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.

Immunohistochemical expression of mTOR negatively correlates with PTEN expression in gastric carcinoma.

The phosphoinositide-3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway is a cellular pathway involved in cell growth, tumorigenesis and cell invasion which is frequently activated in various types of cancer. The downstream effector of the pathway is mTOR which is important in cellular growth and homeostasis and aberrant activation of mTOR has been reported in several types of cancer. The tumor suppressor gene phosphatase and tensin homolog (PTEN) is essential in this pathway for inhibiting tumor invasion and metastasis. However, the involvement of mTOR and PTEN in the progression of human gastric cancer remains to be identified. Immunohistochemical staining was performed to detect the expression of mTOR and PTEN in paraffin-embedded gastric tissue sections obtained from 33 patients with gastric cancer and 30 normal controls. The expressed mTOR was mainly distributed in the cytoplasm, while PTEN was mainly localized to the nucleus. By considering negative mTOR expression with positive PTEN expression as one group and negative PTEN expression with positive mTOR expression as the other, significant statistical differences were observed in various categories, including histological types and metastatic and clinical pathology stages, between the 2 groups (P<0.01 or 0.05). The results indicated that the expression levels of mTOR and PTEN were negatively correlated in the PI3K-AKT-mTOR signaling pathway. Combined detection of mTOR and PTEN expression may be used to evaluate the degree of malignancy in gastric cancer and may be a useful marker for the early diagnosis 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.

miR-373 negatively regulates methyl-CpG-binding domain protein 2 (MBD2) in hilar cholangiocarcinoma.

BACKGROUND: microRNAs (miRNAs) are a class of non-coding, single-stranded RNA molecules that regulate gene expression at the posttranscriptional level. Methyl-CpG-binding domain proteins (MBPs) are transcription repressors through binding to methylated gene promoters. Recent studies have shown that the effect of miRNAs on DNA methylation by targeting DNA methyltransferase (DNMTs) and/or MBPs plays an important role in various human cancers. AIMS: This study focuses on the regulation of MBPs by miR-373 and its downstream effect in hilar cholangiocarcinoma. METHODS: miR-373 was investigated by TaqMan miRNA Assay; mRNA and protein of MBD1, MBD2, and Mecp2 were determined by QuantiTect(®) Primer Assays and Western blotting, respectively; RASSF1A mRNA was measured by SYBR-Green real-time PCR; The targeting at MBD2-3'UTR by miR-373 was evaluated by dual-luciferase reporter gene assay. RESULTS: miR-373 decreased and closely associated with poor cell differentiation, advanced clinical stage, and shorter survival in hilar cholangiocarcinoma; MBD2 exclusively over-expressed and reciprocally related to miR-373; precursor miR-373 inhibited the luciferase activity of MBD2-3'UTR construct; exogenous miR-373 suppressed the expression of MBD2 and enhanced RASSF1A mRNA in QBC(939) cells; anti-miR-373 inhibitor up-regulated the expression of MBD2 and reduced RASSF1A mRNA in HIBEpic cells. CONCLUSIONS: miR-373 is one negative regulator of MBD2. In hilar cholangiocarcinoma, down-expression of miR-373 leads to increase of MBD2, which in turn suppresses the methylation-mediated gene such as RASSF1A.

Methyl-CpG binding protein MBD2 is implicated in methylation-mediated suppression of miR-373 in hilar cholangiocarcinoma.

Aberrant expression of miRNAs is associated with particular cancers showing tissue- and clinical-feature-specificity patterns. Some miRNA genes harboring or being embedded in CpG islands undergo methylation mediated silencing. MBP, methyl CpG binding protein, suppresses transcription through binding to methylated CpG dinucleotides. Expression of miR-373 has been reported to be suppressed in malignant bile duct cell lines. Bioinformatic prediction reveals that the transcription start site (TSS) of miR-373 is implanted in a 402 bp canonical CpG island containing 26 CpG dinucleotides. In this study, we aim to determine the epigenetic regulation of miR-373 gene in hilar cholangiocarcinoma. Taqman microRNA assay shows that down-regulation of miR-373 is closely associated with poor cell differentiation, advanced clinical stage and shorter overall and disease-free survival in hilar cholangiocarcinomas. Methylation analysis shows that the promoter-associated CpG island is hypermethylated which is consistent with the inhibition of miR-373. Chromatin immunoprecipitation (ChIP) assay indicates that down-regulation of miR-373 results from the selective recruitment of MBD2 to methylated CpG islands. In contrast, MBD2 knock-down by use of a specific siRNA promoted the expression of miR-373. Reactivation of miR-373 by pharmacologic induction of 5-aza-CdR and trichostatin A (TSA) led to decreased enrichment of MBD2 at CpG island regions. Enhanced expression of exogenous MBD2 in stable QBC939 cells which stably express pGL4-m373-prom induces strengthened recruitment of MBD2. Our findings suggest that miR-373 is a methylation-mediated gene and the implication of MBD2 in methylation-mediated suppression of miR-373 plays an important role in tumourigenesis and development in hilar cholangiocarcinoma.

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.

Effects of dendritic cells transfected with full length wild type P53 and modified by gastric cancer lysate on immune response.

To investigate the effects of dendritic cells (DCs) transfected with full length wild type p53 and modified by gastric cancer lysates on immune response, the wild type P53 was transducted to DCs with adenovirus, and the DCs were modified by gastric cancer lysates (Lywt-P53DC). The concentration of the surface molecules (B7-1, B7-2, MHC-I , MHC-II) of all DCs was determined by FACS, and the ability of the DCs to induce efficient and specific immunological response in anti-51Cr-labeled target cells studied. BALB/c mice model infected with DCs and Mk28 was established. CTL response in mice immunized with Lywt-p53DC and the effectiveness of Lywt-p53DC in the treatment of tumor-bearing mice was assayed. FACS revealed that the surface molecules of Ly-wt-P53 DC had a high expression: for B7-1 86.70% +/- 0.07%, B7-2 18.77% +/- 0.08%, MHC-I 87.20% +/- 0.05%, MHC-II 56.70%+/-0.07%; The T lymphocytes had a specific CTL lysing ability induced by Lywt-P53DC with the CTL lysis rate being 81%. The immune protective effect of Lywt-p53DC group was more obvious than any other groups (P<0.05). The tumor diameter in Lywt-p53DC group was 3.10+/-0.31 mm, 2.73+/-0.23 mm, 3.70+/-0.07 mm on the day 13, 16 and 19, smaller than DC, wtp53DC and LyDC groups (P<0.05). On the other hand, the growth rate of tumor in Lywt-p53DC group was slower than any other groups (P<0.05). It was suggested that DCs transfected with wild type P53 and modified by gastric cancer lysates had specific CTL killing capability.