Selective lysis of breast carcinomas by simultaneous stimulation of sodium channels and blockade of sodium pumps

Abstract

Sodium influx through voltage-gated sodium channels (VGSCs) coupled with balanced removal of sodium ions via Na⁺, K⁺-ATPase is a major determinant of cellular homeostasis and intracellular ionic concentration. Interestingly, many metastatic carcinomas express high levels of these channels. We hypothesized that if excess VGSCs are activated and Na⁺, K⁺-ATPase is simultaneously blocked, the intracellular Na⁺ concentration should increase, resulting in water movement into the cell, causing swelling and lytic cell death. MDA-MB-231 breast cancer cells over-express VGSCs by 7-fold. To test our hypothesis, we treated these cells in vitro with the Na⁺, K⁺-ATPase blocker, ouabain, and then stimulated with a sublethal electric current. For in vivo histologic and survival studies, MDA-MB-231 xenografts were established in Nu/J mice. Mice injected with saline or ouabain were electrically stimulated with trains of 10 msec 10V DC pulses. Within seconds to minutes, the cells swelled and lysed. MCF-10a cells, which express normal VGSCs levels, were unaffected by this treatment. Cells from the weakly-malignant cell line, MCF-7, which express 3-fold greater VGSCs than MCF-10a cells, displayed an intermediate time-to-lysis. The rate of lysis correlated directly with the degree of sodium channel expression and malignancy. We also demonstrated efficacy in cell lines from prostate, colon and lung carcinomas. Treated MDA-MB-231 xenografts showed 60-80% cell death. In survival studies, TOL-treated mice showed significantly slower tumor growth vs. controls. These results are evidence that this "targeted osmotic lysis" represents a novel method for selectively killing cancer cells and warrants further investigation as a potential treatment for advanced and end-stage breast cancer.

Keywords: cancer; epithelial carcinoma; sodium channels; sodium pumps; targeted osmotic lysis.

Figure 1. VGSC expression in MCF-10a, MCF-7 and MDA-MB-231 cells

The photomicrographs (A) in A depict immunocytochemical labeling of VGSC protein observed in normal breast cells (MCF-10a), weakly-malignant (MCF-7) and highly-malignant (MDA-MB-231) breast cancer cells. The bar graph, (B) depicts the relative pixel counts obtained from 120 cell profiles outlined in confocal images of normal breast cells, weakly-malignant and highly-malignant breast cancer cells grown in culture that were processed with a pan-specific antibody that recognizes a conserved segment of the channel protein. Note that the results of the pixel evaluation of isolated cell profiles assessed in each cell line are consistent with relative field luminosity data depicted in A and the amounts of sodium channel protein directly correlates with the level of malignancy. P values compared to normal MCF-10a; ^*p < 0.05; ^**p < 0.01.

Figure 2. Osmotic lysis of cancerous breast cells

(A) The photomicrographs were taken from best response video recordings that depict interval changes in cellular integrity of MDA-MB-231 cells in culture that were treated with ouabain (100 nM) and then stimulated with 1.0 V DC delivered in 1 msec pulse trains at 200 pps at stimulus onset (time 0) and after 8 sec and 16 sec of continuous stimulation. Note the bulging of cell membranes and the exudation of cytosol (arrows) into the surrounding media that is indicative of cell lysis. (B) The histogram depicts the average time to lysis of MCF-10a, MCF-7 and MDA-MB-231 cells treated with TOL. The results are consistent with the hypothesis that the rate of TOL is dependent on the relative number of sodium channels expressed in each of the cell types. Note that TOL spares the cells derived from normal breast tissue (MCF-10a). ^*p < 0.05; ^**p < 0.01.

Figure 3. Histological verification of cell lysis after TOL

Photomicrographs of malignant breast cancer xenografts that received no treatment (A), ouabain treatment alone (B) and ouabain followed by stimulation of 10 V, 200 pulses per second electric current twice for 1 min allowing 15 min without current between each stimulation (C and D). D includes uninjured muscle tissue (arrow) located adjacent to the tumor shown in C; calibration bars = 50 µm.

Figure 4. Survival and tumor growth after a single treatment with TOL

Groups of male Nu/J mice (n = 6) were injected with MDA-MB-231 cells to establish xenografts. The graph illustrates the growth of xenografts in mice treated with TOL compared to the growth of xenografts in mice treated with saline (Sal), ouabain (10 mg/kg) or stimulation alone. All TOL-treated mice survived to the end of the experiment. One mouse in the saline group and 1 mouse in the Stim group met NIH criteria for euthanasia and were sacrificed early. ^*p < .05 compared to controls.

Figure 5. Photographic depiction of the apparatus used to stimulate cells in vitro

(A) SD9 Grass Stimulator; (B) Narishige Model MN-151 Micromanipulator; (C) Leica Model DMIL Microscope.