[Objectives] To investigate the pharmacologic effects of active components from A. membranaceus on human esophageal cancer HCE-4 cells and its apoptosis mechanism. [Methods] The viabilities of HCE-4 cells were measure...[Objectives] To investigate the pharmacologic effects of active components from A. membranaceus on human esophageal cancer HCE-4 cells and its apoptosis mechanism. [Methods] The viabilities of HCE-4 cells were measured by MTT assay. The induction of active components from A. membranaceus on apoptosis of HCE-4 cells was detected by Annexin V-FITC/PI double staining. The apoptotic-related protein expression levels were determined by Western blotting. [Results] Formononetin and astragaloside IV suppressed the proliferation of HCE-4 cells in a dose-dependent manner. The Annexin V-FITC/PI double staining results showed that formononetin and astragaloside IV could induce HCE-4 cells apoptosis in a time-dependent manner. The Western blotting results showed that formononetin and astragaloside IV could significantly down-regulate p-AKT,pro-caspase-3,and increase cle-caspase-3 protein expression in HCE-4 cells. [Conclusions]Active components from A. membranaceus such as formononetin and astragaloside IV significantly inhibited the proliferation of human esophageal cancer HCE-4 cells by inducing mitochondrial dependent apoptosis via AKT signaling pathway.展开更多
AIM:To assess the prevalence of human papilloma virus(HPV) in esophageal squamous cell carcinoma(ESCC) in the south-eastern region of Poland.METHODS:The study population consisted of 56 ESCC patients and 35 controls.T...AIM:To assess the prevalence of human papilloma virus(HPV) in esophageal squamous cell carcinoma(ESCC) in the south-eastern region of Poland.METHODS:The study population consisted of 56 ESCC patients and 35 controls.The controls were patients referred to our department due to other nonesophageal and non-oncological disorders with no gross or microscopic esophageal pathology as confirmed by endoscopy and histopathology.In the ESCC patients,samples were taken from normal mucosa(56 mucosa samples) and from the tumor(56 tumor samples).Tissue samples from the controls were taken from normal mucosa of the middle esophagus(35 control samples).Quantitative determination of DNA was carried out using a spectrophotometric method.Genomic DNA was isolated using the QIAamp DNA Midi Kit.HPV infection was identified following PCR amplification of the HPV gene sequence,using primers MY09 and MY11 complementary to the genome sequence of at least 33 types of HPV.The sequencing results were computationally analyzed using the basic local alignment search tool database.RESULTS:In tumor samples,HPV DNA was identified in 28 of 56 patients(50%).High risk HPV phenotypes(16 or/and 18) were found in 5 of 56 patients(8.9%),low risk in 19 of 56 patients(33.9%) and other types of HPV(37,81,97,CP6108) in 4 of 56 patients(7.1%).In mucosa samples,HPV DNA was isolated in 21 of 56 patients(37.5%).High risk HPV DNA was confirmed in 3 of 56 patients(5.3%),low risk HPV DNA in 12 of 56 patients(21.4%),and other types of HPV in 6 of 56 patients(10.7%).In control samples,HPV DNA was identified in 4 of 35 patients(11.4%) with no high risk HPV.The occurrence of HPV in ESCC patients was significantly higher than in the controls [28 of 56(50%) vs 4 of 35(11.4%),P < 0.001].In esophageal cancer patients,both in tumor and mucosa samples,the predominant HPV phenotypes were low risk HPV,isolated 4 times more frequently than high risk phenotypes [19 of 56(33.9%) vs 5 of 56(8.9%),P < 0.001].A higher prevalence of HPV was identified in female patients(71.4% vs 46.9%).Accordingly,the high risk phenotypes were isolated more frequently in female patients and this difference reached statistical significance [3 of 7(42.9%) vs 2 of 49(4.1%),P < 0.05].Of the pathological characteristics,only an infiltrative pattern of macroscopic tumor type significantly correlated with the presence of HPV DNA in ESCC samples [20 of 27(74.1%) vs 8 of 29(27.6%) for ulcerative or protruding macroscopic type,P < 0.05].The occurrence of total HPV DNA and both HPV high or low risk phenotypes did not significantly differ with regard to particular grades of cellular differentiation,phases in depth of tumor infiltration,grades of nodal involvement and stages of tumor progression.CONCLUSION:Low risk HPV phenotypes could be one of the co-activators or/and co-carcinogens in complex,progressive,multifactorial and multistep esophageal carcinogenesis.展开更多
AIM： To examine the presence of human papillomavirus （HPV） in esophageal squamous cell carcinoma （ESCC） specimens collected from Colombia and Chile located in the northern and southern ends of the continent, resp...AIM： To examine the presence of human papillomavirus （HPV） in esophageal squamous cell carcinoma （ESCC） specimens collected from Colombia and Chile located in the northern and southern ends of the continent, respectively.METHODS： We examined 47 and 26 formalin-fixed and paraffin-embedded ESCC specimens from Colombia and Chile, respectively. HPV was detected using GP5＋/GP6＋ primer pair for PCR, and confirmed by Southern blot analysis. Sequencing analysis of L1 region fragment was used to identify HPV genotype. In addition, P16^INK4A protein immunostaining of all the specimens was conducted.RESULTS： HPV was detected in 21 ESCC specimens （29%）. Sequencing analysis of L1 region fragment identified HPV-16 genome in 6 Colombian cases （13%） and in 5 Chilean cases （19%）. HPV-18 was detected in i0 cases （21%） in Colombia but not in any Chilean case. Since Chilean ESCC cases had a higher prevalence of HPV-16 （without statistical significance）, but a significantly lower prevalence of HPV-18 than in Colombian cases （P = 0.011） even though the two countries have similar ESCC incidence rates, the frequency of HPV-related ESCC may not be strongly affected by risk factors affecting the incidence of ESCC. HPV-16 genome was more frequently detected in p16 positive carcinomas, although the difference was not statistically significant. HPV-18 detection rate did not show any association with p16 expression. Well-differentiated tumors tended to have either HPV-16 or HPV-18 but the association was not statistically significant. HPV genotypes other than HPV-16 or 18 were not detected in either country.CONCLUSION： HPV-16 and HPV-18 genotypes can be found in ESCC specimens collected from two South American countries. Further studies on the relationship between HPV-16 presence and p16 expression in ESCC would aid understanding of the mechanism underlying the presence of HPV in ESCC.展开更多
BACKGROUND It is well known that an alcohol consumption habit together with inactive heterozygous aldehyde dehydrogenase-2(ALDH2)is an important risk factor for the development of esophageal squamous cell carcinoma(ES...BACKGROUND It is well known that an alcohol consumption habit together with inactive heterozygous aldehyde dehydrogenase-2(ALDH2)is an important risk factor for the development of esophageal squamous cell carcinoma(ESCC).It remains controversial whether human papillomavirus(HPV)infection contributes to the occurrence/development of ESCC.There has been no study in which the relationship between ESCC and HPV in addition to alcohol dehydrogenase-1B(ADH1B)and ALDH2 genotypes was evaluated.AIM To evaluate relationships between HPV infection and development of esophageal cancer,particularly early esophageal cancer,based on ADH1B/ALDH2 polymorphisms.METHODS We conducted an exploratory retrospective study using new specimens,and we enrolled 145 patients who underwent endoscopic resection for superficial ESCC and had been observed for more than two years by both physical examination and endoscopic examination in Hokkaido University Hospital.Saliva was collected to analyze genetic polymorphisms of ADH1B/ALDH2.We performed in situ hybridization for resected specimens to detect HPV by using an HPV type 16/18 probe.RESULTS HPV was detected in 15(10.3%)of the 145 patients with ESCC.HPV-positive rates in inactive ALDH2*1/*2 and ALDH2*1/*1+*2/*2 were 10.8%and 9.8%,respectively(P=1.00).HPV-positive rates in slow-metabolizing ADH1B*1/*1 and ADH1B*1/*2+*2/*2 were 12.0%and 10.0%,respectively(P=0.72).HPV-positive rates in the heavy or moderate alcohol consumption group and the light or rare consumption group were 11.1%and 8.7%,respectively(P=0.68).HPV-positive rates in the heavy smoking group and the light or no smoking group were 11.8%and 8.3%,respectively(P=0.59).The 3-year incidence rates of secondary ESCC or head and neck cancer after initial treatment in the HPV-positive and HPVnegative groups were 14.4%and 21.4%(P=0.22),respectively.CONCLUSION In the present situation,HPV status is considered to be less important than other risk factors,such as alcohol consumption,smoking habit,ADH1B/ALDH2 polymorphisms,and HPV status would therefore have no effect on ESCC risk management.展开更多
AIM:To investigate the association between human papillomavirus(HPV)and esophageal squamous cell carcinoma(ESCC)in southern Brazil.METHODS:We studied 189 esophageal samples from125 patients from three different groups...AIM:To investigate the association between human papillomavirus(HPV)and esophageal squamous cell carcinoma(ESCC)in southern Brazil.METHODS:We studied 189 esophageal samples from125 patients from three different groups:(1)102 biopsies from 51 patients with ESCC,with one sample from the tumor and another from normal esophageal mucosa distant from the tumor;(2)50 esophageal biopsies from 37 patients with a previous diagnosis of head and neck squamous cell carcinoma(HNSCC);and(3)37 biopsies from esophageal mucosa with normal appearance from 37 dyspeptic patients,not exposed to smoking or alcohol consumption.Nested-polymerase chain reaction(PCR)with the MY09/11 and GP5/6 L1primers was used to detect HPV L1 in samples fixed in formalin and stored in paraffin blocks.All PCR reactions were performed with a positive control(cervicovaginal samples),with a negative control(Human Genomic DNA)and with a blank reaction containing all reagents except DNA.We took extreme care to prevent DNA contamination in sample collection,processing,and testing.RESULTS:The histological biopsies confirmed the diagnosis of ESCC in 52 samples(51 from ESCC group and 1 from the HNSCC group)and classified as well differentiated(12/52,23.1%),moderately differentiated(27/52,51.9%)or poorly differentiated(7/52,13.5%).One hundred twenty-eight esophageal biopsies were considered normal(51 from the ESCC group,42 from the HNSCC group and 35 from dyspeptic patients).Nine had esophagitis(7 from the HNSCC and 2 from dyspeptic patients).Of a total of 189 samples,only 6 samples had insufficient material for PCR analysis:1 from mucosa distant from the tumor in a patient with ESCC,3from patients with HNSCC and 2 from patients without cancer.In 183 samples(96.8%)GAPDH,G3PDH and/orβ-globin were amplified,thus indicating the adequacy of the DNA in those samples.HPV DNA was negative in all the 183 samples tested:52 with ESCC,9 with esophagitis and 122 with normal esophageal mucosa.CONCLUSION:There was no evidence of HPV infection in different ESCC from southern Brazil.展开更多
The development of esophageal cancer accompanied by the presence of human papillomavirus (HPV) DNA into the host genome. By evaluating the expression of this virus for tumor cell origin and also their cell grows and m...The development of esophageal cancer accompanied by the presence of human papillomavirus (HPV) DNA into the host genome. By evaluating the expression of this virus for tumor cell origin and also their cell grows and migrations, we examined esophageal cancer clonality in the context of intra-tumor heterogeneity. In this research, we have checked the expression of HPV18 E6 and E7 in different single cell clones by the manual cell picking method in the HPV positive esophageal cancer (EC109), EC109 cell line used as a negative control, and Hela cell line used as the positive control. Quantitative real-time PCR (QRT-PCR) was run to detect the expression levels of HPV E6 and E7, Cell Counting Kit-8 (CCK-8) assay was used to examine cell proliferation, invasion assays performed using Costar chambers and wounding assay to study cell migrations in vitro. We investigated the intra-tumor heterogeneity of HPV E6 and E7 in esophageal cancer and the evaluation of the growth and migrations at the clonal level, using 10 single cell clones. In particular clones, C7 & C10 displayed a highly variable expression in both HPV E6 and E7 and weak in four clones (C1, C3, C4, and C9) consequently, the cell invasion, proliferation, and migration increase with increasing the level of HPV expression and inverse. In conclusion, the resulting based on single cell cloning showed the relationship between HPV and cell growth and migration in esophageal cancer. Future study in HPV DNA integration needed to explore the mains specific integration site of HPV DNA in esophageal cancer and molecular monitoring of the HPV for future prevention researches and also effective therapeutic strategies.展开更多
Marsdenia tenacissima extract(MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal can...Marsdenia tenacissima extract(MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal cancer and other cancers in the digestive tract. In the present study, the potential mechanism for MTE's activity in esophageal cancer was explored. The effects of MTE on the proliferation of human esophageal cancer cells(KYSE150 and Eca-109) were investigated by the MTT assay, the Brd U(bromodeoxyuridine) incorporation immunofluorescence assay, and flow cytometric analysis. MTE inhibited cell proliferation through inducing G0/G1 cell cycle arrest in KYSE150 and Eca-109. Western blot analysis was employed to determine protein levels in the MTE treated cells. Compared with the control cells, the expression levels of the cell cycle regulatory proteins cyclin D1/D2/D3, cyclin E1, CDK2/4/6(CDK: cyclin dependent kinase), and p-Rb were decreased significantly in the cells treated with MTE at 40 mg·m L-1. In addition, MTE had an inhibitory effect on the MAPK(mitogen-activated protein kinase) signal transduction pathway, including ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), and p38 MAPK. Moreover, MTE showed little additional effects on the regulation of cyclin D1/D3, CDK4/6, and p-Rb when the ERK pathway was already inhibited by the specific ERK inhibitor U0126. In conclusion, these data suggest that MTE inhibits human esophageal cancer cell proliferation through regulation of cell cycle regulatory proteins and the MAPK signaling pathways, which is probably mediated by the inhibition of ERK activation.展开更多
基金Supported by the Nature Science Foundation of Heilongjiang Province of China(LC2015036)the Program of Cultivation and Support Projects of Heilongjiang Bayi Agricultural University(XA2015-04)+2 种基金the Research Project of Heilongjiang Bayi Agricultural University(XYB2013-24)the Postdoctoral Scientific Research Foundation of Heilongjiang Province of China(LBH-Q13132)the Scientific Research Innovation Program for College Graduates of Heilongjiang Bayi Agricultural University(YJSCX2017-Y72)
文摘[Objectives] To investigate the pharmacologic effects of active components from A. membranaceus on human esophageal cancer HCE-4 cells and its apoptosis mechanism. [Methods] The viabilities of HCE-4 cells were measured by MTT assay. The induction of active components from A. membranaceus on apoptosis of HCE-4 cells was detected by Annexin V-FITC/PI double staining. The apoptotic-related protein expression levels were determined by Western blotting. [Results] Formononetin and astragaloside IV suppressed the proliferation of HCE-4 cells in a dose-dependent manner. The Annexin V-FITC/PI double staining results showed that formononetin and astragaloside IV could induce HCE-4 cells apoptosis in a time-dependent manner. The Western blotting results showed that formononetin and astragaloside IV could significantly down-regulate p-AKT,pro-caspase-3,and increase cle-caspase-3 protein expression in HCE-4 cells. [Conclusions]Active components from A. membranaceus such as formononetin and astragaloside IV significantly inhibited the proliferation of human esophageal cancer HCE-4 cells by inducing mitochondrial dependent apoptosis via AKT signaling pathway.
基金Supported by Medical University of Lublin,Scientific Research Grant
文摘AIM:To assess the prevalence of human papilloma virus(HPV) in esophageal squamous cell carcinoma(ESCC) in the south-eastern region of Poland.METHODS:The study population consisted of 56 ESCC patients and 35 controls.The controls were patients referred to our department due to other nonesophageal and non-oncological disorders with no gross or microscopic esophageal pathology as confirmed by endoscopy and histopathology.In the ESCC patients,samples were taken from normal mucosa(56 mucosa samples) and from the tumor(56 tumor samples).Tissue samples from the controls were taken from normal mucosa of the middle esophagus(35 control samples).Quantitative determination of DNA was carried out using a spectrophotometric method.Genomic DNA was isolated using the QIAamp DNA Midi Kit.HPV infection was identified following PCR amplification of the HPV gene sequence,using primers MY09 and MY11 complementary to the genome sequence of at least 33 types of HPV.The sequencing results were computationally analyzed using the basic local alignment search tool database.RESULTS:In tumor samples,HPV DNA was identified in 28 of 56 patients(50%).High risk HPV phenotypes(16 or/and 18) were found in 5 of 56 patients(8.9%),low risk in 19 of 56 patients(33.9%) and other types of HPV(37,81,97,CP6108) in 4 of 56 patients(7.1%).In mucosa samples,HPV DNA was isolated in 21 of 56 patients(37.5%).High risk HPV DNA was confirmed in 3 of 56 patients(5.3%),low risk HPV DNA in 12 of 56 patients(21.4%),and other types of HPV in 6 of 56 patients(10.7%).In control samples,HPV DNA was identified in 4 of 35 patients(11.4%) with no high risk HPV.The occurrence of HPV in ESCC patients was significantly higher than in the controls [28 of 56(50%) vs 4 of 35(11.4%),P < 0.001].In esophageal cancer patients,both in tumor and mucosa samples,the predominant HPV phenotypes were low risk HPV,isolated 4 times more frequently than high risk phenotypes [19 of 56(33.9%) vs 5 of 56(8.9%),P < 0.001].A higher prevalence of HPV was identified in female patients(71.4% vs 46.9%).Accordingly,the high risk phenotypes were isolated more frequently in female patients and this difference reached statistical significance [3 of 7(42.9%) vs 2 of 49(4.1%),P < 0.05].Of the pathological characteristics,only an infiltrative pattern of macroscopic tumor type significantly correlated with the presence of HPV DNA in ESCC samples [20 of 27(74.1%) vs 8 of 29(27.6%) for ulcerative or protruding macroscopic type,P < 0.05].The occurrence of total HPV DNA and both HPV high or low risk phenotypes did not significantly differ with regard to particular grades of cellular differentiation,phases in depth of tumor infiltration,grades of nodal involvement and stages of tumor progression.CONCLUSION:Low risk HPV phenotypes could be one of the co-activators or/and co-carcinogens in complex,progressive,multifactorial and multistep esophageal carcinogenesis.
基金Supported by Grants-in-Aid for Scientific Research on Priority Areas of the Ministry of Education, Culture, Sports, Science and Technology, Japan, No. 12218231 and 17015037
文摘AIM： To examine the presence of human papillomavirus （HPV） in esophageal squamous cell carcinoma （ESCC） specimens collected from Colombia and Chile located in the northern and southern ends of the continent, respectively.METHODS： We examined 47 and 26 formalin-fixed and paraffin-embedded ESCC specimens from Colombia and Chile, respectively. HPV was detected using GP5＋/GP6＋ primer pair for PCR, and confirmed by Southern blot analysis. Sequencing analysis of L1 region fragment was used to identify HPV genotype. In addition, P16^INK4A protein immunostaining of all the specimens was conducted.RESULTS： HPV was detected in 21 ESCC specimens （29%）. Sequencing analysis of L1 region fragment identified HPV-16 genome in 6 Colombian cases （13%） and in 5 Chilean cases （19%）. HPV-18 was detected in i0 cases （21%） in Colombia but not in any Chilean case. Since Chilean ESCC cases had a higher prevalence of HPV-16 （without statistical significance）, but a significantly lower prevalence of HPV-18 than in Colombian cases （P = 0.011） even though the two countries have similar ESCC incidence rates, the frequency of HPV-related ESCC may not be strongly affected by risk factors affecting the incidence of ESCC. HPV-16 genome was more frequently detected in p16 positive carcinomas, although the difference was not statistically significant. HPV-18 detection rate did not show any association with p16 expression. Well-differentiated tumors tended to have either HPV-16 or HPV-18 but the association was not statistically significant. HPV genotypes other than HPV-16 or 18 were not detected in either country.CONCLUSION： HPV-16 and HPV-18 genotypes can be found in ESCC specimens collected from two South American countries. Further studies on the relationship between HPV-16 presence and p16 expression in ESCC would aid understanding of the mechanism underlying the presence of HPV in ESCC.
文摘BACKGROUND It is well known that an alcohol consumption habit together with inactive heterozygous aldehyde dehydrogenase-2(ALDH2)is an important risk factor for the development of esophageal squamous cell carcinoma(ESCC).It remains controversial whether human papillomavirus(HPV)infection contributes to the occurrence/development of ESCC.There has been no study in which the relationship between ESCC and HPV in addition to alcohol dehydrogenase-1B(ADH1B)and ALDH2 genotypes was evaluated.AIM To evaluate relationships between HPV infection and development of esophageal cancer,particularly early esophageal cancer,based on ADH1B/ALDH2 polymorphisms.METHODS We conducted an exploratory retrospective study using new specimens,and we enrolled 145 patients who underwent endoscopic resection for superficial ESCC and had been observed for more than two years by both physical examination and endoscopic examination in Hokkaido University Hospital.Saliva was collected to analyze genetic polymorphisms of ADH1B/ALDH2.We performed in situ hybridization for resected specimens to detect HPV by using an HPV type 16/18 probe.RESULTS HPV was detected in 15(10.3%)of the 145 patients with ESCC.HPV-positive rates in inactive ALDH2*1/*2 and ALDH2*1/*1+*2/*2 were 10.8%and 9.8%,respectively(P=1.00).HPV-positive rates in slow-metabolizing ADH1B*1/*1 and ADH1B*1/*2+*2/*2 were 12.0%and 10.0%,respectively(P=0.72).HPV-positive rates in the heavy or moderate alcohol consumption group and the light or rare consumption group were 11.1%and 8.7%,respectively(P=0.68).HPV-positive rates in the heavy smoking group and the light or no smoking group were 11.8%and 8.3%,respectively(P=0.59).The 3-year incidence rates of secondary ESCC or head and neck cancer after initial treatment in the HPV-positive and HPVnegative groups were 14.4%and 21.4%(P=0.22),respectively.CONCLUSION In the present situation,HPV status is considered to be less important than other risk factors,such as alcohol consumption,smoking habit,ADH1B/ALDH2 polymorphisms,and HPV status would therefore have no effect on ESCC risk management.
文摘AIM:To investigate the association between human papillomavirus(HPV)and esophageal squamous cell carcinoma(ESCC)in southern Brazil.METHODS:We studied 189 esophageal samples from125 patients from three different groups:(1)102 biopsies from 51 patients with ESCC,with one sample from the tumor and another from normal esophageal mucosa distant from the tumor;(2)50 esophageal biopsies from 37 patients with a previous diagnosis of head and neck squamous cell carcinoma(HNSCC);and(3)37 biopsies from esophageal mucosa with normal appearance from 37 dyspeptic patients,not exposed to smoking or alcohol consumption.Nested-polymerase chain reaction(PCR)with the MY09/11 and GP5/6 L1primers was used to detect HPV L1 in samples fixed in formalin and stored in paraffin blocks.All PCR reactions were performed with a positive control(cervicovaginal samples),with a negative control(Human Genomic DNA)and with a blank reaction containing all reagents except DNA.We took extreme care to prevent DNA contamination in sample collection,processing,and testing.RESULTS:The histological biopsies confirmed the diagnosis of ESCC in 52 samples(51 from ESCC group and 1 from the HNSCC group)and classified as well differentiated(12/52,23.1%),moderately differentiated(27/52,51.9%)or poorly differentiated(7/52,13.5%).One hundred twenty-eight esophageal biopsies were considered normal(51 from the ESCC group,42 from the HNSCC group and 35 from dyspeptic patients).Nine had esophagitis(7 from the HNSCC and 2 from dyspeptic patients).Of a total of 189 samples,only 6 samples had insufficient material for PCR analysis:1 from mucosa distant from the tumor in a patient with ESCC,3from patients with HNSCC and 2 from patients without cancer.In 183 samples(96.8%)GAPDH,G3PDH and/orβ-globin were amplified,thus indicating the adequacy of the DNA in those samples.HPV DNA was negative in all the 183 samples tested:52 with ESCC,9 with esophagitis and 122 with normal esophageal mucosa.CONCLUSION:There was no evidence of HPV infection in different ESCC from southern Brazil.
文摘The development of esophageal cancer accompanied by the presence of human papillomavirus (HPV) DNA into the host genome. By evaluating the expression of this virus for tumor cell origin and also their cell grows and migrations, we examined esophageal cancer clonality in the context of intra-tumor heterogeneity. In this research, we have checked the expression of HPV18 E6 and E7 in different single cell clones by the manual cell picking method in the HPV positive esophageal cancer (EC109), EC109 cell line used as a negative control, and Hela cell line used as the positive control. Quantitative real-time PCR (QRT-PCR) was run to detect the expression levels of HPV E6 and E7, Cell Counting Kit-8 (CCK-8) assay was used to examine cell proliferation, invasion assays performed using Costar chambers and wounding assay to study cell migrations in vitro. We investigated the intra-tumor heterogeneity of HPV E6 and E7 in esophageal cancer and the evaluation of the growth and migrations at the clonal level, using 10 single cell clones. In particular clones, C7 & C10 displayed a highly variable expression in both HPV E6 and E7 and weak in four clones (C1, C3, C4, and C9) consequently, the cell invasion, proliferation, and migration increase with increasing the level of HPV expression and inverse. In conclusion, the resulting based on single cell cloning showed the relationship between HPV and cell growth and migration in esophageal cancer. Future study in HPV DNA integration needed to explore the mains specific integration site of HPV DNA in esophageal cancer and molecular monitoring of the HPV for future prevention researches and also effective therapeutic strategies.
基金financially supported by National Natural Science Foundation of China(Nos.81302794,81071841,81102853)the Study of Marsdenia tenacissima extract(MTE):Study on quality control of antitumor traditional Chinese medicine Xiao-Ai-Ping injection(No.2011ZX09201-201)
文摘Marsdenia tenacissima extract(MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal cancer and other cancers in the digestive tract. In the present study, the potential mechanism for MTE's activity in esophageal cancer was explored. The effects of MTE on the proliferation of human esophageal cancer cells(KYSE150 and Eca-109) were investigated by the MTT assay, the Brd U(bromodeoxyuridine) incorporation immunofluorescence assay, and flow cytometric analysis. MTE inhibited cell proliferation through inducing G0/G1 cell cycle arrest in KYSE150 and Eca-109. Western blot analysis was employed to determine protein levels in the MTE treated cells. Compared with the control cells, the expression levels of the cell cycle regulatory proteins cyclin D1/D2/D3, cyclin E1, CDK2/4/6(CDK: cyclin dependent kinase), and p-Rb were decreased significantly in the cells treated with MTE at 40 mg·m L-1. In addition, MTE had an inhibitory effect on the MAPK(mitogen-activated protein kinase) signal transduction pathway, including ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), and p38 MAPK. Moreover, MTE showed little additional effects on the regulation of cyclin D1/D3, CDK4/6, and p-Rb when the ERK pathway was already inhibited by the specific ERK inhibitor U0126. In conclusion, these data suggest that MTE inhibits human esophageal cancer cell proliferation through regulation of cell cycle regulatory proteins and the MAPK signaling pathways, which is probably mediated by the inhibition of ERK activation.
