Proliferation Of EC9706 Cells Research Articles

Integrative analysis of bulk and single-cell gene expression profiles to identify tumor-associated macrophage-derived CCL18 as a therapeutic target of esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignancy with poor patient prognosis. Current treatment for ESCC, including immunotherapy, is only beneficial for a small subset of patients. Better characterization of the tumor microenvironment (TME) and the development of novel therapeutic targets are urgently needed.MethodsIn the present study, we hypothesized that integration of single-cell transcriptomic sequencing and large microarray sequencing of ESCC biopsies would reveal the key cell subtypes and therapeutic targets that determine the prognostic and tumorigenesis of ESCC. We characterized the gene expression profiles, gene sets enrichment, and the TME landscape of a microarray cohort including 84 ESCC tumors and their paired peritumor samples. We integrated single-cell transcriptomic sequencing and bulk microarray sequencing of ESCC to reveal key cell subtypes and druggable targets that determine the prognostic and tumorigenesis of ESCC. We then designed and screened a blocking peptide targeting Chemokine C–C motif ligand 18 (CCL18) derived from tumor associated macrophages and validated its potency by MTT assay. The antitumor activity of CCL18 blocking peptide was validated in vivo by using 4-nitroquinoline-1-oxide (4-NQO) induced spontaneous ESCC mouse model.ResultsComparative gene expression and cell–cell interaction analyses revealed dysregulated chemokine and cytokine pathways during ESCC carcinogenesis. TME deconvolution and cell interaction analyses allow us to identify the chemokine CCL18 secreted by tumor associated macrophages could promote tumor cell proliferation via JAK2/STAT3 signaling pathway and lead to poor prognosis of ESCC. The peptide Pep3 could inhibit the proliferation of EC-109 cells promoted by CCL18 and significantly restrain the tumor progression in 4-NQO-induced spontaneous ESCC mouse model.ConclusionsFor the first time, we discovered and validated that CCL18 blockade could significantly prevent ESCC progression. Our study revealed the comprehensive cell–cell interaction network in the TME of ESCC and provided novel therapeutic targets and strategies to ESCC treatment.

ECT2 promotes proliferation and metastasis of esophageal squamous cell carcinoma via the RhoA-ERK signaling pathway.

In this study, the effect of epithelial cell transformation sequence 2 (ECT2) on the proliferation, invasion and migration of esophageal squamous cell carcinoma (ESCC) was investigated by interfering the expression of ECT2. Interfering with the expression level of ECT2 in human squamous cell carcinomas KYSE140 and EC9706 cell lines, the changes of KYSE140 and EC9706 cell proliferation, invasion, and migration were measured using the CCK-8 method, transwell test, and scratch test, respectively. The effects of ECT2 on the Ras homolog gene family, member A-extracellular regulated protein kinases (RhoA-ERK) signaling pathway were also observed. Compared with the control group, the proliferation, migration, and invasion ability of EC9706 and KYSE140 cells after ECT2 knockout were significantly reduced (p <0.05). The knockdown of ECT2 expression in ESCC cell lines suppressed the activation of RhoA-ERK signaling pathway and protein expression of VEGF and MMP9. ECT2 could regulated the expression of VEGF and MMP9 to inhibit cells proliferation, invasion, migration and tumor development through RhoA-ERK signaling pathway. Therefore, ECT2 could be an available marker, and provide a new theoretical basis for the treatment of ESCC.

Combination treatment with artemisinin and oxaliplatin inhibits tumorigenesis in esophageal cancer EC109 cell through Wnt/β-catenin signaling pathway.

BackgroundEsophageal cancer (EC) is a prevalent malignant cancer worldwide. Interestingly, the antimalaria compound artemisinin (ART) is also reported to have anticancer potential, although its underlying mechanism in EC is unclear. In this study, we explored the anticancer role of ART in EC109 and further explored the combination of ART and oxaliplatin (OXA) for their synergetic anticancer functions.MethodsHuman EC cell line EC109 was used. After ART or oxaliplatin (OXA) treatment, cell proliferation, migration, and invasion were measured by MTT, transwell, and scratch wound assays, respectively. Flow cytometry was performed to examine the cell cycle and apoptosis. The mRNA and protein levels were determined using qRT‐PCR and western blotting.ResultsThe migration and invasion abilities of EC109 were suppressed by ART. This was due to the inhibitory effect of ART on the Wnt/β‐catenin signaling pathway. The levels of β‐catenin, c‐myc, and survivin were also downregulated by ART. ART inhibits the proliferation of EC109 cells by arresting the cells in the G1‐phase of cell cycle. By using LiCl, an activator of the Wnt/β‐catenin pathway, we further verified that the inhibition of the Wnt/β‐catenin pathway was indeed due to ART. Remarkably, ART enhanced the anticancer effects of OXA in EC109 cells. OXA combined with ART was found to be more efficient in decreasing tumor growth compared to the individual drugs.ConclusionsART could suppress tumor progression by inhibiting Wnt/β‐catenin signaling pathway, and it may also enhance the antitumor effect of OXA in EC. Thus, ART could be a novel anticancer drug for EC treatment.Key pointsSignificant findings of the studyART could be a novel anticancer drug for esophageal cancer (EC) treatment.What this study addsCombination treatment with artemisinin and oxaliplatin inhibits tumorigenesis in esophageal cancer EC109 cells through the Wnt/β‐catenin signaling pathway.

Effect and mechanism of microRNA-10b on proliferation and invasion of esophageal cancer cells.

MicroRNA (miR)-10b is highly expressed in esophageal cancer tissues and is associated with poor prognosis of esophageal cancer. However, the role and mechanism of miR-10b in esophageal cancer cells remains unclear, therefore, the present study aimed to investigate this. Esophageal cancer cells, TE-1 and EC9706, were transfected with miR-10b mimic, miR-10b inhibitor or incubated with transforming growth factor-β (TGF-β). MTT and EdU assays were used to detect cell proliferation. Flow cytometry was used to determine cell cycle analysis and apoptosis. Cell migration and invasion were also analyzed. Western blot analysis was used to detect protein levels and reverse transcription-quantitative PCR was used to analyze miR-10b expression. The present results demonstrated that, compared with the control group, miR-10b significantly promoted TE-1 and EC9706 cell proliferation. Compared with miR-10b inhibitor group and control group, miR-10b mimic promoted esophageal cancer cell cycle progression, inhibited apoptosis of esophageal cancer cells and promoted the migration and invasion of cells. The proliferation of esophageal cancer cells increased in a dose-dependent manner with TGF-β concentration. TGF-β treatment induced high expression of miR-10b in both cell lines. The miR-10b mimic + TGF-β group further promoted the migration and invasion of esophageal cancer cells. Western blot analysis determined that, compared with the control group, miR-10b mimic increased TGF-β expression. miR-10b mimic also inhibited the expression of phosphatase and tensin homolog (PTEN) in tumor cells. Compared with the control group, TGF-β inhibited the expression of PTEN with the miR-10b mimic + TGF-β group further inhibiting the PTEN. miR-10b inhibitor + TGF-β reversed the effect of TGF-β and miR-10b on PTEN. In conclusion, miR-10b promoted cell cycle progression, inhibited apoptosis and promoted the migration and invasion of esophageal cancer cells. The mechanism may be related to the upregulation of TGF-β and the downregulation of PTEN. The present findings suggested that miR-10b might be a potential therapeutic target for esophageal cancer.

MiR-196a regulates the proliferation, invasion and migration of esophageal squamous carcinoma cells by targeting ANXA1.

MicroRNA (miR)-196a is upregulated in various types of malignancy, including esophageal squamous cell carcinoma (ESCC); however, its role in ESCC is currently unclear. The present study aimed to investigate the biological role and molecular mechanism of miR-196a in ESCC. The expression levels of miR-196a in 25 tumor tissues and adjacent non-tumor tissues from patients with ESCC were measured by reverse transcription-quantitative polymerase chain reaction. In addition, miR-196a expression levels were assessed in the human normal esophageal epithelial cell line Het-1A and the ESCC cell line EC109. The effects of miR-196a on the proliferation, apoptosis, invasion and migration of EC109 cells were determined by MTT, flow cytometry and Transwell assays, respectively. A luciferase reporter assay and western blotting were performed to confirm the target gene of miR-196a, and to explore the molecular mechanism underlying the effects of miR-196a on regulation of ESCC cell phenotypes. The results demonstrated that miR-196a was markedly upregulated in ESCC tissues and EC109 cells. In addition, miR-196a downregulation suppressed EC109 cell proliferation, invasion and migration, but did not affect apoptosis. Annexin A1 (ANXA1) was demonstrated to be a direct target gene of miR-196a. ANXA1 protein knockdown reversed the effects of miR-196a inhibition on EC109 cell proliferation, invasion and migration. Furthermore, alongside the downregulation of miR-196a and the increase in ANXA1 expression, cyclooxygenase 2 (COX2), matrix metalloproteinase (MMP)-2 and Snail were downregulated, and E-cadherin was upregulated in EC109 cells. The results of the present study suggested that miR-196a may act as an oncogene in ESCC, and that miR-196a may regulate the proliferation, invasion and migration of ESCC cells by targeting ANXA1. Subsequently, ANXA1 may further modulate the expression levels of COX2, MMP-2, Snail and E-cadherin. In conclusion, the miR-196a/ANXA1 axis may represent a potential therapeutic target in ESCC.

Effects of zerumbone on proliferation and apoptosis of esophageal cancer cells and on P53 and Bcl-2 expression levels.

The effects of zerumbone on the proliferation and apoptosis of esophagus cancer cells and on the P53 and Bcl-2 expression levels were studied. The esophagus cancer EC-109 cells were cultured and inoculated. The effect of zerumbone on proliferation of EC-109 cells was detected via the Cell Counting Kit-8 (CCK-8) method. Cell apoptosis was detected via TdT-mediated dUTP nick end-labeling (TUNEL) staining. Moreover, the mRNA expression levels of P53 and Bcl-2 were detected via reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression levels of P53 and Bcl-2 were evaluated via western blotting. CCK-8 detection results showed that compared with control group, zerumbone in different concentrations could inhibit the activity of EC-109, and the proliferation inhibition rate was significantly increased in a concentration-dependent manner with the increase of concentration. TUNEL staining showed that cell apoptosis gradually occurred in administration group, and the number of apoptotic cells was increased in a concentration-dependent manner with the increase of concentration. RT-PCR detection results showed that the mRNA expression level of P53 in administration group was significantly increased compared with that in control group, but that of Bcl-2 was significantly decreased. Western blotting showed that the protein expression level of Bcl-2 in administration group in different concentrations was significantly increased with the increase of zerumbone concentration, but that of Bcl-2 was significantly decreased in a concentration-dependent manner. Zerumbone can inhibit the proliferation and induce apoptosis of esophageal cancer EC-109 cells, and its induction of apoptosis may be realized through upregulating the mRNA expression of P53 and downregulating the mRNA expression of Bcl-2, and upregulating the protein expression of P53 and downregulating the protein expression of Bcl-2.

Docosahexaenoic acid enhances the sensitivity of esophageal carcinoma cell line EC9706 to cisplatin via inhibiting endoplasmic reticulum stress

Objective To investigate the effect of docosahexaenoic acid (DHA) on the sensitivity of esophageal carcinoma cell line EC9706 to cisplatin and the mechanism behind this effect. Methods EC9706 cells were randomly divided into 5 groups: control group, DHA group, cisplatin (DDP) group, DHA+ DDP group and endoplasmic reticulum stress (ERS) activation tunicamycin (TM) group (DHA+ DDP+ TM group). MTT method was used to evaluate inhibition ratio of cell proliferation. The apoptotic ratio was examined by flow eytometry.Western blot was used to detect the protein expressions of apoptosis cytokines (caspase-3 and Bcl-2) and ERS cytokines [glucose-regulated protein 78 (GRP78) and inositol-requiring enzyme 1 (IRE-1)]. Results DHA causes concentration-dependent and time-dependent inhibition of the proliferation of EC9706 cells (P=0.00). DHA significantly enhanced the sensitivity of esophageal carcinoma cell line EC9706 to cisplatin. Compared to DDP treatment alone, the inhibition ratio[(60.19±5.05)% vs. (36.72±3.52)%, P=0.02] and apoptotic ratio [(54.88±4.94)% vs. (39.74±4.64)%, P=0.03] of EC9706 cells were enhanced by DHA+ DDP treatment. Western blot showed that the expression of apoptotic factor caspase-3 protein was increased by DHA+ DDP treatment. Meanwhile, the protein expressions of anti-apoptotic factor (Bcl-2) and ERS-related factors (GRP78 and IRE-1) were significantly inhibited by DHA+ DDP treatment (P=0.01). However, the salutary effects of DHA were reversed by ERS activation tunicamycin. Conclusion DHA enhances the sensitivity of esophageal carcinoma cell line EC9706 to cisplatin, the mechanism of which may be the suppression of ERS response. Key words: Esophageal carcinoma cells; Docosahexaenoic acid; Cisplatin; Endoplasmic reticulum stress

Chloroquine affects autophagy to achieve an anticancer effect in EC109 esophageal carcinoma cells in vitro.

Esophageal carcinoma is a malignancy that severely threatens human health, with a high incidence rate and a low 5-year survival rate. Resistance to chemotherapy frequently emerges during its treatment, partly due to the induction of autophagy. Therefore, targeting autophagy may be a promising therapeutic approach for the treatment of esophageal carcinoma. In the present study, it was investigated how chloroquine (CQ) can influence the growth ability and biological behaviors of EC109 esophageal squamous carcinoma cells in vitro, as well as the potential molecular mechanisms behind its activity. It was demonstrated that CQ could suppress the growth and proliferation of EC109 cells in a time- and dose-dependent manner; migration and colony formation abilities were also inhibited by CQ. Furthermore, subsequent to the exposure to CQ, the number of autophagosomes was clearly increased in EC109 cells overexpressing green fluorescent protein tagged-light chain (LC)3 when observed by fluorescence microscopy. Protein expression of the endogenous autophagy markers LC3-II and p62 was elevated subsequent to CQ treatment, whereas the expression of proteins from the protein kinase B/mechanistic target of rapamycin target of rapamycin pathway was inhibited. This suggested that CQ could induce the formation of autophagosomes in the initiation of autophagy, but inhibit the degradation of autophagosomes in a later stage of autophagy. The overall effect was that autophagic cell death was activated by CQ, as confirmed by flow cytometry. Overall, the anticancer effect of chloroquine on EC109 was revealed to be mediated through modulating autophagy, and this may produce promising therapeutic benefits for esophageal carcinoma.

Knockdown of TGIF attenuates the proliferation and tumorigenicity of EC109 cells and promotes cisplatin-induced apoptosis.

A previous study has reported that frequent amplifications of the TG-interacting factor (TGIF) were observed in esophageal squamous cell carcinoma. The aim of the present study was to investigate the potential role of TGIF in the proliferation and tumorigenicity of the esophageal cancer cell line EC109 and cisplatin-induced apoptosis. Stable TGIF-knockdown EC109 cell line was established by infecting short hairpin RNA (shRNA) lentiviral particles. Soft agar and tumor xenograft assays were applied in nude mice. Flow cytometry was employed to evaluate the cell cycle and apoptosis. Western blot analysis was used to detect the expression of proteins. TGIF knockdown suppressed EC109 cell proliferation, colony formation in soft agar and tumor growth in nude mice, induced cell cycle arrest in the G1 phase, and promoted cisplatin-induced apoptosis. In addition, TGIF knockdown significantly reduced the expression of phospho-Rb in EC109 cells. The reduced level of full length PARP expression and the increased level of cleaved caspase-3 expression were observed in EC109 cells with the treatment of cisplatin and TGIF knockdown. The results suggest that knockdown of TGIF attenuated the proliferation and tumorigenicity of EC109 cells, and promoted cisplatin-induced apoptosis.

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer.

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer.

Long non-coding RNA MEG3 induces cell apoptosis in esophageal cancer through endoplasmic reticulum stress.

Long non-coding RNAs(lncRNAs) play important roles in diverse biological processes, such as cell growth, apoptosis and migration. Although downregulation of lncRNA MEG3 has been identified in several cancers, little is known about its role in esophageal squamous cell carcinoma(ESCC). The aim of the present study was to detect MEG3 expression in clinical ESCC tissues, investigate its biological functions and the endoplasmic reticulum(ER) stress-relative mechanism. MEG3 expression levels were detected by qRT-PCR in both tumor tissues and adjacent non-tumor tissues from 28 ESCC patients. PcDNA3.1-MEG3 recombinant plasmids were constructed and transfected to EC109 cells. Cell growth was analyzed by CCK-8 assay. Cell apoptosis was analyzed by fluorescence microscope and AnnexinV/PI assay. The protein expression was determined by western blot analysis. The results showed that MEG3 decreased significantly in ESCC tissues relative to adjacent normal tissues. PcDNA3.1-MEG3 plasmids were successfully constructed and the expression level of MEG3 significantly increased after MEG3 transfection to EC109 cells. Ectopic expression of MEG3 inhibited EC109 cell proliferation and induced apoptosis invitro. MEG3 overexpression increased the expression of ER stress‑related proteins (GRP78, IRE1, PERK, ATF6, CHOP and cleaved‑caspase-3). Our results first demonstrate that MEG3 is downregulated in ESCC tissues. MEG3 was able to inhibit cell growth and induced apoptosis in EC109 cells, most probably via activation of the ER stress pathway.

5-Azacytidine suppresses EC9706 cell proliferation and metastasis by upregulating the expression of SOX17 and CDH1.

5-Azacytidine is a well-known anticancer drug that is clinically used in the treatment of breast cancer, melanoma and colon cancer. It has been reported that 5-azacytidine suppresses the biological behavior of esophageal cancer cells. However, corresponding mechanisms remain unclear. In this study, using Transwell invasion and cell proliferation assays, we demonstrated that 5-azacytidine significantly inhibited the metastasis and proliferation of EC9706 cells, and upregulated the expression of cadherin 1 (CDH1) and SRY-box containing gene 17 (SOX17). Moreover, the inhibition of the metastasis of the 5-azacytidine-treated EC9706 cells was impaired following transfection with siRNA targeting CDH1 (CDH1 siRNA), and the inhibition of cell proliferation was attenuated following the downregulation of SOX17 by siRNA targeting SOX17 (SOX17 siRNA). Furthermore, 5-azacytidine remarkably reduced the CDH1 and SOX17 promoter methylation levels, suggesting that 5-azacytidine upregulates the expression of SOX17 and CDH1 by inhibiting the methylation of the SOX17 and CDH1 promoter. The findings of our study confirm that 5-azacytidine suppresses the proliferation and metastasis of EC9706 esophageal cancer cells by upregulating the expression of CDH1 and SOX17. The expression levels of CDH1 and SOX17 negatively correlate with the promoter methylation levels. CDH1 and SOX17 are potential indicators of the clinical application of 5-azacytidine.

Abstract 3541: Targeting autophagy to enhance paclitaxel sensitivity in esophageal carcinoma

Abstract Esophageal cancer is one of the malignancies threatening human health with high incidence and a very low 5-year survival rate with poor outcomes in the developing countries. Chemotherapy has been played indispensable roles while companied with more and more chemoresistance and side effects. Numerous studies have shown that autophagy may play specific role as one of the drug resistance mechanisms through promoting cancer cell survival via self-digestion. Therefore targeting autophagy might be another promising approach of therapeutics in cancer. Paclitaxel(PTX) is widely used for malignant tumors treatment through inducing apoptosis and autophagy. More findings discovered that chemoresistance is still a major hurdle for PTX due to cytoprotective autophagy. In order to investigate whether targeting autophagy could confer the sensitivity of PTX in esophageal carcinoma, chloroquine (CQ) was applied as an autophagy inhibitor to study the esophageal carcinoma cells EC109 co-treated with PTX in vitro and explore the potential molecular mechanisms. Firstly, CQ and PTX were used separately and also combined to treat EC109 cells at different dosages and time points, and the MTT results showed that CQ could contribute to the suppressive effect of PTX on the growth and proliferation of EC109 cells. Meanwhile the migration and invasion abilities of EC109 were significantly inhibited by co-treatment of PTX and CQ comparing with PTX only through scratch test and cell invasion Transwell chamber test, moreover the combination treatment dramatically suppressed the colony formation on soft agar. Further investigation showed that mTOR pathway was activated in CQ or PTX exposure, which resulted in phosphorylated mTOR(p-mTOR) down-regulation, followed by the decline of phosphorylated p70S6K (p-p70S6K) and phosphorylated p-4EBP1, and the autophagy marker LC3I/II conversion and p62 elevated eventually, which indicated that autophagy was mediated in PTX or CQ treatment in EC109 cells. On the other hand, GFP-LC3 was transfected into EC109 cells to observe the LC3 translocation status after CQ or PTX treatment, which usually be regarded as the morphological maker of autophagy, and the results confirmed the findings in Western Blot. Most importantly, the combination treatment of CQ and PTX resulted in synergistic consequences. For further understanding the mTOR pathway was initiated in CQ or PTX mediated autophagy in this study, we used lentiviral shRNA embedding mTOR to knockdown the expression of mTOR first and then studied the signaling pathway changes after CQ and PTX co-treatment, and we found a decrease trend of LC3-II/ I ratio and p62 also compared with the competent mTOR control. From these results, we confirmed that autophagy would benefit the survival of EC109 cells, and our findings demonstrated that targeting of autophagy using CQ could be an effective and potential strategy to improve PTX treatment outcomes in the management of esophageal carcinoma. Citation Format: Jiajing Cai, Yan Cai, Qiang Ma, Jiang Zou, Binfeng He, Fan Chang, Lei Xu, Xiaolan Guo. Targeting autophagy to enhance paclitaxel sensitivity in esophageal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3541.

Insights into anticancer activity and mechanism of action of a ruthenium(II) complex in human esophageal squamous carcinoma EC109 cells

A ruthenium(II) complex [Ru(p-cymene)(NHC)Cl2] (NHC=1,3-bis(4-(tert-butyl)benzylimidazol-2-ylidene), referred to as L-4, has been designed and synthesized recently in order to look for new anticancer drugs with high efficacy and low side effects. The anticancer activity and mechanism of action of L-4 in human esophageal squamous carcinoma EC109 cells were systematically investigated. The results revealed that L-4 exerted strong inhibitory effect on the proliferation of EC109 cells, and it arrested EC109 cells at G2/M phase, accompanied with the up-regulation of p53 and p21 and the down-regulation of cyclin D1. The results also showed that the reactive oxygen species (ROS)-dependent apoptosis of EC109 can be induced by L-4 via inhibiting the activity of glutathione reductase (GR), decreasing the ratio of glutathione to oxidized glutathione (GSH/GSSG), and leading to the generation of reactive oxygen species. The mitochondria-mediated apoptosis of EC109 induced by L-4 was also observed from the increase of Bax/Bcl-2 ratio, overload of Ca(2+), disruption of mitochondrial membrane potential (MMP), redistribution of cytochrome c, and activation of caspase-3/-9. However, the effects of L-4 on the cell viability, GR activity, GSH/GSSG ratio, reactive oxygen species level, mitochondria dysfunction and apoptosis induction were remarkably attenuated by adding the reactive oxygen species scavenger, NAC. Therefore, it was concluded that L-4 can inhibit the proliferation of EC109 cells via blocking cell cycle progression and inducing reactive oxygen species-dependent and mitochondria-mediated apoptosis. These findings suggested that the ruthenium(II) complex might be a potential effective chemotherapeutic agent for human esophageal squamous carcinoma (ESCC) and worthy of further investigation.

Effect of SNX-2112 on proliferation of esophageal cancer cells via regulation of excision repair cross-complementing 1, epidermal growth factor receptor, and p53 expression.

SNX-2112 is a potential molecular targeted therapeutic drug against esophageal cancer (EC). However, its exact mechanism of action remains to be explained. The aim of this study was to investigate the effect of SNX-2112 on excision repair cross- complementing 1 (ERCC1), epidermal growth factor receptor (EGFR), and p53, to elucidate the mechanism of action of SNX-2112 on EC. Fresh tumor sections were surgically obtained from 65 patients with EC, and the expression of ERCC1, EGFR, and p53 was determined by immunohistochemical staining. Furthermore, the effect of SNX-2112 (0.2 μM) on the proliferation of EC-9706 cells and the expression of ERCC1, EGFR, and p53 in these cells were analyzed by a cell proliferation assay and western blot, respectively. We observed a significant decrease and increase in ERCC1 (P = 0.001) and p53 (P = 0.043) expression, respectively, and no significant difference in EGFR (P = 0.59) expression, with the TNM stage of EC, which suggested that ERCC1 and p53 could be potential markers for the TNM stage of EC. We also observed a significant increase in ERCC1 expression, and decrease in p53 and EGFR expression, in EC-9706 cells treated with SNX-2112 (P < 0.05), indicating the regulation of EC by SNX-2112. Furthermore, SNX-2112 treatment induced a significant decrease in the proliferation of EC-9706, which confirmed the function of SNX- 2112. In summary, SNX-2112 inhibits the proliferation of EC cells by regulating the expression of ERCC1, EGFR, and p53.

Influence of Tanshinone IIA on the Apoptosis of Human Esophageal Ec-109 Cells

The induced-apoptosis effect and mechanism of human esophageal cancer Ec-109 cells via tanshinone IIA was investigated. The Ec-109 cells were cultured in vitro with different concentrations of tanshinone IIA (2 µg/mL, 4 µg/mL, or 8 µg/mL) for 12, 24, 36, and 48 hours. MTT assay was used to evaluate the proliferative inhibition rate of tanshinone IIA on esophageal Ec-109 cells. After 24 hours of culturing in vitro, a control group was assigned. The apoptosis rate was detected by the AO/EB and annexin V-FITC/propidium iodide assay, and the protein levels of Caspase-4 and CHOP were determined by the Western blot technique. MTT data showed that tanshinone IIA could significantly inhibit the proliferation of Ec-109 cells with a dose- and time-dependent mode. Compared with the control group, tanshinone IIA could apparently induce apoptosis of Ec-109 cells, and the level of Caspase-4 and CHOP (p < 0.01) obviously increased. Tanshinone IIA can significantly induce the apoptosis of Ec-109 cells, which may take effect by the stress pathway of the endoplasmic reticulum.

Study on chemotherapeutic sensitizing effect of nimotuzumab on different human esophageal squamous carcinoma cells.

Esophageal cancer is one of the leading causes of mortality worldwide. Although, surgery, radio- and chemotherapy are used to treat the disease, the identification of new drugs is crucial to increase the curative effect. The aim of the present study was to examine the chemotherapeutic sensitizing effect of nimotuzumab (h-R3) and cisplatin cytotoxic drugs cisplatin (DDP) and 5-fluorouracil (5-FU) on esophageal carcinoma cells with two different epidermal growth factor receptor (EGFR) expressions. The expression of EGFR was detected in the human EC1 or EC9706 esophageal squamous cell carcinoma cell line using immunohistochemistry. The inhibitory effect of DDP and 5-FU alone or combined with h-R3 on EC1 or EC9706 cell proliferation was detected using an MTT assay. Flow cytometry and the TUNEL assay were used to determine the effect of single or combined drug treatment on cell apoptosis. The results showed that the expression of EGFR was low in EC1 cells but high in EC9706 cells. The inhibitory effect of the single use of h-R3 on EC1 or EC9706 cell proliferation was decreased. The inhibitory effect between single use of h-R3 alone and combined use of the chemotherapy drugs showed no statistically significant difference (P>0.05) on the EC1 cell growth rate, but showed a statistically significant difference (a=0.05) on EC9706 cell growth rate. The results detected by flow cytometry and TUNEL assay showed that the difference between single use of h-R3 alone and the control group was statistically significant with regard to the EC1 apoptosis rate effect (P<0.05), but not statistically significant for EC9706 (P>0.05). However, statistically significant differences were identified in the apoptotic rate of EC9706 cells between the h-R3 combined chemotherapy group and single chemotherapy group (P<0.05), but not on in the EC1 chemotherapy group (P>0.05). In conclusion, the sensitization effect of h-R3 on chemotherapy drugs is associated with the expression level of EGFR in EC1 or EC9706 cells. The cell killing effect of the combined use of h-R3 with DDP and 5-FU showed no obvious synergistic effect compared to the single-drug group, but only an additive effect.

Effects of silencing the ATP-binding cassette protein E1 gene by electroporation on the proliferation and migration of EC109 human esophageal cancer cells.

In the present study, the gene expression of ATP-binding cassette protein E1 (ABCE1) in the EC109 human esophageal cancer cell line was silenced using electroporation to examine the effect if the ABCE1 gene on the growth migration and cell cycle of cancer cells. The small interference (si)RNA sequence of ABCE1 was designed and synthesized to transfect the EC109 cells by electroporation. The mRNA and protein expression levels of ABCE1 were then detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of the cell cycle and apoptosis was performed using flow cytometry. The effect of silencing the ABCE1 gene on the proliferation, migration and invasive ability of the EC109 human esophageal cancer cells were assessed using a Cell counting kit-8 (CCK-8) and with proliferation, wound-healing and cell invasion assays. The mRNA and protein expression levels of ABCE1 were significantly lower in the experimental group compared with the control group (P<0.05). The apop-totic rate of the experimental group was markedly higher than the control group and blank group (P<0.01). The CCK-8 proliferation assay revealed that, compared with the control and blank groups, the proliferation of the EC109 cells in the experimental group was significantly inhibited (P<0.05). The wound healing assay revealed that the migration capacity of the cells in the experimental group was significantly decreased (P<0.05). The Transwell chamber assay demonstrated that the invasive ability of the EC109 cells in the experimental group was significantly decreased (P<0.01). These results revealed that ABCE1 is closely associated with cell proliferation, invasion and migration in esophageal cancer and silencing the ABCE1 gene by electroporation can significantly reduce the proliferation, invasion and migration capacity of EC109 cells in vitro.

Effect of ECRG2 in combination with cisplatin on the proliferation and apoptosis of EC9706 cells.

The aim of the present study was to explore the effect of esophageal cancer-related gene 2 (ECRG2) protein in combination with cisplatin (DDP) on the proliferation and apoptosis of esophageal cancer cells. A 3-(4, 5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the effects of ECRG2 alone and ECRG2 in combination with DDP on the proliferation of EC9706 esophageal cancer cells. Hoechst 33258 staining was performed to analyze the effects of ECRG2 alone and ECRG2 in combination with DDP on apoptosis in the EC9706 cells. The expression levels of Bcl-2-associated X protein (Bax) mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The results from the MTT assay revealed that ECRG2 inhibited the proliferation of EC9706 cells and that ECRG2 in combination with DDP had a greater inhibitory effect on cell proliferation. The antiproliferative effects were time- and concentration-dependent, within a certain range of concentrations. The Hoechst 33258 staining results demonstrated that the number of apoptotic cells following treatment with ECRG2 in combination with DDP for 24 h was higher than that following treatment with ECRG2 alone for the same duration. Western blot analysis and RT-PCR results revealed that the expression levels of Bax mRNA and protein were upregulated in cells treated with ECRG2 in combination with DDP compared with those in cells treated with ECRG2 alone. Thus, ECRG2 in combination with DDP had an enhanced inhibitory effect on EC9706 cell proliferation compared with that of ECRG2 alone, and an increased inductive effect on EC9706 cell apoptosis, possibly due to the upregulation of the expression of Bax.

TPX2 siRNA regulates growth and invasion of esophageal cancer cells

Observe how specific small RNA interference (siRNA) aimed at TPX2 gene suppresses TPX2 gene expression in esophageal cancer EC9706 cells and the effect on esophageal cancer cell growth and invasion ability. Transfect TPX2 siRNA into EC9706 cells via lipofectamin 2000. The experiments were divided into three groups, a negative control, a blank control and an siRNA interference group (24h, 48h, 72h, 96h). We examined RNA and protein level alteration of the TPX2 gene after TPX2 siRNA transfection by RT-PCR and Western blot analysis. Detection of how TPX2 siRNA influences EC9706 cell proliferation was done by MTT, cell apoptosis monitored through Tunel assay, in vitro invasion ability via Boyden chamber and cell cycle change by flow cytometry. After effective siRNA transfection, TPX2 mRNA and protein expression level in siRNA interference group were (0.31±0.08, 0.39±0.12),72h after transfection, significantly lower than blank control group (1.00±0.01) and negative control group (0.98±0.11), (F=71.182, t1=8.17, t2=7.90, P<0.05); MTT results demonstrated that cell growth and proliferation were inhibited and the inhibition rate was up to 35.4% (P<0.05) compared with the control group. TUNEL results indicated that cell apoptosis index in siRNA interference group was 18.28±0.35, higher than that in blank control group (4.07±0.26)and negative control group (4.13±0.22), (F=244.5, t1=60.61, t2=53.32, P<0.01). Boyden chamber results showed that the transmembrane cell number was 45.30±8.08 in siRNA interference group, less than blank control group (121.90±7.83), (F=122.46, t1=11.81, t2=10.47, P<0.01); besides, in siRNA interference group cell invasion inhibition rate was 71.42±9.12, higher than negative control group (5.65±3.55), (t=14.256, P<0.01). Flow cytometry results illustrated that more EC9706 cells went into apoptosis and cell cycle arrested in S phase. Similar results were obtained by in vivo transplantation, as TPX2 siRNA transfection significantly reduced tumor growth of the xenograft in nude mice. siRNA could effectively inhibit the invasion and metastasis of EC9706 cells, promote the apoptosis of tumor cells and may become a new approach for treatment of esophageal carcinoma.