Develop and submit an initial research proposal

Once you have initiated discussions with an academic contact, you should prepare an initial
research proposal.
Your initial proposal should include the following information:

Title: Working title for the project.

Academic staff contact: Name(s) of the academic staff with whom you have discussed your proposal
and, if relevant a potential supervisor.

Objectives: What aims does the work have?

Synopsis: Briefly describe the key aspects of what you will be investigating.

Background: Describe what research has already been done in relation to your topic.

Significance: Why is the topic important?

Methodology: Approach or methodology to be used in the research.

Resources: Provide details of the resources required for you to carry out your research project.

Work plan: An initial plan for completion with annual milestones.

Doctor of Philosophy (Ph.D.) project entitled: The HAMLET Molecular Species with
Anti-Cancer Properties

Background: The compounds of interest are the HAMLET family of broad-spectrum

anti-cancer and anti-bacterial agents, composed of a protein carrier and the active
ingredient oleic acid HAMLET stands for human -lactalbumin made lethal to
tumors. HAMLET compounds exhibit anti-cancer activity against over 40 cancer cell
lines (Svanborg et al , 2003), including cells resistant to cell death (Hallgren et al,
2006). In humans and rodents, HAMLET and BAMLET (the bovine series of species of
molecule) showed non-toxicity and efficacy in reducing bladder cancer tumour sizes
(Mossberg 2007; Mossberg 2010; Xiao 2013), and showed non-toxicity and efficacy
in the nasopharynx of mice in antibacterial studies (Marks et al; 2012, Marks et al,
2013). BAMLET compounds are deactivated by blood and are expected to be
deactivated when injected intravenously (Likov et al, 2011; Marks et al, 2013). In
the absence of the deactivating blood serum, BAMLET is cytotoxic to blood cells
(Hkansson et al,1995; Brinkmann et al, 2011).

The laboratory of Dr W. Bret Church has shown that BAMLET represents a new type
of protein-lipid structure (Rath et al, 2014) which binds potentially bioactive oleic
acid in a partially unfolded protein conformation. Although many targets of BAMLET
have been identified inside the cell (Khler et al, 1999; Dringer et al, 2003;

Pettersson-Kastberg et al, 2009; Rammer et al, 2010; Trulsson et al, 2011), the
current thinking is that BAMLET initiates cell death by compromising the cell
membrane (Zherelova et al, 2009; Mossberg et al, 2010b; Vukojevi et al, 2010;
Storm et al, 2013) and the cell then dies by whatever mechanism is available to it,
whether that be apoptosis, autophagy or necrosis (Hkansson et al, 1995; Rammer
et al, 2010; Brinkmann et al, 2011). Cancer cells are more sensitive to BAMLET than
their healthy differentiated counterparts (Hkansson et al, 1995; Brinkmann et al,
2011).

Summary: BAMLET is thought to represent a new class of anti-cancer compound

that kills cancer cells by a different mechanism than those used by current
chemotherapies. BAMLET is thus expected to be effective against chemotherapyresistant cancer cells. Resistance to current chemotherapies is responsible for the
high death toll of cancer worldwide. The goal of this research is to realise BAMLET's
anti-cancer potential by overcoming BAMLET's problem of being
deactivated by blood.

Research plan: The activities of this research project will include:

1. Developing and characterising potential delivery systems for BAMLET to

overcome the problem of deactivation by blood.

2. In vitro testing of the efficacy and safety of BAMLET incorporated into the delivery
systems by treating and analysing cancerous and non-cancerous cell lines by cell
death assay, flow cytometry and microscopy, with a particular focus on chemoresistant cell lines.

3. In vivo testing of the efficacy and safety of BAMLET incorporated into the delivery
systems by inducing orthotopic tumours in immunocompetant animals and then
treating with BAMLET. Analysis of results includes histopathology and
immunohistochemistry.

References

Brinkmann et al. (2011) The toxicity of bovine -lactalbumin made lethal to tumor
cells is highly dependent on oleic acid and induces killing in cancer cell lines and
noncancer-derived primary cells. FEBS J 278(11):1955-67.
Dringer et al. (2003) HAMLET interacts with histones and chromatin in tumor cell
nuclei. J Biol Chem 278(43):42131-5.
Hkansson et al. (1995) Apoptosis induced by a human milk protein. PNAS USA
92(17):8064-8.
Hkansson et al. (1999) Multimeric alpha-lactalbumin from human milk induces
apoptosis through a direct effect on cell nuclei. Exp Cell Res 246(2):451-60.
Hallgren et al. (2006) HAMLET triggers apoptosis but tumor cell death is
independent of caspases, Bcl-2 and p53. Apoptosis 11(2):221-33.
Khler et al. (1999) Protease activation in apoptosis induced by MAL. Exp Cell Res
249(2):260-8.
Likov, Kamila (2011) Biologically active complexes of oleic acid and bovine whey
proteins. (PhD Thesis) University College Cork, Ireland.
Marks et al. (2012) The human milk protein-lipid complex HAMLET sensitizes
bacterial pathogens to traditional antimicrobial agents. PLoS One 7(8):e43514.
Marks et al. (2013) Sensitization of Staphylococcus aureus to methicillin and other
antibiotics in vitro and in vivo in the presence of HAMLET. PLoS One 8(5):e63158.
Mossberg et al. (2007) Bladder cancers respond to intravesical instillation of
HAMLET (human alpha-lactalbumin made lethal to tumor cells). Int J Cancer
121(6):1352-9.
Mossberg et al. (2010) HAMLET treatment delays bladder cancer development. J
Urol 183(4):1590-7.
Mossberg et al. (2010b) HAMLET interacts with lipid membranes and perturbs their
structure and integrity. PLoS One 5(2):e9384.
Pettersson-Kastberg et al. (2009) Alpha-lactalbumin, engineered to be nonnative
and inactive, kills tumor cells when in complex with oleic acid: a new biological
function resulting from partial unfolding. J Mol Biol 394(5):994-1010.

Rammer et al. (2010) BAMLET activates a lysosomal cell death program in cancer
cells. Mol Cancer Ther 9(1):24-32.
Rath EM, Duff AP, Hakansson AP, Knott RB, Church WB (2014) Small-angle X-ray
scattering of BAMLET at pH 12: a complex of -lactalbumin and oleic acid.
Proteins 82(7):1400-8.
Storm et al. (2013) A unifying mechanism for cancer cell death through ion channel
activation by HAMLET. PLoS One 8(3):e58578.
Svanborg et al. (2003) HAMLET kills tumor cells by an apoptosis-like mechanism-cellular, molecular, and therapeutic aspects. Adv Cancer Res 88:1-29.
Trulsson et al. (2011) HAMLET binding to -actinin facilitates tumor cell detachment.
PLoS One 6(3):e17179.
Vukojevi et al. (2010) Lipoprotein complex of equine lysozyme with oleic acid
(ELOA) interactions with the plasma membrane of live cells. Langmuir
26(18):14782-7.
Xiao et al. (2013) A molecular complex of bovine milk protein and oleic acid
selectively kills cancer cells in vitro and inhibits tumour growth in an orthotopic
rat bladder tumour model. BJU Int 112(2):E201-10.
Zherelova et al. (2009) Interaction of antitumor alpha-lactalbumin-oleic acid
complexes with artificial and natural membranes. J Bioenerg Biomembr
41(3):229-37.

Synopsis
The HAMLET family of compounds (human alpha-lactalbumin made lethal to
tumours) is composed of the alpha-lactalbumin protein "carrier" and oleic acid.
They show broad spectrum anti-cancer activity in vitro. We have performed
structural studies on the BAMLET compounds (the bovine equivalents with
similar properties to the human versions) as well as a beta-lactoglobulin
analogue. Our work indicates that the majority of the protein is located on the
periphery of the BAMLET and the protein component does not completely fill the
centre. The current evidence is that the oleic acid is the component with the anticancer activity, and we are carrying out further experiments to provide further
specific information on the oleic acid, in addition to the protein component. These
scattering results support a structural model beginning to explain the delivery of
the oleic acid of the broad spectrum anti-cancer activity of the BAMLET family of
compounds - the protein carrier is able solubilise a large number of oleic acid
molecules by carrying them internally in an oversized protein. This model is

consistent with the absence of specific cancer cell binding sites in the action of
BAMLET on cells in vitro, suggesting that if a suitable approach to delivering
HAMLET and HAMLET-like molecules in the in vivo situation can be developed,
cancers against which the therapy is directed would not be able to evolve
resistance to BAMLET treatment by point mutations for instance. We are
continuing our studies pursuing the specifics of these anti-cancer species, as well
as studies to find delivery systems which preserve the salient features of
HAMLET-like molecules with the potential to be effective anti-cancer treatments.
This work is with our collaborators, Drs Anthony Duff and Robert Knott at
ANSTO, as well as Anders Hakansson, University of Buffalo, in the U.S.A.

Additional Information
Techniques with the potential to be used are enzyme and cell-based assays,
molecular biology, bioinformatics, microscopies, X-ray crystallography, small
angle X-ray scattering, small angle neutron scattering, molecular modeling;
aspects may be suitable as Honours projects.

GROUP IN BIOMOLECULAR STRUCTURE AND INFORMATICS