HAMLET (human alpha-lactalbumin made lethal to tumor cells)
Encyclopedia
HAMLET is a complex between alpha-lactalbumin
and oleic acid
that induces apoptosis
in tumor cells, but not in healthy cells.
HAMLET is a novel chemotherapeutic agent with potent tumoricidal properties. Alpha-lactalbumin
is the primary protein component of human milk. In a 1995 study, it was noted that multimeric alpha-lactalbumin (MAL), a compound isolated from a fraction of human milk called casein
, induced what appeared to be apoptosis
in human lung carcinoma cells, pneumococcus bacteria, and other pathogens, while leaving healthy, differentiated cells unaffected. The active component responsible for the tumoricidal activity was finally elucidated in 2000 and found to be a complex of alpha-lactalbumin and oleic acid
.
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.
and o-glycosylated mucin
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. It is interesting to note that cell death is not prevented by caspase inhibitors, or by BCL-2
or p53
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.
As part of its multi-faceted attack, HAMLET also targets the nucleus, where it interacts with histones 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
and leads to activation of p53
. 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, 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
, 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.
s are pre-malignant lesions on the skin or mucosal membranes and are often caused by Human Papilloma Virus infections. In a double-blind study, HAMLET was applied topically to skin papillomas of patients for 3 weeks, and the lesions were monitored over the course of the next two years. The study found papilloma volume reduction in 100% of treated patients and in only 15% of placebo patients. No adverse reactions were reported, and there were no differences observed between immunocompetent and immunocompromised patients.
The effects of HAMLET were also investigated in a mouse xenograft model of human glioblastoma. Glioblastoma is the most common and most aggressive form of brain cancer in humans, with a median survival time of approximately 14 months. Current treatments are invasive and do not have a significant effect on survivability. In vitro studies revealed that HAMLET selectively induced apoptosis in glioblastoma cells and not in differentiated brain cells. Glioblasoma tissue biopsies were then implanted onto mouse brains and were found to infiltrate and form masses in a manner similar to the pathology observed in humans. The brain cavity surrounding the implantation was diffusely perfused with HAMLET solution. Tumor masses of both HAMLET-treated and alpha-lacalbumin-treated control mouse brains were compared and a significant reduction in tumor volume was reported in treatment animals over control. Furthermore, HAMLET was found to delay the onset of other neurological symptoms significantly over the control. TUNEL assay
s of brain tissue sections revealed that HAMLET induced apoptosis in tumor cells, while sparing the surrounding brain tissue. No other neurological side-effects were reported as a result of the treatment.
HAMLET may prove to be a unique weapon for clinicians to selectively target tumor cells in patients whose cancer has shown resistance to other chemotherapeutic treatments and ionizing radiation. In vitro studies have shown preliminary success in HAMLET treatment of carcinomas of the lung, throat, kidney, colon, bladder, prostate, and ovaries, as well as melanomas, glioblastomas, and leukemias. A recent clinical study of bladder cancer, HAMLET therapy was found to cause shedding of TUNEL-positive cancer cells into the urine, with no adverse side-effects on healthy cells.
There exists a possible developmental justification for the success of HAMLET. The stomach of an infant is an acidic environment, which serves to induce protein unfolding. Human breastmilk also contains triglycerides, which are hydrolyzed in the stomach, releasing oleic acid. All of the ingredients for HAMLET production exist endogenously in the human digestive tract. Furthermore, studies have shown that breastfed infants have much lower incidence rates of childhood cancer, especially lymphoma and cancers of rapidly dividing GI tissue. The potential tumoricidal and antiviral properties of HAMLET would make it indispensible for an immune naïve infant.
Alpha-lactalbumin
Lactalbumin, alpha-, also known as LALBA, is a protein that in humans is encoded by the LALBA gene.- Function :α-Lactalbumin is an important whey protein in cow's milk , and is also present in the milk of many other mammalian species...
and oleic acid
Oleic acid
Oleic acid is a monounsaturated omega-9 fatty acid found in various animal and vegetable fats. It has the formula CH37CH=CH7COOH. It is an odorless, colourless oil, although commercial samples may be yellowish. The trans isomer of oleic acid is called elaidic acid...
that induces apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
in tumor cells, but not in healthy cells.
HAMLET is a novel chemotherapeutic agent with potent tumoricidal properties. Alpha-lactalbumin
Alpha-lactalbumin
Lactalbumin, alpha-, also known as LALBA, is a protein that in humans is encoded by the LALBA gene.- Function :α-Lactalbumin is an important whey protein in cow's milk , and is also present in the milk of many other mammalian species...
is the primary protein component of human milk. In a 1995 study, it was noted that multimeric alpha-lactalbumin (MAL), a compound isolated from a fraction of human milk called casein
Casein
Casein is the name for a family of related phosphoprotein proteins . These proteins are commonly found in mammalian milk, making up 80% of the proteins in cow milk and between 60% and 65% of the proteins in human milk....
, induced what appeared to be apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
in human lung carcinoma cells, pneumococcus bacteria, and other pathogens, while leaving healthy, differentiated cells unaffected. The active component responsible for the tumoricidal activity was finally elucidated in 2000 and found to be a complex of alpha-lactalbumin and oleic acid
Oleic acid
Oleic acid is a monounsaturated omega-9 fatty acid found in various animal and vegetable fats. It has the formula CH37CH=CH7COOH. It is an odorless, colourless oil, although commercial samples may be yellowish. The trans isomer of oleic acid is called elaidic acid...
.
Endogenous human alpha-lactalbumin is complexed with a calcium ion and serves as a cofactor
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....
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
Cytotoxicity
Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are a chemical substance, an immune cell or some types of venom .-Cell physiology:...
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
The HAMLET protein has been described as the Hydra of ancient Greek mythology, with many heads that regenerate when cut off, making the creature impossible to kill. 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 phosphatidylserinePhosphatidylserine
Phosphatidylserine is a phospholipid component, usually kept on the inner-leaflet of cell membranes by an enzyme called flippase...
and o-glycosylated mucin
Mucin
Mucins are a family of high molecular weight, heavily glycosylated proteins produced by epithelial tissues in most metazoans. Mucins' key characteristic is their ability to form gels; therefore they are a key component in most gel-like secretions, serving functions from lubrication to cell...
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
Mitochondrion
In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 1.0 micrometers in diameter...
. Electron microscopy has revealed physical damage to the mitochondrial membranes and assays have found cytochrome c
Cytochrome c
The Cytochrome complex, or cyt c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is a highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an...
release and activation of the caspase cascade
Caspase
Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis , necrosis, and inflammation....
, the most notable ones being caspases 2, 3, and 9. It is interesting to note that cell death is not prevented by caspase inhibitors, or by BCL-2
Bcl-2
Bcl-2 is the founding member of the Bcl-2 family of apoptosis regulator proteins encoded by the BCL2 gene. Bcl-2 derives its name from B-cell lymphoma 2, as it is the second member of a range of proteins initially described in chromosomal translocations involving chromosomes 14 and 18 in...
or p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
mutagenesis, indicating that the traditional apoptotic caspase cascade is not the ultimate cause of cell death.
Another target of HAMLET is the proteasome
Proteasome
Proteasomes are very large protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks...
. 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.
As part of its multi-faceted attack, HAMLET also targets the nucleus, where it interacts with histones 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
Nucleosome
Nucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool....
. This interaction irreversibly blocks transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
and leads to activation of p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
. This process has been demonstrated to be similar to histone hyperacetylation and it was found that histone de-acetylase inhibitors
Histone deacetylase inhibitor
Histone deacetylase inhibitors are a class of compounds that interfere with the function of histone deacetylase.HDIs have a long history of use in psychiatry and neurology as mood stabilzers and anti-epileptics...
potentiated the effects of HAMLET.
HAMLET cells showed the physiological characteristics of macroautophagy, 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
Mammalian target of rapamycin
The mammalian target of rapamycin also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1 is a protein which in humans is encoded by the FRAP1 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.
Clinical applications
Recent clinical studies have been remarkably successful in treating both human skin papillomas and xenografts of human glioblastomas with HAMLET. PapillomaPapilloma
Papilloma refers to a benign epithelial tumor growing exophytically in finger-like fronds. In this context papilla refers to the projection created by the tumor, not a tumor on an already existing papilla . When used without context, it frequently refers to infections caused by human...
s are pre-malignant lesions on the skin or mucosal membranes and are often caused by Human Papilloma Virus infections. In a double-blind study, HAMLET was applied topically to skin papillomas of patients for 3 weeks, and the lesions were monitored over the course of the next two years. The study found papilloma volume reduction in 100% of treated patients and in only 15% of placebo patients. No adverse reactions were reported, and there were no differences observed between immunocompetent and immunocompromised patients.
The effects of HAMLET were also investigated in a mouse xenograft model of human glioblastoma. Glioblastoma is the most common and most aggressive form of brain cancer in humans, with a median survival time of approximately 14 months. Current treatments are invasive and do not have a significant effect on survivability. In vitro studies revealed that HAMLET selectively induced apoptosis in glioblastoma cells and not in differentiated brain cells. Glioblasoma tissue biopsies were then implanted onto mouse brains and were found to infiltrate and form masses in a manner similar to the pathology observed in humans. The brain cavity surrounding the implantation was diffusely perfused with HAMLET solution. Tumor masses of both HAMLET-treated and alpha-lacalbumin-treated control mouse brains were compared and a significant reduction in tumor volume was reported in treatment animals over control. Furthermore, HAMLET was found to delay the onset of other neurological symptoms significantly over the control. TUNEL assay
TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids.- Method :...
s of brain tissue sections revealed that HAMLET induced apoptosis in tumor cells, while sparing the surrounding brain tissue. No other neurological side-effects were reported as a result of the treatment.
HAMLET may prove to be a unique weapon for clinicians to selectively target tumor cells in patients whose cancer has shown resistance to other chemotherapeutic treatments and ionizing radiation. In vitro studies have shown preliminary success in HAMLET treatment of carcinomas of the lung, throat, kidney, colon, bladder, prostate, and ovaries, as well as melanomas, glioblastomas, and leukemias. A recent clinical study of bladder cancer, HAMLET therapy was found to cause shedding of TUNEL-positive cancer cells into the urine, with no adverse side-effects on healthy cells.
There exists a possible developmental justification for the success of HAMLET. The stomach of an infant is an acidic environment, which serves to induce protein unfolding. Human breastmilk also contains triglycerides, which are hydrolyzed in the stomach, releasing oleic acid. All of the ingredients for HAMLET production exist endogenously in the human digestive tract. Furthermore, studies have shown that breastfed infants have much lower incidence rates of childhood cancer, especially lymphoma and cancers of rapidly dividing GI tissue. The potential tumoricidal and antiviral properties of HAMLET would make it indispensible for an immune naïve infant.