Betulinic acid impairs metastasis and reduces immunosuppressive cells in breast cancer models.

Breast cancer is the most common female cancer with considerable metastatic potential, explaining the need for new candidates that inhibit tumor metastasis. In our study, betulinic acid (BA), a kind of pentacyclic triterpenoid compound derived from birch trees, was evaluated for its anti-metastasis activity in vitro and in vivo. BA decreased the viability of three breast cancer cell lines and markedly impaired cell migration and invasion. In addition, BA could inhibit the activation of stat3 and FAK which resulted in a reduction of matrix metalloproteinases (MMPs), and increase of the MMPs inhibitor (TIMP-2) expression. Moreover, in our animal experiment, intraperitoneal administration of 10 mg/kg/day BA suppressed 4T1 tumor growth and blocked formation of pulmonary metastases without obvious side effects. Furthermore, histological and immunohistochemical analyses showed a decrease in MMP-9 positive cells, MMP-2 positive cells and Ki-67 positive cells and an increase in cleaved caspase-3 positive cells upon BA administration. Notably, BA reduced the number of myeloid-derived suppressor cells (MDSCs) in the lungs and tumors. Interestingly, in our caudal vein model, BA also obviously suppressed 4T1 tumor pulmonary metastases. These findings suggested that BA might be a potential agent for inhibiting the growth and metastasis of breast cancer.

Bio-Inspired Fragmentations: Rapid Assembly of Indolones, 2-Quinolinones, and (-)-Goniomitine.

Inspired by the biogenetic origin of goniomitine, new synthetic bio-inspired fragmentation strategies for the synthesis of functionalized 2-quinolinones and indolones have been developed. Remarkable synthetic efficiency was achieved by telescoping several transformations into one-pot reactions, allowing for the direct coupling of 2-alkynyl-anilines and diazo ketones. The synthetic utility was demonstrated by the 5-step asymmetric total synthesis of (-)-goniomitine from 2-ethyl-cyclopentanone.

Correlation study of basic Chinese medicine syndromes and neurotransmitter levels in patients with primary insomnia.

OBJECTIVE: To investigate the correlation between basic Chinese medicine (CM) syndromes (deficiency and excess syndromes) and intracranial neurotransmitter levels in primary insomnia (PI), to provide objective indicators and syndrome-based medical evidence for the differentiation of PI. METHODS: A total of 158 patients with PI were recruited for CM syndrome differentiation. Another 30 healthy people without sleep disorders were selected as control group. An encephalofluctuograph analyzer was used to test the levels of intracranial neurotransmitters, including γ- aminobutyric acid (GABA), glutamate (Glu), 5-hydroxytryptamine (5-HT), dopamine (DA), etc., and their relevance were analyzed. RESULTS: The neurotransmitter levels in the basic-deficiency group were lower than those in the healthy-control group, while the basic-excess group had higher levels than the healthy-control and basic-deficiency groups. Among the neurotransmitters, the 5-HT level was higher in the basic-excess group than in the basic-deficiency group (24.20±4.07 vs. 21.13±3.23; P<0.05); for the intermingled deficiency-excess group, the level of GABA was higher than that in the basic-deficiency group (9.48±3.07 vs. 7.23±3.67; P<0.05), Glu level was higher than that in the healthy-control group (7.53±4.10 vs. 5.83±0.99, P<0.05), and 5-HT and DA levels were lower than those in the healthy-control group (19.80±5.68 vs. 22.63±3.31, 5.27±3.79 vs. 6.83±1.58, respectively; P<0.05). CONCLUSIONS: There was a correlation between the basic syndromes and intracranial neurotransmitter levels in patients with PI, which could objectively reflect the CM differentiation in PI. This information could be important for improving CM diagnosis and treatment in PI.

[The Comparison Between Extraction Methods of Exosomes From Ascites of Colorectal Cancer].

OBJECTIVE: To compare the two different methods to isolate the exosome from the ascites of colorectal cancer (CRC) patient and find the efficient one. METHODS: Exosome from the ascites of CRC patient were isolated by two different methods: density gradient exosome isolation (DG-Exo) and Exo-Quick isolation, and followed by identification with transmission electron microscopy observation and Western blot analysis. And then, Nanodrop was used for protein quantification. RESULTS: Exosome were isolated by both of the two methods. The protein concentration of the exosome isolated by the Exo-Quick isolation were higher than that of DG-Exo. CONCLUSION: Exo-Quick isolation can obtain higher purity and more complete exosome from the ascites.

miR-434-3p and DNA hypomethylation co-regulate eIF5A1 to increase AChRs and to improve plasticity in SCT rat skeletal muscle.

Acetylcholine receptors (AChRs) serve as connections between motor neurons and skeletal muscle and are essential for recovery from spinal cord transection (SCT). Recently, microRNAs have emerged as important potential biotherapeutics for several diseases; however, whether miRNAs operate in the modulation of AChRs remains unknown. We found increased AChRs numbers and function scores in rats with SCT; these increases were reduced following the injection of a eukaryotic translation initiation factor 5A1 (eIF5A1) shRNA lentivirus into the hindlimb muscle. Then, high-throughput screening for microRNAs targeting eIF5A1 was performed, and miR-434-3p was found to be robustly depleted in SCT rat skeletal muscle. Furthermore, a highly conserved miR-434-3p binding site was identified within the mRNA encoding eIF5A1 through bioinformatics analysis and dual-luciferase assay. Overexpression or knockdown of miR-434-3p in vivo demonstrated it was a negative post-transcriptional regulator of eIF5A1 expression and influenced AChRs expression. The microarray-enriched Gene Ontology (GO) terms regulated by miR-434-3p were muscle development terms. Using a lentivirus, one functional gene (map2k6) was confirmed to have a similar function to that of miR-434-3p in GO terms. Finally, HRM and MeDIP-PCR analyses revealed that DNA demethylation also up-regulated eIF5A1 after SCT. Consequently, miR-434-3p/eIF5A1 in muscle is a promising potential biotherapy for SCI repair.

Analysis of transcription profile to reveal altered signaling pathways following the overexpression of human desumoylating isopeptidase 2 in pancreatic cancer cells.

Human desumoylating isopeptidase 2 (DESI-2) is a member of the DESI family and contains a conserved PPPDE1 domain. Previous studies have demonstrated that DESI-2 overexpression may induce cell apoptosis. In the present study, differentially expressed genes were analyzed using a transcription microarray in DESI-2 overexpressing PANC-1 pancreatic cancer cells. A total of 45,033 genes were examined by microarray, which identified 1,766 upregulated and 1,643 downregulated genes. A series of altered signaling pathways were analyzed, in which certain essential signaling factors, including retinoid X receptor (RXR), BH3 interacting-domain death agonist, Ras homolog gene family member A (RhoA) and Rho-associated protein kinase, were further investigated at the protein level. The release of cytochrome c and the activation of caspase-3 were also detected by western blot analysis. Immunohistochemistry further revealed the expression features of RXR and RhoA in pancreatic ductal adenocarcinoma tissues with various DESI-2 expression levels. The results serve as a valuable reference for the further elucidation of the functions of DESI-2 in pancreatic cancer.

Metal-Free Ring-Expansion Reaction of Six-membered Sulfonylimines with Diazomethanes: An Approach toward Seven-Membered Enesulfonamides.

A new metal-free, ring-expansion reaction of six-membered N-sulfonylimines with unstable diazomethanes, generated in situ from the N-tosylhydrazones, has been developed. This reaction delivers valuable seven-membered enesulfonamides by a Tiffeneau-Demjanov rearrangement and intramolecular proton transfer tautomerization process. Moreover, this ring-expansion reaction can be carried out in a one-pot fashion and scaled up to the gram scale by using aryl aldehydes, without the need to isolate the N-tosylhydrazone.

Fully human HER2/cluster of differentiation 3 bispecific antibody triggers potent and specific cytotoxicity of T lymphocytes against breast cancer.

The use of a bispecific antibody (BsAb) is a promising and highly specific approach to cancer therapy. In the present study, a fully human recombinant single chain variable fragment BsAb against human epidermal growth factor receptor (HER)2 and cluster of differentiation (CD)3 was constructed with the aim of developing an effective treatment for breast cancer. HER2/CD3 BsAb was expressed in Chinese hamster ovary cells and purified via nickel column chromatography. Flow cytometry revealed that the HER2/CD3 BsAb was able to specifically bind to HER2 and CD3­positive cells. HER2/CD3 BsAb was able to stimulate T-cell activation and induce the lysis of cultured SKBR­3 and BT474 cells in the presence of unstimulated T lymphocytes. HER2/CD3 BsAb efficiently inhibited the growth of breast cancer tissue by activating and inducing the proliferation of tumor tissue infiltrating lymphocytes. Therefore, HER2/CD3 BsAb is a potent tool which may be a suitable candidate for the treatment of breast cancer.

High Phosphorylation Status of AKT/mTOR Signal in DESI2-Reduced Pancreatic Ductal Adenocarcinoma.

Desumoylating isopeptidase 2 (DESI2) is a recently identified protein with unclear functions. In this study, a total of 132 tissue samples of pancreatic ductal adenocarcinoma and 73 samples of pancreatic normal tissues were explored to assess DESI2 expression and its implications to AKT/mTOR signal. Immunohistochemistry showed DESI2 expression is significantly decreased in cancer tissues versus normal tissues, presenting lowest level in poorly differentiated cancer. Unlike DESI2, the key factors in AKT/mTOR pathway including p-AKT, mTOR, p-mTOR and p-P70S6K present high expression in pancreatic cancer. It is notable that p-mTOR is significantly increased in DESI2-lower cancer compared with DESI2-higher cancer, although mTOR presents no difference in the two groups. The relative p-mTOR/mTOR ratio is also significantly elevated in DESI2-lower cancer. Moreover, the samples whose p-AKT and p-mTOR scores both exceed the median are obviously increased in DESI2-lower cancer compared with DESI2-higher cancer. As a downstream molecule of AKT/mTOR pathway, p-P70S6K was found to display higher level in DESI2-lower pancreatic cancer. High phosphorylation status of those proteins in DESI2-reduced pancreatic cancer indicates that there is high activity of AKT/mTOR signal in condition of DESI2 reduction, which could provide clues to reveal the implications of DESI2 in carcinogenesis.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma.

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

Implications of PPPDE1 expression in the distribution of plakoglobin and ß-catenin in pancreatic ductal adenocarcinoma.

Human PPPDE peptidase domain-containing protein 1 (PPPDE1) is a recently identified protein; however, its exact functions remain unclear. In our previous study, the PPPDE1 protein was found to be decreased in certain cancer tissues. In the present study, a total of 96 pancreatic ductal carcinoma tissue samples and 31 normal tissues samples were assessed to investigate the distribution of plakoglobin and ß-catenin under the conditions of various PPPDE1 expression levels by means of immunohistochemistry. Generally, the staining of PPPDE1 was strong in normal tissues, but weak in cancer tissues. Plakoglobin was mainly distributed along the membrane and cytoplasm border in normal cells, but was less evident in the membranes of cancer cells. In particular, a greater percentage of cells exhibited low membrane plakoglobin expression in cancer tissue with low PPPDE1 expression (PPPDE1-low cancer) compared with that in cancer tissue with high PPPDE1 expression (PPPDE1-high cancer). The distribution of ß-catenin in normal tissues was similar to that of plakoglobin. However, ß-catenin was peculiarly prone to invade nucleus in PPPDE1-low cancer compared with PPPDE1-high cancer. Our data suggested potential links between PPPDE1 expression and the distribution of plakoglobin and ß-catenin in pancreatic ductal adenocarcinoma, providing insights into the role of PPPDE1 in the progression of pancreatic cancer.

FIP200 is involved in murine pseudomonas infection by regulating HMGB1 intracellular translocation.

BACKGROUND: FIP200, a critical autophagy initiating protein, can participate in numerous cellular functions including cancer development; however, its functional role in P. aeruginosa infection of alveolar macrophages is unknown. METHODS: To investigate the role of FIP200 in host defense, we transfected murine alveolar macrophage MH-S cells with FIP200 siRNA. Having confirmed that FIP200 knockdown inhibited PAO1-induced autophagosme formation, we sought to characterize the underlying signaling pathways by immunoblotting. Further, we used fip200 KO mice to study the effects of fip200 deficiency on HMGB1 translocation. RESULTS: We showed that Pseudomonas PAO1 strain infection facilitated autophagosome formation, whereas knockdown of FIP200 inhibited autophagosome formation and HMGB1 expression in MH-S cells. Silencing FIP200 impaired the translocation of HMGB1 to cytosol of MH-S cells and almost abolished acetylation of HMGB1 during PAO1 infection. In contrast, FIP200 overexpression facilitated the cytosol translocation of HMGB1 from nuclei and increased acetylation of HMGB1 in PAO1-infected MH-S cells. Importantly, expression and acetylation of HMGB1 were also significantly down-regulated in fip200 KO mice following PAO1 infection. CONCLUSIONS: Collectively, these findings elucidate that FIP200 may regulate expression and translocation of HMGB1 during PAO1 infection, which may indicate novel therapeutic targets to control pulmonary infection.

Phosphorylation and changes in the distribution of nucleolin promote tumor metastasis via the PI3K/Akt pathway in colorectal carcinoma.

Here, we investigated the molecular mechanism underlying the changes in the distribution of nucleolin. Our study identified PI3K/Akt signaling as an essential pathway regulating the distribution of nucleolin. Furthermore, nucleolin can interact with phospho-PI3K-p55, and changes in the distribution of nucleolin were related to its phosphorylation. Subsequently, we analyzed the correlation of VEGF and nucleolin, and found that distribution of nucleolin related to metastatic potential. Finally, blocking cell surface nucleolin influences the process of epithelial-mesenchymal transitions. This indicates that nucleolin may be a novel cancer therapy target and a predictive marker for tumor migration in colorectal carcinoma.

[Clinical observation on the neurotransmitters regulation in patients of insomnia differentiated as yang deficiency pattern treated with warm acupuncture and auricular point sticking therapy].

OBJECTIVE: To evaluate the effect mechanism of warm acupuncture combined with auricular point sticking therapy and its efficacy on insomnia by monitoring the level of brain neurotransmitters in the insomnia patients. METHODS: One hundred and thirty patients with insomnia were randomized into an observation group and a control group, 65 cases in each one. In the observation group, based on the treating principle of warming yang and benefiting qi, acupuncture was applied to Xinshu (BL 15), Pishu (BL 20), Shenshu (BL 23), Yaoyangguan (GV 3), Baihui (GV 20), Neiguan (PC 6) and Shenmen (HT 7). Warm acupuncture was supplemented at Xinshu (BL 15), Pishu (BL 20), Shenshu (BL 23) and Yaoyangguan (GV 3). The treatment was given once every day. In the control group, estazolam tablets, 0. 5 to 1 mg were prescribed for oral administration, 30 min before sleep at night. The treatment of 14 days was taken as one session and 2 sessions were required in the two groups. The encephal of luctuograph technology was used to observe the sleep quality and brain neurotransmitters before and after treatment in the two groups and the efficacy was compared between the two groups. RESULTS: The total effective rate was 83.1% (54/65) in the observation group and 87.7% (57/65) in the control group. The efficacy was similar between the two groups (P > 0.05). In the observation group, after treatment, 5-HT and GABA/Glu were all increased compared with those before treatment (P < 0.05, P < 0.01) and norepinephrine (NE) was reduced compared with that before treatment (P < 0.05). The level of each index did not change significantly before and after treatment in the control group (all P > 0.05). The regulations of 5-HT, GABA/Glu in the observation group were superior to those in the control group (all P < 0.05). CONCLUSION: The combined therapy of warm acupuncture and auricular point sticking method for warming yang and benefiting qi effectively improves brain neurotransmitters and essentially improves sleep quality of the patients with insomnia differentiated as yang deficiency pattern.

The preparation of VEGFR1/CD3 bispecific antibody and its specific cytotoxicity against VEGFR1-positive breast cancer cells.

Bispecific antibody (BsAb) has been proved to be a very effective antitumor approach because of its distinctive advantages of immune-mediated cytotoxicity. To enhance the ability to recruit and activate T lymphocytes for tumor-specific killing, we constructed and prepared a recombinant human single-chain Fv bispecific antibody (BsAb), named VEGFR1/CD3 BsAb, targeting VEGFR1 and CD3. The VEGFR1/CD3 BsAb was expressed in CHO-K1 cells and purified by Ni-NTA affinity chromatography. The CD3 and VEGFR1-binding activity of VEGFR1/CD3 BsAb was confirmed by flow cytometry. T lymphocyte activation and proliferation induced by VEGFR1/CD3 BsAb were also demonstrated in vitro. Notably, our VEGFR1/CD3 BsAb presented a powerful and specific killing effect against VEGFR1-positive human breast cancer cell MDA-MB-231 and MDA-MB-435 through activating T lymphocyte at very low concentrations, indicating that it will be a valuable antibody drug for treatment of VEGFR1-positive cancers in the future.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

Enantioselective [4 + 2] cycloaddition of cyclic N-sulfimines and acyclic enones or ynones: a concise route to sulfamidate-fused 2,6-disubstituted piperidin-4-ones.

A concise route to valuable sulfamate-fused 2,6-disubstituted piperidin-4-ones or 2,3-dihydropyridin-4(1H)-ones in good yield with high diastereo- and enantioselectivity is presented. The combination of chiral primary amine and o-fluorobenzoic acid efficiently promoted an asymmetric [4 + 2] cycloaddition reaction of N-sulfonylimines and enones or ynones. The cycloaddition reaction between cyclic N-sulfonylimines and ynones is first reported.

One-pot synthesis of optically enriched 2-piperidinones from aliphatic aldehydes and cyanoacrylamides.

A highly stereoselective one-pot reaction of aliphatic aldehydes and cyanoacrylamides has been developed. The one-pot reaction includes an organocatalytic Michael addition followed by an intramolecular hemiaminalization. After reduction, optically enriched 2-piperidinones with three contiguous chiral centers were obtained in up to 95% yield and 9:1 dr with 99% ee.

HOCl oxidation-modified CT26 cell vaccine inhibits colon tumor growth in a mouse model.

Despite progress in elucidating mechanisms associated with colorectal cancer and improvement of treatment methods, it remains a frequent cause of death worldwide. New and more effective therapies are therefore urgently needed. Recent studies have shown that immunogenicity of whole ovarian tumor cells and subsequent T cell response were potentiated by oxidation modification with hypochlorous acid (HOCl) in vitro and ex vivo. These results prompted us to investigate the protective antitumor response with an HOCl treated CT26 colorectal cancer cell vaccine in an in vivo mouse model. Administration of HOCl modified vaccine triggered robust antitumor immunity to autologous tumor cells in mice and prolonged survival period significantly. In addition, increased necrosis and apoptosis were found in tumor tissue from the oxidation group. Interestingly, ELISPOT assays showed that specific T cell responses were not elicited in response to the immunizing cellular antigen, in contrast to raising sera antibody titer and antibody binding activity shown by ELISA assay and flow cytometry. Further evaluation of the mechanisms underlying HOCl modified vaccine mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results combined with previous studies suggest that HOCl oxidation modified whole cell vaccine has wide applicability as a cancer vaccine because it can target both T cell- and B cell-specific responses. It may thus represent a promising approach for the immunotherapy of colorectal cancer.

Molecular mechanism of hepatocellular carcinoma-specific antitumor activity of the novel thienopyridine derivative TP58.

Despite progress made in the treatment of hepatocellular carcinoma (HCC), there is no curable treatment. Novel therapies are therefore needed. In our previous study on the design and synthesis of a small molecular inhibitor targeting Aurora kinase, we discovered a novel thienopyridine derivative compound (1g, TP58) which displayed the most potent and relatively specific inhibition of the proliferation of HepG2 human hepatoma cells in vitro. However, the molecular mechanism remains to be elucidated. Herein, in vitro and in vivo studies were conducted to further verify its antitumor activity against HCC. cDNA microarray and two-dimensional protein gel electrophoresis technology were utilized to elucidate the mechanism of HCC-specific inhibition of TP58. Flow cytometry analysis displayed that TP58 can significantly induce G0/G1 arrest in HepG2 cells. Sixteen genes involved in cell cycle regulation were found to be dysregulated following TP58 treatment using microarray technology. Nine proteins whose expression was altered (corresponding to 10 spots identified as differentially expressed) were identified by proteomic analysis. Further study showed that TP58 can modulate the expression of some liver-enriched transcription factors (LETFs) and liver-specific marker genes, such as hepatic nuclear factor (HNF-4) and α-fetoprotein (AFP). These findings may help explain the mechanism of HCC-specific antitumor activity of TP58 and provide some useful insight for anti-HCC drug design and future use of thienopyridine derivatives in HCC therapy.