Tripartite motif-containing 3 (TRIM3) inhibits tumor growth and metastasis of liver cancer

Background:Reduced expression of tripartite motif-containing 3 (TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma (HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines (SK-Hep1,Hep3B,Huh7,HepG2,Bel-7402) and one normal liver cell line (L02) were detected with Western blotting.HepG2 and Bel-7402 cells with IowTRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3 (LV-TRIM3),whereas Huh7 and Hep3B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA (siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect of TRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed thatTRIM3 overexpression induced G0/G1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3B cells by siTRIM3 led to significantly decreased percentages of both cells in the G0/G1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed thatTRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G0/G1 phase.     

MTA1基因表达与人骨肉瘤细胞浸润和转移的关系

Objective： To compare the expression level of metastasis associated-1 （MTA1） in the higher and lower metastasis sublines of human osteosarcoma cells （MG63）, and to investigate the relationship between the expression level of MTA1-EGFP and in vitro invasion and metastasis of human osteosarcoma cells. Methods： The expression level of MTA1 in two sublines of MG63 cells was detected by semi-quantitative RT-PCR, and cell invasion assay and cell proliferation assay were used to evaluate the invasive capacity in vitro in two sublines. The lower metastasis line of MG-63 cells were transfected with MTA1-EGFP full-length cDNA expression plasmid by lipofectamine. The changes of the MTA1-EGFP expression and in vitro invasion potential were measured after transfection. Results： M8 subline expressed significantly higher level of MTA1 than that of M6 subline by RT-PCR. The invasive potentials of low metastasis MG63 cell line were increased after MTA1 gene transfection. Conclusion： There may be a relationship between MTA1 and invasive potentials of human osteosarcoma cells, and MTA1 may play a role in the molecular mechanism of tumor metastases and be a potential target for gene therapy of osteosarcoma. Further studies of MTA1 in human ostersarcoma cell metastasis are needed.     

Suppression of Human Liver Cancer Cell Migration and Invasion via the GABAA Receptor

Objective To investigate the roles of theγ-aminobutyric acid(GABA) in the metastasis of hepatocellular carcinoma(HCC) and to explore the potential of a novel therapeutic approach for the treatment of HCC. Methods The expression levels of GABA receptor subunit genes in various HCC cell lines and patients’ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis.Transwell cell migration and invasion assays were carried out for functional analysis.The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer. Results The mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue.GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABAa receptor as a result of the induction of liver cancer cell cytoskeletal reorganization.Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo. Conclusions These findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system.     

Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells

Objective To explore the interaction of Anxa2 with P-Glycoprotein(P-gp) in the migration and invasion of the multidrug-resistant (MDR) human breast cancer cell line MCF-7/ADR. Methods A pair of short hairpin RNA(shRNA) targeting P-gp was transfected into MCF-7/ADR cells,and monoclonal cell strains were screened.The expression of P-gp was detected by Western blot.Transwell chambers were used to observe the cell migration capacity and invasion ability.The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses. Results P-gp expression was significantly knocked down,and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells(P<0.05).There was a close interaction between Anxa2 and P-gp. Conclusions MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities.The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells.The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells.     

Clinical significance of XRCC1 gene expression in human breast cancer and its cell and molecular mechanisms北大核心CSCD

Objective: To investigate the expression of X-ray repair cross complementing 1 (XRCC1) in human breast cancer and its relationship with the clinical characteristics, and to analyze the effects of XRCC1 over-expression on the proliferation and migration of breast cancer MB-231 cells and the molecular mechanism. Methods: The expression level of XRCC1 mRNA in breast cancer cell lines and human breast cancer tissues was detected by real-time fluorescent quantitative PCR. The expression of XRCC1 protein in human breast cancer tissues was detected by immunohistochemistry. The relationship between the expression of XRCC1 protein and the clinicopathological characteristics of breast cancer patients was analyzed. The pcDNA3.1(+)-Flag-XRCC1 plasmids were transfected into breast cancer MB-231 cells for the overexpression of XRCC 1 gene. Then the proliferation activity was detected by CCK-8 and soft agar plate clone formation assay. The cell cycle and apoptosis were detected by FCM method. The cell migration and invasion were detected by Transwell chamber assay. The expressions of cell cycle-, apoptosis- and migration-related proteins were detected by Western blotting. Results: The expression level of XRCC1 mRNA was significantly decreased in most breast cancer cell lines (all P < 0.001). As compared with the normal mammary epithelium and the paired adjacent breast tissues, the expression levels of XRCC1 mRNA and protein were downregulated in human breast cancer tissues (all P < 0.001). The expression level of XRCC1 mRNA was positively correlated with the prognosis of breast cancer patients (γ 2 =0.052, P =0.046), and XRCC1 protein expression was correlated with tumor diameter, lymph node metastasis, histological grade and TNM stage (all P < 0.05). After the overexpression of XRCC 1 gene, the proliferation, colony formation, invasion and migration of breast cancer MB-231 cells were significantly inhibited (all P < 0.01), the cell cycle was significantly blocked in G1 phase (P < 0.001), and the apoptosis rate was significantly increased (P < 0.001). Furthermore, the expressions of p21, p27, Bax, cleaved caspase-3 and E-cadherin were significantly upregulated (all P < 0.001), while the expressions of cyclin-dependent kinase 4/6 (CDK4/6), cyclin D1, Bcl-2, N-cadherin and vimentin were down-regulated (all P < 0.001) in MB-231 cells with XRCC1 overexpression. Conclusion: XRCC1 expression is down-regulated in breast cancer cell lines and tissues, and its expression level is positively correlated with the prognosis of breast cancer patients. Restoring XRCC 1 gene expression can inhibit cell growth, migration and invasion, and can induce apoptosis. So XRCC1 may be a potential tumor suppressor regulating the occurrence and development of human breast cancer. © 2019 by TUMOR.     

黄芩素对人乳腺癌MDA-MB-231细胞株SATB1表达的影响(英文)

Baicalein had been proved to have anti-cancer activity in vitro and in vivo, including the inhibition of malignant proliferation, migration, adhesion and invasion of many kinds of cancer cells. The special AT-rich sequence binding protein 1 （SATB1） is a tissue-specific expression of nuclear matrix-binding protein and is reported to be a breast cancer ＂gene group organizer＂. Previous studies have shown that SATB1 is involved in the growth, metastasis and prognosis of breast cancer. The present study was aimed to investigate whether baicalein inhibits the proliferation and migration of MDA-MB-231 human breast cancer cells through down-regulation of the SATB1 expression. Methods： MDA-MB-231 cells were treated for 24 h, 48 h and 72 h with various concentrations of baicalein （0, 5, 10, 20, 40 and 80 pM） respectively. Then, the proliferation and migration of MDA-MB-231 cells following treatment with baicalein were determined using colorimetric 3-（4, 5-dimethylthia- zol-2-yl） 2, 5-diphenyltetrazolium bromide （MTT） and wound healing assays. Thereafter, western blot analysis was performed to detect the changes of SATB1 protein expression in MDA-MB-231 cells. Results： Along with the prolongation of time and increase of drug concentration, inhibitory effect of baicalein on proliferation and migration of MDA-MB-231 cells gradually in- creased, in a time.- and dose- dependent manner （P 〈 0.05）. Meanwhile, after treated with baicalein in different concentrations for 48 h, the level of SATB1 protein expression of MDA-MB-231 cells decreased obviously, in a dose-dependent manner （P 〈 0.05）. Conclusion： Baicalein inhibits breast cancer cell proliferation and suppresses its invasion and metastasis by reducing cell migration possibly by down-regulation of the SATB1 protein expression, indicating that baicalein is a potential therapeutic agent for human breast cancer.     

TGF-β1 alters microRNA profile in human gastric cancer cells

Objective: MicroRNAs (miRNAs) are important regulators that play a key role in tumorigenesis and tumor progression. Transforming growth factor-β1 (TGF-β1) is involved in invasion and metastasis in many tumors. In this study, we investigated the microRNAs (miRNA) profiles altered by TGF-β1 in gastric cancer (GC) cells. Methods: We detected the expression profiles of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Migration and invasion, wound-healing assay, prediction of miRNA targets, Western blot and qRT-PCR analysis were carried out to determine the role of one selected miRNA, namely miR-193b, in affecting the biological behaviors of GC BGC823 cells. Results: Among 847 human miRNAs in the microarray, three miRNAs (miR-27a, miR-29b-1 and miR-194) were up-regulated and three (miR-574-3p, miR-193b and miR-130b) were down-regulated in BGC823 cells treated with TGF-β1 compared with control. miR-193b suppressed the invasion and metastasis of GC cells in vivo and in vitro, and down-regulated urokinase-type plasminogen activator (uPA) protein in GC cells. Conclusions: TGF-β1 altered miRNA expression profile in BGC823 cells. Among the altered miRNAs, TGF-β1 induced the down-regulation of miR-193b, which inhibited cell invasion and metastasis in vivo and in vitro, and down-regulated uPA protein in GC cells.     

Experimental study on enhancement of the metastatic potential of portal vein tumor thrombus-originated hepatocellular carcinoma cells using portal vein serum

Objective： Portal vein metastasis of hepatocellular carcinoma（HCC） results in a poor prognosis and seriously affects the survival rate of patients. The mechanism underlying the formation of portal vein tumor thrombus（PVTT） is complex and is not yet fully understood. This study was conducted to investigate the impact of portal vein blood on the proliferation, metastasis, invasion and apoptosis of PVTT cells and to explore its possible mechanisms, which was expected to lay a foundation for ascertaining the mechanism underlying the portal vein metastasis of HCC.
Methods： Peripheral blood and portal vein blood were collected from patients with HCC, and the sera from these two sources were used to culture the PVTT-originated HCC cell line CSQT-2. The cells were collected after 24 h, and flow cytometry was performed to detect cell proliferation, cell cycle stages and apoptosis. Transwell migration and invasion assays were applied to detect the metastasis and invasion of the cells in each group. The changes in the expression of MMP-2 and MMP-9 in cells were detected via Western blotting. The contents of IL-12, IFN-γ, IL-1β, IL-2 and TNF-α in the two groups of sera were quantified using corresponding kits.
Results： Compared with the group of cells cultured with peripheral serum, the cells cultured with portal vein serum showed significantly lower apoptosis（P〈0.01）, significantly enhanced cell metastasis and invasion（P〈0.01）, whereas cell proliferation and the stages of the cell cycle did not differ significantly（P〉0.05）. A significantly increased expression level of MMP-2 has been observed in tumor cells treated portal vein serum. In addition, compared with peripheral serum, the content of IL-12 was significantly decreased in portal vein serum（P〈0.05）, while the contents of IFN-γ, IL-1β, IL-2, and TNF-α did not differ significantly（P〈0.05）.
Conclusions： Portal vein serum from HCC patients could inhibit the apoptosis of PVTT-originated HCC cells and promote cell met     

Expression of heparanase and its clinical significance in human non-small cell lung cancer

Objective： To explore the expression and significance of heparanase in non-small cell lung cancer （NSCLC） regarding prognosis and clinicopathological parameters. Methods： The expression of heparanase was assessed using immunohistochemistry staining and Western blot in 122 paraffin-embedded specimens and 38 freshly taken tissues. The relationship between heparanase expression and the clinicopathological factors was analyzed by Chi-square test, multivariate analysis and Kaplan-Meier method. Results： In the immunoreactive cells, staining was mainly located in cytoplasma and membrane. Human heparanase was highly expressed in lung cancer tissue （78.7%, 96/122） while negative in epithelia of normal lung tissues. The level of heparanase was remarkably higher in NSCLC than that in normal tissue （P=0.043）. Expression of heparanase significantly correlated with TNM stage （P=0.025）, lymphatic metastasis （P=0.002） and vascular invasion （P=0.0003）. The patients with positive heparanase expression had a significantly shorter survival than those with negative heparanase expression （P=0.0006）. In multivariate analysis, only p-TNM stage, lymphatic metastasis and vascular invasion could be considered as prognostic factors. Conclusion： Elevated level of heparanase in human non-small cell lung cancer tissues correlates with the TNM stage, invasion, metastasis and prognosis. However, heparanase expression is not an independent prognostic factor.     

Metadherin regulates proliferation and metastasis via actin cytoskeletal remodelling in non-small cell lung cancer

Background:

Metaderin (MTDH) protein is a novel component part of tight junction complex. The aim of this study was to investigate the correlation between MTDH and prognosis of patients and to explore the role of MTDH on NSCLC development and metastasis.

Methods:

Relative mRNA expression was evaluated by quantitative real-time PCR, and protein expression was detected using immunohistochemistry staining. The role of MTDH in cancer cell proliferation, migration and invasion was studied by modulation of MTDH expression in NSCLC cell lines. These functions of MTDH were further confirmed in vivo.

Results:

In NSCLC, low MTDH protein expression was correlated with lymph node metastasis, TNM stage and decreased OS (P=0.001, 0.011 and 0.013, respectively). Overexpression of MTDH reduced anchorage-independent and -dependent growth through arresting cell cycle, inhibited migration and invasion in vitro and further suppressed tumorigenesis, tumour growth and metastasis in vivo. Knockdown of MTDH expression increased cell invasiveness. MTDH overexpression reversed pro-metastatic actin cytoskeleton remodelling and inhibited EMT, supporting that MTDH has a key role on cancer proliferation and metastasis.

Conclusions:

MTDH has an important role in NSCLC proliferation and metastasis and provides potential in predicting metastasis and prognosis for patients with NSCLC.     

DDX3 as a strongest prognosis marker and its downregulation promotes metastasis in colorectal cancer

Background

Conflicting results regarding the role of DEAD-box polypeptide 3 (DDX3) are seen not only between cancer types but also within the same type of cancer. In this study, we aimed at clarifying the prognostic significance of DDX3 in patients of major cancer types through large cohort survival analysis and further investigated its effects on cancer progression.

Methods

Large cohort survival analysis of 7 cancer types, including colorectal cancer, breast cancer, lung cancer, head and neck cancer, liver cancer, glioblastoma, and ovarian cancer, was performed using public database at RNA level and was further confirmed by IHC analysis at protein level. Phenotype parameters of DDX3 knockdown colon cancer cells and the mechanism of DDX3 regulated cancer progression were investigated in vitro and in vivo.

Results

In large cohort survival analysis, DDX3 had a significant prognostic predictive power in colorectal cancer at both RNA and protein level. Patients with low DDX3 expression had poor prognosis and frequent distant metastasis. Knockdown of DDX3 enhanced the migration and invasion abilities of colon cancer cells and promoted tumor metastasis in vivo. Snail upregulation with decreased membranous E-cadherin expression and reduced cell aggregation were found after DDX3 downregulation.

Conclusions

Our study revealed the strong prognostic effect of DDX3 on colorectal cancer among seven major cancer types through larger cohort survival analysis at RNA and protein level. Low DDX3 expression promotes Snail/E-cadherin pathway mediated cancer metastasis and poor clinical outcome in colorectal cancer patients.     

P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells

Background:

Our previous study demonstrated that extracellular adenosine 5′-triphosphate (ATP) stimulated prostate cancer cell invasion via P2Y receptors. However, the purinergic receptor subtype(s) involved in this process remains unclear. Here we aimed to determine whether P2Y2, one subtype of P2Y receptors, was involved in the invasion and metastasis of prostate cancer cells, and elucidated the underlying mechanism.

Methods:

RNAi was introduced to silence the expression of P2Y2. In vitro invasion and migration assays and in vivo experiments were carried out to examine the role of P2Y2 receptor in cell invasion and metastasis. cDNA microarray was performed to identify the differentially expressed genes downstream of ATP treatment.

Results:

P2Y2 was significantly expressed in the prostate cancer cells. Knockdown of P2Y2 receptor suppressed cell invasion and metastasis in vitro and in vivo. Further experiments identified that ATP could promote IL-8 and Snail expression and inhibit E-cadherin and Claudin-1 expression. Knockdown of P2Y2 receptor affected the expression of these EMT/invasion-related genes in vitro and in vivo.

Conclusion:

P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells via some EMT/invasion-related genes. Thereby, P2Y2 receptor could be a potential therapeutic target for the treatment of prostate cancer.     

Lipocalin 2 promotes lung metastasis of murine breast cancer cells

Background

Lipocalin 2, an iron binding protein, is abnormally expressed in some malignant human cancers and may play an important role in tumor metastasis. However, the roles of lipocalin 2 in breast cancer formation and metastasis have not been clearly shown. This study aimed to investigate the roles of lipocalin 2 in breast tumor metastasis.

Methods

Lipocalin 2 was overexpressed in the metastatic 4T1 murine mammary cancer cells. The effects of lipocalin 2 overexpression on the malignancy of breast cancer cells were examined using cell proliferation assay, migration assay, invasion assay, and soft agar assay in vitro. Tumor formation and metastasis abilities were examined using a well established mouse mammary tumor model in vivo.

Results

Lipocalin 2 overexpression significantly enhanced the migration and invasion abilities of 4T1 cells in vitro, and lung metastasis in vivo. But overexpression of lipocalin 2 in 4T1 cells didn''t affect cell proliferation and anchorage-independent growth in vitro, and primary tumor weight in vivo. Further studies demonstrated that the inhibition of the PI3K/Akt pathway could be a causative mechanism for the promotion of breast cancer migration/invasion induced by lipocalin 2 overexpression.

Conclusion

These results clarified that lipocalin 2 could promote lung metastasis of 4T1 cells through the inhibition of the PI3K/Akt pathway, suggesting that lipocalin 2 was a potential target for therapy of breast cancer.     

FAT4 functions as a tumour suppressor in gastric cancer by modulating Wnt/β-catenin signalling

Background:

FAT4, a cadherin-related protein, was shown to function as a tumour suppressor; however, its role in human gastric cancer remains largely unknown. Here, we investigated the role of FAT4 in gastric cancer and examined the underlying molecular mechanisms.

Methods:

The expression of FAT4 was evaluated by immunohistochemistry, western blotting, and qRT–PCR in relation to the clinicopathological characteristics of gastric cancer patients. The effects of FAT4 silencing on cell proliferation, migration, and invasion were assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay, and migration and invasion assays in gastric cancer cell lines in vitro and in a mouse xenograft model in vivo.

Results:

Downregulation of FAT4 expression in gastric cancer tissues compared with adjacent normal tissues was correlated with lymph-node metastasis and poor survival. Knockdown of FAT4 promoted the growth and invasion of gastric cancer cells via the activation of Wnt/β-catenin signalling, and induced epithelial-to-mesenchymal transition (EMT) in gastric cancer cells, as demonstrated by the upregulation and downregulation of mesenchymal and epithelial markers. Silencing of FAT4 promoted tumour growth and metastasis in a gastric cancer xenograft model in vivo.

Conclusions:

FAT4 has a tumour suppressor role mediated by the modulation of Wnt/β-catenin signalling, providing potential novel targets for the treatment of gastric cancer.     

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis

Background:

TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown.

Methods:

miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC.

Results:

miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G₀/G₁ cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels.

Conclusion:

We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.     

Reply: Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells

Background:

Emerging evidence has demonstrated that lysine-specific demethylase 1 (LSD1) has an important role in many pathological processes of cancer cells, such as carcinogenesis, proliferation and metastasis. In this study, we characterised the role and molecular mechanisms of LSD1 in proliferation and metastasis of colon cancer.

Methods:

We evaluated the correlation of LSD1, CDH-1 and CDH-2 with invasiveness of colon cancer cells, and investigated the roles of LSD1 in proliferation, invasion and apoptosis of colon cancer cells. We further investigated the mechanisms of LSD1-mediated metastasis of colon cancer.

Results:

Lysine-specific demethylase 1 was upregulated in colon cancer tissues, and the high LSD1 expression was significantly associated with tumour-node-metastasis (TNM) stages and distant metastasis. Functionally, inhibition of LSD1 impaired proliferation and invasiveness, and induced apoptosis of colon cancer cells in vitro. The LSD1 physically interacted with the promoter of CDH-1 and decreased dimethyl histone H3 lysine 4 (H3K4) at this region, downregulated CDH-1 expression, and consequently contributed to colon cancer metastasis.

Conclusion:

Lysine-specific demethylase 1 downregulates the expression of CDH-1 by epigenetic modification, and consequently promotes metastasis of colon cancer cells. The LSD1 antagonists might be a useful strategy to suppress metastasis of colon cancer.