Targeted Osmotic Lysis of Highly Invasive Breast Carcinomas Using Pulsed Magnetic Field Stimulation of Voltage-Gated Sodium Channels and Pharmacological Blockade of Sodium Pumps

Abstract

Abstract: Concurrent activation of voltage-gated sodium channels (VGSCs) and blockade of Na⁺ pumps causes a targeted osmotic lysis (TOL) of carcinomas that over-express the VGSCs. Unfortunately, electrical current bypasses tumors or tumor sections because of the variable resistance of the extracellular microenvironment. This study assesses pulsed magnetic fields (PMFs) as a potential source for activating VGSCs to initiate TOL in vitro and in vivo as PMFs are unaffected by nonconductive tissues. In vitro, PMFs (0-80 mT, 10 msec pulses, 15 pps for 10 min) combined with digoxin-lysed (500 nM) MDA-MB-231 breast cancer cells stimulus-dependently. Untreated, stimulation-only, and digoxin-only control cells did not lyse. MCF-10a normal breast cells were also unaffected. MDA-MB-231 cells did not lyse in a Na⁺-free buffer. In vivo, 30 min of PMF stimulation of MDA-MB-231 xenografts in J/Nu mice or 4T1 homografts in BALB/c mice, concurrently treated with 7 mg/kg digoxin reduced tumor size by 60-100%. Kidney, spleen, skin and muscle from these animals were unaffected. Stimulation-only and digoxin-only controls were similar to untreated tumors. BALB/C mice with 4T1 homografts survived significantly longer than mice in the three control groups. The data presented is evidence that the PMFs to activate VGSCs in TOL provide sufficient energy to lyse highly malignant cells in vitro and to reduce tumor growth of highly malignant grafts and improve host survival in vivo, thus supporting targeted osmotic lysis of cancer as a possible method for treating late-stage carcinomas without compromising noncancerous tissues.

Keywords: Na+, K+-ATPase; advanced stage carcinoma; pulsed magnetic fields; sodium channels; sodium pumps; targeted osmotic lysis.

Figure 1

Evidence of tumor lysis following treatment with TOL using electric current stimulation. The photomicrograph depicts an ectopic, MDA-MB-231 xenograft that was removed 24 h after 3 treatments with TOL (10 mg/kg ouabain; 10 V, 1 msec pulses, 15 pps, DC electric current for 5 min). Due to the size and configuration of the stimulating electrodes, the stimulus had been delivered to only the portion of the tumor between the arrows. Note the obvious difference in gross appearance between the stimulated and unstimulated portions of the tumor. The stimulated area is necrotic. Calibration bar = 5 mm.

Figure 2

Stimulus-response curve for pulsed magnetic fields. MDA-MB-231 cells incubated in DMEM with or without 500 nM digoxin were treated with the indicated magnetic field stimulus intensity for 15 min. Subsequent cell counts revealed the stimulus dependence of TOL, with a maximum lysis rate of 95–100% cell death following TOL compared to 3–5% in controls. A two-way ANOVA revealed a main effect for treatment and a treatment X group interaction. * p < 0.05 by Tukey II post-hoc analysis.

Figure 3

TOL dependence on Na⁺. The lysis of cells incubated in normal Ringer’s solution with 500 nM digoxin was 67% compared to the 15–22% lysis of cells in normal Ringer’s solution without digoxin when exposed to an 80 mT PMF. Counts of lysed cells incubated in Na⁺-free Ringer’s with digoxin and stimulated with PMF was comparable to controls. * p < 0.001 by planned orthogonal t-test.

Figure 4

Sodium Channel Expression in MDA-MB-231 and 4T1 Breast Cancer Cells. Cultured cells were stained with a pan-specific anti-body for a conserved segment of the VGSC protein. Immunocytochemical imaging reveals labeling in cells of both MDA-MB-231 (A) and 4T1 (B) cell lines. The labeling depicted in the photomicrographs in Figure 4 was not observed in cells in which the anti-sodium channel antibody was pre-blocked with a 500-fold excess of the peptide antigen to which it was raised. The relative expression of VGSC proteins was confirmed quantitatively with flow cytometric analysis of the cell populations. Calibration bars = 15 µm).

Figure 5

Targeted Osmotic Lysis of 4T1 Mouse Breast Cancer Cells. Cultured 4T1 cells in suspension were incubated for 15 min in DMEM + 500 nM digoxin or in DMEM alone, then stimulated with the pulsed magnetic field for 15 min. Assessment of viability was as with the previous experiment. (A): TOL-treated cells; (B): Vehicle-treated cells (calibration bars = 30 µm); (C): Cell counts comparing relative viability in samples that were treated with digoxin or vehicle and then exposed concurrently to an 80 mT PMF. *: p < 0.01 by t-test.

Figure 6

Tumor viability rating. The photomicrographs depict representative tissue sections selected by the blinded veterinary pathologist to illustrate the morphologic features of tissue samples taken from MDA-MB-231 tumors rated at 1 (A; no damage (there is no necrosis), ×10), 2.5 (B; moderate damage (necrosis with pyknotic cells), ×20) and 4 (C; significant damage (necrosis with cavitations), ×10). To date, we have been unable to reliably achieve complete tumor destruction, a Grade 5 response. The rating system was applied to the evaluation of samples from >200 mice.

Figure 7

Relative viability of homografts. The average viability scores of TOL-treated xenografts compared to controls as determined by a veterinary pathologist who was blinded to the treatment provided are illustrated. Sections taken from 35 mice were used to determine the mean ratings of tumor viability for TOL-treated and control mice. Note that the TOL-treated tumors averaged 20–40% viability compared to 50–60% viability in control tumors. The three control groups were collapsed and compared to the tumors treated with both digoxin and PMF. * Χ² p < 0.05. The necrosis observed in control can be largely attribute to damage seen during the natural history of a rapidly growing tumor.

Figure 8

Morphology of representative tissues taken from the kidney (A; ×10), spleen (B; ×10), skin (C; ×10) and skeletal muscle (‡) adjacent to a homograft tumor (D (*); ×1.25) treated with TOL. The morphology of these tissues were determined to be normal, (although the skin has a nonsignificant lesion) indicating that it is unlikely that they were affected by treatment with TOL.

Figure 9

Growth of MDA-MB-231 xenografts (A) and 4T1 homografts (B) treated with TOL compared to the growth of xenografts that received drug or stimulation alone or vehicle. A. Groups of mice (n = 8) were treated as indicated. None of the mice that were treated with TOL (red curve) met NIH criteria for humane endpoint euthanasia. Three mice in the drug-only group (brown curve), 2 in the stim-only group (blue curve) and 2 in the vehicle-only group (green curve) met humane endpoint criteria and had to be sacrificed. B. The graph shows that the rate of growth of ectopic 4T1 homografts treated with TOL is significantly slower when compared to the growth of homografts that received drug or stimulation alone or vehicle. A one-way ANOVA revealed a main effect. * p < 0.05 compared to each of the controls by Tukey II post-hoc comparisons.

Figure 10

Post-treatment survival. Treating ectopic 4T1 homografts with TOL significantly increased survival when compared to the growth of homografts that received drug or stimulation alone or vehicle. n = 12/group.

Figure 11

Time to 50% mortality (meeting NIH humane endpoint criteria for euthanasia). Treatment with TOL consistently extended the time to 50% survival for mice with 4T1 breast cancer homografts by approximately 1 week when compared to controls. One-way ANOVA revealed a main effect. * = p < 0.01 by Tukey II post-hoc comparisons.