as potential therapeutic method in this distinct NSCLC population.

[Expression and significance of substance P, neurofilament-H in glomus tumors with chronic pain].

OBJECTIVE: To observe the expression and distribution of substance P (SP), neurofilament-H (NFH) in glomus tumors with chronic pain, and to discuss the process of chronic pain and the relationship with pain degree. METHODS: Twenty-seven patients diagnosed as glomus tumor with chronic pain were enrolled as case group, and divided into light pain symptomatic group (LPSG) (n = 12) and severe pain symptomatic group (SPSG) (n = 15) according to clinical manifestations. Control group (CG) were enrolled by 30 patients with amputated extremities or hands after trauma. Immunohistochemical methods were used to determine the expression of SP, NFH which were detected quantitatively by computer graph analysis system too. RESULTS: The positive expression and distribution of SP, NFH existed in all the three groups and SPSG expression level was the highest [Grayscale Value(SP) (143.3 +/- 7.5), Grayscale Value(NFH) (167.7 +/- 4.4)], LPSG followed [Grayscale Value(SP) (156.2 +/- 8.2), Grayscale Value(NFH) (194.8 +/- 4.0)], control group was the third [Grayscale Value(SP) (208.2 +/- 16.6), Grayscale Value(NFH) (225.1 +/- 8.3)]; The difference of expression level among three groups was significant [SPSG vs LPSG (P(SP) = 0.002, P(NFH) < 0.0001), SPSG vs CG (P(SP) < 0.0001, P(NFH) < 0.0001), LPSG vs CG (P(SP) < 0.0001, P(NFH) < 0.0001)]. The findings of Pearson product-moment correlation analysis between quantitative grayscale value of SP, NFH respectively and pain score in all the patients with glomus tumor showed linear negative correlation (r(SP) = -0.8974, P(SP) = 0.000001; r(NFH) = -0.6545, P(NFH) = 0.000212). CONCLUSION: SP is the mainly afferent pain transmitter in the process of chronic pain in glomus tumor, and NFH plays an important role in pain-transmitted activities.