Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo

Environ Toxicol. 2017 Feb;32(2):550-568. doi: 10.1002/tox.22259. Epub 2016 Mar 18.

Abstract

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca2+ release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550-568, 2017.

Keywords: apoptosis; autophagy; leukemia mice; murine leukemia WEHI-3; triptolide.

MeSH terms

  • Animals
  • Antineoplastic Agents, Alkylating / toxicity*
  • Apoptosis / drug effects*
  • Autophagy / drug effects*
  • B-Lymphocytes / cytology
  • B-Lymphocytes / drug effects
  • B-Lymphocytes / metabolism
  • Caspase 3 / metabolism
  • Caspase 8 / metabolism
  • Caspase 9 / metabolism
  • Cell Cycle Checkpoints / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cytochromes c / metabolism
  • DNA Damage / drug effects
  • Diterpenes / toxicity*
  • Endoplasmic Reticulum Chaperone BiP
  • Epoxy Compounds / toxicity
  • Leukemia / metabolism
  • Leukemia / pathology
  • Lymphocyte Activation / drug effects
  • Macrophages / cytology
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Male
  • Medicine, Chinese Traditional
  • Membrane Potential, Mitochondrial / drug effects
  • Mice
  • Mice, Inbred BALB C
  • Microscopy, Confocal
  • Phenanthrenes / toxicity*
  • Reactive Oxygen Species / metabolism
  • Spleen / drug effects
  • Spleen / metabolism
  • Spleen / pathology
  • T-Lymphocytes / cytology
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism
  • Transplantation, Homologous

Substances

  • Antineoplastic Agents, Alkylating
  • Diterpenes
  • Endoplasmic Reticulum Chaperone BiP
  • Epoxy Compounds
  • Hspa5 protein, mouse
  • Phenanthrenes
  • Reactive Oxygen Species
  • triptolide
  • Cytochromes c
  • Caspase 3
  • Caspase 8
  • Caspase 9