Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells
Citations: Biochemical and Biophysical Research Communications
Volume 374, Issue 2, 19 September 2008, Pages 258-263
William Ka Kei Wu, Ya Chun Wu, Le Yu, Zhi Jie Li, Joseph Jao Yiu Sung and Chi Hin Cho
Summary: The ubiquitin-proteasome system and lysosome based autophagy are two major pathways of protein degradation. It has been noticed that proteasome inhibitors are effective against certain cancers and it has been proposed, and in some cases demonstrated, that this due to the role of proteasomes in degrading apoptotic proteins.
The authors hypothesize, based on recent evidence, that it may also be due to the role of autophagy in protein disposal, and that the surpression of proteasomes results in overagressive autophagy leading to apoptosis and cell death.
To this end, the authors used the proteasome inhibitor MG-132 and measured its effect on the HT-29 line of colon cancer cells and the present of autophagioc vacuoles.
After treatment, it was determined by MTT assay that cell mitosis was greatly inhibited, and flow cytometry indicated a buildup of cells stuck at the G2/M checkpoint.
The amount of autophagic vesicles was determined by LC3 antibodies directed and LC3+ autophagic vacuoles. Acridine orange was used to distinguish acidic vesicles. Both assays showed a significant increase in autophagic vesicles in cells treated with MG-132.
Western blotting was done on LC3-I and II, as well as Beclin-1, proteins known to be associated with autophagy. While LC3-I and II levels rose markedly in treated cells, Beclin-1 showed no change.
3-Methyladenine has been used to block Class III phosphoinositide 3-kinases, which have been shown to be involve in autophagy. In cells that received 3-Methyladenine as well as MG-132, autophagic vacuoles and LC3 expression were greatly reduced compared to cells receiving only MG-132. Further, cell proliferation failed to be suppressed beyond the levels that would be due to 3-Methyladenine alone.
Importance: This helps to establish autophagy as a major complementary pathway of protein degradation. Further, studies have been done on the therapeutic potential of proteasome-inhibitors in cancer suppression. The elucidation of the mechanism of their function, and the understanding of the importance of autophagy, will be useful in further development and experimentation.
Crit: I wish they had found a better way of establishing that autophagy was the mechanism than 3-Methyladenine. The fact that 3-Methyladenine itself inhibits cell proliferation raises the possibility that it was masking any autophagy-independent effects of MG-132, since the effects of 3-Methyladenine alone were already greater than the effects of MG-132, based on MTT assay. (See the figures). I would have also liked to see more follow-up on Beclin-1, and perhaps a negative protein control, to show that there was not some sort of (admittedly unlikely) global uptick in protein production. Further, perhaps increased autophagy is due to an uptick in apoptotic proteins? Further investigation is warranted.
6 comments:
In my mind, this paper raises two issues whether the autophagy seen is specific to colon cancer cells and whether it is specific to MG-132. It is noted that ubiquitin proteasome inhibitors are used in the treatment of hematopoietic malignancies. Is autophagy also the mechanism for cellular arrest in blood cancers? If so, why is this process more effective in arresting cancer cells versus normal? Lastly, it seems curious why the authors chose to use only one inhibitor when there are many other similar inhibitors, some of which like Velcade are better characterized and have been clinically approved. Perhaps it may be the case that the induction of autophagy was a compound-specific off-target effect.
I think the hypothesis here is that it would be more general than just colon cancer cells or MG-132, since there's no particular reason to expect it not to be, but that's one of the reasons why more study is warranted.
And did they say it was more effective than normal? What is normal?
And I too am curious why they chose MG-132.
Well, it is possible that a drug like MG-132 is a proteasome inhibitor but that its real effect in autophagy is due to another, understudied "side-effect". Furthermore, it is likely that it's a cell type specific effect. For example, many chemotherapy drugs only work against certain tissues (ie brain cancer drugs cannot be used on liver cancers). That is why I am curious as to whether autophagy leads to cellular arrest in hematopoietic cancers where proteasome inhibitors like Velcade are used.
Ah well, their references about cancer suppression point to Clinical trials about Velcade. My guess is Velcade might be still proprietary so MG-132 is the most similar thing they have to go on.
They should definitely test other cancers, but the fact that Velcade works on many different types of cancers suggests the mechanism they found might be general. (*might*)
Knocking out of the proteosome would have many effects on the cell cycle, and what you said about the cell being stuck at the G2 checkpoint may be explained by inhibitory kinase Wee1. Wee1 blocks activity of and phosphorylates Cdc2, and is normally tagged for degradation during a normal cell cycle. However, with MG-132 inhibiting proteasomic activity, Wee1 cannot be degraded. This in turn leads to CDC2 never being activated, and the CyclinB-CDC2 complex never forming.
Naturally, G2 is not meant to be held for a sustained period of time, especially with a proteosomal inhibitor blocking degradation of normally destroyed substrates. As a result, the cell will need to get rid of these proteins, thus creating autophagic vesicles, as well as set off stress sensors, which are already normally activated by the progression of the cell cycle. These sensors, activated to this point, could easily trigger a cascade that could induce apoptosis. A further experiment could be done by removing intracellular stress sensors and apoptotic pathways.
It seems to me that this paper has a long ways to go in order to prove that autophagy is indeed the primary cause of apoptosis, and that these are not just two symptoms of MG-132.hat is to say, I don't really see much causation here; only correlation.
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