目的研究PI3k/Akt信号通路在肝癌细胞对索拉菲尼耐药中作用机制,评估PI3k抑制剂LY294002逆转肝癌细胞对索拉菲尼耐药作用。方法 Western blot检测不同措施处理后索拉菲尼耐药肝癌细胞PI3k/Akt信号通路蛋白、EMT标记蛋白及凋亡相关蛋白Ba...目的研究PI3k/Akt信号通路在肝癌细胞对索拉菲尼耐药中作用机制,评估PI3k抑制剂LY294002逆转肝癌细胞对索拉菲尼耐药作用。方法 Western blot检测不同措施处理后索拉菲尼耐药肝癌细胞PI3k/Akt信号通路蛋白、EMT标记蛋白及凋亡相关蛋白Bax表达。CCK-8实验及Transwell实验检测低浓度索拉菲尼联合LY294002作用后Huh7R细胞增殖活力及迁移、侵袭能力。结果与亲本Huh7细胞相比,Huh7R的PI3k和p-Akt表达量升高,PTEN表达量下降,同时上皮标记蛋白E-cadherin表达下降,间质标记蛋白N-cadherin、Vimenti、Snail、Slug表达增加。LY294002作用后,EMT标志相关蛋白Snail、Slug、Vimenti、N-cadherin的表达量呈时间依赖性和浓度依赖性下降。低浓度索拉菲尼联合LY294002使Huh7R细胞增殖活力明显下降,同时抑制索拉菲尼耐药肝癌细胞迁移、侵袭能力。结论长期索拉菲尼暴露诱导肝癌细胞PI3k/Akt信号通路活化及促进肝癌细胞上皮间质变;LY294002通过抑制PI3k/Akt信号通路活化从而逆转索拉菲尼耐药肝癌上皮间质变作用;LY294002联合低浓度索拉菲尼可以协同作用逆转肝癌对索拉菲尼耐药,抑制索拉菲尼耐药肝癌细胞的迁移、侵袭能力。展开更多
The prognosis for patients who are diagnosed with advanced stage hepatocellular carcinoma(HCC)is poor because there are few treatment options.Recent research has focused on the identification of novel molecular entiti...The prognosis for patients who are diagnosed with advanced stage hepatocellular carcinoma(HCC)is poor because there are few treatment options.Recent research has focused on the identification of novel molecular entities that can be targeted to inhibit oncogenic signals that are involved in the carcinogenesis,proliferation and progression of HCC.Among all of the pathways that are involved in the development of HCC,Hedgehog(HH)signalling has demonstrated a substantial role in hepatocarcinogenesis and HCC progression.HH plays a physiological role in embryogenesis,through the induction of the differentiation of hepatocytes from endodermal progenitors.The re-activation of the HH pathway in chronic damaged liver is a mechanism of fibrotic degeneration and is implicated in various stages of HCC development.HH activation sustains the subpopulation of immature liver epithelial cells that are involved in the pathogenesis of cirrhosis and HCC,and HH itself is a mediator of the alcohol-derived malignant transformation of liver cells.High levels of expression of HH protein markers in liver tumour tissues are correlated with aggressive histological and biological features and a poor clinical outcome.In vitro and in vivo inhibition models of the HH pathway confirm that HH is essential in maintaining tumour growth,metastasis and a mesenchymal phenotype.展开更多
AIM:To investigate the effect of hyperthermia on hy-poxia-induced epithelial-mesenchymal transition (EMT) in HepG2 hepatocellular carcinoma (HCC) cells, and its mechanism. METHODS:Cells were treated with hyperthermia ...AIM:To investigate the effect of hyperthermia on hy-poxia-induced epithelial-mesenchymal transition (EMT) in HepG2 hepatocellular carcinoma (HCC) cells, and its mechanism. METHODS:Cells were treated with hyperthermia at 43 ℃ for 0.5 h, followed by incubation under hypoxic or normoxic conditions for 72 h. Cell morphology was observed. Expressions of E-cadherin and vimentin were determined by immunofluorescence assay or Western blot. The protein and mRNA expressions of Snail were also determined by Western blot and reverse transcrip-tion-polymerase chain reaction. Cell migratory capacity was evaluated. RESULTS:Hypoxia induced EMT in HepG2 cells, which was evidenced by morphological, molecular and func-tional changes, including the formation of a spindle shape and the loss of cell contact. The expression of E-cadherin was decreased but the expression of vimentin was increased; also, the migratory capability was increased by 2.2 ± 0.20-fold as compared with normoxia. However, those effects were inhibited by hyperthermia pretreatment. Furthermore, protein synthesis and mRNA expression of Snail in the cells were enhanced by hy-poxia as compared with normoxia, and also significantly inhibited by hyperthermia pretreatment. CONCLUSION:Hyperthermia may inhibit hypoxia-induced EMT in HepG2 HCC cells, and the mechanism may involve inhibition of induced expression of Snail.展开更多
To elucidate the effect of expression of doublecortin and CaM kinase-like-1 (DCLK1) in patients with pancreatic ductal adenocarcinoma (PDAC). METHODSTumor specimens were obtained from 136 patients with pancreatic canc...To elucidate the effect of expression of doublecortin and CaM kinase-like-1 (DCLK1) in patients with pancreatic ductal adenocarcinoma (PDAC). METHODSTumor specimens were obtained from 136 patients with pancreatic cancer who had undergone resection without preoperative therapy between January 2000 and December 2013 at the Department of Surgical Oncology, Osaka City University. The resected specimens were analyzed for associations with clinicopathological data, including DCLK1 expression, epithelial mesenchymal transition (EMT) marker expression, and cancer stem cell (CSC) marker expression. Univariate and multivariate survival analyses were performed and we assessed the association between DCLK1 expression and clinicopathological factors, including the EMT marker and CSC marker. RESULTSIn total, 48.5% (66/136) of the pancreatic cancer samples were positive for DCLK1. Patients with DCLK1-positive tumors had significantly shorter survival times than those with DCLK1-negative tumors (median, 18.7 mo vs 49.5 mo, respectively; P < 0.0001). Positive DCLK1 expression correlated with histological grade (P = 0.0290), preoperative CA19-9 level (P = 0.0060), epithelial cell adhesion molecule (EpCAM) expression (P = 0.0235), and the triple-positive expression of CD44/CD24/EpCAM (P = 0.0139). On univariate survival analysis, five factors were significantly associated with worse overall survival: histological grade of G2 to G4 (P = 0.0091), high preoperative serum SPan-1 level (P = 0.0034), R1/2 (P < 0.0001), positive expression of DCLK1 (P < 0.0001) or CD44 (P = 0.0245). On multivariate survival analysis, R1/2 [odds ratio (OR) = 2.019, 95% confidence interval (CI): 1.380-2.933; P = 0.0004] and positive DCLK1 expression (OR = 1.848, 95%CI: 1.2854-2.661; P = 0.0009) were independent prognostic factors. CONCLUSIONDCLK1 expression was found to be an independent prognostic factor and it may play a crucial prognostic role by promoting acquisition of stemness.展开更多
AIM To explore the functional role of cullin 4A(CUL4A), a core subunit of E3 ubiquitin ligase, in perihilar cholangiocarcinoma(PHCC).METHODS The expression of CUL4 A in PHCC cell lines was evaluated by Western blot an...AIM To explore the functional role of cullin 4A(CUL4A), a core subunit of E3 ubiquitin ligase, in perihilar cholangiocarcinoma(PHCC).METHODS The expression of CUL4 A in PHCC cell lines was evaluated by Western blot and quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry(IHC) was adopted to investigate the relationship between CUL4 A expression and clinicopathological characteristics of PHCC. Univariate analysis and multivariate regression analysis were performed to analyze the risk factors related to overall survival(OS) and progression-free survival(PFS) of PHCC patients. Wound healing, Transwell and Matrigel assays were utilized to explore the function of CUL4 A in PHCC metastasis. Furthermore, expression of epithelial to mesenchymal transition(EMT) markers was verified in cells with CUL4 A knockdown or overexpression. The relationship between CUL4 A expression and E-cadherin expression was also analyzed by IHC assay. Finally, the role of ZEB1 in regulating CUL4 A mediated PHCC was detected by IHC, Western blot, Transwell and Matrigel assays.RESULTS CUL4 A overexpression was detected in PHCC cell lines and clinical specimens. Clinicopathological analysis revealed a close correlation between CUL4 A overexpression and tumour differentiation, T, N and TNM stages in PHCC. Kaplan-Meier analysis revealed that high CUL4 A expression was correlated with poor OS and PFS of PHCC patients. Univariate analysis identified the following four parameters as risk factors related to OS rate of PHCC: T, N, TNM stages and high CUL4 A expression; as well as three related to PFS: N stage, TNM stage and high CUL4 A expression. Further multivariate logistic regression analysis identified high CUL4 A expression as the only independent prognostic factor for PHCC. Moreover, CUL4 A silencing in PHCC cell lines dramatically inhibited metastasis and the EMT. Conversely, CUL4 A overexpression promoted these processes. Mechanistically, ZEB1 was discovered to regulate the function of CUL4 A in promoting the EMT and metastasis.CONCLUSION CUL4 A is an independent prognostic factor for PHCC, and it can promote the EMT by regulating ZEB1 expression. CUL4 A may be a potential therapeutic target for PHCC.展开更多
文摘目的研究PI3k/Akt信号通路在肝癌细胞对索拉菲尼耐药中作用机制,评估PI3k抑制剂LY294002逆转肝癌细胞对索拉菲尼耐药作用。方法 Western blot检测不同措施处理后索拉菲尼耐药肝癌细胞PI3k/Akt信号通路蛋白、EMT标记蛋白及凋亡相关蛋白Bax表达。CCK-8实验及Transwell实验检测低浓度索拉菲尼联合LY294002作用后Huh7R细胞增殖活力及迁移、侵袭能力。结果与亲本Huh7细胞相比,Huh7R的PI3k和p-Akt表达量升高,PTEN表达量下降,同时上皮标记蛋白E-cadherin表达下降,间质标记蛋白N-cadherin、Vimenti、Snail、Slug表达增加。LY294002作用后,EMT标志相关蛋白Snail、Slug、Vimenti、N-cadherin的表达量呈时间依赖性和浓度依赖性下降。低浓度索拉菲尼联合LY294002使Huh7R细胞增殖活力明显下降,同时抑制索拉菲尼耐药肝癌细胞迁移、侵袭能力。结论长期索拉菲尼暴露诱导肝癌细胞PI3k/Akt信号通路活化及促进肝癌细胞上皮间质变;LY294002通过抑制PI3k/Akt信号通路活化从而逆转索拉菲尼耐药肝癌上皮间质变作用;LY294002联合低浓度索拉菲尼可以协同作用逆转肝癌对索拉菲尼耐药,抑制索拉菲尼耐药肝癌细胞的迁移、侵袭能力。
文摘The prognosis for patients who are diagnosed with advanced stage hepatocellular carcinoma(HCC)is poor because there are few treatment options.Recent research has focused on the identification of novel molecular entities that can be targeted to inhibit oncogenic signals that are involved in the carcinogenesis,proliferation and progression of HCC.Among all of the pathways that are involved in the development of HCC,Hedgehog(HH)signalling has demonstrated a substantial role in hepatocarcinogenesis and HCC progression.HH plays a physiological role in embryogenesis,through the induction of the differentiation of hepatocytes from endodermal progenitors.The re-activation of the HH pathway in chronic damaged liver is a mechanism of fibrotic degeneration and is implicated in various stages of HCC development.HH activation sustains the subpopulation of immature liver epithelial cells that are involved in the pathogenesis of cirrhosis and HCC,and HH itself is a mediator of the alcohol-derived malignant transformation of liver cells.High levels of expression of HH protein markers in liver tumour tissues are correlated with aggressive histological and biological features and a poor clinical outcome.In vitro and in vivo inhibition models of the HH pathway confirm that HH is essential in maintaining tumour growth,metastasis and a mesenchymal phenotype.
基金Supported by Medical Science and Technology Innovation Foundation of Nanjing Military Command of Chinese People’s Liberation Army, No. 11MA036Natural Science Foundation of China, No. 81000998
文摘AIM:To investigate the effect of hyperthermia on hy-poxia-induced epithelial-mesenchymal transition (EMT) in HepG2 hepatocellular carcinoma (HCC) cells, and its mechanism. METHODS:Cells were treated with hyperthermia at 43 ℃ for 0.5 h, followed by incubation under hypoxic or normoxic conditions for 72 h. Cell morphology was observed. Expressions of E-cadherin and vimentin were determined by immunofluorescence assay or Western blot. The protein and mRNA expressions of Snail were also determined by Western blot and reverse transcrip-tion-polymerase chain reaction. Cell migratory capacity was evaluated. RESULTS:Hypoxia induced EMT in HepG2 cells, which was evidenced by morphological, molecular and func-tional changes, including the formation of a spindle shape and the loss of cell contact. The expression of E-cadherin was decreased but the expression of vimentin was increased; also, the migratory capability was increased by 2.2 ± 0.20-fold as compared with normoxia. However, those effects were inhibited by hyperthermia pretreatment. Furthermore, protein synthesis and mRNA expression of Snail in the cells were enhanced by hy-poxia as compared with normoxia, and also significantly inhibited by hyperthermia pretreatment. CONCLUSION:Hyperthermia may inhibit hypoxia-induced EMT in HepG2 HCC cells, and the mechanism may involve inhibition of induced expression of Snail.
文摘To elucidate the effect of expression of doublecortin and CaM kinase-like-1 (DCLK1) in patients with pancreatic ductal adenocarcinoma (PDAC). METHODSTumor specimens were obtained from 136 patients with pancreatic cancer who had undergone resection without preoperative therapy between January 2000 and December 2013 at the Department of Surgical Oncology, Osaka City University. The resected specimens were analyzed for associations with clinicopathological data, including DCLK1 expression, epithelial mesenchymal transition (EMT) marker expression, and cancer stem cell (CSC) marker expression. Univariate and multivariate survival analyses were performed and we assessed the association between DCLK1 expression and clinicopathological factors, including the EMT marker and CSC marker. RESULTSIn total, 48.5% (66/136) of the pancreatic cancer samples were positive for DCLK1. Patients with DCLK1-positive tumors had significantly shorter survival times than those with DCLK1-negative tumors (median, 18.7 mo vs 49.5 mo, respectively; P < 0.0001). Positive DCLK1 expression correlated with histological grade (P = 0.0290), preoperative CA19-9 level (P = 0.0060), epithelial cell adhesion molecule (EpCAM) expression (P = 0.0235), and the triple-positive expression of CD44/CD24/EpCAM (P = 0.0139). On univariate survival analysis, five factors were significantly associated with worse overall survival: histological grade of G2 to G4 (P = 0.0091), high preoperative serum SPan-1 level (P = 0.0034), R1/2 (P < 0.0001), positive expression of DCLK1 (P < 0.0001) or CD44 (P = 0.0245). On multivariate survival analysis, R1/2 [odds ratio (OR) = 2.019, 95% confidence interval (CI): 1.380-2.933; P = 0.0004] and positive DCLK1 expression (OR = 1.848, 95%CI: 1.2854-2.661; P = 0.0009) were independent prognostic factors. CONCLUSIONDCLK1 expression was found to be an independent prognostic factor and it may play a crucial prognostic role by promoting acquisition of stemness.
文摘AIM To explore the functional role of cullin 4A(CUL4A), a core subunit of E3 ubiquitin ligase, in perihilar cholangiocarcinoma(PHCC).METHODS The expression of CUL4 A in PHCC cell lines was evaluated by Western blot and quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry(IHC) was adopted to investigate the relationship between CUL4 A expression and clinicopathological characteristics of PHCC. Univariate analysis and multivariate regression analysis were performed to analyze the risk factors related to overall survival(OS) and progression-free survival(PFS) of PHCC patients. Wound healing, Transwell and Matrigel assays were utilized to explore the function of CUL4 A in PHCC metastasis. Furthermore, expression of epithelial to mesenchymal transition(EMT) markers was verified in cells with CUL4 A knockdown or overexpression. The relationship between CUL4 A expression and E-cadherin expression was also analyzed by IHC assay. Finally, the role of ZEB1 in regulating CUL4 A mediated PHCC was detected by IHC, Western blot, Transwell and Matrigel assays.RESULTS CUL4 A overexpression was detected in PHCC cell lines and clinical specimens. Clinicopathological analysis revealed a close correlation between CUL4 A overexpression and tumour differentiation, T, N and TNM stages in PHCC. Kaplan-Meier analysis revealed that high CUL4 A expression was correlated with poor OS and PFS of PHCC patients. Univariate analysis identified the following four parameters as risk factors related to OS rate of PHCC: T, N, TNM stages and high CUL4 A expression; as well as three related to PFS: N stage, TNM stage and high CUL4 A expression. Further multivariate logistic regression analysis identified high CUL4 A expression as the only independent prognostic factor for PHCC. Moreover, CUL4 A silencing in PHCC cell lines dramatically inhibited metastasis and the EMT. Conversely, CUL4 A overexpression promoted these processes. Mechanistically, ZEB1 was discovered to regulate the function of CUL4 A in promoting the EMT and metastasis.CONCLUSION CUL4 A is an independent prognostic factor for PHCC, and it can promote the EMT by regulating ZEB1 expression. CUL4 A may be a potential therapeutic target for PHCC.
