Amlodipine inhibits the proliferation and migration of esophageal carcinoma cells through the induction of endoplasmic reticulum stress.

BACKGROUND: L-type calcium channels are the only protein channels sensitive to calcium channel blockers, and are expressed in various cancer types. The Cancer Genome Atlas database shows that the mRNA levels of multiple L-type calcium channel subunits in esophageal squamous cell carcinoma tumor tissue are significantly higher than those in normal esophageal epithelial tissue. Therefore, we hypothesized that amlodipine, a long-acting dihydropyridine L-type calcium channel blocker, may inhibit the occurrence and development of esophageal cancer (EC). AIM: To investigate the inhibitory effects of amlodipine on EC through endoplasmic reticulum (ER) stress. METHODS: Cav1.3 protein expression levels in 50 pairs of EC tissues and corresponding paracancerous tissues were examined. Subsequently, the inhibitory effects of amlodipine on proliferation and migration of EC cells in vitro were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and Transwell assays. In vivo experiments were performed using murine xenograft model. To elucidate the underlying mechanisms, in vitro cell studies were performed to confirm that ER stress plays a role in inhibition proliferation and migration of EC cells treated with amlodipine. RESULTS: The expression level of Cav1.3 in esophageal carcinoma was 1.6 times higher than that in paracancerous tissues. Amlodipine treatment decreased the viability of esophageal carcinoma cells in a dose- and time-dependent manner. In vivo animal experiments also clearly indicated that amlodipine inhibited the growth of EC tumors in mice. Additionally, amlodipine reduces the migration of tumor cells by inhibiting epithelial-mesenchymal transition (EMT). Mechanistic studies have demonstrated that amlodipine induces ER stress-mediated apoptosis and suppresses EMT. Moreover, amlodipine-induced autophagy was characterized by an increase in autophagy lysosomes and the accumulation of light chain 3B protein. The combination of amlodipine with the ER stress inhibitor 4-phenylbutyric acid further confirmed the role of the ER stress response in amlodipine-induced apoptosis, EMT, and autophagy. Furthermore, blocking autophagy increases the ratio of apoptosis and migration. CONCLUSION: Collectively, we demonstrate for the first time that amlodipine promotes apoptosis, induces autophagy, and inhibits migration through ER stress, thereby exerting anti-tumor effects in EC.

[Effects of TOFA on growth of Eca109 and KYSE-450 cells in human esophageal squamous cell carcinoma].

Objective: To investigate the effects of 5-tetradecanoxy 2-furanic acid (TOFA) on cell proliferation, cell cycle and apoptosis of esophageal squamous cell carcinoma (ESCC) cells. Methods: Eca-109 cells and KYSE-450 cells were divided into control group (DMSO) and experimental group (TOFA), respectively. The cells (4×103 cells/100 µl) were inoculated into 96-well plates with 5 multiple wells at each concentration. After 24 h culture, cells were treated with DMSO or different concentrations (1, 3, 5, 10 µg/ ml) of TOFA for 24, 48 and 72 h. Cell proliferation was detected by MTT, cell cycle and apoptosis were detected by flow cytometry, the expression levels of p21 and Cleaved caspase-3 and modification levels of p-Akt, p-mTOR and p-4EBP1 were detected by Western blot, and intracellular free fatty acids were detected by special kits. Results: MTT results showed that TOFA inhibited the proliferation of Eca109 and KYSE-450 cells in a concentration and time dependent manner (all P<0.05), with IC50 of 4.65 µg/ml and 3.93 µg/ml for 48 h, respectively. Flow cytometry results showed that compared with DMSO group, the percentage of cells in G2/M phase was increased and the apoptosis rate was increased in the experimental group. Western blotting results showed that compared with DMSO group, p21 and Cleaved caspase-3 protein expression levels were up-regulated, and p-AKT, p-mTOR and p-4EBP1 protein expression levels were down-regulated (all P<0.05). Conclusion: TOFA inhibits the proliferation, blocks the cycle progression and promotes apoptosis of ESCC, the mechanism may be related to the AKT/mTOR/4EBP1 signaling pathway.

[Influence of Substrate Exposure Level on ANAMMOX Microbial Activity and Biomass].

Substrate exposure levels are vital for the growth and metabolism of ANAMMOX microorganisms, and their effects on growth characteristics of ANAMMOX sludge during the enrichment process have been rarely reported. Using two continuous flow stirred reactors and the process of a gradually developing nitrogen load, the changes in biomass and activity, as well as nitrogen removal efficiency of the reactors were investigated under high substrate exposure level culture mode (R1:effluent NH4+-N and NO2--N concentrations were 40-60 mg·L-1) and low substrate exposure level culture mode (R2:effluent NH4+-N and NO2--N concentrations were 0-20 mg·L-1). The results showed that the high substrate exposure level culture mode was more beneficial to the improvement of nitrogen removal performance of the ANAMMOX reactor. For comparison, the NLR (nitrogen load rate), which was 0.69 kg·(m3·d)-1, and the NRR (nitrogen remove rate), which was up to 0.41 kg·(m3·d)-1, was obtained in the high substrate exposure culture mode. These values were twice as high as those obtained in the low substrate exposure culture mode. Under the culture mode with high substrate exposure level, the sludge concentration (in VSS) and the total gene copy numbers of ANAMMOX reached 1805 mg·L-1 and 4.81×1012 copies, respectively, which was conducive to the rapid enrichment of ANAMMOX microorganisms. In the low substrate exposure level culture mode, ANAMMOX sludge was more active,in N/VSS, 0.27 g·(g·d)-1, which was conducive to the cultivation of ANAMMOX sludge with higher biological activity.

[Inhibitory Effects of Phosphate and Recovery on a Nitrification System].

The effect of phosphate concentration on nitrification was studied by using a stabilization nitrosation system, which was started up in a continuous flow reactor by inoculating sludge from a municipal wastewater treatment plant. The results showed that the nitrification system was started successfully after operating for 14 days. The conversion rate of ammonia nitrogen reached 92.2%, the nitrite accumulation rate was 73.66%, and the nitrite generation rate was 14.42 g·(m3·d)-1. There was no effect of phosphate concentration on the nitrosation system between 10 and 30 mg·L-1; and the conversion rate of ammonia nitrogen was decreased with the continuous increase in phosphate concentration. When the concentration of phosphate was 80 mg·L-1, with an ammonia conversion rate 13.6%, accumulation rate of nitrite of 18.19%, and nitrite generation rate of 0.54 g·(m3·d)-1, the reaction was severely inhibited. After reducing the influent phosphate concentration to 0, with the ammonia nitrogen conversion rate at more than 80%, nitrite accumulation rate improved to 86.96%, and the nitrite generation rate being 15.63 g·(m3·d)-1, the system recovered after operating for 14 days.

[Effect of Temperature and pH on Nitrogen Conversion in Feammox Process].

In recent years, the oxidation of NH4+ using Fe(Ⅲ) as an electron acceptor under anaerobic conditions (Feammox) has received significant research attention. In this study, the effect of pH and temperature on nitrogen conversion during the Feammox process was studied through activity recovery of Feammox sludge acclimated by anaerobic ammonium oxidation (ANAMMOX) sludge. Results showed that after 40 d operation, activity of Feammox sludge was recovered. There was evident ammonia nitrogen conversion and total nitrogen removal from the environment, and the products were mainly nitrate and nitrogen. The concentration of nitrite remained below 2 mg·L-1. pH value and temperature significantly influenced nitrogen transformation during the Feammox process. With pH value of 7 and temperature of 30℃ during the Feammox process, the removal rate of total nitrogen was relatively high (>50%). When the pH value was 6.5, the conversion rate of ammonia nitrogen was 80.2%. During the Feammox reaction process, precipitation of iron ion compounds and coating on the sludge surface were the main interference factors leading to continuous operation of the reactor and exploration of the reaction mechanism.

[Analysis of CANON Process Start-up with Fiber Carrier].

A CANON reactor with fiber carrier was started up by seeding nitrification sludge and ANAMMOX sludge to study the operating characteristics of a fiber carrier. The results showed that total nitrogen removal load rose from 0.09 kg·(m3·d)-1 to 0.9 kg·(m3·d)-1 and remained steady in the 85th day. This indicated that fiber carrier is beneficial to the accumulation of sludge, and the reactor can maintain a higher biomass. The DO in the reactor reached 5 mg·L-1 with the enrichment of microorganisms, biofilm thickening, and the improvement of the reactor's ability. The DO gradient of the biofilm from the outside to the inside was 0.32-0 mg·L-1, which could be obtained by a microelectrode. It was shown that the permeability of oxygen to the biofilm decreased, and the amount of nitrifying microorganisms decreased with biofilm thickening. The quantitative PCR results showed that the abundance of ANAMMOX was an order of magnitude more than before. The abundance of AOB increased slightly, while the abundance of NOB stayed at a relatively low level.

[Granular Characteristics of Anaerobic Ammonia Oxidation Sludge During the Recovery Process].

Operation instability has become one of the factors restricting the application of the anaerobic ammonia oxidation (ANAMMOX) process. Under the condition that the substrate is not suppressed, the effects of the substrate concentration on the granulation and activity of ANAMMOX granular sludge in the recovery process were studied by restoring the activity of ANAMMOX sludge, which was derived from early-stage operation instability of the continuous stirred tank reactor (CSTR). The results show that the activity of ANAMMOX sludge was recovered and the denitrification capacity increased significantly after 126 days of operation. When the NH4+-N and NO2--N concentrations were 450 mg·L-1 and 560 mg·L-1, respectively, the nitrogen removal was achieved in both the high-and low-substrate concentration reactors and the maximum NRR was 16.97 kg·(m3·d)-1 and 14.43 kg·(m3·d)-1, respectively. With the improvement of the nitrogen removal capacity of the reactor (the granular diameter of the sludge is increased), the extracellular polymeric substance (EPS) content increased in both reactors from 34.45 to 77.52 and to 94.18 mg·g-1, respectively, and the PN/PS increased from 1.89 to 6.25 and 6.84, respectively. To a certain extent, the increase of PN/PS is conducive to the granulation of ANAMMOX sludge, but a too large PN/PS would lead to the instability of granular sludge and sludge loss.

[Ammoniac Waste Gas in-situ Treatment Based on ANAMMOX Process].

The feasibility of ammoniac waste gas in-situ treatment by partial nitrification(PN)-anaerobic ammonia oxidation(ANAMMOX) reactor was studied. The PN-ANAMMOX reactor was successfuly started up after 60 d operation in conditions of low dissolved oxygen control(0.2 mg·L-1), pH 7.9-8.2 and temperature(30-35℃). The total nitrogen removal efficiency reached 88% and the nitrogen removal rate increased from 0.05 kg·(m3·d)-1 to 0.7 kg·(m3·d)-1. In the treatment of ammoniac waste gas, the excess oxygen in it would lead to massive growth of nitrifying bacteria, when the ammonia concentration was lower than 2.59%. The ammonia and oxygen in waste gas met the requirement of the reactor for nitrogen removal, when the ammonia concentration stayed between 2.59% and 4.2%. Extra air flow was required for reactor oxygen supplement when the ammonia concentration was higher than 4.2%. The ammonia removal efficiency reached 100%, the total nitrogen removal efficiency reached 90.06%, while the total nitrogen removal rate was 0.51 kg·(m3·d)-1 after 60 day operation for ammoniac waste gas purification. It is indicated that the integrated reactor based on PN-ANAMMOX process can achieve stable removal of ammonia waste gas.

[Removal of Nitrogen from Alcohol Wastewater by PN-ANAMMOX].

An integrated partial nitrification anaerobic ammonia oxidation reactor was used to explore the feasibility of nitrogen removal from recycled ethanol wastewater. The results show that the integrated partial nitrification-anaerobic ammonia oxidation (PN-ANAMMOX) reactor was started successfully after 40 d under the conditions of pH 7.8±0.5, temperature 30-35℃, and aerobic ORP value 120-150 mV. The total nitrogen removal rate of 0.125 kg·(m3·d)-1 increased to 0.75 kg·(m3·d)-1, Inoculation of mature nitrosated biofilms and anaerobic ammonium oxide granules can accelerate the start of the reactor. The effects of alcohol wastewater on the PN-ANAMMOX reactor were mainly caused by biodegradable TOC, The biodegradable TOC concentration of 100mg·L-1 in alcohol wastewater can reduce the removal rate of total nitrogen from 0.75 kg·(m3·d)-1 to 0.25 kg·(m3·d)-1,this inhibition can be restored. Different concentrations of alcohol wastewater were dosed into the PN-ANAMMOX reactor to acclimate the bacteria. The total nitrogen removal rate first decreased and then increased, as the influent concentration gradient increased, which was beneficial for improving the efficiency of nitrogen removal by extending the HRT and increasing the dissolved oxygen in the PN stage. Finally, the nitrogen removal rate stabilized at 0.65 kg·(m3·d)-1. These results show that PN-ANAMMOX can be used for the treatment of alcohol wastewater.

[Start-up and Capacity Enhancement of a Partial Nitrification Pilot Reactor in Continuous Flow].

The continuous flow reactor was used to treat simulated ammonia nitrogen wastewater by inoculating the sludge after filtration and adding a suspended filler. Regulations of free ammonia (FA), free nitrous acid (FNA), and dissolved oxygen (DO) in the reactor were the key to achieving a successful start-up of the pilot scale nitrosation reactor. The results show that the enrichment of ammonium oxidizing bacteria (AOB) and the elimination of nitrite oxidizing bacteria (NOB) are achieved by adjusting the operational mode of high DO, low DO, FA, and FNA in the reactor operation. The nitrite production rate (NPR) in the reactor was 1.27 kg·(m3·d)-1 and the nitrogen accumulation rate (NAR) was stable at 98% at the end of the start-up period. qPCR was used to study the difference in the functional microorganisms (AOB, NOB) between the beginning and the end of the start-up period. The results show that the copy number of microbial AOB grew from 5.3×109 copies·mL-1 to 1.6×1011 copies·mL-1. The copy number of NOB decreased from 1.1×1010 copies·mL-1 to 1.2×109 copies·mL-1, because of the joint regulation of DO, FA, FNA to suppress NOB.

[Influence of Phosphate on Nitrogen Removal Efficiency of ANAMMOX Sludge].

In this study, the effect of phosphate concentration on the nitrogen removal efficiency of ANAMMOX sludge was researched. The inhibition kinetic parameters were fitted and the abundance of ANAMMOX bacteria in the reactor before and after phosphate inhibition was analyzed by Real-time PCR. The short-term experiments showed that there was no significant effect on the nitrogen removal efficiency of ANAMMOX sludge when phosphate concentration was less than 30mg·L-1. With the increase of influent phosphate concentration, the removal rate of nitrogen decreased rapidly. When the phosphate concentration was raised to 200mg·L-1, the activity of ANAMMOX sludge reached a complete inhibition state. The parameters of phosphate inhibition were fitted by Haldane inhibition model. The half-inhibition constant was 70.1 mg·L-1. The long-term results showed that when the phosphate concentration was less than 50 mg·L-1, the effect on the nitrogen removal efficiency of ANAMMOX sludge was not significant. When the phosphate concentration reached 70-90 mg·L-1, The activity of ANAMMOX sludge was obviously affected, and the stability could be restored after a certain period. Increasing the phosphate concentration had a more significant effect and resulted in a longer recovery time. Finally, when the concentration of phosphate reached 100 mg·L-1, the nitrogen removal efficiency of ANAMMOX was seriously inhibited. The rate of nitrogen removal decreased from 158.33 g·(m3·d)-1 to 60.17 g·(m3·d)-1, resulting in 62% inhibition. The results of real-time PCR showed that the concentration of ANAMMOX was decreased from (9.97±0.86)×107 cells·mL-1 to (8.26±0.54)×107 cells·mL-1 in the restraining sludge system, and there was a relatively reduced trend.

Berberine displays antitumor activity in esophageal cancer cells in vitro.

AIM: To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS: Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS: Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 µmol/L berberine for 48 h, the number of cells in G2/M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION: Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.

[Incorporation of TV regularization in deconvolution for partial volume correction in PET imaging].

OBJECTIVE: We propose a method using total variation (TV) regularization in deconvolution for partial volume correction in PET imaging. In the degraded image model, we used TV regularization procedure in Van Cittert (VC) and Richardson-Lucy (RL) deconvolution algorithms. These methods were tested in simulated NCAT images and images of NEMA NU4-2008 IQ phantom and tumor-bearing mouse scanned by Simens Invoen microPET. The simulated experiment and tumor-bearing mouse experiment showed that the algorithms using TV regularization provided superior qualitative and quantitative appearance compared with traditional VC and RL algorithms. When the mean intensity of the tumor increased by (10±1.8)%, the SD increase percentage was decreased from 49.98% to 14.26% and from 42.76% to 4.70%, suggesting the efficiency of the proposed algorithms for reducing PVEs in PET.

High DEPTOR expression correlates with poor prognosis in patients with esophageal squamous cell carcinoma.

OBJECTIVE: The disheveled, Egl-10, and pleckstrin (DEP) domain containing mammalian target of rapamycin (mTOR)-interacting protein (DEPTOR) is a binding protein containing mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2), and an endogenous mTOR inhibitor. DEPTOR shows abnormal expressions in numerous types of solid tumors. However, how DEP-TOR is expressed in esophageal squamous cell carcinoma (ESCC) remains elusive. METHODS: The expression of DEPTOR in 220 cases of ESCC and non-cancerous adjacent tissues was detected by immunohistochemistry. DEPTOR levels in ESCC and paired normal tissue were quantified using reverse transcription-polymerase chain reaction and Western blot analysis to verify the immunohistochemical results. The relationship between DEPTOR expression and the clinicopathological features of ESCC was analyzed based on the results of immunohistochemistry. Finally, we analyzed the relationship between DEPTOR expression and the prognosis of patients with ESCC. RESULTS: Immunohistochemical staining showed that the expression rate of DEPTOR in ESCC tissues was significantly increased. DEPTOR mRNA and protein expression was significantly higher in ESCC tissues than in normal adjacent esophageal squamous tissues. High DEPTOR expression was significantly correlated with regional lymph node status in the TNM stage of patients with ESCC. Kaplan-Meier survival curves showed that the rate of overall survival was significantly lower in patients with high DEPTOR expression than in those with low DEPTOR expression. Additionally, high DEPTOR expression was an independent prognostic predictor for ESCC patients. CONCLUSION: High DEPTOR expression is an independent prognostic biomarker indicating a worse prognosis for patients with ESCC.