Three Neolignan Glycosides from the Root of Nothopanax davidii.

Three neolignan glycosides, including a new compound (7S,8R)-dihydro-3'-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1'-benzofuranpropanol-9-O-ß-D-xylopyranoside (1), were isolated from the root of Nothopanax davidii. Their structures were determined by extensive spectroscopic analyses, particularly NMR, HR-ESI-MS, and ECD experiments, and the absolute configuration of 2 was first definitively determined. The anti-tumor activity was assessed on four tumor cells by MTT assay, the anti-inflammatory activity was determined by inhibition of NO production in LPS reduced RAW264.7 cells, and the interaction with iNOS was predicted by molecular docking. At the dose of 100 µM, the three neolignan glycosides showed no cytotoxic activity against HepG2, HCT116, HeLa and A549 human tumor cells, but significantly inhibited LPS induced NO generation in RAW264.7 cells with inhibition rates of 31.53 %, 23.95 %, and 20.79 %, respectively, showing weak anti-inflammatory activity, possibly due to their binding to key residues of iNOs involved in inhibitor binding.

Four New Triterpenoid Saponins from the Root of Ilex centrochinensis.

One new siaresinolic acid saponin (1) and three new rotundic acid saponins (2-4) were isolated from the roots of Ilex centrochinensis. Their structures were confirmed by detailed analysis of standard spectroscopic data (IR, MS, 1D and 2D NMR). Compounds 1-4 exhibited anti-inflammatory activity by inhibiting nitric oxide production in a lipopolysaccharide-induced RAW264.7 cell inflammatory model. However, they showed no significant lipid-lowering activity against the production of triglycerides in the lipid-accumulation model of HepG2 cells induced by oleic acid.

A population pharmacokinetic study of tacrolimus in healthy Chinese volunteers and liver transplant patients.

AIM: To develop a population pharmacokinetic (PopPK) model of tacrolimus in healthy Chinese volunteers and liver transplant recipients for investigating the difference between the populations, and for potential individualized medication. METHODS: A set of 1100 sparse trough concentration data points from 112 orthotopic liver transplant recipients, as well as 851 dense data points from 40 healthy volunteers receiving a single dose of tacrolimus (2 mg, p.o.) were collected. PopPK model of tacrolimus was constructed using the program NONMEM. Related covariates such as age, hepatic and renal functions that were potentially associated with tacrolimus disposition were evaluated. The final model was validated using bootstrapping and a visual predictive check. RESULTS: A two-compartment model of tacrolimus could best describe the data from the two populations. The final model including two covariates, population (liver transplant recipients or volunteers) and serum ALT (alanine aminotransferase) level, was verified and adequately described the pharmacokinetic characteristics of tacrolimus. The estimates of V2/F, Q/F and V3/F were 22.7 L, 76.3 L/h and 916 L, respectively. The estimated CL/F in the volunteers and liver transplant recipients was 32.8 and 18.4 L/h, respectively. Serum ALT level was inversely related to CL/F, whereas age did not influence CL/F. Thus, the elderly (≥65 years) and adult (<65 years) groups in the liver transplant recipients showed no significant difference in the clearance of tacrolimus. CONCLUSION: Compared with using the sparse data only, the integrating modeling technique combining sparse data from the patients and dense data from the healthy volunteers improved the PopPK analysis of tacrolimus.

Human CD133-positive hematopoietic progenitor cells initiate growth and metastasis of colorectal cancer cells.

The tumour-specific 'pre-metastatic niche' has emerged as a potential driving force for tumour metastasis and has been confirmed using mouse models of cancer metastasis. Vascular endothelial growth factor receptor-1(+) hematopoietic progenitor cells (HPCs) have been shown to play an important role in metastasis, forming a 'pre-metastatic niche' at designated sites for distant tumour progression. Here, CD133+ human umbilical hematopoietic progenitor cells (HUHPCs) were purified from human umbilical cord blood and expanded in vitro. We studied the effects of CD133+ HUHPCs on the growth and metastasis of four colorectal cancer (CRC) cell lines by using cell-to-cell co-culture. Our results revealed that CD133+ HUHPCs promoted the proliferation and invasion of CRC cells in vitro and enhanced tumour growth and metastasis in vivo. Moreover, CD133+ HUHPCs were observed in the pre-metastatic liver tissue using immunohistochemical analysis after co-injection of SW480/EGFP(+) cells and HUHPCs. Further experiments were therefore conducted to uncover the molecular mechanisms by which CD133+ HUHPCs influenced colon carcinogenesis and cancer progression. Extracted proteins were separated using the two-dimensional difference in gel electrophoresis technology. Among the differentially expressed proteins, mitogen-activated protein 4 kinase 4, stromal cell-derived factor-1, matrix metallopeptidase 9, calumenin, peripherin, leucine zipper, putative tumour suppressor 1 and guanidinoacetate methyltransferase attracted our attention. Western blot analysis further confirmed the differential expression of these proteins. Altogether, these results suggest that CD133+ HUHPCs may induce proliferation or metastasis of CRC cells and impact their derived proteins by providing a pre-metastatic microenvironment.

SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1.

Slingshot phosphatase 3 (SSH3) is a member of the SSH phosphatase family that regulates actin filament dynamics. However, its role in cancer metastasis is relatively unclear compared to that of SSH1. Here, we showed that SSH3 was upregulated in colorectal cancer (CRC). Of note, SSH3 was upregulated in the tumor thrombus and lymph node metastasis compared with that in paired primary CRC tissues. High SSH3 expression was associated with the aggressive phenotype of CRC and may be an independent prognostic factor for the poor survival of patients with CRC. SSH3 significantly enhanced the invasion and metastasis of CRC cells in vitro and in vivo. Moreover, SSH3 regulated the remodeling of actin, which is involved in the cytoskeleton signaling pathway, through its interaction with LIMK1/Rac1 and subsequently promoted CRC cell invasion and metastasis. Our data elucidate an important role for SSH3 in the progression of CRC, and SSH3 may be considered a potential therapeutic target for CRC.

[Effect of propofol on gastric mucosal injury after hemorrhagic shock and reperfusion in rabbits].

OBJECTIVE: To study the therapeutic effect and its mechanisms of propofol on gastric mucosal injury after hemorrhagic shock with reperfusion in rabbits. METHODS: Seventy-five New Zealand healthy adult male rabbits were randomly divided into 5 groups of 15 rabbits each: control group (S group), model group (M group), pre-ischemia group (P1 group), pre-reperfusion group (P2 group) and post-reperfusion group (P3 group). The hemorrhage reperfusion-gastric mucosal injury (HR-GMI) model was reproduced. In P1, P2 and P3 groups, propofol 5 mg/kg was injected intravenously 10 minutes before ischemia, reperfusion and 20 minutes after reperfusion respectively, then propofol 20 mgxkg(-1)xh(-1) was infused. In control and model groups, propofol was replaced with equal volume of normal saline. The ultrastructure change in gastric mucosa was examined, and the activity of superoxide dismutase (SOD) and contents of malondialdehyde (MDA) in serum and gastric mucosa were determined. RESULTS: (1) In M group, the most of the cristae and membranes of mitochondria in chief cells of gastric mucosa were fused or disappeared, with formation of vacuoles which induced vacuolization, and a part of membrane blended or disappeared, and degranulation appeared in rough endoplasmic reticulum in these cells. These changes in mitochondria and rough endoplasmic reticulum were mitigated in all propofol groups, especially in P1 group. (2) The activity of SOD was significantly decreased and the contents of MDA were obviously increased in serum and gastric mucosa in M group compared with those in S group (all P<0.01). The activity of SOD was significantly increased and the content of MDA was decreased in serum and gastric mucosa in all propofol groups compared with those in M group (P<0.05 or P<0.01). The activity of SOD was higher and the content of MDA was lower in P1 group than those in P3 group (both P<0.05). CONCLUSION: Propofol can attenuate the gastric mucosa injury by eliminating oxygen free radical and preventing its generation caused by hemorrhagic shock and reperfusion.

Hypermethylation of NDN promotes cell proliferation by activating the Wnt signaling pathway in colorectal cancer.

The progression of CRC is a multistep process involving several genetic changes or epigenetic modifications. NDN is a member of the MAGE family, encoding a protein that generally suppresses cell proliferation and acting as a transcriptional repressor. Immunohistochemical staining revealed that the expression of NDN was significantly down-regulated in CRC tissues compared with normal tissues and the down-regulation of NDN in CRC could reflect the hypermethylation of the NDN promoter. Treatment of the CRC cell line SW480 with the demethylating agent 5-Aza-CdR restored the NDN expression level. The down-regulation of NDN was closely related to poor differentiation, advanced TNM stage and poor prognosis of CRC. The inhibition of NDN promoted CRC cell proliferation by enriching cells in the S phase. Furthermore, we observed that NDN binds to the GN box in the promoter of LRP6 to attenuate LRP6 transcription and inhibit the Wnt signaling pathway in CRC. In conclusion, our study revealed that the hypermethylation of NDN promotes cell proliferation by activating the Wnt signaling pathway through directly increasing the transcription of LRP6 in CRC. These findings might provide a new theoretical basis for the pathogenesis of CRC and facilitate the development of new therapeutic strategies against CRC.

Identification of NCK1 as a novel downstream effector of STAT3 in colorectal cancer metastasis and angiogenesis.

Signal transducer and activator of transcription 3 (STAT3) is known to activate targets associated with invasion, proliferation, and angiogenesis in a wide variety of cancers. The adaptor protein NCK1 is involved in cytoskeletal movement and was identified as a STAT3-associated target in human tumors. However, the underlying molecular mechanism associated with colorectal cancer (CRC) metastasis is not yet completely understood. In this study, we report a novel STAT3 to NCK1 signaling pathway in colorectal cancer (CRC). We investigated the expression of NCK1 and its potential clinical and biological significance in CRC. NCK1 was noticeably up-regulated in human CRC tissues. NCK1 was also significantly associated with serosal invasion, lymph metastasis, and tumor-node-metastasis classification but was inversely correlated with differentiation. Gain-of-function and loss-of-function studies have shown that ectopic expression of NCK1 enhanced metastasis and angiogenesis in CRC cells. By gene expression analyses, we revealed a high co-overexpression of STAT3 and NCK1 in CRC tissues. Ectopic overexpression of STAT3 in CRC cells induced the expression of NCK1, whereas STAT3 knockdown decreased the expression of NCK1. Promoter activation and binding analyses demonstrated that STAT3 promoted the expression of NCK1 via direct action on the NCK1 promoter. The knock down of NCK1 partially reduced the CRC cell metastasis and angiogenesis promoted by STAT3. Additionally, by co-immunoprecipitation assays, we verified that NCK1 interacted with PAK1, which resulted in the activation of the PAK1/ERK pathway. STAT3 induced the transcription of NCK1 and triggered a PAK1/ERK cascade in CRC. These findings suggest a novel STAT3 to NCK1 to PAK1/ERK signaling mechanism that is potentially critical for CRC metastasis and angiogenesis.

[Effect of miR-101 on biological characteristics of colorectal cancer cell line SW620].

OBJECTIVE: To investigate the effect of miR-101 on biological behaviors of colorectal cancer cell line SW620. METHODS: CCK-8 method, colony formation assay, cell cycle analysis and apoptosis analysis were applied to assess the effects of miR-101 on cell proliferation, invasion and apoptosis of SW620 cells. RESULTS: Over-expression of miR-101 in SW620 cells significantly suppressed the cell proliferation and attenuated the colony-forming ability of the cells. Flow cytometry showed that over-expression of miR-101 in SW620 cells caused obvious cell cycle arrest in G2/M and 1/ phases, and significantly increased the cell apoptosis rate. CONCLUSION: Over-expression of miR-101 can inhibit the proliferation, cause cell cycle arrest and promote apoptosis of colorectal cancer SW620 cells.

Novel inflammatory markers associated with cognitive performance: Singapore Longitudinal Ageing Studies.

We identified and validated several novel inflammatory markers of cognitive performance in community-living older persons. An exploratory study (n = 83) correlated 177 inflammatory markers assayed by Luminex with the Mini-Mental State Examination (MMSE) and identified 8 inflammatory markers for enzyme-linked immunosorbent assay (ELISA) and correlations with MMSE, Montreal Cognitive Assessment (MoCA), and cognitive impairment in the validation study (n = 139). The validation study replicated the significant associations of soluble interleukin-2 receptor alpha chain (sIL-2Rα; p = 0.050), soluble tumor necrosis factor receptor 2 (sTNFR2; p = 0.002) and soluble glycoprotein 130 (sgp130; p = 0.026) with MMSE, and sIL-2Rα (p = 0.019) and sgp130 (p < 0.001) with MoCA. Significant trends of associations of tertiles of sgp130, sIL-2Rα, and sTNFR2 were found with cognitive impairment. Highly elevated estimates of association of high versus low tertiles were obtained for sgp130 (odds ratio [OR] = 4.24, 95% confidence interval [CI] 0.96-18.8), sIL-2Rα (OR = 3.94, 95% CI 0.83-18.7), and sTNFR2 (OR = 7.58, 95% CI 1.19-48.1). sgp130, sTNFR2, and sIL-2Rα are promising inflammatory markers of low cognitive performance for further investigation.

Loss of TINCR expression promotes proliferation, metastasis through activating EpCAM cleavage in colorectal cancer.

Long non-coding RNAs (lncRNAs) are involved in kinds of human diseases, including colorectal cancer (CRC). TINCR, a 3.7 kb long non coding RNA, was associated with cell differentiation in keratinocyte and gastric cancer cells. However, little is known about the role of TINCR in regulation CRC progression. Here, we showed that lncRNA TINCR was associated with CRC proliferation and metastasis. TINCR was statistically downregulated in CRC tissues and metastatic CRC cell lines compared with their counterparts. TINCR was reversely correlated with CRC progression and promoted tumor cells growth, metastasis in vivo and in vitro. While overexpression of TINCR had opposite effect. In addition, we also found that TINCR specifically bound to EpCAM through RNA IP and RNA pull down assays. Loss of TINCR promoted hydrolysis of EpCAM and then released EpICD, subsequently, activated the Wnt/ß-catenin pathway. Further studies shown that c-Myc repressed the expression of TINCR through repressing sp1 transcriptive activity, which established a positive feedback loop controlling c-Myc and TINCR expression. These findings elucidate that loss of TINCR expression promotes proliferation and metastasis in CRC and it could be considered as a potential cancer suppressor gene.

Regulation of colorectal carcinoma stemness, growth, and metastasis by an miR-200c-Sox2-negative feedback loop mechanism.

PURPOSE: To elucidate a novel mechanism of miR-200c in the regulation of stemness, growth, and metastasis in colorectal carcinoma (CRC). EXPERIMENTAL DESIGN: Quantitative reverse transcription PCR was used to quantify miR-200c expression in CRC cell lines and tissues. A luciferase assay was adopted for the target evaluation. The functional effects of miR-200c in CRC cells were assessed by its forced or inhibited expression using lentiviruses. RESULTS: MiR-200c was statistically lower in CRC clinical specimens and highly metastatic CRC cell lines compared with their counterparts. Sox2 was validated as a target for miR-200c. The knockdown of miR-200c significantly enhanced proliferation, migration, and invasion in CRC cell lines, whereas the upregulation of miR-200c exhibited an inverse effect. Moreover, rescue of Sox2 expression could abolish the effect of the upregulation of miR-200c. In addition, the reduction of miR-200c increased the expression of CRC stem cell markers and the sphere-forming capacity of CRC cell lines. Further study has shown that miR-200c and Sox2 reciprocally control their expression through a feedback loop. MiR-200c suppresses the expression of Sox2 to block the activity of the phosphoinositide 3-kinase (PI3K)-AKT pathway. CONCLUSION: Our findings indicate that miR-200c regulates Sox2 expression through a feedback loop and is associated with CRC stemness, growth, and metastasis.

[Long-term culture and identification of CD133⁺ hematopoietic progenitor cells from human umbilical cord blood].

OBJECTIVE: To isolate CD133(+) hematopoietic progenitor cells from human umbilical cord blood and optimize the culture condition for maintaining their stem cell characteristics. METHODS: CD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system, and the cells were detected by flow cytometry. Four methods were used for culturing cells. After 8 weeks' culture, cytomorphology, flow cytometry, immunocytochemistry and immunofluorescence assay were used to identify the characteristics of the stem cells. RESULTS: Over 80% of CD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system. The cells were effectively expanded using optimized serum-free medium after 8 weeks of cell culture, whereas the cells in other media differentiated into adherent cells in a poor state. CONCLUSION: The optimized serum-free medium allows effective expansion of CD133(+) hematopoietic progenitor cells that maintain stem cell characteristics after a long-term culture.

[Construction of has-miR-335 lentiviral vector and verification of the target gene of miR-335].

OBJECTIVE: To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335. METHODS: The precursor sequence of miR-335 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. Real-time quantitative RT-PCR was used to detect miR-335 and RASA1 expression in different colorectal cancer cell lines. A recombinant vector psiCHECK-2-RASA1 containing RASA1 3'UTR was constructed followed by site-directed mutagenesis of RASA1 3'UTR to establish the vector psiCHECK-2-RASA1-Mut. Co-transfection of hsa-mir-335 or a NC with these recombined vectors in HEK293A and SW480 cells was performed, and dual-luciferase reporter assay was utilized to examine the changes in luciferase activities. The recombinant PLVTHM-miR335 plasmid was packaged into mature lentivirus by 293FT cells and used to infect SW620 cells. Flow cytometry was employed for sorting the GFP+ cells. The expression of miR-335 and RASA1 were determined by qRT-PCR, and Western blotting was used to detect the expression of RASA1 protein in SW620 cell lines. RESULTS: The recombinant lentiviral vector PLVTHM-miR335, psiCHECK-2-RASA1 and the mutation expression vector psiCHECK-2-RASA1-Mut were successfully constructed. Dual-luciferase reporter assay showed that miR-335 decreased luciferase activity in cells co-transfected with psiCHECK-2-RASA1. The expression of miR-335 in SW620 cells infected with the lentivirus PLVTHM-miR335 was significantly increased, but the expression of RASA1 showed only slight changes. Overexpression of miR-335 suppressed the expression of RASA1 protein in SW620 cells. CONCLUSION: We have successfully constructed the lentiviral vector containing mir-335 gene and a SW620 cell line with miR-335 overexpression. MiR-335 can suppress RASA1 gene expression by targeting the specific sequence of RASA1 3'UTR.

[Selection and identification of specific-binding peptides for cancer stem cell surface marker CD133].

OBJECTIVE: To select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7>(TM) phage peptide library. METHODS: With a biotinylated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein, the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning. The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA. The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis, and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique. RESULTS: After 4 rounds of liquid phase selection, the phages capable of specific binding with human CD133 were effectively enriched, with an enrichment ratio of 388 times compared to that at the fourth and first rounds. Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones, among which 11 had identical amino acid sequence of TISWPPR, and 2 had the sequence of STTKLAL, and the former sequence showed a stronger binding specificity to CD133. CONCLUSION: We have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7(TM) phage peptide library, demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.