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Volume 17,Issue 3,2010 Table of Contents
2010, 17(3):243-249.
DOI: 10.3872/j.issn.1007-385X.2010.3.001
Abstract:
As a promising new strategy for cancer biotherapy, combined therapy of molecular targeted agents and adoptive cellular immunotherapy is established based on the biological characteristics of NK cell activating signal pathway (natural killer group 2 member Dnatural killer group 2 member D ligand, NKG2DNKG2DLs), especially its regulable ligand and receptor theory. Molecular targeted agents play dual functions: in addition to their toxic effects against tumor cells, they can also induce expression of NKG2D ligands on tumor cells, resulting in enhanced immune eradication of NK cells. As a pivotal activating receptor for NK cells, NKG2D and its ligands play important roles in the antitumor immune system, especially adoptive cellmediated immune response. NKG2D is mainly expressed by NK cells, CD8+T cells, γδ T cells and activated macrophages. As for NKG2D ligands, they have restricted expressions in normal tissues, but are frequently expressed on primary tumors. Therefore, expressions of these ligands are closely related to the antitumor effect of immune cells, without damage to the normal tissues. NKG2D ligands can be regulated by multiple stimuli including molecular targeted agents. The immunoregulation induced by molecular targeted agents through NKG2DNKG2DLs pathway has been shown valuable both for its distinguished targeting effects and for its wellestablished regulating effects. In summary, combination of molecular targeted agents and adoptive cellular immunotherapy may represent a bright future for tumor biotherapy.
As a promising new strategy for cancer biotherapy, combined therapy of molecular targeted agents and adoptive cellular immunotherapy is established based on the biological characteristics of NK cell activating signal pathway (natural killer group 2 member Dnatural killer group 2 member D ligand, NKG2DNKG2DLs), especially its regulable ligand and receptor theory. Molecular targeted agents play dual functions: in addition to their toxic effects against tumor cells, they can also induce expression of NKG2D ligands on tumor cells, resulting in enhanced immune eradication of NK cells. As a pivotal activating receptor for NK cells, NKG2D and its ligands play important roles in the antitumor immune system, especially adoptive cellmediated immune response. NKG2D is mainly expressed by NK cells, CD8+T cells, γδ T cells and activated macrophages. As for NKG2D ligands, they have restricted expressions in normal tissues, but are frequently expressed on primary tumors. Therefore, expressions of these ligands are closely related to the antitumor effect of immune cells, without damage to the normal tissues. NKG2D ligands can be regulated by multiple stimuli including molecular targeted agents. The immunoregulation induced by molecular targeted agents through NKG2DNKG2DLs pathway has been shown valuable both for its distinguished targeting effects and for its wellestablished regulating effects. In summary, combination of molecular targeted agents and adoptive cellular immunotherapy may represent a bright future for tumor biotherapy.
HUANG Yuxian
,
WANG Yang
,
LI Yu-hua
,
SONG Chao-yang
,
CHEN Jin-zhang
,
HE Yan-Jie
,
ZHOU Xue-yun
,
GUO Kun-yuan
2010, 17(3):250-255.
DOI: 10.3872/j.issn.1007-385X.2010.3.002
Abstract:
Objective:To investigate molecular targeted agentinduced expressions of natural killer group 2 member D ligands (NKG2D ligands, NKG2DLs) in multidrug resistant nasopharyngeal carcinoma cell line CNE2/DDP with high or low ATPbinding cassette superfamily G member 2 expression (ABCG2 highCNE2/DDP cells or ABCG2lowCNE2/DDP cells) and its effects on their cytotoxic sensitivities to NK cells. Methods: ABCG2highCNE2/DDP cells, ABCG2lowCNE2/DDP cells and NK cells were isolated by magnetic activated cell sorting. Purity of the isolated cells and expression rates of NKG2DLs on ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cells before and after treatment with different molecular targeted agents (bortezomib, sorafenib, sunitinib) were examined by flow cytometry (FCM). Cytotoxic sensitivities of ABCG2highCNE2/DDP and ABCG2 lowCNE2/DDP cells were measured by LDH releasing assay. Results: Expression rates of ABCG2 on ABCG2 highCNE2/DDP and ABCG2lowCNE2/DDP cells were (91.40±2.32)% and (1.70±024)%, respectively. More than 90% of the isolated NK cells were CD3-CD16+CD56+ cells. Expressions of 5 types of NKG2DLs (MICA, MICB, ULBP1, ULBP2 and ULBP3) on untreated ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cells were relatively low, but their expressions were significantly upregulated after treatment with different molecular targeted agents (bortezomib, sorafenib, sunitinib), with the highest NKG2DLs expressions found in the sunitinibtreated group cells. In addition, cytotoxic sensitivities of ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cells to NK cells were increased with the upregulated expressions of NKG2DLs. Conclusion: Different molecular targeted agents can upregulate NKG2DLs expressions in human multidrug resistant nasopharyngeal carcinoma cells, with sunitinib showing the highest induction ability. A linear correlation exists between NKG2DLs expressions and tumor cell cytotoxic sensitivities to NK cells.
Objective:To investigate molecular targeted agentinduced expressions of natural killer group 2 member D ligands (NKG2D ligands, NKG2DLs) in multidrug resistant nasopharyngeal carcinoma cell line CNE2/DDP with high or low ATPbinding cassette superfamily G member 2 expression (ABCG2 highCNE2/DDP cells or ABCG2lowCNE2/DDP cells) and its effects on their cytotoxic sensitivities to NK cells. Methods: ABCG2highCNE2/DDP cells, ABCG2lowCNE2/DDP cells and NK cells were isolated by magnetic activated cell sorting. Purity of the isolated cells and expression rates of NKG2DLs on ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cells before and after treatment with different molecular targeted agents (bortezomib, sorafenib, sunitinib) were examined by flow cytometry (FCM). Cytotoxic sensitivities of ABCG2highCNE2/DDP and ABCG2 lowCNE2/DDP cells were measured by LDH releasing assay. Results: Expression rates of ABCG2 on ABCG2 highCNE2/DDP and ABCG2lowCNE2/DDP cells were (91.40±2.32)% and (1.70±024)%, respectively. More than 90% of the isolated NK cells were CD3-CD16+CD56+ cells. Expressions of 5 types of NKG2DLs (MICA, MICB, ULBP1, ULBP2 and ULBP3) on untreated ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cells were relatively low, but their expressions were significantly upregulated after treatment with different molecular targeted agents (bortezomib, sorafenib, sunitinib), with the highest NKG2DLs expressions found in the sunitinibtreated group cells. In addition, cytotoxic sensitivities of ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cells to NK cells were increased with the upregulated expressions of NKG2DLs. Conclusion: Different molecular targeted agents can upregulate NKG2DLs expressions in human multidrug resistant nasopharyngeal carcinoma cells, with sunitinib showing the highest induction ability. A linear correlation exists between NKG2DLs expressions and tumor cell cytotoxic sensitivities to NK cells.
HUANG Yu-xian
,
WANG Yang
,
LI Yu-hua
,
YANG Yu-lian
,
ZHAO Tong-feng
,
HE Qing-mei
,
LU Hui-fang
,
HE Yan-Jie
,
HUANG Rui
,
GUO Kun-yuan
2010, 17(3):256-261.
DOI: 10.3872/j.issn.1007-385X.2010.3.003
Abstract:
Objective: To investigate the mec hanism by which unitinib induces upregulation of NKG2D ligands(NKG2DLs) expressions in nasopharyngeal carcinoma CNE2/DDP cells with high or low ABCG2 expression (ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells). Methods: Caspase8 activity and mitochondrial membrane potential were detected by caspase8 activity kit and mitochondrial membrane potential assay kit in ABCG2highCNE2/DDP cells or ABCG2low CNE2/DDP cells after cocultured with NK cells. Expressions of signal molecules involved in DNA damage and repair system were detected by RTPCR in ABCG2highCNE2/DDP cells and ABCG2lowCNE2/DDP cells before and after sunitinib treatment. Results: Caspase8 activities in ABCG2highCNE2/DDP cells and ABCG2lowCNE2/DDP cells were ignificantly increased after cocultured with in NK cells. After treatment with sunitinib, caspase8 activities in the co culture system were 1-1.5 times higher than those in the untreated ABCG2high and ABCG2lowCNE2/DDP cells (P<0.01). Sunitinib inhibited mitochondrial membrane potentials of ABCG2high and ABCG2lowCNE2/DDP cells in NK cell coculture systems, with the potentials in two kinds of sunitinibtreated CNE2/DDP cells decreased to (76.58±2.32)% and (73.11±1.93)%, respectively, which were markedly lower than those in the untreated ABCG2high and ABCG2low CNE2/DDP cells (P<0.05). Sunitinib could increase mRNA expressions of ATR, CHK1 and CHK2 in ABCG2high and ABCG2 lowCNE2/DDP cells, and induce P53 and NFκB mRNA expressions. Conclusion: Sunitinib can upregulate NKG2DLs expressions in CNE2/DDP cells by activating signaling molecules related to DNA damage and repair system and NFκB, and enhance NK cellinduced apoptosis of tumor cells through death receptor and mitochondrial pathways.
Objective: To investigate the mec hanism by which unitinib induces upregulation of NKG2D ligands(NKG2DLs) expressions in nasopharyngeal carcinoma CNE2/DDP cells with high or low ABCG2 expression (ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells). Methods: Caspase8 activity and mitochondrial membrane potential were detected by caspase8 activity kit and mitochondrial membrane potential assay kit in ABCG2highCNE2/DDP cells or ABCG2low CNE2/DDP cells after cocultured with NK cells. Expressions of signal molecules involved in DNA damage and repair system were detected by RTPCR in ABCG2highCNE2/DDP cells and ABCG2lowCNE2/DDP cells before and after sunitinib treatment. Results: Caspase8 activities in ABCG2highCNE2/DDP cells and ABCG2lowCNE2/DDP cells were ignificantly increased after cocultured with in NK cells. After treatment with sunitinib, caspase8 activities in the co culture system were 1-1.5 times higher than those in the untreated ABCG2high and ABCG2lowCNE2/DDP cells (P<0.01). Sunitinib inhibited mitochondrial membrane potentials of ABCG2high and ABCG2lowCNE2/DDP cells in NK cell coculture systems, with the potentials in two kinds of sunitinibtreated CNE2/DDP cells decreased to (76.58±2.32)% and (73.11±1.93)%, respectively, which were markedly lower than those in the untreated ABCG2high and ABCG2low CNE2/DDP cells (P<0.05). Sunitinib could increase mRNA expressions of ATR, CHK1 and CHK2 in ABCG2high and ABCG2 lowCNE2/DDP cells, and induce P53 and NFκB mRNA expressions. Conclusion: Sunitinib can upregulate NKG2DLs expressions in CNE2/DDP cells by activating signaling molecules related to DNA damage and repair system and NFκB, and enhance NK cellinduced apoptosis of tumor cells through death receptor and mitochondrial pathways.
HUANG Yu-xian
,
WANG Yang
,
LI Yu-hua
,
SONG Chao-yang
,
CHEN Tu-zhen
,
ZHOU Xuan
,
TU San-fang
,
HE Ying-zhi
,
GUO Kun-yuan
2010, 17(3):262-267.
DOI: 10.3872/j.issn.1007-385X.2010.3.004
Abstract:
Objective:To investigate the effect of sunitinib on NKG2D ligands (NKG2DLs) expressions and its influences on antitumor effect of NK cells. Methods: ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cellimplanted mouse tumor models were established and were divided into the following 8 groups. A, E: inoculated with ABCG2highCNE2/DDP or ABCG2low CNE2/DDP cells; B, F: inoculated with sunitinibstimulated ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells; C, G: inoculated with ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells and NK cells;and D, H: inoculated with sunitinibstimulated ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells and NK cells.Tumor formation times and rates, tumor volumes, and tumor inhibitory rates were observed in different groups. NKG2DLs expressions in implanted tumor tissues were examined by immunohistochemistry assay. Results: Tumor formation times in A, B,C, D, E, F, G, and H groups were (5.43±1.00), (8.50±0.35), (11.10±1.25), (13.56±1.23), (900±100), (12.30±0.78),(14.50±0.50), and (17.25±0.77) d, respectively, with those in sunitinib and NK cell combination groups (D and H groups) being the longest ones (P<0.01). Tumor masses in A, B, C, D, E, F, G, and H groups were (2.63±0.89), (1.00±0.03), (0.65±0.08), (0.21±0.27), (279±0.83), (1.18±0.77), (0.96±0.50), and (0.86±0.82) g, respectively, with those in sunitinib and NK cell combination groups (D and H groups) being the lightest ones (P<0.01); the tumor inhibitory rates in sunitinib and NK cell combination groups (D and H groups) were 62% and 69%. Sunitinib upregulated NKG2DLs expressions in implanted tumor tissues, with those in ABCG2highCNE2/DDP cells higher than those in ABCG2lowCNE2/DDP cells. Conclusion: unitinib can upregulate NKG2DLs expressions in CNE2/DDP cellimplanted tumor tissues in vivo and enhance antitumor effect of NK cells.
Objective:To investigate the effect of sunitinib on NKG2D ligands (NKG2DLs) expressions and its influences on antitumor effect of NK cells. Methods: ABCG2highCNE2/DDP and ABCG2lowCNE2/DDP cellimplanted mouse tumor models were established and were divided into the following 8 groups. A, E: inoculated with ABCG2highCNE2/DDP or ABCG2low CNE2/DDP cells; B, F: inoculated with sunitinibstimulated ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells; C, G: inoculated with ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells and NK cells;and D, H: inoculated with sunitinibstimulated ABCG2highCNE2/DDP cells or ABCG2lowCNE2/DDP cells and NK cells.Tumor formation times and rates, tumor volumes, and tumor inhibitory rates were observed in different groups. NKG2DLs expressions in implanted tumor tissues were examined by immunohistochemistry assay. Results: Tumor formation times in A, B,C, D, E, F, G, and H groups were (5.43±1.00), (8.50±0.35), (11.10±1.25), (13.56±1.23), (900±100), (12.30±0.78),(14.50±0.50), and (17.25±0.77) d, respectively, with those in sunitinib and NK cell combination groups (D and H groups) being the longest ones (P<0.01). Tumor masses in A, B, C, D, E, F, G, and H groups were (2.63±0.89), (1.00±0.03), (0.65±0.08), (0.21±0.27), (279±0.83), (1.18±0.77), (0.96±0.50), and (0.86±0.82) g, respectively, with those in sunitinib and NK cell combination groups (D and H groups) being the lightest ones (P<0.01); the tumor inhibitory rates in sunitinib and NK cell combination groups (D and H groups) were 62% and 69%. Sunitinib upregulated NKG2DLs expressions in implanted tumor tissues, with those in ABCG2highCNE2/DDP cells higher than those in ABCG2lowCNE2/DDP cells. Conclusion: unitinib can upregulate NKG2DLs expressions in CNE2/DDP cellimplanted tumor tissues in vivo and enhance antitumor effect of NK cells.
HUANG Yu-xian
,
WANG Yang
,
LI Yu-hua
,
CHEN Jin-zhang
,
QIAN Min
,
WU Bing-yi
,
SUN Cai-xia
,
DENG Lan
,
GUO Kun-yuan
2010, 17(3):268-273.
DOI: 10.3872/j.issn.1007-385X.2010.3.005
Abstract:
Objective: To investigate the effects of cetuximab on NKG2D ligands (NKG2DLs) expressions in multidrug resistant nasopharyngeal carcinoma CNE2/DDP cells and IFNγ production in NK cells. Methods: EGFR expressions on CNE2/DDP cells with high and low ABCG2 expression (ABCG2highCNE2/DDP cells and ABCG2lowCNE2/DDP cells) and NKG2DLs expressions on ABCG2high and ABCG2lowCNE2/DDP cells before and after cetuximab treatment were detected by flow cytomertry. ABCG2high and ABCG2lowCNE2/DDP cells were cocultured with NK cells before and after cetuximab treatment, and then IFNγ levels in the supernatants of different groups were detected by ELISA. Cytotoxicity of NK cells against CNE2/DDP cells was measured by LDH releasing assay in different groups. Results: EGFR expressions in ABCG2 high and ABCG2lowCNE2/DDP cells were (43.60±2.01)% and (47.20±2.07)%, respectively. The expressions of MICA, MICB, ULBP1, and ULBP2 on ABCG2hgh and ABCG2lowCNE2/DDP cells were upregulated by cetuximab stimulation, while ULBP3 expression on ABCG2highCNE2/DDP cells was downregulated by cetuximab stimulation. IFNγ levels in coculture systems were significantly increased after ABCG2low and ABCG2highCNE2/DDP cells were treated with cetuximab (P<0.01). Cetuximab enhanced cytotoxic sensitivities of ABCG2high and ABCG2lowCNE2/DDP cells in response to NK cells (P<0.01). Conclusion: Cetuximab exerts a dual immunological regulation by upregulating NKG2DLs expressions on nasopharyngeal carcinoma CNE2/DDP cells and stimulating IFNγ production by NK cells indirectly.
Objective: To investigate the effects of cetuximab on NKG2D ligands (NKG2DLs) expressions in multidrug resistant nasopharyngeal carcinoma CNE2/DDP cells and IFNγ production in NK cells. Methods: EGFR expressions on CNE2/DDP cells with high and low ABCG2 expression (ABCG2highCNE2/DDP cells and ABCG2lowCNE2/DDP cells) and NKG2DLs expressions on ABCG2high and ABCG2lowCNE2/DDP cells before and after cetuximab treatment were detected by flow cytomertry. ABCG2high and ABCG2lowCNE2/DDP cells were cocultured with NK cells before and after cetuximab treatment, and then IFNγ levels in the supernatants of different groups were detected by ELISA. Cytotoxicity of NK cells against CNE2/DDP cells was measured by LDH releasing assay in different groups. Results: EGFR expressions in ABCG2 high and ABCG2lowCNE2/DDP cells were (43.60±2.01)% and (47.20±2.07)%, respectively. The expressions of MICA, MICB, ULBP1, and ULBP2 on ABCG2hgh and ABCG2lowCNE2/DDP cells were upregulated by cetuximab stimulation, while ULBP3 expression on ABCG2highCNE2/DDP cells was downregulated by cetuximab stimulation. IFNγ levels in coculture systems were significantly increased after ABCG2low and ABCG2highCNE2/DDP cells were treated with cetuximab (P<0.01). Cetuximab enhanced cytotoxic sensitivities of ABCG2high and ABCG2lowCNE2/DDP cells in response to NK cells (P<0.01). Conclusion: Cetuximab exerts a dual immunological regulation by upregulating NKG2DLs expressions on nasopharyngeal carcinoma CNE2/DDP cells and stimulating IFNγ production by NK cells indirectly.
2010, 17(3):274-279.
DOI: 10.3872/j.issn.1007-385X.2010.3.006
Abstract:
To isolate and identify lung cancer stemlike cells from large cell lung cancer specimens, so as to lay a foundation for immunotherapy targeting lung cancer stem cells. Methods: Cancer cells were isolated and cultured from large cell lung cancer specimens using serumfree medium, and a large cell lung cancer cell line was established. Lung cancer subset cells expressing different degrees of CD44 and (or) CD90 were sorted by flow cytometry. Biologic characters of the different subsets were studied by single cell clone formation assay, plat colony formation assay, cell sphere formation assay, and Xray radiation sensitive assay. Results: Highly purified primary cancer cells were successfully cultured from large cell lung cancer specimens using primary cell culture technology, and the cells, named LC006, could be passaged for more than 30 generations. Flow cytometry results showed that 1.0% of LC006 cells expressed stronger CD44（CD44++LC006 cells）than the main population, which was weakly positive for CD44 (CD44+LC006 cells). CD44++LC006 cells had stronger colongy formation ability and could form holoclones and cell spheres; however, CD4+LC006 cells formed neither holoclones nor cell spheres, indicating that the CD44++LC006 cells contained cancer stem cells. When CD44 and CD90 antibodies were costained, LC006 cells could be individuated into 4 subsets, i.e. CD44+CD90+ (12.8%), CD44+CD90-(86.3%), CD44++CD90+(0.4%), and CD44++CD90-(0.5%) LC006 cells; CD44++CD90+LC006 cells showed strongest cell sphere formation ability and Xray resistant ability than the other three subsets. These results indicated that CD90 might be used as another cancer stemlike cell marker for large cell lung cancer. Conclusion: CD44++CD90+LC006 cells are successfully isolated and identified from large cell lung cancer, which may be the cancer stemlike cells of large cell lung cancer.
To isolate and identify lung cancer stemlike cells from large cell lung cancer specimens, so as to lay a foundation for immunotherapy targeting lung cancer stem cells. Methods: Cancer cells were isolated and cultured from large cell lung cancer specimens using serumfree medium, and a large cell lung cancer cell line was established. Lung cancer subset cells expressing different degrees of CD44 and (or) CD90 were sorted by flow cytometry. Biologic characters of the different subsets were studied by single cell clone formation assay, plat colony formation assay, cell sphere formation assay, and Xray radiation sensitive assay. Results: Highly purified primary cancer cells were successfully cultured from large cell lung cancer specimens using primary cell culture technology, and the cells, named LC006, could be passaged for more than 30 generations. Flow cytometry results showed that 1.0% of LC006 cells expressed stronger CD44（CD44++LC006 cells）than the main population, which was weakly positive for CD44 (CD44+LC006 cells). CD44++LC006 cells had stronger colongy formation ability and could form holoclones and cell spheres; however, CD4+LC006 cells formed neither holoclones nor cell spheres, indicating that the CD44++LC006 cells contained cancer stem cells. When CD44 and CD90 antibodies were costained, LC006 cells could be individuated into 4 subsets, i.e. CD44+CD90+ (12.8%), CD44+CD90-(86.3%), CD44++CD90+(0.4%), and CD44++CD90-(0.5%) LC006 cells; CD44++CD90+LC006 cells showed strongest cell sphere formation ability and Xray resistant ability than the other three subsets. These results indicated that CD90 might be used as another cancer stemlike cell marker for large cell lung cancer. Conclusion: CD44++CD90+LC006 cells are successfully isolated and identified from large cell lung cancer, which may be the cancer stemlike cells of large cell lung cancer.
2010, 17(3):280-284.
DOI: 10.3872/j.issn.1007-385X.2010.3.007
Abstract:
Objective:To investigate the structural domains and sites of P73 which can be phosphorylated by polo like kinases 3 (Plk3), and to analyze the effect of Plk3 on P73mediated apoptosis. Methods: Coimmunoprecipitation experiment was used to examine the interaction between Plk3 and P73. Immunofluorescence was used to examine the localization of Plk3 and P73 in cells. Different deletion mutants of GSTP73 fusion protein were prepared. A sitemutation plasmid of GSTP73 (1130) was constructed by converting threonine86 to alanine86 (T86A) and was named GSTP73 (1130) (T86A). The phosphorylated domains and sites of P73 by Plk3 were determined by in vitro phosphorylation assay. The effect of Plk3 on P73mediated apoptosis of HeLa cells was examined by cleaved PARP detection. Results: Plk3 could interact with P73; Plk3 and P73 colocated in the cell nuclei. Different deletion mutants of GSTP73 fusion protein were successfully prepared, and Plk3 phosphorylated P73 at Nterminal 63113 amino residues. Point mutation (T86A) of GSTP73 (1130) fusion protein could not influence the phosphorylation status of P73 by Plk3. Furthermore, Plk3 inhibited P73mediated apoptosis in HeLa cells. Conclusion: Plk3 can interact with and phosphorylate P73 at Nterminal 63113 amino residues (but not at the 86 threonine), thereby inhibiting P73mediated apoptosis of HeLa cells.
Objective:To investigate the structural domains and sites of P73 which can be phosphorylated by polo like kinases 3 (Plk3), and to analyze the effect of Plk3 on P73mediated apoptosis. Methods: Coimmunoprecipitation experiment was used to examine the interaction between Plk3 and P73. Immunofluorescence was used to examine the localization of Plk3 and P73 in cells. Different deletion mutants of GSTP73 fusion protein were prepared. A sitemutation plasmid of GSTP73 (1130) was constructed by converting threonine86 to alanine86 (T86A) and was named GSTP73 (1130) (T86A). The phosphorylated domains and sites of P73 by Plk3 were determined by in vitro phosphorylation assay. The effect of Plk3 on P73mediated apoptosis of HeLa cells was examined by cleaved PARP detection. Results: Plk3 could interact with P73; Plk3 and P73 colocated in the cell nuclei. Different deletion mutants of GSTP73 fusion protein were successfully prepared, and Plk3 phosphorylated P73 at Nterminal 63113 amino residues. Point mutation (T86A) of GSTP73 (1130) fusion protein could not influence the phosphorylation status of P73 by Plk3. Furthermore, Plk3 inhibited P73mediated apoptosis in HeLa cells. Conclusion: Plk3 can interact with and phosphorylate P73 at Nterminal 63113 amino residues (but not at the 86 threonine), thereby inhibiting P73mediated apoptosis of HeLa cells.
GUO Xiao-ling
,
WEI Ya
,
ZHU Ping
,
NIU Zhi-yun
,
CAI Sheng-xin
,
ZHANG Gai-ling
,
DA Wan-ming
,
PAN Ling
2010, 17(3):285-291.
DOI: 10.3872/j.issn.1007-385X.2010.3.008
Abstract:
Objective:To construct a DCCIK (cytokineinduced killer cell) coculture system using peripheral blood mononuclear cells (PBMCs) derivedDC from hematopoietic stem cell transplantation (HSCT) patients with posttransplant lympholiferative disorder (PTLD) after pulsed with EBVspecial peptides, so as to lay a foundation for new adoptive immunotherapy of patients with PLTD after HSCT. Methods: PBMCs were obtained from patients with PTLD after HSCT; DC was induced from adherent cells; and CIK was induced from suspension cells. DC was further pulsed with EBVspecial peptides and cocultured with CIK to establish the DCCIK coculture system; the immunophenotype of cells in DCCIK system before and after coculture were determined by FACS, IFNγ secretion was assayed by ELISA, and TCRβ genealogy was examined by genetic analyzer. Results: The ratio of HLADR+CD86+DC increased from 125% to 91.17% after cytokine stimulation. After coculture with DC for 14 d, the numbers of CIK in two patients with PTLD increased to 5.3 and 6.8 times,respectively. The ratios of CD3+, CD8+, CD3+CD8+, and CD3+CD56+ cells were significantly increased after DCCIK coculture. IFNγ level in peptidepulsed DCCIK group was significantly higher than that in peptideunpulsed DCCIK group（\[1 332.6±92.38\] pg/ml vs \[693.42±62.41\] pg/ml，P<0.05\]）; TCRβ genealogy assay found the clone expansion peak of 5.2 TCRβ subfamily in DCCIK coculture system. Conclusion: EBV peptidepulsed DC can induce CD3+CD8+ and CD3+CD56+ cell expansion in DCCIK coculture system with high level of IFNγ. DCCIK can be used as a new adoptive immunotherapy to HSCT patients with EBV infection and PTLD.
Objective:To construct a DCCIK (cytokineinduced killer cell) coculture system using peripheral blood mononuclear cells (PBMCs) derivedDC from hematopoietic stem cell transplantation (HSCT) patients with posttransplant lympholiferative disorder (PTLD) after pulsed with EBVspecial peptides, so as to lay a foundation for new adoptive immunotherapy of patients with PLTD after HSCT. Methods: PBMCs were obtained from patients with PTLD after HSCT; DC was induced from adherent cells; and CIK was induced from suspension cells. DC was further pulsed with EBVspecial peptides and cocultured with CIK to establish the DCCIK coculture system; the immunophenotype of cells in DCCIK system before and after coculture were determined by FACS, IFNγ secretion was assayed by ELISA, and TCRβ genealogy was examined by genetic analyzer. Results: The ratio of HLADR+CD86+DC increased from 125% to 91.17% after cytokine stimulation. After coculture with DC for 14 d, the numbers of CIK in two patients with PTLD increased to 5.3 and 6.8 times,respectively. The ratios of CD3+, CD8+, CD3+CD8+, and CD3+CD56+ cells were significantly increased after DCCIK coculture. IFNγ level in peptidepulsed DCCIK group was significantly higher than that in peptideunpulsed DCCIK group（\[1 332.6±92.38\] pg/ml vs \[693.42±62.41\] pg/ml，P<0.05\]）; TCRβ genealogy assay found the clone expansion peak of 5.2 TCRβ subfamily in DCCIK coculture system. Conclusion: EBV peptidepulsed DC can induce CD3+CD8+ and CD3+CD56+ cell expansion in DCCIK coculture system with high level of IFNγ. DCCIK can be used as a new adoptive immunotherapy to HSCT patients with EBV infection and PTLD.
2010, 17(3):292-296.
DOI: 10.3872/j.issn.1007-385X.2010.3.009
Abstract:
Objective:To study the effects of JWA (ADPribosylationlike factor 6 interacting protein 5, ARL6IP5) gene on expression and function of Pglycoprotein in tumor cells. Methods: JWAshRNA and controlshRNA plasmids were transfected into human choriocarcinoma cell line JAR and human breast cancer cell line MCF7,and flagJWA and flagcontrol plasmids were transfected into etoposide (VP16)resistant JAR cells (JAR/VP16) by liposomemediated transfection assay. JWA and Pglycoprotein expressions were examined by Western blotting analysis. Intercellular retention of rhodamine 123 (Rh123) was determined by FCM. Results: After JWA expression was downregulated by JWAshRNA transfection, Pglycoprotein expression was increased in JAR and MCF7 cells, and Rh123 retention was decreased. After JWA was overexpressed by flagJWA transfection, Pglycoprotein expression was decreased in JAR /VP16 cells and Rh123 retention was increased. Conclusion: JWA gene can regulate the expression and transportation of Pglycoprotein in tumor cells.
Objective:To study the effects of JWA (ADPribosylationlike factor 6 interacting protein 5, ARL6IP5) gene on expression and function of Pglycoprotein in tumor cells. Methods: JWAshRNA and controlshRNA plasmids were transfected into human choriocarcinoma cell line JAR and human breast cancer cell line MCF7,and flagJWA and flagcontrol plasmids were transfected into etoposide (VP16)resistant JAR cells (JAR/VP16) by liposomemediated transfection assay. JWA and Pglycoprotein expressions were examined by Western blotting analysis. Intercellular retention of rhodamine 123 (Rh123) was determined by FCM. Results: After JWA expression was downregulated by JWAshRNA transfection, Pglycoprotein expression was increased in JAR and MCF7 cells, and Rh123 retention was decreased. After JWA was overexpressed by flagJWA transfection, Pglycoprotein expression was decreased in JAR /VP16 cells and Rh123 retention was increased. Conclusion: JWA gene can regulate the expression and transportation of Pglycoprotein in tumor cells.
ZHANG Huan-ling
,
WANG Jun-xia
,
YOU Hong-yu
,
LIU Jian-min
,
ZHENG Long
,
LIAN Wei-guang
,
LIU Fu-ying
2010, 17(3):297-301.
DOI: 10.3872/j.issn.1007-385X.2010.3.010
Abstract:
Objective:To observe the targeting proapoptotic effect of expression vector containing AFPregulation sequence on AFP positive hepatoma cells. Methods: AFP promoter, silencer and the most remote enhancer Ⅲ were ligated to construct a 12 kb AFP regulation sequence, which was then used to construct a pAFPEGFP plasmid. The pAFPEGFP was used to transfect human hepatoma HepG2 (AFP positive), human hepatoma SMMC7721 (AFP negative) and human cervical carcinoma HeLa (AFP negative) cells; the fluorescent protein expression intensities were observed under fluorescence microscope. A pAFPP53EGFP plasmid was further constructed by inserting P53 gene into pAFPEGFP, which was then transfected into HepG2, SMMC7721 and HeLa cells. P53 protein expressions were detected by Western blotting analysis in different groups; apoptosis rates and cell cycles were examined by flow cytometry. Results: pAFPEGFP and pAFPP53EGFP recombinant plasmids were successfully constructed. The expression of EGFP fluorescent protein in pAFPEGFPtransfected AFP positive HepG2 cells was significant higher than those in AFP negative SMMC7721 and HeLa cells; P53 protein expression in HepG2 cells transfected with pAFPP53EGFP was also significantly higher than those in SMMC7721 and HeLa cells. The G1 phase proportion and apoptosis rate of pAFPP53EGFPtransfected HepG2 cells were significantly higher than those of SMMC7721 and HeLa cells (\[66.7±0.25\]% vs \[50.5±0.18\]%, \[51.0±020\]%, P<0.05; \[2.65±0.08\]% vs \[0.42±0.03\]%, \[0.39±0.02\]%, P<0.05), but S phase proportion of pAFPP53EGFPtransfected HepG2 cells was significantly lower than those of SMMC7721 and HeLa cells (\[20.1±022\]% vs \[29.8±0.18\]%, \[37.8±0.21\]%, P<0.05). Conclusion: The pAFPP53EGFP plasmid containing AFP regulation sequence can specifically target AFP positive hepatoma cells, inducing cell cycle arrest and apoptosis of hepatoma cells.
Objective:To observe the targeting proapoptotic effect of expression vector containing AFPregulation sequence on AFP positive hepatoma cells. Methods: AFP promoter, silencer and the most remote enhancer Ⅲ were ligated to construct a 12 kb AFP regulation sequence, which was then used to construct a pAFPEGFP plasmid. The pAFPEGFP was used to transfect human hepatoma HepG2 (AFP positive), human hepatoma SMMC7721 (AFP negative) and human cervical carcinoma HeLa (AFP negative) cells; the fluorescent protein expression intensities were observed under fluorescence microscope. A pAFPP53EGFP plasmid was further constructed by inserting P53 gene into pAFPEGFP, which was then transfected into HepG2, SMMC7721 and HeLa cells. P53 protein expressions were detected by Western blotting analysis in different groups; apoptosis rates and cell cycles were examined by flow cytometry. Results: pAFPEGFP and pAFPP53EGFP recombinant plasmids were successfully constructed. The expression of EGFP fluorescent protein in pAFPEGFPtransfected AFP positive HepG2 cells was significant higher than those in AFP negative SMMC7721 and HeLa cells; P53 protein expression in HepG2 cells transfected with pAFPP53EGFP was also significantly higher than those in SMMC7721 and HeLa cells. The G1 phase proportion and apoptosis rate of pAFPP53EGFPtransfected HepG2 cells were significantly higher than those of SMMC7721 and HeLa cells (\[66.7±0.25\]% vs \[50.5±0.18\]%, \[51.0±020\]%, P<0.05; \[2.65±0.08\]% vs \[0.42±0.03\]%, \[0.39±0.02\]%, P<0.05), but S phase proportion of pAFPP53EGFPtransfected HepG2 cells was significantly lower than those of SMMC7721 and HeLa cells (\[20.1±022\]% vs \[29.8±0.18\]%, \[37.8±0.21\]%, P<0.05). Conclusion: The pAFPP53EGFP plasmid containing AFP regulation sequence can specifically target AFP positive hepatoma cells, inducing cell cycle arrest and apoptosis of hepatoma cells.
2010, 17(3):302-307.
DOI: 10.3872/j.issn.1007-385X.2010.3.011
Abstract:
Objective:To study the effects of survivinpromoterregulated survivinIGF1RmiR30 (surIGFIRmiR30) lentiviral vector on the IGF1R expression and proliferation of hepatoma Hep3B cells. Methods：Survivin promoter was amplified by PCR and surpPRIME plasmid was constructed. Interference sequence targeting IGF1R gene was synthesized and cloned into surpPRIME plasmid, named surIGF1RmiR30. SurIGF1RmiR30, psPAX2, and pMD2G were cotransfected into 293T cells to amplify lentivirus, and the lentivirus titer was examined. IGF1R expression in Hep3B cells was detected by RTPCR and Western blotting analysis, and the proliferation of Hep3B cells was evaluated by CCK8 assay. Results：SurIGF1RmiR30 lentiviral vector regulated by survivin promoter were successfully constructed, and the virus titer was 4.58×109 PFU/ml. Fluorescent protein after surIGF1RmiR30 infection was expressed in Hep3B cells, but not in L02 cells. SurIGF1RmiR30 infection inhibited IGF1R mRNA and protein expressions in Hep3B cells and the proliferation of Hep3B cells, with the inhibitory rate being 60％ at 7 d (P<0.05). Conclusion: SurIGF1RmiR30 lentiviral vector can inhibit IGF1R expression and hepatoma cell proliferation.
Objective:To study the effects of survivinpromoterregulated survivinIGF1RmiR30 (surIGFIRmiR30) lentiviral vector on the IGF1R expression and proliferation of hepatoma Hep3B cells. Methods：Survivin promoter was amplified by PCR and surpPRIME plasmid was constructed. Interference sequence targeting IGF1R gene was synthesized and cloned into surpPRIME plasmid, named surIGF1RmiR30. SurIGF1RmiR30, psPAX2, and pMD2G were cotransfected into 293T cells to amplify lentivirus, and the lentivirus titer was examined. IGF1R expression in Hep3B cells was detected by RTPCR and Western blotting analysis, and the proliferation of Hep3B cells was evaluated by CCK8 assay. Results：SurIGF1RmiR30 lentiviral vector regulated by survivin promoter were successfully constructed, and the virus titer was 4.58×109 PFU/ml. Fluorescent protein after surIGF1RmiR30 infection was expressed in Hep3B cells, but not in L02 cells. SurIGF1RmiR30 infection inhibited IGF1R mRNA and protein expressions in Hep3B cells and the proliferation of Hep3B cells, with the inhibitory rate being 60％ at 7 d (P<0.05). Conclusion: SurIGF1RmiR30 lentiviral vector can inhibit IGF1R expression and hepatoma cell proliferation.
2010, 17(3):308-312.
DOI: 10.3872/j.issn.1007-385X.2010.3.012
Abstract:
Objective: To explore the regulatory effect of autocrined VEGF on membranebound complement regulatory proteins (mCRPs) expression in lung cancer A549 cells and the involved mechanisms. Methods: mRNA expressions of CD46, CD55,CD59, VEGF and their receptors (KDR and FLT1), and IL8 and its receptors (CXCR1 CXCR2) in A549 cells were detected by RTPCR. The effects of antiVEGF antibody and antiIL8 antibody on the proliferation and mCRPs expression in A549 cells were examined by MTT assay and flow cytometry, respectively. The effects of antiVEGF antibody on the expression of transcription factor KLF2 and phosphoNFκB p65 were examined by Western blotting analysis. Results: In addition to membrane CD46, CD55 and CD59 mRNA, both VEGF, IL8 and their receptors (KDR, FLT1; CXCR1, CXCR2) mRNA were expressed in A549 cells. AntiVEGF antibody significantly inhibited the proliferation of A549 cells (P<0.05). CD55 and CD59 mRNA and protein levels in A549 cells were decreased after treatment with 0.1 μg/ml antiVEGF antibody for 72 h (P<0.05). The relative values of KLF2 in cytoplasm and nuclear decreased from 0.63 and 0.88 to 0.42 and 0.66 after treatment with antiVEGF antibody, while those of phosphoNFκB p65 decreased from 0.44 and 0.28 to 0.32 and 019. Conclusion: VEGF may enhance CD55 and CD59 expressions in A549 cells through upregulating expressions of NFκB P65 and KLF2 transcription factors in an autocrine manner.
Objective: To explore the regulatory effect of autocrined VEGF on membranebound complement regulatory proteins (mCRPs) expression in lung cancer A549 cells and the involved mechanisms. Methods: mRNA expressions of CD46, CD55,CD59, VEGF and their receptors (KDR and FLT1), and IL8 and its receptors (CXCR1 CXCR2) in A549 cells were detected by RTPCR. The effects of antiVEGF antibody and antiIL8 antibody on the proliferation and mCRPs expression in A549 cells were examined by MTT assay and flow cytometry, respectively. The effects of antiVEGF antibody on the expression of transcription factor KLF2 and phosphoNFκB p65 were examined by Western blotting analysis. Results: In addition to membrane CD46, CD55 and CD59 mRNA, both VEGF, IL8 and their receptors (KDR, FLT1; CXCR1, CXCR2) mRNA were expressed in A549 cells. AntiVEGF antibody significantly inhibited the proliferation of A549 cells (P<0.05). CD55 and CD59 mRNA and protein levels in A549 cells were decreased after treatment with 0.1 μg/ml antiVEGF antibody for 72 h (P<0.05). The relative values of KLF2 in cytoplasm and nuclear decreased from 0.63 and 0.88 to 0.42 and 0.66 after treatment with antiVEGF antibody, while those of phosphoNFκB p65 decreased from 0.44 and 0.28 to 0.32 and 019. Conclusion: VEGF may enhance CD55 and CD59 expressions in A549 cells through upregulating expressions of NFκB P65 and KLF2 transcription factors in an autocrine manner.
2010, 17(3):313-317.
DOI: 10.3872/j.issn.1007-385X.2010.3.013
Abstract:
Objective:To study the efficacy of Schisandra Chinensis (SC) extract in prevention of lymphocyte reduction caused by radiation and to explore the related mechanism. Methods: A total of 48 BALB/c mice were divided into 4 groups: SC treated group (SC+ray), NS control group (NS+ray), SC control group (SC), and normal control group (NS). After BALB/c mice were radiated with 6 Gy, the WBC and lymphocyte numbers were counted; T lymphocyte subsets in peripheral blood were detected by immunofluorescence cell staining; morphology of thymocytes was observed by Gimsa staining; lymphocyte subsets and apoptosis rate of thymocytes were examined by flow cytometry; Bcl2 and Fas mRNA expressions in thymus tissues were measured by RTPCR. Results: In NS+ray group, the quantities of WBC, lymphocytes, and lymphocyte subsets were significantly decreased after radiation (P<0.01), but in SC+ray group, the numbers of WBC and lymphocytes were significantly increased compared with those in NS+ray group (P<0.01). The percentages of CD3+, CD4+, and CD8+ T cells in peripheral blood were significantly increased in SC+ray group compared with those in NS+ray group (P<0.01). The quantity of thymocytes was decreased and apoptosis of thymocytes was increased in NS+ray group; cell apoptosis rate in SC+ray group was significantly increased than that in NS+ray group (\[2.87±103\]% vs \[21.32±2.56\]%, P<0.01). The expression of Bcl2 mRNA was significantly downregulated and Fas mRNA was upregulated in NS+ray group (P<0.01); Bcl2 mRNA was upregulated and Fas mRNA was significantly downregulated in SC+ray group compared with those in NS+ray group (P<0.01). Conclusion: SC can prevent radiationinduced lymphocyte reduction through regulating apoptosis of thymocytes, which is mediated by upregulation of Bcl2 and downregulation of Fas.
Objective:To study the efficacy of Schisandra Chinensis (SC) extract in prevention of lymphocyte reduction caused by radiation and to explore the related mechanism. Methods: A total of 48 BALB/c mice were divided into 4 groups: SC treated group (SC+ray), NS control group (NS+ray), SC control group (SC), and normal control group (NS). After BALB/c mice were radiated with 6 Gy, the WBC and lymphocyte numbers were counted; T lymphocyte subsets in peripheral blood were detected by immunofluorescence cell staining; morphology of thymocytes was observed by Gimsa staining; lymphocyte subsets and apoptosis rate of thymocytes were examined by flow cytometry; Bcl2 and Fas mRNA expressions in thymus tissues were measured by RTPCR. Results: In NS+ray group, the quantities of WBC, lymphocytes, and lymphocyte subsets were significantly decreased after radiation (P<0.01), but in SC+ray group, the numbers of WBC and lymphocytes were significantly increased compared with those in NS+ray group (P<0.01). The percentages of CD3+, CD4+, and CD8+ T cells in peripheral blood were significantly increased in SC+ray group compared with those in NS+ray group (P<0.01). The quantity of thymocytes was decreased and apoptosis of thymocytes was increased in NS+ray group; cell apoptosis rate in SC+ray group was significantly increased than that in NS+ray group (\[2.87±103\]% vs \[21.32±2.56\]%, P<0.01). The expression of Bcl2 mRNA was significantly downregulated and Fas mRNA was upregulated in NS+ray group (P<0.01); Bcl2 mRNA was upregulated and Fas mRNA was significantly downregulated in SC+ray group compared with those in NS+ray group (P<0.01). Conclusion: SC can prevent radiationinduced lymphocyte reduction through regulating apoptosis of thymocytes, which is mediated by upregulation of Bcl2 and downregulation of Fas.
2010, 17(3):318-321.
DOI: 10.3872/j.issn.1007-385X.2010.3.014
Abstract:
Objective:To study the effect of polypeptide A28, which was designed by computer aided drug designing system, on the cytotoxic effect of cisplatin against colon cancer cells. Methods: Colon cancer cell line HCT116 and human umbilical vein endothelial cells (HUVEC) were used in the present study. The concentration of polypeptide A28 was 20 μmol /L and those of cisplatin were 10, 30 and 90 μmol/L. The effects of polypeptide A28 combined with cisplatin on the growth of HCT116 and HUVEC cells were measured by MTT; their effects on the apoptosis of HCT116 cells were examined by flow cytometry. Results：Cisplatin dosedependently inhibited proliferation of HCT116 cells; A28 further enhanced the inhibitory effect of cisplatin on HCT116 cells and increased apoptosis induction effect of cisplatin on HCT116 cells, with the growth inhibition rate of the combination group being (43.3±0.03)%, which was significantly higher than that of the cisplatin single group (15.6±0.10)% (P<0.01). In combination group, when cisplatin concentrations (30, 90 μmol/L) were increased, the inhibitory effects on HCT116 cells were not increased compared with the 10 μmol/L cisplatin combination group (P>0.05). A28 combined with cisplatin (1.1, 3.3, 10, 30, or 90 μmol/L) induced apoptosis of more HCT116 cells than cisplatin single group did (P<0.01). Csplatin at 10 μmol/L combined with A28 at 20 μmol/L effectively killed HCT116 cells, whereas with less toxic effect on HUVEC cells. Conclusion：Polypeptide A28 can enhance the cytotoxic effect of cisplatin on colon cancer cell line HCT116 and decrease its lethal effect on HUVEC.
Objective:To study the effect of polypeptide A28, which was designed by computer aided drug designing system, on the cytotoxic effect of cisplatin against colon cancer cells. Methods: Colon cancer cell line HCT116 and human umbilical vein endothelial cells (HUVEC) were used in the present study. The concentration of polypeptide A28 was 20 μmol /L and those of cisplatin were 10, 30 and 90 μmol/L. The effects of polypeptide A28 combined with cisplatin on the growth of HCT116 and HUVEC cells were measured by MTT; their effects on the apoptosis of HCT116 cells were examined by flow cytometry. Results：Cisplatin dosedependently inhibited proliferation of HCT116 cells; A28 further enhanced the inhibitory effect of cisplatin on HCT116 cells and increased apoptosis induction effect of cisplatin on HCT116 cells, with the growth inhibition rate of the combination group being (43.3±0.03)%, which was significantly higher than that of the cisplatin single group (15.6±0.10)% (P<0.01). In combination group, when cisplatin concentrations (30, 90 μmol/L) were increased, the inhibitory effects on HCT116 cells were not increased compared with the 10 μmol/L cisplatin combination group (P>0.05). A28 combined with cisplatin (1.1, 3.3, 10, 30, or 90 μmol/L) induced apoptosis of more HCT116 cells than cisplatin single group did (P<0.01). Csplatin at 10 μmol/L combined with A28 at 20 μmol/L effectively killed HCT116 cells, whereas with less toxic effect on HUVEC cells. Conclusion：Polypeptide A28 can enhance the cytotoxic effect of cisplatin on colon cancer cell line HCT116 and decrease its lethal effect on HUVEC.
2010, 17(3):322-326.
DOI: 10.3872/j.issn.1007-385X.2010.3.015
Abstract:
Objective:To investigate the effect of COX2 inhibitor celecoxib on induction of apoptosis of hepatoma carcinoma SMMC7721 cell line and the possible mechanism. Methods: SMMC7721 cells were treated with 10, 25, 50, 75 and 100 μmol/L celecoxib; their proliferation was examined by MTT assay; typical apoptotic morphology was studied by Hoechst 33342/PI double staining; and cell cycle and apoptosis rate were detected by flow cytometry. Fas and Bcl2 mRNA expressions were examined by RTPCR, and their protein expressions were detected by Western blotting analysis. Results: Celecoxib dosedependently inhibited the growth of SMMC7721 cells. SMMC7721 cells showed typical apoptotic morphology features, such as nuclear chromatin condensation and nuclear membrane rupture, after celecoxib treatment. Apoptotic rates of SMMC7721 cells treated with 25, 50, and 100 μmol/L celecoxib were (16.32±2.32)%, (38.05±247)%, and (71.17±3.19)%, respectively;celecoxib induced cell cycle arrest of SMMC7721 cells in G0/G1 phase. Fas mRNA and protein expressions in celecoxibtreated SMMC7721 cells were higher than those in the untreated cells (P<001); the expression of Bcl2 mRNA was not affected by celecoxib stimulation (P>0.05), but Bcl2 protein expression was significantly downregulated (P<0.01). Conclusion：Celecoxib can inhibit the proliferation and induce apoptosis of SMMC7721 cells, possibly by regulating the expression of apoptosis related genes.
Objective:To investigate the effect of COX2 inhibitor celecoxib on induction of apoptosis of hepatoma carcinoma SMMC7721 cell line and the possible mechanism. Methods: SMMC7721 cells were treated with 10, 25, 50, 75 and 100 μmol/L celecoxib; their proliferation was examined by MTT assay; typical apoptotic morphology was studied by Hoechst 33342/PI double staining; and cell cycle and apoptosis rate were detected by flow cytometry. Fas and Bcl2 mRNA expressions were examined by RTPCR, and their protein expressions were detected by Western blotting analysis. Results: Celecoxib dosedependently inhibited the growth of SMMC7721 cells. SMMC7721 cells showed typical apoptotic morphology features, such as nuclear chromatin condensation and nuclear membrane rupture, after celecoxib treatment. Apoptotic rates of SMMC7721 cells treated with 25, 50, and 100 μmol/L celecoxib were (16.32±2.32)%, (38.05±247)%, and (71.17±3.19)%, respectively;celecoxib induced cell cycle arrest of SMMC7721 cells in G0/G1 phase. Fas mRNA and protein expressions in celecoxibtreated SMMC7721 cells were higher than those in the untreated cells (P<001); the expression of Bcl2 mRNA was not affected by celecoxib stimulation (P>0.05), but Bcl2 protein expression was significantly downregulated (P<0.01). Conclusion：Celecoxib can inhibit the proliferation and induce apoptosis of SMMC7721 cells, possibly by regulating the expression of apoptosis related genes.
2010, 17(3):327-332.
DOI: 10.3872/j.issn.1007-385X.2010.3.016
Abstract:
Objective:To investigate the antitumor activity of cytokineinduced killer cells (CIKs) against cervical cancer cell lines, CasKi and HeLa in vitro and in vivo. Methods: The CIKs were induced from peripheral blood mononuclear cells (PBMCs) of patients with cervical cancer using an improved method, which only used IL2 and antiCD3 antibody, without IFNγ; CIKs were sorted using FACS. The levels of IFNγ, IL2 and TNFα in culture supernatants of CIKs were determined by ELISA. The antitumor activities of CIKs against CasKi and HeLa cells were determined by LDH assay. The nude mouse xenograft models of cervical cancer cell lines, CasKi or HeLa cells, were established, and 1×106 or 1×107 CIKs were administered intravenously once a week for three weeks, then the tumor volumes and weights were measured.Results: CIKs were successfully induced from PBMCs of cervical cancer patients by IL2 and antiCD3 antibody, with CD3+CD56+ cells greatly expanded. CIKs produced significant amounts of IFNγ and TNFα, but few IL2, after PHA stimulation. CIKs dosedependently killed CasKi and Hela cells with a maximum killing rate reaching 43% and 46%, respectively. In addition, the in vivo antitumor experiments demonstrated that CIKs remarkably inhibited the growth of subcutaneous tumors in nude mice (P<0.01). Conclusion: The improved CIKs expansion method used in our study is feasible and the resultant CIKs have remarkable cytotoxicity against Caski and HeLa cells both in vivo and in vitro.
Objective:To investigate the antitumor activity of cytokineinduced killer cells (CIKs) against cervical cancer cell lines, CasKi and HeLa in vitro and in vivo. Methods: The CIKs were induced from peripheral blood mononuclear cells (PBMCs) of patients with cervical cancer using an improved method, which only used IL2 and antiCD3 antibody, without IFNγ; CIKs were sorted using FACS. The levels of IFNγ, IL2 and TNFα in culture supernatants of CIKs were determined by ELISA. The antitumor activities of CIKs against CasKi and HeLa cells were determined by LDH assay. The nude mouse xenograft models of cervical cancer cell lines, CasKi or HeLa cells, were established, and 1×106 or 1×107 CIKs were administered intravenously once a week for three weeks, then the tumor volumes and weights were measured.Results: CIKs were successfully induced from PBMCs of cervical cancer patients by IL2 and antiCD3 antibody, with CD3+CD56+ cells greatly expanded. CIKs produced significant amounts of IFNγ and TNFα, but few IL2, after PHA stimulation. CIKs dosedependently killed CasKi and Hela cells with a maximum killing rate reaching 43% and 46%, respectively. In addition, the in vivo antitumor experiments demonstrated that CIKs remarkably inhibited the growth of subcutaneous tumors in nude mice (P<0.01). Conclusion: The improved CIKs expansion method used in our study is feasible and the resultant CIKs have remarkable cytotoxicity against Caski and HeLa cells both in vivo and in vitro.
2010, 17(3):333-335.
DOI: 10.3872/j.issn.1007-385X.2010.3.017
Abstract:
Objective:To investigate the antitumor activity of cytokineinduced killer cells (CIKs) against cervical cancer cell lines, CasKi and HeLa in vitro and in vivo. Methods: The CIKs were induced from peripheral blood mononuclear cells (PBMCs) of patients with cervical cancer using an improved method, which only used IL2 and antiCD3 antibody, without IFNγ; CIKs were sorted using FACS. The levels of IFNγ, IL2 and TNFα in culture supernatants of CIKs were determined by ELISA. The antitumor activities of CIKs against CasKi and HeLa cells were determined by LDH assay. The nude mouse xenograft models of cervical cancer cell lines, CasKi or HeLa cells, were established, and 1×106 or 1×107 CIKs were administered intravenously once a week for three weeks, then the tumor volumes and weights were measured.Results: CIKs were successfully induced from PBMCs of cervical cancer patients by IL2 and antiCD3 antibody, with CD3+CD56+ cells greatly expanded. CIKs produced significant amounts of IFNγ and TNFα, but few IL2, after PHA stimulation. CIKs dosedependently killed CasKi and Hela cells with a maximum killing rate reaching 43% and 46%, respectively. In addition, the in vivo antitumor experiments demonstrated that CIKs remarkably inhibited the growth of subcutaneous tumors in nude mice (P<0.01). Conclusion: The improved CIKs expansion method used in our study is feasible and the resultant CIKs have remarkable cytotoxicity against Caski and HeLa cells both in vivo and in vitro.
Objective:To investigate the antitumor activity of cytokineinduced killer cells (CIKs) against cervical cancer cell lines, CasKi and HeLa in vitro and in vivo. Methods: The CIKs were induced from peripheral blood mononuclear cells (PBMCs) of patients with cervical cancer using an improved method, which only used IL2 and antiCD3 antibody, without IFNγ; CIKs were sorted using FACS. The levels of IFNγ, IL2 and TNFα in culture supernatants of CIKs were determined by ELISA. The antitumor activities of CIKs against CasKi and HeLa cells were determined by LDH assay. The nude mouse xenograft models of cervical cancer cell lines, CasKi or HeLa cells, were established, and 1×106 or 1×107 CIKs were administered intravenously once a week for three weeks, then the tumor volumes and weights were measured.Results: CIKs were successfully induced from PBMCs of cervical cancer patients by IL2 and antiCD3 antibody, with CD3+CD56+ cells greatly expanded. CIKs produced significant amounts of IFNγ and TNFα, but few IL2, after PHA stimulation. CIKs dosedependently killed CasKi and Hela cells with a maximum killing rate reaching 43% and 46%, respectively. In addition, the in vivo antitumor experiments demonstrated that CIKs remarkably inhibited the growth of subcutaneous tumors in nude mice (P<0.01). Conclusion: The improved CIKs expansion method used in our study is feasible and the resultant CIKs have remarkable cytotoxicity against Caski and HeLa cells both in vivo and in vitro.
2010, 17(3):336-338.
DOI: 10.3872/j.issn.1007-385X.2010.3.018
Abstract:
目的：检测肿瘤相关抗原calponin2对肝细胞癌(hepatocellular carcinoma, HCC)的特异性。方法：应用SEREX （serological identification of antigens by recombinant expression loning)技术检测calponin2抗原在肝癌、肺癌、胃癌、直肠癌、肝炎、肝硬化患者（各31例）及正常健康者（32人）血清的抗体阳性率。结果：相应抗体主要见于HCC患者血清，其血清阳性反应率为54.8% ；肝炎、肝硬化患者血清阳性反应率均为3.2%；肺癌、直肠癌、胃癌患者的血清阳性反应率分别是3.2%、97%和6.5%;正常人血清阳性反应率为3.1%。Calponin2抗体在肝癌血清中的阳性率最高（P<001）。Calponin2阳性率与AFP阳性率及含量、癌组织病理分级、患者年龄及γ谷氨酰转肽酶（γGT）水平无相关性。使用calponin2诊断肝癌的灵敏度、特异性和准确度分别为54.8%、95.2%和89.4%。结论: Calponin2在HCC中有一定的特异性，具有作为新的HCC血清学诊断潜在标志物的意义。
目的：检测肿瘤相关抗原calponin2对肝细胞癌(hepatocellular carcinoma, HCC)的特异性。方法：应用SEREX （serological identification of antigens by recombinant expression loning)技术检测calponin2抗原在肝癌、肺癌、胃癌、直肠癌、肝炎、肝硬化患者（各31例）及正常健康者（32人）血清的抗体阳性率。结果：相应抗体主要见于HCC患者血清，其血清阳性反应率为54.8% ；肝炎、肝硬化患者血清阳性反应率均为3.2%；肺癌、直肠癌、胃癌患者的血清阳性反应率分别是3.2%、97%和6.5%;正常人血清阳性反应率为3.1%。Calponin2抗体在肝癌血清中的阳性率最高（P<001）。Calponin2阳性率与AFP阳性率及含量、癌组织病理分级、患者年龄及γ谷氨酰转肽酶（γGT）水平无相关性。使用calponin2诊断肝癌的灵敏度、特异性和准确度分别为54.8%、95.2%和89.4%。结论: Calponin2在HCC中有一定的特异性，具有作为新的HCC血清学诊断潜在标志物的意义。
2010, 17(3):339-343.
DOI: 10.3872/j.issn.1007-385X.2010.3.019
Abstract:
CCR4, a member of CCR (CC chemokine receptor) family, is expressed in many kinds of lymphocytes. Its high affinity ligands include thymus, activation regulated chemokine (TARC/CCL17) and macrophagederived chemokine (MDC/CCL22/STCP1). CCR4 exerts its immune activities by CCR4＋Treg cells and CCR4+Th2 cells. High expression of CCR4 is associated with infiltration and prognosis of many hematological and solid malignancies; the binding of CCR4 with its ligands TARC and MDC in Treg cells may be responsible for the chemotaxis of Treg cells, the resulting immune tolerance and worse clinical outcomes. The researches of malignant tumor metastatic models further revealed the relationship between CCR4 expression and metastasis of malignant solid tumors. The study of KW0761, a defucosylated humanized antiCCR4 antibody,has already been in the phase Ⅱ clinical trial. Therefore, blockage TARC/MDCCCR4 signal pathway might be a novel therapy strategy for malignant tumors.
CCR4, a member of CCR (CC chemokine receptor) family, is expressed in many kinds of lymphocytes. Its high affinity ligands include thymus, activation regulated chemokine (TARC/CCL17) and macrophagederived chemokine (MDC/CCL22/STCP1). CCR4 exerts its immune activities by CCR4＋Treg cells and CCR4+Th2 cells. High expression of CCR4 is associated with infiltration and prognosis of many hematological and solid malignancies; the binding of CCR4 with its ligands TARC and MDC in Treg cells may be responsible for the chemotaxis of Treg cells, the resulting immune tolerance and worse clinical outcomes. The researches of malignant tumor metastatic models further revealed the relationship between CCR4 expression and metastasis of malignant solid tumors. The study of KW0761, a defucosylated humanized antiCCR4 antibody,has already been in the phase Ⅱ clinical trial. Therefore, blockage TARC/MDCCCR4 signal pathway might be a novel therapy strategy for malignant tumors.
2010, 17(3):344-348.
DOI: 10.3872/j.issn.1007-385X.2010.3.020
Abstract:
Nuclear factor of activated T cell (NFAT) is an important factor in cellular signal pathways and plays a key role in the immune system as well as tumor development and progression. NFAT is activated mainly by calcium flux;activatedNFAT translocates to nucleus and interacts with targetDNA, regulating the expression of target genes by working together with other coactivators. Different NFAT subtypes have various regulatory effects on tumor cell transformation and proliferation, increasing tumor angiogenesis and infiltration or migration. Further understanding of NFAT expression in tumors, its acting mechanisms, and its roles in tumor development and progression will help to find potential tumor targets for tumor clinical therapy.
Nuclear factor of activated T cell (NFAT) is an important factor in cellular signal pathways and plays a key role in the immune system as well as tumor development and progression. NFAT is activated mainly by calcium flux;activatedNFAT translocates to nucleus and interacts with targetDNA, regulating the expression of target genes by working together with other coactivators. Different NFAT subtypes have various regulatory effects on tumor cell transformation and proliferation, increasing tumor angiogenesis and infiltration or migration. Further understanding of NFAT expression in tumors, its acting mechanisms, and its roles in tumor development and progression will help to find potential tumor targets for tumor clinical therapy.
2010, 17(3):349-353.
DOI: 10.3872/j.issn.1007-385X.2010.3.021
Abstract:
Biotherapy is especially beneficial for Ewing’s sarcoma with bad prognosis. The therapy includes gene therapy, immunotherapy, antiangiogenesis therapy, etc.. Gene therapy mainly refers to the application of antisense nucleic acid technique; immunotherapy refers to antibody therapy, T cell therapy, dendritic cell therapy, and tumor vaccine,etc.; and antiangiogenesis therapy refers to inhibition of tumor angiogenesis, thus suppressing tumor growth and metastasis. With the deeper understanding of Ewing’s sarcoma, changes in the telomere length, microsomal glutathione Stransferase 1 (MGST1) expression, the expression of tumor metastasis and multidrug resistance genes, and papillomavirus binding factor might serve as novel predictors for the prognosis of Ewing’s sarcoma.
Biotherapy is especially beneficial for Ewing’s sarcoma with bad prognosis. The therapy includes gene therapy, immunotherapy, antiangiogenesis therapy, etc.. Gene therapy mainly refers to the application of antisense nucleic acid technique; immunotherapy refers to antibody therapy, T cell therapy, dendritic cell therapy, and tumor vaccine,etc.; and antiangiogenesis therapy refers to inhibition of tumor angiogenesis, thus suppressing tumor growth and metastasis. With the deeper understanding of Ewing’s sarcoma, changes in the telomere length, microsomal glutathione Stransferase 1 (MGST1) expression, the expression of tumor metastasis and multidrug resistance genes, and papillomavirus binding factor might serve as novel predictors for the prognosis of Ewing’s sarcoma.
2010, 17(3):354-358.
DOI: 10.3872/j.issn.1007-385X.2010.3.022
Abstract:
Biotherapy is especially beneficial for Ewing’s sarcoma with bad prognosis. The therapy includes gene therapy, immunotherapy, antiangiogenesis therapy, etc.. Gene therapy mainly refers to the application of antisense nucleic acid technique; immunotherapy refers to antibody therapy, T cell therapy, dendritic cell therapy, and tumor vaccine,etc.; and antiangiogenesis therapy refers to inhibition of tumor angiogenesis, thus suppressing tumor growth and metastasis. With the deeper understanding of Ewing’s sarcoma, changes in the telomere length, microsomal glutathione Stransferase 1 (MGST1) expression, the expression of tumor metastasis and multidrug resistance genes, and papillomavirus binding factor might serve as novel predictors for the prognosis of Ewing’s sarcoma.
Biotherapy is especially beneficial for Ewing’s sarcoma with bad prognosis. The therapy includes gene therapy, immunotherapy, antiangiogenesis therapy, etc.. Gene therapy mainly refers to the application of antisense nucleic acid technique; immunotherapy refers to antibody therapy, T cell therapy, dendritic cell therapy, and tumor vaccine,etc.; and antiangiogenesis therapy refers to inhibition of tumor angiogenesis, thus suppressing tumor growth and metastasis. With the deeper understanding of Ewing’s sarcoma, changes in the telomere length, microsomal glutathione Stransferase 1 (MGST1) expression, the expression of tumor metastasis and multidrug resistance genes, and papillomavirus binding factor might serve as novel predictors for the prognosis of Ewing’s sarcoma.
2010, 17(3):359-362.
DOI: 10.3872/j.issn.1007-385X.2010.3.023
Abstract:
Ironcarbon nanoparticles include ironcarbon nanoparticles, carboncoated iron nanoparticles, etc., which can be prepared by mechanical trituration, carbon arc, gas phase deposition, pyrolysis and explosion methods. Ironcarbon nanoparticles have good adsorptive and magnetic effects, and they are perfect carriers for chemotherapeutic drugs, such as adriamycin, mitomycin and carboplatin, etc. Ironcarbon nanoparticle complex has highdrug loading and stable release abilities, thus can maintain sufficient drug concentration for inhibition of tumor cell proliferation. In addition, they can greatly aggregate in target organs, reducing the adverse and toxic effects to nontarget organs. Moreover, ironcarbon nanoparticles have magnetic induction ability and can kill tumor cells by thermogenic effects, whose intensity is correlated to the amount, concentration and action time of induced electric field. Great progress has been made in the research of ironcarbon nanoparticles as magnetic carriers for chemotherapeutic drugs in vitroand in vivo, and it is believed to have great values in clinical tumor therapy in future.
Ironcarbon nanoparticles include ironcarbon nanoparticles, carboncoated iron nanoparticles, etc., which can be prepared by mechanical trituration, carbon arc, gas phase deposition, pyrolysis and explosion methods. Ironcarbon nanoparticles have good adsorptive and magnetic effects, and they are perfect carriers for chemotherapeutic drugs, such as adriamycin, mitomycin and carboplatin, etc. Ironcarbon nanoparticle complex has highdrug loading and stable release abilities, thus can maintain sufficient drug concentration for inhibition of tumor cell proliferation. In addition, they can greatly aggregate in target organs, reducing the adverse and toxic effects to nontarget organs. Moreover, ironcarbon nanoparticles have magnetic induction ability and can kill tumor cells by thermogenic effects, whose intensity is correlated to the amount, concentration and action time of induced electric field. Great progress has been made in the research of ironcarbon nanoparticles as magnetic carriers for chemotherapeutic drugs in vitroand in vivo, and it is believed to have great values in clinical tumor therapy in future.
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- 《中国肿瘤生物治疗杂志》
- 1994年创刊
- 主办单位：Chinese Society of Immunology, Chinese Anti-cancer Association
- 邮编：200433
- 电话：021-81871002-22
- 电子邮箱：cjcb@biother.cn
- 网址:http://www.biother.cn
- 刊号：ISSN 1007-385X
- CN 31-1725/R
- 国内定价： ￥20元/册
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.