Enhanced breast cancer therapy with nsPEFs and low concentrations of gemcitabine

Abstract

Background: Chemotherapy either before or after surgery is a common breast cancer treatment. Long-term, high dose treatments with chemotherapeutic drugs often result in undesirable side effects, frequent recurrences and resistances to therapy.

Methods: The anti-cancer drug, gemcitabine (GEM) was used in combination with pulse power technology with nanosecond pulsed electric fields (nsPEFs) for treatment of human breast cancer cells in vitro. Two strategies include sensitizing mammary tumor cells with GEM before nsPEF treatment or sensitizing cells with nsPEFs before GEM treatment. Breast cancer cell lines MCF-7 and MDA-MB-231 were treated with 250 65 ns-duration pulses and electric fields of 15, 20 or 25 kV/cm before or after treatment with 0.38 μM GEM.

Results: Both cell lines exhibited robust synergism for loss of cell viability 24 h and 48 h after treatment; treatment with GEM before nsPEFs was the preferred order. In clonogenic assays, only MDA-MB-231 cells showed synergism; again GEM before nsPEFs was the preferred order. In apoptosis/necrosis assays with Annexin-V-FITC/propidium iodide 2 h after treatment, both cell lines exhibited apoptosis as a major cell death mechanism, but only MDA-MB-231 cells exhibited modest synergism. However, unlike viability assays, nsPEF treatment before GEM was preferred. MDA-MB-231 cells exhibited much greater levels of necrosis then in MCF-7 cells, which were very low. Synergy was robust and greater when nsPEF treatment was before GEM.

Conclusions: Combination treatments with low GEM concentrations and modest nsPEFs provide enhanced cytotoxicity in two breast cancer cell lines. The treatment order is flexible, although long-term survival and short-term cell death analyses indicated different treatment order preferences. Based on synergism, apoptosis mechanisms for both agents were more similar in MCF-7 than in MDA-MB-231 cells. In contrast, necrosis mechanisms for the two agents were distinctly different in MDA-MB-231, but too low to reliably evaluate in MCF-7 cells. While disease mechanisms in the two cell lines are different based on the differential synergistic response to treatments, combination treatment with GEM and nsPEFs should provide an advantageous therapy for breast cancer ablation in vivo.

Keywords: Apoptosis; Breast cancer; Clonogenics; Gemcitabine; MCF-7; MDA-MB-231; Nanosecond pulsed electric fields (nsPEFs); Necrosis (necroptosis); Non-thermal; Synergism.

Figure 1

Dose Responses for gemcitabine – The survival fractions are shown for MCF-7cells (A) and MDA-MB-231 cells (B) treated with indicated concentrations of gemcitabine for 24 h (black squares) and 48 h (red circles). IC₅₀ values for 48 h treatment are indicated in the text.

Figure 2

MCF-7 cell survival after treatment with gemcitabine, nsPEFs and combination – Experiments were carried out with MCF-7 cells as described in Methods for survival 24 h (A) and 48 h (B) after nsPEF treatment. Synergism quotients (SQ) are shown for both treatment orders. The statistical significance among groups of nsPEFs, GEM and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t test.

Figure 3

MDA-MB-231 cell survival after gemcitabine, nsPEFs and combined treatment - Experiments were carried out with MDA-MB-231 cells as described in Methods for survival 24 h (A) and 48 h (B). Synergism quotients (SQ) are shown for both treatment orders. The statistical significance among groups of nsPEFs, GEM and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t test.

Figure 4

Clonogenic survival for MCF-7 cell and MDA-MB-231 after gemcitabine, nsPEFs and combined treatment – Clonogenic survival assays and statistics are described in Methods for MCF-7 cells (A) and MDA-MB-231 cells (B). Synergism quotients (SQ) are shown for both treatment orders.

Figure 5

Typical figures analyzing Annexin-V-FITC binding and propidium iodide (PI) staining for MCF-7 and MDA-MB-231 cells treated with gemcitabine, nsPEFs and combinations. Experiments were carried out as described in Methods and shown with GEM alone (0.38 μM), nsPEF alone at indicated electric fields and GEM after treatment with nsPEFs. Panel A shows an experiment with MCF-7 cells and panel B show an experiment with MDA-MB-231 cells. Cell populations are shown in quadrants with Annexin-V-FITC levels indicated on the X-axis and PI staining indicated on the Y-axis. Cells with Annexin-V-FITC labeling only are considered apoptotic (lower right quadrant) and cells with Annexin-V-FITC and PI labeling are considered necrotic (upper right quadrant). Synergism quotients are shown for each cell death type in the respective quadrants. A typical experiment is shown for each cell line.

Figure 6

Cell apoptosis and necrosis for MCF-7 cell after gemcitabine, nsPEF and combined treatment – Experiments were carried out as described in Methods. Panel A shows MCF-7 cell responses for apoptosis (PI-/FITC+) and panel B shows MCF-7 cell response for necrosis (PI+/FITC+). GEM was used at 0.38 μM. Data show the apoptotic or necrotic percentages in 10,000 cells analyzed by flow cytometry.

Figure 7

Cell apoptosis and necrosis for MDA-MB-231 cell after gemcitabine, nsPEF and combined treatment - Experiments were carried out as described in Methods. Panel A shows MDA-MB-231 cell responses for apoptosis (PI-/FITC+) and panel B shows MDA-MB-231 cell response for necrosis (PI+/FITC+). GEM was used at 0.38 μM. Data show the apoptotic or necrotic percentages in 10,000 cells analyzed by flow cytometry.