Growth Of Esophageal Carcinoma Cells Research Articles

Baitouweng decoction suppresses growth of esophageal carcinoma cells through miR-495-3p/BUB1/STAT3 axis.

Esophageal carcinoma (EC) is one of the most prevalent cancers in human populations worldwide. Baitouweng decoction is one of the most important Chinese medicine formulas, with the potential to treat cancer. To investigate the role and mechanism of Baitouweng decoction on EC cells. Differentially expressed genes (DEGs) in EC tissues and normal tissues were screened by the cDNA microarray technique and by bioinformatics methods. The target genes of microRNAs were predicted based on the TargetScan database and verified by dual luciferase gene reporter assay. We used Baitouweng decoction to intervene EC cells, and detected the activity of EC9706 and KYSE150 cells by the MTT method. Cell cycle and apoptosis were measured by flow cytometry. The expression of BUB1 mRNA and miR-495-3p was measured by qRT-PCR. The protein levels of BUB1, STAT3, p-STAT3, CCNB1, CDK1, Bax, Caspase3, and Caspase9 were measured by Western blot analysis. The migration and invasion abilities of the cells were measured by wound-healing assay and Transwell invasion assay, respectively. DEGs identified are involved in biological processes, signaling pathways, and network construction, which are mainly related to mitosis. BUB1 was the key hub gene, and it is also a target gene of miR-495-3p. Baitouweng decoction could upregulate miR-495-3p and inhibit BUB1 expression. In vitro experiments showed that Baitouweng decoction significantly inhibited the migration and invasion of EC cells and induced apoptosis and G2/M phase arrest. After treatment with Baitouweng decoction, the expression of Bax, Caspase 3, and Caspase 9 in EC cells increased significantly, while the expression of BUB1, CCNB1, and CDK1 decreased significantly. Moreover, the STAT3 signaling pathway may play an important role in this process. Baitouweng decoction has a significant inhibitory effect on EC cell growth. BUB1 is a potential therapeutic target for EC. Further analysis showed that Baitouweng decoction may inhibit the growth of EC cells by upregulating miR-495-3p targeting the BUB1-mediated STAT3 signal pathway.

EIF6 is potential diagnostic and prognostic biomarker that associated with 18F-FDG PET/CT features and immune signatures in esophageal carcinoma

BackgroundAlthough eukaryotic initiation factor 6 (eIF6) is a novel therapeutic target, data on its importance in the development of esophageal carcinoma (ESCA) remains limited. This study evaluated the correlation between eIF6 expression and metabolic analysis using fluorine-18 fluorodeoxyglucose (18F-FDG) -Positron emission tomography (PET) and immune gene signatures in ESCA.MethodsThis study employed The Cancer Genome Atlas (TCGA) to analyze the expression and prognostic value of eIF6, as well as its relationship with the immune gene signatures in ESCA patients. The qRT-PCR and Western blot analyses were used to profile the expression of eIF6 in ESCA tissues and different ESCA cell lines. The expression of tumor eIF6 and glucose transporter 1 (GLUT1) was examined using immunohistochemical tools in fifty-two ESCA patients undergoing routine 18F-FDG PET/CT before surgery. In addition, the cellular responses to eIF6 knockdown in human ESCA cells were assessed via the MTS, EdU, flow cytometry and wound healing assays.ResultsOur data demonstrated that compared with the normal esophageal tissues, eIF6 expression was upregulated in ESCA tumor tissues and showed a high diagnostic value with an area under curve of 0.825 for predicting ESCA. High eIF6 expression was significantly correlated with shorter overall survival of patients with esophagus adenocarcinoma (p = 0.038), but not in squamous cell carcinoma of the esophagus (p = 0.078). In addition, tumor eIF6 was significantly associated with 18F-FDG PET/CT parameters: maximal and mean standardized uptake values (SUVmax and SUVmean) and total lesion glycolysis (TLG) (rho = 0.458, 0.460, and 0.300, respectively, p < 0.01) as well as GLUT1 expression (rho = 0.453, p < 0.001). A SUVmax cutoff of 18.2 led to prediction of tumor eIF6 expression with an accuracy of 0.755. Functional analysis studies demonstrated that knockdown of eIF6 inhibited ESCA cell growth and migration, and fueled cell apoptosis. Moreover, the Bulk RNA gene analysis revealed a significant inverse association between eIF6 and the tumor-infiltrating immune cells (macrophages, T cells, or Th1 cells) and immunomodulators in the ESCA microenvironment.ConclusionOur study suggested that eIF6 might serve as a potential prognostic biomarker associated with metabolic variability and immune gene signatures in ESCA tumor microenvironment.

Construction and investigation of \u03b23GNT2-associated regulatory network in esophageal carcinoma

BackgroundGlycosyltransferases play a crucial role in various cancers. β1, 3-N-acetylglucosaminyltransferase 2, a polylactosamine synthase, is an important member of the glycosyltransferase family. However, the biological function and regulatory mechanism of β3GNT2 in esophageal carcinoma (ESCA) is still poorly understood.MethodsThe Cancer Genome Atlas and Genotype-Tissue Expression databases were used for gene expression and prognosis analysis. Quantitative real-time PCR, Western blot, and immunohistochemistry were performed to detect the expression of β3GNT2 in ESCA cell lines and tissues. In vitro assays and xenograft tumor models were utilized to evaluate the impact of β3GNT2 on ESCA progression. The downstream effectors and upstream regulators of β3GNT2 were predicted by online software and verified by functional experiments.ResultsWe found that β3GNT2 was highly expressed in ESCA tissues and positively correlated with poor prognosis in ESCA patients. β3GNT2 expression was closely associated with the tumor size, TNM stage, and overall survival of ESCA patients. Functionally, β3GNT2 promoted ESCA cell growth, migration, and invasion in vitro, as well as tumorigenesis in vivo. Mechanistically, β3GNT2 knockdown decreased the expression of the polylactosamine on EGFR. Knockdown of β3GNT2 also inhibited the JAK/STAT signaling pathway. Meanwhile, the JAK/STAT inhibitor could partly reverse the biological effects caused by β3GNT2 overexpression. Moreover, β3GNT2 expression was positively regulated by CREB1 and negatively regulated by miR-133b. Both CREB1 and miR-133b was involved in the β3GNT2-mediated ESCA progression.ConclusionsOur study, for the first time, reveals the importance of β3GNT2 in ESCA progression and offers a potential therapeutic target for ESCA.

USP53 activated by H3K27 acetylation regulates cell viability, apoptosis and metabolism in esophageal carcinoma via the AMPK signaling pathway.

Esophageal carcinoma (ESCA) is a leading cause of cancer death worldwide, despite an overall decline in the incidence of new cases. However, knowledge of gene expression signatures for risk and prognosis stratification of ESCA is inadequate. Thus, identifying novel molecular biomarkers and therapeutic targets for ESCA might improve its prognosis and treatment. The current study investigated the role of ubiquitin-specific peptidase 53 (USP53), a member of the USP family that exhibits deubiquitinating activity, in ESCA and showed that USP53 is downregulated in ESCA tissues, indicating poor prognosis. USP53 suppresses the proliferation and growth of ESCA cells in vitro and in vivo, whereas its knockdown exerts opposite effects. AMP-activated protein kinase inhibitor reverses the effects of USP53 knockdown. USP53 also inhibits glycolysis, oxidative metabolism and mitochondrial dynamics. H3K27 acetylation increases USP53 expression by binding to its promoter region. Our study reveals that USP53 is activated by H3K27 acetylation and suppresses ESCA progression by regulating cell growth and metabolism. USP53 is therefore a promising target for ESCA treatment.

Upregulation of deubiquitinase USP7 by transcription factor FOXO6 promotes EC progression via targeting the JMJD3/CLU axis

Esophageal carcinoma (EC) is recognized as one of the most frequently occurring malignancies worldwide, and its high morbidity rate motivates efforts to identify potential therapeutic targets. Notably, forkhead box (FOX) family genes are highlighted as possible biomarkers for diagnostics, prognostics, and therapeutics of various malignancies, including EC. Our present study was performed to investigate the underlying mechanism of FOXO6 on the development of EC. We observed a significant upregulation of FOXO6 in EC tissues, contributing to the migration and proliferation in EC cells through gain- and loss-of-function assays. FOXO6 directly interacted with the ubiquitin-specific processing protease 7 (USP7) gene promoter and enhanced its transcriptional activity, which resulted in suppressed cancer cell apoptosis as revealed by chromatin immunoprecipitation (ChIP)-qPCR. USP7 enhanced the ubiquitination of Jumonji domain-containing protein D3 (JMJD3), elevated JMJD3-promoted growth of EC cells, and transcriptionally activated clusterin (CLU) expression at the promoter region via histone H3 lysine 27 tri-methyl (H3K27me3) demethylation, according to immunoprecipitation and ubiquitination assays. Finally, we verified that FOXO6 mediated effects on the USP7/JMJD3/CLU axis to exert an oncogenic role in vivo, which was blocked by USP7 and JMJD3 inhibitor. Our findings demonstrate an important role of the FOXO6/USP7/JMJD3/CLU pathway in EC progression and thus provide attractive potential therapeutic targets for EC patients.

Gallic acid from Terminalia chebula inhibited the growth of esophageal carcinoma cells by suppressing the Hippo signal pathway.

Objective(s):To explore the molecular mechanism of gallic acid (GA) from Terminalia chebula in suppressing the growth of esophageal carcinoma (EC).Materials and Methods:Human EC cells (EC9706 and KYSE450) were treated with different concentrations of GA (10, 20, and 40 μg/ml), which were subjected to 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, plate clone formation assay, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, and Western blotting. EC mice were divided into Model, 0.3% GA, and 1% GA groups to observe the tumor volume and the expressions of YAP, TAZ, Ki-67, and Caspase-3 in tumor tissues. Results:GA decreased cell viability and colony formation of EC9706 and KYSE450 cells, which was more obvious as the concentration increased. In addition, GA promoted cell apoptosis in a concentration-dependent manner with the up-regulation of pro-apoptotic proteins (Bax, cleaved caspase-3, and cleaved caspase-9) and nuclear YAP/TAZ, as well as the down-regulation of anti-apoptotic protein Bcl-2 and the levels of p-YAP and p-TAZ. Moreover, GA decreased the growth of xenograft tumor in vivo, with the reduction in the tumor volume and the reduction of YAP and TAZ expressions in the tumor tissues. In addition, Ki-67 expression in GA groups was lower than those in the Model group, with the increase in caspase-3 expression in the tumor tissues. Changes aforementioned were obviously shown in the 0.3% GA group. Conclusion:GA blocked the activity of the Hippo pathway to suppress cell proliferation of EC and facilitate cell apoptosis, which is expected to be a novel strategy for treatment of EC.

Abstract 1854: Oridonin inhibits cell growth of esophageal carcinoma cells via induction of apoptosis

Abstract Esophageal Cancer estimated leading deaths are more than 400,000 every year in worldwide, while 80% due to esophageal squamous cell carcinoma. In worldwide, esophageal cancer leading death ranks in sixth among cancer leading causes of death, while the 5-year survival rate of esophageal cancer is 19%. Oridonin, a tetracyclic diterpenes compound, has been indicated the anti-tumor activity for digestive system tumors. To assess the anticancer activity of oridonin on esophageal cancer, we performed cell proliferation experiment of oridonin on esophageal squamous cell carcinoma cell line TE-8 using SRB assay. We found that IC50 of oridonin on TE-8 treated for 72h is 3.0μM, which is better than positive control Gemcitabine treatment on TE-8 for 72h (5.7 μM), as well as TE-8 cells shrunk and floated with increasing concentration on oridonin. At the same time, oridonin induced a dose-dependent inhibition of cell colony formation of esophageal cancer cell line. Colony numbers of TE-8 cell line treated with 1μM and 2μM oridonin are 111 and 54, respectively, which are significantly different (p&amp;lt;0.05). Accompanying with marked morphological changes, flow cytometer results showed that oridonin triggered sub-G0/G1 cell cycle arrest in a dose-dependent manner. After treatment by 10μM and 20μM oridonin for 24h, percentage of sub-G0/G1 cells were 25.5 and 55.3%, respectively, which are significantly different (p&amp;lt;0.05). The apoptosis results showed that oridonin treatment led to the accumulation of cells in early-(Annexin V+/PI-) and late-stage (Annexin V+/PI+) apoptosis in a dose-dependent manner. Citation Format: Xiaojun Zhang, Mengtao Xing, Yangcheng Ma, Xiao Wang, Jianying Zhang. Oridonin inhibits cell growth of esophageal carcinoma cells via induction of apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1854.

Betulinic acid enhanced the chemical sensitivity of esophageal cancer cells to cisplatin by inducing cell pyroptosis and reducing cell stemness.

Betulinic acid (BA) is a lupine pentacyclic triterpene compound derived from the bark of the white mulberry tree, which has a variety of pharmacological properties. The purpose of this study was to investigate the effects of BA combined with cisplatin on the proliferation, stemness, pyroptosis, and xenograft growth of esophageal carcinoma cells in a nude mouse xenograft model. The cell survival rate was detected by CCK-8 method. TE-11 cells were treated with 3μM of BA and 15 μM of cisplatin. The cells were randomly divided into four groups: the control group, the BA group, the cisplatin group, and the BA + Cisplatin group. Western blotting was used to detect the expression levels of Ki67, PCNA, SOX2, OCT4, ASC, and Caspase-1. The xenograft model of nude mice was constructed to detect tumor volume, and the positive expression rates of Ki67 and Caspase-1 were detected by immunohistochemistry. Compared with the control group, Ki67, PCNA, SOX2, and OCT4 levels in the BA and cisplatin groups were significantly lower (P<0.05), while ASC and Caspase-1 levels were significantly higher (P<0.05). Compared with the BA group, Ki67, PCNA, SOX2, and OCT4 levels in the BA + cisplatin group were significantly lower (P<0.05), while ASC and Caspase-1 levels were significantly higher (P<0.05). In the nude mouse xenograft model, compared with the control group, the tumor volume of the BA and cisplatin groups was significantly decreased (P<0.05), the expression rate of Ki67 was significantly decreased (P<0.05), and the expression rate of Caspase-1 was significantly increased (P<0.05). Compared with the BA group, the levels of ASC and Caspase-1 in the BA + cisplatin group were significantly lower (P<0.05), the positive expression rate of Ki67 was significantly lower (P<0.05), and the positive expression rate of Caspase-1 was significantly higher (P<0.05). BA enhances the chemical sensitivity of esophageal cancer cells to cisplatin by inhibiting cell proliferation, reducing cell stemness, and inducing pyroptosis.

Abstract 5862: YAP1 mediated CDK6 activation confers radiation resistance in esophageal cancer: rationale for the combination of YAP1 and CDK4/6 inhibitors in EC

Abstract Background: Esophageal carcinoma (EC) is a lethal disease with high incidence globally and often exist therapy resistance. Alterations (either overexpression or amplification) of YAP1 and CDK4/6 were found frequently in esophageal cancer. Deregulation of these pathways may represent key elements for resistance in esophageal cancer. Methods: Expression of YAP1 and CDK4/6 were examined in esophageal tumor tissues as well as cell lines using immunohistochemistry and immunoblotting. Inducible YAP1 overexpression in EC cells by lentivirus system was performed to test YAP1 mediated CDK4/6 expression and activation and association with radiation resistance. YAP inhibitor CA3 and CDK4/6 inhibitor Lee001 were used to test their antitumor activities in vitro and in vivo. Cell proliferation assay (MTS), Flow cytometry and immunofluorecence and tumor sphere formation assay were performed to test cell cycle distribution, cancer stem cell (CSC) population maintenance and over growth and therapy resistance. Results: We demonstrate that overexpressed YAP1 is positively associated with CDK4/6 expression in resistant tumor tissues and EC cell lines. Overexpression YAP1 by inducible lentivirus system in EC cells up-regulates expression of CDK4/6 at level of its transcription, while knock down YAP1 in JHESO EC cells dramatically decreased CDK6 expression as well as its transcription. CA3, a novel YAP1 inhibitor, was able to decrease activation of CDK4/6 and phosphorylation RB and cell cycle progression induced by YAP1. Interestingly, we found that inducible YAP1 high EC cells are resistant to radiation resistance and CDK6 knockout by Lenti-Crisp sensitize radiation resistant in EC cells induced by YAP1. Further, we demonstrated radiation resistant cells are enriched CSC population and demonstrated increased level of both YAP1 and CDK4/6, while blocking the YAP and CDK4/6 pathway by using their inhibitor CA3 or Lee001 reduced EC cell growth and CSCs properties especially in EC resistant cells. Most importantly, we found combination of Lee001 and CA3 exert a synergistic anticancer effect in radiation resistant EC cells with high YAP1 and CDK4/6 in Vitro and in Vivo. Conclusions: Our data provide evidence that YAP1 mediated CDK4/6 up-regulation plays an important role in conferring radiation resistance in esophageal cancer cells. Targeting both YAP1 and CDK4/6 may provide novel therapeutic strategies in EC. Corresponding Authors: Shumei Song, Tel:713-834-6144; Email: ssong@mdanderson.org; or Jaffer A. Ajani, Tel: 713-792-3685; Email: jajani@mdanderson.org Citation Format: Fan Li, Xiaoquan Dong, Haitao Zhu, Ailing W. Scott, Lang Ma, Jiankang Jin, Jeannelyn Santiano Estrella, Heath Skinner, Randy L. Johnson, Shumei Song, Jaffer A. Ajani. YAP1 mediated CDK6 activation confers radiation resistance in esophageal cancer: rationale for the combination of YAP1 and CDK4/6 inhibitors in EC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5862. doi:10.1158/1538-7445.AM2017-5862

Clinical implications of vasohibin-1 in esophageal carcinoma cells: Inhibition of cell growth and migration.

As one of the first-established negative feedback regulators of angiogenesis, mesenchymal vasohibin-1 (VASH1) serves important roles in the progression and prognosis of various types of tumor. However, the clinical implications of VASH1 in esophageal carcinoma (EC) cells have not been reported and the direct effects of VASH1 on EC cells remain unknown. In the present study, the expression of VASH1 in EC cells was observed using immunohistochemistry and western blotting; a χ2test was used to analyze the correlation of VASH1 with clinical parameters, and it was observed that VASH1 was negatively-correlated with tumor size (r=‑0.399; P<0.01) and invasion depth (r=‑0.318; P<0.01). Survival analysis demonstrated that VASH1 was positively‑correlated with increased overall survival (P=0.039) and disease free survival (P=0.012). The direct effects of VASH1 on EC cells were analyzed by altering VASH1 expression, and it was observed that downregulation of VASH1 increased proliferation, clone formation and the migratory ability of EC9706 cells, whereas upregulation of VASH1 inhibited proliferation, clone formation and the migratory ability of EC1 cells. The results of the present study demonstrated that VASH1 in EC cells was negatively‑correlated with progression and poor prognosis of patients with EC. VASH1 was able to directly inhibit the growth and migration of EC cells.

Effects of RNA silencing of matrix metalloproteinase-2 on the growth of esophageal carcinoma cells in vivo.

Esophageal carcinoma is one of the most common malignancies in China. Previous studies reported that matrix metalloproteinases (MMPs) have important roles in the progression and invasion of numerous types of solid tumors. Among the MMPs, MMP-2 has been closely associated with tumor growth and invasion. In the present study, a short hairpin RNA (shRNA) lentiviral expression vector targeting the MMP-2 gene was constructed in order to observe the inhibitory effect of MMP-2 gene silencing on the growth of the KYSE150 esophageal carcinoma cell line in vivo. Three small hairpin RNA sequences targeting MMP-2 were designed and cloned into lentiviral vectors. Following transfection of the lentiviral vectors into KTSE150 cells, MMP-2 mRNA and protein expression levels were examined by reverse transcription-quantitative polymerase chain reaction and western blotting, and the growth rate of cells was analyzed by MTT assays. Subsequently, tumor growth was assessed in nude mice. Lentivirus-mediated RNA interference effectively inhibited the expression of MMP-2 mRNA and protein in KYSE150 esophageal carcinoma cells, and suppressed the growth of esophageal carcinoma cells in vivo. The results of the present study suggested that lentivirus-mediated gene therapy targeting MMP-2 may be an attractive strategy for the treatment of esophageal carcinoma and justifies the performance of further studies on the application of lentivirus vectors to cancer gene therapy.

Abstract 1253: Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells

Abstract The long-term goal of our research is to investigate the molecular carcinogenesis of esophageal squamous cell carcinoma and to develop strategies for esophageal cancer prevention. Previous studies in our laboratory demonstrated an association between activation of PI3K/AKT/mTOR pathways and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. The objective of current study is to evaluate the effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, and MK2206, an AKT inhibitor, alone or in combination in esophageal cancer cell lines, KYSE-70, -150, -270 and -410. The cancer cell lines were treated with different doses of NVP-BEZ235 (1, 10, 50, 100, 1000 nMol/L) and MK2206 (0.5, 1, 3, 10, 20 uMol/L), or NVP-BEZ235 100 nMol/L plus MK2206 3 uMol/L. Western blot was performed to assess the activation of PI3K/AKT/mTOR pathways. Cell proliferation, apoptosis and cell cycle were analyzed by WST1 and flow cytometry. We found that the combination of NVP-BEZ235 and MK2206 significantly reduced the phosphorylation of p-AKTT308, p-AKTS473, p-mTORS2448, p-S6S235/236 and p-4EBP1T37/46, when compared to NVP-BEZ235 (1000 nMol/L) and MK2206 (20 uMol/L) alone (p&amp;lt;0.05). The cell proliferation rate was reduced by NVP-BEZ235 1000 nMol/L, MK2206 20 uMol/L, or NVP-BEZ235 plus MK2206 to 20% (p&amp;lt;0.01), 24% (p&amp;lt;0.01) and 17% (p&amp;lt;0.01), respectively. The NVP-BEZ235 1000 nMol/L and MK2206 10 uMol/L induced apoptosis in esophageal cancer cells to 30% and 20% (p&amp;lt;0.01), respectively. NVP-BEZ235 plus MK2206 induced apoptosis in esophageal cancer cells to 45% (p&amp;lt;0.01). The combination treatment increased the proportion of cells at G0/G1 phase compared to cells treated with NVP-BEZ235 or MK2206 alone. Our results indicate that co-targeting the PI3K/mTOR and AKT signaling pathways produces synergistic activity against esophageal carcinoma cell growth. This in-vitro study provides evidence to test the efficacy of NVP-BEZ235 in combination with MK2206 in inhibition of esophageal tumor development in vivo. (Supported by NIH/NCI R01 CA131073-01A1). Citation Format: Hao Yu, Ni Shi, Zui Pan, Tong Chen. Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1253. doi:10.1158/1538-7445.AM2014-1253

Preliminary study on the biological functions of interferon-λ in human esophageal carcinoma cells

Objective To investigate the biological function of IFN-λ in 7 human esophageal carcinoma cells. Methods The gene expression of IL-28α, IL-10β and antiviral molecule was examined with PCR. The MHC molecules expression and the profiles of cell cycle were analyzed with flow cytometer. Cell proliferation was evaluated with MTT assay. Results All of esophageal carcinoma cells express the gene of II-28α and IL-10β. IFN-λ induced or augmented the gene expression of antiviral molecules, 2′5′-OAS and MxA. IFN-λ enhanced the MHC class Ⅰ molecule expression. IFN-λ inhibited the growth of esophageal carcinoma cells through the regulation of cell cycle distribution. Conclusion Esophageal carcinoma cells express the IFN-λ receptor complex. IFN-λ has the antiviral, anti-proliferative and immunoregulation activity. Key words: IFN-λ; Antiviral activity ; Antiproliferative activity; Immunoregulation

Inhibition of α‐mannosidase Man2c1 gene expression suppresses growth of esophageal carcinoma cells through mitotic arrest and apoptosis

To study the effects of suppressed alpha-mannosidase Man2c1 gene expression on EC9706 human esophageal carcinoma cells, the cells were treated with short interfering RNA. Growth inhibition of EC9706 cells was observed when Man2c1 expression was inhibited in this way. Flow cytometric analysis showed accumulation of cells in S and G(2)-M phases, as well as cell apoptosis. The mitotic index test showed cell-cycle arrest at the M checkpoint. Although the percentage of cells in (pro)metaphase increased, the proportion of cells in anaphase and telophase decreased. Apoptosis was trigged by mitotic arrest. Furthermore, microtubules in EC9607 cells were examined by means of fluorescence staining of alpha-tubulin. Although control cells showed a nest-like microtubule network, the microtubule network in experimental cells was vague and condensed at the perinuclear region. Some cells with Man2c1 suppression had large protrusions of cytoplasm, some of which linked with the main body through a long, thin connection. Western blotting showed that tubulin polymerization was inhibited. The data imply that induction of mitotic arrest and consequent apoptosis resulted from microtubule disorganization, which appears to be one of the major cellular mechanisms by which suppressed expression of the Man2c1 gene causes growth inhibition of EC9706 esophageal carcinoma cells. In addition, Man2c1 suppression results in upregulation of E-cadherin, alpha-catenin, and beta-catenin expression in cells.

Downregulation of survivin by RNAi inhibits the growth of esophageal carcinoma cells

Esophageal squamous cell carcinoma ranks among one of the most frequent cause of cancer death in the world. Understanding of the molecular mechanisms involved in the pathogenesis of esophageal cancer becomes critical to develop more effective treatments. Elevated expression of survivin in esophageal carcinoma has been reported before and suppression of survivin expression leads to many tumor cells growth inhibition. We hypothesized that down-regulation of survivin would inhibit the growth of human esophageal cancer cells. RNA interference directed against survivin was introduced into a human esophageal squamous cell carcinoma cell line KYSE510. Stable clones were selected and western blot analysis was performed to detect the protein level of survivin. Tumor cell growth in vitro and in vivo was assessed by trypan blue exclusion and nude mice experiments. Annexin ¢?/propidium iodide staining followed by flow cytometric analysis and TUNEL assay were used to detect apoptosis in cell culture and in nude mice. We found that RNA interference could efficiently and stably suppress survivin expression in KYSE510 cells. Down-regulation of survivin resulted in significantly inhibition of tumor growth in vitro and in vivo. The mechanism appears to be increased induction of apoptosis. Our results suggest a potential role for the targeting of survivin in the treatments of esophageal carcinoma.

Inhibitory effects of extracellular adenosine triphosphate on growth of esophageal carcinoma cells

To study the growth inhibitory effects of ATP on TE-13 human squamous esophageal carcinoma cells in vitro. MTT assay was used to determine the inhibition of proliferation of ATP or adenosine (ADO) on TE-13 cell line. The morphological changes of TE-13 cells induced by ATP or ADO were observed under fluorescence light microscope by acridine orange (AO)/ethidium bromide (EB) double stained cells. The internucleosomal fragmentation of genomic DNA was detected by agarose gel electrophoresis. The apoptotic rate and cell cycle after treatment with ATP or ADO were determined by flow cytometry. ATP and ADO produced inhibitory effects on TE-13 cells at the concentration between 0.01 and 1.0 mmol/L. The IC(50) of TE-13 cells exposed to ATP or ADO for 48 and 72 h was 0.71 or 1.05, and 0.21 or 0.19 mmol/L, respectively. The distribution of cell cycle phase and proliferation index (PI) value of TE-13 cells changed, when being exposed to ATP or ADO at the concentrations of 0.01, 0.1, and 1 mmol/L for 48 h. ATP and ADO inhibited the cell proliferation by changing the distribution of cell cycle phase via either G(0)/G(1) phase (ATP or ADO, 1 mmol/L) or S phase (ATP, 0.1 mmol/L) arrest. Under light microscope, the tumor cells exposed to 0.3 mmol/L ATP or ADO displayed morphological changes of apoptosis. A ladder-like pattern of DNA fragmentation was obtained from TE-13 cells treated with 0.1-1 mmol/L ATP or ADO in agarose gel electrophoresis. ATP and ADO induced apoptosis of TE-13 cells in a dose-dependent manner at the concentration between 0.03 and 1 mmol/L. The maximum apoptotic rate of TE-13 cells exposed to ATP or ADO for 48 h was 16.63% or 16.9%, respectively. ATP and ADO inhibit cell proliferation, arrest cell cycle, and induce apoptosis of TE-13 cell line.

Down-regulation of beta catenin inhibits the growth of esophageal carcinoma cells

Introduction Esophageal cancer remains a highly lethal malignancy, with therapeutic options of limited efficacy in the majority of patients. Understanding the molecular events involved in the pathogenesis of esophageal cancer offers insight into potential targets for treatment. Beta catenin and Wnt signaling abnormalities are involved in the development of both adenocarcinoma and squamous carcinoma of the esophagus. We hypothesized that down-regulation of beta catenin would inhibit the growth of human esophageal cancer. Methods A human esophageal squamous cell carcinoma cell line (TE10) was treated with phosphorothioate antisense oligonucleotides to beta catenin. The cells were subsequently assayed for beta catenin mRNA and protein by real-time polymerase chain reaction and Western blot. Beta catenin transcriptional activity was determined by TOPFlash assay. Cell viability and growth was assessed by methyl-thiazol-diphenyl-tetrazolium assay and trypan blue exclusion. A colorimetric assay was employed to assess caspase 3 activity, and flow cytometry was done to determine percentage of cells in a given phase of the cell cycle. Results Following antisense treatment, beta catenin mRNA and protein concentration were decreased. There was corresponding decrease in beta catenin–transcription factor–dependent transcription. Treatment with beta catenin antisense resulted in significantly decreased cell viability and proliferation. The mechanism appears to be increased induction of apoptosis. Conclusions These data suggest a potential role for the targeting of beta catenin in the treatment of esophageal cancer.

Inhibition of adenovirus-mediated p27kip1 gene on growth of esophageal carcinoma cell strain.

To investigate the inhibition of p27kip1 gene on the growth of esophageal carcinoma cell strain (EC9706). Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effect on p27kip1 expression, the growth of esophageal carcinoma cell, DNA replication, protein synthesis, cell multiplication and apoptosis were explored by means of cell growth count, 3H-TdR, 3H-Leucine incorporation, flow cytometry, DNA fragment analysis and TUNEL. Recombinant adenovirus Ad-p27kip1 was successfully constructed with a virus titer of 1.24 X 10(12) pfu/ml. p27kip protein expression increased markedly after EC-9706 transfection, while incorporation quantity of 3H-TdR and 3H-Leucine decreased significantly. The growth of esophageal carcinoma cell was inhibited obviously. Testing of flow cytometry displayed a typical apoptosis peak, and DNA gel electrophoresis showed a typical apoptosis ladder. TUNEL showed the apoptosis rate of Ad-p27kip1 group and control group to be 37.3% and 1.26% (P<0.001) respectively. Ad-p27kip1 can inhibit the growth and multiplication of esophageal carcinoma cells and induce apoptosis. Therefore, enhanced p27kip1 expression may be a new way to treat esophageal carcinoma.