cucurbitacin-d and Leukemia-Lymphoma--Adult-T-Cell

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

Topics: Animals; CD4 Antigens; CD5 Antigens; Cell Line, Tumor; Cell Survival; Extracellular Signal-Regulated MAP Kinases; Humans; Interferon-gamma; Interleukins; JNK Mitogen-Activated Protein Kinases; Leukemia-Lymphoma, Adult T-Cell; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Triterpenes

Human T-cell leukemia is an aggressive malignancy of T lymphocytes. T-cell leukemia has a very poor prognosis, even with intensive chemotherapy, indicating the need for development of new drugs to treat the disease. Triterpenoid cucurbitacins have been shown to have antitumor activity, but the mechanism of this activity is not fully understood.. The effects of cucurbitacin D on the proliferation and apoptotic induction of T-cell leukemia cells using the Cell viability assay and Annexin V staining were evaluated. To investigate the mechanisms of apoptosis, antiapoptotic protein, NF-κB, and the proteasome activity of leukemia cells treated with cucurbitacin D were evaluated by Western blotting both in vitro and in vivo.. In this study, cucurbitacin D was found to inhibit proliferation and to induce apoptosis of T-cell leukemia cells. Constitutively activated NF-κB was inhibited by cucurbitacin D in the nucleus, which resulted in accumulation of NF-κB in the cytoplasm, leading to down-regulation of the expression of antiapoptotic proteins Bcl-xL and Bcl-2. Furthermore, cucurbitacin D induced the accumulation of inhibitor of NF-κB (IκB)α by inhibition of proteasome activity. Low doses of cucurbitacin D synergistically potentiated the antiproliferative effects of the histone deacetylase inhibitor VPA. Finally, the proapoptotic and proteasome inhibitory activities of cucurbitacin D also were demonstrated using SCID mice in an in vivo study.. Cucurbitacin D induced apoptosis through suppression of proteasome activity both in vitro and in vivo, making cucurbitacin D a promising candidate for clinical applications in the treatment of T-cell leukemia.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Female; Histone Deacetylase Inhibitors; Humans; Leukemia-Lymphoma, Adult T-Cell; Mice; Mice, SCID; NF-kappa B; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Triterpenes; Ubiquitination