Gibco Cell Culture Heroes

Cell Culture Heroes

Abstract

Webinar

Get to know Sophie

Sophie Prosolek, PhD candidate

Quadram Institute Bioscience, University of East Anglia, UK

Sophie Prosolek, PhD candidate

Connect with Sophie

@sophieprosolek

@InfraRedRum

Biography

Sophie is a BBRSC funded PhD student studying the role of dietary compounds in the genetic control of cell metabolism at the Quadram Institute, UK. Aside from her research duties, Sophie is a keen science communicator. As city coordinator for the 'Pint of Science' outreach festival and chair/founder of the UEA Science Communication society, Sophie manages various public engagement initiates which she advertises through local TV/Radio and print media streams. By combining her scientific expertise with a fierce passion for adults' education and lifelong learning, Sophie hopes to integrate her research career with media production for mass education post-PhD.

Learn about Sophie's research

Title: Role of PAPOLG gene in metabolic homeostasis

Learning objectives:

  • To understand the role of broccoli-derived bioactives in metabolic regulation
  • To understand the role of RNA turnover in acute metabolic regulation
  • To explore the use of dietary compounds in the prevention of disease

There is significant epidemiological evidence to suggest that the consumption of a hgh-broccoli diet is associated with a reduced risk of cancer and cardiovascular disease. Human intervention studies have shown that a broccoli-rich diet can reduce cholesterol levels and rebalance central cell metabolism. Our studies also show that the effect of diet is also influenced by the allelic status of a gene known as PAPOLG, an RNA poly(A) polymerase not previously associated metabolic homeostasis. Through the use of in vitro cell models, high throughput metabolomics and live cell energy phenotyping, the nutrigenetic relationship between PAPOLG and the broccoli-rich diet can be explored. In this webinar I will expand upon the link between broccoli bioactives and metabolic homeostasis, highlight the potential role of RNA turnover in metabolic control, and offer insights into the potential implications for the dietary prevention of disease.

Watch the webinar

Presenter:  Sophie Prosolek - Diet & Gene Interactions in the Control of Central Cell Metabolism

00:00 – Slide 0

Moderator:  Hello, everyone and welcome to today's live broadcast, Diet & Gene Interactions in the Control of Central Cell Metabolism presented by Sophie Prosolek, PhD student, Quadram Institute Bioscience,  University of East Anglia.  I'm Alexis Corrales of LabRoots and I'll be your moderator for today's event.  Today's educational web seminar is brought to you by LabRoots and sponsored by Thermo Fisher Scientific.  For more information on our sponsor, please visit Thermofisher.com.   (00:34)

Now, let's get started.  Before I begin I would like to remind everyone that this event is interactive.  We encourage you to participate by submitting as many questions as you want at any time you want during the presentation.  To do so simply type them into the "Ask a Question" box and click on the send button.  We'll answer as many questions as we have time for at the end of the presentation.  If you have trouble seeing or hearing the presentation, please click on the support tab found at the top right of the presentation window or report your problem by clicking on the answer a question box located on the far left of your screen.  (01:10)  This presentation is educational and, thus, offers continuing education credits.  Please click on the Continuing Education Credits tab located at the top right of the presentation window and follow the process to obtain your credits.

I'd like to now introduce our presenter, Sophie Prosolek.  Sophie is a BBRSC-funded PhD student studying the role of dietary compounds in the genetic control of cell metabolism at the Quadram Institute U.K.  Aside from her research duties, Sophie is a keen science communicator as City Coordinator for the Pint of Science Outreach Festival and chair founder of the UEA Science Communication Society. (01:49)  Sophie manages various public engagement initiatives which she advertises through local TV radio and print media streams.  By combining her scientific expertise with the fierce passion for adult education and lifelong learning, Sophie hopes to integrate her research career with media production for mass education post-PhD.  Sophie, you may now begin your presentation.

02:16 – Slide 1

Hello.  Thank you for the introduction.  (02:20)  Welcome to my webinar entitled Diet and Gene Interactions in the Control of Central Cell Metabolism.  I'm Sophie Prosolek, based at the Quadram Institute in Norwich.

2:30 – Slide 2

In today's webinar, I'm going to first give you an introduction into our research group here, what we do, and our expertise.  I'm going to talk about bioactive compounds from broccoli; what those bioactive compounds are, and, more importantly, how we analyze the metabolic effects of those broccoli bioactives.  (02:51)  I'm also going to talk a little bit about a gene called PAPOLG and its implication with diet and health.  I'm going to first introduce what PAPOLG is, and how we explore its role in cancer metabolism.  Then I'm going to end with the question, could we cure cancer with broccoli?  And that's not quite as crazy as it sounds.

03:14 – Slide 3

So, I've promised a little bit about me.  (03:20)  I'm a third year PhD student based at the Quadram Institute in Norwich in the U.K.  I started my PhD in 2015, and my current project, surprise-surprise, focuses on the role of broccoli bioactives and RNA turnover in metabolic control.  So, I studied for my BSC in biomedicine here in Norwich at the University of East Anglia, and decided to stay here for my PhD.  In my spare time, I'm a passionate science communicator, a city coordinator for the Point of Science Outreach Festival.  (03:56)  Basically, I get scientists to come into the pub and speak to the general public about their research.  I've been doing that since 2017, and I'm also an activist for the Protection and Inclusion of Neuro Diversity in Research.  So, follow me on Twitter if you want to find out about any of that. I'm quite an active tweeter and I love to hear from you, your thoughts on my webinar, and just general chitchat, really.

04:23 - Slide 4

So, also, a little bit about our group.  We're a mixed group of human biologists, clinicians, plant biologists, and we're supported by a fantastic bioinformatician and two analytical chemists.  We're very lucky here in Norwich that we have a very broad range of expertise across the Norwich Research Park. We work with clinicians from the Norfolk and Norwich University Hospital and plant biologists from the John Innes Centre which we're, luckily, based just across the road from.  (04:57)  And this sort of perfect storm of expertise has made our group particularly unique when it comes to studying bioactive compounds in foods, particularly crops, and the sort of clinical effects and outcomes of cancer research.  So, we investigate the role of broccoli bioactives in cell metabolism and cancer interception. So, we use novel broccoli cell lines developed by our group.  (05:26) As I mentioned, we work very closely with the John Innes Center, who are world famous for crop science.  And we use, as I say, these novel broccoli cell lines developed by Richard Mithen, who conventionally bred these broccoli lines which are high in levels of glucosinolates, particularly glucoraphanin.  He bred these broccoli lines by conventional breeding techniques, using ancestral broccoli lines and modern crops to enhance these levels of glucosinolates, which we use in human intervention studies.  (06:08)  Like I say, these broccoli crops were bred to contain high levels of certain bioactive metabolites, and we basically turn them into soup, broccoli and Stilton soup, which are great for human intervention studies. So, one thing that's really unique about our group is that we use these human intervention studies to inform in-vitro cell culture work.  (06:33)  Usually when you think of translational biomedicine or translational research, you think of a bench-to-bedside approach.  Well, we employ a bedside-to-bench approach so we can keep relevant, and we find this is a really useful approach to cancer research using human intervention data to inform basic science research into diet and health.  (06:55)  It keeps us relevant and it means that this sort of cross-disciplinary expertise can be sort of maximized to get the most we can out of our research.

07:08 - Slide 5

So, this one isn't a hard sell.  A broccoli diet is associated with a reduced risk of cancer.  We all know broccoli is good for us because it's green.  Broccoli is rich in fiber, vitamins, minerals, but it also contains health-promoting bioactive compounds.    So, compounds are biologically active. (07:30)  A diet rich in broccoli is associated with a reduced risk of multiple cancers, including prostate, breast, lung.  If there's a type of cancer for that sort of tissue there's probably a study looking at broccoli bioactives and that kind of cancer.  This is quite widely studied.  But anti-cancer properties of broccoli have largely been attributed to one class of compounds in particular called glucosinolates, in particular glucoraphanin, and its metabolite sulforaphane.  (08:02)  So, broccoli and other brassica vegetables, might I add, contain high levels of glucosinolates, but broccoli contains, perhaps, the most. So, we ingest these glucosinolates or glucoraphanin, to name one, when we eat broccoli.  And upon mastication of the broccoli, the plant material, the vacuoles of the plant are broken, and enzymes which can convert glucoraphanin to its metabolites, sulforaphane are released. (08:37)  So, there's some conversion of glucoraphanin to sulforaphane in the mouth, but also by our gut microbiota.  A lot of glucoraphanin is converted by the gut microbiota into sulforaphane, which can be absorbed in the lower gut, sulforaphane and to systemic circulation, and can have all kinds of bioactive effects throughout the body. (9:02)  So, sulforaphane is a potent activator of the NRF2 antioxidant response.  So, I could give an entire webinar, to be honest, on NRF2 and the antioxidant response, but that's kind of going into too much detail there.  It's beyond the scope of this particular webinar.  But basically what's sulforaphane does is it conjugates to glutathione.  Glutathione would usually mop up reactive oxygen species.  (09:30)  So, the binding of sulforaphane to glutathione changes the redox status of the cell.  Initially the pro-oxidant, but this attunes the antioxidant response, the NRF2 antioxidant response to be specific, and what regulates the expression of detoxifying genes in the cell.  So, overall sulforaphane, though, acting initially as a pro-oxidant is an antioxidant, and it's this antioxidant effect that we think is enough to push cancer over the edge.  (10:05)  Sulforaphane has been shown as a promising novel therapy for cancer interception.  We do run our own human intervention studies, but  I'll just mention that in a second.  But there's a particularly interesting paper by Ho, et al that was published earlier this year detailing the use of a broccoli diet in a targeted treatment of colorectal cancer in mice. (10:38)  I really recommend.  If you're interested in diet and cancer interception, go and give that a read because it's a really exciting paper.

10:49 - Slide 6

So, I promised I'd mention one of our human intervention studies, and that was the ESCAPE study.  So, ESCAPE stands for somehow exploring broccoli diet in prostate cancer interception.  I can't give you too many details on this because it's currently in review, but all I can say is look out for it, it's going to be exciting.  (11:11)  So, men with prostate cancer were recruited to this intervention study where they were asked to consume one portion of 400 grams of broccoli and Stilton soup per week.  So, we looked at standard broccoli and then the high glucoraphanin broccoli that was developed by our group.  And we had a look at, in the prostate biopsy, in blood and urine, gene expression and metabolite concentration, and also cancer grade or WHO grade.  (11:44)  So, you can find more details of this trial on clinicaltrials.gov and the link is in the corner below.  And, like I say, this one's going to be an exciting one when it comes out.  So, keep an eye out for that.

12:00 - Slide 7

So, as I mentioned, broccoli contains high levels of these glucosinolates, particularly glucoraphanin, which we know it's bioactive against cancer, but broccoli also contains other bioactive compounds.  (12:15) In addition to glucoraphanin, broccoli contains other sulphur metabolites which are particularly abundant in in brassica crops or broccoli crops.  And they're the compounds that, if you've ever been cooking broccoli, they give it the distinct broccoli smell, the sulphurous smell that you get from cooking brassica; cooking cabbage; cooking broccoli.  (12:41)  And these self-contained metabolites can be found in the prostate biopsies of men consuming the broccoli intervention diet such as those on the ESCAPE study that I mentioned.  And these are the broccoli metabolites, one of which I have an example on the right-hand side here, can be broken down by the microbiota just like glucoraphanin and sulforaphane.  (13:06) This example here, S-methyl-L-cysteine sulfoxide, can be broken down to daughter compounds, MMTSI and MMTSO, which literature details at high levels can be used as a bioactive to inform protein studies.  Literature also report anti-mutagenic and anti-diabetic effect of these various broccoli sulphur metabolites in addition to sulforaphane.  (13:37)  But most of the studies on broccoli bioactives use supraphysiological concentrations.  Much higher concentrations than you would get from a standard broccoli diet.  Certainly higher concentrations than you'd get from that 400 grams per week dosage that we give to the intervention volunteers.  So, they use quite a reductionist model, really, looking at, perhaps, sulforaphane only which, like I say, is widely studied.  (14:13)  And looking at these supraphysiological concentrations, you have to kind of consider how relevant some of the current literature is if we're talking about diet and cancer interception.

14:23 - Slide 8

So, it's really important for us to define our cell models to keep relevant to whatever we're studying.  We have to choose carefully and, in our case, we chose PNT1A immortalized human prostate epithelial cell lines and attached it to moralized human hepatocellular carcinoma cell line.  (14:47) Really, the value of of using two cell lines is so that you can assess those mechanisms which are conserved amongst different tissues and those mechanisms which are divergent from tissue to tissue. So, you're able to compare and contrast.  And, as well, an important decision for us in looking at liver cells, whilst we were researching prostate cancer, using a liver model helps us think about systemic metabolism, or it helps think about, we can use varying glucose concentrations to mimic hyper or hypoglycemic diabetic states, and this allows us to test metabolic pathways in a way which we wouldn't probably do using just a prostate cell model.  (15:41)  We also have normal human hepatocytes, Thle-2 cells, which are immortalized cell line or mouse primary hepatocyte available.  And we do dip in and out of using these models where we need a little bit of confirmation or backup to something that we may have already seen in another cell line.  So, as I say, using multiple cell lines means that you're able to compare and contrast across tissues and across cell types.  (16:09)  But these two cell lines that we've chosen, HepG2 and PNT1A, are both highly glycolytic.  So, we can assess those mechanisms which are conserved, but also those which are divergent.  And by challenging these cells in different glucose environments we're able to simulate a diet or metabolic syndrome scenario.

16:34 - Slide 9

So, next we need to define our treatments, because we must answer a relevant biological question.  We need to think about the toxicity of these broccoli bioactives that we're treating ourselves with.  Some of these bioactives cause toxicity in in vitro when used at really high concentrations, and that certainly is not physiologically relevant.  (16:59)  So, we need to investigate that before any sort of cell studies.  So, we culture cells and we measure metabolic activity with a colorimetric change in WST-1 reagent, like this example that I have on the right here.  So, we treat with increasing concentrations or an increasing dosage of our compound of interest, and then look at colorimetric change in WST-1 reagent which is indicative of metabolic activity, and a decrease in viability or decrease in metabolic activity indicates, as I have an example here, in viability.  (17:41)  Now, you have to take this with a bit of a pinch of salt because we are looking at metabolically active compounds here.  But certainly, when you have a metabolic activity which is decreased to sort of 10 percent of the original capacity, and this can be considered indicative of cell death.  This is also really revealing in terms of which compounds we choose to study because the plant metabolites themselves are not always directly responsible for their associated biological effect.  (18:18)  As I mentioned earlier, glucoraphanin is converted to sulforaphane; SMCSO is converted to MMTSO and MMTSI.  And it's not always a parent compound which is bioactive, sometimes it's the daughter compound.  And we've shown that these daughter metabolites are toxic beyond 125 micromolar concentrations in in vitro whereas the parent compound shows no toxic effect at all.  (18:47)  And to show no toxic effect really suggests no biological activity at these sort of supraphysiological concentrations.

18:58 - Slide 10

So, we've defined our cell model, we've defined our cell treatment.  So, next, the question is, do sulphur metabolites from broccoli, do these broccoli bioactives alter metabolism?  And, well, they do, because I'm speaking to you here now.  (19:14)  So, we can use a variety of techniques, but our first port of call is really to look at a generalist approach to analyzing metabolism in these cell lines.  So, we culture cells in microplates and treat with a physiological concentration of our compound, which is informed by our human intervention studies, prostate biopsies, blood and urine samples.  (19:43)  We can estimate physiological concentrations of these compounds, treat our cells in vitro, and then we use a machine called the Seahorse Extracellular Flux Analyzer to measure metabolic rate in live cells.  So, I love this machine, I think it's fantastic.  It's really great at getting an overall snapshot of energy metabolism in live cells.  (20:07)  Through the injection of drug compounds, oligomycin, FCCP, Rotenone/Antimycin we can really test metabolic capacity.  So, you culture your cells in these microplates, inject drug compounds over the course of the assay, and using a fluorescent probe, you measure extracellular acidification which is indirect measurement of glycolysis and oxygen consumption rate, which is indirect measurement of mitochondrial respiration.  (20:36)  Again, this is another thing that could have an entire webinar on itself, but information about this sort of assay can be found in the manufacturer's website.  And it's really great, I think, for offering an overall snapshot of live cell metabolism.

20:52 - Slide 11

So, what does the Seahorse assay look like?  It looks like this.  So, sulphur metabolites were shown to increase glycolytic rate, as you can see by ECAR on the left-hand side, and maximum mitochondrial capacity on the right-hand side in PNT1A cells.  (21:11)  So, compound treated cells in red show an increased glycolytic rate, and it increased maximum mitochondrial capacity compared to control-treated cells, which is denoted in blue.

21:29 - Slide 12

So, from there, we've had an overall snapshot of metabolism through this live cell energy phenotype.  I  didn't mention there that these measurements are taken in live cells of metabolism as it's happening, which is really cool.  You can see this real-time change.  But then, once we've seen that, we want to have a look, more specifically, at what's going on with specific enzymes and prove metabolism a little bit more in a bit more of a detailed kind of way.  (21:59)  So, from there we go to RNA-level analysis.  Again, with cultured cells, treat them with our compound of interest, extract RNA, and then we can choose to do qRT-PCR for target gene expression or whole genome RNA sequencing.  We do both.  I have just sent some samples off to Macrogen for sequencing.  But also the individual gene expressions.  (22:27) And looking at these differential gene expressions helps us identify pathways and networks which are affected by our compound treatment.  So, I did some qRT-PCR for glycolytic enzymes, again, in prostate epithelial immortalized cell lines, PNT1A cells treated with compound, and we saw a dose-dependent increase in these Proteolytic enzymes.  So, it appears from our Seahorse assay and from our targeted gene expression analysis that our compound of interest, our sulphur metabolite, is increasing glycolysis in PNT1A cells.  (23:04)  So, by increasing glycolysis in these cells, could this compound support cancer interception?  Well, to answer that we need to understand a little bit about the pathogenesis of prostate cancer.

23:17 - Slide 13

So, cancer is a largely metabolic disease, and cancer cells exhibit the Warburg effect.  So, unhealthy metabolism, glucose is converted to pyruvate, and then that's shunted into the TCA cycle and oxidative phosphorylation to generate lots of ATP.  (23:36)  This is normal under conditions with oxygen, but the Warburg effect basically means that in those same oxygenated conditions, cells are preferentially metabolizing in an anaerobic kind of way.  Cells preferentially respire with non-mitochondrial respiration even in the presence of oxygen.  (24:02)  And mitochondrial metabolism is perturbed in the Warburg effect.  So, cells become sort of addicted to glycolytic substrates like glucose or glutamine.  And these high rates of glycolysis are thought to generate metabolites needed for anabolic processes and notably cell growth in cancer.  But, interestingly prostate cancer does not exhibit the Warburg effect.  (24:27)  Prostate epithelial tissue does utilize glycolysis to generate citrate, an important constituent of seminal fluid.  And this is a normal, healthy function of prostate epithelial tissue.

24:44 - Slide 14

So, by increasing glycolysis, are we in fact increasing the normal, healthy functions of this prostate epithelial tissue and contributing to cancer interception; is that possible?  So, we also have to think about systemic metabolic health as well as cellular metabolic health in cancer prevention because obesity and poor metabolic health is also a risk factor for cancer pathogenesis.  (25:13)  And surprise, surprise, broccoli diet is associated with improved metabolic health.   This is another one of our studies, our human intervention studies where we use, again, high-glucoraphanin broccoli, and this delivers increased levels of dietary sulforaphane.  And we saw that volunteers on this diet were showing improved markers of metabolic health, but they showed, also, a clustered metabolic response correlated with polymorphisms in this gene known as PAPOLG.  (25:46)  So, could PAPOLG influence the cellular and systemic metabolism, and be causing these custom metabolic responses that we saw in our intervention study?

25:55 - Slide 15

Well, we need to know first what PAPOLG is, and that's been a challenge.  PAPOLG is a poly(A) polymerase, poly(A) polymerase gamma to be specific, and it's largely understudied, to be honest.  It catalyzes the sequential addition of adenine residues to RNA transcript.  (26:15)  So, it's denoted here on the right-hand side as poly(A) polymerase and it forms part of this cleavage and polyadenylation complex which is responsible for post translational modifications of RNA transcript.  So, it confers stability or signals transcript for degradation depending on the poly(A) tail length or poly(A) pattern.  (26:42)  And this potentially affects a range of pathways within the cell.  And one thing that's interesting about PAPOLG is that it's over expressed in many cancers, many cancers.  And as we've demonstrated from our human intervention study, snips within this gene appear to be correlated with metabolic health.  So, what's the metabolic role of PAPOLG?  To investigate this, we used HepG2 and PNT1A cells again, transfected with SiRNA, small interfering RNA.  (27:18)  So, these are small RNA nucleotides of about 25 nucleotides in length, and they complimentary base pair to your gene interest and signal it for degradation.  So, the overall effect of using siRNA is that the gene of interest is abrogated, the expression is abrogated.  (27:41)  It's not knocked down completely, but it's significantly reduced, and you can then run comparative assays on your cell population to antagonize what the effect of this gene might be.

27:55 - Slide 16

So, we did exactly that.  We cultured cells and prepared lysate of cultures treated with siRNA, and then we looked at targeted analysis of metabolite levels using LCMS.  (28:09)  As I said, we're supported here by some very talented chemists who helped very much with with protocol development and the LCMS side of things, but then there's also the option of high-throughput metabolomics.  And we have we have sent off samples to metabolite in the past.  (28:33)  So, we measured multiple metabolic intermediates in a sample of either cells or media by LCMS, and we can show that when normalized to cell number, there's a decrease in metabolite concentrations following gene knockdown.  This was representative of various metabolites across TCA cycle.  (29:03)  So, this helps us identify where there might be enzymatic bottlenecks in the pathway.  And, as I mentioned earlier, sulforaphane, for example, affects the redox status of a cell.  So, these are often redox sensitive enzymes.

29:21 - Slide 17

So, to summarize, we've demonstrated that there are multiple bioactives that can be derived from a broccoli diet and that glucoraphanin, as an example, it's already well studied, but there are other sulphur-containing metabolites that, perhaps, warrant further study.  We've reported on the bioactivity of certain sulphur-containing metabolites, and identifying that it's often the daughter compound, not the parent compound that is responsible for the biological effects.  (29:58)  And that these metabolites fundamentally alter energy metabolism in vitro of both normal and cancer cell lines.  We've demonstrated that this broccoli diet may interact with genotype, notably the genotype of PAPOLG to cause inter-individual variation in the response to a broccoli diet.  And, of course, we've identified a novel metabolic role for an RNA polymerase that had not previously been associated with metabolic control.

30:33 - Slide 18

So, this really tells us that we need a multi-dimensional approach to cell-based research.  Broccoli contains multiple potent bioactives which could have a synergistic effect in in vivo, and many in vitro studies have not taken this into account.  They've taken a reductionist approach focusing on one bioactive only, but our research demonstrates the pitfalls of that in nutrition research because, of course, we are looking at whole food matrix and human intervention studies.  We need to consider all the bioactives within that food matrix along with consumer genotype, of course, because we're all different.  (31:12)  So, by translating our research sort of from bedside-to-bench we can narrow down a physiological scope of our research, maintaining clinical relevance even at the level of cell culture studies.   So, we go from clinical trial to in vitro experimental design, and then we can take a targeted nutrigenetic approach.  And this all supports the potential role of broccoli diet in cancer interception.

31:39 - Slide 19

So, I'm going to leave you with the question.  Could we cure cancer with broccoli?  Well, I mentioned at the beginning of the talk, in that paper by Ho, et al in which they look at sulforaphane in colorectal cancer interception in mice.  Well, could we also do the same with other sulphur-derived and broccoli metabolites — sulphur-containing broccoli metabolites, I should say.  Could we take the same approach?  Could the approach be enhanced by looking at multiple metabolites and the cumulative synergistic effect?  (32:13)  Of course, diet is a way that everyone, in theory, can maintain their own health and keep metabolic diseases at bay.  So, could we cure cancer with broccoli?  We'll see; wait for the ESCAPE paper.

32:35 - Slide 20

Any questions?

Moderator:  Thank you, Sophie, for your informative presentation. We will now start the live Q&A portion of the webinar.  If you have a question you'd like to ask, please do so now.  Just click on the answer a question box located on the far left of your screen.  We'll answer as many of your questions as we have time for.  So, let's get started.  (33:05)  Our first question is, there have been many cell studies using sulforaphane treatments to model the benefits of a broccoli diet.  How relevant are these studies, now we know there are multiple bioactives which may act synergistically?

Right.  So, I think that those sulforaphane treatments and cell studies are still extremely relevant as much of what we understand about the health benefits of broccoli are based upon what we learned about sulforaphane in cell studies.  (33:42)  It's still extremely relevant.  However, I think it's really important, as research progresses, that we consider the whole food matrix and that means multiple bioactives when considering a dietary intervention because, of course, the person is consuming the whole food.

Moderator:  Now, Sophie, why did you use siRNA to create a pebbled knockdown?  Was there a CRISPR alternative available?

(34:12)

So, this is quite a good question because CRISPR has gained a lot of publicity in recent years about being superior to siRNA in knockdown experiments, but I think it really depends what question you're asking.  Really, do you want to spend a lot of money on an expensive CRISPR system which might be really great if you're looking at multiple knockdowns or doing this in a high-throughput scenario?  (34:39)  But I think when taking a knockdown that hasn't really been studied before, of course, you don't know if it's going to be toxic to your cell.  An siRNA is sufficient just to abrogate expression and give you an insight before you sort of take the plunge with more committal CRISPR steps.

Moderator:  And what level of POPOLG knockdown did you achieve with your siRNA?

So, we managed to get about 40 percent of initial expression.  So, the gene was knocked down by about 60 percent in both the cell lines that we use the siRNA in.

(35:20)

Moderator:  And, Sophie, our next question.  What are the challenges of designing a long-term human intervention study with food?

That's another good question because there are many challenges.  Of course, the first being compliance.  People need to, if this is a long-term study, adhere to a diet long-term.  And, yeah, that's sometimes difficult.  In the example of our ESCAPE study, we were giving patients one portion of broccoli soup per week.  (35:56)  So, it really has to be something that's doable.  If we'd ask them to consume, I don't know, a portion a day, we might have really not had very good compliance.  And, of course, the way you check compliance is usually through food diaries, and people tell fibs, to be honest.  People don't want to tell you that they've had, you know, six portions of chips a week.  They want to tell you that they've eaten apples and all the healthy things.  So, sometimes the food diaries can be a little bit misleading.  (36:28)  And, of course, the controls.  How do you control for broccoli without it being broccoli?  So, this is one of the real strengths of our group in that we have the high glucosinolate broccoli and standard broccoli.  So, we have a standard control which is sometimes difficult with some foods.

(36:53)

Moderator:  We are getting some great questions here, Sophie.  Let's look at this one.  Sophie, have you watched The Magic Pill?  It promises that eating the correct food can be superior to drug intervention, the phrase "you are what you eat."  So, what is your opinion on diet as preventing, as well as treating, disease?

Well, that's a good one.  (37:17)  Of course, diet keeps your body healthy.  Healthy diet means a healthy body in many circumstances.  Of course, that's not the case for everybody.  But I think it really is important to focus on prevention rather than treatment.  And sort of particularly, you know, as people are living longer, and there's an increased burden on the health service, and the focus tends toward healthy aging, I think diet is crucial.  (37:49)  Not just for sort of the purse strings of the health service, but also for the psyche of the patient or to-be-patient.  You want to avoid medicalizing people where possible, and I think this is where dietary intervention has real power.

Moderator:  Now, Sophie, what are the challenges of designing a long-term human intervention?  Oh, excuse me, we did already asked that one.  Yes, we did.  All right.  Well, let's move on to our next one which is, are animal models always relevant as an intermediate between self-studies and human intervention studies?

(38:27)

Now, this is another one that depends very much on the experimental design and the variable which you're experimenting.  We're lucky in that we study broccoli and, like I said, in my presentation that's not a hard sell.  You're not going to do any damage with one portion of broccoli soup a week in a human.  So, we're lucky in that we can translate our cell studies, pretty much, straight into humans.  (38:54)  But, of course, it depends exactly on what you're looking at.  I don't think it's always relevant as an intermediate, but it does depend what you're looking at and the nature of your study design.

Moderator:  And it looks like we are going to be getting a couple more questions in.  Our next one.  Is broccoli classed as a superfood?  And what are the other foods in this category for cancer?

(39:17)

Ooh, well, of course, our research group has developed this high glucoraphanin broccoli which I would consider a superfood. I don't know if superfood is a more like a marketing term.  But there's numerous foods that you can consider superfood, broccoli perhaps being one.  But others are foods which contain high levels of anthocyanins, for example.  (39:45)  Anthocyanin is a generally purple-colored compound which are found in like aubergine skins, to an extent in tomatoes.  There's a study in collaboration with the Quadram Institute and the John Innes Centre, here in Norwich, which looks at high anthocyanin in tomatoes, for example, in cardiovascular disease risk.  So, pepper foods are good for you.  They can be considered superfoods, broccoli.  Yeah, yeah.

(40:20)

Moderator:  As a little bit of a piggyback to that question, what about blackberries?

Ooh, blackberries, I'm pretty sure, do contain anthocyanins, but I'm not sure how many — what volume of anthocyanin they contain.  Like I say, it's generally purple compounds, but purple foods generally contain varying levels of anthocyanin.  So, yeah, I'm not sure, but I know they contain anthocyanin, but I'm not sure at what level.

(40:52)

Moderator:  All right.  It looks like we do have another question right here.  Does sulforaphane elevate the oxidative stress level in both of the tested cell lines?

Pardon?  Sorry.

Moderator:  It's okay.

Ah, is sulforaphane — right.  Is sulforaphane elevating the oxidative stress level in both the tested cell lines?

Moderator:  Yes.

So, this is how sulforaphane are actually works within the cell.  (41:23)  What it does is it combines with glutathione which means the less glutathione available to mop-up reactive oxygen species.  So, initially it sort of increases the oxidative stress level a little bit which is enough to tune the antioxidant response.  This is the general action of sulforaphane within any cell, as far as I understand, that it initially acts as a little bit of a pro-oxidant, but its overall effect within various cell lines is antioxidant.

(41:58)

Moderator:  All right.  Let's see if we have any more questions here.  Okay.  We do have another one.  Are other greens beneficial, for example, spinach, kale or arugula?

So, that's an interesting one, because spinach and kale are sort of in the same scale, certainly is in the same family of vegetable as broccoli and cauliflower, Brussels sprouts, Chinese cabbage, I think.  (42:33)  Yeah,  they're all within the same sort of brassica family and they all contain glucosinolates, but broccoli contains the most.  So, that's why we choose to study broccoli.

Moderator:  Here's a question for you, Sophie.  What are your next steps in your research?

Ooh, that's an interesting one.  So, the next steps in my research, so I'm looking at other sulphur metabolites from broccoli and I have some indication that some may affect NRF2 signaling.  (43:10)  I might want to explore that.  And, also, I want to explore how these sulphur compounds affect protein structures and whether they promote this sort of antioxidant response from other sulphur metabolites as well as sulforaphane.  So, that's something I'm quite interested in also.  Yeah, there's always a million directions you can take.

(43:37)

Moderator:  We do have another question.  In response to our audience, if there are questions that we do not have time for today Sophie will be able to answer them via e-mail after this presentation is over.  So, Sophie, our next question.  What is the effect of sulforaphane on NRF2?

Ooh, this is a good one.  Sulforaphane is the most potent activator of NRF2.  There's a lot of literature on that.  If you Google that, you'll come up with so much literature you won't know what to read first.  (44:09)  Any cell line, any tissue, you name it, there's been studies on sulforaphane and NRF2.

Moderate:  All right, Sophie, let's look and see if we have any more questions.  Let's go ahead and,  again, like I said, to our audience who may have put in some questions, that we just did not have time for.  (44:37)  Sophie will answer questions after our presentation with the the contact information that you provided at the beginning of the presentation.  So, thank you, again, Sophie.  Do you have any final comments for our audience?

Final comments, feel free to give me an e-mail or even tweet me if you've got another question that comes up after afterwards.  Like I say, I'm a very prolific tweeter and happy to respond to any questions via e-mail.

Moderator:  Before we go, I'd like to thank the audience for joining us today and for their interesting questions.  (45:15)  Questions we do not have time for today and those submitted during the on-demand period will be addressed by the speaker via the contact information you provided at the time of the registration.  We would like to thank LabRoots and our sponsor, Thermo Fisher Scientific, for underwriting today's educational webcast.  This webcast can be viewed on-demand through December of 2018.  LabRoots will alert you via e-mail when it's available for replay.  We encourage you to share that e-mail with your colleagues who may have missed today's live event. Until next time, goodbye.

End Presentation: 45:45

Get to know Sophie

Why did you choose cancer research?
Cancer isn’t just a disease, it’s a model of many cellular processes gone wrong. By studying cancer we gain a weath of knowledge about the fundamental workings of a cell; this is what excited me into cancer research.

What motivates you to succeed in your field? 
I’m motivated simply by my desire to learn something new each day

What are your top 3 favorite things to do outside of the lab?
Outside the lab my top three interests are:

  • Music: I enjoy an eclectic music taste and am a keen multi-instrumentalist and singer/songwriter.
  • The outdoors: I love being outdoors and I love spending time around animals, particularly horses as I’ve always loved horse-riding.
  • Food: of course, working in diet and health I cant help but love to cook and share good food with good company!

What role have the mentors you’ve had in your passion for basic research?
I had a very talented and approachable mentor during my first summer internship in basic research. She taught me some very good habits and technical skills which have stuck with me and continue to influence the way I work. She still remains a great inspiration to me today.

Which scientist current or past would you most like to meet and why?
There are three scientists I’d really love to meet–Elsie Widdowson. I’d be surprised if many people have heard of Elsie; she was a British chemist who pioneered of the scientific study of nutrition. She helped create the state-recommended diet during the Second World War and condicted pioneering resreach on vitamins. I’d love to meet Elsie because she really utilized her research to save a nation, and that must be one hell of an exciting story to tell!

Is outreach/STEM important to you? Why?
It’s incredibly important to me. I grew up in a deprived area with little opportunity where few of my peers left school with a basic level of education. I don’t think I would have been able to go to university at all if I hadn’t learned from outreach initiatives what my school struggled to teach. I feel very grateful to have had the incredible opportunities life has presented me with and I am very keen to give something back through participating in STEM initiatives

Why did you become a scientist?
After my BSc I didn’t feel ‘done’ with education; I wanted to feel intellectually stimulated, to keep learning in a dynamic and exciting way and contribute to the wider community. The natural option was to continue studying for a PhD and become a scientist.  

What are some small things that make your day better?

  • A good cup of coffee makes catching up on the latest literature feel like self-care
  • The smell of fresh paper when I open a new copy of my favorite science magazine inspires me to write
  • Going to a lunchtime gym classes with my friend and colleague really helps me draw a line under the mornings’ work and feel revitalized for an afternoon in the lab

What would be your first question after waking up from being cryogenically frozen for 100 years?
“Turn the radio up?” Of course one of the first things I’d want to hear is the news headlines, but I’d also want to hear which directions music had taken over those 100 years!

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