Mechanisms of BAMLET (bovine α-lactalbumin made lethal to tumor cells) in cancer cell death

Abstract

Human alpha-lactalbumin made lethal to tumor cells (HAMLET) is a complex formed by human alpha-lactalbumin and oleic acid that displays tumoricidal activity but does not kill healthy, differentiated cells. As the sequence of alpha-lactalbumin is conserved among mammals, lactalbumins from other species are expected to form oleic acid complexes with similar activity. This study investigated the tumoricidal activity of Bovine alpha-lactalbumin made lethal to tumor cells (BAMLET). Bovine alpha-lactalbumin was shown to form a complex with oleic acid, which exhibited tumoricidal properties similar to HAMLET against several cancer cell lines. The cellular uptake of BAMLET was quantified, using confocal imaging and Western blots. A rapid internalization pattern was identified within 30 min. Effects on cell viability were also rapid as shown by ATP lite and Presto Blue assays. Long-term cell death was confirmed using the colony formation assay. The cancer cells response to BAMLET was affected by different media compositions, providing tools to further study the mechanisms of cell death and how to maintain the activity of BAMLET under different conditions.

Popular Abstract

Mechanism of BAMLET in cancer cell death

Human alpha-lactalbumin made lethal to tumor cells (HAMLET) is the first member of a family of protein-lipid complexes with broad tumoricidal effects against cancer cells of different origins. The complex of HAMLET consists of partially unfolded alpha lactalbumin from human breast milk and oleic acid. Bovine alpha lactalbumin has 85% homology with human alpha lactalbumin and has been shown to form oleic acid complexes with tumoricidal activity. This study examined the properties of BAMLET (Bovine alpha-lactalbumin made lethal to tumor cells).

This study first compared the tumoricidal effects of BAMLET to HAMLET and the peptide-oleate complex alpha1-oleate. BAMLET killed cancer cells in a dose dependent manner with an efficacy similar to HAMLET, as shown by the ATPlite, PrestoBlue and Clonogenic assay. About 90% cell death was recorded at the highest concentration, after 1hour treatment. In contrast, there was no significant tumoricidal effect by bovine alpha-lactalbumin or oleic acid alone.

To further examine the tumoricidal effect of BAMLET, on different cancer cell lines, lung carcinoma cells (A549) and two colorectal adenocarcinoma cell lines (DLD1 and HT29) were examined. The cells were treated with different concentrations of BAMLET and all cell lines were sensitive as shown by a reduction in ATP and PrestoBlue. The colony forming ability of cells decreased with increasing concentrations of BAMLET.

Furthermore, Western blot and live cell imaging were used to analyze the uptake of BAMLET by the different cell lines. A time- and dose-dependent uptake of BAMLET was detected in A549, DLD1 and HT29 cells. By live cell imaging rapid aggregation of BAMLET around the cell membrane was detected, followed by cytoplasmic uptake with a diffuse cytoplasmic localization pattern and finally BAMLET was shown to translocate into the nuclei of tumor cells.

The results confirm that BAMLET resembles HAMLET in terms of its tumoricidal activity. BAMLET will therefore be further explored as a potential prophylactic or therapeutic agent.

Master’s Degree Project in Molecular Biology 30 credits 2019
Department of Biology, Lund University

Advisor: Hien Tran and Catharina Svanborg.
Advisors Unit/Department: BMC/ Division of Microbiology, Immunology and Glycobiology - MIG

• author: Esmaeili, Parisa
• supervisor: Thi Hien Tran; Catharina Svanborg
• organization: Degree Projects in Molecular Biology
• course: MOBM02 20191
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9006842
• date added to LUP: 2020-03-18 14:46:33
• date last changed: 2020-03-18 14:46:33