Grouppe Kurosawa: HIV - Caffeine Inhibits the Synthesis of the HIV Virus (print)

Caffeine Inhibits the Synthesis of the HIV Virus
In T Cells by 94%

You think the title of this essay is some kind of advertising “lead in”, don’t you. We, in industrialized countries, have been so numbed by ridiculous advertising claims over our lifetimes that we instinctively stop listening to anything that even remotely sounds “too good to be true”. The parable of the “boy (shepherd) who cried wolf too many times” comes immediately to mind. On the one occasion that the wolf actually appeared, no one would respond to his cries. The villagers had become desensitized to his previous, false, cries for help. They didn’t listen to him any longer. Every person who lives in an industrialized society is bombarded with advertising messages and promises that rarely live up to expectations. So they stop listening, or, as one advertising sage once responded, “they only listen, partially, with one ear”. Remember, the fall of the Berlin wall and the breakup of the Soviet Union was once thought to be “too good to be true”. They happened anyway. What we are about to tell you will seem too good to be true. It isn’t. The Berlin Wall of infectious disease, AIDS, is about to come down. Not all at once, mind you. But the cracks are forming and the collapse is imminent as long as people, for once, listening to a story that is not “too good to be true”.

Caffeine does indeed block the transcription of the HIV viral gene in infected CD4 T cells by 94%. This is not idle speculation. The biochemical pathway by which caffeine inhibits HIV synthesis is known. The scientific study in question was conducted in the laboratory using cell cultures (1). No one has ever tried to treat AIDS or early HIV infections in humans with caffeine or other methylxanthine class drugs. However, some significant studies have been conducted in mice. Theophylline, a synthetic methylxanthine drug commonly used to treat asthma, was administered to mice two weeks after a usually fatal injection of a retrovirus similar to the HIV virus. This virus induces an immune hyperactivity and wasting syndrome very similar to AIDS. Scientists call the syndrome murine (mouse) AIDS or MAIDS. As long as theophylline was in the drinking water, the mice did not develop any symptoms over a four-month test period (2). The authors, citing previous scientific studies with theophylline, concluded that theophylline induced apoptosis or programmed death in the infected cells. This may be true, but they provided no direct evidence for their conclusion. Another explanation may be more viable.

Caffeine and theophylline are both weak inhibitors of A1 and A2 adenosine receptor activity. Large amounts of adenosine, produced during inflammation or in cases where the catabolic enzyme adenosine deaminase is inhibited, can activate cyclic AMP and block IL-2 synthesis in lymphocytes, resulting in immunosuppression. Methylxanthines can also increase the level of cyclic AMP in the cell by inhibiting the activity of cyclic AMP phosphodiesterase. Increasing or decreasing the level of cyclic AMP in an HIV infected cell by methylxanthines, however biologically significant as an explanation for immunosuppression, cannot account for a 94% reduction in the synthesis of the HIV virus in a cell culture assay. A more likely explanation, and one favored by numerous studies over the last 10 years, is that caffeine abolishes the mammalian G2/M DNA checkpoint in the cell cycle. It does this by inhibiting the activity of ataxia-telangiectasia-mutated kinase (ATM), and a related kinase called ATR. ATM is an enzyme that phosphorylates and activates another enzyme called Chk2/Cds1. Upon activation, Chk2 migrates into the nucleus and phosphorylates Cdc25c on a serine residue. Non-phosphorylated Cdc25 is an active phosphatase that controls the cell cycle by dephosphorylating the G2/M checkpoint enzyme Cdc2. When certain phosphate residues are removed from Cdc2, the Cdc2 enzyme becomes activated and allows the cell to complete the cell cycle without becoming blocked at the G2/M checkpoint. On the other hand, if Chk2 phosphorylates and inactivates Cdc25, Cdc2 becomes hyper-phosphorylated and inactive. As a result, the cells cannot complete the normal cell cycle and accumulate at the G2/M checkpoint. Confused? You think we aren’t? We’ll go over this again and why this information is important, but first some additional background information needs to be provided.

The normal cell cycle takes approximately 24 hours to complete. The cell cycle is an immensely complicated process of activation and deactivation of numerous enzymes in specific phases of the 24-hour cycle. See the diagram below.

G0..G1………………x1…….S………………………..G2…………x2..M……G0

Cells remain at rest in the G0 phase of the cell cycle. Nothing much is happening. Upon activation, they move into the G1 phase where they increase in size. The G1 phase is characterized by rapid protein synthesis. At checkpoint 1 or x1, the cell senses its surroundings to determine if all the necessary biochemical pathways have been activated or deactivated before entry into S phase. If not, the cell pauses at the x1 checkpoint. At this point, the cell may or may not self-destruct depending on the reason for the G1 pause. This self-destruct process is called apoptosis or programmed cell death, and it is an absolutely critical process that prevents damaged cells from dividing. Without checkpoints or cell-cycle sensors, damaged cells could evolve into cancer cells—cells that divide without restraint and fulfill no functional purpose. The DNA of the cell replicates in S phase. Replicating DNA is very susceptible to damage by chemicals, UV light, enzymes, and just about everything else. If there is damage to the DNA, it is detected during G2 at the x2 checkpoint. This is called the G2/M interface. The x2 checkpoint again senses its surroundings to determine if the cell cycle can be allowed to proceed to completion. If the DNA has been damaged during the S phase, a prolonged pause in G2 allows DNA repair enzymes to repair the damage before the cells actually divide into two separate cells. Frankly, considering all the complicated biochemical processes that has to occur in order for a cell to divide, it is miraculous that a cell makes it all the way through the cycle at all.

Caffeine prevents cells from pausing at x2. It doesn’t affect x1, to our knowledge. One of the biological roles of the G2 checkpoint is to pause the cell long enough for damaged DNA to be repaired. During G2, DNA repair enzymes are synthesized or activated. People who have a genetic defect in the synthesis of specific DNA repair enzymes cannot, ever, be exposed to UV light. They will develop skin cancers all over their body.
One of the goals of cancer chemotherapy or radiation therapy is to damage the DNA of cancer cells to the extent that they literally fall apart. If the cancer cell is allowed to pause at the G2 checkpoint, it can repair the damaged DNA and defeat the efficacy of the chemotherapy/radiation treatment and proceed to M or mitosis, the phase where the cell actually divides into two separate cells. Caffeine blocks the x2 checkpoint thereby preventing any DNA repair from occurring. The cancer cell with its damaged DNA proceeds to G1 where it usually dies at the x1 checkpoint. Caffeine’s ability to sensitize cancer cells to chemotherapy and radiation therapy has been known for 10 years. Until recently, the biochemical pathway by which caffeine blocks the G2 checkpoint was unknown.

Caffeine, theophylline and other methylxanthines inhibit the ATM/ATR enzymes in the cytoplasm of the cell. This sets off a biochemical chain reaction that eventually dephosphorylates (activates) Cdc2 in the nucleus, thereby allowing cell division to occur.
What does this process have to do with AIDS? In a word, everything! The HIV virus produces a number of accessory proteins that have a pronounced effect on the virulence or toxicity of the virus. One of these proteins is called VPR. VPR activates the glucocorticoid receptor (GR) (the receptor for the anti-inflammatory steroid hormone hydrocortisone) in the cytoplasm of infected and non-HIV cells and induces the receptor to migrate into the nucleus. Once in the nucleus, the glucocorticoid receptor activates many genes and inactivates even more, such as pro-inflammatory, anti-HIV immune hormones. This single act, the activation of the GR by the viral protein VPR, has a pronounced negative effect on the ability of the body to eliminate the virus because it induces a state of immunosuppression in the sites of greatest viral replication, such as the lymph nodes, spleen and thymus. This is bad news. We’ll discuss this process in another essay. VPR does something else of significance. It increases, by ten-fold, the synthesis of the HIV virus in infected T cells. VPR does this by acting synergistically with TAT, another immunosuppressive HIV encoded accessory protein. Both TAT and VPR are well known to activate the HIV viral gene. Until recently, it was not known how dependent TAT and VPR are on one another. TAT induces HIV synthesis in the S and G2 phases of the cell cycle. VPR also activates the HIV LTR; more important, it blocks the cell cycle at x2 of the G2/M interface. In doing so, it allows itself and TAT additional time to increase HIV synthesis before the entire process is shut down in M phase. Caffeine blocks the ability of VPR to induce a x2 block. In doing so, it prevents the cell cycle from slowing down and antagonizes the ability of TAT to induce the synthesis of greater amounts of virus. As stated previously, caffeine reduces the synthesis of HIV in T cells by 94%. Impressive.

When VPR induces a prolonged G2 block in T cells, two biochemical pathways are activated. First, large amounts of virus will be synthesized and released. Second, the cell will probably die from apoptosis. The induction of apoptosis in HIV infected cells is the Holy Grail of immunologists. Eliminating the sites of viral production, by immunological or other means, is or should be the number one priority of any anti-HIV therapy protocol. If this is the case, why would we want caffeine to prevent this process? Blocking an HIV infected cell at x2 induces both viral synthesis AND cell death. The two events are highly correlated. Caffeine allows the HIV infected cell to proceed through the cell cycle, where it will continue to release a low amount of virus over the lifetime of the cell. Caffeine, quite obviously, is an important therapeutic tool that can drastically reduce the amount of HIV produced in T cells, but it will not eliminate the virus producing cells from the body. Fortunately, other simple compounds, used in conjunction with caffeine or other methylxanthines, can do so. The intrigue builds.

The G1 checkpoint, x1, will be the focus of our attention now. If an HIV infected cell is not blocked in G2, it can continue to cycle and produce more virus. However, if the infected cell is blocked in G1, it can be induced to self-destruct WITHOUT stimulating any viral synthesis. The virus is ONLY synthesized in the S and G2 phases of the cycle. This is the basis for the treatment protocol we (Grouppe Kurosawa) are promoting. Caffeine prevents the G2 block, while other natural agents, such as resveratrol from grape skins, and phytochemicals from ginseng and green tea, induce a G1 block. The HIV infected cell can be tricked into dying without producing additional virus. As a general rule, one never wants to induce a state of viral latency. Latency shuts down viral synthesis, but it does not eliminate the viral producing cells from the body. If virus is not produced, cytotoxic T lymphocytes cannot recognize the infected cells as harboring virus and target them for destruction. In a state of latency, the cells are not making any virus so they can escape destruction by the immune system. The therapeutic induction of latency is certainly “better than nothing”, but it is a short-term gain, at best. Some biological event inevitably will reactive the cells harboring the virus and the virus producing cycle will begin anew. Since the immune response to HIV infected cells is unpredictable, it is best to circumvent the immune response all together. Simple natural products can be used to trick the HIV infected cells into self-destruction. Some things “too good to be true” are actually true.

One of the most important proteins in a damaged cell is called p53. In the scientific literature, p53 is known as a “tumor suppressor”. When a cell is stressed by environmental factors, e.g. it is exposed to a high concentration of free oxygen radicals, extreme cold/heat, or chemicals/radiation/UV light that can damage DNA, p53 is induced by cell factors called sensors. P53 is a wonderful protein. It can shut down the cell cycle at both the G1 and G2 checkpoints. Picture a bicycle wheel slowly revolving. Now throw two sticks into the spokes in different places. The wheel will come to an abrupt stop. This is what happens to the cell cycle when p53 is induced. The cell rapidly self-destructs. Invariably, cancer cells have mutations in p53 which neutralize its ability to induce programmed cell death. The mutations in p53 release the brake, so to speak, and allow the cancer cells to continue their replication cycle.

The synthesis of the HIV virus places an extreme stress on infected cells, yet they do not necessary die. P53 can be induced in infected cells, but the viral protein TAT neutralizes its activity by a number of different mechanisms, including physically interacting with p53 as it is made. In the absence of p53, a major inducer of programmed cell death is eliminated, thereby providing a safe haven for further viral synthesis. Interestingly, p53 and the glucocorticoid receptor (hydrocortisone) physically interact in cells, and antagonize each other’s activity. When TAT binds and neutralizes p53, the glucocorticoid receptor is free to interact with the other viral protein VPR and to enter the nucleus. Both TAT and VPR can be released from infected cells where they can induce cell death in bystander, non-infected lymphoid cells. VPR is also present in high concentrations in viral particles, and is released when the particles fall apart in the blood and tissue spaces. We will discuss the relationship between TAT, VPR and the hydrocortisone receptor in another essay. The point to understand about the HIV virus is that the accessory proteins it produces in order to enhance its own synthesis also have important and damaging effects of their own on the immune system.

If the G1 checkpoint senses a problem, p53 is induced. P53, in turn, induces the synthesis of a host of additional proteins, including a protein called p21Cip1/Waf1 (don’t you just love scientific terminology?). P21 shuts down the cell cycle in G1. If certain other events occur, this cell cycle block will induce programmed cell death. If p53 is inactivated by mutation (cancer) or by interacting with another protein (HIV), it is reasonable to assume that p21 cannot be induced and the G1 block can not be activated. Never assume anything. P21 can be transcriptionally induced (at the gene) in the absolute absence of p53 by a number of common plant derived chemicals. These compounds include resveratrol from grapes, saponins from ginseng, and EGCG (epigallocatechin-3-gallate) from green tea. There are probably many other natural compounds that can induce the synthesis of the p21Cip1/Waf1 protein, and, ultimately, the death of HIV infected cells.

At this point, our anti-HIV therapy consists of two components. Caffeine or theophylline prevent a block in G2. This reduces the amount of HIV synthesized in T cells by 94%. Resveratrol, EGCG, and/or ginseng saponins block the infected cells in G1 preventing them from further viral synthesis. Cells that are blocked in G1 do not necessarily die. This is particularly true of lymphocytes. For example, if lymphocytes are forced to divide in the absence of the immune hormone IL-2 (Interleukin 2), they will pause at the G1 checkpoint but they do not die. This process is called anergy and it is fundamental to an immunological process called tolerance. A reduction in the synthesis of IL-2 is common in HIV infections. If these G1 paused lymphocytes are infected with the HIV virus, they aren’t going to divide or produce virus, but they won’t necessarily die either. We want these virally infected cells to self-destruct so they can be eliminated from the body. As flippant as this may seem, this is easy. An activation pathway called phosphatidylinositol-3 kinase—Akt is activated shortly after cells are stimulated to divide. If this pathway is activated normally, a cell can still be induced to pause at G1 yet remain alive. If this pathway is blocked, the activated cells will reach G1 and immediately die by the process of programmed cell death. Numerous plant products can inhibit this activation pathway and contribute to the death of HIV infected cells.

This protocol may be able to eliminate the T cell viral reservoirs from the body, but HIV infected macrophages are another story. Macrophages are white blood cells that scavenge debris and microorganisms that they find in the blood and tissues. They also play an active role in activating T and B lymphocytes. In the case of AIDS, macrophages also play a significant role in producing virus and inducing the rapid depletion of CD4 T cells in the body. Macrophage-tropic HIV virus (viruses that like to infect macrophages over T cells) usually predominate early in an infection. Both macrophage and T cell HIV viruses contribute to the decline in CD4 T cells in AIDS. We can block HIV synthesis in T cells, but can we use the same compounds to block HIV synthesis in macrophages? The answer is no! Macrophages do not divide once they mature. However, there are other treatments that can not only reduce viral synthesis in macrophages, but prevent macrophages from killing non-infected CD4 T cells as well. This is the topic of yet another forthcoming essay. Please read the posted essay “The Day AIDS Ceased to be Fatal”. It contains some hopeful information about stopping the spread of the virus—within days upon the initiation of treatment.

In addition to the use of G1 and G2 blockers, other simple compounds can also be used to reduce the synthesis of the HIV virus. These include the minerals selenium and zinc, and the anti-oxidant alpha lipoic acid. Virtually all HIV infected people are deficient in selenium and zinc—two very important minerals that control the level of oxidant stress in cells. Oxidant stress increases the synthesis of the HIV virus. All HIV infected cells produce a large amount of free radicals, molecules that rapidly react and damage other molecules. Anti-oxidants such as vitamin C and vitamin E control the spread of these radicals, but not as effectively as alpha lipoic acid (ALA). ALA, alone, can completely shut down the synthesis of the HIV virus in T cells by blocking the activation of an important genetic control factor called nuclear factor kappa beta (NF-kappaB). Green tea, and resveratrol can also block the activation of this factor. These studies were conducted in a laboratory culture dish. In the real world, the human body, it is unlikely that ALA, alone, could control the spread of the HIV virus. However, it is highly likely that ALA, used in conjunction with caffeine and G1 blockers, could have a potent anti-HIV effect in the body.

There is one major caveat to this treatment protocol. Caffeine is not harmless if taken in large amounts. In order for these compounds to be effective against the HIV virus, they must reach specific concentrations in the blood and lymph fluid. Although the thought of treating AIDS with red wine and green tea (which contains a great deal of caffeine) sounds attractive, it is an unrealistic treatment protocol. Orally ingested caffeine, red wine, ginseng, or green tea will not deliver the therapeutic phytochemicals into the blood in high enough concentrations to be therapeutically useful against someone with even a moderate viral load. The phytochemicals must be injected subcutaneously (under the skin) or absorbed through the skin. We prefer the latter for reasons to be discussed in yet another essay. In this forthcoming essay, we will teach the method by which these compounds can be prepared and introduced into the lymph fluid that bathes every cell in the body. This fluid flows through the lymphatic ducts to the lymph nodes, the primary site of HIV viral synthesis. The transdermal introduction of these compounds into the body will allow them to stay in the body for longer periods of time and at higher concentrations than if they were ingested or injected intravenously. If you want an example, consider the stability of ibuprofen administered orally or through the skin in the form of a gel. If 600 mg ibuprofen is given orally, it is completely eliminated from the body in 24 hours. When topically applied, 99.5% of the ibuprofen remains in the tissues after 24 hours. Only 0.5% can be detected in urine. The topical delivery of drugs and natural phytochemicals is one of Grouppe Kurosawa’s greatest technical strengths. On a good day, we actually believe we know what we are doing.

One further comment. It should be obvious that the p53 mutation that exists in most cancers could be overcome by phytochemicals that induce the synthesis of the G1 blocker p21Cip1. This is true. Resveratrol, ginseng, EGCG and a host of other phytochemicals can all induce apoptosis in a wide diversity of cancer cells, including breast and prostate cancer. Preparing these compounds so they can be used therapeutically is another story and a topic for another essay.

Stay tuned to this web site for further information. The next scheduled essay will discuss how quercetin, a common flavonoid found in many foods (and sold in semi-purified form in health food stores), can inhibit the activity of the VPR protein (thereby preventing widespread immunosuppression), reduce the activity of HIV reverse transcriptase by 100% (thereby PREVENTING infection of new cells) at concentrations of 1-2 micrograms/ml, and reduce overall HIV synthesis by 80% in cell lines in concentrations as low as 40 microM.

Key References

1. Zhu, Y., Gelbard, HA, Roshal, M., Pursell, S, Jamieson, BD, Planelles, V. Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes. J. Virology 75:3791, 2001.

This article is available in its entirety on Medline.

2. Liang, B, Jiang, S., Zhang, Z., Inserra, P., Lee, J., Solkoff, D., Watson, RR. Anti-inflammatory effects of theophylline: modulation of immune functions during murine leukemia virus infection. Immunopharmacol. Immunotoxicol. 23:307, 2001.

This article is available in abstract form on Medline.