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Volume 20,Issue 4,2013 Table of Contents
2013, 20(4):383-390.
DOI: 10.3872/j.issn.1007-385X.2013.04.001
Abstract:
Chimeric antigen receptor (CAR)-engineered T cell is a newly developed strategy of adoptive immunotherapy. Its unique theoretical superiority and attractive application prospects open up a promising arena for anticancer therapy. CAR combines single chain variable fragment (scFv) antibody recognizing tumor-associated antigen with T cell activation motif, which endows T cells with tumor-orientated targeting ability, stronger killing activity, and prolonged survival by genetic modification. Since first proposed by Dr. Eshhar in 1989, CAR has been developed from the first generation to the second and the third generations containing costimulatory molecular. The clinical trials in leukemia, lymphoma, and melanoma have obtained exciting results. However, the off-target effect, cytokine strom, and graft-versus-host disease are potential challenges for clinical use. Future research will focus on designing safer CAR of the fourth generation, selecting good therapeutic T cell subsets, optimizing clinical scheme of administration, and improving pre-clinical models. It is believed that the obstacles from bench to clinic will be cleared and that CAR will become one of the main cancer therapies with breakthroughs in immunology, gene therapy and cell engineering.
Chimeric antigen receptor (CAR)-engineered T cell is a newly developed strategy of adoptive immunotherapy. Its unique theoretical superiority and attractive application prospects open up a promising arena for anticancer therapy. CAR combines single chain variable fragment (scFv) antibody recognizing tumor-associated antigen with T cell activation motif, which endows T cells with tumor-orientated targeting ability, stronger killing activity, and prolonged survival by genetic modification. Since first proposed by Dr. Eshhar in 1989, CAR has been developed from the first generation to the second and the third generations containing costimulatory molecular. The clinical trials in leukemia, lymphoma, and melanoma have obtained exciting results. However, the off-target effect, cytokine strom, and graft-versus-host disease are potential challenges for clinical use. Future research will focus on designing safer CAR of the fourth generation, selecting good therapeutic T cell subsets, optimizing clinical scheme of administration, and improving pre-clinical models. It is believed that the obstacles from bench to clinic will be cleared and that CAR will become one of the main cancer therapies with breakthroughs in immunology, gene therapy and cell engineering.
Zhang Yanjun
,
Li Shuangjing
,
Jiang Linlin
,
Liu Rong
,
Gao Xue
,
Yuan Xiangfei
,
Qi Huaifeng
,
Fan Dongmei
,
Miao Qingfang
,
Zhen Yongsu
,
Xiong Dongsheng
2013, 20(4):391-397.
DOI: 10.3872/j.issn.1007-385X.2013.04.002
Abstract:
Objective: To construct a fusion protein IL-3-lidamycin(IL-3-LDM) with an IL-3 guide and a LDM warhead, and to investigate its targeting cytotoxicity on CD123+ leukemia cells in vivo. Methods: IL-3-LDP (interlekin 3-lidamycin) fusion protein was obtained in a prokaryotic system, and further assembled with active enediyne (AE) to get IL-3-LDM. The expression of CD123 in six leukemia cell lines (KG1-a, TF-1, M07e, HL-60, K562, Raji) was detected by flow cytometry and the binding ability of IL-3-LDM with different leukemia cell lines was examined. The cytotoxicity of IL-3-LDM fusion protein on leukemia cells with different CD123 expression levels was detected by CCK-8. Results: The purity of recombinant protein IL-3-LDM was more than 90% after assembling with AE. The results showed that the CD123 expression ratio was 88.9% on AML (acute myeloid leukemia) KG-1a cells, ＞75% on MO7e and TF-1 cells, 7.8% on HL-60 cells, and negative on K562 and Raji cells. The expression ratio of CD123 on leukemia cells （KG-1a, MO7e, TF-1 and HL-60）was positively related to its binding ability and sensitivity to IL-3-LDM in vitro. The cytotoxicity of LDM on KG-1a cells which expressed the highest level of CD133 was 1 415.8 fold stronger than that of adriamjcin (ADR), and the cytotoxicity of IL-3-LDM was 9.6 fold than that of LDM. Conclusion: IL-3-LDM fusion protein can effectively target cytotoxic drug LDM to kill CD123+ leukemia cells.
Objective: To construct a fusion protein IL-3-lidamycin(IL-3-LDM) with an IL-3 guide and a LDM warhead, and to investigate its targeting cytotoxicity on CD123+ leukemia cells in vivo. Methods: IL-3-LDP (interlekin 3-lidamycin) fusion protein was obtained in a prokaryotic system, and further assembled with active enediyne (AE) to get IL-3-LDM. The expression of CD123 in six leukemia cell lines (KG1-a, TF-1, M07e, HL-60, K562, Raji) was detected by flow cytometry and the binding ability of IL-3-LDM with different leukemia cell lines was examined. The cytotoxicity of IL-3-LDM fusion protein on leukemia cells with different CD123 expression levels was detected by CCK-8. Results: The purity of recombinant protein IL-3-LDM was more than 90% after assembling with AE. The results showed that the CD123 expression ratio was 88.9% on AML (acute myeloid leukemia) KG-1a cells, ＞75% on MO7e and TF-1 cells, 7.8% on HL-60 cells, and negative on K562 and Raji cells. The expression ratio of CD123 on leukemia cells （KG-1a, MO7e, TF-1 and HL-60）was positively related to its binding ability and sensitivity to IL-3-LDM in vitro. The cytotoxicity of LDM on KG-1a cells which expressed the highest level of CD133 was 1 415.8 fold stronger than that of adriamjcin (ADR), and the cytotoxicity of IL-3-LDM was 9.6 fold than that of LDM. Conclusion: IL-3-LDM fusion protein can effectively target cytotoxic drug LDM to kill CD123+ leukemia cells.
Liu Qingchi
,
Zhang Huimin
,
Zhang Yuna
,
Pang Yuhui
,
Ma Chuanbao
,
Yang Huitao
,
Wang Rongxiao
,
Xiong Jianjun
,
Gao Xihua
,
Ma Yahui
,
Hu Xiaodong
,
Liang Chungeng
,
Feng Xinwang
,
Wu Dayong
,
Wu Weihai
2013, 20(4):398-403.
DOI: 10.3872/j.issn.1007-385X.2013.04.003
Abstract:
Objective: To evaluate the clinical efficacy and safety of allogenic dendritic cells (DCs) and cytokine-induced killer (CIK) cells combined with chemotherapy for eliminating minimal residual leukemia (MRL). Methods: Forty-eight acute leukemia patients with morphological complete remission (CR) but molecular complete remission (CRm), or patients with minimal residual leukemia (MRL) were selected from Ping’an Hospital of Shijiazhuang during Jan. 2009 to Jun. 2011. According to the patients’ will, 48 patients were divided into combined group and chemotherapy group, with 24 each. All the patients were in the comparable general data and disease level. The combined group was treated with DC-CIK and consolidation chemotherapy, and the chemotherapy group was treated with consolidation chemotherapy. PBMCs were collected from healthy donors (the patient’s parents or children) to prepare DC-CIK cells. DC-CIK cells were intravenous injected into patients once every 15 days, a total of 4-6 times infusion. Expression of leukemia specific and related genes were detected by Real-time PCR. Changes of peripheral lymphocyte subsets and MRL immunophentotype in patients were detected by flow cytometry. Adverse reactions were examined. Results: All the patients were followed up to Jun. 2012. Compared with the chemotherapy group, the CRm rate of combined group was significantly raised (45.8%\[11/24\] vs 8.3% \[2/24\]; χ2=855, P<0.01); the four-color combination flow cytometric immunophenotype of minimal residual leukemia (CFIM) negative conversion rate of patients in the combined group was significantly raised (66.7% vs 25.0%, χ2=8.39, P<001); the negative conversion rate of MRL was significant higher in the combined group (66.7% vs 25.0%, χ2=8.39, P<0.01); the complete remission rate (CCR) of patients in the combined group after 3 years was significantly raised (79.2% vs 45.8%; χ2=569, P<0.05). After treatment the ratio of CD4+/CD8+ lymphocyte was significant increased in the combined group (1.3±0.4 vs 0.8±0.4, P<0.05). No serious adverse reactions were observed after DC-CIK infusion. Conclusion: DC-CIK combined with chemotherapy can inhibit leukemia related gene, promote the negative conversion of CFIM, facilitate the clear of MRL, improve immune function and prolong remission of the patients. No serious adverse reactions are found in patients receiving DC-CIK infusion.
Objective: To evaluate the clinical efficacy and safety of allogenic dendritic cells (DCs) and cytokine-induced killer (CIK) cells combined with chemotherapy for eliminating minimal residual leukemia (MRL). Methods: Forty-eight acute leukemia patients with morphological complete remission (CR) but molecular complete remission (CRm), or patients with minimal residual leukemia (MRL) were selected from Ping’an Hospital of Shijiazhuang during Jan. 2009 to Jun. 2011. According to the patients’ will, 48 patients were divided into combined group and chemotherapy group, with 24 each. All the patients were in the comparable general data and disease level. The combined group was treated with DC-CIK and consolidation chemotherapy, and the chemotherapy group was treated with consolidation chemotherapy. PBMCs were collected from healthy donors (the patient’s parents or children) to prepare DC-CIK cells. DC-CIK cells were intravenous injected into patients once every 15 days, a total of 4-6 times infusion. Expression of leukemia specific and related genes were detected by Real-time PCR. Changes of peripheral lymphocyte subsets and MRL immunophentotype in patients were detected by flow cytometry. Adverse reactions were examined. Results: All the patients were followed up to Jun. 2012. Compared with the chemotherapy group, the CRm rate of combined group was significantly raised (45.8%\[11/24\] vs 8.3% \[2/24\]; χ2=855, P<0.01); the four-color combination flow cytometric immunophenotype of minimal residual leukemia (CFIM) negative conversion rate of patients in the combined group was significantly raised (66.7% vs 25.0%, χ2=8.39, P<001); the negative conversion rate of MRL was significant higher in the combined group (66.7% vs 25.0%, χ2=8.39, P<0.01); the complete remission rate (CCR) of patients in the combined group after 3 years was significantly raised (79.2% vs 45.8%; χ2=569, P<0.05). After treatment the ratio of CD4+/CD8+ lymphocyte was significant increased in the combined group (1.3±0.4 vs 0.8±0.4, P<0.05). No serious adverse reactions were observed after DC-CIK infusion. Conclusion: DC-CIK combined with chemotherapy can inhibit leukemia related gene, promote the negative conversion of CFIM, facilitate the clear of MRL, improve immune function and prolong remission of the patients. No serious adverse reactions are found in patients receiving DC-CIK infusion.
2013, 20(4):404-408.
DOI: 10.3872/j.issn.1007-385X.2013.04.004
Abstract:
Objective: To investigate the possibility of inducing dendritic cells (DCs) by interferon-α (IFN-α) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) from peripheral blood mononuclear cells (PBMCs) in gastric cancer patients. Methods: PBMCs from 10 gastric cancer patients were cultivated using granulocyte macrophage colony stimulating factor (GM-CSF) 100 ng/ml combined with IFN-α 500 IU/ml (named IFN-α DC) or IL-4 50 ng/ml (named IL-4 DCs) and then CD40L and LPS were added to induce DC maturation. The morphologic features of IFN-α DCs and IL-4 DCs were observed by Giemsa staining. The expressions of CD1a, CD80, CD83, CD86 and HLA-DR on the surface of IFN-α DCs and IL-4 DCs were assayed by flow cytometry. The abilities of IFN-α DCs and IL-4 DCs to induce the proliferation of allogenic T cells were determined by mixed lymphocyte reaction (MLR). Results: Both IFN-α DCs and IL-4 DCs displayed typical DC features in morphology. The expressions of CD1a, CD80, CD83, CD86 and HLA-DR in IFN-α DCs and IL-4 DCs were achieved at high levels at 3 d and 5 d after induced. Mature IFN-α DCs expressed a higher value of CD83 (\[78.25±15.36\]% vs \[50.14±10.24\]%, P<0.05) and CD86 (\[84.84±1012\]% vs \[62.93±15.12\]%, P<0.05) than mature IL-4 DCs. Mature IFN-α DCs was stronger than immature IFN-α DCs on the ability to induce proliferation of allogenic T cells (P<0.05). At the ratios of DCs〖DK〗∶T cell being 1∶40 and 1∶20, mature IFN-α DCs had a stronger ability to induce proliferation of allogeneic T cells than did mature IL-4 DCs (\[39.43±9.21\]% vs \[27.34±10.63\]%, P<0.05; \[60.31±7.86\]% vs \[48.63±625\]%, P<0.05). Conclusion: IFN-α combined with GM-CSF can induce the differentiation of DCs from PBMCs of gastric cancer patients, which have a shorter culture period and stronger ability to induce the proliferation of allogenic T cells than traditional DCs induced by IL-4 and GM-CSF. It may result from the up-regulation of CD83 and CD86 expressions on IFN-α DCs.
Objective: To investigate the possibility of inducing dendritic cells (DCs) by interferon-α (IFN-α) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) from peripheral blood mononuclear cells (PBMCs) in gastric cancer patients. Methods: PBMCs from 10 gastric cancer patients were cultivated using granulocyte macrophage colony stimulating factor (GM-CSF) 100 ng/ml combined with IFN-α 500 IU/ml (named IFN-α DC) or IL-4 50 ng/ml (named IL-4 DCs) and then CD40L and LPS were added to induce DC maturation. The morphologic features of IFN-α DCs and IL-4 DCs were observed by Giemsa staining. The expressions of CD1a, CD80, CD83, CD86 and HLA-DR on the surface of IFN-α DCs and IL-4 DCs were assayed by flow cytometry. The abilities of IFN-α DCs and IL-4 DCs to induce the proliferation of allogenic T cells were determined by mixed lymphocyte reaction (MLR). Results: Both IFN-α DCs and IL-4 DCs displayed typical DC features in morphology. The expressions of CD1a, CD80, CD83, CD86 and HLA-DR in IFN-α DCs and IL-4 DCs were achieved at high levels at 3 d and 5 d after induced. Mature IFN-α DCs expressed a higher value of CD83 (\[78.25±15.36\]% vs \[50.14±10.24\]%, P<0.05) and CD86 (\[84.84±1012\]% vs \[62.93±15.12\]%, P<0.05) than mature IL-4 DCs. Mature IFN-α DCs was stronger than immature IFN-α DCs on the ability to induce proliferation of allogenic T cells (P<0.05). At the ratios of DCs〖DK〗∶T cell being 1∶40 and 1∶20, mature IFN-α DCs had a stronger ability to induce proliferation of allogeneic T cells than did mature IL-4 DCs (\[39.43±9.21\]% vs \[27.34±10.63\]%, P<0.05; \[60.31±7.86\]% vs \[48.63±625\]%, P<0.05). Conclusion: IFN-α combined with GM-CSF can induce the differentiation of DCs from PBMCs of gastric cancer patients, which have a shorter culture period and stronger ability to induce the proliferation of allogenic T cells than traditional DCs induced by IL-4 and GM-CSF. It may result from the up-regulation of CD83 and CD86 expressions on IFN-α DCs.
2013, 20(4):409-413.
DOI: 10.3872/j.issn.1007-385X.2013.04.005
Abstract:
Objective: To investigate the inhibitoty effects of recombinant adenovirus-p53 (rAd-p53) on the growth of lung adenocarcinoma H1299 cells (wtP53-/-) in vitro and in vivo, and observe the treatment feasibility of lung adenocarcinoma with tail intravenous injection of rAd-p53. Methods: MTT assay was performed to detect the inhibitory effect of rAd-p53 on the proliferation of H1299 cells. After transfected by rAd-p53 with multiplicity of infection (MOI)=500, the expression of p53 mRNA in H1299 cells was detected by RT-PCR at 24 h; the expression of P53 protein in H1299 cells and the apoptosis of H1299 cells were detected at 72 h by Western blotting and flow cytometry, respectively. BALB/c nude mice were injected subcutaneously with H1299 cells to establish a lung adenocarcinoma nude mice model and then the mice were intravenously administrated by rAd-p53; the tumor growth was observed and tumor growth curve was drawn. Results: H1299 cells were infected by rAd-p53with MOI=500; after infection for 24 h, wild-type p53mRNA was expressed in rAd-p53 group, and at 72 h, wt P53 protein was detected in rAd-p53group. rAd-p53infection could significantly inhibit the proliferation of H1299 cells, the cell proliferation ratio of rAd-p53 group was significant lower than that of the control group (2.8±0.4 vs 6.1±0.5, P<005). The apoptotic rates of H1299 cells in rAd-p53group were increased with time, which were significantly higher than those in the control group (\[27.6±0.05\]% vs \[4.9±0.09\]%, P＜0.01) after infection for 48 h. H1299 tumor-bearing nude mice were successfully established, and the tumor volume of rAd-p53 group was significantly smaller than that of the control group even two weeks after tail intravenous injection (\[0.875±0253\] cm3 vs \[0.479±0.215\] cm3,P<0.05). Conclusion: Tail intravenous infection of rAd-p53 could up-regulate the protein expression of P53 in H1299 cells, then restrain the growth of H1299 cells, promote the apoptosis and significantly inhibit the growth of H1299 cell xenograft tumors in nude mice.
Objective: To investigate the inhibitoty effects of recombinant adenovirus-p53 (rAd-p53) on the growth of lung adenocarcinoma H1299 cells (wtP53-/-) in vitro and in vivo, and observe the treatment feasibility of lung adenocarcinoma with tail intravenous injection of rAd-p53. Methods: MTT assay was performed to detect the inhibitory effect of rAd-p53 on the proliferation of H1299 cells. After transfected by rAd-p53 with multiplicity of infection (MOI)=500, the expression of p53 mRNA in H1299 cells was detected by RT-PCR at 24 h; the expression of P53 protein in H1299 cells and the apoptosis of H1299 cells were detected at 72 h by Western blotting and flow cytometry, respectively. BALB/c nude mice were injected subcutaneously with H1299 cells to establish a lung adenocarcinoma nude mice model and then the mice were intravenously administrated by rAd-p53; the tumor growth was observed and tumor growth curve was drawn. Results: H1299 cells were infected by rAd-p53with MOI=500; after infection for 24 h, wild-type p53mRNA was expressed in rAd-p53 group, and at 72 h, wt P53 protein was detected in rAd-p53group. rAd-p53infection could significantly inhibit the proliferation of H1299 cells, the cell proliferation ratio of rAd-p53 group was significant lower than that of the control group (2.8±0.4 vs 6.1±0.5, P<005). The apoptotic rates of H1299 cells in rAd-p53group were increased with time, which were significantly higher than those in the control group (\[27.6±0.05\]% vs \[4.9±0.09\]%, P＜0.01) after infection for 48 h. H1299 tumor-bearing nude mice were successfully established, and the tumor volume of rAd-p53 group was significantly smaller than that of the control group even two weeks after tail intravenous injection (\[0.875±0253\] cm3 vs \[0.479±0.215\] cm3,P<0.05). Conclusion: Tail intravenous infection of rAd-p53 could up-regulate the protein expression of P53 in H1299 cells, then restrain the growth of H1299 cells, promote the apoptosis and significantly inhibit the growth of H1299 cell xenograft tumors in nude mice.
2013, 20(4):414-418.
DOI: 10.3872/j.issn.1007-385X.2013.04.006
Abstract:
Objective: To observe the effect of NDRG2 (N-myc downstream-regulated gene 2) overexpression on invasion and metastasis of human bladder cancer T24 cell in vitro. Methods: The T24 cells were infected with lentivirus carrying NDRG2 gene (Lenti-NDRG2) to construct a stable cell line overexpression NDRG2.Western blotting was performed to examine the expression of NDRG2, matrix metalloproteinase (MMP)-2 and MMP-9 in T24 cells. The effects of NDRG2 overexpression on the invasion and migration of T24 cells were examined by the transwell invasion and migration assays, respectively. Results: The T24 cell line stable overexpression NDRG2was successfully constructed. Compared with the LEN-LacZ group and the control group, the NDRG2 expression was efficiently increased in the Lenti-NDRG2 group (\[301±1.27\] vs \[9.9±1.24\], \[8.2±1.28\]; P<0.01); the MMP-2 (\[13.5±1.32\] vs \[30.7±1.29\], \[28.8±1.30\]; P<0.01) and MMP-9 expressions (\[11.7±1.27\] vs \[25.2±1.28\], \[26.4±1.31\]; P<0.01) in T24 cells of Lenti-NDRG2 group were significantly suppressed; the capabilities of the invasion (\[18.1±3.4\] vs \[88.5±4.2\], \[90.2±4.1\]; P<0.01）and migration (\[20.1±3.5\] vs \[109.4±5.6\], \[113.0±4.9\]; P<0.01) of T24 cells were significantly suppressed in the Lenti-NDRG2 group. Conclusion: The lentivirus-mediated NDRG2 overexpression can suppress the invasion and migration of human bladder cancer T24 cells.
Objective: To observe the effect of NDRG2 (N-myc downstream-regulated gene 2) overexpression on invasion and metastasis of human bladder cancer T24 cell in vitro. Methods: The T24 cells were infected with lentivirus carrying NDRG2 gene (Lenti-NDRG2) to construct a stable cell line overexpression NDRG2.Western blotting was performed to examine the expression of NDRG2, matrix metalloproteinase (MMP)-2 and MMP-9 in T24 cells. The effects of NDRG2 overexpression on the invasion and migration of T24 cells were examined by the transwell invasion and migration assays, respectively. Results: The T24 cell line stable overexpression NDRG2was successfully constructed. Compared with the LEN-LacZ group and the control group, the NDRG2 expression was efficiently increased in the Lenti-NDRG2 group (\[301±1.27\] vs \[9.9±1.24\], \[8.2±1.28\]; P<0.01); the MMP-2 (\[13.5±1.32\] vs \[30.7±1.29\], \[28.8±1.30\]; P<0.01) and MMP-9 expressions (\[11.7±1.27\] vs \[25.2±1.28\], \[26.4±1.31\]; P<0.01) in T24 cells of Lenti-NDRG2 group were significantly suppressed; the capabilities of the invasion (\[18.1±3.4\] vs \[88.5±4.2\], \[90.2±4.1\]; P<0.01）and migration (\[20.1±3.5\] vs \[109.4±5.6\], \[113.0±4.9\]; P<0.01) of T24 cells were significantly suppressed in the Lenti-NDRG2 group. Conclusion: The lentivirus-mediated NDRG2 overexpression can suppress the invasion and migration of human bladder cancer T24 cells.
Luo Hongbin
,
Liu Weinan
,
Lin Jianhua
,
Cheng Yuanrong
,
Zhang Li
,
Huang Jie
,
Wu Zhaoyang
,
Lin Jinluan
,
Lan Wenbin
2013, 20(4):419-424.
DOI: 10.3872/j.issn.1007-385X.2013.04.007
Abstract:
Objective: To investigate the effect of phosphorus sirolimus derivatives FIM-A, a new mammalian mammalia target of rapamycin (mTOR) inhibitor, on the proliferation and apoptosis of human MG-63 osteosarcoma cell line. Methods: Human MG-63 osteosarcoma cells and hF-OB1.19 osteoblasts were cultured in vitro and incubated with different concentrations of FIM-A (1×10-9-1×10-5 mol/L) for 24 hours. CCK-8 assay was used to evaluate the cell proliferation. The cell cycle and apoptosis were analyzed using flow cytometry. ELISA was used to detect the secretions of vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). The expressions of mTOR, p70S6 kinase protein (p70s6k) and 4E-binding protein 1 (4E-BP1) mRNA and protein were detected by RT-PCR and Western blotting, respectively. Results: The expressions of mTOR, p70s6k and 4E-BP1 mRNA in MG-63 osteosarcoma cells were significantly higher than that in the hF-OB1.19 osteoblasts (P<0.05). The proliferation of the MG-63 osteosarcoma cells were significantly inhibited after FIM-A treatment. The proliferation inhibition rate of MG-63 cells was significantly higher than that of the negative control group after the treatment of 1×10-7 mol/L FIM-A (\[37.64±2.07\]% vs 0, P<0.05), and the cell proliferation inhibition rate increased along with FIM-A concentrations in a dose-dependent manner (r=0940, P<0.01). After the treatment of 1×10-6 mol/L FIM-A for 24 hours, the proportion of MG-63 cells in G0/G1 phase was significantly increased compared with the control group (\[56.4±3.2\]% vs \[43.4±6.9\]%, P<005). No obvious changes were found in the apoptotic rate of MG-63 cells compared with the control group. The expression levels of HIF-1 α and VEGF in MG-63 cells were significantly lower than those of the control group after the treatment of different concentrations of FIM-A for 24 hours (P<005), and as concentrations increased, the level of HIF-1 α (r=0.988, P<001) and VEGF (r=0.998, P<0.01) decreased gradually in a dose-dependent manner. Meanwhile, the inhibitory effect of FIM-A on phosphorylations of p-mTOR (r=-0.919, P<0.01), p-p70s6k (r=-0.843, P<0.01) and p-4EBP1 (r=-0.818, P<0.01) proteins in MG-63 cells was also in a dose-dependent manner. Conclusion: FIM-A can significantly inhibit human MG-63 osteosarcoma cells and induce G0/G1 phase cell cycle arrest, and its anti-tumor effect was probably through the intervention of mTOR pathway.
Objective: To investigate the effect of phosphorus sirolimus derivatives FIM-A, a new mammalian mammalia target of rapamycin (mTOR) inhibitor, on the proliferation and apoptosis of human MG-63 osteosarcoma cell line. Methods: Human MG-63 osteosarcoma cells and hF-OB1.19 osteoblasts were cultured in vitro and incubated with different concentrations of FIM-A (1×10-9-1×10-5 mol/L) for 24 hours. CCK-8 assay was used to evaluate the cell proliferation. The cell cycle and apoptosis were analyzed using flow cytometry. ELISA was used to detect the secretions of vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). The expressions of mTOR, p70S6 kinase protein (p70s6k) and 4E-binding protein 1 (4E-BP1) mRNA and protein were detected by RT-PCR and Western blotting, respectively. Results: The expressions of mTOR, p70s6k and 4E-BP1 mRNA in MG-63 osteosarcoma cells were significantly higher than that in the hF-OB1.19 osteoblasts (P<0.05). The proliferation of the MG-63 osteosarcoma cells were significantly inhibited after FIM-A treatment. The proliferation inhibition rate of MG-63 cells was significantly higher than that of the negative control group after the treatment of 1×10-7 mol/L FIM-A (\[37.64±2.07\]% vs 0, P<0.05), and the cell proliferation inhibition rate increased along with FIM-A concentrations in a dose-dependent manner (r=0940, P<0.01). After the treatment of 1×10-6 mol/L FIM-A for 24 hours, the proportion of MG-63 cells in G0/G1 phase was significantly increased compared with the control group (\[56.4±3.2\]% vs \[43.4±6.9\]%, P<005). No obvious changes were found in the apoptotic rate of MG-63 cells compared with the control group. The expression levels of HIF-1 α and VEGF in MG-63 cells were significantly lower than those of the control group after the treatment of different concentrations of FIM-A for 24 hours (P<005), and as concentrations increased, the level of HIF-1 α (r=0.988, P<001) and VEGF (r=0.998, P<0.01) decreased gradually in a dose-dependent manner. Meanwhile, the inhibitory effect of FIM-A on phosphorylations of p-mTOR (r=-0.919, P<0.01), p-p70s6k (r=-0.843, P<0.01) and p-4EBP1 (r=-0.818, P<0.01) proteins in MG-63 cells was also in a dose-dependent manner. Conclusion: FIM-A can significantly inhibit human MG-63 osteosarcoma cells and induce G0/G1 phase cell cycle arrest, and its anti-tumor effect was probably through the intervention of mTOR pathway.
2013, 20(4):425-431.
DOI: 10.3872/j.issn.1007-385X.2013.04.008
Abstract:
Objective: To prepare recombinant immunotoxin targetting a cryptic epitope (287-302 residues) in epithelial growth factor receptor (EGFR) and to explore its biological properties. Methods: Prokaryotic expression vector pET-22b-806scFv-PE38KDEL encoding anti-EGFR (287-302) 806 single-chain antibody (806scFv) fused with PE38KDEL, a truncated form of pseudomonas exotoxin A (PEA), via a flexible peptide was constructed. The immunotoxin fusion protein (806scFv-PE38KDEL) was expressed in E. coli strain BL21 (DE3) and purified. The binding capacity of the immunotoxin to EGFR was detected by ELISA and flow cytometry. The internalization of immunotoxin was showed via indirect immuno-fluorescent assay. The cytotoxicity effect of the immunotoxin on human glioma cell U87MG and U87MG-EGFRvⅢ, epidermis tumor cell A431, breast cancer cell MDA-MB-468 and tongue cancer CAL-27 cell lines were assessed by CCK-8 assay. Results: The recombinant immunotoxin 806scFv-PE38KDEL was constructed successfully. The induced expression product 806scFv-PE38KDEL existed in a form of inclusion body and the purity was above 95% after purification. The protein was identified by SDS-PAGE and Western blotting. 806scFv-PE38KDEL can bind to the protein of EGFRvⅢ extracellular domain and also the cancer cells with exogenous EGFRvⅢ or with a endogenous EGFR overexpression but not to the cancer cells with a low level of endogenous EGFR. The immuno-fluorescent assay showed that the internalization of immunotoxin was mediated by 806scFv. 806scFv-PE38KDEL showed the cytotocicity on targeted cells with EGFRvⅢ overexpression such as U87MG-EGFRvⅢ cells with IC50 values of (5.85±0.03) ng/ml (P<0.01) and EGFR overexpression cancer cells such as MDA-MB-468, A431 and CAL-27 cells with IC50 values of (162.80±0.06) ng/ml, (75.72±0.04) ng/ml, (123.70±0.03) ng/ml, respectively. 806scFv-PE38KDEL almost completely inhibited the growth of cancer cells such as U87MG-EGFRvⅢ, MDA-MB-468, A431 and CAL-27 cells compared the control group, and the inhibitory rates significantly increased (\[98.67±0.07\]% vs \[2.45±2.85\]%, \[8626±1.01\]% vs \[0.48±1.76\]%, \[96.72±0.16\]% vs \[1.33±1.31\]%, \[96.29±0.30\]% vs \[2.00±060\]%; P<0.01) but no effect on U87MG cells (\[3.59±2.09\]% vs \[0.19±0.95\], P>005). Conclusion: Recombinant immunotoxin 806scFv-PE38KDEL that targeted to EGFR (287-302) epitope prepared in this study is a candidate cancer therapeutic agent which can selectively bind and significantly inhibit the growth of the cancer cells with EGFRvⅢ or EGFR overexpression.
Objective: To prepare recombinant immunotoxin targetting a cryptic epitope (287-302 residues) in epithelial growth factor receptor (EGFR) and to explore its biological properties. Methods: Prokaryotic expression vector pET-22b-806scFv-PE38KDEL encoding anti-EGFR (287-302) 806 single-chain antibody (806scFv) fused with PE38KDEL, a truncated form of pseudomonas exotoxin A (PEA), via a flexible peptide was constructed. The immunotoxin fusion protein (806scFv-PE38KDEL) was expressed in E. coli strain BL21 (DE3) and purified. The binding capacity of the immunotoxin to EGFR was detected by ELISA and flow cytometry. The internalization of immunotoxin was showed via indirect immuno-fluorescent assay. The cytotoxicity effect of the immunotoxin on human glioma cell U87MG and U87MG-EGFRvⅢ, epidermis tumor cell A431, breast cancer cell MDA-MB-468 and tongue cancer CAL-27 cell lines were assessed by CCK-8 assay. Results: The recombinant immunotoxin 806scFv-PE38KDEL was constructed successfully. The induced expression product 806scFv-PE38KDEL existed in a form of inclusion body and the purity was above 95% after purification. The protein was identified by SDS-PAGE and Western blotting. 806scFv-PE38KDEL can bind to the protein of EGFRvⅢ extracellular domain and also the cancer cells with exogenous EGFRvⅢ or with a endogenous EGFR overexpression but not to the cancer cells with a low level of endogenous EGFR. The immuno-fluorescent assay showed that the internalization of immunotoxin was mediated by 806scFv. 806scFv-PE38KDEL showed the cytotocicity on targeted cells with EGFRvⅢ overexpression such as U87MG-EGFRvⅢ cells with IC50 values of (5.85±0.03) ng/ml (P<0.01) and EGFR overexpression cancer cells such as MDA-MB-468, A431 and CAL-27 cells with IC50 values of (162.80±0.06) ng/ml, (75.72±0.04) ng/ml, (123.70±0.03) ng/ml, respectively. 806scFv-PE38KDEL almost completely inhibited the growth of cancer cells such as U87MG-EGFRvⅢ, MDA-MB-468, A431 and CAL-27 cells compared the control group, and the inhibitory rates significantly increased (\[98.67±0.07\]% vs \[2.45±2.85\]%, \[8626±1.01\]% vs \[0.48±1.76\]%, \[96.72±0.16\]% vs \[1.33±1.31\]%, \[96.29±0.30\]% vs \[2.00±060\]%; P<0.01) but no effect on U87MG cells (\[3.59±2.09\]% vs \[0.19±0.95\], P>005). Conclusion: Recombinant immunotoxin 806scFv-PE38KDEL that targeted to EGFR (287-302) epitope prepared in this study is a candidate cancer therapeutic agent which can selectively bind and significantly inhibit the growth of the cancer cells with EGFRvⅢ or EGFR overexpression.
9 Inhibitory effect of triptolide on human pancreatic cancer PANC-1 cells and its possible mechanism
2013, 20(4):432-437.
DOI: 10.3872/j.issn.1007-385X.2013.04.009
Abstract:
Objective: To observe the inhibitory effect of triptolide (TPL) on the proliferation and apoptosis of human pancreatic cancer PANC-1 cells in vitro and in vivo, and to analyze its impact on Toll-like receptor 4 (TLR4), vascular endothelial cell growth factor (VEGF) expression and tumor angiogenesis. Methods: PANC-1 cells were treated with 0, 20, 40 and 80 ng/ml TPL in vitro. MTT and flow cytometry were performed to detect the proliferation and apoptosis of PANC-1 cells treated by TPL, respectively. TLR4 and VEGF protein expression in PANC-1 cells were evaluated by Western blotting. PANC-1 tumor-bearing nude mice were established and randomly divided into two groups: TPL group and PBS group. The tumor volume was examined, and all the tumors were taken out at 35 days after treatment. The expression of TLR4, VEGF and CD31 in the xenograft tumors was detected by immunohistochemical staining, and the microvessel density (MVD) was calculated. Results: Compared with 0 ng/ml group, after treated with 20, 40 and 80 ng/ml TPL 24 h, the apoptosis rate of PANC-1 cells was significantly increased (\[4.7±1.0\]%, \[10.5±2.0\]%, \[21.1±4.2\]% vs \[26±0.5\]%，P<0.05 or P<0.01); after 48 h, the cell proliferation rate of PANC-1 cells was significantly decreased (\[68.0±5.3\]%, \[59.6±5.0\]%, \[51.6±4.2\]% vs \[99.6±5.2\]%; all P<0.01), and was also decreased compared with that in 24 h treated with the same dose. The expression of TLR4( \[20.2±4.7\]% vs \[57.5±63\]%，P<001) and VEGF( \[35.8±4.0\]% vs \[92.1±8.3\]%，P<0.01) in PANC-1 cells treated by 80 ng/ml TPL was decreased significantly than those of the untreated group. On day 34, the tumor volume of the TPL treatment group were reduced significantly than that of the PBS control group (\[510.9±79.8\] vs \[1 220.6 ±127.2\] mm3, P<001). The expression of TLR4 (P<0.01) and VEGF in the xenograft tumor tissues of the TPL group were significantly lower than those of the PBS group (\[3.2±0.6\] vs \[6.7±1.1\], \[3.7±0.7\] vs 7.1±1.2; all P<0.01)，also the MVD within the transplanted tumor in the TPL group was significantly decreased compared with the PBS group (\[12.2±4.0\] vs \[227±5.6\], P<0.01). Conclusion: TPL can inhibit the growth of pancreatic cancer PANC-1 cells and its xenografts tumor in nude mice, and induce apoptosis of PANC-1 cells. Its mechanism may be related to the inhibitory effect on TLR4 and VEGF expression and tumor angiogenesis.
Objective: To observe the inhibitory effect of triptolide (TPL) on the proliferation and apoptosis of human pancreatic cancer PANC-1 cells in vitro and in vivo, and to analyze its impact on Toll-like receptor 4 (TLR4), vascular endothelial cell growth factor (VEGF) expression and tumor angiogenesis. Methods: PANC-1 cells were treated with 0, 20, 40 and 80 ng/ml TPL in vitro. MTT and flow cytometry were performed to detect the proliferation and apoptosis of PANC-1 cells treated by TPL, respectively. TLR4 and VEGF protein expression in PANC-1 cells were evaluated by Western blotting. PANC-1 tumor-bearing nude mice were established and randomly divided into two groups: TPL group and PBS group. The tumor volume was examined, and all the tumors were taken out at 35 days after treatment. The expression of TLR4, VEGF and CD31 in the xenograft tumors was detected by immunohistochemical staining, and the microvessel density (MVD) was calculated. Results: Compared with 0 ng/ml group, after treated with 20, 40 and 80 ng/ml TPL 24 h, the apoptosis rate of PANC-1 cells was significantly increased (\[4.7±1.0\]%, \[10.5±2.0\]%, \[21.1±4.2\]% vs \[26±0.5\]%，P<0.05 or P<0.01); after 48 h, the cell proliferation rate of PANC-1 cells was significantly decreased (\[68.0±5.3\]%, \[59.6±5.0\]%, \[51.6±4.2\]% vs \[99.6±5.2\]%; all P<0.01), and was also decreased compared with that in 24 h treated with the same dose. The expression of TLR4( \[20.2±4.7\]% vs \[57.5±63\]%，P<001) and VEGF( \[35.8±4.0\]% vs \[92.1±8.3\]%，P<0.01) in PANC-1 cells treated by 80 ng/ml TPL was decreased significantly than those of the untreated group. On day 34, the tumor volume of the TPL treatment group were reduced significantly than that of the PBS control group (\[510.9±79.8\] vs \[1 220.6 ±127.2\] mm3, P<001). The expression of TLR4 (P<0.01) and VEGF in the xenograft tumor tissues of the TPL group were significantly lower than those of the PBS group (\[3.2±0.6\] vs \[6.7±1.1\], \[3.7±0.7\] vs 7.1±1.2; all P<0.01)，also the MVD within the transplanted tumor in the TPL group was significantly decreased compared with the PBS group (\[12.2±4.0\] vs \[227±5.6\], P<0.01). Conclusion: TPL can inhibit the growth of pancreatic cancer PANC-1 cells and its xenografts tumor in nude mice, and induce apoptosis of PANC-1 cells. Its mechanism may be related to the inhibitory effect on TLR4 and VEGF expression and tumor angiogenesis.
2013, 20(4):438-443.
DOI: 10.3872/j.issn.1007-385X.2013.04.010
Abstract:
Objective: To study the impact of CD133 on biological characteristics of human glioma U251 cells by construction a U251 cell line stably overexpression human CD133 gene. Methods: The full length human CD133 cDNA was sub-cloned into retroviral expressing vector pEGZ-Term to obtain recombinant pEGZ-Term-CD133 retrovirus. Then, U251 cells were infected by pEGZ-Term-CD133 retrovirus. The expression of CD133 on infected U251 cells was detected by flow cytometry and real-time PCR. By cell counting and neurosphere formation analysis, the impact of CD133 overexpression on the proliferation and neurosphere formation of U251 cells in vitro was determined. Tumorigenicity was evaluated by subcutaneous injection of CD133 infected U251 cells into the nude mice. Results: The pEGZ-Term-CD133 retrovirus expression vector was constructed successfully and a CD133 stably infected U251 cell line was obtained. Compared with U251-mock and U251 cells, the expressions of CD133 mRNA (\[7 400.2±5 003.4\] vs \[2.0±1.1, 1.0±2.2\]; all P=0.0007) and CD133 protein were significantly increased in U251-CD133 cells. pEGZ-Term-CD133 retrovirus infection showed no effect on the proliferation of U251 cells (P>0.05). However, the neurosphere formation of U251-CD133 cells was obviously higher than U251-mock and U251 cells in the presence of serum free neurosphere medium (\[34.0±7.5\] vs \[146±23\], \[11.5±13\]; all P<0.01). Compared with the U251-mock cells, the tumor formation time of U251-CD133 cells was shorter (32 d vs 38 d), the tumorigenesis ratio was higher (100% vs 30%) and the tumor volume was significantly larger (\[180.3±146.8\] vs \[4.0±0.0\] mm3, P=0.003) at 41 d when subcutaneous inoculated 1×105 cells. Conclusion: CD133 has no influence on proliferation of glioma U251 cells, but could enhance neurosphere formation and tumorigenicity of U251 cells.
Objective: To study the impact of CD133 on biological characteristics of human glioma U251 cells by construction a U251 cell line stably overexpression human CD133 gene. Methods: The full length human CD133 cDNA was sub-cloned into retroviral expressing vector pEGZ-Term to obtain recombinant pEGZ-Term-CD133 retrovirus. Then, U251 cells were infected by pEGZ-Term-CD133 retrovirus. The expression of CD133 on infected U251 cells was detected by flow cytometry and real-time PCR. By cell counting and neurosphere formation analysis, the impact of CD133 overexpression on the proliferation and neurosphere formation of U251 cells in vitro was determined. Tumorigenicity was evaluated by subcutaneous injection of CD133 infected U251 cells into the nude mice. Results: The pEGZ-Term-CD133 retrovirus expression vector was constructed successfully and a CD133 stably infected U251 cell line was obtained. Compared with U251-mock and U251 cells, the expressions of CD133 mRNA (\[7 400.2±5 003.4\] vs \[2.0±1.1, 1.0±2.2\]; all P=0.0007) and CD133 protein were significantly increased in U251-CD133 cells. pEGZ-Term-CD133 retrovirus infection showed no effect on the proliferation of U251 cells (P>0.05). However, the neurosphere formation of U251-CD133 cells was obviously higher than U251-mock and U251 cells in the presence of serum free neurosphere medium (\[34.0±7.5\] vs \[146±23\], \[11.5±13\]; all P<0.01). Compared with the U251-mock cells, the tumor formation time of U251-CD133 cells was shorter (32 d vs 38 d), the tumorigenesis ratio was higher (100% vs 30%) and the tumor volume was significantly larger (\[180.3±146.8\] vs \[4.0±0.0\] mm3, P=0.003) at 41 d when subcutaneous inoculated 1×105 cells. Conclusion: CD133 has no influence on proliferation of glioma U251 cells, but could enhance neurosphere formation and tumorigenicity of U251 cells.
2013, 20(4):444-448.
DOI: 10.3872/j.issn.1007-385X.2013.04.011
Abstract:
Objective: To observe the inhibitory effect of sachalin rhodiola rhizome extract (SRR) on regulatory T cells (Tregs) in xenograft tumors of Lewis lung cancer bearing mice and primarily discuss its mechanism of suppressing tumor growth. Methods: Lewis lung cancer-bearing mice were established and randomly divided into 3 groups: SRR group, paclitaxel (PTX) positive control group and PBS group. The changes of tumor volume were recorded in different groups, the tumor inhibition rates were calculated and the survival time of Lewis-bearing mice was observed. The proportion of CD4+CD25+Foxp3+Tregs in the xenograft tumor tissues was detected by flow cytometry. The mRNA expression levels of Foxp3 and TGF-β in the tumor tissues were detected by real-time PCR. Results: On day 20 after the establishment of the Lewis-bearing mouse model, the tumor volume of mice in the SRR group was significantly smaller than that in the PBS group (\[719.6±2.4\] vs \[1 030.5±3.1\] mm3, P<0.05), and showed no significant difference with the PTX positive control group (P>0.05). Compared with the PBS group, the survival time of mice in the SRR group was significantly prolonged (\[36.0±1.0\] vs \[22.0±2.0\] d, P<0.05), and showed no significant difference with the PTX group (P>005). The proportion of CD4+CD25+Foxp3+Tregs in CD4+T cells of the tumor tissues in the SRR group was significantly lower than that of the PBS group (\[8.5±0.3\]% vs \[11.2±0.2\]%, P<0.01), and no significant difference was observed between the SRR group and the PTX group (P>0.05). The mRNA expressions of Foxp3 (\[1.2±0.2\] vs \[21±0.2\], P<0.05) and TGF-β (\[1.2±0.2\] vs \[2.1±0.2\], P<0.05) in SRR group were significantly lower than that in the PBS group, and no significant difference was observed between the SRR group and the PTX group (P>005). Conclusion: SRR may enhance the antitumor immune response by down-regulating the proportion of CD4+CD25+Tregs and the mRNA expressions of Foxp3 and TGF-β in the tumor tissues.
Objective: To observe the inhibitory effect of sachalin rhodiola rhizome extract (SRR) on regulatory T cells (Tregs) in xenograft tumors of Lewis lung cancer bearing mice and primarily discuss its mechanism of suppressing tumor growth. Methods: Lewis lung cancer-bearing mice were established and randomly divided into 3 groups: SRR group, paclitaxel (PTX) positive control group and PBS group. The changes of tumor volume were recorded in different groups, the tumor inhibition rates were calculated and the survival time of Lewis-bearing mice was observed. The proportion of CD4+CD25+Foxp3+Tregs in the xenograft tumor tissues was detected by flow cytometry. The mRNA expression levels of Foxp3 and TGF-β in the tumor tissues were detected by real-time PCR. Results: On day 20 after the establishment of the Lewis-bearing mouse model, the tumor volume of mice in the SRR group was significantly smaller than that in the PBS group (\[719.6±2.4\] vs \[1 030.5±3.1\] mm3, P<0.05), and showed no significant difference with the PTX positive control group (P>0.05). Compared with the PBS group, the survival time of mice in the SRR group was significantly prolonged (\[36.0±1.0\] vs \[22.0±2.0\] d, P<0.05), and showed no significant difference with the PTX group (P>005). The proportion of CD4+CD25+Foxp3+Tregs in CD4+T cells of the tumor tissues in the SRR group was significantly lower than that of the PBS group (\[8.5±0.3\]% vs \[11.2±0.2\]%, P<0.01), and no significant difference was observed between the SRR group and the PTX group (P>0.05). The mRNA expressions of Foxp3 (\[1.2±0.2\] vs \[21±0.2\], P<0.05) and TGF-β (\[1.2±0.2\] vs \[2.1±0.2\], P<0.05) in SRR group were significantly lower than that in the PBS group, and no significant difference was observed between the SRR group and the PTX group (P>005). Conclusion: SRR may enhance the antitumor immune response by down-regulating the proportion of CD4+CD25+Tregs and the mRNA expressions of Foxp3 and TGF-β in the tumor tissues.
Cai Kai
,
Ai Yueqin
,
Zhang Chuang
,
Gao Yanrong
,
Guo Shulin
,
Jiang Longwei
,
Zhang Yan
,
Jia Shaochang
2013, 20(4):449-455.
DOI: 10.3872/j.issn.1007-385X.2013.04.012
Abstract:
Objective: To investigate the safety and efficiency of dendritic cells (DCs) combined with cytokine-induced killer (CIK) cells in the treatment of local advanced and advanced pancreatic cancer. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 24 pancreatic cancer patients in Ⅲ-Ⅳ stage from the department of tumor bio-therapy of No.81 Hospital of PLA during Jul. 2011 to May. 2012 and were cultured in vitro to obtain DC and CIK cells. DCs were sensitized by PANC1 (pancreatic cancer cell line) cell lysates and then transfused to pancreatic cancer patients combined with CIK cells. The changes in peripheral blood lymphocyte subsets, serum tumor markers and clinical efficiency were evaluated before and after DC-CIK cell treatment. Results: The proportions of CD3+ T cells, CD8+T cells and CD4+CD25+Treg were significantly decreased after DC-CIK cell treatment for 3 months (all P<0.05), while the ratio of CD4+/CD8+ cells was significantly increased (1.1±0.7 vs 1.5±0.9, P<0.05). The serum tumor marker CA19-9 level declined 1 month after-treatment (382.8±277.7 vs 213.8±214.6, P<0.05) and 3 months after-treatment (2138±214.6 vs 1540±118.2, P<0.01). In all the 24 patients, there was no case of complete response, 3 cases of partial response, 4 cases of stable disease, 17 cases of progressive disease; the clinical response rate was 12.5% and the disease control rate was 29.2%; the median survival time was 5.7 months; the 6- and 9-month overall survival rates were 33% and 27%, respectively. No grade 3-4 adverse events were observed in all cases during treatment. Conclusion: DCs combined with CIK cell treatment on local advanced and advanced pancreatic cancer patients are safe and feasible. DC-CIK treatment could increase patient's immune function and show clinical benefits.
Objective: To investigate the safety and efficiency of dendritic cells (DCs) combined with cytokine-induced killer (CIK) cells in the treatment of local advanced and advanced pancreatic cancer. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 24 pancreatic cancer patients in Ⅲ-Ⅳ stage from the department of tumor bio-therapy of No.81 Hospital of PLA during Jul. 2011 to May. 2012 and were cultured in vitro to obtain DC and CIK cells. DCs were sensitized by PANC1 (pancreatic cancer cell line) cell lysates and then transfused to pancreatic cancer patients combined with CIK cells. The changes in peripheral blood lymphocyte subsets, serum tumor markers and clinical efficiency were evaluated before and after DC-CIK cell treatment. Results: The proportions of CD3+ T cells, CD8+T cells and CD4+CD25+Treg were significantly decreased after DC-CIK cell treatment for 3 months (all P<0.05), while the ratio of CD4+/CD8+ cells was significantly increased (1.1±0.7 vs 1.5±0.9, P<0.05). The serum tumor marker CA19-9 level declined 1 month after-treatment (382.8±277.7 vs 213.8±214.6, P<0.05) and 3 months after-treatment (2138±214.6 vs 1540±118.2, P<0.01). In all the 24 patients, there was no case of complete response, 3 cases of partial response, 4 cases of stable disease, 17 cases of progressive disease; the clinical response rate was 12.5% and the disease control rate was 29.2%; the median survival time was 5.7 months; the 6- and 9-month overall survival rates were 33% and 27%, respectively. No grade 3-4 adverse events were observed in all cases during treatment. Conclusion: DCs combined with CIK cell treatment on local advanced and advanced pancreatic cancer patients are safe and feasible. DC-CIK treatment could increase patient's immune function and show clinical benefits.
2013, 20(4):456-460.
DOI: 10.3872/j.issn.1007-385X.2013.04.013
Abstract:
Objective: To detect the expression level of early growth response gene-1 (EGR-1) protein in human lung squamous cell carcinoma tissues, and to investigate the relationship of EGR-1 protein expression with clinicopathologic factors and prognosis of lung squamous cell carcinoma. Methods: Carcinoma and corresponding paracancerous tissues were obtained from 83 lung squamous cell carcinoma patients (Qilu Hospital of Shandong University during Jan. 2007 to Dec. 2007). Immunohistochemical staining (SP method) was performed to detect the expression level of EGR-1 in carcinoma and paracancerous tissues. χ2 test was used to analyze the relationship between EGR-1 protein expression and patients' clinicopathologic factors, Kaplan-Meier method was used to calculate the 5-year survival rate of patients, Log-rank test was used to compare survival differences, and Cox regression analysis was used to determine the independent prognostic factor. Results: The expression of EGR-1 protein in lung squamous cell carcinoma tissues was significantly lower than that in paracancerous tissues (\[4.12±0.35\] vs \[6.90±4.58\], P<0.01). EGR-1 protein low expression was not significantly associated with patient’s age (P=0.912) and gender (P=0.429), and the differentiation stage of tumor (P=0.289), but significantly associated with patient’s smoking history (P=0.025), tumor size (P=0.013), lymph node metastasis (P=0.003) and TNM stage of tumor (P=0.028). The patients with a low expression of EGR-1 protein had a significantly low overall survival (2.5% vs 15.9%, P=0.04) at 5 years after operation compared with patients with a high expression of EGR-1 protein. TNM stage (P=0.020) and low expression of EGR-1 protein (P=0.035) were independent prognosticators of patients with lung squamous cell carcinoma. Conclusion: EGR-1 protein is low expressed in lung squamous cell carcinoma tissues, and is associated with tumor progression and prognosis.
Objective: To detect the expression level of early growth response gene-1 (EGR-1) protein in human lung squamous cell carcinoma tissues, and to investigate the relationship of EGR-1 protein expression with clinicopathologic factors and prognosis of lung squamous cell carcinoma. Methods: Carcinoma and corresponding paracancerous tissues were obtained from 83 lung squamous cell carcinoma patients (Qilu Hospital of Shandong University during Jan. 2007 to Dec. 2007). Immunohistochemical staining (SP method) was performed to detect the expression level of EGR-1 in carcinoma and paracancerous tissues. χ2 test was used to analyze the relationship between EGR-1 protein expression and patients' clinicopathologic factors, Kaplan-Meier method was used to calculate the 5-year survival rate of patients, Log-rank test was used to compare survival differences, and Cox regression analysis was used to determine the independent prognostic factor. Results: The expression of EGR-1 protein in lung squamous cell carcinoma tissues was significantly lower than that in paracancerous tissues (\[4.12±0.35\] vs \[6.90±4.58\], P<0.01). EGR-1 protein low expression was not significantly associated with patient’s age (P=0.912) and gender (P=0.429), and the differentiation stage of tumor (P=0.289), but significantly associated with patient’s smoking history (P=0.025), tumor size (P=0.013), lymph node metastasis (P=0.003) and TNM stage of tumor (P=0.028). The patients with a low expression of EGR-1 protein had a significantly low overall survival (2.5% vs 15.9%, P=0.04) at 5 years after operation compared with patients with a high expression of EGR-1 protein. TNM stage (P=0.020) and low expression of EGR-1 protein (P=0.035) were independent prognosticators of patients with lung squamous cell carcinoma. Conclusion: EGR-1 protein is low expressed in lung squamous cell carcinoma tissues, and is associated with tumor progression and prognosis.
2013, 20(4):461-467.
DOI: 10.3872/j.issn.1007-385X.2013.04.014
Abstract:
Objective: To systematically assess the therapeutic effectiveness of adoptive immunotherapy combined with chemo/radio therapy compared with chemo/radio therapy alone in non-small cell lung cancer (NSCLC) patients. Methods: A systematic search of Pubmed, Medline, EMBASE, Cochrane Library, CNKI and VIP database from January 1995 to September 2012 was performed to identify the eligible randomized controlled trials (RCTs) and non-randomized concurrent controlled trails (NRCCTs). The improper documents were excluded. The soft-ware Revman5.0 was used for data synthesis. Results: A total of 10 studies involving 1 326 patients were identified. The result of Meta-analyses showed that adoptive immunotherapy combined with radio/chemo therapy improved the 2-year progression-free survival (PFS) (OR=2.20, 95%CI：1.44-3.36, P=0.0003) and 2-year overall survival (OR=2.69, 95%CI：1.92-3.78, P<000001). Both early-stage and advanced NSCLC patients benefited from adoptive immunotherapy (OR=3.24\[1.65-6.35\]; OR=2.86\[1.37-5.98\]). The adverse events were self limiting, including fever, chill, nausea, and fatigue. No severe toxicity was observed. Conclusion: Adoptive immunotherapy combined with chemo/radio therapy can decrease the risk of recurrence and improve the overall survival of NSCLC patients; early-stage patients receiving adoptive immunotherapy may benefit more.
Objective: To systematically assess the therapeutic effectiveness of adoptive immunotherapy combined with chemo/radio therapy compared with chemo/radio therapy alone in non-small cell lung cancer (NSCLC) patients. Methods: A systematic search of Pubmed, Medline, EMBASE, Cochrane Library, CNKI and VIP database from January 1995 to September 2012 was performed to identify the eligible randomized controlled trials (RCTs) and non-randomized concurrent controlled trails (NRCCTs). The improper documents were excluded. The soft-ware Revman5.0 was used for data synthesis. Results: A total of 10 studies involving 1 326 patients were identified. The result of Meta-analyses showed that adoptive immunotherapy combined with radio/chemo therapy improved the 2-year progression-free survival (PFS) (OR=2.20, 95%CI：1.44-3.36, P=0.0003) and 2-year overall survival (OR=2.69, 95%CI：1.92-3.78, P<000001). Both early-stage and advanced NSCLC patients benefited from adoptive immunotherapy (OR=3.24\[1.65-6.35\]; OR=2.86\[1.37-5.98\]). The adverse events were self limiting, including fever, chill, nausea, and fatigue. No severe toxicity was observed. Conclusion: Adoptive immunotherapy combined with chemo/radio therapy can decrease the risk of recurrence and improve the overall survival of NSCLC patients; early-stage patients receiving adoptive immunotherapy may benefit more.
15 A Meta-analysis of Kang’ai injection combined with chemotherapy in the treatment of gastric cancer
2013, 20(4):468-474.
DOI: 10.3872/j.issn.1007-385X.2013.04.015
Abstract:
Objective: To investigate whether Kang’ai injection combined with chemotherapy can improve the clinical efficiency of patients with gastric cancer treated with chemotherapy alone. Methods: Relevant randomized controlled trials (RCTs) of Kang’ai injection combined with chemotherapy on gastric cancer patients were searched in the PubMed, CJFD database, VIP database and CBMdisc database published in 2005-2012. Stata software was used for data processing and Meta analysis. Results: Fourteen RCTs were included in the Meta analysis. Meta analysis results suggested that compared with chemotherapy alone, the combination therapy containing Kang’ai injection significantly improved the quality of life (QOL) (P=0.000), white blood cell (WBC) reduction (P=0.000), gastrointestinal reaction (P=0.000), liver injury (P=0.047), peripheral neuritis (P=0.000), weight lose (P=0.002) and pain (P=0.017) of patients with gastric cancer. However, the Kang’ai injection combined with chemotherapy showed no significant difference on clinical efficiency (P=0.093) and other side effects compared with chemotherapy alone. Conclusion: From the existing clinical evidence, Kang’ai injection combined with chemotherapy can improve the life quality of patients with gastric cancer and alleviate some common side effects of chemotherapy.
Objective: To investigate whether Kang’ai injection combined with chemotherapy can improve the clinical efficiency of patients with gastric cancer treated with chemotherapy alone. Methods: Relevant randomized controlled trials (RCTs) of Kang’ai injection combined with chemotherapy on gastric cancer patients were searched in the PubMed, CJFD database, VIP database and CBMdisc database published in 2005-2012. Stata software was used for data processing and Meta analysis. Results: Fourteen RCTs were included in the Meta analysis. Meta analysis results suggested that compared with chemotherapy alone, the combination therapy containing Kang’ai injection significantly improved the quality of life (QOL) (P=0.000), white blood cell (WBC) reduction (P=0.000), gastrointestinal reaction (P=0.000), liver injury (P=0.047), peripheral neuritis (P=0.000), weight lose (P=0.002) and pain (P=0.017) of patients with gastric cancer. However, the Kang’ai injection combined with chemotherapy showed no significant difference on clinical efficiency (P=0.093) and other side effects compared with chemotherapy alone. Conclusion: From the existing clinical evidence, Kang’ai injection combined with chemotherapy can improve the life quality of patients with gastric cancer and alleviate some common side effects of chemotherapy.
Luo Guanghua
,
Ma Ming
,
Guo Lili
,
Duan Yuqing
,
Wang Tingting
,
Jia Yunlong
,
Han Hengli
,
Su Zhengjun
,
Liu Lihua
2013, 20(4):475-477.
DOI: 10.3872/j.issn.1007-385X.2013.04.016
Abstract:
目的： 分析接受细胞因子诱导的杀伤（cytokine-induced killer，CIK）细胞治疗后肿瘤患者外周血免疫细胞亚型的变化，探讨CIK细胞治疗对肿瘤患者免疫功能的影响。 方法： 采集2012年3月7日至2012年8月30日期间河北医科大学第四医院生物治疗科收治的60例肿瘤患者（肺癌25例、胃癌8例、直肠癌6例、食管癌11例、黑素瘤7例和乳腺癌3例）外周血及对照组20名健康志愿者外周血，分离外周血单个核细胞，常规方法体外扩增CIK细胞，分3次回输患者体内，流式细胞术检测CIK细胞治疗前后肿瘤患者外周血淋巴细胞亚型的变化。 结果： 与治疗前相比，经1次CIK细胞治疗后，CD3+CD4+T细胞比例升高（P=0.016）、CD4+/CD8+细胞比值升高（P=0.013）且均达到正常水平，CD3+CD8+细胞比例降低（P=0109）但仍高于对照组（P=0.048)；3次CIK细胞治疗后相比1次治疗后无显著变化（P>0.05）。CD4+CD25+Treg比例经1次CIK治疗后较治疗前显著下降（P=0.007），达到正常水平；3次治疗后持续下降（P=0.005）。CIK治疗对CD3-CD19+B细胞和CD3-CD56+自然杀伤（natural killer，NK）细胞水平无显著影响（P>0.05）。 结论： CIK细胞治疗能够降低肿瘤患者外周血Treg比例，升高CD3+CD4+T细胞比例及CD4+/CD8+细胞比值，从而减轻或消除肿瘤患者的免疫抑制状态。
目的： 分析接受细胞因子诱导的杀伤（cytokine-induced killer，CIK）细胞治疗后肿瘤患者外周血免疫细胞亚型的变化，探讨CIK细胞治疗对肿瘤患者免疫功能的影响。 方法： 采集2012年3月7日至2012年8月30日期间河北医科大学第四医院生物治疗科收治的60例肿瘤患者（肺癌25例、胃癌8例、直肠癌6例、食管癌11例、黑素瘤7例和乳腺癌3例）外周血及对照组20名健康志愿者外周血，分离外周血单个核细胞，常规方法体外扩增CIK细胞，分3次回输患者体内，流式细胞术检测CIK细胞治疗前后肿瘤患者外周血淋巴细胞亚型的变化。 结果： 与治疗前相比，经1次CIK细胞治疗后，CD3+CD4+T细胞比例升高（P=0.016）、CD4+/CD8+细胞比值升高（P=0.013）且均达到正常水平，CD3+CD8+细胞比例降低（P=0109）但仍高于对照组（P=0.048)；3次CIK细胞治疗后相比1次治疗后无显著变化（P>0.05）。CD4+CD25+Treg比例经1次CIK治疗后较治疗前显著下降（P=0.007），达到正常水平；3次治疗后持续下降（P=0.005）。CIK治疗对CD3-CD19+B细胞和CD3-CD56+自然杀伤（natural killer，NK）细胞水平无显著影响（P>0.05）。 结论： CIK细胞治疗能够降低肿瘤患者外周血Treg比例，升高CD3+CD4+T细胞比例及CD4+/CD8+细胞比值，从而减轻或消除肿瘤患者的免疫抑制状态。
2013, 20(4):478-484.
DOI: 10.3872/j.issn.1007-385X.2013.04.017
Abstract:
近年来个体化治疗理念在肿瘤领域已被广泛接受，基因检测和个体化治疗方案制定是实施个体化治疗的重要步骤。在肿瘤个体化治疗领域中，非小细胞肺癌（non-small-cell lung cancer，NSCLC）的靶标基因研究最为深入和广泛。在早期NSCLC中靶标基因的相关研究主要集中在术后辅助治疗效果预测、生存预测及复发预测等方面，相关的靶标检测目前尚未进入临床应用；在晚期NSCLC中，主要集中在化疗和靶向治疗药物疗效预测等方面，部分相关的靶标基因检测已应用于NSCLC的临床治疗和预后。目前，研究较为深入的化疗疗效相关靶标主要包括 ERCC1、RRM1、TUBB3、STMN1、ERCC2和XRCC1等基因；靶向药物疗效相关靶标主要包括 EGFR、ALK、Ros1、K-Ras、BRAF、PI3-KCA、HER2和MET 等基因。靶标基因检测能够识别NSCLC患者的个体差异，预测早期NSCLC患者的术后复发、生存及其对辅助治疗的获益情况，可为晚期NSCLC患者的药物选择及治疗方案的制定提供直观、科学的依据。
近年来个体化治疗理念在肿瘤领域已被广泛接受，基因检测和个体化治疗方案制定是实施个体化治疗的重要步骤。在肿瘤个体化治疗领域中，非小细胞肺癌（non-small-cell lung cancer，NSCLC）的靶标基因研究最为深入和广泛。在早期NSCLC中靶标基因的相关研究主要集中在术后辅助治疗效果预测、生存预测及复发预测等方面，相关的靶标检测目前尚未进入临床应用；在晚期NSCLC中，主要集中在化疗和靶向治疗药物疗效预测等方面，部分相关的靶标基因检测已应用于NSCLC的临床治疗和预后。目前，研究较为深入的化疗疗效相关靶标主要包括 ERCC1、RRM1、TUBB3、STMN1、ERCC2和XRCC1等基因；靶向药物疗效相关靶标主要包括 EGFR、ALK、Ros1、K-Ras、BRAF、PI3-KCA、HER2和MET 等基因。靶标基因检测能够识别NSCLC患者的个体差异，预测早期NSCLC患者的术后复发、生存及其对辅助治疗的获益情况，可为晚期NSCLC患者的药物选择及治疗方案的制定提供直观、科学的依据。
2013, 20(4):485-492.
DOI: 10.3872/j.issn.1007-385X.2013.04.018
Abstract:
固有免疫是人体防御外来病原体侵袭的第一道防线，也是适应性免疫的基础和启动者，在生物体内发挥重要作用，可以维持宿主免疫反应和保护感染组织间的平衡，该过程必须被精细识别与调控。近年来，固有免疫在分子水平上的识别及调控机制越来越受到关注。哺乳动物的固有免疫识别及调控主要通过一系列胚系编码的模式识别受体（pattern recognition receptor，PRR）识别病原微生物上表达的保守的病原体相关分子模式（pathogen associated molecular pattern，PAMP）来实现，这种形式让机体不但可以发现入侵的病原体，而且能够识别其类型，并通过一系列信号途径活化效应分子，识别自我与非我，激活与调控固有免疫应答，并且相互协同或互相调节以形成调控网络，从而控制并清除病原体，在固有免疫中发挥独特的功能。随着分子技术的发展，固有免疫识别与调控在分子水平的研究成果转化将对包括肿瘤在内的多种免疫相关疾病治疗产生重大的影响。但目前对于固有免疫信号转导途径的研究还不是很完善，信号通路中各个分子的具体作用机制还需要深入研究。
固有免疫是人体防御外来病原体侵袭的第一道防线，也是适应性免疫的基础和启动者，在生物体内发挥重要作用，可以维持宿主免疫反应和保护感染组织间的平衡，该过程必须被精细识别与调控。近年来，固有免疫在分子水平上的识别及调控机制越来越受到关注。哺乳动物的固有免疫识别及调控主要通过一系列胚系编码的模式识别受体（pattern recognition receptor，PRR）识别病原微生物上表达的保守的病原体相关分子模式（pathogen associated molecular pattern，PAMP）来实现，这种形式让机体不但可以发现入侵的病原体，而且能够识别其类型，并通过一系列信号途径活化效应分子，识别自我与非我，激活与调控固有免疫应答，并且相互协同或互相调节以形成调控网络，从而控制并清除病原体，在固有免疫中发挥独特的功能。随着分子技术的发展，固有免疫识别与调控在分子水平的研究成果转化将对包括肿瘤在内的多种免疫相关疾病治疗产生重大的影响。但目前对于固有免疫信号转导途径的研究还不是很完善，信号通路中各个分子的具体作用机制还需要深入研究。
2013, 20(4):493-497.
DOI: 10.3872/j.issn.1007-385X.2013.04.019
Abstract:
Hedgehog（Hh）信号通路可以调控细胞的增殖、迁移和分化等许多过程，并且在胚胎发育时期起重要作用。根据Hh信号通路激活后是否依赖Gli蛋白发挥生物学效应，可分为经典的Hh/Gli信号通路和非经典的Hh信号通路。其中，非经典的Hh信号通路可通过小G蛋白Rho家族信号和钙离子依赖的信号通路调控细胞骨架形态、细胞增殖、凋亡及迁移；经典与非经典的Hh信号通路形成相互联系的信号通路网络介导Hh信号的功能。Hh信号通路的异常持续的激活是许多肿瘤发生、发展的原因之一，Hh信号通路还与肿瘤干细胞的调控、肿瘤血管形成和肿瘤的侵袭转移密切相关，对Hh信号通路的深入研究有利于以Hh信号通路蛋白为靶点的抗肿瘤药物的研发与应用。
Hedgehog（Hh）信号通路可以调控细胞的增殖、迁移和分化等许多过程，并且在胚胎发育时期起重要作用。根据Hh信号通路激活后是否依赖Gli蛋白发挥生物学效应，可分为经典的Hh/Gli信号通路和非经典的Hh信号通路。其中，非经典的Hh信号通路可通过小G蛋白Rho家族信号和钙离子依赖的信号通路调控细胞骨架形态、细胞增殖、凋亡及迁移；经典与非经典的Hh信号通路形成相互联系的信号通路网络介导Hh信号的功能。Hh信号通路的异常持续的激活是许多肿瘤发生、发展的原因之一，Hh信号通路还与肿瘤干细胞的调控、肿瘤血管形成和肿瘤的侵袭转移密切相关，对Hh信号通路的深入研究有利于以Hh信号通路蛋白为靶点的抗肿瘤药物的研发与应用。
2013, 20(4):498-505.
DOI: 10.3872/j.issn.1007-385X.2013.04.020
Abstract:
MicroRNA（miRNA）是非蛋白编码的短RNAs，可在转录或转录后水平调节mRNA的表达。大量研究表明，miRNA在细胞增殖、分化、凋亡和代谢方面发挥着重要作用，并参与了癌基因和抗癌基因的信号调控。在多种人类恶性肿瘤中已经检测到异常表达的miRNA，并且发现miRNA与肿瘤的发生、发展、预测、诊断、治疗和预后有关，因此，miRNA作为癌症诊断、预测和治疗的潜在标记物，具有广阔的临床应用前景。miRNA在非小细胞肺癌中扮演着多重角色，包括在肺癌发生过程中发挥促癌基因或抑癌基因的作用，调控肺癌细胞的增殖和分化，参与肺癌的侵袭和转移，影响肺癌对放、化疗的敏感性等。因而，miRNA可以作为一种标志物，在血液或痰液标本中为临床提供诊断依据，在肺癌的化学治疗及放射治疗中成为疗效预测指标，还可能提示肺癌组织的分化程度和患者预后，同时还可以作为肺癌治疗潜在靶点
MicroRNA（miRNA）是非蛋白编码的短RNAs，可在转录或转录后水平调节mRNA的表达。大量研究表明，miRNA在细胞增殖、分化、凋亡和代谢方面发挥着重要作用，并参与了癌基因和抗癌基因的信号调控。在多种人类恶性肿瘤中已经检测到异常表达的miRNA，并且发现miRNA与肿瘤的发生、发展、预测、诊断、治疗和预后有关，因此，miRNA作为癌症诊断、预测和治疗的潜在标记物，具有广阔的临床应用前景。miRNA在非小细胞肺癌中扮演着多重角色，包括在肺癌发生过程中发挥促癌基因或抑癌基因的作用，调控肺癌细胞的增殖和分化，参与肺癌的侵袭和转移，影响肺癌对放、化疗的敏感性等。因而，miRNA可以作为一种标志物，在血液或痰液标本中为临床提供诊断依据，在肺癌的化学治疗及放射治疗中成为疗效预测指标，还可能提示肺癌组织的分化程度和患者预后，同时还可以作为肺癌治疗潜在靶点
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.