HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a complex between

alpha-lactalbumin Lactalbumin, alpha-, also known as LALBA, is a protein that in humans is encoded by the ''LALBA'' gene. Overview α-Lactalbumin is a protein that regulates the production of lactose in the milk of almost all mammalian species. In primates, alp ...

and

oleic acid Oleic acid is a fatty acid that occurs naturally in various animal and vegetable fats and oils. It is an odorless, colorless oil, although commercial samples may be yellowish. In chemical terms, oleic acid is classified as a monounsaturated omeg ...

that has been shown in cell culture experiments to induce

cell death Cell death is the event of a biological cell ceasing to carry out its functions. This may be the result of the natural process of old cells dying and being replaced by new ones, as in programmed cell death, or may result from factors such as d ...

in tumor cells, but not in healthy cells. HAMLET is a possible

chemotherapeutic agent This is a list of chemotherapeutic agents, also known as cytotoxic agents or cytostatic drugs, that are known to be of use in chemotherapy for cancer. This list is organized by type of agent, although the subsections are not necessarily definitive ...

with the ability to kill cancer cells.

Alpha-lactalbumin Lactalbumin, alpha-, also known as LALBA, is a protein that in humans is encoded by the ''LALBA'' gene. Overview α-Lactalbumin is a protein that regulates the production of lactose in the milk of almost all mammalian species. In primates, alp ...

is the primary protein component of

human milk Breast milk (sometimes spelled as breastmilk) or mother's milk is milk produced by mammary glands located in the breast of a human female. Breast milk is the primary source of nutrition for newborns, containing fat, protein, carbohydrates (lacto ...

. In a 1995 study, it was discovered by Swedish scientist Anders Håkansson (Anders Hakansson) that multimeric alpha-lactalbumin (MAL), a compound isolated from a fraction of human milk called

casein Casein ( , from Latin ''caseus'' "cheese") is a family of related phosphoproteins ( αS1, aS2, β, κ) that are commonly found in mammalian milk, comprising about 80% of the proteins in cow's milk and between 20% and 60% of the proteins in hum ...

, induced what appeared to be apoptosis in human lung carcinoma cells, pneumococcus bacteria, and other pathogens, while leaving healthy, differentiated cells unaffected. It has been the perfect cure in this case. The active component responsible for the tumoricidal activity was found in 2000 and found to be a complex of alpha-lactalbumin and

. Endogenous human alpha-lactalbumin is complexed with a calcium ion and serves as a cofactor in lactose synthesis, but has no tumoricidal properties. The alpha-lactalbumin must be partially unfolded to allow for release of the calcium ion and replacement with an oleic acid molecule. The partially folded conformation is essential to the cytotoxicity of HAMLET, as mutagenesis studies have shown that completely unfolded alpha-lacalbumin does not retain the functional properties of HAMLET. The oleic acid is necessary for stabilizing this molecule in this partially unfolded state. Over the past several years, additional work has further characterized the structure and function of HAMLET and its clinical applications are currently under investigation. However, in order to develop effective therapies, more must be known about the mechanism of action of HAMLET.

Mechanism of action

HAMLET carries out independent attacks on many distinct cell organelles, including mitochondria, proteasomes, and histones, and interferes with cell processes such as macroautophagy. It has been shown that HAMLET binds to the cell surface and rapidly invades cells, with tumor cells taking up far more protein than healthy, differentiated cells. The mechanism of its entry is poorly understood, but recent studies indicate that the oleic acid in the HAMLET complex interacts with

phosphatidylserine Phosphatidylserine (abbreviated Ptd-L-Ser or PS) is a phospholipid and is a component of the cell membrane. It plays a key role in cell cycle signaling, specifically in relation to apoptosis. It is a key pathway for viruses to enter cells via ap ...

and o-glycosylated

mucin Mucins () are a family of high molecular weight, heavily glycosylated proteins (glycoconjugates) produced by epithelial tissues in most animals. Mucins' key characteristic is their ability to form gels; therefore they are a key component in most ...

on the plasma membrane, both of which are expressed in greater amounts on the plasma membrane of tumor cells, possibly providing for HAMLET’s specificity. One of the most prominent targets of HAMLET once inside the cell is the mitochondrion. Electron microscopy has revealed physical damage to the mitochondrial membranes and assays have found cytochrome c release and activation of the caspase cascade, the most notable ones being caspases 2, 3, and 9. Cell death is not prevented by caspase inhibitors, or by

BCL-2 Bcl-2 (B-cell lymphoma 2), encoded in humans by the ''BCL2'' gene, is the founding member of the Bcl-2 family of regulator proteins that regulate cell death (apoptosis), by either inhibiting (anti-apoptotic) or inducing (pro-apoptotic) apoptosi ...

p53 p53, also known as Tumor protein P53, cellular tumor antigen p53 (UniProt name), or transformation-related protein 53 (TRP53) is a regulatory protein that is often mutated in human cancers. The p53 proteins (originally thought to be, and often s ...

mutagenesis, indicating that the traditional apoptotic caspase cascade is not the ultimate cause of cell death. Another target of HAMLET is the proteasome. 26S proteasomes are activated in response to large quantities of unfolded HAMLET protein in the cytoplasm, but degradation of HAMLET by the proteasome is unusually slow. Furthermore, in vitro studies have shown that HAMLET is capable of binding the catalytic 20S subunit of the proteasome and disabling its enzymatic activity, an effect that has never before been demonstrated for any protein. However, proteasome inhibition alone does not seem to be responsible for HAMLET-induced cell death, as proteasome inhibitors have been shown to reduce the cytotoxicity of HAMLET. HAMLET also targets the nucleus, where it interacts with

histones In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn ar ...

to interfere with transcriptional processes. Studies have shown that HAMLET is mostly localized to the nucleus within one hour of invading a tumor cell. Hamlet has been shown to bind with high affinity to individual histone proteins, to be specific H2a, H2b, H3, and H4, as well as entire nucleosomal units. This interaction irreversibly blocks

transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...

and leads to activation of

. This process has been demonstrated to be similar to histone hyperacetylation and it was found that histone de-acetylase inhibitors potentiated the effects of HAMLET. HAMLET cells showed the physiological characteristics of

macroautophagy Autophagy (or autophagocytosis; from the Ancient Greek , , meaning "self-devouring" and , , meaning "hollow") is the natural, conserved degradation of the cell that removes unnecessary or dysfunctional components through a lysosome-dependent re ...

, a process in which cellular components are sequestered in double membrane-bound vesicles that fuse with lysosomes for degradation. Cells also showed decreased levels of

mTOR The mammalian target of rapamycin (mTOR), also referred to as the mechanistic target of rapamycin, and sometimes called FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), is a kinase that in humans is encoded by the ''MTOR'' gene. ...

, a known inhibitor of macroautophagy. HAMLET cells and cells under conditions of amino acid starvation (a known initiator of macroautophagy) showed similar expression patterns of autophagocytotic proteins and responded equally well to addition of macroautophagy inhibitors.

Research

Antibiotic adjuvant

Whil
HAMLET
on its own is not active against most bacteria, when present together with antibiotics, HAMLET may help. Specifically, HAMLET can make

MRSA Methicillin-resistant ''Staphylococcus aureus'' (MRSA) is a group of Gram-positive bacteria that are genetically distinct from other strains of ''Staphylococcus aureus''. MRSA is responsible for several difficult-to-treat infections in humans. ...

bacteria sensitive against methicillin, vancomycin, gentamicin and erythromycin

Tumors

Research is being conducted to determine if this could be a possible treatment for cancer. Animal models of

glioblastoma Glioblastoma, previously known as glioblastoma multiforme (GBM), is one of the most aggressive types of cancer that begin within the brain. Initially, signs and symptoms of glioblastoma are nonspecific. They may include headaches, personality ...

have been studied with tentative success. The first human trial of the therapy was on benign skin growths known as

warts Warts are typically small, rough, hard growths that are similar in color to the rest of the skin. They typically do not result in other symptoms, except when on the bottom of the feet, where they may be painful. While they usually occur on the ...

and showed positive outcomes without any side effects. It is being studied in carcinomas of the lung, throat, kidney, colon, bladder, prostate, and ovaries, as well as melanomas, glioblastomas, and leukemias. A study of bladder cancer in a mouse found it caused shedding of TUNEL-positive cancer cells into the urine, with no adverse side-effects on healthy cells.

References

External links