Research progress on high throughput discovery and functional verification of functional micropeptides.

With the rapid development of computational biology and deep sequencing technology, more and more studies have shown that a large number of non-classical open reading frames that have not been annotated and hidden in non-coding RNA can encode functional micropeptide. This article reviewed the current research status and technology strategy of gene sources, biological properties, predicted methods and functional verification of micropeptide, providing theoretical and reference basis for the subsequent discovery of micropeptides, research on regulatory mechanisms and development of novel targets and biomarkers.

[Progress on sORF-encoded micropeptides].

Existing research has shown that there are a large amount of non-coding RNAs (ncRNAs) in organisms. Short open reading frames (sORFs) abundantly exist in molecular sequences inaccurately annotated as ncRNAs. Several sORFs can be transcribed and translated into evolutionarily conserved micropeptides, which were ignored in previous studies due to short sequence lengths and the limitations of research techniques. To date, sORF-encoded micropeptides with various functions have been found to play important roles in regulating vital biological activities. This article reviews the functional micropeptides which have been found in recent years, introduces the new micropeptide designated as MIAC that we have discovered and describes the related technologies for mining potential micropeptides, thereby providing insights and references for new micropeptide discovery for researchers.

Antiangiogenic activity of terpenoids from Euphorbia neriifolia Linn.

Angiogenesis is known to serve an important role in embryonic development, wound healing, tissue regeneration, and growth. Two new abietane-type diterpenoids (3, 5), a new lanosterol triterpenoid (8) and seven known compounds haven been isolated from the Euphorbia neriifolia Linn. The structures of all compounds were elucidated by spectroscopic analysis and comparing their NMR data with reported data. Furthermore, we found that compounds 6 and 9 had the antiangiogenic effects in vitro. They could inhibit HUVEC migration and microvessel sprouting in rat aortic rings. Moreover, compound 6 inhibited VEGFR and phosphorylation of Akt, but compound 9 only shown inhibitory effect on phosphorylation of Akt. Taken together, these results suggest that inhibition of VEGF signaling and downstream pathways may be responsible for the antiangiogenic activity of compounds 6 and 9.

The wound healing potential of collagen peptides derived from the jellyfish Rhopilema esculentum.

PURPOSE: Wound represents a major health challenge as they consume a large amount of healthcare resources to improve patient's quality of life. Many scientific studies have been conducted in search of ideal biomaterials with wound-healing activity for clinical use and collagen has been proven to be a suitable candidate biomaterial. This study intended to investigate the wound healing activity of collagen peptides derived from jellyfish following oral administration. METHODS: In this study, collagen was extracted from the jellyfish--Rhopilema esculentum using 1% pepsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fourier transform infrared (FTIR) were used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. Wound scratch assay (in vitro) was done to determine migration potential of human umbilical vein endothelial cells (HUVEC) covering the artificial wound created on the cell monolayer following treatment with collagen peptides. In vivo studies were conducted to determine the effects of collagen peptides on wound healing by examining wound contraction, re-epithelialization, tissue regeneration and collagen deposition on the wounded skin of mice. Confidence level (p < 0.05) was considered significant using GraphPad Prism software. RESULTS: The yield of collagen was 4.31%. The SDS-PAGE and FTIR showed that extracted collagen from jellyfish was type I. Enzymatic hydrolysis of this collagen using collagenase II produced collagen peptides (CP1) and hydrolysis with alkaline proteinase/papain resulted into collagen peptides (CP2). Tricine SDS-PAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25 kDa. Wound scratch assay showed that there were significant effects on the scratch closure on cells treated with collagen peptides at a concentration of 6.25 µg/mL for 48 h as compared to the vehicle treated cells. Overall treatment with collagen peptide on mice with full thickness excised wounds had a positive result in wound contraction as compared with the control. Histological assessment of peptides treated mice models showed remarkable sign of re-epithelialization, tissue regeneration and increased collagen deposition. Immunohistochemistry of the skin sections showed a significant increase in ß-fibroblast growth factor (ß-FGF) and the transforming growth factor-ß1 (TGF-ß1) expression on collagen peptides treated group. CONCLUSION: Collagen peptides derived from the jellyfish-Rhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future.

[Common cancer genetic analysis methods and application study based on TCGA database].

The development of second-generation sequencing (NGS) technology is providing numerous data which shifts the focus of cancer research from the sequencing of multi-species to the analysis and comparison of select data via high-throughput sequencing. The NGS also facilitates the diversity of available genetic data analysis methods, the constant optimization and innovation of analytical approaches for high-throughput genomics as well as the rapid development of genetic data mining and analysis models. The Cancer Genome Atlas (TCGA) database is a direct result of this work. The TCGA database provides a comprehensive record of genetic data collected from a tumor patient's sample, including its DNA sequence, transcriptional information, epigenetic modification and related. This review elaborates the latest progress in both the mining algorithm and analysis methods for tumor genomics. Specially, we introduce and review the TCGA database and data analysis approaches while demonstrating its applicability using representative cases. This review may shed light on new tumor-related targets discovery for researchers by means of bid data.

A New 1,5-Dihydroxy-4-methoxyisoquinoline from Scolopendra subspinipes mutilans.

A new isoquinoline, 1,5-dihydroxy-4-methoxyisoquinoline (1), was obtained from Scolopendra subspinipes mutilans. Compound 1 showed moderate cytotoxicity on tumour cells with IC50 values ranging from 13 to 26 µm against five esophageal squamous cancer cells whereas low cytotoxicity against normal human esophageal epithelial cells. Isoquinoline ring oxidized at C(1), C(4), and C(5) can enhance its cytotoxicity. In addition, compound 1 showed potent inhibitory effect (inhibition rate > 50% at 13 µm) on cell migration in human umbilical vein endothelial cells. This article mainly studies the structure and activity of 1, and more modification of 1 as a potential anticancer agent.

Novel perspectives of long non-coding RNAs in esophageal carcinoma.

Esophageal carcinoma (EC) is one of the most aggressive cancer types worldwide. However, the underlying genomic events of EC are not fully understood. It is becoming evident that long non-coding RNAs (lncRNAs) play vital roles in tumorgenesis, metastasis, prognosis and diagnosis. Accumulating EC-related lncRNAs have been verified to involve in various biological processes through diverse functions including signal, decoy, scaffold and guide. However, the molecular mechanism of lncRNAs in EC has not been fully explored. In this review, we outline the functions and underlying mechanism of EC-related lncRNAs to pave the way for identification of novel potential biomarkers for EC.

The peptide Acein promotes dopamine secretion through clec-126 to extend the lifespan of elderly C. elegans.

Dopamine plays a crucial role in regulating brain activity and movement and modulating human behavior, cognition and mood. Regulating dopamine signaling may improve cognitive abilities and physical functions during aging. Acein, a nonapeptide of sequence H-Pro-Pro-Thr-Thr-Thr-Lys-Phe-Ala-Ala-OH is able to stimulate dopamine secretion in the brain. By using genetic editing and lifespan investigation in C. elegans, we showed that the lack of the C-type lectin domain-containing protein clec-126 significantly suppressed the aging phenotype and prolonged lifespan, while overexpression of clec-126 promoted aging-related phenotypes and accelerated the aging process. We examined the aging phenotype of C. elegans and showed that Acein could induce a decrease in clec-126 expression, prolonging the lifespan of aged C. elegans. The mechanism proceeds through the Acein-induced stimulation of dopamine secretion that ameliorates motor function decline and extends the healthy lifespan of aged C. elegans. In addition, we also observed an increase in brood number. Our study has shown that Acein regulates dopamine secretion and has good antiaging activity by decreasing clec-126 expression.

RGD-modified endostatin fragments showed an antitumor effect through antiangiogenesis.

EDSM, an endostatin-derived synthetic polypeptide, contains the amino acids 6-48 of endostatin from its N-terminus, which could inhibit human umbilical vein endothelial cell (HUVEC) migration and tumor growth. To increase the targeted delivery of EDSM to tumors and further enhance its antiangiogenic activity, the RGD sequence (Arg-Gly-Asp) was introduced into EDSM and two peptides were obtained: EDSM-X with RGD on the N-terminus of EDSM and EDSM-Y with RGD on the C-terminus. Both modified peptides showed a significant antiangiogenic activity in the HUVEC migration assay, the HUVEC tube formation assay, and the murine aortic ring formation assay in vitro. In agreement with the in-vitro data, EDSM-X and EDSM-Y also showed a significant antitumor activity in vivo. From the cell adhesion assay, it was confirmed that the molecular target of the modified peptides on HUVECs was integrin αvß3, rather than integrin α5ß1. Furthermore, EDSM-Y exhibited more potent antiangiogenic activity and antitumor activity than EDSM-X in vitro and in vivo, and this phenomenon was attributed to the difference in the two modified peptides in their three-dimensional structure modeling and their molecular dockings with integrin αvß3.

Molecular mechanisms of (R,R)ZX-5 on NO synthesis and its anti-angiogenic effect.

(R,R)ZX-5 is a NO regulatory compound, which could significantly increase choroidal blood flow in New Zealand rabbit. The aim of this paper is to investigate the molecular mechanism of (R,R)ZX-5 promoting NO production. Besides this, we also investigated the antiangiogenic activity of (R,R)ZX-5. Analysis of Western blot showed that (R,R)ZX-5 up-regulated the expression of Akt, p-Akt (Thr473), eNOS and p-eNOS (Ser1177), down-regulated the expression of Cyclin D1 in human retinal endothelial cells and escalated the intracellular free Ca(2+) concentration. Additionally, (R,R)ZX-5 inhibited the growth of blood vessels in the chick chorioallantoic membrane model. It is concluded that (R,R)ZX-5 promotes choroidal blood flow through PI3K/Akt-eNOS and Akt-Ca(2+)-eNOS pathways. Additionally, (R,R)ZX-5 can inhibit angiogenesis.

Centranthera grandiflore alleviates alcohol-induced oxidative stress and cell apoptosis.

Alcohol liver disease (ALD) has become a global threat to human health. It is associated with a wide range of liver diseases including alcohol fatty liver, steatosis, fibrosis and cirrhosis, and finally leads to liver cancer and even death. Centranthera grandiflora is a traditional Chinese medicinal herb commonly used to treat ALD, but no research about its mechanism is available. This study evaluated the hepatoprotective effect and mechanism of C. grandiflora against alcohol-induced liver injury in mice. We found that the ethanol extracts of C. grandiflora (CgW) alleviated the alcohol-induced liver injury, enhanced the levels of antioxidant enzymes, and reduced the amount of lipid peroxides. CgW also affected cell apoptosis by inhibiting the activity of Bax, cleaved-caspase 3 and cleaved-caspase 9, and increasing the activity of Bcl-2. In conclusion, the results showed that CgW can effectively improve ALD through alleviating oxidative stress and inhibiting cell apoptosis for the first time. This study suggested that C. grandiflora is a promising herbal medicine for ALD treatment.

In vivo anti-tumor activity of polypeptide HM-3 modified by different polyethylene glycols (PEG).

HM-3, designed by our laboratory, is a polypeptide composed of 18 amino acids. Pharmacodynamic studies in vivo and in vitro indicated that HM-3 could inhibit endothelial cell migration and angiogenesis, thereby inhibiting tumor growth. However, the half-life of HM-3 is short. In this study, we modified HM-3 with different polyethylene glycols (PEG) in order to reduce the plasma clearance rate, extend the half-life in the body, maintain a high concentration of HM-3 in the blood and increase the therapeutic efficiency. HM-3 was modified with four different types of PEG with different molecular weights (ALD-mPEG(5k), ALD-mPEG(10k), SC-mPEG(10k) and SC-mPEG(20k)), resulting in four modified products (ALD-mPEG(5k)-HM-3, ALD-mPEG(10k)-HM-3, SC-mPEG(10k)-HM-3 and SC-mPEG(20k)-HM-3, respectively). Anti-tumor activity of these four modified HM-3 was determined in BALB/c mice with Taxol as a positive control and normal saline as a negative control. Tumor weight inhibition rates of mice treated with Taxol, HM-3, ALD-mPEG(5k)-HM-3, ALD-mPEG(10k)-HM-3, SC-mPEG(10k)-HM-3 and SC-mPEG(20k)-HM-3 were 44.50%, 43.92%, 37.95%, 31.64%, 20.27% and 50.23%, respectively. Tumor inhibition rates in the Taxol, HM-3 and SC-mPEG(20k)-HM-3 groups were significantly higher than that in the negative control group. The efficiency of tumor inhibition in the SC-mPEG(20k)-HM-3 group (drug treatment frequency: once per two days) was better than that in the HM-3 group (drug treatment frequency: twice per day). In addition, tumor inhibition rate in the SC-mPEG(20k)-HM-3 group was higher than that in the taxol group. We conclude that SC-mPEG(20k)-HM-3 had a low plasma clearance rate and long half-life, resulting in high anti-tumor therapeutic efficacy in vivo. Therefore, SC-mPEG(20k)-HM-3 could be potentially developed as new anti-tumor drugs.

Effect of RGD-4C position is more important than disulfide bonds on antiangiogenic activity of RGD-4C modified endostatin derived synthetic polypeptide.

ES-2 (IVRRADRAAVP), an endostatin-derived synthetic polypeptide, contains the amino acids 50-60 of endostatin from its N terminus, and it had no inhibitory effects on tumor growth in vivo. In order to increase the targeted delivery of ES-2 to tumors and further enhance the activity, the polypeptide RGD-4C (ACDCRGDCFC) was introduced into ES-2, and the effects of RGD-4C position and RGD-4C disulfide bonds on polypeptides activity were investigated. When RGD-4C polypeptides (with or without disulfide bonds) were introduced to the N-terminals of synthesized ES-2, the modified ES-2 showed significant antitumor activity in vivo. Cell proliferation and chicken chorioallantoic membrane (CAM) assay results showed that disulfide bonds had no significant effects on RGD-4C-modified ES-2 antiangiogenic activity. Furthermore, the target of modified peptides was integrin alpha5beta1, rather than integrin alphavbeta3 as previous studies mentioned.

The mechanism of (R,R) ZX-5 on increasing NO release.

(R,R) ZX-5 has been proven to have positive effects on choroidal blood flow without affecting the sclera and ciliary bodies in New Zealand white rabbits. This study was designed to investigate the mechanisms of (R,R) ZX-5 on improving the choroidal blood flow and promoting NO production. HUVECs (human umbilical vein endothelial cells) were used to determine the production of eNOS, p-eNOS, AKT and Erk1/2 by Western blot analysis. iNOS and eNOS mRNA levels were investigated by RT-PCR and the effect of (R,R) ZX-5 on NO production were determined by eNOS activity assay. We found (R,R) ZX-5 upregulated protein expression of eNOS and iNOS, increased NO production, and reduced ERK and Akt protein level. Therefore, (R,R) ZX-5 may promote the choroidal blood flow in New Zealand white rabbits without affecting the blood flow in the iris or ciliary bodies via increasing NO production. These results suggest that (R,R) ZX-5 may function to cure and prevent Age-related macular degeneration (AMD).

Studies of poly(ethylene glycol) modification of HM-3 polypeptides.

An RGD modified endostatin-derived synthetic peptide, named HM-3, is a polypeptide angiogenesis inhibitor previously synthesized in our laboratory. Its robust inhibitory effects on endothelial cell migration and tumor growth have been demonstrated by in vivo and in vitro activity assays. The RGD integrin recognition sequence enables the selective binding of HM-3 and its specific targeting to tumor cells that express high levels of integrin. However, the drug has relatively short half-life in vivo, thus requiring administration twice a day to achieve its optimal in vivo antitumor efficacy. In the current study designed to prolong HM-3 half-life, we used methoxy-poly(ethylene glycol)-aldehyde (mPEG-ALD) to specifically modify its N terminus and optimized the reaction condition via monitoring the modification by reverse-phase high-performance liquid chromatograph (RP-HPLC) under varying stoichiometric ratios (n(mPEG10k-ALD):n(HM-3)), reaction times, and pH values. The maximal modification rate was achieved in a reaction when substrates mPEG10k-ALD and HM-3 were mixed at the molar ratio of 2:1 in a pH 6 phosphate buffer after 4 h incubation at room temperature. The reaction product of this optimal reaction was purified to 96% purity by RP-HPLC. Compared with HM-3, the newly modified PEG(10k)-HM-3 was shown to be more active in the inhibition of angiogenesis in the chorioallantoic membrane of chick embryos (CAM), its rate of in vitro degradation in serum was markedly reduced, and its in vivo half-life was prolonged by 5.86-fold relative to unmodified HM-3 after intravenous injection into male SD rats.

Facile Synthesis and Cytotoxicity of Phenazine-Chromene Hybrid Molecules Derived from Phenazine Natural Product.

AIM AND OBJECTIVE: Small molecule targeted drugs can effectively reduce the toxicity and side effects of drugs, and improve the efficacy of drugs by their specific antitumor activity. Hence, the development of small molecular targeted drugs for cancer has important significance. This study was undertaken to design and synthesize novel phenazine-chromene hybrid molecules in order to optimize the structure and improve the efficacy of this kind of hybrids. MATERIALS AND METHODS: O-diaminobenzene was used as starting material to synthesize twentyfour heterocyclic compounds designed as hybrid molecules of phenazine and 4H-chromene pharmacophores by facile methods. The structures of the compound were confirmed by 1H NMR, 13C NMR and HRMS. Furthermore, the synthesized compounds were evaluated for in vitro activity against four human cancer cell lines and two non-cancer cell lines by MTT test. RESULTS: Some compounds showed strong cytotoxic activities against HepG2 and A549 cancer lines (IC50 = 5-10 µM). Comparing 2i with 2l, the introduction of hydrophilic groups on the phenazine core could not improve the antiproliferative activity significantly. Except 2d and 3c, compounds owning chlorine substituent on the 4H-chromene pharmacophore seemingly contribute to enhance the compounds' antiproliferative activity. Specially, compound 3c showed highest cytotoxicity against A549 cells with IC50 values of 3.3±0.4 µM. Furthermore, all compounds showed low or no cytotoxicity against HUVEC and L02 non-cancer cells in vitro. CONCLUSION: Compound 3c may be used as potential lead molecule against A549 cancer cells.

An RGD-modified endostatin-derived synthetic peptide shows antitumor activity in vivo.

It has been reported that an endostatin-derived synthetic peptide, named ES-2, that contains the amino acids 60-70 of endostatin from its N terminus, efficiently inhibits basic fibroblast growth factor-induced directional migration and tubular morphogenesis of microvascular endothelial cells. We found that the peptide had no effects on tumor growth in vivo. However, when the peptide Arg-Gly-Asp (RGD) was introduced into ES-2, the modified ES-2 showed significant antitumor results in animal models. Histochemical and immunohistochemical analysis showed that RGD-modified ES-2 induced large areas of continuous necrosis within tumors and significantly reduced the vessel density compared to control. Furthermore, only the peptides with RGD were able to bind tumor cells in vitro, suggesting that additional RGD domains may help in improving the receptor-binding ability and pharmacokinetic properties of ES-2 and preventing organic clearance, as well as enzymatic degradation of the peptide, thus enabling a greater fraction of the administered dose to be biologically available.

Valproic acid inhibits the growth of cervical cancer both in vitro and in vivo.

Valproic acid (VPA), a well-known anti-convulsant, is currently under extensive evaluation as an anti-cancer agent. It is known to exert its anti-cancer effect mainly by inhibiting the enzyme histone deacetylase I. In our study, we investigated the effects of VPA on cervical cancer both in vitro and in vivo cancer models. We examined the effects of acute VPA (0, 1.2, 2.4, 5.0 mM) treatment on cell proliferation in cervical cancer cell lines HeLa, SiHa and Ca Ski and histone acetylation, p21 and p53 gene expression in HeLa cell line. We also investigated the effect of chronic VPA administration in tumour xenograft growth studies. Our results show that with acute treatment, VPA can increase the expression of net histone H3 acetylation and up-regulate p21 expression with no effect on p53 expression. Chronic administration of VPA had a net cytostatic effect that resulted in a statistically significant reduction of tumour growth and improved survival advantages in tumour xenografts studies. Furthermore, we also demonstrated that VPA has a direct anti-angiogenic effect in tumour studies and could potentially be a promising candidate for further cervical cancer trails.

New arbutin derivatives from the leaves of Heliciopsis lobata with cytotoxicity.

Heliciopsis lobata is a medicinal plant, which is exclusively used to treat tumor in Li folk region. Two new arbutin derivatives, 6'-((E)2-methoxy-5-hydroxycinnamoyl) arbutin (1) and 2'-((E)2, 5-dihydroxycinnamoyl) arbutin (2) along with five known compounds (3-7), were isolated from the leaves of Heliciopsis lobata. Their structures were elucidated on the basis of extensive spectroscopic interpretations. They were evaluated for their potential anticancer activity. Compounds 6 and 7 exhibited cytotoxicity against MGC-803 cells with IC50 values being 44.1 and 11.3 µg·mL-1, respectively. Additionally, compounds 1, 2 and 5-7 exhibited a moderate inhibition of MGC-803 cells invasion; compound 2 at 20 µg·mL-1 inhibited the invasion of MGC-803 cells by 43.0%, compared with the controls.

Two new isoquinoline alkaloids from Scolopendra subspinipes mutilans induce cell cycle arrest and apoptosis in human glioma cancer U87 cells.

Two new isoquinoline alkaloids 1-2 and seven known compounds 3-9 were isolated from the ethanol extract of centipede Scolopendra subspinipes mutilans L. Koch. The structures were elucidated by a combination of spectroscopic analyses including 1D and 2D NMR, and HRESIMS. Compounds 1-2 exhibited good cytotoxicity with IC50 values ranging from 1.19 to 31.28µM against six human cancer cell lines and low cytotoxicity against human normal liver L-02 cell lines, suggesting that compounds 1-2 had high specific cytotoxicity on human cancer cell lines. Further analyses showed that compounds 1-2 inhibited U87 cells proliferation by arresting cell cycle progress at G0/G1 phase and inducing apoptosis through loss of mitochondrial membrane potential (MMP), activation of caspase 9/3 and down-regulation of the Bcl-2/Bax protein ratio. The results suggest that compounds 1-2 induce apoptosis in U87 cells through the mitochondria apoptosis pathway, and they deserve further research as potential anti-glioma cancer agents.