Experimental Study On Apoptosis And Its Mechanism In Hela Cells Induced By Intense Picosecond Pulsed Electric Field

Targeted non-invasive treatment of tumor is the world's mostpromising area of medical research. The application of pulsed electricfields (PEF) is emerging as a new technique for tumor therapy. Accordingto the pulse duration, PEF can be classified into millisecond (ms),microsecond (s), nanosecond (ns), picosecond, et al. Most researchersfocused on the ms, s and ns pulse range for a more in-depth study.However, the application of ms, s and ns PEF still needs to use theinvasive or minimally invasive needle or plate electrodes, to guide thepuncture of tumor tissue, which to some extent limit the clinical applicationof this method.Picosecond PEF (psPEF) has a wealth of ultra-broadband spectrum,with a high time and spatial resolution, and low signal distortion. It can betransferred to target deep tissue non-invasively and precisely withwideband antennas. But to our knowledge, the research of the biologicaleffect of psPEF on cells remains rare.Electric field possesses parameters related different biophysical effects, that is, the impact of electric field pulses on cells has a certain windoweffect. Ms or s PEF targets outer membrane mainly, and there is littleinfluence to cell nucleus, mitochondrion, and other organelles; thus, itcauses electroporation to the outer membrane. As the pulse durationdecreases, the electroporation effect changes gradually from the outermembrane to intracellular organelle membrane. Submicrosecond PEF caninduce significant voltages across both the inner and outer membranes,therefore, causing damage to both the inner and outer membranes.While these effects of PEF continue to be explored, a new domain ofpulsed electric field interactions with cell structures and functions opens upwhen the pulse duration is reduced to values such that membrane chargingbecomes negligible. For mammalian cells, this holds for a pulse duration ofone nanosecond or less. We dare to assume from the rules above, when theelectrical pulses duration is shorter than one nanosecond, PEF can inducelarger voltage across the inner membrane and acts mostly on intracellularsubstructures. According to cell biology and electromagnetic theory, themitochondrial membrane charging time constant (a few hundreds ofpicoseconds) is much shorter than nuclear membrane and the membranecharging time constant (tens of nanoseconds, and hundreds of nanoseconds,respectively). Under the action of the intense psPEF, the mitochondrialmembrane will charge fast, at a time when the nuclear membrane and themembrane had no chance to respond, so the mitochondria transmembrane potential will be changed. We can speculate that intense psPEF target themitochondria and lead to changes in transmembrane potential, release ofCyt c and AIF etc., activation of caspase9and caspase3, and finallyapoptosis.Cervical cancer is one of the most common gynecologicalmalignancies. Its incidence in young women is increasing in recent years.The traditional surgical treatment often leads to genital tract severe damageand affect patients'sexual function and fertility. Despite of advances insurgical techniques, conservative treatment such as radical trachelectomyhas appeared, it still has a great impact on patients'fertility. Non-invasivetreatment with preserved fertility is the expectation for both doctors andpatients.In this study, we tested the hypothesis that intense psPEF can inducecell apoptosis through mitochondrial path. Human cervical cancer cells,Hela cells were chosen to be exposed to psPEF. This research can not onlyenrich the biological effects of picosecond pulsed field theory, but alsoprovide cervical cancer patients a new non-invasive treatment of preservingfertility.Objective: To investigate the apoptosis-related factors Caspase-3and caspase-9activity and gene expression in HeLa cells after exposed tointense picosecond pulsed electric fields.Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups.6hours and12hours after exposed to intense psPEF, Caspase protein activity kit wasused to test the Caspase-3and Caspase-9activity.2hours and6hours afterexposed to intense psPEF, RT-PCR was used to test the Caspase-3andCaspase-9gene expression.Results: Intense picosecond PEF could significantly enhanced theCaspase-3and Caspase-9activity and gene expression in a dose-dependentmanner, the protein activity increased in parallel with pulse numbers andelectric field amplitude. Each treated group showed significant difference incomparison to the control group (all P<0.05). The caspase activity increased2hours after pulses and could reach a maximum level6hours afterexposed to intense psPEF.Conclusion: psPEF could generate activation of caspase-3andcaspase-9in Hela cells, and the effect was in a dose-dependent manner. Theactivity increased in parallel with pulse numbers and electric fieldamplitude. Objective: To investigate the Mechanism of apoptosis in Hela cellsthrough a mitochondrial mediated pathway induced by intense psPEF.Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups. Aftertreated with psPEF of different doses,[Ca2+]i was marked by Fluo3-AM andanalyzed by laser confocal scanning microscope.2h,6h, and12h afterexposed to intense psPEF, mitochondrial membrane potential was markedwith Rh123and detected by laser confocal scanning microscope.2h,6h afterexposed to intense psPEF, Western blot was used to measure the proteinlevels of Cyt C and AIF.Results:[Ca2+]i elevated significantly shortly after treated with psPEF.Mitochondrial membrane potential declined2hours after pulses, andreached a minimum level6hours after pulses. The protein expression ofCyt C and AIF increased significantly2hours after pulses. The effect wasin a dose-dependent manner. Each treated group showed significantdifference in comparison to the control group (all P<0.05).Conclusion: Intense psPEF could target the mitochondria and lead tochanges in transmembrane potential, release of Cyt C and AIF and inducecells apoptosis through mitochondrial pathway. Objective: To study the effects of intense psPEF on regulation ofapoptosis in Hela cells.Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups.Immunocytochemistry was used to observe the bax and bcl-2proteinexpression after exposed to psPEF.2h,6h after exposed to intense psPEF,the mRNA expression of bax, bcl-2and p53was detected with RT-PCR.Results: Immunocytochemistry of Bax: the cytoplasm of the controlgroup was stained brown, and the treated cells showed the same distribution,but were stained more deeply. The average level of the control group andtreatment group were86.27±9.32and110.75±10.23individually, thedifference was statistically significant (P<0.05). Immunocytochemistry ofBcl-2: the cytoplasm of the control group was stained brown, and treatedcells showed the same distribution, but were stained less deeply. Theaverage level of the control group and treatment group were102.36±10.45and69.45±9.38individually, the difference was statistically significant(P<0.05).The mRNA expression of bax and p53increased significantly2hours after pulses, and bcl-2decreased2hours after pulses. The effect wasin a dose-dependent manner. Each treated group showed significantdifference in comparison to the control group (all P<0.05).Conclusion: Imbalance of Bax and Bcl-2, p53were involved in theprocess of apoptosis induced by intense psPEF.