Good evening and good afternoon to those of us here in the United States who are on this call right now, and to those around the world, good morning, good night, wherever you are listening, we really appreciate you being here. This is Fertility and Sterility Journal Club Global, and this is a special one. This is our first of 2023, and so we're excited to be back.
This is our first fellowship program that we've had host at Journal Club in about six months or more, so we're really happy to have the University of Texas at San Antonio led by Program Director Winifred Mack here, and all the fellows and staff, so thank you, and then we're highlighting Fertility and Sterility Science, so one of the four in our family of journals, and our one primary basic science journal club, so we are excited to be here tonight. Daylen James will be our host, the media editor for Fertility and Sterility Science. If you're our listeners, on the right-hand side you see handouts.
You can download the article for free, so please do that and read it and look through it for questions. If you want to give us questions, type them into the questions function on the right-hand side of your screen, or tweet us at FertSTERT. The Twitter account is open, and I'll be receiving questions by Twitter, so Daylen, without further ado, I will turn it over to you, sir.
All right, thanks, Michael. Like you said, I'm Daylen James, media editor for FNS Science, and I'm so delighted to take part in my first Journal Club Global with such a great group of physician-scientists. Today, we're going to be discussing an article from FNS Science entitled Curcumin Inhibits Human Lyomyoma Xenograft Tumor Growth and Induces Dissolution of the Extracellular Matrix.
Joining us will be the first and last authors, Mini Malik and Bill Caterino, also our guest expert, Ayman Al-Hendi, and the REI Fellowship that is guest hosting this episode, the gang from UT San Antonio, made up of Fellows Srini Reddy, Arlene Goh, and Catherine Kostrin, Fellowship Director Winifred Mack, Division Director Belinda Yeager, Lab Director Art Chang, and Faculty Yetunde Ibrahim. Incidentally, my father-in-law has been pushing the health benefits of turmeric and curcumin on me for years now, so I can't wait to get into this, but first, Bill, as Editor-in-Chief of FNS Science, why don't you tell us about the journal and why people should publish their work there? Fantastic. Thank you, Daylen.
Obviously, one of our greatest crown jewels is Dr. Daylen James, who is our Media Editor and does a wonderful job helping to advance our media presence, and it's important to highlight this before we even get into the journal. One of the strongest aspects of being in fertility and sterility science is being part of the fertility and sterility family, because it goes well beyond impact factor at that point. When you're talking about the number of times an article has been cited determining an impact factor, we now talk to how many times your article has been seen and viewed in social media and other aspects, and we have a tremendous presence on all social aspects, well, maybe not Truth Central, but all major social media forums, and we have the ability to get information out, such as we're doing now with an article, to be able to share it in a different way than simply presenting it as a written piece of work, so it really provides a tremendous opportunity to get both feedback, to get collaborative efforts that you wouldn't otherwise get in the absence of just being part of another journal set, so fertility and sterility really provides some tremendous value in with regard to that.
Now, fertility and sterility science is specifically focused on basic and translational research that is advancing our field. The goal is to publish those articles that are going to be the seminal articles 5, 10, 20, 50 years from now. We're really trying to get those major advances that are there, and it has some distinct challenges and distinct advantages.
Clearly, our focus is not on case reports or on issues that are directly relatable today, because our goal is to advance the field and advance it in a major step by better understanding mechanistic aspects of the disease process, of the methods that we use, of the other areas that are involved in reproductive medicine, so it allows us to think about things and consider different topics that give us a greater understanding of these disease processes. By not focusing on the day-to-day typical care that you are typically providing, it allows you to break away from that for a moment, to step back to your times, and for some of you, it may be very fun, for some of you, it may be less fun, but have your research time and your fellowships or other times in which you've had the opportunity to kind of allow yourself the ability to kind of think through these mechanisms, and in that sense, it's great for the reader to be able to enjoy that, and the topics are tremendously relatable to the disease processes that you care about. Then beyond that, for the person who is submitting it, this is a great way to reach the specific audience that you're trying to reach.
If you're interested in gamete biology, in ovarian or testicular biology, uterine biology, you want to understand disease processes, and you want to get the real cutting-edge manuscripts out there that allow people to see this, it's a fantastic way to be able to reach that audience as well. I encourage you to submit your articles to FNS Science. It's very straightforward, and they've made it very, very easy to do that, and if you have any questions at any time with any problems, please feel free to contact me directly, and I'll be happy to assist you.
Thanks. Yes, as media editor, I'm clearly biased, but I have to say, as someone who's read the articles very closely for over a year now, it strikes a very fine balance between the basic hard science and the clinical relevance, and I think everyone will get a lot out of having a look at the journal and spreading your work that way, but enough of the promotions. Let's get to it.
Why don't you guys start, fellows, why don't you start off with a brief synthesis of the manuscript before we start the discussion, and your key takeaways from the article. Hi, I'm Katherine Kostra. I'm a first-year fellow.
Again, I'll start with the article. So curcumin is a flavonoid derived from the spice turmeric, and its use is known in many herbal supplements currently. Curcumin is highly pleiotropic and is known to interact in many cellular pathways.
Prior in vitro studies by these authors have shown that curcumin inhibits proliferation of human leiomyoma cell lines, increases apoptosis, and decreases matrix protein production. This basic science animal study sought to evaluate if curcumin may be utilized for prevention of fibroid formation or growth as a nutritional supplement in those at risk of symptomatic uterine leiomyomas as a low-cost and highly tolerated option. Next slide, please.
In this study, 30 over-reactomized mice were randomized into two groups. One group was given the standard lab diet, and the second group was fed a 2% curcumin fortified diet. Combined estrogen and progesterone pellets were implanted in the rats during the study.
Immortalized leiomyoma and myometrial cell lines were grown in rat collagen matrix in 3D form for 7 to 10 days before implantation. The leiomyoma and myometrial cells were then placed subcutaneously in each flank of the mouse or in a single uterine horn. Serums curcumin levels were measured at baseline and termination of experiment by liquid chromatography mass spectrometry.
After 4 to 6 weeks of exposure, the xenografts were measured and then excised. H&E stains were performed for histopathologic assessment. Further immunohistochemistry was performed to evaluate for fibronectin, furcican, and total collagen.
MGPY staining was performed to study apoptosis. Enzymography was used to evaluate for matrix metalloproteinase 2 activity. Xenograft volume was then calculated and the result reported as percent difference from original tumor size.
Statistical analysis was performed by student t-tests and one-way ANOVAs with a p-value of less than 0.5 considered significant. Hi, I'm Srini. I'm the third year fellow.
I'm going to go through the results. If you look at the left two plots here, the total volume of leiomyoma xenograft in mice on curcumin, which is labeled as L-C-U-R, decreased more than 50 percent as compared to the volume of xenografts in controlled diet labeled L-C-O-N after 4 to 6 weeks of curcumin exposure. The median leiomyoma tumor volume on the curcumin diet was 4 millimeters cubed compared to 10 millimeters cubed on the control diet.
That's a 60 percent decrease and the difference was statistically significant. If you look at the right two plots, we see that the volume of myometrial xenografts on the control diet labeled M-C-O-N versus on the curcumin diet labeled M-C-U-R were not statistically significantly different in size. The median volumes of M-C-U-R and M-C-O-N xenografts were 8.3 and 8.5 millimeters cubed respectively.
This indicates that curcumin plays a role in reducing the size of leiomyomas but does not affect neighboring myometrial tissue. Next slide please. Looking at figure 3 on the left, the leiomyoma xenografts in control mice expressed a compact extracellular matrix that was located adjacent to the mouse skeletal muscle layer.
But in figure 4 on the right, which shows the xenografts obtained from the curcumin diet mice, we see the dissolution of this extracellular matrix structure. Next slide please. In these figures, blue staining indicates new collagen formation.
The left figure shows the control xenografts with intense blue staining indicating new collagen formation. The right figure shows the curcumin diet xenografts with diminished blue staining indicating decreased total collagen production. Next slide please.
Figure 6a shows strong fibronectin staining of the control xenograft mice indicating actively growing leiomyoma xenografts. Whereas figures 6b and 6c show very weak staining for fibronectin in the curcumin diet xenografts. 6d shows positive versicans staining in the control xenografts as opposed to 6e and 6f which show a decrease in versicans staining in the curcumin diet xenografts.
Next slide please. Figure 8 shows apoptotic cells with condensed nuclei and eosinophilic cytoplasm in the leiomyoma xenografts of curcumin treated mice. The images on the right here in 8b and 8d show this more clearly at a higher magnification.
The black arrows point to the apoptotic leiomyoma cells where the blue arrows point to the myometrial cells of the mice with no changes to the nucleus or cytoplasm in contrast to the apoptotic changes observed in the human leiomyoma xenograft. This indicates that curcumin induces apoptosis in leiomyoma xenograft cells but does not induce apoptosis in healthy mouse tissue. Next slide.
MGPY stains the condensed chromatin of apoptotic cells and appears as a red stain. Figure 9a on the top left is of controlled diet xenografts which show no MGPY staining indicating no apoptosis. 9b shows this same control group at a higher magnification with still no apoptosis noted.
However, when we look at the second row which shows leiomyoma cells in curcumin diet mice, there is significant deep red staining indicating apoptosis. Black arrows in 9d point to these areas of apoptosis. Blue arrows again point to healthy mouse myometrial cells without any apoptosis which is again consistent with the previous slide demonstrating that curcumin only affects leiomyoma cells and has no detrimental effect on surrounding mouse tissue.
To summarize the author's conclusions, they observed that the mice on curcumin diet demonstrated decreased growth of the leiomyoma xenografts, dissolution of the extracellular matrix, and increased cell apoptosis. This suggests that curcumin may be used as a nutritional supplement for women at risk of symptomatic uterine leiomyomas to treat uterine fibroids, but of course further human studies are required first. And just to get the discussion going, we had a couple questions for the authors to start things off.
So Dr. Malik, first for you, what motivated this study and what do you feel like was the most interesting in terms of findings? Dr. Malik, you're muted. Yep, so it all started as you had covered that in 2008 we had done the in vitro work and we had seen some apoptosis, increased apoptosis. So there was this big gap where we were developing the 3D cultures because we wanted to understand what really was happening in the matrix because in 2D cultures when you're growing the cells and plates you really don't have a matrix to study the changes that are taking place.
And that also led to the development of the 3D CDX which is cell line derived xenografts. So the question in my mind always had been, and right now I'd like to clarify that I'm a plant biologist by my degree, and was that the bioavailability of most of the plant products, whether they are flavonoids like curcumin or they are polyphenolics, the red color in your wine that I'm sure you all are going to have after the this meeting, is the bioavailability of these compounds is very low. So they are taken up very quickly, they get metabolized very quickly, and they get excreted out very quickly.
So always the question was are we ever going to have serum levels enough so that the tumors that are tumors like uterine fibroids or breast cancer tumors will be affected or not. So with most of the plant products the tumors that are found in the GI tract, they are influenced much more quickly because there the plant extracts or the plant products are coming directly in contact with the cells or the mucosa lining or the colon cancer. The experiments which I had done early in 2000s, you see a very positive effect.
But with uterine fibroids, I kept going back to reading the clinical trials trying to find out if is it even worth doing the experiments or not. So this was two years ago when I decided to do a small pilot study. And at the end of the study, I saw the mice are on the diet before we do the implantation of the xenografts.
So after I collected the serum and I separated out the serum, the serum was dark yellow in color. And that made me think, uh-oh, something's gone wrong. Why is the serum yellow in color? So I shut down everything, stored it in minus 80, and I said I have to do another pilot study and see if this is true.
So a couple of months later, almost really six months later, I set up another small study. And these were just five mice. Collected the serum at the start of the study, middle of the study, and at the end of the study, and the serum was dark yellow in color.
That is when I got in touch with Dr. Sean Moran, who's the LC-MS expert. And we did a lot more searches and we precipitated out the proteins, thinking maybe it's the proteins that are there to which the curcumin is binding. And that's why you're having the yellow color.
So a lot of proteins were precipitated out, but you still saw that yellow color. And that is what triggered this, the 15 mice study that we did a year later. And that was the first interesting thing that we found in this study.
After that, Dr. Britton, who is the pathologist, and one day just called us. I don't know if you were there, Bill, or not. She called and she said, I want you guys to come to the conference room and I want to show you some slides.
So we go there and she says, what do you see here? And we said, oh, the matrix looks funny. So maybe the xenograft is not what it is supposed to be. And at that time, I know Bill and I had this conversation.
We were talking about the fact that could there be something wrong with the 3D cultures that we implanted in that. So we went back to older studies. Dr. Britton just took out that and the control mice from the other studies that we had done in the mouse study did not show any dissolution of the matrix.
The matrix was always compact, but all the mice from the pilot study mice, as well as this large study mice, all along the periphery of the compact matrix, there was this dissolution of the matrix taking place. The cells were much more closer together and at higher magnification, you could see that the collagen was more stringy compared to what you would see in a compact matrix. So that was a second very interesting observation we had.
And that led to the study of the metalloproteinases because anything that was breaking down, not only the matrix that the cells were forming, but also the matrix in which the cells were already growing. These are actively growing cells in a matrix that are implanted into the mice and that was breaking apart. So that led to the metalloproteinases study and we saw that there was an increase in MMP2 and MMP9.
So I haven't added the data for that. Now, this was the study that we have. If I go over the time, Dr. James, you can stop me anytime.
But this was not the only study we did. We did the diet study because that is what we were interested in. But we had a study where we were even you know, injecting the curcumin injections were being given to mice.
But that's a completely different story and if anybody's interested, I can always go over those also. So those were our interesting observations that really made me think because a lot of people would ask me and Dr. Alhundy will agree with that. A lot of people used to ask me, it's a huge gap from 208 to 222 where, you know, and nobody else published.
So that also always put this question in my mind, what's going on? So I agree, extremely interesting results and we are very proud of it. That's good news. Thank you.
And then I wanted to give Dr. Caterino a chance to add anything in terms of kind of other motivating factors and highlight the most, their most interesting findings as well. Sure. So if you think about it, I mean, from a clinical perspective, we're always struggling with how do you treat fibroids.
And way back in the day, and Ayman has heard this a thousand times, I would always say, listen, if we come up with a compound, if I came up with a drug that killed every single cancer cell and left all the other cells alone, I deserve a Nobel Prize. If I come up with a compound that kills every fibroid cell and leaves the rest of the fibroid there, I haven't really helped her that much. It won't continue to grow, but that fibroid is still there because it's a big scarred mass.
So when we're looking at therapies, we need to try to identify therapies that actually don't just affect the cell, but affect that extracellular matrix. And that's why, that's what motivated us to develop the 3D model and the in vivo model. Now, at the same time, we're slowly getting therapies for patients who have symptomatic fibroids.
But what do you do about the woman who's 24, has a three centimeter intramural fibroid, and her mother or sister aunt all had hysterectomies in their early thirties. Now, she's not symptomatic. You're not going to give her any therapy that's currently for symptomatic fibroids, but you don't really have a lot of options.
And Ayman has done wonderful work on vitamin D. And we started looking at curcumin, other groups looked at resveratrol and other compounds. And that's, we started to say, is there something that we can give that has a very high therapeutic index, meaning that it can, you can give large doses and the patient is going to really be all that symptomatic. Is there a compound that we can give that you can add to your, either take it as a tablet or add to your food that would allow you to treat these fibroids so that you're not at risk of having that fibroid continue to grow.
And it's always been kind of that holy grail of trying to identify a really good therapy that isn't going to harm the patient. All fields look for this type of therapy, obviously, because you really want to limit symptoms and help these patients. So one of the challenges as we're setting up some of the later studies, and one of the things that was important is that instead of injecting curcumin, it needed to be in the diet.
And the reason it needed to be in the diet is if we were unable, as Dr. Malik pointed out, that if you give it to patients or give it to patients, give it to mice, and it has a very low bioavailability, very little is absorbed and very little is active. And because of that, you're never going to get blood levels that are sufficient to treat the fibroid. So we needed to know whether it was possible.
So one of the first points that you see in the paper is that we give it to the animals in their chow, and the question was, do they hate it? Do they refuse to eat? Are they losing weight? You know, is it that it's just unbearable and they just can't, they can't take it? And the answer is no. They eat their chow just as much. It's not like we have lots of leftover chow.
It's not like they're losing weight. Okay, that's encouraging. Then we look at the blood levels.
Hey, it's getting into the blood. Then we looked at what it's doing to the fibroid. Guess what? It's decreasing fibroid size.
We look at the myometrium, and it's patient-matched myometrial cultures. So genetically, they're similar. They're from the same patient.
And we don't see those changes. That's very encouraging. Then we say, okay, well, how is it doing it? And that's where we start getting into these really interesting aspects where we see it breaking through that matrix.
And this is where you start to get a sense of this has real potential. And then in the end, this is what we're trying to do is identify the compounds that we can ultimately say, what do we talk to with patients? That 24-year-old with that three centimeter fibroid. Right now, we would do watchful waiting.
That's the best we could do. We really don't have therapies for her. But if we can say, here are some things that might provide value, that's encouraging.
Ultimately, and as you all pointed out, this would have to be ultimately assessed in patients who are also able to do the studies that we're describing now. Is it that they could tolerate a sufficient dose? Can we get appropriate blood levels? Does it have an impact on their fibroid without causing them symptoms? Because remember, we're talking about asymptomatic patients. So this is a very exciting first step in trying to move curcumin from an in vitro world into an in vivo world where it provides that value.
Thank you. And we'll definitely bring up some questions to try and more clinical relevance. I just wanted to give Dr. Alhundy a chance maybe to lend some expertise specifically in discussing strengths and limitations of the paper before we dive into the more clinical relevance.
Sure. Thanks. And thank you for a very nice presentation of data.
So I'm Alhundy. I'm a gynecologist, minimally invasive surgeon here in Chicago. I'm also a scientist.
Our team has been funded by the NIH for the last 21 years continuously to study fibroid. And I'm a tenured professor here at the University of Chicago. So I'm just so happy to be here discussing this very nice, interesting paper.
I love the paper for all the reasons that Dr. Malik and Dr. Catharina talked about. It's such an important disease. And as Dr. Catharina said, fibroid is a proliferating cell and lots of extracellular matrix.
So when you address a disease, you have to address both, exactly as you heard. This also address a very important unmet need in the field. As you heard earlier, we have some new medications, FDA approved, in a golex with advect therapy, golex with advect therapy have been FDA approved in the last two, three years.
So we have the new tool for the first time, probably since Lupron, which is, you know, like 30 years ago. But all of these are hormonal. Everything to do with fibroid in the last 30, 40 years has been hormonal treatment.
So there's a huge unmet need and gap of knowledge in terms of non-hormonal treatment of fibroid. So this paper start to address that. And why is that important, especially for you guys? I'm telling you, you're all RAIs and many people on the call are RAIs.
Because what do we do now with many patients with fibroid? Yes, they might be symptomatic with heavy bleeding, et cetera, but they're also pursuing a pregnancy. And short of doing a myomectomy, what we do now, we tell them, okay, we're going to treat you with something to shrink your fibroid and so on. But while you are on that, because they're all hormonal, you cannot pursue pregnancy.
All right? So you have to put your plans, ambition to get pregnant on hold, you know, for six months or even a year to shrink your fibroid as much as you can. And then we're going to stop this. You're going to start ovulating.
You already need to get pregnant very quickly because your fibroid is going to grow again. This is not really a viable way to treat a disease, a human disease. It's very frustrating to the patient, to the doctor and so on.
So we really need fertility-friendly medications. And then this being a natural product, although this paper didn't touch on that, and I included this among the potential weakness or missed opportunity, but natural products tend to be very fertility-friendly. They don't tend to be benign on the ovary, et cetera.
So this is very exciting. So this paper has a lot of strength. This team, Dr. Malik, Dr. Fino, really has been leading the field on what's happening with the extracellular matrix in fibroid, just understand the biology, and now developing new treatment options.
And they also, I congratulate them also in taking the in vitro. Many people do in vitro studies with many different products. But really, until you do the in vivo in a reliable mouse model, you really don't know if this even have any chance of working in humans.
And many teams stop at that point, don't do the in vivo. So I want to really applaud them for taking this further into a mouse model, a great mouse model. Also, as you mentioned in the presentation, they also actually innovated the mouse model.
So it's not only subcutaneous, but they also injected the leiomyoma inside the wall of the uterus. And those who work with mice, they know it's not easy. The wall of the uterus in mice is so thin.
So I'm really curious, and I'll ask Dr. Malik when I see her at SRI, how did she do that? So really, this paper also have some technical innovations and so on. You know, like any paper, there are potential limitation, potential weakness, really very few. I would say like the bioavailability, major challenge to a lot of these nutritional products is they are not available in the serum and reach the uterus in sufficient amount.
This paper, yes, they did show efficacy, but I'd be curious to know if those they use in the mouse is translatable or can be extrapolated to the human safely. And speaking of safely, also this paper, maybe it's coming in the next paper or so, but there was really no assessment of safety. I know curcumin is a natural product, probably very benign, but I think there are some reports high dose of curcumin can also be negative on the liver.
So I hope this yellow serum, Dr. Malik is not showing this or anything. Unlikely, because the mouse, you said they were healthy and so on, but I think that we should also evaluate safety even of natural products. Also, some of the observations in the figures were more qualitative.
Yes, I can appreciate the difference. It looks really different treatment versus control. It would be nice to quantify things to avoid any subjectivity and so on.
But also the last thing is this stain, the MGPY, I have to look it up. It's not used the commonly for an old stain, really designed to differentiate DNA from RNA. There's a lot more reliable apoptosis staining like tunnel, like staining for caspase-3, caspase-8 and so on.
But overall, I love this paper. I think it's an important addition to the field and it's highly translational. And I would love to see the next step and maybe even a clinical trial.
If I can step in just to allay some of Dr. Alhandy's concerns. One of the challenges that we're always going to have, I think it's true, is our own success. So one of the problems that we run into is we put the xenograft in, we allow it to grow, and then we treat it, and it gets smaller.
So we don't have a lot of tissue to work with. We would have loved to do Westerns. We would have loved to do these other things.
But because of the very limited amount of tissue that we had, literally cells that we could count, because it was working, it put us in a situation where we had to come up with novel ways of answering this. So Dr. Alhandy's exactly right. The studies that he's talking about as far as quantitation, as far as truly defining apoptosis is exactly correct.
These would be the best options to choose. Our hands were tied because the masses were going down. One thing I can say with regard to liver, which I think is a really good point, is Dr. Britton is a trained pathologist and she would do autopsies on all of our animals.
And so we would get slides and she would show us sections. In fact, she saved us a tremendous amount of time and effort by being able to identify the reasons that some of our mice were dying and allowed us to change practices such that it moved us forward very quickly. So we have actually looked at it.
Now, you're exactly right that in order to move this forward, we need to do more careful toxicology type studies. But the encouraging news is we didn't see a lot of harm to tissues in the liver, lung, kidney, other areas that you'd expect it was really toxic. We should see some.
Excellent. Thank you for those answers. Now we'll turn it over to some of our faculty.
They have a couple of questions as well to kick off discussion. Hi. Great paper.
I really enjoyed it. So I have a couple of questions. So I think Dr. Alhindi had sort of sort of alluded to this is I was just wondering how you decided to use a 2 percent curcumin diet versus like a lower dose or a higher dose.
And how does that translate the 2 percent? Is that how much would you have to add to your food, I guess? So 2 percent. This was mainly by literature search. So 2 percent was somewhere in middle what people have been using for their animal studies.
It ranges anywhere from point. So 500 milligram per kg to people have been using up to four. So we decided to start with 2 percent.
And it kind of relates back to in humans as it has been FDA labels it as grass, which is really it's generally recognized as safe material to be ingested by humans. And people have gone up to eight grams. And in many of the studies, eight grams of curcumin per kg body weight.
It relates to about almost two micromolar in the serum. So a lot of biochemistry on curcumin has been done in relation to clinical trials that have been taking place. So people have been taking if you up to almost what is recommended is three to five milligrams.
That is what ultimately the grass allows as has defined curcumin to be taken in. So three to five milligrams per kg body weight per day is what you can safely take without any side effects. There have been like said there have been some toxicity studies that have been done by clinical trials.
It depends. It has. Correct me if you're wrong.
You all would know anti-plasticity plus what is it? Platelet. Sorry. And the come on, you guys should know this.
You are all MDs. So does the platelet formation. That is where the toxicity comes in.
And I'm not sure which cancer you can relate it to. But that is the only toxicity paper that I came across. But overall, I was quite surprised that as to how much amount you can take per day for curcumin.
But as I said, if you read most of the clinical trial results or you read the review articles, they are extremely effective as anti-inflammatory because of their antioxidant activity. And they are also very effective for the GI tract tumors more than. And I did come across one paper, which I was trying to find again, is that triple breast cancer, it has an opposite effect.
So it increases the invasiveness of the tumors. So you have to be really careful. One thing you have to realize that turmeric is made of three curcuminoids and that is curcumin by itself.
It's keto form. It's a demethoxy curcumin and this is the methoxy curcumin. The methoxy groups are removed.
So if you look at the first figure, you will see that curcumin by itself is around 300 nanograms per ml, whatever you're seeing. But the other two curcuminoids are much higher in concentration. So that led to the question is, is it really curcumin as a curcuminoid, which is more effective? Or is it the other two curcuminoids that are found in turmeric, which might be much more effective? So there are people working on that.
We've also decided to work with that. Also the reduction components of the reduced forms of curcumin, these hydrogenated forms of curcumin, which also are effective, both as anti-carcinogenic compounds, but also some of them at certain concentrations can have an opposite effect. And this is something you will find very much in, I'll call them the natural compounds, but even the polyphenolics, if you read these papers, a plant product as a whole can be very effective anti-carcinogenic compound or have anti-gasogenic activity.
You separate out these compounds as, for example, in the polyphenols, you separate out these compounds and you'll be very surprised. Some of them have carcinogenic activity. Some of them might have anti-carcinogenic activity.
So I think the field is taking baby steps right now. We have a long way to go for this. I hope I answered the question or did I just... No, that was great.
I think I just actually opened up. I got an ad about curcumin as being sold as something people should take. So I was like, oh, how much should we take? Is it going to be a pill? So that's great.
Thank you so much. One thing to also remember is that when you're getting it, you can go to Amazon or you can go to a natural food place and you have no idea what's inside of that tablet. So you really need to have groups that are serious about providing you with what is stated on the bottle.
And it's important. I think it's true for all compounds or anything that you're taking, that it comes from a place that takes this seriously and isn't simply trying to take advantage of the interest in this particular therapy or any particular nutraceutical. So I'll add something here is nowadays you have the nano formulations of curcumin that are coming out.
These are curcumin in nano formations. It's liposomal compounds. And basically people are starting work with that now to see if we can increase the bioavailability.
These are available and they are also available in surprisingly in food stores and all that. So I would say you have to realize it doesn't really matter much is to feel you're not taking very high doses, but if you're taking the doses that you're having in this thing, it's a 0.01% of it is going to be absorbed in. And that will affect your even go to the tissues.
It's really a very low concentration that is absorbed in the body. But it is effective. All right.
How about another question from the faculty for the authors? So one question I have is just a little bit more about the technical aspects of it. So as you mentioned, these are very small amounts of tissue that you were working with during your collection of the xenografts. How did you ensure that you were just getting the xenograft to study and separating it from the surrounding tissue and things like that? So if you look at figure one or is it figure two where you see the zoomed up picture of the Lyomyoma tumors, those they are very clear.
I mean, you can literally see that you insert a one millimeter to two millimeter size and it's grown up to 11 millimeters in size. But it really grows up as like the fibroid. It's not too much flat or anything, but it's a nice rounded structure that you will see.
So it becomes we usually take it when we collect it. We collect it with the mouse tissue because we really like to see how it is between which layers it is present and all that. So we do take that.
But as Dr. Cafferino had said, Dr. Britton is a trained pathologist and we have done, we have published where we've shown that it is easy to distinguish using mouse specific proteins and human specific proteins. You can always tell the difference as to which is the mouse tissue and where the human tissue starts. It is and right now I'd like to say for the in uterine, the uterine horn localization, most of the credit goes to Dr. Anthony DeAngelis.
He is an expert surgeon. That's all I can say. At the end of the study, after a few weeks, you couldn't even see the incision site.
We really had to make sure we had the right mouse, but they did not grow as fast as we had anticipated because subcutaneously they grow really by fast. I don't mean like tumors, but they grow by four weeks to eight weeks to 12 weeks. We are able to see a nice fibroid formation, whereas in four weeks it was very difficult to locate the fibroid xenograft in the uterine horn.
The only way we could do it was we were looking for the cells. We did the ECM, which are human specific and the protein antibodies. So we looked at those and that is how we were able to distinguish.
And the fact that the NGY-PY stain is, and that was the only way we could really see the bright red color of the thing. I mean, we were thinking of doing tunnel. We were, we lacked the tissue.
Now we are developing, going further on to develop the better, develop the model system. And I'm sure we'll be able to do that. But most of the credit goes to the surgeons or the fellows who work with us in really putting the xenograft in the uterine horns.
A quick, a quick comment about this intramural model, because it's really intriguing. And if it's really can be used and it's feasible to be done, not by only that person, then I think would be an important tool because you put the fibroids in the natural environment. Really, most of them, human made models for fibroid, aside from the ECHA rat, really, they are all subq or intraperitoneal, which is not the natural place for a fibroid.
But I'm curious, because you said after a while you couldn't see it, like, you know, there are some human, because obviously it integrate and remodel with the mouse myometrium. There are some proteins that are only human specific, like vimentin or certain RNA primers, like alloprimers and so on. You can do RNA scope, things like that to help you, you know, find the human tissue, even in the ocean of mouse tissue.
It seemed like you relied on the brilliant pathologist you have and her experience, but that might not be available to everyone. That's true. She unfortunately retired in December.
But you're right. We are still developing the model system. The reason why we put that in there was the fact that, compared to the subcutaneous apoptosis results, the ones in the interuterine horn showed intense apoptosis.
It was as if the tumors just didn't grow in there. And it was not only in one mice, it was in all the mice, the intensity of apoptosis of the cells that was taking place, not in the surrounding mouse tissue, but the human tissue was much more intense than what we were seeing sub-Q. The other thing we knew why it was happening was that if you take, we had one control uterine horn and we had one uterine horn that's in the supplementary figure where you see the very implanted xenograft is the picture that we didn't show is that the blood supply from the sides, the blood supply that goes in the normal uterine horn where there was the control uterine horn, it was very normal.
It was not red. Whereas the intensity of the blood supply to the, please put it in the medical terms as you all want, but the intensity of the blood supply in the uterine horn in which the implant was there, the xenograft was there, was much more. So we knew that something was going on and that could be the reason that the intensity of apoptosis of the cells that was taking place in intrauterine was much higher than what you were seeing in subcutaneously.
And one other, so with regard to assessment, I think there's a couple of things. Minnie brought up the fact that you could see it grossly in the subcutaneous, even though you're seeing that angiogenic process where the blood supply is supporting it. And Dr. Britton actually made it very clear that you didn't need a state.
She could identify very clear where the human cells were and where mouse cells were based upon the shape of the nucleus, all the things that you've learned in pathology, she brought to bear and showed that there are clearly different cell types, different areas, you could identify them just visually and the stain just confirmed it. But the fact of the matter is that you could identify it. One of the, and this is inherently a challenge that we have, where we have more control tissue because it can grow a bit more and our treatment is decreasing in size, but the ability to identify which ones were human cells grossly and microscopically, we've actually spent a good amount of time trying to come up with complementary assays to make sure that we feel comfortable that we're actually calling this, you know, a real human cell-like mass.
Plus remember, we also had some myometrial cell-like to compare it to, that allows us to say we know what human looks like and it's less impacted, really wouldn't see much of an impact at all. All right, let me just hijack the conversation here as we're getting toward the end and ask about next steps. This is a question for you, Dr. Catharino.
You know, this isn't your first dance with curcumin and myomas, you did a FNS story almost 15 years ago. I'm aging a bit there, sorry. But looking at the positive therapeutic effect in vitro, and I think that was a really big move here with this story is that you elevated it, right? You took it in vivo, but where do you go from here? I mean, one argument would be maybe you circle back, take the lessons that you got from this in vivo study, the matrix metalloproteinase, or whatever you think is underlying the dissolution of the ECM there, and look back at a more higher resolution level in your in vitro cells, or a comment from Dr. Alhandy about, do you go to patient-specific lines? In the chat, he was saying, do you go to patient-specific lines to kind of obviate the in vitro artifact of these immortalized cells? What are your thoughts moving forward, how you can elevate this even more? You know, beyond just the idea of therapeutic levels and delivery of curcumin, whether we could get into patients, how can we understand the biology and mechanism of this therapeutic effect? Where would you start asking that question? Oh my God.
Okay, so you're hitting a lot of different topics. So clearly, you need to work in both directions. From a patient-centered focus, because we are a clinical department, we're a clinical field, we really need to be addressing the fact of, if this is an effective therapy, what are the steps that are required to be able to confidently move forward with human trials? And there's a slew of them.
I presented a talk on moving a drug forward, and there's many steps that we would need to do to move in that direction. With regard to mechanisms, which is also fascinating, we've opened the door a little bit. Dr. Mallett pointed out the fact that we had several, both the curcumin main and then the metabolites that we identified, and it's certainly possible that the metabolites may or may not have differing effects.
And if we identify that one of them actually has a primary effect, and perhaps the others do not, that would help us hone that in. And how curcumin works is a challenging grab bag, because it does so many things. The one thing that we can say is that we can see the effects on this particular tissue type, but we can't necessarily yet identify what mechanism leads to that effect.
And that would certainly allow us to potentially create an even more directed therapy, or perhaps one that has greater bioavailability that allows us to do lower concentrations. So there's quite a bit of work to be done. We're happy to have people join us in this way.
We're thrilled that Dr. Alhendi is working on his same thing, because we're all here for the same reasons. We're all here to provide therapies that give women better opportunities. And one thing that I'd like to do is ask the fellows, let's assume that one of these therapies, since we're talking about curcumin, we can say curcumin, but one of these therapies is available for your patients, and your patient says, I've got fibroids, I'd like to take this, but I'd also like to get pregnant.
How do you counsel such a patient in that scenario? I think I'd focus on the safety profile in pregnant women. I don't know if that's been evaluated, but that's something I would mention, as well as the efficacy in a woman who's pregnant, and possibly revisit based on the data after they're done being pregnant. Quick comment on that, because I think that's really important.
Curcumin has been shown to, in addition to all the wonderful things that Dr. Malik talked about, you know, anti-oxidative, anti-inflammatory, but it has been shown to actually stimulate the contraction of a smooth muscle. So I think that's the kind of things that we need to have in trial, because, I mean, a patient with fibroid, you already have dysmenorrhea, you don't want to give them more dysmenorrhea, and certainly if they're going to continue in pregnancy, you need to think about, you know, miscarriage, preterm labor. So definitely we need a lot of safety studies and so on, and some of these studies, honestly, unfortunately, you cannot do it unless you try a primate or so, but sometimes you just have to do it in a phase one trial.
All right, so we got future directions from Dr. Catharina, we got a cautionary note from Dr. Alhandy. Let's go around just to finish up here and get some major takeaways in a minute or so. Starting with you, Dr. Mack, what's your takeaway from this story, if you had to synthesize in a minute? Sorry, yeah, so I think that overall, I think it's definitely something that definitely we should evaluate the safety, but I think the in vitro study and the in vivo in the mouse are really promising, and I think it's really exciting work.
All right, how about the rest of the group from UT San Antonio, one by one, you want to give us your takeaway? Sure, I think, you know, I just, I want to know, you know, how this study helps you plan the next trial in humans in terms of dosing of curcumin and how you're going to measure the side effects in humans. So, you know, I think it's very promising what we've seen in this study, and I think, you know, using the information that you all provided in terms of what the allowable doses that, you know, patients are using over the counter right now, I'd be interested in knowing how you're actually going to determine the dose you're going to use in actual human studies to measure the safety and efficacy of this curcumin. Sorry, I joined late, so I'm curious, you know, if there's some natural, you know, kind of situations where this could be looked at.
I know curcumin is, you know, used in a lot higher, you know, amount in certain areas of the world. You know, is there, you know, an opportunity for some natural experiments that are already happening from a dietary standpoint, measuring serum levels and then actually looking at prevalence of fibroids that, you know, would kind of form the trials that would need to be done, you know, in the future. So, I'm curious about that.
So, I'm going to interrupt here and say in most of the Asian countries where not only curcumin, but the effects of soy and curcumin and other polyphenolics, which are used in cooking, starts in utero. I mean, they are exposed much earlier. There was a study where they had done in Asian women's, they were looking at the fibroids, if there was less fibroids or more fibroids or something.
I'm sorry, it's right now, I don't remember the authors in that study, but the study was done in a province in China, and it was also done in one of the hospitals in India. And overall, the conclusion, it was inconclusive. They could not say whether the exposure of most of these compounds in utero was resulting in decreased uterine fibroids or any of the other cancers.
There was a review article I read, I'll try to find it again. And so, and you also have to remember that curcumin or soy, which is taken in most of the Asian countries, these are cooked, this is put in meals, so they are cooked, so they lose a lot of their potency anyway when they are being cooked. So, it would be nice to see what the serum levels of curcumin throughout most of the countries where people are taking it almost every day, versus what we get, for example, as a cautionary tale is this soy is now in almost every food material that you have here in US.
And there was this young, I hope you have time, but this young girl in the University of Maryland came and she had calculated how much soy her mother was taking every day, soy milk, soy bars, soy this, everything, everything. And she calculated and she came and said, this is the amount of soy my mother takes in a day. And do you think this is safe? And this was Food and Nutrition Department in University of Maryland, College Park.
And so, she wrote a grant proposal for Howard Hughes Fellowship. She got the fellowship and she did the experiment with us and she worked with rats. So, young, old, mature and old rats.
And surprisingly, for colon cancer, it was induced by azoxymethane. And we found that the ACF decreased in young and mature mice, but most, we lost 80% of our older mice, which was 22 months old mice because of unexplained bleeding. So, the pathologist said that they were getting a lot of bleeding.
So, that was very high concentration, almost a very high concentration of soy that was given the papers published. But things like that happen. People, the public just gets excited about something and they just start taking it.
And the companies that sell it don't, even though they will put it in their applications as to how much they're going to put, nobody really looks at how much they are taking. So, that is something that has to be a precautionary measure that public needs to be aware of, which you don't see much out there. So, the patients, many of the people at ASRM who came up to me and said, Dr. Al-Handeed, they came up to me and they said, well, if you're recommending our patients for vitamin D, should we also tell them to start taking curcumin? And I was like, just tell them to consult their physicians.
I have no idea. That is where you all come in. You have that precautionary thing you have to tell your patients because public just doesn't know and they just go overboard.
All right. Well, in that time is closing in on us. I think we have time for one minute for the senior author here, Dr. Catherine, to give us our final impact statement.
What do you want the readers of the journal and the reader of this article to take away from this story? All right. So, the main thing is it's important to recognize that we have a lot of work to do with uterine fibroids. They're clearly very prevalent.
They're very symptomatic for a lot of patients. And patients are really suffering. This is something that we're really, it's an important topic and one in which we can make progress.
And we have made a substantial progress. When I first got in this field, all we had were pathological specimens. Now you look at the opportunities of the various model systems and the interest is growing in novel therapeutic ways of addressing uterine fibroids.
This is one of many and one exciting area where we have the opportunity to advance and provide a totally new, as Dr. Al-Handy pointed out, something that's non-hormonal but provides a significant benefit. Is curcumin though the study? Am I recommending everybody run out and get it? Of course not. But I am saying here is an exciting finding.
And we should continue to press on to identify how this plays out, such that we can provide novel therapies and advance both our understanding of how fibroids develop, but also how we can better treat our patients. Yeah. I mean, listening to this, I have to say, I'm all about scientific rigor, but there's this highly unscientific theory I like to cite called the Lindy effect.
And it postulates that the future life expectancy of any idea or approach, any non-perishable thing is directly proportional to its age. For example, like a book like The Odyssey that's been read for a thousand years, it's likely to stay around for another thousand years because it has that longevity. And when I see treatments like curcumin, which has been in practice for centuries in Ayurvedic medicine, I think of the Lindy effect and I believe that there's got to be some biological influence that can be leveraged therapeutically.
But the science is murky, right? Because it's chemically unstable, water insoluble, highly metabolized, minimally bioavailable, all these things we've talked about by a diet, meaning the distribution is going to be poor and variable. In the gut it's there, in the ovary maybe not, in the uterus maybe not. And the compounds metabolites are really promiscuous also.
We haven't talked a lot about that, but all those metabolites, they have many different targets. And so you see all this different activity, antibacterial, antimicrobial, anti-inflammatory, antioxidant, anti-aging, anti-carcinogenic, anti-everything. The great thing about this study, in my view, is it delivers on critical points, which Dr. Catharino really highlighted earlier.
It incorporates a careful and controlled design. It demonstrates bioavailability of curcumin and the metabolites in the mice. It shows a clear influence of the curcumin diet on the xenograft volume.
And it delves into the molecular mechanism for reduced tissue volume that's supported, I think, by previous experiments, both by Catharino's group and elsewhere. So if my father-in-law, who's always pushing the turmeric on me and or the linear effect have anything to say about it, I don't think it's going anywhere. In the short term, and we need work like this to parse out the influence at an organ, and perhaps tumor or any cell-specific resolution.
And I think, as you described, the future directions there are going to be the first steps along that path in a real way, in a way that's clinically translational. So I applaud you for the work. And I'm really grateful just to be a part of this discussion.
You guys really have brilliant minds, and thanks for taking part. Mike, I'll hand it over to you. Great.
Thank you, Daylen. Fantastic discussion and great summary. You know, when we sweated figuring who we would get as media editor for Bill's journal for science, like it was easy for reviews and reports, get a young guy, social media person that loves doing that.
But for science, how do we get someone that's facile in the science as a PhD that's leading the way, but also knows social media is just cool on the microphone and has a bigger podcast than our own? Like, that's what we went after, and that's what we got in Daylen. So thank you, Daylen. You actually asked Bill one of the harder questions I feel he was asked tonight.
So I like seeing impressed like that. I actually asked people on Twitter, if Bill was ever your thesis examiner for your ABOG orals and you want revenge, this is your time. If you have PTSD, tweet me a question.
No one was up for the challenge, Bill. So I want to thank especially the fellows from San Antonio. You are our future.
Thank you, Winifred, for leading that fellowship. Thank you, retired Colonel Belinda Yeager, one of my best friends and mentors in life. Thank you for leading that division there.
Thank you for Bill and Minnie for doing this. Daylen, I'm going to give you the last word. So we have two podcasts through FNS.
Well, we actually have a third, which is when we do the journal clubs. The first is FNS On Air. That's me, Pietro, Eve Feinberg, and Kurt Barnhart.
But we also have another one called FNS Unplugged that I think you also lead your own wildly successful podcast. So you close us, tell us about your two podcasts and where we can get them and why we should be listening to those. Well, I'll first tell you that you won't find me on social media.
So you might have to find someone else if you want someone who's facile on that. But I'm privileged enough to have been a part of a podcast called the STEM Cell Podcast that's focused on stem and progenitor cells. And the greater gift, I think, is to be invited into this sphere, talking to people who are really moving the field clinically, and I think the most quick moving science and medicine, which is assisted reproductive technologies.
So yeah, please listen to our unplugged podcast, FNS Unplugged, if not for the content where we break down an article from all the three sister journals of FNS. If not for the content, then for the theme song. And if you're curious about that, you're just going to have to go check it out.
It's a very strong baseline from Dr. Evans, who's a co-host on the podcast. I invite you to listen to that and to the content. And of course, I hope to take part in some of these Journal Club global events again soon.
This has really been quite a treat for me. Thank you guys.
