Metformin (03) In-vivo experiments require non-lethal dose rates!

Just before I move on to metformin-induced substrate oxidation changes in healthy volunteers, I think it's worth looking at this neoplasia paper in a little detail. It's fairly typical of the work done on metformin as an anti-cancer agent and focuses on the highly reproducible inhibitory effect of metformin on complex I.

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.

Most of this work is very clever and very carefully done, but lives with the problem that the experiments usually use concentrations of metformin in-vitro which would be lethal in-vivo because, well, everybody does it and there is no effect if you don't... However the mouse xenograft studies have to use clinically relevant therapeutic doses of metformin otherwise the mice would be, well, a bit dead. There are other problems which will become apparent as we work through the data.

The figure I'd like to focus on is supplementary data section three of figure seven.

Graphs B and C look like this:

This is what they did to generate them. They took A549 tumour cells and injected them in to immuno-incompetent mice then measured the growth of the resulting tumour.  A549 cells are highly sensitive to metformin, so graph B comes as no surprise. Graph C is much, much cleverer. They wanted to prove that metformin was actually working on complex I. So they destroyed complex I with a shRNA targeting NDUSF3, an essential subunit of this complex. To keep the cell line functional they replaced complex I with our old friend the yeast derived NADH dehydrogenase NDI1. This enzyme does not bind metformin nor pump protons but does reduce NADH to NAD+ and does feed electrons to the CoQ couple and the downstream complexes. You can see from graph C that replacing complex I with NDI1 protects the A549 cell derived tumours from the growth slowing effects of metformin.

Look at B. Look at C. Protection from metformin in C. Yes?

Now, you have to ask: What is the effect of knocking down complex I in cancer cells? If you cannot reduce NADH to NAD+ then the TCA cannot turn. Citrate cannot be metabolised to alpha ketoglutarate so is exported from the mitochondria and can be used for tumour anabolism. The tumour becomes highly aggressive. Like this:

Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation, Metastasis by Mediating Mitochondrial Metabolism

or this, blogged about many years ago:

Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression

This illustrates my marked discomfort with accepting complex I blockade as the mechanism of anti-cancer action of metformin. Blockading complex I will admittedly decrease ATP supply from oxidative phosphorylation but at the cost of supplying a large amount of citrate to the cytoplasm ready for anabolic processes, while glycolysis continues unabated, supplying cytoplasmic NADH and ATP.

So in the current paper, by knocking down NDUSF3, they should have generated an aggressive phenotype. They didn't, because they also engineered-in NDI1, which will reduce cytoplasmic NADH to NAD+ very effectively. Dropping the NADH to NAD+ ratio suppresses tumour aggressiveness in the above papers.

Does the engineered A549 NDUSF3 + NDI1 tumour in nude mice show reduced or increased aggressiveness compared to the A549 unmodified tumour? We are looking to compare the top line in graph B above (dark squares) with the pale squares in graph C. By eyeball they actually look pretty much the same.

Except for the x axes. Graph B is 40 weeks, graph C is 50 weeks. Hard to compare the two... But if we stretch graph C so that weeks 10-40 align with weeks 10-40 of graph B, then superimpose the two graphs we can generate the following, rather more informative, image:

It looks to me as if inserting NDI1 in to the mitochondria of a cell line, (probably) made aggressive by knockdown of NDUSF3, renders the in-vivo tumour growth rate much lower than the natural tumour cell line and remarkably similar to that of metformin treated natural tumour cell line. Probably by reducing the NADH:NAD+ ratio.

This doesn't automatically suggest that metformin might be acting by reducing the NADH:NAD+ ratio, though it might be, but it does illustrate how nicely you can still pull interesting snippets out of papers full of experiments with metformin at lethal concentrations.

The difference between isolated mitochondrial preparations and mouse models is that the mouse models have a supply of insulin, glycerol-3-phosphate and the enzyme to use cytoplasmic NADH to reduce the CoQ couple, facilitating insulin signalling and so cancer growth. This is much more likely to be the process which we can block with metformin at therapeutic concentrations.

Peter