Cyclopamine Suppresses Human Esophageal Carcinoma Cell Growth by Inhibiting Glioma-Associated Oncogene Protein-1, a Marker of Human Esophageal Carcinoma Progression.

BACKGROUND Esophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth. MATERIAL AND METHODS Gli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression. RESULTS Gli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly (P<0.05) positive correlations with age, lymphatic metastasis, tumor recurrence, and stage. At 12 h (F=214.57), 24 h (F=76.832), 48 h (F=236.90), and 72 h (F=164.55), the higher the concentration of cyclopamine, the higher the inhibition rate of suppressing EC9706 proliferation, and this effect was significant (P<0.05). The number of early-apoptosis cells increased as the concentration of cyclopamine increased. Morphology of EC9706 cells appeared as round with rough edges, karyopyknosis, and karyorrhexis. After 48 h, apoptosis rates of EC9706 cells treated with different concentrations of cyclopamine were (7.73±1.25)% at 2.5 µM, (13.37±1.42)% at 5.0 µM, (22.3±2.92)% at 10.0 µM, and (33.57±1.75)% at 20.0 µM, and the effect was dose-dependent. Gli-1 was obviously reduced after cyclopamine treatment and the effect was dose-dependent. CONCLUSIONS Gli-1 is highly expressed in human esophageal carcinoma, and could be a marker for use in assessing tumor stage and the deciding on treatment target.

Rho/ROCK Pathway Regulates Migration and Invasion of Esophageal Squamous Cell Carcinoma by Regulating Caveolin-1.

BACKGROUND Esophageal squamous cell carcinoma (ESCC) is a common cancer with poor prognosis. Caveolin-1 (Cav1) and Rho/ROCK pathway play important roles in tumor metastasis, separately. However, less research was focused on the relationship between Cav1 and Rho/ROCK in ECSS metastasis. Therefore, we investigated the relationship between Cav1 and Rho/ROCK pathway in ESCC metastasis. MATERIAL AND METHODS Cav1 and phosphorylated Cav1 (PY14Cav1) were examined in ESCC and in adjacent and non-tumorous tissues from ESCC patients by immunohistochemistry (IHC). Small interfering RNA (siRNA) targeting Cav1 or Rho/ROCK inhibitor was used to treat EC109, Eca109, TE1, and TE13 cells. Western blotting (WB) was used to detect Cav1 and PY14Cav1 expression. The wound healing scratch test and transwell assays were used to assess migration and invasion. RESULTS Cav1 and PY14Cav1 were gradually expressed at higher levels in ECSS than in adjacent and non-tumor tissues as ESCC stage and lymphatic metastasis increased, and this difference was significant (P<0.05). Cav1 was expressed at higher levels in TE1 and TE13 than in EC109 and Eca109, while PY14Cav1 was enhanced in TE1 and TE13 cells but not in EC109 and Eca109, and the difference was significant (P<0.05). TE1 and TE13 had significantly (P<0.05) stronger motility, migratory, and invasion abilities than EC109 and Eca109 cells. Silencing Cav1 decreased PY14Cav1 expression in TE1 and TE13 cells, as well as suppressing the migration and invasion of all ECSS cells, and these differences were significant (P<0.05). Suppressing the Rho/ROCK pathway obviously inhibited Cav1 and PY14Cav1 expressions, as well as significantly (P<0.05) decreasing migration and invasion of ESCC cells. CONCLUSIONS Cav1 and PY14Cav1 were positively correlated with ESCC lymphatic metastasis and cancer stages. Rho/ROCK pathway activation promoted ESCC metastasis by regulating Cav1.

Discovery and validation of combined biomarkers for the diagnosis of esophageal intraepithelial neoplasia and esophageal squamous cell carcinoma.

Early diagnosis and intervention of esophageal squamous cell carcinoma (ESCC) can improve the prognosis. The purpose of this study was to identify biomarkers for ESCC and esophageal precancerous lesions (intraepithelial neoplasia, IEN). Based on the proteomic and genomic data of esophageal tissue including previously reported data, up-regulated proteins with copy number amplification in esophageal cancer were screened as candidate biomarkers. Five proteins, including KDM2A, RAD9A, ECT2, CYHR1 and TONSL, were confirmed by immunohistochemistry on ESCC and normal esophagus (NE). Then, we investigated the expression of 5 proteins in 236 participants (60 NEs, 93 IENs and 83 ESCCs) which were randomly divided into training set and test set. When distinguishing ESCC from NE, the area under curve (AUC) of the multiprotein model was 0.940 in the training set, while the lowest AUC of a protein was 0.735. In the test set, the results were similar. When distinguishing ESCC from IEN or distinguishing IEN from NE, the diagnostic efficiency of the multi-protein models were also improved compared with that of single protein. Our findings suggest that combined detection of KDM2A, RAD9A, ECT2, CYHR1 and TONSL can be used as potential biomarkers for the early diagnosis of ESCC and precancerous lesion development prediction. SIGNIFICANCE: Candidate biomarkers including KDM2A, RAD9A, ECT2, CYHR1 and TONSL screened by integrating genomic and proteomic data from the esophagus can be used as potential biomarkers for the early diagnosis of esophageal squamous cell carcinoma and precancerous lesion development prediction.

Self-Expandable Metal Stent as a Bridge to Surgery for Left-Sided Acute Malignant Colorectal Obstruction: Optimal Timing for Elective Surgery.

Objectives: This randomized, single-center, retrospective, comparative cohort study is aimed at investigating the optimal time interval from self-expandable metal stent (SEMS) placement to surgery and potential risk factors for complications in patients with acute malignant colorectal obstruction. Methods: A total of 64 patients with left-sided acute malignant colorectal obstruction treated with SEMS placement and subsequent surgery between January 2013 and September 2020 were enrolled and allocated to a case group (SEMS placing time ≤ 14 days; n = 19 patients) and a control group (SEMS placing time > 14 days; n = 45 patients). The primary outcome was the difference in baseline information, patients' conditions during surgery, and postoperative conditions between the two groups. The secondary outcome included potential risk factors of postoperative complications. The propensity score matching (PSM) and super learner (SL) methods were used to eliminate multiple confounding factors of baseline data. A cohort of 21 samples was used for external validation, comprising 6 cases and 15 controls. Results: A significant difference was observed between the two groups in intraoperative blood loss (P = 0.009), postoperative hospital stay (P = 0.002), postoperative complications (Clavien-Dindo grading ≥ II) (P < 0.001), stoma creation (P < 0.001), and primary anastomosis (P < 0.001). After a 1 : 3 PSM analysis, no statistically significant differences between eight confounding variables of the two groups were observed (P > 0.05). Caliper set as 0.2 multiple logistic regression analysis showed that the potential risk factor for postoperative complications was SEMS placing time (RR = 0.109, 95% confidence interval (CI) = 0.028-0.433; P = 0.002), indicating that SEMS placing time > 14 days was an independent risk factor for postoperative complications in bridge-to-surgery (BTS) setting. The area under the AUC curve was 76.7% and validated using the validation cohort. Conclusions: Long duration of SEMS placement (>14 days) may not influence surgical difficulty but could increase the risk of postoperative complications.

Decreased plasma riboflavin is associated with poor prognosis, invasion, and metastasis in esophageal squamous cell carcinoma.

BACKGROUND: Riboflavin deficiency confers a predisposition for esophageal cancer. The role of plasma riboflavin levels in development and prognosis of individuals with digestive tract inflammation and ulcer (DTIU), digestive tract polyps (DTPs), and ESCC is not well understood. METHODS: We performed a cross-sectional study, including 177 DTIU, 80 DTP, and 324 ESCC cases, to measure the plasma riboflavin levels among the three populations. Correlation between plasma riboflavin levels (categorized as ≥31.8, 6.5-31.8 and ≤6.5 nmol/L groups) and clinical characteristics, as well as survival of ESCC patients (556 cases) was analyzed. RESULTS: There was no difference in plasma riboflavin levels between DTIU, DTP, and ESCC cases (P > 0.05). Plasma riboflavin levels were inversely correlated with invasive depth (correlation coefficient = -0.09, P = 0.026) and lymph node metastasis (correlation coefficient = -0.11, P = 0.010) of ESCC, and ESCC patients with low riboflavin levels had poor recurrence-free survival (P = 0.035) and overall survival (P = 0.003). Decreased riboflavin was a prognostic factor for poor overall survival (HR = 1.91, 95% CI = 1.19-3.07, P = 0.007). CONCLUSIONS: Plasma riboflavin levels in DTIU, DTP, and ESCC patients are similar. Plasma riboflavin levels are associated with the development and prognosis of ESCC.

In vivo anti-tumor effect of hybrid vaccine of dendritic cells and esophageal carcinoma cells on esophageal carcinoma cell line 109 in mice with severe combined immune deficiency.

AIM: To develop a fusion vaccine of esophageal carcinoma cells and dendritic cells (DC) and observe its protective and therapeutic effect against esophageal carcinoma cell line 109 (EC109). METHODS: The fusion vaccine was produced by fusing traditional polyethyleneglycol (PEG), inducing cytokine, sorting CD34+ magnetic microbead marker and magnetic cell system (MACS). The liver, spleen and lung were pathologically tested after injection of the fusion vaccine. To study the therapeutic and protective effect of the fusion vaccine against tumor EC109, mice were divided immune group and therapeutic group. The immune group was divided into P, E, D and ED subgroups, immunized by phosphate buffered solution (PBS), inactivated EC109, DC and the fusion vaccine respectively, and attacked by EC109 cells. The tumor size, weight, latent period and mouse survival period were recorded and statistically analyzed. The therapeutic group was divided into four subgroups: P, inactivated EC109, D and ED subgroups, which were attacked by EC109 and then treated with PBS, inactivated EC109, DC, and EC109-DC respectively. Pathology and flow cytometry were also used to study the therapeutic effect of the fusion vaccine against EC109 cells. RESULTS: Flow cytometry showed that the expression of folate receptor (FR), EC109 (C), DCs (D) in human nasopharyngeal carcinoma cell line (HNE1) (B) was 78.21%, 89.50%, and 0.18%, respectively. The fusion cells (C) were highly expressed. No tumor was found in the spleen, lung and liver after injection of the fusion vaccine. Human IgG was tested in peripheral blood lymphocytes (PBL). In the immune group, the latent period was longer in EC109-DC subgroup than in other subgroups, while the tumor size and weight were also smaller than those in ED subgroup. In the therapeutic group, the tumor size and weight were smaller in ED subgroup than in P, inactivated EC109 and DC subgroups. CONCLUSION: Fusion cells are highly expressed not only in FR but also in CD80. The fusion vaccine has a distinctive protective effect against tumor EC109 and can inhibit the growth of tumor in mice, and its immune protection against tumor attack is more significant.

Decreased expression of serum miR-424 correlates with poor prognosis of patients with hepatocellular carcinoma.

BACKGROUND: MicroRNAs (miRNAs) play important roles in many important cellular processes and deregulation of miRNAs is linked to many human diseases including cancer. Although miR-424 has been demonstrated to inhibit progression of hepatocellular carcinoma (HCC), its expression level in serum samples and the potential clinical values remain unknown. MATERIALS AND METHODS: The expression level of miR-424 in the serum clinical samples from HCC patients and healthy volunteers were determined by qRT-PCR. Then the association of serum miR-424 expression level with various important clinicopathological parameters and survival rates was evaluated. Multivariate Cox regression analysis was used to identify the independent risk factors for HCC. RESULTS: The expression level of serum miR-424 was significantly decreased in patients with HCC compared with the healthy volunteers (P<0.01). Reduced expression of serum miR-424 was associated with serum AFP (P=0.048), vein invasion (P=0.006) and TNM stage (P=0.003). In addition, survival analysis showed that HCC patients with lower serum miR-424 expression suffered poorer overall survival (P=0.018) and disease free survival (P=0.008). Moreover, serum miR-424 was demonstrated to be an independent risk factor for HCC. CONCLUSIONS: Our findings provide the compelling evidence that the decreased expression of serum miR-424 may serve as a novel biomarker to predict the unfavorable prognosis of HCC patients.

Expression levels of matrix metalloproteinase-9 in human gastric carcinoma.

The present report investigated the correlation between the expression levels of matrix metalloproteinase (MMP)-9 in gastric carcinoma patients and the clinicopathological characteristics. Forty-five samples of gastric carcinoma and distal gastric mucosa tissue, and 10 samples of healthy gastric mucosa tissue were analyzed using semi-quantitative polymerase chain reaction, as well as immunohistochemical and hematoxylin and eosin staining. MMP-9 protein levels in serum samples from the same patients were quantified by enzyme-linked immunosorbent assay. The present report identified that MMP-9 expression was markedly higher in the gastric carcinoma tissue (86.67%) than in the adjacent healthy tissue (10.00%). A positive association was identified between the level of MMP-9 protein expression and the depth of cancer invasion (P<0.05). Furthermore, the preoperative serum levels of the MMP-9 protein in the gastric carcinoma tissue were correlated with the tumor-node-metastasis stage and occurrence of lymph node metastasis (P<0.01). Data from the present report indicates that MMP-9 may be key in gastric carcinoma malignancy, and implies that MMP-9 may serve as a novel biomarker in the diagnosis and prognosis of gastric carcinoma.

Reactive oxygen species and mitochondrial membrane potential are modulated during CDDP-induced apoptosis in EC-109 cells.

cis-Diamminedichloroplatinum (CDDP), commonly know as cisplatin, is a well known DNA-damaging agent, which is highly active in suppressing the proliferation of tumor cells. However, it is not clear that CDDP can induce growth inhibition of esophagus cancer cells. Using the cell line EC-109 from the esophagus, we found that CDDP would induce apoptotic responses. The addition of CDDP to cells led to the inhibition of growth in a time- and dose-dependent manner. CDDP generated reactive oxygen species (ROSs) in cells, which brought about a reduction in the intracellular mitochondrial transmembrane potential (Deltapsim), leading to apoptosis. Our findings demonstrate that ROSs, and the resulting oxidative stress, play a pivotal role in apoptosis. Preincubation of EC-109 cells with the hydrogen-peroxide-scavenging enzyme catalase partially inhibited the following: (i) the production of ROS; (ii) the disruption of the Deltapsim; and (iii) apoptosis. These results indicate that the enhancement of the generation of ROS and the disruption of Deltapsim are events involved in the apoptotic pathway of EC-109 induced by CDDP.

[The role of reactive oxygen species in cisplatin-induced apoptosis of esophageal cancer cell line EC-109].

BACKGROUND & OBJECTIVE: Reactive oxygen species (ROS), in vivo oxygen metabolites and important signaling molecules, play a vital role in cell apoptosis. This study was to investigate the role of ROS in cisplatin (DDP)-induced apoptosis of esophageal cancer cell line EC-109, and explore the mechanism. METHODS: EC-109 cells were treated with different concentrations (0, 1, 5, 10, and 15 microg/ml) of DDP. MTT assay was used to evaluate the influence of DDP on cell proliferation. Flow cytometry was used to test ROS levels, intracellular mitochondrial transmembrane potential (Delta psi m), and hypodiploid apoptosis peak in EC-109 cells. Cell apoptosis after pretreatment with hydrogen peroxide-scavenging enzyme catalase (CAT) was also detected. RESULTS: DDP obviously suppressed proliferation of EC-109 cells. When treated with 0, 1, 5, 10, 15 microg/ml of DDP for 2 h, ROS levels were (3.3+/-1.0)%, (21.6+/-2.0)%, (32.6+/-3.2)%, (44.7+/-2.2)%, and (53.1+/-3.6)%, respectively; when treated for 12 h, Delta psi m were (97.2+/-1.9)%, (90.6+/-1.9)%, (85.5+/-1.4)%, (67.8+/-2.0)%, and (62.4+/-3.0)%, respectively; when treated for 24 h, cell apoptotic rates were (3.4+/-1.2)%, (16.2+/-2.3)%, (28.1+/-1.5)%, (33.2+/-3.9)%, and (45.5+/-3.8)%, respectively. Pretreatment with CAT significantly rescued cells from apoptosis (P<0.05). CONCLUSION: DDP generates ROS in esophageal cancer EC-109 cells, which causes mitochondrial membrane permeabilization and Delta psi m decrease, therefore, leads to apoptosis of EC-109 cells.

Antitumor activity of a fusion of esophageal carcinoma cells with dendritic cells derived from cord blood.

The aim of this experiment was to develop a cytotoxic cancer vaccine (EC109-DC) prepared by fusions of esophageal carcinoma cells with dendritic cells derived from cord blood and to study the biological characteristics and resultant induction of antitumor immunity. CD34+ hematopoietic stem cells were isolated from cord blood using a CD34+ Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS). CD34+ cells were incubated with rhGM-CSF, rhTNF-alpha and rhSCF for 2 weeks as DC (dendritic cells), and then by PEG-3600 to fuse with an esophageal carcinoma cell line. Selection with MACS marked with HLA-DR MicroBeads generated EC109-DC. Phenotypes and proliferation were analyzed by flow cytometry and cell culture in vitro. The lymphocyte proliferation reaction and CTL cytotoxicity were examined by MTT assay. The EC109-DC cells could proliferate slowly in vitro and highly expressed CD80, CD83 and CD86. The lymphocyte proliferation reaction and specific cytotoxicity against EC109 induced by EC109-DC cells were significantly higher than in control groups (p < 0.05). EC109-DC cells obtained by PEG fusion acquired the immuno-stimulating phenotype and could significantly stimulate the lymphocyte proliferation reaction and CTL activity. The results of this research provide the basis for materials to develop the DC-based vaccine against esophageal carcinoma.