Monthly Archives: November 2011

Of Platelets, cancer cells, EMT and Metastasis.

Hello again!

This time I’ll be writing about a paper that recently appeared in the journal Cancer Cell. The paper is something of a landmark because it has showed how the mere interaction of platelets with cancer cells is sufficient to induce activation of EMT (Epithelial Mesenchymal Transition). It details how the presence of a protein called TGFß derived from platelets, in combination with direct contact between cancer cells and platelets, can activate the NF-kB and Smad signalling pathways that confer invasiveness to cancer cells, thus making metastasis possible.

TGF-beta signalling pathway,courtesy Cell Signaling Technologies.

NF-kB Signalling, courtesy Cell Signaling Technologies.


The paper (see journal reference at the bottom for a link) is elegant because of the way critical questions were asked and addressed… the main questions were if platelets could stimulate metastasis and if so, by what means and methods.

The first question was quite easy to address. They took colon carcinoma cells from a cell line called MC38GFP and breast cancer cells from a cell line called Ep5, they grouped cells from each of those lines into two groups each. One of each group was treated with platelets while the other wasn’t, and the cells were then injected into mice to look for the frequency of metastasis. and bingo, the platelet treated cells showed a higher frequency of metastasis.

 

Number of metastatic foci.

Treatment with Platelets tends to vastly increase the number of metastatic foci compared to untreated cells.

There you go, first question answered. The researchers then examined whether platelet treatment induced an EMT-like phenotype, and they did this by looking at the mRNA and protein concentrations of various genes and their products that are markers of EMT, such as MMP-9 (a matrix metalloproteinase) and found that the expression of such marker genes and proteins is highly upregulated. Some of the markers used were Snail, Vimentin, Fibronectin and PAI-1 for a mesenchymal phenotype, and E-cadherin expression as a marker of epithelial phenotypes (or the loss thereof).

(D) Relative fold change in mRNA expression in MC38GFP or Ep5 cells treated with buffer or platelets for 40 hr (n = 3). Values are normalized to Gapdh expression. (E) Detection of E-cadherin protein levels by immunoblotting of lysates ofMC38GFPorEp5cells treated as in (D). Amounts of platelets equal to those used to treat cells were also loaded as control (no cells). b-tubulin was used as loading control. (F) Zymography for MMP-9 in the conditioned medium of MC38GFP or Ep5 cells treated as in (D). Amounts of platelets equal to those used to treat cells were also loaded as control (no cells). (G) MC38GFP and Ep5 cells were added at the top of transwells coated with Matrigel and treated with buffer or platelets. The total number of cells that invaded to the bottom of the transwell was counted after 48 hr (n = 3). For (A), (B), (D), and (G) bars represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 were determined by Student’s t test.

The question then would be how platelets could induce EMT, could TGFß be to blame? To find out they carried out bioinformatics analysis of data from microarray experiments looking for differentially expressed genes when cells were treated with platelets. They found that expression of EMT genes was upregulated and there was increased expression of genes in the TGFß pathway.

Gene upregulation data from microarray analysis when platelet stimulation is induced. Gene enrichment analysis using Gene Ontology functional categorization is presented in the table. You can see how the upregulated genes are all associated with EMT and with TGF-beta dependent activation processes.

This indicated to them that platelets induced the EMT phenotype through the TGFß pathway. They then wanted to find out if TGFß alone was responsible or if direct contact with platelets also had something to do with said upregulation.

To do this, they set up a rather cute experiment where they used a PAI-1 reporter gene to assay TGFß activation (this is possible because PAI-1 is downstream in that pathway and can be used as a surrogate marker)

They assayed levels of activation when platelets AND TGFß were added as opposed to TGFß alone. They observed more PAI-1 activation in the former than the latter, thus indicating that the presence of platelets and TGFß together act synergistically in activating the pathways involved. The central role of TGFß in the process was confirmed by blocking TGFß using an inhibitory antibody and a small molecule, which led to loss of PAI-1 expression.

The question then, of course, was why the presence of both platelets and TGFß would have more effect in EMT activation compared to TGFß alone. What other pathways would be activated such that the added effect could be accounted for?

To test this, they used reporter based assays for multiple pathways involved in cancer, and found that the releasate from platelets (platelet free) activated the JNK pathway, while the presence of cells and the releasate activated the JNK and NF-kB pathways.

Now this is where things got really interesting. Firstly, they confirmed whether NF-kB was actually able to account for the results of cell-inclusive treatment experiments by using Ep5 cells with mutant NF-kB and a reporter with reference to a control with a normal NF-kB and the corresponding reporter.

They found that mutant cells didn’t show NF-kB activation when treated with platelets, and that these cells had the same metastatic potential as cells that were treated with TGFß alone, thus fully implicating this pathway in platelet contact induced EMT.

These results were further validated when treatment with an NF-kB inhibitor also downregulated the expression of several key markers of EMT, such as MMP-9, in cells with intact NF-kB signalling.

They found that inhibiting cells with the NF-kB inhibitor switched off a reporter in that pathway, but not one in the other pathway, indicating that TGFß activation was independent of NF-kB activation, while the effects were synergistic.

And finally, here’s the piece de resistance of the paper; they showed that blocking TGFß secretion in megakaryocytes and platelets or wrecking the TGFß pathway was enough to prevent metastasis, they did this using mice in which TGFß expression was knocked out where seeding with cancer cells prevented metastasis to the lungs. They also showed that even cells pre-treated with wild type platelets for a while before introduction into TGFß fl/fl mice wasn’t enough to trigger metastasis.

This basically confirms that platelet derived TGFß signalling is absolutely necessary, while NF-kB signalling in synergy with it renders tumour cells potentially more metastatic, but isn’t per se sufficient for metastasis.

They note that this could have therapeutic implications, since TGFß inhibition in platelets doesn’t have physiological effects on normal cells, and if this could be replicated in humans it might serve as a brilliant therapeutic strategy.

However, as always, blocking metastasis, it would appear, would only really be useful in tumours where metastasis hasn’t occurred, how established metastatic disease should be dealt with is still a very open, and problematic question.

Journal Reference: Labelle et al, Direct Signaling between Platelets and Cancer Cells Induces an Epithelial-Mesenchymal-Like Transition and Promotes Metastasis, Cancer Cell. http://www.sciencedirect.com/science/article/pii/S1535610811003564

That is all from me this time round.

Cheers,
Ankur.

 

 

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In which my appreciation of science art continues.

Just another quick one today. I would like to introduce y’all to some rather brilliant scientific artwork by a friend.
Lucy Walsh is an artist who specializes in painting pictures of galaxies and nebulae and I think they look quite gorgeous. Her work is usually acrylic on canvas and has a crisp and vivid feel to it.

Some samples of her work follow…

An abstract piece called “Fusion”
Fusion

V838 Monocerotis Eruptive Star.

V838

Pluto and its 3 moons.

Pluto and its 3 moons

Flame Nebula

Flame nebula.

Arp 24
Two Galaxies

If you like her work, please do check out her official page on DeviantArt Gallery at http://lucyjain.deviantart.com/
or her official facebook page at https://www.facebook.com/pages/Lucyjains-Space-Art/130069343727953

Quite a few of these pieces have prints available from DeviantArt, too.

That is all from me this time round.

Cheers.
Exploreable.

In which I admire stunning glass sculptures – part un.

OK, this is a bit of a tangent compared to the usual serious business that is characteristic of this blog. However, it is still extremely science related, and focuses on the aesthetics of what science may reveal to us.

I am not really talking about Charlie Murphy’s glass casts of naughty bits on display at the Wellcome Collection in London, either, fascinating as they are… Instead, I am talking of Luke Jerram’s Glass Sculptures.

Luke Jerram is an artist from Bristol who produces installation art and glass sculptures, and it is his glass sculptures that I am going to be focusing on. He has basically created some rather stunning glass sculptures of viruses and bacteria and I intend to present some photographs for your edification below.

I went to the Wellcome Collection a few weeks ago and I found this sculpture of the Swine Flu Virus that he’d concocted.

Swine Flu Virus Sculpture by Luke Jerram, top view. Photo taken at the Wellcome Collection by Exploreable.

Same sculpture as above, side view.

That isn’t all there is to it, though, he’s produced a whole set of sculptures under the aegis of the “Glass Microbiology” collection (unfortunately, all of those aren’t at the Wellcome Collection) and as a result I will have to use the good old internet to supply thee with the eye candy thou crave…

The following are from the Glass Microbiology website at http://www.lukejerram.com/glass/gallery

E.coli sculpture

E.coli again.

Those two are of E.coli.

Swine-Flu (Oval) - Close-up.

Swine Flu (Oval) Full unit

Those two are of the Swine Flu Virus.

HPV closeup

HPV from a distance

Those two are of the Human Papilloma Virus

Malaria Sculpture

That one is of Plasmodium vivax, the organism that causes malaria.

What follows is a collection of videos featuring those sculptures

Here’s a BBC interview clipwhere Luke speaks about what motivated him to produce the collection. I think it is excellent, except for the gaffe where E.coli has been labelled as a virus.

Finally, here’s a clip that shows some of the making of Jerram’s HIV sculpture.

If you get a chance to go to one of his exhibitions, then please do!

In the future, I may try to do a post on Annie Cattrell’s work, which is also quite exquisite.

Cheers,
Exploreable.

Examining the cytotoxicity of metallic nanoparticles…a little blast from the past.

This is just a little experiment I did with a classmate during my undergraduate years to see if metallic nanoparticles could have an effect on bacteria and yeast, which could perhaps be used to address the use of nanoparticles in a microbicidal capacity.

Here goes the report.

Abstract

Introduction

An investigation was carried out on the cytotoxic properties of metallic nanoparticles on both Prokaryotic and Eukaryotic model organisms using qualitative assays for cytotoxicity.

Methodology & Results.

Metallic nanoparticles of Copper, Silver and Iron were prepared using Turkevich synthesis, these were then screened for cytotoxicity using well diffusion assays using a reference set of four bacterial species, results indicated that Silver nanoparticles were potently cytotoxic against bacteria, being capable of inducing substantial zones of inhibition in the medium into which said particles could
diffuse.

The test was repeated for Eukaryotes using Saccharomyces cerevisiae as the model organism and turbidimetry for evaluation. Values were obtained as a function of varying concentration vs varying turbidity. Results indicated a direct statistical relationship between the strength of nanoparticle
solutions of Silver as opposed to the other two.

Conclusions
The cytotoxicity of nanoparticles of silver was demonstrated in both prokaryotes and eukaryotes, this backed up the previously reported instances of nanosilver toxicity in the scientific literature & highlighted the potential concerns associated with environmental contamination of the environment
with metallic nanoparticles. It also raised questions about the potential use of silver nanoparticles in conjugates as a form of cytotoxic therapy, which may be worth investigating.

Materials and Methods

Turkevich Synthesis

Materials Required

0.1 M Silver Nitrate solution, 0.1 M Copper Sulphate Solution, 0.1 M Ferrous sulphate solution, 38.8 M Sodium Citrate solution (aqueous) , deionized water, standard glassware.

Principle

Turkevich synthesis uses a reducing agent to first reduce metal salts to nanoparticles, the same agent then inhibits re-coagulation.

Procedure

Solutions of the aforementioned nature were prepared from raw salts using deionized water, then reaction mixtures were prepared using metal salt solutions and sodium citrate in the ratio 2:1.

A water bath was heated to 100’C and the reaction mixtures were boiled until colour changed , this change in colour is often attributed to the Surface Plasmon Effect, which is due to changes in the dielectric properties of synthesized nanoparticles.

The solutions, once colour had changed, were gradually cooled back to room temperature and stored in reagent bottles, estimation of the concentrations of the nanoparticles and shape-size characterization weren’t carried out due to the lack of access to a Scanning Electron Microscope.

The production of nanoparticles was verified by cross-corroborated change in colour due to the surface plasmon effect.

Assay for Prokaryotic Cytotoxicity

Materials Required

Bacterial broth cultures, 24-48 hours old , of Staphylococcus aureus, Bacillus subtilis, Enterobacter aerogenes and Escherichia coli, cork borer, nutrient agar/blood agar base medium, laminar airflow unit, swabs, micropipettes and pipette tips, Ethanol (70%), standard glass labware.

Procedure

All non-heat labile materials were sterilized by autoclaving under standard conditions, 121’C at 15psi, 20 minutes, heat labile materials such as pipettes were sterilized using ultraviolet irradiation. Sterile, aseptic conditions were established for carrying out the experiment

Swab cultures were carried out and a well diffusion assay was set-up using 75 microliters of the nanoparticle dispersions in the first round and a much higher concentration of 200 microliters in the second phase of the experiment, in the preliminary, first round, just E.coli and S.aureus were used, and the well diffusion assay took into account dilutions of nanoparticles (x, x/10 & x/100) , it was found that preliminary antimicrobial activity of any significant potency was only shown by the dispersions at their original concentrations, therefore this was the only concentration chosen for further corroborative investigations.

Results of prokaryotic cytotoxicity assay. Zones of inhibition are clearly apparent.

The process was repeated the second time with all four of the aforementioned cultures, with only original concentrations of the nanoparticle dispersions being used.

Nansosilver at the original concentration showed the maximum amount of microbicidal activity, which by inference makes it cytotoxic to prokaryotes, data about ZOI diameters is available in the appendix that follows this report.

Assay for Eukaryotic Cytotoxicity

While the initial plans included the use of trypan blue cell exclusion method for corroboration, the inconsistency of the yeast dispersion used meant that the results were practically unusable, and therefore data had to be tentatively evaluated using turbidimetry alone, we recommend caution before jumping to conclusions on the basis of this experiment alone and suggest further corroboration and verification.

Materials

yeast dispersion, 1 pellet in 50 ml of sucrose broth (approximately 0.5 g added) , nanoparticle dispersions with dilutions (x , x/10 and x/100) respectively, photocolorimeter (a spectrophotometer was preferable but not available)

Procedure.

Control and test solutions were created for each set of nanoparticle dispersions, for all three metallic nanoparticles, the controls consisted of 1 ml of the nanoparticle dispersions, and 1 ml of uninoculated sucrose broth and 8 ml distilled water, while the test samples contained 8 ml distilled water, 1 ml nanoparticle solutions and 1 ml of inoculated sucrose broth, the tubes were plugged with cotton and incubated overnight at room temperature (25-27’C)

Readings for optical density were then taken after 24 hours of culture, to examine if there was a trend between dilutions and rate of cell growth, a direct relationship between the two variables would yield an inversely proportional relationship.

A trend of cytotoxicity was found to occur for treatment with nanosilver solutions, this has been corroborated by studies which have illustrated the ability of silver nanoparticles to induce mutations and cell death (P V Asharani et al 2008 Nanotechnology) The other two nanoparticle dispersions showed no cytotoxicity.

The raw data and the graphical analysis of the data from this experiment is also available in the appendix at the end, a baseline correction factor was included to make presenting information in the graphs easier, the correction was applied by adding an equal amount to all derived results.

Results and Conclusions

Turkevich synthesis can be carried out in simple facilities to produce nanoparticle dispersions for further investigation.

Silver nanoparticles are potently cytotoxic against all the organisms tested against in the prokaryote cytotoxicity assay, regardless of whether they are gram +ve or gram –ve, and as such can be used as microbicides.

However, silver nanoparticles have been shown to be cytotoxic against Eukaryotes too, this means that unregulated efflux into the environment can lead to disastrous ecological consequences, and therefore strict regulatory norms are imperative.This cytotoxicity, however could have biomedical applications in the form of immunoconjugates against cancer specific receptors, for instance,

The other nanoparticle dispersions (Copper and Iron) , at the tested concentrations, were found not to be cytotoxic , as were silver dispersions at less than x/10 dilution, this could indicate a preliminary statistic for hazard levels and an association with the concentration, while clearing the other two for free usage at the investigated concentrations.

However, for the data to become fully valid for policymaking, an accurate method such as mass spectrometry must be used to evaluate precisely the concentration of the nanoparticles in their respective dispersions.

To sum up, our data is in agreement with research findings that silver is cytotoxic, and these studies so far point towards iron and copper not being cytotoxic, however, further research needs to be carried to account for any imperfections in the study itself; these results are a rough starting point, at best.

References

P.K. Khanna, Narendra Singh, Deepti Kulkarni, S. Deshmukh, Shobhit Charan, P.V. Adhyapak, Water based simple synthesis of re-dispersible silver nano-particles.

Iron nanoparticles: Synthesis and Applications in surface enhanced Raman Scattering and electrocatalysis, Guo et al, Physical Chemistry Chemical Physics, 2001.

An antibiotic assay by agar well diffusion method, Perez et al, Acta Biol. Med. Exp, 1990

Trypan Blue Exclusion Test of Cell Viability , Strober, Current Protocols in Immunology,

Toxicity of silver nanoparticles in zebrafish models
P V Asharani et al 2008 Nanotechnology 19 255102 (8pp) doi: 10.1088/0957-4484/19/25/255102

Appendix A : Turbidimetry Data and Graphs

Results from turbidimetry experiment.

Appendix B : Prokaryotic Cytotoxicity Assay.

Prokaryote zone of inhibition data.

Appendix C: Tentative estimates of concentration.

Silver Nanoparticle dispersion.

100 ml of AgNO3 solution contained 10.8 mg of Silver
3 ml of the finally prepared solution contained 0.324 mg of Silver nanoparticles

Solutions for the Yeast assay, contained 0.108 mg (x) , 0.0108 mg (x/10) and 0.00108 mg (x/100) of
Silver & 200 Microlitre aliquots, used for the prokaryote assay, contained 0.0216 mg of silver

Iron solution.

100 ml of FeSO4 solution contained 5.6 mg of Iron

3 ml of the prepared nanosolution contained 0.168 mg of Iron Nanoparticles
1 ml, for the yeast assay, contained 0.056 mg (x) , 0.0056 mg (x/10) , 0.00056 (x/100)

200 Microlitre aliquots, for prokaryote assays, contained 0.0112 mg of Iron nanoparticles

 

Copper solution.

100 ml of CuSO4 solution contained 6.35 mg

1 ml of the prepared solution contained 0.0635 mg (yeast assay) , the other two quantities used were 0.00635 mg and 0.000635 mg.

200 Microlitre aliquots contained 0.0127 mg

The p53 barcode – a brief introduction.

Notice – to view images at full size,please right click on the image and click “view image”.

p53 may be one of the most famous proteins in all of oncology, with in excess of a million hits on Google scholar for a strict keyword search. This, perhaps, is due to the myriad of interactions it has with signalling pathways in cells. It can modulate a variety of responses, ranging from growth arrest in the case of DNA damage, to cellular senescence, to apoptosis. This happens in response to cellular stress signals.

p53 responses and core network.

Now, the presence of a multitude of responses immediately begins to raise questions; what might trigger one response as opposed to another? Could the level of free p53 be one determinant? Apparently, this is one of the mechanisms that does play a role, however, it would appear that it is far from the sole determinant. The explanation offered for how this might take place hinges on promoter affinity, if p53 levels are low, it might roughly tend to preferentially bind to high-affinity promoters, which might be associated with cell cycle arrest, and if levels are high an apoptotic response may be preferred. See here for a reference.

The same paper also does note that the aforementioned roles aren’t strictly followed. What else, then, might determine how p53 functions? A combination of post-translational modifications and the trigger that sets off the p53 response to stress signals are good candidates, and it is these that will be the focus of this article.

Now, writing a thesis on p53 post-translational modification is not what I’m going to do; I’m just going to mention that there are a multitude of these modifications and these can have significantly different effects on what p53 does, cue an illustration…

p53 map of posttranslational modifications.

Image source – http://www.bnl.gov/biology/cellbio/human_p53.asp

Some of these post-translational modifications act by changing promoter selectivity/affinity. An illustration from the primary reference for this article follows.

p53 PTM - Modifiers, modifications, and target genes.

Then there are some proteins and protein interactions that tend to affect promoter-selectivity directly…

Factors that affect p53 promoter selectivity directly.

Some other modifications can affect p53 by controlling where in the cell it is localized. Yet other factors include proteins that serve as cofactors that can enhance the transcriptional activation that p53 executes on target genes, and yet some others, most notably Mdm2, can directly bind to p53 and ubiquitylate it, breaking it down. To put it simply, there is a lot of flexibility in p53 activity that enables it to tweak the response it elicits to different inducers.

So, how would one go about representing this? One solution is a p53 barcode. This is far from ideal because it doesn’t really map out the fine details of what is going on, but it does simplify the representation of the processes involved.

Here’s one more illustration.

p53 is activated by an array of cellular stresses and responds by activating various signalling pathways that are involved in diverse cellular mechanisms, from apoptosis to DNA repair. The protein level, localization, post-translational modifications and the cofactors of p53 are crucial to the function and regulation of p53. We propose that each individual aspect of p53 regulation represents a bar from a barcode that directs p53 activity. Different combinations of bars form different barcodes, and the barcode dictates which response p53 induces, be it apoptosis, cell-cycle arrest or senescence. Importantly, this allows p53 to be activated in a manner that is stress and cell-type dependent. The diagram shows a range of p53 regulations that control p53 activity and, ultimately, determine the cellular response. This response may be transcription dependent or independent. Each regulator is illustrated with its own bars. The number and width of the bars was assigned arbitrarily and has no relevance to the importance of each aspect in the regulation of p53 activity. From the main reference (see end of post)

The reason I think that this is particularly elegant is simple; the actual network is rather dizzying in its apparent complexity and this form of representation can really highlight what variations exist in terms of the activity of the protein. As an aside, at this juncture, I wish to introduce one or two papers that describe how p53, through miRNA, can suppress metastasis. http://jem.rupress.org/content/208/5/875.short & http://jcb.rupress.org/content/early/2011/10/19/jcb.201103097.short

Finally, if you fancy taking a peek at the various pathways that p53 is involved in, you might find this collection from the Nature-NCI Pathway Interaction Database useful.

Reference : Zmijewski et al A complex barcode underlies the heterogeneous response of p53 to stress. Nature Reviews Molecular Cell Biology 9, 702-712 (September 2008). The paper is behind a paywall, but using Google Scholar you may be able to find an institution that has a PDF copy online somewhere.

That is all from me this time round.