cancer cell negative charge

This journey started with a request to look up the charge on different types of cancer cells. This request may have started with a layperson’s understanding of chemistry and charge. Donglu Shi of the University of Cincinnati postulated that the folic acid receptor might be a common tumor cell target whose charge may change when confronted with excess lactic acid [1] from glycolysis not coupled to the TCA cycle and electron transport chain, aka the Warburg Effect. We will conclude that micro domains of the folic acid receptor is where CopperOne might work its therapeutic effects.

The idea is for the supramaramagnetic nanoparticles with the positive surface chare to capture cancer cells for both diagnostic and therapeutic purposes.

The strategy is to separate tumor cells from normal cells that have less of a negative charge, perhaps because of the lactate production, from sialic acid, or from a combination of the two. These are HeLa cells. Breast cancer cells may be in the range 20–30 m, while the typical dimension of the nanoprobe is on the order of 200–300 nm.

Douglas Shi and his laboratory at the University of Cincinnati have designed magnetic nanoparticles that bind to charged surfaces on cells. Dr Shi reminds us that one of the hallmarks of cancer cells is their over reliance on glycolysis and the overproduction of lactic acid/lactate. Numerous sources tell us that the tumor micro environment is acidic due to lactic acid. How can this be so? If we have lactic acid, the tendency would be to donate some of its protons to negatively charged amino acid side chains like glutamate and aspartate. The export system for cancer cells is mono carboxylate transporter 4, or MCT4, or MOT4. This transporter not only removes lactate in the “conjugate base” form from the cell but also releases H⁺. Lactate is then left to “steal” H⁺ from uncharged glutamic acid and aspartic acid side chains leaving them with negative charges of their conjugate bases glutamate and aspartate.

The strategy is to separate tumor cells from normal cells that have less of a negative charge, perhaps because of the lactate production, from sialic acid, or from a combination of the two. Breast cancer cells may be in the range 20–30 m, while the typical dimension of the nanoprobe is on the order of 200–300 nm.

Close to 100% of some cancer cell lines are isolated on these magnetic beads with positively charged groups. (PMC) We can hypothesize that cells that are close to 100% captured like HepG2 cells (liver cancer) have a greater negative surface charge than say LM-3 cells with less than 50% captured. The other intriguing twist is that the normal cells, turquoise lettering, are not captured by nanoparticles with positive surface charges. Normal liver cells are weakly captured by negatively charged nanoparticles. Most interestingly to CopperOne is the evidence for large positive charge on peripheral mononuclear cells (PMN). Granulocytes are part of the PMC family of white blood cells. When granulocytes encounter bacteria that secrete positively charged peptides called defensins.The following image is from the second Shi Laboratory review [2]

Another research group used a slightly different technique to measure surface charge on just one particularly aggressive cancer cell type.

Zeta Potential and cell surface charge

Perhaps another review on zeta potential is in order. The Wikipedia authors have compiled an excellent review that is probably beyond most lay readers. Suffice it to say, the Stern double layer is the layer of counter ion charges associated with a charged surface.

A illustration of zeta potential and the electrophoresis technique used to measurement. Note: Surface charge on any cell will not be uniform as illustrated for this sphere with a uniform negative charge with a collection of positive counter ions.

The slipping plane is the place where charged solutes associated surface start to resemble the bulk solution. This is the zeta potential. For particles with charged surfaces, such as cancer cells, the potential is measured by electrophoresis, that is movement through a solution in an electric field between two electrodes.

A Slovakian and Polish group used this approach to measure the surface charge of a glioblasoma multiforme cell line LN-229. GBM is a particularly hard cancer to teat because of its heterogeneity and inaccessibility. Naturally occurring plant phenolics cinnamic acid and ferulic acid were explored as treatments. Electrophoretic mobility was used to measure the surface charge density of LN-229 cells. Treatment of both phenolics caused a decrease in negative charges at high pH and a decrease in positive charges at low pH. PubChem lists two very similar values for the pKa of ferulic acid: 4.42 and 4.58. The pKa is the pH of which half of the groups with an exchangeable H⁺ are in the protonated form. Pubchem lists two experimental values for the pKa of lactic acid: 3.79 and 3.82.

Based on electrophoretic mobility values, the surface charge density δ was determined according
to the above equations in which: η—the viscosity of the solution, u—the electrophoretic mobility, d—the diffuse layer thickness. The diffuse layer thickness was determined using the formula in which R is the gas constant, T is the temperature, F is the Faraday constant, I is the ionic strength of 0.9% NaCl, and ε and ε ₀ refer to the permeability of the electric medium. The pH in these experiments was adjusted with NaOH and HCl. It wasn’t clear if the cells were rinsed after being treated with the ferulic acid or if the indicated concentration of ferulic acid was added to the electrophoresis medium in which it becomes the main buffering agent. Lines have been drawn from the approximate pKa of ferulic acid and the corresponding charge density in Coulombs per square meter. [3]

In their discussion Naumowicz and coauthors listed factors that may affect zeta potential of the entire cells: temperature, pH, ionic strength, and solvent viscosity (also very low). Naumowicz and coauthors listed factors that are different in tumor cells that might affect surface charge: increased sialic acid, free fatty acids/phospholipids, alterations in acidic/basic functional groups, and a decrease in integral membrane protein levels. Some aspects of charge/charge neutralization were discussed. This would occur above the pKa of ferulic acid. This is in fact what they observed.

Starting with the negatively charged bacterium or cancer cell
Ionic strength was high enough to keep the cells from lysing. Physiological solutions also contain divalent cations. Would these form a counter ion cloud to shield the bacterium from the immune cell?
Would viscosity inducing small and large molecules keep the bacterium or cancer cell from electrostatically interacting with the immune cell?
Electrical permeability. Many biological molecules have dipole moments such they line up between the bacterium/cancer cell and the immune cell.

Many scientists are convinced that charge is important. Looking at the literature, it is complicated on the level of a whole cancer cell or even a much smaller whole bacterium. Let’s go back to the mention of the folic acid receptor by Dr Shi. [1]. If Cu⁺ can make it’s way to the active site of this transporter, surely this is where charge is important on cancer cells.

Folic acid receptor aside

Indeed, the human folic acid receptor alpha, FRα, is expressed in a wide variety of tumor cells that need more folic acid for the synthesis of nucleotide involved in rapid cell division. [4] The group that solved this crystal structure stated that The folate pteroate moiety is buried inside the receptor, whereas its glutamate moiety is solvent-exposed and sticks out of the pocket entrance, allowing it to be conjugated to drugs without adversely affecting FRα binding. [4] This might be where the Shi laboratory was coming from when they stated that tumor cells develop a negative charge because they have increased ability to bind their cancer targeting positively charged group conjugted Fe₃O₄@Cu_2-xS nano particles. [1]

Image of the FRα color coded by charge. Red is negative; blue is positive. These charges electrostatically attract the charged groups on the D-folic acid molecule. [3]

This post may leave the reader “head scratching” as to the importance of overall surface charge on cancer cells. The Shi [1,2] and Kotynsa [3] laboratories used isolated cells. Real cancer cells, as both groups admitted, live in crowded, acidic tumors. The Shi Lab was also concentrating their efforts on isolating these cancer cells that had escaped the tumors.

References

Deng Z, Lin J, Bud’ko SL, Webster B, Kalin TV, Kalinichenko VV, Shi D. Dual Targeting with Cell Surface Electrical Charge and Folic Acid via Superparamagnetic Fe₃O₄@Cu_2-xS for Photothermal Cancer Cell Killing. Cancers (Basel). 2021 Oct 21;13(21):5275. PMC free article
Shi D. (2017) Cancer cell surface negative charges: A bio-physical manifestation of the warburg effect. Nano LIFE. 2017;7:1771001.
Naumowicz M, Kusaczuk M, Zając M, Gál M, Kotyńska J. Monitoring of the Surface Charge Density Changes of Human Glioblastoma Cell Membranes upon Cinnamic and Ferulic Acids Treatment. Int J Mol Sci. 2020 Sep 22;21(18):6972. PMC free article
Chen C, Ke J, Zhou XE, Yi W, Brunzelle JS, Li J, Yong EL, Xu HE, Melcher K. Structural basis for molecular recognition of folic acid by folate receptors. Nature. 2013 Aug 22;500(7463):486-9. PMC free article