We are all autophagic – and that is good. All the time, our cells are self digesting and self renovating, breaking down and recycling proteins by autophagy, a biological mechanism. Previously regarded merely as a process cellular death, this form of selective self-destruction of cell components now appears to be an artifice for the survival of organisms: only when no further mending is possible do cells become extinguished. As autophagy can apparently be speeded up or slowed down, it has become a novel strategy to fight diseases and extend cell life, the interior of which should reflect as much movement as the works of the painter Jackson Pollock.
At present, autophagy is opening up possibilities for new applications of old drugs. For instance, lithium, used to treat people with bipolar disorder, which is characterized by sudden mood switches from euphoria to deep depression, may be useful to stop Alzheimer’s disease, a form of neuron degeneration that tends to worsen with age. Chloroquine, besides placating malaria, may help to fight tumors. Rapamycin, an antibiotic used to avoid transplant rejection, extended the life of a group of mice, as compared to a control group, whose aging progressed normally.
“Establishing the safety of use and determining new application dosages for medication that is already approved is far easier than starting from scratch”, argues Soraya Soubhi Smaili, a professor from the Federal University of São Paulo (Unifesp). She heads one of the few groups doing research into this area in Brazil. Cláudia Bincoletto, who also teaches at Unifesp and is a researcher on Soraya’s team, showing why this strategy for finding new drugs could be convenient for countries that have limited financial resources, such as Brazil, adds: “New drugs are far more expensive than old ones.”
There is also room for research into new drugs. At Unifesp, Cláudia studies the promising effects of compounds of the chemical element palladium upon autophagy to fight tumors. She has been working on the possibility of regulating autophagy by means of chemical compounds, which might be a path to increase the efficiency of antitumor compounds, reducing the dose and the undesirable side effects upon other cells.
In a newly completed study conducted at the University of São Paulo (USP), Renato Massaro, under the guidance of Silvya Maria-Engler, tested a compound extracted from the roots and leaves of a Mata Atlântica (Atlantic Seaboard Rainforest) shrub called pariparoba (Piper umbellatum), against a strain of human brain tumor cells grown in a suitable lab culture environment . The results indicated that this compound, 4-nerolidylcatechol or 4-NC, may stimulate autophagy in this type of tumor, called glioma, and activate biochemical paths leading not only to recycling, but also to cell death. Gliomas originate in the so-called neuroglia cells, found in the brain in far greater numbers than neurons.
Massaro observed that 4-NC also diminished the capacity of tumor cells to invade the space of healthy cells. This was a good sign. The problem is that other research groups had already indicated that the tumor cells might become resistant to death inducing stimuli. One of the typical features of tumor cells is precisely their capacity to escape from genetically programmed cell death.
As apoptosis and autophagy are related, each stimulating or holding back the other, Massaro adopted the inverse strategy: he added an autophagy blocking compound, 3-methyladenine or 3-MA, to the human tumor cell culture. The 3-MA enhanced the effect of the 4-NC and tumor death rose by 30%, probably stimulating another cell death mechanism relative to cells treated only with 4-NC. At Unifesp, with other compounds, Cláudia Bincoletto reached similar results, indicating that autophagy does not lead to cell death but to cell survival. Therefore, when one inhibits autophagy, antitumor compounds become more effective. “This strategy has been advocated by many groups that are seeking new tumor treatments”, commented Soraya.
‘Our challenge now is to find the dosage that only eliminates tumor cells, without damaging the normal ones”, says Silvya. According to her, changing the normal autophagy levels in healthy cells might give rise to unbalanced genetic processes, or to unwanted inflammatory responses. The USP team had already indicated in 2008 that 4-NC might stimulate apoptosis in skin tumor cells or melanoma cells kept in lab cultures.
At the Federal University of Rio Grande do Sul (UFRGS), Guido Lenz’s team has been studying the effects of resveratrol, a natural compound found in the skin of grapes and in red berries and peanuts, upon cell death and life. Under his guidance, Eduardo Chiela compared the effects of resveratrol and temozolomide, one of the main glioma drugs that, as we already know, can induce death by autophagy. The study, currently in the final stages of being written up, indicated that the compound in the skin of grapes (especially of the dark ones) stimulates two cell death mechanisms, autophagy and apoptosis, in human tumor cell cultures.
In an earlier study, Lauren Zamin, Guido Lenz and other researchers from UFRGS assessed the effects of resveratrol and of quercetin, another compound found in grapes and in other fruit: grape skin contains some 50 to 100 micrograms of resveratrol per gram and 40 micrograms of quercetin; red wine, some 7 to 13 micrograms of resveratrol and 7.4 of quercetin. A combination of these two substances led rat glioma cells to enter senescence, an irreversible aging process that may culminate in autophagy and that normal cells employ to avoid becoming cancerous. Under the effect of these two substances, the tumor cells became gigantic and then burst.
Animal testing is proceeding and highlighting the dual role of resveratrol, as it may, inversely, have an anti -aging effect on healthy cells. “Resveratrol seems to know whether a cell is healthy or tumoral”, notes Lenz. “It won’t be easy, but we’re very interested in pursuing the research, in so far as actionable results appear positive, toward applications for humans”. Other studies have already described resveratrol as a compound that can hold back other types of tumor, stimulate autophagy and halt aging.
“Autophagy offers a promising approach for the treatment of melanoma (skin cancer), commented Damià Tormo, a researcher from the Spanish Center of Cancer Research in Madrid, during a presentation he made at USP in January. He has coordinated the construction of a synthetic RNA (ribonucleic acid) structure that activates specific proteins and fosters autophagy, as described in an article in the journal Cancer Cell in 2009. Tormo also works at his start up company, BiOnco Tech, to further the development of this molecule, which has been shown to be effective in halting the growth of skin cancers, which often become drug resistant, in the first experiments conducted with cell cultures and with genetically modified mice.
Even with new substances that seem to be promising in terms of their effect and have low toxicity, it will be difficult to advance. First, because of how difficult it is to develop new medication in Brazil. Second, because of the double role of authophagy, which can aid the survival or the elimination of normal and tumoral cells. Several studies, comments Guido Kroemer, a researcher from the Gustaf Rouassy Institute in Paris, have shown that autophagy may have different functions, depending on the type of cell. In neurons, heart cells or other types of cells that are reproducing normally, this mechanism might be an aid to cleaning up, eliminating residues and preparing cells for death through apoptosis. In cells that are multiplying with no control, i.e., that have the potential for forming tumors, it might encourage survival and therefore eventual resistance to compounds or external stimuli deployed against them.
Recognized in the 1970’s by Daniel Klionsky, a researcher from the University of Michigan, in the USA, autophagy was regarded for almost three decades as merely one way, of no great importance, for cells to get rid of themselves. Therefore, it was named type 2 programmed cell death, to differentiate it from apoptosis, or type 1 death, which has been studied to a far greater extent. “One can say that autophagy precedes cell death or that it crosses cell death, but now it is no longer correct to state that autophagy is a form of cell death”, comments Soraya.
Identification of the genes that control autophagy began in 1997. At first, it only involved yeasts, the unicellular organisms used to make bread, wine, beer and fuel alcohol. Starting with the yeast genes, experts learnt which proteins drive this flexible cell component recycling mechanism and how they interact. Besides dismantling whatever is not working properly, autophagy fulfills other tasks during the course of cell development and does not always lead to death. It is necessary, for instance, for yeasts to reproduce and for insect larvae to turn into pupae.
“Today, we see that autophagy is geared mainly to survival and resistance rather than to cell death”, observes Soraya. “In the face of an aggressive stimulus or a cell defect, the cell can become autophagic in an attempt to fix matters. Only when fixing is no longer an option does the cell death process set in”. Several studies have suggested that the genes and proteins that stimulate autophagy may block apoptosis, or the contrary, starting with highly defined stimuli, thereby establishing crossed communication between these two phenomena.
When they get internal or external stimuli, the two dozen autophagy controlling genes that have already been identified activate the production of proteins that little by little join together to form membranes around those cellular components that are to be dismantled before they start causing problems. Then, driven by other specific proteins, the membrane merges with the lysosomes, which are cell compartments rich in the enzymes that routinely fragment proteins.
The lysosomes digest defective cell proteins more slowly than another cell cleaning mechanism, the proteasomes. Though slower, the lysosomes can eliminate larger cell structures when these are damaged or deficient, especially the mitochondria, cell compartments that turn the energy obtained from food into ATP molecules, fundamental for cell maintenance. At Unifesp, under the guidance of Soraya, Juliana Terashima irrigated cells with a compound known by the acronym FCCF, which is highly toxic for mitochondria. In response, the cells entered a state of autophagy that, once activated, helped to remove the mitochondria that had been damaged by the compound.
By being involved with cell dismantling, lysosomes enable cells to build new molecules even when they have not been supplied with their habitual raw materials, which are provided by food. The merging of the membranes with the lysosomes leads to the formation of large sacs, called autosomal vacuoles, which pursue the transformation of waste into raw material for new molecules. According to Lenz, it is apparently the number of mitochondria eliminated by these vacuoles that marks the moment at which the cell moves on from the recycling phase into the total destruction phase. The problem is finding this limit. In practical terms, this means discovering how many mitochondria a cell must lose – a cell has 200 of them on average – to enter irreversibly along the path of cell death.
The understanding of this cell disassembly line, as it grew, began raising possibilities, now more concrete, of intervening in this chain of biochemical reactions in order to extend healthy cell life and reduce tumor cell life. In a study published in February 2008 in the journal PNAS, Italian researchers showed that lithium, given for 15 months to a group of 44 people, might delay the progress of amyotrophic lateral sclerosis, a neurodegenerative disease.
One month earlier, a team from Cambridge University showed in Human Molecular Genetics the possibility of using lithium and rapamycin combined to treat Huntington’s disease, another illness involving continuous loss of neuron functionality. “Autophagy seems to remove the malformed protein aggregates, which get in the way of the functioning of nerve cells and are found in neurodegenerative diseseases such as Huntington’s, Parkinson’s and Alzheimer’s”, observes Soraya. According to her, studies conducted in her laboratory with cells from patients with Huntingtons showed that stimulating autophagy might postpone cell death by apoptosis.
A cell that has cleaned itself up by autophagy may live longer, according to a study conducted in the US and published in Nature in July 2009. To reach this conclusion, the researchers looked after some 3,000 elderly mice, whose age was equivalent to 60 years among humans. Some of the animals were given rapamycin, an autophagy-stimulating compound. The researchers then waited for all of them to die naturally, five to seven months later. The mice that had been given rapamycin lived 28% to 38% longer than those that had not.
This experiment was impressive because of its grand scale, since the number of animals used in research is rarely this high, but it did not elicit a consensus of acceptance, many researchers arguing that the mice may have lived longer for other reasons or that this result is insufficient to link the control of autophagy to extending cell life. In any event, the mechanisms of the workings of authophagy became clearer. Other experiments suggested that merely depriving cells of nutrients could stimulate this type of cell cleaning. “If it gets less glucose”, comments Soraya, “the cell will produce less energy via its usual metabolic paths, but it will also produce less waste, which speeds up aging; this will also stimulate autophagy, which can remove malformed mitochondria and proteins.”
In an article published in 2006 in Cancer Cell, Melanie Hippert, Patrick O’Toole and Andrew Thorburn, from the University of Colorado, in Denver, USA, acknowledged that the manipulation of autophagy might be useful to hold back the progress of tumors and to improve the efficiency of cancer treatments. The problem is that authophagy has a dual role: it can inhibit or it can foster tumor growth, depending on the circumstances. Therefore, autophagy could perhaps be stimulated to avoid the formation of tumors in people who are at risk for cancer, but reduced if a tumor has already become manifest in the organism.
After finding a suitable compound, the next challenge will be to determine the best dosage, so that only tumor cells die. Chi Dang, from John Hopkins University, USA, explained in January 2008, in the Journal of Clinical Investigation, that chloroquinin, an anti-malaria drug, may help to prevent tumor evolution. He warned, however, that using this compound for a long time might lead to unforeseen side effects, as the drug inhibits autophagy and stimulates apoptosis and because our understanding of cell balance is still rudimentary.
“I don’t believe that new antitumor drugs will only stimulate autophagy”, comments Lenz. “It would be risky. The solution might be something like resveratrol, which can have multiple targets and activate more than one biochemical process leading to tumor death, including autophagy”. Even if new compounds do not appear in the near future, the capacity to induce or to block cell death should become one of the characteristics of drugs in general, helping to explain how they operate within the body; incidentally, many antitumor drugs already used may induce autophagy. It might also help to resume a lot of research that was suspended. “Pharmaceutical compounds that failed in clinical trials should perhaps be revisited”, ponders Silvya Stuchi Maria-Engler, from USP, “because they might turn out to be excellent if used in conjunction with other drugs, capable of inducing or inhibiting autophagy.”
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Hippert, M. M. et al. Autophagy in cancer: good, bad, or both? Cancer Research. V. 66, n. 19, p. 9 349-51. 2006.
Harrison, D. E. et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. V. 460, p. 392-5. 2009.
TORMO, D. et al. Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells. Cancer Cell. V. 16, n. 2, p. 103-14. 2009.