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Volume 19,Issue 4,2012 Table of Contents
2012, 19(4):343-347.
DOI: 10.3872/j.issn.1007-385X.2012.4.001
Abstract:
Cancer stem cells (CSCs) with self-renewal, proliferation and defective differentiation capacities are the seeds leading to tumor occurrence and metastasis. Due to chemo-resistance, radio-resistance and immuno-resistance, CSC is responsible for tumor relapse. It is pivotal to eliminate CSC so as to improve outcome of tumor therapy. Recognizing surface biomarkers and stemness-related essential molecules of CSC is favorable for attacking CSC efficiently and distinctively. Immunotherapy with characteristics of tumor antigen recognition and spatial and temporal dependent reactive effects lays the foundation of targeting CSC. It is practical and challenging to eliminate CSC through immunotherapy mainly relying on monoclonal antibody and sensitized-immune cells.
Cancer stem cells (CSCs) with self-renewal, proliferation and defective differentiation capacities are the seeds leading to tumor occurrence and metastasis. Due to chemo-resistance, radio-resistance and immuno-resistance, CSC is responsible for tumor relapse. It is pivotal to eliminate CSC so as to improve outcome of tumor therapy. Recognizing surface biomarkers and stemness-related essential molecules of CSC is favorable for attacking CSC efficiently and distinctively. Immunotherapy with characteristics of tumor antigen recognition and spatial and temporal dependent reactive effects lays the foundation of targeting CSC. It is practical and challenging to eliminate CSC through immunotherapy mainly relying on monoclonal antibody and sensitized-immune cells.
2012, 19(4):348-354.
DOI: 10.3872/j.issn.1007-385X.2012.4.002
Abstract:
Objective： To observe the immunological enhancement effect of interferon-α (IFN-α) on anti-leukemia activity of eosinophils granulocytes (EOSs) in patients with chronic myelogenous leukemia (CML) after hydroxycarbmide (OHU) therapy, and to investigate the related immunologic mechanism. Methods: Forty-six BCR-ABL positive CML patients (from Jan. 2010 to Feb. 2012) admitted in the 148th Hospital of PLA were included in this study, and were divided into OHU group (20 cases) and OHC combined IFN-α group (26 cases) with a balance in sex ratio and age range. Meanwhile, specimens of 30 healthy volunteers from the Regular Physical Examination Center of the hospital were collected as control. ELISA was used to determine the concentrations of cytokines IL-6, IL-12, IL-17 and IFN-γ in serum of the CML patients. Cytochemistry staining was used to observe the peroxydase (POX) expression of immunocyte in bone marrow. Immunofluorescence (IF) staining was used to observe cytokines IL-12 and IL-17A expression levels as well as the expression of mannose receptor (MR). Results: The serum concentrations of IL-6, IL-12 and IL-17 were increased significantly in CML patients compared with the healthy control group \[(116.13?15.16) vs (90.98?12.32) pg/ml; (189.26?22.14) vs (96.60?4.92) pg/ml; (34.42?2.16) vs (23.74?1.36) pg/ml, P<0.05\]. After OHU treatment, the serum concentrations of IL-6, IL-12 and IL-17 in the CML patients decreased to (87.14?13.37) , (60.22?20.16) and (17.03?2.16) pg/ml, respectively (P<0.05). Compared with the OHU treatment group, the serum IL-6, IL-12 and IL-17 concentrations in the OHU combined IFN-α treatment group were significantly up-regulated \[(122.04?1025), (101.12?27.16) and (40.16?4.11) pg/ml, P<0.05 or P<0.01\]. EOS in bone marrow of CML patients expressed IL-12, IL-17A and MR. OHU treatment combined with IFN-α could decrease immunosuppressive effect of OHU therapy, the quantity of EOS with positive IL-12, IL-17A and MR expressions was increased obviously, and the POX activity of EOS was also increased. Conclusion: EOS can secrete cytokines IL-12 and IL-17 and express MR. OHU treatment combined with IFN-α can enhance the anti-leukemia immunological effect of EOS and decrease the immunosuppressive effect of OHU therapy on EOS.
Objective： To observe the immunological enhancement effect of interferon-α (IFN-α) on anti-leukemia activity of eosinophils granulocytes (EOSs) in patients with chronic myelogenous leukemia (CML) after hydroxycarbmide (OHU) therapy, and to investigate the related immunologic mechanism. Methods: Forty-six BCR-ABL positive CML patients (from Jan. 2010 to Feb. 2012) admitted in the 148th Hospital of PLA were included in this study, and were divided into OHU group (20 cases) and OHC combined IFN-α group (26 cases) with a balance in sex ratio and age range. Meanwhile, specimens of 30 healthy volunteers from the Regular Physical Examination Center of the hospital were collected as control. ELISA was used to determine the concentrations of cytokines IL-6, IL-12, IL-17 and IFN-γ in serum of the CML patients. Cytochemistry staining was used to observe the peroxydase (POX) expression of immunocyte in bone marrow. Immunofluorescence (IF) staining was used to observe cytokines IL-12 and IL-17A expression levels as well as the expression of mannose receptor (MR). Results: The serum concentrations of IL-6, IL-12 and IL-17 were increased significantly in CML patients compared with the healthy control group \[(116.13?15.16) vs (90.98?12.32) pg/ml; (189.26?22.14) vs (96.60?4.92) pg/ml; (34.42?2.16) vs (23.74?1.36) pg/ml, P<0.05\]. After OHU treatment, the serum concentrations of IL-6, IL-12 and IL-17 in the CML patients decreased to (87.14?13.37) , (60.22?20.16) and (17.03?2.16) pg/ml, respectively (P<0.05). Compared with the OHU treatment group, the serum IL-6, IL-12 and IL-17 concentrations in the OHU combined IFN-α treatment group were significantly up-regulated \[(122.04?1025), (101.12?27.16) and (40.16?4.11) pg/ml, P<0.05 or P<0.01\]. EOS in bone marrow of CML patients expressed IL-12, IL-17A and MR. OHU treatment combined with IFN-α could decrease immunosuppressive effect of OHU therapy, the quantity of EOS with positive IL-12, IL-17A and MR expressions was increased obviously, and the POX activity of EOS was also increased. Conclusion: EOS can secrete cytokines IL-12 and IL-17 and express MR. OHU treatment combined with IFN-α can enhance the anti-leukemia immunological effect of EOS and decrease the immunosuppressive effect of OHU therapy on EOS.
2012, 19(4):355-362.
DOI: 10.3872/j.issn.1007-385X.2012.4.003
Abstract:
Objective: To investigate the effect of lentivirus-mediated integrin α6 (ITGA6) shRNA on growth and invasion of human non-small cell lung cancer (NSCLC) H460SM cells. Methods: The expression levels of ITGA6 in 9 human NSCLC cell lines were evaluated by quantitative-PCR (Q-PCR) and Western blotting. Lentiviral ITGA6 shRNA were transfected into H460SM cells and ITGA6expression was detected by Q-PCR and Western blotting; the cellular morphology change was observed under a microscope; cell proliferation was detected by MTS assay; anchorage-independent growth was determined by colony formation assay in soft agar; apoptosis and cell cycle were analyzed by flow cytometry; and cell invasion and migration were determined by invasion and migration assay. Results: ITGA6 was expressed in 7 NSCLC cell lines. A significantly higher level of ITGA6 expression was seen in H460SM cells compared with the parental H460 cells (\[8.75±0.09\] vs \[5.78±0.26\], P<0.01). After H460SM cells were stably transfected with lentiviral ITGA6 shRNA (H460SM-75, H460SM-76 cells), a significant down-regulation of ITGA6 expression was observed in H460SM-75 and H460SM-76 cells compared with negative control H460SM-NS cells (\[1.00±0.01\] vs \[0.11±0.04\], \[0.22±004\], P<0.01). The cell viability of H460SM-75 and H460SM-76 cells had no difference with H460SM-NS cells (P>0.05), as well as the apoptotic rate, cell proliferative index and proportion of G0/G1 phase cells as compared with the negative control (P>0.05). The colony formation rate of H460SM-75 and H460SM-76 cells was significantly decreased as compared with H460SM-NS cells (\[20.81±1.38\]% vs \[18.87±1.47\]%, \[18.85±1.11\]%, P<0.05), and the migration rate (\[100.00±0.50\]% vs \[43.92±0.41\]%, \[24.10±0.33\]%, P<0.01) and invasion rate (\[100.00±6.74\]% vs \[7.04±2.96\]%, \[4.68±0.27\]%, P<0.01) were also significantly decreased. Conclusion: Knockdown of ITGA6 expression can inhibit anchorage-independent cell growth, migration and invasion of NSCLC cells, but have no effect on cell proliferation and apoptosis.
Objective: To investigate the effect of lentivirus-mediated integrin α6 (ITGA6) shRNA on growth and invasion of human non-small cell lung cancer (NSCLC) H460SM cells. Methods: The expression levels of ITGA6 in 9 human NSCLC cell lines were evaluated by quantitative-PCR (Q-PCR) and Western blotting. Lentiviral ITGA6 shRNA were transfected into H460SM cells and ITGA6expression was detected by Q-PCR and Western blotting; the cellular morphology change was observed under a microscope; cell proliferation was detected by MTS assay; anchorage-independent growth was determined by colony formation assay in soft agar; apoptosis and cell cycle were analyzed by flow cytometry; and cell invasion and migration were determined by invasion and migration assay. Results: ITGA6 was expressed in 7 NSCLC cell lines. A significantly higher level of ITGA6 expression was seen in H460SM cells compared with the parental H460 cells (\[8.75±0.09\] vs \[5.78±0.26\], P<0.01). After H460SM cells were stably transfected with lentiviral ITGA6 shRNA (H460SM-75, H460SM-76 cells), a significant down-regulation of ITGA6 expression was observed in H460SM-75 and H460SM-76 cells compared with negative control H460SM-NS cells (\[1.00±0.01\] vs \[0.11±0.04\], \[0.22±004\], P<0.01). The cell viability of H460SM-75 and H460SM-76 cells had no difference with H460SM-NS cells (P>0.05), as well as the apoptotic rate, cell proliferative index and proportion of G0/G1 phase cells as compared with the negative control (P>0.05). The colony formation rate of H460SM-75 and H460SM-76 cells was significantly decreased as compared with H460SM-NS cells (\[20.81±1.38\]% vs \[18.87±1.47\]%, \[18.85±1.11\]%, P<0.05), and the migration rate (\[100.00±0.50\]% vs \[43.92±0.41\]%, \[24.10±0.33\]%, P<0.01) and invasion rate (\[100.00±6.74\]% vs \[7.04±2.96\]%, \[4.68±0.27\]%, P<0.01) were also significantly decreased. Conclusion: Knockdown of ITGA6 expression can inhibit anchorage-independent cell growth, migration and invasion of NSCLC cells, but have no effect on cell proliferation and apoptosis.
2012, 19(4):363-368.
DOI: 10.3872/j.issn.1007-385X.2012.4.004
Abstract:
Objective：To explore the inhibitory effect and mechanism of sequential or combined treatment of gefitinib (G) and paclitaxel (P) on three-dimensional non-small cell lung cancer (NSCLC) cell lines. Methods: The human NSCLC cell lines A427 and Calu-3 were cultured in the ultralow attachment plates and the three-dimensional cell clusters were formed; the inhibitory effect of paclitaxel followed by gefitinib (P-G), gefitinib followed by paclitaxel (G-P), and paclitaxel combined with gefitinib (P+G) on the monolayer and three-dimensional cell lines were detected using sulforhodamine B (SRB) and Cell Titer-Blue assay, respectively; the cell cycle was detected by flow cytometry; and the expression of total EGFR and Akt protein and their phosphorylation were detected using Western blotting. Results: In the monolayer cells, the survival rates of A427 cells in the group of P-G, G-P, and P+G were (39.5±0.07)%, (57.7±003)% and (53.7±0.05)% respectively. Those of Calu-3 cells in the group of P-G, G-P and P+G were (23.9±002)%, (58.2±0.05)% and (48.8±0.07)% respectively. The inhibitory effect of P-G was the strongest (P<005). In the three-dimensional cells, the survival rates of A427 cells in the group of P-G, G-P and P+G were (19.9±2.89)%, (43.2±8.64)% and (36.6±9.79)% respectively. Those of Calu-3 cells in the group of P-G, G-P and P+G were (10.2±0.76)%, (50.0±3.45)%, (31.4±6.15)% respectively. The inhibitory effect of P-G was the strongest (P<0.05). In addition, the inhibitory effects of P-G on A427 and Calu-3 in the three-dimensional cells were stronger than those in the monolayer cells (P<0.05). The sequence of P-G resulted in an increase of subG1 proportion and arrested monolayer cells in G1 phase. The phosphorylation of EGFR and Akt was decreased in subsequent exposure to P-G. Conclusion: The sequence of P-G seems to be superior to the reverse schedule or combination in the inhibition of proliferation of NSCLC cells, based on the factors of G1 arrest and the decrease of phosphorylation of Akt and EGFR in three-dimensional cells.
Objective：To explore the inhibitory effect and mechanism of sequential or combined treatment of gefitinib (G) and paclitaxel (P) on three-dimensional non-small cell lung cancer (NSCLC) cell lines. Methods: The human NSCLC cell lines A427 and Calu-3 were cultured in the ultralow attachment plates and the three-dimensional cell clusters were formed; the inhibitory effect of paclitaxel followed by gefitinib (P-G), gefitinib followed by paclitaxel (G-P), and paclitaxel combined with gefitinib (P+G) on the monolayer and three-dimensional cell lines were detected using sulforhodamine B (SRB) and Cell Titer-Blue assay, respectively; the cell cycle was detected by flow cytometry; and the expression of total EGFR and Akt protein and their phosphorylation were detected using Western blotting. Results: In the monolayer cells, the survival rates of A427 cells in the group of P-G, G-P, and P+G were (39.5±0.07)%, (57.7±003)% and (53.7±0.05)% respectively. Those of Calu-3 cells in the group of P-G, G-P and P+G were (23.9±002)%, (58.2±0.05)% and (48.8±0.07)% respectively. The inhibitory effect of P-G was the strongest (P<005). In the three-dimensional cells, the survival rates of A427 cells in the group of P-G, G-P and P+G were (19.9±2.89)%, (43.2±8.64)% and (36.6±9.79)% respectively. Those of Calu-3 cells in the group of P-G, G-P and P+G were (10.2±0.76)%, (50.0±3.45)%, (31.4±6.15)% respectively. The inhibitory effect of P-G was the strongest (P<0.05). In addition, the inhibitory effects of P-G on A427 and Calu-3 in the three-dimensional cells were stronger than those in the monolayer cells (P<0.05). The sequence of P-G resulted in an increase of subG1 proportion and arrested monolayer cells in G1 phase. The phosphorylation of EGFR and Akt was decreased in subsequent exposure to P-G. Conclusion: The sequence of P-G seems to be superior to the reverse schedule or combination in the inhibition of proliferation of NSCLC cells, based on the factors of G1 arrest and the decrease of phosphorylation of Akt and EGFR in three-dimensional cells.
2012, 19(4):369-373.
DOI: 10.3872/j.issn.1007-385X.2012.4.005
Abstract:
Objective: To observe the effect of PI3K/Akt/mTOR pathway inhibitors wortmannin or rapamycin on the proliferation and PHLPP (PH domain leucine-rich repeat protein phosphatase) protein expression of leukemia cell lines. Methods: Human leukemia cell lines K562 and HL-60 were treated with different concentrations of wortmannin or rapamycin. The proliferation of K562 and HL-60 cells was examined by WST-1 assay and the apoptosis of K562 and HL-60 cells was detected by Annexin Ⅴ-FITC double staining flow cytometry. The expressions of PHLPP, phosphorate-Akt (p-Akt) and total Akt protein were detected by Western blotting. Results: Wortmannin inhibited the proliferation of K562 and HL-60 cells in a dose- and time-dependent manner, with the IC50 value of 48 h being (187.6±48.4) nmol/L and (185.5±48.1) nmol/L (P<0.05). After treating K562 cells with 100 nmol/L wortmannin and HL-60 with 50 nmol/L wortmannin for 12 and 24 h, the apoptosis rates were significantly higher than those in the control group (\[12.4±0.7\]%, \[176±2.3\]% vs \[5.0±0.6\]%, P<0.05; \[11.0±0.2\]%, \[17.9±1.6\]% vs \[6.8±0.4\]%, P<0.05). After treating K562 and HL-60 cells with wortmannin for 12，24 and 36 h，the protein expressions of p-Akt and PHLPP were significantly reduced. And rapamycin also down-regulated the protein expression of PHLPP in K562 and HL-60 cells. Conclusion: The inhibitor of PI3K/Akt/mTOR signaling pathway inhibit the proliferation as well as PHLPP protein expression of leukemia cell lines.
Objective: To observe the effect of PI3K/Akt/mTOR pathway inhibitors wortmannin or rapamycin on the proliferation and PHLPP (PH domain leucine-rich repeat protein phosphatase) protein expression of leukemia cell lines. Methods: Human leukemia cell lines K562 and HL-60 were treated with different concentrations of wortmannin or rapamycin. The proliferation of K562 and HL-60 cells was examined by WST-1 assay and the apoptosis of K562 and HL-60 cells was detected by Annexin Ⅴ-FITC double staining flow cytometry. The expressions of PHLPP, phosphorate-Akt (p-Akt) and total Akt protein were detected by Western blotting. Results: Wortmannin inhibited the proliferation of K562 and HL-60 cells in a dose- and time-dependent manner, with the IC50 value of 48 h being (187.6±48.4) nmol/L and (185.5±48.1) nmol/L (P<0.05). After treating K562 cells with 100 nmol/L wortmannin and HL-60 with 50 nmol/L wortmannin for 12 and 24 h, the apoptosis rates were significantly higher than those in the control group (\[12.4±0.7\]%, \[176±2.3\]% vs \[5.0±0.6\]%, P<0.05; \[11.0±0.2\]%, \[17.9±1.6\]% vs \[6.8±0.4\]%, P<0.05). After treating K562 and HL-60 cells with wortmannin for 12，24 and 36 h，the protein expressions of p-Akt and PHLPP were significantly reduced. And rapamycin also down-regulated the protein expression of PHLPP in K562 and HL-60 cells. Conclusion: The inhibitor of PI3K/Akt/mTOR signaling pathway inhibit the proliferation as well as PHLPP protein expression of leukemia cell lines.
2012, 19(4):374-380.
DOI: 10.3872/j.issn.1007-385X.2012.4.006
Abstract:
Objective ：To study the anti-tumor activity of cytokine-induced killer cells (CIK cells) against HeLa-luc cells (cervical cancer HeLa cells labeling luciferase) in vivo and in vitro, and to analyze the distribution characteristics of CIK cells in different organs in a mouse xenograft model of cervical cancer nude. Methods: CIK cells were induced from peripheral blood mononuclear cells of health volunteers and cultured in vitro. The phenotype of CIK cells were determined by flow cytometry. The expression of IFN-γ mRNA in CIK cells was detected by RT-PCR assay. The killing activity of CIK cells on HeLa-luc cells was determined by MTT assay and Wright-Giemsa’s staining. HeLa-luc cell-bearing BALB/c nude mouse model was established. Tumor size changes and treatment effect were detected using in vivo Xenogen IVIS Imaging System. The distribution characteristics of CIK cells in different organs were detected by confocal microscopy. Results: CIK cells grew up to the peak after being cultured for 14-21 d. The percentage of CD3+CD56+T cells was increased more 50 times than that of the beginning. The expression level of IFN-γ mRNA in CIK cells was also increased to the peak. When the ratios of effect to target were 20 ∶1, and 40 ∶1, the cytotoxicity of CIK cells on HeLa-luc cells was (5116±2.64)% and (72.14%±4.21)%, respectively, and was significantly higher than that of the control group (\[1633±3.09\]%, \[21.26±2.71\]%, respectively, P<0.05). The inhibitory rate of CIK cells on the tumor was 47.18% and 64.38% at the fifth week and the eighth week, respectively. The level of IFN-γ mRNA in the CIK experiment group (61.92±6.49) pg/ml was significantly higher than that in the control group (34.30±1.78) pg/ml (P<005). Green fluorescence can be observed in the lung, liver, spleen, peripheral blood and tumor tissues under the confocal microscope 3 h after CFSE-labeled CIK cells injection via peritoneal cavity and tumor adjacent. 24 h after injection via peritoneal cavity, the highest concentration of CIK cells was 20.56% in the tumor tissues, and 3 h after injection via tumor adjacent, the highest concentration of CIK cells was 25.75% in the tumor tissues. Conclusion: CIK cells show a strong cytotoxicity on cervical cancer HeLa-luc cells in vivo and in vitro. The CIK cells are extensively distributed into different organs after injection via peritoneal cavity or tumor adjacent. The concentration of CIK cells in different organ is closely related to the injection route and time.
Objective ：To study the anti-tumor activity of cytokine-induced killer cells (CIK cells) against HeLa-luc cells (cervical cancer HeLa cells labeling luciferase) in vivo and in vitro, and to analyze the distribution characteristics of CIK cells in different organs in a mouse xenograft model of cervical cancer nude. Methods: CIK cells were induced from peripheral blood mononuclear cells of health volunteers and cultured in vitro. The phenotype of CIK cells were determined by flow cytometry. The expression of IFN-γ mRNA in CIK cells was detected by RT-PCR assay. The killing activity of CIK cells on HeLa-luc cells was determined by MTT assay and Wright-Giemsa’s staining. HeLa-luc cell-bearing BALB/c nude mouse model was established. Tumor size changes and treatment effect were detected using in vivo Xenogen IVIS Imaging System. The distribution characteristics of CIK cells in different organs were detected by confocal microscopy. Results: CIK cells grew up to the peak after being cultured for 14-21 d. The percentage of CD3+CD56+T cells was increased more 50 times than that of the beginning. The expression level of IFN-γ mRNA in CIK cells was also increased to the peak. When the ratios of effect to target were 20 ∶1, and 40 ∶1, the cytotoxicity of CIK cells on HeLa-luc cells was (5116±2.64)% and (72.14%±4.21)%, respectively, and was significantly higher than that of the control group (\[1633±3.09\]%, \[21.26±2.71\]%, respectively, P<0.05). The inhibitory rate of CIK cells on the tumor was 47.18% and 64.38% at the fifth week and the eighth week, respectively. The level of IFN-γ mRNA in the CIK experiment group (61.92±6.49) pg/ml was significantly higher than that in the control group (34.30±1.78) pg/ml (P<005). Green fluorescence can be observed in the lung, liver, spleen, peripheral blood and tumor tissues under the confocal microscope 3 h after CFSE-labeled CIK cells injection via peritoneal cavity and tumor adjacent. 24 h after injection via peritoneal cavity, the highest concentration of CIK cells was 20.56% in the tumor tissues, and 3 h after injection via tumor adjacent, the highest concentration of CIK cells was 25.75% in the tumor tissues. Conclusion: CIK cells show a strong cytotoxicity on cervical cancer HeLa-luc cells in vivo and in vitro. The CIK cells are extensively distributed into different organs after injection via peritoneal cavity or tumor adjacent. The concentration of CIK cells in different organ is closely related to the injection route and time.
2012, 19(4):381-386.
DOI: 10.3872/j.issn.1007-385X.2012.4.007
Abstract:
Objective：To study the mechanism of the differentiation of mouse melanoma B16 cells induced by ethanol extract of cochinchina momordica seed (CMSEE). Methods: After treated with CMSEE, morphological changes of B16 cells were observed by Giemsa staining, and melanin level was assessed by colorimetric method. Western blotting was used to measure the expression changes of phosphorylation p38 (p-p38) and ERK (p-ERK) in B16 cells after treatment with CMSEE and p38, ERK pathway inhibitors, SB203580 and SD98059. Results: CMSEE could induce the differentiation of B16 cells. The melanin level in B16 cells was significantly increased after CMSEE treatment (P<0.01), with D value being (0.057±0.007), (0.173±0.005) and (0.249±0.002) for 10, 20 and 40 μg/ml CMSEE and (0.037±0002) for control group. 20 μg/ml CMSEE elevated the level of p-p38 protein and blocked the expression of p-ERK (P<0.01) in B16 cells, and the expression level of p-p38 protein was 5.6 fold and p-ERK was 0.25 fold that of the control group after treatment with CMSEE for 60 min. SB203580, the inhibitor of p38 pathway, blocked CMSEE-induced B16 cell differentiation, and PD98059, the inhibitor of ERK pathway, elevated CMSEE-induced B16 cell differentiation. Conclusion: p38 and ERK signal pathway activations are involved in the differentiation of B16 melanoma cells induced by CMSEE.
Objective：To study the mechanism of the differentiation of mouse melanoma B16 cells induced by ethanol extract of cochinchina momordica seed (CMSEE). Methods: After treated with CMSEE, morphological changes of B16 cells were observed by Giemsa staining, and melanin level was assessed by colorimetric method. Western blotting was used to measure the expression changes of phosphorylation p38 (p-p38) and ERK (p-ERK) in B16 cells after treatment with CMSEE and p38, ERK pathway inhibitors, SB203580 and SD98059. Results: CMSEE could induce the differentiation of B16 cells. The melanin level in B16 cells was significantly increased after CMSEE treatment (P<0.01), with D value being (0.057±0.007), (0.173±0.005) and (0.249±0.002) for 10, 20 and 40 μg/ml CMSEE and (0.037±0002) for control group. 20 μg/ml CMSEE elevated the level of p-p38 protein and blocked the expression of p-ERK (P<0.01) in B16 cells, and the expression level of p-p38 protein was 5.6 fold and p-ERK was 0.25 fold that of the control group after treatment with CMSEE for 60 min. SB203580, the inhibitor of p38 pathway, blocked CMSEE-induced B16 cell differentiation, and PD98059, the inhibitor of ERK pathway, elevated CMSEE-induced B16 cell differentiation. Conclusion: p38 and ERK signal pathway activations are involved in the differentiation of B16 melanoma cells induced by CMSEE.
2012, 19(4):387-391.
DOI: 10.3872/j.issn.1007-385X.2012.4.008
Abstract:
Objective：To investigate the effects of tautomycetin on the proliferation and apoptosis of human breast cancer cell line MCF-7/ADR and the related mechanism. Methods: The effect of tautomycetin on the proliferation of MCF-7/ADR cells was examined by MTT assay; its effect on apoptosis of MCF-7/ADR cells was assessed by flow cytometry; and its effects on expressions of caspase-related proteins, Bcl-2, cytochrome C (Cyto-C), P53 and Akt in MCF-7/ADR cells were detected by Western blotting. Results: Tautomycetin inhibited the proliferation of MCF-7/ADR cells in a dose-dependent manner （0.01~100 μmol/L，P<0.05), with the IC50 value of (1.26±0.12) μmol/L. Compared with the control group, the early apoptosis rate of MCF-7/ADR cells after 1 μmol/L tautomycetin treatment was increased from (0.67±0.18)% to (17.2±3.8)%, and the late apoptosis rate from (0.96±0.23)% to (28.4±5.7)%, (P<005); tautomycetin activated caspase-9 and caspase-7, decreased Bcl-2 expression, promoted Cyto-C secretion and decreased p-Akt levels in MCF-7/ADR cells, while showed no obvious effect on caspase-8 and P53 expressions. Conclusion: Tautomycetin can inhibit the phosphorylation of Akt, and induce the Cyto-C-mediated apoptosis of MCF-7/ADR cells in a P53-independent pathway.
Objective：To investigate the effects of tautomycetin on the proliferation and apoptosis of human breast cancer cell line MCF-7/ADR and the related mechanism. Methods: The effect of tautomycetin on the proliferation of MCF-7/ADR cells was examined by MTT assay; its effect on apoptosis of MCF-7/ADR cells was assessed by flow cytometry; and its effects on expressions of caspase-related proteins, Bcl-2, cytochrome C (Cyto-C), P53 and Akt in MCF-7/ADR cells were detected by Western blotting. Results: Tautomycetin inhibited the proliferation of MCF-7/ADR cells in a dose-dependent manner （0.01~100 μmol/L，P<0.05), with the IC50 value of (1.26±0.12) μmol/L. Compared with the control group, the early apoptosis rate of MCF-7/ADR cells after 1 μmol/L tautomycetin treatment was increased from (0.67±0.18)% to (17.2±3.8)%, and the late apoptosis rate from (0.96±0.23)% to (28.4±5.7)%, (P<005); tautomycetin activated caspase-9 and caspase-7, decreased Bcl-2 expression, promoted Cyto-C secretion and decreased p-Akt levels in MCF-7/ADR cells, while showed no obvious effect on caspase-8 and P53 expressions. Conclusion: Tautomycetin can inhibit the phosphorylation of Akt, and induce the Cyto-C-mediated apoptosis of MCF-7/ADR cells in a P53-independent pathway.
2012, 19(4):392-396.
DOI: 10.3872/j.issn.1007-385X.2012.4.009
Abstract:
Objective：To explore the effects of small interference RNA (siRNA) targeting livin on the proliferation, apoptosis and invasion of human colon cancer cell line HT-29. Methods: Chemically synthetic double-strand siRNA targeting livin (livin-siRNA) was transfected into HT-29 cells, and then RT-PCR and Western blotting were used to detect the expression of livin mRNA and protein in HT-29 cells. MTT assay was performed to analyze the proliferation of HT-29 cells. The cell apoptosis and cell cycle distribution were analyzed by flow cytometry. The invasion assay and caspase-3 detective kit were used to detect the change of invasion and caspase-3 activity in HT-29 cells. Results: Forty-eight hours after transfection, there was a significant decrease in the expressions of both livin mRNA (0.073±0007 vs 0.395±0082, 0.423±0.025, 0.418±0.032, P<0.05) and livin protein (0.106±0.003 vs 0.456±0065, 0.473±0078、0.491±0.045, P<0.05) in the livin-siRNA group, compared with the blank and negative control and liposome groups. Ninety-six hours after transfection, the growth of HT-29 cells in the livin-siRNA group was significantly lower than that in the control and liposome groups (0.564±0.102 vs 0.833±0.127, 0.860±0.153，P<005), and the rate of apoptosis was obviously increased (\[16.5±2.8\] % vs \[2.4 ±0.5\]%, \[3.7±1.0\] %, P<005). The invasion assay demonstrated that the number of the migration cells was lower in the livin-siRNA group than in the control and liposome groups (31.3±4.5 vs 101.3±8.6, 97.4±7.8, P<0.05). The activity of caspase-3 in the livin-siRNA group was decreased compared with that in the control group (0.160 ±0.023 vs 0.347± 0.058, P<0.05). Conclusion: The siRNA silencing livin expression in HT-29 cells can suppress the proliferation, induce the apoptosis and inhibit the invasion of HT-29 cells.
Objective：To explore the effects of small interference RNA (siRNA) targeting livin on the proliferation, apoptosis and invasion of human colon cancer cell line HT-29. Methods: Chemically synthetic double-strand siRNA targeting livin (livin-siRNA) was transfected into HT-29 cells, and then RT-PCR and Western blotting were used to detect the expression of livin mRNA and protein in HT-29 cells. MTT assay was performed to analyze the proliferation of HT-29 cells. The cell apoptosis and cell cycle distribution were analyzed by flow cytometry. The invasion assay and caspase-3 detective kit were used to detect the change of invasion and caspase-3 activity in HT-29 cells. Results: Forty-eight hours after transfection, there was a significant decrease in the expressions of both livin mRNA (0.073±0007 vs 0.395±0082, 0.423±0.025, 0.418±0.032, P<0.05) and livin protein (0.106±0.003 vs 0.456±0065, 0.473±0078、0.491±0.045, P<0.05) in the livin-siRNA group, compared with the blank and negative control and liposome groups. Ninety-six hours after transfection, the growth of HT-29 cells in the livin-siRNA group was significantly lower than that in the control and liposome groups (0.564±0.102 vs 0.833±0.127, 0.860±0.153，P<005), and the rate of apoptosis was obviously increased (\[16.5±2.8\] % vs \[2.4 ±0.5\]%, \[3.7±1.0\] %, P<005). The invasion assay demonstrated that the number of the migration cells was lower in the livin-siRNA group than in the control and liposome groups (31.3±4.5 vs 101.3±8.6, 97.4±7.8, P<0.05). The activity of caspase-3 in the livin-siRNA group was decreased compared with that in the control group (0.160 ±0.023 vs 0.347± 0.058, P<0.05). Conclusion: The siRNA silencing livin expression in HT-29 cells can suppress the proliferation, induce the apoptosis and inhibit the invasion of HT-29 cells.
2012, 19(4):397-401.
DOI: 10.3872/j.issn.1007-385X.2012.4.010
Abstract:
Objective To explore the regulatory effects of Krüppel-like factors 4 (KLF4) on chemotherapy and photodynamic therapy on HepG2 cells. Methods: Lentiviral pWPTS-KLF4 vector containing human KLF4 was constructed. The expression of KLF4 mRNA and protein in HepG2 cells after pWPTS-KLF4 infection was analyzed by RT-PCR and Western blotting; the tolerance of HepG2 cells to cisplatin, cyclophosphamide and fluorouracil and the sensitivity of HepG2 cells to photodynamic therapy were evaluated by MTT assay; the change of mitochondrial membrane potential in HepG2 cells was examined by Rhodamine 123 staining. Results: Lentiviral pWPTS-KLF4 vector was successfully constructed. Treated with cisplatin, cyclophosphamide or fluorouracil for 72 h, the vialble HepG2 cells were significantly increased after pWPTS-KLF4 infection (\[43.43±4.78\]% vs \[18.09±1.02\]%；\[110.51±4.58\]% vs \[75.23±592\]%；\[34.55±293\]% vs \[19.16±1.32\]%, P<0.01); Treated with ALA mediated photodynamic therapy for 24 h, vialble HepG2 cells were significantly decreased after pWPTS-KLF4 infection (\[37.16±3.26\]% vs \[57.24±801\]%, P<0.01), and mitochondrial membrane potential was significantly decreased. Conclusion: Recombinant human KLF4 can increase the tolerance of HepG2 cells to chemotherapy and the sensitivity of HepG2 cells to photodynamic therapy.
Objective To explore the regulatory effects of Krüppel-like factors 4 (KLF4) on chemotherapy and photodynamic therapy on HepG2 cells. Methods: Lentiviral pWPTS-KLF4 vector containing human KLF4 was constructed. The expression of KLF4 mRNA and protein in HepG2 cells after pWPTS-KLF4 infection was analyzed by RT-PCR and Western blotting; the tolerance of HepG2 cells to cisplatin, cyclophosphamide and fluorouracil and the sensitivity of HepG2 cells to photodynamic therapy were evaluated by MTT assay; the change of mitochondrial membrane potential in HepG2 cells was examined by Rhodamine 123 staining. Results: Lentiviral pWPTS-KLF4 vector was successfully constructed. Treated with cisplatin, cyclophosphamide or fluorouracil for 72 h, the vialble HepG2 cells were significantly increased after pWPTS-KLF4 infection (\[43.43±4.78\]% vs \[18.09±1.02\]%；\[110.51±4.58\]% vs \[75.23±592\]%；\[34.55±293\]% vs \[19.16±1.32\]%, P<0.01); Treated with ALA mediated photodynamic therapy for 24 h, vialble HepG2 cells were significantly decreased after pWPTS-KLF4 infection (\[37.16±3.26\]% vs \[57.24±801\]%, P<0.01), and mitochondrial membrane potential was significantly decreased. Conclusion: Recombinant human KLF4 can increase the tolerance of HepG2 cells to chemotherapy and the sensitivity of HepG2 cells to photodynamic therapy.
2012, 19(4):402-407.
DOI: 10.3872/j.issn.1007-385X.2012.4.011
Abstract:
Objective:To study the effect of NF-κB signaling pathway for hepatitis B virus X protein (HBX) on the apoptosis of different hepatocyte lines. Methods: To establish the normal hepatic cell LO2 and hepatic cancer cell HepG2 stably transfected with PEGFP-N1-HBX plasmid (L02/HBX or HepG2/HBX cells). NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) was used to cut off NF-κB signal transduction in LO2 and HepG2 cells. Flow cytometry was applied to study the cell cycle and apoptosis of LO2 and HepG2 cells before and after PEGFP-N1-HBX transfection as well as before and after PDTC treatment. Western blotting was used to examine the expression of NF-κB. Results: LO2/HBX cells and HepG2/HBX cells stably transfected with PEGFP-N1-HBX were established successfully. The apoptosis of L02/HBX cells significantly increased compared with the control L02 cells \[(31.31±0.51)% vs (14.05±009)%, P<0.05\], and the proportion of cells in G0/G1 stage increased with cells in S and G2/M stage decreased. The apoptosis of HepG2/HBX cells significantly decreased compared with the control HepG2 cells (\[1.21±0.04\]% vs \[1026±0.10\]%, P<0.05), and the proportion of cells in G0/G1 stage decreased with cells in S and G2/M stage increased. After PDTC treatment, the proportion of L02/HBX/PDTC cells in G0/G1 phase increased significantly (\[40.33±0.07\]%), while that in S and G2/M phase decreased remarkably, and the apoptosis rate (\[5.45±007\]%) was at a significantly higher level compared with L02/HBX cells. The apoptosis and the proportion of cells in G0/G1 phase of HepG2/HBX/PDTC were increased significantly, and decreased remarkably in S and G2/M phase, while the apoptosis rate was still lower than the HepG2 cells. The expression of NF-κB protein was significantly decreased in L02/HBX cells but increased in HepG2/HBX cells compared with the control cells. There was almost no expression of NF-κB protein in L02/HBX/PDTC and HepG2/HBX/PDTC cells. Conclusion: HBX can retard the cell cycle of normal hepatic L02 cells and facilitate their apoptosis through down-regulating the expression of NF-κB protein. HBX can accelerate the cell cycle of hepatic cancer HepG2 cells and suppress the apoptosis through up-regulating the expression of NF-κB protein.
Objective:To study the effect of NF-κB signaling pathway for hepatitis B virus X protein (HBX) on the apoptosis of different hepatocyte lines. Methods: To establish the normal hepatic cell LO2 and hepatic cancer cell HepG2 stably transfected with PEGFP-N1-HBX plasmid (L02/HBX or HepG2/HBX cells). NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) was used to cut off NF-κB signal transduction in LO2 and HepG2 cells. Flow cytometry was applied to study the cell cycle and apoptosis of LO2 and HepG2 cells before and after PEGFP-N1-HBX transfection as well as before and after PDTC treatment. Western blotting was used to examine the expression of NF-κB. Results: LO2/HBX cells and HepG2/HBX cells stably transfected with PEGFP-N1-HBX were established successfully. The apoptosis of L02/HBX cells significantly increased compared with the control L02 cells \[(31.31±0.51)% vs (14.05±009)%, P<0.05\], and the proportion of cells in G0/G1 stage increased with cells in S and G2/M stage decreased. The apoptosis of HepG2/HBX cells significantly decreased compared with the control HepG2 cells (\[1.21±0.04\]% vs \[1026±0.10\]%, P<0.05), and the proportion of cells in G0/G1 stage decreased with cells in S and G2/M stage increased. After PDTC treatment, the proportion of L02/HBX/PDTC cells in G0/G1 phase increased significantly (\[40.33±0.07\]%), while that in S and G2/M phase decreased remarkably, and the apoptosis rate (\[5.45±007\]%) was at a significantly higher level compared with L02/HBX cells. The apoptosis and the proportion of cells in G0/G1 phase of HepG2/HBX/PDTC were increased significantly, and decreased remarkably in S and G2/M phase, while the apoptosis rate was still lower than the HepG2 cells. The expression of NF-κB protein was significantly decreased in L02/HBX cells but increased in HepG2/HBX cells compared with the control cells. There was almost no expression of NF-κB protein in L02/HBX/PDTC and HepG2/HBX/PDTC cells. Conclusion: HBX can retard the cell cycle of normal hepatic L02 cells and facilitate their apoptosis through down-regulating the expression of NF-κB protein. HBX can accelerate the cell cycle of hepatic cancer HepG2 cells and suppress the apoptosis through up-regulating the expression of NF-κB protein.
LI Zhou
,
FANG Su-zhen
,
HAN Shuai
,
CUI Da-xiang
,
LI Qiang
,
CAI Zhai
,
ZHU Hui-juan
,
HUANG Zong-hai
2012, 19(4):408-413.
DOI: 10.3872/j.issn.1007-385X.2012.4.012
Abstract:
Objective:To investigate the possibility of polyamidoaminedendrimer (PAMAM)-liposome for survivin antisense oligonucleotide (survivin-asODN) delivery system and explore the effects of PAMAM-liposome-survivin-asODN on survivin expression and apoptosis of human hepatic cancer cell line SMMC-7721. Methods: The liposome modified PAMAM (PAMAM-liposome) was synthesized with liposome and PAMAM. Survivin-asODN was combined with the PAMAM-liposome or PAMAM to form PAMAM-liposome-survivin-asODN or PAMAM-survivin-asODN complexes. The shape and size of the two complexes were observed under a transmission electron microscope and their zeta potentials were measured with a zeta analytical tool. The encapsulating efficiency and DNA loading level were determined by ultraviolet spectrophotometer using a centrifuging method. PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN were transfected into SMMC-7721 cells, and the transfection efficiency was measured. The protein expression of survivin in SMMC-7721 cells was measured by Western blotting, and the apoptosis of SMMC-7721 cells was assessed by flow cytometry. Results: PAMAM-liposome, PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN were successfully established. No significant difference appeared in diameter between PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN (\[189.33±15.42\] vs \[181.83±13.67\] nm, P>0.05), as well as the encapsulating efficiency and drug loading level, but the zeta potential of PAMAM-liposome-survivin-asODN was higher than that of PAMAM-survivin-asODN (\[42.83±7.14\] vs \[32.33±5.57\] mV, P<0.05). The transfection efficiency of PAMAM-liposome-survivin-asODN was higher than that of PAMAM-survivin-asODN (\[73.33±9.29)% vs \[60.67±7.81\]%, P<0.05) in SMMC-7721 cells. The expression of survivin protein in SMMC-7721 cells of PAMAM-liposome-survivin-asODN group was less than that of PAMAM-survivin-asODN group (24.67±11.74 vs 43.17±11.63, P<0.05), while the apoptosis rate was higher than that of PAMAM-survivin-asODN (\[73.31±12.59\]% vs \[52.67±12.19\]%, P<0.05). Conclusion: The PAMAM-liposome can delivery survivin-asODN into SMMC-7721 cells effectively and induce SMMC-7721 cell apoptosis.
Objective:To investigate the possibility of polyamidoaminedendrimer (PAMAM)-liposome for survivin antisense oligonucleotide (survivin-asODN) delivery system and explore the effects of PAMAM-liposome-survivin-asODN on survivin expression and apoptosis of human hepatic cancer cell line SMMC-7721. Methods: The liposome modified PAMAM (PAMAM-liposome) was synthesized with liposome and PAMAM. Survivin-asODN was combined with the PAMAM-liposome or PAMAM to form PAMAM-liposome-survivin-asODN or PAMAM-survivin-asODN complexes. The shape and size of the two complexes were observed under a transmission electron microscope and their zeta potentials were measured with a zeta analytical tool. The encapsulating efficiency and DNA loading level were determined by ultraviolet spectrophotometer using a centrifuging method. PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN were transfected into SMMC-7721 cells, and the transfection efficiency was measured. The protein expression of survivin in SMMC-7721 cells was measured by Western blotting, and the apoptosis of SMMC-7721 cells was assessed by flow cytometry. Results: PAMAM-liposome, PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN were successfully established. No significant difference appeared in diameter between PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN (\[189.33±15.42\] vs \[181.83±13.67\] nm, P>0.05), as well as the encapsulating efficiency and drug loading level, but the zeta potential of PAMAM-liposome-survivin-asODN was higher than that of PAMAM-survivin-asODN (\[42.83±7.14\] vs \[32.33±5.57\] mV, P<0.05). The transfection efficiency of PAMAM-liposome-survivin-asODN was higher than that of PAMAM-survivin-asODN (\[73.33±9.29)% vs \[60.67±7.81\]%, P<0.05) in SMMC-7721 cells. The expression of survivin protein in SMMC-7721 cells of PAMAM-liposome-survivin-asODN group was less than that of PAMAM-survivin-asODN group (24.67±11.74 vs 43.17±11.63, P<0.05), while the apoptosis rate was higher than that of PAMAM-survivin-asODN (\[73.31±12.59\]% vs \[52.67±12.19\]%, P<0.05). Conclusion: The PAMAM-liposome can delivery survivin-asODN into SMMC-7721 cells effectively and induce SMMC-7721 cell apoptosis.
ZHANG Ye
,
ZHU Shou-xing
,
SHEN Xiao-su
,
ZHU Wei-min
,
MA Su-juan
,
SHI Hong-zhen
,
SHI Yang
,
ZHU Chen-yao
,
LI Wei
2012, 19(4):414-420.
DOI: 10.3872/j.issn.1007-385X.2012.4.013
Abstract:
Objective:To investigate the efficacy of polypeptide-loaded dendritic cells (DCs) in combination with cytokine-induced killer cells (CIKs) against hormone refractory metastatic prostate cancer (HRPC) patients. Methods: Twenty-six HLA-A2+ patients with HRPC were enrolled from the Department of Chinese Traditional and Western Medicine of the Wuxi No.4 People’s Hospital. Peripheral blood mononuclear cells (PBMCs) were separated, and the adherent cells were induced into DCs by GM-CSF and IL-4. Then DCs were loaded with three peptides (prostate specific antigen, PSA; prostatic acid phosphatase, PAP; prostate specific membrane antigen, PSMA) to prepare DC vaccine and were injected intracutaneously. The un-adherent cells of PBMCs were induced into CIK by IFN-γ, IL-2, CD3 monoclonal antibody and IL-1, and were injected intravenously. Delayed type hypersensitivity (DTH) was detected one week after treatment, and cytokine and PSA in serum were determined before and after treatment. The short-term efficacy was evaluated 4 weeks after treatment. Results: DC-CIK therapy was well tolerated in 26 HRPC patients. The serum IL-2, IL-12, and IFN-γ levels after therapy were significantly increased (increased 65.07%, 67.69% and 12538%, P<0.05 or P<001), while TNF-α and IL-10 levels were unchanged. The positive rate of DTH was 43.5% (10/23). The proportion of CD8+IFNγ+ cells after therapy was significantly increased (\[8.95±2.74\]% vs \[0.39±015\]%, P<0.01). A decrease of PSA (13% to 66%) was observed in 8 of 26 patients. The short-term efficacy of 26 HRPC patients was evaluated, with 3 PR, 4 PD, and 19 SD after treatment, and no severe adverse reaction was observed. Conclusion: The polypeptide-loaded DC in combination with CIK therapy can elicit specific immune responses in HRPC patients, and induce type I cytokine secretion with well short-term clinical efficacy, indicating that DC-CIK therapy is a safe treatment for HRPC.
Objective:To investigate the efficacy of polypeptide-loaded dendritic cells (DCs) in combination with cytokine-induced killer cells (CIKs) against hormone refractory metastatic prostate cancer (HRPC) patients. Methods: Twenty-six HLA-A2+ patients with HRPC were enrolled from the Department of Chinese Traditional and Western Medicine of the Wuxi No.4 People’s Hospital. Peripheral blood mononuclear cells (PBMCs) were separated, and the adherent cells were induced into DCs by GM-CSF and IL-4. Then DCs were loaded with three peptides (prostate specific antigen, PSA; prostatic acid phosphatase, PAP; prostate specific membrane antigen, PSMA) to prepare DC vaccine and were injected intracutaneously. The un-adherent cells of PBMCs were induced into CIK by IFN-γ, IL-2, CD3 monoclonal antibody and IL-1, and were injected intravenously. Delayed type hypersensitivity (DTH) was detected one week after treatment, and cytokine and PSA in serum were determined before and after treatment. The short-term efficacy was evaluated 4 weeks after treatment. Results: DC-CIK therapy was well tolerated in 26 HRPC patients. The serum IL-2, IL-12, and IFN-γ levels after therapy were significantly increased (increased 65.07%, 67.69% and 12538%, P<0.05 or P<001), while TNF-α and IL-10 levels were unchanged. The positive rate of DTH was 43.5% (10/23). The proportion of CD8+IFNγ+ cells after therapy was significantly increased (\[8.95±2.74\]% vs \[0.39±015\]%, P<0.01). A decrease of PSA (13% to 66%) was observed in 8 of 26 patients. The short-term efficacy of 26 HRPC patients was evaluated, with 3 PR, 4 PD, and 19 SD after treatment, and no severe adverse reaction was observed. Conclusion: The polypeptide-loaded DC in combination with CIK therapy can elicit specific immune responses in HRPC patients, and induce type I cytokine secretion with well short-term clinical efficacy, indicating that DC-CIK therapy is a safe treatment for HRPC.
14 Clinical efficiency of radiotherapy combined with self-immune cell therapy for cervical carcinoma
ZHU Yue-hua
,
LIU Jun-quan
,
CAO Hui
,
CHEN Hui-ping
,
CHEN Fu-xing
,
CHEN Ling
,
ZHANG Song
,
L Xiao-ting
2012, 19(4):421-427.
DOI: 10.3872/j.issn.1007-385X.2012.4.014
Abstract:
Objective:To compare changes of cellular immune function and survival of cervical carcinoma patients receiving radiotherapy combined with self-immune cell therapy with those receiving radiotherapy alone. Methods: Sixty-eight cases of cervical carcinoma were divided into 2 groups: 38 cases in radiotherapy group; 30 cases in combination group (radiotherapy combined with self-immune cell therapy). Peripheral blood mononuclear cells (PBMCs) were separated, and then CD3AK cells, cytokine-induced killer cells (CIKs), dendritic cells (DCs), γδT cells and NK cells were induced and cultured. CD3+, CD4+, CD8+, CD19+, CD16+CD56+ and γδT cell numbers and ratios as well as the rate of positive expression of perforin, granzyme B (GraB), and CD107a of PBMCs were evaluated by FCM before and after treatment. The patients were followed by 5 years follow up study. Results: In most patients of the combination group, the quality of life was improved. The scores of Karnofsky in the combination group were significantly higher than those in the radiotherapy group (P<0.05). The immune cell numbers in the combination group were significantly higher than those in the radiotherapy group (P<005). The rates of the positive expression of perforin, GraB and CD107a of PBMCs were higher in the combination group than in the radiotherapy group correspondingly (P<0.05). The over-all 1-, 2- and 5-year survival rates of Ⅰb-Ⅳ stage patients were 93.3%(28/30), 83.3%(25/30) and 76.6%(23/30) in the combination group, and were 86.82%(33/38), 68.4%(26/38) and 57.9%(22/38) in the radiotherapy group, respectively (P<0.05) with Ⅱb and Ⅲ stage patients showing the most significance. Conclusion: Radiotherapy combined with self-immune cell therapy for cervical carcinoma can enhance the immune function and increase the survival.
Objective:To compare changes of cellular immune function and survival of cervical carcinoma patients receiving radiotherapy combined with self-immune cell therapy with those receiving radiotherapy alone. Methods: Sixty-eight cases of cervical carcinoma were divided into 2 groups: 38 cases in radiotherapy group; 30 cases in combination group (radiotherapy combined with self-immune cell therapy). Peripheral blood mononuclear cells (PBMCs) were separated, and then CD3AK cells, cytokine-induced killer cells (CIKs), dendritic cells (DCs), γδT cells and NK cells were induced and cultured. CD3+, CD4+, CD8+, CD19+, CD16+CD56+ and γδT cell numbers and ratios as well as the rate of positive expression of perforin, granzyme B (GraB), and CD107a of PBMCs were evaluated by FCM before and after treatment. The patients were followed by 5 years follow up study. Results: In most patients of the combination group, the quality of life was improved. The scores of Karnofsky in the combination group were significantly higher than those in the radiotherapy group (P<0.05). The immune cell numbers in the combination group were significantly higher than those in the radiotherapy group (P<005). The rates of the positive expression of perforin, GraB and CD107a of PBMCs were higher in the combination group than in the radiotherapy group correspondingly (P<0.05). The over-all 1-, 2- and 5-year survival rates of Ⅰb-Ⅳ stage patients were 93.3%(28/30), 83.3%(25/30) and 76.6%(23/30) in the combination group, and were 86.82%(33/38), 68.4%(26/38) and 57.9%(22/38) in the radiotherapy group, respectively (P<0.05) with Ⅱb and Ⅲ stage patients showing the most significance. Conclusion: Radiotherapy combined with self-immune cell therapy for cervical carcinoma can enhance the immune function and increase the survival.
2012, 19(4):428-432.
DOI: 10.3872/j.issn.1007-385X.2012.4.015
Abstract:
棘皮动物微管相关蛋白样4（echinoderm microtubule associated protein like 4， EML4 ）间变性淋巴瘤激酶（anaplastic lymphoma kinase，ALK）融合基因（EML4-ALK）是近年来新发现的癌变驱动基因，该融合基因阳性的非小细胞肺癌（non-small cell lung cancer，NSCLC）患者有其独特的临床特征，可能与表皮生长因子受体（epidermal growth factor receptor，EGFR）酪氨酸激酶抑制剂（tyrosine kinase inhibitor，TKI）耐药相关。针对EML4-ALK基因突变的新靶向药物——ALK抑制剂crizotinib，现已经进入Ⅲ期临床试验。Ⅰ期及Ⅱ期临床试验均证实，crizotinib治疗EML4-ALK阳性晚期NSCLC患者有效，能够改善肿瘤患者症状，患者的无进展生存期（progression free survival，PFS）延长，总体有效率（overall response rate，ORR）提高。且crizotinib的毒性作用较小，与传统化疗相比，患者耐受性较好。与其他TKI一样，crizotinib也存在获得性耐药现象，其耐药机制有待进一步研究。本文就crizotinib从基础研究向治疗EML4-ALK阳性晚期NSCLC患者临床应用的转化过程作一回顾。
棘皮动物微管相关蛋白样4（echinoderm microtubule associated protein like 4， EML4 ）间变性淋巴瘤激酶（anaplastic lymphoma kinase，ALK）融合基因（EML4-ALK）是近年来新发现的癌变驱动基因，该融合基因阳性的非小细胞肺癌（non-small cell lung cancer，NSCLC）患者有其独特的临床特征，可能与表皮生长因子受体（epidermal growth factor receptor，EGFR）酪氨酸激酶抑制剂（tyrosine kinase inhibitor，TKI）耐药相关。针对EML4-ALK基因突变的新靶向药物——ALK抑制剂crizotinib，现已经进入Ⅲ期临床试验。Ⅰ期及Ⅱ期临床试验均证实，crizotinib治疗EML4-ALK阳性晚期NSCLC患者有效，能够改善肿瘤患者症状，患者的无进展生存期（progression free survival，PFS）延长，总体有效率（overall response rate，ORR）提高。且crizotinib的毒性作用较小，与传统化疗相比，患者耐受性较好。与其他TKI一样，crizotinib也存在获得性耐药现象，其耐药机制有待进一步研究。本文就crizotinib从基础研究向治疗EML4-ALK阳性晚期NSCLC患者临床应用的转化过程作一回顾。
2012, 19(4):433-436.
DOI: 10.3872/j.issn.1007-385X.2012.4.016
Abstract:
目的： 研究同种异体自然杀伤（natural killer，NK）细胞对人急性髓系白血病细胞株KG1A的杀伤率，探讨供者NK细胞表面杀伤细胞免疫球蛋白样受体2DL1（killer immunoglobulin-like receptor 2DL1，KIR2DL1）表达差异对NK 细胞杀伤KG1A细胞活性的影响。 方法： 自9名健康志愿供者分离外周血NK细胞，流式细胞术检测NK细胞表面KIR2DL1的表达率，LDH释放法测定NK细胞在效靶比为20〖DK〗∶1时对KG1A细胞的杀伤活性。 结果： NK细胞表面KIR2DL1的表达率存在个体差异，9名供者的表达范围为（33.20±1.95）%~（92.69±1.47）%；9名健康供者NK细胞对KG1A细胞的杀伤活性不同，杀伤率范围为（44.40±1.97）%~（93.70±1.12）%。不同个体NK细胞KIR2DL1表达率与其对KG1A细胞的杀伤率呈负相关（r =-1.00，P=0.00）。 结论： 不同个体NK细胞其表面KIR2DL1 表达率与NK细胞对KG1A细胞的杀伤率呈负相关。
目的： 研究同种异体自然杀伤（natural killer，NK）细胞对人急性髓系白血病细胞株KG1A的杀伤率，探讨供者NK细胞表面杀伤细胞免疫球蛋白样受体2DL1（killer immunoglobulin-like receptor 2DL1，KIR2DL1）表达差异对NK 细胞杀伤KG1A细胞活性的影响。 方法： 自9名健康志愿供者分离外周血NK细胞，流式细胞术检测NK细胞表面KIR2DL1的表达率，LDH释放法测定NK细胞在效靶比为20〖DK〗∶1时对KG1A细胞的杀伤活性。 结果： NK细胞表面KIR2DL1的表达率存在个体差异，9名供者的表达范围为（33.20±1.95）%~（92.69±1.47）%；9名健康供者NK细胞对KG1A细胞的杀伤活性不同，杀伤率范围为（44.40±1.97）%~（93.70±1.12）%。不同个体NK细胞KIR2DL1表达率与其对KG1A细胞的杀伤率呈负相关（r =-1.00，P=0.00）。 结论： 不同个体NK细胞其表面KIR2DL1 表达率与NK细胞对KG1A细胞的杀伤率呈负相关。
2012, 19(4):437-441.
DOI: 10.3872/j.issn.1007-385X.2012.4.017
Abstract:
目的： 观察负载抗原的树突状细胞（dendritic cell，DC）与细胞因子诱导的杀伤细胞（cytokine-induced killer cell，CIK）对高表达P-糖蛋白（P-glycoprotein，P-gp）的多药耐药（multidrug resistance，MDR）人乳腺癌MCF-7/ADR细胞的杀伤作用。 方法： 提取健康人外周血单个核细胞，常规诱导出CIK及DC；制备MCF-7/ADR细胞冻融抗原后冲击DC，并与CIK共培养作为实验组（冻融物DC-CIK组），未负载抗原的DC与CIK共培养作为对照组（DC-CIK组），同时设单独培养的CIK组或DC组作为空白对照组。流式细胞术分析细胞的表型及P-gp表达，ELISA法测定细胞上清中IL-12、IFN-γ水平，MTT法检测对MCF-7/ADR及MCF-7细胞株的杀伤活性。 结果： 冻融物DC-CIK组、DC-CIK组细胞增殖活性均大于CIK组（ P<0.05）。冻融物DC-CIK组对MCF-7/ADR、MCF-7细胞的杀伤活性在效靶比10〖DK〗∶1、20〖DK〗∶1、40〖DK〗∶1时分别为（44.29 ±1.39）%、（58.24 ±3.52）%、（68.9 ±2.83）%和（33.51 ±2.18）、（40.43 ±2.3）%、（44.62 ±1.19）% ，均高于DC-CIK组及CIK组（P<0.05）；冻融物DC-CIK组对MCF-7/ADR耐药细胞株的杀伤活性高于非耐药细胞株MCF7（P<0.05）；而对于非耐药株MCF-7细胞的杀伤活性，冻融物DC-CIK组和DC-CIK组之间差异无统计学意义（P>0.05）。 结论： DC与CIK共培养细胞增殖活性和细胞毒活性均强于CIK，经冻融抗原冲击的DC与CIK共培养可以显著提高对MCF-7/ADR耐药细胞株的杀伤活性。
目的： 观察负载抗原的树突状细胞（dendritic cell，DC）与细胞因子诱导的杀伤细胞（cytokine-induced killer cell，CIK）对高表达P-糖蛋白（P-glycoprotein，P-gp）的多药耐药（multidrug resistance，MDR）人乳腺癌MCF-7/ADR细胞的杀伤作用。 方法： 提取健康人外周血单个核细胞，常规诱导出CIK及DC；制备MCF-7/ADR细胞冻融抗原后冲击DC，并与CIK共培养作为实验组（冻融物DC-CIK组），未负载抗原的DC与CIK共培养作为对照组（DC-CIK组），同时设单独培养的CIK组或DC组作为空白对照组。流式细胞术分析细胞的表型及P-gp表达，ELISA法测定细胞上清中IL-12、IFN-γ水平，MTT法检测对MCF-7/ADR及MCF-7细胞株的杀伤活性。 结果： 冻融物DC-CIK组、DC-CIK组细胞增殖活性均大于CIK组（ P<0.05）。冻融物DC-CIK组对MCF-7/ADR、MCF-7细胞的杀伤活性在效靶比10〖DK〗∶1、20〖DK〗∶1、40〖DK〗∶1时分别为（44.29 ±1.39）%、（58.24 ±3.52）%、（68.9 ±2.83）%和（33.51 ±2.18）、（40.43 ±2.3）%、（44.62 ±1.19）% ，均高于DC-CIK组及CIK组（P<0.05）；冻融物DC-CIK组对MCF-7/ADR耐药细胞株的杀伤活性高于非耐药细胞株MCF7（P<0.05）；而对于非耐药株MCF-7细胞的杀伤活性，冻融物DC-CIK组和DC-CIK组之间差异无统计学意义（P>0.05）。 结论： DC与CIK共培养细胞增殖活性和细胞毒活性均强于CIK，经冻融抗原冲击的DC与CIK共培养可以显著提高对MCF-7/ADR耐药细胞株的杀伤活性。
2012, 19(4):442-446.
DOI: 10.3872/j.issn.1007-385X.2012.4.018
Abstract:
目的： 研究表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-TKI)吉非替尼联合环氧合酶-2（cyclooxygenase-2，COX-2）抑制剂塞来昔布对肺腺癌A549细胞增殖的抑制作用及其可能机制。 方法： 实验设空白对照组、吉非替尼组、塞来昔布组、吉非替尼+塞来昔布组。倒置相差显微镜下观察A549细胞形态变化，MTT法检测A549细胞增殖，流式细胞术检测A549细胞周期及凋亡，Western blotting检测A549细胞中EGFR、p-EGFR、COX-2的表达。 结果： 吉非替尼和塞来昔布均可抑制A549细胞的增殖，可见细胞活性下降，G1期细胞阻滞及细胞凋亡增加；联合用药较单药组A549细胞活性下降更明显，G1期阻滞细胞增加，且细胞凋亡增加。吉非替尼或塞来昔布作用前后A549细胞EGFR表达无变化，但联合用药组p-EGFR及COX-2的表达较单药组显著下降。 结论： 吉非替尼联合塞来昔布可抑制人肺腺癌A549细胞的增殖，其机制可能与抑制EGFR的活化及下调COX-2的表达有关。
目的： 研究表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-TKI)吉非替尼联合环氧合酶-2（cyclooxygenase-2，COX-2）抑制剂塞来昔布对肺腺癌A549细胞增殖的抑制作用及其可能机制。 方法： 实验设空白对照组、吉非替尼组、塞来昔布组、吉非替尼+塞来昔布组。倒置相差显微镜下观察A549细胞形态变化，MTT法检测A549细胞增殖，流式细胞术检测A549细胞周期及凋亡，Western blotting检测A549细胞中EGFR、p-EGFR、COX-2的表达。 结果： 吉非替尼和塞来昔布均可抑制A549细胞的增殖，可见细胞活性下降，G1期细胞阻滞及细胞凋亡增加；联合用药较单药组A549细胞活性下降更明显，G1期阻滞细胞增加，且细胞凋亡增加。吉非替尼或塞来昔布作用前后A549细胞EGFR表达无变化，但联合用药组p-EGFR及COX-2的表达较单药组显著下降。 结论： 吉非替尼联合塞来昔布可抑制人肺腺癌A549细胞的增殖，其机制可能与抑制EGFR的活化及下调COX-2的表达有关。
2012, 19(4):447-450.
DOI: 10.3872/j.issn.1007-385X.2012.4.019
Abstract:
弥漫性大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）是成人中发病率最高的中高度恶性淋巴瘤，其在免疫学特性和临床表现上具有很大异质性。治疗性单克隆抗体的出现为包括 DLBCL在内的恶性B细胞淋巴瘤的治疗带来了根本性的变化。针对B细胞CD20抗原，继1997年美国FAD首次批准单克隆抗体 Rituximab应用于治疗B细胞淋巴瘤并取得了良好的临床效果之后，又陆续研发出第2代抗体Ofatumumab和Veltuzumab 以及第3代抗体GA-101。同时，一些针对B细胞其他靶位的单克隆抗体，包括Epratuzumab和CMC-544（抗CD22）、 Medi-551（抗CD19）、Dacetuzumab（抗CD40）、Milatuzumab（抗CD74），以及Apolizumab（抗 HLA DR）等，均已进入临床试验阶段。
弥漫性大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）是成人中发病率最高的中高度恶性淋巴瘤，其在免疫学特性和临床表现上具有很大异质性。治疗性单克隆抗体的出现为包括 DLBCL在内的恶性B细胞淋巴瘤的治疗带来了根本性的变化。针对B细胞CD20抗原，继1997年美国FAD首次批准单克隆抗体 Rituximab应用于治疗B细胞淋巴瘤并取得了良好的临床效果之后，又陆续研发出第2代抗体Ofatumumab和Veltuzumab 以及第3代抗体GA-101。同时，一些针对B细胞其他靶位的单克隆抗体，包括Epratuzumab和CMC-544（抗CD22）、 Medi-551（抗CD19）、Dacetuzumab（抗CD40）、Milatuzumab（抗CD74），以及Apolizumab（抗 HLA DR）等，均已进入临床试验阶段。
2012, 19(4):451-456.
DOI: 10.3872/j.issn.1007-385X.2012.4.020
Abstract:
Caspase信号通路包括内源性和外源性凋亡信号通路，目前，caspase成员活性调控的研究己取得了一定的进展，包括凋亡调控相关蛋白（IAP、Bcl-2等）、有关的信号通路（JNK、PI3K-Akt、ERK等）和一系列具有调控作用的小RNA（microRNA，miRNA）。 miRNA是一类最新发现的内源性非编码RNA，参与调节神经系统发育、细胞的分化与增殖，以及疾病的发生、发展等。miRNA可以作为一种“抑癌”或“促癌”基因，参与caspase凋亡过程的调控。参与内源性凋亡信号通路调节的有miR-15a/16-1、miR-181a、miR-21等；参与外源性凋亡信号通路调节的有miR-14、miR-221和miR-222等。有理由相信，干预这些凋亡相关的miRNA，促使癌细胞凋亡，将为癌症的治疗提供新的思路。
Caspase信号通路包括内源性和外源性凋亡信号通路，目前，caspase成员活性调控的研究己取得了一定的进展，包括凋亡调控相关蛋白（IAP、Bcl-2等）、有关的信号通路（JNK、PI3K-Akt、ERK等）和一系列具有调控作用的小RNA（microRNA，miRNA）。 miRNA是一类最新发现的内源性非编码RNA，参与调节神经系统发育、细胞的分化与增殖，以及疾病的发生、发展等。miRNA可以作为一种“抑癌”或“促癌”基因，参与caspase凋亡过程的调控。参与内源性凋亡信号通路调节的有miR-15a/16-1、miR-181a、miR-21等；参与外源性凋亡信号通路调节的有miR-14、miR-221和miR-222等。有理由相信，干预这些凋亡相关的miRNA，促使癌细胞凋亡，将为癌症的治疗提供新的思路。
2012, 19(4):457-460.
DOI: 10.3872/j.issn.1007-385X.2012.4.021
Abstract:
微核糖核酸（microRNA，miRNA）是一类内源性非编码单链小RNA，可在转录后调节靶标mRNA的剪切或抑制mRNA翻译。研究发现，miRNA在多种病理生理过程中发挥重要作用，如细胞增殖、干细胞分化、肿瘤形成等。肿瘤干细胞常含有异常的miRNA，其中某些miRNA的异常结构或表达可影响肿瘤发展。根据miRNA表达异常对肿瘤发生、发展的不同影响，可把miRNA分为癌基因性miRNA与抑癌基因miRNA两类。随着对miRNA了解的深入，发现部分miRNA的表达还具有促进和抑制肿瘤的双向性作用。因此，通过区别miRNA对肿瘤干细胞的不同作用，可利用miRNA靶点治疗肿瘤，即利用抗miRNA疗法阻滞癌基因性miRNA；利用miRNA mimics或慢病毒恢复抑癌基因性miRNA的功能，从而抑制肿瘤发展。相信随着miRNA与肿瘤干细胞的特异性及其作用机制等方面研究的深入，miRNA将会作为一种新的肿瘤调控因子，加快肿瘤治疗的研究进展。
微核糖核酸（microRNA，miRNA）是一类内源性非编码单链小RNA，可在转录后调节靶标mRNA的剪切或抑制mRNA翻译。研究发现，miRNA在多种病理生理过程中发挥重要作用，如细胞增殖、干细胞分化、肿瘤形成等。肿瘤干细胞常含有异常的miRNA，其中某些miRNA的异常结构或表达可影响肿瘤发展。根据miRNA表达异常对肿瘤发生、发展的不同影响，可把miRNA分为癌基因性miRNA与抑癌基因miRNA两类。随着对miRNA了解的深入，发现部分miRNA的表达还具有促进和抑制肿瘤的双向性作用。因此，通过区别miRNA对肿瘤干细胞的不同作用，可利用miRNA靶点治疗肿瘤，即利用抗miRNA疗法阻滞癌基因性miRNA；利用miRNA mimics或慢病毒恢复抑癌基因性miRNA的功能，从而抑制肿瘤发展。相信随着miRNA与肿瘤干细胞的特异性及其作用机制等方面研究的深入，miRNA将会作为一种新的肿瘤调控因子，加快肿瘤治疗的研究进展。
2012, 19(4):461-465.
DOI: 10.3872/j.issn.1007-385X.2012.4.022
Abstract:
乳腺癌的发病率近年呈上升趋势，生物治疗是继手术、放疗和化疗之后治疗乳腺癌的又一手段。曲妥珠单抗作为一线治疗药物，联合化疗治疗血清Her-2+的转移性或晚期乳腺癌患者有显著的临床效果，可以在疾病恶化之前提升总反应率和疾病进展时间；但该单抗药物有心脏毒性，心脏病患者应谨用，如左心室射血分数≤50%应考虑停药。一种新的曲妥珠单抗与细胞毒药物的复合制剂可以增加疗效，降低不良反应。正在研究的乳腺癌生物治疗方法包括树突状细胞治疗、溶瘤病毒治疗、细胞因子治疗、干细胞治疗和基因治疗等。
乳腺癌的发病率近年呈上升趋势，生物治疗是继手术、放疗和化疗之后治疗乳腺癌的又一手段。曲妥珠单抗作为一线治疗药物，联合化疗治疗血清Her-2+的转移性或晚期乳腺癌患者有显著的临床效果，可以在疾病恶化之前提升总反应率和疾病进展时间；但该单抗药物有心脏毒性，心脏病患者应谨用，如左心室射血分数≤50%应考虑停药。一种新的曲妥珠单抗与细胞毒药物的复合制剂可以增加疗效，降低不良反应。正在研究的乳腺癌生物治疗方法包括树突状细胞治疗、溶瘤病毒治疗、细胞因子治疗、干细胞治疗和基因治疗等。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.