Grouppe Kurosawa: HIV - Green Tea as a Non-toxic Substitute (print)

Green Tea as a Non-toxic Substitute for HIV Protease Inhibitors

No, this isn’t a joke. In our essay identifying caffeine as an inhibitor of HIV synthesis in T cells, we mentioned that green tea could be used to block HIV infected cells in the G1 phase of their cell cycle, thereby promoting their death by a process called apoptosis or programmed cell death. In this essay, we will explain how green and black teas can be used as a treatment for HIV infections. In addition, we will present evidence that the HIV protease inhibitors presently on the market do not function as intended by their manufacturers.

Many viruses encode a protease or protein-cleaving enzyme in their genetic code. This enzyme is necessary to process newly made viral proteins so they can be packaged properly in the viral particles. When an infected cell starts producing virus, many of the viral proteins are made as pre or proproteins. These proteins must be cleaved into smaller, biologically active fragments in order for the viral particle to be infectious. Many biotech and pharmaceutical companies are developing protease inhibitors specific for a host of viruses, including the common cold virus, hepatitis B and C, and of course HIV. The rationale is that a viral particle released from an infected without proper proteolytic cleavage or processing is non-infectious and therefore innocuous. This is true for most viruses, but it is completely untrue for the HIV virus.

Some years ago, a small scientific study was conducted on the infectious nature of the HIV virus, and published in the international scientific journal Science (1). 66 patients with Centers for Disease Control stage I to IVC1 infections were studied by quantitative competitive polymerase chain reaction (QC-PCR) assays and endpoint dilution cultures. As estimated by the highly sensitive PCR technique, the scientists found the HIV-1 RNA ranged from 100 to 22,000,000 copies per milliliter. These numbers correspond to 50 to 11,000,000 viral particles per milliliter. In the second part of the experiment, they took the infected blood (the serum) and conducted what is called a limiting dilution. This is a simple procedure whereby serum is diluted by a factor of 2, 5, 10 (whatever) into small test tubes. For example, a small amount of serum, say 1 milliliter or CC, is mixed into 1 milliliter of cell growth media. When properly mixed, 1 milliliter of this solution, which now contains half the amount of virus as the original serum sample, is added to 1 milliliter of cell growth media. This process is repeated over and over until the amount of virus in the last dilution is theoretically very small. This is a 2 fold dilution because the virus concentration is being reduced by 50% at each step. Assume the scientists had 100 tubes containing virus. In order to detect actual live (infectious virus), they take each tube of growth media/virus and add it to a separate culture dish containing human lymphoid cells. Scientists can’t tell if a virus is alive or dead by looking at it. In fact, the virus is so small they have to kill it in order to see it in a high power electron microscope. If the cultured cells start making virus, it means at least one viral particle was alive when the culture media was added to the cells. If a serum sample contained a large amount of infectious virus, the scientists may have had to dilute the serum sample a thousand times before they found a dilution that finally contained no live virus. In this experiment, they found only 1 viral particle in 60,000 was actually infectious. The other viral particles were probably mutated to the extent that they could not replicate in the human cells. This is a particularly important observation because the scientists confirmed that the level or titer of viral RNA in the blood correlated with both the CDC disease stage and the CD4 T cell count. Yet, in the sample that contained 11,000,000 viral particles/milliliter, only 183 viral particles would be expected to be infectious. These numbers are surprising, but not that difficult to believe. The viral enzyme reverse transcriptase that converts viral RNA into DNA is very, very error prone, probably more so than ever expected. This study and its conclusions speak volumes about the nature of the immunosuppression induced by the HIV virus. HIV is probably the only virus that is as dangerous “dead” as it is alive.

Scientists have traditionally assumed that inhibitors of the HIV viral protease would produce non-infectious virus. Since non-infectious viruses are “theoretically” harmless because they cannot reproduce, these inhibitors were considered the “best bet” for designing an effective HIV treatment protocol. They were wrong on all counts. If a protease inhibitor was highly specific, in that it only inhibited the HIV protease and no other enzyme, it would be relatively worthless as a treatment for AIDS. The previous study found that only 1 in 60,000 viral particles were infectious anyway (this study was conducted in 1993 before protease inhibitors were available). Yet, we cannot escape the fact these protease inhibitors do reduce the viral RNA content in the blood to very low levels. In addition, the protease inhibitors dramatically increased the number of CD4 cells in the blood. These results were not expected by the scientists conducting the early protease inhibitor studies. A protease inhibitor should not prevent infected cells from secreting virus. It simply insures that each secreted viral particle is non-infectious. Since every particle, alive or not, contains RNA, the concentration of viral RNA should not decrease in the blood. Yet, this is exactly what happens. The increase in CD4 T cells was also confusing and set off a heated scientific debate about the turnover of CD4 cells in the body. The initial interpretation of the data was that the CD4 T cell population was turning over rapidly because the HIV virus was rapidly killing cells. When viral synthesis stopped, there was a rebound effect of newly produced CD4 T cells. Other studies found this was not true—the CD4 T cell population was not turning over abnormally. Finally, there is an explanation for all this confusing data.

The HIV protease inhibitors may indeed inhibit the HIV protease, but this is therapeutically irrelevant. These inhibitors are not HIV-specific. They also inhibit the chymotrypsin enzyme activity of the proteasome complex in infected and non-infected cells (2-4). This complex is a critically important regulator of protein turnover in the cell. When it is inhibited, proteins that should have been degraded remain active. Also, proteins that depend on the proteasome enzymes for proper processing are unable to do so. Some examples. First, it is estimated that 30% of all proteins made in a cell are defective. These defective proteins must be degraded as soon as possible. This degradation is done by the proteasome. Second, many proteins are designed to have a very short life span in a cell. The p21 G1 cell cycle blocker discussed in our caffeine/HIV essay is a case in point. This protein is rapidly induced if cellular conditions dictate it, and equally rapidly degraded thereafter by the proteasome. The same is true of p53, and p27, other cell cycle control proteins. If the proteasome is inhibited, these proteins will accumulate and destroy the cell. If the cell is a cancer cell or an HIV infected cell, this is good. However, if the cell is a normal muscle cell in the heart, you have a serious problem on your hands. Third, the proteasome processes normal and viral proteins by cleaving them into smaller pieces, much like the HIV protease. If the proteasome is inhibited, these proteins cannot be activated and this can have an equally deleterious effect on normal cells. Many proteasome inhibitors are now in development. As a short term treatment for a specific disease, such as cancer, proteasome inhibitors are promising drugs. For long term care, as in the treatment of HIV, they are extremely toxic.

A few years ago, scientists found that when HIV protease inhibitors inhibited the chymotrypsin activity of the proteasome, the HIV virus could no longer be secreted from infected cells (5). Apparently, the proteasome processes some of the viral proteins and allows them to mature. When the proteasome is inhibited, the viral particles do not mature and are not released from the cell. This data now explains why HIV protease inhibitors reduce the level of viral RNA in the blood. At least, this is one explanation. Proteasome inhibited cells probably also die from apoptosis or programmed cell death (6). This is an excellent way to eliminate the HIV viral reservoirs from the body. Those cells that remain intact are unable to make virus because transcription or gene activating factors, such as NF-kappaB, cannot enter the nucleus to bind DNA. The inhibitor of NF-kappaB, IkappaBalpha, which normally binds and sequesters NF-kappaB in the cytoplasm or interior of the cell, is normally degraded by the proteasome. This liberates NF-kappaB from its constraints and allows it to migrate into the nucleus. If the proteasome is inhibited, this isn’t going to occur. In addition to activating the HIV gene, NF-kappaB also activates numerous pro-inflammatory hormones, such as tumor necrosis factor-alpha (TNF-alpha), an extremely proinflammatory immune hormone that is thought to “drive” the progression of the HIV infection. In addition to activating genes, NF-kappaB can also turn off important genes, such as those coding for proteins that INHIBIT apoptosis or programmed cell death. When NF-kappaB activation is prevented by an inhibitor of the cellular proteasome complex, the HIV virus gene becomes less active, proinflammatory immune hormones cannot be synthesized, and the infected cell dies by the process of programmed cell death. If you are trying to kill a virally infected cell or a cancer cell, this is an ideal treatment protocol as long as the treatment does not continue long term. Remember, HIV proteasome inhibitors are inhibiting the proteasome complex in normal as well as infected cells. This cannot be allowed to continue long term or the normal cells will become dysfunctional and die.

Now, how do HIV proteasome inhibitors increase the level of CD4 T cells in the blood? The answer probably also lies with inhibition of the proteasome complex. Numerous HIV viral proteins bind the CD4 molecule in the infected cell and cause it to be degraded in the proteasome (7). When the proteasome is inhibited, this isn’t going to occur. In reality, the increase in CD4 T cells that is found shortly after initiation of treatment with protease inhibitors may not reflect an actual increase in production of new cells. It may simply be an increase in the density of CD4 molecules on the membranes of previously existing cells.

People who have been treated with HIV protease inhibitors for one or more years develop a host of additional medical problems, including hypertension, diabetes, heart disease, high cholesterol levels, bone deterioration, kidney and liver diseases, and disorders of fat metabolism. It is highly likely that all these problems can be traced to inhibition of the proteasome complex in normal, non-HIV infected cells. Many of these defects are characteristic of Cushings Disease, a disease characterized by high levels of the anti-inflammatory hormone hydrocortisone in the blood. One of the consequences of long term proteasome inhibition is an increase in hydrocortisone sensitivity in virtually every cell in the body. The fail-safe mechanisms built into the human body are so complex and varied that they make the space shuttle appear like an amateur science project. Glucocorticoid (hydrocortisone) excess is a case in point. When a human is stressed, his/her hydrocortisone concentration in the blood increases dramatically. Chronically high levels of hydrocortisone, such as that manifested by chronic stress, are dangerous. Hydrocortisone is a catabolic hormone—it breaks down tissues in response to stress situations. In order to protect against chronically elevated concentrations of hydrocortisone, Nature has evolved a unique method of limiting the danger posed by excessive hydrocortisone concentrations. Hydrocortisone breaks down tissues by activating a series of enzymes, one of which is the proteasome complex. Hydrocortisone stimulates the synthesis of various proteins that enhance proteasome activity dramatically. Hydrocortisone breaks down bone, muscle and lymphoid tissues by activating their respective cellular proteasomes. The degraded proteins are released into the blood as peptides and amino acids where they can be used by other tissues. One of the proteins degraded by the proteasome is the glucocorticoid receptor itself (8). This is a classic feedback inhibition response. Elevated concentrations of hydrocortisone activate the proteasome, which degrades the glucocorticoid receptor. Since hydrocortisone can’t do much in the body without a cellular receptor, this limits the potential cellular damage that hydrocortisone can cause. HIV protease inhibitors, acting as inhibitors of the proteasome complex, block this feedback inhibition. As a result, normal concentrations of hydrocortisone in the blood can have an enhanced catabolic effect on every tissue in the body because these tissues cannot terminate the hydrocortisone response. The brake petal has been disabled, and the car is building up speed. If a person taking HIV protease inhibitors finds himself/herself under stress, the elevated hydrocortisone levels in their blood will have a tremendously enhanced ability to destroy their tissues even though the actual concentration of hydrocortisone in the blood is not excessively high. The result is hypertension, diabetes, the breakdown of bone and muscle, the formation of abnormal fat deposits, and, of course, continued immune dysfunction. The HIV protease inhibitors stopped the progression of the viral disease only to impose a host of new chronic diseases upon the drug recipients. You just can’t win. Or can you?

What does green tea have to do with all this? In a word, everything. In the caffeine/HIV essay, we said that the polyphenol EGCG found in green and black tea can block a cell in G1 and induce programmed cell death. We suggested that green tea, which also contains a great deal of caffeine, might be a good treatment for HIV. This sounds ridiculous, and too good to be true, but it is neither. EGCG and similar molecules inhibit the proteasome complex at concentrations that can be attained by drinking tea (9). Tea is a fascinating plant from many different perspectives. Epidemiological studies have shown that drinking tea has a protective effect against human cancers. Animal studies have also found that tea polyphenols can suppress the formation and growth of skin, lung, liver, esophagus, and stomach cancer. This suppression is directly related to the ability of EGCG to inhibit, in intact cells, the proteasome at a concentration of 1-10 microM. As we have stated before, inhibiting the proteasome blocks the cell cycle in the G1 phase and induces programmed cell death in actively growing tissues, such as cancer cells and HIV infected cells. Green tea has never been found to be toxic in any way to normal tissues. Although perhaps not as potent as synthetic proteasome inhibitors (which is good), tea may be able to stop the progression of the infection while simultaneously inducing HIV infected cells to die by programmed cell death. Tea could be combined with other natural compounds, such as quercetin, that also inhibits the synthesis of the HIV virus in addition to blocking the biological activity of the immunosuppressive VPR protein. These natural medicines are safe and potentially very effective if consumed on a regular basis. Synthetic medicines, regardless of their toxicity, are of no use to most of the HIV infected people in the world. They can’t afford modern medicine, but they can certainly afford traditional medicines such as tea, quercetin, resveratrol and caffeine. What other choice do they have?

To date, no scientific studies have been conducted on the use of natural anti-HIV medicines on humans. We believe it is time these studies were initiated.

Key References

1. Piatak, M., Saag, MS, Yang, LC, Clark, SJ, Kappes, JC, Luk, KC, Hahn, BH, Shaw, GM, Lifson, JD. High Levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 262:1585, 1993.
2. Andre, P, Groettrup, M, Klenerman, P, de Giuli, R, Booth, BL, Cerundolo, V, Bonneville, M, Jotereau, F, Zinkernagel, RM, Lotteau, V. An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proc. Natl. Acad. Sci. 95:13120, 1998.
3. Schmidtke, G, Holzhutter, HG, Bogyo, M, Kairies, N, Groll, M, de Giuli, R, Emch, S, Groettrup, M. How an inhibitor of the HIV-1 protease modulates proteasome acitivity. J. Biol. Chem. 274:35734, 1999.
4. Liang, JS, Distler, O, Cooper, DA, Jamil, H, Deckelbaum, RJ, Ginsberg, HN, Sturley, SL. HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: a potential mechanism for protease inhibitor-induced hyperlipidemia.. Nature Medicine 7:1327, 2001.
5. Schubert, U, Ott, DE, Chertova, EN, Welker, R, Tessmer, U, Princiotta, MF, Bennink, JR, Krausslich, HG, Yewdell, JW. Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. 97:13057, 2000.
6. Jeremias, I, Kupatt, C, Baumann, B, Herr, I, Wirth, T, Debatin, KM. Inhibition of Nuclear Factor kB activation attenuates apoptosis resistance in lymphoid cells. Blood 91:4624, 1998.
7. Schubert, U, Anton, LC, Bacik, I, Cox, JH, Bour, S, Bennink, JR, Orlowski, M, Strebel, K, Yewdell, JW. CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway. J. Virology 72:2280, 1998.
8. Wallace, AD, Cidlowski, JA. Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. J. Biol. Chem. 276:42714, 2001.
9. Nam, S, Smith, DM, Dou, QP. Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J. Biol. Chem. 276:13322, 2001.