EGFR及E-cadherin表达与食管癌转移的关系

目的探讨分析食管癌组织中表皮生长因子受体(EGFR)的表达与上皮型钙黏附素(E-cadherin)的表达与食管癌转移的关系。方法采用免疫组织化学LSAB法对42例食管癌标本的EGFR和E-cadherin表达进行研究。结果EGFR在远处转移组和局部淋巴结转移组的表达率(分别为47.62%、56.52%)均高于相应对照组(27.08%、26.32%)(P<0.05);在组织学分级Ⅰ、Ⅱ、Ⅲ级3组中的表达率分别为16.67%、42.86%、71.43%,3组间差异有显著性(P<0.05)。E-cadherin在远处转移组和局部淋巴结转移组的表达率(分别为33.33%、26.09%)均低于相应对照组(54.17%、57.89%)(P<0.01,P<0.05);在组织学分级Ⅰ、Ⅱ、Ⅲ级3组中表达率分别为71.43%、47.62%、14.29%,3组间差异有显著性(P<0.05)。结论EGFR及E-cadherin与食管癌的浸润和转移相关。     

Serological aspects of rat tumour xenograft growth in athymic nude mice.

The serum of athymic nude mice bearing rat tumour xenografts has been examined for tumour-specific antigen. With a sarcoma and a hepatoma, tumour-specific antigen expression continued in xenograft growths, and sera of tumour-bearing mice contained free antigen, assayed by its ability to neutralise reactivity of tumour-immune rat sera against tumour target cells in an indirect membrane-immunofluorescence test. In contrast, no anti-rat antibody was detectable in sera of mice bearing the xenografts, or rejecting cells injected in admixture with BCG.     

KG-135, enriched with selected ginsenosides,inhibits the proliferation of human prostate cancer cells in culture and inhibits xenograft growth in athymic mice

Sun ginseng (SG) was recently developed as a heat-processed form of ginseng. The Rg3, Rk1, and Rg5 ginsenosides are its main ginsenoside components. SG has been reported to have more potent pharmacological activities than red ginseng (RG), where these pharmacological activities include vasodilatory, anti-oxidant and anti-tumorigenic effects. In the present study, we investigated KG-135, the ginsenoside-rich fraction of SG and demonstrated that this fraction inhibits proliferation of human prostate cancer cells both in vitro and in vivo. KG-135 caused a significant growth inhibition of DU145 and PC-3 human prostate cancer cells. KG-135 induced cell cycle arrest in the G1 phase and caused an associated increase in the p21^Cip1 protein levels. When KG-135 was fed to mice that had been xenografted with DU145 tumors, a time-dependent inhibition of tumor growth was noted without any observed toxicity. Immunohistochemical analysis of the tumor tissues showed that KG-135 led to a decrease in the expression of proliferating cell nuclear antigen (PCNA). Microarray analysis of the tumors revealed that KG-135 inhibited tumor growth and also caused changes in the expression levels of multiple cancer-related genes. These data suggest that KG-135 effectively inhibits prostate cancer cell proliferation. Its mechanism of action likely involves cyclin inhibition and regulation of the expression of the TNFRSF25 and ADRA2A genes.     

Identification of EGFR mutations in esophageal cancer

Background

It is well known that the prognosis for esophageal cancer is worse than for other digestive cancers in spite of multimodality treatment, and there is an urgent need to improve this situation. The epidermal growth factor receptor (EGFR) inhibitor, gefitinib, was approved in Japan to treat advanced non-small cell lung cancer patients and several papers have since reported that the successfully treated patients had genetic mutations in EGFR.

Purpose

The aim of this study was to investigate the existence of EGFR mutations in esophageal cancer cell lines and primary lesions, and also to explore the possibility of treating esophageal cancer using gefitinib.

Materials and Methods

Nineteen esophageal cancer cell lines were cultured and DNA was extracted using an ultracentrifugation method. Fifty cases of primary cancer and corresponding normal tissue samples were obtained and DNA was extracted using the same protocol. Nested PCR and DNA sequencing targeting exons 18, 19, 20 and 21 of EGFR were performed to investigate the presence of mutations in esophageal cancer cell lines and primary tumors.

Results

Three of the 19 cell lines had the same silent mutation at nucleotide 2607, a G-to-A substitution in exon 20. One of the 50 patients had an EGFR mutation in codon 719, resulting in an amino acid substitution from glycine to aspartic acid.

Conclusion

EGFR mutations in esophageal carcinoma are rare but do exist, and thus gefitinib could be included in esophageal cancer treatment regimens by selecting those patients who possess such mutations.     

抑制USP9x表达增强食管癌Ec9706-R细胞的放射敏感性

背景与目的：泛素特异性蛋白酶9x（ubiquitin-specific protease 9x,USP9x）与多种肿瘤的发生、发展以及肿瘤细胞的放射抗拒相关。研究发现,USP9x的表达与食管鳞状细胞癌的浸润深度和淋巴结转移相关,但其食管癌细胞放射抗拒作用尚未见报道。探究USP9x对放射抗拒食管癌Ec9706-R细胞放射敏感性的作用及其机制。方法：首先通过实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR）和蛋白质印迹法（Western blot）检测放射线照射后Ec9706-R细胞及其亲本细胞Ec-9706中USP9x和抗髓样细胞白血病-1（myeloid cell leukemia-1,Mcl-1）mRNA表达和蛋白水平。然后将Ec9706-R细胞随机分成3组：放射（irradiation,IR）组、IR+对照siRNA组（IR+si-NC组,转染Control siRNA）和IR+USP9x siRNA组（IR+si-USP9x组,转染USP9x siRNA）,各组细胞均使用一定量的6 MV-X射线照射。噻唑蓝（methyl thiazolyl tetrazolium,MTT）比色法检测不同剂量（0、2、4、6和8 Gy）6 MV-X射线照射下各组细胞的活力。Transwell、流式细胞术、RTFQ-PCR和Western blot分别检测3组细胞在6 Gy照射下的细胞迁移、凋亡、Mcl-1 mRNA表达和蛋白水平以及增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）和DNA损伤修复相关基因核苷酸切除修复交叉互补基因1（excision repair cross-complementing gene 1,ERCC1）蛋白水平。结果：放射后Ec9706-R和Ec-9706细胞中USP9x和Mcl-1 mRNA表达和蛋白水平均增加,Ec9706-R细胞尤为显著（P<0.05）。与IR组相比,IR+si-USP9x组中细胞活力、迁移细胞数目、PCNA、ERCC1和Mcl-1的表达均降低,细胞凋亡增加（P<0.05）。但是与IR组相比,IR+si-NC组中上述指标均无显著变化（P>0.05）。结论：抑制USP9x表达能够增强放射抗拒食管癌Ec9706-R细胞的放射敏感性,这可能是通过下调Mcl-1的表达发挥作用的。     

Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice

Endocrine therapy with estrogen deprivation or with antiestrogens results in tumor regression in a subset of patients with advanced breast cancer. To better understand the mechanisms by which estrogens and antiestrogens modulate breast cancer growth in vivo, we have studied the effects of endocrine manipulation on the development and growth of tumors derived from cultured human breast cancer cells in the athymic nude mouse. MCF-7 breast cancer cells were inoculated into 6-week-old female BALB/c athymic nude mice. Tumor growth did not occur in ovariectomized mice. Cells remained viable, however, since estrogen supplementation more than 30 days later resulted in tumor formation. Minimal tumor growth was observed in intact female nude mice which have low circulating estrogen levels. Tumor development and growth in ovariectomized or intact mice supplemented with 17 beta-estradiol in the form of a s.c. pellet were dose dependent; growth rates increased with estrogen doses ranging from 0.01 to 0.5 mg. Antiestrogen treatment with either tamoxifen or LY156758 caused transient stimulation of tumor growth, followed by a prolonged stationary phase. Growth resumed with estrogen supplementation. Treatment of mice bearing established MCF-7 tumors with estrogen withdrawal (removal of estrogen pellet) resulted in cessation of tumor growth, but not in tumor regression. Growth inhibition was also observed with antiestrogens and was dose dependent. However, tumor regression did not occur, even in mice treated with high doses of tamoxifen (serum concentration of 1.0 microM) for as long as 60 days. Tumor growth was restored in these mice with estrogen replenishment. Tumor cells also remained viable histologically despite prolonged (1 month) estrogen deprivation or antiestrogen therapy, although the mitotic index was markedly reduced. Similar observations were made with mice inoculated with the hormone-responsive ZR75-1 human breast cancer cells, but not with hormone-independent MDA-231 cells which were not influenced by estrogen or antiestrogen treatment. In summary, development and growth of MCF-7 and ZR75-1 tumors in nude mice are estrogen dependent. Endocrine therapy by estrogen deprivation or antiestrogen treatment inhibits tumor cell proliferation in nude mice, but does not cause tumor regression or loss of cell viability.     

RNA干扰抑制UHRF1基因表达对食管癌细胞放射增敏作用的研究

目的 研究RNA干扰抑制UHRF₁表达对食管癌细胞系(TE-1)放射敏感性的影响及作用机制。方法 以慢病毒感染方法将UHRF₁基因的短发夹状RNA (shRNA)转入TE-1细胞,并分为未转染组、转染NC-shRNA组、转染UHRF₁-shRNA组,前二者为对照组。采用 RT-PCR和蛋白印记法检测转染前后细胞UHRF₁的mRNA和蛋白表达,成克隆法、流式细胞术及蛋白印记法检测转染UHRF₁-shRNA联合X线照射对TE-1细胞放射敏感性、周期、凋亡及DNA损伤标识蛋白γ-H₂AX的影响。     

人胃癌裸鼠移植瘤中标记滞留细胞的初步鉴定(英文)

Objective:This study has investigated the existence of label-retaining cell and its distribution in gastric cancer,in the hope that this information will assist investigations on gastric cancer stem cells.Methods:The gastric carcinoma cell line BGC-823 was labeled with BrdU in vitro and then engrafted into the right axilla of nude mice,which developed tumors.Label-retaining cells were quantified by immunohistochemical methods.Results:BrdU positive cells constituted about 96%of the cells in xenograft tumors after 10 days.Subsequently,BrdU positive cells gradually decreased,at the 80th day,labelretaining cells steadily occupied about 0.5%.This set of population cell localized in the margin of cancer nests,which had no difference in cellular morpha.Conclusion:The study demonstrates the presence of label-retaining cells in human gastric cancer xenografts in nude mice and the label-retaining cells may be related with cancer stem cells,which are most likely the cause for spread,metastasis and recurrence.     

Antineoplastic effect of erbstatin on human mammary and esophageal tumors in athymic nude mice

The growth of MCF-7, a human mammary carcinoma, in athymic nude mice was inhibited by intraperitoneal administration of erbstatin for 14 days in combination with an iron chelator, foroxymithine, which inhibits the decomposition of erbstatin. Another human mammary carcinoma, Br-10, was not affected. Foroxymithine alone had no anti-tumor activity. In four esophageal tumors, erbstatin retarded tumor growth. There were no side-effects in any erbstatin-treated group. Levels of epidermal growth factor receptors were not changed throughout treatment with erbstatin at any dose. Erbstatin, a tyrosine kinase inhibitor, may have an antineoplastic effect against human mammary and esophageal tumors.     

EZH2 inhibition enhances the efficacy of an EGFR inhibitor in suppressing colon cancer cells

Metastatic colon cancer has a 5-year survival of less than 10% despite the use of aggressive chemotherapeutic regimens. As signaling from epidermal growth factor receptor (EGFR) is often enhanced and epigenetic regulation is often altered in colon cancer, it is desirable to enhance the efficacy of EGFR-directed therapy by co-targeting an epigenetic pathway. We showed that the histone methyltransferase EZH2, which catalyzes methylation of histone H3 lysine 27 (H3K27), was upregulated in colon cancers in The Cancer Genome Atlas (TCGA) database. Since co-inhibition of both EGFR and EZH2 has not been studied in colon cancer, we examined the effects of co-inhibition of EGFR and EZH2 on 2 colon cancer cell lines, HT-29 and HCT-15. Co-inhibition of EZH2 and EGFR with the small molecules UNC1999 and gefitinib, led to a significant decrease in cell number and increased apoptosis compared to inhibition of either pathway alone, and similar results were noted after EZH2 shRNA knockdown. Moreover, co-inhibition of EZH2 and EGFR also significantly induced autophagy, indicating that autophagy may play a role in the observed synergy. Together, these findings suggest that inhibition of both EZH2 and EGFR serves as an effective method to increase the efficacy of EGFR inhibitors in suppressing colon cancer cells.     

microRNA-155在食管癌中的表达及其对食管癌细胞增殖凋亡的影响

目的 探讨miRNA-155在食管癌中的表达及其对食管癌EC9706细胞增殖及凋亡的影响.方法 采用TaqMan探针实时定量PCR(qRT-PCR)方法检测50例食管癌患者中新鲜食管癌组织和其相应癌旁正常组织中miRNA-155的表达情况;使用脂质体LipofectamineTM2000转染EC9706细胞上调miRNA-155的表达,利用四甲基偶氮唑盐比色法(MTT法)检测转染后细胞的增殖变化,流式细胞仪检测其细胞凋亡率的变化.结果 食管癌组织中miRNA-155的表达水平明显低于与其相应的癌旁正常组织(P=0.000);转染miRNA-155后,miRNA-155转染组EC9706细胞中miRNA-155的表达水平高于对照组和NC组(P﹤0.05);细胞的增殖明显受到抑制,且细胞的凋亡率明显升高.结论 miRNA-155在食管癌组织中呈低表达,上调其表达能够明显抑制食管癌EC9706细胞增殖和诱导其凋亡.     

Lack of AKT activation in lung cancer cells with EGFR mutation is a novel marker of cetuximab sensitivity

Epidermal growth factor receptor (EGFR) mutation is the best marker of sensitivity to the EGFR tyrosine kinase inhibitor gefitinib, but a marker for the anti-EGFR antibody cetuximab has not been identified in lung cancer. The present study investigated markers for sensitivity to cetuximab. Sensitivity to cetuximab and gefitinib was compared with EGFR expression, EGFR and KRAS mutation, and EGFR gene copy numbers in lung cancer cell lines. We also studied the effect of these agents on the activation of EGFR, ERK, AKT, and STAT3 in cetuximab-sensitive and -resistant cell lines. We found one cetuximab-sensitive cell line with EGFR mutation among 19 lung cancer cell lines. Analysis of molecules downstream from EGFR revealed that AKT phosphorylation was suppressed in this cell line. Augmentation of AKT phosphorylation by transfection of a plasmid induced resistance to cetuximab. Acquisition of cetuximab resistance was associated with AKT activation in this cell line, while pharmacological inhibition of AKT markedly enhanced the growth inhibitory effect of cetuximab. Dephosphorylation of AKT in association with EGFR mutation is a candidate marker for sensitivity to cetuximab, and combined use of an AKT pathway inhibitor with cetuximab could be a novel therapeutic strategy for lung cancer.     

Treatment effects of the EGFR pathway drugs on head and neck cancer stem cells

(1) Head and neck cancer (HNC) is the sixth most common cancer worldwide and show low survival rates and drug resistance, which can be due to the presence of cancer stem cells (CSCs), a small cell population with metastatic potential, invasion and self-renewal ability. (2) Here, seven tumor cells were sorted as CD44+/CD117+/CD133+ or ALDH+, considered as HNC stem cells (HNCSCs), and as CD44-/CD117-/CD133- or ALDH-, considered non-HNCSCs after both cells sorted criteria was compared to evaluate cell migration, invasion, and colony forming assays. These subpopulations were treated with Cetuximab, Paclitaxel, or a combination of both drugs and evaluated for cell viability. Quantitative PCR and western blot were performed to evaluate EGFR, TRKB, KRAS and HIF-1α gene and protein expression. (3) HNCSCs presented more colonies and appeared to be more sensitive to the drug combination when compared with non-HNCSCs, regardless cells sorted criteria and primary tumor subsite. The EGFR, TRKB, KRAS and HIF-1α genes and proteins were upregulated in CSCs compared with non-HNCSCs, thus explaining the drug resistance. (4) This study contributes to the better development of specific therapeutic protocols based on Cetuximab and Paclitaxel drugs in the treatment of HNC in the presence of CSCs and cell proliferation biomarkers.     

SNX10对人结直肠癌细胞增殖和EGFR表达的影响

目的:探讨SNX10过表达对人结直肠癌细胞增殖和EGFR表达的影响.方法:应用基因转染方法将SNX10质粒导入结直肠癌细胞LOVO细胞中,蛋白质印迹法方法鉴定转染后SNX10在细胞中的表达.利用四甲基偶氮唑盐(MTT)法检测SNX10对结直肠癌细胞增殖的影响,利用蛋白质印迹法和免疫荧光法观察EGFR表达量和定位情况.结果:蛋白质印迹法结果显示,SNX10质粒成功导入LOVO细胞中,SNX10的过表达使结直肠癌细胞增殖能力明显降低(F=16.76,P＜0.01),EGF刺激后,过表达SNX10的细胞EGFR表达明显低于对照组,荧光定位显示SNX10和EGFR存在共定位关系.结论:SNX10通过调控EGFR表达使结直肠癌细胞增殖能力明显下降,SNX10可能是结直肠癌的增殖抑制基因.     

Trichostatin A enhances radiosensitivity and radiation-induced DNA damage of esophageal cancer cells

BackgroundTrichostatin A (TSA) is emerging as a potential component of anticancer therapy. In this study, we aimed to identify the radiosensitizing effects of TSA in esophageal squamous carcinoma cell lines and identify the genomic alteration of histone acetylation associated with TSA treatment.MethodsEC109 and KYSE450 cells were pretreated with TSA (0.1 µM) for 12 hours prior to irradiation, and the cell viability, flow cytometry, and comet assays were performed to analyze cell growth, cell apoptosis, and DNA damage, respectively. Chromatin immunoprecipitation sequencing (ChIP-Seq) was performed to identify the acetylation sites of histone H3 lysine 9 (H3K9), which was altered by TSA.ResultsOur data showed that TSA could sensitize esophageal cancer cells to radiation by inducing cell cycle arrest and increasing cell apoptosis. DNA damage induced by radiation was enhanced by TSA treatment. In addition, a total of 105 differential peak-related genes were found to be associated with TSA treatment, which was identified using ChIP-Seq with specific antibodies against acetylated histone H3K9.ConclusionsOur data suggest that pretreatment with TSA can enhance ionizing radiation-induced DNA damage of esophageal cancer cells, which was associated with the altered histone modification of whole genome. TSA has potential implications for clinical use in increasing the anticancer efficacy of radiation.