INTRODUCTION
Electric fields with pulse duration of nanoseconds to milliseconds affect the integrity of intracellular organelles and the cellular membrane.[12] Millisecond-pulsed electric fields have been used for gene transfection and cell fusion,[34] because they impact on the cell membrane inducing pore formation (electroporation).[56789] Pores are usually reversible and the opening time depends on the power of electric field applied. This modality is used for chemotherapy to enhance the influx of chemotherapeutic agents into cells, thereby improving the efficacy (i.e., electochemotherapy).[101112]
Electroporation may be irreversible if the power is high enough. When the strength of voltage or the number of pulses reaches a threshold, the membrane is broken down permanently thereby inducing cell death. IRE can be applied to ablate target tissues without affecting the surrounding normal tissue, if used correctly. Therefore, IRE has been considered as a potential therapeutic technique for cancers.[13]
IRE has been shown to have some advantages.[14] It can rapidly create a lesion but promptly resolve it in the tissues; thus, it can ablate the focus of the electrical field completely and reserve blood vessels. IRE can kill malignant cells directly and thereby eliminate the side effects associated with chemotherapy. To date, IRE has been tested in vitro and in vivo.[141516171819] A clinical trial on prostate cancer showed that IRE was as effective at destroying the lesion with fewer side effects, maintaining regional nerve and urethral integrity.[20]
IRE ablation is a non-thermal method to eliminate tumor cells in the body,[20] but the mechanisms remain unclear. To date, researchers in the field have thought that IRE breaks down the lipid bilayer of the membrane to rupture it directly or makes micropores in the surface, which then allows ions and macromolecules from the extracellular stroma entering into cells promoting cell death. High-voltage IRE leads to cell necrosis and thereafter, cellular and tissue debris triggers local or systemic inflammation and immunoreaction. In addition, macro necrosis can induce severe hemorrhage and infection, and these events are not appropriate in anticancer therapy. Nevertheless, low-voltage IRE induces cancer cells to undergo apoptosis rather than necrosis,[2122] which could be a favorable alternative to high-voltage electric fields. But it is unclear if IRE induced by low-voltage can eliminate cancer cells as effective as high-voltage.
In this study, we compared the effects of low- and high-voltage treatments on induction of necrosis and apoptosis in HeLa cells. Our data showed that both low-voltage pulses with more numbers and high-voltage pulse with fewer numbers can efficiently deactivate cancer cells. But the procedure of cell death is different. The adequate combination of these parameters for electric field pulses could ablate malignant tissues partly through apoptosis.
MATERIALS AND METHODS
Human cervical adenocarcinoma HeLa cells (SIBS, China) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from HyClone, USA) at 37°C and 5% CO 2 .
HeLa cells were grown and washed thrice with phosphate buffered saline (PBS, HyClone, USA). Cells were digested with 0.25% Trypsin-EDTA solution (Gibco, USA) and centrifuged at (1000 rpm for 5 min). Cells were then resuspended in PBS to a final concentration of 2 × 106 /mL, 760 μL aliquot was placed in a parallel aluminum plated Gene Pulser Cuvette (Bio-Rad, USA) with 4 mm gaps. Electroporation was then performed on the cell suspension with an electric pulses therapeutic system (State Key Laboratory of Power Transmission Equipment and System Security and New Technology, Chongqing University, China) at a frequency of 1 Hz, voltage of 100 to 2000 V, and the pulse duration of 100 μs. The voltage and wavelength of electric pulses was monitored throughout the procedures with a TDS3032B oscilloscope (Tektronix, USA).
To determine the effects of electric field intensity on HeLa cell viability, we exposed the cells to 8, 100-ms pulses of square wave at a frequency of 1 Hz, with voltages of 100 to 1000 V. Due to the electroporation cuvette with 4 mm gaps, the electric field intensities were calculated using the formula 'E (V/cm) = Voltage/0.4'.
Cell viability was determined with Kit-8 assay (CCK-8; Dojindo Laboratories, Japan). Twenty microliters of the cell suspension after electroporation were added into 80 μL RPMI-1640 and seeded into 96-wells plates. Cells were then incubated for 24 h in a 5% CO 2 humidified incubator at 37 °C. Twenty-four hours later, 10 μL of CCK-8 were added to each well and the plates were then incubated for an additional 2 h. Optical density (OD) was then measured with an ELX800 absorbance microplate reader (Bio-Tek, USA) at a wavelength of 450 nm. The cell viability was calculated relative to the control ([experimental group OD-zeroing OD]/[control group OD-zeroing OD]). The experiments were repeated 3 times with similar results.
Apoptosis was identified by flow cytometry with an apoptosis detection kit (Becton Dickinson). After electroporation, the cell suspensions containing about 8 × 105 cells were centrifuged and resuspended in combined buffer solution and then were double-stained with fluorescein isothiocyanate-labeled-Annexin V (Annexin V-FITC) and propidium iodide (PI). By binding to phosphatidylserine exposed on the cell membrane, Annexin V-FITC is an early indicator of apoptosis, while PI is a viability dye that stains only late apoptotic and necrotic cells. The samples were analyzed with flow cytometry (BD FACSVantage SE, USA). Early apoptotic cells were stained only with Annexin V-FITC, and cells positive for both Annexin V-FITC and PI were considered late apoptotic or necrotic.
Cell morphology, apoptosis, and necrosis were determined by using transmission electron microscopy (TEM). Briefly, the cell suspensions containing about 8 × 106 cells after electroporation were collected and centrifuged at 800 rpm for 5 min and then at 1200 rpm for 10 min. Cells were fixed with 4% glutaraldehyde and 1% osmic acid, embedded in epoxy resin Epon 618. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope (H-7500, Hitachi Japan). 100 cells were sampled for each group.
Total cellular protein was isolated from the cells after electroporation and then subjected to Western blot analysis for caspase-3 (CASP3) and caspase-8 (CASP8) proteins. Briefly, 80 μg of proteins from each treatment were separated in 12% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were then incubated in 5% skim milk solution in Tris-buffered-saline with Tween (TBST) containing 20 mM Tris-HCl, 140 mM NaCl, pH 7.5, and 0.05% Tween 20 for 2 h at room temperature. After being washed with TBST, the membranes were incubated with a monoclonal rabbit anti-CASP3 or anti-CASP8 antibody (diluted 1:200, Abcam) overnight at 4 °C. In the next day, the membranes were washed 3 times with TBST for 10 min each and then incubated with a goat anti-rabbit IgG antibody (diluted 1:2000, Abcam) for 2 h at room temperature. Immunoreactivity was detected with BeyoECL Plus kit (Beyotime, China) and visualized by autoradiography. Anti-β-actin or glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was used as a loading control. The data were analyzed with Quantity One software (Bio-Rad, USA).
Statistical analysis was carried out using SPSS version 11.0 software. The data were presented as the mean ± standard deviation (SD). A P value of < 0.05 was considered statistically significant.
RESULTS
In order to determine the effects of electroporation with different voltages on tumor cell viability, we performed cell viability assay. Specifically, 24 h after electroporation (100V-1000V), cell viability was analyzed by CCK-8 kit. Compared to the control group, the survival rates of HeLa cells exposed to electric fields decreased gradually with increasing voltage [Figure 1]. Viability of Group 4 (400 V) cells was 82.71 ± 12.32%, which was significantly lower than Group 1 (100V; P < 0.05). The survival rate of HeLa cells was lowest at an electric field intensity of 2250 V/cm in Group 9 (900 V). The survival rate of HeLa cells of Group 9 was 62.25 ± 11.17%, indicating that there was no significant difference between Group 9 and Group 10 (1000V; 60.50 ± 12.08%; P > 0.05). Thus, 400 V and 900 V were selected as the standard parameters for low- and high-voltage, respectively.
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Figure 1: HeLa cell viability after exposure to electric fi eld pulses. Cells were grown and exposed to one set of pulses, 8 pulses/set, and 24 h later, a CCK-8 kit was used to evaluate the survival rate of the tumor cells. The 96-well plate was analyzed using a spectrophotometer. The data are summarized as mean ± SD of 5 separate experiments
Next, we investigated whether these two voltages could ablate HeLa cells equally well as the number of pulses increased. When the sets of pulses (8 pulses/set) increased from 1 to 5 (i.e., 8 to 40 pulses in total), the survival rates at 400 V were 79.26 ± 7.43%, 60.52 ± 10.02%, 31.45 ± 2.75%, 17.55 ± 4.71%, 6.35 ± 1.09%, respectively. The survival rates at 900 V were 59.43 ± 4.92%, 5.94 ± 1.83%, 2.20 ± 1.66%, 1.53 ± 1.45%, 1.50 ± 1.54% respectively [Figure 2]. Furthermore, 5 sets of pulses at 400 V could ablate the HeLa cells almost as effectively as 2 sets of pulses at 900 V; both programs could significantly eliminate HeLa cells. The morbidity rates were approximately 94% at 24 h after electroporation.
Figure 2: Viability of HeLa cells after exposure to electric pulses of 400 or 900 V. Tumor cells were grown and exposed to 1, 2, 3, 4 or 5 sets of 400- or 900-V electric pulses, 8 pulses/set. Cell survival was determined by CCK-8 kit after 24 h. The data are summarized as mean ± SD of 5 separate experiments
The data showed that viability of HeLa cells was significantly altered up to 24 h after electroporation, as detected by CCK-8 kit [Figure 3].After treatment with 2 sets of 900 V pulses, the viability of HeLa cells was obviously suppressed within 2 h (the survival rate was 11.21 ± 3.44% at 2 h after electroporation). Four hours after the treatment, the survival rate was near the lowest (5.56 ± 1.37%). However, it was quite different for the group of 5 sets of 400 V, in which cell viability decreased gradually. The loss of cell viability was time-dependent until 12 h after treatment, especially from 6 h to 8 h after electroporation when the HeLa cell survival rate decreased from 40.74 ± 8.25% to 13.69 ± 6.84%.
Figure 3: Time-course of cell viability changes after ablation of HeLa cells by two different treatments. HeLa cells were grown and treated with 5 sets of 400 V pulses (8 pulses/set) or 2 sets of 900 V pulses.The cell viability was determined with a CCK-8 kit at 2, 4, 6, 8, 10, 12 and 24 h post-treatment. The data are summarized as mean ± SD of 5 separate experiments
Necrosis and apoptosis were detected by double staining with Annexin V-FITC and PI following electric pulse treatments and flow cytometric analysis. Figure 4 shows the plots of Annexin V-FITC and PI staining data. The cell viability of both the 400 V and 900 V groups reduced significantly compared to the control group. Electroporation at 400 V induced apoptosis in 21.83 ± 3.47% compared to 2.23 ± 0.22% for the control cells, while electroporation at 900 V resulted in 8.62 ± 4.71% apoptotic cells. In contrast, the necrosis rates of HeLa cells were 50.56 ± 8.77% and 90.69 ± 8.67% after exposure to 400 V and 900 V, respectively. The necrosis rate of the untreated control cells was only 4.87 ± 1.42%. The necrotic and apoptotic ratios in both the 400 V and 900 V groups were significantly different (P < 0.05).
Figure 4: Induction of apoptosis after the administration of electric field pulses to HeLa cells. The tumor cells were grown and exposed to 5 sets of 400-V pulses or 2 sets of 900-V pulses and analyzed by flow cytometry after annexin V and PI staining. (A) Untreated control cells; (B) 5 sets of 400 V pulses; (C) 2 sets of 900 V pulses. Both (-) were considered intact cells; annexin V (+) and PI (-) were considered apoptotic cells; both (+) were considered necrotic cells. The datasheet shows the apoptosis and necrosis rate in each group. Data are summarized as mean ± SD of 4 separate experiments
Cells treated with higher voltage were severely destroyed. Most cells showed incomplete cytoplasmic membranes or the absence of membranes. There were also some fragments of necrotic cells. However, the cells in the lower voltage group were shrunken and pyknotic, at various phases of chromatin condensation and fragmentation within their nuclei. These are distinctive features of apoptosis. Apoptotic bodies were also observed [Figure 5].
Figure 5: Ultrastructure of HeLa cells exposed to pulse treatment. TEM illustrates the effect of irreversible electroporation on HeLa cells 6 h after exposure to pulse treatment. (A) Control cells; (B) Treatment of 5 pulse sets of 400 V: Apoptotic HeLa cell with several apoptotic bodies (#); (C) Treatment of 2 pulse sets of 900 V: HeLa cell cytoplasmic membranes(*) were defi cient and absent
After that, we analyzed the activity of CASP3 and CASP8 after these treatments using Western blot. The lower voltage (400 V) group increased significantly in the activity of both CASP3 and CASP8, evident by the appearance of the cleaved forms of CASP3 and CASP8, compared to that in the control cells and higher voltage (900V) [Figure 6]. The increase was statistically significant (P < 0.05).
Figure 6: Western blot analysis of CASP3 and CASP8 expression. HeLa cells were grown and treated with 400 V or 900 V pulse and 6 h later, the total cellular protein was isolated and subjected to Western blot analysis. (A) Control cells; (B) 900 V pulse; and (C) 400 V pulse treatment. The data are summarized as mean ± SD of 4 separate experiments
DISCUSSION
Exposure of tumor cells to electric pulses with microsecond duration has been considered as a novel therapeutic method. Such electric pulses can induce apoptosis or necrosis by causing the electroporation of the cell membrane, after which the cell collapses and dies.[23] Indeed, our current data demonstrates that the viability of HeLa cells was significantly reduced after application of a single set of pulses. Mild electric fields and few pulses may not induce irreversible electroporation of HeLa cells and not affect cell viability. Pulses of 400 V (an electric field intensity of 1000 V/cm) and higher significantly reduced tumor cell viability compared to the control cells.
A previous study demonstrated that 1000 V/cm (400 V) was the threshold for irreversible electroporation of HeLa cells.[24] But only about 40% of HeLa cells were deactivated after being treated with a single strong electric field pulse of 900 V or 1000 V. The power of the electric field can be enhanced by increasing the voltage or number of pulses. We found that 400 V and 900 V were optimal conditions for further study. Our data found that these two voltage settings were almost equally able to suppress viability of HeLa cells at different numbers of pulses. These optimized voltages and numbers of pulses significantly reduced HeLa cell viability and induced them to undergo either apoptosis or necrosis.
Next, we tried to find out the adequate parameters by applying to HeLa cells 1, 2, 3, 4, or 5 sets of electric pulses, 8 pulses/set. We found that the cell viability of both groups decreased as the number of pulses rose from 1 to 5 sets. However, 24 h after electroporation, there was no significant difference between 5 sets of 400 V pulses and 2 sets of 900 V pulses on HeLa cell viability.. This indicated that we could achieve similar effects in killing HeLa cells using either high-voltage or low-voltage by varying the number of pulses. This coincides with data from a previous study on prostate cancer cells.[25] It now remains to be determined by further study if these two pulse treatments are able to differentially reduce tumor and normal cells.
In addition, the viability of the HeLa cells was judged at different time points after these two treatments. Although the survival rates were equal in 24 h post-treatment, but mechanism of cell death is different. For the treatment parameters of 2 sets of 900 V voltage pulses, most cells died within 2 h, and cell viability decreased maximally by 4 h. These results suggest that lethality to HeLa cells under these conditions is very quick. However, the equal killing effect required approximately 12 h when the treatment was 5 sets of 400 V pulses, and here the cells lost viability gradually, especially from 6 to 8 h. The higher voltage pulses caused severe and irreversible electroporation of cell membranes, which disintegrated or broke into fragments. The lower voltage (400 V) pulses increased membrane permeability gently, causing the loss of cell homeostasis, as intracellular macromolecules and ions were lost and those normally extracellular leaked in. Cell death was thus the result of apoptosis.
In fact, data from the present experiments suggest that milder-voltage electric fields could also induce cancer cell apoptosis. Noriaki applied 200 pulses with an electric field intensity of 75 V/cm to head and neck cancer SCC9 cells, and found that the rate of apoptosis was approximately 6% with induction of CASP3 activity, although under such pulse parameters the inhibition rate of cell viability was only about 20%.[26] Thus, this treatment was not sufficient to eliminate cancer cells. Therefore, in our current study, we varied the number of pulses at lower voltage to determine if HeLa cells could be ablated as effectively as with the high voltage. We then determined the apoptosis rates associated with these treatments.
Apoptosis does not induce inflammation and immunoreaction in the human body and has been used as a cancer therapy strategy. We therefore distinguished between electric field-induced apoptosis and necrosis after electroporation using flow cytometry and TEM. We showed that 6 h after electroporation, almost all of the cells treated with 2 sets of 900 V pulses were necrotic, whereas the HeLa treated with 400 V were a simultaneous mix of alive, necrotic, and apoptotic cells. The ultramicrographs confirmed the presence of many apoptotic cells in the HeLa samples after the 400 V pulse treatments, whereas the plasmalemma of cells in the 900 V-pulse group were altered in integrity, and most cells were disaggregated. We also found an outflow of cytoplasm and organelles. These data indicate that the pathways leading to cell death are definitely distinct between these 2 treatments, i.e., higher voltage with fewer pulses (900 V, 2 sets of pulses) ablate the cells immediately by severely destroying the integrity of the cell membrane, while the lower voltage with more pulses (400 V, 5 sets of pulses) induce apoptosis as well as destruction of the plasmalemma.
Indeed, increase in caspase activity is a hallmark for induction of apoptosis. In the current study, we observed that activities of both CASP3 and CASP8 increased in the low-voltage pulse group compared to the control and the high-voltage pulse group. Activation of CASP8 protein is the first step in the cascade of apoptotic events induced by the cell-surface receptor Fas (APO-1/CD95). When we performed 5 sets of 400 V pulses on HeLa cells, some signals might stimulate Fas at the same time that micropores are formed. After CASP8 was upregulated, apoptosis was triggered following activation of a series of apoptosis-inducing factors such as CASP3. In this study, our experiments are proof of the principle behind IRE-induced apoptosis. Future studies will define the molecular pathways that trigger apoptosis by IRE.
In summary, this study demonstrated differential effects of the low- and high voltage IRE on the Hela cells. The high-voltage with fewer pulses induced irreversibly electroporated effects on HeLa cells but caused necrotic mass in the cells after the treatment. In contrast, the low-voltage IRE with more pulses was a favorable alternative, which induced the tumor cells to undergo apoptosis and eliminated HeLa cells effectively. Thus, the data from this study provided useful information for future translation of IRE into a novel treatment for cervical cancer.
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