Effects of Bufalin on Human Esophageal Cancer Cells Proliferation and Mechanism Study / 中国药学杂志

OBJECTIVE: To investigate the effect of bufalin on the proliferation of esophageal cancer and its possible molecular mechanism. METHODS: The effects of bufalin on the activity of esophageal cancer cell line KYSE-70 were determined by MTT assay and LDH assay kit. Colony-forming and EdU (5-ethynyl-2-deoxyuridine) assay were performed to detect the inhibitory effect of bufalin on the proliferation of KYSE-70 cells. DAPI staining, TUNEL and flow cytometry were used to assess the effects of bufalin on the apoptosis of KYSE-70 cells. Quantitative real-time PCR (qPCR) and Western blotting were used to determine the relevant expression changes in mRNA and protein of apoptosis-related factors Bcl-2, Bax, NF-κBp65, NF-κBpp65. The mitochondrial function changes of KYSE-70 cells were studied by using JC-1 fluorescent probe kit and ATP detection kit after bufalin treatment. Finally, the effect of bufalin on esophageal cancer proliferation in vivo was studied by xenograft model in nude mice. RESULTS: The results of MTT and LDH assay shown that bufalin inhibited the activity of KYSE-70 cells. Colony-forming and EdU assay showed that bufalin significantly suppressed KYSE-70 cells proliferation. DAPI staining showed that chromatin heterogeneity, nuclear concentration and fragmentation were observed after bufalin treatment. It was found that bufalin treatment significantly promoted KYSE-70 cells apoptosis by TUNEL staining and flow cytometry assay of qPCR and Western blot showed that Bcl-2 expression was down-regulated, Bax expression was up-regulated and Bax/ Bcl-2 expression ratio was increased after bufalin treatment. The mitochondrial membrane potential and the ATP production of KYSE-70 cells were significant reduced after bufalin treatment. In vivo, the growth of xenograft tumors was significantly inhibited in bufalin group. CONCLUSION: Bufalin markedly inhibits KYSE-70 cells proliferation by promoting apoptosis, and the possible mechanism of apoptosis may be related to mitochondrial pathway. Our results indicate that bufalin may be a potential therapeutic agent for esophageal cancer.

Docosahexaenoic acid enhances the sensitivity of esophageal carcinoma cell line EC9706 to cisplatin via inhibiting endoplasmic reticulum stress / 中华临床营养杂志

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.

Effect of recombinant human endostatin on the radiosensitivity of esophageal carcinoma cells and underlying mechanism / 中华放射医学与防护杂志

Objective To study the effect of recombinant human endostatin (rhES)on the radiosensitivity of esophageal squamous cells KYSE-150 and its preliminary mechanism.Methods Cells were divided into four groups:control group without treatment,rhES group treated with recombinant human endostatin,radiation alone group exposed with X-rays,and combination group exposed with X-rays plus endostatin.Colony formation assay was used to measure cell survival fraction.A single-hit multi-target model was used to fit cell survival curve and calculate the sensitive enhancement ratio (SER).Influence of rhES combined with X-ray radiation on cell cycle and apoptosis was measured by flow cytometry.Expressions of Cyclin B1,Cyclin D1,Bcl-2 and Bax mRNAs were determined by RT-PCR.Protein expressions of HIF-1α,VEGF,and VEGFR were determined by Western blot.Results D0,Dq and SF2 value of KYSE-150 cells decreased along with the concentration of rhES.At D0dose,the SER for 100 and 200 μg/ml rhES was 1.14 and 1.27,respectively.Compared with the radiation alone group,the apoptosis rate and bax expression increased,while the expressions of VEGF and HIF-1α decreased in the combination group (t =7.97,3.02,117.55,7.22,P ＜ 0.05).Conclusions rhES has radiosensitive effect on esophageal carcinoma cells KYSE-150 in vitro by inhibiting the expressions of HIF-1α and VEGF,regulating bax expression,and inducing apoptosis.

Autophagy Induced by NGAL Protein in Esophageal Carcinoma Cells / 生物化学与生物物理进展

Previous studies suggest that NGAL (neutro phil gelatinase-associated lipocalin) is involved in the transformation and development of esophageal carcinoma. Alteration of NGAL expression can trigger the change of cellular morphology in esophageal carcinoma cells. However, the mechanisms remain unclear. To get a better understanding of NGAL function in esophageal carcinoma, NGAL protein was expressed in methylotrophic yeast, Pichia pastoris, and purified by chromatography. EC1.71 cells expressed high levels of NGALR (NGAL receptor) and EC109 cells expressed low levels of NGALR were used as cells model. The trafficking and the possible function of NGAL protein were then analyzed in the esophageal carcinoma cells. The results showed that 5-FAM-labeled recombinant NGAL protein could internalize into the EC1.71 and EC109 cells. Furthermore, the internalized NGAL protein could induce the alteration of cellular morphology, resulting in generation of autophagosome, transcriptional up-regulation of genes associated with autophagy and increase of phospho-ERK1/2 (p-ERK1/2). Interestingly, the treatment with the NGAL protein did not affect the intracellular iron level. These data indicate that induced autophagy by exogenous NGAL protein is a mechanism that internalized NGAL plays important roles in esophageal carcinoma cells, independent with NGAL-mediated iron transport process, while ERK1/2 signal pathway is involved in activation of autophagy by exogenous NGAL protein.