Anyhow, the article that I've picked is called COX-2 dependent regulation of mechanotransduction in human breast cancer cells, by A-Rum Yoon, Ioannis Stasinopoulos, Jae Hun Kim, Hwan Mee Yong, Onur Kilic, Denis Wirtz, Zaver M Bhujwalla and Steven S An. I'm not going to read the abstract just yet because some random on Facebook said that sometimes reading the abstract can bias the way you read the rest of the article. Of course, you shouldn't just go around believing everything randoms on Facebook say, but I don't see the harm in trying this.
On to the introduction...
Introduction
Breast cancer sucks. Metastasis doubly sucks because then it's like two cancers for the price of one, which is a deal that you wouldn't want to take up, but it's not like you have any choice in the matter because not enough is known about how and why cancers metastasise. This is why we do research!
This article's going to focus on the metastasising part, so let's sum up very briefly what we know about it, or at least what we think we know about it. Essentially some traits accumulate, allowing cells to escape, travel through the extracellular matrix (ECM) and enter the systemic circulation. The cells then travel to distant organs, where they burrow their way into the ECM and set up camp in a new tumour. Also according to this introduction inflammation is currently assumed to play a part in all of these processes, but there's still a lot of fuzziness and grey area to explore here.
This is where COX-2 comes in! COX-2 stands for "cyclooxygenase-2," which I mentioned before when I wrote about structure-activity relationships and the coxibs. COX-2 mediates inflammation and converts arachidonic acid into biologically active lipids known as prostaglandins (notably prostaglandin E2, or PGE2). The interesting part is that COX-2 has been found to be expressed at higher levels in malignancies, and PGE2 has also been implicated in metastasis. Previous studies have also shown that inhibition of COX-2 synthesis by RNA interference (a process in which RNA molecules inhibit the gene expression, by destroying the mRNA or otherwise) reduce the expression of ECM metalloproteinases (metalloproteinases are protein-destroying enzymes that require metal to work properly; those in the matrix have been related to invasion of a cancer). This, in turn, was shown to decrease the ability of metastasising cells to invade a new tissue.
Well that's all interesting, but what's the actual aim of this study? To my understanding, the aim of the study was to find out whether COX-2 and mechanotransduction (sensing an external force and turning it into some kind of response from the cell) are related, but I could be wrong: I didn't find it particularly clear what the actual aim was from the introduction alone. But maybe I'm just dumb. Idk.
Methods
The "methods" section was stuck right at the end, because let's face it, unless you're trying to critique a paper or whatever that tends to be the first part you're going to skip. But I'm actually trying to go over the paper here, so let's have a look at what's here.
Cell lines and culture materials
This section just listed off the ingredients of the cell media, as well as the temperature and percentage of CO2. Not sure if that's particularly relevant to me unless I want to replicate the experiment or double check that they used the right media for the cells (not that I know anyway :P).
Preparation of elastic matrix
As with many papers involving mechanotransduction, they've made an elastic polyacrylamide hydrogel out of acrylamide and bisacrylamide. Acrylamide basically makes long chains, whereas bisacrylamide crosslinks these chains. The more crosslinks there are (i.e. a higher percentage of bisacrylamide), the more rigid the gel will be. They made two similar mixtures: one without any extra stuff added, and one with fluorescent microbeads added so that traction force microscopy could be done: more on this in a bit.
Unfortunately, cells themselves can't adhere directly to the matrix, which is why they also coated both gel blocks with type I collagen. Type I collagen can't adhere directly to the matrix either, though, so a cross-linking particle called sulfo-sanpah was used.
Fourier transform traction microscopy (FTTM)
From my understanding, this procedure was used to quantify the contractile stress between the cell and synthetic ECM (i.e. the elastic matrix and other stuff on top). This used the gel with the fluorescent microbeads. Essentially a cell was put onto the gel block and allowed to stabilise. Pictures of the fluorescent microbeads were taken at various times. Afterwards, they used trypsin to detach the cells and then took another picture as the reference. The distance the beads move can be related to the force that the cells exert on the matrix. I don't really know what Fourier transform traction cytometry is about, and finding out seems to involve reading another paper, and this just reeks of looking up words in my Chinese dictionary only to have to look up definitions for words in the definition and so forth.
Magnetic twisting cytometry (MTC)
Okay, I've never heard of this one before, plus I've never done physics, so here's my really basic understanding of how it works. Essentially they get a magnetic bead and anchor it to the cytoskeleton through cell surface integrin receptors and the tripeptide RGD (arginine-glycine-aspartic acid). This bead is magnetised and twisted. As it twists, the cell resists. A camera can be used to detect the lateral bead displacements, and algorithms used to work out the stiffness of the cells. Or something.
Detection of COX-2 activity
Enzyme immunoassays (EIA) were used to detect the concentration of PGE2, which in turn was used to assess COX-2 activity (remember, COX-2 converts arachidonic acid into PGE2). I feel that I don't know as much about how EIA works as I should, but thankfully I found a handy video:
In the case of this paper, they appear to have quantified the amount of binding by using a spectrophotometer, which quantifies the intensity of the signal (i.e. how much the colour changed). They would have presumably also used a standard curve, which takes samples that have the substance at known concentrations, for comparison. (Or at least I'm just assuming that because that's what we've done in labs at uni.)
Fluorescence-activated cell-sorting (FACS) analysis
This was used to quantify the cell surface expression levels of beta 1 and beta 3 integrins. Essentially some cells were divided into two groups and incubated with specific FITC-conjugated antibodies. One group received antibodies against integrin beta 1, whereas the other received antibodies against integrin beta 3. FITC is a green fluorescent dye. Its fluorescence can be detected and quantified by a machine known as a flow cytometer.
Statistical analysis
The main statistical tests they used were Student's t-test and ANOVA (Analysis of Variance). I don't know much about either of these tests to be honest, and after typing up all that stuff above, I don't really feel like looking them up right now either. These can potentially be a topic for a later post.
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