Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

Long non-coding RNA <Emphasis Type="Italic">HOTAIR</Emphasis> regulates proliferation and invasion via activating Notch signalling pathway in retinoblastoma

Retinoblastoma is the most frequently occurring tumour in the eyes in early childhood. Novel targets that are important for the diagnosis or treatment of retinoblastoma could be valuable in increasing the survival rate of patients affected by this disease. Long non-coding RNAs (lncRNAs) are a recently discovered type of RNAs with no protein-coding function; yet it has become increasingly clear that lncRNAs are responsible for important gene regulatory functions in various diseases. In this study, the expression of lncRNA HOTAIR was measured by qRT-PCR, and HOTAIR expression was found to be significantly upregulated in human retinoblastomas tissues as compared with that in paracancerous tissues. Knockdown of HOTAIR restricted the proliferation and invasion of the more invasive retinoblastoma Y79 cells, and led to G0/G1 arrest, possibly through inhibiting phospho-RB1, RB1 and CCNE. Furthermore, we found that the Notch signalling pathway was activated abnormally in retinoblastoma cell lines, while knockdown of HOTAIR attenuated the endogenous Notch signalling pathway in vitro and in vivo. In addition, knockdown of HOTAIR could inhibit the tumour progression in a xenograft model of retinoblastoma. In summary, our findings indicate that HOTAIR may play important roles in retinoblastoma progression via Notch pathway. HOTAIR has the potential to enhance the development of novel targeted diagnostic and therapeutic approaches for retinoblastoma.     

Thymoquinone induces cytotoxicity and reprogramming of EMT in gastric cancer cells by targeting PI3K/Akt/mTOR pathway

Gastric cancer is one of the lethal causes of cancer-related deaths worldwide. The incidence and mortality rates of this disease is comparatively higher in China. In the current study, we evaluated the anticancer effects of Thymoquinone (TQ) against gastric cancer cells (MGC80-3 and SGC-7901) and normal noncancerous GES-1 cells and attempted to investigate the underlying mechanism. Our results indicated that TQ exhibited significant growth inhibitory effects on gastric cancer cells (MGC80-3 and SGC-7901). However, lower cytotoxicity was observed against normal GES-1 cells. Moreover, TQ could inhibit the colony formation potential of MGC80-3 and SGC-7901 cells in a dose-dependent manner. TQ also inhibited cell migration ability of the gastric cancer cells and down-regulated the expression of the mesenchymal genes such as N-cadherin, Vimentin, and TWIST. However, the epithelial markers such as E-cadherin and cytokeratin-19 were distinctly up-regulated in TQ-treated gastric cancer cells. Since PI3K/Akt/ mTOR plays an important role in progression and tumorigenesis, we also investigated the effect of TQ on PI3K/Akt/mTOR signalling pathway in gastric cancer cells. It was observed that TQ down-regulated the expression of some of the key proteins of this pathway. Taken together, we conclude that TQ may prove lead molecule for the treatment of gastric cancer.     

Organic two-photon nanoparticles modulate reactive oxygen species,intracellular calcium concentration,and mitochondrial membrane potential during apoptosis of human gastric carcinoma SGC-7901 cells

Objectives

To investigate the biocompatibility of human gastric carcinoma cells (SGC-7901) with organic two-photon nanoparticles (NPs).

Results

Different concentrations of NPs were incubated with SGC-7901 cells for different times. The levels of cell apoptosis, reactive oxygen species (ROS), intracellular calcium, and mitochondrial membrane potential (MMP) were measured by staining the SGC-7901 cells with Annexin V-FITC/PI, 2′,7′-dichlorofluorescin diacetate, Fluo-3 AM, and Rhodamine 123, followed by the flow cytometry assay. NPs at <4 µg/ml, did not have any significant effect on apoptosis, necrosis, generation of ROS, increase of intracellular Ca²⁺ concentration or decrease of MMP in SGC-7901 cells, but >4 µg/ml had a major effects on all the above mentioned parameters.

Conclusion

2,5,2′,5′-Tetra(4-N,N-diphenylamine styryl) biphenyl NPs can be used at an appropriate concentration as a safe drug carrier or imaging marker and may serve as an effective tool for developing a photodynamic cancer therapy.

     

Inhibition of breast cancer cell proliferation and tumorigenesis by long non-coding RNA <Emphasis Type="Italic">RPPH1</Emphasis> down-regulation of miR-122 expression

Background

Recent studies showed that long non-coding RNA (lncRNA) plays an important role in many life activities. RPPH1 is one of the lncRNA genes that are expressed differently between breast cancer and normal tissues by the lncRNA gene chip. Our study was conducted to examine the regulation of lncRNA RPPH1 in breast cancer.

Methods

Two cell lines, MCF-7 and MDA-MB-231, were selected to be the research objects in this study; RPPH1 overexpression and knockdown models were established by transforming vectors. Real-time polymerase chain reaction, MTT assay, clone formation and cell flow cytometer assay were used to test the function of RPPH1. Dual-luciferase assay was used to detect a target relationship between RPPH1 and miR-122.

Results

RPPH1 overexpression promoted cell cycle and proliferation and increased colony formation. In the RPPH1 overexpression model, there was a target relationship between RPPH1 and miR-122, and some of the downstream genes of miR-122, including ADAM10, PKM2, NOD2 and IGF1R, were increased. Moreover, we found that lentivirus-mediated interference of lncRNA RPPH1 inhibited tumour growth in nude mice.

Conclusion

Breast cancer progression can be promoted by directly targeting miR-122 through lncRNA RPPH1. This study provided evidence that can serve as the molecular basis for improving treatment options for patients.

     

MYO18B promotes hepatocellular carcinoma progression by activating PI3K/AKT/mTOR signaling pathway

Background

MYO18B has been identified as a novel tumor suppressor gene in several cancers. However, its specific roles in the progression of hepatocellular carcinoma (HCC) has not been well defined.

Methods

We firstly identified the expression and prognostic values of MYO18B in HCC using TCGA cohort and our clinical data. Then, MYO18B knockdown by RNA inference was implemented to investigate the effects of MYO18B on HCC cells. Quantitative RT-PCR and Western blot were used to determine gene and protein expression levels. CCK-8 and colony formation assays were performed to examine cell proliferation capacity. Wound healing and transwell assays were used to evaluate the migration and invasion of HepG2 cells.

Results

MYO18B was overexpressed and correlated with poor prognosis in HCC. MYO18B expression was an independent risk factor for overall survival. Knockdown of MYO18B significantly inhibited the proliferation, migration and invasion of HepG2 cells. Meanwhile, MYO18B knockdown could effectively suppress the phosphorylation of PI3K, AKT, mTOR and P70S6K, suggesting that MYO18B might promote HCC progression by targeting PI3K/AKT/mTOR signaling pathway.

Conclusions

MYO18B promoted tumor growth and migration via the activation of PI3K/AKT/mTOR signaling pathway. MYO18B might be a promising target for clinical intervention of HCC.

     

G蛋白偶联胆汁酸受体1促进胃癌细胞的增殖、迁移和侵袭的实验研究

目的:探讨G蛋白偶联胆汁酸受体1(G-protein coupled bile acid receptor 1,GPBAR1/TGR5)对胃癌细胞增殖、迁移和侵袭的影响。方法:免疫组织化学染色方法(Immunohistochemistry,IHC)检测胃癌及癌旁组织芯片中TGR5表达情况;qRT-PCR及Western blot检测胃癌细胞系中TGR5表达水平;小干扰RNA处理AGS、MKN-45胃癌细胞后构建TGR5敲减细胞系,慢病毒载体转染胃癌SGC-7901细胞构建TGR5过表达细胞系;CCK-8实验、平板克隆形成实验、裸鼠皮下移植瘤实验检测TGR5对细胞增殖的影响;流式细胞仪检测TGR5对细胞周期及凋亡的影响;Tanswell实验检测TGR5对胃癌细胞迁移及侵袭的影响;Western blot检测上皮间充质转化(Epithelial-mesenchymal transition,EMT)相关分子β-连环蛋白(β-catenin)、锌脂蛋白转录因子(Snail)、E盒结合锌指蛋白(Zinc finger E-box binding homeobox 1,ZEB)1在AGS、MKN-45及SGC-7901胃癌细胞中的表达。结果:TGR5在胃癌及癌旁组织中均有表达,胃癌组织TGR5高表达率(41.0%)显著高于癌旁组织(9.5%),伴肠化生癌旁组织TGR5高表达率(50%)显著高于不伴肠化生的癌旁组织(0%),胃癌组织TGR5表达与肿瘤大小相关。TGR5在正常人胃上皮永生化细胞株GES-1及各胃癌细胞系中均有表达。TGR5表达敲低的AGS和MKN-45细胞增殖能力减弱、凋亡率显著升高、侵袭和迁移能力显著降低。过表达TGR5的SGC-7901细胞增殖能力增强、克隆形成能力提高、凋亡率明显减低、侵袭和迁移能力显著升高。此外,TGR5过表达显著上调了间质细胞标志物β-catenin、Snail、ZEB1的表达水平。结论:TGR5能够增强胃癌细胞增殖及迁移能力,并抑制细胞凋亡。TGR5可能通过EMT途径介导胃癌细胞转移。     

Tumour suppressive function of HUWE1 in thyroid cancer

HUWE1 (the HECT, UBA, and WWE domain-containing protein 1) is an ubiquitin E3 ligase which plays an important role in coordinating diverse cellular processes. It has been found to be dysregulated in various cancer type and its functions in tumorigenesis remain controversial. The potential tumour suppressive role of HUWE1 in thyroid cancer development was investigated by knocking down HUWE1 in three authentic thyroid cancer cell lines, WRO, FTC133 and BCPAP, followed by various functional assays, including cell proliferation, scratch wound healing and invasion assays. Xenograft experiment was performed to examine in vivo tumour suppressive properties of HUWE1. Small-interfering RNA mediated knockdown of HUWE1 promoted cell proliferation, cell migration and invasion in thyroid cancer cells. Overexpression of HUWE1 conferred partial sensitivity to chemo drugs interfering with DNA replication in these cells. Moreover, HUWE1 was found to be down-regulated in human thyroid cancer tissues compared with matched normal thyroid tissues. In addition, overexpression of HUWE1 significantly inhibited tumour growth in vivo using xenograft mouse models. Mechanistic investigation revealed that HUWE1 can regulate p53 protein level through its stabilization. HUWE1 functions as a tumour suppressor in thyroid cancer progression, which may represent a novel therapeutic target for prevention or intervention of thyroid cancer.     

TIGAR cooperated with glycolysis to inhibit the apoptosis of leukemia cells and associated with poor prognosis in patients with cytogenetically normal acute myeloid leukemia

Background

Cancer cells show increased glycolysis and take advantage of this metabolic pathway to generate ATP. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects tumor cells from intracellular reactive oxygen species (ROS)-associated apoptosis. However, the function of TIGAR in glycolysis and survival of acute myeloid leukemia cells remains unclear.

Methods

We analyzed TIGAR expression in cytogenetically normal (CN-) AML patients and the correlations with clinical and biological parameters. In vivo and in vitro, we tested whether glycolysis may induce TIGAR expression and evaluated the combination effect of glycolysis inhibitor and TIGAR knockdown on human leukemia cell proliferation.

Results

High TIGAR expression was an independent predictor of poor survival and high incidence of relapse in adult patients with CN-AML. TIGAR also showed high expression in multiple human leukemia cell lines and knockdown of TIGAR activated glycolysis through PFKFB3 upregulation in human leukemia cells. Knockdown of TIGAR inhibited the proliferation of human leukemia cells and sensitized leukemia cells to glycolysis inhibitor both in vitro and in vivo. Furthermore, TIGAR knockdown in combination with glycolysis inhibitor 2-DG led leukemia cells to apoptosis. In addition, the p53 activator Nutlin-3α showed a significant combinational effect with TIGAR knockdown in leukemia cells. However, TIGAR expression and its anti-apoptotic effects were uncoupled from overexpression of exogenous p53 in leukemia cells.

Conclusions

TIGAR might be a predictor of poor survival and high incidence of relapse in AML patients, and the combination of TIGAR inhibitors with anti-glycolytic agents may be novel therapies for the future clinical use in AML patients.

     

Cytotoxic and toxicogenomic effects of silibinin in bladder cancer cells with different <Emphasis Type="Italic">TP53</Emphasis> status

Silibinin is a natural phenol found in the seeds of the milk thistle plant. Recent data have shown its effectiveness for preventing/treating bladder tumours. Therefore, in this study we investigated the cytotoxic and toxicogenetic activity of silibinin in bladder cancer cells with different TP53 statuses. Two bladder urothelial carcinoma cell lines were used: RT4 (wild-type TP53 gene) and T24 (mutated TP53 gene). Cell proliferation, clonogenic survival, apoptosis rates, genotoxicity and relative expression profile of FRAP/mTOR, FGFR3, AKT2 and DNMT1 genes and of miR100 and miR203 were evaluated. Silibinin promoted decreased proliferation and increased late apoptosis in TP53 mutated cells. Increased early apoptosis rates, primary DNA damage, and decrease of cell colonies in the clonogenic survival assay were detected in both RT4 and T24 cell lines. Down-regulation of FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 expression occurred in RT4 cells. Modulation of miR203 was observed in both cell lines. In conclusion, despite the reduction of clone formation in both cell lines, the toxicogenomic effect of silibinin on FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 was dependent on the TP53 status. Taken together, the data confirmed the role of silibinin as an antiproliferative compound, whose mechanism of action was related to the TP53 status.     

In vitro virulence characteristics of rare serovars of <Emphasis Type="Italic">Salmonella enterica</Emphasis> isolated from sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis> L.)

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.     

RNA binding protein QKI contributes to WT1 mRNA and suppresses apoptosis in ST cells

The RNA binding protein quaking (QKI), a key member of the STAR family, as an upstream gene could involve in much process including cell proliferation, apoptosis, differentiation and so on. However, the roles of QKI in germ cell, especially in swine testis (ST) cells, was not clear currently. And apoptosis plays important roles in the growth and development. The purpose of the present study was to clarify the relationship between QKI and apoptosis in ST cells. Firstly, our results showed that pEF1α-QKI and shQKI3 have clear effects on expression levels of QKI. Secondly, we established that QKI directly binds to WT1 3′UTR by binding with QRE-1 (2046–2052 bp, ACTAAC) only. Furthermore, QKI overexpression significantly increased the expression levels of WT1 and Bcl-2. QKI also has the effect on delaying the degradation of WT1 mRNA. In addition, we verified that QKI had a significantly suppressed apoptosis in ST cells. Finally, pBI-WT1 could make up for shQKI3-induced decrease in WT1, Bcl-2 mRNA levels and suppress apoptosis in ST cells. The results demonstrated that QKI was an important regulatory factor that affects apoptosis by targeting WT1 gene.     

Galectin 3 acts as an enhancer of survival responses in <Emphasis Type="Italic">H. pylori</Emphasis>-infected gastric cancer cells

Galectin 3 (Gal-3) is upregulated in gastric epithelial cells as a host response to Helicobacter pylori infection. However, the significance of Gal-3 expression in H. pylori-infected cells is not well established. We analyzed Gal-3 intracellular expression, localization, and its effects in H. pylori-infected gastric epithelial cells. The predominantly nuclear confined Gal-3 was shown to be upregulated and exported out to the cytoplasm in H. pylori-infected AGS cells. The nuclear export was channeled through CRM-1 (exportin-1) protein. Interestingly, knock down of Gal-3 expression led to reduced NF-κB promoter activity and interleukin-8 (IL-8) secretion, suggesting its pro-inflammatory roles. Furthermore, Gal-3 was found to be pro-proliferative and anti-apoptotic in nature, as its knock down caused a reduction in cell proliferation and an increase in apoptosis, respectively. Taken together, our data suggest the expression and upregulation of Gal-3 as a critical endogenous event in H. pylori infection that interferes with various intracellular events, causing prolonged cell survival, which is characteristic in carcinogenesis.     

Upregulation of p72 enhances malignant migration and invasion of glioma cells by repressing Beclin1 expression

p72 is the member of the DEAD-box RNA helicase family, which can unwind double-stranded RNA and is efficient for microRNA (miRNA, miR) processing. However, its specific role in glioma has not been elucidated. First, the expression of p72 in glioma cell lines and tissues was explored using Western blot. To explore the role of p72 on glioma progression, adenovirus inhibiting p72 was transfected into A172 and T98G cells. Cell autophagy was determined using GFPLC3 dots, and cell apoptosis was determined using flow cytometry. The effect of Beclin1 was explored using GFP-LC3 dots, flow cytometry, and colony formation. The possible miRNAs that target the 3′-untranslated region (3′-UTR) of Beclin1 were predicted using TargetScan. Dual luciferase reporter assay was applied to determine whether these miRNAs bind to the 3′-UTR of Beclin1. The expression of p72 was significantly increased in glioma cell lines and tissues. Autophagy-related protein Beclin1 was found to be significantly enhanced when p72 was inhibited. The accumulation of GFP-LC3 dots was significant in cells transfected with ad-sh-p72 compared with ad-con. Colony formation capacity and cell apoptosis were also found to be significantly decreased with p72 inhibition. Furthermore, upregulation of Beclin1 contributes to A172 cell autophagy, invasion, and apoptosis. Overexpression of p72 induces increased miR-34-5p and miR-5195-3p expression in A172 and T98G cells. Beclin1 was the target gene of miR-34-5p and miR-5195-3p. In conclusion, we found for the first time that overexpression of p72 decreased Beclin1 expression partially by increasing miR-34-5p and miR-5195-3p expression in A172 and T98G cells.