Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human ␣-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from ␣-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n ؍ 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and ␣-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all ␣-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm 3 ). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.
SummaryThis study describes an a-lactalbumin folding variant from human milk with bactericidal activity against antibiotic-resistant and -susceptible strains of Streptococcus pneumoniae. The active complex precipitated with the casein fraction at pH 4.6 and was puri®ed from casein by a combination of anion exchange and gel chromatography. Unlike other casein components, the active complex was retained on the ion-exchange matrix and eluted only with high salt. The eluted fraction showed N-terminal and mass spectrometric identity with human milk a-lactalbumin, but native alactalbumin had no bactericidal effect. Spectroscopic analysis demonstrated that the active form of the molecule was in a different folding state, with secondary structure identical to a-lactalbumin from human milk whey, but¯uctuating tertiary structure. Native a-lactalbumin could be converted to the active bactericidal form by ion-exchange chromatography in the presence of a cofactor from human milk casein, characterized as a C18:1 fatty acid. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram-negative and other Gram-positive bacteria were resistant. The folding variant of a-lactalbumin is a new example of naturally occurring molecules with antimicrobial activity.
As the result of a typesetting error, Conversion of a-lactalbumin to the active form requires a cofactor.A. Ion-exchange chromatography using a caseinconditioned column. Native or apo-a-lactalbumin was subjected to ion-exchange chromatography on a column previously exposed to human casein. More than 95% of the apo protein (red line) was retained on the column and eluted as a sharp peak at 1 M NaCl. In contrast, most of the native a-lactalbumin eluted in the void volume (blue line) with only a small fraction eluting at 1 M NaCl.B. Near-UV CD spectrum of material eluted from the casein-conditioned column. Apo (dashed black line) and native (solid black line) a-lactalbumin controls are as in Fig. 1. The apo-a-lactalbumin eluate from the casein-conditioned column (red line) had a spectrum similar to that of the apo control. The native a-lactalbumin eluate from the caseinconditioned column (blue line) had a spectrum similar to that of the native control.C. ANS fluorescence spectrum of material eluted from the casein-conditioned column. Apo (dashed black line) and native (solid black line) a-lactalbumin controls are as in Fig. 1. The apo-alactalbumin eluate (red line) bound ANS, but the native a-lactalbumin eluate (blue line) did not.
Histone deacetylase inhibitors (HDIs) and HAMLET (human Alactalbumin made lethal to tumor cells) interact with histones, modify the structure of chromatin, and trigger tumor cell death. This study investigated how the combination of HDIs and HAMLET influences cell viability, histone acetylation, and DNA integrity. The pretreatment of tumor cells with HDIs was shown to enhance the lethal effect of HAMLET and the histone hyperacetylation response to HDIs increased even further after HAMLET treatment. HDIs and HAMLET were shown to target different histone domains as HAMLET bound tailless core histones, whereas HDIs modify the acetylation of the histone tail. DNA damage in response to HAMLET was increased by HDIs. The DNA repair response (p21WAFI expression) was induced by both agonists but abolished when the two agonists were combined. The results suggest that the synergy of HDIs and HAMLET is based on different but converging death pathways, both involving chromatin alterations. We speculate that HAMLET and HDIs might be combined to promote tumor cell death in vivo.
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