High-voltage pulsed electric field plus photodynamic therapy kills breast cancer cells by triggering apoptosis

Abstract

This study evaluated the effects and mechanism of action of combining irreversible electroporation (IRE) and photodynamic therapy (PDT) in breast cancer cells in vitro and in vivo. Jin's formula was used to assess killing efficacy of different IRE+PDT dosing combinations in breast cancer MCF-7 cells. Flow cytometry, high-content imaging, and confocal laser scanning microscopy were used to detect apoptosis. qRT-PCR and western blotting were used to evaluate expression of apoptosis-related genes and proteins. IRE+PDT combination therapy was administered to BALB/C mice with breast cancer tumors in vivo; tumor size was used to assess treatment efficacy. Killing mechanisms were examined using transmission electron microscopy and immunohistochemistry. We found that IRE+PDT combination therapy produced significant synergistic killing effects in breast cancer cells (highest Jin q value of 1.32). Early apoptosis rates were significantly higher in the IRE+PDT group (16.0%) than in IRE-alone (7.6%) and PDT-alone (4.6%) groups (P<0.05). qRT-PCR showed higher Caspase-1, -3, -5, -6, -7, -8, and -9 and TNFRSF1A expression with IRE+PDT than with control. Western blots showed increased cleaved Caspase-3, -7, and -9, and PARP levels in the IRE+PDT group. In vivo tumor suppression rate for IRE (1200 V)+PDT (10 mg/kg) was 68.3%. Combination therapy produced the most obvious apoptosis effects. Compared with controls, the IRE+PDT group exhibited lower new blood vessel (VEGF, CD31), metastasis (TGF-β), and cell proliferation (Ki-67) indicators and higher inflammation indicator (TNF-α) 1 day post-treatment. Thus, combining IRE and PDT enhanced their anti-tumor effects in breast cancer, and apoptosis played a key role in this process.

Keywords: High-voltage pulsed electric field; apoptosis; breast cancer; combination therapy; photodynamic therapy.

Figure 1

Detecting the survival rate of cells with different therapeutic doses of irreversible electroporation (IRE) and photodynamic therapy (PDT). A: Killing effects in MCF-7 cells exposed to IRE of different strengths. *P<0.05 versus 400 V/cm group. B: Killing effects in MCF-7 cells of PDT at different PSD-007 doses. *P<0.05 versus 3.125 µg/mL group. C: Killing effects in MCF-7 cells of IRE alone, PDT alone, and IRE+PDT. Data represent mean values of three independent experiments (n=3). *P<0.05 versus 380 V+12.5 µg/mL group.

Figure 2

Effects of electric pulses on photosensitizer PSD-007 emission spectrum and cellular absorption. A: Emission spectrum changes before (1) and after (2) electric pulses. B: Cellular PSD-007 absorption at different times with (bottom) or without (top) irreversible electroporation (IRE). The blue signal is DNA marked by Hoechst 3342. Photosensitizer PSD-007 excited by 405-nm wavelength laser emitted 630-nm wavelength red light, causing the cells to appear red. C: Fluorescence intensity changes within cells over time. Data represent mean values of three independent experiments (n=3). *P<0.05 versus PSD-007 (PSD-alone) group.

Figure 3

Qualitative and quantitative apoptosis detection. A: MCF-7 cells treated with irreversible electroporation (IRE), photodynamic therapy (PDT), or both (IRE+DPT) using two doses underwent detection for apoptosis by the high-content system after 24 h (10 × amplification). For IRE+PDT, dose I and II are 380 V+12.5 µg/mL and 380 V +10 µg/mL, respectively; for IRE, dose I and II are 630 V and 580 V, respectively; and for PDT, dose I and II are 32 µg/mL and 29.5 µg/mL, respectively. B: MCF-7 apoptosis, as detected by flow cytometry at 0, 2, and 6 h after treatment. C: MCF-7 apoptosis, as detected by confocal microscopy 2 h after treatment, shown at 63 × magnification. The blue signal is DNA marked by Hoechst 3342. The green signal represents eversion of phosphatidylserine in the molecular membrane marked with Annexin-FITC, which indicates early apoptosis. The red signal represents PI entry into the nuclei due to increased membrane permeability during the later stages of apoptosis. IRE, irreversible electroporation treatment alone; P DT, photodynamic therapy alone; IRE+PDT, treatment with irreversible electroporation and photodynamic therapy. IRE stands for the group solely treated with IRE, PDT for the group solely treated with PDT and IRE+PDT for the group treated with the combination of IRE and PDT.

Figure 4

Bar chart of the percentage of viable and apoptotic cells in control and treated MCF-7 cell populations at 0 h (A), 2 h (B), and 6 h (C) after treatment. Data represent the mean values of three independent experiments (n=3). *P<0.05 versus control. IRE, irreversible electroporation treatment alone; PDT, photodynamic therapy alone; IRE+PDT, treatment with irreversible electroporation and photodynamic therapy.

Figure 5

Changes in expression of apoptosis-related genes and proteins after treatment with irreversible electroporation (IRE), photodynamic therapy (PDT), and IRE+PDT. A: Apoptosis-related gene expression 2 h after MCF-7 cells were treated. B: Cleaved Caspase-3, Caspase-7, and Caspase-9 and PARP levels 6 h after MCF-7 cells were treated. C: The relative gray date from WB experiments. Data represent the mean values of three independent experiments (n=3). *P<0.05 versus control.

Figure 6

Mouse treatment procedure and tumor suppression rates after treatment. A: Irreversible electroporation (IRE) process for mice with breast cancer. B: Photodynamic therapy (PDT) process for mice with breast cancer. C: Left, changes in tumor size with treatments at different doses; right, tumors in mice treated with the combination method changed at different times. Data represent the mean values of three independent measurements (n=3). *P<0.05 for the IRE (1200 V)+PDT (10 mg/kg) group versus control.

Figure 7

Ultrastructural and histopathological changes of various groups at different times. A: Changes in tumor tissues on the first day after treatment, as viewed under the electron microscope (TEM). B: Histopathological changes in tumor tissues at days 1, 7, and 14 after treatment, as viewed by light microscopy (LM) at 400 × magnification.

Figure 8

Changes in percentages of positively-stained cells for tumor-related factors in the various irreversible electroporation (1000V and 1200V), photodynamic therapy (PDT), and IRE+PDT (1000V+PDT and 1200V+PDT) groups, as determined by immunohistochemical staining. A: Left, VEGF expression; right, tissue changes in the control and 1200V+PDT groups. B: Left, CD31 expression; right, tissue changes in the control and 1200V+PDT groups. C: Left, TGF-β expression; right, tissue changes in the control and 1200V+PDT groups. D: Left, Ki-67 expression; right, tissue changes in the control and 1200V+PDT groups. E: Left, TNF-α expression; right, tissue changes in the control and 1200V+PDT groups. Light microscopy images are shown at 400x magnification. Data represent the mean values from three independent measurements (n=3). *P<0.05 versus control.

Figure 9

Expression pathways of apoptosis-related genes in the different treatment groups. IRE, irreversible electroporation treatment alone; PDT, photodynamic therapy alone; IRE+PDT, treatment with irreversible electroporation and photodynamic therapy.