Norcantharidin alleviates cyclophosphamide-induced immunosuppression via circBCL2L1/miR-30c-3-3p/TRAF6 axis

Main Article Content

Guochuan Wang
Yali Zhang
Xiaolu Zhou
Mei Yang
Xiaoyu Ma
Xin Liu


norcantharidin, cyclophosphamide, immunosuppression, circBCL2L1, miR-30c-3-3p, TRAF6


Cyclophosphamide is a widely used antitumor drug, with induced adverse effects, such as intestinal mucosal injury and immunosuppression. Norcantharidin possesses anticancer activity through enhancement of antitumor immunity. We investigated the role of norcantharidin in cyclophosphamide-induced immunosuppression. Mice were treated with cyclophosphamide, and exposed to norcantharidin. Enzyme-linked-immunosorbent serologic assay was performed to assess the levels of immunoglobulin and cytokines in serum, and the splenic T lymphocytes were analyzed by immunohistochemistry. Incubation with norcantharidin increased the serum levels of immunoglobulin G (IgG), interleukin (IL)-12, interferon-gamma (IFN-γ), and IL-6, and enhanced the percentage of CD4+ and CD8+ T lymphocytes in cyclophosphamide-induced mice. Expression of circBCL2L1 was down-regulated in the spleen of cyclophosphamide-induced mice, while up-regulated by norcantharidin incubation. Norcantharidin attenuated cyclophosphamide-induced up-regulation of miR-30c-3-3p and down-regulation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in mice. Over-expression of circBCL2L1 increased serum levels of immunoglobulin and cytokines, and enhanced the percentage of splenic CD4+ and CD8+ T lymphocytes in cyclophosphamide-induced mice. Moreover, over-expression of circBCL2L1 increased TRAF6 in cyclophosphamide-induced mice through down-regulation of miR-30c-3-3p. Knockdown of TRAF6 attenuated norcantharidin-induced increase of serum levels of IgG, IL-12, IFN-γ, and IL-6, and up-regulation of CD4+ and CD8+ T lymphocytes in cyclophosphamide-induced mice. Norcantharidin exhibited protective effect against cyclophosphamide-induced immunosuppression in mice through regulation of circBCL2L1/miR-30c-3-3p/TRAF6 axis.

Abstract 249 | PDF Downloads 330 HTML Downloads 215 XML Downloads 7


Ahlmann, M. and Hempel, G., 2016. The effect of cyclophosphamide on the immune system: implications for clinical cancer therapy. Cancer Chemotherapy and Pharmacology 78: 661–671. 10.1007/s00280-016-3152-1

Audia, S., Nicolas, A., Cathelin, D., Larmonier, N., Ferrand, C., Foucher, P., et al. 2007. Increase of CD4+ CD25+ regulatory T cells in the peripheral blood of patients with metastatic carcinoma: a phase I clinical trial using cyclophosphamide and immunotherapy to eliminate CD4+ CD25+ T lymphocytes. Clinical and Experimental Immunology 150: 523–530. 10.1111/j.1365-2249.2007.03521.x

Chen, Y.-N., Chen, J.-C., Yin, S.-C., Wang, G.-S., Tsauer, W., Hsu, S.-F., et al. 2002. Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells. International Journal of Cancer (Journal International du Cancer) 100: 158–165. 10.1002/ijc.10479

Chen, X.-T., Li, J., Wang, H.-L., Cheng, W.-M., Zhang, L. and Ge, J.-F., 2006. Immunomodulating effects of fractioned polysaccharides isolated from Yu-Ping-Feng powder in cyclophosphamide-treated mice. American Journal of Chinese Medicine 34: 631–641. 10.1142/S0192415X06004168

Dainichi, T., Matsumoto, R., Mostafa, A. and Kabashima, K., 2019. Immune control by TRAF6-mediated pathways of epithelial cells in the EIME (epithelial immune microenvironment). Frontiers in Immunology 10: 1107–1107. 10.3389/fimmu.2019.01107

Delcambre, G. H., Liu, J., Herrington, J. M., Vallario, K. and Long, M. T., 2016. Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue. PeerJ 4: e1601. doi: 10.7717/peerj.1601

Emadi, A., Jones, R.J. and Brodsky, R.A., 2009. Cyclophosphamide and cancer: golden anniversary. Nature Reviews Clinical Oncology 6: 638–647. 10.1038/nrclinonc.2009.146

Fang, Z., Jiang, C. and Li, S., 2021. The potential regulatory roles of circular RNAs in tumor immunology and immunotherapy. Frontiers in Immunology 11: 617583.. 10.3389/fimmu.2020.617583

Gonzalez, H., Hagerling, C. and Werb, Z., 2018. Roles of the immune system in cancer: from tumor initiation to metastatic progression. Genes & Development 32: 1267–1284. 10.1101/gad.314617.118

Huang, X.-Y., Zhang, P.-F., Wei, C.-Y., Peng, R., Lu, J.-C., Gao, C., et al. 2020. Circular RNA circMET drives immunosuppression and anti-PD1 therapy resistance in hepatocellular carcinoma via the miR-30-5p/snail/DPP4 axis. Molecular Cancer 19: 92–92. 10.1186/s12943-020-01213-6

Lin, C.-L., Chen, C.-M., Lin, C.-L., Cheng, C.-W., Lee, C.-H. and Hsieh, Y.-H., 2017. Norcantharidin induces mitochondrial-dependent apoptosis through Mcl-1 inhibition in human prostate cancer cells. Biochimica et biophysica acta. Molecular Cell Research 1864: 1867–1876. 10.1016/j.bbamcr.2017.07.015

Liu, Y., Wu, X., Jin, W. and Guo, Y., 2020. Immunomodulatory effects of a low-molecular weight polysaccharide from enteromorpha prolifera on RAW 264.7 macrophages and cyclophosphamide-Induced immunosuppression mouse models. Marine Drugs 18: 340. 10.3390/md18070340

Lu, S., Gao, Y., Huang, X. and Wang, X., 2014. Cantharidin exerts anti-hepatocellular carcinoma by miR-214 modulating macrophage polarization. International Journal of Biological Sciences 10: 415–425. 10.7150/ijbs.8002

Mo, L., Zhang, X., Shi, X., Wei, L., Zheng, D., Li, H., et al. 2018. Norcantharidin enhances antitumor immunity of GM-CSF prostate cancer cells vaccine by inducing apoptosis of regulatory T cells. Cancer Science 109: 2109–2118. 10.1111/cas.13639

Nicholson, L.B., 2016. The immune system. Essays in Biochemistry 60: 275–301. 10.1042/EBC20160017

Noh, E.-M., Kim, J.-M., Lee, H.Y., Song, H.-K., Joung, S.O., Yang, H.J., et al. 2019. Immuno-enhancement effects of Platycodon grandiflorum extracts in splenocytes and a cyclophosphamide-induced immunosuppressed rat model. BMC Complementary and Alternative Medicine 19: 322–322. 10.1186/s12906-019-2724-0

Qi, Q., Dong, Z., Sun, Y., Li, S. and Zhao, Z., 2018. Protective effect of bergenin against cyclophosphamide-induced immunosuppression by immunomodulatory effect and antioxidation in Balb/c mice. Molecules 23: 2668. 10.3390/molecules23102668

Qiu, S., Feng, Y., LeSage, G., Zhang, Y., Stuart, C., He, L., et al. 2015. Chronic morphine-induced microRNA-124 promotes microglial immunosuppression by modulating P65 and TRAF6. Journal of Immunology (Baltimore, MD, 1950) 194: 1021–1030. 10.4049/jimmunol.1400106

Walsh, M.C., Lee, J. and Choi, Y., 2015. Tumor necrosis factor receptor-associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system. Immunological Reviews 266: 72–92. 10.1111/imr.12302

Whiteside, T., 2006. Immune suppression in cancer: effects on immune cells, mechanisms and future therapeutic intervention. Seminars in Cancer Biology 16: 3–15. 10.1016/j.semcancer.2005.07.008

Yan, H., Lu, J., Wang, J., Chen, L., Wang, Y., Li, L., Miao, L., et al. 2021. Prevention of cyclophosphamide-induced immunosuppression in mice with traditional Chinese medicine Xuanfei Baidu decoction. Frontiers in Pharmacology 12: 730567. 10.3389/fphar.2021.730567

Yun, L., Wu, T., Li, Q. and Zhang, M., 2018. Dietary supplementation with purified wheat germ glycoprotein improve immunostimulatory activity in cyclophosphamide-induced Balb/c mice. International Journal of Biological Macromolecules 118: 1267–1275. 10.1016/j.ijbiomac.2018.06.199

Zheng, J., Du, W., Song, L.-J., Zhang, R., Sun, L.-G., Chen, F.-G., et al. 2014. Norcantharidin induces growth inhibition and apoptosis of glioma cells by blocking the Raf/MEK/ERK pathway. World Journal of Surgical Oncology 12: 207. 10.1186/1477-7819-12-207

Zheng, W., Sun, L., Yang, L. and Xu, T., 2021. The circular RNA circBCL2L1 regulates innate immune responses via microRNA-mediated downregulation of TRAF6 in teleost fish. Journal of Biological Chemistry 297: 101199. 10.1016/j.jbc.2021.101199