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Volume 21,Issue 4,2014 Table of Contents
2014, 21(4):359-365.
DOI: 10.3872/j.issn.1007-385X.2014.4.001
Abstract:
A biosimilar product is defined as a biologic medical item that is highly similar to and clinically not different from the originator or reference product in terms of safety, purity, and potency. Given their structure complexity and manufacturing process variability, a biosimilar can only be defined as similar, but not identical. To support a demonstration of biosimilarity, a series of tests, including physicochemical analysis, pre-clinical studies and clinical trials, should be taken between the biosimilar and the originator, as well as approval by the authority before being marketed. The biosimlar of low molecular weight biologics was marketed in 2006, the new class of biosimilars, monoclonal antibodies (mAbs), was not approved by the European Medicines Agency (EMA) until 2013, next decade will be the fastigium of biosimilar development. In this paper, we will outline the EMA guidelines for monoclonal antibody biosimilar products and review the clinical trials of trastuzumab, an anti-HER2 monoclonal antibody indicated for breast cancer. A particular focus of discussion will be the clinical trial-related issues including trial design, selection and recruitment of sensitive study populations, extrapolation of indications, interchangeability with the reference product, post-authorization pharmacovigilance and final product labeling. Taking the EMA guidelines in account together with the lessons from previous clinical trials of trastuzumab, we strongly suggest that more precautions should be taken when biosimilar drugs are subject to evaluation and approval to ensure the efficacy and safety profiles are highly similar to those of their reference products for the safety of patients, particularly those with life-threatening diseases, such as cancer.
A biosimilar product is defined as a biologic medical item that is highly similar to and clinically not different from the originator or reference product in terms of safety, purity, and potency. Given their structure complexity and manufacturing process variability, a biosimilar can only be defined as similar, but not identical. To support a demonstration of biosimilarity, a series of tests, including physicochemical analysis, pre-clinical studies and clinical trials, should be taken between the biosimilar and the originator, as well as approval by the authority before being marketed. The biosimlar of low molecular weight biologics was marketed in 2006, the new class of biosimilars, monoclonal antibodies (mAbs), was not approved by the European Medicines Agency (EMA) until 2013, next decade will be the fastigium of biosimilar development. In this paper, we will outline the EMA guidelines for monoclonal antibody biosimilar products and review the clinical trials of trastuzumab, an anti-HER2 monoclonal antibody indicated for breast cancer. A particular focus of discussion will be the clinical trial-related issues including trial design, selection and recruitment of sensitive study populations, extrapolation of indications, interchangeability with the reference product, post-authorization pharmacovigilance and final product labeling. Taking the EMA guidelines in account together with the lessons from previous clinical trials of trastuzumab, we strongly suggest that more precautions should be taken when biosimilar drugs are subject to evaluation and approval to ensure the efficacy and safety profiles are highly similar to those of their reference products for the safety of patients, particularly those with life-threatening diseases, such as cancer.
2014, 21(4):366-370.
DOI: 10.3872/j.issn.1007-385X.2014.4.002
Abstract:
治疗性肿瘤疫苗的作用机制不同于细胞毒药物，其临床研究操作规范不能完全套用细胞毒药物的模式，因此迫切需要制定适用于治疗性肿瘤疫苗临床试验的操作规范。2011年11月，美国FDA正式公布了有关治疗性肿瘤疫苗临床试验的行业指南，对早期（Ⅰ、Ⅱ期）和晚期（Ⅲ期）肿瘤治疗临床试验中需注意的问题提出了一系列指导性意见，其内容主要包括肿瘤疫苗临床试验对受试者的选择、临床免疫反应的监测、佐剂的使用、疫苗的延迟效应、伴随治疗和后续治疗的影响、起始剂量和给药方案、剂量递增方案、统计学处理、终点评价指标等。本文尝试对该指南进行解读，将FDA有关肿瘤疫苗临床试验的一系列指导性意见介绍给国内读者，希望引起国内的肿瘤疫苗研究者、临床试验设计者、临床医生以及业务监管部门的重视，并在实际工作中加以借鉴，从而推动我国肿瘤疫苗的临床研究。
治疗性肿瘤疫苗的作用机制不同于细胞毒药物，其临床研究操作规范不能完全套用细胞毒药物的模式，因此迫切需要制定适用于治疗性肿瘤疫苗临床试验的操作规范。2011年11月，美国FDA正式公布了有关治疗性肿瘤疫苗临床试验的行业指南，对早期（Ⅰ、Ⅱ期）和晚期（Ⅲ期）肿瘤治疗临床试验中需注意的问题提出了一系列指导性意见，其内容主要包括肿瘤疫苗临床试验对受试者的选择、临床免疫反应的监测、佐剂的使用、疫苗的延迟效应、伴随治疗和后续治疗的影响、起始剂量和给药方案、剂量递增方案、统计学处理、终点评价指标等。本文尝试对该指南进行解读，将FDA有关肿瘤疫苗临床试验的一系列指导性意见介绍给国内读者，希望引起国内的肿瘤疫苗研究者、临床试验设计者、临床医生以及业务监管部门的重视，并在实际工作中加以借鉴，从而推动我国肿瘤疫苗的临床研究。
2014, 21(4):371-375.
DOI: 10.3872/j.issn.1007-385X.2014.4.003
Abstract:
Objective: To develop and evaluate the methodology for biological assessment of anti-HER2-MCC-DM1, an antibody-drug-conjugate (ADC) consisting of an anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody conjugated to the maytansinoid emtansine (DM1) via nonreducible thioether linkage (MCC) in breast cancer cells. Methods: Representative samples were collected from three batches of bulk drug and three batches of final products, along with the manufacturer’s validated reference standard. Breast cancer BT-474 cells were treated with the test samples and reference standard respectively. Five days after treatment, cell proliferation inhibition and cell viability were determined by colorimetric assay using a commercial cell counting kit and dual fluorescent staining analysis. Experiments were repeated three times. Results: Both test samples and reference standard of anti-HER2-MCC-DM1 inhibited BT-474 cell proliferation in a dose-dependent manner. The variation coefficient of percent inhibition within the three experiments was less than 15% for both test samples and reference standard. Compared with the nude anti-HER2 antibody, the bulk drug and final product increased BT-474 cell proliferation inhibition by （326.72±21.58）% and （315.76±34.90）% respectively. HER2-MCC-DM1 effectively induced BT-474 cell shrinkage and death as revealed by both light microscopy and fluorescence microscopy. Conclusion: The biological assessment of anti-HER2-MCC-DM1 in breast cancer BT-474 cells in vitro, developed in this study, is highly reproducible and accurate, thus offering a promising method for quality control and evaluation of ADCs.
Objective: To develop and evaluate the methodology for biological assessment of anti-HER2-MCC-DM1, an antibody-drug-conjugate (ADC) consisting of an anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody conjugated to the maytansinoid emtansine (DM1) via nonreducible thioether linkage (MCC) in breast cancer cells. Methods: Representative samples were collected from three batches of bulk drug and three batches of final products, along with the manufacturer’s validated reference standard. Breast cancer BT-474 cells were treated with the test samples and reference standard respectively. Five days after treatment, cell proliferation inhibition and cell viability were determined by colorimetric assay using a commercial cell counting kit and dual fluorescent staining analysis. Experiments were repeated three times. Results: Both test samples and reference standard of anti-HER2-MCC-DM1 inhibited BT-474 cell proliferation in a dose-dependent manner. The variation coefficient of percent inhibition within the three experiments was less than 15% for both test samples and reference standard. Compared with the nude anti-HER2 antibody, the bulk drug and final product increased BT-474 cell proliferation inhibition by （326.72±21.58）% and （315.76±34.90）% respectively. HER2-MCC-DM1 effectively induced BT-474 cell shrinkage and death as revealed by both light microscopy and fluorescence microscopy. Conclusion: The biological assessment of anti-HER2-MCC-DM1 in breast cancer BT-474 cells in vitro, developed in this study, is highly reproducible and accurate, thus offering a promising method for quality control and evaluation of ADCs.
2014, 21(4):376-382.
DOI: 10.3872/j.issn.1007-385X.2014.4.004
Abstract:
Objective: To investigate the expression, methylation status and functional role of miR-203 in pathogenesis of ESCC. ethods: Eighty-three patients diagnosed with ESCC in Hebei Medical University-Affiliated Fourth Hospital between 2008 and 2011 were recruited. Biopsy specimens were collected from primary tumors and the corresponding adjacent tissues. Quantitative real-time RT-PCR (qRT-PCP) and methylation specific PCR (MSP) were used to respectively detect the mRNA abundance and methylation status of miR-203 gene in the collected specimens. Five esophageal cancer cell lines (TE1, TE13, T.TN, Yes-2, and EC109) were treated with DNA methyltransferase inhibitor 5-Aza-2’-detoxycytidine (5-Aza-dC) Levels of CpG methylation of the miR-203 gene and miR-203 were assessed by qRT-PCR and MSP, respectively, 72 h after 5-Aza-dC treatment. Results： Relatively low levels of miR-203 mRNA and hypermethylation were detected in all the five untreated esophageal cancer cell lines. After 5-Aza-dC treatment, miR-203 mRNA was increased in all five cell lines studied and the methylation level of miR-203 was decreased in YES-2 cells and complete miR-203 unmethylation occurred in TE1, TE13, T.TN, and EC109 cells. The abundance of miR-203 mRNA was significantly lower (0.54±0.11 vs 1.00±0.01, P<0.05) and the methylation frequency of miR-203 promoter was significantly higher (62.65%vs 7.23%,P<0.05) in ESCC tissues than in corresponding tissues. Both miR-203 mRNA abundance and methylation frequency were all correlated with TNM stage and pathological differentiation (P<0.05). The expression of miR-203 in ESCC with miR-203 methylation was significantly lower than that in ESCC without miR-203 methylation (P<0.05). Conclusion： Aberrantly low expression of miR-203 is closely related to the development and progression of ESCC and promoter DNA methylation is one of the possible mechanisms underlying miR-203 inactivationin ESCC.
Objective: To investigate the expression, methylation status and functional role of miR-203 in pathogenesis of ESCC. ethods: Eighty-three patients diagnosed with ESCC in Hebei Medical University-Affiliated Fourth Hospital between 2008 and 2011 were recruited. Biopsy specimens were collected from primary tumors and the corresponding adjacent tissues. Quantitative real-time RT-PCR (qRT-PCP) and methylation specific PCR (MSP) were used to respectively detect the mRNA abundance and methylation status of miR-203 gene in the collected specimens. Five esophageal cancer cell lines (TE1, TE13, T.TN, Yes-2, and EC109) were treated with DNA methyltransferase inhibitor 5-Aza-2’-detoxycytidine (5-Aza-dC) Levels of CpG methylation of the miR-203 gene and miR-203 were assessed by qRT-PCR and MSP, respectively, 72 h after 5-Aza-dC treatment. Results： Relatively low levels of miR-203 mRNA and hypermethylation were detected in all the five untreated esophageal cancer cell lines. After 5-Aza-dC treatment, miR-203 mRNA was increased in all five cell lines studied and the methylation level of miR-203 was decreased in YES-2 cells and complete miR-203 unmethylation occurred in TE1, TE13, T.TN, and EC109 cells. The abundance of miR-203 mRNA was significantly lower (0.54±0.11 vs 1.00±0.01, P<0.05) and the methylation frequency of miR-203 promoter was significantly higher (62.65%vs 7.23%,P<0.05) in ESCC tissues than in corresponding tissues. Both miR-203 mRNA abundance and methylation frequency were all correlated with TNM stage and pathological differentiation (P<0.05). The expression of miR-203 in ESCC with miR-203 methylation was significantly lower than that in ESCC without miR-203 methylation (P<0.05). Conclusion： Aberrantly low expression of miR-203 is closely related to the development and progression of ESCC and promoter DNA methylation is one of the possible mechanisms underlying miR-203 inactivationin ESCC.
2014, 21(4):383-388.
DOI: 10.3872/j.issn.1007-385X.2014.4.005
Abstract:
Objective: To investigate the role of microRNA-10a (miR-10a) in hepatocellular carcinoma (HCC) growth. Methods: Paired HCC and adjacent non-tumor tissue specimens were surgically collected from 144 patients who were diagnosed with primary HCC in Guangxi Medical University-Affiliated Tumor Hospital between October 2001 and July 2005. HCC QGY-7701, Huh7, and PCL/PRF/5 cells were transfected with miR-10a mimics or scramble control miRNA. The abundance of miR-10a in both tissue specimens and transfected cells was quantified by real-time PCR and E2F3 protein in transfected cells was assessed by Western blotting. Proliferation of the transfectants was assessed by a colorimetric cell counting assay. Cell cycle progression and apoptosis of the transfectants were assessed by FACS. Results: The abundance of miR-10a mRNA was significantly lower in HCC tissue specimens than in normal tissue specimens (-9.89±168 vs -784±1.97, P =0.0001). HCC cells transfected with miR-10a mimics had miR-10a abundance 16 times higher than both wild-type HCC cells and HCC cells transfected with the control miRNA with scrambled sequences. Overexpression of miR-10a resulted in significant increases in suppression of HCC cell proliferation ( P <0.05） and G 1 phase arrest. In contrast, overexpression of miR-10a had no influence on apoptosis of HCC cells. Bioinformatics suggested that transcription factor E2F3 might be a downstream target of miR-10a and the expression of E2F3 in HCC cells transfected with miR-10a was significantly lower than in wild-type HCC cells and HCC cells transfected with the control miRNA (050±0.12 vs 0.79±0.21, P <0.05). Conclusion: MiR-10a may suppress HCC cell proliferation through G 1 phase arrest in an E2F3-dependent mechanism.
Objective: To investigate the role of microRNA-10a (miR-10a) in hepatocellular carcinoma (HCC) growth. Methods: Paired HCC and adjacent non-tumor tissue specimens were surgically collected from 144 patients who were diagnosed with primary HCC in Guangxi Medical University-Affiliated Tumor Hospital between October 2001 and July 2005. HCC QGY-7701, Huh7, and PCL/PRF/5 cells were transfected with miR-10a mimics or scramble control miRNA. The abundance of miR-10a in both tissue specimens and transfected cells was quantified by real-time PCR and E2F3 protein in transfected cells was assessed by Western blotting. Proliferation of the transfectants was assessed by a colorimetric cell counting assay. Cell cycle progression and apoptosis of the transfectants were assessed by FACS. Results: The abundance of miR-10a mRNA was significantly lower in HCC tissue specimens than in normal tissue specimens (-9.89±168 vs -784±1.97, P =0.0001). HCC cells transfected with miR-10a mimics had miR-10a abundance 16 times higher than both wild-type HCC cells and HCC cells transfected with the control miRNA with scrambled sequences. Overexpression of miR-10a resulted in significant increases in suppression of HCC cell proliferation ( P <0.05） and G 1 phase arrest. In contrast, overexpression of miR-10a had no influence on apoptosis of HCC cells. Bioinformatics suggested that transcription factor E2F3 might be a downstream target of miR-10a and the expression of E2F3 in HCC cells transfected with miR-10a was significantly lower than in wild-type HCC cells and HCC cells transfected with the control miRNA (050±0.12 vs 0.79±0.21, P <0.05). Conclusion: MiR-10a may suppress HCC cell proliferation through G 1 phase arrest in an E2F3-dependent mechanism.
Qian Li
,
Lu Jiahui
,
Qin Hongchao
,
Rui Chenglei
,
Chen Wenyan
,
Jia Xiaoqin
,
Fu Yi
,
Gong Weijuan
,
Tian Fang
,
Hu Maozhi
,
Ji Mingchun
2014, 21(4):389-395.
DOI: 10.3872/j.issn.1007-385X.2014.4.006
Abstract:
Objective: To study the effects of murine bone marrow-derived pro-B (BaF3) cells overexpressing membrane-bound form of IL-15 (mbIL-15) and retinoic acid early transcript 1ε (RAE1ε), either each alone or two in combination, on the expression of functional molecules in murine interferon producing killer dendritic cells (IKDCs) in vitro . Methods: Clones of BaF3 cells overexpressing mbIL-15 , RAE1ε or both mbIL-15 and RAE1ε were established and inactivated by mitomycin. Mouse bone marrow mononuclear cells were isolated and induced into mature DCs by GM-CSF and IL-4. Mature DCs were co-cultured with the three mitomycin inactivated-BaF3 derivatives respectively. At 24 and 72 hours after co-culture, the percentage of CD11clow B220+NK1.1+ IKDCs and levels of CD40 and CD80 in DCs and levels of CD40, CD80, CD86, NKG2D, MHC class Ⅱ molecule, CD107a and FasL in IKDCs were determined by flow cytometry. Results: BaF3 cells overexpressing both mbIL-15 and RAE were more effective than BaF3 cells overexpressing mbIL-15 or RAE alone to stimulate the DC to IKDC conversion (\[50.0±5.6\]% vs \[30.3±8.2\]%, \[36.0±4.6\]%, P <0.05). IKDCs co-cultured with BaF3 cells overexpressing mbIL-15 and RAE had higher levels of CD40 (180.1±282 vs 44.7±7.8, 36.0±3.1, P <0.01) and FasL ( P <0.05) as compared to IKDCs co-cultured with mbIL-15- or RAE-overexpressing BaF3 cells. IKDCs co-cultured with BaF3 cells overexpressing mbIL-15 and RAE had higher levels of CD80 ( P <0.05) as compared to IKDCs co-cultured with BaF3 cells or mbIL-15-overexpressing BaF3 cells. In contrast, co-culture with BaF3 derivatives showed no effects either on CD40 and CD80 expression in DCs ( P >0.05)or on CD86, MHC class Ⅱ molecule, NKG2D and CD107a expression in IKDCs. Conclusion: IL-15 combined with RAE1ε may promote the proliferation and CD40 and FasL expression in IKDCs.
Objective: To study the effects of murine bone marrow-derived pro-B (BaF3) cells overexpressing membrane-bound form of IL-15 (mbIL-15) and retinoic acid early transcript 1ε (RAE1ε), either each alone or two in combination, on the expression of functional molecules in murine interferon producing killer dendritic cells (IKDCs) in vitro . Methods: Clones of BaF3 cells overexpressing mbIL-15 , RAE1ε or both mbIL-15 and RAE1ε were established and inactivated by mitomycin. Mouse bone marrow mononuclear cells were isolated and induced into mature DCs by GM-CSF and IL-4. Mature DCs were co-cultured with the three mitomycin inactivated-BaF3 derivatives respectively. At 24 and 72 hours after co-culture, the percentage of CD11clow B220+NK1.1+ IKDCs and levels of CD40 and CD80 in DCs and levels of CD40, CD80, CD86, NKG2D, MHC class Ⅱ molecule, CD107a and FasL in IKDCs were determined by flow cytometry. Results: BaF3 cells overexpressing both mbIL-15 and RAE were more effective than BaF3 cells overexpressing mbIL-15 or RAE alone to stimulate the DC to IKDC conversion (\[50.0±5.6\]% vs \[30.3±8.2\]%, \[36.0±4.6\]%, P <0.05). IKDCs co-cultured with BaF3 cells overexpressing mbIL-15 and RAE had higher levels of CD40 (180.1±282 vs 44.7±7.8, 36.0±3.1, P <0.01) and FasL ( P <0.05) as compared to IKDCs co-cultured with mbIL-15- or RAE-overexpressing BaF3 cells. IKDCs co-cultured with BaF3 cells overexpressing mbIL-15 and RAE had higher levels of CD80 ( P <0.05) as compared to IKDCs co-cultured with BaF3 cells or mbIL-15-overexpressing BaF3 cells. In contrast, co-culture with BaF3 derivatives showed no effects either on CD40 and CD80 expression in DCs ( P >0.05)or on CD86, MHC class Ⅱ molecule, NKG2D and CD107a expression in IKDCs. Conclusion: IL-15 combined with RAE1ε may promote the proliferation and CD40 and FasL expression in IKDCs.
2014, 21(4):396-401.
DOI: 10.3872/j.issn.1007-385X.2014.4.007
Abstract:
Objective: To elucidate the putative involvement of epithelial membrane protein-1 (EMP1) in the pathogenesis of colorectal carcinoma (CRC). Methods: Tumor ( n =63) and peri-tumor ( n =31) tissue specimens were collected from 63 patients with CRC. EMP1 protein content in these specimens was assessed by immunohistochemistry and Western blotting. The relationship between EMP1 protein content in the tumor tissue and the pathologic grade of the lesions was analyzed. To further evaluate the putative role for EMP1 in the development of CRC, we also performed analysis in vitro . To this end, CRC SW-480 cells were transfected with an EMP1 lentiviral vector pLenti6-EMP1 or a control vector plenti6/V5-DEST. EMP1 mRNA abundance and protein content in transfected cells were determined by quantitative real-time PCR and Western blotting respectively. Effects of EMP1 overexpression on the proliferation, apoptosis and invasion of SW-480 cells were evaluated by methyl thiazolyl tetrazoliun (MTT) assay, flow cytometry (FCM) and transwell invasion assays, respectively. Results: EMP1 protein content was significantly lower in CRC tissue than in peritumor tissues ( P <0.05). The level of EMP1 protein was not correlated with gender, age, and tumor location ( P >0.05) but was positively correlated with lymph node metastasis, clinic stage and histological grade of the lesions ( P <0.05). Compared with SW-480 cells transfected with the control vector, SW-480 cells overexpressing EMP1 had a lower survival fraction (\[60.94±4.04\]% vs \[100.00±0.00\]%， P <0.05), a higher cell apoptosis rate (\[12.10±1.30\]% vs \[3.10±060\]%， P <0.05), a decreased invasive capacity (\[178.00±21.00\] vs \[87.00±12.00\], P <0.05), higher caspase-9 protein content (0.764±0.073 vs 0.231±0.029, P <0.05) and lower VEGFC protein content (0.663±0.065 vs 0.185±0022, P <0.05). Conclusion: EMP1 protein content is significantly lower in the CRC tissue than in the non-CRC tissue. Overexpression of EMP1 in CRC cells in vitro results in a significant inhibition of invasive activity through regulating the expression of caspase-9 and VEGF-C. These findings suggest that EMP1 is involved in the pathogenesis of colorectal carcinoma.
Objective: To elucidate the putative involvement of epithelial membrane protein-1 (EMP1) in the pathogenesis of colorectal carcinoma (CRC). Methods: Tumor ( n =63) and peri-tumor ( n =31) tissue specimens were collected from 63 patients with CRC. EMP1 protein content in these specimens was assessed by immunohistochemistry and Western blotting. The relationship between EMP1 protein content in the tumor tissue and the pathologic grade of the lesions was analyzed. To further evaluate the putative role for EMP1 in the development of CRC, we also performed analysis in vitro . To this end, CRC SW-480 cells were transfected with an EMP1 lentiviral vector pLenti6-EMP1 or a control vector plenti6/V5-DEST. EMP1 mRNA abundance and protein content in transfected cells were determined by quantitative real-time PCR and Western blotting respectively. Effects of EMP1 overexpression on the proliferation, apoptosis and invasion of SW-480 cells were evaluated by methyl thiazolyl tetrazoliun (MTT) assay, flow cytometry (FCM) and transwell invasion assays, respectively. Results: EMP1 protein content was significantly lower in CRC tissue than in peritumor tissues ( P <0.05). The level of EMP1 protein was not correlated with gender, age, and tumor location ( P >0.05) but was positively correlated with lymph node metastasis, clinic stage and histological grade of the lesions ( P <0.05). Compared with SW-480 cells transfected with the control vector, SW-480 cells overexpressing EMP1 had a lower survival fraction (\[60.94±4.04\]% vs \[100.00±0.00\]%， P <0.05), a higher cell apoptosis rate (\[12.10±1.30\]% vs \[3.10±060\]%， P <0.05), a decreased invasive capacity (\[178.00±21.00\] vs \[87.00±12.00\], P <0.05), higher caspase-9 protein content (0.764±0.073 vs 0.231±0.029, P <0.05) and lower VEGFC protein content (0.663±0.065 vs 0.185±0022, P <0.05). Conclusion: EMP1 protein content is significantly lower in the CRC tissue than in the non-CRC tissue. Overexpression of EMP1 in CRC cells in vitro results in a significant inhibition of invasive activity through regulating the expression of caspase-9 and VEGF-C. These findings suggest that EMP1 is involved in the pathogenesis of colorectal carcinoma.
2014, 21(4):402-407.
DOI: 10.3872/j.issn.1007-385X.2014.4.008
Abstract:
Objective: To evaluate the effect of cecropin-XJ in itself or in combination with one of the commonly used chemotherapeutic agents on the proliferation and apoptosis of human esophageal carcinoma Eca109 cells in vitro. Methods：Eca109 cells were treated with cecropin-XJ, either in itself or in combination with adriamycin (ADM), cis-diamminedichloride platinum II (DDP) or cyclophosphamide (CTX). Twenty-four hours after treatment, cell viability was assessed by MTT assays, and apoptosis, reactive oxygen species (ROS) levels and mitochondrial membrane potential were assessed by flow cytometry. Results：Cecropin-XJ alone significantly inhibited Eca109 cell proliferation at the doses used (range from 1 to 50 μmol/L) (P<0.05). Compared with the cecropin-XJ mono-treatment, the combined treatment of cecropin-XJ with ADM, DDP or CTX were significantly more effective to induce Eca109 cell proliferation inhibition, apoptosis and reactive oxygen species production, and to decrease Eca109 cell mitochondrial membrane potential (P<0.05). In contrast, the combination of cecropin-XJ with CBP showed an antagonistic effect. Conclusion：Cecropin-XJ combined with ADM, DDP, and CTX respectively may inhibit proliferation and induce apoptosis of human esophageal carcinoma cells more effectively than cecropin-XJ in itself. The synergistic effect may be associated with changes in the intracellular ROS production and mitochondrial membrane potential.
Objective: To evaluate the effect of cecropin-XJ in itself or in combination with one of the commonly used chemotherapeutic agents on the proliferation and apoptosis of human esophageal carcinoma Eca109 cells in vitro. Methods：Eca109 cells were treated with cecropin-XJ, either in itself or in combination with adriamycin (ADM), cis-diamminedichloride platinum II (DDP) or cyclophosphamide (CTX). Twenty-four hours after treatment, cell viability was assessed by MTT assays, and apoptosis, reactive oxygen species (ROS) levels and mitochondrial membrane potential were assessed by flow cytometry. Results：Cecropin-XJ alone significantly inhibited Eca109 cell proliferation at the doses used (range from 1 to 50 μmol/L) (P<0.05). Compared with the cecropin-XJ mono-treatment, the combined treatment of cecropin-XJ with ADM, DDP or CTX were significantly more effective to induce Eca109 cell proliferation inhibition, apoptosis and reactive oxygen species production, and to decrease Eca109 cell mitochondrial membrane potential (P<0.05). In contrast, the combination of cecropin-XJ with CBP showed an antagonistic effect. Conclusion：Cecropin-XJ combined with ADM, DDP, and CTX respectively may inhibit proliferation and induce apoptosis of human esophageal carcinoma cells more effectively than cecropin-XJ in itself. The synergistic effect may be associated with changes in the intracellular ROS production and mitochondrial membrane potential.
2014, 21(4):408-412.
DOI: 10.3872/j.issn.1007-385X.2014.4.009
Abstract:
Objective: To investigate the inhibitory effect of microRNA-129-2(miR-129-2) on xenografted human nasopharyngeal carcinoma development and SRY-related HMG-box transcription factor (SOX4) expression in nude mice. Methods: Nude mice were injected with human nasopharyngeal carcinoma CNE1 cells. Animals with confirmed tumor lesions were randomized into 4 treatment groups (n=8): saline control (subcutaneous injection of saline 0.2 ml/d), blank plasmid control (50 μg each mice in 0.2 ml daily), cisplatin (DDP) (1 mg/kg in 0.2 ml daily ) and miR-129-2 (50 μg each mice in 0.2 ml daily). At 5 weeks after treatment, body weight and tumor volume and weight were measured, the proportion of cells at S/G2-M arrest in xenografted tumor cells was assessed by flow cytometry, and SOX4 protein content in xenograft tumors was assessed by immunohistochemical analysis and Western blotting. Results: In nude mice that developed nasopharyngeal carcinoma lesions after grafting of CNE1 cells, miR-129-2 led a significant decrease, comparable to that resulted from DDP treatment, in tumor volume and weight (P<0.01) whereas the control plasmid showed no effect (P>005), as compared with the non-treatment control 22 days after treatment. Mice in the miR-129-2 group were significantly heavier than all other three groups of animals (P<0.01). The proportion of cells at S/G2-M arrest was (37.95±151)% in the non-treatment group, which was not different from the control plasmid group (36.75±1.48)% but significantly decreased (P<0.01) in the miR-129-2 group (31.81±1.45)% and the DDP treatment group (32.34±1.67)%. SOX4 protein content was significantly higher in tumors than in peritumoral tissues (P<0.05). Both miR-129-2 and DDP significantly lowered SOX4 protein content in tumors (P<0.01), but the effect of miR-129-2 was more pronounced. Conclusion: The small non-coding RNA molecule miR-129-2 is capable of suppressing the growth of xenografted human nasopharyngeal carcinoma through down-regulation of SOX4 expression in nude mice, thus possessing a therapeutic potential for nasopharyngeal carcinoma.
Objective: To investigate the inhibitory effect of microRNA-129-2(miR-129-2) on xenografted human nasopharyngeal carcinoma development and SRY-related HMG-box transcription factor (SOX4) expression in nude mice. Methods: Nude mice were injected with human nasopharyngeal carcinoma CNE1 cells. Animals with confirmed tumor lesions were randomized into 4 treatment groups (n=8): saline control (subcutaneous injection of saline 0.2 ml/d), blank plasmid control (50 μg each mice in 0.2 ml daily), cisplatin (DDP) (1 mg/kg in 0.2 ml daily ) and miR-129-2 (50 μg each mice in 0.2 ml daily). At 5 weeks after treatment, body weight and tumor volume and weight were measured, the proportion of cells at S/G2-M arrest in xenografted tumor cells was assessed by flow cytometry, and SOX4 protein content in xenograft tumors was assessed by immunohistochemical analysis and Western blotting. Results: In nude mice that developed nasopharyngeal carcinoma lesions after grafting of CNE1 cells, miR-129-2 led a significant decrease, comparable to that resulted from DDP treatment, in tumor volume and weight (P<0.01) whereas the control plasmid showed no effect (P>005), as compared with the non-treatment control 22 days after treatment. Mice in the miR-129-2 group were significantly heavier than all other three groups of animals (P<0.01). The proportion of cells at S/G2-M arrest was (37.95±151)% in the non-treatment group, which was not different from the control plasmid group (36.75±1.48)% but significantly decreased (P<0.01) in the miR-129-2 group (31.81±1.45)% and the DDP treatment group (32.34±1.67)%. SOX4 protein content was significantly higher in tumors than in peritumoral tissues (P<0.05). Both miR-129-2 and DDP significantly lowered SOX4 protein content in tumors (P<0.01), but the effect of miR-129-2 was more pronounced. Conclusion: The small non-coding RNA molecule miR-129-2 is capable of suppressing the growth of xenografted human nasopharyngeal carcinoma through down-regulation of SOX4 expression in nude mice, thus possessing a therapeutic potential for nasopharyngeal carcinoma.
2014, 21(4):413-418.
DOI: 10.3872/j.issn.1007-385X.2014.4.010
Abstract:
Objective: To assess differences in erlotinib resistance between EGFR-mutated and EGFR wildtype non-small lung cancer (NSCLC) cells following hepatocyte growth factor (HGF) in vitro. Methods: EGFR-mutated NSCLC PC9 cells and EGFR-wild type NSCLC H292 and A549 cells were left untreated (control) or treated with HGF, erlotinib or HGF plus erlotinib. Cell viability was assessed by MTT assays, cell appotosis and cell cycle progression by flow cytomety and protein contents of c-Met, EGFR and ErbB3 by Western blotting. Results: Erlotinib resulted in a significant proliferation inhibition in all three types of NSCLS cells in a dose-dependent manner and HGF effectively attenuated the erlotinib-induced proliferation inhibition. In all three types of cells studied, the apoptosis rate in the combination treatment (HGF plus erlotinib) group was lower than that in the erlotinib group (P<0.05), and erlotinib induced G1 arrest. In H292 and A549 cells, the proportion of cells at G0/G1 phase was significantly lower in the combination treatment group than in the erlotinib group (P<0.05). Protein content of c-Met was significantly higher in the combination treatment than in the erlotinib group (P<0.05) whereas protein contents of p-EGFR and p-ErbB3 were not different between treatment groups (P>0.05) in all three types of cells. Conclusion: HGF may induce erlotinib resistance differentially in EGFR wildtype and EGFR-mutated NSCLC cells, possibly an Met activation-dependent mechanism.
Objective: To assess differences in erlotinib resistance between EGFR-mutated and EGFR wildtype non-small lung cancer (NSCLC) cells following hepatocyte growth factor (HGF) in vitro. Methods: EGFR-mutated NSCLC PC9 cells and EGFR-wild type NSCLC H292 and A549 cells were left untreated (control) or treated with HGF, erlotinib or HGF plus erlotinib. Cell viability was assessed by MTT assays, cell appotosis and cell cycle progression by flow cytomety and protein contents of c-Met, EGFR and ErbB3 by Western blotting. Results: Erlotinib resulted in a significant proliferation inhibition in all three types of NSCLS cells in a dose-dependent manner and HGF effectively attenuated the erlotinib-induced proliferation inhibition. In all three types of cells studied, the apoptosis rate in the combination treatment (HGF plus erlotinib) group was lower than that in the erlotinib group (P<0.05), and erlotinib induced G1 arrest. In H292 and A549 cells, the proportion of cells at G0/G1 phase was significantly lower in the combination treatment group than in the erlotinib group (P<0.05). Protein content of c-Met was significantly higher in the combination treatment than in the erlotinib group (P<0.05) whereas protein contents of p-EGFR and p-ErbB3 were not different between treatment groups (P>0.05) in all three types of cells. Conclusion: HGF may induce erlotinib resistance differentially in EGFR wildtype and EGFR-mutated NSCLC cells, possibly an Met activation-dependent mechanism.
2014, 21(4):419-422.
DOI: 10.3872/j.issn.1007-385X.2014.4.011
Abstract:
Objective: To study the effect of norcantharidin on apoptosis of esophageal cancer Ec9706 cells and the putative mechanism(s) underlying the effect. Methods: Ec9706 cells were treated with norcantharidin at 0, 5, 10, 20 or 40 μg/ml. Cell viability was determined by MTT assays and apoptosis, caspase-3 and survivin protein levels in the treated Ec9706 cells were assessed by flow cytometry, respectively, at 12, 24 and 48 h after treatment. Results: Norcantharidin inhibited the growth of Ec9706 cells in a dose- and time-dependent manner; the growth inhibition rate was (80.00±215)% at 40 μg/ml for 48 h. Norcantharidin induced apoptosis, significantly increased caspase-3 protein level (P<0.05) and significantly decreased survivin protein level (P<0.05) in Ec9706 cells in a dose- and time-dependent manner; the apoptosis rate was 38.57±1.76%at 20 μg/ml for 48h. Conclusion: Norcantharidin may induce apoptosis of esophageal cancer Ec9706 cells through downregulation of survivin expression and upregulation of caspase-3 expression.
Objective: To study the effect of norcantharidin on apoptosis of esophageal cancer Ec9706 cells and the putative mechanism(s) underlying the effect. Methods: Ec9706 cells were treated with norcantharidin at 0, 5, 10, 20 or 40 μg/ml. Cell viability was determined by MTT assays and apoptosis, caspase-3 and survivin protein levels in the treated Ec9706 cells were assessed by flow cytometry, respectively, at 12, 24 and 48 h after treatment. Results: Norcantharidin inhibited the growth of Ec9706 cells in a dose- and time-dependent manner; the growth inhibition rate was (80.00±215)% at 40 μg/ml for 48 h. Norcantharidin induced apoptosis, significantly increased caspase-3 protein level (P<0.05) and significantly decreased survivin protein level (P<0.05) in Ec9706 cells in a dose- and time-dependent manner; the apoptosis rate was 38.57±1.76%at 20 μg/ml for 48h. Conclusion: Norcantharidin may induce apoptosis of esophageal cancer Ec9706 cells through downregulation of survivin expression and upregulation of caspase-3 expression.
2014, 21(4):423-427.
DOI: 10.3872/j.issn.1007-385X.2014.4.012
Abstract:
Objective: To construct an optimized human telomerase reverse transcriptase (hTERT) gene-specific RNAi and to evaluate its effect on human colon cancer cell proliferation in vitro. Method: Three hTERT-specific RNAi sequences and a negative control (NC) or scrambled sequence were cloned, respectively, into a pGPU6/GFP/Neo vector to generate pGPU6-GFP-hTERT-1, pGPU6-GFP-hTERT-2, pGPU6-GFP-hTERt-3 and pGPU6-GFP-NC. Human colon cancer SW480 cells were transfected with these vectors respectively. At 24, 48 and 72 h after transfection, hTERT mRNA abundance was assessed by RT-PCR and cell viability by MTT assay. Results: The 3 hTERT-specific RNAi vectors constructed were all effective to silence the hTERT gene; hTERT mRNA abundance in SW480 cells transfected with pGPU6-GFP-hTERT-3 was significantly lower than that in SW480 cells transfected with pGPU6-GFP-NC (0.347±0.028 vs 0513 ± 0.032,P<0.01). All the three hTERT sequence-specific RNAi vectors were effective to inhibit the proliferation of SW480 cells; cellular proliferation inhibition rate in SW480 cells of pGPU6-GFP-hTERT-3 group was significantly increased than that of blank contro, liposomal and NC group (\[50.08±0.43\]% vs \[4.11±0.39\]%, \[3.88±035\]% and \[3.38±0.35\]%; P<0.05). Conclusion: RNAi-mediated hTERT gene silencing results in colon cancer cell growth inhibition and may offer a novel therapy for colon cancer.
Objective: To construct an optimized human telomerase reverse transcriptase (hTERT) gene-specific RNAi and to evaluate its effect on human colon cancer cell proliferation in vitro. Method: Three hTERT-specific RNAi sequences and a negative control (NC) or scrambled sequence were cloned, respectively, into a pGPU6/GFP/Neo vector to generate pGPU6-GFP-hTERT-1, pGPU6-GFP-hTERT-2, pGPU6-GFP-hTERt-3 and pGPU6-GFP-NC. Human colon cancer SW480 cells were transfected with these vectors respectively. At 24, 48 and 72 h after transfection, hTERT mRNA abundance was assessed by RT-PCR and cell viability by MTT assay. Results: The 3 hTERT-specific RNAi vectors constructed were all effective to silence the hTERT gene; hTERT mRNA abundance in SW480 cells transfected with pGPU6-GFP-hTERT-3 was significantly lower than that in SW480 cells transfected with pGPU6-GFP-NC (0.347±0.028 vs 0513 ± 0.032,P<0.01). All the three hTERT sequence-specific RNAi vectors were effective to inhibit the proliferation of SW480 cells; cellular proliferation inhibition rate in SW480 cells of pGPU6-GFP-hTERT-3 group was significantly increased than that of blank contro, liposomal and NC group (\[50.08±0.43\]% vs \[4.11±0.39\]%, \[3.88±035\]% and \[3.38±0.35\]%; P<0.05). Conclusion: RNAi-mediated hTERT gene silencing results in colon cancer cell growth inhibition and may offer a novel therapy for colon cancer.
2014, 21(4):428-436.
DOI: 10.3872/j.issn.1007-385X.2014.4.013
Abstract:
Objective: The purpose of our study was to analyze the expression of melanoma antigen genes MAGE-A9 and MAGE-A11 in benign and malignant breast cancer specimens and in breast cancer cell lines following drug treatment. Methods: Tissue specimens were obtained from tumor-free breast (n=60), benign breast cancer (n=60) and malignant breast cancer (n=60), respectively. The mRNA abundance and protein content of MAGE-A9 and MAGE-A11 in these specimens were determined by RT-PCR and immunohistochemistry respectively and were analyzed for their associations with the major demographic, physiologic and pathologic variables. To examine the influence of chemotherapeutic drugs on MAGEs in breast cancer, MCF-7 and MDA-MB-231 cells were treated with 5-Aza-2’-deoxycytidine (5-Aza-CdR), a DNA methylase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, either each alone or both together, and mRNA levels of MAGE-A9 and MAGE-A11 were assessed by RT-PCR. Results：MAGE-A9 and MAGE-A11 transcripts were detected in 45% and 66.7% of breast cancer specimens respectively and MAGE-A9 and MAGE-A11 proteins in 433% and 63.3% of cancer specimens, respectively. MAGE-A9 and MAGE-A11 expression was positively correlated with the expression of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER-2) in cancer specimens (P<0.05). Neither transcripts nor proteins of MAGE-A9 and MAGE-A11 were detected in normal control specimens. While 5-Aza-CdR alone induced the expression of MAGE-A9 and MAGE-A11 in the test cell lines, TSA alone showed no effects. However, TSA was able to enhance 5-Aza-CdR-induced MAGE-A transcription (P<0.01).Conclusion：MAGE-A9 and MAGE-A11 are tumor-specific antigens. Both DNA hypermethylation and histone deacetylation are possible mechanisms underlying MAGE-A9 and MAGE-A11 gene silencing.
Objective: The purpose of our study was to analyze the expression of melanoma antigen genes MAGE-A9 and MAGE-A11 in benign and malignant breast cancer specimens and in breast cancer cell lines following drug treatment. Methods: Tissue specimens were obtained from tumor-free breast (n=60), benign breast cancer (n=60) and malignant breast cancer (n=60), respectively. The mRNA abundance and protein content of MAGE-A9 and MAGE-A11 in these specimens were determined by RT-PCR and immunohistochemistry respectively and were analyzed for their associations with the major demographic, physiologic and pathologic variables. To examine the influence of chemotherapeutic drugs on MAGEs in breast cancer, MCF-7 and MDA-MB-231 cells were treated with 5-Aza-2’-deoxycytidine (5-Aza-CdR), a DNA methylase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, either each alone or both together, and mRNA levels of MAGE-A9 and MAGE-A11 were assessed by RT-PCR. Results：MAGE-A9 and MAGE-A11 transcripts were detected in 45% and 66.7% of breast cancer specimens respectively and MAGE-A9 and MAGE-A11 proteins in 433% and 63.3% of cancer specimens, respectively. MAGE-A9 and MAGE-A11 expression was positively correlated with the expression of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER-2) in cancer specimens (P<0.05). Neither transcripts nor proteins of MAGE-A9 and MAGE-A11 were detected in normal control specimens. While 5-Aza-CdR alone induced the expression of MAGE-A9 and MAGE-A11 in the test cell lines, TSA alone showed no effects. However, TSA was able to enhance 5-Aza-CdR-induced MAGE-A transcription (P<0.01).Conclusion：MAGE-A9 and MAGE-A11 are tumor-specific antigens. Both DNA hypermethylation and histone deacetylation are possible mechanisms underlying MAGE-A9 and MAGE-A11 gene silencing.
2014, 21(4):437-443.
DOI: 10.3872/j.issn.1007-385X.2014.4.014
Abstract:
Objective: To assess the expression of Ras-association domain family 7 (RASSF7) gene in esophageal squamous cell carcinoma (ESCC) in association with the disease pathogenesis and progression and to evaluate the effect of DNA methylation on RASSF7 expression. Methods: Sixty-nine patients diagnosed with ESCC in Hebei Medical University-Affiliated Fourth Hospital between 2011 and 2012 were recruited. Carcinoma and its surrounding non-carcinoma tissue specimens were collected from 69 of these patients. Four esophageal cancer cell lines (TE13, T.Tn, YES-2, and Ec109) were treated with DNA methyltransferase inhibitor 5-aza-2’-detoxycytidine (5-Aza-dC). RASSF7 mRNA abundance in the 69 tissue specimens and 4 esophageal cancer cell lines after 5-Aza-dC treatment were determined by RT-PCR and methylation specific polymerase chain reaction (MSP). RASSF7 protein content in the tissue specimens were assessed by immunohistochemical staining. Results：RASSF7 mRNA was detected in TE13, T.TN, Yes-2 cells but not in Ec109 cells. Treatment with 5-Aza-dC induced RASSF7 mRNA but significantly decreased RASSF7 mRNA levels in TE13, T.Tn, and Yes-2 cells. No aberrant RASSF7 methylation was detected in any of the four esophageal cancer cell lines studied, regardless of 5-aza-dC treatment. Carcinoma tissue specimens had significantly higher abundance of RASSF7 mRNA (0.63±008 vs 0.42±0.20, P<0.01) and a significantly higher rate of positive immunohistochemical staining for RASSF7 protein 56(81.2% vs 53.6%, P<0.01) as compared with the non-carcinoma normal tissue specimens, and both RASSF7 mRNA and protein were associated with lymph node metastasis and histological grade of ESCC. Methylation was detected neither in ESCC specimens nor in the corresponding non-carcinoma tissue specimens. Conclusion：The differential expression of RASSF7 in different esophageal cancer cell lines, ESCC tumor tissue and non-carcinoma tissues is not related to methylation status of RASSF7. Increased expression of RASSF7 in the ESCC tissue suggests a role for RASSF7 in the pathogenesis and metastasis of ESCC.
Objective: To assess the expression of Ras-association domain family 7 (RASSF7) gene in esophageal squamous cell carcinoma (ESCC) in association with the disease pathogenesis and progression and to evaluate the effect of DNA methylation on RASSF7 expression. Methods: Sixty-nine patients diagnosed with ESCC in Hebei Medical University-Affiliated Fourth Hospital between 2011 and 2012 were recruited. Carcinoma and its surrounding non-carcinoma tissue specimens were collected from 69 of these patients. Four esophageal cancer cell lines (TE13, T.Tn, YES-2, and Ec109) were treated with DNA methyltransferase inhibitor 5-aza-2’-detoxycytidine (5-Aza-dC). RASSF7 mRNA abundance in the 69 tissue specimens and 4 esophageal cancer cell lines after 5-Aza-dC treatment were determined by RT-PCR and methylation specific polymerase chain reaction (MSP). RASSF7 protein content in the tissue specimens were assessed by immunohistochemical staining. Results：RASSF7 mRNA was detected in TE13, T.TN, Yes-2 cells but not in Ec109 cells. Treatment with 5-Aza-dC induced RASSF7 mRNA but significantly decreased RASSF7 mRNA levels in TE13, T.Tn, and Yes-2 cells. No aberrant RASSF7 methylation was detected in any of the four esophageal cancer cell lines studied, regardless of 5-aza-dC treatment. Carcinoma tissue specimens had significantly higher abundance of RASSF7 mRNA (0.63±008 vs 0.42±0.20, P<0.01) and a significantly higher rate of positive immunohistochemical staining for RASSF7 protein 56(81.2% vs 53.6%, P<0.01) as compared with the non-carcinoma normal tissue specimens, and both RASSF7 mRNA and protein were associated with lymph node metastasis and histological grade of ESCC. Methylation was detected neither in ESCC specimens nor in the corresponding non-carcinoma tissue specimens. Conclusion：The differential expression of RASSF7 in different esophageal cancer cell lines, ESCC tumor tissue and non-carcinoma tissues is not related to methylation status of RASSF7. Increased expression of RASSF7 in the ESCC tissue suggests a role for RASSF7 in the pathogenesis and metastasis of ESCC.
2014, 21(4):444-449.
DOI: 10.3872/j.issn.1007-385X.2014.4.015
Abstract:
Objective:To investigate the expression of voltage-gated sodium channels (VGSCs) alpha subunit, neonatal Nav1.5 (nNav1.5), in association with human glioma cell migration and invasion. Methods: Biopsy tumor specimens were collected from 68 patients who were diagnosed with glioma in China Medical University Hospital. Human glioma U251 cells were transfected transiently with an expression vector encoding nNav1.5. The mRNA abundance and protein content of nNav1.5 in both biopsy specimens and transfected U251 cells were assessed by real-time PCR and Western blotting respectively. The metastasis and invasion capacities of transfected U251 cells were assessed by wound healing assay and transwell invasion assay, respectively. Results: Neonatal Nav1.5 was positive in 72.6% of human glioma tissue specimens but only in 23.0% of normal tissue specimens (P<0.01). The expression of nNav1.5 was positively associated with the severity of glioma; 85.8% of WHO grade Ⅲ-Ⅳ gliomas were nNav1.5-positive and 52.9% of WHO grade Ⅰ-Ⅱ gliomas were nNav1.5-positive (P<0.01). Neonatal Nav1.5 silencing resulted in robust decreases in nNav1.5 mRNA and protein in U251 cells and significant inhibition of U251migration and invasion. Conclusion: Neonatal Nav1.5 is highly expressed in human glioma where it promotes glioma cell invasion and migration. Our findings suggest that nNav1.5 may serve as a novel diagnostic marker and/or therapeutic target for human glioma.
Objective:To investigate the expression of voltage-gated sodium channels (VGSCs) alpha subunit, neonatal Nav1.5 (nNav1.5), in association with human glioma cell migration and invasion. Methods: Biopsy tumor specimens were collected from 68 patients who were diagnosed with glioma in China Medical University Hospital. Human glioma U251 cells were transfected transiently with an expression vector encoding nNav1.5. The mRNA abundance and protein content of nNav1.5 in both biopsy specimens and transfected U251 cells were assessed by real-time PCR and Western blotting respectively. The metastasis and invasion capacities of transfected U251 cells were assessed by wound healing assay and transwell invasion assay, respectively. Results: Neonatal Nav1.5 was positive in 72.6% of human glioma tissue specimens but only in 23.0% of normal tissue specimens (P<0.01). The expression of nNav1.5 was positively associated with the severity of glioma; 85.8% of WHO grade Ⅲ-Ⅳ gliomas were nNav1.5-positive and 52.9% of WHO grade Ⅰ-Ⅱ gliomas were nNav1.5-positive (P<0.01). Neonatal Nav1.5 silencing resulted in robust decreases in nNav1.5 mRNA and protein in U251 cells and significant inhibition of U251migration and invasion. Conclusion: Neonatal Nav1.5 is highly expressed in human glioma where it promotes glioma cell invasion and migration. Our findings suggest that nNav1.5 may serve as a novel diagnostic marker and/or therapeutic target for human glioma.
2014, 21(4):450-454.
DOI: 10.3872/j.issn.1007-385X.2014.4.016
Abstract:
Objective: To evaluate the efficacy of recombinant human adenovirus p53 (rAd-p53) combined with chemotherapy in patients with recurrent epithelial ovarian carcinoma (REOC). Methods: Forty-nine patients with REOC were assigned into a combined-treatment group (n=25) or a mono-chemotherapy group (n=24). Patients in the combined-treatment group underwent chemotherapy plus 2 cycles of rAd-p53 (once a week for 8 weeks), while those in the mono-chemotherapy group received chemotherapy only. Patient outcomes and adverse events were compared between the two groups. Results：The combined-treatment resulted in a significant decrease in CA-125 levels after 3 cycles chemotherapy compared to the monotherapy (P=0.02). There was no significantly difference in the disease control rate (92.0% vs 87.5%, P=0.59), overall survival (39.6 versus 32.5 months, P=0.13) between the two treatment regimens. No serious adverse events were observed in either of the two groups. Conclusion：Patients with REOC may benefit from chemotherapy combined with administration of rAd-p53, without concerns over adverse effects. This benefit needs to be further evaluated in more prospective trials.
Objective: To evaluate the efficacy of recombinant human adenovirus p53 (rAd-p53) combined with chemotherapy in patients with recurrent epithelial ovarian carcinoma (REOC). Methods: Forty-nine patients with REOC were assigned into a combined-treatment group (n=25) or a mono-chemotherapy group (n=24). Patients in the combined-treatment group underwent chemotherapy plus 2 cycles of rAd-p53 (once a week for 8 weeks), while those in the mono-chemotherapy group received chemotherapy only. Patient outcomes and adverse events were compared between the two groups. Results：The combined-treatment resulted in a significant decrease in CA-125 levels after 3 cycles chemotherapy compared to the monotherapy (P=0.02). There was no significantly difference in the disease control rate (92.0% vs 87.5%, P=0.59), overall survival (39.6 versus 32.5 months, P=0.13) between the two treatment regimens. No serious adverse events were observed in either of the two groups. Conclusion：Patients with REOC may benefit from chemotherapy combined with administration of rAd-p53, without concerns over adverse effects. This benefit needs to be further evaluated in more prospective trials.
2014, 21(4):455-458.
DOI: 10.3872/j.issn.1007-385X.2014.4.017
Abstract:
Objective:To determine the expression of macrophage migration inhibitory factor (MIF) and vascular endothelial growth factor (VEGF) in gastric carcinoma in association with clinicopathological features of the carcinoma lesions. Methods: Eighty-nine tumor tissue specimens were collected between 2008 and 2013 and four pairs of tumor tissue and corresponding adjacent tissue specimens were collected in 2013 from patients who were diagnosed with gastric carcinoma in Henan University-Affiliated Hospital. Contents of MIF and VEGF proteins in gastric carcinoma and adjacent tissue specimens were assessed by immunohistochemical staining and Western blotting. The correlation of MIF and VEGF levels with clinicopathological features of gastric carcinoma was analyzed by statistical analysis. Results: MIF and VEGF were detected in 88.8% and 50.6% of gastric carcinoma respectively, but were undetectable in normal tissue specimens. In the four tissue pairs, gastric carcinoma tissue had significantly higher levels of MIF (0.87±0.29 vs 0.23±0.14, P<0.05) and VEGF (0.89±0.23 vs 0.34±0.21, P<0.05) compared with the corresponding adjacent tissue. The expression of MIF and VEGF was not associated with age and sex but was positively correlated with the histological grade and TNM stage of gastric carcinoma (P<0.05). Conclusion: MIF and VEGF are highly expressed in gastric cancer and the expression level is correlated with the cancer staging.
Objective:To determine the expression of macrophage migration inhibitory factor (MIF) and vascular endothelial growth factor (VEGF) in gastric carcinoma in association with clinicopathological features of the carcinoma lesions. Methods: Eighty-nine tumor tissue specimens were collected between 2008 and 2013 and four pairs of tumor tissue and corresponding adjacent tissue specimens were collected in 2013 from patients who were diagnosed with gastric carcinoma in Henan University-Affiliated Hospital. Contents of MIF and VEGF proteins in gastric carcinoma and adjacent tissue specimens were assessed by immunohistochemical staining and Western blotting. The correlation of MIF and VEGF levels with clinicopathological features of gastric carcinoma was analyzed by statistical analysis. Results: MIF and VEGF were detected in 88.8% and 50.6% of gastric carcinoma respectively, but were undetectable in normal tissue specimens. In the four tissue pairs, gastric carcinoma tissue had significantly higher levels of MIF (0.87±0.29 vs 0.23±0.14, P<0.05) and VEGF (0.89±0.23 vs 0.34±0.21, P<0.05) compared with the corresponding adjacent tissue. The expression of MIF and VEGF was not associated with age and sex but was positively correlated with the histological grade and TNM stage of gastric carcinoma (P<0.05). Conclusion: MIF and VEGF are highly expressed in gastric cancer and the expression level is correlated with the cancer staging.
2014, 21(4):459-463.
DOI: 10.3872/j.issn.1007-385X.2014.4.018
Abstract:
Objective: To optimize the construction of eukaryotic expression vectors encoding p70/p85 ribosomal protein S6 kinase 1 (S6K1)and to evaluate the function of the constructed vectors in human breast cancer MCF-7 cells. Methods: Fragments of p70 S6K1 and p85 S6K1 cDNAs with restriction endonucleases sites were amplified by PCR with pRK7-HA-S6K1 as a template and cloned into an eukaryotic expression vector with a flag tag, pcDNA3.1 (-)-flag. MCF-7 cells were transfected with the constructed vectors, pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1. At 24 h after transfection, protein contents of p70 S6K1 and p85 S6K1 were assessed by Western blotting using anti-flag and anti-p70/85 S6K1 antibodies and cell death following induction with 1 mmol/L hydrogen peroxide for another 36 h was analyzed by microscopy. Results: Eukaryotic expression vectors pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 were successfully constructed; the full-length open reading frames were confirmed by DNA sequencing. Overexpression of S6K1 and S6K1 was detected in MCF-7 cells transfected with pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 respectively. Overexpression of p85 S6K1 but not p70 S6K1 enhanced MCF-7 cell death induced by 1 mmol/L hydrogen peroxide. Conclusion: Expression vectors pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 were constructed successfully. Overexpression of p85 S6K1 may enhance H2O2-induced breast cancer cell death.
Objective: To optimize the construction of eukaryotic expression vectors encoding p70/p85 ribosomal protein S6 kinase 1 (S6K1)and to evaluate the function of the constructed vectors in human breast cancer MCF-7 cells. Methods: Fragments of p70 S6K1 and p85 S6K1 cDNAs with restriction endonucleases sites were amplified by PCR with pRK7-HA-S6K1 as a template and cloned into an eukaryotic expression vector with a flag tag, pcDNA3.1 (-)-flag. MCF-7 cells were transfected with the constructed vectors, pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1. At 24 h after transfection, protein contents of p70 S6K1 and p85 S6K1 were assessed by Western blotting using anti-flag and anti-p70/85 S6K1 antibodies and cell death following induction with 1 mmol/L hydrogen peroxide for another 36 h was analyzed by microscopy. Results: Eukaryotic expression vectors pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 were successfully constructed; the full-length open reading frames were confirmed by DNA sequencing. Overexpression of S6K1 and S6K1 was detected in MCF-7 cells transfected with pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 respectively. Overexpression of p85 S6K1 but not p70 S6K1 enhanced MCF-7 cell death induced by 1 mmol/L hydrogen peroxide. Conclusion: Expression vectors pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 were constructed successfully. Overexpression of p85 S6K1 may enhance H2O2-induced breast cancer cell death.
2014, 21(4):464-467.
DOI: 10.3872/j.issn.1007-385X.2014.4.019
Abstract:
胸腺瘤是来源于胸腺上皮组织的肿瘤，长期以来主要治愈手段为手术治疗，放化疗多用于无法手术切除的患者。随着近年来肿瘤分子生物学研究的深入，肿瘤靶向治疗日益深入人心。近年来多家研究机构对胸腺肿瘤的分子靶点如EGFR、KIT和IGF2IR等进行了基因表达和突变等方面的研究，临床研究结果显示EGFR抑制剂和KIT抑制剂等对胸腺瘤有一定的抑制活性。但总的来说，由于该病发病率相对较低，胸腺瘤的分子靶向治疗还处于起步阶段。然而，肿瘤治疗已经进入分子靶向治疗的新时代，胸腺瘤的个体化靶向治疗也必将发挥越来越重要的作用。
胸腺瘤是来源于胸腺上皮组织的肿瘤，长期以来主要治愈手段为手术治疗，放化疗多用于无法手术切除的患者。随着近年来肿瘤分子生物学研究的深入，肿瘤靶向治疗日益深入人心。近年来多家研究机构对胸腺肿瘤的分子靶点如EGFR、KIT和IGF2IR等进行了基因表达和突变等方面的研究，临床研究结果显示EGFR抑制剂和KIT抑制剂等对胸腺瘤有一定的抑制活性。但总的来说，由于该病发病率相对较低，胸腺瘤的分子靶向治疗还处于起步阶段。然而，肿瘤治疗已经进入分子靶向治疗的新时代，胸腺瘤的个体化靶向治疗也必将发挥越来越重要的作用。
2014, 21(4):468-472.
DOI: 10.3872/j.issn.1007-385X.2014.4.020
Abstract:
近年来，基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionization time of flight mass spectrometry，MALDI-TOF-MS)作为一种新的质谱技术成功检测出许多与疾病相关的新型生物标志物，尤其在肿瘤的诊治方面，MALDI-TOF-MS可以快速检测出癌症患者体内特异的蛋白表达、辅助制定患者特异性的疗法、预测患者的治疗效果以及是否有复发的可能等。近年来，国内外学者开展了多项MALDI-TOF-MS在宫颈癌、子宫内膜癌、卵巢癌三大女性生殖道恶性肿瘤中的早期筛查诊断研究，其应用于宫颈癌的蛋白组学诊断已被证实具有实验可行性；其可以检测固态、液态等多种形态样本的特点，为寻求无创早期诊断的子宫内膜癌研究者提供了新的研究方法；其在卵巢癌差异蛋白检测、化疗耐药相关分子检测中发挥了重要作用。但仍存在如何提高MALDI-TOF-MS检测的灵敏度以及满足蛋白质组学研究高通量的需要等问题。
近年来，基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionization time of flight mass spectrometry，MALDI-TOF-MS)作为一种新的质谱技术成功检测出许多与疾病相关的新型生物标志物，尤其在肿瘤的诊治方面，MALDI-TOF-MS可以快速检测出癌症患者体内特异的蛋白表达、辅助制定患者特异性的疗法、预测患者的治疗效果以及是否有复发的可能等。近年来，国内外学者开展了多项MALDI-TOF-MS在宫颈癌、子宫内膜癌、卵巢癌三大女性生殖道恶性肿瘤中的早期筛查诊断研究，其应用于宫颈癌的蛋白组学诊断已被证实具有实验可行性；其可以检测固态、液态等多种形态样本的特点，为寻求无创早期诊断的子宫内膜癌研究者提供了新的研究方法；其在卵巢癌差异蛋白检测、化疗耐药相关分子检测中发挥了重要作用。但仍存在如何提高MALDI-TOF-MS检测的灵敏度以及满足蛋白质组学研究高通量的需要等问题。
2014, 21(4):473-476.
DOI: 10.3872/j.issn.1007-385X.2014.4.021
Abstract:
细胞因子诱导的杀伤（cytokine induced killer，CIK）细胞免疫治疗作为一种毒副作用较轻、前景良好的过继性细胞免疫治疗方法，其联合化疗对胃癌的治疗已进行了一定的研究。虽然，目前所报道的临床研究已经证实CIK细胞治疗的安全性，但尚不足以充分证明CIK细胞联合化疗治疗胃癌的有效性，因为这些试验设计方案均存在一些缺陷，如为非随机对照的临床研究、样本量较小、未明确胃癌类型、未标准化的CIK细胞制备方法和治疗方案、无用于预测CIK细胞治疗效果的特异性生物标志物等。因此，迫切需要解决这些问题方能促进CIK细胞用于胃癌临床研究的发展。
细胞因子诱导的杀伤（cytokine induced killer，CIK）细胞免疫治疗作为一种毒副作用较轻、前景良好的过继性细胞免疫治疗方法，其联合化疗对胃癌的治疗已进行了一定的研究。虽然，目前所报道的临床研究已经证实CIK细胞治疗的安全性，但尚不足以充分证明CIK细胞联合化疗治疗胃癌的有效性，因为这些试验设计方案均存在一些缺陷，如为非随机对照的临床研究、样本量较小、未明确胃癌类型、未标准化的CIK细胞制备方法和治疗方案、无用于预测CIK细胞治疗效果的特异性生物标志物等。因此，迫切需要解决这些问题方能促进CIK细胞用于胃癌临床研究的发展。
2014, 21(4):477-481.
DOI: 10.3872/j.issn.1007-385X.2014.4.022
Abstract:
目的：Exosomes是活细胞分泌的具有脂质双分子层结构、直径在40~100 nm之间的纳米级囊泡。肿瘤细胞来源的Exosomes含有mRNA、microRNA（miRNA)和蛋白质，其通过与受体（靶）细胞互动对话（cross talk），将其携带的mRNA、miRNA和蛋白质传送至受体细胞，促进细胞间信息的交流和传递，进而调控受体细胞的生物学行为。肿瘤细胞来源的Exosomes不仅对肿瘤免疫、肿瘤的侵袭与转移及肿瘤耐药具有重要的调节作用，在肿瘤的诊断与治疗方面也具有重要价值。本文就近来肿瘤细胞来源的Exosomes研究进展进行阐述。
目的：Exosomes是活细胞分泌的具有脂质双分子层结构、直径在40~100 nm之间的纳米级囊泡。肿瘤细胞来源的Exosomes含有mRNA、microRNA（miRNA)和蛋白质，其通过与受体（靶）细胞互动对话（cross talk），将其携带的mRNA、miRNA和蛋白质传送至受体细胞，促进细胞间信息的交流和传递，进而调控受体细胞的生物学行为。肿瘤细胞来源的Exosomes不仅对肿瘤免疫、肿瘤的侵袭与转移及肿瘤耐药具有重要的调节作用，在肿瘤的诊断与治疗方面也具有重要价值。本文就近来肿瘤细胞来源的Exosomes研究进展进行阐述。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.