HAMLET (human alpha-lactalbumin made lethal to tumor cells)

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) 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.

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 phosphatidylserine 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.

Clinical applications
Recent clinical studies have been remarkably successful in treating both human skin papillomas and xenografts of human glioblastomas with HAMLET. Papillomas 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 assays 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.