Song-Tao Yu

H-2Kb–Restricted CTL Epitopes from Mouse Heparanase Elicit an Antitumor Immune Response In vivo

The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancer-specific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumor-associated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K(b)-restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptide-binding assay to determine the affinity of the predicted epitopes with the H-2K(b) molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K(b), and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K(b) (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumor-bearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K(b)-restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies.

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.

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

Noninvasive and real-time monitoring of the therapeutic response of tumors in vivo with an optimized hTERT promoter

Telomerase is commonly recognized as an effective anticancer target. The human telomerase reverse transcriptase (hTERT), the rate‐limiting component of telomerase, is expressed in most malignant tumors, but it is not found in most normal somatic cells. Here, we report a real‐time and noninvasive method to monitor tumor response to a lentivirus‐based hTERT‐conditional suicidal gene therapy.

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.

hTERT mediates gastric cancer metastasis partially through the indirect targeting of ITGB1 by microRNA-29a.

Human telomerase reverse transcriptase (hTERT) plays a key role in tumor invasion and metastasis, but the mechanism of its involvement in these processes is not clear. The purpose of this study is to investigate the possible molecular mechanism of hTERT in the promotion of gastric cancer (GC) metastasis. We found that the up-regulation of hTERT in gastric cancer cells could inhibit the expression of miR-29a and enhance the expression of Integrin β1 (ITGB1). In addition, the invasive capacity of gastric cancer cells was also highly increased after hTERT overexpression. Our study also found that the restoration of miR-29a suppressed the expression of ITGB1 and inhibited GC cell metastasis both in vitro and in vivo. Taken together, our results suggested that hTERT may promote GC metastasis through the hTERT-miR-29a-ITGB1 regulatory pathway.

Inhibition of tankyrase 1 in human gastric cancer cells enhances telomere shortening by telomerase inhibitors.

Telomere stability is believed to be related to aging and tumorigenesis. Besides telomerase, telomere length is also regulated by several telomere-specific binding proteins. Tankyrase 1, a telomeric poly(ADP-ribose) polymerase (PARP), elongates telomere length by inhibiting TRF1 binding to telomeres. In order to study the synergistic action of tankyrase 1 and telomerase in the maintenance of telomere length in mammalian cells, we constructed anti-sense tankyrase 1 (aTNKS) eukaryotic expression vectors and then transfected them into the SGC-7901 human gastric cancer cell line, as well as SGC-7901 cells that had been transfected with antisense hTR (7901-ahTR) and antisense hTERT (7901-ahTERT) with DOTAP liposomes. The activity of telomerase, telomere length and telomerase-associated protein activities were measure by TRAP-ELISA, Southern blot and western blot analysis, respectively, in aTNKS transfected and untransfected cells. The results demonstrated that telomere length was significantly shorter in cells with concomitant tankyrase 1 and telomerase inhibition than by either tankyrase 1 or telomerase inhibition alone, in SGC-7901 cells. We also found that aTNKS had no effect on telomerase activity. These results reveal that inhibition of tankyrase 1 could shorten telomere length and play a synergistic role with telomerase inhibitors in telomere length shortening in the SGC-7901 gastric cancer cell line. Co-inhibition of tankyrase 1 and telomerase activity may be a rational strategy for telomere-directed gastric cancer therapeutics.

An Optimized Telomerase-Specific Lentivirus for Optical Imaging of Tumors

Advances in medical imaging techniques, such as ultrasound, computed tomography, magnetic resonance imaging, and positron emission tomography, have made great progress in detecting tumors. However, these imaging techniques are unable to differentiate malignant tumors from benign ones. Recently developed optical imaging of tumors in small animals provides a useful method to distinguish malignant tumors from their surrounding normal tissues. Human telomerase reverse transcriptase (hTERT) is normally inactivated in most somatic cells, whereas it is commonly reactivated in many cancer cells. In this study, we constructed a lentiviral vector that expresses green fluorescent protein (GFP) driven by an optimized hTERT promoter to create a noninvasive tumor-specific imaging methodology. The activity of this optimized hTERT promoter was found to be equal to the activity of SV40 and cytomegalovirus promoters. In vitro experiments showed that GFP was only expressed in telomerase-positive tumor cells infected with this lentivirus, whereas there was no GFP expression in telomerase-negative tumor cells or normal somatic cells. We also found that subcutaneous telomerase-positive tumors could be visualized 24 hours after an intratumoral injection with this lentivirus by using a charge-coupled device (CCD) camera. In contrast, telomerase-negative tumors could not be imaged after an intratumoral injection even for 30 days. These results suggest that infection with lentivirus containing this optimized hTERT promoter might be a useful diagnostic tool for the real-time visualization of macroscopically invisible tumor tissues using a highly sensitive CCD imaging system.

Responsiveness to aspirin in patients with unstable angina pectoris by whole blood aggregometry.

Aims:  To evaluate aspirin responsiveness in patients with unstable angina pectoris (UAP) by whole blood aggregometry. Another goal was to differentiate aspirin‐resistant patients into pharmacokinetic or pharmacodynamic type.

MR molecular imaging of tumors based on an optimal hTERT promoter tyrosinase expression system.

The early diagnosis and treatment of tumors is of vital significance to increase patient survival. Therefore, we constructed a lentiviral vector expressing tyrosinase (TYR) driven by an optimized human telomerase reverse transcriptase (hTERT) promoter or a cytomegalovirus(CMV) promoter in the hopes of performing noninvasive and real-time tumor-specific imaging. First, hTERT-TYR and CMV-TYR were constructed to infect cancer cell lines (telomerase-negative cell line: U2OS; telomerase-positive cell lines: SGC-7901, SW480 and HepG2). Subsequently, stable tyrosinase-expressing cell lines were sorted by flow cytometry out of these infected cancer cell lines. Then, the mRNA and protein levels of tyrosinase were analyzed. Thetyrosinase activity, melanin production and ferric ion adsorption were measured followed by an MR scan. Consequently the results showed that tyrosinase was only expressed in telomerase-positive tumor cells infected by hTERT-TYR, whereas tyrosinase was expressed in both telomerase-negative and telomerase-positive tumor cells infected by CMV-TYR. Finally, we performed in vivo tumor MR using a clinical 3T MR scanner and found increased signals at T1W1 from telomerase-positive cells infected by hTERT-TYR, which revealed that MR scanning could distinguish cells with hTERT -positive cells from hTERT-negative cells infected with the optimized lentivirus. The mechanism underlying this effect is that tyrosinase promotes melanin production and ferric ion adsorption only in hTERT-expressing cells. Taken together, these data show that this optimized hTERT promoter-driving tyrosinase expression system might be a useful diagnostic tool for the detection of tumors using MR imaging.