The opposite of what we knew…

February 20, 2010 / / 3 Comments

Do you remember all the hoopla caused by a June 2009 study on green tea and bortezomib? If not, please click here: http://margaret.healthblogs.org/life-with-myeloma/discovery-of-curcumin/curcumin-and-bortezomib/egcg-and-bortezomib That study came as very bad news for devoted green-tea-drinking patients on Velcade, even though, as Dr. Durie pointed out during a patient seminar that I attended last fall, those patients can still have their cuppa…just not on their Velcade days…

Now for today’s topic. Not too long ago, a blog reader (thanks!) reminded me of a November 2009 study that I had read in December but hadn’t posted about… Sherlock, grazie!, sent me the full study, whose main result is spelled out in the abstract (http://tinyurl.com/ydyaqca): EGCG is synergistic with bortezomib (=Velcade) against the KM3 multiple myeloma cell line. SYNERGISTIC??? That means that EGCG and Velcade are more efficient myeloma cell assassins when used together…

Wait a sec…in June we were told that EGCG antagonizes bortezomib (see: http://tinyurl.com/yfe24cl, full text also available for free)…this group of researchers found that not only was EGCG not toxic or (at higher concentrations) only mildly toxic to myeloma cells, but also it prevented bortezomib from doing its job. In other words, based on the June study, EGCG actually protected the myeloma cells…from bortezomib.

When I first read and posted about the June study, I don’t remember being bothered by the occasionally arrogant language used by the authors…especially in this excerpt about EGCG: this “miracle herb” extract is also consumed by many cancer patients who follow popular trends and self-medicate with complementary and alternative medicine (CAM) in hopes to support their conventional therapy or to lessen the burden of side effects—sometimes without the knowledge of their health care provider. “Popular trrrends”??? I say, I am feeling quite offended right now…how did I miss that condescending tone when I first read the study last year? Well, I suppose I was more focused on the importance of the issue at hand, i.e., warning Velcade-users against taking EGCG or even drinking green tea…

I also (!) didn’t question the finding that EGCG, when used alone, wasn’t able to kill even one miserable little myeloma cell, even at concentrations that are much greater than the typical concentrations achieved in humans. This is contrary, e.g., to the results of an authoritative 2006 study (see: http://tinyurl.com/yzsdu38)… Makes me wonder…

One more thing. I would like to point out that, incredibly, the online media paid no attention whatsoever to the November pro-EGCG study, whereas the complete opposite is true of the June anti-EGCG study…in fact, if you do a quick online search, you will still find warnings, even recent ones, about drinking green tea with bortezomib…even if you type the words “EGCG” “bortezomib” and “synergy.”

What you will not find (at least I did not) is the slightest mention of the November 2009 study, the one with the synergy results. That story just wasn’t picked up, for…some reason (I can only guess…). Well, today I decided to fill the gap…not because I think that the November study is a better one, that is not for me to judge!, but because I don’t think it fair that this study be so blatantly ignored…

Okay, for reasons of simplicity, from now Study A will be the June anti-EGCG with bortezomib study, and Study B will be the November pro-EGCG with bortezomib study.

Just a quick glance at the two studies showed that there were differences in caspase activation…In Study A, EGCG blocked the activation of caspase-7, which essentially stopped bortezomib from exterminating the myeloma cells. In Study B, instead, EGCG activated different caspases, specifically caspase-3, -8 and -9, leading to the death of the myeloma cells. Some day I should really look into this caspase business…until then I will not be able to figure out how, why or if this might be significant (I’d be glad for some help, here!)…

For lack of time, sorry, I am going to jump to the Discussion part of Study B. As I had hoped, this is where the authors compare their own results to the ones of Study A (identified as “they” in the following excerpts):

-They use a relative [sic] lower concentrations of EGCG (10 mM) and bortezomib (10 nM), whereas we used a relative [sic] high concentration of EGCG (25, 50, and 100 mM) and bortezomib (20 nM). So let’s see…Study A used a lower dose of EGCG and bortezomib, Study B a higher one. Okay, that is certainly a difference.

-We focused on the mechanism of EGCG inhibiting myeloma cell growth and inducing cell apoptosis potentiated by bortezomib, whereas they focused on the effect of EGCG on bortezomib in myeloma cells. So, we found that EGCG inhibits myeloma cell growth and induces cell apoptosis potentiated by bortezomib. This part wasn’t clearly worded, in my opinion (but it is true that I just got over a cold…is my mind still fogged by a bit of congestion?). In fact, I don’t see any difference at all…Mental note: I need to find the time to compare how the experiments were carried out in both studies.

-This may be because of different drug concentrations or different cell lines. Study A and B (and even the above-mentioned 2006 study, by the way) tested EGCG and bortezomib on different myeloma cell lines. And different doses were used in both studies. Is that enough to explain their differing results? Possibly.

I found myself wondering about the different myeloma cell lines used in different studies. Interesting topic. I had no idea that there were so many myeloma cell lines. I began doing some research but had to give up. Too complicated and time-consuming…

Well, I admit, I am still baffled. I suppose that I would still be cautious about taking green tea or EGCG with Velcade. I would follow Dr. Durie’s suggestion. Best to be cautious. Still, Study B gives us some (green) tea for thought and shows how much we still have to learn…

P.S. Here is a list of things that Velcade patients should avoid taking: http://tinyurl.com/yk2tclg

Velcade and dormant myeloma cells

April 4, 2009 / / 10 Comments

Sherlock (grazie!) sent me the full study mentioned in yesterday’s post. It’s “only” 8 pages long, so I thought I would give it a whirl.

In the Introduction, we can read that the use of prolonged bortezomib therapy has lead to the development of drug resistance. The subsequent paragraphs and quite a bit of online research added a bit to my understanding of how this process occurs.

In order to retain my sanity while going through this complex study, I tried to visualize the dormancy process. For this, I needed a hibernating creature. After considering polar bears, toads and snakes, I went back to my original example: ants. If there are any ant-lovers out there, I sincerely apologize in advance for comparing them to myeloma cells.

Let’s imagine a northern European State at the beginning of a very harsh Velcade winter. As it starts getting colder, the ants aka myeloma cells get cold, too, but quite a number of them have enough forewarning and are able to escape the increasingly frigid, Velcade killer temperatures by hiding inside their underground nests. Their metabolism starts slowing down. In this inactive state, they require no food. It’s clearly a good survival mechanism.

The stubborn ones that remain above ground don’t have a chance of surviving (not in my visualization, at least). As soon as the weather starts warming up, though, the dormant ants wake up again. If I sound obsessed with ants, yes, I suppose I am. Every spring, I find myself battling fiercely with an ant colony that has established itself in my front yard. I don’t mind their being there, don’t get me wrong, but at this time of year some of them desperately and stubbornly want to march through my house, I suppose in order to reach the back yard. So right now, grrr!, I don’t feel badly about identifying them as myeloma cells. But I digress…

Now for a more, er, scientific approach.

First, though, a look at proteasomes. These are large protein complexes that are found in both normal and cancer cells. The relevant (for us) information about proteasomes is that cancer cells depend on these proteins in order to proliferate, metastasize and survive. And, in fact, increased numbers of proteasomes can be found in the blood of myeloma patients.

So one way to kill cancer cells is to inhibit the cancer-friendly activity of proteasomes. Probably the best-known proteasome inhibitor is bortezomib (marketed as Velcade). When Velcade is injected into a patient’s body, it disrupts the proteasome’s activities. As a consequence (simply put), cancer growth is inhibited, and the cancer cells die.

Now we get to our full Velcade-dormant myeloma cell study. Proteasome inhibitors provoke what is called an “ER (which, unfortunately, has nothing to do with the dashing George Clooney but stands for “endoplasmic reticulum”) stress response” in myeloma cells. This type of “stress” kills between 50 and 70 % of them, according to the study…but at the same time it sends out certain survival signals to 30-50 % of these cells that are thus able to avoid apoptosis by slipping into a deep sleep (or, more scientifically, by becoming quiescent).

They basically stop growing. And, since Velcade attacks cells that are in the process of dividing rapidly (=typical of cancer cells), this is an excellent survival strategy, which, incidentally, is used also by other types of cancer cells, such as head and neck squamous carcinoma cells.

At any rate, these quiescent cells can be annihilated, the researchers discovered, by adding to Velcade another drug called salubrinal. The actual experiment is rather neat, so I will attempt to summarize the parts that I understand in a comprehensible fashion. The researchers identified the myeloma cells that survived the Velcade attack and washed them to remove the drug. I confess, I was a bit amused by the image of myeloma cells being washed…anyway, the researchers realized that these cells had stopped growing. They were fast asleep…the little buggers. Velcade was no threat to them.

Now, there is a tremendous amount of detail in the study. I am not interested in the intricate mechanisms that show exactly how myeloma cells avoid death, mechanisms that, to be quite honest, I can barely understand, such as CHOP induction and XBP-1 splicing. So, skip skip skip.

Let’s get to the part that describes what happens once salubrinal enters the picture. After 24 hours, treatment with salubrinal of the Velcade-surviving myeloma cells resulted in a more or less a 10-fold reduction in the number of viable cells. Hey, not bad! And even after 5 days, the cells that are still dormant were highly affected by salubrinal. Ah, an important titbit: salubrinal does not affect the control population.

And now we finally (wiping our sweaty brows) reach the Discussion part where we discover that, after administration of Velcade, 30-50 % of myeloma cells escape death by becoming inactive. The researchers argue that this is more likely due to treatment adaptation than to selection of a cell population genetically predisposed to undergo quiescence. But the fact that so many myeloma cells survive treatment with Velcade is the probable and unfortunate cause of disease recurrence.

The researchers also found that survival of the residual quiescent cells hinges on the down-regulation of eIF2a phosphorylation. Phospho-whaaat? Okay, let’s not get too bogged down by the details of this phospho-thingie process. What we need to know is that the strong phosphorylation of eIF2a is associated with apoptosis. In the Velcade-surviving myeloma cells, you see, eIF2a was attenuated, so that is probably a mechanism whereby the blasted surviving cells are able to avoid apoptosis. Well, let us leave it at that…for now.

In conclusion, the solution to the Velcade-caused “dormancy” problem, as we have seen, lies in the addition of salubrinal to the mix. Salubrinal can virtually eliminate the fraction of quiescent MM cells surviving proteasome inhibition by enhancing the above-mentioned, er, eIF2a phosphorylation.

Quick note: this combination treatment will apparently benefit only myeloma patients. It doesn’t work against other forms of cancer, such as chronic myeloid leukemia.

The study’s conclusion: In summary, we report that the induction of MM tumor cell quiescence and survival can be an undesirable side effect of proteasome inhibition. We also show that, by blocking eIF2a dephosphorylation, proteasome inhibitor efficiency can be maximized during acute treatment and that residual cells can be eliminated by nontoxic doses of salubrinal as a monotherapy for MM minimal residual disease after proteasome inhibition.

Now I need a few days to digest all this stuff…

Some dietary flavonoids inhibit Bortezomib

August 8, 2008 / / Leave a comment

Sherlock (grazieeeee!) sent me a study titled “Dietary flavonoids inhibit the anti-cancer effects of the proteasome inhibitor Bortezomib” (see abstract: http://tinyurl.com/5pdxtu), published in the July 16 2008 issue of “Blood.” This is an important study for those who are taking Velcade (Bortezomib) and supplements at the same time, so I decided to read, and post about, the full study. It’s quite a long post, sorry about that!

Quercetin is the most mentioned compound in this study. As you can read on my Quercetin Page, quercetin is a plant chemical, specifically a flavonol, found in apples and red onions and a variety of other foods such as capers, broccoli and red grapes.

In the abstract we can read that quercetin inhibited the killing effect of Bortezomib on primary CLL and malignant B-cell lines. Interestingly, though, when inorganic boric acid (a low toxic substance derived from boron, an element found in rocks, soil and water) was added to the quercetin-Bortezomib combination, this inhibitory effect was diminished. Let me highlight that the cell lines under scrutiny were CLL (=chronic lymphocytic leukaemia) and other malignant B-cell ones.

And what about myeloma cell lines? Well, the researchers found that quercetin had a direct effect on myeloma cells, which sounds like very good news. Indeed, the researchers write, At high doses, quercetin itself induced tumor cell death. Death to myeloma cells? Yippeedadoodee! (Except that I recall reading that we should not take high doses of quercetin, I don’t remember why right at the moment, but I have never taken more than 1.5 grams/day of this supplement, so please be cautious…).

Let’s go on to the full study now. I might as well fess up right at the beginning that I didn’t understand all of it. So I will concentrate on the bits that I did understand. An interesting titbit is that Bortezomib kills CLL cells in vitro but does not display substantial anti-tumor activity in patients with CLL. So Bortezomib has anti-cancer activity in a lab setting but not when administered to CLL patients. Just goes to show that sometimes, unfortunately, in vitro anti-cancer activity cannot necessarily be reproduced in vivo.

The researchers then tell us that Quercetin is one of the most abundant flavonoids in the human diet and is a potent anti-oxidant. In fact, and this is something I did not know, quercetin is the chief dietary flavonol present in the blood. It is also a proteasome inhibitor, as is Bortezomib.

Let’s get to some results. As the abstract anticipated, there is definitely interference between quercetin and Bortezomib as far as CLL cells and other malignant B-cell lines are concerned. Not a good thing, clearly.

Two specific myeloma cell lines were also tested. In order to fight the killing activity of Bortezomib, however, these cells had to be treated with a very high dose of quercetin: Quercetin also had the same inhibitory effect on Bortezomib-induced apoptosis in two myeloma cell lines, U266 and RPMI-8226. However, the myeloma cell lines required higher levels of quercetin (40 micro-M) for maximal inhibition of Bortezomib-induced apoptosis, as compared to 20 micro-M for primary CLL, HRC57 and DoHH2 cells.

Then we read that In addition, myeloma cell lines were more susceptible to quercetin-mediated G2/M arrest compared with the B-lymphoma cell lines. So, in sum, when myeloma cells are “pre-incubated” with a not-too-high-dose of quercetin, their sensitivity to Bortezomib increases, which is a good thing. And another thing: myeloma cells can apparently be killed outright by quercetin, if I understood this part correctly. Of course, in vitro effects may not be the same in vivo, as we know. Proceed with caution, as always.

At any rate, further on we can read a possible explanation for this peculiar occurrence. When quercetin and Bortezomib were added at the same time, the former had an inhibitory effect on the latter. But when myeloma cells were pre-incubated with quercetin, the inhibitory effect was less evident: However, in both myeloma cells lines, pre-incubation with quercetin led to less inhibition on Bortezomib-induced killing, suggesting that myeloma cells are more active in their uptake and utilization of quercetin, thereby reducing the chemical binding between quercetin and Bortezomib. So myeloma cells, unlike CLL cells, gobble up quercetin, which is how the chemical bonding between the two substances can be avoided. Interesting.

I will skip the technical part about Bax activation and go on to a more comprehensible section. (Anyone interested in that part, though, can drop me a line; I would be glad to forward the study.)

The researchers tested other dietary flavonoids, such as myricetin (found in grapes, berries, fruit, veggies, herbs and walnuts), apigenin (in parsley and celery) and kaempferol (in broccoli, tea and grapefruit) , which, like quercetin, are antioxidant compounds possessing anti-proteasome activity. Of these, only myricetin had an inhibitory effect on Bortezomib. Interestingly, its chemical structure is similar to that of quercetin. Makes sense.

The researchers conclude that there are many dietary flavonoids which have similar structures with quercetin or myricetin, so the intake of dietary flavonoids may reduce the killing activity of Bortezomib on circulating leukemic cells.

Other findings: EGCG has an inhibitory effect on Bortezomib, so I probably wouldn’t drink any green tea if I were taking Velcade. I also wouldn’t drink any Cabernet Sauvignon or eat cranberries or Concord grapes, which contain a compound called delphinidin that also inhibits Bortezomib. As does cyanidin, found in many types of berries including grapes, blackberries, blueberries, cranberries, raspberries and cherries; and also in apples and plums.

But there is some GOOD NEWS: curcumin and resveratrol were also tested and found to be okay when taken together with Bortezomib, which confirms what I had read in other studies.

In the Discussion part I found another interesting titbit that is a bit on the technical side (sorry about that!)…here is the full quote: Quercetin and Bortezomib share similarities with respect to proteasome inhibition and the induction of apoptosis. They both can be used in the treatment of cancer. However, the differences between these two compounds are (1) Bortezomib is a boronic dipeptide acid and quercetin is a flavonoid; (2) Bortezomib is a ROS-inducing agent and quercetin is an anti-oxidant. The question is whether they are synergistic or counteract each other in combination. We observed that quercetin prevented Bortezomib-induced ROS generation and apoptosis in primary CLL cells. However, quercetin also blocked Bortezomib-induced apoptosis in B-cell lines in which ROS generation was not evoked. I repeat, this inhibitory effect of quercetin on Bortezomib is true for CLL and other malignant B-cells. Not for myeloma cells.

Further on, we read that, while Bortezomib does not seem to work well for CLL patients, it is highly active in myeloma, another B-lymphoid malignancy, suggesting differential sensitivity to Bortezomib according to tumor type. Also, and this sort of repeats what I have written earlier, myeloma cells pre-incubated with quercetin were not as protected as CLL cells from the apoptotic effect of Bortezomib. The researchers suggest that quercetin (at 20 micro-M) has a direct effect on myeloma cells, but not CLL, HRC57 or DoHH2 cells, to increase sensitivity to, and/or synergize with, Bortezomib.

The researchers add that it is not known if dietary quercetin (i.e., not in supplement form) would interfere with Bortezomib. So CLL patients don’t necessarily have to stop eating apples and onions (the easiest thing to do is ask your doctor). And they also remind us that Not all dietary flavonoids can inhibit Bortezomib. Myricetin showed a similar effect to quercetin on the inhibition of Bortezomib, but kaempferol and apigenin did not.

The study ends as follows: The differential in vivo activity of Bortezomib seen in myeloma and CLL may partly be attributable to the effect of dietary flavonoids: quercetin primes myeloma cells, but not CLL cells, such that they become more sensitive to Bortezomib-induced killing. Further work will elucidate the in vivo significance of these findings, which in turn will inform the need for dietary advice on the intake of flavonoids, as well as drug manipulation of flavonoid activity.

Hmmm, just as I had written a few concluding sentences and was about to publish this post on my blog, I did a quick online search just for the heck of it and came across a pertinent June 2008 “Haematologica” report (http://tinyurl.com/6hzqjg) whose findings made me add the following paragraph.

A group of Korean researchers tested six polyphenols—rutin, quercetin, caffeic acid, gallic acid, EGCG, and tannic acid—discovering that they all inhibited the anti-cancer activity of Bortezomib. Well, we already knew about quercetin and EGCG, so that is nothing new.

Now, the following is mere speculation on my part, but I have the feeling, based on what I understood from the July “Blood” study, that these compounds may have been added to the myeloma cell lines at the same time as Bortezomib, and that might explain the interference.

My point: the Korean study results might have been different if these substances had been added an hour or so before Bortezomib, which would have given the myeloma cells enough time to gobble them up, thus diminishing the interference potential. Pre-incubation with these polyphenols, in other words, might have made the myeloma cells more sensitive to the killing effect of Bortezomib, which would agree with the “Blood” study results. Again, since I have not seen the full Korean report, this is pure speculation on my part.

After reading this abstract, though, I would be concerned if I were taking Velcade and any of the above-mentioned and possibly interfering compounds (not curcumin or resveratrol, I repeat). So, here is my advice to Velcade-takers: please consult your doctor before adding anything to your protocol. Best to be on the safe side.

Margaret's Corner

Tag: Velcade