Apoptosis Of Eca109 Cells Research Articles

Study on Optimization of Liquid Fermentation Medium and Antitumor Activity of the Mycelium on Phyllopora lonicerae.

Phylloporia lonicerae is an annual fungus that specifically parasitizes living Lonicera plants, offering significant potential for developing new resource food and medicine. However, wild resources and mycelium production of this fungus is limited, and its anti-tumor active ingredients and mechanisms remain unclear, hampering the development of this fungus. Thus, we optimized the fermentation medium of P. lonicerae and studied the anti-tumor activity of its mycelium. The results indicated that the optimum fermentation medium consisted of 2% sucrose, 0.2% peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.16% Lonicera japonica petals, 0.18% P fungal elicitor, and 0.21% L. japonica stem. The biomass reached 7.82 ± 0.41 g/l after 15 days of cultivation in the optimized medium, a 142% increase compared with the potato dextrose broth medium, with a 64% reduction in cultivation time. The intracellular alcohol extract had a higher inhibitory effect on A549 and Eca-109 cells than the intracellular water extract, with half-maximal inhibitory concentration values of 2.42 and 2.92 mg/ml, respectively. Graded extraction of the alcohol extract yielded petroleum ether phase, chloroform phase, ethyl acetate phase, and n-butanol phase. Among them, the petroleum ether phase exhibited a better effect than the positive control, with a half-maximal inhibitory concentration of 113.3 μg/ml. Flow cytometry analysis indicated that petroleum ether components could induce apoptosis of Eca-109 cells, suggesting that this extracted component can be utilized as an anticancer agent in functional foods. This study offers valuable technical support and a theoretical foundation for promoting the comprehensive development and efficient utilization of P. lonicerae.

Erianin Impedes the Proliferation and Metastatic Migration Through Suppression of STAT-3 Phosphorylation in Human Esophageal Cancer Cells.

In this study, we have investigated erianin, a natural phenolic drug that impedes proliferation and metastatic migration through suppression of STAT-3 phosphorylation in human esophageal cancer cells. Eca-109 cells were treated with different concentrations of erianin (4, 8, 12 µM) for 24h, and then cell proliferation, apoptosis, and metastatic markers were evaluated. Erianin-induced cytotoxicity and cell proliferation were examined using MTT and crystal violet staining techniques. The measurement of reactive oxygen species (ROS) and the study of apoptotic changes were conducted through flow cytometry. Furthermore, protein expression analyses via western blotting included an evaluation of JAK-STAT3, cell survival, cell cycle, proliferation, and apoptosis-related proteins. Moreover, erianin treatment-associated MMP expressions were studied by RT-PCR. In this study, erianin treatment induces substantial cytotoxicity and ROS production based on the concentrations in Eca-109 cells. Moreover, erianin inhibits the MAPK phosphorylation, proliferation, and metastatic protein in Eca-109 cells. STAT-3 is a crucial transcriptional factor that regulates numerous downstream proteins, such as proliferation, anti-apoptosis, and metastatic proteins. In this study, erianin treatment inhibited the protein expression of IL-6, IL-10, JAK-1, and p-STAT-3 expressions leading to induce apoptosis in Eca-109 cells. Moreover, erianin inhibited the expression of proliferation, metastatic, and anti-apoptotic markers in Eca-109 cells. Hence, erianin suppressed the JAK/STAT-3 signaling pathway and demonstrates potential as a chemotherapeutic agent for the treatment of esophageal cancer.

MiR-302a-3p reduces cisplatin resistance of esophageal squamous cell carcinoma cells by targeting EphA2

Platinum-based chemotherapy is a common clinical treatment for esophageal squamous cell carcinoma (ESCC), and chemoresistance is a major leading reason for cancer treatment failure. MiR-302a-3p is involved in the development of many diseases. Here, we investigated the role of miR-302a-3p in the cisplatin resistance of ESCC cells and explored its potential mechanism via molecular techniques. The expression of miR-302a-3p was significantly reduced, while the expressions of EphA2 were increased in ESCC tumor tissues and cells. EphA2 was one target gene of miR-302a-3p, and was negatively regulated by miR-302a-3p. By regulating EphA2, miR-302a-3p reduced the viability and promoted the apoptosis of ECA109 cells treated with cisplatin, suggesting that miR-302a-3p could enhance the sensitivity of ECA109 cells to cisplatin treatment by targeting EphA2. MiR-302a-3p plays an important role in reducing cisplatin resistance by inhibiting EphA2, suggesting that it may be a promising therapeutic strategy for cisplatin resistance in ESCC in the future.

Essential oil from Saussurea costus inhibits proliferation and migration of Eca109 cells via mitochondrial apoptosis and STAT3 signaling

Objective: To investigate the effect and its underlying molecular mechanisms of essential oil from Saussurea costus in esophageal cancer cell line Eca109. Methods: The chemical composition of essential oil from Saussurea costus was investigated by gas chromatography-mass spectrometry (GC-MS). The anti-proliferative, anti-migrative, and apoptotic effects of essential oil from Saussurea costus against Eca109 cells were analyzed. Moreover, the expression of proteins associated with cell cycle, metastasis, and apoptosis was determined. Results: GC-MS analysis showed that essential oil from Saussurea costus was predominantly comprised of sesquiterpenes. Saussurea costus essential oil inhibited the viability of Eca109 cells in a dose-and time-dependent manner with IC50 values of (24.29±1.49), (19.16±2.27) and (6.97±0.86) μg/mL at 12, 24, and 48 h, respectively. The expression levels of target proteins in the cell cycle (phase G1/S), including cyclin D1, p21, and p53, were affected by Saussurea costus essential oil. The essential oil also downregulated the expression of metastasis-related proteins MMP-9 and MMP-2. Moreover, it induced apoptosis of Eca109 cells through the mitochondrial pathway, as well as inhibition of STAT3 phosphorylation. Conclusions: The essential oil from Saussurea costus exhibited anti-proliferative, anti-migrative, and apoptotic effects on Eca109 cells, and could be further explored as a potential anti-esophageal cancer agent.

Sparfloxacin - Cu(II) - aromatic heterocyclic complexes: synthesis, characterization and in vitro anticancer evaluation.

Two new copper(II) complexes of sparfloxacin (sf), [Cu(Hsf)(HPB)(H2O)](ClO4)2 (1) and [Cu(Hsf)(PBT)(H2O)](ClO4)2 (2) (where HPB = 2-(2'-pyridyl)benzimidazole and PBT = 2-(4'-pyridyl) benzothiazole), have been synthesized and characterized by physicochemical and spectroscopic techniques. The oil-water partition coefficient (log P) values of complexes 1 and 2 were 1.47 and 1.71, respectively. By studying the interaction between the complexes and DNA, it was found that the complexes could bind to DNA through an intercalation mode. Moreover, both complexes were evaluated for antitumor activity, revealing that the complexes displayed good inhibitory activity toward the tested cancer cell lines (human lung carcinoma A549 cells, human hepatocellular carcinoma Bel-7402 cells and human esophageal carcinoma Eca-109 cells), but showed relatively low toxicity against normal human hepatic LO2 cells. In particular, the antitumor mechanism of the complexes on Eca-109 cells was investigated by morphological analysis, apoptosis analysis and determination of cell cycle arrest, mitochondrial membrane potential, reactive oxygen species (ROS) levels, and release of cytochrome c and Ca2+. The results demonstrated that the complexes could induce loss of intracellular mitochondrial functions and increase of ROS levels, which led to an increase of Ca2+ levels and the release of cytochrome c into the cytoplasm. In addition, the cell cycle was arrested in the G2/M phase, and western blot analysis showed that the caspase family was activated. These results fully proved that the complexes could induce apoptosis through DNA damage and loss of mitochondrial functions, accompanied by the regulation of endogenous proteins.

MTOR inhibitor PP242 increases antitumor activity of sulforaphane by blocking Akt/mTOR pathway in esophageal squamous cell carcinoma.

Sulforaphane (SFN) is a kind of isothiocyanate from cruciferous vegetables with extensive anti-tumor activity. Esophageal squamous cell carcinoma (ESCC) is a popular malignancy in East Asia, East and South Africa, while the more efficient medicines and therapeutic strategies are still lack. This study aims to explore the anti-tumor activity of SFN alone and combined with Akt/mTOR pathway inhibitors as well as the potential molecular mechanism in ESCC. Cell proliferation, migration, cell cycle phase, apoptosis and protein expression were detected with MTT assay, clone formation experiment, wound healing assays, flow cytometry and Western blot, respectively, after ESCC cells ECa109 and EC9706 treated with SFN alone or combined with Akt/mTOR inhibitors. Xenograft models were used to evaluate the efficiency and mechanism of SFN combined with PP242 in vivo. The results showed that SFN significantly inhibited the viability and induced apoptosis of ECa109 and EC9706 cells by increasing expression of Cleaved-caspase 9. SFN combined with PP242, but not MK2206 and RAD001, synergetic inhibited proliferation of ESCC cells. Moreover, compared to SFN alone, combination of SFN and PP242 had stronger inhibiting efficiency on clone formation, cell migratory, cell cycle phase and growth of xenografts, as well as the more powerful apoptosis-inducing effects on ESCC. The mechanism was that PP242 abrogated the promoting effects of SFN on p-p70S6K (Thr389) and p-Akt (Ser473) in ESCC. Our findings demonstrate that PP242 enhances the anti-tumor activity of SFN by blocking SFN-induced activation of Akt/mTOR pathway in ESCC, which provides a rationale for treating ESCC using SFN combined with Akt/mTOR pathway inhibitors.

Downregulation of miR-135b-5p Suppresses Progression of Esophageal Cancer and Contributes to the Effect of Cisplatin.

Esophageal cancer (EC) is one of the commonest human cancers, which accompany high morbidity. MicroRNAs (miRNAs) play a pivotal role in various cancers, including EC. Our research aimed to reveal the function and mechanism of miR-135b-5p. Our research identified that miR-135b-5p was elevated in EC samples from TCGA database. Correspondingly real-time PCR assay also showed the miR-135b-5p is also higher expressed in Eca109, EC9706, KYSE150 cells than normal esophageal epithelial cells (Het-1A). CCK8, Edu, wound healing, Transwell assay, and western blot demonstrated miR-135b-5p inhibition suppresses proliferation, invasion, migration and promoted the apoptosis in Eca109 and EC9706 cells. Moreover, the miR-135b-5p inhibition also inhibited xenograft lump growth. We then predicted the complementary gene of miR-135b-5p using miRTarBase, TargetScan, and DIANA-microT. TXNIP was estimated as a complementary gene for miR-135b-5p. Luciferase report assay verified the direct binding site for miR-135b-5p and TXNIP. Real-time PCR and western blot assays showed that the inhibition of miR-135b-5p remarkably enhanced the levels of TXNIP in Eca109 and EC9706 cells. Furthermore, cisplatin (cis-diamminedichloroplatinum II, DDP) decreased miR-135b-5p expression and increased TXNIP expression. Enhanced expression of miR-135b-5p attenuated the inhibitory ability of cisplatin (cis-diamminedichloroplatinum II, DDP) in Eca109 cells, accompanied by TXNIP downregulation. In conclusion, the downregulation of miR-135b-5p suppresses the progression of EC through targeting TXNIP. MiR-135b-5p/TXNIP pathway contributes to the anti-tumor effect of DDP. These findings may provide new insight into the treatment of EC.

CALM1 promotes progression and dampens chemosensitivity to EGFR inhibitor in esophageal squamous cell carcinoma

BackgroundCalmodulin1 (CALM1) has been identified as one of the overexpression genes in a variety of cancers and EGFR inhibitor have been widely used in clinical treatment but it is unknown whether CALM1 and epidermal growth factor receptor (EGFR) have a synergistic effect in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to explore the synergistic effects of knock-out CALM1 combined with EGFR inhibitor (Afatinib) and to elucidate the role of CALM1 in sensitizing the resistance to Afatinib in ESCC.MethodImmunohistochemistry (IHC) and qRT-PCR were used to examine the expression of CALM1 and EGFR in ESCC tissues. Kaplan–Meier survival analysis was used to analyze the clinical and prognostic significance of CALM1 and EGFR expression in ESCC. Furthermore, to evaluate the biological function of CALM1 in ESCC, the latest gene editing technique CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats)was applied to knockout CALM1 in ESCC cell lines KYSE150, Eca109 and TE-1. MTT, flow cytometry, Transwell migration, scratch wound-healing and colony formation assays were performed to assay the combined effect of knock-out CALM1 and EGFR inhibitor on ESCC cell proliferation and migration. In addition, nude mice xenograft model was used to observe the synergistic inhibition of knock-out CALM1 and Afatinib.ResultsBoth CALM1 and EGFR were found to be significantly over-expressed in ESCC compared with paired normal control. Over-expressed CALM1 and EGFR were significantly associated with clinical stage, T classification and poor overall prognosis, respectively. In vitro, the combined effect of knock-out CALM1 mediated by the lentivirus and EGFR inhibitor was shown to be capable of inhibiting the proliferation, inducing cell cycle arrest at G1/S stage and increasing apoptosis of KYSE-150 and Eca109 cells; invasion and migration were also suppressed. In vivo, the results of tumor weight and total fluorescence were markedly reduced compared with the sgCtrl-infected group and sgCAML1 group.ConclusionOur data demonstrated that knock-out of CALM1 could sensitize ESCC cells to EGFR inhibitor, and it may exert oncogenic role via promotion of EMT. Taken together, CALM1 may be a tempting target to overcome Afatinib resistance.

Dexmedetomidine represses proliferation and promotes apoptosis of esophageal cancer cells by regulating C-Myc gene expression via the ERK signaling pathway.

The aim of this study was to investigate the effects of dexmedetomidine (DEX) on proliferation and apoptosis of esophageal cancer (EC) cells, and to explore the possible underlying mechanism. EC cells (Eca109) were randomly divided into two groups, namely, Control group and DEX group. The viability, proliferation, and apoptosis of Eca109 cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-Ethynyl-2'-deoxyuridine (EdU) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. Meanwhile, the messenger ribonucleic acid (mRNA) and protein expression levels of extracellular signal-regulated kinase (ERK) 1/2 and c-Myc in Eca109 cells were measured by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting, respectively. The viability of Eca109 cells was remarkably weakened in DEX group when compared with Control group (p<0.05). DEX could significantly inhibit the proliferation and promote the apoptosis of Eca109 cells (p<0.05). Moreover, the mRNA and protein levels of ERK1/2 and c-Myc in Eca109 cells declined notably (p<0.05). DEX represses the proliferation and facilitates the apoptosis of Eca109 cells prominently. The possible underlying mechanism may be associated with the inhibition of c-Myc gene expression through the ERK signaling pathway.

MiR-495 Inhibits Cisplatin Resistance and Angiogenesis in Esophageal Cancer by Targeting ATP7A.

Background: Cancer resistance to chemotherapy is closely associated with changes in transporter systems. In this study, we investigated the possible regulation of 1 copper ion transporter (ATP7A; ATPase copper transporting alpha) by microRNA miR-495 and its implications in cisplatin resistance and angiogenesis in esophageal cancer. Methods: MiR-495 and ATP7A mRNA expression in clinical tissue samples and 2 cancer cell lines (Eca-109 and TE1) were detected by quantitative real-time polymerase chain reaction. The levels of miR-495 and ATP7A expression in Eca-109 and TE1 cells were increased by transfection with miR-495 mimics and ATP7A-overexpression vectors. Cell proliferation, apoptosis, and angiogenesis were assessed by CCK-8, flow cytometry, and tube formation assays, respectively. The levels of TNF-α and VEGF in cell culture supernatants were detected by enzyme linked immunosorbent assay, and in situ expression of NLRP3 was measured by immunofluorescence. The binding of miR-495 to ATP7A sequences was verified by dual luciferase reporter assays. Results:ATP7A expression was significantly increased, while miR-495 expression was decreased in the cancer tissues of esophageal cancer patients. MiR-495 mimics decreased the proliferation and promoted the apoptosis of cisplatin-resistant Eca-109 and TE1 cells. Furthermore, tube formation by human umbilical vein endothelial cells, TNF-α and VEGF secretion, and the levels of MRP1, ABCG1, ABCA1, and NLRP3 expression in cisplatin-resistant Eca-109 and TE1 cells were all reduced by miR-495 mimics. MiR-495 was shown to directly bind to ATP7A gene sequences to repress ATP7A expression in Eca-109 and TE1 cells. ATP7A overexpression substantially abrogated the changes in proliferation, apoptosis, angiogenesis, and above-mentioned gene expression in cisplatin-resistant Eca-109 and TE1 cells. Conclusions: MiR-495 suppressed cisplatin resistance and angiogenesis in esophageal cancer cells by targeting ATP7A gene expression.

Inhibitory effects of Bombyx mori antimicrobial peptide cecropins on esophageal cancer cells

Bombyx mori antimicrobial peptides (BmAMPs) are important effectors in silkworm immune system. They can inhibit and kill a variety of bacteria and fungi. Recent studies have shown that some kinds of BmAMPs exert strong inhibitory effects on a variety of tumor cells. In the present study, the antitumor activity of BmAMP Cecropin A (BmCecA) and BmAMP Cecropin D (BmCecD) was investigated against human esophageal cancer cells and their antitumor mechanism preliminary explored. Cell Counting Kit-8 and colony formation assays indicated that BmCecA and BmCecD suppressed cell proliferation and reduced colony formation of both Eca109 and TE13 cells in a dose-dependent manner, but exhibited no inhibitory effect on normal human embryonic kidney 293T cells. Wound healing and invasion experiments indicated that both BmCecA and BmCecD inhibited migration and invasion of Eca109 and TE13 cells in vitro. Annexin V/propidium iodide staining and flow cytometry detection suggested that BmCecA induced the apoptosis of Eca109 cells in a dose-dependent manner. RT-qPCR and western blot analysis showed that BmCecA induced apoptosis of Eca109 cells through the activation of a mitochondria-mediated caspase pathway, the upregulation of B-cell lymphoma 2 (Bcl-2)-associated X protein and the downregulation of Bcl-2. In addition, BmCecA significantly inhibited the growth of xenograft tumors in Eca109-bearing mice. These results suggested that BmCecA and BmCecD might serve as potential therapeutic agents for the treatment of cancer in the future.

Limonoid compounds from Xylocarpus granatum and their anticancer activity against esophageal cancer cells

BackgroundTo investigate the anticancer effects of limonoid compounds that were isolated and purified from Xylocarpus granatum fruits on human esophageal cancer (EC) cells. A structure‐activity relationship experiment was designed to identify the functional moiety of limonoid compounds identified as being critical for its anticancer activity.MethodsEca109 cells were cultured in RPMI1640 medium and treated with limonoid compounds. Cell proliferation was determined by the MTT assay in vitro. Eca109 cells apoptosis was analyzed by by flow cytometry after being treated with xylogranatin C. The expression of p53, Bax, bcl‐2, caspase‐3 and GRP78 in Eca109 cells after xylogranatin C treatment was examined by western blot assay.ResultsFour linonoid compounds strongly inhibited the cellular proliferation of Eca109 cells. Xylogranatin C was the strongest inhibitor, whose inhibitory effect was comparable to that of the well‐known chemotherapeutic agent, cisplatin. Furthermore, xylogranatin C might induce Eca109 cell apoptosis through joint effects on multiple pathways, including the death receptor and endoplasmic reticulum pathways. Additionally, xylogranatin C suppressed tumor cell proliferation by upregulating miR‐203a expression in Eca109 cells.ConclusionsXylogranatin C induced Eca109 cellular apoptosis and exerted antitumor activity. Xylogranatin C suppressed tumor cell proliferation by upregulating miR‐203a expression in Eca109 cells.

Limonoid compounds from Xylocarpus granatum and their anti-cancer activity against esophageal cancer cells.

e16516 Background: We aimed to study the anti-cancer effects of limonoid compounds that were isolated and purified from Xylocarpus granatum fruits on human esophageal cancer cells. A structure-activity relationship experiment was designed to identify the functional moiety of limonoid compounds identified as being critical for its anti-cancer activity. Methods: Esophageal cancer Eca109 cells were treated with limonoid compounds. Cell proliferation was determined by the MTT assay in vitro and cellular apoptosis by flow cytometry and Western Immunolotting. Results: Four linonoid compounds strongly inhibited the cellular proliferation of Eca109 cells. Xylogranatin C was the strongest inhibitor, whose inhibitory effect was comparable to that of the well-known chemotherapeutic agent, cisplatin. Furthermore, xylogranatin C might induce Eca109 cell apoptosis through joint effects on multiple pathways, including the death receptor and endoplasmic reticulum pathways. Additionally, xylogranatin C suppressed tumor cell proliferation by up-regulating miR-203a expression in Eca109 cells. Conclusions: Inhibitory effects of xylogranatin C were more potent than cisplatin. Xylogranatin C induced Eca109 cellular apoptosis and exerted anti-tumor activity. Xylogranatin C might have induced Eca109 cellular apoptosis via joint effects on multiple pathways, including the death receptor and mitochondria-dependent signaling pathways. Additionally, xylogranatin C suppressed tumor cell proliferation by up-regulating miR-203a expression in Eca109 cells.

Knockdown of MTA1 inhibits the proliferation and apoptosis of esophageal cancer cells

Objective: To investigate the effects of metastasis associated gene 1 (MTA1) expression on the proliferation and apoptosis of human esophageal cancer Eca109 cells. Methods: MTA1 siRNA was transfected into human esophageal cancer Eca109 cells, and the control group and blank group were set up. The expression of MTA1 in Eca109 cells with different treatment was detected by real-time fluorescent quantitative PCR (RT-PCR) and western blot. The proliferation of Eca109 cells was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The cloning formation ability of Eca109 cells was detected by plate cloning assay. The apoptosis of Eca109 cells were detected by flow cytometry. The expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related proteins, including cleaved caspase-3 and total caspase-3 protein in Eca109 cells were detected by western blot. Results: After 48 hours of transfection, RT-PCR result showed that the relative expression levels of MTA1 mRNA in Eca109 cells in the blank group, control group, and siRNA group were 1.00±0.10, 0.98±0.09 and 0.21±0.03, respectively. The expression of MTA1 mRNA in siRNA group was significantly inhibited (P<0.05), while no significant difference of MTA1 mRNA expression between the blank group and the control group has been found (P>0.05). Western blot results were consistent with those of RT-PCR. MTT array results showed that, compared with the blank group and transfection group, the absorbance values of Eca109 cells in siRNA group were dramatically reduced at 48, 72, and 96 h (all P<0.05). There were no significant differences of absorbance values between the blank group and the control group at 48, 72, and 96 h (all P>0.05). The results of the plate colony formation test showed that the number of colony formation in the blank group and control group were 58.64±6.86 and 60.02±7.04, respectively, significantly higher than 18.10±3.16 in siRNA group (P<0.05), while there was no significant difference between the blank group and control group (P>0.05). Flow cytometry results showed that the apoptosis rates in the blank group and control group were (2.13±0.54)% and (2.27±0.61)%, respectively, significantly lower than (32.61±5.28)% in siRNA group (P<0.05), while there was no significant difference between blank group and control group (P>0.05). Western blot results showed that the expression of PCNA protein was down-regulated while cleaved caspase-3 protein expression was upregulated in siRNA group, compared to the control group and blank group (P<0.05). Conclusions: Inhibition of MTA1 expression can inhibit the proliferation of Eca109 cells and induce apoptosis. This process may be related to the down-regulation of PCNA protein expression and activation of caspase-3 protein expression.

Downregulation of SPARC Expression Decreases Cell Migration and Invasion Involving Epithelial-Mesenchymal Transition through the p-FAK/p-ERK Pathway in Esophageal Squamous Cell Carcinoma.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) is an extracellular glycoprotein overexpressed in various malignancies, including esophageal squamous cell carcinoma (ESCC), and is involved in tumor development and progression. This study was initially designed to investigate the biological roles of SPARC in ESCC cell lines by silencing SPARC expression.Methods: The expression of SPARC was examined in eight human ESCC cell lines. Eca109 and HKESC cell lines with high SPARC expression were selected and transiently transfected with SPARC-targeted small interfering RNAs (siRNAs) and subsequently evaluated its impact on cell proliferation, migration and invasion in vitro, as well as the underlying mechanism.Results: Knockdown of SPARC by the specified siRNAs in Eca109 and HKESC cell lines resulted in dramatically downregulation of SPARC expression, and significantly decreased cell migration and invasion involving epithelial-mesenchymal transition (EMT) in vitro. Moreover, SPARC-targeted siRNA reduced the activation of phosphorylated focal adhesion kinase (p-FAK) and extracellular regulated protein kinase (p-ERK). Furthermore, downregulation of either FAK or SPARC expression with specified siRNAs inhibited the phosphorylation of ERK and inhibited cell migration and invasion. However, decreased SPARC expression showed no impact on cell proliferation, survival or apoptosis of Eca109 and HKESC cells when comparing to control transfected groups.Conclusions: These results demonstrated that downregulation of SPARC could decrease cell migration and invasion involving EMT via the p-FAK/p-ERK pathway that might serve as a novel therapeutic target against ESCC.

An alcohol-soluble polysaccharide from Atractylodes macrocephala Koidz induces apoptosis of Eca-109 cells

In this study, polysaccharides from Atractylodes macrocephala Koidz (APA) which were soluble in alcohol were prepared, purified, analyzed the structure and investigated the antitumor activity in vitro cell experiment. Results of high-performance gel permeation chromatography (HPGPC), fourier-transform infrared spectroscopy (FT-IR), and gas chromatography (GC) showed that APA was a 2.1KDa neutral hetero polysaccharide composed of arabinose and glucose (molar ratio, 1.00:4.57) with pyranose rings and α-type and β-type glycosidic linkages. Results by MTT experiments showed that the proliferation inhibition was 74.63% in Eca109 cells treated with 2 mg/mL dose of APA. Annexin V/PI assay, Hoechst 33,258 staining, cell cycle distribution, rhodamine 123 dye assay and western blot assay clarified that APA could accelerate the apoptosis of Eca109 cells by mitochondrial pathway and stocked cells at S phase. These data indicated that APA is a promising potential candidate for therapeutic treatment of esophageal cancer.

Downregulation of MicroRNA-135b-5p Inhibits Esophageal Cancer Cell Progression via Targeting GNG7

Objective: MicroRNAs (miRNAs) have emerged as the critical modulators of the tumorigenesis and tumor progression. Guanine nucleotide binding (G) protein gamma 7 (GNG7) has been shown to be closely associated with the occurrence of tumors. We aimed to investigate the effects of microRNA-135b-5p (miR-135b-5p) mediated GNG7 knockdown on the biological behavior of esophageal carcinoma (EC) cells. Material and Methods: The expression of miR-135b-5p and GNG7 in 33 cases of EC clinical tissues and EC cell lines, including TE-1, ECA109 and EC9706, were examined by PCR and western blotting assays to select the optimum cell line for subsequent experiments. After ECA109 cells were transfected with miR-135b-5p mimics or inhibitor, siRNA GNG7 and NC plasmids, the cell viability, migration invasion, proliferation and apoptosis of ECA109 cells were evaluated by MTT, wound healing, Transwell, colony formation and flow cytometry assays, respectively. The expression of relevant molecules (p53 and VEGF-A) was determined by western blot analysis. Results: The results indicated that the levels of miR-135b-5p were upregulated while GNG7 were downregulated in EC tissues in comparison with the corresponding normal tissues, and miR-135b-5p mimics and knockdown of GNG7 sharply increased cell viability, migration, invasion and colony formation of ECA109 cells, and inhibited cell apoptosis. The luciferase reporter assay identified that miR-135b-5p decreased the level of GNG7 via binding to the 3′-UTR of GNG7 gene. In addition, p53 expression were decreased and VEGFA expression was up-regulated in ECA109 cells after transfected with miR-135b-5p mimics and GNG7 siRNA. However, inhibition of miR-135b-5p suppressed biological behavior abilities and promoted apoptosis of ECA109 cells that was promoted by miR-135b-5p mimics and siRNA GNG7. Conclusions: In conclusion, miR-135b-5p is identified as a potential tumor-promoting miRNA likely through the regulation of GNG7 expression, which could be developed as a new therapeutic target for EC.

Long non-coding RNA colon cancer-associated transcript 2 may promote esophageal cancer growth and metastasis by regulating the Wnt signaling pathway.

The aim of this study was to investigate how long non-coding (lnc)RNA colon cancer-associated transcript 2 (CCAT2) regulates the proliferation, invasion and metastasis of esophageal cancer cells via the Wnt signaling pathway. The expression of lncRNA CCAT2 was quantified by reverse transcription-quantitative PCR in four esophageal cancer cell lines (Eca-109, EC9706, KYSE150 and TE-1) and normal human esophageal epithelial cells (HEECs). The effect of silencing CCAT2 (si-CCAT2) and inhibiting Wnt signaling (using the inhibitor FH535) on the proliferation, migration and invasion of Eca-109 cells was measured by MTT, wound-healing and Transwell invasion assays. Flow cytometry was used to evaluate apoptosis in si-CCAT2 Eca-109 cells. The expression of β-catenin and proliferating cell nuclear antigen (PCNA) proteins was detected by immunohistochemistry. The pro-apoptotic protein Bax, cyclin D1 and Wnt target proteins, including c-Myc and adenomatous polyposis coli (APC), were detected by western blotting. LncRNA CCAT2 was highly expressed in the four esophageal cancer cell lines compared with the HEEC cells. The expression of CCAT2 was significantly decreased in si-CCAT2 Eca-109 cells. Treatment with si-CCAT2 and FH535 alone or in combination significantly inhibited the proliferation, migration and invasion of Eca-109 cells. The treatments also promoted apoptosis, upregulated the expression of Bax and APC proteins, and downregulated β-catenin, PCNA, cyclin D1 and c-Myc proteins. In summary, lncRNA CCAT2 is upregulated in esophageal cancer cells and the knockdown of lncRNA CCAT2 inhibits their proliferation, migration and invasion via the Wnt signaling pathway.

PSMD4 regulates the malignancy of esophageal cancer cells by suppressing endoplasmic reticulum stress.

Proteasome 26S subunit non-ATPase 4 (PSMD4) is an important proteasome ubiquitin receptor and plays a key role in endoplasmic reticulum stress (ERS). However, the study of PSMD4 in esophageal cancer (EC) is relatively rare. Here, we found that the expression of PSMD4 was markedly enhanced in EC tissues and cell lines. The cell counting kit-8 (CCK-8) assay showed that overexpression of PSMD4 significantly enhanced Eca109 cell viability, while inhibition of PSMD4 reduced Eca109 cell viability. Knockdown of PSMD4 induced Eca109 cell apoptosis and cell cycle arrest. More importantly, knockdown of PSMD4 significantly enhanced the expression of glucose regulated protein 78, activating transcription factor 6, and p-protein kinase R-like ER kinase, indicating an enhanced ERS response in esophageal cancer cells. Compared with the control cells, brefeldin A significantly inhibited the expression of PSMD4 and increased the expression of p53-upregulated modulator of apoptosis. However, such effects were largely reversed after overexpressing PSMD4 in Eca109 cells, suggesting that silencing PSMD4 could enhance ERS-induced cell apoptosis. In summary, upregulation of PSMD4 promoted the progression of esophageal cancer mainly by reducing ERS-induced cell apoptosis.

Influence of luteolin on the apoptosis of esophageal cancer Eca109 cells and its mechanism of action

The present study was conducted to verify the influence of luteolin on apoptosis of Eca109 cells and to further investigate the possible mechanisms underlying its effect on apoptosis. The cells were exposed to different concentrations of luteolin (0, 40, 80, 120, 160, 200, 240μM) for 24, 48, and 72h respectively. The influence of luteolin on proliferation of Eca109 cells was detected using MTT assay. Eca109 cells were then treated with luteolin (0, 40, 160, 240μM) for 24h. The effect of luteolin on cell cycle progression and apoptosis was assayed by using flow cytometry (FCM). Expression of caspase9 and caspase3 mRNA and protein was analyzed by real-time PCR and Western blot respectively. The results showed that luteolin could inhibit the proliferation of Eca109 cells at all concentrations in a time-dependent manner and the relative inhibition rate showed an inverted U-shaped association with the concentration of luteolin. Further, the cell cycle was arrested in the S phase following treatment with luteolin. Apoptosis analysis indicated that luteolin could induce the apoptosis of Eca109 cells across the three concentration groups, which exhibited a trend of first promotional and then inhibitory with the increases in luteolin concentration. The effect of luteolin on the mRNA and protein expression of caspase 9 and caspase3 first manifested as promotion, then inhibition. Therefore, luteolin may serve a role in promoting cell apoptosis by inducing Eca109 cell apoptosis that involves the expression of caspase3, caspase9 mRNA and protein. This study provides theoretical basis for further study and clinical application of luteolin. The specific mechanism has not yet been clarified and the other activation pathways inducing apoptosis need to be further studied.