Multiple Signal Pathways Involved in Crocetin-Induced Apoptosis in KYSE-150 Cells.

BACKGROUND: Crocetin is a carotenoid extracted from the traditional Chinese medical herb saffron. Previous studies have demonstrated that crocetin possesses anticancer properties that are effective against various cancers. As an extension of our earlier study, the present study explored the underlying mechanisms in crocetin's anticancer effect on KYSE-150 cells. The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), Mitogen-activated protein kinases (MAPK), and p53/p21 signal pathways play an important role in carcinogenesis, progression, and metastasis of carcinoma cells. Thus, we investigated crocetin's effects on the PI3K/AKT, MAPK, and p53/p21 pathways in esophageal squamous carcinoma cell line KYSE-150 cells. METHODS: KYSE-150 cells were treated with various concentrations of crocetin. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide assay, Annexin V/PI stain as well as Rh123 stain were used to evaluate the cell viability, apoptosis, and MMP. Western blot was used to detect the expression of PI3K, AKT, ERK1/2, p38, c-Jun NH-terminal kinase (JNK), P53, P21, Bcl-2, Bax, and cleaved caspase-3, which were associated with cell proliferation and apoptosis. RESULTS: Our results showed that crocetin significantly inhibited the proliferation of KYSE-150 cells in a dose- and time-dependent manner. Crocetin also markedly induced cell apoptosis. Furthermore, we have found that crocetin not only inhibited the activation of PI3K/AKT, extracellular signal-regulated kinase-1/2 (ERK1/2), and p38 but also upregulated the p53/p21 level. These regulations ultimately triggered the mitochondrial-mediated apoptosis pathway with an eventual disruption of MMP, increased levels of Bax and cleaved caspase-3, and decreased levels of Bcl-2. CONCLUSIONS: These findings suggested that crocetin interfered with multiple signal pathways in KYSE-150 cells. Therefore, this study suggested that crocetin could potentially be used as a therapeutic candidate for the treatment of esophageal cancer.

Synergistic anticancer effect of combined crocetin and cisplatin on KYSE-150 cells via p53/p21 pathway.

BACKGROUND: More than 400,000 patients die from esophageal cancer annually. Considerable efforts have been made to develop new and effective treatments, one of which is directed toward herbal medication. Crocetin is a natural carotenoid dicarboxylic acid isolated from the Chinese herb saffron. We recently reported on the anticancer effects of saffron. This study aimed to determine whether crocetin combined cisplatin has synergistic effect in KYSE-150 cells and explore the underlying mechanism. METHODS: KYSE-150 cells were treated with crocetin and/or cisplatin. The effects on cell viability, cell apoptosis, mitochondrial membrane potential (MMP), as well as the expression levels of PI3K/AKT, MAPKs, p53/p21, and apoptosis-related protein were evaluated. MTT assay, Annexin V-FITC/PI staining, Rh123 staining, and Western blot analysis were used. RESULTS: The cell proliferation significantly decreased and cell apoptosis was induced with combined crocetin and cisplatin, compared with either crocetin only or cisplatin only. The outcome suggested that crocetin combined cisplatin has synergistic effects on inhibition of cell proliferation and pro-apoptotic effect of cisplatin on KYSE-150 cells. Disruption of MMP, upregulation of cleaved caspase-3 expression, and downregulation of Bcl-2 occurred in the group treated with combined treatment. No significant differences in p-PI3K, p-AKT, and MAPKs activity were indicated between combined treatment group and the individual treatment group. However, the expression levels of p53 and p21 were markedly higher in the combined treatment group than in the individual treatment group. The wild-type p53 inhibitor, PFT-α suppressed the overexpression of p53/p21 and the synergistic effect induced by the combination of crocetin and cisplatin. CONCLUSIONS: We concluded that crocetin combined with cisplatin exerts a synergistic anticancer effect by up-regulating the p53/p21 pathway.

Identification of microRNAs involved in the radioresistance of esophageal cancer cells.

Radioresistance is considered as the most important reason for local tumour recurrence. This study investigates the role of miRNAs in radioresistant human esophageal cancer cells. Human miRNA microarray has been used to detect the differential expressed microRNAs between radioresistant esophageal cell line KYSE-150R and the parental cell line KYSE-150. The relative expression of some candidate miRNAs was measured by quantitative real-time PCR (qRT-PCR). Potential mRNA targets were analysed bioinformatically. Significant upregulation of 10 microRNAs and downregulation of 25 microRNAs were detected. The statistical significance of downregulation in hsa-miR-301a, hsa-miR-141 and hsa-miR-18b expression (P < 0.05) were confirmed by qRT-PCR. The correlation of the predicted microRNA target genes to apoptosis (63 genes), cell cycle (67 genes), DNA damage and repair (18 genes) were confirmed by functional annotation. The downregulation of hsa-miR-301a promoted radioresistance in KYSE-150R through the upregulation of wnt1, indicating that wnt/ß-catenin signal pathway might be important in radioresistance. In conclusion, a unique set of miRNAs and their expression profiles in radiation resistance have been identified, providing a solid basis for future studies to investigate the target genes of these miRNAs and their function.

Antitumor effect of recombinant human interferon-ß adenovirus on esophageal squamous cell cancer in vitro.

Interferon (IFN)-ß has efficient antitumor effect both in vitro and in vivo, but its clinical implication is limited by short half-life and systemic toxicities. Gene therapy could be the choice to avoid the defects. Adenovirus vector containing human IFN-ß gene was transfected into esophageal squamous cell carcinoma KYSE150 cells. Expression of human (h)IFN-ß was detected by reverse transcription polymerase chain reaction and immunocytochemistry in KYSE150 cells. Cell growth and clonogenic assays, and flow cytometry were used to observe the antiproliferation effect and apoptosis on tumor cells, respectively. Reverse transcription polymerase chain reaction and immunocytochemistry showed obvious hIFN-ß expression in KYSE150 cells after transfection and the tumor cell proliferation was obviously inhibited through cell proliferation and clonogenic assays. Flow cytometry analysis showed 27.3% cell apoptosis in adenovirus vector containing human IFN-ß gene transfection group compared with 1.12% in empty vector control group. These findings indicate that hIFN-ß gene mediated by recombinant adenovirus may have antitumor activity against human esophageal carcinoma cell by inducing apoptosis in vitro.

Matrine-loaded Nano-liposome Induces Apoptosis in Human Esophageal-squamous Carcinoma KYSE-150 Cells.

INTRODUCTION: Esophageal-Squamous Cell Carcinoma (ESCC) is often diagnosed at the middle or late stage, thus requiring more effective therapeutic strategies. Pharmacologically, the anti-tumor activity of the principal active constituent of Sophora flavescens, matrine (MA), has been explored widely. Notwithstanding, it is significant to nanotechnologically enhance the anti-tumor activity of MA in view of its potential to distribute non-tumor cells. METHODS: Herein, MA-loaded Nano-Liposomes (MNLs) were prepared to enhance the effect of anti-ESCC. The MNL showed a smaller sized particle (25.95 ± 1.02 nm) with a low polydispersed index (PDI = 0.130 ± 0.054), uniform spherical morphology, good solution stability, and encapsulated efficiency (65.55% ± 2.47). Furthermore, we determined the characteristics of KYSE-150 cells by cell viability assay, IC50, Mitochondrial Membrane Potential (MMP), Western blot, and apoptotic analysis, which indicated that MNLs down-regulated the cell viability and IC50 in a concentration-dependent manner and induced a significant change in JC-1 fluorescence from red to green. RESULTS: The above observations resulted in increased Bax and Caspase-3 levels, coupled with a substantial decrease in Bcl-2 and apoptotic promotion at the advanced stage compared with MA. CONCLUSION: Based on these results, MNLs may serve as a more effective and promising therapeutic option for ESCC.

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells.

In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R2 = 0.9981-0.9999), precision (interday precision of 1.6%-19.0% and intraday precision of 1.4%-19.8%) and accuracy (83.4%-115.7%). Moreover, the recovery assessments in tissues (82.5%-107.3%) and in cells (98.1%-118.9%) with GSH-13C2, 15N, and Cys-15N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.

miR-488-5p promotes esophageal squamous cell carcinoma progression by suppressing the P53 pathway.

BACKGROUND: miR-488-3p has been reported to play an important role in cancer progression and metastasis. The protein 53 (P53) gene serves as a mediator and biomarker of esophageal squamous cell carcinoma (ESCC). However, the molecular mechanism underlying miR-488-5p in the pathology of ESCC through the P53 pathway has not been examined. METHODS: The expression levels of miR-488-5p were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cytological experiments were performed to evaluate the biological functions of miR-488-5p. A bioinformatics analysis was performed to determine the pathways and key miR-488-5p targets associated with ESCC. Correlations between miR-488-5p and P53 signaling pathways were validated by western blotting and the dual luciferase reporter gene system. Finally, the expression level of miR-488-5p was regulated and tumor formation experiments were performed in nude mice. RESULTS: The qRT-PCR analysis showed that MiR-488-5p expression was more upregulated in the KYSE-150 group than the HEEC group. In the KYSE-150 cells, the colony formation assay and flow cytometry analysis indicated that the miR-488-5p inhibitor inhibited cell viability and increased cell apoptosis; however, these effects were recovered by P53 knockdown (KD). In addition, cell invasion and cell migration were inhibited by the miR-488-5p inhibitor, but were also improved by P53 KD. Similarly, the miR-488-5p inhibitor induced the expression of P53 and P21 than normal control (NC) group in which miR-488-5p expression was normal, while P53 KD prevented the effects of the miR-488-5p inhibitor in KYSE-150 cells. Additionally, we found that tumor size was obviously smaller in miR-488-5p overexpression (OE)+ P53 OE mice than miR-488-5p OE mice. Hematoxylin and eosin and immunohistochemistry staining also revealed similar results. CONCLUSIONS: Our results suggest that miR-488-5p promotes ESCC progression by suppressing the P53 pathway. These findings should provide novel ideas for ESCC therapies.

LINC00152 Knock-down Suppresses Esophageal Cancer by EGFR Signaling Pathway.

AIM: This study aims to explain the role and mechanism of lncRNA LINC00152 in esophageal cancer. METHODS: The 30 pairs of esophageal cancer and adjacent normal tissues were collected and measuring the lncRNA LINC00152 expression by ISH and RT-qPCR assay. In the next cell experiment, Eca 109 and Kyse 150 cells were divided into 3 groups: NC group were treated with non-treatment; BL group were transfected with empty vector and lncRNA group were transfected with lncRNA LINC00152. The cells proliferation were measured by MTT assay; the cells apoptosis and cell cycle were evaluated by flow cytometry. The relative proteins expressions were measured by WB assay. RESULTS: Compared with NC groups, the cell proliferation rate of lncRNA groups were significantly suppressed (P<0.05, respectively); the cell apoptosis and G1 phase rates were significantly enhanced in the lncRNA groups (P<0.05, respectively). In the proteins expressions, the EGFR, PI3K and AKT proteins expressions of lncRNA group were significantly inhibited and the P21 proteins expressions were significantly stimulated in the lncRNA groups compared with those of NC groups in Eca 109 and Kyse 150 cells. CONCLUSION: The lncRNA LINC00152 had anti-tumor effects on esophageal cancer in the Eca 109 and Kyse 150 cells, the mechanisms were relative with EGFR pathway.

lncRNA MIAT promotes cell invasion and migration in esophageal cancer.

Long non-coding RNAs (lncRNAs) serve crucial roles in carcinogenesis. Myocardial infarction-associated transcript (MIAT), originally isolated as a candidate gene for myocardial infarction, has been revealed to serve as an oncogene in chronic lymphocytic leukaemias and neuroendocrine prostate cancer. However, little is known about its expression pattern, biological function and underlying mechanism in esophageal cancer. Cell lines of esophageal cancer were used in the current study. The results of the present study revealed that MIAT knockdown decreased cell viability, migration, invasion and cell cycle arrest in the G1 phase. Mechanistic assessment revealed that MIAT interacts with histone methyltransferase mixed-lineage leukemia (MLL). The relative proteins expressions were measured by western blotting assay. MIAT knockdown suppressed cell invasion and migration by regulation MMP-2/9 protein expressions. The results of the current study indicated that MIAT expression was associated with esophageal cancer and may serve as a critical target in the progression and metastasis in esophageal cancer.

Downregulation of H19 decreases the radioresistance in esophageal squamous cell carcinoma cells.

Background: Radiotherapy is one of the most common treatments for esophageal squamous cell carcinoma (ESCC). Radioresistance is a major obstacle that limits the efficacy of radiotherapy. H19 has been considered as a factor affecting radioresistance, whereas the specific mechanism of H19 in ESCC radioresistance remains to be further elucidated. Purpose: The objective of this study was to identify the relationship between H19 and radioresistance. The findings are expected to provide new insights into the treatment of radioresistant ESCC. Methods: The expression levels of H19 in ESCC was analyzed using the online database starBase. The Oncomine database was used to further verify the association between H19 expression and patient age, gender, and tumor stage. The overall survival rates of ESCC patients were analyzed using the KM plotter database. Clonogenic survival was conducted to identify the value of survival fraction. The optical density values were obtained via MTS assays. Cells migration and stemness were observed through Transwell and sphere formation assays. The expression levels of H19, miR-22-3p and WNT1 were analyzed using qPCR. Results: In our study, we firstly screened the H19 according to the online database starBase, and then the Oncomine database and KM plotter database showed that H19 expression was significantly upregulated in the ESCC tissues and associated with poor prognosis. Secondly, an ESCC radioresistant cell line, KYSE150R was established. Clonogenic survival showed that radiation decreased the value of survival fraction. MTS assays suggested that optical density values in KYSE150R cells were significantly higher than that in KYSE150 cells. Transwell and sphere formation assays showed radiation enhanced cell migration and stemness in ESCC cells. In addition, qPCR showed that H19 was upregulated in KYSE150R cells, and survival fraction assays showed that knockdown of H19 decreased the survival fraction values. MTS assays, migration and invasion assays suggested that H19 inhibited cells proliferation, migration and stemness in radioresistant KYSE150 cells. Moreover, qPCR assay showed that miR-22-3p expression levels was downregulated, but WNT1 was upregulated in KYSE150R cells as well as protein levels. Luciferase activity assay further showed that miR-22-3p inhibits the WNT1 expression. Conclusion: Our results demonstrate that H19 knockdown downregulates the WNT1 via upregulating miR-22-3p expression, which leads to the inhibition of cells proliferation, migration and stemness in the radioresistant ESCC cells.

Chinese herb medicine matrine induce apoptosis in human esophageal squamous cancer KYSE-150 cells through increasing reactive oxygen species and inhibiting mitochondrial function.

Matrine, as a natural alkaloid isolated from the traditional herb medicine sophora flavescens, has been proved to possess excellent biological activities, including anticancer effects. Now, this research aims to assess the anticancer activities and the mechanism of matrine against esophageal cancer cells, we investigated the proliferative inhibition, apoptosis induction, as well as the underlying mechanism of matrine on esophageal cancer KYSE-150 cells. It was found that matrine could suppress KYSE-150 cell proliferation and significantly mediate cell apoptosis in a dose-dependent relation by increasing intracellular reactive oxygen species level and triggering mitochondrial membrane potential disruption. More precise mechanism studies demonstrated that matrine could up-regulate the expression of Bax proteins and down-regulate the expression of Bcl-2 proteins, as well as the activation about caspase-3, 8 and 9 in KYSE-150 cells. The morphological analysis of KYSE-150 cells exhibited that matrine could destroy the F-actin and nuclei structures and induce morphological damage with increased surface height distribution and roughness of cell membrane. These results not only demonstrated the potential anticancer activity mechanism of matrine at nanoscale, but also provide preliminary guidance for the treatment of esophageal cancer using matrine.

Anticancer effects of crocetin in human esophageal squamous cell carcinoma KYSE-150 cells.

Crocetin is the main pharmacologically-active component of saffron and has been considered as a promising candidate for cancer chemoprevention. The purpose of the present study was to investigate the anticancer effects of crocetin and the possible mechanisms of these properties in the esophageal squamous cell carcinoma cell line KYSE-150. The KYSE-150 cells were cultured in Dulbecco's modified Eagle's medium and incubated with 0, 12.5, 25, 50, 100 or 200 µmol/l crocetin for 48 h. Cell proliferation was measured using an MTT assay. Hoechst 33258 staining and observation under fluorescent microscopy were used to analyze the proapoptotic effects of crocetin. The migration rate was assessed by a wound-healing assay. The cell cycle distribution was analyzed using flow cytometry analysis subsequent to propidium iodide staining. The expression of B-cell lymphoma-2-associated X protein (Bax) and cleaved caspase 3 was determined by western blot analysis. It was found that treatment of KYSE-150 cells with crocetin for 48 h significantly inhibited the proliferation of the cells in a concentration-dependent manner, and the inhibition of proliferation was associated with S phase arrest. Crocetin was also found to induce morphological changes and cell apoptosis in a dose-dependent manner through increased expression of proapoptotic Bax and activated caspase 3. In addition, crocetin suppressed the migration of KYSE-150 cells. The present study provides evidence that crocetin exerts a prominent chemopreventive effect against esophageal cancer through the inhibition of cell proliferation, migration and induction of apoptosis. These findings reveal that crocetin may be considered to be a promising future chemotherapeutic agent for esophageal cancer therapy.