Angiopoietin-like protein 3 promotes colorectal cancer progression and liver metastasis partly via the mitogen-activated protein kinase 14 pathway.

Colorectal cancer (CRC) remains one of the most common malignancies worldwide, and liver metastasis represents a considerable challenge during CRC treatment. Aberrant expression of angiopoietin-like protein 3 (ANGPTL3) has been reported in several human cancer types. However, the function and mechanism of ANGPTL3 in CRC remain unclear. In this study, we first explored ANGPTL3 expression profiles in CRC datasets from ONCOMINE and in local samples from patients with CRC. We then elucidated the function of ANGPTL3 via knockdown and overexpression experiments. Bioinformatic analyses were performed to investigate the biological function and associated molecular mechanisms of ANGPTL3 in CRC oncogenesis and development. Finally, a xenograft model of liver metastasis was used to determine the role of ANGPTL3 in CRC metastasis. Our findings indicated that ANGPTL3 expression was upregulated in human CRC tissues, with high ANGPTL3 expression significantly correlated with poor survival of patients with CRC. ANGPTL3 overexpression promoted the proliferation and migration of CRC cells partially through mitogen-activated protein kinase 14 (MAPK14), while ANGPTL3 silencing had the opposite effect. Moreover, ANGPTL3 downregulation suppressed tumor growth and liver metastasis in xenograft mice. Collectively, the results presented here indicate that ANGPTL3 promotes cell proliferation and liver metastasis partly via MAPK14, suggesting that ANGPTL3 plays a tumor-promoting role in CRC progression and thus may represent a therapeutic target for CRC treatment.

ARHGAP17 enhances 5-Fluorouracil-induced apoptosis in colon cancer cells by suppressing Rac1.

Colon cancer is a common cause of death in the world, and its main cause of therapy failure is chemoresistance. Apoptosis is de-regulated in colon cancer and is one key mechanism of cancer treatment. We recently reported that reduced expression of ARHGAP17, a Rho GTPase activating protein, correlated with a poor prognosis of colon cancer patients. Here we investigated the role of ARHGAP17 in apoptosis induced by 5-fluorouracil (5-FU) in human colon cancer cells and in mouse xenograft tumor model. We observed a decreased protein level of ARHGAP17 in 5-FU resistant colon cancer cells (HCT116/5-FU and HCT8/5-FU). While ARHGAP17 knockdown attenuated apoptosis upon 5-FU treatment in HCT116 and HCT8, and ARHGAP17 overexpression in HCT116/5-FU and HCT8/5-FU cells increased apoptosis induced by 5-FU. We also found that ARHGAP17 knockdown led to a high level of active Rac1 in HCT116 and HCT8, but ARHGAP17 overexpression reduced active Rac1 in HCT116/5-FU and HCT8/5-FU cells. However, Rac1 inhibitor abolished the effect of ARHGAP17 knockdown, and Rac1 overexpression diminished the effect of ARHGAP17 overexpression on apoptosis induced by 5-FU. Apoptosis was also confirmed by cleaved Caspase-3 and cleaved PARP. Further, we observed that overexpression of ARHGAP17 promoted 5-FU-induced apoptosis and attenuated tumor growth in vivo. Collectively, our data indicate that ARHGAP17 sensitizes chemotherapy-resistant colon cancer cells to apoptosis induced by 5-FU, which is in part through suppressing Rac1.

N6­methyladenosine upregulates miR­181d­5p in exosomes derived from cancer­associated fibroblasts to inhibit 5­FU sensitivity by targeting NCALD in colorectal cancer.

Resistance to 5­Fluorouracil (5­FU) is a frequent occurrence in patients with colorectal cancer (CRC). MicroRNAs (miRNAs) from cancer­associated fibroblasts (CAFs)­secreted exosomes have been associated with 5­FU sensitivity. The potential molecular mechanism of CAFs­exosomal miRNAs in CRC remains unclear. The aim of the present study was to elucidate the role of exosomal miRNAs in 5­FU sensitivity in CRC. Exosomes derived from CAFs were extracted. Exosomal miR­181d­5p was identified as a miRNA associated with 5­FU sensitivity. The putative function of exosomal miR­181d­5p was evaluated by ethynyl­2­deoxyuridine staining, flow cytometry, RNA immunoprecipitation, luciferase reporter assay, tumor xenograft formation, reverse transcription­quantitative PCR and western blot analysis. Modification of miR­181d­5p by the RNA N6­methyladenosine (m6A) methyltransferase like (METTL)3 was examined by m6A methylation analysis. The results indicated that m6A modification and METTL3 expression were upregulated in CRC patients. METTL3­dependent m6A methylation promoted the miR­181b­5p process by DiGeorge Syndrome Critical Region 8 (DGCR8) in CAFs. CAFs­derived exosomes inhibited 5­FU sensitivity in CRC cells through the METTL3/miR­181d­5p axis. A mechanistic study revealed that miR­181d­5p directly targeted neurocalcin δ (NCALD) to inhibit the 5­FU sensitivity of CRC cells. Patients with higher NCALD levels exhibited a higher survival rate. Taken together, METTL3­dependent m6A methylation was upregulated in CRC to promote the processing of miR­181d­5p by DGCR8. This led to increased miR­181d­5p expression, which inhibited the 5­FU sensitivity of CRC cells by targeting NCALD. The results of the present study provided novel insight into exosomal microRNAs in 5­FU sensitivity in CRC cells. Furthermore, exosomal miR­181d­5p may represent a potential prognostic marker for CRC.

CPNE1 Enhances Colorectal Cancer Cell Growth, Glycolysis, and Drug Resistance Through Regulating the AKT-GLUT1/HK2 Pathway.

INTRODUCTION: Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide. Copines-1 (CPNE1) has been shown to be overexpressed in various cancers; however, the role of CPNE1 in CRC remains unknown. Therefore, it is of great importance to elucidate the role of CPNE1 in CRC and its underlying mechanism of action. METHODS: CPNE1 expression in CRC tissues was measured by quantitative real-time PCR and immunohistochemical (IHC) staining. CPNE1 was knocked down (KD) or overexpressed using small inferring RNAs or lentiviral transduction in CRC cells. The proliferation, apoptosis, glycolysis, and mitochondrial respiration of CRC cells were assessed by cell counting kit-8, flow cytometry, and Xfe24 extracellular flux analyzer assays, respectively. The role of CPNE1 in tumor growth and chemoresistance was further confirmed in xenograft and patient-derived tumor xenograft models, respectively. RESULTS: CPNE1 mRNA and protein were upregulated in CRC tissues. CPNE1 promoted proliferation, inhibited apoptosis, increased mitochondrial respiration, enhanced aerobic glycolysis by activating AKT signaling, upregulated glucose transporter 1 (GLUT1) and hexokinase 2 (HK2), and downregulated the production of cleaved Caspase-3 (c-Caspase 3). CPNE1 also contributed to chemoresistance in CRC cells. CPNE1 KD inhibited tumor growth and increased the sensitivity of tumors to oxaliplatin in vivo. CONCLUSION: CPNE1 promotes CRC progression by activating the AKT-GLUT1/HK2 cascade and enhances chemoresistance.

General Identifiability Condition for Network Topology Monitoring with Network Tomography.

Accurate knowledge of network topology is vital for network monitoring and management. Network tomography can probe the underlying topologies of the intervening networks solely by sending and receiving packets between end hosts: the performance correlations of the end-to-end paths between each pair of end hosts can be mapped to the lengths of their shared paths, which could be further used to identify the interior nodes and links. However, such performance correlations are usually heavily affected by the time-varying cross-traffic, making it hard to keep the estimated lengths consistent during different measurement periods, i.e., once inconsistent measurements are collected, a biased inference of the network topology then will be yielded. In this paper, we prove conditions under which it is sufficient to identify the network topology accurately against the time-varying cross-traffic. Our insight is that even though the estimated length of the shared path between two paths might be "zoomed in or out" by the cross-traffic, the network topology can still be recovered faithfully as long as we obtain the relative lengths of the shared paths between any three paths accurately.

TRIM52 promotes colorectal cancer cell proliferation through the STAT3 signaling.

BACKGROUND: The tripartite motif (TRIM) family proteins are implicated in the pathogenesis of various human malignancies. The up-regulation and oncogenic roles of TRIM52 have been reported in hepatocellular carcinoma. In the current study, we aimed to examine its expression and possible function in colorectal cancer (CRC). METHOD: Immunohistochemical staining or immunoblotting analysis was carried out to detect protein expression. Cell proliferation and apoptosis was evaluated by Cell Counting Kit-8 (CCK-8) and flow cytometry assay, respectively. RESULTS: TRIM52 expression was increased in 67.5% of CRC tissues (54/80) compared to matched normal colonic mucosa. TRIM52 expression was closely related with tumor size (p = 0.0376), tumor stage (p = 0.0227) and overall survival (p = 0.0177). Short hairpin RNAs (shRNAs) targeting TRIM52 had the potential anti-proliferative effects on CRC cell lines, SW480 and LoVo, by inducing cell apoptosis. In addition, an in vivo xenograft experiment confirmed the in vitro results. In addition, TRIM52 shRNAs decreased the phosphorylation of STAT3, but increased the protein expression of SHP2, a negative regulator of STAT3 phosphorylation. TRIM52 formed a complex with SHP2 and promoted the ubiquitination of SHP2. Furthermore, inhibition of the STAT3 signaling by AG490 in RKO cells significantly abolished the effects of TRIM52 overexpression on cell proliferation, apoptosis and STAT3 activation. CONCLUSIONS: TRIM52 might exert oncogenic role in CRC via regulating the STAT3 signaling pathway.

Decreased expression of ARHGAP15 promotes the development of colorectal cancer through PTEN/AKT/FOXO1 axis.

Copious evidence demonstrates the crucial role of Rho GTPase-activating proteins in human malignancies. The downregulation of Rho GTPase-activating protein 15 (ARHGAP15), a Rac1-specific GAP, has been observed in glioma and pancreatic ductal adenocarcinoma. The present study explored the expression in colorectal cancer (CRC) by quantitative real-time PCR and immunohistochemistry analysis. The possible function of ARHGAP15 in CRC was investegated in vitro and in vivo. We found that ARHGAP15 expression was obviously lower in CRC specimens than in normal colonic mucosa. ARHGAP15 expression was significantly correlated with clinical stage, tumor size metastasis, vital status, and overall survival of CRC patients. ARHGAP15 overexpression inhibited cell growth, migration, and invasion of HT29 and RKO cells in vitro, whereas opposite results were observed in ARHGAP15-silenced LoVo cells. Mechanically, we found that PTEN (phosphatase and tensin homology deleted on chromosome 10) signaling pathway was closely correlated with ARHGAP15 expression by Gene set enrichment analysis with The Cancer Genome Atlas CRC data set. Increased PTEN and Forkhead box protein O1 (FOXO1, a downstream transcription factor of AKT), and decreased phosphorylation of AKT were observed in ARHGAP15-overexpressed HT29 and RKO cells. In addition, ARHGAP15 overexpression increased p21, which was responsible for the accelerated cell growth and S phase arrest, but decreased the protein levels of MMP-2 and MMP-9, which were stimuli for cell metastasis. Notably, upregulating PTEN expression, FOXO1 overexpression and interdicting the activation of AKT pathway with MK2206 suppressed the proliferation and the metastatic ability of ARHGAP15-silenced LoVo cells. In addition, FOXO1 overexpression markedly enhanced the expression and the promoter activity of ARHGAP15. Furthermore, ARHGAP15 overexpression significantly decelerated the pace of tumor growth and metastasis in the lung in vivo. In summary, these results suggest that ARHGAP15 might serve as a tumor suppressor during CRC progression and metastasis through PTEN/AKT/FOXO1-signaling pathway.

Tumor Suppressive Role of ARHGAP17 in Colon Cancer Through Wnt/ß-Catenin Signaling.

BACKGROUND/AIMS: A few Rho GTPase activating proteins (RhoGAPs) have been identified as tumor suppressors in a variety of human cancers. ARHGAP17, a member of RhoGAPs, has been reported to be involved in the maintenance of tight junction and epithelial barrier. The present study aimed to explore its expression in colon cancer and the possible function in colonic carcinogenesis. METHODS: The mRNA and protein expression was assessed by realtime PCR and immunoblotting, respectively. Cell Counting Kit-8 (CCK-8) and Transwell assays were performed to evaluate cell proliferation and invasion, respectively. RESULTS: We found that ARHGAP17 expression was obviously lower in colon cancer specimens than in normal colonic mucosa. ARHGAP17 expression was associated with tumor stage, size and differentiation. In vitro analysis demonstrated that ARHGAP17 overexpression inhibited cell growth and invasion of HCT-8 and HCT-116 cells. In addition, an in vivo experimental metastasis model showed that ARHGAP17 overexpression restricted cancer metastasis to the lung. Mechanically, we found that Wnt signaling contributed to the functions of ARHGAP17 in colon cancer cells. Gene set enrichment analysis (GSEA) in The Cancer Genome Atlas dataset showed that the Wnt signaling pathway was negatively associated with ARHGAP17 expression. The mRNA expression of ß-catenin (an important signaling transducer of canonical Wnt signaling) gene (CTNNB1) was negatively correlated with ARHGAP17 expression. Immunoblot analysis of downstream effectors of ß-catenin (c-Myc/p27 and MMP7) in ARHGAP17 overexpressing colon cancer cells and metastatic tumors within the lung also validated the GSEA result. ARHGAP17 overexpression increased the phosphorylation of glycogen synthetase kinase 3ß, and decreased ß-catenin nuclear localization and transcriptional activity. Furthermore, inhibition of Wnt signaling by Wnt Inhibitor Factor-1 (WIF-1) in HIEC cells with ARHGAP17 knockdown significantly attenuated the promotion effects of ARHGAP17 knockdown on cell proliferation, invasion and the activation of ß-catenin. CONCLUSION: these results suggest that ARHGAP17 might serve as a tumor suppressor in colon cancer progression and metastasis through Wnt/ß-catenin signaling pathway.

KIT performed as a driver gene candidate affecting the survival status of patients with stomach adenocarcinoma.

Stomach adenocarcinoma is estimated to cause 10,000 deaths in the US in 2016 and is the third most deadly cancer in China. We aim to identify the proteins and the genes that have impact on the prognosis of patients with stomach adenocarcinoma. Data of patients with stomach adenocarcinoma were retrieved from The Cancer Genome Atlas (TCGA). Proteins whose expression levels were highly correlated with survival status of patients were figured out. The expression levels of their mRNAs and their roles in the pathway were used to determine the driver gene candidates. The effects of mutations on the genes encoding KIT on mRNA expressions were carried. Ten antibodies were figured out to have significant correlation with stomach cancer prognosis. The coefficients of COXPH models matches their roles in the previous studies. The expression levels of mRNAs versus proteins suggested that KIT might act as a driver gene, which was also the central in the pathway of other selected proteins. The missense mutations on the gene encoding KIT led to the low expression of its mRNAs and there were much fewer nonsense mutations compared with other genes. It suggested that the important role of KIT as an oncogene in the progression of cancer, as well as a tyrosine-protein kinase during the normal activity. Ten antibodies, corresponding to fifteen proteins, were highly correlated with patients' survival time, within which KIT played a critical roles. It suggested that KIT might be used as biomarker or as target of cancer therapies.

The effect of Piper laetispicum extract (EAE-P) during chronic unpredictable mild stress based on interrelationship of inflammatory cytokines, apoptosis cytokines and neurotrophin in the hippocampus.

BACKGROUD: The Piper laetispicum C.DC. (Piperaceae) is a traditionally used herb in China for invigorating circulation and reducing stasis, detumescence and analgesia, which is distributed in the southern part of China and the southeastern part of Asia. Previous studies demonstrated that the ethyl acetate extract (EAE-P) of P. laetispicum possesses a significant antidepressant-like effect at doses higher than 60 mg/kg in Kunming (KM) mice, and this effect was not due to an increase in locomotive activity. METHODS: To research this mechanism, in the present study, the chronic unpredictable mild stress (CUMS) model in Sprague-Dawley rats was used to further elucidate behavioral changes and corresponding changes in inflammatory cytokines (TNF-α, IL-6, IL-10), apoptosis cytokines (P53, Bax, Bcl2, caspase-3) and neurotrophin (BDNF) in the hippocampus of EAE-P treatment animals. RESULTS AND CONCLUSIONS: The results suggest that EAE-P is beneficial to the behavioral outcome of the CUMS model animals, and decreased amounts of inflammatory cytokine IL-6 contributed to the antidepressant-like activation of EAE-P in every dosage group (15, 30, 60 mg/kg). In the low dosage group, down-regulated apoptosis cytokine p53 is associated with EAE-P effect, but it is inflammatory cytokine TNF-α that is related to the effect of EAE-P in the high dosage group. Meanwhile, the P53-dependent antiapoptotic effect of EAE-P may not be through Bcl-2 and Bax modulation. Furthermore, EAE-P showed up-regulated expression of brain-derived neurotrophic factor (BDNF) mRNA and down-regulated apoptosis cytokine caspase-3 mRNA, which was the same change tendency as with Fluoxetine.

Reactivation of HIC-1 gene by saRNA inhibits clonogenicity and invasiveness in breast cancer cells.

Hypermethylated in cancer 1 (HIC-1) is a tumor suppressor gene, which is epigenetically silenced in breast cancer. It is known that the loss of HIC-1, caused by promoter hypermethylation, is associated with tumor aggression and poor survival in breast carcinoma. It has been shown that small activating RNA (saRNA) targeting promoter sequences may induce gene re-expression. In the current study, saRNA was used to restore HIC-1 expression, and the effects on colony formation, invasiveness and the cell cycle in breast cancer cells were explored. dsHIC1-2998, an saRNA, exhibited activating efficacy on MCF-7 and MDA-MB-231 cancer cell lines. A clonogenicity assay showed that evident colony inhibition was induced via saRNA-mediated re-expression of HIC-1 in the two cancer cell lines. Reactivation of HIC-1 significantly inhibited cell migration and invasion, resulting in G0/G1 cell cycle arrest in these cell lines. These findings suggest that HIC-1 may be a potential target in gene therapy for the treatment of breast cancer. saRNA may function as a therapeutic option for upregulating tumor suppressor genes in breast cancer.

Small activating RNA restores the activity of the tumor suppressor HIC-1 on breast cancer.

HIC-1 is a gene that is hypermethylated in cancer, and commonly downregulated in human breast cancer. However, the precise mechanisms and molecular pathways regulated by HIC-1 remain unclear. We assessed HIC-1 expression on a tissue microarray containing 80 cases of breast cancer. We also analyzed its biological function by restoring HIC-1 expression using 5-aza-2' deoxycytidine (5-CdR) and small-activating RNAs for the reversal of HIC-1 tumor suppressive effects on MCF-7 and MDA-MB-231 cell lines. An Agilent Q44h global expressing microarray was probed after restoring the expression of HIC-1. Data demonstrated that HIC-1 expression was reduced significantly in breast cancer tissues. HIC-1 immunohistochemistry resulted in mean staining scores in cancer tissue and normal ductal epithelia of 3.54 and 8.2, respectively (p<0.01). 5-CdR partially reversed HIC-1 expression, and modulated cell growth and apoptosis. dsHIC1-2998, an saRNA, showed activating efficacy in breast cancer cells. A group of differentially expressed genes were characterized by cDNA microarray. Upon saRNA treatment, genes upregulated included those involved in immune activation, cell cycle interference, the induction of apoptosis, anti-metastasis, and cell differentiation. Downregulated genes included oncogenes and those that play roles in cell invasion, cell growth, and cell division. Our findings may provide valuable resources not only for gene functional studies, but also for potential clinical applications to develop novel drug targets.

Structure-activity relationship of 39 analogs of laetispicine with antidepressant properties.

The natural product Laetispicine ( N -isobutyl-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienoamide), was isolated from the Piper laetispicum C. DC and screened, for its antidepressant activity and antinociceptive effects. Structure-functional activities of five natural products indicated that biological activity is dependent on double bonds present within the benzene ring and a conjugated double bond located at positions 2-3 and 4-5 in the molecular structure. To further understand the structural-activity relationship of Laetispicine as a new potent and safe antidepressant, the structural-activity relationship of 39 analogs of Laetispicine were synthetized and tested in forced swimming tests in mice whilst also in protective effects against glutamate or H 2 O 2 induced apoptosis in PC12 cells. The results show that the compound 30 - N -isobutyl-11-(4-chlorophenyl) undeca-2E,4E,9E-trienamide exhibited the same activity as the parental compound Laetispicine, and furthermore, the effective dose of this compound is lower than Laetispicine. Therefore, the compound 30 might be a potentially useful therapy in the treatment of depression. For structure, the conjugated double bonds located at 2-3, 4-5 and isolated double bonds from benzene ring are necessary for the antidepressant activities no matter the different length of carbon chain; the isobutyl connected with acylamino also are necessary; and the benzodioxole moiety is replaceable, the halogen atom in phenyl ring at the para-position could enhance this kind of activity.

Inactivation of tumor suppressor gene HIC1 in gastric cancer is reversed via small activating RNAs.

HIC1 is a tumor suppressor gene that is down-expressed in different malignancies, in part, because of promoter hypermethylation. However, the biological function of HIC1 in gastric cancer remains unclear. It is known that small double-stranded RNAs can induce gene expression by targeting promoter sequences. In the present study, we examined the expression levels of HIC1 in gastric cancer tissue. Several pieces of small double-stranded RNAs were used for the activation of HIC1. Tissue microarray analysis of gastric cancer indicated that down-regulation of HIC1 in gastric cancer tissue was dramatic compared with the adjacent gastric mucosa. Gastric cancer cell lines also showed down-regulated HIC1 expression compared with a human immortalized gastric mucosa cell line. One out of four dsRNAs produced activation of HIC1 as assessed by real-time PCR and Western blotting. Use of a cell counting kit 8 and clonogenicity assays indicated that dsRNA-mediated re-expression of HIC1 inhibited cell proliferation and clonogenicity in gastric cancer. Reactivation of HIC1 suppressed cell migration and induced cell cycle arrest in the G0/G1 phase, as well as induced apoptosis. These results suggest that HIC1 is a potential target of gene therapy against gastric cancer, and that dsRNAs could function as a therapeutic option for up-regulating tumor suppressor genes in gastric cancer and other malignancies.

Three new xanthones from the resin of Garcinia hanburyi.

Three new xanthones, 22,23-dihydroxydihydrogambogenic acid (1), 12-hydroxygambogefic acid A (2), and hanburixanthone (3), along with thirteen known compounds were isolated from the resin of Garcinia hanburyi. The structures of the new compounds were determined by detailed analysis of 1D and 2D NMR spectra and by comparison with related model compounds. All compounds were tested for their cytotoxicities against A549, HCT116, and MDA-MB-231, and most of them showed significant effects on the cell lines.

Cytotoxic alkoxylated xanthones from the resin of Garcinia hanburyi.

Three new xanthones, garcinolic acid (1), 10α-ethoxy-9,10-dihydromorellic acid (2), and 10α-ethoxy-9,10-dihydrogambogenic acid (3), along with six known compounds were isolated from the resin of Garcinia hanburyi. These compounds were tested for their cytotoxicities against A549, HCT116, SK-BR-3 and HepG2, and showed high inhibitory effects on the cell lines.

3-Eth-oxy-carbonyl-2-hy-droxy-6-meth-oxy-4-methyl-benzoic acid.

The title compound, C(12)H(14)O(6), a substituted isophthalic acid monoester which was isolated from the lichen Thamnolia vermicularis var. subuliformis, displays intra-molecular carbox-yl-meth-oxy O-H⋯O and hy-droxy-carboxyl O-H⋯O hydrogen-bonding inter-actions. The terminal methyl group of the ethyl ester is disordered over two sets of sites with occupancies of 0.599 (19) and 0.401 (19).

Total synthesis and antidepressant activities of laetispicine and its derivatives.

The first total synthesis of laetispicine (1a), an amide alkaloid isolated from the stems of Piper laetispicum C.DC (Piperaceae), and the synthesis of some of its derivatives were described. Based on the evaluation of antidepressant activities in the forced swimming test, compounds 1h and 1i were identified as potent and safe antidepressant lead compounds.

A new lignan and other constituents from Zanthoxylum armatum DC.

A new lignan (1), named armatumin, along with 25 known compounds, was isolated from Zanthoxylum armatum. The structure of compound 1 was elucidated using spectroscopic methods. Compounds 7, 8, 11, 13, 16, 20 and 21 were first isolated from Z. armatum and compounds 14 and 19 were isolated for the first time from Rutaceae.