The Experimental Investigation On Picosecond Pulsed Electric Fields Induce Apoptosis In Hela Cells And Its Mechanism

In recent years, the application of pulsed electric field (PEF) inbiomedical fields was given more and more attention from scholars athome and abroad. Previous studies have showed that microsecond andnanosecond pulsed electric fields produced significant killing effects intumor cells. However, the application of ms, μs and ns PEF still needs touse the invasive or minimally invasive needle or plate electrodes, to guidethe puncture of tumor tissue, which to some extent limit the clinicalapplication of this method. Therefore, picosecond pulsed electric fieldscame into being, which can be transferred to target deep tissuenon-invasively and reach the surface of tumor precisely.Matching the impulse radiating antenna (IRA), picosecond pulsedelectric fields with excellent directivity and fine handling, would become anew method in the field of the non-invasive treatment in tumors. IRA canbe carried out energy within the particular scope. It can converge theelectromagnetic pulse on a designated area, or even destroy the target.Radiation energy is concentrated both in time and in space. According tothis feature, picosecond pulsed electric field was transported into the tumortissue by IRA. And the non-invasive treatment of tumors can come true.But the research of the biological effects of psPEF on cells is limited.According to the window effect of pulses electric fields on cells, the electroporation effect of microsecond pulses and the intracellularelectromanipulations effect of nanosecond pulses, the main role ofpicosecond pulsed electric fields should be in the organelles. In addition,previous experiments of our group found that the picosecond pulsedelectric fields cause changes in mitochondrial membrane potential of tumorcells. So, in this study we will further investigate the influence of thepicosecond pulsed electric field on the function of another organelle-endoplasmic reticulum.Cervical carcinoma, the most common malignant tumor of femalereproductive system, is a serious threat to global women’s physical andmental health. Its incidence is gradually getting younger and younger inrecent years. The traditional surgical treatment means the loss of fertilityand affects patients’ sexual function, which is very difficult to accept foryoung patients. Therefore, non-invasive treatment with preserved fertilityhas increasingly become the expectation for both doctors and patients.Although the cervical is not in the surface, it can be fully exposed by asimple method, which provides convenient conditions for the application ofpicosecond pulsed electric field (psPEF).Based on this, human cervical carcinoma HeLa cells were exposed topsPEF to investigate the underlying mechanisms of apoptosis in this study.Treatment with psPEF led to marked cell apoptosis and cell cycle arrest atG2/M phase. In addition, psPEF impacted the phosphorylation levels ofendoplasmic reticulum sensors and up-regulated the expression ofGlucose-regulated protein78(GRP78), Glucose-regulated protein94(GRP94) and C/EBP homologous protein (CHOP). These changes wereaccompanied by the elevation of Ca2+concentrations in intracellular.Furthermore, activation of caspase-12, caspase-9, caspase-3, increasing therelease of cytochrome c and up-regulation of Bax, and down-regulation of Bcl-2were observed in HeLa cells. Taken together, our findings suggestthat psPEF is an efficient apoptosis-inducing agent for HeLa cells, whichexerts its effects, at least partially, via the endoplasmic reticulum stress andcaspase-dependent signaling pathways.Part1Picosecond pulsed electric fields induced apoptosis in HeLa cellsChapter1The apoptosis phenomena of HeLa cells under Picosecondpulsed electric fieldsObjective: To observe the occurrence of apoptosis of HeLa cells aftertreated with picosecond pulsed electric fields.Methods: HeLa cells were divided into control group and fourexperimental groups (different amplitudes of picosecond pulsed electricfields treated groups). The parameters of experimental groups are asfollows: Fixed pulse width800ps, frequency3Hz, the number of pulses2000, three different electric field strength (200kV/cm,400kV/cm,600kV/cm). After treated with pulsed electric fields, the cell was culturedfor12h. Flow cytometry was used to detect changes in the rate of apoptosisand cell cycle; Hoechst33258staining was used to observe thecharacteristic changes in apoptosis; The ultrastructures of cells of thecontrol and experimental groups were observed by transmission electronmicroscopy.Results: After treated with psPEF, the apoptosis rate of theexperimental groups was significantly increased than the control group.The cell cycle distribution was assessed by monitoring intensity of PI fluorescence. Results indicated that psPEF blocked cells cleavage in phaseG2/M of the cell cycle. After treated with different amplitudes of psPEFand cultured for12h, typical apoptotic morphological changes of HeLacells were detected by Hoechst33258staining. The quantity of positivecells of the experimental groups increased significantly compared to thecontrol group. The experimental group cells showed typical apoptoticmorphology and the mitochondria and endoplasmic reticulum swollenunder effect of psPEF.Conclusion: The picosecond pulsed electric fields induced apoptosisof HeLa cells, and the cell cycle was arrested at the G2/M phase.Chapter2Caspase activity in HeLa cells exposed to picosecond pulsedelectric fieldsObjective: To explore the activity of apoptosis-related factorscaspase-12, caspase-9and caspase-3after exposed to picosecond pulsedelectric fields.Methods: After treated with different amplitudes of psPEF andcultured for12h, the total RNA and proteins of HeLa cells was extracted.The level of mRNA of caspase-12, caspase-9and caspase-3was detectedby RT-PCR. And the level of proteins of pro-caspase-12, pro-caspase-9,pro-caspase-3and PARP was detected by Western Blot.Results: Compared with the control group, the level of transcriptionof caspase-12, caspase-9and caspase-3was upregulated in theexperimental groups. And as the field strength increases, the relativemRNA levels of caspase gradually increased. In the levels of protein, theexpression of inactive pro-caspase-12, pro-caspase-9and pro-caspase-3 was reduced with the increase of the field strength. In addition, the activityof PARP (Cleaved PARP,89kDa) significantly increased with the fieldstrength increasing.Conclusion: The caspase cascade of events was activated whenpsPEF induced the apoptosis of HeLa cells. The apoptosis of HeLa cellsunder psPEF is caspase-dependent.Part2Mechanism of apoptosis through the endoplasmic reticulumpathway in Hela cells induced by psPEFObjective: To investigate the mechanism of apoptosis in Hela cellsvia endoplasmic reticulum stress induced by intense psPEF.Methods: After treated with different amplitudes of psPEF andcultured for12h, cells were loaded with the calcium probe Fluo-3-AM andthen examined by immunofluorescence using confocal microscopy andFlow cytometry. The phosphorylation of PERK and eIF2α and theactivation of ATF6were analyzed by Western blot. The expression of themolecular chaperone GRP78and GRP94and the pro-apoptotictranscription factor CHOP was analyzed by Real Time-PCR and Westernblot from the transcriptional and translational levels.Results: The green fluorescence intensity increased after the pulses,which indicated the elevation of Ca2+concentrations. The expression ofGRP78, GRP94and CHOP had a significant increase12h after the pulses,regardless of the level of gene or protein, compared with the control group.p-PERK, p-eIF2α and ATF6fragments increased, whereas their normalforms did not change.Conclusion: After psPEF exposure, PERK and eIF2α were phosphorylated; ATF6was cleaved to the activated form ATF6fragmentation; CHOP has a significant expression up-regulation; thechaperone proteins GRP78and GRP94were up-regulated, which showedthat the apoptosis of HeLa cells under psPEF was related to endoplasmicreticulum stress.Part3Effects of psPEF on Bax, Bcl-2and Cyt c in HeLa cellsObjective: To investigate the effects of psPEF on Bax, Bcl-2and Cytc, which are associated with both endoplasmic reticulum stress andmitochondrial dysfunction.Methods: After treated with different amplitudes of psPEF andcultured for12h, the total RNA and proteins of HeLa cells was extracted.The level of mRNA of Bax and Bcl-2was detected by RT-PCR. And thelevel of proteins of Bax, Bcl-2and Cyt c was detected by Western Blot.Results: Treatment with psPEF resulted in an increase of cytosoliccytochrome c in a dose dependent manner in HeLa cells. RT-PCR andWestern blot analysis revealed that the expression of Bax was up-regulated.In contrast, the expression of Bcl-2was significantly down-regulated.Conclusion: psPEF induced the up-regulation of Bax expression,down-regulation of Bcl-2expression and the movement of Cyt c from themitochondria into the cytoplasm. Combined with the results of previousexperiments, it can be concluded: psPEF induced apoptosis of HeLa cells,which at least partially, by the induction of mitochondrial dysfunction. Twoapoptotic pathways could be linked by Bcl-2. And there may be part of thecollaborative relationship between the two pathway of the mitochondriadysfunction and endoplasmic reticulum stress.