Triptolide Downregulates Treg Cells and the Level of IL-10, TGF-β, and VEGF in Melanoma-Bearing Mice

Abstract

Regulatory T cells play a key role in suppressing tumor immunity. Triptolide, a major active component isolated from the Chinese medicinal herb Tripterygium wilfordii, has been proven to possess multiple antitumor activities. Here, we investigated the effect of triptolide on regulatory T cells and on the level of IL-10, transforming growth factor-β, and vascular endothelial growth factor in tumor-bearing mice. Fifty male C57BL/6 mice were randomly grouped as follows: normal control group, model group with B16-F10 cells implanted, and three treatment groups with cyclophosphamide, triptolide-high dose, triptolide-low dose. The proportion of regulatory T cells in the spleen and axillary lymph nodes was evaluated by flow cytometric analysis. Production of cytokines IL-10, transforming growth factor-β, and vascular endothelial growth factor in serum was measured using enzyme-labeled immunosorbent assay kits. The mRNA levels of Foxp3, IL-10, and transforming growth factor-β in the spleen and vascular endothelial growth factor in tumor tissue were detected by real-time PCR. The results showed that triptolide significantly decreased the proportion of regulatory T cells and lowered the Foxp3 level in the spleen and axillary lymph nodes of tumor-bearing mice. Production of IL-10 and transforming growth factor-β in peripheral blood, and the mRNA level of IL-10 and transforming growth factor-β in the spleen were also decreased. Additionally, triptolide could remarkably inhibit production of vascular endothelial growth factor in tumor-bearing mice. In conclusion, our study demonstrated that triptolide might inhibit tumor growth by inhibiting regulatory T cells and some cytokines such as IL-10 and transforming growth factor-β, as well as production of vascular endothelial growth factor.

Key words

triptolide - regulatory T cells - melanoma - cytokines - Tripterygium wilfordii - Celastraceae

• References

• 1 Antonoff MB, Chugh R, Borja-Cacho D, Dudeja V, Clawson KA, Skube SJ, Sorenson BS, Saltzman DA, Vickers SM, Saluja AK. Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo . Surgery 2009; 146: 282-290
• 2 Liu J, Jiang Z, Xiao J, Zhang Y, Lin S, Duan W, Yao J, Liu C, Huang X, Wang T, Liang Z, Wang R, Zhang S, Zhang L. Effects of triptolide from Tripterygium wilfordii on ERalpha and p 53 expression in two human breast cancer cell lines. Phytomedicine 2009; 16: 1006-1013
• 3 Chugh R, Sangwan V, Patil SP, Dudeja V, Dawra RK, Banerjee S, Schumacher RJ, Blazar BR, Georg GI, Vickers SM, Saluja AK. A preclinical evaluation of Minnelide as a therapeutic agent against pancreatic cancer. Sci Transl Med 2012; 4: 156ra139
• 4 Kiviharju TM, Lecane PS, Sellers RG, Peehl DM. Antiproliferative and proapoptotic activities of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells. Clin Cancer Res 2002; 8: 2666-2674
• 5 Zhu W, Ou Y, Li Y, Xiao R, Shu M, Zhou Y, Xie J, He S, Qiu P, Yan G. A small-molecule triptolide suppresses angiogenesis and invasion of human anaplastic thyroid carcinoma cells via down-regulation of the nuclear factor-kappa B pathway. Mol Pharmacol 2009; 75: 812-819
• 6 He MF, Huang YH, Wu LW, Ge W, Shaw PC, But PP. Triptolide functions as a potent angiogenesis inhibitor. Int J Cancer 2010; 126: 266-278
• 7 Zhao H, Yang Z, Wang X, Zhang X, Wang M, Wang Y, Mei Q, Wang Z. Triptolide inhibits ovarian cancer cell invasion by repression of matrix metalloproteinase 7 and 19 and upregulation of E-cadherin. Exp Mol Med 2012; 44: 633-641
• 8 Yinjun L, Jie J, Yungui W. Triptolide inhibits transcription factor NF-kappaB and induces apoptosis of multiple myeloma cells. Leuk Res 2005; 29: 99-105
• 9 Qiu D, Zhao G, Aoki Y, Shi L, Uyei A, Nazarian S, Ng JC, Kao PN. Immunosuppressant PG490 (triptolide) inhibits T-cell interleukin-2 expression at the level of purine-box/nuclear factor of activated T-cells and NF-kappaB transcriptional activation. J Biol Chem 1999; 274: 13443-13450
• 10 Wang Y, Lu JJ, He L, Yu Q. Triptolide (TPL) inhibits global transcription by inducing proteasome-dependent degradation of RNA polymerase II (Pol II). PloS One 2011; 6: e23993
• 11 Ménétrier-Caux C, Gobert M, Caux C. Differences in tumor regulatory T-cell localization and activation status impact patient outcome. Cancer Res 2009; 69: 7895-7898
• 12 Lindau D, Gielen P, Kroesen M, Wesseling P, Adema GJ. The immunosuppressive tumour network: myeloid-derived suppressor cells, regulatory T cells and natural killer T cells. Immunology 2013; 138: 105-115
• 13 Miller AM, Lundberg K, Ozenci V, Banham AH, Hellstrom M, Egevad L, Pisa P. CD4+CD25high T cells are enriched in the tumor and peripheral blood of prostate cancer patients. J Immunol 2006; 177: 7398-7405
• 14 Petersen RP, Campa MJ, Sperlazza J, Conlon D, Joshi MB, Harpole jr. DH, Patz jr. EF. Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 2006; 107: 2866-2872
• 15 Huang Y, Wang FM, Wang T, Wang YJ, Zhu ZY, Gao YT, Du Z. Tumor-infiltrating FoxP3+ regs and CD8+ T cells affect the prognosis of hepatocellular carcinoma patients. Digestion 2012; 86: 329-337
• 16 Wang Y, Ma Y, Fang Y, Wu S, Liu L, Fu D, Shen X. Regulatory T cell: a protection for tumour cells. J Cell Mol Med 2012; 16: 425-436
• 17 Beyer M, Schultze JL. Regulatory T cells in cancer. Blood 2006; 108: 804-811
• 18 Liu Y, Chen Y, Liu FQ, Lamb JR, Tam PK. Combined treatment with triptolide and rapamycin prolongs graft survival in a mouse model of cardiac transplantation. Transpl Int 2008; 21: 483-494
• 19 Zhang G, Liu Y, Guo H, Sun Z, Zhou YH. Triptolide promotes generation of FoxP3+ T regulatory cells in rats. J Ethnopharmacol 2009; 125: 41-46
• 20 Facciabene A, Motz GT, Coukos G. T-regulatory cells: key players in tumor immune escape and angiogenesis. Cancer Res 2012; 72: 2162-2171
• 21 Cohen AD, Diab A, Perales MA, Wolchok JD, Rizzuto G, Merghoub T, Huggins D, Liu C, Turk MJ, Restifo NP, Sakaguchi S, Houghton AN. Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity. Cancer Res 2006; 66: 4904-4912
• 22 Ghiringhelli F, Menard C, Puig PE, Ladoire S, Roux S, Martin F, Solary E, Le Cesne A, Zitvogel L, Chauffert B. Metronomic cyclophosphamide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer Immunol Immunother 2007; 56: 641-648
• 23 Sutmuller RP, van Duivenvoorde LM, van Elsas A, Schumacher TN, Wildenberg ME, Allison JP, Toes RE, Offringa R, Melief CJ. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 2001; 194: 823-832
• 24 Rasku MA, Clem AL, Telang S, Taft B, Gettings K, Gragg H, Cramer D, Lear SC, McMasters KM, Miller DM, Chesney J. Transient T cell depletion causes regression of melanoma metastases. J Transl Med 2008; 6: 12
• 25 Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003; 4: 330-336
• 26 Liang J, Chen H, Pan W, Xu C. Puerarin inhibits caspase-3 expression in osteoblasts of diabetic rats. Mol Med Rep 2012; 5: 1419-1422
• 27 Yu Z, Li W. Induction of apoptosis by puerarin in colon cancer HT-29 cells. Cancer Lett 2006; 238: 53-60
• 28 Tao Y, Zhang ML, Ma PC, Sun JF, Zhou WQ, Cao YP, Li LJ. Triptolide inhibits proliferation and induces apoptosis of human melanoma A375 cells. Asian Pac J Cancer Prev 2012; 13: 1611-1615
• 29 Lu N, Liu J, Liu J, Zhang C, Jiang F, Wu H, Chen L, Zeng W, Cao X, Yan T, Wang G, Zhou H, Lin B, Yan X, Zhang XK, Zeng JZ. Antagonist effect of triptolide on AKT activation by truncated retinoid X receptor-alpha. PLoS One 2012; 7: e35722
• 30 Manzo SG, Zhou ZL, Wang YQ, Marinello J, He JX, Li YC, Ding J, Capranico G, Miao ZH. Natural product triptolide mediates cancer cell death by triggering CDK7-dependent degradation of RNA polymerase II. Cancer Res 2012; 72: 5363-5373
• 31 Lu L, Kanwar J, Schmitt S, Cui QC, Zhang C, Zhao C, Dou QP. Inhibition of tumor cellular proteasome activity by triptolide extracted from the Chinese medicinal plant ʼthunder god vineʼ. Anticancer Res 2011; 31: 1-10
• 32 Miyara M, Sakaguchi S. Natural regulatory T cells: mechanisms of suppression. Trends Mol Med 2007; 13: 108-116
• 33 Jankovic D, Kullberg MC, Feng CG, Goldszmid RS, Collazo CM, Wilson M, Wynn TA, Kamanaka M, Flavell RA, Sher A. Conventional T-bet(+)Foxp3(−) Th1 cells are the major source of host-protective regulatory IL-10 during intracellular protozoan infection. J Exp Med 2007; 204: 273-283
• 34 Dercamp C, Chemin K, Caux C, Trinchieri G, Vicari AP. Distinct and overlapping roles of interleukin-10 and CD25+ regulatory T cells in the inhibition of antitumor CD8 T-cell responses. Cancer Res 2005; 65: 8479-8486
• 35 Strauss L, Bergmann C, Szczepanski M, Gooding W, Johnson JT, Whiteside TL. A unique subset of CD4+CD25highFoxp3+ T cells secreting interleukin-10 and transforming growth factor-beta1 mediates suppression in the tumor microenvironment. Clin Cancer Res 2007; 13: 4345-4354
• 36 Powrie F, Carlino J, Leach MW, Mauze S, Coffman RL. A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells. J Exp Med 1996; 183: 2669-2674
• 37 Massague J. TGFbeta in cancer. Cell 2008; 134: 215-230
• 38 Javelaud D, Alexaki VI, Mauviel A. Transforming growth factor-beta in cutaneous melanoma. Pigment Cell Melanoma Res 2008; 21: 123-132
• 39 Meulmeester E, Ten Dijke P. The dynamic roles of TGF-β in cancer. J Pathol 2011; 223: 205-218
• 40 Guise T. Examining the metastatic niche: targeting the microenvironment. Semin Oncol 2010; 37: S2-S14
• 41 Yamagiwa S, Gray JD, Hashimoto S, Horwitz DA. A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J Immunol 2001; 166: 7282-7289
• 42 Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003; 198: 1875-1886
• 43 Beck C, Schreiber H, Rowley D. Role of TGF-beta in immune-evasion of cancer. Microsc Res Tech 2001; 52: 387-395
• 44 Leek RD. The prognostic role of angiogenesis in breast cancer. Anticancer Res 2001; 21: 4325-4331
• 45 Grothey A, Allegra C. Antiangiogenesis therapy in the treatment of metastatic colorectal cancer. Ther Adv Med Oncol 2012; 4: 301-319
• 46 Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003; 9: 669-676
• 47 Ning H, Shao QQ, Ding KJ, Gao DX, Lu QL, Cao QW, Niu ZH, Fu Q, Zhang CH, Qu X, Lü JJ. Tumor-infiltrating regulatory T cells are positively correlated with angiogenic status in renal cell carcinoma. Chin Med J (Engl) 2012; 125: 2120-2125
• 48 Terme M, Pernot S, Marcheteau E, Sandoval F, Benhamouda N, Colussi O, Dubreuil O, Carpentier AF, Tartour E, Taieb J. VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer. Cancer Res 2013; 73: 539-549
• 49 Hu KB, Liu ZH, Guo XH, Liu D, Li LS. Triptolide inhibits vascular endothelial growth factor expression and production in endothelial cells. Acta Pharmacol Sin 2001; 22: 651-656
• 50 Johnson SM, Wang X, Evers BM. Triptolide inhibits proliferation and migration of colon cancer cells by inhibition of cell cycle regulators and cytokine receptors. J Surg Res 2011; 168: 197-205