48 replies to this topic

[Turnbuckle]

  Inhibiting p53 with SIRT-1 (or resveratrol) appears to revert these cells to a normal non-senescent state...

 

 

Are you sure about that?

 

We propose a model that resveratrol-activated ERKs and p38 kinase bind to p53 molecules to form a complex, leading to phosphorylation of p53 at Ser 15 or other phosphorylation sites, thereby enhancing its functional activities. Our data are in agreement with the recent proposal that to achieve optimal activity, p53 NH2-terminal sites may be phosphorylated by a p53-associated complex containing several kinases (65).

http://cancerres.aac.../61/4/1604.long

 

 

[RWhigham]

Are you sure about that?

Never can be sure, but if the 2001 article referenced above is correct then resveratrol should cause apoptosis of senescent cells.

 

The 2017 article that inspired this Longecity thread tested resveratrol in numerous cultures, Resveratrol and its analogues reduced the number of senescent cells, but the experimenters could not find any products of apoptosis produced in their cultures.  However, only a limited number of senescent cells in the cultures reversed, and they did not make sure the entire culture was in replicative senescence. This leaves the door open to question whether the cells reversed were in replicative senescence or not.  The cells reversed may very well not have been in replicative senescence. This seems probable to me. The cells in replicative senescence have already completed the preliminaries for apoptosis and are waiting for the final step in which nuclear p53 releases cytochrome-c from the mitochondria. This seems difficult to reverse--but who knows?

 

Review: Senescent cells with functional telomeres but with nuclear damage are maintained in a non-replicative (senescent) state by p53 while waiting for repair.  Inhibiting p53 reverts these cells to non senescence. I think there is some evidence that this non-replicative state may also be induced by adjacency to cells in replicative senescence.  So I would expect a certain fraction of "senescent" cells to be in this reversible state.

Edited by RWhigham, 30 May 2018 - 02:41 PM.

[Turnbuckle]

 

Review: Senescent cells with functional telomeres but with nuclear damage are maintained in a non-replicative (senescent) state by p53 while waiting for repair.  Inhibiting p53 reverts these cells to non senescence. I think there is some evidence that this non-replicative state may also be induced by adjacency to cells in replicative senescence.  So I would expect a certain fraction of "senescent" cells to be in this reversible state.

 

Expect senescent cells to progress to apoptosis given a sufficient level of fission, and apigenin induces fission. So talking apigenin and quercetin will fission mitochondria and upregulate p53, thereby inducing apoptosis. Both quercetin and resveratrol promote apoptosis via p53. One might also add IP6, which upregulates p53, though it has a short half life of around 20 minutes.

 

Quercetin--

In summary, our results indicate that quercetin, an

ubiquitous bioactive plant flavonoid, induced p53-

mediated cell cycle arrest and apoptosis in HepG2

through the stabilization of p53 mRNA and protein.

These findings will provide new insight into the cancer

chemoprevention properties of plant flavonoids.

https://www.jstage.j...0/_pdf/-char/en

 

 

 

IP6--

These results suggested that topically applied IP6 directly induces apoptotic machinery by modulating the expression of mt p53, Bcl-2, and caspase activity.

https://www.ncbi.nlm.../?term=18652568

 

 

[RWhigham]

 

Expect senescent cells to progress to apoptosis given a sufficient level of fission, and apigenin induces fission. So talking apigenin and quercetin will fission mitochondria and upregulate p53, thereby inducing apoptosis. Both quercetin and resveratrol promote apoptosis via p53. One might also add IP6, which upregulates p53, though it has a short half life of around 20 minutes.

 

Stimulating more p53 production might be an alternative to inhibiting FOX04, assuming the FOX04 can be "used up" temporarily. Perhaps enough p53 could overwhelm the FOX04 and unblock apoptosis.

 

Heliocare is a brand name for Polypodium leucotomos (calaguala) extract.  Heliocare is a powerful p53 agonist. If I take enough heliocare then the increased p53 may overwhelm the FOX04 inhibition and allow apoptosis to proceed. I have 4 bottles on order.

 

 

 

[Turnbuckle]

Stimulating more p53 production might be an alternative to inhibiting FOX04, assuming the FOX04 can be "used up" temporarily. Perhaps enough p53 could overwhelm the FOX04 and unblock apoptosis.

 

Heliocare is a brand name for Polypodium leucotomos (calaguala) extract.  Heliocare is a powerful p53 agonist. If I take enough heliocare then the increased p53 may overwhelm the FOX04 inhibition and allow apoptosis to proceed. I have 4 bottles on order.

 

 

I think you've made this too complicated. You don't need any "unblocking." All you need is sufficient fission and p53, which you obviously got with apigenin and quercetin in the OP. Or wasn't being "wiped out" sufficient?

[RWhigham]

I think you've made this too complicated. You don't need any "unblocking." All you need is sufficient fission and p53, which you obviously got with apigenin and quercetin in the OP. Or wasn't being "wiped out" sufficient?

• Have you read the FOX04_DRI article?
• I cannot tell that I cleared any replicative senescent cells.
• We need some objective measure that can be used at home. Any suggestions?

Edited by RWhigham, 30 May 2018 - 04:19 PM.

[Turnbuckle]

Have you read the FOX04_DRI article?

 

They don't even mention fission. But fission is required for apoptosis.

 

We need some objective measure that can be used at home. Any suggestions?

 

 

I am presently using a protocol to increase the pool of stem cells via fusion/C60, and then removing senescent cells via fission/p53. Senescent cells would typically have an epigenetic age much older than the average while stem cells that will replace them will start off with zero epigenetic age. So by doing this for some time and getting one's epigenetic age tested before and after should give one some feedback. If it works, the tests should show a decline in age.

Edited by Turnbuckle, 30 May 2018 - 04:29 PM.

[RWhigham]

Mitochondrial control of apoptosis: the role of cytochrome c "Mitochondrial cytochrome c (cyt c) has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Through interaction with apoptotic protease activating factors (Apaf), cyt c can initiate the activation cascade of caspases once it is released into the cytosol."  The FOX04_DRI article indicates that replicative senescent cells are poised on the edge of apoptosis, only needing p53 to  release cytochrome-c.  Can you explain how fission fits in?

Edited by RWhigham, 30 May 2018 - 04:30 PM.

[Turnbuckle]

Mitochondrial control of apoptosis: the role of cytochrome c "Mitochondrial cytochrome c (cyt c) has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Through interaction with apoptotic protease activating factors (Apaf), cyt c can initiate the activation cascade of caspases once it is released into the cytosol."  The FOX04_DRI article indicates that replicative senescent cells are poised on the edge of apoptosis, only needing p53 to  release cytochrome-c.  Can you explain how fission fits in?

 

 

That is an old paper that doesn't even mention fission. However, there are exceptions. For instance--

 

Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.

 

https://www.ncbi.nlm...les/PMC1636857/

 

So again, I expect that fission from apigenin and a p53 stimulant is all most people will need. It certainly gave you an effect in the OP that was far beyond what you would have expected from either supplement alone, am I right?

Edited by Turnbuckle, 30 May 2018 - 04:41 PM.

[QuestforLife]

Never can be sure, but if the 2001 article referenced above is correct then resveratrol should cause apoptosis of senescent cells.

 

The 2017 article that inspired this Longecity thread tested resveratrol in numerous cultures, Resveratrol and its analogues reduced the number of senescent cells, but the experimenters could not find any products of apoptosis produced in their cultures.  However, only a limited number of senescent cells in the cultures reversed, and they did not make sure the entire culture was in replicative senescence. This leaves the door open to question whether the cells reversed were in replicative senescence or not.  The cells reversed may very well not have been in replicative senescence. This seems probable to me. The cells in replicative senescence have already completed the preliminaries for apoptosis and are waiting for the final step in which nuclear p53 releases cytochrome-c from the mitochondria. This seems difficult to reverse--but who knows?

 

Review: Senescent cells with functional telomeres but with nuclear damage are maintained in a non-replicative (senescent) state by p53 while waiting for repair.  Inhibiting p53 reverts these cells to non senescence. I think there is some evidence that this non-replicative state may also be induced by adjacency to cells in replicative senescence.  So I would expect a certain fraction of "senescent" cells to be in this reversible state.

 

I've read this paper many times and contributed to that thread, and it is my reading that the rejuvenated cells were still proliferation competent (so not yet irreversibly senescent (seen by the fact they were Ki67 positive). What is more interesting is that  resveratrol re-elongates telomeres in (near) senescent cells; this is an older paper showing the same is possible in endothelial progenitor cells:

 

www.ncbi.nlm.nih.gov/pubmed/18587418

 

The mechanism is likely through the TERT gene becoming available because of the removal of the telomere tail (through shortening). See;

 

http://journals.plos...al.pbio.2000016

 

Resveratrol or its analogues then adjust RNA splicing factors (as per the paper you reference; another telomerase repression mechanism) to permit expression of telomerase. Presumably lengthening of the telomere then halts this process because TERT once again is epigenetically suppressed. Only 5uM of resveratrol was required for this to occur (over 24 hours).

 

Apoptosis of cancer cells took both a greater concentration 25-150uM) and longer exposure (48-72h) to achieve apoptosis in cancer cells, according to this paper:

 

www.ncbi.nlm.nih.gov/pmc/articles/PMC4337473

 

It is doubtful we could achieve such concentrations in the blood using resveratrol, though it might be possible with a more bioavailable analogue such as pterostilbene. I have been experimenting with this for some time. In any case, this would suggest resveratrol is not ideal for Turnbuckle's Turning Back the Clocks protocol.

 

(Could we cross (post this to that thread, moderators?)
 

Edited by QuestforLife, 31 May 2018 - 02:25 PM.

[MikeDC]

Inhibiting p53 will prevent apoptosis. The reason you use Foxo4-DRI is to free p53 from FOXO4.

[Rocket]

I've read this paper many times and contributed to that thread, and it is my reading that the rejuvenated cells were still proliferation competent (so not yet irreversibly senescent (seen by the fact they were Ki67 positive). What is more interesting is that  resveratrol re-elongates telomeres in (near) senescent cells; this is an older paper showing the same is possible in endothelial progenitor cells:

 

www.ncbi.nlm.nih.gov/pubmed/18587418

 

The mechanism is likely through the TERT gene becoming available because of the removal of the telomere tail (through shortening). See;

 

http://journals.plos...al.pbio.2000016

 

Resveratrol or its analogues then adjust RNA splicing factors (as per the paper you reference; another telomerase repression mechanism) to permit expression of telomerase. Presumably lengthening of the telomere then halts this process because TERT once again is epigenetically suppressed. Only 5uM of resveratrol was required for this to occur (over 24 hours).

 

Apoptosis of cancer cells took both a greater concentration 25-150uM) and longer exposure (48-72h) to achieve apoptosis in cancer cells, according to this paper:

 

www.ncbi.nlm.nih.gov/pmc/articles/PMC4337473

 

It is doubtful we could achieve such concentrations in the blood using resveratrol, though it might be possible with a more bioavailable analogue such as pterostilbene. I have been experimenting with this for some time. In any case, this would suggest resveratrol is not ideal for Turnbuckle's Turning Back the Clocks protocol.

 

(Could we cross (post this to that thread, moderators?)
 

 

Why can't we achieve those levels? Especially with liposomal resveratrol available and it's better bio-availability.
 

[QuestforLife]

Why can't we achieve those levels? Especially with liposomal resveratrol available and it's better bio-availability.

I'd be interested to see the figures on how bioavailable liposomal resveratrol is. We know it helps with absorption and bypassing the liver first time round. But depending on how quickly it's absorbed into tissues it may simply be metabolized by the liver on the next pass. The problem with resveratrol is not absorption, it's rapid metabolism. So we need to have a liposome that goes into the required tissue rapidly. It may or may not do this. But I doubt you'll get days and resveratrol just hanging about, doing it's thing, even within cells. That's why I've bet pterostilbene is a much better choice. But honestly, it's just educated guesswork.

[Rocket]

I'd be interested to see the figures on how bioavailable liposomal resveratrol is. We know it helps with absorption and bypassing the liver first time round. But depending on how quickly it's absorbed into tissues it may simply be metabolized by the liver on the next pass. The problem with resveratrol is not absorption, it's rapid metabolism. So we need to have a liposome that goes into the required tissue rapidly. It may or may not do this. But I doubt you'll get days and resveratrol just hanging about, doing it's thing, even within cells. That's why I've bet pterostilbene is a much better choice. But honestly, it's just educated guesswork.

I know from some bodybuilding forums that guys were injecting resveratrol and getting results. But sirt1 activation was causing weight loss which is the antithesis of bodybuilding so guys jumped off of it.

Maybe injections is the way to go.

[Rocket]

I have recently learned that leucine activates sirt1 after adding it to my protein shakes. I wonder how much leucine and how much sirt1? I am taking about 20g daily.

[Invicta Immortalem]

@RWhigham

 

Maybe it is worth also considering the micronized form of resveratrol, which can boost the absorption by 220%: 

 

http://microhealth.b...0mg-120-vcaps/ 

Edited by Invicta Immortalem, 25 July 2018 - 06:07 PM.

[HBRU]

Define your "go big" numbers. I took three consecutive rounds of 140mg dasatinib combined with three rounds of 400mg EMIQ on days 5, 6, and 7 of a ten day water/coffee only fast. I felt nothing out of the ordinary of a usual ten day water fast. Maybe I didn't go big enough. Don't kill myself yet. My urine ketone lev measured >80. Who knows, I'm speculating blindly I was also in some magical mysterious states of macroautophagy; but since "we" can't measure cell death very well here in our consumer hell of self-experimental desert, wtf knows. One day we'll have some more mouse answers, I'm sure, just wait five more years as if, right. Tell us about your go big.

 

We try to kill the less metabolically active cells, right ?

I think that during senolytic protocols is better not to fast, stress and such... and even I'd like to suppress SIRT1 activity with long span Niacin.

Othervise we may stimulate autophagy, and that "saving state" protects senesent cells from death.
 

[HBRU]

Copied  from a post of Fafner55

FOX04 is modulated by SIRT1. The activity of FOX04 is suppressed by SIRT1 inhibitors, such as nicotinamide, and enhanced by SIRT1  activators, such as resveratrol.

“SIRT1 is critical regulator of FOXO-mediated transcription in response to oxidative stress” (2005) https://www.ncbi.nlm.nih.gov/pubmed/16012755?dopt=Abstract

From this view, improvements to senolytic treatments could follow from

• Not fasting
• Not taking NR, resveratrol or pterostilbene
• Not engaging in vigorous exercise
• Inhibiting SIRT1 by large doses of nicotinamide, such as 2 to 3 gm.

 Nicotinamide is a potent inhibitor of SIRT1. I would expect it to decrease FOX04 but don't have support.

1. https://www.researchgate.net/post/How_long_does_it_take_for_Niacinamide_to_inhibit_SIRT1_and_how_long_until_it_stops
2. “Nicotinamide-mediated inhibition of SIRT1 deacetylase is associated with the viability of cancer cells exposed to antitumor agents and apoptosis” (2013) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789038/

[HBRU]

I think that nicotinammide could be really much more usefull than fasting, resveratrol and such for senolytic periods...

Also the antiinfiammatory properties are usefull to damper a bit the inevitable inflamation that follows....

 

Augmented SIRT1 expression was observed only at low concentrations (>80% cell viability) and the inhibition of SIRT1 deacetylase by NAM decreased the viability of the cancer cells exposed to low concentrations of antitumor agents. NAM induced typical apoptosis in the MCF-7 tumor cells, accompanied by the activation of the caspase cascade. SIRT1 promotes cellular survival at certain stress levels by its deacetylase function. The SIRT1 deacetylase inhibitor, NAM, triggers the activation of the caspase cascade and induces typical apoptosis in MCF-7 cells.

 

https://www.ncbi.nlm...les/PMC3789038/