AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasi...AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.展开更多
In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells,a new murine ascites hepatocarcinoma cell line wi...In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells,a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(Hca-P/L_(6))was established and the effect of curcumin on biological behavior of Hca-P/L_(6) was observed.Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential(Hca-P)was subcutaneously inoculated into the medioventral line of a mouse 615 and thefirst generation of metastatic tumor cells of inguinal lymph node(Hca-P/L_(1))was obtained.Then,Hca-P/L_(1) was screened by the route of mouse foot pad subcutaneously!lymph node!scale-up culture in vitro!mouse foot pad subcuta-neously forfive times consecutively.The sensitivity of two murine ascites hepatocarcinoma cell lines(Hca-P and Hca-P/L_(6))and two anchorage-dependent human hepato-carcinoma cell lines(SMC7721 and HepG_(2))to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay after these cells had been pretreated by curcumin at the concentration of 15–240μmol/L for 48 h.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15μmol/L in vitro,the effect of curcumin against cell proliferation of Hca-P and Hca-P/L6 was observed by inverted micro-scope,cell growth curve and cell population doubling time;the effects of curcumin on cell cycles of Hca-P/L6 and Hca-P were studied byflow cytometry(FCM).The results showed Hca-P/L_(6) spreading to the lymph nodes at multiple sites in mice was screened from Hca-P.The lymph node metastatic rate was 100%.Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner(P<0.05).At concentrations of 30–120μmol/L,curcu-min had more inhibition on murine ascites hepatocarci-noma cell lines than on human anchorage-dependent hepatocarcinoma cell lines.At concentrations of 60–240μmol/L,curcumin had more inhibition on Hca-P/L_(6) with the 50%inhibitory concentration(IC50)of 51.48μmol/L than on Hca-P with IC50 of 90.87μmol/L.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days,the proliferations of Hca-P/L_(6) and Hca-P were inhibited(P<0.05)in a time-dependent manner(P<0.01)and the population doubling time of Hca-P/L6 and Hca-P was prolonged(P<0.01),and curcumin had more inhibition on Hca-P/L6 than on Hca-P(P<0.05).After pretreatment by 15μmol/L curcumin for 48 h,the morphous of Hca-P/L_(6) was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L6 being blocked in the S phase and Hca-P in the S and G_(2)/M phases.Hca-P/L_(6) was validated to be more sensitive to curcumin than Hca-P.Hca-P/L_(6) is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.展开更多
There was no detectable expression of c-fos,but a little c-myc,high c-fms and mederate high IGF-ⅡmRNA in the untreated human hepatocarcinoma cell SMMC- 7721.After treatment with 10 μmol/L retinoic acid or 0.5 mmol/L...There was no detectable expression of c-fos,but a little c-myc,high c-fms and mederate high IGF-ⅡmRNA in the untreated human hepatocarcinoma cell SMMC- 7721.After treatment with 10 μmol/L retinoic acid or 0.5 mmol/L dibutyryl cyclic-3',5'adenosine monophosphate(db-cAMP),the c-fos was transiently expressed within 20- 60mins.If the treatment of RA or db-cAMP prolonged to 1-5 days, the transcriptions of c- myc were increased,reaching the highest level on the 2nd and 4th day.Simultaneously the transcriptions of c- fms and IGF- Ⅱwere gradually decreased.On the 5th day of the treatment,c-fms and IGF-ⅡmRNA were decreased to 32% and 14%respectively of the control (untreated cell) value by RA,and 35% and 22%respectively by db-cAMP.The biological significance of the above mentioned results was discussed.展开更多
Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect o...Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30370403the Key Project of Chinese Ministry of Education, No. 705046the Doctoral Foundation of Xi’an Jiaotong University, grants No. DFXJTU2005-05
文摘AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.
基金The study was supported by the National Natural Science Foundation of China(Grant No.90209048)and the Knowledge Innovation Program of the Chinese Academy of Sciences(Nos.KSCX2-SW-329 and KGCX2-SW-213).
文摘In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells,a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(Hca-P/L_(6))was established and the effect of curcumin on biological behavior of Hca-P/L_(6) was observed.Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential(Hca-P)was subcutaneously inoculated into the medioventral line of a mouse 615 and thefirst generation of metastatic tumor cells of inguinal lymph node(Hca-P/L_(1))was obtained.Then,Hca-P/L_(1) was screened by the route of mouse foot pad subcutaneously!lymph node!scale-up culture in vitro!mouse foot pad subcuta-neously forfive times consecutively.The sensitivity of two murine ascites hepatocarcinoma cell lines(Hca-P and Hca-P/L_(6))and two anchorage-dependent human hepato-carcinoma cell lines(SMC7721 and HepG_(2))to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay after these cells had been pretreated by curcumin at the concentration of 15–240μmol/L for 48 h.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15μmol/L in vitro,the effect of curcumin against cell proliferation of Hca-P and Hca-P/L6 was observed by inverted micro-scope,cell growth curve and cell population doubling time;the effects of curcumin on cell cycles of Hca-P/L6 and Hca-P were studied byflow cytometry(FCM).The results showed Hca-P/L_(6) spreading to the lymph nodes at multiple sites in mice was screened from Hca-P.The lymph node metastatic rate was 100%.Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner(P<0.05).At concentrations of 30–120μmol/L,curcu-min had more inhibition on murine ascites hepatocarci-noma cell lines than on human anchorage-dependent hepatocarcinoma cell lines.At concentrations of 60–240μmol/L,curcumin had more inhibition on Hca-P/L_(6) with the 50%inhibitory concentration(IC50)of 51.48μmol/L than on Hca-P with IC50 of 90.87μmol/L.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days,the proliferations of Hca-P/L_(6) and Hca-P were inhibited(P<0.05)in a time-dependent manner(P<0.01)and the population doubling time of Hca-P/L6 and Hca-P was prolonged(P<0.01),and curcumin had more inhibition on Hca-P/L6 than on Hca-P(P<0.05).After pretreatment by 15μmol/L curcumin for 48 h,the morphous of Hca-P/L_(6) was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L6 being blocked in the S phase and Hca-P in the S and G_(2)/M phases.Hca-P/L_(6) was validated to be more sensitive to curcumin than Hca-P.Hca-P/L_(6) is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.
文摘There was no detectable expression of c-fos,but a little c-myc,high c-fms and mederate high IGF-ⅡmRNA in the untreated human hepatocarcinoma cell SMMC- 7721.After treatment with 10 μmol/L retinoic acid or 0.5 mmol/L dibutyryl cyclic-3',5'adenosine monophosphate(db-cAMP),the c-fos was transiently expressed within 20- 60mins.If the treatment of RA or db-cAMP prolonged to 1-5 days, the transcriptions of c- myc were increased,reaching the highest level on the 2nd and 4th day.Simultaneously the transcriptions of c- fms and IGF- Ⅱwere gradually decreased.On the 5th day of the treatment,c-fms and IGF-ⅡmRNA were decreased to 32% and 14%respectively of the control (untreated cell) value by RA,and 35% and 22%respectively by db-cAMP.The biological significance of the above mentioned results was discussed.
文摘Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.
