EC9706 Cells Research Articles

Trichostatin A promotes esophageal squamous cell carcinoma cell migration and EMT through BRD4/ERK1/2-dependent pathway.

BackgroundHistone deacetylases (HDACs) have been demonstrated to be aberrantly activated in tumorigenesis and cancer development. Thus, HDAC inhibitors (HDACIs) are considered to be promising anti‐cancer therapeutics. However, recent studies have shown that HDACIs promote the migration of many cancer cells. Therefore, there is a need to elucidate the underlying mechanisms of HDACIs on cancer cell migration to establish a combination therapy that overcomes HDACI‐induced cell migration.MethodsKYSE‐150 and EC9706 cells were treated differently. Effects of drugs and siRNA treatment on tumor cell migration and cell signaling pathways were investigated by transwell migration assy. Gene expression for SNAI2 was tested by RT‐qPCR. Western blot analysis was employed to detect the level of E‐cadherin, β‐catenin, vimentin,Slug，ERK1/2, H3, PAI‐1 and BRD4. The effect of drugs on cell morphology was evaluated through phase‐contrast microscopic images.ResultsTSA promotes epithelial‐mesenchymal transition (EMT) in ESCC cells by downregulating the epithelial marker E‐cadherin and upregulating mesenchymal markers β‐catenin, vimentin, Slug, and PAI‐1. Knockdown of Slug by siRNA or inhibition of PAI‐1 clearly suppressed TSA‐induced ESCC cell migration and resulted in the reversal of TSA‐triggered E‐cadherin, β‐catenin, and vimentin expression. However, no crosstalk between Slug and PAI‐1 was observed in TSA‐treated ESCC cells. Blocking ERK1/2 activation also inhibited TSA‐induced ESCC cell migration, EMT, and upregulation of Slug and PAI‐1 levels in ESCC cells. Interestingly, inhibition of BRD4 suppressed TSA‐induced ESCC cell migration and attenuated TSA‐induced ERK1/2 activation and upregulation of Slug and PAI‐1 levels.ConclusionsOur data indicate the existence of at least two separable ERK1/2‐dependent signaling pathways in TSA‐mediated ESCC cell migration: an ERK1/2–Slug branch and an ERK1/2‐PAI‐1 branch. Both branches of TSA‐induced ESCC cell migration appear to favor the EMT process, while BRD4 is responsible for two separable ERK1/2‐dependent signaling pathways in TSA‐mediated ESCC cell migration.

HPV16 infection promotes an M2 macrophage phenotype to promote the invasion and metastasis of esophageal squamous cell carcinoma.

High-risk human papillomavirus (HR-HPV) is an important risk factor for esophageal cancer. Macrophages constitute a crucial immune medium for regulating HPV-related tumors; however, the specific regulatory mechanisms remain unknown. Therefore, the purpose of our current study was to investigate the mechanism by which HPV16E6 regulates macrophages to promote the invasion and metastasis of esophageal cancer. HPV16E6 infection was detected by polymerase chain reaction. Immunohistochemistry was used to verify the distribution of tumor-associated macrophages (TAMs) and MMP-9 expression in esophageal squamous cell carcinoma tissues (ESCCs), and cancer adjacent normal tissues (CANs) from Kazakh patients. ESCC cells were transfected with a plasmid over-expressing HPV16E6 and non-contact cocultured with macrophages. The infection rate of HPV16E6 in Kazakh ESCCs was clearly higher than that in CANs (P < 0.05). The density of CD163-positive TAMs was significantly positively correlated with HPV16E6 infection in ESCCs (P < 0.05). After coculturing macrophages and EC9706 cells transfected with the HPV16E6 plasmid, the phenotype of macrophages transformed into M2 macrophages. The migration and invasion ability of ESCC cells were higher in the HPV16E6-transfected and coculture group than in the HPV16E6 empty vector-transfected and non-cocultured HPV16E6-transfected groups (all P < 0.05). The density of M2-like TAMs in ESCCs was positively correlated with the level of MMP-9 expression. MMP-9 expression in the HPV16E6-ESCC coculture macrophages group was substantially higher than that in controls (all P < 0.05). HPV16 infection mediates tumor-associated macrophages to promote ESCC invasion and migration.

Effects of Qingre Huatan Huoxue prescription on biological characteristics and cell cycle proteins of esophageal cancer stem cells.

e16052 Background: Esophageal cancer (EC) stem cells characterized with immature differentiation, high invasion, high tumorigenesis and other biological characteristics, which are the root of the occurrence, development, recurrence, metastasis of EC. In this study, its purpose is to select and identify EC stem cells from serum-free cultured EC9706 cells, explore the effect of Chinese herbal prescription and its separated prescriptions on the biological characteristics of EC9706 stem cells, and to reveal the cytological and molecular mechanisms of Chinese herbal prescription anti-cancer. Methods: Firstly, MTT method was applied to explore the serum-free culture conditions of EC9706 stem cells in diameter of 60 mm low adhesion culture dish, at the same time, applying flow cytometry to identify the p75NTR mark ratio of each passage of cell to screenEC9706 stem cells. Next, Plotting the growth curve of EC9706 stem cells, detecting the proliferation inhibition rate EC9706 stem cells by cisplatin, soft agar clone formation, cell cycle to identify the characteristics of EC9706 stem cells by MTT method, soft agar assay, flow cytometry. Then applying MTT method and orthogonal test to screen and optimize Qingre Huatan Huoxue prescription (QHHP), using flow cytometry and western blot to test the effects of QHHP and its separated prescriptions on EC9706 stem cells appraisal indicators and Cell Cycle Proteins. Results: EC9706 cells with concentration of 8×104 cells/dish inoculateed on diameter of 60mm low adhesion culture dish, with adding 5 ml DMEM/F12 medium, a daily supplement of 20ng EGF, 10ng bFGF, per 10 to 14 days to passage one generation. After 5 generations of cell passage, the expression rate of p75NTR was 4-5%, was selected as the experimental object.EC9706 stem cells with the biological characteristics of slower adhesive growth, stronger cisplatin resistance, higher soft agar clone formation and p75NTR expression rate than that of EC9706 cells, and The cell cycle distribution was concentrated in the G0/G1 phase. 6 Chinese herbs were screened out ( Coptis coptidis, Rhizoma officinalis, Cucurbita pedicle, Toona bark, Cantharidopsis cantharidis, Augustia japonica) and compose QHHP. QHHP and its separated prescriptions could change the morphology, colony formation ability and cycle distribution of EC9706 stem cells in serum-free culture condition, which were related to the BMI1/CyclinB1 signaling pathway. Conclusions: Serum-free cell culture is a method to enrich stem cell-like cells in EC9706 cells. Qingre Huatan Huoxue prescription and its separated prescriptions inhibited the proliferation of EC9706 stem cells and change esophageal cancer stem cells characters. The mechanism of QHHP and its separated prescriptions on stem cells in EC9706 cells was related to BMI1 /CyclinB1 signaling pathway.

Effects of formula composed of active monomers of removing heat and phlegm prescription on malignant phenotypes and growth signaling in esophageal carcinoma cells.

e16051 Background: Esophageal carcinoma (EC) ranks seventh in the incidence of malignant tumors and the sixth mortality worldwide. Removing heat and phlegm prescription(RHPP, composed of Rhizoma Dioscoreae Nipponicae, Rhizoma Paridis, Saponin, Rhizoma Bolbostemmae) was an effective formula screened from 531 Chinese herbs for treating EC in our previous study. The purpose of this study is to further track, isolate, purify and identify the active monomers of RHPP, study the effect of each active monomer on Eca109, EC9706, EC-1, TE-1 four EC cells and the signaling mechanism, then optimize and compose new active monomers formula and study its mechanism on EC cells. Methods: The active monomer of RHPP was extracted, separated and identified by ethanol extractioncolumn, chromatography, recrystallization, HPLC, spectroscopic method,etc. The effects of four kinds of active monomers and their composed formula on the proliferation, migration, clone formation, cell cycle and signal pathway proteins expression of Eca109 cell, EC9706 cell and TE-1 cell were investigated by MTT assay, RTCA assay, soft agar assay, flow cytometry and western blot analysis. Results: Four active monomers were identified from Rhizoma Dioscoreae Nipponicae (CSL), Saponin (ZJ), Rhizoma Bolbostemmae (TBM), Rhizoma Paridis (CL) respectively with molecular weights of 868, 1003, 1365, 854.5, and corresponding molecular formulas of C45H72O16, C50H82O20, C64H100O31 and C44H70O16(China patient No. ZL202010136431.6). Four active monomers significantly affected the cell morphology, proliferation, migration, cloning ability, cell cycle of four EC cells. The IC50 values of the active monomer of CSL, ZJ, TBM and CL for Eca109 were 1.29±0.12, 2.42±0.09, 1.93±0.09, 3.04±0.28μg/ml respectively; for EC9706 were 1.41±0.02, 5.94±0.45, 1.97±0.09, 0.63±0.04μg/ml respectively; for TE-1 were 3.72±0.28, 35.58±0.58, 7.90±0.41, 1.85±0.09μg/ml respectively; for EC-1 were 1.11±0.14, 3.56±0.28, 1.23±0.02, 0.61±0.02μg/ml respectively. Four active monomers could downregulate EGFR, PLC-γ1, and PKCα protein expression. Using the baseline proportional increase and decrease design method to compose new removing heat prescription(RH), removing phlegm prescription(RP) and removing heat and phlegm prescription (RHPP), the ratio of each drug monomer in RH, RP and RHPP were mCSL: mCL = 6: 4, mZJ: mTBM = 3: 7, mCSL: mCL: mZJ: mTBM = 48: 32: 6: 14. RH, RP and RHPP all could inhibit the proliferation, migration, cloning ability, cell cycle of four EC cells. Conclusions: The four active monomers and new composed RHPP all can inhibit the proliferation of EC9706, Eca109, TE-1, EC-1 four EC cells, which have close relationship with EGFR, PLC-γ1, and PKCαproteins expression. This study provides new formula and basis for the development of anti-esophageal carcinoma drugs in traditional Chinese medicine.

WITHDRAWN: Discovery of indoline derivatives as anticancer agents via inhibition of tubulin polymerization

Human esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers in human digestive system. It is necessary to discover novel antitumor agents for the treatment of esophageal cancers because of its poor prognosis. Indoline has been reported as an efficient anticancer fragment to design novel anticancer agents. In this work, indoline derivatives were designed, synthesized and explored their anticancer activity. Compound 9d, which exhibited potent antiproliferative activity with IC50 values of 1.84μM (MGC-803 cells), 6.82μM (A549 cells), 1.61μM (Kyse30 cells), 1.49μM (Kyse450 cells), 2.08μM (Kyse510 cells) and 2.24μM (EC-109 cells), respectively. The most active compound 9d was identified as a tubulin inhibitor targeting colchicine binding site with an IC50 value of 3.4µM. Compound 9d could strongly suppress the tubulin polymerization in Kyse450 cells. The results of molecular docking also suggested compound 9d could tightly bind into the colchicine binding site of β-tubulin. Besides, compound 9d inhibited the growth of KYSE450 cells in time and dose-dependent manners. All the results suggest that the indoline derivatives might be a class of novel tubulin inhibitors with potential anticancer activity and is worthy of further study.

Discovery of indoline derivatives as anticancer agents via inhibition of tubulin polymerization

Human esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers in human digestive system. It is necessary to discover novel antitumor agents for the treatment of esophageal cancers because of its poor prognosis. Indoline has been reported as an efficient anticancer fragment to design novel anticancer agents. In this work, indoline derivatives were designed, synthesized and explored their anticancer activity. Compound 9d, which exhibited potent antiproliferative activity with IC50 values of 1.84 μM (MGC-803 cells), 6.82 μM (A549 cells), 1.61 μM (Kyse30 cells), 1.49 μM (Kyse450 cells), 2.08 μM (Kyse510 cells) and 2.24 μM (EC-109 cells), respectively. The most active compound 9d was identified as a tubulin inhibitor targeting colchicine binding site with an IC50 value of 3.4 µM. Compound 9d could strongly suppress the tubulin polymerization in Kyse450 cells. The results of molecular docking also suggested compound 9d could tightly bind into the colchicine binding site of tubulin. Besides, compound 9d inhibited the growth of KYSE450 cells in a time and dose-dependent manner. All the results suggest that the indoline derivatives may be a class of novel tubulin inhibitors with potential anticancer activity, and which is worthy of further study.

Beclin-1 is a Promising Prognostic Biomarker in a Specific Esophageal Squamous Cell Carcinoma Population.

The effects of autophagy and apoptosis in the prognostic assessment and treatment of Esophageal squamous cell carcinoma (ESCC) remain to be elucidated. Here, we conducted a retrospective study on the histopathology of ESCC, investigated the expression of Beclin-1 and Bcl-2 proteins (both autophagy- and apoptosis-related) in esophageal cancer tissue, and analyzed the significance of these proteins for the prognosis of ESCC. In the present study, the expression level of Beclin-1 in ESCC was significantly lower than that in adjacent tissues (p < 0.01), whereas the expression level of Bcl-2 showed the opposite pattern (p < 0.01). Furthermore, low expression of Beclin-1 was associated with more advanced ESCC stages and lymph node metastasis. However, high expression of Bcl-2 was associated with more advanced ESCC stages, deeper tumor invasion, and lymph node metastasis. Moreover, the relationship between Bcl-2 expression and OS was not significant (p > 0.05), whereas Beclin-1 expression was significantly associated with OS (p < 0.05). Subgroup analysis showed that Beclin-1 expression was significantly associated with OS in the high-Bcl-2-expression group but not in the low-Bcl-2-expression group. Importantly, Beclin-1 upregulation or downregulation significantly upregulated or downregulated invasion, respectively, in EC9706 cells in combination with high expression but not low expression of Bcl-2. These findings reveal that differences in autophagy and apoptotic states and their activities may promote malignant tumor differentiation, which could lead to a more aggressive esophageal squamous cell phenotype and a worse survival prognosis. Here, Beclin-1 was shown to be a promising prognostic biomarker and therapeutic target for patients with ESCC in the high-Bcl-2-expression population.

MiR-493 by regulating of c-Jun targets Wnt5a/PD-L1-inducing esophageal cancer cell development.

BackgroundEsophageal cancer is one of the most common cancers across the globe; the 5‐year survival of esophageal cancer patients is still low. MicroRNA (miRNA) dysregulation has been implicated in cancer development, and the miRNAs play a pivotal role in esophageal cancer pathogenesis. It is urgently needed to find out how miRNA dysregulation was involved in esophageal cancer (EC) development.MethodsThrough experiments in vivo and in vitro, we explored potential signaling pathways, miR‐493/Wnt5A/c‐JUN loop, in EC. Their mechanistic roles in EC cell proliferation, migration, and invasion were investigated through multiple validation steps in EC9706 and TE13 cell lines and EC specimens.ResultsOverexpression of miR‐493 attenuates esophageal cancer cell proliferation, migration, and invasion in vivo and in vitro. Moreover, miR‐493 downregulation is an unfavorable factor in EC and negatively correlated with Wnt5A. The existence of miR‐493 is also an important attribute of metabolism. Based on mechanism analyses, we show that miR‐493 inhibits the activity of c‐JUN and p‐PI3K/p‐AKT with enhanced p21 and directly regulates Wnt5A expression and function, whereas c‐JUN binds the promoter region of miR‐493 and suppressed the expression of miR‐493, forming a negative feedback loop.ConclusionsThe miR‐493/Wnt5A/c‐JUN loop is a molecular feedback loop that refers to the development of esophageal cancer cells and a potential target for the treatment of esophageal cancer.

TRPC1 Inhibits Cell Proliferation/Invasion and Is Predictive of a Better Prognosis of Esophageal Squamous Cell Carcinoma.

Background and ObjectivesIn China, over 90% of esophageal cancer (EC) cases are esophageal squamous cell carcinoma (ESCC). ESCC is a frequently malignant tumor with poor prognosis despite the development of comprehensive therapeutic strategies, for which there is still a lack of effective prognostic factors. Previous studies found that the abnormal expression of TRPC1 is closely related to the proliferation, invasion, metastasis, and differentiation of various tumors. However, the relationship between TRPC1 and ESCC is currently unclear. The present study aimed to clarify the clinical significance of TRPC1 and to preliminarily assess the molecular mechanism by which TRPC1 regulates cell proliferation, migration, and invasion in ESCC.Materials and MethodsImmunohistochemistry (IHC) was used to determine the expression of TRPC1 and Ki-67 in 165 cases of ESCC. The correlations between TRPC1 expression and clinicopathological characteristics were determined, and both univariate and multivariate analyses were utilized to quantify the impact of TRPC1 expression on patient survival. Cell Counting Kit-8, scratch wound healing, and transwell assays were used to determine the effects of TRPC1 on proliferation, migration, and invasion in ESCC in vitro, respectively.ResultsThe positive expression rate of TRPC1 showed significantly decreased in ESCC (45.50%) compared with the levels in normal esophageal mucosa (NEM; 80.80%) and high-grade intraepithelial neoplasia (HGIEN; 63.20%) (P<0.001). Higher expression rate of TRPC1 was associated with low lymph node metastasis (P<0.001), high differentiation (rs = 0.232, P=0.003), and low Ki-67 (rs = −0.492, P<0.001). We further revealed that low expression of TRPC1 was associated with poor prognosis (Disease-free survival, DFS: 95% CI=0.545–0.845, P=0.001; Overall survival, OS: 95% CI=0.553–0.891, P=0.004). Furthermore, we showed that downregulation of TRPC1 promoted the proliferation, migration, and invasion of human esophageal squamous cell carcinoma cell line EC9706 in vitro. In contrast, overexpression of TRPC1 inhibited the proliferation, migration, and invasion of human esophageal squamous cell carcinoma cell line KYSE150 (P<0.01), in a manner at least in part mediated through the AKT/p27 pathway.ConclusionTRPC1 inhibited the proliferation, migration, and invasion of EC9706 and KYSE150 cells, at least, in part mediated through the AKT/p27 pathway in vitro. The downregulation of TRPC1 may be one of the most important molecular events in the malignant progression of ESCC. TRPC1 could be a new candidate tumor suppressor gene and a new prognostic factor of ESCC.

Red light triggered photodynamic-chemo combination therapy using a prodrug caged by photosensitizer.

Development of the drug with high therapeutic efficacy and low toxicity is crucial to cancer ablation. In this study, we have demonstrated a red light-responsive prodrug BDP-TK-CPT by connecting the chemotherapeutic agent camptothecin with a boron dipyrromethene (BDP)-based photosensitizer via a reactive oxygen species (ROS)-labile thioketal chain. Since camptothecin is modified by a BDP-based macrocycle at the active site, the formed prodrug displays an extremely low toxicity in dark. However, upon illumination by red light, it can efficiently generate ROS leading to cell death by photodynamic therapy. Meanwhile, the ROS generated can destroy thioketal group to release free camptothecin which further results in local cell death by chemotherapy. The combined antitumor effects of the prodrug have been verified in HepG2, EC109, and HeLa cancer cells and mice bearing H22 tumors. This study may provide an alternative strategy for stimuli-responsive combination treatment of tumors by conjugation of ROS-activatable prodrugs with photosensitizing agents.

Development of a Novel Highly Spontaneous Metastatic Model of Esophageal Squamous Cell Carcinoma Using Renal Capsule Technology.

PurposeIncreasing evidence has demonstrated that animal models are imperative to investigate the potential molecular mechanism of metastasis and discover anti-metastasis drugs; however, efficient animal models to unveil the underlying mechanisms of metastasis in esophageal squamous cell carcinoma (ESCC) are limited.MethodsESCC cell EC9706 with high invasiveness was screened by repeated Transwell assays. Its biological characteristics were identified by flow cytometry as well as by the wound healing and CCK-8 assays. Besides, the levels of epithelial–mesenchymal transition-related markers were examined using Western blotting. Parental (EC9706-I0) and subpopulation (EC9706-I3) cells were employed to establish the renal capsule model. Next, the tumor growth was detected by a live animal imaging system, and hematoxylin and eosin staining was applied to evaluate the metastatic status in ESCC.ResultsEC9706-I3 cells showed rapid proliferation ability, S phase abundance, and high invasive ability; obvious upregulation in N-cadherin, Snail, Vimentin, and Bit1; and downregulation in E-cadherin. EC9706-I3 cells were less sensitive to the chemotherapy drug 5-fluorouracil than EC9706-I0 cells; however, both cell lines reached a tumorigenesis rate of 100% in the renal capsule model. The live animal imaging system revealed that the tumors derived from EC9706-I0 cells grew more slowly than those from EC9706-I3 cells at weeks 3–14. The EC9706-I3 xenograft model displayed a spontaneous metastatic site, including kidney, heart, liver, lung, pancreas, and spleen, with a distant metastatic rate of 80%.ConclusionOur data suggested that the metastatic model was successfully established, providing a novel platform for further exploring the molecular mechanisms of metastasis in ESCC patients.

Human esophageal fibroblast-derived exosomal miR-21 reduced the cisplatin sensitivity to esophageal carcinoma EC9706 cells.

The objective of this study was to investigate the effect of human esophageal fibroblast-derived exosomal miR-21 on cisplatin sensitivity against esophageal squamous EC9706 cells. EC9706 cells were co-cultured indirectly with human esophageal fibroblasts (HEF) or miR-21 mimics transfected-HEF in the transwell system. The exosomes in HEF-culture conditioned medium were extracted by differential ultracentrifugation. EC9706 cells were co-cultured with HEF-derived exosomes directly. The cisplatin sensitivity against EC9706 cells was revealed via half maximal inhibitory concentration (IC50) values using MTT assay. The expressions of miR-21, programmed cell death 4 (PDCD4) mRNA, and gene of phosphate and tension homology deleted on chromosome ten (PTEN) mRNA were determined by qRT-PCR. The changes of the protein level were detected using western blot assay. IC50 values of cisplatin against EC9706 cells were increased after EC9706 cells were co-cultured with either HEF or exosomes derived from miR-21 mimics-transfected HEF. Following the increased level of miR-21, the mRNA expression and protein levels of PTEN and PDCD4 were decreased in EC9706 cells. The cisplatin sensitivity to EC9706 cells was reduced by HEF-derived exosomal miR-21 through targeting PTEN and PDCD4. This study suggested that non-tumor cells in the tumor micro-environment increased the tumor anti-chemotherapy effects through their exosomes.

Transferred by exosomes-derived MiR-19b-3p targets PTEN to regulate esophageal cancer cell apoptosis, migration and invasion.

The present study aims to investigate the relationship between miR-19b-3p and esophageal cancer (ESCA), and to detect the effects of miR-19b-3p transferred by exosomes on the phenotype of EC9706 cells. The expression of miR-19b-3p was detected by starBase analysis and real-time quantitative PCR (RT-qPCR). The target genes of miR-19b-3p were predicted by TargetScan and further verified by luciferase analysis. The mRNA and protein expression levels of PTEN and EMT-related genes were detected by RT-qPCR and Western blotting. The effects of miR-19b-3p transferred by exosomes and its target genes on the apoptosis, migration and invasion of EC9706 cells were studied by establishing a co-culture model of donor cells. The expression of miR-19b-3p in ESCA plasma, cells and exosomes was significantly up-regulated. miR-19b-3p transferred by exosomes could significantly reduce EC9706 cells apoptosis rate, promote cell migration and invasion, and could target the inhibition of PTEN expression. PTEN overexpression promoted apoptosis, inhibited cell migration and invasion, down-regulated the expression of MMP-2 and vimentin, and up-regulated E-cadherin expression; however, these effects could be partially reversed by miR-19b-3p. In summary, our results reveal that miR-19b-3p transferred by exosomes can target PTEN to regulate ESCA biological functions in the receptor EC9706 cells.

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.

Mechanism of autophagy regulating chemoresistance in esophageal cancer cells

The current study aimed to explore the mechanism of autophagy-regulating chemoresistance in esophageal cancer (EC) cells. Methods: 45 cases of esophageal cancer cell tissue and 25 cases of adjacent normal tissue excised in the surgical resection were collected from the tumor pathology department of our hospital from March to November 2017. The above cancer cells and paracancerous cells were cultured according to the cell culture procedures. The autophagy was induced by cisplatin in human esophageal cancer EC9706 cells line. The effect of autophagy on the survival of EC9706 cells was observed by autophagy inhibitor 3-MA. Cell viability was also measured by cell counting kit-8 (CCK-8). Apoptosis and cell cycle were detected by flow cytometry. Furthermore, monodansylcadaverine (MDC) was used to detect autophagy. Western blot was applied to determine the molecular changes during treatment. Diketopyrrolopyrrole (DPP) is able to inhibit cell proliferation, induce cell death and cell cycle arrest in the S phase. In addition, autophagy was activated through PI3K-III pathway. Results: 3-MA inhibitor plus 10% fetal bovine serum were added for culture, and the cell culture temperature and humidity were the best conditions. There were few autophagic vesicles in the stationary cells, where their brightness was weakened. There were more and brighter green fluorescent particles in the DPP group without a 3-MA inhibitor, indicating that autophagic parameters actually exist in this process. The apoptosis rate of DDP-induced cell death was not found to be the best, but was higher than that of the control group (P<0.05). The combination of DDP and 3-MA had a more obvious catalytic effect on apoptosis, and the apoptosis rate was much higher than that of single DDP (P<0.05), indicating that DDP was capable of inducing significant apoptosis after inhibiting autophagy. The combination of DDP and 3-MA had an obvious catalytic effect on apoptosis, and the apoptosis rate was higher than that of DDP alone (P < 0.05), suggesting that DDP could significantly improve the ability to induce apoptosis after inhibiting autophagy. The expression level of autophagy-related proteins was also detected by Western blotting. Our findings indicated that autophagy may be a self-protective mechanism of esophageal cancer cells induced by DDP, and its inhibition may be a new strategy for adjuvant chemotherapy in esophageal cancer.

MiR-15b, a diagnostic biomarker and therapeutic target, inhibits oesophageal cancer progression by regulating the PI3K/AKT signalling pathway.

MicroRNA (miR)-15b is an important regulator in several types of cancer, such as gastric cancer, colorectal cancer and oesophageal squamous cell carcinoma. The PI3K/AKT signalling pathway has been implicated in the growth and metastasis of oesophageal cancer (EC). The aim of the present study was to investigate the biological effects of miR-15b in EC, as well as the underlying mechanism involving the PI3K/AKT signalling pathway. The present study included 74 patients with EC and 74 healthy volunteers. The expression of miR-15b in peripheral blood mononuclear cells (PBMCs) and EC cell lines was evaluated via reverse transcription-quantitative PCR. The receiver operating characteristic curve was plotted to determine the diagnostic significance of miR-15b. EC cell viability, apoptosis, migration and invasion were analysed by conducting MTT, flow cytometry and transwell assays, respectively. Protein expression levels were analysed via western blotting. The results indicated that PBMCs isolated from patients with EC had lower miR-15b expression levels compared with PBMCs isolated from healthy volunteers. In patients with EC, miR-15b expression was strongly associated with tumour size, lymph node metastasis, TNM stage, fibrous membrane invasion and histologic grade. The results of the gain/loss-of-function in vitro experiments indicated that miR-15b inhibited EC cell viability, migration and invasion, facilitated EC cell apoptosis and attenuated the PI3K/AKT signalling pathway in EC109 and TE10 cells. Treatment of EC cells with the PI3K/AKT pathway agonist recilisib displayed the opposite effects, blocking the inhibitory function of miR-15b mimic on EC cell viability, migration and invasion. In summary, the results indicated that miR-15b suppressed EC cell viability, migration and invasion, and promoted EC cell apoptosis by inhibiting the PI3K/AKT signalling pathway.

Synthesis and anticancer activity of new spirooxindoles incorporating[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine moiety

A series of new spirooxindole derivatives (4a-4l) were designed, synthesized and characterized, in which the structure of compound 4f was further confirmed by single X-ray diffraction.Their antiproliferative activities were evaluated in vitro by MTT assay, the results indicated that most of the prepared compounds exhibited moderate to potent antiproliferative activities against four cancer cell lines, DU145, EC109, MGC803, and MCF-7. Particularly, compound 4d showed 3.0, 1.6, 2.7 and 1.3 times more active than positive control 5-fluorouracil (IC50 = 24.29±0.04 μM, 10.38±0.01 μM, 25.54±0.05 μM, 22.46±0.03 μM) in inhibiting DU145, EC109, MGC803, and MCF-7 cell proliferation with IC50 values of 8.02±0.64 μM, 6.62±0.89 μM, 9.49±0.78 μM, and 17.65±0.82 μM, respectively. These encouraging results should provide important information for the development of new anticancer agents.

GPR120 promotes radiation resistance in esophageal cancer via regulating AKT and apoptosis pathway.

The aim of the study is to investigate the role of GPR120 on the biological behavior of esophageal cancer cells in the setting of radiation and explore the mechanism. GPR120 knockdown was fulfilled by siRNA-mediated effects in two esophageal cancer cell lines Eca109 and EC9706. Colony formation, survival fraction calculation, viable cell evaluation by cell counting kit-8 assay and cell apoptosis analysis by phycoerythrin annexin V and 7-amino-actinomycin (7-AAD) staining and the flow cytometry examination was evaluated in Eca109 and EC9706 under the treatment of different radiation dosage. The mechanisms were explored by the evaluation of the Akt pathway and apoptosis protein level. Significantly decreased GPR120 mRNA and protein after GPR120 siRNA treatment compared to control siRNA treatment. Significantly decreased colony formation was found in GPR120 siRNA-treated Eca109 and EC9706 cells compared to control siRNA-treated cells at the radiation dosage of 2, 4, 6 and 8 Gy. Moreover, decreased survival fraction number with increased sensitive enhancing ratio was also found in GPR120 siRNA-treated Eca109 and EC9706 cells compared to control siRNA-treated cells. Decreased cell viability and increased cell apoptosis in GPR120 siRNA-treated esophageal cancer cells. GPR120 siRNA decreased the Akt phosphorylation and anti-apoptotic Bcl-2 expression level, but increased pro-apoptotic Bim expression level in esophageal cancer cell lines. GPR120 regulated the biological behavior of the esophageal cancer cells via affecting Akt pathway and apoptosis molecules. Moreover, GPR120 siRNA combined radiation treatment could be a therapeutic choice for esophageal cancer.

Long non‑coding RNA MEG3 suppresses epithelial‑to‑mesenchymal transition by inhibiting the PSAT1‑dependent GSK‑3β/Snail signaling pathway in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer in China, and the prognosis of patients remains poor mainly due to the occurrence of lymph node and distant metastasis. The long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been shown to have tumor-suppressive properties and to play an important role in epithelial-to-mesenchymal transition (EMT) in some solid tumors. However, whether MEG3 is involved in EMT in ESCC remains unclear. In the present study, the MEG3 expression level and its association with tumorigenesis were determined in 43 tumor tissues of patients with ESCC and in ESCC cells using reverse transcription-quantitative PCR analysis. Gene microarray analysis was performed to detect differentially expressed genes (DEGs). Based on the functional annotation results, the effects of ectopic expression of MEG3 on cell growth, migration, invasion and EMT were assessed. MEG3 expression level was found to be markedly lower in tumor tissues and cells. Statistical analysis revealed that MEG3 expression was significantly negatively associated with lymph node metastasis and TNM stage in ESCC. Fluorescence in situ hybridization assay demonstrated that MEG3 was expressed mainly in the nucleus. Ectopic expression of MEG3 inhibited cell proliferation, migration, invasion and cell cycle progression in EC109 cells. Gene microarray results demonstrated that 177 genes were differentially expressed ≥2.0 fold in MEG3-overexpressing cells, including 23 upregulated and 154 downregulated genes. Functional annotation revealed that the DEGs were mainly involved in amino acid biosynthetic process, mitogen-activated protein kinase signaling, and serine and glycine metabolism. Further experiments indicated that the ectopic expression of MEG3 significantly suppressed cell proliferation, migration, invasion and EMT by downregulating phosphoserine aminotransferase 1 (PSAT1). In pathological tissues, PSAT1 and MEG3 were significantly negatively correlated, and high expression of PSAT1 predicted poor survival. Taken together, these results suggest that MEG3 may be a useful prognostic biomarker and may suppress EMT by inhibiting the PSAT1-dependent glycogen synthase kinase-3β/Snail signaling pathway in ESCC.

MicroRNA‑216a‑5p suppresses esophageal squamous cell carcinoma progression by targeting KIAA0101.

The KIAA0101 protein (also referred to as NS5ATP9 or Paf15) is overexpressed in esophageal squamous cell carcinoma (ESCC) and is associated with disease progression and poor patient survival, but how KIAA0101 expression is regulated remains unknown. The relationship between tumor miR-216a-5p expression and prognosis in patients with ESCC was revealed by survival analyses. Quantitative reverse-transcriptase PCR and western blot analysis were used to evaluate miR-216a-5p and KIAA0101 expression in human ESCC tissues and cell lines. The targeting of KIAA0101 by miR-216a-5p was verified by dual-luciferase reporter assays. The EC9706 and TE1 cell lines were transfected with miR-216a-5p mimics and inhibitor, or KIAA0101-specific shRNA and KIAA0101-expressing plasmids, in order to evaluate the effect of manipulating miR-216a-5p and KIAA0101 expression on ESCC cell proliferation, cell cycle progression, migration, and invasion. miR-216a-5p was lowly expressed and inversely correlated with KIAA0101 protein expression in ESCC tissues and cell lines. Lower miR-216a-5p expression was associated with worse prognosis in patients with ESCC. miR-216a-5p negatively regulated KIAA0101 expression by directly targeting the 3′-untranslated region of the KIAA0101 mRNA. Overexpression of miR-216a-5p suppressed the proliferation, migration, and invasion of the ESCC cell lines, whereas inhibition of miR-216a-5p had the opposite effects. Meanwhile, KIAA0101 promoted ESCC migration and invasion, and its overexpression abolished the antitumor effects of miR-216a-5p mimics. As a tumor suppressor, miR-216a-5p targets KIAA0101 to inhibit the proliferation, migration, and invasion of ESCC. Therefore, the miR-216a-5p/KIAA0101 axis may be a potential target for ESCC treatment.