lactoferrin and Leukemia--T-Cell

Lactoferrin (Lf) has been shown to control the proliferation of a variety of mammalian cells. Recently, we reported that human Lf induces apoptosis via a c-Jun N-terminal kinases (JNK)-associated Bcl-2 pathway that stimulates programmed cell death. In order to gain insight into the mechanism underlying Lf-triggered apoptotic features, we attempted to determine the mechanisms whereby the Lf-induced Bcl-2 family proteins exert their pro- or anti-apoptotic effects in Jurkat leukemia T lymphocytes. Treatment of the cells with high concentrations of Lf resulted in a significant reduction in in vitro growth and cell viability. As the levels of Lf increased, greater quantities of CDK6 and hyper-phosphorylated retinoblastoma protein were produced, resulting in the induction of E2F1-dependent apoptosis. Simultaneously, PARP and caspases were efficiently cleaved during Lf-induced apoptosis. The E2F1-induced apoptotic process occurred preferentially in p53-deficient Jurkat leukemia cells. Therefore, we attempted to determine whether E2F1-regulated Bcl-2 family proteins involved in the apoptotic process were relevant to Lf-induced apoptosis. We found that Lf increased the interaction of Bcl-2 with the pro-apoptotic protein Bad, whereas the total protein levels did not change significantly. Our results, collectively, suggest that Lf exploits the control mechanism of E2F1-regulated target genes or Bcl-2 family gene networks involved in the apoptotic process in Jurkat human leukemia T lymphocytes.

Topics: Apoptosis; E2F1 Transcription Factor; Humans; Jurkat Cells; Lactoferrin; Leukemia, T-Cell; Proto-Oncogene Proteins c-bcl-2; Structure-Activity Relationship; T-Lymphocytes; Tumor Cells, Cultured

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide with potent cytotoxic activity against cancer cells. The antimicrobial activity of LfcinB resides in its RRWQWR amino acid sequence (referred to here as LfcinB6); however, the anticancer activity of LfcinB6 is not known. Here, we show that free LfcinB6 did not kill T-leukemia or breast cancer cells but LfcinB6 was strongly cytotoxic when delivered to the cytosolic compartment by fusogenic liposomes. LfcinB6 bound weakly to isolated mitochondria but, unlike LfcinB, did not permeabilize mitochondria or cause cytochrome c to be released. Cathepsin B and caspase activity were important for cytotoxicity caused by intracellular LfcinB6 whereas reactive oxygen species were not involved. The mechanism of LfcinB6-induced cytotoxicity is therefore different from that of LfcinB. We suggest that LfcinB6, in combination with a fusogenic liposome delivery system that selectively targets malignant cells, has potential as a novel anticancer agent.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Antineoplastic Agents; Breast Neoplasms; Cattle; Drug Delivery Systems; Humans; Jurkat Cells; Lactoferrin; Leukemia, T-Cell; Liposomes

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

Topics: Amidohydrolases; Animals; Antimicrobial Cationic Peptides; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cattle; Cell Line, Tumor; Ceramidases; Ceramides; Drug Evaluation, Preclinical; Drug Synergism; Enzyme Inhibitors; Glucosyltransferases; Humans; Jurkat Cells; Lactoferrin; Leukemia, T-Cell