Combination of taxol and Bcl‐2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion,angiogenesis and tumour growth

Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti‐apoptotic molecule Bcl‐2 is up‐regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti‐apoptotic Bcl‐2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl‐2 siRNA or both agents together for 72 h. Knockdown of Bcl‐2 potentiated efficacy of taxol for cell death. Fluorescence‐activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl‐2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl‐2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl‐2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl‐2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.     

Cell‐bound exopolysaccharide from probiotic bacteria induces autophagic cell death of tumour cells

Aim: Lactic acid bacteria (LAB) are beneficial micro‐organisms that have been associated with several probiotic effects in both humans and animals. Here, using proteome analysis, we investigate the antitumour effects of cell‐bound exopolysaccharides (cb‐EPS) isolated from Lactobacillus acidophilus 606 on colon cancer cells and explore the proteins critical for their antitumour activity. Methods and Results: cb‐EPS inhibited the proliferation of HT‐29 colon cancer cells by directly affecting cell morphology and not the cell cycle. Using two‐dimensional polyacrylamide gel electrophoresis coupled with matrix‐assisted laser desorption‐ionization time‐of‐flight mass spectrometry (MALDI‐TOF/MS) and immunoblot analysis, we found that cb‐EPS dramatically induced Beclin‐1 and GRP78, and affected Bcl‐2 and Bak regulation. Conclusions: The results of this study indicate that cb‐EPS are antitumourigenic against HT‐29 colon cancer cells and that this activity is because of the activation of autophagic cell death promoted directly by the induction of Beclin‐1 and GRP78, as well as indirectly through the induction of Bcl‐2 and Bak. Significance and Impact of the Study: These results may contribute to understanding the novel mechanisms by which probiotic bacteria induce tumour cell death via autophagy.     

The deubiquitinase Usp27x stabilizes the BH3‐only protein Bim and enhances apoptosis

Bim is a pro‐apoptotic Bcl‐2 family member of the BH3‐only protein subgroup. Expression levels of Bim determine apoptosis susceptibility in non‐malignant and in tumour cells. Bim protein expression is downregulated by proteasomal degradation following ERK‐dependent phosphorylation and ubiquitination. Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK‐dependent phosphorylation and can upregulate its expression levels. Overexpression of Usp27x reduces ERK‐dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA‐stimulated cells, as well as in tumour cells with a constitutively active Raf/ERK pathway. Loss of endogenous Usp27x enhances the Bim‐degrading activity of oncogenic Raf. Overexpression of Usp27x induces low levels of apoptosis in melanoma and non‐small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling. Finally, deletion of Usp27x reduces apoptosis in NSCLC cells treated with an EGFR inhibitor. Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti‐apoptotic effects of ERK activity, and therefore acts as a tumour suppressor.     

Curcumin inhibits adverse psychological stress-induced proliferation and invasion of glioma cells via down-regulating the ERK/MAPK pathway

Curcumin is a natural polyphenol extracted from the rhizome of Curcuma that has an important antitumour effect, but its effect on adverse psychological stress-induced tumour proliferation and invasion has not been reported to date. Here, we found that curcumin not only inhibited the growth of xenografts in chronically stressed nude mice, but also decreased the expression of matrix metalloproteinase (MMP)-2/9 and CD147 in tumour tissues. Exogenous norepinephrine (NE) was used to stimulate glioma cells to simulate the stress environment in vitro, and it was found that curcumin inhibited the NE-induced proliferation and invasion of glioma cells in a dose-dependent manner. Further research found that the effects of NE on glioma cells could lead to the activation of the mitogen-activated protein kinase (MAPK) signalling pathway through β-adrenergic receptor, while curcumin suppressed the level of extracellular signal–regulated kinase (ERK)1/2 phosphorylation. In addition, blocking ERK1/2 expression with U0126 resulted in the down-regulated expression of CD147, which further led to the decreased expression of MMP-2 and MMP-9. Curcumin could also inhibit the expression of cyclin D1/CDK4/6 and anti-apoptotic protein Bcl-2/Bcl-XL induced by NE, and induced cell cycle changes and increased apoptosis. Therefore, curcumin may be a potential candidate drug for preventing and treating the progression of glioma induced by adverse psychological stress.     

LncRNA ZEB1‐AS1 down‐regulation suppresses the proliferation and invasion by inhibiting ZEB1 expression in oesophageal squamous cell carcinoma

Multiple studies have unveiled that long non‐coding RNAs (lncRNAs) play a pivotal role in tumour progression and metastasis. However, the biological role of lncRNA ZEB1‐AS1 in oesophageal squamous cell carcinoma (ESCC) remains under investigation, and thus, the current study was to investigate the functions of ZEB1‐AS1 in proliferation and invasion of ESCC. Here, we discovered that ZEB1‐AS1 and ZEB1 were markedly up‐regulated in ESCC tissues and cells relative to their corresponding normal control. ZEB1‐AS1 and ZEB1 overexpressions were both related to TNM staging and lymph node metastasis as well as poor prognosis in ESCC. The hypomethylation of ZEB1‐AS1 promoter triggered ZEB1‐AS1 overexpression in ESCC tissues and cells. In addition, ZEB1‐AS1 knockdown mediated by siRNA markedly suppressed the proliferation and invasion in vitro in EC9706 and TE1 cells, which was similar with ZEB1 siRNA treatment, coupled with EMT alterations including the up‐regulation of E‐cadherin level as well as the down‐regulation of N‐cadherin and vimentin levels. Notably, ZEB1‐AS1 depletion dramatically down‐regulated ZEB1 expression in EC9706 and TE1 cells, and ZEB1 overexpression obviously reversed the inhibitory effects of proliferation and invasion triggered by ZEB1‐AS1 siRNA. ZEB1‐AS1 shRNA evidently inhibited tumour growth and weight, whereas ZEB1 elevation partly recovered the tumour growth in ESCC EC9706 and TE1 xenografted nude mice. In conclusion, ZEB1‐AS1 overexpression is tightly involved in the development and progression of ESCC, and it exerts the antitumour efficacy by regulating ZEB1 level in ESCC.     

Loss of PPM1F expression predicts tumour recurrence and is negatively regulated by miR‐590‐3p in gastric cancer

Objectives

MicroRNAs (miRNAs) as small non‐coding RNA molecules act by negatively regulating their target genes. Recent studies have shown that protein phosphatase Mg2+/Mn2+‐dependent 1F (PPM1F) plays a critical role in cancer metastasis. But, the regulation mechanisms of PPM1F by miRNAs in gastric cancer (GC) remain undefined.

Methods

The correlation of PPM1F or miR‐590‐3p (miR‐590) expression with clinicopathological features and prognosis of the patients with GC was analysed by TCGA RNA‐sequencing data. The miRNAs that target PPM1F gene were identified by bioinformatics and Spearman correlation analysis, and the binding site between miR‐590 and PPM1F 3′UTR was confirmed by dual luciferase assay. MTT and Transwell assays were conducted to evaluate the effects of miR‐590 or (and) PPM1F on cell proliferation and invasion.

Results

We found that PPM1F expression was downregulated in GC tissues and cell lines and was correlated with tumour recurrence in patients with GC. The decreased expression of PPM1F was attributed to the dysregulation of miR‐590 expression rather than its genetic or epigenetic alterations. Overexpression of miR‐590 promoted cell proliferation and invasion capability of GC cells, while knockdown of miR‐590 reversed these effects. Moreover, PPM1F was validated as a direct target of miR‐590 and counteracted the tumour‐promoting effects caused by miR‐590. The expression of miR‐590 presented the negative correlation with PPM1F expression and acted as an independent prognostic factor for tumour recurrence in patients with GC.

Conclusion

PPM1F may function as a suppressive factor and is negatively regulated by miR‐590 in GC.

     

Curcumin inhibits the growth of triple‐negative breast cancer cells by silencing EZH2 and restoring DLC1 expression

Enhancer of zeste homolog 2 (EZH2), an oncogene, is a commonly up‐regulated epigenetic factor in human cancer. Hepatocellular carcinoma deletion gene 1 (DLC1) is an antioncogene that is either expressed at low levels or not expressed in many malignant tumours. Curcumin is a promising anticancer drug that has antitumour effects in many tumours, but its mechanism of action is unclear. Our research demonstrated that EZH2 was up‐regulated in breast cancer (BC) tissues and cells, whereas DLC1 was down‐regulated, and the expression of EZH2 and DLC1 was negatively correlated in BC. By analysing the characteristics of clinical cases, we found that positive expression of EZH2 and negative expression of DLC1 may be predictors of poor prognosis in patients with triple‐negative breast cancer (TNBC). Moreover, knockdown of EZH2 expression restored the expression of DLC1 and inhibited the migration, invasion and proliferation, promoted the apoptosis, and blocked the cell cycle of MDA‐MB‐231 cells. Furthermore, we found that curcumin restored the expression of DLC1 by inhibiting EZH2; it also inhibited the migration, invasion and proliferation of MDA‐MB‐231 cells, promoted their apoptosis and blocked the cell cycle. Finally, xenograft tumour models were used to demonstrate that curcumin restored DLC1 expression by inhibiting EZH2 and also inhibited the growth and promoted the apoptosis of TNBC cells. In conclusion, our results suggest that curcumin can inhibit the migration, invasion and proliferation, promote the apoptosis, block the cycle of TNBC cells and restore the expression of DLC1 by inhibiting the expression of EZH2.     

Long non‐coding RNA LINC01225 promotes proliferation,invasion and migration of gastric cancer via Wnt/β‐catenin signalling pathway

Emerging evidence has classified the aberrant expression of long non‐coding RNAs (lncRNAs) as a basic signature of various malignancies including gastric cancer (GC). LINC01225 has been shown to act as a hepatocellular carcinoma‐related gene, with its expression pattern and biological function not clarified in GC. Here, we verified that LINC01225 was up‐regulated in tumour tissues and plasma of GC. Analysis with clinicopathological information suggested that up‐regulation of LINC01225 was associated with advanced disease and poorer overall survival. Receiver operating characteristic (ROC) analysis showed that plasma LINC01225 had a moderate accuracy for diagnosis of GC. In addition, knockdown of LINC01225 led to retardation of cell proliferation, invasion and migration, and overexpression of LINC01225 showed the opposite effects. Mechanistic investigations showed that LINC01225 silencing inhibited epithelial‐mesenchymal transition (EMT) process and attenuated Wnt/β‐catenin signalling of GC. Furthermore, ectopic expression of Wnt1 or suppression of GSK‐3β abolished the si‐LINC01225‐mediated suppression against EMT, thereby promoting cell proliferation, invasion and migration of GC. In conclusion, LINC01225 promotes the progression of GC through Wnt/β‐catenin signalling pathway, and it may serve as a potential target or strategy for diagnosis or treatment of GC.     

microRNA-95 knockdown inhibits epithelial–mesenchymal transition and cancer stem cell phenotype in gastric cancer cells through MAPK pathway by upregulating DUSP5

This study investigated the role of microRNA-95 (miR-95) in gastric cancer (GC) and to elucidate the underlying mechanism. Initially, bioinformatic prediction was used to predict the differentially expressed genes and related miRNAs in GC. miR-95 and DUSP5 expression was altered in GC cell line (MGC803) to evaluate their respective effects on the epithelial–mesenchymal transition (EMT) process, cellular processes (cell proliferation, migration, invasion, cell cycle, and apoptosis), cancer stem cell (CSC) phenotype, as well as tumor growth ability. It was further predicted in bioinformatic prediction and verified in GC tissue and cell line experiments that miR-95 was highly expressed in GC. miR-95 negatively regulated DUSP5, which resulted in the MAPK pathway activation. Inhibited miR-95 or overexpressed DUSP5 was observed to inhibit the levels of CSC markers (CD133, CD44, ALDH1, and Lgr5), highlighting the inhibitory role in the CSC phenotype. More important, evidence was obtained demonstrating that miR-95 knockdown or DUSP5 upregulation exerted an inhibitory effect on the EMT process, cellular processes, and tumor growth. Together these results, miR-95 knockdown inhibited GC development via DUSP5-dependent MAPK pathway.     

Tumor necrosis factor-α-mediated suppression of dual-specificity phosphatase 4: crosstalk between NFκB and MAPK regulates endothelial cell survival

We investigated the effects of tumor necrosis factor-α (TNF-α) exposure on mitogen-activated protein kinase signaling in human microvascular endothelial cells. TNF-α caused a significant suppression of a dual specificity phosphatase, DUSP4, that regulates ERK1/2 activation. Thus, we hypothesized that suppression of DUSP4 enhances cell survival by increasing ERK1/2 signaling in response to growth factor stimulation. In support of this concept, TNF-α pre-exposure increased growth factor-mediated ERK1/2 activation, whereas overexpression of DUSP4 with an adenovirus decreased ERK1/2 compared to an empty adenovirus control. Overexpression of DUSP4 also significantly decreased cell viability, lessened recovery in an in vitro wound healing assay, and decreased DNA synthesis. Pharmacological inhibition of NFκB activation or a dominant negative construct of the inhibitor of κB significantly lessened TNF-α-mediated suppression of DUSP4 expression by 70–84 % and attenuated ERK activation, implicating NFκB-dependent pathways in the TNF-α-mediated suppression of DUSP4 that contributes to ERK1/2 signaling. Taken together, our findings show that DUSP4 attenuates ERK signaling and reduces cell viability, suggesting that the novel crosstalk between NFκB and MAPK pathways contributes to cell survival.     

miR‐124 interacts with the Notch1 signalling pathway and has therapeutic potential against gastric cancer

Aberrant Notch signalling plays an important role in cancer progression. However, little is known about the interaction between miRNA and the Notch signalling pathway and its role in gastric cancer (GC). In this study, we found that miR‐124 was down‐regulated in GC compared with adjacent normal tissue. Forced expression of miR‐124 inhibited GC cell growth, migration and invasion, and induced cell cycle arrest. miR‐124 negatively regulated Notch1 signalling by targeting JAG1. miR‐124 levels were also shown to be inversely correlated with JAG1 expression in GC. Furthermore, we found that the overexpression of the intracellular domain of Notch1 repressed miR‐124 expression, promoted GC cell growth, migration and invasion. Conversely, blocking Notch1 using a γ‐secretase inhibitor up‐regulated miR‐124 expression, inhibited GC cell growth, migration and invasion. In conclusion, our data demonstrates a regulatory feedback loop between miR‐124 and Notch1 signalling in GC cells, suggesting that the miR‐124/Notch axis may be a potential therapeutic target against GC.     

Spondin 2 promotes the proliferation,migration and invasion of gastric cancer cells

Spondin 2 (SPON2), a member of the Mindin F‐Spondin family, identifies pathogens, activates congenital immunity and promotes the growth and adhesion of neurons as well as binding to their receptors, but its role in promoting or inhibiting tumour metastasis is controversial. Here, we investigated its expression levels and mechanism of action in gastric cancer (GC). Western blotting and GC tissue arrays were used to determine the expression levels of SPON2. ELISAs were performed to measure the serum levels of SPON2 in patients with GC. Two GC cell lines expressing low levels of SPON2 were used to analyse the effects of regulating SPON2 expression on proliferation, migration, invasion, the cell cycle and apoptosis. The results revealed that SPON2 was highly expressed in GC tissues from patients with relapse or metastasis. The levels of SPON2 in sera of patients with GC were significantly higher compared with those of healthy individuals and patients with atrophic gastritis. Knockdown of SPON2 expression significantly inhibited the proliferation, migration and invasion of GC cells in vitro and in vivo. Down‐regulation of SPON2 arrested the cell cycle in G1/S, accelerated apoptosis through the mitochondrial pathway and inhibited the epithelial‐mesenchymal transition by blocking activation of the ERK1/2 pathway. In summary, this study suggests that SPON2 acts as an oncogene in the development of GC and may serve as a marker for the diagnosing GC as well as a new therapeutic target for GC.     

Regulation of Survivin and Bcl‐2 in HepG2 Cell Apoptosis Induced by Quercetin

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in a variety of human cancer cells. However, the regulation of survivin and Bcl‐2 on the quercetin‐induced cell‐growth inhibition and apoptosis in cancer cells remains unclear. In the present study, we report that quercetin can inhibit proliferation and induce apoptosis in HepG2 cells in dose‐ and time‐dependent manner. Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) staining showed that HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to quercetin. Cell‐cycle analysis reveals a significant increase of the proportion of cells in G₀/G₁ phase. We also demonstrate that the levels of survivin and Bcl‐2 protein expression in HepG2 cells decreased concurrently, and the levels of p53 protein increased significantly after treatment with quercetin by immunocytochemistry analysis. Relative activity of caspase‐3 and caspase‐9 increased significantly. These data clearly indicate that quercetin‐induced apoptosis is associated with caspase activation, and the levels of survivin and Bcl‐2. Our results indicate that the expression of survivin may be associated with Bcl‐2 expression, and the inhibition expression of survivin, in conjunction with Bcl‐2, might cause more pronounced apoptotic effects. Together, concurrent down‐regulated survivin and Bcl‐2 play an important role in HepG2 cell apoptosis induced by quercetin.     

TFPI‐2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour‐stromal cell interactions

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α₁ integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.     

miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.     

Quercetin protected against isoniazide‐induced HepG2 cell apoptosis by activating the SIRT1/ERK pathway

Isoniazid (INH) is one of the most commonly used antituberculosis drugs, but its clinical applications have been limited by severe hepatic toxicity. Quercetin (Que), a natural flavonoid, has been proved to have many medicinal properties. This study aimed to clarify the possible protective effects of Que against INH‐induced hepatotoxicity using HepG2 cells. Our results indicated that Que significantly increased cell viability, superoxide dismutase, and GSH levels, while decreased alanine aminotransferase/aspartate aminotransferase levels. Besides, Que significantly abrogated INH‐induced cell apoptosis by upregulating the expression levels of Bcl‐2 and decreasing the levels of Bax, cleaved caspase‐3, and cleaved caspase‐9. Furthermore, Que obviously reversed the inhibition of INH on Sirtuin 1 (SIRT1) expression and extracellular signal‐regulated kinase (ERK) phosphorylation. Next, the SIRT1 inhibitor EX527 blocked the enhancement of Que upon ERK phosphorylation. Notably, EX527 partially abolished the beneficial effects of Que. In brief, our results provided the first evidence that Que protected against INH‐induced HepG2 cells by regulating the SIRT1/ERK pathway.