Human Esophageal Cancer Cell Lines Research Articles

Radiosensitization of HER2-positive esophageal cancer cells by pyrotinib.

Radiation therapy is a widely used treatment for esophageal cancer. However, radiation resistance might result in a poor prognosis. Overexpression of HER2 has been related to adaptive radiation resistance. Pyrotinib is a HER2 inhibitor that shows an anti-tumor effect in breast cancer. The present study aims to explore the influence of pyrotinib combined with radiotherapy on HER2-positive esophageal cancer cells and explore the underlying mechanism. We screened two cell lines (TE-1 and KYSE30) that highly express HER2 from several human esophageal cancer cell lines. Cells were treated with pyrotinib or/and radiation. Cell proliferation, cell cycle distribution, and cell migration were measured. The protein levels involved in cell cycle and DNA repair were measured by Western blot. Results showed that pyrotinib inhibited HER2 activation and exerted an anti-proliferative effect in TE-1 and KYSE30 cells. Furthermore, it enhanced the anti-proliferative effect of radiation in these two cell lines. These effects might be via inhibiting HER2 phosphorylation, inducing G0/G1 arrest, and reducing EMT and DNA repair. Our results indicated that pyrotinib sensitivitied HER2 positive esophageal cancer cells to radiation treatment through various mechanisms. These findings may provide a new therapeutic strategy for treating HER2 positive esophageal cancer.

FOXM 1 induces Vasculogenic mimicry in esophageal cancer through \u03b2-catenin /Tcf4 signaling

ObjectiveTo investigate the role of FOXM1, β-catenin and TCF4 in esophageal cancer (EC) and their relationship to VM (Vasculogenic Mimicry).MethodsCCK-8 were performed to examine EC cell proliferation in FOXM1 silenced cells. EC cell migration and invasion were investigated through wound healing and Transwell assays, respectively. The formation of pipe like structures were assessed in 3D cultures. The expression of Foxm1, β-catenin, Tcf4 and E-cadherin were investigated through western blot, RT-qPCR and immunohistochemistry (IHC) staining. The relationship between FOXM1 expression, clinic-pathological features, and overall survival (OS) were further analyzed.ResultsA loss of FOXM1 expression correlated with the OS of ESCC patients. FOXM1 silencing led to a loss of cell growth and suppressed cell migration and invasion in ESCC cells. VM structures were identified in ESCC tissues and human EC cell lines. Mechanistically, FOXM1 was found to promote tumorigenesis through the regulation of β-catenin, Tcf4, and E-cadherin in EC cells, leading to the formation of VM structures.ConclusionsThese findings highlight FoxM1 as a novel therapeutic target in ESCC.

In vitro antitumor and immunotropic activity of carrageenans from red algae Chondrus armatus and their low-molecular weight degradation products.

Antitumor and immunotropic effects of κ-, λ-carrageenan from red marine algae Chondrus armatus and their low-molecular weight (LMW) degradation products were explored. Effects on human esophageal cancer cell lines KYSE30 and FLO1 viability and ability to induce production of pro- and anti-inflammatory cytokines by human monocytes was assessed. All polysaccharides demonstrated antimetabolic and cytostatic activity towards cancer lines, with high-molecular weight carrageenans possessing higher antimetabolic and lower cytostatic activity than their LMW degradation products. All carrageenans induced monocytes to produce pro-inflammatory cytokines IL1β, IL6, IL18, and TNFα. However, secretion of anti-inflammatory cytokine IL10 was induced only by LMW λ-carrageenan, which exhibited the highest cytokine production inducing efficacy overall. We demonstrate that LMW carrageenan degradation products not only retain biological activity of their precursors, but also increase their efficacy in type-dependent manner, allowing for their future development for pharmacological practice.

Primary impact of Gli1 on radioresistance in esophageal cancer.

Radioresistance is the primary cause for the low efficacy of radiotherapy in the treatment of esophageal cancer (EC). Increasing evidence has demonstrated that the Sonic Hedgehog (Shh) signaling pathway may be involved in the pathology of various tumors, including EC. The present study aimed to examine the association between radioresistance in EC and the Sonic Hedgehog pathway, and to determine whether a downstream transcription factor of the Shh pathway, glioma-associated oncogene family zinc finger 1 (Gli1), serves a primary role in radioresistance. The radiation-resistant cell line Eca109R was established by repeated low dose (cumulative dose 60 Gy) irradiation of the human EC cell line Eca109. The level of cell radiosensitivity was determined by colony formation assay, and the localization of Gli1 was detected using immunofluorescence. Western blotting was used to determine the protein expression levels of Gli1, Shh, patched 1 (Ptch) and smoothened frizzled class receptor (Smo) in the two cell lines. Significantly higher levels of Gli1 were identified in the Eca109R cell line compared with those inEca109 cells (P<0.05). Additionally, western blotting analysis demonstrated an increased expression level of the Gli1, Shh, Ptch and Smo proteins in Eca109R, compared with Eca109 cells (P<0.05). Overexpression of Gli1 in the parental cell line led to decreased levels of radiosensitivity and radiosensitivity of the radioresistant cell line was restored through knockdown of Gli1. The present study demonstrated that Gli1 may be associated with the development of radioresistance in EC.

Synthesis and anticancer evaluation of benzo-N-heterocycles transition metal complexes against esophageal cancer cell lines

Three novel transition metal complexes, Cu(p-2-bmq)Cl2 (1), Zn(p-2-bmq)Cl2 (2) and [Co(p-2-bmq)Cl2]2 (3) (where p-2-bmq = 2-((1-(pyridin-2-yl)-1H-benzoimidazol-2-yl)methyl) quinolone, have been synthesized. The complexes were detected for their cytotoxicity in vitro against four human esophageal cancer cell lines (SMMC7721, BGC823, HCT116 and HT29) by MTT assay. The results showed that they all have anti-tumor cell proliferation activity. E specially, complex 1 exhibited significant cytotoxicity with IC50 value of 15.89 μM against SMMC7721 cells for 72 h. The morphological changes of nuclei by fluorescence staining methods proved that complex 1 could induce intracellular DNA damage. The flow cytometry analysis revealed that the treatment of SMMC7721 cells with complex 1 induced intracellular ROS increased, mitochondrial potential collapse, G2/M-phase arrest, and even apoptosis. These studies should highly valuable for the development of transition metal-based compounds to the potential anticancer medicinal applications.

Facile fabrication of Cu9-S5 loaded core-shell nanoparticles for near infrared radiation mediated tumor therapeutic strategy in human esophageal squamous carcinoma cells nursing care of esophageal cancer patients

Copper chalcogenides have been exhibited to be an encouraging photothermal operator because of their great photothermal transformation proficiency, engineered effortlessness, and ease. Notwithstanding, the hydrophobic and low biocompatibility attributes related with their manufactured procedures hamper broadly natural applications. An elective methodology for improve hydrophilic nature and biocompatibility to coating into the copper-based chalcogenide nanostructures containing core shell silica materials. In this manuscript, the level headed planning configuration results in effective covering silica nanostructures onto the synthesized Cu9S5 to form Cu9S5@MS core-shell nanostructures. The structural formation and nanostructures of prepared nanomaterials with core shell structure were confirmed via analysis of transmission microscopic and particles distribution investigates, which infers that Cu9S5@MS has been organized by nano level with high stability. Also, the formation of Cu9S5@MS was confirmed by UV–Visible and X-ray techniques. As-prepared Cu9S5@MS nanovesicles display good biocompatibility, and are successfully utilized for photothermal removal of disease cells and NIR therapy. Additionally, the mode of cell death in esophageal squamous carcinoma cells were monitored various staining techniques (AO and EB, nuclear staining and flowcytometry). Further, we evaluated by the human esophageal squamous cancer cell lines to observe cell cycle arrest ability. Significantly, we demonstrate the combination of photothermal and chemotherapeutic techniques through the prepared nanovesicles exhibits outstanding impacts in the treatment of esophageal cancer therapies in vitro and in vivo.

Garcinol inhibits esophageal cancer metastasis by suppressing the p300 and TGF-β1 signaling pathways

Metastasis causes the main lethality in esophageal cancer patient. Garcinol, a natural compound extracted from Gambogic genera, is a histone acetyltransferase (HAT) inhibitor that has shown anticancer activities such as cell cycle arrest and apoptosis induction. In this study, we investigated the effects of garcinol on the metastasis of esophageal cancer in vitro and in vivo. We found that garcinol (5–15 μM) dose-dependently inhibited the migration and invasion of human esophageal cancer cell lines KYSE150 and KYSE450 in wound healing, transwell migration, and Matrigel invasion assays. Furthermore, garcinol treatment dose-dependently decreased the protein levels of p300/CBP (transcriptional cofactors and HATs) and p-Smad2/3 expression in the nucleus, thus impeding tumor cell proliferation and metastasis. Knockdown of p300 could inhibit cell metastasis, but CBP knockdown did not affect the cell mobility. It has been reported that TGF-β1 stimulated the phosphorylation of Smad2/3, which directly interact with p300/CBP in the nucleus, and upregulating HAT activity of p300. We showed that garcinol treatment dose-dependently suppressed TGF-β1-activated Smad and non-Smad pathway, inhibiting esophageal cancer cell metastasis. In a tail vein injection pulmonary metastasis mouse model, intraperitoneal administration of garcinol (20 mg/kg) or 5-FU (20 mg/kg) significantly decreased the number of lung tumor nodules and the expression levels of Ki-67, p300, and p-Smad2/3 in lung tissues. In conclusion, our study demonstrates that garcinol inhibits esophageal cancer metastasis in vitro and in vivo, which might be related to the suppression of p300 and TGF-β1 signaling pathways, suggesting the therapeutic potential of Garcinol for metastatic tumors.

Abstract LB-310: Overcoming resistance of conventional therapies by targeting cancer-associated fibroblasts (CAFs) with near-infrared photoimmunotherapy (NIR-PIT)

Abstract Esophageal cancer is known to one of the most aggressive malignant tumors, however, the effect of conventional chemotherapy and radiotherapy is insufficiently. Cancer-associated fibroblasts (CAFs) are thought to play an essential role in cancer invasion, migration, metastasis, immunosuppression, and resistance to anticancer drugs. Previously, we have developed a novel near-infrared photoimmunotherapy (NIR-PIT) targeting CAFs, which induces the selective destruction by targeting specific surface protein of CAFs; fibroblast activation protein (FAP). The aim of this study is to analyze the influence of CAFs for human esophageal cancer and to evaluate the improvement of conventional therapeutic resistance using NIR-PIT targeting CAFs.We produced the IR700-FAP antibody for this study. Human esophageal cancer cell lines (TE4 and OE19) and FEF3 human fibroblasts were used. At first, we demonstrated that cancer cells stimulated by CAFs acquired resistance to conventional therapies by cell viability assay. Furthermore, invasion, migration, and colony-formation assays demonstrated the CAF-stimulated cancer cells had a more malignant phenotype. Western blotting analysis showed that E-cadherin expression was decreased and vimentin expression was increased in CAF-stimulated cancer cells, indicating the epithelial mesenchymal transition induction. In vivo experiments demonstrated that subcutaneous tumors co-injected with CAFs were more refractory to chemotherapy than the tumors without CAFs as same as observed in vitro. Secondary, we found that acquired therapeutic resistance in stimulated cancer cells were restored by excluding continuous stimulation of CAFs. Finally, we investigated whether depleting CAFs by NIR-PIT could affect the chemo-resistance of cancer cells in vitro and in vivo. To evaluate the combination effect of conventional chemotherapy and NIR-PIT in vivo, xenograft models co-injected TE4 cells with CAFs were treated with 5-FU plus NIR-PIT, 5-FU alone, or PBS as control. 5-FU alone therapy could not suppress tumor growth, however combination therapy as 5-FU plus NIR-PIT could suppress tumor growth compared to control significantly. These results demonstrated that CAFs gave human esophageal cancer cells higher malignant and resistant phenotype, which could be overcome by using NIR-PIT targeting CAFs. Targeting fibroblast itself is a unique strategy and can be clinically promising as combination targeting cancer cells and their fundamental microenvironment, CAFs. Citation Format: Satoshi Komoto, Kazuhiro Noma, Ryoichi Katsube, Takuya Kato, Toshiaki Ohara, Hiroaki Sato, Toru Narusaka, Noriyuki Nisiwaki, Hiroshi Tazawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Overcoming resistance of conventional therapies by targeting cancer-associated fibroblasts (CAFs) with near-infrared photoimmunotherapy (NIR-PIT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-310.

Diverse Polyphenols from Hypericum faberi

Six new polyphenols with different isoprenylated xanthones, isoprenylated acylphloroglucinols, and chromone architectures, hyperfaberols A–F (1–6), were isolated from the whole plants of Hypericum faberi along with seven other related known compounds. In which hyperfaberols A/B (1/2) and 12–13 were isoprenylated xanthones, hyperfaberols C–E (3–5) and 8–11 were seven isoprenylated acylphloroglucinol derivatives, while 6–7 were two chromones. Their structures were elucidated by comprehensive analysis of their spectroscopic data as well as detailed comparison with the literature data. Compounds 1 and 11 showed cytotoxities against the human esophageal cancer cell line (ECA-109) and the pancreatic tumor cell line (PANC-1) in vitro, respectively.Graphical

Investigation of programmed death-ligand 1 expression and its contribution to the epithelial-to-mesenchymal transition in human esophageal cancer cell line Eca-109

Objective To investigate the contribution of programmed death-ligand 1 (PD-L1) on epithelial-to-mesenchymal transition (EMT) in esophageal cancer cell line Eca-109. Methods We manipulated PD-L1 molecule in Eca-109 by using lentiviral transfection to construct LV-PD-L1-short hairpin RNA (shRNA), PD-L1 over-expression (LV-PD-L1-WT-OE), PD-L1 truncated for cytoplasmic fragment overexpression (LV-PD-L1-TN-OE), LV-NC and LV-Vector-Ctrl cell lines of PD-L1 deletion type overexpression (LV-PD-L1-TN-OE). The expression of E-cadherin (E-cad), N-cadherin (N-cad), Zinc Finger E-Box Binding Homeobox 1 (Zeb1) and Vimentin (Vim) were detected by Real-time polymerase chain reaction (PCR) and the protein expression levels were detected by Western blotting. Results LV-PD-L1-shRNA, LV-PD-L1-WT-OE, LV-PD-L1-TN-OE, LV-NC and LV-Vector-Ctrl cell lines were successfully established. When we compared LV-PD-L1-shRNA to LV-NC cells, the mRNA level of E-cad (P<0.05) was significantly increased while N-cad (P<0.01), Zeb1 (P<0.01) and Vim (P<0.01) were significantly decreased. When we compared LV-PD-L1-WT-OE to LV-Vector-Ctrl, the mRNA level of E-cad (P<0.01) was significantly decreased while N-cad (P<0.01), Zeb1 (P<0.01) and Vim (P<0.01) were significantly increased. When we compared LV-PD-L1-TN-OE to LV-PD-L1-WT-OE, the mRNA level of E-cad (P<0.01) was significantly increased while N-cad (P<0.01), Zeb1 (P<0.01) and Vim (P<0.01) were significantly decreased. Furthermore, we used PD-1 fusion protein to stimulate the cell lines such as LV-PD-L1-shRNA, LV-PD-L1-WT-OE, LV-PD-L1-TN-OE, LV-NC and LV-Vector-Ctrl at 5μg/ml. When we compared LV-PD-L1-shRNA to LV-NC, the mRNA as well as the protein levels of N-cad (P<0.05) and Zeb1 (P<0.05) were significantly decreased. When we compared LV-PD-L1-WT-OE to LV-Vector-Ctrl, both mRNA and protein levels of N-cad (P<0.05) and Zeb1 (P<0.05) were significantly increased. When we compared LV-PD-L1-WT-OE to LV-PD-L1-TN-OE, both mRNA and protein levels of N-cad (P<0.05) and Zeb1 (P<0.05) were also significantly increased. Conclusion Abnormal expression of PD-L1 in esophageal cancer cells closely related to EMT. The intracellular domain of PD-L1 plays an important role in EMT of esophageal cancer cells. Key words: Programmed death-ligand 1; Esophageal cancer; Epithelial-to-mesenchymal transition

Inhibitory Effects of the Extracts of Juglans sigillata Green Husks on the Proliferation, Migration and Survival of KYSE150 and EC9706 Human Esophageal Cancer Cell Lines

This study evaluated the antitumor activity of the extracts of green husks of Juglans sigillata Dode on esophageal cancer. KYSE150 EC9706 cells were treated with different concentrations of six components of the extracts of J. sigillata green husks. Cell viability was measured by MTT. Cell migration and cell invasion were measured by wound-healing assays and transwell assays, respectively. Cell apoptosis and cycle were measured by flow cytometry. The expression of cell migration, cell cycle and cell apoptosis regulatory proteins was analyzed by Western blotting. Only the three constituents, including EtOH extractives, EtOAc soluble fraction and gallic acid (GA), exhibited inhibitory effects on the cell viability, migration and invasion by decreasing MMP2 and MMP9 expression (all P < 0.05). Flow cytometry revealed that these three constituents also induced cell apoptosis by increasing Bax and cleaved caspase-3 but decreasing Bcl-2 in KYSE150 and EC9706 cells. Furthermore, these constituents arrested the cell cycle at G0/G1 by downregulating the expression of Cyclin D1 but upregulating p53 and phospho-p53 expression in KYSE150 cells. In conclusion, the green husks of J. sigillata may act as a potential inhibitor on esophageal cancer growth. GA was the major single active constituent of the extracts.

Genome-Wide Profiling of the Toxic Effect of Bortezomib on Human Esophageal Carcinoma Epithelial Cells.

Objectives:Bortezomib has been widely used to treat multiple myeloma and other hematological malignancies. However, not much is known about its effect on solid tumors. The aim of this study was to study the effect of Bortezomib on human esophageal cancer cell lines and investigate the potential target pathways.Methods:Two human esophageal cancer cell lines, TE-1 and KYSE-150, were used in this study. Cell viability, cell cycle distribution, and apoptosis after Bortezomib treatment was detected by Cell Counting Kit-8, flow cytometry, and Annexin V/propidium iodide staining, respectively. The genes targeted by Bortezomib were analyzed at the messenger RNA level by microarray chips and quantitative real-time polymerase chain reaction.Results:The proliferation of human esophageal cancer cell lines was inhibited by Bortezomib in a dose- and time-dependent manner. Bortezomib treatment led to G2/M arrest and apoptosis. Microarray chips revealed multiple signaling pathways targeted by Bortezomib, including proteasome, endoplasmic reticulum, Wnt-, and calcium-mediated pathway. The expression patterns of 4 representative genes UBD, CUL3, HDAC6, and GADD45A were verified by quantitative real-time polymerase chain reaction and showed consistency with the microarray assay.Conclusion:Bortezomib could suppress cell viability, cause G2/M arrest, and induce apoptosis in human esophageal cancer cells, with possible targets including UBD, CUL3, HDAC6, and GADD45A.

Grape Seed Proanthocyanidin Extract Inhibits Human Esophageal Squamous Cancerous Cell Line ECA109 via the NF-κB Signaling Pathway.

Esophageal squamous cell carcinoma is the most common type of squamous cell carcinoma. Grape seed proanthocyanidin extract (GSPE) is considered to exhibit anticancer activity against several different types of cancer. We aimed to determine whether GSPE inhibited esophageal squamous cancerous cells and the possible involvement of NF-κB in this process. The human esophageal squamous cancer cell line ECA109 was treated with GSPE (0–80 μg/mL) and BAY11-7082 (10 μmol/L) for 12, 24, and 48 h. The MTT assay was used to determine cell proliferation; alterations in cell apoptosis were detected by flow cytometry; levels of inflammatory factors interleukin-6 and cyclooxygenase-2 and apoptotic proteins Bax/Bcl-2 were measured by ELISA; qRT-PCR and western blots were used to examine the activation of caspase-3 and NF-κB signaling. GSPE inhibited the proliferation of ECA109 cells and induced cellular apoptosis in a time- and dose-dependent manner. ELISA results showed that GSPE and BAY11-7082 reduced the secretion of inflammatory cytokines interleukin-6 and cyclooxygenase-2. The results of PCR and western blotting indicated that GSPE and BAY11-7082 activated caspase-3 and attenuated the activation of the NF-κB signaling pathway. GSPE induced apoptosis in ECA109 cells and inhibited ECA109 cell proliferation via a reduction in the secretion of inflammatory cytokines. This mechanism may be related to the attenuation of NF-κB activity and the sensitization of caspase-3.

Radiosensitization of esophageal carcinoma cells by knockdown of HMGB1 expression.

Radiotherapy (RT) is a traditional and important treatment for carcinoma of the esophagus along with surgery and chemotherapy. High mobility group box 1 (HMGB1) plays a crucial part in inhibiting the apoptosis of cancer cells after irradiation treatment. The present study, was designed to analyze the function of HMGB1 in esophageal cancer progression and elucidate the effects of HMGB1 on the radiosensitivity of human esophageal cancer cell lines. In the present study, an immunohistochemical evaluation of HMGB1 was performed on 77 biopsies, and the results revealed that HMGB1 overexpression was positively correlated with gross tumor volume (GTV), tumor‑node‑metastasis (TNM) stage, T classification, distant metastasis, and relapse and negatively correlated with patient survival rates, suggesting that HMGB1 acts as a key factor in the development of esophageal cancer. An shRNA targeting HMGB1 was designed for the knockdown of HMGB1 in ECA109 and TE13 cells, and the transfection efficiency of the shRNA was assessed using quantitative real‑time reverse transcription polymerase chain reaction and western blot analysis. CCK‑8 and clonogenic assays were used to analyze the effect of HMGB1 on the proliferation and radiosensitivity, respectively, of esophageal cancer cells in vitro. The influence of HMGB1 on radiation‑induced changes in the migration, invasion, and cell cycle as well as apoptosis of tumor cells was examined by wound‑healing and Transwell assays and flow cytometry, respectively. In addition, xenograft tumor models were constructed to observe the effect of HMGB1 on tumor growth in vivo. The results of the study in vitro revealed that the proliferation of the HMGB1‑shRNA group decreased after irradiation, and the radiation treatment reduced the tumor volume of the xenograft model which was more marked in HMGB1‑shRNA group. Moreover, HMGB1 was involved in the phosphorylation of H2AX after irradiation, and HMGB1 knockdown blocked the cell cycle in the G0/G1 phase and increased apoptosis. HMGB1 deficiency was also correlated with the upregulation of p16, Bax and caspase‑9 and the downregulation of MMP‑2, MMP‑9, cyclin D1, CDK4, γH2AX and Bcl‑2. These data indicated that the overexpression of HMGB1 prior to treatment was correlated with poor clinical outcome in esophageal carcinoma and that knockdown HMGB1 expression in human esophageal cancer cell lines increased their radiosensitivity by allowing the induction of apoptosis and G0/G1 arrest after exposure to radiation.

Casticin inhibits esophageal cancer cell proliferation and promotes apoptosis by regulating mitochondrial apoptotic and JNK signaling pathways.

Casticin, a flavonoid isolated from Vitex species, has been found to have anti-tumor property in multiple human cancers. The present study aimed to investigate the effect of casticin on the proliferation and apoptosis of esophageal cancer (EC) cells, and further illustrate the underlying mechanisms. In in vitro studies, human EC cell lines TE-1 and ECA-109 were treated with various concentrations of casticin (low-, middle-, and high-dose groups). The results showed that casticin dose-dependently inhibited the proliferation and clonogenicity of EC cells and induced cell cycle arrest in sub-G1 and G2 phases. Furthermore, casticin markedly enhanced EC cell apoptosis as detected by flow cytometry and Hoechst 33342 staining. The level of anti-apoptotic Bcl-2 protein was decreased, while the levels of pro-apoptotic Bax, cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP were conversely increased in casticin-treated TE-1 and ECA-109 cells. Moreover, casticin decreased the mitochondrial membrane potential and increased the release of mitochondrial cytochrome C into cytoplasm. In addition, the JNK signaling pathway was involved in casticin-medicated anti-proliferation and pro-apoptosis. Cells pretreated with SP600125, a JNK pathway inhibitor, partially abolished the effect of casticin. Finally, the anti-tumor property of casticin was confirmed in in vivo xenograft models. Overall, we provided both in vitro and in vivo evidences that casticin inhibited the proliferation and induced apoptosis of EC cells, and the anti-tumor action of casticin was mediated, in part, by the mitochondrial-dependent apoptosis and the activation of JNK signaling pathway.

Artesunate enhances radiosensitivity of esophageal cancer cells by inhibiting the repair of DNA damage

Radiotherapy plays an important therapeutic role in esophageal cancer (EC). However, acquired radioresistance impairs the efficacy of radiotherapy, often leading to treatment failure. Therefore, it is important to develop novel radiosensitizers to enhance the clinical treatment of EC. The purpose of this study was to investigate the role of artesunate (ART) on radiosensitivity of human EC cell line TE-1. We found that ART inhibited the proliferation of EC cells and enhanced the radiosensitivity of TE-1 cells (SER = 1.24). In vivo tumor growth of xenografts was inhibited markedly by irradiation (IR) combined with ART, with a tumor inhibition rate of 53.76% in IR + ART group vs. 41.13% in IR-alone group. Pretreatment with ART significantly prompted cell apoptosis and reversed the IR-induced G2/M arrest. ART treatment could aggravate DNA damage of EC cells and prolong the formation of γ-H2AX foci induced by IR. ART up-regulated P21 and down-regulated the expression of cyclin D1, RAD51, RAD54, Ku70 and Ku86 protein of irradiated TE-1 cells. These findings support that ART induce radiosensitivity of TE-1 cells in vitro and in vivo, and may prove to be a promising radiosensitizer for EC treatment.

Knockdown of USP28 enhances the radiosensitivity of esophageal cancer cells via the c-Myc/hypoxia-inducible factor-1 alpha pathway.

Acquired radioresistance is a major clinical obstacle in the treatment of esophageal cancer (EC). Ubiquitin-specific protease 28 (USP28) has been implicated in tumor growth in various cancer types. However, the role of USP28 and its underlying mechanisms of radioresistance in EC remain unknown. In the current study, we found that USP28 and c-Myc levels were upregulated in EC tissues and EC cell lines. The mRNA expression levels of USP28 and c-Myc were increased in the radioresistant human EC cell line (ECA109R) compared with those in ECA109 cells. In addition, the expression levels of USP28 and c-Myc were increased with increase in culture time after irradiation. Meanwhile, overexpression of USP28 decreased the radiosensitivity of ECA109 cells. In contrast, USP28 knockdown enhanced the radiosensitivity of ECA109R cells. Moreover, USP28 positively regulated the protein level of c-Myc, and c-Myc negatively regulated the radiosensitivity of ECA109 and ECA109R cells. Furthermore, c-Myc reversed the inhibitory effect of USP28 on the radiosensitivity of EC cells. Additionally, c-Myc enhanced the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α) at the posttranscriptional level, and the reinforcing effect of c-Myc silencing on the radiosensitivity of EC cells could be reversed by HIF-1α overexpression. Besides, knockdown of USP28 blocked the effect of c-Myc on activation of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related DNA damage checkpoint after irradiation. In conclusion, knockdown of USP28 enhanced the radiosensitivity of EC cells by destabilizing c-Myc and enhancing the accumulation of HIF-1α. Therefore, USP28 may serve as a novel therapeutic target to overcome EC radioresistance.

Long non-coding RNA FAL1 regulated cell proliferation through Akt pathway via targeting PDK1 in esophageal cancer cells.

Esophageal cancer (EC) is one of the most common cancers in the world. Long non-coding RNA focally amplified lncRNA on chromosome 1 (FAL1) is an oncogene which is frequently and focally amplified in human cancers. However, the role of FAL1 in EC remains unknown. The aim of the present study was to evaluate the effect of FAL1 on EC cell lines and explore the underlying mechanism. The expression levels of FAL1 in EC tissues and cell lines were detected by RT-PCR. Then, Eca109 cell, a human esophageal cancer cell line, was transfected with FAL1-overexpressing vector or FAL1-siRNA or empty vector. The cell proliferation was measured by MTT assay. The cell apoptosis was assessed by TUNEL assay. The cell invasion was determined by transwell assay. The interaction effect between FAL1 and 3-phosphoinositide-dependent protein kinase 1 (PDK1) was evaluated by chromatin immunoprecipitation (ChIP) assay. FAL1 was significantly up-regulated in EC tissues and human EC cell lines including Eca109, KYSE150, Eca9706, Kyse30, and TE-1 cells. Overexpression of FAL1 promoted cell proliferation, invasion ability, and cell cycle, but it inhibited cell apoptosis in EC cell lines. Overexpressed FAL1 activated Akt pathway via interacting with PDK1 in EC cell lines. FAL1 regulated cell proliferation through Akt pathway via targeting PDK1 in EC cells. These findings revealed that FAL1 exhibited oncogenic activity in EC, and inhibiting FAL1 might be useful for preventing the progression of EC.

Abstract 5066: Dual-targeting photoimmunotherapy (NIR-PIT) for esophageal cancer cells and cancer-associated fibroblasts (CAFs)

Abstract Background: Esophageal cancer has been reported to express HER family, especially EGFR and HER2, of which expressed cases have been reported to show poor prognosis. However, although there are some clinical trials that target those markers, using cetuximab (Cet), panitumumab (Pan), and trasutuzumab (Tra), the result is not sufficient. On the other hand, cancer-associated fibroblasts (CAFs) are one of the major components of tumor microenvironment and suggested to be associated with those chemoprevention, leading to poor prognosis. Thus, to target both cancer cells and CAFs, we focused on near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT is a new molecular targeted therapy, which is based on a near-infrared (NIR) photosensitizer IR700, conjugated to monoclonal antibody (mAb) targeting particular molecules. We developed the CAFs-targeting PIT targeted on fibroblast activated protein (FAP) and have confirmed the efficacy to FAP-expressed CAFs. In this study, we hypothesized that the effect of cancer-targeting PIT is decreased under CAF-rich condition as like other antibody-based therapy. Here we firstly evaluated the effect of cancer-targeting PIT in esophageal cancer cells and furthermore the possibility of additional effect by “dual-targeting PIT” for CAFs and cancer cells. Materials and Methods: Human esophageal cancer cell lines and human esophageal fibroblasts (FEF3) were used in this study. FEF3 stimulated with conditioned medium (CM) of cancer cells defined as CAFs. We used cetuximab and panitumumab for anti-EGFR antibody and trastuzumab for anti-HER2 antibody. The expression of EGFR, HER2 in esophageal cancer cells was confirmed by Western blot (WB). Cell viability for quantitative evaluation was determined using an XTT Cell Proliferation Kit II. Result: WB demonstrated that HER2 expression was increased in TE4 and OE19, and EGFR was increased in TE8. FEF3 stimulated with CM was increased FAP expression compared with control. XTT assay demonstrated that the effect of cancer-targeting PIT using Pan-IR700, Tra-IR700 is efficient. Additionally, we confirmed that CAFs-targeting PIT worked in co-culture condition with cancer cells as well as monoculture condition. Furthermore, “dual-targeting PIT” showed rapid cell death of both in co-culture condition, and XTT assay showed the additive effects in vitro, as expected. Discussion: In this study, dual-targeting therapy has shown cellular specific cell death by targeting to cell-specific antigen and induced both cells death simultaneously. NIR-PIT can be novel effective therapy for esophageal cancer, and also for CAFs in tumor microenvironment. Thus, “dual-targeting PIT," which is combined with both mAb-IR700 for cancer cells and CAFs, can regulate tumor stroma as well as cancer cells simultaneously and can be highly expected to have strong antitumor effect by remodeling tumor microenvironment. Citation Format: Hiroaki Sato, Kazuhiro Noma, Toru Narusaka, Satoshi Komoto, Yuki Katsura, Takuya Kato, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Yasuhiro Shirakawa, Hisataka Kobayashi, Toshiyoshi Fujiwara. Dual-targeting photoimmunotherapy (NIR-PIT) for esophageal cancer cells and cancer-associated fibroblasts (CAFs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5066.

MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4.

Although microRNA (miR) 199a-3p functions as a tumor suppressor in multiple malignancies, its expression and role in esophageal cancer have not been studied. Based on our previous observation that miR-199a-3p is markedly downregulated in esophageal cancer cell lines relative to esophageal epithelial cells, we examined the function of miR-199a-3p in these cells. MiR-199a-3p is predicted to bind with high affinity to the mRNA of p21 activated kinase 4 (PAK4). This kinase has been shown to be overexpressed in several malignancies and to modulate proliferation and motility. The current study is designed to determine whether miR-199a-3p regulates the expression of PAK4 in esophageal cancer cells and to understand the functional consequences of this interaction. Herein, we demonstrate reduced expression of miR-199a-3p in human esophageal cancer specimens and cell lines compared to esophageal epithelial cells, with associated increased expression of PAK4. Forced expression of miR-199a-3p decreases expression of PAK4 in esophageal cancer cell lines. Mechanistic studies reveal that miR-199a-3p binds to the 3’UTR of PAK4 mRNA. This interaction results in reduced levels of PAK4 mRNA due to decreased mRNA stability. Downregulation of PAK4 leads to decreased cyclin D1 (CD1) transcription and protein expression, resulting in markedly impaired cellular proliferation. When PAK4 expression is rescued, both CD1 transcription and protein return to baseline levels. Our results show that miR-199a-3p functions as a tumor suppressor in esophageal cancer cells through repression of PAK4. These findings suggest that both miR-199a-3p and PAK4 may be novel therapeutic targets in the treatment of esophageal cancer.