Diclofenac exhibits cytotoxic activity associated with metabolic alterations and p53 induction in ESCC cell lines and decreases ESCC tumor burden in vivo.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC.

microRNA-497-mediated Smurf2/YY1/HIF2α axis in tumor growth and metastasis of esophageal squamous cell carcinoma.

Aberrant expression of microRNA-497 (miR-497) is associated with tumor progression, but the molecular mechanisms in tumorigenesis remain largely unknown. Here, we report that miR-497 expression is downregulated in esophageal squamous cell carcinoma (ESCC) clinical samples. Consistently, upregulation of miR-497 inhibits ESCC cell malignant properties and tumor growth in vivo. Importantly, we uncovered that miR-497 upregulation suppressed ESCC cell growth and tumor growth by inhibiting Smurf2. Mechanistically, we showed that Smurf2 was a target of miR-497, and mediated YY1 expression to elevate HIF2α expression, thereby enhancing the malignancy of ESCC cells. Together, our study uncovered the role of the miR-497-mediated Smurf2/YY1/HIF2α axis in tumor growth and metastasis, which might provide potential therapeutic targets for human ESCC.

Potential Chemotherapeutic Effect of Selenium for Improved Canceration of Esophageal Cancer.

Esophageal squamous cell carcinoma is the most common type of esophageal cancer and accounts for 5% of malignant tumor deaths. Recent research suggests that chronic inflammation and DNA damage may drive the onset of esophageal squamous cell carcinoma, implying that lowering chronic inflammation and DNA damage compounds may provide chemo-prevention. According to epidemiological and experimental evidence, selenium is linked to a lower risk of several malignancies, including esophageal squamous cell carcinoma. However, its exact mechanism is still unclear. In the present study, we used cell lines and a 4-NQO mice model to explore the anti-cancer mechanism of four types of selenium. Our findings indicated that selenium inhibited the proliferation, colony formation, and ROS level of ESCC cell lines in a time-dependent manner. Intriguingly, selenium treatment impeded 4-NQO-induced high-grade intraepithelial neoplasia and reduced the number of positive inflammatory cells by preserving DNA from oxidative damage. In addition, selenium significantly decreased the expression of Ki-67 and induced apoptosis. This study demonstrates that selenium has a significant chemo-preventive effect on ESCC by reducing high-grade dysplasia to low-grade dysplasia. For the first time, selenium was shown to slow down the progression of esophageal cancer by lowering inflammation and oxidative DNA damage.

Role of Nutritional Status in the Treatment Outcome for Esophageal Squamous Cell Carcinoma.

Undernourishment is reported to impair treatment response, further leading to poor prognosis for cancer patients. We aimed to investigate the role of nutritional status on the prognosis of squamous cell carcinoma (SCC) of the esophagus, and its correlation with anticancer immune responsiveness. We retrospectively reviewed 340 esophageal-SCC patients who completed curative treatment and received a nutrition evaluation by the Patient-Generated Subjective Global Assessment (PGSGA) score at the beginning and completion of neoadjuvant treatment at our hospital. The correlation between the nutritional status and various clinicopathological parameters and prognosis were examined. In addition, the role of nutritional status in the regulation of the anticancer immune response was also assessed in cancer patients and in a 4-nitroquinoline 1-oxide (4NQO)-induced esophageal tumor model. Our data revealed that malnutrition (patients with a high PGSGA score) was associated with advanced stage and reduced survival rate. Patients in the group with a high PGSGA score were correlated with the higher neutrophil-to-lymphocyte ratio, higher proportion of myeloid-derived-suppressor cells (MDSC) and increased IL-6 level. Furthermore, surgical resection brought the survival benefit to patients in the low PGSGA group, but not for the malnourished patients after neoadjuvant treatment. Using a 4NQO-induced tumor model, we found that nutrition supplementation decreased the rate of invasive tumor formation and attenuated the immune-suppressive microenvironment. In conclusion, malnutrition was associated with poor prognosis in esophageal-SCC patients. Nutritional status evaluated by PGSGA may be useful to guide treatment decisions in clinical practice. Nutritional supplementation is suggested to improve prognosis, and it might be related to augmented anticancer immune response.

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.

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.

Isoliquiritigenin Suppressed Esophageal Squamous Carcinoma Growth by Blocking EGFR Activation and Inducing Cell Cycle Arrest.

Isoliquiritigenin (ILQ) is a natural product isolated from licorice root which has served as traditional Chinese medicine for a long time. Recently, the antitumor effects of ILQ have been widely studied in various cancers, but the role and related mechanisms of ILQ in esophageal squamous carcinoma cells (ESCC) are still poorly understood. In our studies, ILQ showed profound antitumor activities in ESCC cells. In vitro, ILQ substantially inhibited cell proliferation and anchorage-independent growth in a panel of human ESCC cells. Mechanism studies showed that EGFR signaling pathway played an important role for ILQ to exert its antitumor activity in ESCC. Exposure to isoliquiritigenin substantially decreased EGF-induced EGFR activation and its downstream Akt and ERK1/2 signaling pathway. EGFR knockdown with shRNA in ESCC cell significantly reduced the sensitivity of cancer cells to ILQ. Moreover, it was found that ILQ had a significantly inhibitory effect on AP-1 family, the protein of Jun and Fos subfamilies was substantially downregulated, and the transcriptional activity of AP-1 family was dramatically suppressed by ILQ. By reducing the expression of cyclin D1, ESCC cells were induced G0/G1 arrest, and cell division was substantially blocked. Finally, the antitumor potency of ILQ was validated in xenograft models and the tumor growth was prominently restrained by ILQ. Briefly, our study showed that ILQ, or its analogue, appeared to be a promising new therapeutic agent for ESCC management.

Retrochalcone Echinatin Triggers Apoptosis of Esophageal Squamous Cell Carcinoma via ROS- and ER Stress-Mediated Signaling Pathways.

Esophageal squamous cell carcinoma (ESCC) is a poor prognostic cancer with a low five-year survival rate. Echinatin (Ech) is a retrochalone from licorice. It has been used as a herbal medicine due to its anti-inflammatory and anti-oxidative effects. However, its anticancer activity or underlying mechanism has not been elucidated yet. Thus, the objective of this study was to investigate the anti-tumor activity of Ech on ESCC by inducing ROS and ER stress dependent apoptosis. Ech inhibited ESCC cell growth in anchorage-dependent and independent analysis. Treatment with Ech induced G2/M phase of cell cycle and apoptosis of ESCC cells. It also regulated their related protein markers including p21, p27, cyclin B1, and cdc2. Ech also led to phosphorylation of JNK and p38. Regarding ROS and ER stress formation associated with apoptosis, we found that Ech increased ROS production, whereas its increase was diminished by NAC treatment. In addition, ER stress proteins were induced by treatment with Ech. Moreover, Ech enhanced MMP dysfunction and caspases activity. Furthermore, it regulated related biomarkers. Taken together, our results suggest that Ech can induce apoptosis in human ESCC cells via ROS/ER stress generation and p38 MAPK/JNK activation.

Photoimmunotherapy for cancer-associated fibroblasts targeting fibroblast activation protein in human esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) are strongly implicated in tumor progression, including in the processes of tumorigenesis, invasion, and metastasis. The targeting of CAFs using various therapeutic approaches is a novel treatment strategy; however, the efficacy of such therapies remains limited. Recently, near-infrared photoimmunotherapy (NIR-PIT), which is a novel targeted therapy employing a cell-specific mAb conjugated to a photosensitizer, has been introduced as a new type of phototherapy. In this study, we have developed a novel NIR-PIT technique to target CAFs, by focusing on fibroblast activation protein (FAP), and we evaluate the treatment efficacy in vitro and in vivo. Esophageal carcinoma cells exhibited enhanced activation of fibroblasts, with FAP over-expressed in the cytoplasm and on the cell surface. FAP-IR700-mediated PIT showed induced rapid cell death specifically for those cells in vitro and in vivo, without adverse effects. This novel therapy for CAFs, designed as local control phototherapy, was safe and showed a promising inhibitory effect on FAP+ CAFs. PIT targeting CAFs via the specific marker FAP may be a therapeutic option for CAFs in the tumor microenvironment in the future.

Tetrahydrocurcumin, Curcumin, and 5-Fluorouracil Effects on Human Esophageal Carcinoma Cells.

BACKGROUND: Esophageal cancer responds poorly to traditional therapies, and novel treatments are needed. The phytochemical curcumin is a potential treatment for Esophageal Squamous Cell Carcinoma (ESCC). A curcumin metabolite, tetrahydrocurcumin (THCUR), has anti-cancer effects and greater bioavailability than curcumin. OBJECTIVE: Evaluate THCUR as an anti-cancer agent relative to curcumin and a standard cancer drug, 5-fluorouracil (5-FU), along with treatment interactions. MATERIALS AND METHODS: Assay cell proliferation and viability following individual and combined delivery of the compounds to three ESSC cell lines (TE-1, TE-8, and KY-5) that have different percentages of Cancer Stem Cells (CSCs). RESULTS: Curcumin was significantly more effective than 5-FU in all three cell lines. It also had the greatest effect on KY-5 cells, which have the highest CSC properties, consistent with the ability of curcumin to target CSCs. Effects on ESCC cell proliferation were not detected from 40µM THCUR, a dosage above the IC50 of curcumin and 5-FU. However, THCUR at this dosage in combination with 5-FU significantly suppressed TE-1 cell proliferation, but 5-FU alone did not. As TE-1 has low CSC properties relative to the two other cell lines, it was expected to have the least resistance to chemotherapeutic treatments. Surprisingly, TE-1 was the most resistant to inhibition by 5-FU. CONCLUSION: These results and the greater stability and water solubility of THCUR than curcumin support further testing of THCUR in combination with standard treatments, particularly for chemoresistant ESCC. In contrast to concerns that curcuminoids taken by patients through diet or diet supplements might interfere with chemotherapy, suppression of 5-FU efficacy by curcumin was not observed.

p-Hydroxylcinnamaldehyde slows the progression of 4NQO-induced oesophageal tumourigenesis via the RhoA-MAPK signaling pathway.

p-Hydroxylcinnamaldehyde isolated from the Cochinchina momordica seed (CMSP) has been identified to inhibit growth and metastasis in oesophageal squamous cell carcinoma (ESCC) by inducing differentiation. The aim of the present study was to evaluate the effect and underlying mechanism of CMSP on 4-nitroquinoline 1-oxide (4NQO)-induced oesophageal tumourigenesis. In the present study, a mouse model of oesophageal preneoplastic lesions was established by providing 4NQO-containing drinking water to C57BL/6 mice. The effect of CMSP on tumourigenesis induced by the chemical mutagen and the effect of CMSP on immune function were investigated. The results showed that the incidence and pathological stage of atypical hyperplasia in oesophageal tissues were significantly reduced in CMSP-treated mice compared with untreated mice. Immunohistochemistry and pull-down assay results revealed that the expression levels of p-ERK1/2, p-SAPK/JNK, and GTP-RhoA were significantly decreased in the oesophageal tissue of CMSP-treated mice. In addition, the proportions of CD4+ T cells, CD8+ T cells, and NK cells were increased, while the proportion of CD4+ CD25+ regulatory T cells (Tregs) was decreased, in the peripheral blood of CMSP-treated mice. These results indicated that CMSP could hamper 4NQO-induced oesophageal tumourigenesis by regulating the RhoA-ERK/JNK signaling pathway and promoting immune system function, thus providing a new potential strategy for treating preneoplastic lesions of the oesophagus.

Tocopherols inhibit esophageal carcinogenesis through attenuating NF-κB activation and CXCR3-mediated inflammation.

Esophageal cancer is one of the common causes of cancer mortality in the world. The predominant histological subtype, esophageal squamous cell carcinoma (ESCC), often results in poor prognosis due to the lack of effective approaches for the early diagnosis and treatment, highlighting the need for preventive intervention against this disease. Here we report that dietary tocopherols significantly prevents esophageal carcinogenesis by inhibiting the activation of NF-κB and the subsequent interaction of chemokine CXCL9/10/11 with their receptor CXCR3 in ESCC induced by N-nitrosomethylbenzylamine (NMBA) in murine models. Dietary supplementation with 0.15% α-tocopherol (α-T), δ-tocopherol (δ-T), or γ-tocopherol rich mixture (γ-TmT) markedly suppressed the production of pro-inflammatory cytokines, as well as the induction of CXCR3+ effector T cells (CD4+ Th1 and CD8+ CTLs) infiltration, especially at the early stage of carcinogenesis. In experiments in vivo and in vitro, these events were tightly correlated with the blockade of NF-κB activation. Our results show that tocopherols decrease carcinogenesis through inhibiting NF-κB and CXCR3 signaling, as well as related inflammation in early premalignant lesions. This pathway may offer a novel target for chemoprevention of esophageal cancer.

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.