[Show abstract][Hide abstract] ABSTRACT: Main conclusion:
The present study documented the action of a potential allelochemical, narciclasine, on auxin transport in Arabidopsis by mainly affecting subcellular trafficking of PIN and AUX1 proteins and through interfering actin cytoskeletal organization. Narciclasine (NCS), an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs, has potential allelopathic activity and affects auxin transport. However, little is known about the cellular mechanism of this inhibitory effect of NCS on auxin transport. The present study characterizes the effects of NCS at the cellular level using transgenic Arabidopsis plants harboring the promoters of PIN, in combination with PIN-GFP proteins or AUX1-YFP fusions. NCS treatment caused significant reduction in the abundance of PIN and AUX1 proteins at the plasma membrane (PM). Analysis of the subcellular distribution of PIN and AUX1 proteins in roots revealed that NCS induced the intracellular accumulation of auxin transporters, including PIN2, PIN3, PIN4, PIN7 and AUX1. However, other PM proteins, such as PIP2, BRI1, and low temperature inducible protein 6b (LTI6b), were insensitive to NCS treatment. NCS-induced PIN2 compartments were further defined using endocytic tracer FM 4-64 labeled early endosomes and suggested that this compound affects the endocytosis trafficking of PIN proteins. Furthermore, pharmacological analysis indicated that the brefeldin A (BFA)-insensitive pathway is employed in the cellular effects of NCS on PIN2 trafficking. Although NCS did not alter actin dynamics in vitro, it resulted in the depolymerization of the actin cytoskeleton in vivo. This disruption of actin filaments by NCS subsequently influences the actin-based vesicle motility. Hence, the elucidation of the specific role of NCS is useful for further understanding the mechanisms of allelopathy at the phytohormone levels.
[Show abstract][Hide abstract] ABSTRACT: Objective
To examine the effects of TRIB3 on endoplasmic reticulum stress induced β-cell apoptosis and to investigate the mechanism with a specific emphasis on the role of NF-κB pathway.
We investigated the effects of TRIB3 on ER stress-induced β-cell apoptosis in INS-1 cells and primary rodent islets. The potential role of TRIB3 in thapsigargin (Tg)-induced β-cell apoptosis was assessed using overexpression and siRNA knockdown approaches. Inducible TRIB3 β-cells, regulated by the tet-on system, were used for sub-renal capsule transplantation in streptozotocin (STZ)-diabetic mice, to study the effects of TRIB3 on ER stress-induced β-cell apoptosis in vivo. Apoptosis was determined by TUNEL staining both in vivo and in vitro, while the molecular mechanisms of NF-κB activation were investigated.
TRIB3 was induced in ER-stressed INS-1 cells and rodent islets, and its overexpression was accompanied by increased β-cell apoptosis. Specifically, TRIB3 overexpression enhanced ER stress inducer thapsigargin (Tg)-induced INS-1 derived β-cell apoptosis both in vitro and in sub-renal capsular transplantation animal model. Additionally, knockdown of Trib3 blocked Tg-induced apoptosis. Mechanistically, the induction of TRIB3 during ER stress resulted in the activation of NF-κB and aggravated INS-1 derived β-cell apoptosis, while inhibiting the NF-κB pathway significantly abrogated this response and prevented β-cell apoptosis, both in vitro and in sub-renal capsular transplantation animal model.
TRIB3 mediated ER stress-induced β-cell apoptosis via the NF-κB pathway.
[Show abstract][Hide abstract] ABSTRACT: Chronic exposure to free fatty acids (FFAs) may induce β cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger β cell apoptosis is still unclear. Tribbles homolog 3 (TRB3) is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 β cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC) δ was enhanced by upregulation of TRB3. Preventing PKCδ nuclear translocation and PKCδ selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 β cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.
PLoS ONE 05/2014; 9(5):e96089. DOI:10.1371/journal.pone.0096089 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atherosclerotic plaque vulnerability is associated with cerebrovascular events in patients with carotid atherosclerosis. The aim of this study was to investigate the expression of inflammatory factors in carotid artherosclerotic plaques in order to explore its clinical significance in patients with carotid stenosis. Forty three patients with carotid stenosis were divided into symptomatic group (n=24) and asymptomatic group (n=19) based on clinical manifestation. All patients were treated with selective standard carotid endarterectomy (CEA); the carotid atherosclerotic plaques were removed surgically and studied pathologically to investigate the expression of nuclear factor-kappa κ (NF-κB), CD68 and CD105. The plaques were grouped into stable and unstable plaques based on thickness of the fibrous cap and the area of lipid-rich core in the plaques. The proportion of unstable plaques were significantly higher in symptomatic group than in asymptomatic group (70.8% vs. 63.2%, P=0.026). Results of immunohistochemisty staining showed that the expression of NF-κB, CD68 and CD105 in unstable plaques was higher than stable plaques (P<0.001). The association of the higher expression of these factors with instability of carotid plaque needs to be clarified in future study.
[Show abstract][Hide abstract] ABSTRACT: Background
Survivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of mitosis and programmed cell death. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. The aim of this study was to determine the antitumor activity of YM155 in SK-NEP-1 cells.
SK-NEP-1 cell growth in vitro and in vivo was assessed by MTT and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis in cell culture. Then gene expression profile of tumor cells treated with YM155 was analyzed with real-time PCR arrays. We then analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis tool.
YM155 treatment resulted in inhibition of cell proliferation of SK-NEP-1cells in a dose-dependent manner. Annexin V assay, cell cycle, and activation of caspase-3 demonstrates that YM155 induced apoptosis in SK-NEP-1 cells. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ± 0.77 cm3; YM155 10 mg/kg: 0.95 ± 0.55 cm3) compared to DMSO group (DMSO: 3.70 ± 2.4 cm3) or PBS group cells (PBS: 3.78 ± 2.20 cm3, ANOVA P < 0.01). YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ± 0.24 g; YM155 10 mg/kg: 0.72 ± 0.17 g) compared to DMSO group (DMSO: 2.06 ± 0.38 g) or PBS group cells (PBS: 2.36 ± 0.43 g, ANOVA P < 0.01). Real-time PCR array analysis showed between Test group and control group there are 32 genes significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment. Ingenuity pathway analysis (IPA) showed cell death was the highest rated network with 65 focus molecules and the significance score of 44. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to cell death, cellular function maintenance, cell morphology, carbohydrate metabolism and cellular growth and proliferation. Death receptor signaling (3.87E-19), TNFR1 signaling, induction of apoptosis by HIV1, apoptosis signaling and molecular mechanisms of cancer came out to be the top four most significant pathways. IPA analysis also showed top molecules up-regulated were BBC3, BIRC3, BIRC8, BNIP1, CASP7, CASP9, CD5, CDKN1A, CEBPG and COL4A3, top molecules down-regulated were ZNF443, UTP11L, TP73, TNFSF10, TNFRSF1B, TNFRSF25, TIAF1, STK17A, SST and SPP1, upstream regulator were NR3C1, TP53, dexamethasone , TNF and Akt.
The present study demonstrates that YM155 treatment resulted in apoptosis and inhibition of cell proliferation of SK-NEP-1cells. YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors. Real-time PCR array analysis firstly showed expression profile of genes dyes-regulated after YM155 treatment. IPA analysis also represents new molecule mechanism of YM155 treatment, such as NR3C1 and dexamethasone may be new target of YM155. And our results may provide new clues of molecular mechanism of apoptosis induced by YM155.
BMC Cancer 12/2012; 12(1):619. DOI:10.1186/1471-2407-12-619 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The calcium-regulated transcription co-activator CREST (Ca2+-responsive transactivator) was expressed in pancreatic beta-cells. Moreover, CREST expression became significantly increased in pancreatic islets isolated from hyperglycemic Goto-Kakizaki (GK) rats as compared to normoglycemic Wistar controls. In addition, culture of beta-cells in the presence of high glucose concentrations also increased CREST expression in vitro. To further investigate the role of this transactivator in the regulation of beta-cell function, we established a stable beta-cell line with inducible CREST expression. Hence, CREST over-expression mimicked the glucotoxic effects on insulin secretion and cell growth in beta-cells. Moreover, high glucose-induced apoptosis was aggravated by up-regulation of the transactivator, but inhibited when CREST expression was partially silenced by siRNA technology. The further investigation found that up-regulation of Bax and down-regulation of Bcl-2 was indeed induced by its expression, especially under high glucose condition. In addition, as two causing factors leading to beta-cell apoptosis under diabetic conditions, endoplasmic reticulum (ER) stress and high free fatty acid mimicked the high glucose effects on CREST up-regulation and generation of apoptosis in beta-cells and these effects were specifically offset by the siRNA knockdown of CREST. These results indicated that CREST is implicated in beta-cell apoptosis induced by culture in high glucose, and hence that CREST may become a potential pharmacological target for the prevention and treatment of type 2 diabetes mellitus.
Journal of Endocrinology 11/2012; 216(2). DOI:10.1530/JOE-12-0286 · 3.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Elevated extracellular free fatty acids (FFAs) can induce pancreatic beta cell apoptosis, thereby contributing to the pathogenesis of type 2 diabetes mellitus (T2D). Mitochondrial dysfunction has been implicated in FFA-induced beta cell apoptosis. However, molecular mechanisms linking mitochondrial dysfunction and FFA-induced beta cell apoptosis are not clear. Dynamin-related protein 1 (DRP-1) is a mitochondrial fission modulator. In this study, we investigated its role in FFA-induced INS-1 beta cell apoptosis. DRP-1 protein was promptly induced in INS-1 cells and rat islets after stimulation by FFAs, and this DRP-1 upregulation was accompanied by increased INS-1 cell apoptosis. Induction of DRP-1 expression significantly promoted FFA-induced apoptosis in DRP-1 WT (DRP-1 wild type) inducible INS-1-derived cell line, but not in DRP-1K38A (a dominant negative mutant of DRP-1) inducible INS-1-derived cell line. To validate these in vitro results, we transplanted DRP-1 WT or DRP-1 K38A cells into renal capsules of streptozotocin (STZ)-treated diabetic mice to study the apoptosis in xenografts. Consistent with the in vitro results, the over-expression of DRP-1 led to aggravated INS-1-derived cell apoptosis triggered by FFAs. In contrast, dominant-negative suppression of DRP-1 function as represented by DRP-1 K38A significantly prevented FFA-induced apoptosis in xenografts. It was further demonstrated that mitochondrial membrane potential decreased, while cytochrome c release, caspase-3 activation, and generation of reactive oxygen species (ROS) were enhanced by the induction of DRP-1WT, but prevented by DRP-1 K38A in INS-1-derived cells under FFA stimulation. These results indicated that DRP-1 mediates FFA-induced INS-1-derived cell apoptosis, suggesting that suppression of DRP-1 is a potentially useful therapeutic strategy for protecting against beta cell loss that leads to type 2 diabetes.
PLoS ONE 11/2012; 7(11):e49258. DOI:10.1371/journal.pone.0049258 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study aimed to identify novel molecular markers for the early detection of colorectal cancer liver metastasis.
Genes related to hepatic metastasis were screened from the Oncomine database. The candidate markers were tested by immunohistochemistry, and their predictive accuracy was assessed by the cross-validation method and an independent test set.
We got three datasets containing 2,973 genes that were highly expressed in primary colon cancer tissues compared with non-metastatic colon cancer tissues and identified 7 candidate molecules for immunohistochemical validation. A total of 213 colorectal cancer samples were randomly divided into a training set (113 cases) and a blind testing set (100 cases). In the training set, immunohistochemical analysis showed that HP, OPN, and PTGIS expression were significantly higher in the hepatic metastasis group than in the non-metastasis group. Logistic regression analysis showed that HP and OPN levels in primary tumors and lymph node metastasis status were the only significant (P<0.05) parameters for detecting liver metastasis. The predictive accuracy of markers was assessed by the cross-validation method and an independent test set. In leave-one-out cross-validation, the two markers combined with clinicopathologic features resulted in 91.2% sensitivity and 96.4% specificity for hepatic metastasis detection. In an independent test set, the combination achieved 94.5% sensitivity and 88.9% specificity for predicting the hepatic metastasis of colorectal cancer.
Our results suggest that combined HP and OPN expression levels are significantly related to liver metastasis and prognosis, and, if this is validated, they could be used as accurate predictors of liver metastasis in colorectal cancer.
[Show abstract][Hide abstract] ABSTRACT: Treatment failure for breast cancer is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in breast cancer cells in vitro and in vivo. Identification of new targets will facilitate the developmental pace of new techniques in screening and early diagnosis. Improved abilities to predict progression and metastasis, therapeutic response and toxicity will help to increase survival of breast cancer patients.
Differential protein expression in two breast cancer cell lines, one with high and the other with low metastatic potential, was analyzed using two-dimensional liquid phase chromatographic fractionation (Proteome Lab PF 2D system) followed by matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI-TOF/MS).
Up regulation of α-subunit of ATP synthase was identified in high metastatic cells compared with low metastatic cells. Immunohistochemical analysis of 168 human breast cancer specimens on tissue microarrays revealed a high frequency of ATP synthase α-subunit expression in breast cancer (94.6%) compared to normal (21.2%) and atypical hyperplasia (23%) breast tissues. Levels of ATP synthase expression levels strongly correlated with large tumor size, poor tumor differentiation and advanced tumor stages (P < 0.05). ATP synthase α-subunit over-expression was detected on the surface of a highly invasive breast cancer cell line. An antibody against the ATP synthase α-subunit inhibited proliferation, migration and invasion in these breast cancer cells but not that of a non-tumor derived breast cell line.
Over-expression of ATP synthase α-subunit may be involved in the progression and metastasis of breast cancer, perhaps representing a potential biomarker for diagnosis, prognosis and a therapeutic target for breast cancer. This finding of this study will help us to better understand the molecular mechanism of tumor metastasis and to improve the screening, diagnosis, as well as prognosis and/or prediction of responses to therapy for breast cancer.
Journal of Translational Medicine 12/2011; 9(1):211. DOI:10.1186/1479-5876-9-211 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pancreatic β-cell dysfunction is a critical component in the pathogenesis of diabetes. Endoplasmic reticulum (ER) stress is one of the factors that induces pancreatic β-cell dysfunction, but the underlying mechanisms have not been well elucidated. In this study, we report that a mitochondrial fission modulator, dynamin-related protein 1 (DRP-1), plays an important role in ER stress-induced β-cell apoptosis. Induction of DRP-1 expression significantly promoted ER stress-induced apoptosis in the DRP-1 WT (DRP-1 wild- type) inducible β-cell line, but not in the DRP-1 K38A (a dominant negative mutant of DRP-1) inducible β-cell line. We further demonstrated that the mitochondrial membrane potential decreased, and that cytochrome c release, caspase-3 activation and generation of reactive oxygen species (ROS) were enhanced by induction of DRP-1 WT, but prevented by DRP-1 K38A in pancreatic β-cells under ER stress conditions. These results indicate that DRP-1 mediates ER stress-induced pancreatic β-cell apoptosis.
International Journal of Molecular Medicine 08/2011; 28(2):161-9. DOI:10.3892/ijmm.2011.684 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To explore the protective effect of KLF4 against cytotoxicity induced by cisplatin and its possible mechanisms.
The expression levels of KLF4 were detected by RT-PCR and western blot in cancer stem-like cells derived from hepatocarcinoma (T3A-A3) and the hepatocarcinoma cell line HepG2. KLF4 was knocked down in T3A-A3 by infection of pLVTHM-shKLF4 lentivirus and ectopic expressed in HepG2 by infection of pWPTS-KLF4 lentivirus. The MTT assay was carried out to determine the impact of KLF4 on cell survival in response to cisplatin. Cisplatin-induced DNA damage was measured by TUNEL staining. Glutathione content was measured by enzymatic assay. Buthionine sulfoximine was used to deplete the content of glutathione. The expression of γ-glutamylcysteine synthetase was analyzed by RT-PCR in HepG2 cells ectopic expressed KLF4.
With a higher level of KLF4, T3A-A3 cells were found to be more resistant to cisplatin than HepG2 cells. KLF4 knockdown was found to reduce cisplatin resistance in T3A-A3 cells. Ectopic expression of KLF4 in HepG2 cells was found to be associated with heightened resistance to DNA damage after exposure to cisplatin. Furthermore, the content of glutathione was found to be higher in T3A-A3 cells than in HepG2 cells. A nearly twofold increase in the cellular level of glutathione was identified in HepG2 cells with ectopic expression of KLF4. This was accompanied by heightened resistance to cisplatin. KLF4-mediated resistance to cisplatin in HepG2 cells was found to be completely abrogated by treatment with buthionine sulfoximine, an inhibitor of glutathione synthesis, which did not affect the expression of KLF4. Moreover, the mRNA expression of γ-glutamylcysteine synthetase, a rate-limiting enzyme of glutathione synthesis was up-regulated by KLF.
We conclude that KLF4 regulates the cellular sensitivity to cisplatin in hepatocarcinoma stem-like cells and hepatocarcinoma cells by elevating intracellular glutathione levels.
Cancer Chemotherapy and Pharmacology 07/2011; 69(2):377-85. DOI:10.1007/s00280-011-1708-7 · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: P-cadherin belongs to the family of classic cadherins, which is important for maintaining cellular localization and tissue integrity. Recently, it has become evident that P-cadherin contributes to the oncogenesis of many tumor types, including melanoma, prostate, breast, and colon carcinomas. Although cadherin switching is a crucial step in metastasis, the role of P-cadherin in colon cancer metastasis to the liver is unknown. In this study, we performed gene expression analysis and found that the level of P-cadherin was higher in tissue from liver metastases of colon cancer than in the corresponding primary colon cancer tissues. IHC analysis also showed that P-cadherin expression was significantly higher in liver metastases than in paired primary colorectal cancer tumors. Knockdown of P-cadherin in colon cancer cells inhibited wound healing, proliferation, and colony formation and resulted in developing fewer liver metastatic foci and reducing the tumor burden in vivo. Inhibition of P-cadherin expression also induced the up-regulation of E-cadherin and the down-regulation of β-catenin and its downstream target molecules, including survivin and c-Myc. In summary, these results uncover a novel function of P-cadherin in the regulation of colon cancer metastasis to the liver, suggesting that blocking the activity of P-cadherin or its associated signaling may be a valuable target for the treatment of hepatic metastases of colon carcinomas.
American Journal Of Pathology 07/2011; 179(1):380-90. DOI:10.1016/j.ajpath.2011.03.046 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the in vitro effect of tanshinone IIA on leukocyte-associated hypoxia-reoxygenation injury of human brain-blood barrier (BBB), we established the BBB model by culturing purified primary human brain microvascular endothelial cells (HBMVEC) to confluence on cell culture insert. BBB was identified by tight junction, transendothelial electrical resistance (TEER) and the permeability of BBB to horseradish peroxidase (HRP). The effect of tanshinone IIA on the permeability of BBB was tested at 2 h after hypoxia and 1h after reoxygenation with or without the supernatants of activated leukocytes. The effect of tanshinone IIA on leukocytes activation was analyzed by detection of MMP-9, cytokines and reactive oxygen species. The results showed that BBB formed by confluent HBMVECs had no cellular gap. Immunofluorescent staining for ZO-1 confirmed that the cells were connected by tight junction. Moreover, the BBB model had a higher TEER and a lower permeability for HRP than confluent HUVECs. The permeability of BBB for HRP was enhanced by hypoxia-reoxygenation and further greatly enhanced by adding the supernatants of activated leukocytes before reoxygenation. But such an effect was reversed by addition of tanshinone IIA before hypoxia. Moreover, tanshinone IIA could decrease the levels of MMP-9, TNF-α, IL-1α, IL-2, IFN-γ and reactive oxygen species in leukocytes. In conclusion, tanshinone IIA can protect BBB against leukocyte-associated hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the injury effects of leukocytic products. Tanshinone IIA may be a novel therapeutic agent for cerebral ischemia-reperfusion injury.
European journal of pharmacology 12/2010; 648(1-3):146-52. DOI:10.1016/j.ejphar.2010.08.040 · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the relevant molecular mechanisms of platelet in promoting metastasis of tumor cell. The adhesion of fluorescence dye labeled-platelet to human liver sinusoidal endothelial cell (LSEC) line and tumor cell lines were detected by fluorescence microscope and fluorescence plate reader or laser scanning confocal microscope. The relevant adhesion molecules were analyzed by the antibody blockage experiment. The immune colloidal gold transmission electron microscope (TEM), flow cytometry and dye transfer were used to decipher the adhesion and fusion of platelet and LSEC. The tumor cells adhesion to vessels in ischemia condition was analyzed on mouse mesenteric vessels and the metastasis and neovascularization of metastatic foci in pulmonary tissue were also detected after tumor cells injected into nude mice via tail veil. After hypoxia-reoxygenation, tumor cell or LSEC markedly increased its adhesion with platelet, which could be blocked by different antibodies to platelet adhesion molecules. Platelet increased adhesion of tumor cell to LSEC in dose-dependent manner. The fusion of platelet and LSEC was demonstrated by translocation of fluorescent dye from platelet into the adherent LSEC; gpIIb emerged on the LSEC; and confirmed by TEM. The morphological examination found platelet presented between tumor cell and LSEC. Animal experiment indicated that the tumor adhesion to vessels was seldom in normal condition, but increased in ischemia-reperfusion condition, and further significantly enhanced by platelets. The number of tumor metastatic foci and the density of blood vessels within metastatic foci in lung were markedly increased by tumor cell pre-adhered with platelet. The adhesion or fusion of platelet to endothelial cell mediated by platelet surface adhesion molecules, which could promote the adhesion of tumor cell with endothelial cells and the tumor metastasis.
[Show abstract][Hide abstract] ABSTRACT: Increasing evidence suggests that microRNAs are involved in human carcinogenesis as tumor suppressors or oncogenes. A growing number of reports has shown that an interest has been sparked in aberrant microRNA expression and how they can be used to treat human diseases, including cancer. However, their precise biological role remains largely unknown. In the present study, we aimed to identify micro-RNA species involved in the regulation of tumor growth. By performing quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, we demonstrated that mir-663 was downregulated in human gastric cancer cell lines unlike in normal cells. A transient introduction of mir-663 into the human gastric cancer cell lines BGC823 and SNU5 induced morphology changes and suppression of proliferation of these cells. In addition, mir-663 alters the DNA content and induces phenotypes of mitotic catastrophe in tumor cells. Moreover, the liposome-mediated delivery of mir-663 suppressed the in vivo growth of the BGC823 and SNU5 cells. Western blot analyses performed after the introduction of mir-663 revealed upregulation of cyclin B following transfection with mir-663. Our results provide evidence that downregulation of mir-663 in tumor cells may contribute to aberrant cell hyperplasia, leading to the development of gastric cancer. Therefore, mir-663 might function as a potent suppressor of tumor growth.
[Show abstract][Hide abstract] ABSTRACT: This study aimed to identify novel biological markers for the prediction of colorectal cancer liver metastasis.
We established two models that mimicked the interactions between colorectal tumor cells and the liver microenvironment. From these models we established subcell lines that had an enhanced ability to metastasize to the liver. Genes that related to hepatic metastasis were screened by microarray. The candidate markers were tested by immunohistochemistry, and their predictive accuracy was assessed by the cross-validation method and an independent test set.
Highly metastatic colon cancer cell sublines SW1116p21 and SW1116v3 were established from the tumor cell-microenvironment interaction models. Seven of the up-regulated genes in the sublines were selected as candidate markers for predicting metastatic potential. A total of 245 colorectal cancer samples were divided into a training set containing 117 cases and a test set containing 128 cases. In the training set, immunohistochemical analysis showed CCL2 and SNCG expression was higher in the hepatic metastasis group than in the nonmetastasis group, and was correlated with poor survival. Logistic regression analysis revealed that CCL2 and SNCG levels in primary tumors, serum carcinoembryonic antigen level, and lymph node metastasis status were the only significant (P < 0.05) parameters for detecting liver metastasis. In leave-one-out-cross-validation, the two markers, when combined with clinicopathologic features, resulted in 90.5% sensitivity and 90.7% specificity for hepatic metastasis detection. In an independent test set, the combination achieved 87.5% sensitivity and 82% specificity for predicting the future hepatic metastasis of colorectal cancer.
Our results suggest that these models are able to mimic the interactions between colorectal cancer cells and the liver microenvironment, and may represent a promising strategy to identify metastasis-related genes. CCL2 and SNCG, combined with clinicopathologic features, may be used as accurate predictors of liver metastasis in colorectal cancer.
Clinical Cancer Research 08/2009; 15(17):5485-93. DOI:10.1158/1078-0432.CCR-08-2491 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate invasion- and metastasis-related genes in gastric cancer. To this end, we used the transwell system to select a highly invasive subcell line from minimally invasive parent cells and compared gene expression in paired cell lines with high- and low-invasive potentials. Lysyl oxidase-like 2 (LOXL2) was overexpressed in the highly invasive subcell line. Immunohistochemical analysis revealed that LOXL2 expression was markedly increased in carcinoma relative to normal epithelia, and this overexpression in primary tumor was significantly associated with depth of tumor invasion, lymph node metastasis and poorer overall survival. Moreover, LOXL2 expression was further increased in lymph node metastases compared with primary cancer tissues. RNA interference-mediated knockdown and ectopic expression of LOXL2 showed that LOXL2 promoted tumor cell invasion in vitro and increased gastric carcinoma metastasis in vivo. Subsequent mechanistic studies showed that LOXL2 could activate both the Snail/E-cadherin and Src kinase/Focal adhesion kinase (Src/FAK) pathways. However, secreted LOXL2 induced gastric tumor cell invasion and metastasis exclusively via the Src/FAK pathway. Expression correlation analysis in gastric carcinoma tissues also revealed that LOXL2 promoted invasion via the Src/FAK pathway but not the Snail/E-cadherin pathway. We then evaluated secreted LOXL2 as a target for gastric carcinoma treatment and found that an antibody against LOXL2 significantly inhibited tumor growth and metastasis. Overall, our data revealed that LOXL2 overexpression, a frequent event in gastric carcinoma progression, contributes to tumor cell invasion and metastasis, and LOXL2 may be a therapeutic target for preventing and treating metastases.
[Show abstract][Hide abstract] ABSTRACT: Esophageal cancer is characterized by rapid clinical progression and poor prognosis, due to early-stage invasion of adjacent tissues and metastasis. Tissue factor pathway inhibitor-2 (TFPI-2) has been implicated as a metastasis-associated gene in many types of tumors. Here we describe the potential involvement of TFPI-2 in the development of esophageal carcinoma. Western blotting revealed that TFPI-2 was downregulated in 75% of esophageal carcinomas and in most esophageal carcinoma cell lines. Immunohistochemistry revealed that TFPI-2 was significantly downregulated in tumor tissues and in lymph node metastases. Experimental overexpression of TFPI-2 in KYSE450, KYSE510, YES2, and EC9706 cells significantly inhibited their invasive ability. Overexpression of TFPI-2 in EC9706 cells inhibited xenograft tumor growth and invasion into surrounding tissues, as well as reduced lung metastasis. Further studies demonstrated that recombinant TFPI-2 protein significantly inhibited the activity of matrix metalloproteinases and tumor-related angiogenesis. Parenteral treatment with recombinant TFPI-2 protein significantly suppressed xenograft growth and metastasis. Together, these data indicate that TFPI-2 inhibits tumor invasion and angiogenesis both in vitro and in vivo, and suggest a potentially important therapeutic role for recombinant TFPI-2 in the treatment of malignant esophageal carcinomas.
Human gene therapy 01/2009; 20(1):41-9. DOI:10.1089/hum.2008.129 · 3.76 Impact Factor