leupeptins and Mesothelioma

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Apoptosis; Arabinonucleosides; bcl-X Protein; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Clofarabine; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Humans; Leupeptins; Lung Neoplasms; M Phase Cell Cycle Checkpoints; Mesothelioma; Mesothelioma, Malignant; Myeloid Cell Leukemia Sequence 1 Protein; Resveratrol; RNA Interference; RNA, Messenger; RNA, Small Interfering; Stilbenes

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of tumor necrosis factor family and it is important for ligand induced apoptosis in tumor cells. TRAIL has been shown to be synergistic with a variety of chemotherapies and targeted agents. In the study, a combination of TRAIL and a histone deacetylase inhibitor LBH589 was studied in mesothelioma cell lines.. Five mesothelioma cell lines and two normal cell lines were tested for cell growth inhibition and apoptosis using high-throughput assays in the presence of LBH589, TRAIL and a combination of the two. Caspase induction was studied and levels of X-linked inhibitor of apoptosis (XIAP) were tested using Western blotting. A combination of a direct inhibitor of XIAP was also tested in combination with TRAIL.. In mesothelioma cell lines, a combination of LBH589 and TRAIL markedly increased cell growth inhibition and apoptosis when compared with the effect on normal cell lines. LBH589 and TRAIL appeared to induce higher levels of caspase 3 and 7 and this appeared to be closely related to ability of LBH589 to degrade XIAP. In addition, a direct inhibitor of XIAP was also sensitized cells to TRAIL apoptosis, providing an indirect confirmation for XIAP degradation as a possible mechanism of synergy.. In mesothelioma cell lines, LBH589 increases the sensitivity to TRAIL. In addition, at least partly, the mechanism of this induction of TRAIL sensitivity is due to LBH589 related degradation of XIAP. These results provide initial evidence for testing this combination in clinical trials.

Topics: Acetylation; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Indoles; Leupeptins; Mesothelioma; Panobinostat; Poly(ADP-ribose) Polymerases; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; X-Linked Inhibitor of Apoptosis Protein