Corynoline inhibits esophageal squamous cell carcinoma growth via targeting Pim-3.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is an aggressive and deadly malignancy characterized by late-stage diagnosis, therapy resistance, and a poor 5-year survival rate. Finding novel therapeutic targets and their inhibitors for ESCC prevention and therapy is urgently needed. METHODS: We investigated the proviral integration site for maloney murine leukemia virus 3 (Pim-3) protein levels using immunohistochemistry. Using Methyl Thiazolyl Tetrazolium and clone formation assay, we verified the function of Pim-3 in cell proliferation. The binding and inhibition of Pim-3 by corynoline were verified by computer docking, pull-down assay, cellular thermal shift assay, and kinase assay. Cell proliferation, Western blot, and a patient-derived xenograft tumor model were performed to elucidate the mechanism of corynoline inhibiting ESCC growth. RESULTS: Pim-3 was highly expressed in ESCC and played an oncogenic role. The augmentation of Pim-3 enhanced cell proliferation and tumor development by phosphorylating mitogen-activated protein kinase 1 (MAPK1) at T185 and Y187. The deletion of Pim-3 induced apoptosis with upregulated cleaved caspase-9 and lower Bcl2 associated agonist of cell death (BAD) phosphorylation at S112. Additionally, binding assays demonstrated corynoline directly bound with Pim-3, inhibiting its activity, and suppressing ESCC growth. CONCLUSIONS: Our findings suggest that Pim-3 promotes ESCC progression. Corynoline inhibits ESCC progression through targeting Pim-3.

YY1 modulates the radiosensitivity of esophageal squamous cell carcinoma through KIF3B-mediated Hippo signaling pathway.

Radiotherapy is an important strategy in the comprehensive treatment of esophageal squamous cell carcinoma (ESCC). However, effectiveness of radiotherapy is still restricted by radioresistance. Herein, we aimed to understand the mechanisms underlying ESCC radioresistance, for which we looked into the potential role of YY1. YY1 was upregulated in radioresistant tissues and correlated with poor prognosis of patients with ESCC. YY1 depletion enhanced the radiosensitivity of ESCC in vitro and in vivo. Multi-group sequencing showed that downregulation of YY1 inhibited the transcriptional activity of Kinesin Family Member 3B (KIF3B), which further activated the Hippo signaling pathway by interacting with Integrin-beta1 (ITGB1). Once the Hippo pathway was activated, its main effector, Yes-associated protein 1 (YAP1), was phosphorylated in the cytoplasm and its expression reduced in the nucleus, thus enhancing the radiosensitivity by regulating its targeted genes. Our study provides new insights into the mechanisms underlying ESCC radioresistance and highlights the potential role of YY1 as a therapeutic target for ESCC.

PLK1 regulating chemoradiotherapy sensitivity of esophageal squamous cell carcinoma through pentose phosphate pathway/ferroptosis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the most common pathological type of esophageal cancer in China, accounting for more than 90 %. Most patients were diagnosed with advanced-stage ESCC, for whom new adjuvant therapy is recommended. Therefore, it is urgent to explore new therapeutic targets for ESCC. Ferroptosis, a newly discovered iron-dependent programmed cell death, has been shown to play an important role in carcinogenesis by many studies. This study explored the effect of Polo like kinase 1 (PLK1) on chemoradiotherapy sensitivity of ESCC through ferroptosis METHODS: In this study, we knocked out the expression of PLK1 (PLK1-KO) in ESCC cell lines (KYSE150 and ECA109) with CRISPR/CAS9. The effects of PLK1-knock out on G6PD, the rate-limiting enzyme of pentose phosphate pathway (PPP), and downstream NADPH and GSH were explored. The lipid peroxidation was observed by flow cytometry, and the changes in mitochondria were observed by transmission electron microscopy. Next, through the CCK-8 assay and clone formation assay, the sensitivity to cobalt 60 rays, paclitaxel, and cisplatin were assessed after PLK1-knock out, and the nude mouse tumorigenesis experiment further verified it. The regulation of transcription factor YY1 on PLK1 was evaluated by dual luciferase reporter assay. The expression and correlation of PLK1 and YY1, and their impact on prognosis were analyzed in more than 300 ESCC cases from the GEO database and our center. Finally, the above results were further proved by single-cell sequencing. RESULTS: After PLK1 knockout, the expression of G6PD dimer and the level of NADPH and GSH in KYSE150 and ECA109 cells significantly decreased. Accordingly, lipid peroxidation increased, mitochondria became smaller, membrane density increased, and ferroptosis was more likely to occur. However, with the stimulation of exogenous GSH (10 mM), there was no significant difference in lipid peroxidation and ferroptosis between the PLK1-KO group and the control group. After ionizing radiation, the PLK1-KO group had higher lipid peroxidation ratio, more cell death, and was more sensitive to radiation, while exogenous GSH (10 mM) could eliminate this difference. Similar results could also be observed when receiving paclitaxel combined with cisplatin and chemoradiotherapy. The expression of PLK1, G6PD dimer, and the level of NADPH and GSH in KYSE150, ECA109, and 293 T cells stably transfected with YY1-shRNAs significantly decreased, and the cells were more sensitive to radiotherapy and chemotherapy. ESCC patients from the GEO database and our center, YY1 and PLK1 expression were significantly positively-correlated, and the survival of patients with high expression of PLK1 was significantly shorter. Further analysis of single-cell sequencing specimens of ESCC in our center confirmed the above results. CONCLUSION: In ESCC, down-regulation of PLK1 can inhibit PPP, and reduce the level of NADPH and GSH, thereby promoting ferroptosis and improving their sensitivity to radiotherapy and chemotherapy. Transcription factor YY1 has a positive regulatory effect on PLK1, and their expressions were positively correlated. PLK1 may be a target for predicting and enhancing the chemoradiotherapy sensitivity of ESCC.

Epinodosin suppresses the proliferation, invasion, and migration of esophageal squamous cell carcinoma by mediating miRNA-143-3p/Bcl-2 axis.

Epinodosin has shown antibacterial and antitumor biological characteristics in the documents. We found that Epinodosin has an effective inhibitory effect on esophageal squamous cell carcinoma (ESCC). However, the potential roles and mechanisms of Epinodosin in ESCC remain unclear. We performed many experiments to clarify the effect and mechanism of Epinodosin on ESCC. In this study, cell viability, invasion, migration, and apoptosis were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,-diphenytetrazoliumromide (MTT), Transwell, and flow cytometry. The differentially expressed miRNAs were screened through RNA transcriptome sequencing. The expression levels of miRNA-143-3p and some proteins were measured by real-time polymerase chain reaction (PCR) and Western blot. The anticancer effects of Epinodosin in vivo were determined by a nude mouse model. Epinodosin suppressed cell proliferation/invasion/migration and induced ESCC cell apoptosis. Epinodosin remarkably affected the protein expression of mitogen-activated protein kinase (MAPK) signaling pathway. The animal experiments demonstrated that Epinodosin could attenuate the growth of ESCC tumors in nude mice. The expression of p53, Bim, and Bax was upregulated, while that of Bcl-2 was downregulated in tumor tissues. In conclusion, Epinodosin suppresses cell viability/invasion/migration, while induces ESCC cell apoptosis by mediating miRNA-143-3p and Bcl-2, and can markedly attenuate the growth of ESCC tumors in nude mice.

SLC43A2 and NFκB signaling pathway regulate methionine/cystine restriction-induced ferroptosis in esophageal squamous cell carcinoma via a feedback loop.

Studies have indicated dietary restriction of methionine/cystine provided a therapeutic benefit in diseases such as cancer. However, the molecular and cellular mechanisms that underlie the interaction between methionine/cystine restriction (MCR) and effects on esophageal squamous cell carcinoma (ESCC) have remained elusive. Here, we discovered the dietary restriction of methionine/cystine has a large effect on cellular methionine metabolism as assayed in a ECA109 derived xenograft model. RNA-seq and enrichment analysis suggested the blocked tumor progression was affected by ferroptosis, together with the NFκB signaling pathway activation in ESCC. Consistently, GSH content and GPX4 expression were downregulated by MCR both in vivo and in vitro. The contents of Fe2+ and MDA were negatively correlated with supplementary methionine in a dose-dependent way. Mechanistically, MCR and silent of SLC43A2, a methionine transporter, diminished phosphorylation of IKKα/ß and p65. Blocked NFκB signaling pathway further decreased the expression of SLC43A2 and GPX4 in both mRNA and protein level, which in turn downregulated the methionine intake and stimulated ferroptosis, respectively. ESCC progression was inhibited by enhanced ferroptosis and apoptosis and impaired cell proliferation. In this study, we proposed a novel feedback regulation mechanism underlie the correlation between dietary restriction of methionine/cystine and ESCC progression. MCR blocked cancer progression via stimulating ferroptosis through the positive feedback loop between SLC43A2 and NFκB signaling pathways. Our results provided the theoretical basis and new targets for ferroptosis-based clinical antitumor treatments for ESCC patients.

The effect of Andrographis paniculata water extract on palliative management of metastatic esophageal squamous cell carcinoma-A phase II clinical trial.

Patients with metastatic esophageal squamous cell carcinoma (ESCC) have a grave prognosis with limited life expectancy. Here, a phase II clinical trial was conducted to investigate the effect of Andrographis paniculata (AP) on the palliative care of patients with metastatic ESCC. Patients with metastatic or locally advanced ESCC deemed unfit for surgery, and who have already completed palliative chemotherapy or chemoradiotherapy or are not fit for these treatments, were recruited. These patients were prescribed AP concentrated granules for 4 months. They also received clinical and quality of life assessments for clinical response, as well as positron emission tomography-computed tomography at 3 and 6 months after AP treatment for the assessment of tumor volume. Furthermore, the change in gut microbiota composition after AP treatment was studied. From the results, among the 30 recruited patients, 10 completed the entire course of AP treatment, while 20 received partial AP treatment. Patients who completed the AP treatment achieved significantly longer overall survival periods with the maintenance of the quality of life during the survival period when compared to those who could not complete AP treatment. The treatment effect of AP also contributed to the shift of the overall structure of gut microbiota for ESCC patients towards those of healthy individuals. The significance of this study is the establishment of AP as a safe and effective palliative treatment for patients with squamous cell carcinoma of the esophagus. To the best of our knowledge, this is the first clinical trial of AP water extract in esophageal cancer patients demonstrating its new medicinal use.

Implication of changes in PD-L1 expression during neoadjuvant chemotherapy with docetaxel, cisplatin, and 5-fluorouracil (DCF) regimen in esophageal squamous cell carcinoma.

BACKGROUND: Neoadjuvant docetaxel plus cisplatin and 5-FU (NAC-DCF) and adjuvant nivolumab monotherapy are the standard care for locally advanced resectable esophageal squamous cell carcinoma (ESCC). However, no effective biomarkers have been found in perioperative setting. We investigated how programmed death-ligand 1 (PD-L1) changes before and after NAC-DCF and how it relates to the therapeutic effect of NAC-DCF in resectable ESCC. METHODS: PD-L1 expression in paired diagnostic biopsy and surgically resected tissues from ESCC patients who underwent surgical resection after receiving two or three NAC-DCF cycles was evaluated. PD-L1 positivity was defined as a combined positive score (CPS) of 10% ≤ . Gene expression analysis was conducted using samples before NAC-DCF. RESULTS: Sixty-six paired samples from 33 patients were included in PD-L1 expression analysis, and 33 Pre-NAC samples acquired by diagnostic biopsy were included in gene expression analysis. Pretreatment, 3 (9%), 13 (39%), and 17 (52%) patients harbored tumors with CPS ranges of < 1%, 1%-10%, and 10% ≤ , respectively. After NAC-DCF, 5 (15%), 15 (45%), and 13 (39%) tumors presented CPS ranges of < 1%, 1%-10%, and 10% ≤ , respectively. The concordance rate between Pre-and Post-NAC-DCF samples was 45%. Patients with PD-L1-negative tumors both before and after NAC-DCF (n = 9) had shorter survival and different gene expression profile characterized by upregulation in WNT signaling or neutrophils. CONCLUSIONS: A substantial PD-L1 expression alteration was observed, resulting in low concordance rate before and after NAC-DCF. Tumors persistently lacking PD-L1 had distinct gene expression profile with worse clinical outcomes, raising the need for further investigation.

ypN0 in Patients With Definitive cN-positive Status After Preoperative Treatment Is a Prognostic Factor in Esophageal Cancer.

BACKGROUND: Histopathological tumor regression grade is applied not to lymph nodes but primary tumors modified by preoperative treatments. This study focused on patients whose pathological examination at the time of surgery showed no residual tumor after chemo(radio)therapy in the primary lesion (ypT0) or lymph nodes (ypN0). PATIENTS AND METHODS: A total of 87 patients with clinical stage II/III thoracic esophageal cancer underwent esophagectomy following preoperative treatments to evaluate significances between pathological response and clinical outcomes; 51 patients with clinically definitive lymph node metastasis (cN+) were analyzed as a subgroup. RESULTS: ypT0 rates were 20.7% and 23.5%, and ypN0 rates were 47.1% and 27.5% in the whole cohort and in the cN+ subgroup, respectively. Disease-free survival, from surgery to relapse or death, was significantly influenced by ypN status (p=0.035) but not by ypT status in the 51 patients with definitive cN+ disease. Preoperative chemoradiation was an independent favorable factor for achievement of ypN0 in the 51 patients (odds ratio=0.09; p=0.007). CONCLUSION: ypN status was a predictive factor for DFS in patients treated with docetaxel plus low-dose 5-fluorouracil and cisplatin combined chemotherapy, superior to ypT status, especially in patients with definitive cN+ disease.

Genomic and epigenomic evolution of acquired resistance to combination therapy in esophageal squamous cell carcinoma.

BACKGROUNDTargeted arterial infusion of verapamil combined with chemotherapy (TVCC) is an effective clinical interventional therapy for esophageal squamous cell carcinoma (ESCC), but multidrug resistance (MDR) remains the major cause of relapse or poor prognosis, and the underlying molecular mechanisms of MDR, temporal intratumoral heterogeneity, and clonal evolutionary processes of resistance have not been determined.METHODSTo elucidate the roles of genetic and epigenetic alterations in the evolution of acquired resistance during therapies, we performed whole-exome sequencing on 16 serial specimens from 7 patients with ESCC at every cycle of therapeutic intervention from 3 groups, complete response, partial response, and progressive disease, and we performed whole-genome bisulfite sequencing for 3 of these 7 patients, 1 patient from each group.RESULTSPatients with progressive disease exhibited a substantially higher genomic and epigenomic temporal heterogeneity. Subclonal expansions driven by the beneficial new mutations were observed during combined therapies, which explained the emergence of MDR. Notably, SLC7A8 was identified as a potentially novel MDR gene, and functional assays demonstrated that mutant SLC7A8 promoted the resistance phenotypes of ESCC cell lines. Promoter methylation dynamics during treatments revealed 8 drug resistance protein-coding genes characterized by hypomethylation in promoter regions. Intriguingly, promoter hypomethylation of SLC8A3 and mutant SLC7A8 were enriched in an identical pathway, protein digestion and absorption, indicating a potentially novel MDR mechanism during treatments.CONCLUSIONOur integrated multiomics investigations revealed the dynamics of temporal genetic and epigenetic inter- and intratumoral heterogeneity, clonal evolutionary processes, and epigenomic changes, providing potential MDR therapeutic targets in treatment-resistant patients with ESCC during combined therapies.FUNDINGNational Natural Science Foundation of China, Science Foundation of Peking University Cancer Hospital, CAMS Innovation Fund for Medical Sciences, Major Program of Shenzhen Bay Laboratory, Guangdong Basic and Applied Basic Research Foundation, and the third round of public welfare development and reform pilot projects of Beijing Municipal Medical Research Institutes.

Dandelion root extract affects ESCC progression via regulating multiple signal pathways.

Dandelion, a medicinal and edible plant, exhibits anti-inflammatory activity. The purpose of the present study was to investigate the inhibitory effectiveness of the aqueous dandelion root extract (DRE) on esophageal squamous cell carcinoma (ESCC). The in vitro cell proliferation, migration, invasion and apoptosis and the in vivo tumor growth were evaluated. The effects of DRE on PI3K/Akt and Ras/Raf/ERK pathways, which are important signaling pathways related to the development and progression of esophageal squamous cell carcinoma, were studied. The effects of DRE on the expression of apoptosis-related proteins BCL2 and BAX were also investigated. Meanwhile, the role of a cystathionine-ß-synthase (CBS)/H2S system in ESCC cells and the effects of DRE on the CBS/H2S system were assessed. The results showed that DRE selectively inhibited cell growth, proliferation, migration and invasion and induced cell apoptosis in ESCC cells. Moreover, the oral administration of DRE retarded the growth of tumors in human ESCC xenograft models. The DRE treatment led to a dose-dependent reduction in the levels of PI3K, p-Akt, Ras, Raf and pERK1/2 proteins in ESCC cells. DRE also caused a decrease in the anti-apoptotic protein BCL2 and an increase in the pro-apoptotic protein BAX. The data also showed that the CBS/H2S system implicated in the process of ESCC and DRE inhibited the CBS/H2S system. Moreover, the CBS knockdown weakened the cancer cell-inhibiting effectiveness of DRE. Therefore, DRE may affect ESCC progression through the regulation of PI3K/Akt and Ras/Raf/ERK signal pathways as well as the endogenous CBS/H2S system, and consequently, serve as an effective anti-cancer alternative for human ESCC treatment.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: QGF could prevent invasion and metastasis of esophageal cancer by inhibiting the Gas6/Axl signaling pathway.

The 3-deoxysappanchalcone induces ROS-mediated apoptosis and cell cycle arrest via JNK/p38 MAPKs signaling pathway in human esophageal cancer cells.

BACKGROUND: The 3-deoxysappanchalcone (3-DSC), a chemical separated from Caesalpinia sappan L, has been substantiated to display anti-inflammatory, anti-influenza, and anti-allergy activities according to previous studies. However, the underlying mechanisms of action on esophageal cancer remain unknown. PURPOSE: The present research aims to survey the action mechanisms of 3-DSC in esophageal squamous cell carcinoma (ESCC) cells in vitro. METHODS: Evaluation of cytotoxicity was determined by MTT tetrazolium salt assay and soft agar assay. Cell cycle distribution, apoptosis induction, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), and multi-caspases activity were appreciated by Muse™ Cell Analyzer. The expressions of cell cycle- and apoptosis-related proteins were presented using Western blotting. RESULTS: 3-DSC blocked cell growth and colony formation ability in a concentration-dependent manner and invoked apoptosis, G2/M cell cycle arrest, ROS production, MMP depolarization, and multi-caspase activity. Furthermore, Western blotting results demonstrated that 3-DSC upregulated the expression of phospho (p)-c-jun NH2-terminal kinases (JNK), p-p38, cell cycle regulators, pro-apoptotic proteins, and endoplasmic reticulum (ER) stress-related proteins whereas downregulated the levels of anti-apoptotic proteins and cell cycle promoters. The effects of 3-DSC on ROS induction were counteracted by pretreatment with N-acetyl-L-cysteine (NAC). Also, our results indicated that p38 (SB203580) and JNK (SP600125) inhibitor slightly inhibited 3-DSC-induced apoptosis. These results showed that 3-DSC-related G2/M phase cell cycle arrest and apoptosis by JNK/p38 MAPK signaling pathway in ESCC cells were mediated by ROS. CONCLUSION: ROS generation by 3-DSC in cancer cells could be an attractive strategy for apoptosis of cancer cells by inducing cell cycle arrest, ER stress, MMP loss, multi-caspase activity, and JNK/p38 MAPK pathway. Our findings suggest that 3-DSC is a promising novel therapeutic candidate for both prevention and treatment of esophageal cancer.

Pseudolaric Acid B Inhibits Proliferation, Invasion, and Angiogenesis in Esophageal Squamous Cell Carcinoma Through Regulating CD147.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor of the digestive system. Studies have shown that pseudolaric acid B (PAB) has several pharmacological effects like anti-microtubule, anti-angiogenesis, and antitumor functions, while the effect and mechanism of PAB on esophageal cancer are still unclear. This study was designed to investigate the effects of PAB on ESCC. METHODS: To study the effects of PAB on the biological function through a series of in vitro and in vivo experiments. RESULTS: The results revealed that PAB inhibited the proliferation, invasion, and migration, but promoted the apoptosis of ESCC. Moreover, PAB restrained the growth of cancer cells in vivo and inhibited the angiogenesis of HUVEC in mice with ESCC. CD147 expression was increased in the esophageal squamous cell lines, and interference with CD147 hindered the proliferation, invasion, and migration of ESCC cells, and inhibited the growth and angiogenesis of the esophageal squamous cell line. PAB reduced the expression of CD147 in vivo and in vitro. The expression of MMP2, 3, and 9 was increased after overexpression of CD147, which provided the opportunity to reverse the role of PAB in inhibiting proliferation, invasion, migration, and angiogenesis of ESCC. DISCUSSION: The results revealed that PAB inhibited the proliferation, invasion, migration, and angiogenesis of ESCC in vitro and in vivo by CD147. PAB is a promising monomer for therapy of ESCC, providing references for future research on ESCC treatment.

Deoxypodophyllotoxin, a Lignan from Anthriscus sylvestris, Induces Apoptosis and Cell Cycle Arrest by Inhibiting the EGFR Signaling Pathways in Esophageal Squamous Cell Carcinoma Cells.

Deoxypodophyllotoxin (DPT) derived from Anthriscus sylvestris (L.) Hoffm has attracted considerable interest in recent years because of its anti-inflammatory, antitumor, and antiviral activity. However, the mechanisms underlying DPT mediated antitumor activity have yet to be fully elucidated in esophageal squamous cell carcinoma (ESCC). We show here that DPT inhibited the kinase activity of epidermal growth factor receptor (EGFR) directly, as well as phosphorylation of its downstream signaling kinases, AKT, GSK-3ß, and ERK. We confirmed a direct interaction between DPT and EGFR by pull-down assay using DPT-beads. DPT treatment suppressed ESCC cell viability and colony formation in a time- and dose-dependent manner, as shown by MTT analysis and soft agar assay. DPT also down-regulated cyclin B1 and cdc2 expression to induce G2/M phase arrest of the cell cycle and upregulated p21 and p27 expression. DPT treatment of ESCC cells triggered the release of cytochrome c via loss of mitochondrial membrane potential, thereby inducing apoptosis by upregulation of related proteins. In addition, treatment of KYSE 30 and KYSE 450 cells with DPT increased endoplasmic reticulum stress, reactive oxygen species generation, and multi-caspase activation. Consequently, our results suggest that DPT has the potential to become a new anticancer therapeutic by inhibiting EGFR mediated AKT/ERK signaling pathway in ESCC.

Inhibition of Endoplasmic Reticulum Stress-Mediated Autophagy Enhances the Anticancer Effect of Iodine-125 Seed Radiation on Esophageal Squamous Cell Carcinoma.

Autophagy has been reported to play a radioresistance role in high-dose-rate irradiation. However, its mechanisms and roles in continuous low-dose-rate (CLDR) irradiation have not been clearly understood. Iodine-125 (I-125) seed brachytherapy is a modality of CLDR irradiation and has been used in the treatment of various cancers. In this study, we investigated the mechanisms and roles of autophagy induced by I-125 seed radiation in human esophageal squamous cell carcinoma (ESCC) cell lines (Eca-109 and EC-109) and a xenograft mouse model. The results of this work showed that I-125 seed radiation induced a dose-dependent increase in autophagy in both cell lines. In Eca-109 cells, I-125 seed radiation-induced endoplasmic reticulum (ER) stress, manifesting as the increased levels of intracellular Ca2+ and Grp78/BiP, and activated PERK-eIF2α, IRE1, and ATF6 pathways of the unfolded protein response. Knockdown of PERK led to the decreased expression of autophagy marker, LC3B-II. Inhibition of autophagy by chloroquine or knockdown of ATG5 enhanced I-125 seed radiation-induced cell proliferation inhibition and apoptosis. Interestingly, chloroquine did not aggravate ER stress but promoted apoptosis via the mitochondrial pathway. The animal experiment showed that inhibition of autophagy by chloroquine improved the efficacy of I-125 seed radiation. In summary, our data demonstrate that I-125 seed CLDR radiation induces ER stress-mediated autophagy in ESCC. Autophagy plays a pro-survival role in I-125 seed CLDR irradiation, and chloroquine is a potential candidate for use in combination therapy with I-125 seed radiation treatment to improve efficacy against ESCC.

Ganoderic acid D induces synergistic autophagic cell death except for apoptosis in ESCC cells.

ETHNOPHAMACOLOGICAL RELEVANCE: Ganoderma lucidum has been used as a medicinal mushroom for more than 2000 years in China. Ganoderic acid D (GAD) as a representative active triterpenoid from Ganoderma lucidum is known to possess anticancer activity. However, the mechanism involved in its anticancer cell process is still largely elusive. AIM OF THE STUDY: Our study aimed to investigate the anticancer effects of GAD on the esophageal squamous cell carcinoma (ESCC) cells and the underlying mechanisms at the cell level. MATERIALS AND METHODS: EC9706 and Eca109 cells were treated with GAD (0, 10, 20, 40 µM) for 24 h. The cell viability, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis rate, caspase-3 activity, autophagic flux, lysosomal function were examined. Cell cycle, apoptotic, autophagy and mTOR signal pathway related proteins such as P53, Cyclin B1, CytoC, PARP, Beclin-1, P62, LC3, PI3K, AKT and mTOR were analyzed by Western blot approach. RESULTS: GAD inhibited cell proliferation and induced both apoptosis and autophagic cell death. In particular, we found that in the early stage of the autophagic process, GAD could initiate and enhance the autophagy signal while in the late stage it on the contrary could block the autophagic flux by impairing the autophagosome-lysosome fusion and inhibited the lysosomal degradation. Besides the autophagic cell death, GAD also induced the apoptosis mediated by caspase-related process in parallel. The mechanism involved for the synergistic apoptotic and autophagic cell death was also explored. We found that GAD down-regulated the expression of PI3K, AKT and mTOR phosphorylated proteins in the mTOR signaling pathway which thus led to the synergistic effect on apoptosis and autophagic cell death in the ESCC cells. CONCLUSIONS: In summary, this study has documented that GAD may inhibit cell proliferation through the mTOR pathway in ESCC cells, and induce synergistic apoptosis and autophagic cell death by disrupting the autophagic flux. This work therefore also suggests that GAD may be used as an efficient anticancer adjuvant for ESCC cancer therapy.

YY1-induced upregulation of FOXP4-AS1 and FOXP4 promote the proliferation of esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) belongs to one of the most common malignant tumors worldwide and possesses high mortality. Long non-coding RNAs (lncRNAs) have been demonstrated to be essential biological participants in the progression of ESCC. On the basis of bio-informatics prediction, forkhead box P4 antisense RNA 1 (FOXP4-AS1) and forkhead box P4 (FOXP4) were upregulated in esophageal carcinoma samples and were positively correlated with each other. The present study aimed to explore the function of FOXP4-AS1 and FOXP4 in ESCC cells. Function assays disclosed that knockdown of FOXP4-AS1 or FOXP4 efficiently suppressed cell proliferation and induced cell apoptosis. Moreover, FOXP4-AS1 positively regulated FOXP4 by interacting with insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) to stabilize FOXP4 messenger RNA. In addition, FOXP4-AS1 could upregulate the expression of FOXP4 by sponging miR-3184-5p. Finally, we found that Yin Yang 1 (YY1) is a transcription factor that can transcriptionally activate both FOXP4-AS1 and FOXP4 in ESCC cells. In a word, YY1-induced upregulation of FOXP4-AS1 and FOXP4 promote the proliferation of ESCC cells.

Risk Factors of Esophageal Squamous Cell Cancer Specific for Different Macroscopic Types.

Objective: To further explore risk factors of esophageal squamous cell cancer specific for different macroscopic types.Methods: A total of 423 patients and 423 age (±3 years) and gender matched controls were recruited. Multinomial logistic regression and multivariable logistic regression analysis were used to evaluate the risk factors of ESCC specific for different macroscopic types.Results: In this study, we found that drinking hot tea (OR = 1.98, 95% CI:1.14-3.43) and higher intake of hard food (OR = 1.64, 95% CI:1.05-2.58) positively associated with ulcerative type of ESCC, but not with medullary type or other types. Although alcohol drinking and lower intake of fresh vegetable appeared to be more harmful to the ulcerative-type ESCC, the discrepant risks were not significantly different in ulcerative type and medullary type. Moreover, tobacco smoking, intake of hot food, spicy food, fresh fruit, scallion and garlic were related to ESCC risk, whereas no significant difference in magnitude of their associations with respect to macroscopic type was observed. Furthermore, significant multiplicative interaction between tobacco smoking and alcohol drinking was found in ulcerative-type and medullary-type ESCC.Conclusion: Drinking hot tea and higher intake of hard food were associated with increased risk of ulcerative type of ESCC. However, the mechanism for this difference needs to be further studied.

Janus kinase 2 inhibition by Licochalcone B suppresses esophageal squamous cell carcinoma growth.

Esophageal cancer (EC) is one of the leading causes to cancer death in the worldwide and major population of EC is esophageal squamous cell carcinoma (ESCC). Still, ESCC-targeted therapy has not been covered yet. In the present study we have identified that Licochalcone B (Lico B) inhibited the ESCC growth by directly blocking the Janus kinase (JAK) 2 activity and its downstream signaling pathway. Lico B suppressed KYSE450 and KYSE510 ESCC cell growth, arrested cell cycle at G2/M phase and induced apoptosis. Direct target of Lico B was identified by kinase assay and verified with in vitro and ex vivo binding. Computational docking model predicted for Lico B interaction to ATP-binding pocket of JAK2. Furthermore, treatment of JAK2 clinical medicine AZD1480 to ESCC cells showed similar tendency with Lico B. Thus, JAK2 downstream signaling proteins phosphorylation of STAT3 at Y705 and S727 as well as STAT3 target protein Mcl-1 expression was decreased with treatment of Lico B. Our results suggest that Lico B inhibits ESCC cell growth, arrests cell cycle and induces apoptosis, revealing the underlying mechanism involved in JAK2/STAT3 signaling pathways after Lico B treatment. It might provide potential role of Lico B in the treatment of ESCC.

Abrogation of esophageal carcinoma development in miR-31 knockout rats.

MicroRNA-31 (miR-31) is overexpressed in esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary Zn deficiency and inflammation. In a Zn deficiency-promoted rat ESCC model with miR-31 up-regulation, cancer-associated inflammation, and a high ESCC burden following N-nitrosomethylbenzylamine (NMBA) exposure, systemic antimiR-31 delivery reduced ESCC incidence from 85 to 45% (P = 0.038) and miR-31 gene knockout abrogated development of ESCC (P = 1 × 10-6). Transcriptomics, genome sequencing, and metabolomics analyses in these Zn-deficient rats revealed the molecular basis of ESCC abrogation by miR-31 knockout. Our identification of EGLN3, a known negative regulator of nuclear factor κB (NF-κB), as a direct target of miR-31 establishes a functional link between oncomiR-31, tumor suppressor target EGLN3, and up-regulated NF-κB-controlled inflammation signaling. Interaction among oncogenic miR-31, EGLN3 down-regulation, and inflammation was also documented in human ESCCs. miR-31 deletion resulted in suppression of miR-31-associated EGLN3/NF-κB-controlled inflammatory pathways. ESCC-free, Zn-deficient miR-31-/- rat esophagus displayed no genome instability and limited metabolic activity changes vs. the pronounced mutational burden and ESCC-associated metabolic changes of Zn-deficient wild-type rats. These results provide conclusive evidence that miR-31 expression is necessary for ESCC development.