Human Esophageal Carcinoma Cell Line Research Articles

Human esophageal cancer stem-like cells escape the cytotoxicity of natural killer cells via down-regulation of ULBP-1

BackgroundCancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity.MethodsCSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry.ResultsWe observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity.ConclusionsThis study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.

Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma

BackgroundIncreasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins.MethodsThe GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms.ResultsPDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription.ConclusionsOur research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

Circ_0003855 involvement of esophageal cancer progression through miR-622/FLOT1.

To confirm the relationship between Circ_0003855 and EC, we purchased the Human esophageal carcinoma cell line Eca109 and normal human esophageal epithelial cells HEEC, and the expression levels of Circ_0003855, miR-622, and FLOT1 were detected. The results show that Circ_0003855 and FLOT1 were highly expressed in Eca109 cells, while miR-622 was lowly expressed (p < 0.05). Subsequently, Circ_0003855 small interfering RNA (si-Circ_0003855) and its negative control (si-NC) were used to detect changes in cellular biological behaviors. We found that the activity of Eca109 cells was reduced after interfering with the expression of Circ_0003855, and miR-622 expression was elevated, while FLOT1 was decreased (p < 0.05). Additionally, si-Circ_0003855 and miR-622 inhibitor sequence (miR-622-inhibition) were co-transfected into cells with miR-622-inhibition alone, and untreated Eca109 cells were used as a control to detect the expression of FLOT1. Co-transfection of si-Circ_0003855 and miR-622-inhibition showed no significant difference in FLOT1 expression compared to the control cells (p > 0.05). Synthesizing the results of these experiments above, we believe that interfering with the expression of Circ_0003855 can inhibit the activity of EC cells, and its mechanism is related to miR-622 and FLOT1.

The expression of annexin-A1 in esophageal squamous cell carcinoma and its association with the biological behavior of the primary human esophageal squamous carcinoma cell line.

This study aimed to observe the differential expression of Annexin-A1 in esophageal squamous cell carcinoma (ESCC) and explored the effect of small interfering ribonucleic acid (RNAi)-Annexin-A1 on the biological behavior of CE81T-0 cells. An immunohistochemical approach was used to detect the expression of Annexin-A1 in 86 pairs of ESCC samples. Quantitative reverse transcription polymerase chain reaction was used to detect the expression of Annexin-A1 in CE81T-0 and CE81T-4 cells, and the expression of Annexin-A1 in CE81T-0 cells was knocked out by RNAi. A methyl-thiazolyl-tetrazolium assay was used to observe the effect of Annexin-A1 on cell proliferation, and flow cytometry was conducted to analyze its effect on cell cycles and apoptosis. A scratch assay and a Transwell chamber were used to detect changes in cell migration and invasion. From the results, compared with the Annexin-A1 expression rate of 59.3% in para-carcinoma tissues, the expression of Annexin-A1 in cancer was reduced to only 32.6% in ESCC cells. Annexin-A1 was strongly expressed in highly differentiated ESCC cells without lymphatic metastasis and highly expressed in the CE81T-0 cell group with low metastasis. Annexin-A1 gene silencing promoted cell proliferation and inhibited apoptosis, blocked cells in the S-phase, and increased cell migration, leading to an increase in the number of invaded cells. Above all, Annexin-A1 could reflect the differentiation degree and lymph node metastasis of ESCC cells to some extent and was involved in the invasion, metastasis, proliferation, and other biological behaviors of ESCC cells, indicating an experimental basis for Annexin-A1 as a molecular marker in the early diagnosis of ESCC and the prediction of cell metastasis, invasion, and differentiation degree.

Effects of irradiation with different fractionations on radioresistant esophageal cancer cells and lung tissues

BackgroundLocal recurrence is the main cause of treatment failure for esophageal cancer and a large part of them require re-irradiation which needs to face the two major challenges of radioresistance and radiation-induced lung injury (RILI). This study aimed to evaluate the effects of irradiation with different fractionations on inhibition in radioresistant esophageal cancer cells in vitro and RILI in vivo. MethodsEC109R was selected from the human esophageal squamous carcinoma cell line EC109 by multiple exposures and verified to be radioresistant through colony forming assay and cell proliferation assay. EC109R was irradiated with different fractionations (conventional radiotherapy, CR: 2 Gy/d, 5 d; pulsed low dose-rate radiotherapy, PLDR: 0.2 Gy x 10 pulses with 3-min intervals/d, 5 d; hypo-fractionated radiotherapy, HR: 5 Gy/d, 2 d) to verify their inhibition by CCK-8. The protection of PLDR on RILI was assessed by H&E-staining paraffin sections and mice serum TGF-β1 levels. ResultsEC109R exhibited much better radioresistance (p < 0.0001) than EC109 in colony forming and cell proliferation assays. For EC109R, PLDR showed slightly lower inhibition by detection of optical density than HR (p = 0.0025) but similar to CR (p = 0.1155). In addition, pathological sections of lung tissues from PLDR group showed less alveolar structural damage and inflammatory cells infiltration, and serum TGF-β1 levels were significantly lower than those in the other two fractionations (vs CR: p = 0.0092; vs HR: p = 0.0045). ConclusionPLDR could effectively inhibit the proliferation of radioresistant esophageal cancer cells in vitro and mitigate RILI in vivo.

Inhibition of APLN suppresses cell proliferation and migration and promotes cell apoptosis in esophageal cancer cells in vitro, through activating PI3K/mTOR signaling pathway.

Esophageal cancer is the sixth leading cause of cancer mortalities globally with a high incidence rate. Apelin (APLN) plays regulatory roles in different organs. However, its role in esophageal cancer remains unknown. Therefore, our study aims to explore the effect of APLN on esophageal cancer. One hundred and eighty-four (184) esophageal tumor tissues samples from patients with esophageal cancer, and 11 esophageal tissues samples from healthy volunteers were analyzed for the expression of APLN. APLN was highly expressed in the tumor of patients with esophageal cancer and esophageal cancer cells. Patients with high expressions of APLN had a lower survival rate than the ones with low to medium expressions of APLN. Human esophageal carcinoma cell lines, TE-1 and ECA-109 cells were transfected with APLN siRNA to knockdown APLN, or transfected with pcDNA-APLN to overexpress APLN. Inhibition of APLN by siRNA-APLN reduced proliferative, migrative, and invasive abilities of esophageal cancer cells and promoted cell apoptosis, which could be all restored by pcDNA-APLN. Moreover, knocking down APLN by siRNA-APLN suppressed the PI3K/mTOR signaling pathway. These findings identify that APLN inhibition might ameliorate esophageal cancer through activating the PI3K/mTOR signaling pathway, thus APLN could be a potential target for esophageal cancer.

Abstract 5808: Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions

Abstract Esophageal cancer is one of the most aggressive forms of human malignancy with a 5-yearsurvival rate of less than 20%. Mitochondria are dynamic organelles that play essential roles in various cellular processes, including energy metabolism, redox homeostasis, and apoptotic cell death. Alterations in mitochondrial biology are associated with esophageal carcinogenesis and esophageal cancer cell response to therapy. As the anti-inflammatory drug diclofenac (DCF)induces mitochondrial dysfunction, we hypothesized that DCF may inhibit esophageal carcinogenesis by affecting mitochondrial processes. In the human esophageal squamous cell carcinoma (ESCC) cell line TE11, we performed MTT assay and Annexin-V/Propidium Iodide (PI) to evaluate the impact of DCF on proliferation and cell death, respectively. RNA sequencing and Ingenuity Pathway Analysis (IPA) identified differentially expressed genes (DEGs) and altered canonical pathways in DCF-treated TE11 cells. To assess metabolic function, we measured the levels of pyruvate, lactate, and ATP. Mitochondrial membrane potential and ROS production were assessed by flow cytometry for MitoTracker red/green and MitoSox red, respectively. Mice bearing subcutaneous syngeneic ESCC tumors were treated with DCF.DCF inhibited proliferation of TE11 in dose dependent manner with an IC50 concentration of 76.7µM. By contrast, the IC50 concentration of DCF in normal esophageal keratinocytes was 876.2µM, supporting selectivity of DCF for esophageal cancer cells. In TE11, 200 µM DCF induced apoptosis in 60% of cells. RNA-sequencing identified that DCF significantly altered expression of3287 genes (FC ≥ 1.50) in TE11, including the top cancer-associated genes: PLK1, MCM2, MCM3,MCM7, MCM10 and SKP2. IPA analysis revealed that DCF activated p53 signaling while inhibited Gluconeogenesis I, Glycolysis I and Oxidative Phosphorylation. DCF significantly inhibited glycolysis as demonstrated by reduction in concentrations of pyruvate (1.76-fold) and lactate (7.30-fold) and downregulation of the glycolysis- associated genes PKM2, PFKM, LDHA, and PKM1. A decreased production of ATP (5-fold) was also noted in DCF-treated TE11 cells. In addition, DCF depleted mitochondrial membrane potential (2.5-fold) and increased mitochondrial ROS production (5-fold). A reduction in ROS by mitochondrial antioxidant MitoTempo increased viability of the DCF-treated TE11 cells, indicating that ROS contributes to the anticancer activity of DCF. Consistent with antitumor activity of DCF in TE11, the drug significantly decreased the tumor volume in syngeneic ESCC tumors in vivo. Our preclinical findings indicate that DCF may limit esophageal cancer cell growth through the inhibition of mitochondrial functions, identifying a novel experimental therapeutic for ESCC. Future studies will define the precise molecular mechanisms through which DCF promotes cell death in vitro and in vivo. Citation Format: Mohammad Faujul Kabir, Adam Karami, Anbin Mu, Don-Gerard Conde, Kelly A. Whelan. Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5808.

Identification and function of circular RNA hsa_circ_0071106: A novel biomarker for differentiation degree of esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) has a high degree of malignancy, and there is currently no effective treatment. Circular RNA is a novel endogenous noncoding RNA with a stable loop structure. Several theories have been proposed regarding its biogenesis and usefulness as a biomarker in various cancers. Here we explore the predictive value and potential functions of a candidate circRNA (hsa_circ_0071106) in ESCC. MethodsThe altered expression of hsa_circ_0071106 was validated in clinical tissue samples from 64 patients with ESCC. The correlation between the clinical characteristics of these patients and the expression of hsa_circ_0071106 was analyzed by the chi-square test. The receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to evaluate the diagnostic value of hsa_circ_0071106. The human esophageal carcinoma cell lines ECA109 and ECA9706 and the normal human esophageal epithelial cell line (HET-1A) were applied for functional analysis of hsa_circ_0071106. qRT-PCR, sanger sequencing, agarose gel electrophoresis, RNase R digestion assay, Actinomycin D inhibition assay, nucleoplasm separation assay, and FISH assays were utilized to verify the circular characteristics and subcellular localization of hsa_circ_0071106. SiRNA and circRNA interference lentivirus was constructed to inhibit the expression of hsa_circ_0071106 in the ECA109 and ECA9706 cell lines. We used the EdU assay, Transwell assay, and flow cytometry to detect changes in cell proliferation, invasion, migration, and apoptosis. ResultsWe identified that hsa_circ_0071106 showed significantly decreased expression levels in ESCC cells (P < 0.001) and tissue samples (P < 0.001). The correlation analysis of clinicopathological features and gene expression revealed that low expression of hsa_circ_0071106 was related to poor differentiation grade (P < 0.05). ROC curves indicated that hsa_circ_0071106 might have a diagnostic value for ESCC (AUC = 0.826, 95% CI: 0.748–0.904) and a predictive value for a high degree of histological differentiation (AUC = 0.730, 95% CI: 0.607–0.854). Hsa_circ_0071106 has a stable circular structure and was primarily localized in the cytoplasm. A transient knockdown of hsa_circ_0071106 by siRNA promotes proliferation, migration, and invasion and inhibits apoptosis of ESCC cells. A steady knockdown of hsa_circ_0071106 by a lentivirus knockout vector significantly promoted cell migration and invasion in ECA109 and ECA9706 cell lines. ConclusionHsa_circ_0071106 was a downregulated circRNA related to ESCC and promoted ESCC progression by regulating cell migration and invasion. These findings suggest that hsa_circ_0071106 could be a promising diagnostic biomarker and potential therapeutic target and might predict ESCC’s histological differentiation degree.

The Anti-Proliferative and Apoptotic Effects of Rutaecarpine on Human Esophageal Squamous Cell Carcinoma Cell Line CE81T/VGH In Vitro and In Vivo.

The overall five-year survival rate for patients with esophageal cancer is low (15 to 25%) because of the poor prognosis at earlier stages. Rutaecarpine (RTP) is a bioalkaloid found in the traditional Chinese herb Evodia rutaecarpa and has been shown to exhibit anti-proliferative effect on tumor cells. However, the mechanisms by which RTP confer these effects and its importance in esophageal squamous cell carcinoma treatment remain unclear. Thus, in the present study, we first incubated human esophageal squamous cell carcinoma cell line, CE81T/VGH, with RTP to evaluate RTP’s effects on tumor cell growth and apoptosis. We also performed a xenograft study to confirm the in vitro findings. Furthermore, we determined the expression of p53, Bax, bcl-2, caspase-3, caspase-9, and PCNA in CE81T/VGH cells or the tumor tissues to investigate the possible mechanisms. All the effects of TRP were compared with that of cisplatin. The results showed that RTP significantly inhibits CE81T/VGH cell growth, promotes arrest of cells in the G2/M phase, and induces apoptosis. Consistently, the in vivo study showed that tumor size, tumor weight, and proliferating cell nuclear antigen protein expression in tumor tissue are significantly reduced in the high-dose RTP treatment group. Furthermore, the in vitro and in vivo studies showed that RTP increases the expression of p53 and Bax proteins, while inhibiting the expression of Bcl-2 in cancer cells. In addition, RTP significantly increases the expression of cleaved caspase-9 and cleaved caspase-3 proteins in tumor tissues in mice. These results suggest that RTP may trigger the apoptosis and inhibit growth in CE81T/VGH cells by the mechanisms associated with the regulation of the expression of p53, Bax, Bcl-2, as well as caspase-9 and caspase-3.

Polysaccharide of Atractylodis Macrocephalae Rhizoma inhibits expression of immune checkpoint PD-L1 by targeting miR-34a in esophageal carcinoma cells

The immune checkpoint programmed cell death-ligand 1(PD-L1)-mediated immunosuppression is among the important features of tumor. PD-L1, an immunosuppressant, can induce T cell failure by binding to programmed cell death-1(PD-1). Thus, the key to restoring the function of T cells is inhibiting the expression of PD-L1. The Chinese medicinal Atractylodis Macrocephalae Rhizoma(AMR) has the anti-tumor, anti-inflammatory, antioxidant, and hypoglycemic activities, and the polysaccharide in AMR(PAMR) plays a crucial role in immunoregulation, but the influence on the immune checkpoints which are closely related to immunosuppression has not been reported. MicroRNA-34 a(miR-34 a) expression in esophageal carcinoma tissue is significantly lower than that in normal tissue. This study aims to investigate the inhibitory effect of PAMR on esophageal carcinoma cells, and the relationship between its inhibitory effect on PD-L1 expression and miR-34 a, which is expected to clarify the anti-tumor mechanism of PAMR. Firstly, different human esophageal carcinoma cell lines(EC9706, EC-1, TE-1, EC109 cells) were screend out, and expression of PD-L1 was determined. Then, EC109 cells, with high expression of PD-L1, were selected for further experiment. The result showed that PAMR suppressed EC109 cell growth. According to the real-time quantitative PCR(qPCR) and Western blot, it significantly suppressed the mRNA and protein expression of PD-L1, while promoting the expression of tumor suppressor miR-34 a. The confocal microscopy and luci-ferase assay proved that PAMR alleviated the inhibitory effect of PD-L1 while blocked miR-34 a. Additionally, the expression of PD-L1 was controlled by miR-34 a, and the combination of miR-34 a inhibitor with high-dose PAMR reversed the inhibitory effect of PAMR on PD-L1 protein expression. Thus, the PAMR may inhibit PD-L1 by increasing the expression of miR-34 a and regulating its downstream target genes. In conclusion, PAMR inhibits the expression of PD-L1 mainly by inducing miR-34 a.

Developing a Mathematical Model of Intracellular Calcium Dynamics for Evaluating Combined Anticancer Effects of Afatinib and RP4010 in Esophageal Cancer.

Targeting dysregulated Ca2+ signaling in cancer cells is an emerging chemotherapy approach. We previously reported that store-operated Ca2+ entry (SOCE) blockers, such as RP4010, are promising antitumor drugs for esophageal cancer. As a tyrosine kinase inhibitor (TKI), afatinib received FDA approval to be used in targeted therapy for patients with EGFR mutation-positive cancers. While preclinical studies and clinical trials have shown that afatinib has benefits for esophageal cancer patients, it is not known whether a combination of afatinib and RP4010 could achieve better anticancer effects. Since TKI can alter intracellular Ca2+ dynamics through EGFR/phospholipase C-γ pathway, in this study, we evaluated the inhibitory effect of afatinib and RP4010 on intracellular Ca2+ oscillations in KYSE-150, a human esophageal squamous cell carcinoma cell line, using both experimental and mathematical simulations. Our mathematical simulation of Ca2+ oscillations could fit well with experimental data responding to afatinib or RP4010, both separately or in combination. Guided by simulation, we were able to identify a proper ratio of afatinib and RP4010 for combined treatment, and such a combination presented synergistic anticancer-effect evidence by experimental measurement of intracellular Ca2+ and cell proliferation. This intracellular Ca2+ dynamic-based mathematical simulation approach could be useful for a rapid and cost-effective evaluation of combined targeting therapy drugs.

Mannose enhances the radio-sensitivity of esophageal squamous cell carcinoma with low MPI expression by suppressing glycolysis

BackgroundTo investigate the effect of mannose on radio-sensitivity of human esophageal squamous cell carcinoma (ESCC) cell line and its possible mechanism.MethodsThe expression of mannose phosphate isomerase (MPI) in human esophageal cancer cell lines were detected by Western blot. The inhibitory effect of mannose on human esophageal cancer cell lines were observed by MTT assay. Plate clone formation assay was performed to investigate the efficacy of mannose on radio-sensitivity of human esophageal cancer cells. The apoptosis rates of tumor cells treated with mannose and/or radiation therapy was calculated by flow cytometry. Furthermore, we analyzed intracellular metabolites using liquid chromatography mass spectrometry to identify selective sugar metabolites.ResultsMPI expression was various in human esophageal cancer cells. KYSE70 cells was associated with the highest MPI expression whereas KYSE450 cells had the lowest MPI expression level. When administrated with 11.1 mM/L mannose, the same inhibitory effect was observed in both KYSE70 and KYSE450 cell lines. Moreover, the inhibitory effect was significant on KYSE450 cell lines with an increased mannose concentration. The application of 11.1 mM/L mannose could significantly enhance the radio-sensitivity of KYSE450 cell line; and tumor cell apoptosis rate was also increased. However, there was limited efficacy of mannose on the radio-sensitivity and apoptosis rate of KYSE70 cell line. Additionally, intracellular metabolites analyzation revealed that glycolysis could be disturbed by mannose when combined with radiation therapy in esophageal cancer cells.ConclusionIn esophageal cancer cell lines with low MPI expression, the administration of mannose was associated with enhanced radio-sensitivity.

Identification of ion channel/transporter expression profiles in digestive cancer stem cells for novel targeting therapy

Recent evidence suggests that the targeting of membrane transporters specifically activated in cancer stem cells (CSCs) is an important strategy for cancer therapy. The objectives of the present study were to investigate the ion channel expression profiles in digestive CSCs. Cells strongly expressing CSC markers, such as ALDH1A1 and CD44, were separated from the human esophageal squamous cell carcinoma, gastric cancer, and pancreatic cancer cell lines using fluorescence-activated cell sorting, and CSCs were identified based on tumorsphere formation. Messenger RNA levels of CSC markers were higher in CSCs than in non-CSCs. These CSCs also exhibited resistance to anticancer agents. The microarray analysis revealed that the expression of transient receptor potential vanilloid 2 (TRPV2), voltage-gated calcium channels (VGCCs), and voltage-gated potassium channels (Kv) were upregulated in esophageal, gastric, and pancreatic CSCs, respectively, compared with non-CSCs. The TRPV2 inhibitor tranilast, VGCCs inhibitors amlodipine and verapamil, and Kv inhibitor 4-aminopyridine exhibited greater cytotoxicity in CSCs compared with non-CSCs, and their inhibitory effects were also confirmed in a xenograft model in nude mice. Taking these results, phase I/II study to investigate clinical safety and efficacy of neoadjuvant combination chemotherapy of tranilast in advanced esophageal squamous cell carcinoma (TNAC study) is ongoing. These researches identified a role of ion channels in the persistence of CSCs and suggested that their inhibitors may have potential as a therapeutic agent for digestive cancers.

Bivalent β-Carbolines Inhibit Colorectal Cancer Growth through Inducing Autophagy.

In this study, a series of alkyl diamine linked bivalent β-carbolines was synthesized and evaluated as antitumor agent. The results demonstrated that most compounds displayed good antiproliferative activities with IC50 value lower than 10 µM against a panel of human tumor cell lines, and compound 8 was found to be the most potent antiproliferative agent with IC50 value of 1.39, 1.96, 1.42, 1.49, 1.32, 1.96 and 1.63 µM against human breast cancer cell line (MCF-7), human adenocarcinoma cell line (769-P), human malighant melanoma cell line (A375), human ovarian cancer cell line (SK-OV-3), human colon carcinoma cell line (HCT-116), human gastric cancer cell line (BGC-823) and human esophageal squamous carcinoma cell line (Eca-109), respectively. Further investigations on mechanism of action of this class of compound demonstrated that the representative compound 8 inhibited colorectal cancer growth through inducing autophagy.

Ultrasound-stimulated microbubbles contributes to radiotherapy in esophageal carcinoma.

This study aimed to explore the effect of ultrasound-stimulated microbubbles (USMBs) on tumor radiosensitivity in esophageal carcinoma (EC). The human EC cell line KYSE-510 and human umbilical vein endothelial cells (HUVECs) were exposed to radiation alone or in combination with USMBs. CCK-8, colony formation, and EdU assays were used to determine cell viability and proliferation. Cell apoptosis was assessed using flow cytometry. Cell migration and invasion were examined by wound healing and transwell assays. Western blotting showed that the protein levels were associated with apoptosis, epithelial-mesenchymal transition (EMT), and angiogenesis. An endothelial tube-forming assay was used to detect the angiogenic activity of HUVECs. Xenograft experiments were used to examine the effect of USMBs on EC radiosensitivity in vivo. The expression of Ki-67 in tumors was detected using immunohistochemistry. USMBs enhanced the suppressive effect of radiation on proliferation, migration, invasion, and EMT, and promoted radiation-induced apoptosis in EC cells in vitro. Angiogenesis in EC was suppressed by radiation and further inhibited by the combination of radiation and USMBs. In vivo experiments revealed that USMBs increased the radiosensitivity of ECs to tumor growth. Collectively, USMBs enhanced the effects of radiotherapy in esophageal carcinoma.

Carvacrol Induces Cell Apoptosis in Human Esophageal Squamous Carcinoma the Human Esophageal Squamous Carcinoma Cell Line (KYSE-150) Cells

Carvacrol is a natural phenol with antioxidant, antimicrobials, and anti-cancer activities. The present study aimed to explore the anti-cancer activity of carvacrol in human esophageal squamous carcinoma KYSE-150 cells. Cell proliferation and apoptosis were measured using WST-1 assay and cell death detection ELISA kit respectively. Caspase activities were determined with caspase fluorometric assay kits. WST-1 assay revealed that carvacrol suppressed KYSE-150 cell proliferation in both dose and time-dependent manner. Carvacrol could increase cell apoptosis in a dose-dependent manner through caspase-3 and caspase-9 activation. In addition, Bcl-2 mRNA levels were significantly decreased by carvacrol treatment. This study delivers that carvacrol exerts a favorable anti-cancer action against esophageal cancer via inhibiting cell proliferation and inducing cell apoptosis. These discoveries suggest that carvacrol is deserved to be further studied and developed as a chemotherapeutic agent for esophageal cancer.

Network analysis of miRNA targeting m6A-related genes in patients with esophageal cancer.

BackgroundWe investigated the miRNA-m6A related gene network and identified a miRNA-based prognostic signature in patients with esophageal cancer using integrated genomic analysis.MethodsWe obtained expression data for m6A-related genes and miRNAs from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Survival analysis was conducted to identify potential prognostic biomarkers. LASSO Cox regression was performed to construct the overall survival (OS) associated prediction signature. We used the Kaplan–Meier (K–M) curve and receiver operating characteristic (ROC) curves to explore the signature’s efficiency and accuracy. Interactions between the m6A-related genes and miRNAs were identified in starBase3.0 and used to construct the miRNA-m6A related gene network.ResultsWe found that HNRNPC, YTHDF, ZC3H13, YTHDC2, and METTL14 were dysregulated in esophageal cancer tissues. Multivariate Cox regression analysis revealed that HNRNPC may be an independent risk factor for OS. Five hundred twenty-two potential upstream miRNAs were obtained from starBase3.0. Four miRNAs (miR-186, miR-320c, miR-320d, and miR-320b) were used to construct a prognostic signature, which could serve as a prognostic predictor independent from routine clinicopathological features. Finally, we constructed a key miRNA-m6A related gene network and used one m6A-related gene and four miRNAs associated with the prognosis. The results of our bioinformatics analysis were successfully validated in the human esophageal carcinoma cell lines KYSE30 and TE-1.ConclusionOur study identified a 4-miRNA prognostic signature and established a key miRNA-m6A related gene network. These tools may reliably assist with esophageal cancer patient prognosis.

Effect of silencing C-erbB-2 on esophageal carcinoma cell biological behaviors by inhibiting IGF-1 pathway activation

ObjectiveC-erbB-2 has been confirmed to be an oncogene that participates in cell growth, differentiation and division of tumors. We are wondered if its silenced expression can exert an anti-tumor effect. Therefore, this study is conducted to investigate the mechanism of C-erbB-2 silencing and IGF-1 pathway on esophageal carcinoma (EC) cell biological behaviors.MethodsThe objects of study were 84 EC patients from Heping Hospital Affiliated to Changzhi Medical College, with the collection of EC tissue and adjacent normal tissue (> 5 cm away from cancer tissue). C-erbB-2 protein expression in EC tissues was detected by immunohistochemistry. Human EC cell line Eca-109 was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Based on different transfection protocols, EC cells with logarithmic growth phase of 3–5 passages were divided into blank control group, oe-C-erbB-2 NC group, siRNA C-erbB-2 NC group, oe-C-erbB-2 group, siRNA C-erbB-2 group, OSI-906 group, Rg5 group, Rg5 + siRNA C-erbB-2 NC group and Rg5 + siRNA C-erbB-2 group. Cell proliferation was detected by MTT assay; cell cycle distribution and apoptosis by flow cytometry; C-erbB-2, IGF-1, IGF-1R and Akt mRNA and protein expressions by qRT-PCR and western blot; and cell invasion and migration by Transwell assay and scratch test. Tumor growth was observed in male BALB/c nude mice (Shanghai Experimental Animal Center) based on Eca109 cell implantation, raising, and measurement.ResultsC-erbB-2, IGF-1, IGF-1R and Akt expression were higher in EC tissues than those in adjacent tissues (all P < 0.05). Compared with blank control group, both si-C-erbB-2 and OSI-906 groups had decreased IGF-1, IGF-1R and Akt mRNA and protein expressions, decreased cell proliferation, migration and invasion, prolonged G0/G1 phase, shortened S phase, increased cell apoptosis, and inhibited tumor growth (all P < 0.05); while opposite trends were detected in C-erbB-2 vector and Rg5 groups (all P < 0.05), without statistical differences in siRNA C-erbB-2 + Rg5 group (all P > 0.05).ConclusionSilencing C-erbB-2 expression may inhibit EC cell proliferation, promote cell apoptosis and block cell cycle progression by inhibiting IGF-1 pathway activation. The beneficial effect of silencing C-erbB-2 expression can be reversed by promoting the activation of IGF-1 pathway. Findings in our study may provide potential reference for understanding the molecular mechanism of EC and supply possible axis for preventing the development of EC from the perspective of molecular biology.

Qigefang Inhibits Migration, Invasion, and Metastasis of ESCC by Inhibiting Gas6/Axl Signaling Pathway.

In recent years, there is an increasing interest in using Traditional Chinese Medicine (TCM) and their patents for the treatment of cancers. Qigefang (QGF) is a TCM formula and has been used for the treatment of metastatic esophageal cancer in China. However, its therapeutic effect on tumors and its mechanism of action is largely unknown. The aim of this study is to explore the role of QGF in the treatment of metastasis of Esophageal Squamous Cell Carcinoma( ESCC). Human esophageal carcinoma cell line KYSE150 was used for this study. CCK-8 assay was used to determine the cytotoxicity of QGF. The KYSE150 cells were treated with QGF to determine its effect on cell migration (cell scratch assay and imaging) and invasion (Transwell system based with Matrigel assay). Western blotting was used to investigate the effect of QGF on relevant molecules of signaling pathways. A mouse model of lung metastasis of esophageal cancer was established by injecting the KYSE150-Luc cells through the tail vein. A small animal imaging system was used to observe tumor metastasis in the mice. QGF reduced cell migration and invasion of KYSE150 cells. QGF significantly inhibited lung metastasis in nude mice. Further study revealed that the expression of Growth arrest-specific 6 (Gas6), Anexelekto (Axl), N-Nuclear factor-kappa B (NF-κB) and matrix metalloproteinase-9 (MMP-9) proteins were decreased both in vitro and in vivo upon treatment with QGF. QGF could prevent invasion and metastasis of esophageal cancer by inhibiting the Gas6/Axl signaling pathway.