Shi-Ming Yang

Alterations of telomerase activity and terminal restriction fragment in gastric cancer and its premalignant lesions

Aims: In order to explore the role of alterations of telomerase activity and terminal restriction fragment (TRF) length in the development and progression of gastric cancer.

Programmed Cell Death 4 (PDCD4) Enhances the Sensitivity of Gastric Cancer Cells to TRAIL-Induced Apoptosis by Inhibiting the PI3K/Akt Signaling Pathway

AbstractObjective: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is thought to be a promising anti-neoplastic agent because of its ability to selectively induce apoptosis in cancer cells. However, some cancer cells are resistant to TRAIL. The mechanisms underlying this resistance are unclear. The aim of this study was to explore the role of programmed cell death 4 (PDCD4) in regulating TRAIL sensitivity in gastric cancer cells.n Methods: PDCD4 complementary DNA and PDCD4-specific short-hairpin RNA (shRNA) fragments were transfected into TRAIL-sensitive and -resistant gastric cancer cells. Expression of PDCD4 and Akt was detected via western blot. Cell survival and apoptosis were measured using 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry (FCM) assays.n Results: We found that upregulation of PDCD4 enhanced TRAIL sensitivity in gastric cancer cells. Downregulation of PDCD4 decreased TRAIL sensitivity. Inhibition of Akt by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 induced PDCD4 activity and enhanced TRAIL sensitivity in TRAIL-resistant gastric cancer cells.n Conclusion: We demonstrated that PDCD4 regulates TRAIL sensitivity in gastric cancer cells by inhibiting the PI3K/Akt signaling pathway.

Dendritic Cells Reconstituted with a Human Heparanase Gene Induce Potent Cytotoxic T-Cell Responses against Gastric Tumor Cells in vitro

Background and Aims: Dendritic cell-based tumor vaccination is a promising approach in the treatment of cancer. Strategies to modify dendritic cells (DCs) with tumor-associated antigens (TAAs) can elicit specific immune responses against tumors. Heparanase is overexpressed in gastric cancer, especially in invasive and metastatic cells, but is downregulated in differential normal tissue. Therefore, heparanase is a potential target in immunotherapy for patients with advanced gastric cancer who are not candidates for surgery. The present paper was designed to investigate the immune response of a heparanase gene-modified DC-based vaccine against gastric cancer cell lines in vitro. Methods: DCs from peripheral blood mononuclear cells of healthy HLA-A2-positive donors were transfected with recombinant adenovirus containing the full-length cDNA of heparanase (rAd-Hpa) to generate heparanase gene-modified DC vaccine. T lymphocytes from the same donors were repeatedly activated by genetically modified DC vaccine to generate heparanase-specific cytotoxicity T lymphocytes (CTLs). CTL-mediated cell lysis of gastric cancer cells lines (KATO-III and SGC-7901) was analyzed in vitro by a standard 51Cr releasing assay. IFN-γ secretion was measured by ELISA in heparanase-specific CTLs cocultured with those gastric cancer cell lines. Results: Our results showed that the expression of heparanase in DCs transfected with rAd-Hpa was significantly increased.Furthermore, DCs transfected with rAd-Hpa could induce heparanase-specific CTLs against HLA-matched and heparanase-positive gastric cancer cells in vitro, while there were no killing effects on autologous lymphocytes. Meanwhile, these rAd-Hpa-modified DCs could increase IFN-γ secretion of effector cells when cocultured with KATO-III cells. Conclusions: These findings demonstrate for the first time that the transduction of DCs with rAd-Hpa can induce CTLs that specifically lyse heparanase-positive gastric cancer cells and increase IFN-γ secretion in an MHC-restricted fashion. Heparanase gene-modified DC vaccine offers a great opportunity for immunotherapy in patients with advanced gastric cancer and possibly also with other malignancies.

Antisense human telomerase reverse transcriptase could partially reverse malignant phenotypes of gastric carcinoma cell line in vitro.

Telomerase activity is detected in more than 90% of examined tumors but not in most normal somatic cells. Among three subunits of human telomerase, human telomerase reverse transcriptase (hTERT) is the rate-limiting component for telomerase activity. Therefore, targeting hTERT represents a promising approach for diminishing telomerase function that will probably not cause substantial side effects on telomerase negative somatic cells. To explore the effects of antisense hTERT (ahTERT) on the malignant phenotypes of human SGC-7901 gastric cancer cell line in vitro, an antisense eukaryotic expression vector of hTERT was constructed by gene recombinant technology. Telomerase activity by telomeric repeat amplification protocol–ELISA, mRNA of telomerase subunits, c-myc and bcl-2 by reverse transcript-PCR, terminal restriction fragment (TRF) by Southern blot, cell cycle distribution by flow cytometry and protein expression of hTERT, c-myc and bcl-2 by Western blot were analyzed in SGC-7901 cells before and after transfection. Cloning efficiency assay in soft agar and tumorigenesis in nude mice were also examined and evaluated in the above cells. The results demonstrated that after ahTERT transfection, the proliferation of SGC-7901 cells was significantly inhibited. Further study showed that telomerase activity, telomere length, the mRNA and protein expression of hTERT, bcl-2 and c-myc were decreased in ahTERT-transfected cells. There were, however, no obvious effects on transcription of human telomerase RNA (hTR) and human telomerase associated protein1 (TP1) in both transfected and untransfected cells. Flow cytometric analysis displayed an accumulation of G0/G1 phase and a decreasing proliferation index (PI) in ahTERT-transfected cells. Moreover, no tumorigenicity was found after subcutaneous injection of ahTERT-transfected cells in nude mice, whereas palpable tumors were observed in mice injected with control cells. Our study indicates that exogenous ahTERT can inhibit proliferation and partially reverse malignant phenotypes of SGC-7901 cells via the suppression of telomerase activity, hTERT, c-myc and bcl-2 expression. Antisense technology targeted hTERT strategy might be a potential approach for gastric cancer therapy.

Heparanase: a universal immunotherapeutic target in human cancers

Heparanase has been identified as a particularly important player in metastasis, and its expression directly correlates with the metastatic spread of various tumors. Ideal targets for immunotherapy are gene products that are silenced in normal tissues but overexpressed in cancer, and that are directly involved in tumor cell survival and progression. Metastasis is the culmination of neoplastic progression. The importance of the role of heparanase in metastasis implies that immune escape by downregulation of heparanase expression could reduce the mortality of the cancer. These characteristics of heparanase make it an attractive universal target for cancer immunotherapy. Here, we review current knowledge about heparanase and its involvement in tumor metastasis, with an emphasis on recent results from heparanase-targeted cancer immunotherapy studies.

Programmed cell death 4 (PDCD4) mediates the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by down-regulation of FLIP expression.

Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL.

Induction of anti-tumour immunity by dendritic cells transduced with hTERT recombinant adenovirus in mice.

Dendritic cells (DCs) transfected with recombinant, replication‐defective adenovirus (Ad) vectors encoding the human telomerase reverse transcriptase (hTERT) are potent inducers of cytotoxic T lymphocytes (CTLs) and anti‐tumour immunity. However, previous studies have mostly been in vitro. In this study, we sought to determine whether DCs transfected with hTERT (DC/Ad‐hTERT) could elicit a potent anti‐tumour immunogenic response in vivo. We found that murine DCs transfected with recombinant adenovirus encoding the hTERT gene (DC/Ad‐hTERT) induced hTERT‐specific CTLs in vivo effectively, compared with Ad‐LacZ‐transduced DC (DC/Ad‐LacZ) controls. These hTERT‐specific CTLs lysed various tumour cell lines in an hTERT‐specific and MHC‐I molecule‐restricted fashion. We also found that DC/Ad‐hTERT could increase antigen‐specific T‐cell proliferation and augment the number of IFN‐γ secreting T‐cells in mice. These data suggest that the DC/Ad‐hTERT vaccine may induce anti‐tumour immunity against tumour cells expressing hTERT in an MHC‐I molecule‐restricted fashion in vivo through the augmentation of the hTERT‐specific CTL response. The DC/Ad‐hTERT vaccine may thus be used as an efficient DC‐based tumour vaccine in clinical applications. Copyright

Cytotoxic T lymphocyte epitopes from human heparanase can elicit a potent anti-tumor immune response in mice

Heparanase is expressed in almost all advanced tumors, and therefore it may serve as a potential target for tumor therapy. Our previous study has shown that heparanase can serve as a potential universal tumor-associated antigen (TAA) for the immunotherapy of advanced tumors. Further study demonstrated that the HLA-A*0201-restricted Cytotoxic T lymphocytes (CTL) epitopes Hpa525 (PAFSYSFFV), Hpa277 (KMLKSFLKA) and Hpa405 (WLSLLFKKL) from human heparanase could induce a potent anti-tumor immune response in vitro. The present study was designed to investigate whether the above peptides could induce immune responses in mice. Our results demonstrated that the effectors from heparanase peptide-immunized mice could effectively lyse various tumor cells that were heparanase positive and HLA-A*0201 matched. We also found that these peptide-specific CTLs did not lyse autologous lymphocytes that had low heparanase activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-γ-producing T cells as compared to a negative peptide. These results suggest that Hpa525, Hpa277, and Hpa405 peptides are novel HLA-A*0201-restricted CTL epitopes capable of inducing heparanase-specific CTLs in mice. Because heparanase is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy of patients with advanced tumors.

E2F1: A potential negative regulator of hTERT transcription in normal cells upon activation of oncogenic c-Myc

Summary Previous studies have revealed that the link between c-Myc and E2F1 pathway plays a pivotal role in regulating cell growth and death. Human telomerase reverse transcriptase (hTERT), activation of which is required for cell immortalization and transformation, has been confirmed to be a direct transcriptional target of c-Myc. It is of note that E2F1, which is also a direct transcriptional target of c-Myc, can bind the hTERT promoter and repress its expression. Thus, although oncogene c-Myc can be activated in normal cells, for the subsequent induction of E2F1, it may still be insufficient to trigger the expression of hTERT. This negative feedback regulation, if it exists, may be another mechanism for normal cells to control the transmission of c-Myc-mediated oncogenic signals. In this article, we reviewed current knowledge about the crosstalk among c-Myc, E2F1 and hTERT, with an emphasis on the hypothesis that E2F1 negatively regulates c-Myc-induced hTERT transcription. Additionally, we postulated that the miR-17-92 cluster-mediated regulation of c-Myc and E2F1 expression may be of particular importance for the repression of hTERT transcription.

PinX1 Inhibits Telomerase Activity in Gastric Cancer Cells Through Mad1/c-Myc Pathway

IntroductionThe aim of this study was to investigate the role of Mad1/c-Myc in telomerase regulation in gastric cancer cells in order to gain insight into telomerase activity and to evaluate PinX1 as a putative inhibitor of gastric cancer.MethodsPinX1 and PinX1siRNA eukaryotic expression vectors were constructed by recombinant technology and transfected into gastric carcinoma cells using Lipofectamine™ 2000. Telomerase activity was measured by the telomeric repeat amplification protocol. Apoptosis of gastric cancer cells was analyzed by flow cytometry and transmission electron microscopy. Reverse transcription-polymerase chain reaction and Western blotting were used to assess the expression levels of PinX1 and Mad1/c-Myc.ResultsWe found that PinX1-negative gastric cancer cells showed significantly higher telomerase activity than did the PinX1-postive cells. PinX1-transfection reduced telomerase activity in PinX1-negative gastric cancer cells and exhibited an upregulation of Mad1 and downregulation of c-Myc expression. Pinx1 RNAi treatment led to downregulation of Mad1 and upregulation of c-Myc.ConclusionSuppression of telomerase activity mediated by PinX1 is involved in the Mad1/c-Myc pathway.