It's paper time!!! I told you I was leading up to something. And here it is!
"Changes in Proteasome Structure and Function Caused by HAMLET in Tumor Cells" was published in PLOSone April 2009. It's free, open access so go and read it! Or if you want, read on and I'll try to take you through it.
So we established that HAMLET is a variation of the alpha*-lactalbumin protein. HAMLET is formed when alpha-lactalbumin (encoded by the LALBA gene, remember that one?) is partially unfolded, loosing a Ca2+, and hooking around a fatty acid called oleic acid. This can occur in acidic environments, like say, in the stomach. Let's recap that: a major protein found in human breast milk changes to a cancer-fighting molecule when it passes through the stomach. In a clinical study, topical HAMLET killed off skin cancer cells. In humans with bladder cancer, an infusion of HAMLET caused tumor cells to be shed and tumors shrank. Interesting.
So this team, coming from the Lund University in Sweeden and the A*STAR group from Singapore, wanted to look at how HAMLET might be killing tumor cells. First they showed in cell culture that HAMLET selectively kills off several type of cancer cells but not normal differentiated cells. Also, alpha-lactalbumin does not kill either type of cell. They also showed through fluorescent-labeled HAMLET that 50x more HAMLET gets inside tumor cells than alpha-lactalbumin. So, somehow HAMLET is selectively entering and killing tumor cells, but not normal cells.
Next they turned to the proteasome. They showed in vitro (not in cells, in cell lysate and chemistry experiments) that HAMLET binds to the 20S proteasome. They also did co-immunoprecipitation and used antibodies specific to HAMLET to pull it out of a cell slurry, and they showed that particles of the 20S proteasome were attached to HAMLET. Finally they used immunofluorescence in cultured tumor cells to show that HAMLET and the 20S proteasome were located very close to one another. These data are not water-tight but are suggestive that HAMLET and the proteasome are interacting.
So, HAMLET interacts with the proteasome and also selectively picks off the tumor cells. What could it be doing? The group next showed that HAMLET is resistant to proteasomal degradation, by showing it is resistant to the enzymes trypsin and chymotrypsin, and it also is a partial proteasome inhibitor, by showing proteasomes from HAMLET treated tumor cells don't degrade other proteins. Using mass spectrometry, they see some changes to subunits of the proteasome in HAMLET treated tumor cells: namely, structural subunits, a nuclear localization signal, and some catalytic subunits. This suggests HAMLET gums up the normal workings of the proteasome. They also show this on protein immunoblot; proteasome subunits from HAMLET treated tumor cells end up on a different part of the gel, suggesting the size or composition of those subunits is altered. Finally, they show increased fluorescent staining of proteasomes in HAMLET treated tumor cells, again suggesting the trash can is broken.
Why tumor cells and not normal cells? Well, they didn't do any of their proteasome studies in normal cells, and they only looked at differentiated normal cells. Would it have an effect on proliferating normal cells?
And what is this doing coming from breast milk? It's great that HAMLET has anti-tumor effects, but what is it doing for babies? Is it just a random by-product or is it somehow offering early protection for our babes in the early years? There exist a few anecdotal stories out there of women providing breast milk to cancer-striken loved ones, and they loved ones recover. Just anecdote, or did the conversion of alpha-lactalbumin to HAMLET help these cancer patients?
Related - does it have to be HAMLET from human milk, or does bovine alpha-lactalbumin converted to HAMLET do the same thing?
Well, just thought that was a pretty little study. As a nursing mom and a cancer researcher (on hiatus) it's things like this that fascinate me and make me glad I'm doing what I'm doing.
P.S. Sorry this one was long-winded and kinda jargon-y.
P.P.S You can email comments/questions/concerns/suggestions here!
* anyone know how to make the alpha symbol on the Mac using keyboard strokes?