Proliferative And Functional Effects Of Cucurbitacin B On Lymphocyte And Jurkat T Cells

Aim:1. To investigate the effects of cucurbitacin B (CuB) on the activation, proliferation,apoptosis and synthesis of interferon-γ (IFN-γ) of mouse lymphocytes, in order to elucidatethe mechanism of the immunomodulatory effects of CuB.2. To investigate the effects of cucurbitacin B (CuB) on the proliferation, autophagyand apoptosis on human T-cell leukemia Jurkat T cells, in order to explore the anti-tumoreffect and action mechanism of CuB.Methods:1. Lymphocytes were stimulated with concanavalin A (Con A,5μg/mL) or phorbolester (PDB,10–7mol/L) plus ionomycin (Ion,0.5μg/mL) in the presence or absence ofdifferent concentrations CuB, and then the proliferation, expression of CD69and IFN-γ,sub-G0/G1peaks and mitochondrial membrane potential (ΔΨm) and activation of caspase-3in activated lymphocytes were determined.2. Leukemia Jurkat T cells were treated with different concentrations of CuB. Cellproliferation was measured by MTS assay. Cell cycle distribution, mitochondrial membranepotential and annexin V staining were analyzed using flow cytometry. The expression ofLC3-II was determined by immunofluorescent microscopy. Western blotting was used todetermine the levels of apoptosis-and autophagy-related proteins (LC3-II, Bcl-2, Caspase-3and PARP) and cytoskeleton-related proteins (G-actin, F-actin and cofilin).Results:1. Analysis by counting the total numbers of cells revealed that CuB inhibited theproliferation of the activated lymphocytes in a dose-dependent manner. DNA contentanalysis showed that cell numbers in the apoptotic peak (sub-G0/G1) was elevated with theincrease of CuB concentration. Moreover, in the CuB-treated groups, syntheses of CD69(the marker of early activation) and IFN-γ in T lymphocytes were inhibited in a dose-dependent manner. The JC-1staining revealed that CuB (10μmol/L) couldsignificantly reduce the ΔΨm of lymphocytes (P <0.01). Western blotting analysis showedthat CuB (10μmol/L) treatment for24h resulted in the activation of caspase-3.2. After treated with different concentrations of CuB for48h, the proliferation ofJurkat T cells was measured by MTS assay, the results indicated that CuB inhibited theproliferation of Jurkat T cells in a dose-dependent manner. The IC50value was1.58±0.17μmol/L. Cell cycle analysis demonstrated that CuB treatment significantly inducedG2/M-phase arrest and formation of tetraploid cells in Jurkat T cells. Flow cytometryanalysis revealed that CuB induced decrease of mitochondrial membrane potential (ΔΨm)and apoptosis in Jurkat T cells. Results of immunofluorescent microscopy and westernblotting showed that autophagy-related protein LC3-II was markedly upregulated andcytoskeleton-related proteins Cofilin was activated in CuB-treated cells. The proportion ofapoptotic cells was elevated by suppressing the CuB-induced autophagy.Conclusion:1. CuB inhibited the proliferation or cytokine synthesis of Con-A-orPDB+Ion-activated lymphocytes in a dose-dependent manner. CuB (10μmol/L) inhibitedthe expression of CD69and induced apoptosis in lymphocytes. These results indicated thatCuB could inhibit the proliferation, activation and expression of IFN-γ and inducedapoptotic cell death in lymphocytes significantly. These may be the mechanism ofanti-inflammatory activities and immunomodulatory effects of CuB.2. CuB could inhibit the proliferation of Jurkat T cells in a dose-dependent manner andinduce G2/M-phase arrest. Moreover, CuB induced decrease of mitochondrial membranepotential (ΔΨm) and apoptosis in Jurkat T cells. CuB activated the Cofilin and induceddisruption of cytoskeleton in Jurkat T cells. These experiments demonstrated that CuBexhibited anti-tumor activity in Jurkat T cells through inhibition of cell proliferation andinduction of apoptosis which were at least partly due to the disruption of actin dynamics.