- Open Access
CpG oligonucleotides suppress HepG2 cells-induced Jurkat cell apoptosis via the Fas-FasL-mediated pathway
© Zheng et al; licensee BioMed Central Ltd. 2011
- Received: 9 February 2011
- Accepted: 3 May 2011
- Published: 3 May 2011
To explore the potential role of CpG motif-containing oligonucleotides (CpG-ODN) in modulating the expression of FasL in HepG2 and Fas in Jurkat cells in vitro, and to examine the effect of CpG-ODN treatment on the HepG2 cells-mediated Jurkat cell apoptosis in vitro.
The expressions of FasL in HepG2 and Fas in Jurkat cells were examined by real time PCR and flow cytometry (FCM). HepG2 and Jurkat cells were co-cultured, and the frequency of apoptotic Jurkat cells and levels of activated caspase-3 were determined by FCM.
Treatment with CpG-ODN down-regulated the expression of FasL in HepG2 cells in a dose- and time-dependent manner. In addition, treatment with CpG-ODN down-regulated the Fas mRNA transcription and protein expression in Jurkat cells. Treatment of HepG2 cells or Jurkat cells with FasL-neutralizing antibody NOK-2 remarkably inhibited the HepG2-medaited Jurkat cell apoptosis. Pre-treatment of HepG2 or Jurkat cells with CpG-ODN significantly reduced the frequency of HepG2-mediated apoptotic Jurkat cells and inhibited the activation of caspase-3 in Jurkat cells in vitro.
Our data indicated that treatment with CpG-ODN inhibited the HepG2 cells-mediated Jurkat cell apoptosis by modulating the Fas/FasL pathway. Apparently, CpG-ODN treatment may be a potential therapeutic reagent for HCC.
- hepatocellular carcinoma
Tumors escape immune surveillance through multiple mechanisms. For example, tumors can produce inhibitory factors, such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), leading to the reduced dendritic cell activation and impaired tumor-specific T cell immunity . Tumor cells can up-regulate some of the functional surface molecules, including FasL, which can actively induce the apoptosis of the Fas-expressing activated T lymphocytes, while others can down-regulate the expression of other molecules, such as MHC class I and Fas [2, 3]. Although the mechanisms by which tumor cells evade immune surveillance are not well understood, the selective induction of tumor cell apoptosis has been thought to be a valuable strategy for tumor therapy. CpG-ODN can function as a Th-1 adjuvant  and is able to activate dendritic cells . Accordingly, CpG-ODN has been used as an adjuvant for the induction of anti-tumor immune responses [6–8].
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, particularly in China. Accumulating evidences have suggested that several mechanisms contribute to the carcinogenesis of HCC [9, 10]. The relative resistance to apoptosis triggering and the strong proliferation in HCC cells have been thought as predominant factors contributing to the development of HCC . Recently, high levels of FasL have been found in HCC tumor cells . Given that Fas is highly expressed by activated T cells, HCC may trigger the apoptosis of activated T cells through the Fas/FasL pathway, escaping from immune surveillance. However, little is known whether CpG-ODN could modulate the expression of FasL in HCC cells and Fas in human T cells as well as the HCC-triggered human T cell apoptosis.
This study aimed at exploring the potential effect of CpG-OND treatment on the HepG2-induced Jurkat cell apoptosis. We found that treatment with CpG-ODN down-regulated the expression of FasL in HepG2 cells and Fas in Jurkat cells, and inhibited the HepG2-mediated Jurkat cell apoptosis in vitro. We discussed the implication of our findings.
The CpG-ODN-M362  used in the experiment was synthesized by Invitrogen (Invitrogen Inc, Shanghai, China). Oligonucleotides were dissolved in TE-buffer (pH 8.0) containing 10 mM Tris-HCl and 1 mM EDTA at a concentration of 100 μM, which were then aliquoted and stored at -20°C until use. RPMI-1640 medium was obtained from Invitrogen Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from GIBCO BRL (Grand Island, NY, USA). Monoclonal antibody against human FasL, NOK-2, was purchased from BD Pharmingen (San Diego, CA, USA).
Human hepatocellular carcinoma cell line, HepG2 and lymphoma cell line, Jurkat were maintained in our laboratory and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in 25 cm2 polystyrene flasks at 37°C in a humidified atmosphere of 5% CO2 incubator. Routine passage was carried out every 2 or 3 days.
Flow cytometry analysis
HepG2 cells at 5 × 105 cells/well were treated in duplicate with 10-4 to 5 μM CpG-ODN in 10% FBS RPMI1640 in 12-well plates for 48 h to determine the optimal dosage of CpG-ODN for modulating the FasL expression. In addition, HepG2 cells at 5 × 105 cells/well were treated in duplicate with 1 μM CpG-ODN for 0-48 h. The cells were harvested and stained with phycoerythrin (PE) anti-human FasL antibody and isotype control (eBioscience, San Diego, CA, USA). The frequency of Fas-expressing HepG2 cells were determined by flow cytometry analysis. Approximately, 10,000 cells from each sample were analyzed by flow cytometry on a FACS Calibur instrument (Becton Dickinson, San Jose, CA, USA).
Jurkat cells at 5 × 105 cells/well were treated in duplicate with 1 μM CpG-ODN for 24 h and cultured in medium alone as controls. The cells were harvested and stained with PE-anti-human Fas antibody or isotype control (eBioscience). The frequency of Fas-expressing cells was determined by flow cytometry analysis. Data were analyzed using CellQuest software.
HepG2 and Jurkat cells coculture
HepG2 cells at 2 × 106 cells/well were cultured in 10% FBS RPMI1640 alone or treated with 1 μM CpG-ODN or 10 μg/ml anti-FasL antibody NOK-2 in RPMI1640 for 24 h to prepare the inducers. Jurkat cells at 2 × 106 cells/well were cultured 10% FBS RPMI1640 alone or treated with 1 μM CpG-ODN or 10 μg/ml anti-FasL antibody NOK-2 in RPMI1640 for 24 h to prepare the target cells. These cells were cultured as the untreated HepG2 (2 × 106) and Jurkat cells (4 × 105) for 24 h (controls); the NOK-2-treated HepG2 and untreated Jurkat cells; the untreated HepG2 and the NOK-2-treated Jurkat cells; the CpG-ODN-treated HepG2 and untreated Jurkat cells; and the untreated HepG2 and the CpG-ODN-treated Jurkat cells, respectively. Subsequently, the suspended Jurkat cells were collected and stained with FITC-Annexin V and PI. The apoptotic Jurkat cells were determined by flow cytometry analysis. Data were analyzed using CellQuest software.
In addition, the unmanipulated Jurkat cells or the CpG-ODN-treated Jurkat cells were harvested after co-culture with unmanipulated HepG2 or the CpG-ODN-treated HepG2 cells. The cells were stained with PE-anti-activated caspase-3 using the PE-conjugated active caspase-3 apoptosis kit (BD Pharmingen), and the activation of capsase-3 was determined by flow cytometry analysis.
the sequences of primers.
Annealing temperature (°C)
Data were expressed as means ± S.E.M. Statistical significance was assessed using either Student's t-test or one-way ANOVA followed by post hoc Dunnett, SNK test. A value of p < 0.05 was considered significantly different.
CpG-ODN downregulated the expression of FasL in HepG2 cells in a dose- and time-dependent manner
Effect of CpG-ODN on the Fas expression in Jurkat cells
Effect of CpG-ODN on the HepG2-mediated Jurkat cell apoptosis
More interestingly, co-culture of the CpG-ODN-treated HepG2 cells with unmanipulated Jurkat cells or unmanipulated HepG2 with the CpG-ODN-treated Jurkat cells significantly reduced the frequency of apoptotic Jurkat cells, particularly following treatment of Jurkat cells with CpG-ODN. These data indicated that down-regulation of FasL and Fas expression by CpG-ODN in either HepG2 or Jurkat cells inhibited the HepG2 cell-mediated Jurkat cell apoptosis in vitro.
Caspase-3 activity analysis
The up-regulated expression of FasL has been found in various types of tumors, including melanoma, lymphoma, gastric carcinoma, and breast carcinoma . It has been reported that high levels of FasL expression are associated with the presence of tumor-infiltrating lymphocytes (TIL), leading to high susceptibility of activated T cells in tumor tissues to apoptosis triggers due to high levels of Fas expression by activated T cells . Indeed, engagement of Fas by the FasL can promote the formation of death-inducing signaling complex, resulting in activated T cell apoptosis. This may partially contribute to tumor cells escaping from immune surveillance and leading to tumor progression.
Due to the important role of Fas in the tumor progression and metastasis, the Fas-mediated apoptosis might be a target for cancer therapy. Notably, the apoptotic cascade is a sequential process of many events that can be regulated at different stages. Several agents have been found to directly or indirectly inhibit cellular apoptosis. The arsenic trioxide and tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL) can modulate the intrinsic and extrinsic pathways, respectively . The caspase activators can regulate the common pathway, and ONY-015 can regulate modulators of the apoptosis pathways . CpG-ODN can activate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activated protein 1 through the Toll-like receptor (TLR) sigaling pathway , and has been thought to act as a potent adjuvant for inducing Th1 response. The NF-κB can regulate the expression of the FasL gene, exhibiting both anti-apoptotic and pro-apoptotic functions . In this study, we examined the effects of CpG-ODN treatment on the HepG2 cell-induced Jurkat cell apoptosis. We found that CpG-ODN inhibited the expression of FasL in HepG2 in a dose- and time-dependent manner (Figure 1). Treatment with CpG-ODN at 1 μM for 24 h greatly inhibited the expression of FasL in HepG2 cells in vitro. Furthermore, we found that treatment with CpG-ODN effectively down-regulated the expression of Fas in human Jurkat cells (Figure 2). Jurkat cells are derived from human T lymphocyte leukemia cells, mimic the activated T lymphocyte cells, and have been widely used as experimental models to study the functions of T cells . In addition, co-culturing the unmanipulated HepG2 cells with Jurkat cells triggered a high frequency of Jurkat cells undergoing apoptosis, which was effectively abrogated by pre-treatment of either HepG2 or Jurkat cells with anti-FasL antibody. These data indicated that HepG2 cells induced Jurkat cell apoptosis via the Fas/FasL pathway. More importantly, pre-treatment of Jurkat cells or HepG2 cells with CpG-ODN efficiently inhibited the HepG2-mediated Jurkat cell apoptosis (Figure 3) and the caspase activation in Jurkat cells (Figure 4). CpG-ODN can suppress apoptosis of macrophages via TLR9 through PKB/Akt/FOXO pathway , since macrophages and T cells play an important role in anti-tumor immune, our study showed CpG-ODN suppresses apoptosis through FasL/Fas pathway, maybe PKB/Akt/FOXO is another way in anti-apoptosis anti-cancer therapeutic strategies of CpG-ODN.
Currently, treatment of HCC relies on surgery, conventional chemotherapy, and radiation therapy at clinic. Other therapeutic strategies, such as an antibody targeting the specific molecules, are currently in trials. DNA-based drugs, such as CpG-ODN and antisense ODN, are regarded as a new alternative therapy for the brain tumors . The regulation of the complex signaling pathways in tumors has been a new strategy for the rational design of anticancer strategies. Escaping from immune surveillance and being resistant to apoptosis triggers play an important role in the progression and metastasis of tumors. Our results indicated that CpG-ODN down-regulated the FasL expression in HepG2 cells and Fas in Jurkat cells, and suppressed the HepG2 cells-mediated caspase-dependent apoptosis of Jurkat cells. Conceivably, CpG-ODN treatment may be a promising strategy for the intervention of HCC.
We thank Dr. Lihua Hu, Department of Laboratory & Institute of Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, for her helpful comments on this manuscript.
- Vicari AP, Caux C, Trinchieri G: Tumour escape from immune surveillance through dendritic cell inactivation. Semin Cancer Biol. 2002, 12: 33-42. 10.1006/scbi.2001.0400.View ArticlePubMedGoogle Scholar
- Gratas C, Tohma Y, Barnas C, Taniere P, Hainaut P, Ohgaki H: Up-regulation of Fas (APO-1/CD95) ligand and down-regulation of Fas expression in human esophageal cancer. Cancer Res. 1998, 58: 2057-62.PubMedGoogle Scholar
- Wu JD, Higgins LM, Steinle A, Cosman D, Haugk K, Plymate SR: Prevalent expression of the immunostimulatory MHC class I chain-related molecule is counteracted by shedding in prostate cancer. J Clin Invest. 2004, 114: 560-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Roman M, Martin-Orozco E, Goodman JS, et al: Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat Med. 1997, 3: 849-54. 10.1038/nm0897-849.View ArticlePubMedGoogle Scholar
- Sparwasser T, Vabulas RM, Villmow B, Lipford GB, Wagner H: Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins. Eur J Immunol. 2000, 30: 3591-7. 10.1002/1521-4141(200012)30:12<3591::AID-IMMU3591>3.0.CO;2-J.View ArticlePubMedGoogle Scholar
- Heckelsmiller K, Beck S, Rall K, et al: Combined dendritic cell- and CpG oligonucleotide-based immune therapy cures large murine tumors that resist chemotherapy. Eur J Immunol. 2002, 32: 3235-45. 10.1002/1521-4141(200211)32:11<3235::AID-IMMU3235>3.0.CO;2-J.View ArticlePubMedGoogle Scholar
- Okamoto M, Sato M: Toll-like receptor signaling in anti-cancer immunity. J Med Invest. 2003, 50: 9-24.PubMedGoogle Scholar
- Wooldridge JE, Weiner GJ: CpG DNA and cancer immunotherapy: orchestrating the antitumor immune response. Curr Opin Oncol. 2003, 15: 440-5. 10.1097/00001622-200311000-00006.View ArticlePubMedGoogle Scholar
- Thorgeirsson SS, Teramoto T, Factor VM: Dysregulation of apoptosis in hepatocellular carcinoma. Semin Liver Dis. 1998, 18: 115-22. 10.1055/s-2007-1007148.View ArticlePubMedGoogle Scholar
- Lau SH, Guan XY: Cytogenetic and molecular genetic alterations in hepatocellular carcinoma. Acta Pharmacol Sin. 2005, 26: 659-65. 10.1111/j.1745-7254.2005.00126.x.View ArticlePubMedGoogle Scholar
- Park YN, Chae KJ, Kim YB, Park C, Theise N: Apoptosis and proliferation in hepatocarcinogenesis related to cirrhosis. Cancer. 2001, 92: 2733-8. 10.1002/1097-0142(20011201)92:11<2733::AID-CNCR10126>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
- Hou L, Li Y, Jia YH, et al: Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice. World J Gastroenterol. 2001, 7: 532-6.PubMedGoogle Scholar
- Hartmann G, Battiany J, Poeck H, et al: Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-alpha induction in plasmacytoid dendritic cells. Eur J Immunol. 2003, 33: 1633-41. 10.1002/eji.200323813.View ArticlePubMedGoogle Scholar
- Ramakers C, Ruijter JM, Deprez RH, Moorman AF: Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett. 2003, 339: 62-66. 10.1016/S0304-3940(02)01423-4.View ArticlePubMedGoogle Scholar
- Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H: Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression's C (T) difference" formula. J Mol Med. 2006, 84: 901-10. 10.1007/s00109-006-0097-6.View ArticlePubMedGoogle Scholar
- Kim R, Emi M, Tanabe K, Uchida Y, Toge T: The role of Fas ligand and transforming growth factor beta in tumor progression: molecular mechanisms of immune privilege via Fas-mediated apoptosis and potential targets for cancer therapy. Cancer. 2004, 100: 2281-91. 10.1002/cncr.20270.View ArticlePubMedGoogle Scholar
- Muppidi JR, Siegel RM: Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nat Immunol. 2004, 5: 182-9. 10.1038/ni1024.View ArticlePubMedGoogle Scholar
- Lam HK, Li K, Chik KW, et al: Arsenic trioxide mediates intrinsic and extrinsic pathways of apoptosis and cell cycle arrest in acute megakaryocytic leukemia. Int J Oncol. 2005, 27: 537-45.PubMedGoogle Scholar
- Ghobrial IM, Witzig TE, Adjei AA: Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin. 2005, 55: 178-94. 10.3322/canjclin.55.3.178.View ArticlePubMedGoogle Scholar
- Takeda K, Akira S: TLR signaling pathways. Semin Immunol. 2004, 16: 3-9. 10.1016/j.smim.2003.10.003.View ArticlePubMedGoogle Scholar
- O'Connell J, O'Sullivan GC, Collins JK, Shanahan F: The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med. 1996, 184: 1075-82. 10.1084/jem.184.3.1075.View ArticlePubMedGoogle Scholar
- Lim EJ, Park DW, Lee JG, et al: Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression. Exp Mol Med. 2010, 42 (10): 712-20. 10.3858/emm.2010.42.10.070.PubMed CentralView ArticlePubMedGoogle Scholar
- Guo LH, Schluesener HJ: Binding and uptake of immunostimulatory CpG oligodeoxynucleotides by human neuroblastoma cells. Oligonucleotides. 2004, 14: 287-98. 10.1089/oli.2004.14.287.View ArticlePubMedGoogle Scholar
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