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Volume 21,Issue 3,2014 Table of Contents
2014, 21(3):237-244.
DOI: 10.3872/j.issn.1007-385X.2014.03.001
Abstract:
To assess the efficacy and safety profiles of vaccination with autologous DC loaded with apoptotic heat-shocked autologous tumor cell antigens in the treatment of triple-negative breast cancer (TNBC). Methods: A total of 168 patients with TNBC were recruited from three hospitals located, respectively, in Nanjing, Changzhou and Wuxi and randomized to a control group (n=56) and a treatment group (n=112). Patients in the treatment group were treated with DC vaccination for three cycles (one week each at an interval of one month) whereas those in the control group received placebo. The primary outcome measures were disease progression time (DPT) and progression-free survival rate (PFSR) at the end of 2-year follow up. The secondary outcome measures were side effects, tolerance to DC vaccination, tumor specific immune responses (i.e., changes in IL-2, IL-10, IL-12, TNF-α and IFN-γ concentrations）, the percentage of specific CD8+IFN-γ+T lymphocytes in peripheral blood and delayed type Ⅳ hypersensitivity reaction (DTH) before and after DC vaccinations. Results： No more than level Ⅱ side effects were observed in any of the participants. After one cycle of vaccination, there were significant and sustained increases in serum levels of Th1 type cytokines IL-2 (P=0038), TNF-α (P=0.024) and IFN-γ (P=0.022) in patients with stage I to stage Ⅲ lesions. The number of tumor-specific CD8+IFN-γ+T lymphocytes in the peripheral blood was increasing slowly and gradually over the course of vaccination therapy in patients with stage I to stage Ⅲ lesions; by the third cycle of DC vaccination the number became significantly higher as compared with the placebo (P<0.05). Overall, the rate of DTH was positively correlated with the vaccination cycle (r=0.973, P<0.05). When disease stages were compared, the rate of DTH was significantly higher in patients with stage I to stage Ⅲ disease than that in patients with stage Ⅳ lesions. The average DPT was 669 days in patients with stage Ⅰ to stage Ⅲ lesions and 656 days in stage Ⅳ patients in the treatment group, significantly longer than that in patients with stage I to stage Ⅲ (618 days) and stage Ⅳ (573 days) lesions in the control group (P<0.001). The average PFSR of stage Ⅲ and stage Ⅳ patients was 71.43% in the treatment group, but only 32.73% in the corresponding patients in the control group (P<0.001). Moreover, the average PFSR of DTH-positive patients was 87.30%, significantly higher than that of DTH-negative patients (51.02%). Conclusion: Autologous DC loaded with heat-shocked apoptotic autologous tumor cells may be effective in both eliciting the non-specific immune response of Th1 cells and tumor-specific CTL responses and delaying disease progression and improving survival in triple-negative breast cancer patients.
To assess the efficacy and safety profiles of vaccination with autologous DC loaded with apoptotic heat-shocked autologous tumor cell antigens in the treatment of triple-negative breast cancer (TNBC). Methods: A total of 168 patients with TNBC were recruited from three hospitals located, respectively, in Nanjing, Changzhou and Wuxi and randomized to a control group (n=56) and a treatment group (n=112). Patients in the treatment group were treated with DC vaccination for three cycles (one week each at an interval of one month) whereas those in the control group received placebo. The primary outcome measures were disease progression time (DPT) and progression-free survival rate (PFSR) at the end of 2-year follow up. The secondary outcome measures were side effects, tolerance to DC vaccination, tumor specific immune responses (i.e., changes in IL-2, IL-10, IL-12, TNF-α and IFN-γ concentrations）, the percentage of specific CD8+IFN-γ+T lymphocytes in peripheral blood and delayed type Ⅳ hypersensitivity reaction (DTH) before and after DC vaccinations. Results： No more than level Ⅱ side effects were observed in any of the participants. After one cycle of vaccination, there were significant and sustained increases in serum levels of Th1 type cytokines IL-2 (P=0038), TNF-α (P=0.024) and IFN-γ (P=0.022) in patients with stage I to stage Ⅲ lesions. The number of tumor-specific CD8+IFN-γ+T lymphocytes in the peripheral blood was increasing slowly and gradually over the course of vaccination therapy in patients with stage I to stage Ⅲ lesions; by the third cycle of DC vaccination the number became significantly higher as compared with the placebo (P<0.05). Overall, the rate of DTH was positively correlated with the vaccination cycle (r=0.973, P<0.05). When disease stages were compared, the rate of DTH was significantly higher in patients with stage I to stage Ⅲ disease than that in patients with stage Ⅳ lesions. The average DPT was 669 days in patients with stage Ⅰ to stage Ⅲ lesions and 656 days in stage Ⅳ patients in the treatment group, significantly longer than that in patients with stage I to stage Ⅲ (618 days) and stage Ⅳ (573 days) lesions in the control group (P<0.001). The average PFSR of stage Ⅲ and stage Ⅳ patients was 71.43% in the treatment group, but only 32.73% in the corresponding patients in the control group (P<0.001). Moreover, the average PFSR of DTH-positive patients was 87.30%, significantly higher than that of DTH-negative patients (51.02%). Conclusion: Autologous DC loaded with heat-shocked apoptotic autologous tumor cells may be effective in both eliciting the non-specific immune response of Th1 cells and tumor-specific CTL responses and delaying disease progression and improving survival in triple-negative breast cancer patients.
Liu Honglin
,
Peng Liang
,
Wang Zai
,
Xu Shiqing
,
Lou Jinning
,
Ran Yuliang
,
Yang Zhihua
,
Wang Peigang
,
Zhang Wenjian
2014, 21(3):245-250.
DOI: 10.3872/j.issn.1007-385X.2014.03.002
Abstract:
To investigate the multilineage differentiation potential of single-cell-cloned liver cancer stem cells (LCSCs). Methods: Single cell cloned LCSCs were generated by limiting dilution cloning and confirmed by RT-PCR analysis of stem cell markers. Phenotype-confirmed LCSCs were induced to differentiate into osteoblasts, chondrocytes and adipocytes, respectively, by culturing them in corresponding differentiation-inducing media for 3 weeks. Both before and after the differentiation induction culture, the expression of markers specific for osteoblasts, chondrocytes and adipocytes was comparatively analyzed by real-time PCR and cell type-specific staining techniques.Results: Stem cell markers including CD133, CD34, ATP-binding cassette sub-family G member 2 (ABCG2), nestin, stem cell factor (SCF) and stem cell growth factor receptor (C-kit) were detected in undifferentiated single cell cloned LCSCs. After three weeks of differentiation induction, cells cultured in the osteoblast-specific medium formed orange calcium nodules as demonstrated by Alizarin red staining; cells cultured in the chondrocyte-specific medium showed blue proteoglycan depositions as revealed by Alcian blue staining; and cells cultured in the adipocyte-specific medium showed Oil Red O stained lipid droplets. In the three corresponding differentiation induction cultures, LCSCs acquired the expression of markers for osteoblast (osteocalcin and collagen), chondrocyte (aggrecan and collagen type Ⅱ), and adipocyte (adiponectin and peroxisome proliferator-activated receptor) respectively. All the differences were significant (P<0.01) except for collagen type Ⅰand collagen type Ⅱ (P<0.05). Conclusion: These preliminary observations suggest that single cell cloned LCSCs possess a plasticity and may potentially differentiate into mesenchymal-like cells under specific microenvironments.
To investigate the multilineage differentiation potential of single-cell-cloned liver cancer stem cells (LCSCs). Methods: Single cell cloned LCSCs were generated by limiting dilution cloning and confirmed by RT-PCR analysis of stem cell markers. Phenotype-confirmed LCSCs were induced to differentiate into osteoblasts, chondrocytes and adipocytes, respectively, by culturing them in corresponding differentiation-inducing media for 3 weeks. Both before and after the differentiation induction culture, the expression of markers specific for osteoblasts, chondrocytes and adipocytes was comparatively analyzed by real-time PCR and cell type-specific staining techniques.Results: Stem cell markers including CD133, CD34, ATP-binding cassette sub-family G member 2 (ABCG2), nestin, stem cell factor (SCF) and stem cell growth factor receptor (C-kit) were detected in undifferentiated single cell cloned LCSCs. After three weeks of differentiation induction, cells cultured in the osteoblast-specific medium formed orange calcium nodules as demonstrated by Alizarin red staining; cells cultured in the chondrocyte-specific medium showed blue proteoglycan depositions as revealed by Alcian blue staining; and cells cultured in the adipocyte-specific medium showed Oil Red O stained lipid droplets. In the three corresponding differentiation induction cultures, LCSCs acquired the expression of markers for osteoblast (osteocalcin and collagen), chondrocyte (aggrecan and collagen type Ⅱ), and adipocyte (adiponectin and peroxisome proliferator-activated receptor) respectively. All the differences were significant (P<0.01) except for collagen type Ⅰand collagen type Ⅱ (P<0.05). Conclusion: These preliminary observations suggest that single cell cloned LCSCs possess a plasticity and may potentially differentiate into mesenchymal-like cells under specific microenvironments.
2014, 21(3):251-256.
DOI: 10.3872/j.issn.1007-385X.2014.03.003
Abstract:
To analyse T4 phage-induced M1 re-polarization of tumor-associated M2 type macrophages, and to evaluate the ability of re-polarized M1 macrophages on apoptosis and metastasis of mouse Lewis lung cancer cells in vitro. Methods: Mouse macrophage RAW264.7 cells were induced into M2 type macrophages by interleukin-4 (IL-4), which were subsequently induced to re-polarize with T4-bacteriophage. In both IL-4-induced M2 macrophages and T4 repolariezed-macrophages, mRNA levels of 〖STBX〗IL-12, TNF-α, Arg-1, TGF-β, IL-10〖STBZ〗 and iNOS were analyzed by Real-time PCR, and protein levels of iNOS and Arg-1 were determined by Western blotting. The effect of T4-phage-induced re-polarization on the apoptosis and metastasis of Lewis lung cancer cells were evaluated by the flow cytometry and transwell assays, respectively. Results: RAW264.7 cells were successfully induced into an M2 phenotype with a significant increase in mRNA levels of 〖STBX〗Arg-1〖STBZ〗 and 〖STBX〗IL-10〖STBZ〗 (161.2 and 120.3 folds, respectively), together with a significant decrease in iNOS and 〖STBX〗IL-12〖STBZ〗 mRNA levels (3.3 and 7.8 folds, respectively). Both wild-type and the soc-hoc-T4 bacteriophages effectively induce M1 re-polarization of M2 type macrophages, but the induction activity of the soc-hoc-T4 phages was significantly stronger than that of the wild-type (P<0.05). Both wild-type and polarized M1 type macrophages more effectively induced apoptotic cell death ([35.3±2.44]%, [39.1±2.08]% vs [4.68±0.56]%, P<0.01) and suppressed the invasive capability in Lewis lung cancer cells ([43.8±7.51]%, [23.2±4.33]% vs [177.5±12.33]%, P<0.01), as compared with M2 macrophages. Conclusion: T4 bacteriophages can induce M1 re-polarization of M2 type macrophages, which results in an increase in apoptotic cell death and a decrease in invasive activity in Lewis lung cancer cells in vitro.
To analyse T4 phage-induced M1 re-polarization of tumor-associated M2 type macrophages, and to evaluate the ability of re-polarized M1 macrophages on apoptosis and metastasis of mouse Lewis lung cancer cells in vitro. Methods: Mouse macrophage RAW264.7 cells were induced into M2 type macrophages by interleukin-4 (IL-4), which were subsequently induced to re-polarize with T4-bacteriophage. In both IL-4-induced M2 macrophages and T4 repolariezed-macrophages, mRNA levels of 〖STBX〗IL-12, TNF-α, Arg-1, TGF-β, IL-10〖STBZ〗 and iNOS were analyzed by Real-time PCR, and protein levels of iNOS and Arg-1 were determined by Western blotting. The effect of T4-phage-induced re-polarization on the apoptosis and metastasis of Lewis lung cancer cells were evaluated by the flow cytometry and transwell assays, respectively. Results: RAW264.7 cells were successfully induced into an M2 phenotype with a significant increase in mRNA levels of 〖STBX〗Arg-1〖STBZ〗 and 〖STBX〗IL-10〖STBZ〗 (161.2 and 120.3 folds, respectively), together with a significant decrease in iNOS and 〖STBX〗IL-12〖STBZ〗 mRNA levels (3.3 and 7.8 folds, respectively). Both wild-type and the soc-hoc-T4 bacteriophages effectively induce M1 re-polarization of M2 type macrophages, but the induction activity of the soc-hoc-T4 phages was significantly stronger than that of the wild-type (P<0.05). Both wild-type and polarized M1 type macrophages more effectively induced apoptotic cell death ([35.3±2.44]%, [39.1±2.08]% vs [4.68±0.56]%, P<0.01) and suppressed the invasive capability in Lewis lung cancer cells ([43.8±7.51]%, [23.2±4.33]% vs [177.5±12.33]%, P<0.01), as compared with M2 macrophages. Conclusion: T4 bacteriophages can induce M1 re-polarization of M2 type macrophages, which results in an increase in apoptotic cell death and a decrease in invasive activity in Lewis lung cancer cells in vitro.
Mei Weiqun
,
Li Xiaoya
,
Wang Weiguo
,
Ma Juming
,
Ji Weidan
,
Hu Huizhen
,
Song Qizhe
,
Su Changqing
,
Wu Mengchao
2014, 21(3):257-262.
DOI: 10.3872/j.issn.1007-385X.2014.03.004
Abstract:
To evaluate the efficacy of cell-penetrating peptide-fused HSP70 gene therapy in combination with the use of cytokine-induced killer (CIK) cells for gastric cancer in a xenograft nude mouse model. Methods: CMV promoter-driven adenoviral vectors expressing wild-type HSP70 (AdCMV-HSP70) and HSP70 fused with a cell-penetrating peptide of 11 arginines (AdCMV-HSP70s) were constructed. In in vitro experiments, human gastric cancer SGC-7901 and GES-1 cells were infected with these two viral vectors respectively. At 48 h after infection, cell viability was assessed by MTT assays and HSP70 protein content by Western blotting. In in vivo experiments, Sgc-7901 cells were injected subcutaneously into BALB/c nude mice. Cancer cell-challenged mice were treated every 48 h for a total of five times with AdCMV-HSP70 or AdCMV-HSP70s, either each alone or in combination with a single dose of 1×107 CIK cells (tail vein injection) prepared from normal BALB/c mice. Thirty-five days after treatment, animals were sacrificed. The number and size of tumors formed were assessed. Results: HSP70 protein content after AdCMV-HSP70s infection was significantly higher than that after AdCMV-HSP70 infection in both SGC cells (360.72±20.89 ng/ml vs 121.01±15.94 ng/ml, P<0.05) and GES-1 cells (188.62±10.82 ng/ml vs 135.00±13.96 ng/ml, P<0.05). Fusion of 11 arginine peptide into HSP70 led to significant inhibition of gastric cancer cell proliferation; at low multiplicity of infection (MOI) of 100 pfu/ml, the cell viability was (66.33±4.33)% in AdCMV-HSP70s-infected SGC-7901 cells, significantly lower than that (101.33±7.64%) in AdCMV-HSP70-infected SGC-7901 cells (P<0.05). CIK cell administration resulted in immune reconstruction in nude mice carrying xenograft gastric cancer cells. Adenoviral delivery of HSP70 enhanced infiltration of CD3+ T cells into tumor tissues and induce antitumor immune response. CIK cells in combination with AdCMV-HSP70s showed a significantly higher tumor inhibition rate (66.5%) than CIK cells in combination with AdCMV-HSP70 (43.6%) in nude mince challenged with gastric cancer cells (P<0.05). Conclusion: In nude mice carrying human gastric cancer cells, HSP70 may not only inhibit gastric cancer cell proliferation but also induce antitumor immune response. These effects can be enhanced by fusion of a cell-penetrating peptide with the HSP70 molecule and implantation of cytokine-induced killer cells.
To evaluate the efficacy of cell-penetrating peptide-fused HSP70 gene therapy in combination with the use of cytokine-induced killer (CIK) cells for gastric cancer in a xenograft nude mouse model. Methods: CMV promoter-driven adenoviral vectors expressing wild-type HSP70 (AdCMV-HSP70) and HSP70 fused with a cell-penetrating peptide of 11 arginines (AdCMV-HSP70s) were constructed. In in vitro experiments, human gastric cancer SGC-7901 and GES-1 cells were infected with these two viral vectors respectively. At 48 h after infection, cell viability was assessed by MTT assays and HSP70 protein content by Western blotting. In in vivo experiments, Sgc-7901 cells were injected subcutaneously into BALB/c nude mice. Cancer cell-challenged mice were treated every 48 h for a total of five times with AdCMV-HSP70 or AdCMV-HSP70s, either each alone or in combination with a single dose of 1×107 CIK cells (tail vein injection) prepared from normal BALB/c mice. Thirty-five days after treatment, animals were sacrificed. The number and size of tumors formed were assessed. Results: HSP70 protein content after AdCMV-HSP70s infection was significantly higher than that after AdCMV-HSP70 infection in both SGC cells (360.72±20.89 ng/ml vs 121.01±15.94 ng/ml, P<0.05) and GES-1 cells (188.62±10.82 ng/ml vs 135.00±13.96 ng/ml, P<0.05). Fusion of 11 arginine peptide into HSP70 led to significant inhibition of gastric cancer cell proliferation; at low multiplicity of infection (MOI) of 100 pfu/ml, the cell viability was (66.33±4.33)% in AdCMV-HSP70s-infected SGC-7901 cells, significantly lower than that (101.33±7.64%) in AdCMV-HSP70-infected SGC-7901 cells (P<0.05). CIK cell administration resulted in immune reconstruction in nude mice carrying xenograft gastric cancer cells. Adenoviral delivery of HSP70 enhanced infiltration of CD3+ T cells into tumor tissues and induce antitumor immune response. CIK cells in combination with AdCMV-HSP70s showed a significantly higher tumor inhibition rate (66.5%) than CIK cells in combination with AdCMV-HSP70 (43.6%) in nude mince challenged with gastric cancer cells (P<0.05). Conclusion: In nude mice carrying human gastric cancer cells, HSP70 may not only inhibit gastric cancer cell proliferation but also induce antitumor immune response. These effects can be enhanced by fusion of a cell-penetrating peptide with the HSP70 molecule and implantation of cytokine-induced killer cells.
2014, 21(3):263-268.
DOI: 10.3872/j.issn.1007-385X.2014.03.005
Abstract:
To investigate the effect of (human calcium/calmodulin-dependent protein kinase Ⅱ inhibitory alpha (hCaMKⅡN-α) on the production of immunosuppressive factors in colon cancer cells and the mechanisms underlying the effect in vitro. Methods: Overexpression and silencing of the hCaMKⅡN-α gene in human colon adenocarcinoma (LoVo, SW620 and HT29) cells were achieved by transfection with a hCaMKⅡN-α-expressing plasmid (pKⅡN-α) and an siRNA (si-KⅡN-α ) vector, respectively. Messenger RNA levels of interleukin-8 (IL-8), interleukin-10 (IL-10) and vascular endothelial cell growth factor (VEGF) in LoVo cells transfected with pKⅡN- were analyzed by RT-PCR. Protein levels of IL-8, IL-10 and VEGF in SW620 and LoVo cells transfected with pKⅡN- and in HT29 cells transfected with si-KⅡN- were determined by ELISA. The differences in IL-8 and VEGF protein levels in HT29 cells transfected with pKIN-α in the presence or absence of U0126 (10 M), a selective ERK1/2 inhibitor, were analyzed to elucidate the role of ERK1/2 in hCaMKⅡN-α-mediated IL-8 and VEGF production. Results: Overexpression of hCaMKⅡN- significantly decreased the mRNA abundance and protein levels of VEGF and IL-8 (P<0.05) but not PGE2 (P>0.01). Silencing of hCaMKⅡN- by siRNA significantly increased the secretion of VEGF and IL-8 in HT29 cells, but had no effect on the secretion of PGE2 and IL-10. U0126 treatment resulted in a complete reversion of increased IL-8 secretion but only a partial reversion of increased VEGF secretion in HT29 cells overexpressing hCaMKⅡ-α. Conclusion: Our observations suggest that hCaMKⅡ- may inhibit the secretion of VEGF and IL-8 and thus down-regulate the immune response in rectal tumor cells through an ERK signaling pathway-dependent mechanism.
To investigate the effect of (human calcium/calmodulin-dependent protein kinase Ⅱ inhibitory alpha (hCaMKⅡN-α) on the production of immunosuppressive factors in colon cancer cells and the mechanisms underlying the effect in vitro. Methods: Overexpression and silencing of the hCaMKⅡN-α gene in human colon adenocarcinoma (LoVo, SW620 and HT29) cells were achieved by transfection with a hCaMKⅡN-α-expressing plasmid (pKⅡN-α) and an siRNA (si-KⅡN-α ) vector, respectively. Messenger RNA levels of interleukin-8 (IL-8), interleukin-10 (IL-10) and vascular endothelial cell growth factor (VEGF) in LoVo cells transfected with pKⅡN- were analyzed by RT-PCR. Protein levels of IL-8, IL-10 and VEGF in SW620 and LoVo cells transfected with pKⅡN- and in HT29 cells transfected with si-KⅡN- were determined by ELISA. The differences in IL-8 and VEGF protein levels in HT29 cells transfected with pKIN-α in the presence or absence of U0126 (10 M), a selective ERK1/2 inhibitor, were analyzed to elucidate the role of ERK1/2 in hCaMKⅡN-α-mediated IL-8 and VEGF production. Results: Overexpression of hCaMKⅡN- significantly decreased the mRNA abundance and protein levels of VEGF and IL-8 (P<0.05) but not PGE2 (P>0.01). Silencing of hCaMKⅡN- by siRNA significantly increased the secretion of VEGF and IL-8 in HT29 cells, but had no effect on the secretion of PGE2 and IL-10. U0126 treatment resulted in a complete reversion of increased IL-8 secretion but only a partial reversion of increased VEGF secretion in HT29 cells overexpressing hCaMKⅡ-α. Conclusion: Our observations suggest that hCaMKⅡ- may inhibit the secretion of VEGF and IL-8 and thus down-regulate the immune response in rectal tumor cells through an ERK signaling pathway-dependent mechanism.
Xu Gaoya
,
Ji Weidan
,
Yan Yan
,
Bao Longlong
,
Shen Shuwen
,
Gu Lei
,
Fu Xiaohui
,
Jiang Xiaoqing
,
Su Changqing
,
Wu Mengchao
2014, 21(3):269-274.
DOI: 10.3872/j.issn.1007-385X.2014.03.006
Abstract:
To investigate the possibility of enhance the sensitivity of breast cancer MCF-7 cells to the PARP inhibitor AZD2281 by up-regulate the expression of hSulf-1. Methods: MCF-7 cells were infected with Ad5-hSulf1 or Ad5-EGFP. Transfectants were treated with different concentrations of AZD2281 and the most optimal concentration was determined. In further experiments, both Ad5-hSulf-1-overexpressing MCF-7 cells and Ad5-EGFP-expressing control MCF-7 cells were treated with AZD2281 at the optimal concentration determined. After treatment for 24 h, cell cycle progression was assessed by flow cytometry (FCM), formation ability of MCF-7 cells by colony formation assay, protein levels of cyclin dependent kinase 4 (CDK4) and phosphorylated protein kinase B (p-AKT) Western blotting, cell migration by Transwell assay, and proliferative ability by MTT assay. Results: AZD2281 showed the peak inhibitory activity at a concentration of 7 μmol/L. When this concentration was used,Ad5-EGFP-expressing MCF-7 cells showed significant increased in the proportion of G2/M phase cells ([22.15±0.17]% vs [17.44±0.57]%, P<0.01), the colony formation ability ([21.43±1.52]% vs [49.43±1.44]%, P<0.01 ), levels of cell cycle protein CDK4 (0.67±0.02 vs 0.72±002, P<0.01) and p-AKT (0.17±0.003 vs 0.42±0.02, P<0.01), and the rateof migration ([57.69±483]% vs [79.35±5.44]%, P<0.01) and proliferation (10.33±1.53 vs 50.67±2.31, P<0.01), as compared with MCF-7 cells expression Ad5- hSulf-1. Conclusion: The overexpression of hSulf-1 may significantly increase the chemosensitivity of MCF-7 cells to AZD2281, induce ell cycle arrest at G2/M-phase and inhibit cell proliferation and migration capacities, possibly through regulation of CDK4 expression and AKT phosphorylation.
To investigate the possibility of enhance the sensitivity of breast cancer MCF-7 cells to the PARP inhibitor AZD2281 by up-regulate the expression of hSulf-1. Methods: MCF-7 cells were infected with Ad5-hSulf1 or Ad5-EGFP. Transfectants were treated with different concentrations of AZD2281 and the most optimal concentration was determined. In further experiments, both Ad5-hSulf-1-overexpressing MCF-7 cells and Ad5-EGFP-expressing control MCF-7 cells were treated with AZD2281 at the optimal concentration determined. After treatment for 24 h, cell cycle progression was assessed by flow cytometry (FCM), formation ability of MCF-7 cells by colony formation assay, protein levels of cyclin dependent kinase 4 (CDK4) and phosphorylated protein kinase B (p-AKT) Western blotting, cell migration by Transwell assay, and proliferative ability by MTT assay. Results: AZD2281 showed the peak inhibitory activity at a concentration of 7 μmol/L. When this concentration was used,Ad5-EGFP-expressing MCF-7 cells showed significant increased in the proportion of G2/M phase cells ([22.15±0.17]% vs [17.44±0.57]%, P<0.01), the colony formation ability ([21.43±1.52]% vs [49.43±1.44]%, P<0.01 ), levels of cell cycle protein CDK4 (0.67±0.02 vs 0.72±002, P<0.01) and p-AKT (0.17±0.003 vs 0.42±0.02, P<0.01), and the rateof migration ([57.69±483]% vs [79.35±5.44]%, P<0.01) and proliferation (10.33±1.53 vs 50.67±2.31, P<0.01), as compared with MCF-7 cells expression Ad5- hSulf-1. Conclusion: The overexpression of hSulf-1 may significantly increase the chemosensitivity of MCF-7 cells to AZD2281, induce ell cycle arrest at G2/M-phase and inhibit cell proliferation and migration capacities, possibly through regulation of CDK4 expression and AKT phosphorylation.
2014, 21(3):275-281.
DOI: 10.3872/j.issn.1007-385X.2014.03.007
Abstract:
To evaluate the synergic antitumor effects of chemotherapeutic agents and melanoma differentiation-associated gene-7-expressing adenoviruses (Ad-MDA-7) in esophageal carcinoma cells in vitro. Methods: Human esophageal carcinoma TE-11 and YES-5 cells and human fibroblasts (control) underwent Ad-MDA-7 infection and chemotherapy, either each alone or in combination. Changes in mRNA levels of the IL-24 receptor complexes before and after treatment were assessed by RT-PCR. Cell viability was determined by MTT assays. Cell cycle progression was analyzed by flow cytometry. Results: Transcripts for IL-24 receptor complex components, IL-20R2, IL-20R1 and IL-22R1, were detected in both TE-11 and YES-5 cells but only IL-20R2 mRNA was detected in fibroblasts. TE-11 and YES-5 cells were susceptible were to Ad-MDA-7-mediated cytotoxicity in a dose-dependent manner; at 3×104 VP/cell, cytotoxicity was >80% and >50%, respectively, in TE-11 and YES 5 cells. In contrast, fibroblasts were resistant to Ad-mad-7. The cytotoxicity of 5-fluorouracil, cisplatin, mitomycin C or etoposide, each in combination with Ad-MDA-7 infection was significantly higher than that of these therapeutic agents and Ad-MDA-7, each alone. Increases in G2/M-phase and S-phase arrests were observed in cells, respectively, infected with Ad-MDA-7 and treated with 5-FU . The combination of Ad-mad-7 and 5-FU augmented sub-G1 populations. Compared with 5-FU alone, the combination regimen resulted in increases in caspase-8, -9, -3 expression and Akt phosphorylation and a decrease in IκB-α level. Conclusion: These data collectively suggest that adenovirus delivery of melanoma differentiation-associated gene-7 may enhance the sensitivity of esophageal carcinoma cells to chemotherapeutic agents through Akt activation.
To evaluate the synergic antitumor effects of chemotherapeutic agents and melanoma differentiation-associated gene-7-expressing adenoviruses (Ad-MDA-7) in esophageal carcinoma cells in vitro. Methods: Human esophageal carcinoma TE-11 and YES-5 cells and human fibroblasts (control) underwent Ad-MDA-7 infection and chemotherapy, either each alone or in combination. Changes in mRNA levels of the IL-24 receptor complexes before and after treatment were assessed by RT-PCR. Cell viability was determined by MTT assays. Cell cycle progression was analyzed by flow cytometry. Results: Transcripts for IL-24 receptor complex components, IL-20R2, IL-20R1 and IL-22R1, were detected in both TE-11 and YES-5 cells but only IL-20R2 mRNA was detected in fibroblasts. TE-11 and YES-5 cells were susceptible were to Ad-MDA-7-mediated cytotoxicity in a dose-dependent manner; at 3×104 VP/cell, cytotoxicity was >80% and >50%, respectively, in TE-11 and YES 5 cells. In contrast, fibroblasts were resistant to Ad-mad-7. The cytotoxicity of 5-fluorouracil, cisplatin, mitomycin C or etoposide, each in combination with Ad-MDA-7 infection was significantly higher than that of these therapeutic agents and Ad-MDA-7, each alone. Increases in G2/M-phase and S-phase arrests were observed in cells, respectively, infected with Ad-MDA-7 and treated with 5-FU . The combination of Ad-mad-7 and 5-FU augmented sub-G1 populations. Compared with 5-FU alone, the combination regimen resulted in increases in caspase-8, -9, -3 expression and Akt phosphorylation and a decrease in IκB-α level. Conclusion: These data collectively suggest that adenovirus delivery of melanoma differentiation-associated gene-7 may enhance the sensitivity of esophageal carcinoma cells to chemotherapeutic agents through Akt activation.
2014, 21(3):282-287.
DOI: 10.3872/j.issn.1007-385X.2014.03.008
Abstract:
To investigate the effect and underlying mechanisms of p-hydroxylcinnamaldehyde (PHD) isolated from the cochinchina momordica seed on the differentiation, proliferation and metastasis of mouse melanoma B16 cells in vitro. Methods: B16 cells were treated with PHD. At the designated time points after treatment, proliferation inhibition was assessed by the Suphrhodamine B assay, colony formation by plate colony assay, morphological changes by Giemsa staining, melanin content and tyrosinase activity by colorimetric analysis, metastasis by wound healing assays, and protein levels of Tyr, Trp1, t-proteins, p-p38, p-JNK and p-ERK1/2 by Western blotting. Results: PHD inhibited B16 cell proliferation in a dose-dependent manner (P<0.05). The inhibition rate at 20 μmol/L (37.70±2.28%), 40 μmol/L (42.17±4.18%) was even higher as compared with forskelin treatment (22.00±1.13%, P<0.05). B16 cells treated with PHD at 40 μmol/L for 24, 48, 72 h showed a dendrite-like morphology, indicative of differentiation. PHD (10 to 40 μmol/L) significantly increased melanin amount at 24 h (0.097±0.02), 48 h (0.13±0.04), and 48 h (0.15±005) as compared with non-treatment control (0.085±0.003, P<0.05) and tryosinase activity at 12 h (1.11±031), 24 h (1.43±0.05), and 48 h (1.67±0.49) as compared with control (0.64±0.10, P<0.05). PHD treatment effectively attenuated metastasis (P<0.05) and remarkably reduced colony-forming capacity (P<0.05) in B16 cells. Moreover, PHD significantly increased the protein content of Tyr, Trp1 and enhanced phosphorylation of P38 and but not ERK. Conclusion: P-hydroxylcinnamaldehyde may inhibit melanoma growth and metastasis, possibly through activating P38 and JNK signaling pathways.
To investigate the effect and underlying mechanisms of p-hydroxylcinnamaldehyde (PHD) isolated from the cochinchina momordica seed on the differentiation, proliferation and metastasis of mouse melanoma B16 cells in vitro. Methods: B16 cells were treated with PHD. At the designated time points after treatment, proliferation inhibition was assessed by the Suphrhodamine B assay, colony formation by plate colony assay, morphological changes by Giemsa staining, melanin content and tyrosinase activity by colorimetric analysis, metastasis by wound healing assays, and protein levels of Tyr, Trp1, t-proteins, p-p38, p-JNK and p-ERK1/2 by Western blotting. Results: PHD inhibited B16 cell proliferation in a dose-dependent manner (P<0.05). The inhibition rate at 20 μmol/L (37.70±2.28%), 40 μmol/L (42.17±4.18%) was even higher as compared with forskelin treatment (22.00±1.13%, P<0.05). B16 cells treated with PHD at 40 μmol/L for 24, 48, 72 h showed a dendrite-like morphology, indicative of differentiation. PHD (10 to 40 μmol/L) significantly increased melanin amount at 24 h (0.097±0.02), 48 h (0.13±0.04), and 48 h (0.15±005) as compared with non-treatment control (0.085±0.003, P<0.05) and tryosinase activity at 12 h (1.11±031), 24 h (1.43±0.05), and 48 h (1.67±0.49) as compared with control (0.64±0.10, P<0.05). PHD treatment effectively attenuated metastasis (P<0.05) and remarkably reduced colony-forming capacity (P<0.05) in B16 cells. Moreover, PHD significantly increased the protein content of Tyr, Trp1 and enhanced phosphorylation of P38 and but not ERK. Conclusion: P-hydroxylcinnamaldehyde may inhibit melanoma growth and metastasis, possibly through activating P38 and JNK signaling pathways.
2014, 21(3):288-292.
DOI: 10.3872/j.issn.1007-385X.2014.03.009
Abstract:
To investigate the methylation status in the promoter region of the retinoblastoma protein-interacting zinc finger1 (RIZ1) gene in four human glioblastoma (GBM) cell lines as well as the influence of methyltransferase inhibitor 5-Aza-CdR on RIZ1 gene transcription and proliferation in GBM cells with a hypermethylated RIZ1 promoter. Methods: The methylation status in the promoter region of RIZ1 in four human GBM cell lines, U87, U251, A172 and T98, were analyzed by methylation-specific polymerase chain reaction. The cell line with RIZ1 promoter hypermethylation was treated with 5-Aza-Cdr. In the treated cells, RIZ1 mRNA was assessed by real-time PCR and viability by MTT assays. Results: Promoter methylation of the RIZ1 gene was detected in GBM cell lines U87 and U251. The abundance of RIZ1 mRNA was significantly increased in U87 after treatment with 5-Aza-CdR. Treatment of U87 cells with 5-Aza-CdR resulted in a time- and concentration-dependent proliferation inhibition. Conclusions: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human GBM cells.
To investigate the methylation status in the promoter region of the retinoblastoma protein-interacting zinc finger1 (RIZ1) gene in four human glioblastoma (GBM) cell lines as well as the influence of methyltransferase inhibitor 5-Aza-CdR on RIZ1 gene transcription and proliferation in GBM cells with a hypermethylated RIZ1 promoter. Methods: The methylation status in the promoter region of RIZ1 in four human GBM cell lines, U87, U251, A172 and T98, were analyzed by methylation-specific polymerase chain reaction. The cell line with RIZ1 promoter hypermethylation was treated with 5-Aza-Cdr. In the treated cells, RIZ1 mRNA was assessed by real-time PCR and viability by MTT assays. Results: Promoter methylation of the RIZ1 gene was detected in GBM cell lines U87 and U251. The abundance of RIZ1 mRNA was significantly increased in U87 after treatment with 5-Aza-CdR. Treatment of U87 cells with 5-Aza-CdR resulted in a time- and concentration-dependent proliferation inhibition. Conclusions: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human GBM cells.
2014, 21(3):293-297.
DOI: 10.3872/j.issn.1007-385X.2014.03.010
Abstract:
To examine the effect of curcumin on mTOR signaling, proliferation and apoptosis in nasopharyngeal carcinoma C666-1 cells in vitro. Methods: C666-1 cells were stimulated by curcumin at increasing concentrations (0, 10, 25, 50, and 100 μmol/L) for 24 h or at a concentration of 50 μmol/L for 0, 6, 12 h and 24 h. Cell proliferation was assessed with the Dojindo’s Cell Counting Kit-8 (CCK-8), cell apoptosis by a TUNEL-based assay and levels of AMPK, S6K, S6 proteins by Western blotting. Results: Curcumin inhibited C666-1 cell proliferation and induced C666-1 cell apoptosis in dose-and time-dependent manners. In parallel with changes in cell proliferation and apoptosis, levels of phosphorylated AMPK were increasing but levels of phosphorylated S6K1 and S6 proteins were decreasing with the dose of curcumin used; against the baseline levels arbitrarily set as 1 in C666-1 cells in the non-treatment control group, the fold of changes in levels of phosphorylated AMPK, S6K1 and S6 proteins was 3.87±1.38, 0.19±0.16 and 0.39±024, respectively, in cells treated with 50 μmol/L for 24 h and was 4.34±1.34, 0.059±0.043 and 0.11±0.095, respectively, in cells treated with 100 μmol/L for 24 h. Conclusion: Curcumin may inhibit nasopharyngeal carcinoma cell proliferation and induce nasopharyngeal carcinoma cell apoptosis through an mTOR signaling pathway-dependent mechanism.
To examine the effect of curcumin on mTOR signaling, proliferation and apoptosis in nasopharyngeal carcinoma C666-1 cells in vitro. Methods: C666-1 cells were stimulated by curcumin at increasing concentrations (0, 10, 25, 50, and 100 μmol/L) for 24 h or at a concentration of 50 μmol/L for 0, 6, 12 h and 24 h. Cell proliferation was assessed with the Dojindo’s Cell Counting Kit-8 (CCK-8), cell apoptosis by a TUNEL-based assay and levels of AMPK, S6K, S6 proteins by Western blotting. Results: Curcumin inhibited C666-1 cell proliferation and induced C666-1 cell apoptosis in dose-and time-dependent manners. In parallel with changes in cell proliferation and apoptosis, levels of phosphorylated AMPK were increasing but levels of phosphorylated S6K1 and S6 proteins were decreasing with the dose of curcumin used; against the baseline levels arbitrarily set as 1 in C666-1 cells in the non-treatment control group, the fold of changes in levels of phosphorylated AMPK, S6K1 and S6 proteins was 3.87±1.38, 0.19±0.16 and 0.39±024, respectively, in cells treated with 50 μmol/L for 24 h and was 4.34±1.34, 0.059±0.043 and 0.11±0.095, respectively, in cells treated with 100 μmol/L for 24 h. Conclusion: Curcumin may inhibit nasopharyngeal carcinoma cell proliferation and induce nasopharyngeal carcinoma cell apoptosis through an mTOR signaling pathway-dependent mechanism.
2014, 21(3):298-302.
DOI: 10.3872/j.issn.1007-385X.2014.03.011
Abstract:
To predict and identify the target genes of microRNA-486-5p (miRNA-486-5p) in human gastric cancer SGC7901 cells. Methods: Possible target genes of miRNA-486-5p were predicted by bioinformatics techniques and accordingly miRNA-486-5p over-expressing plasmid (GV214-miR) against the identified target gene, neuropilin-2 (NRP-2) was constructed. SGC7901 cells were transfected with a control miRNA and an NRP-2-specific miRNA-486-5p. In the transfectants and non-transfected control cells, miRNA-486-5p and NRP-2 mRNA levels and NRP-2 protein levels were analyzed by real-time PCR and Western blotting respectively, and the NRP-2 promoter activity was evaluated by a dual luciferase reporter assay. Results: The expression of miRNA-486-5p in miRNA-486-5p-transfected SGC7901 cells (SGC7901-miR cells) was significantly up-regulated compared with that in the control group (8.21±118 vs 1.02±026, P<0.01). No significant difference in NRP2 mRNA abundance was observed (P>0.05). However, the NRP2 protein level was significantly reduced in SGC7901-miR cells (0.36±0.06) as compared with SGC7901 cells transfected with the control plasmid (0.76±0.05, P<0.05). Dual luciferase reporter assay demonstrated that miRNA-486-5p directly targeted the 3’-untranslated region (UTR) of the NRP2 gene, resulting in inhibition of the post-transcriptional translation of NRP2. Conclusion: Sequence-specific miRNA-486-5p may suppress the expression of NRP2 at the protein level in human gastric cancer cells by binding to NRP2 mRNA 3’UTR directly.
To predict and identify the target genes of microRNA-486-5p (miRNA-486-5p) in human gastric cancer SGC7901 cells. Methods: Possible target genes of miRNA-486-5p were predicted by bioinformatics techniques and accordingly miRNA-486-5p over-expressing plasmid (GV214-miR) against the identified target gene, neuropilin-2 (NRP-2) was constructed. SGC7901 cells were transfected with a control miRNA and an NRP-2-specific miRNA-486-5p. In the transfectants and non-transfected control cells, miRNA-486-5p and NRP-2 mRNA levels and NRP-2 protein levels were analyzed by real-time PCR and Western blotting respectively, and the NRP-2 promoter activity was evaluated by a dual luciferase reporter assay. Results: The expression of miRNA-486-5p in miRNA-486-5p-transfected SGC7901 cells (SGC7901-miR cells) was significantly up-regulated compared with that in the control group (8.21±118 vs 1.02±026, P<0.01). No significant difference in NRP2 mRNA abundance was observed (P>0.05). However, the NRP2 protein level was significantly reduced in SGC7901-miR cells (0.36±0.06) as compared with SGC7901 cells transfected with the control plasmid (0.76±0.05, P<0.05). Dual luciferase reporter assay demonstrated that miRNA-486-5p directly targeted the 3’-untranslated region (UTR) of the NRP2 gene, resulting in inhibition of the post-transcriptional translation of NRP2. Conclusion: Sequence-specific miRNA-486-5p may suppress the expression of NRP2 at the protein level in human gastric cancer cells by binding to NRP2 mRNA 3’UTR directly.
2014, 21(3):303-308.
DOI: 10.3872/j.issn.1007-385X.2014.03.012
Abstract:
To investigate the effects and underlying mechanisms of miR-125b overexpression on proliferation and apoptosis of endometrial carcinoma (EC) HEC-1B cells in vitro. Methods: Paired EC tissue and adjacent tissue specimens were collected from 30 patients with EC who were treated in the Department of Gynecology and Obstetrics, Sichuan Women’s and Children's Hospital between November 2012 and November 2013. Levels of miR-125b mRNA in these specimens were assessed by real-time PCR. To elucidate the effect and mechanisms of miR-125b overexpression on EC cell death/survival, HEC-1B cells were transfected with an expression vector containing a mimic fragment of miR-125b and a control vector with a sequenced-scrambled fragment, respectively, using lipofectamine were, and cell proliferation, cell cycle progression/apoptosis, and protein levels of PIK3CD, p-AKT and total Akt were assessed by cell counting kit-8 (CCK-8) assays, flow cytometery and Western blotting analysis, respectively, in the transfectants and wild type HEC-1B cells. Results: In all 30 paired specimens, miR-125b mRNA abundance was significantly lower in the endometrial cancer tissue than in the adjacent normal tissue (P<0.05). Transfection of HEC-1B cells with the miR-125b vector resulted in an increase in miR-125b mRNA by more than 820 times. The proliferation index was 0.53±0.06 in HEC-1B cells overexpressing miR-125b, significantly higher (P<0.05) than that in HEC-1B cells overexpressing the sequence-scrambled fragment (0.82±0.07) and wild-type HEC-1B cells (0.89±0.08). The rate of apoptosis with G1 phase arrest was significantly higher in miR-125b-transfected HEC-1B cells as compared with control vector-transfected and wild-type HEC-1B cells ([21.5±3.2]% vs [14.2±2.3]% and [13.5±2.1]%, P<0.01). While no significant difference was observed in total Akt protein content among the three groups of HEC-1B cells (P>0.05), protein contents of PIK3CD and p-AKT were significantly reduced in HEC-1B cells transfected with miR-125b compared with cells transfeced with the control vector and untreated HEC-1B cells (P<0.01). Conclusion: The expression of miR-125b is significantly decreased in the EC tissue. Overexpression of miR-125b may suppress cell proliferation and cell cycle progression and induce cell apoptosis , possibly through suppressing the PI3K/AKT signaling pathway, in HEC-B cells in vitro.
To investigate the effects and underlying mechanisms of miR-125b overexpression on proliferation and apoptosis of endometrial carcinoma (EC) HEC-1B cells in vitro. Methods: Paired EC tissue and adjacent tissue specimens were collected from 30 patients with EC who were treated in the Department of Gynecology and Obstetrics, Sichuan Women’s and Children's Hospital between November 2012 and November 2013. Levels of miR-125b mRNA in these specimens were assessed by real-time PCR. To elucidate the effect and mechanisms of miR-125b overexpression on EC cell death/survival, HEC-1B cells were transfected with an expression vector containing a mimic fragment of miR-125b and a control vector with a sequenced-scrambled fragment, respectively, using lipofectamine were, and cell proliferation, cell cycle progression/apoptosis, and protein levels of PIK3CD, p-AKT and total Akt were assessed by cell counting kit-8 (CCK-8) assays, flow cytometery and Western blotting analysis, respectively, in the transfectants and wild type HEC-1B cells. Results: In all 30 paired specimens, miR-125b mRNA abundance was significantly lower in the endometrial cancer tissue than in the adjacent normal tissue (P<0.05). Transfection of HEC-1B cells with the miR-125b vector resulted in an increase in miR-125b mRNA by more than 820 times. The proliferation index was 0.53±0.06 in HEC-1B cells overexpressing miR-125b, significantly higher (P<0.05) than that in HEC-1B cells overexpressing the sequence-scrambled fragment (0.82±0.07) and wild-type HEC-1B cells (0.89±0.08). The rate of apoptosis with G1 phase arrest was significantly higher in miR-125b-transfected HEC-1B cells as compared with control vector-transfected and wild-type HEC-1B cells ([21.5±3.2]% vs [14.2±2.3]% and [13.5±2.1]%, P<0.01). While no significant difference was observed in total Akt protein content among the three groups of HEC-1B cells (P>0.05), protein contents of PIK3CD and p-AKT were significantly reduced in HEC-1B cells transfected with miR-125b compared with cells transfeced with the control vector and untreated HEC-1B cells (P<0.01). Conclusion: The expression of miR-125b is significantly decreased in the EC tissue. Overexpression of miR-125b may suppress cell proliferation and cell cycle progression and induce cell apoptosis , possibly through suppressing the PI3K/AKT signaling pathway, in HEC-B cells in vitro.
2014, 21(3):309-313.
DOI: 10.3872/j.issn.1007-385X.2014.03.013
Abstract:
To determine mRNA and protein levels of sentrin-specific protease 1 (SENP-1) in human non-tumor hepatic cells and various types of human hepatocellular carcinoma (HCC) cells with different invasive and metastatic capacities. Methods: Low invasive HCC cell lines MHCC97L, high invasive HCC cell lines MHCC97H and HCCLM3, non-invasive HCC cell lines SMMC-7721and HepG2 and immortalized normal liver cell line HL-7702 were utilized. Their levels of SENP-1 mRNA and protein were determined by RT-PCR andquantitative real-time PCR and Western blotting respectively while the intracellular location of SENP-1 assessed by immunofluorescence staining. Results: RT-PCR and real-time PCR analyses showed that SENP-1 mRNA abundance was significantly different between the cell lines evaluated (F=5.658；P=0.042) and increased with the incisive capacity of the cells. protein (F=88.909, P=0.000) in HCC cell lines were increased significantly. Western blotting analysis showed that SENP-1 protein levels were significantly higher in all five HCC cell lines tested as compared the immortalized normal liver cell line (F=88.9, P=0.0001). Immumofluorescence staining revealed that SENP1 protein was localized predominantly in cytolymph and in a small quantity in the nucleus. Conclusion: The higher the invasive and metastatic capacities of hepatocellular carcinoma cells, the higher the SENP-1 mRNA and protein levels. This observation suggests that SENP-1 may promote hepatocellular carcinoma cell invasion and metastasis．
To determine mRNA and protein levels of sentrin-specific protease 1 (SENP-1) in human non-tumor hepatic cells and various types of human hepatocellular carcinoma (HCC) cells with different invasive and metastatic capacities. Methods: Low invasive HCC cell lines MHCC97L, high invasive HCC cell lines MHCC97H and HCCLM3, non-invasive HCC cell lines SMMC-7721and HepG2 and immortalized normal liver cell line HL-7702 were utilized. Their levels of SENP-1 mRNA and protein were determined by RT-PCR andquantitative real-time PCR and Western blotting respectively while the intracellular location of SENP-1 assessed by immunofluorescence staining. Results: RT-PCR and real-time PCR analyses showed that SENP-1 mRNA abundance was significantly different between the cell lines evaluated (F=5.658；P=0.042) and increased with the incisive capacity of the cells. protein (F=88.909, P=0.000) in HCC cell lines were increased significantly. Western blotting analysis showed that SENP-1 protein levels were significantly higher in all five HCC cell lines tested as compared the immortalized normal liver cell line (F=88.9, P=0.0001). Immumofluorescence staining revealed that SENP1 protein was localized predominantly in cytolymph and in a small quantity in the nucleus. Conclusion: The higher the invasive and metastatic capacities of hepatocellular carcinoma cells, the higher the SENP-1 mRNA and protein levels. This observation suggests that SENP-1 may promote hepatocellular carcinoma cell invasion and metastasis．
He Sai
,
Zheng Jianbao
,
Sun Xuejun
,
Chen Nanzheng
,
Zhou Peihua
,
Wei Guangbing
,
Wang Hui
,
Yao Jianfeng
,
Zhang Li
,
Jia Pengbo
2014, 21(3):314-319.
DOI: 10.3872/j.issn.1007-385X.2014.03.014
Abstract:
Homebox transcription factor 2 (CDX2) has been described as a colorectal tumor suppressor. This study aimed to evaluate the effect of overexpression of the hypoxia response element enhancer (HRE) sequence-inserted CDX2 gene under the control of the human telomerase reverse transcriptase (hTERT) promoter on proliferation of human colon adenocarcinoma LoVo cells in vitro. Methods: Stable clones of LoVo cells expressing pLVX-hTERTp-CDX2-3FLAG (hC), pLVX-5HRE-hTERTp-3FLAG (5Hh) and pLVX-5HRE-hTERTp-CDX2-3FLAG (5HhC), respectively, were generated. LoVo cells stably expressing hC, 5Hh, and 5HhC, respectively, were cultured under hypoxia control (CoCl2 200 μmol/L) and normoxia, respectively. At the designated time points of culture, cell viability was assessed by MTT assays, colony-forming capacity by trypan blue staining and cell cycle progression by flow cytometry. Result: Viability was significantly decreased in hC- and 5HhC-expressing LoVo cells as compared with wild-type LoVo cells and 5Hh-expressing LoVo cells. The proliferation rates under normoxia and hypoxiarespectively were (48.62±3.32)% and (36.81±2.83)% at 5 days (P<0.05) and (56.44±2.28)% and (38.51±3.21)% at 7 days (P<0.05) in 5HhC-expressing cells. LoVo cells stably expressing hC and 5HhC, respectively, formed significantly less clones than cells stably expressing 5Hhunder a hypoxic condition (44.2±3.5 vs 90.8±9.3, P<0.05). The proportion of cells at G1 phase arrest was (63.59±0.55)% and (64.82±2.22)% in cells stably expressing hC and 5HhC respectively, significantly higher (P<0.05) than that in wild type LoVo cells and cells stably expression 5Hh (51.38±0.70 and 51.59±0.38) under normoxia. Under a hypoxic condition, as high as 71.38±3.02 5HhC-expressing LoVo cells were arrested at G1 phase. Conclusion: Overexpression of the CDX2 gene carrying the HRE sequence driven by the hTERT promoter may significantly inhibit proliferation and colony formation of human colon adenocarcinoma LoVo cells in vitro, through inducing G1 phase arrest. These effects were more significant under hypoxia.
Homebox transcription factor 2 (CDX2) has been described as a colorectal tumor suppressor. This study aimed to evaluate the effect of overexpression of the hypoxia response element enhancer (HRE) sequence-inserted CDX2 gene under the control of the human telomerase reverse transcriptase (hTERT) promoter on proliferation of human colon adenocarcinoma LoVo cells in vitro. Methods: Stable clones of LoVo cells expressing pLVX-hTERTp-CDX2-3FLAG (hC), pLVX-5HRE-hTERTp-3FLAG (5Hh) and pLVX-5HRE-hTERTp-CDX2-3FLAG (5HhC), respectively, were generated. LoVo cells stably expressing hC, 5Hh, and 5HhC, respectively, were cultured under hypoxia control (CoCl2 200 μmol/L) and normoxia, respectively. At the designated time points of culture, cell viability was assessed by MTT assays, colony-forming capacity by trypan blue staining and cell cycle progression by flow cytometry. Result: Viability was significantly decreased in hC- and 5HhC-expressing LoVo cells as compared with wild-type LoVo cells and 5Hh-expressing LoVo cells. The proliferation rates under normoxia and hypoxiarespectively were (48.62±3.32)% and (36.81±2.83)% at 5 days (P<0.05) and (56.44±2.28)% and (38.51±3.21)% at 7 days (P<0.05) in 5HhC-expressing cells. LoVo cells stably expressing hC and 5HhC, respectively, formed significantly less clones than cells stably expressing 5Hhunder a hypoxic condition (44.2±3.5 vs 90.8±9.3, P<0.05). The proportion of cells at G1 phase arrest was (63.59±0.55)% and (64.82±2.22)% in cells stably expressing hC and 5HhC respectively, significantly higher (P<0.05) than that in wild type LoVo cells and cells stably expression 5Hh (51.38±0.70 and 51.59±0.38) under normoxia. Under a hypoxic condition, as high as 71.38±3.02 5HhC-expressing LoVo cells were arrested at G1 phase. Conclusion: Overexpression of the CDX2 gene carrying the HRE sequence driven by the hTERT promoter may significantly inhibit proliferation and colony formation of human colon adenocarcinoma LoVo cells in vitro, through inducing G1 phase arrest. These effects were more significant under hypoxia.
15 Expression and clinical significance of Livin and Caspase-7 protein in human osteosarcoma tissues
2014, 21(3):320-324.
DOI: 10.3872/j.issn.1007-385X.2014.03.015
Abstract:
The aim of this study was to analyse livin and caspase-7 expression at the protein level in correlation with the clinical diagnosis, treatment and prognosis of osteosarcoma. Method: Paraffin-embedded 51 osteosarcoma and 12 normal bone tissue specimens were prepared by the Orthopedics Department of our hospital between February 2001 and December 2012. Livin and caspase-7 proteins in the specimens were assessed by immunohistochemical staining. The correlations of livin and caspase-7 protein levels with the clinicopathological features and prognosis of osteosarcoma were analyzed. Results: Compared with normal bone tissue, Osteosarcoma tissue had a significantly higher percentage of livin-positive cells (66.7% vs 8.3%, P<0.01) but a significantly lower percentage of caspase-7-positive cells (49.0% vs 100.0%, P<0.01), as compared with the normal bone tissue. The proportions of livin- and caspase-7-positive cells were significantly correlated with tumor size, Enneking stage and the 5-year survival rate (P<0.01), but was independent of age, gender, pathological type and tumor site (P>0.05). The expression of livin was negatively correlated with the expression of caspase-7 (r=-0.305, P=0.029). Conclusion: Livin expression is positively correlated but caspase-7 expression is negatively correlated with the severity and prognosis of osteosarcoma , suggest that these two proteins may potential diagnostic and prognostic significance for osteosarcoma.
The aim of this study was to analyse livin and caspase-7 expression at the protein level in correlation with the clinical diagnosis, treatment and prognosis of osteosarcoma. Method: Paraffin-embedded 51 osteosarcoma and 12 normal bone tissue specimens were prepared by the Orthopedics Department of our hospital between February 2001 and December 2012. Livin and caspase-7 proteins in the specimens were assessed by immunohistochemical staining. The correlations of livin and caspase-7 protein levels with the clinicopathological features and prognosis of osteosarcoma were analyzed. Results: Compared with normal bone tissue, Osteosarcoma tissue had a significantly higher percentage of livin-positive cells (66.7% vs 8.3%, P<0.01) but a significantly lower percentage of caspase-7-positive cells (49.0% vs 100.0%, P<0.01), as compared with the normal bone tissue. The proportions of livin- and caspase-7-positive cells were significantly correlated with tumor size, Enneking stage and the 5-year survival rate (P<0.01), but was independent of age, gender, pathological type and tumor site (P>0.05). The expression of livin was negatively correlated with the expression of caspase-7 (r=-0.305, P=0.029). Conclusion: Livin expression is positively correlated but caspase-7 expression is negatively correlated with the severity and prognosis of osteosarcoma , suggest that these two proteins may potential diagnostic and prognostic significance for osteosarcoma.
2014, 21(3):325-333.
DOI: 10.3872/j.issn.1007-385X.2014.03.016
Abstract:
Antibody therapy has become the first-line treatment strategy for cancer patients. As of 2013, several antibodies including Cetuximab, Trastuzumab, Rituximab, Bevacizumab, and Ipilimumab have been approved by the Food and Drug Administration (FDA) of the United States for use in clinics to treat cancer patients. It is believed that use of cancer therapeutic antibodies in clinics is the most successful example of translational medicine. Moreover, development of novel cancer therapeutic antibodies has become the spotlight of research in recent years, and several potential antibodies, like MDX-1106 and BMS-936558 targeting PD-1, have entered into clinical trials. In this paper, we aim to review the performance the FDA-approved therapeutic antibodies targeting EGFR, HER2, CD20, VEGF, and CTLA-4 in clinical settings and the progress on the novel therapeutic antibodies under clinical trials.
Antibody therapy has become the first-line treatment strategy for cancer patients. As of 2013, several antibodies including Cetuximab, Trastuzumab, Rituximab, Bevacizumab, and Ipilimumab have been approved by the Food and Drug Administration (FDA) of the United States for use in clinics to treat cancer patients. It is believed that use of cancer therapeutic antibodies in clinics is the most successful example of translational medicine. Moreover, development of novel cancer therapeutic antibodies has become the spotlight of research in recent years, and several potential antibodies, like MDX-1106 and BMS-936558 targeting PD-1, have entered into clinical trials. In this paper, we aim to review the performance the FDA-approved therapeutic antibodies targeting EGFR, HER2, CD20, VEGF, and CTLA-4 in clinical settings and the progress on the novel therapeutic antibodies under clinical trials.
2014, 21(3):334-336.
DOI: 10.3872/j.issn.1007-385X.2014.03.017
Abstract:
检测转染吲哚胺2,3-双加氧酶（IDO）的MFC胃癌细胞移植瘤中E-cadherin、N-cadherin及IL-6的表达水平。方法：建立MFC胃癌细胞移植瘤模型，分为MFC胃癌细胞组、pcDNA3.1- MFC细胞（空质粒）组、pcDNA3.1-IDO- MFC细胞组，免疫组化方法检测3组的E-cadherin 和N-cadherin表达情况，酶联免疫吸附试验检测3组荷瘤小鼠血清液中IL-6的表达水平。结果：pcDNA3.1-IDO- MFC组移植瘤组织中，E-cadherin的表达较空质粒组和MFC细胞组明显减少（P<0.05），N-cadherin的表达较其他两组明显增多（P<0.05）。pcDNA3.1-IDO- MFC细胞组荷瘤小鼠血清IL-6水平较其他两组明显增高（P<0.05）。 结论：IDO转染导致胃癌细胞移植瘤中E-cadherin水平下调、N-cadherin和IL-6水平上调，该结果为IDO参与肿瘤细胞的侵袭和转移提供实验依据。
检测转染吲哚胺2,3-双加氧酶（IDO）的MFC胃癌细胞移植瘤中E-cadherin、N-cadherin及IL-6的表达水平。方法：建立MFC胃癌细胞移植瘤模型，分为MFC胃癌细胞组、pcDNA3.1- MFC细胞（空质粒）组、pcDNA3.1-IDO- MFC细胞组，免疫组化方法检测3组的E-cadherin 和N-cadherin表达情况，酶联免疫吸附试验检测3组荷瘤小鼠血清液中IL-6的表达水平。结果：pcDNA3.1-IDO- MFC组移植瘤组织中，E-cadherin的表达较空质粒组和MFC细胞组明显减少（P<0.05），N-cadherin的表达较其他两组明显增多（P<0.05）。pcDNA3.1-IDO- MFC细胞组荷瘤小鼠血清IL-6水平较其他两组明显增高（P<0.05）。 结论：IDO转染导致胃癌细胞移植瘤中E-cadherin水平下调、N-cadherin和IL-6水平上调，该结果为IDO参与肿瘤细胞的侵袭和转移提供实验依据。
2014, 21(3):337-341.
DOI: 10.3872/j.issn.1007-385X.2014.03.018
Abstract:
肾癌是泌尿系统最常见的恶性肿瘤之一，晚期肾癌预后差且对放化疗不敏感。随着肿瘤生物治疗手段的不断发展，抑癌基因逐渐成为重要的研究对象，其表达下调和失活与肿瘤的发生发展密切相关。以往的研究发现了希佩尔林道（von Hippel-Lindau，VHL）基因、P16，P53等一批经典的抑癌基因，近年来，这些基因在肾癌的调控中的作用有一些新的发现，为肾癌的生物治疗提供了更广阔的思路。同时，一些新的肾癌抑癌基因也被不断发现，如：UNC5Hs，Chmp1A，KISS-1，CADM2等等，为肾癌的基因靶向治疗提供了新的靶点。本文主要就肾癌中抑癌基因的种类、作用以及调控机制等做一综述，为肾癌基因诊断治疗开拓新的思路。
肾癌是泌尿系统最常见的恶性肿瘤之一，晚期肾癌预后差且对放化疗不敏感。随着肿瘤生物治疗手段的不断发展，抑癌基因逐渐成为重要的研究对象，其表达下调和失活与肿瘤的发生发展密切相关。以往的研究发现了希佩尔林道（von Hippel-Lindau，VHL）基因、P16，P53等一批经典的抑癌基因，近年来，这些基因在肾癌的调控中的作用有一些新的发现，为肾癌的生物治疗提供了更广阔的思路。同时，一些新的肾癌抑癌基因也被不断发现，如：UNC5Hs，Chmp1A，KISS-1，CADM2等等，为肾癌的基因靶向治疗提供了新的靶点。本文主要就肾癌中抑癌基因的种类、作用以及调控机制等做一综述，为肾癌基因诊断治疗开拓新的思路。
2014, 21(3):342-347.
DOI: 10.3872/j.issn.1007-385X.2014.03.019
Abstract:
近年来，放疗、化疗及手术联合治疗成为了头颈部鳞癌的主要治疗手段。然而，由放化疗带来的毒性作用不可避免地影响了治疗的依从性。分子靶向治疗作为一种新的生物治疗模式，在治疗头颈部肿瘤方面具有毒性作用小的特点，越来越受到关注。头颈部肿瘤的靶向治疗药物主要包括西妥昔单抗和贝伐单抗等，目前西妥昔单抗等靶向治疗药物已被FDA批准用于头颈部鳞癌的治疗。本文以西妥昔单抗和贝伐单抗为主，同时介绍一些正处于研究中的靶向治疗药物，探讨靶向药物在头颈部肿瘤治疗中的应用和进展。
近年来，放疗、化疗及手术联合治疗成为了头颈部鳞癌的主要治疗手段。然而，由放化疗带来的毒性作用不可避免地影响了治疗的依从性。分子靶向治疗作为一种新的生物治疗模式，在治疗头颈部肿瘤方面具有毒性作用小的特点，越来越受到关注。头颈部肿瘤的靶向治疗药物主要包括西妥昔单抗和贝伐单抗等，目前西妥昔单抗等靶向治疗药物已被FDA批准用于头颈部鳞癌的治疗。本文以西妥昔单抗和贝伐单抗为主，同时介绍一些正处于研究中的靶向治疗药物，探讨靶向药物在头颈部肿瘤治疗中的应用和进展。
20 Research progress of PI3K/AKT/mTOR signing pathway inhibitors in the treatment of colorectal cancer
2014, 21(3):348-352.
DOI: 10.3872/j.issn.1007-385X.2014.03.020
Abstract:
PI3K/AKT/mTOR通路的异常活化在结直肠癌的发生发展中起到重要作用，以此通路为靶点的药物已成为结直肠癌治疗的研究热点，临床前和临床试验研究证明，针对PI3K/AKT/mTOR通路的多种抑制剂具有抗肿瘤活性。越来越多的临床数据显示，PTEN缺乏或PIK3CA基因突变对PI3K/AKT/mTOR通路抑制剂敏感，KRAS突变则预示着耐药；寻找针对这一通路抑制剂敏感的优势人群也成为结直肠癌的研究热点；此外，PI3K/AKT/mTOR通路也会影响常规治疗的疗效，因此PI3K/AKT/mTOR通路抑制剂联合细胞毒治疗方案在结直肠癌中可能起到协同作用。
PI3K/AKT/mTOR通路的异常活化在结直肠癌的发生发展中起到重要作用，以此通路为靶点的药物已成为结直肠癌治疗的研究热点，临床前和临床试验研究证明，针对PI3K/AKT/mTOR通路的多种抑制剂具有抗肿瘤活性。越来越多的临床数据显示，PTEN缺乏或PIK3CA基因突变对PI3K/AKT/mTOR通路抑制剂敏感，KRAS突变则预示着耐药；寻找针对这一通路抑制剂敏感的优势人群也成为结直肠癌的研究热点；此外，PI3K/AKT/mTOR通路也会影响常规治疗的疗效，因此PI3K/AKT/mTOR通路抑制剂联合细胞毒治疗方案在结直肠癌中可能起到协同作用。
2014, 21(3):353-356.
DOI: 10.3872/j.issn.1007-385X.2014.03.021
Abstract:
人类嗅素蛋白（olfactomedin 4, OLFM4）属于olfactomedin相关家族成员，它是一种糖蛋白，正常表达于人的多种器官和组织。OLFM4在肿瘤组织中的表达具有特异性，它在胃癌、胰腺癌、结肠癌、宫颈癌等肿瘤中高表达。OLFM4有促进细胞增殖、调节细胞黏附转移、抑制细胞凋亡和免疫防御等多种功能。OLFM4与消化系统肿瘤的关系成为近年来的研究热点。OLFM4已被证明为胃癌发生、发展和分化的一个重要的标志物，但其具体调控机制目前仍处于研究中，抑制OLFM4基因表达和抗癌药物的联合应用可能成为胃癌的治疗策略。OLFM4在胰腺癌中的高表达率提示其或许可成为胰腺癌新的标志物，OLFM4是通过作用于DNA合成的S期到有丝分裂的G2/M期来促进胰腺癌细胞增殖。OLFM4基因最早是因为其在结肠癌细胞中的高表达被发现，OLFM4在结直肠癌的黏附转移中发挥着一定作用。OLFM4在早期消化系统肿瘤中的敏感性比其他肿瘤标志物高，是否能作为早期诊断消化系统肿瘤的标志物，有待于进一步研究。
人类嗅素蛋白（olfactomedin 4, OLFM4）属于olfactomedin相关家族成员，它是一种糖蛋白，正常表达于人的多种器官和组织。OLFM4在肿瘤组织中的表达具有特异性，它在胃癌、胰腺癌、结肠癌、宫颈癌等肿瘤中高表达。OLFM4有促进细胞增殖、调节细胞黏附转移、抑制细胞凋亡和免疫防御等多种功能。OLFM4与消化系统肿瘤的关系成为近年来的研究热点。OLFM4已被证明为胃癌发生、发展和分化的一个重要的标志物，但其具体调控机制目前仍处于研究中，抑制OLFM4基因表达和抗癌药物的联合应用可能成为胃癌的治疗策略。OLFM4在胰腺癌中的高表达率提示其或许可成为胰腺癌新的标志物，OLFM4是通过作用于DNA合成的S期到有丝分裂的G2/M期来促进胰腺癌细胞增殖。OLFM4基因最早是因为其在结肠癌细胞中的高表达被发现，OLFM4在结直肠癌的黏附转移中发挥着一定作用。OLFM4在早期消化系统肿瘤中的敏感性比其他肿瘤标志物高，是否能作为早期诊断消化系统肿瘤的标志物，有待于进一步研究。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.