NF-kB in CLL

For those of you who swear by the beneficial effects of nutritional supplements, here is an article to gladden your heart.

The first of the abstracts cited below comes from M. D. Anderson. I will try to summarize the science behind it, and then go on to describe how it validates some of the food based phytochemicals we have discussed several times before on this forum.

Nuclear factor- kappa B (or NF-kB) is a most important protein complex. It is typically present in its inactive state in the cytoplasm (outer portions of the cell). It is held in this inactive state by another protein called IkB which inhibits the function of the NF-kB. What makes NF-kB interesting is this: It is possible for the complex of NF-kB and the inhibitory protein IkB to be stimulated by a wide variety of carcinogens, UV radiation, viruses, tumor promoters, inflammatory cytokines etc. Once the complex is activated, NF-kB breaks free from the inhibitory IkB, and the fragments then travel to the inside of the cell, where they enter the nucleus of the cell, and bind to the DNA.

It is now well understood that this binding of the activated NF-kB fragments to the DNA is needed for cell survival, cell proliferation, tumor invasion, and resistance to chemotherapy drugs. Activation of NF-kB regulates a whole slew of genes that are involved in cancer growth, and prevention of cell death (apoptosis). You have heard of some of these gene products before, for example Survivin, Cyclin D1, Bcl-2, VEGF etc.

Bottom line is this: NF-kB is normally inactive in resting cells. When it is activated, it can cause the cells to develop survival advantage, resist cell death, and resist chemotherapy drugs. Many types of cancer cells, including CLL cells, are shown to have very high levels of activated NF-kB, supporting this view of its importance in promoting malignancy.

I have made all this ridiculously simple, nothing about cellular chemistry is either simple or easy to understand. Those of you who are willing to spend the extra time wading through the intricacies, Here is a full text article that you may want to read.

OK. Now for the interesting stuff. Unfortunately the abstract from Bharti and Aggarwal below does not list the agents that block activation of NF-kB, and thereby provide some protection from the initiation and/or progression of cancer. The full article does, and I thought I would share some of the items on that list with you guys, save you the $30 purchase price of the article. Here are some of the more well known food based supplements that are effective in blocking the activation of NF-kB:

There are others on the list, I am just listing some of the better known ones that we have discussed before on CLL Topics.

We are all aware CLL is an indolent disease, it does not progress as fast as some of the other cancers. This is because the rate at which new cancer cells are added is only slightly higher than the rate at which they die. If the disease were more aggressive, the rate of birth would be much higher than the rate of death. Now imagine what will happen if you are able to decrease, even slightly, the rate of birth of new CLL cells, or to look at the other side of the coin, you are able to increase the rate at which CLL cells already present are induced to commit suicide. Either of these would decrease the overall rate of accumulation of the cancer cells. Because we are talking about an indolent disease, small changes can pay off big time in controlling the progression of the disease. By all means, I would recommend incorporating some of these food elements into your diet. There is also good epidemiological evidence to support their incorporation into a healthy diet. Many of them are widely used in ethnic foods that seems to reduce incidence of cancer in those regions of the world.

What makes the whole topic even more interesting is that we seem to be on the verge of discovering much more potent compounds that will be effective in blocking NF-kB activation, compounds that have little or no toxicity of any kind. The day may not be too far away when majority of newly diagnosed patients with early stage disease and few health complications are put on daily maintenance dose of a non-toxic compound that effectively keeps their CLL cell population virtually unchanged over the remainder of their natural life. Not a "cure" for CLL, but not a bad deal if you do not have to do anything more onerous than pop a pill a day to keep it under control? Sounds good to me. I expect to focus more on this in the months ahead.

Abstract:

Nuclear factor-kappa B and cancer: its role in prevention and therapy.

Cytokine Research Section, Department of Bioimmunotherapy, M. D. Anderson Cancer Center, University of Texas, Box 143, 1515 Holcomb Boulevard, Houston, TX 77030, USA.

Cancer is a hyperproliferative disorder in which invasion and angiogenesis lead to tumor metastasis. Several genes that mediate tumorigenesis and metastasis are regulated by a nuclear transcription factor, nuclear factor kappa B (NF-kappaB). A heterotrimeric complex consisting of p50, p65, and IkappaBalpha, NF-kappaB is present in its inactive state in the cytoplasm. When NF-kappaB is activated, IkappaBalpha is degraded and p50-p65 heterodimer is translocated to the nucleus, binds the DNA (at the promoter region), and activates gene. Research within the last few years has revealed that NF-kappaB is activated by carcinogens, tumor promoters, inflammatory cytokines, and by chemotherapeutic agents. The activation of NF-kappaB can suppress apoptosis, thus promoting chemoresistance and tumorigenesis. Interestingly, however, most chemopreventive agents appear to suppress the activation of the NF-kappaB through inhibition of NF- kappaB signaling pathway. These chemopreventive agents also sensitize the tumors to chemotherapeutic agents through abrogation of NF-kappaB activation. Overall, these observations suggest that NF-kappaB is an ideal target for chemoprevention and chemosensitization. This article reviews evidence supporting this hypothesis.

The role of NF-kappaB/IkappaB proteins in cancer: implications for novel treatment strategies.

Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555, USA.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors are involved in multiple cellular processes, including cytokine gene expression, cellular adhesion, cell cycle activation, apoptosis and oncogenesis. Constitutive activation of NF-kappaB has been described in a number of solid tumors and this activation appears to affect cancer cell survival. Inhibition of NF-kappaB has been shown to enhance the sensitivity of some cancer cell lines to antineoplastic- or radiation-induced apoptosis. Furthermore, suppression of NF-kappaB results in attenuation of cancer cachexia in a mouse tumor model. Studies are underway to further delineate the role of NF-kappaB in cancer cell survival, growth and resistance to standard chemotherapy and radiation regimens. Moreover, the effects of novel therapeutic agents which specifically target NF-kappaB proteins are currently being assessed in experimental models of cancer cell growth both in vitro and in vivo. In this review, we discuss the possible involvement of NF-kappaB in the growth of various solid tumors and potential future treatment strategies based on NF-kappaB inhibition.

NF-kB Pathway

11/15/02

The NF-kB pathway and the manner in which it interacts with cellular mechanisms is one heck of a complex area, and much jargon to get through before one can pick up little nuggets of information. Here is an article that has a lot of the information I used in trying to understand some of the salient points. Especially useful are the later sections of the article, you might want to read those sections.

Basically, NF-kB is a complex of very potent proteins that is kept in an inactive state in the cytoplasm (outer skin of the cell) under normal conditions. When the cell is stimulated in some way, that makes it necessary for the cell to protect itself from DNA damage (such as UV radiation, heat, attack by toxic chemicals etc), this is the defense mechanism of the cell that kicks into action. The NF-kB is broken free of its inhibitory tether on the cytoplasm, migrates into the nucleus of the cell, where it triggers up-regulation of anti- apoptosis genes, and down-regulation of pro-apoptosis genes. In other words, it becomes a lot harder to kill, and refuses to hear signals to commit suicide. Activated NF-kB fragments also trigger cell differentiation, so that the cell goes from a resting state to a proliferating state, making more copies of itself. All of which is necessary under normal conditions, when the cell is trying to survive an attack, and trying to make more copies of itself to go on the offensive against what ever virus, dangerous chemical and the like is attacking it.

Problem is that leukemia and lymphoma are cancers of the very immune system that is supposed to protect the body. Activation of NF-kB is out of kilter in cancer cells, a big majority of them are locked into an activated state without any real need to be in such a state. In fact, the vast majority of CLL cells have activated NF-kB pathway, and this gives them survival advantages (down regulated pro-apoptosis genes and up-regulated anti-apoptosis genes), and as a result of NF-kB activation, they also tend to get kicked into a higher rate of proliferation. Particularly interesting is that activation of NF-kB makes the cells less susceptible to "dangerous" chemicals, in this case chemotherapy drugs that we want them to react to!

1. Since simple, easily available drugs such as aspirin, Sulindac and other NSAIDs are known to inhibit the activation of NF-kB pathway, barring contra-indications such as low platelet counts and stomach ulcers, should we be taking maintenance regimes of these drugs to slow down the growth of the cancer cells and make them more prone to apoptosis?

2. Should these drugs be administered concurrently with more standard chemotherapy drugs to increase the potency of the latter? Is this why glucocorticoids, such as Dexamethasone and prednisone, anti- inflammatory drug that stop NF-kB pathway from activating, have such a dramatic effect when they are co-administered with other chemotherapy drugs or monoclonals such as Rituxan?

3. Does systemic inhibition of NF-kB pathways put the patient at risk of the infections, since the body is handicapped in not being able to fight legitimate invaders that does require an inflammatory response, and which needs the activation of the NF-kB pathway?

4. Are anti-viral medications more effective in combination with NF-kB inhibitors, especially in the case of DNA viruses such as EBV, HSV, HCV and HPV, since blocking the NF-kB pathway inhibits cell proliferation, something that these replicating viruses need?

5. The blocking of the NF-kB pathway can be done at a number of points, by different drugs. For example, NSAIDs and glucocorticoidal drugs attack this pathway at different points. Does a multi-pronged approach have a better chance of shutting down this pathway?

6. Does a maintenance therapy of NSAIDs and nutritional supplements to effectively shut down NF-kB pathway, in combination with low-dose metronomic administration of chemotherapy drugs present a relatively non-toxic method of maintaining long term control over cancer and its secondary effects?

I could think up a dozen more questions if I sat down to it, but this would be a good start. If I am getting across to you guys that there is a real buzz about this approach (inhibition of NF-kB pathway) in recent months, I have done my job. Just get to PubMed, type in NF-kB and cancer. You get literally thousands of hits, and especially interesting is that most of the publications are less than a couple of years old. This is hot stuff! More on it later, as I find out.

NF-kB Reference

11/19/02

Here is the website to end all websites on the subject of NF-kB. For those of you who have become intrigued by this area of research ( I am most certainly one of the intrigued folks), this site has excellent information. It is funded in part by the Lymphoma and Leukemia society, so some of your dollar donations went to work here. Do browse, if you have a little time:www.nf-kb.org
This link may also be accessed at http://people.bu.edu/gilmore/nf-kb/ .
If the links provided here prove inaccessible, you may want to conduct a Google search on Dr. Thomas D Gilmore Biology Department, Boston University. This resource appears to suffer from server variability but it is worth seeking out.

Nsaids - With a Twist

6/15/03

Would you like to have a small, cheap, totally non-toxic pill that you pop once a day, and know that it will keep your CLL from progressing for a long, long time? Not a cure mind you, but the next best thing. If newly diagnosed and early stage patients can stay that way indefinitely, and patients who have been through frontline therapies that got us a good remission can maintain that remission for years and years, what is not to like about that? Read on, some interesting new developments suggest this is not such a pipe dream, clinical trials based on this concept are likely to start in the next couple of months.

But, as always, I need to walk you through some of the background science before we can get to the main point. A couple of you wrote and complained that I force people to wade through all this science stuff, why can't I just give the punch line in a nice sound bite? Guilty as charged. I belong to the old school where learning is considered an active process, and real learning does not happen in passive spoon-feeding of sound bites. Fortunately for long winded people like me, you guys are pretty much a captive audience, and there is little doubt of the sincerity of your motivation! Much of the science below has been covered in detail in previous articles, it may be well worth your while to dig them out and refresh your memory. Some of the keywords you may wish to use in your search are: NSAID, Cox-1, Cox-2, angiogenesis, NF-kB, CD23.

NSAID background:

We are all familiar with NSAIDs (Non-Steroidal Anti-Inflammatory Drugs), the most common one of the family being good old aspirin. Almost all of us pop a few of these from time to time, to relieve pain and reduce inflammation.

We have learnt in recent times that a there is a strong relationship between chronic inflammation and cancers of many sorts. Inflammation is one of the body's defenses when it is under attack; for example the site of a bug bite gets inflamed and red, the inflammation is the signal for the body to get the immune system cells activated and rush to the site of the attack, to deal with the foreign proteins injected into your skin by the bug that bit you. But if the inflammation is chronic and continues for a long time, there can be negative consequences. As in the story of the boy who cried "Wolf!" too many times, pretty soon the immune system stops reacting appropriately when the alarm is sounded. The inappropriate response can be either getting lazy and not getting out the troops and killing the enemy; or the reverse of the coin, going berserk and attacking perfectly good cells of the body for no cause. The first is an example of how a confused and lazy ("anergic") immune system lets cancer cells establish a foothold, without killing them on the spot. The second is an example of immune system attacking the body's own cells in various forms of autoimmune diseases, such as AIHA (autoimmune hemolytic anemia), where the red blood cells are attacked and killed by the immune system. This then is the connection between chronic inflammation and cancer, the subversion of the finely tuned machinery of the immune system.

Study of inflammation and how it influences different systems in the body has therefore become an important aspect of oncology. We have learned a great deal about why NSAIDs like aspirin, Ibuprofen etc are so effective in reducing inflammation and associated pain. In this context, Cox-1 and Cox-2 are two enzymes that have become famous in the popular press. Cox-1 is expressed through out the body, all the time, and it is an important enzyme for protecting things like the lining of your stomach. Cox-2, on the other hand, is expressed under special circumstances only, such as when there is inflammation. This is the basis of the popularity of new drugs like Celebrex and Vioxx, over much cheaper and older drugs like plain aspirin. Aspirin inhibits both Cox-1 and Cox-2 enzymes, and therefore if you are in the habit of popping aspirins all the time (never mind if they are "safety-coated"), you are running a significant risk of stomach ulcers caused by inhibition of the protective effects of Cox-1 on the lining of your stomach. Celebrex and Vioxx are the new generation of NSAIDs, the so-called selective Cox-2 inhibitors. The idea is that by selectively inhibiting only Cox-2 enzyme, they can be used safely over long periods of time for inflammatory conditions such as arthritis, since it is the Cox-2 that is important in controlling inflammation.

There have been recent reports that the "selective" Cox-2 inhibitors are not so selective after all, that they too mess with the Cox-1 enzyme to some degree and therefore you are not completely protected against stomach ulcers and the like. Both Cox-1 and Cox-2 are enzymes that control a complex cascade of "prostaglandins", and scientists are still trying to figure out all the details.

Much more specifically, the part of inflammation that interests us as cancer patients is something called the NF-kB ("Nuclear Factor kappa B") pathway, which is triggered when the body goes into an inflammatory mode. Basically, NF-kB is a complex of very potent proteins that is kept in an inactive state in the cytoplasm (the body of the cell outside the nucleus) under normal conditions. When the cell is attacked in some way, that makes it necessary for the cell to protect itself from DNA damage (such as UV radiation, heat, attack by toxic chemicals etc), this is the defense mechanism of the cell that kicks into action. The NF-kB is broken free of its inhibitory tether on the cytoplasm, migrates into the nucleus of the cell, where it triggers all sorts of defenses against dying. In other words, a cell whose NF-kB pathway has been turned on is a lot harder to kill, and it refuses to hear signals from the rest of the body to commit suicide.

Activated NF-kB fragments also trigger cell differentiation, so that the cell goes from a resting state to a proliferating state, making more copies of itself. All of which is necessary under normal conditions, when the cell is trying to survive an attack, and trying to make more copies of itself to go on the offensive against what ever virus, dangerous chemical and the like is attacking it.

But it now known that the switch for this pathway is stuck in the "on" position for large majority of cancer cells, including CLL cells, making it possible for the cancer cells to survive attack by chemicals (such as chemotherapy drugs) and death signals from the body (apoptosis). It also contributes to greater proliferation rates for the cancer cells. This is definitely the case with CLL, where a vast majority of the malignant cells exhibit activated, turned on, NF-kB pathway. This gives them survival advantages (down regulated pro-apoptosis genes and up-regulated anti-apoptosis genes), and as a result of NF-kB activation, they also tend to get kicked into a higher rate of proliferation. Particularly interesting is that activation of NF-kB makes the cells less susceptible to "dangerous" chemicals, in this case the very chemotherapy drugs that we want them to react to and die!

A number of drugs are known to inhibit the NF-kB pathway. Among them are the NSAIDs like aspirin and Celebrex, chemotherapy drugs like Prednisone and Thalidomide, "neutraceuticals" like curcumin from turmeric, catechols from green tea, resveratrol from red wine etc. The trick is finding a potent and strong NF-kB pathway inhibitor that does not have dangerous side effects. Long term use of NSAIDs can cause stomach ulcers, others such as thalidomide are also anti-angiogenesis drugs that can interfere with wound healing, steroids such as prednisone have many side effects, and neutraceuticals such as curcumin are not easy to get going through out the body, they have poor "bioavailability profile", for most of them it is basically in at the mouth and out at the other end. Now for the punch line:

If you have read "Through the looking glass", or heck just preened in front of the mirror recently, you are aware that objects and their mirror images are not exactly identical, they are, well, mirror images of each other. The same is true of chemical molecules as well. Sufficiently complex molecules have mirror image versions of themselves that are almost, but not quite identical. The two forms are called "R" and "S" enantiomers. There are "R-Ibuprofen" and "S-Ibuprofen" molecules, for example. The two are identical in every way, except for the fact that they are each other's mirror image. Almost always, the drug you get when you buy Ibuprofen is a mixture of the R and S forms, since it costs too much to separate them out. And since the two forms are so similar, they are expected to behave the same way, so why bother.

As it turns out, not quite the same behavior. It is found that the "S" form of many NSAID drugs is what is responsible for the Cox inhibition, and control of the prostaglandin cascade processes initiated by these enzymes. The "R" form of the drugs does very little Cox-1 or Cox-2 inhibition. But, and here is the punch line, what the R-NSAIDs do very well is inhibit NF-kB pathway, a whole lot better than the mixture of R + S versions in the original NSAID. Aha! An effective way to control the specific pathway that would otherwise makes tumor cells hard to kill, and allows them to multiply like rabbits! Now we are cooking!

I would expect that not all R-NSAID drugs are equally good as cancer therapy drugs. In addition to Ibuprofen I mentioned a couple of time above, another NSAID which is getting some attention is "R-Flurbiprofen". It is already in clinical trials for prostate cancer. (Why prostate cancer? Some one told me this is an "easy cancer" to monitor, since all they have to do is a blood test and measure the PSA number, to get a good fix on the state of the cancer. No need for bone marrow biopsies, CAT scans to measure size of lymph nodes, spleen etc. Sigh... Not only did we have to get cancer in the first place, we had to get one that is tough to measure, so we are never the first ones to get a shot at the new developments). PubMed abstracts for both R-Ibuprofen and R-Flurbiprofen are below, suggesting the unique behavior of these particular mirror image molecules in controlling NF-kB activation. Also below are URLs to some useful reference articles, as well as the URL to the website of Myriad Pharmaceuticals, the owner of R-Flurbiprofen. Rumor has it this particular one may make it into CLL clinical trials real soon.

URLs:

1) Here is a terrific article with lots of detail on the connection between chronic inflammation and cancer. The full article is available, free of charge: Chronic Inflammation and Cancer.

3) Here is a link to an article in which I summarize the connections between viral drivers, cancer proliferation, NF-kB pathway, etc.: Anti-Inflammatory Pain Relievers and Viruses.

Abstracts:

Inhibition of NF-kappaB and AP-1 activation by R- and S-flurbiprofen.

Tegeder I, Niederberger E, Israr E, Guhring H, Brune K, Euchenhofer C, Grosch S, Geisslinger G. Zentrum der Pharmakologie, Johann Wolfgang Goethe-Universitat, Frankfurt, 60590 Frankfurt am Main, Germany.

R-flurbiprofen is considered the 'inactive' isomer of the nonsteroidal anti-inflammatory drug (NSAID), flurbiprofen, because it does not inhibit cyclooxygenase (COX) activity. However, previous studies have revealed that it has antinociceptive and antitum or effects not due to epimerization to the cyclooxygenase-inhibiting S-isomer. Here, we show that R-flurbiprofen has additional anti-inflammatory activity comparable with that of dexamethasone in the zymosan-induced paw inflammation model in rats. Different criteria suggest that the observed effects are mediated at least in part through inhibition of NF-kB activation: R-flurbiprofen inhibited i) LPS-induced NF-kB DNA binding activity in RAW 264.7 macrophages, ii) translocation of the p65 subunit of NF-kB into the nucleus of these cells, and iii) zymosan-induced NF-kB-dependent gene transcription in the inflamed paw and spinal cord of rats. S-flurbiprofen produced similar effects but was less potent. In addition, R-flurbiprofen inhibited DNA binding activity of AP-1, another key regulatory transcription factor in inflammatory processes. Because R-flurbiprofen does not cause gastrointestinal mucosal damage or other side effects associated with long-term NSAID or glucocorticoid use, it might be a useful drug in inflammatory or other diseases in which increased or constitutive NF-kB and AP-1 activation are involved in the pathophysiological processes.

Modulation of transcription factor NF-kappaB by enantiomers of the nonsteroidal drug ibuprofen.

Scheuren N, Bang H, Munster T, Brune K, Pahl A. Department of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nurnberg, Erlangen, Germany.

1. The nonsteroidal drug ibuprofen exists as an R(-)- and S(+)-enantiomer. Only the S(+)-enantiomer is an effective cyclo-oxygenase inhibitor, while the R(-)-enantiomer is inactive in this respect. Thus the molecular mechanism by which R(-)-ibuprofen exerts its anti-inflammatory and antinociceptive effects remains unknown. 2. In this study the effects of the enantiomers of ibuprofen on modulation of transcription factors have been examined with electrophoretic mobility-shift assay (EMSA), transient transfection experiments, confocal immunofluorescence and nuclear import experiments, to determine their selectivity and potency as inhibitors of the activation of transcription factor nuclear factor-kappaB (NF-kappaB). 3. R(-)-ibuprofen (IC50: 121.8 microM) as well as the S(+)-enantiomer (IC50: 61.7 microM) inhibited the activation of NF-kappaB in response to T-cell stimulation. The effect of ibuprofen was specific because, at concentrations up to 10 mM, ibuprofen did not affect the heat shock transcription factor (HSF) and the activation of NF-kappaB by prostaglandin E2 (PGE2). Very high concentrations of ibuprofen (20 mM) did not prevent NF-kappaB binding to DNA in vitro. Immunofluorescence and nuclear import experiments indicate that the site of ibuprofen action appeared to be upstream of the dissociation of the NF-kappaB-IkappaB-complex. 4. Our data raise the possibility that R(-)-ibuprofen exerts some of its effects by inhibition of NF-kappaB activation.

NSAIDs - Involvement of the NF-kB Pathway

6/15/03

I would like to follow up the preceding article with some additional references which may have the effect of expanding the circle of involvement and importance of the NF-kB pathway.

Recently, the ZAP-70 gene and its associated protein of the same name have been addressed as a prognostic factor. A fellow member mentioned in her message on ZAP-70 that “One of the things that I find interesting (and maybe I'm just being negative!) is that within ten years of initial diagnosis, 40% of the Zap-70 negative (mutated) had had treatment.” She further added a disclaimer that Terry Hamblin said that “only a small percentage of mutated patients would need treatment.” Consistent with the mysteries of CLL, we have the waterfront covered with two widely divergent views of the merits of ZAP-70 as a prognostic indicator. The question is: why is this so?

Based on the work of researchers in the US and Switzerland there may be an answer that is linked to the NF-kB pathway. Celgene has a division called Signal, and on their website at signalpharm are four pathways diagrammed out so we can wee the relationships that are of interest to Signal. The NF-kB pathway and the T-Cell pathway show the NF-kB detaching from the cytoplasm and going into the cell with the resulting regulation of the cytokine genes. Chaya referred to this when she wrote: “trigger [s] all sorts of defenses against dying.” One of the cytokines triggered in the NF-kB pathway is IL-6, which is implicated in the production of malignant lymphocytes in the bone marrow, something that is at the root of our condition. Also, COX-2 is triggered - something else you referenced in her article.

Looking at the T-Cell pathway, two receptors shown are the CD3 and CD28 TCRs (T-Cell Receptors). These are the same receptors that Xcyte Therapies uses in its Xcellerate product to boost the activity of the T-Cells. In the described Signal pathway, though, is p56Lck, a protein tyrosine kinase which regulates the signal transduction of the TCRs after engagement of the TCRs with the pathogen. Where p56Lck is deficient, the immune response against pathogens is dramatically impaired. In other words, the TCRs can’t function properly against the pathogen because a needed helper (p56Lck) is missing or damaged. This impairment of the TCRs apparently alerts the ZAP-70 protein (something like the signals sent out by a wounded swimmer in the water that a shark picks up and homes in on) along with JNK1,2 – see below, and the IKK2 kinase to degrade the Kappa B protein, which otherwise keeps NF-kB in the cytoplasm, and allows the NF-kB to migrate into the cell and to start producing its cytokines and inflicting damage on us CLL’ers..

In Switzerland, a researcher named Margot Thome and her colleagues are investigating a similar track. The web site for her work is Thome.html which describes the identification and characterization of a family of proteins called vE10, Bcl10 and Carma1, which they believe play an important role in the regulation of NF-kB induction in lymphocytes. In her analysis she also links up the sequence of p56Lck, the JNK pathway (1,2) and ZAP-70 as triggering NF-kB, but what she found additionally was the protein Carma 1 can act with Bcl10 and other signaling proteins to activate the NF-kB pathway (i.e., cause NF-kB become active) when the TCR engages the pathogen. Same plot as the Celgene approach, but with additional actors. In the graphic on her site she fills in some of the pieces missing in the Celgene pathway graphic. So how does this relate to ZAP-70 and NF-kB and Zap-70’s use as a prognostic indicator? From the looks of it ZAP-70 isn’t alone in the cascade of cells and signaling processes, so depending on what else is going on in the system, ZAP-70 can either be implicated in treatment or not, as we read earlier.

Now back to a key point. In a healthy person CD3 and CD28 TCRs are supposed to attack the pathogen and kill it; but in a CLL patient there is the situation where CD3 and CD28 TCRs activate the NF-kB pathway through improper signal transduction (both through the down regulation of p56Lck and the collection of proteins (vE10, Bcl10 and Carma1 among others) which allow for the presence of NF-kB, (most likely through the degradation of Kappa B) , causing the patient’s immune system to do exactly the opposite of what the patient wants. In an article Chaya posted on March 20th, the authors wrote “Inflammatory cells secrete a large number of cytokines and chemokines that can promote the outgrowth of neoplastic cells.. Examples of tumor cell cytokine dependence in human disease are the growth dependence of .. B-cell leukemias on the inflammatory cytokines IL-6 and IL-15.. T cells are major sources of cytokines that promote outgrowth of preneoplastic and malignant cells”.

Inflammation is using two pathways to activate the NF-kB gene-signaling pathway to beat up on us. The first pathway is inducing a cascade of chemicals to inhibit the Kappa B protein and thereby activate the NF-kB gene-signaling pathway. This pathway does not use the T-Cell’s chemistry; the second pathway uses the T-Cell’s chemistry to inducing a cascade of chemicals that result in the activation of the NF-kB gene-signaling pathway. Both pathways down regulate the Kappa B inhibitor protein IKK-2.

So, it seems that there is ample evidence, although not fully understood but under investigation and becoming better understood, that reducing the production of selected inflammatory factors such as cytokines, prostaglandins will permit the T-Cells and TCRs to do their work in a better fashion, thereby short-circuiting the NF-kB activation. Given the choice, it may be possible that following a reduced inflammation approach, the patient can use his or her immune system to fight the malignancy. Perhaps antibody therapy will be needed to reduce the tumor load in certain circumstances. But, contrasted with other approaches involving immuno- and myelosuppression, the payoff may be worth it if only to keep one’s options open for a longer period.

On a related note, Salmedix’s anti-inflammatory drug SDX-101 trial at UCLA (Dr. Peter Rosen, PI) should have early trial data ready near the end of September 2003.

Singulair - Effect on CLL

6/22/03

Three months ago my WBC was 70.5. At that time I started taking Singulair which is an NSAID which works by inhibiting leukotriene (lipooxygenase pathway) production and prostaglandin PGE2 (cyclooxygenase pathway) production. (While the COX-2 inhibitors are the usual means to mediate the cyclooxygenase pathway, I am told that the leukotrienes do mediate PGE2. If anyone can shed further light on this situation you will have my full attention) Yesterday’s blood results showed a WBC of 62.7 – a decrease of circulating lymphocytes of 11%. The only drawback appears to be a reduced platelet count at 127 down from 139. Since I have had a previous platelet misreading from the lab used by my local onc, and since I have been taking an anti-histamine (over the counter Claritin-D) which can induce lowered platelets (so can NSAIDs), and since my clotting process appears to be fine (had two minor surgeries with stitches two days ago which are healing nicely), at this moment I cannot say for sure that the platelet situation is an issue or if it is, what its source(s) might be. Will need another 90 days and another blood draw to collect more data.

A few more thoughts on why Singulair might be the cause for the reduction in lymphocytes as opposed to something else. The readings given by the lab for monocytes, basophils, and hematocrit are at their highest levels since diagnosis. Why is this important and what makes the focus on it worth while?

Monocytes ingest dead or damaged cells through phagocytosis and provide immunological defenses against many infectious organisms. They later develop into macrophages and ingest bacteria as part of the immune response to infection. We know that reduced macrophage activity may decrease immune surveillance and thereby allow nascent tumor cells to escape detection by the immune system. If my circulating lymphocyte count is down, does that mean that some of these tumor cells have been detected by a slowly improving immune system, which is now able to start killing them? At the least, the immune system is better able to respond to infectious agents than it was 90 days ago.

Basophils enter tissues only when they are recruited into inflammatory sites. When basophils are triggered they release: histamines and serotonins, which are enzymes that can destroy tissue or cleave complement components (good complement dependent response is essential for a good Rituxan response; see Chaya’s article Complement Dependent Response in Rituxan Therapy); cytokines and leukotrienes that aggravate inflammation, and importantly, are made from arachidonic acid in surrounding tissues. Arachidonic acid is thought to be proapoptotic. So, by using the arachidonic acid to help fight off (or maybe kill) the CLL cells instead of using it to produce a biological response that protects or encourages the CLL cells to keep collecting in the peripheral blood supply, and by avoiding the damage to complement components, we have the makings of a virtuous circle of immunological response instead of a vicious circle of immunological response.

Low levels of hematocrit (too low means anemia) are associated with leukemia. There have been numerous articles on CLL patients who have anemia as a result of their CLL or treatments for it.

So, at the moment it looks as if, for this data point of one (me), and for the data gathered to date, there is a favorable verdict being developed for this particular NSAID for the kind of CLL I have. Thanks again to Chaya for drawing back the curtain on inflammation and to Gary Searching for his info on Dr. Keating’s use of Singulair on his patients. With this good news I decided to go fishing for a week.

Life After Rituxan Therapy - How to Keep the Party Going

8/3/03

We have discussed the pros and cons of Rituxan therapy, who would be a good candidate for it as frontline monotherapy and who would not, the various mechanisms involved in how the B-cells are killed after they are tagged by Rituxan, is there zero risk with this particular therapy.

In my next article I will discuss how a hypothetical Joe Patient could try to improve the quality of his response to Rituxan; how he could try and get around the slight resistance his B-cells might have developed to Rituxan from several prior sessions, because of increased levels of complement inhibitory proteins; how he can add an immunomodulatory agent like GM-CSF to the Rituxan therapy, to improve the assist that his own immune system needs to provide in the process of killing the cells tagged by the Rituxan; why Joe thought this was necessary, since he is always a little low in neutrophils.

Putting the cart before the horse, this article is about what happens after the Rituxan infusions are over. The morning after the big date, as it were. Let us develop the post Rituxan scenario, again for my favorite character, the hypothetical Joe Patient who exists only in my imagination. Any resemblance between Joe and any real person is strictly coincidental, you had too many beers and are just imagining the resemblance, and I bet you can't prove otherwise.

Joe was lucky this time around. Unlike his previous bouts of Rituxan, the addition of GM-CSF (granulocyte macrophage colony stimulating factor) ahead of time meant he went into the Rituxan therapy with good solid numbers of neutrophils and macrophages. And the beta-glucan he took during Rituxan therapy helped make these effector cells primed and ready to kill any B-cell that has been tagged by Rituxan, and as a result, has been opsonized by complement. Joe got a nice deep response this time. One month after he finished his four infusions of Rituxan his absolute lymphocyte count in the peripheral blood was a mere 1.0K, down from more than 40K before start of therapy. Most of his lymph nodes had melted away, and the few that could be felt by careful probing were probably just the remnants of the old lymph node architecture. Hotels still left standing, as it were, not yet razed to the ground, but all the guests long dead and gone, especially if their names started with the letter "B". Soon enough, unless new guests arrived to occupy the rooms, the local municipality would get around to sending in the bull dozers, cart away the rubble of the now no longer needed accommodations. No word on the status of his bone marrow, but the fact that his hemoglobin, red blood cells etc came bouncing back up into the 'pink' probably meant the bone marrow was also doing well, cleaned out to some degree, but probably not entirely free of CLL cells. That would be too good to be true, and Joe is too pragmatic to fall for that old chestnut.

Now, the million dollar question is this: what can Joe do to try and make his brand new remission lasts as long as possible? Rituxan therapy was not hard to take, and it helps that Joe thinks there is zero risk (!?!) of toxicity from this monoclonal antibody. But it is still a pain in the a$$ to go through these infusions, and having to repeat them every six months is a real drag. Is there anything Joe can do to prolong the period of remission? Joe has been around the block enough times to know that is what he has, hopefully a long remission. But not a cure. There are still CLL cells lurking around, and they will grow back over time. But it sure would be nice to have them grow back to levels where Joe may have to do something about them in say, 12 months or longer, rather than go back for a retread every 6-7 months, as he had to do on previous occasions. As it turns out, there is something Joe can do to try and prolong his remission. And that is what we will discuss now.

Joe is starting out with his brand new remission, and our scenario has him with 1.0K lymphocytes in his blood. Just to make this discussion easy, let us not worry about the lymph nodes for now, let us assume they are squeaky clean, devoid of all CLL cells. As for the bone marrow, who knows. Joe works on the "don't ask, don't worry" basis as far as the bone marrow is concerned.

This is how the 1.0K lymphocytes in the peripheral blood broke out into three different groups:

T-cells	0.80K
Good, regular B-cells	0.02K
Leftover CLL Cells	0.18K
Total	1.0K

As you can see, most of the 1.0K in absolute lymphocyte count was actually just T-cells (remember, both T-cells and B-cells together make up "lymphocytes"). Of the B-cells left over, there are nine times as many of the nasty CLL cells as there are good B-cells. Makes sense, Joe had a lot more CLL cells than good B-cells going into Rituxan therapy. As we know by now, Rituxan is selective only to the tune of tagging only mature B-cells, not other cell lines. But it does tag all mature B-cells, as long as they have CD20 marker on them. It does not distinguish between a good B-cell and a bad CLL cell. Both are mature B-cells and that is all that matters for them to display CD20 marker, and therefore get tagged by Rituxan, and therefore get killed.

The problem with CLL cells left over after therapy is two fold: they multiply faster than normal B-cells, and they don't die as quickly. Net result, their populations will grow faster than that of normal cells. If Joe is to have any hope of making this remission last a nice long time, he has to slow down the proliferation rate, and take away as well some of the survival advantages of CLL cells.

We have discussed something called the NF-kB pathway in the past. Never mind the details, just think of it as a mechanism that somehow lets cells breed faster, and live longer. This is probably one of the most important pathways our body has, to make immune system cells quickly, on demand. Normally, this pathway is not activated. Everything is quiet, chugging along nicely. Then imagine a sudden invasion of bacteria, some infection or the other. The immune system has to gear up and produce troops quickly. NF-kB pathway is activated on the existing immune system cells, and this makes the cells more robust, harder to kill. They also multiply faster, and thereby increase the total number of fighting troops the body is able to muster to fend off the invaders. You can see why this is a nice trick our bodies have learned along the way.

Problem with CLL cells (and many other types of cancers) is that they NF-kB pathway is stuck in the "on" position, all the time. Rather than switching on and off as needed, this pathway is in a state of "chronic activation", never switching off. This makes the CLL cells harder to kill, gives them a survival advantage, and they also multiply more rapidly. There have been several studies that show pretty conclusively that indeed the NF-kB pathway is in a chronically activated form in CLL cells, but not so on regular, good, B-cells.

Is there way some way of jimmying the NF-kB switch on CLL cells so that it is not stuck in the "on" mode all the time? Yes, there are several materials that are good at inhibiting the activation of NF-kB pathway. We have discussed most of them in the past, so I will not go into the details. Here is a short list of common every day materials that block NF-kB pathway. You would have thought of most of these as "anti-inflammation" compounds, and you would be right. That is exactly what they do, reduce inflammation, and they do that by shutting down the NF-kB pathway, which is what causes inflammation.

Curcumin (extract from the spice turmeric. The spice itself has only 4-5% or so of curcumin. You would need to eat a lot of turmeric)

Catechols from Green Tea (quite a few cups per day)

Resveratrol found in red wine (now, don't over do it!)

Most NSAIDs like aspirin, ibuprofen etc. (you might need them, after reading this article).

What makes this even more interesting is that in the absence of an attack of invading bacteria, under normal circumstances that do not call for emergency measures, regular, good B-cells do not use NF-kB pathway to make up depleted populations. NF-kB route is normally used only in emergencies, as a quick way to build up the troops. Under normal conditions, "homeostatic proliferation" is nice and gradual increase in cell population using an entirely different mechanism dependent on tyrosine kinases. Never mind the jargon, just remember that CLL cells are prone to multiply and survive via the "emergency" NF-kB activation pathway, and normal B-cells in the absence of an emergency do not use this pathway. In other words, it may be possible for Joe to down-regulate the NF-kB by using one or more of the NF-kB blockers listed above, and thereby put a spoke in the wheel of the CLL cell proliferation and survival advantage, without necessarily preventing the good regular B-cells from growing in a nice orderly fashion.

Joe is a diligent reader of Topics, and knows all about these compounds and NF-kB inhibition. But Joe hates the taste of curry, especially if it is hot, and he can't stand green tea. So he opts for capsules of curcumin (with small amounts of piperine, from pepper, that is supposed to make the curcumin more readily absorbed) and green tea extract capsules. The red wine he can handle by itself, especially if it is a pinot noir, and now he has an excuse. As for the NSAIDs, he tends to have high stomach acidity and does not want to risk getting an ulcer by taking aspirin on a daily basis for any length of time. He decides to wait to hear more about that R-Flurbiprofen that Chaya was discussing on Topics, an optical isomer of regular Flurbiprofen (very much like ibuprofen) that is supposed to be a good NF-kB blocker without the side effects of aspirin. He looked it up on the company website, http://www.myriad.com/pharma/prostate.htm , looked like they were getting some good results with it for other cancers that also used the NF-kB pathway. There were rumors that Anderson may soon be starting clinical trials for CLL patients in remission, using R-Flurbiprofen. Joe decided to wait for that one.

So, is this a slam-dunk, the CLL cells are stopped dead in their tracks, never to multiply because the NF-kB pathway is shut down? Will the good B-cells multiply slowly and methodically, since they don't need NF-kB pathway? Will the tortoise win the race and leave the hare behind in its dust?

Not quite. First, it is impossible to shut down NF-kB activation 100% with these types of compounds. And second, you would not want to, even if you could. Many other immune system cell lines also depend upon the NF-kB pathway as a way of proliferating quickly. And this is still a necessary function, in the event of an attack by invading pathogens, or your body will not be able to mount an effective counter-attack. Even for B-cells, the tyrosine kinase pathway used by good B-cells under normal conditions gives a population of naive B-cells. These are not the mature, antigen tested B-cells that are an effective fighting force. That step, the conversion of naive B-cells into antigen specific and mature B-cells, does need the NF-kB pathway. So this is the trade Joe makes, shut down the NF-kB pathway sufficiently to slow down the accumulation of the CLL cells, but not all the way, so that the pathway is still available for other cell lines and the antigen-dependent maturation of B-cells.

True, letting the NF-kB pathway open only a dribble, as it were, does mean a less effective fighting force in the event of an attack from the outside. There will be fewer mature, antigen savvy B-cells, and more of the newbie naive B-cells. But this is not the end of the world, there are other units in the immune system army that can cover for the untested troops of the B-cell unit. T-cells, macrophages, neutrophils, complement, all of these are also available resources in defending the body against pathogen attacks. It is possible to get by with less than full strength B-cell capabilities.

(Just think about it, Joe was ready to go for six month maintenance schedule of Rituxan, regular as clockwork. Basically whack the B-cell population way back as soon as it showed any signs of recovering. In other words, he was prepared to live with very few B-cells of any kind, for extended periods of time. Obviously, he would not have chosen to do that, if he did not have this little problem called CLL to deal with in the first place. We all make the bargains we must, choose the lesser of two evils).

There is one advantage of having fewer mature, antigen specific B-cells in the pool, and more of the naive variety of B-cells. Naive B-cells, once they leave the bone marrow, don't live all that long. They have to grow up, become antigen specific mature B-cells, or die in a few days, to be replaced by more naive B-cells from the bone marrow. But once a B-cell has met its antigen in a lymph node, and matured appropriately, it lives a lot longer. It leaves the lymph node, circulating in the blood for part of the time, sitting around in the lymph nodes or the bone marrow part of the time. Guess what it does while it is sitting around. Yup, it is making babies, copies of itself. This is one of the reasons why lymph nodes swell up with all the billions of useless mature CLL B-cells, and why the bone marrow gets clogged up with the same crowd. If there are fewer long lived mature B-cells, there is less traffic back to the bone marrow, less chance of it getting clogged up.

I will be following Joe closely (just a figure of speech, since he is only a hypothetical patient, a figment of my imagination) and report back to you how his nice deep remission lasted this time around, did the NF-kB blockers really help slow down the rate of proliferation of CLL cells and thereby increase the duration of the response this time around, did he think he had more sniffles and infections because of the choke hold put on the NF-kB pathway, and the reduced repertoire or good but mature B-cells. Too bad we will not be able to figure out too much about his bone marrow, how it fared during this round of "goosed-up" Rituxan therapy and hopefully extended remission. Joe hates bone marrow biopsies, so we have no before-and-after information on that front.

Below are three abstracts. The first one talks about how regular B-cells do not use the NF-kB pathway under normal circumstance, while this pathway is stuck in the chronically "on" mode for CLL cells, as described in the second abstract. The third abstract talks about naive B-cells versus mature B-cells, and their differences in life span, effectiveness as fighters, and migration patterns to the bone marrow.

Do remember, this is just a story about my hypothetical Joe Patient, I am not suggesting in any way or form that any of you real people out there try this at home, especially without careful adult supervision and consultation with your medical team.

Abstracts:

Naive B lymphocytes undergo homeostatic proliferation in response to B cell deficit.

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

Naive peripheral B cells are maintained in sufficient numbers and diversity to mount effective immune responses against infectious agents. However, the size and repertoire of this B cell pool is constantly diminished by normal cell turnover and Ag activation. Homeostatic (Ag-independent) proliferation in response to B cell depletion is one mechanism to compensate for this cell loss. We have used purified CFSE-labeled B cells and an adoptive transfer model system to show that immature and mature B cells divide in a variety of B cell-deficient (scid, xid, IL-7(-/-), and sublethally irradiated) hosts. Homeostatic B cell proliferation is T cell independent, and B cells that have replicated by this mechanism retain the antigenic phenotype of naive B cells. Replication is significantly reduced in B cell-sufficient normal or B cell-reconstituted immunodeficient recipients by the action of competing mature follicular B cells. Using xid mice and transcription factor knockouts, we show that the activation signal(s) that lead to homeostatic B cell proliferation require Bruton's tyrosine kinase; however, c-Rel, a Bruton's tyrosine kinase-induced NF-kappaB/Rel transcription factor critical for Ag and mitogen stimulation, is dispensable, indicating the uniqueness of this activation pathway. Survival and replication signals can also be separated, because the transcription factor p50 (NF-kappaB1), which is required for the survival of peripheral B cells, is not necessary for homeostatic replication. Homeostatic B cell proliferation provides an Ag-independent mechanism for the maintenance and expansion of naive B cells selected into the mature B cell pool.

Cytokine modulation of nuclear factor-kappaB activity in B-chronic lymphocytic leukemia.

OBJECTIVE: Dysregulation of the apoptotic mechanisms plays a key role in the accumulation of malignant B-chronic lymphocytic leukemia (B-CLL) cells. The transcription nuclear factor (NF)-kappaB is important for cell survival by regulating the expression of anti-apoptotic genes. Several cytokines can modulate leukemic growth and apoptosis in B-CLL. The aim of this study was to determine whether cytokine-mediated regulation of apoptosis occurs via modulation of NF-kappaB activity in peripheral blood mononuclear cells from B-CLL patients. PATIENTS AND METHODS: We evaluated NF-kappaB activity in peripheral blood mononuclear cells from 15 untreated B-CLL patients and 11 controls in resting conditions and in the presence of phorbol-12-myristate-13-acetate (PMA) and different cytokines by electrophoretic mobility shift assay. Apoptosis was studied by spectrophotometric analysis of DNA fragmentation. RESULTS: We found a constitutive high NF-kappaB activity not induced by PMA in B-CLL patients, in contrast with a normal inducible NF-kappaB activity in controls. In B-CLL cultures, addition of interleukin (IL)-4 and IL-13 increased, whereas transforming growth factor (TGF)-beta reduced NF-kappaB activity compared with unstimulated cultures. Accordingly, IL-4 and IL-13 decreased, whereas TGF-beta increased DNA fragmentation compared with unstimulated cultures. IL-13 and IL-4 production was increased, whereas TGF-beta was reduced in PMA-stimulated and unstimulated cultures from B-CLL patients compared with controls. CONCLUSIONS: B-CLL patients have a constitutive high NF-kappaB activity, which is modulated by cytokines. In particular, TGF-beta displays a pro-apoptotic activity, whereas IL-4 and IL-13 have opposite effects. These cytokine alterations could be responsible for a positive autocrine circuit that maintains leukemic cells in a pre-apoptotic state.
Copyright 2003 International Society for Experimental Hematology

An intact NF-kappa B signaling pathway is required for maintenance of mature B cell subsets.

Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

Members of the NF-kappaB/Rel transcription factor family are expressed constitutively during B cell development and are further induced by mitogen activation. Mice harboring germline disruptions in individual NF-kappaB subunits exhibit distinct defects in B lymphocyte activation and survival. However, the role of NF-kappaB in the production and maintenance of B cell subsets has been difficult to dissect in these knockout animals due to functional impairment of other immune cells. To directly address the cell autonomous requirements for NF-kappaB in humoral immune compartments, transgenic mice were generated that express a transdominant form of Ikappa-Balpha in B lineage cells. Whereas expression of the inhibitor had only modest effects on basal or LPS-induced levels of NF-kappaB, transgenic B cells were significantly impaired for cellular proliferation and NF-kappaB induction in response to B cell receptor (BCR) crosslinking. Furthermore, the trans-dominant inhibitor produced a dose-dependent reduction in the population of mature splenic B cells. This cellular defect was more pronounced in long-lived B lymphocyte subsets that recirculate to the adult bone marrow. Together, these results indicate that BCR-mediated signaling must maintain NF-kappaB levels above a stringent threshold for proper regulation of B cell homeostasis.

In the above discussion, I deliberately dodged the issue of dosages for Joe Patient. What I can do for you is suggest some issues to consider as you reach your own conclusions, to be discussed with your doctors prior to taking action on them.

1. Abstract below from M. D. Anderson suggests there is no dose-limiting toxicity to curcumin. I spoke at length with the author, Dr. Aggarwal, about six months back. His very clear advice was that curcumin, in combination with small amount of piperine to improve bioavailability of the curcumin, is the best thing one can do to slow down the rate of proliferation of many different types of cancer. He saw little downside risk in this supplement. PubMed has hundreds of citations on curcumin.

2. Turmeric, the spice, is an inconvenient source for curcumin, since the percentage of curcumin in turmeric is quite low. I suppose one would look for commercial products with guaranteed levels of curcumin, in combination with piperine. Personally, I am too lazy to remember to take anything more than three times a day, so that would pretty much set me up on my personal "maximum tolerated dosage", purely on the basis of convenience. There is also some reason to keep a stable concentration of these compounds within the body, with the doses set apart in equal time periods.

3. There have been reports that curcumin (and many other anti-oxidant phytochemicals) may interfere with some chemotherapy drugs. I strongly advice all members that you should not be taking any (repeat, **ANY**) herbal supplements or over the counter drugs without first discussing with your doctor, especially if you are about to enter chemotherapy. It takes a while for some of these compounds to clear your body, as much as one month in some case, bear that in mind as you schedule.

4. There have been some interesting reports (see the second abstract below) that suggests there is synergy between curcumin and green tea extracts, that the two in combination are more effective than either by itself. That was my reason for getting "Joe" to focus on these two particular NF-kB blockers. That, and their generally accepted minimal toxicity. Again, green tea catechols are potent anti-oxidants, not to be taken without consultation with your doctor during period of chemotherapy.

5. If you like green tea, go for the gusto, drink as many cups as you wish. If you are from the Indian subcontinent, like P.C, and your wife has a green thumb and can grow fresh turmeric (not the awful dried roots or powder), you may enjoy eating various relishes and jams made from fresh turmeric. It has a refreshing taste, like a very mild form of ginger, with a characteristic but
mild flavor. You can also throw it into soups and stir-fry's. My grandmother would have told you, no harm in eating as much of the fresh turmeric relish as you want, it is good for all sorts of ailments. If you are a standard issue meat-and-potatoes type, and don't like all these strange foods, you are stuck with getting capsules from one or the other more reputable companies.

Abstracts:

Anticancer potential of curcumin: preclinical and clinical studies.

Cytokine Research Section, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 143, Houston, TX, USA.

Curcumin (diferuloylmethane) is a polyphenol derived from the plant Curcuma longa, commonly called turmeric. Extensive research over the last 50 years has indicated this polyphenol can both prevent and treat cancer. The anticancer potential of curcumin stems from its ability to suppress proliferation of a wide variety of tumor cells, down-regulate transcription factors NF-kappa B, AP-1 and Egr-1; down- regulate the expression of COX2, LOX, NOS, MMP-9, uPA, TNF, chemokines, cell surface adhesion molecules and cyclin D1; down-regulate growth factor receptors (such as EGFR and HER2); and inhibit the activity of c-Jun N-terminal kinase, protein tyrosine kinases and protein serine/threonine kinases. In several systems, curcumin has been described as a potent antioxidant and anti-inflammatory agent. Evidence has also been presented to suggest that curcumin can suppress tumor initiation, promotion and metastasis. Pharmacologically, curcumin has been found to be safe. Human clinical trials indicated no dose-limiting toxicity when administered at doses up to 10 g/day. All of these studies suggest that curcumin has enormous potential in the prevention and therapy of cancer. The current review describes in detail the data supporting these studies.

Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells.

Department of Surgery, Head and Neck Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

An in vitro model for oral cancer was used to examine the growth inhibitory effects of chemopreventive agents when used singly and in combination. The model consists of primary cultures of normal oral epithelial cells, newly established cell lines derived from dysplastic leukoplakia and squamous cell carcinoma. Two naturally occurring substances, (-)-epigallocatechin-3-gallate (EGCG) from green tea and curcumin from the spice turmeric were tested. Cells were treated singly and in combination and effects on growth determined in 5-day growth assays and by cell cycle analysis. Effective dose 50s and the combination index were calculated with the computerized Chou-Talalay method which is based on the median-effect principle. Agents were shown to differ in their inhibitory potency. EGCG was less effective with cell progression; the cancer cells were more resistant than normal or dysplastic cells. In contrast, curcumin was equally effective regardless of the cell type tested. Cell cycle analysis indicated that EGCG blocked cells in G1, whereas curcumin blocked cells in S/G2M. The combination of both agents showed synergistic interactions in growth inhibition and increased sigmoidicity (steepness) of the dose-effect curves, a response that was dose and cell type dependent. Combinations allowed for a dose reduction of 4.4-8.5-fold for EGCG and 2.2-2.8-fold for curcumin at ED50s as indicated by the dose reduction index (DRI). Even greater DRI values were observed above ED50 levels. Our results demonstrate that this model which includes normal, premalignant and malignant oral cells can be used to analyse the relative potential of various chemopreventive agents. Two such naturally-occurring agents, EGCG and curcumin, were noted to inhibit growth by different mechanisms, a factor which may account for their demonstrable interactive synergistic effect.

Too Much of a Good Thing: NF-kB Blockade

8/4/03

My recent article reporting on how "Joe Patient" plans to party on & enjoy his brand new remission using Rituxan got a few personal emails from members. Some speculated that a high dose combination of every available NF-kB blocker out there may shut down this pathway completely, for all practical purposes, and make sure the CLL has no chance of growing back. Is this a possibility, can we take this approach to getting closer to that elusive "cure"??

My answer, in one word, is: No!! Not on your life, you have to be crazy to do something like that. (O.K, that's more than one word, I am busted).

I would not recommend "heavy-duty" NF-kB blockade for CLL patients, or anyone for that matter. NF-kB is a necessary and vital pathway in our bodies, and you would be very foolish if you tried to push it too far out of its natural equilibrium, in either direction.

For example, total suppression of NF-kB in mice studies show that they are not able to mount sufficiently vigorous immune response when subjected to attack by bacterial infections or other pathogens. As we discussed before, NF-kB is the emergency pathway that the body uses to produce fighting troops quickly, when faced with an immune emergency. Mice that have Nf-KB pathway blocked are also more at risk of death from sepsis.

For example, in addition to controlling the functioning of the immune system, NF-kB activity also regulates the proper functioning of our skin. This is the single biggest barrier between ourselves and the rest of the universe out there, some of it decidedly not friendly to us. Mice whose NF-kB pathway has been genetically altered, so that it is shut down to a very large degree have a much higher incidence of squamous cell carcinoma. Even the "heartbreak of psoriasis" is thought to be caused by insufficient NF-kB activation.

NF-kB modulates TNF-alpha (tumor necrosis factor alpha) and many other cytokines. Left unchecked, TNF alpha can do serious damage, as we saw in my recent piece on Crohn's disease. Uncontrolled production of TNF-alpha can cause rheumatoid arthritis, and it can also make you understand the real misery of B-symptoms, as discussed in a recent article on night sweats.

NF-kB activation to some degree is a necessary function in many neural processes, involved in memory, learning and retention. On the other hand, over stimulation of NF-kB is now considered to be one of the root causes of Alzheimer's, and in fact the NF-kB blocker R-Flurbiprofen that I discussed in an earlier article is now in clinical trials for Alzheimer's Disease. Look up this clinical trial at the URL address given for Myriad Pharmaceuticals in my previous Joe Patient article.

Too much food is bad for you, makes you fat and unhealthy, it can create life threatening cardiac problems, increase the risk of diabetes and cancer. There have been a number of excellent studies indicating that slightly reduced calorie intake, keeping the body on tight rations, can increase our life span significantly. But total "blockade of food" will surely kill you of starvation. Messing with NF-kB pathway is sort of like that. Inhibiting it too much is decidedly not a good thing.

Every system in our body is on a push-pull, feedback control mechanism, not a static set-point. That's just engineer talk (we have our jargon too, even if we wear hard hats and not white coats), all it means is that our body systems are more like a pendulum swinging back and forth, constantly changing position and adjusting, never static in one fixed position. Moreover, there is a lot of very complex inter-connections between the various systems, and multiple redundancies that we are just beginning to learn.

Scientists are learning that in general, it really does not help to whack the heck out of any one system, because that is just setting us up for a loophole that the body develops, one that is harder to treat later on. The same theme is reflected in the multiple drug resistance piece I did. Start flooding that old basement with water, and pretty soon the householder goes out and gets himself a nice sump pump to pump out the water. Flood the cancer cells with chemotherapy too many times, and the cancer cells will find a way of pumping out the drug, develop resistance to that particular drug and a bunch more besides.

That is probably why I like immunotherapy approaches like monoclonal antibodies, such as Rituxan. Rituxan therapy uses much of the body's own immune systems to deal with the problem. Sometimes patients are upset that it takes a while for the effects to show up, as much as a couple of months before the full effect of the therapy can be seen, and the kill rate is rarely 100%, even in the peripheral blood. Well, that is also the reason why there is less chance of the CLL developing / transforming into a more refractory cancer, one that no longer responds to this particular drug. Even with Rituxan, my main concern would be development of a CLL clone that has up regulation of complement inhibitory proteins to a degree that makes Rituxan less effective next time around. Necessity is the mother of invention, and this is true for cancer cells as well. It makes sense not to give the body a reason to invent a new mechanism for subverting what you are trying to do.

We are finding out that the subtle detail and complexity of our bodies means we have to use equally subtle and complex therapies to counter cancer. Very few cancers are treated any more with just one chemotherapy drug, but a variety of drugs in a cocktail, in an effort to try and block all the avenues of escape. Just a few years ago, there was a story on the New York Times front page, that a single angiogenesis drug will soon starve all cancers to death, and a cure is just around the corner. We know now that the body has literally dozens of ways of getting around that single puny road block. Dr. Judah Folkman is a pioneer, and my personal hero for this unique and elegant piece of research. But hubris does not last very long in the field of cancer research.

CLL can not (repeat, can not) be cured by NF-kB inhibition. If you tweak the proliferation rate of CLL cells so that it is just a tad slower than otherwise, and you also tweak the ease with which they commit suicide (apoptosis) to be just a teeny bit higher, net result is that the rate of accumulation of the CLL cells (rate of birth, minus rate of death) will slow down, just a little. That means the ALC (absolute lymphocyte count) will not climb as fast as it would otherwise.

How much is "just a little"? I am guessing, but I would be happy if the rate at which the CLL grows back is reduced by only a modest 10%. CLL is an indolent disease, which means the cancer cells do not accumulate too fast. So we can expect to see real differences in length of remissions, even with small changes in the rate of its growth. Even if you had the capability to shut down NF-kB pathway completely, 100%, you would not want to do this, and try to cut the rate of accumulation of CLL cells to zero (i.e., "cure" the CLL). If you do, two things can and will most likely happen: (1) the CLL finds some other way to grow, a redundant system that we have not considered, and keep growing, probably even faster than before. (2) you would be dead because some other truly vital system in your body comes to a screeching halt because NF-kB is shut down 100%. CLL may or may not be cured, but the patient is dead. Not a good scenario.

As in most things in life, moderation is a virtue. In cancer therapy, this is particularly so. One of the reasons why I had my imaginary Joe Patient focus on curcumin and green tea is that both of these are food based products, with a long history of use in ethnic cuisine. They probably are not as potent as synthetic compounds that would do a better job of blocking Nf-KB, and probably safer because of that very fact. But even there, moderation and patience is the watch word.

Extending Remissions with NF-kB Inhibitors

8/5/03

A question came up on the effect of NF-kB inhibitors on bone marrow infiltration by CLL cells.

There are two parts to the answer to this question.

How well a given patient responds to Rituxan depends on all the issues we have discussed before: his phenotype, tumor burden, prior chemotherapy history, complement levels, quantity and quality of his effector cells (to help in the cell kill of the CD20 positive cells tagged by the Rituxan) etc. At this point, there is no way of predicting ahead of time with a high degree of certainty how well a given patient will respond to Rituxan therapy. I mentioned DISC assay type of testing in a previous article. DISC assay is not very satisfactory even for chemotherapy drugs, it is especially not so for biologic agents like Rituxan. So much of the real action going on in Rituxan therapy is dependent on the actual environment of the human body, the complex interaction between the different systems of the body, something that is impossible to duplicate in a test-tube. We can make some educated guesses as to who would be a good candidate and who would not, along the lines discussed in previous articles, but this would not be a definitive answer.

There is now enough information to suggest that bioavailability of Rituxan in bulky lymph nodes, enlarged spleen / liver, or heavily infiltrated bone marrow is of real concern in CLL patients. This is one of the reasons why my preference for using this monoclonal as a frontline monotherapy would be in early stages, when the tumor load is not as high, and the disease is not as bulky, and the chemo naive patient has relatively intact immune system. There are no documented cases that I know of, where Rituxan by itself as a single agent has delivered 100% squeaky clean bone-marrow, PCR negative status. The more relevant question is not whether or not there are still CLL cells in the bone marrow after Rituxan monotherapy (you can safely bet there are still CLL cells there), the more important question is **how much** of a cleaning out was accomplished. Obviously, we would like the clean-out to be as high as possible.

Even some degree of bone marrow clean-out would improve function of this critical site. If your bone marrow was 90% shot before Rituxan, and it was struggling to make sufficient numbers of red blood cells, and Rituxan cleaned it out so that now it is only 60% infiltrated, that is a huge improvement. You will see the effect almost immediately, there is not as much of a struggle to make the red blood cells and other cell lines. You will notice an immediate improvement in fatigue and your athletic performance will no longer be constrained by insufficient oxygen transport. You would be able to tell your bone marrow has improved after therapy. Exactly how much has it improved, that can be discovered only by a bone marrow biopsy. Even there, the result is not totally reliable, it depends on the location from which the bone marrow sample happened to be collected. Other locations of the bone marrow could be quite different . A reasonable question is why bother with the bone marrow biopsy, if the answer is still uncertain.

So. Rituxan monotherapy will not give you a clean slate, but hopefully a slate that is significantly cleaner than the one you started with. The second part of the answer has to do with keeping the slate as clean as possible, for as long as possible. Again, you would be quite safe to bet that the slate will eventually get dirty again. There are no documented cases of indefinitely long remissions, i.e., a "cure", with Rituxan-only therapy in CLL. All patients **will** relapse, sooner or later. Clearly, patients would prefer later.

CLL cells left over in the bone marrow, lymph nodes, even peripheral blood will grow back, you can bet on that. The real question is can one do anything about slowing down the rate at which they grow back. And that is where NF-kB inhibition may play a role. Inhibition, not total blockade of NF-kB pathway, I discussed the dangers of overkill in my previous article. And bear in mind we are talking about slowing down the rate at which the CLL grows back, not stopping it dead in its tracks. The issue here is one of extending the period of remission, not trying to boot-strap the system into a "cure".

I have no reason to think systemic KF-kB inhibitors will only influence CLL cells in one location. I would think the effects of NF-kB inhibition would be felt by all the CLL cells through out the body, as well as by all the other systems that use NF-kB pathway. In any case, the CLL cells don't stay put in one place, they are constantly milling around going from one location to the other. So my best guess would be that the efficacy of NF-kB inhibition would be felt at all locations. There are no easy or quick answers, I am afraid. Recovery of performance and improved red blood cell and hemoglobin levels do not prove a totally cleaned out bone marrow after Rituxan, just that there was some degree of improvement in bone marrow function. "Normal" peripheral blood counts do not prove the CLL has vanished from other locations as well, in fact it would be safe to bet it has not, that there is still significant level of CLL cells left over, especially in the bone marrow and solid organs. NF-kB inhibition, carried out with prudence and moderation, may slow down the rate at which the disease grows back to full strength, and thereby increase the time of remission. There is every reason to hope that this benefit is felt in the bone marrow as well, that the NF-kB inhibition will also slow down the rate at which the CLL will begin to take over the bone marrow once again.

This is not a home run, just a bunt. But hey, I will take every little bunt, they do add up in the long run. Malcolm would be the first one to admit, even though he has few side effects from Rituxan therapy, that having to go back for retreads once in every 12 months or so sure beats having to do it every six months.

Prolonging Watch and Wait with NF-kB Inhibition

8/6/03

Two members came up with this question: "What about the NF-kB pathway before treatment and how can it be used proactively?"

This is an excellent question. These are the kinds of issues we should be discussing with our oncologists, make the discussions two-way information flow.

There is no question in my mind, if we can slow down the rate of progression of the disease, stay in early stages longer, be able to defer therapy for a while more without being foolish about it, that can only be good. I think of it as buying time. There is a real explosion of new research and information coming down the turnpike. There are choices available now that were not there just a couple of years back. If it is possible to defer therapy, by means of keeping the CLL from progressing as fast as it would otherwise, you will give yourself additional time. Time in which you can learn more, have access to better choices, and make better decisions. Hopefully, you will also use the time to get yourself into better shape, that will make a real difference in how you will respond to therapy.

As I said, I spoke with Dr. Aggarwal on the phone for over an hour. He is very clear that modest and prudent down-regulation of NF-kB pathway has a huge impact on rate of proliferation of all kinds of cancer cells, without harming other systems in the body. He sees this as a "chemoprevention" and "cancer control" techniques, a way of preventing, controlling and slowing down cancers. I have attached a couple more abstracts below, the first one is authored by Dr. Aggarwal.

As for the choice of specific NF-kB blockers, you would have to do your own reading and come to your own conclusions. My own preference would be to give more weight to toxicity and safety issues, even if the choice is then not as effective as some other material. Food "phytochemicals" have the benefit of being part of normal food consumption patterns, as long as one does not go too far out on a limb on the dosage levels. NSAIDs do a lot more than just suppress NF-kB, and there is also the issue of stomach ulcers. I am waiting to get more information on the R-Flurbiprofen from Myriad, since this is an optical isomer of a commonly used NSAID, but one that has none of the COX effects of normal NSAIDs. It is getting good press in Alzheimer's clinical trials, as well as in prostate cancer.

Abstract:

Nuclear factor-kappa B and cancer: its role in prevention and therapy.

Cytokine Research Section, Department of Bioimmunotherapy, M. D. Anderson Cancer Center, University of Texas, Box 143, 1515 Holcomb Boulevard, Houston, TX 77030, USA.

Cancer is a hyperproliferative disorder in which invasion and angiogenesis lead to tumor metastasis. Several genes that mediate tumorigenesis and metastasis are regulated by a nuclear transcription factor, nuclear factor kappa B (NF-kappaB). A heterotrimeric complex consisting of p50, p65, and IkappaBalpha, NF-kappaB is present in its inactive state in the cytoplasm. When NF-kappaB is activated, IkappaBalpha is degraded and p50-p65 heterodimer is translocated to the nucleus, binds the DNA (at the promoter region), and activates gene. Research within the last few years has revealed that NF-kappaB is activated by carcinogens, tumor promoters, inflammatory cytokines, and by chemotherapeutic agents. The activation of NF-kappaB can suppress apoptosis, thus promoting chemoresistance and tumorigenesis. Interestingly, however, most chemopreventive agents appear to suppress the activation of the NF-kappaB through inhibition of NF-kappaB signaling pathway. These chemopreventive agents also sensitize the tumors to chemotherapeutic agents through abrogation of NF-kappaB activation. Overall, these observations suggest that NF-kappaB is an ideal target for chemoprevention and chemosensitization. This article reviews evidence supporting this hypothesis.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced NF-kappaB activation by tea polyphenols, (-)-epigallocatechin gallate and theaflavins.

The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA.

(-)-Epigallocatechin gallate (EGCG) and theaflavins are believed to be the key active components in tea for the chemoprevention of cancer. However, the molecular mechanisms by which EGCG and theaflavins block carcinogenesis are not clear. In the JB6 mouse epidermal cell line a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which causes cell transformation at high frequency, markedly induced NF-kappaB activation. We found that EGCG and theaflavins inhibited TPA-induced NF-kappaB activity in a concentration-dependent manner. These polyphenols blocked TPA-induced phosphorylation of IkappaBalpha at Ser32 in the same concentration range. Moreover, the NF-kappaB sequence-specific DNA-binding activity induced by TPA was also inhibited by these polyphenols. These results suggest that inhibition of NF-kappaB activation is also important in accounting for the anti-tumor promotion effects of EGCG and theaflavins.

Inhibition of Nuclear Factor Kappa B (NF-B): An Emerging Theme in Anti-Inflammatory Therapies

Section of Immunobiology and Department of Molecular Biophysics and Biochemistry Howard Hughes Medical Institute Yale University School of Medicine New Haven, CT 06510

The application of anti-inflammatory therapies began thousands of years ago with the use of readily available natural resources. It is only recently, however, that the cellular and molecular mechanisms of inflammation have been appreciated sufficiently to design anti-inflammatory strategies with limited side effects. For example, salicylates and glucocorticoids, two widely used anti-inflammatory drug classes, are now known to inhibit the activation of NF-

B, a transcription factor that regulates the inducible expression of a wide range of proinflammatory mediators. New generations of NF-

B–targeting anti-inflammatory agents that are specific, efficacious, and cost-effective may therefore complement or replace current therapies. In this review, we describe various classes of NF-

B inhibitors and discuss important unresolved issues regarding their use.
_______________

: Prolonging Watch and Wait with NF-kB Inhibition

: Extending Remissions with NF-kB Inhibitors

: Too Much of a Good Thing: NF-kB Blockade

: Life After Rituxan Therapy - How to Keep the Party Going

: Singulair - Effect on CLL

: NSAIDs - Involvement of the NF-kB Pathway

: Nsaids - With a Twist

: NF-kB Reference

: NF-kB Pathway

: Nuclear Factor - Kappa B (NF-kB)

11/13/02

Abstract:

Nuclear factor-kappa B and cancer: its role in prevention and therapy.

The role of NF-kappaB/IkappaB proteins in cancer: implications for novel treatment strategies.

11/15/02

11/19/02

6/15/03

NSAID background:

URLs:

Abstracts:

Inhibition of NF-kappaB and AP-1 activation by R- and S-flurbiprofen.

Modulation of transcription factor NF-kappaB by enantiomers of the nonsteroidal drug ibuprofen.

6/15/03

6/22/03

8/3/03

Abstracts:

Naive B lymphocytes undergo homeostatic proliferation in response to B cell deficit.

Cytokine modulation of nuclear factor-kappaB activity in B-chronic lymphocytic leukemia.

An intact NF-kappa B signaling pathway is required for maintenance of mature B cell subsets.

Abstracts:

Anticancer potential of curcumin: preclinical and clinical studies.

Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells.

8/4/03

8/5/03

8/6/03

Abstract:

Nuclear factor-kappa B and cancer: its role in prevention and therapy.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced NF-kappaB activation by tea polyphenols, (-)-epigallocatechin gallate and theaflavins.

Inhibition of Nuclear Factor Kappa B (NF-B): An Emerging Theme in Anti-Inflammatory Therapies