Cell Lines KYSE150 Research Articles

Interleukin-8 and Interleukin-6 Are Biomarkers of Poor Prognosis in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common type of cancer characterized by fast progression and high mortality rates, which generally implies a poor prognosis at time of diagnosis. Intricate interaction networks of cytokines produced by resident and inflammatory cells in the tumor microenvironment play crucial roles in ESCC development and metastasis, thus influencing therapy efficiency. As such, cytokines are the most prominent targets for specific therapies and prognostic parameters to predict tumor progression and aggressiveness. In this work, we examined the association between ESCC progression and the systemic levels of inflammatory cytokines to determine their usefulness as diagnostic biomarkers. We analyzed the levels of IL-1β, IL-6, IL-8, IL-10, TNF-α e IL-12p70 in a group of 70 ESCC patients and 70 healthy individuals using Cytometric Bead Array (CBA) technology. We detected increased levels of IL-1β, IL-6, IL-8, and IL-10 in ESCC patients compared to controls. However, multivariate analysis revealed that only IL8 was an independent prognostic factor for ESCC, as were the well-known risk factors: alcohol consumption, tobacco usage, and exposure to pesticides/insecticides. Importantly, patients with low IL-6, IL-8, TNM I/II, or those who underwent surgery had a significantly higher overall survival rate. We also studied cultured Kyse-30 and Kyse-410 cells in mice. We determined that the ESCC cell line Kyse-30 grew more aggressively than the Kyse-410 cell line. This enhanced growth was associated with the recruitment/accumulation of intratumoral polymorphonuclear leukocytes. In conclusion, our data suggest IL-8 as a valuable prognostic factor with potential as a biomarker for ESCC.

RIG-I Promotes Tumorigenesis and Confers Radioresistance of Esophageal Squamous Cell Carcinoma by Regulating DUSP6.

We investigated the expression and biological function of retinoic acid inducible gene I (RIG-I) in esophageal squamous cell carcinoma (ESCC). Materials and methods: An immunohistochemical analysis was performed on 86 pairs of tumor tissue and adjacent normal tissue samples of patients with ESCC. We generated RIG-I-overexpressing ESCC cell lines KYSE70 and KYSE450, and RIG-I- knockdown cell lines KYSE150 and KYSE510. Cell viability, migration and invasion, radioresistance, DNA damage, and cell cycle were evaluated using CCK-8, wound-healing and transwell assay, colony formation, immunofluorescence, and flow cytometry and Western blotting, respectively. RNA sequencing was performed to determine the differential gene expression between controls and RIG-I knockdown. Tumor growth and radioresistance were assessed in nude mice using xenograft models. RIG-I expression was higher in ESCC tissues compared with that in matched non-tumor tissues. RIG-I overexpressing cells had a higher proliferation rate than RIG-I knockdown cells. Moreover, the knockdown of RIG-I slowed migration and invasion rates, whereas the overexpression of RIG-I accelerated migration and invasion rates. RIG-I overexpression induced radioresistance and G2/M phase arrest and reduced DNA damage after exposure to ionizing radiations compared with controls; however, it silenced the RIG-I enhanced radiosensitivity and DNA damage, and reduced the G2/M phase arrest. RNA sequencing revealed that the downstream genes DUSP6 and RIG-I had the same biological function; silencing DUSP6 can reduce the radioresistance caused by the overexpression of RIG-I. RIG-I knockdown depleted tumor growth in vivo, and radiation exposure effectively delayed the growth of xenograft tumors compared with the control group. RIG-I enhances the progression and radioresistance of ESCC; therefore, it may be a new potential target for ESCC-targeted therapy.

Raltitrexed enhanced antitumor effect of anlotinib in human esophageal squamous carcinoma cells on proliferation, invasiveness, and apoptosis

BackgroundAnlotinib is a multi-targeted receptor tyrosine kinase inhibitor (TKI) which has exhibited encouraging clinical activity in advanced non-small cell lung cancer (NSCLC) and soft tissue sarcoma. Raltitrexed is well known to be effective in the treatment of colorectal cancer in China. The present study aims to investigate the combinatory antitumor effect of anlotinib and raltitrexed on human esophageal squamous carcinoma cells and further explore the molecular mechanisms in vitro.MethodsHuman esophageal squamous cell lines KYSE-30 and TE-1 were treated with anlotinib or raltitrexed, or both, then cell proliferation was measured by MTS and colony formation assay; cell migration and invasion were detected by wound-healing and transwell assays; cell apoptosis rate was studied by flow cytometry and the transcription of apoptosis-associated proteins were monitored by quantitative polymerase chain reaction (qPCR) analysis. Finally, western blot was performed to check phosphorylation of apoptotic proteins after treatment.ResultsTreatment with raltitrexed and anlotinib showed enhanced inhibitory effects on cell proliferation, migration and invasiveness compared with raltitrexed or anlotinib monotherapy. Meanwhile, raltitrexed combined with anlotinib strongly increased cell apoptosis percentage. Moreover, the combined treatment down-regulated mRNA level of the anti-apoptotic protein Bcl-2 and invasiveness-associated protein matrix metalloproteinases-9 (MMP-9), while up-regulated pro-apoptotic Bax and caspase-3 transcription. Western blotting showed that the combination of raltitrexed and anlotinib could inhibit the expression of phosphorylated Akt (p-Akt), Erk (p-Erk) and MMP-9.ConclusionsThis study indicated that raltitrexed enhanced the antitumor effects of anlotinib on human ESCC cells by down-regulating phosphorylation of Akt and Erk, providing a novel treatment option for patients with esophageal squamous cell carcinoma (ESCC).

A1 adenosine receptor antagonist induces cell apoptosis in KYSE-30 and YM-1 esophageal cancer cell lines.

Adenosine A1 receptor (AA1R) has been shown to have an inhibitory effect on cell growth in several cancers; however, its function in esophageal cancer is still unclear. In this study, we examined the effect of AA1R on cell growth and apoptosis in esophageal cancer cells. In this study, YM-1 and KYSE-30 esophageal cancer cell lines were cultured. AA1R gene expression was determined by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). As well, the AA1R antagonist (DPCPX) effect on cell viability was evaluated by the MTT assay. Moreover, apoptosis was assessed by annexin-V and propidium iodide staining, and the caspase-3/7 activity assay kit. qRT-PCR results indicated that the AA1R was expressed in YM-1 and KYSE-30 cells. In addition, DPCPX significantly decreased cell proliferation in both cell lines. Furthermore, the A1AR antagonist induced apoptosis in KYSE-30 and YM-1 cells. After treatment of both cell lines with DPCPX, the caspase 3/7 activity was increased. Our finding indicates the AA1R antagonist induces apoptosis through caspase 3/7 activation and can be considered a potential target in esophageal cancer therapy.

LncRNA TPT1-AS1 promotes cell migration and invasion in esophageal squamous-cell carcinomas by regulating the miR-26a/HMGA1 axis.

lncRNA TPT1-AS1 plays an oncogenic role in ovarian and cervical cancers. However, its involvement in the pathological progress of esophageal squamous-cell carcinomas (ESCCs) is unclear. lncRNA TPT1-AS1 was mainly localized in the cytoplasm of ESCC cells and interacted with miR-26a. In ESCC tissues, lncRNA TPT1-AS1 level was obviously increased, while miR-26a level was decreased. Interestingly, lncRNA TPT1-AS1 level was not significantly correlated with miR-26a level but was positively correlated with HMGA1 mRNA, a target of miR-26a. In ESCC cell lines KYSE510 and KYSE-30, lncRNA TPT1-AS1 overexpression enhanced HMGA1 expression, while it had no effect on miR-26a expression. Cell migration and proliferation assays indicated that lncRNA TPT1-AS1 and HMGA1 overexpression promoted ESCC cell migration and invasion, while their effects were alleviated by miR-26a overexpression. The migration and invasion of ESCC cells were suppressed by lncRNA TPT1-AS1 knockdown. In conclusion, lncRNA TPT1-AS1 plays an oncogenic role in ESCC and might function by upregulating HMGA1 via sponging miR-26a.

MiR-139 Affects Radioresistance in Esophageal Cancer by Targeting the PDK1/AKT/Cyclin D1 Signaling Pathway.

We explored the mechanism by which miR-139 modulates radioresistance of esophageal cancer (EC). The radioresistant cell line KYSE150R was obtained from the parental KYSE150 cell line by fractionated irradiation (15×2 Gy; total dose of 30 Gy). The cell cycle was assessed by flow cytometry. A gene profiling study was conducted to detect the expression of genes related to the radioresistance of EC. In the KYSE150R line, flow cytometry revealed increased number of G1-phase cells and decreased number of G2-phase cells; the expression of miR-139 increased. Knockdown of miR-139 decreased radioresistance and changed the distribution of cell cycle phases in KYSE150R cells. Western blotting showed that miR-139 knockdown increased the expression levels of cyclin D1, p-AKT, and PDK1. However, PDK1 inhibitor GSK2334470 reversed this effect for p-AKT and cyclin D1 expression. A luciferase reporter assay indicated that miR-139 directly bound to the PDK1 mRNA 3'-UTR. Analysis of the clinical data from 110 patients with EC showed an association of miR-139 expression with the TNM stage and the effect of therapy. MiR-139 expression significantly correlated with EC and progression-free survival. In conclusion, miR-139 enhances the radiosensitivity of EC by regulating the cell cycle through the PDK1/Akt/Cyclin D1 signaling pathway.

Anti-cancer, Anti-collagenase and Anti-elastase Potentials of Some Natural Derivatives: In vitro and in silico Studies.

The anti-cancer activities of the compounds were evaluated against KYSE-150, KYSE-30, and KYSE-270 cell lines and also on investigated esophageal line HET 1 A as a standard. Modified inhibitory impact on enzymes of collagenase and elastase were used Thring and Moon methods, respectively. Among both compounds, both of them recorded impact on cancer cells being neutral against the control, both had IC50 lower than 100 µM and acted as a potential anticancer drug. The chemical activities of Skullcapflavone I and Skullcapflavone II against elastase and collagenase were investigated utilizing the molecular modeling study. IC50 values of Skullcapflavone I and Skullcapflavone II on collagenase enzyme were obtained 106.74 and 92.04 µM and for elastase enzyme were 186.70 and 123.52 µM, respectively. Anticancer effects of these compounds on KYSE 150, KYSE 30, and KYSE 270 esophageal cancer cell lines studied in this work. For Skullcapflavone I, IC50 values for these cell lines were obtained 14.25, 19.03, 25.10 µM, respectively. Also, for Skullcapflavone II were recorded 20.42, 34.17, 22.40 µM, respectively. The chemical activities of Skullcapflavone I and Skullcapflavone II against some of the expressed surface receptor proteins (CD44, EGFR, and PPARγ) in the mentioned cell lines were assessed using the molecular docking calculations. The calculations showed the possible interactions and their characteristics at an atomic level.

Inhibition of bromodomain-containing protein 4 enhances the migration of esophageal squamous cell carcinoma cells by inducing cell autophagy.

Esophageal squamous cell carcinoma (ESCC), the predominant type of esophageal cancer, has a 5-year survival rate less than 20%. Although the cause of poor prognosis is the high incidence and mortality of ESCC, the high rate of metastasis after esophageal cancer surgery is the main cause of death after the surgery. Bromodomain-containing protein 4 (BRD4), an epigenetic reader of chromatin-acetylated histones in tumorigenesis and development, plays an essential role in regulating oncogene expression. BRD4 inhibition and BRD4 inhibition-based treatment can potentially suppress ESCC growth. However, the effects and mechanisms of action of BRD4 on ESCC cell migration remain unclear. To explore the effect of BRD4 on cell migration of ESCC in vitro and its possible molecular mechanism. Human ESCC cell lines KYSE-450 and KYSE-150 were used. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was performed to examine cell proliferation, and the transwell migration assay was conducted to test ESCC cell migration. JQ1, a BRD4 inhibitor, was applied to cells, and BRD4 siRNA was transfected into ESCC cells to knockdown endogenous BRD4. GFP-RFP-LC3 adenovirus was infected into ESCC cells to evaluate the effect of JQ1 on autophagy. Western blotting was performed to determine the protein levels of BRD4, E-cadherin, vimentin, AMP-activated protein kinase (AMPK), and p-AMPK. BRD4 was either downregulated by small interfering RNA or pretreated with JQ1 in ESCC cells, leading to increased tumor migration in ESCC cells in a dose- and time-dependent manner. Inhibition of BRD4 not only significantly suppressed cell proliferation but also strongly increased cell migration by inducing epithelial-mesenchymal transition (EMT). The protein expression of vimentin was increased and E-cadherin decreased in a dose-dependent manner, subsequently promoting autophagy in KYSE-450 and KYSE-150 cells. Pretreatment with JQ1, a BRD4 inhibitor, inhibited BRD4-induced LC3-II activation and upregulated AMPK phosphorylation in a dose-dependent manner. Additionally, an increased number of autophagosomes and autolysosomes were observed in JQ1-treated ESCC cells. The autophagy inhibitor 3-methyladenine (3-MA) reversed the effects of BRD4 knockdown on ESCC cell migration and blocked JQ1-induced cell migration. 3-MA also downregulated the expression of vimentin and upregulation E-cadherin. BRD4 inhibition enhances cell migration by inducing EMT and autophagy in ESCC cells via the AMPK-modified pathway. Thus, the facilitating role on ESCC cell migration should be considered for BRD4 inhibitor clinical application to ESCC patients.

TWIST1 Plays Role in Expression of Stemness State Markers in ESCC

Stemness markers play critical roles in the maintenance of key properties of embryonic stem cells (ESCs), including the pluripotency, stemness state, and self-renewal capacities, as well as cell fate decision. Some of these features are present in cancer stem cells (CSCs). TWIST1, as a bHLH transcription factor oncogene, is involved in the epithelial-mesenchymal transition (EMT) process in both embryonic and cancer development. Our aim in this study was to investigate the functional correlation between TWIST1 and the involved genes in the process of CSCs self-renewal in human esophageal squamous cell carcinoma (ESCC) line KYSE-30. TWIST1 overexpression was enforced in the ESCC KYSE-30 cells using retroviral vector containing the specific pruf-IRES-GFP-hTWIST1 sequence. Following RNA extraction and cDNA synthesis, the mRNA expression profile of TWIST1 and the stem cell markers, including BMI1, CRIPTO1, DPPA2, KLF4, SOX2, NANOG, and MSI1, were assessed using relative comparative real-time PCR. Ectopic expression of TWIST1 in KYSE-30 cells resulted in an increased expression of TWIST1 compared to control GFP cells by nearly 9-fold. Transduction of TWIST1-retroviral particles caused a significant enhancement in BMI1, CRIPTO1, DPPA2, KLF4, and SOX2 mRNA expression, approximately 4.5-, 3.2-, 5.5-, 3.5-, and 3.7-folds, respectively, whereas this increased TWIST1 expression caused no change in the mRNA expression of NANOG and MSI1 genes. TWIST1 gene ectopic expression in KYSE-30 cells enhanced the level of cancer stem cell markers' mRNA expression. These results may emphasize the role of TWIST1 in the self-renewal process and may corroborate the involvement of TWIST1 in the stemness state capacity of ESCC cell line KYSE-30, as well as its potential as a therapeutic target.

TWIST1 activates cancer stem cell marker genes to promote epithelial-mesenchymal transition and tumorigenesis in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide. Overexpression of EMT master transcription factors can promote differentiated cells to undergo cancer reprogramming processes and acquire a stem cell-like status.MethodsThe KYSE-30 and YM-1 ESCC cell lines were transduced with retroviruses expressing TWIST1 or GFP and analyzed by quantitative reverse transcription PCR (qRT-PCR), chromatin immunoprecipitation (ChIP), and immunostaining to investigate the correlation between TWIST1 and stemness markers expression. Cells expressing TWIST1 were characterized for mRNA candidates by qRT-PCR and for protein candidates by Flow cytometry and Immunocytochemistry. TWIST1-ESCC cells were also evaluated for apoptosis and drug resistance.ResultsHere we identify a role for TWIST1 in the establishment of ESCC cancer stem cell (CSC)-like phenotype, facilitating the transformation of non-CSCs to CSCs. We provide evidence that TWIST1 expression correlates with the expression of CSC markers in ESCC cell lines. ChIP assay results demonstrated that TWIST1 regulates CSC markers, including CD44, SALL4, NANOG, MEIS1, GDF3, and SOX2, through binding to the E-box sequences in their promoters. TWIST1 promoted EMT through E-cadherin downregulation and vimentin upregulation. Moreover, TWIST1 expression repressed apoptosis in ESCC cells through upregulation of Bcl-2 and downregulation of the Bax protein, and increased ABCG2 and ABCC4 transporters expression, which may lead to drug resistance.ConclusionsThese findings support a critical role for TWIST1 in CSC-like generation, EMT progression, and inhibition of apoptosis in ESCC. Thus, TWIST1 represents a therapeutic target for the suppression of esophageal cell transformation to CSCs and ESCC malignancy.

Isolation and characterization of head and neck cancer-derived peritumoral and cancer-associated fibroblasts.

Head and neck squamous cell carcinomas (HNSCC) are characterized by strong cellular and molecular heterogeneity and treatment resistance entailing poor survival. Besides cell-intrinsic properties, carcinoma cells receive important cues from non-malignant cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a major component of the TME that impact on the molecular make-up of malignant cells and have a decisive function in tumor progression. However, the potential functionality of fibroblasts within tumor-adjacent, macroscopically normal tissue remains poorly explored. Here, we isolated primary peritumoral fibroblasts (PtFs) from macroscopically normal tissue in vicinity of primary human papillomavirus-negative and -positive oropharyngeal HNSCC and compared their phenotype and functionality with matched CAFs (n = 5 pairs) and with human oral fibroblasts (hOFs). Expression patterns of CD90, CD73, CD105, smooth muscle actin, Vimentin, and S100A4 were comparable in PtFs, CAFs, and hOFs. Cell proliferation and doubling times of CAFs and PtFs were heterogeneous across patients (n =2 PtF>CAF; n = 1 CAF>PtF; n = 2 CAF=PtF) and reflected inferior growth than hOFs. Furthermore, PtFs displayed an reduced heterogeneity in cell size compared to matched CAFs, which were characterized by the presence of single large cells. Overall, conditioned supernatants from CAFs had more frequently growth-promoting effects on a panel of carcinoma cell lines of the upper aerodigestive tract carcinoma cell lines (Cal27, Cal33, FaDu, and Kyse30), whereas significant differences in migration-inducing effects demonstrated a higher potential of PtFs. Except for Kyse30, CAFs were significantly superior to hOFs in promoting proliferation, while PtFs induced stronger migration than hOFs in all carcinoma lines tested. Analysis of soluble factors demonstrated significantly increased VEGF-A production in CAFs (except in pat.8), and significantly increased PDGF-BB production in PtFs of two patients. Tube formation assays confirmed a significantly enhanced angiogenic potential of conditioned supernatants from CAFs compared to hOFs on human umbilical vascular endothelial cells (HUVECs) in vitro. Hence, matched CAFs and PtFs present in HNSCC patients are heterogeneous in their proliferation-, migration-, and angiogenesis-promoting capacity. Despite this heterogeneity, CAFs induced stronger carcinoma cell proliferation and HUVEC tube formation overall, whereas PtFs promoted migration of tumor cells more strongly.

MIR503HG promotes esophageal squamous cell carcinoma cell proliferation, invasion and migration via hsa-miR-503 pathway

Objective: To explore the function and mechanism of long non-coding RNA MIR503HG in esophageal squamous cell carcinoma (ESCC). Methods: The MIR503HG expression data in 60, 119 and 23 cases of ESCC and their paired adjacent tissues were chosen from three ESCC datasets GSE53622, GSE53624 and GSE130078, respectively. The expression data of MIR503HG in 81 ESCC tissues and 271 unpaired normal esophageal tissues were screened from the combined dataset of Cancer Genome Atlas and Genotype-Tissue Expression Database (TCGA+ GTEx). The MIR503HG knockdown plasmid was constructed, packaged into lentivirus. The lentivirus was used to infect with esophageal squamous cell carcinoma cell lines KYSE30 and KYSE510 to screen out the stable MIR503HG knockdown cell lines. ESCC cell line KYSE30 was transiently transfected with miRNA mimics to overexpress hsa-miR-503-3p and hsa-miR-503-5p.The expression levels of MIR503HG, hsa-miR-503-3p and hsa-miR-503-5p were detected by quantitative real-time polymerase chain reaction. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The invasion and migration ability of the cells were detected by Transwell assay. Cell cycle was detected by flow cytometry. The effect of MIR503HG on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Results: Both GEO and TCGA+ GTEx databases showed that the expression of MIR503HG in ESCC tissues was higher than that in adjacent tissues and normal esophageal tissues (P<0.01). Compared with shNC group, the proliferation rates of KYSE30 and KYSE510 cells after knockdown of MIR503HGwere significantly inhibited (P<0.001). The colony formation numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were (2.00±1.41) and (1.33±0.47), respectively, significantly lower than that of the shNC group (P=0.002). The clone formation numbers of KYSE510 cells in shMIR503HG1 group and shMIR503HG2 group were (174.67±15.97) and (80.33±6.34), respectively, significantly lower than that of the shNC group (P<0.001). The invasive numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 75.33±6.02 and 45.67±7.59, significantly lower than that of the shNC group(P<0.001). The migrating number of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 244.00±10.23 and 210.67±13.52, significantly lower than that of the shNC group(P<0.001), and the cell cycle was arrested in G(0)/G(1) phase. The xenograft experiment showed that the subcutaneous tumor in shMIR503HG group was significantly smaller than that in shNC group, and the tumor weight in shMIR503HG group was (0.097±0.026) g, which was lower than (0.166±0.021) g in shNC group (P<0.001). After knockdown of MIR503HG, the relative expression levels of hsa-miR-503-3p in KYSE30 cells of shMIR503HG1 group and shMIR503HG2 group were 0.66±0.02 and 0.58±0.00, respectively, the relative expression levels of hsa-miR-503-5p were 0.64±0.00 and 0.68±0.03, respectively, which were all lower than those in shNC group (P<0.01). After knockdown of MIR503HG, overexpression of hsa-miR-503-3p and hsa-miR-503-5p attenuated the inhibitory effects of knockdown of MIR503HG on proliferation (P<0.001), invasion (P<0.01) and migration (P<0.001) of KYSE30 cells. Conclusions: MIR503HG promotes the proliferation, invasion and migration of ESCC cells by regulating hsa-miR-503 pathway and can be used as a new potential target for targeted therapy of ESCC.

Artemisinin inhibits the development of esophageal cancer by targeting HIF-1α to reduce glycolysis levels.

China has a high incidence of esophageal cancer (EC), mainly squamous cell carcinoma, which is a serious threat to human life. Previous studies have shown that artemisinin can inhibit the proliferation and metastasis of cancer cells, thus inhibiting the progression of cancer. Aerobic glycolysis plays an important role in the uncontrolled growth of tumor cells. However, there are still different opinions on the anti-cancer mechanism, and there have been few studies involving EC. Our pre-experiment found that artemisinin can inhibit the progression of EC by directly regulating aerobic glycolysis. The EC cell lines KYSE-150 and KYSE-170 were used to detect the effects of artemisinin on cell viability, proliferation, metastasis, and aerobic glycolysis. Network pharmacology technology was used to explore the potential molecular mechanism of artemisinin inhibiting the development of EC through aerobic glycolysis and the findings were verified by molecular docking. Artemisinin could inhibit the proliferation, metastasis, and glycolysis of esophageal squamous cell carcinoma (ESCC), and this was verified by the expression of key metastatic proteins (N-cadherin) and key enzymes of glycolysis [hypoxia-inducible factor-1α (HIF-1α), pyruvate kinase M2 (PKM2)]. Through network pharmacology, we found the potential therapeutic target of artemisinin, HIF-1α. The results of molecular docking showed that artemisinin could directly target HIF-1α and promote its degradation. Artemisinin can target HIF-1α to reduce the level of glycolysis and inhibit the development of EC, which may become a targeted drug for the treatment of EC.

CBX4 contributes to radioresistance by regulating autophagic activity in esophageal squamous cell carcinoma.

BackgroundThe function of Chromobox 4 (CBX4) function has attracted attention in many cancer types due to its unique biological role; however, its mechanism in esophageal squamous cell carcinoma (ESCC) under radiotherapeutic treatment has not yet been investigated.MethodsSilencing of CBX4 was carried out in TE-13 and KYSE-150 cell lines. Cell proliferation, radiosensitivity, DNA damage, apoptosis, and cell cycle distribution were determined by Cell Counting Kit-8 (CCK-8), colony formation assay, immunofluorescence, flow cytometry, and immunoblot in vitro. In vivo xenograft models were also used to assess tumor cell growth and radioresistance. The underpinning mechanisms were explored based on pathway analysis and confirmed by rescue experiments, detecting cellular autophagy.ResultsKnockdown of CBX4 resulted in reduced tumor growth and enhanced radio-response in vivo and in vitro. Down-regulating CBX4 increased DNA damage, apoptotic rate, and G2/M arrest induced by radiation in ESCC cell lines. Gene Set Enrichment Analysis (GSEA) revealed that CBX4 was associated with cellular autophagy regulation. Enhanced radiosensitivity in ESCC cells silenced for CBX4 was partially blocked by autophagy inhibition (P<0.05). Beclin 1 was upregulated at the gene and protein levels in ESCC cells with CBX4 knockdown after irradiation, and overexpressing Beclin 1 reversed the radiosensitivity of ESCC cells with CBX4 knockdown (P<0.05).ConclusionsBy regulating autophagic activity, CBX4 contributes to radioresistance. Targeting CBX4 might constitute an efficient approach for increasing radiosensitivity in ESCC.

Intracellular glutathione levels affect the outcomes of verteporfin-mediated photodynamic therapy in esophageal cancer cells

Photodynamic therapy (PDT) induces cancer cell death by generating reactive oxygen species (ROS). In this process, photosensitizers accumulate in cancer cells irradiated by laser light of a specific wavelength, leading to ROS generation. Verteporfin (VP), a second-generation photosensitizer, is used in PDT for age-related macular degeneration. However, the antitumor effects of VP-PDT remain poorly defined. This study investigated the antitumor effects of VP-PDT on esophageal cancer (EC) cell lines in vitro. Two types of EC cell lines, the KYSE30 cell line, derived from highly differentiated esophageal carcinoma, and the KYSE170 cell line, derived from moderately differentiated carcinoma, were used in this study. VP-PDT exerted effective anticancer effects in both cell lines. Our results revealed that the low-density lipoprotein receptor, albumin receptor, and heme carrier protein-1 in VP uptake were not involved in VP uptake. However, cells rich in intracellular glutathione were resistant to VP-PDT. Our study outcomes suggest that lowering intracellular glutathione via a glutathione synthesis inhibitor or sulfasalazine can increase the effectiveness of VP-PDT-mediated anticancer effects.

Synergistic effects of BAY606583 on docetaxel in esophageal cancer through modulation of ERK1/2.

Docetaxel (DTX) is a taxane chemotherapy agent used to treat many types of cancers, including esophageal squamous cell carcinoma. Adenosine is a purinergic signaling molecule that contributes to cancercellproliferation via A2B adenosine receptor (A2BAR) activation. Extracellular signal-regulated protein kinase (ERK) plays a crucial role in cell proliferation in various types of cancers. Stimulation of A2BAR involves a regulated ERK signaling pathway, and might provide a fascinating approach for treatment, leading to decreased proliferation in certain tumors that express A2BAR. Recent studies demonstrated that DTX and A2BAR have anticancer effects. The current study was designed to investigate the synergistic effect of the A2BAR agonist (BAY606583) on DTX in inducing antiproliferation effects on esophageal squamous cells carcinoma (ESCCs). The cell viability was assessed using the MTT assay in KYSE-30 and Ym-1 cells. In addition, the synergistic effect of DTX on the A2BAR agonist was evaluated. Subsequently, apoptosis was assessed by Annexin-V and propidium iodide staining, and Bcl-2, Bax, and ERK1/2 protein-level expressions were evaluated by Western blot. Use of BAY606583 and cotreatment of DTX and BAY606583 significantly decreased cell proliferation in KYSE-30 and Ym-1 cell lines. The use of BAY606583 and cotreatment of DTX with the A2BAR agonist induced apoptosis in KYSE-30 and Ym-1 cells. Western blot analysis revealed that the use of the A2BAR agonist and cotreatment of DTX with the A2BAR agonist inhibited the expression of apoptotic regulatory proteins as well as the expression of ERK1/2 proteins. Our findings suggested that use of BAY606583 and cotreatment of BAY606583/DTX have an antiproliferative effect on ESCC cell lines through ERK signaling pathway inhibition. BAY606583 has a synergistic effect on DTX, which could be used as an adjuvant for esophageal cancer therapy.

An In Vivo Study of Lactobacillus rhamnosus (PTCC 1637) as a New Therapeutic Candidate in Esophageal Cancer.

Objective This study is aimed at investigating the effect of probiotic Lactobacillus rhamnosus on esophageal cancer in vivo and in vitro. Methods and Results In this study, the cytotoxicity effects of L. rhamnosus supernatant and whole-cell culture on a cancer cell line (Kyse30) compared to 5fu were evaluated by the MTT assay. The real-time PCR method was used to analyse the L. rhamnosus supernatant effect on the expression of Wnt signaling pathway genes. An in vivo investigation in nude mice was done to assess the anti-tumor activity of L. rhamnosus supernatant and whole-cell culture. Both supernatant and whole-cell culture of L. rhamnosus reduced cell survival (Kyse30) P < 0.001. The supernatant of this bacterium significantly reduced the expression of Wnt signaling pathway genes. Administration of supernatant and whole-cell culture of L. rhamnosus expressively reduced tumor growth compared to the control group. The effects of this bacterium on tumor necrosis were quite evident, pathologically P < 0.01. Conclusion This study is the first report that assessed the potential impact of L. rhamnosus, especially its supernatant on esophageal cancer and Wnt signaling pathway genes. Therefore, this bacterium can be a harmless candidate for esophageal cancer therapy.

Construction and Quantitative Evaluation of a Tissue-Specific Sleeping Beauty by EDL2-Specific Transposase Expression in Esophageal Squamous Carcinoma Cell Line KYSE-30.

Gene delivery to esophageal tissue could provide novel treatments for diseases, such as cancer. The Sleeping Beauty (SB) transposon system, as a natural and non-viral tool, is efficient at transferring transgene into the human genome for human cell genetic engineering. The plasmid-based SB transposon can insert into chromosomes through an accurate recombinase-mediated mechanism, providing long-term expression of transgene integrated into the target cells. In this study, we aimed to investigate the activity of ED-L2 tissue-specific promoter that was engineered from the Epstein-Barr Virus (EBV) and combined with the hyperactive SB100X transposase to achieve the stable expression of T2-Onc3 transposon in esophageal squamous epithelial cells. Here we constructed an SB transposon-based plasmid system to obtain the stable expression of transposon upon introduction of a hyperactive SB transposase under the control of tissue-specific ED-L2 promoter via the lipid-based delivery method in the cultured esophageal squamous cell carcinoma cells. Among established human and mouse cell lines, the (ED-L2)-SB100X transposase was active only in human esophageal stratified squamous epithelial and differentiated keratinocytes derived from skin (KYSE-30 and HaCaT cell lines), where it revealed high promoter activity. Data offered that the 782bp sequence of ED-L2 promoter has a key role in its activity in vitro. The (ED-L2)-SB100X transposase mediated stable integration of T2-Onc3 in KYSE-30 cells, thereby providing further evidence of the tissue specificity of ED-L2 promoter. The KYSE-30 cells modified with the SB system integrate on average 187 copies of the T2-Onc3 transposon in its genome. In aggregate, the (ED-L2)-SB100X transposase can be efficiently applied for the tissue-specific stable expression of a transgene in human KYSE-30 cells using SB transposon.

Therapeutic benefits of niraparib tosylate as radio sensitizer in esophageal squamous cell carcinoma: an in vivo and in vitro preclinical study

PurposeEsophageal squamous cell carcinoma is associated with high morbidity and mortality rate for which radiotherapy is the main treatment modality. Niraparib, a Poly (ADP-ribose) polymerase 1 inhibitors (PARPi) was previously reported to confer radiosensitivity in different malignancies including non-small cell lung cancer. In this study, we assessed the in vivo ability of niraparib in conferring radiosensitivity to esophageal squamous cell carcinoma cells.Materials and methodsIn this study, KYSE-30 and KYSE-150 cell lines were selected as in vivo esophageal squamous cell carcinoma models. The experimental groups were: niraparib tosylate alone, radiotherapy alone, control (no intervention), and combination therapy (radiotherapy + niraparib tosylate). Cell cytotoxicity assay, colony formation assay, flow cytometry, immunofluorescence, Western blotting, immunohistochemistry, lentivirus transfection analysis, and xenograft models were used for confirming radiosensitizing ability of niraparib and to investigate the possible cellular mechanism involved in radiosensitization.ResultsThe colony formation efficiency of the combination group was significantly much lower than that of the single radiation group (P < 0.01). Cell cytotoxicity assay demonstrated a significant reduction in proliferation of irradiated cells after treatment with niraparib tosylate compared to niraparib tosylate alone (P < 0.01). Cell apoptosis significantly increased in the combination group compared to either niraparib tosylate or radiotherapy alone (P < 0.01). Rate of tumor suppression rate was significantly high in the combined treatment group (P < 0.01) but, significantly decreased in nude mice. Western blot and lentivirus infection model suggested overexpression of FANCG genes to confer radiosensitivity.ConclusionThese results suggest that the synergistic effect of niraparib tosylate and radiation may be related to the down-regulation of FANCG.

Serine 727 phosphorylation is necessary to induce the STAT3-mediated transcription of LINC00184 in oesophageal squamous cell carcinoma.

LINC00184 has been suggested to be associated with cancer prognosis and has been implicated in cancer glycolysis; however, its role in oesophageal squamous cell carcinoma (ESCC) remains poorly understood. Herein, to understand the expression and the biological roles of LINC00184 in ESCC, in situ hybridization (ISH) and quantitative PCR (qPCR) were performed to detect the expression of LINC00184 in tissue blocks and in fresh tissues, respectively. Furthermore, with an in vitro cell culture system, LINC00184 was stably knocked down in ESCC cell lines KYSE-150 and Eca109, followed by determining alterations in their proliferation and motility relative to control. To gain insight into the regulation of LINC00184, STAT3 was bioinformatically identified as a transcription factor of LINC00184, which was further corroborated by chromatin-immunoprecipitation (CHIP) assay. The dephosphorylation of STAT3 with NSC74859 was shown to be unable to suppress the expression of LINC00184 in vivo in a xenograft mouse model. Moreover, STAT3, once phosphorylated at serine 727, tended to translocate into the mitochondria to promote LINC00184 expression in ESCC cells. Together, these data strongly support the oncogenic role of LINC00184 in ESCC.