Interferon-gamma induces autophagy-associated apoptosis through induction of cPLA2-dependent mitochondrial ROS generation in colorectal cancer cells.

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in females and the third in males. In this work, we aim to investigate the possible anti-cancer effects of interferon-gamma (IFN-γ) in CRC cells. We observed that IFN-γ induced mitochondria-derived reactive oxygen species (ROS) production in a time-dependent manner in SW480 and HCT116 cell lines. The IFN-γ-induced mitochondrial ROS generation was dependent on the activation of cytosolic phospholipase A2 (cPLA2). In addition, a mitochondria-targeted antioxidant SS31 and/or cPLA2 inhibitor AACOCF3 abolished the IFN-γ-induced ROS production and subsequent autophagy and apoptosis. Moreover, suppression of autophagy by CQ was able to reduce IFN-γ-induced cell apoptosis. Beclin-1 gene silencing resulted in caspase-3 inactivation, decreased Bax/Bcl-2 ratio and less population of apoptotic cells. Collectively, our results suggested that IFN-γ induces autophagy-associated apoptosis in CRC cells via inducing cPLA2-dependent mitochondrial ROS production.

Dendritic cells with CED-3 and CED-4 siRNA is effective in prolonging DC lifetime and suppresses tumor growth in gastric cancer.

BACKGROUND/AIMS: We investigated effects of CED-3 and CED-4-siRNA on prolonging dendritic cell life in vivo and in vitro. METHODOLOGY: The DCs were divided into three groups: pure-DC, siRNA and CED-3 and CED-4-siRNA. we performed anti-apoptosis assays for DCs with flow cytometry. The assay for cytotoxicity assay was performed in vitro by a standard chromium assay at various effector/target ratios. Percent-specific lysis was calculated. We injected three kinds of DCs from tail vein every 3 days, we calculated tumor volume control rate and tumor weight control rate with formula. RESULTS: The DC percentages of apoptosis of CED-3 and CED-4 siRNA group were (12.09±1.14)%. Tumor-specific CTL activity showed 82.1% specific lysis for CED-3 and CED-4-siRNA DC group and 39.4% and 40.2% specific lysis for pure DC and siRNA DC group respectively. The lysis of CED-3 and CED-4-siRNA group was higher than the any other groups (p<0.05).The experiment of transplantation tumor in BALB/C mice showed that CED-3 and CED-4-siRNA DC can inhibit mice with tumors in volume and weight. CONCLUSIONS: We found that vaccination with CED-3 and CED-4-siRNA was capable of prolonging the survival of antigen-expressing DCs, and generated a strong therapeutic effect in the treatment of gastric cancer.

A new development of FG-CC' siRNA blocking interaction of Tim-1 and Tim-4 can enhance DC vaccine against gastric cancer .

BACKGROUND/AIMS: Dendritic cells (DCs) are professional antigen-presenting cells responsible for initiating of immune response. However, because of immune tolerance, it is difficult to induce long-term tumor-specific immune response in humans. This is probably because DCs, which combine with Th2 in the Tim-1/Tim-4 pathway, will induce Th2 to proliferation. METHODOLOGY: We have transfected siRNA of FG-CC' gene into DCs stimulated by gastric cancer lysate (lysate-FG-CC-siRNA group), FG-CC-siRNA will block FG-CC' loop,which plays an important role in interaction between Tim-1 and Tim-4. Their potential effect on gastric cancer immunotherapy is assessed by an experimental model. RESULTS: It was observed that lysate-FG-CC-siRNA had the strongest ability of adjusting balance on the Thl/Th2, as a result, these DCs can inhibit gastric cancer growth. In order to test the ability of FG-CC-siR-NA DCs to inhibit tumor growth, we immunized mice subcutaneously with DCs transfected with FG-CC-siR-NA plus tumor antigen. Compared with the control group, a significant inhibition of tumor growth was obvious for the group of lysate-FG-CC-siRNA DC. CONCLUSIONS: We have shown that FG-CC-siRNA blocks FG-CC'loop and significantly enhances the anti-tumor immunity in vitro and in vivo.

Combination DLL4 with Jagged1-siRNA can enhance inhibition of the proliferation and invasiveness activity of human gastric carcinoma by Notch1/VEGF pathway.

BACKGROUND/AIMS: To evaluate the effects of adenovirus- mediated gene transfer of DLL4 and Jagged1 siRNA on proliferation and invasion of SGC7901 cells by Notch/ VEGFR pathway. METHODOLOGY: Plasmid of DLL4 and Jagged1 siRNA were constructed and transfected into SGC7901 cells. siRNA and endostatin (VEGF inhibitor) were designed as the control group. The mRNA and protein expressions of DLL4 and Jagged1 were respectively detected with RT-PCR and western blotting. In order to find out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB/C nude mice with DLL4 and Jagged1-siRNA, and tumor control rate (%) in nude mice was calculated. RESULTS: DLL4 and Jagged1 siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 cells. The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines were on the decline after treatment of Ad-DLL4- Jagged1-siRNA (12.23±3.12 vs. 78.38±17.38, p<0.05). The values of 490nm wavelength light absorption were different in the five groups. The number of alive cells in the group of DLL4-Jagged1-siRNA was lower than others in the 6th d (0.77±0.01 vs. 3.00±0.11 p<0.05). The apoptosis rate of transfected DLL4 and Jagged1 group with FACS were 18.07%±0.98±1.78 and there were significant differences between treated and control groups (18.07%±0.98 vs. 1.08%±0.23, p<0.01). The tumor transplantation experiment in BALB/C nude mice showed that intratumoral injection of DLL4 and Jagged1 siRNA could inhibit tumor growth. CONCLUSIONS: DLL4 and Jagged1 siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a Notch/VEGFR pathway. These results provided a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.

RhoA and RhoC -siRNA inhibit the proliferation and invasiveness activity of human gastric carcinoma by Rho/PI3K/Akt pathway.

AIM: To evaluate the effects of adenovirus-mediated gene transfer of RhoA siRNA and RhoC siRNA on proliferation and invasion of SGC7901 cells by Rho/PI3K/Akt pathway. METHODS: Plasmid of RhoA siRNA and RhoC siRNA were constructed and transfected into SGC7901 cells. siRNA and LY294002 (PI3K inhibitor) were designed as the control group. The mRNA and protein expressions of RhoA and RhoC were respectively detected with RT-PCR and western blotting. In order to find out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB/C nude mice with RhoA and RhoC-siRNA, and tumor control rate (%) in nude mice was calculated. RESULTS: RhoA and RhoC siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 Cells. The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines are on the decline after treatment of Ad-RhoA and RhoC-siRNA (12.64 +/-3.27 vs 87.38 +/- 17.38, P < 0.05). The values of 490 nm wavelength light absorption were different in the five groups. The number of alive cells in the group of RhoA and RhoC-siRNA was lower than others in the 6(th) d (0.71 +/- 0.01 vs 3.82 +/- 0,11 P < 0.05). The apoptosis rate of transfected RhoA and RhoC-siRNA group with FACS were 19.07% +/- 1.78 and there were significant differences between treated and control groups (19.07 +/- 1.78% vs 1.23 +/- 0.11%, P < 0.01). The tumor transplantation experiment in BALB/C nude mice showed intratumoral injection of RhoA or RhoC siRNA can inhibit tumor growth. CONCLUSION: RhoA and RhoC siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a PI3K/Akt pathway. These results provide a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.

[Study of immunological effect of dendritic cell transfected with survivin gene on the specific anti-alimentary tract tumor].

OBJECTIVE: To investigate the effects of dendritic cells (DCs) transfected with survivin gene, and to observe the effective and specific anti-tumor immunological effect induced by modified DC in vitro. METHODS: Survivin gene was transfected to DCs with liposomes. Survivin expression could be detected both in DCs cells and in cell culture with method of Western blot. Cytokines as well as cellular surface molecule such as IL-12, TNF-alpha, CD1 alpha, CD83, MHCII, CD80 and CD86 were detected. The competence of inducing human specific cytotoxic T lymphocyte (CTLs) was also detected with MTT. RESULTS: Survivin expression could be detected both in DCs which were transfected with survivin cDNA and in cell culture superior. The IL-12 and TNF-alpha level was (265.2 +/- 32.7), (437.1 +/- 83.5) pg/ml, and much higher in transgened DC cells than blank DC cells (P < 0.05). CD1 alpha, CD83, MHCII, CD80 and CD86 was high expressed in survivin-DC cells, however, it was low expressed in blank DC cells. The lyse rate to gastric cancer cell, colon cancer cell and bile duct cancer cell was 65%, 77%, and 85% respectively, and these were much higher than those of blank DC cells. CONCLUSIONS: DCs transfected with survivin gene could induce specific cytotoxic T lymphocytes and strikingly raised DC cell's antigen present function, and have specific CTL killing activity.

Effects of dendritic cells transfected with full length wild-type p53 and modified by bile duct cancer lysates on immune response.

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells and are actively used in cancer immunotherapy. Wild-type p53 can be recognized as an antigen and can induce specific cytotoxic T lymphocytes (CTLs) in the host body. The aim of this study was to investigate the effects of DCs transfected with full length wild-type p53 and modified by bile duct lysates on immune response. METHODS: The wild-type p53 was transducted to DCs with adenovirus, which were modified by bile duct lysates (Lywtp53DC). The concentration of the surface molecules (B7-1, B7-2, MHC-I, MHC-II) of all DCs was detected with fluorescence activated cell sorter (FACS), and the ability of the DCs to induce efficient and specific immunological response in anti-(51)Cr-labeled target cells was studied. BALB/c mice infected with the DCs and QBC939 were used. CTL response in mice immunized with Lywtp53DC and treatment of tumor-bearing mice with Lywtp53DC and CTL response in these mice were studied. RESULTS: The surface molecules of Lywtp53DC had a high expression B7-1 (86.70%+/-0.07%), B7-2 (18.77%+/-0.08%), MHC-I(87.20%+/-0.05%), MHC-II(56.70%+/-0.07%) with FACS. The T lymphocytes had a specific CTL lysing ability induced by Lywtp53DC, with a CTL lysis rate of 81%. The immune protection of Lywtp53DC group was obvious, and the tumor diameter of the Lywtp53DC group was 3.10+/-0.31 mm, 2.73+/-0.23 mm, 3.70+/-0.07 mm on days 13, 16 and 19, smaller than those of any control groups (P<0.05), DC, wtp53DC and LyDC. On the other hand, the growth rate of tumor of the Lywtp53DC group was slower than that of any other groups (P<0.05). CONCLUSION: Dendritic cells transfected with wild-type p53 and modified by bile duct lysates have specific CTL killing capability.

Effects of dendritic cells transfected with full-length wild-type p53 and stimulated by gastric cancer lysates on immune response.

AIM: To investigate the effects of dendritic cells (DCs) transfected with full-length wild-type p53 and stimulated by gastric cancer lysates on immune response. METHODS: The wild-type p53 was transduced to DCs with adenovirus, and the DCs were stimulated by gastric cancer lysates. The surface molecules (B7-1, B7-2, MHC-I, MHC-II) of all DCs were detected by FACS, and the ability of the DCs to induce efficient and specific immunological response in anti-51Cr-labeled target cells was studied. BALB/c mice injected with DCs and Mk28 were established, and CTL response in mice immunized with Lywt-p53DC was evaluated. Tumor-bearing mice were treated with Lywt-p53DC. RESULTS: The surface molecules of Lywt-p53DC had a high expression of B7-1 (86.70 +/- 0.07%), B7-2 (18.77 +/- 0.08%), MHC-I (87.20 +/- 0.05%) and MHC-II (56.70 +/- 0.07%); T lymphocytes had a specific CTL lysis ability induced by Lywt-p53DC; the CTL lysis rate was as high as 81%. The immune protection of Lywtp-53DC was obvious, the tumor diameter in Lywtp-53DC group was 3.10 +/- 0.31 mm, 2.73 +/- 0.23 mm, 3.70 +/- 0.07 mm on d 13, 16 and 19, respectively, which were smaller than control, DC, wtp53DC and LyDC group (P<0.05). Tumor growth rate in Lywtp53DC group was slower than that in other groups (P<0.05). CONCLUSION: DCs transfected with wild-type p53 and stimulated by gastric cancer lysates have specific CTL killing activity.

Inhibitory effect of methylation inhibitor 5-aza-2-deoxycytidine on bile duct cancer cell line in vivo and in vitro.

BACKGROUND: Since the resection rate is low for bile duct cancer and the drugs used for chemotherapy are less effective, we studied the inhibitory effects of 5-aza-2-deoxycytidine (ZdCyd) on bile duct cancer cell line QBC939 in vivo and in vitro and its possibility in clinical treatment. METHODS: The survival and apoptosis rates of QBC939 after treatment with different dose of ZdCyd were detected by methyl thiazoy tetrazolium (MTT) and flow cytometry. The cooperative effect of ZdCyd with other chemotherapeutic drugs was also studied with MTT. The cancer cells were transplanted into nude mice, which were pre-treated with ZdCyd after tumor occurrence. RESULTS: ZdCyd decreased the cell survival rate, blocked the cell cycle at G1 phase, and increased the apoptosis rate. These effects were dose and time-dependent. ZdCyd also increased the anti-tumor effects of other chemotherapeutic drugs when used in combination. The tumor occurrence rate was lower in the ZdCyd pre-treated cells than in the untreated cells in nude mice, and ZdCyd was found to inhibit tumor growth. CONCLUSION: ZdCyd can inhibit the growth of QBC939 in vivo and in vitro through induction of cell apoptosis and has the cooperative effect on bile duct cancer cell when it is used with other chemotherapeutic drugs.