Low electric field parameters required to induce death of cancer cells

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1 ISSN: (print), (electronic) Electromagn Biol Med, 2014; 33(2): ! 2014 Informa Healthcare USA, Inc. DOI: / ORIGINAL ARTICLE Low electric field parameters required to induce death of cancer cells Mamdouh M. Shawki 1 and Adel Farid 2 1 Bio-Medical Physics Department and 2 Cell Culture Unit, Medical Technology Centre, Medical Research Institute, Alexandria University, Alexandria, Egypt Abstract Irreversible electroporation (IRE) is a novel technique that deals with killing undesirable cells, mainly cancer cells, directly without using any cytotoxic drugs. Commonly in this technique very high electric field up to 1000 V/cm is used but for very short exposure time (nanoseconds). Low electric fields (LEFs) are used before to internalize molecules and drugs inside the cells (electroendocytosis) but mainly not in killing the cells. The aim of this work is to determine the ability of using LEFs to kill cancer cells (Hela cells). The Physics idea is in making LEFs energy equivalent to IRE energy. Four IRE protocols were selected to represent very high, high, moderate and mild voltages IRE, then we make equivalent energy for each of these protocols using different LEFs parameters of different amplitudes (7, 10, 14 and 20 V), different pulse numbers (40, 80, 160 and 320 pulses), different frequencies from 0.5 to Hz and different pulse widths from 9.38 to 2000 ms. Each of the calculated LEF equivalent to IRE was applied on Hela cell line. The results show complete destruction of the cancer cells for all the tested exposure protocols. This damage was not due to thermal effect because the measured temperature was not changed before and after the exposure. The possible effect mechanism is discussed. It was concluded that the lethal effect on the cancer cells can be achieved using LEFs if the same energy equivalent to IRE is used. This work will help in using low-risk drug-free techniques in cancer treatment. Introduction Killing cancer cells with the aid of electric field has been developed in the previous years. The external electric field to which the tissue is exposed is the primary parameter affecting the transmembrane potential (TMP), the potential difference across the plasma membrane, and induces instabilities in the polarized cell membrane lipid bilayer (Davalos et al., 2005). As a function of the TMP, the pulse can either have no effect on the cell membrane (Rubinsky, 2007), reversibly open the cell membrane after which cells can survive (reversible electroporation) (Sersa et al., 2003) or irreversibly open the cell membrane, after which the cells die (irreversible electroporation) (Arena et al., 2011). The induced increase in the TMP is dependent on the electric pulse (e.g. strength, duration, repetition rate, shape and number) and physical configuration of the electrodes used to deliver the pulses (Garcia et al., 2011). Reversible electroporation has shown great promise in biotechnology and in medicine as a cancer therapy when combined with chemotherapeutic agents Address correspondence to Dr. Mamdouh M. Shawki, Bio-Medical Physics Department, Medical Research Institute, Alexandria University, Alexandria, Egypt. Tel: Fax: mamdouh971@hotmail.com Keywords Equivalent electric energy, Hela cell line, irreversible electroporation, low electric field History Received 24 December 2012 Accepted 14 April 2013 Published online 31 May 2013 (Electrochemotherapy) (Gehl et al., 2011; Mir, 2001) or plasmid DNA (Electrogenetherapy) (Daud et al., 2008). Irreversible electroporation (IRE) results if excess current is applied; the unstable membrane then alters its shape, forming aqueous pathways that possibly are nanoscale pores through the membrane that lead to necrotic cell death (Axel et al., 2007). IRE is a new technique for the focal ablation of undesirable tissue using high-voltage, short duration electric pulses (Davalos et al., 2005). IRE has recently emerged as a non-thermal treatment modality to destroy tumors and other non-cancerous pathologies (Heish et al., 2011). Exposure of cells to short, low voltage electric fields (LVEF) in the range of V has been shown to lead to electric field-driven enhancement of adsorption of macromolecular solutes of different chemical nature to the cell membrane. The intermediate domain of low electric fields, which may lead to the induction of TMP alteration in the range of mv, has been routinely applied in electrophysiological studies of ion transport through channels, and entrance of external solutes to the cell through electroendocytosis mechanism (Antov et al., 2005). Previous studies reported that a moderate electric field induced apoptosis (controlled cell death) (Matsuki et al., 2008). Furthermore, it was previously demonstrated that a relatively low-intensity electric field, with a membrane potential approximately half that of the membrane 2014

2 160 M. M. Shawki and A. Farid Electromagn Biol Med, 2014; 33(2): breakdown threshold of mammalian cells, induced a higher apoptosis ratio (apoptosis/necrosis) than high-intensity electric fields (Matsuki et al., 2010). The main purpose of using LVEF was to induce endocytosis to incorporate molecules to the cell without killing the cells, but cells death percentage was reported as a side effect; this percentage varied from one study to another (Entin et al., 2003). So, there is a threshold obtained at certain electrical parameters to change mechanism of action from induced endocytosis to induced apoptosis. And despite the mechanism of cell death from necrosis in high-voltage IRE to apoptosis in cell death by LVEF, the common electric parameter that can be used to achieve the cell death effect is to make equivalent pulse energy. Similar outcomes of the experiment can also be obtained by using equivalent pulse parameters. Indeed this idea was used in high-voltage electroporation; different high-voltages electroporation protocols are used as instead of using a number of short duration, high-voltage pulses, one can either use longer pulses with lower voltages or adjust the number of pulses by keeping the amplitude or duration of the pulses unchanged (Pucihar et al., 2011). The aim of this article is to determine the electric parameters (pulse amplitudes, pulse numbers and frequency) required to surpass the electroendocytosis threshold to reach to apoptotic conditions for low-voltage range, which was used before in electroendocytosis researches (7 20 V) (Antov et al., 2005; Entin et al., 2003) depending on making equivalent pulse energy between LVEF and IRE parameters. However, finding a suitable combination of pulse parameters proved to be a difficult task because simple relations, such as keeping the same energy of the pulses, turned out to be insufficient (Canatella et al., 2001; Rols and Teissíe, 1998). For this we could not adopt a certain IRE protocol; instead we made LVEF pulse protocols equivalent for four IRE protocols as mentioned below in theory and calculation section to find out the optimum parameters. Theory and calculation There are popular energy equivalent IRE protocols that were used in previous researches to kill cancer cells, with approximately the same effect. Very high-voltage IRE as using 10 KV, high-voltages IRE as using 1000 V, moderate high-voltages IRE as using 575 V and mild pulse amplitude IRE as using of 100 V were commonly used. These Four IRE protocols have the same equivalent electric energy by controlling the exposure time (pulse width) and the number of pulses (Pucihar et al., 2011). To make low-voltage pulse parameters to induce cell death, the idea is to make LVEF protocols equivalent to those IRE protocols. To perform this idea, we depended on the principle of the energy dissipation from the pulses which can be calculated as: E ¼ PNt ð1þ where P is Pulse power, N is the number of pulses and t is the pulse duration time. Substituting P ¼ V 2 =R ð2þ where V is the pulse amplitude (in volts) and R is the resistance of the medium (in Ohms), E ¼ V 2 =R N t ð3þ If we want to make equivalent energy of IRE parameters and LVEF parameters, V 2 h =R Nh t h ¼ V 2 l =R Nl t l ð4þ Here, V h, N h and t h are pulse height, number of pulses and pulse width for IRE, respectively, while V l, N l and t l are pulse height, number of pulses and pulse width for LVEF, respectively. R is constant in both cases. So, V 2 h N h t h ¼ V 2 l N l t l ð5þ Depending on Equation (5) equivalent LVEF and IRE protocols can be made by controlling pulse width and number of pulses. At pulse duty cycle of approximately 100%, t ¼ 1=F where F is the frequency in Hertz, V 2 h N h =Fh ¼ V 2 l N l =Fl ð7þ ð6þ Using Equation (7), equivalent LVEF and IRE protocols can be made depending on the pulse frequencies and number of pulses (Tables 1 4). Table 1. LVEF parameters equivalent to very high IRE voltages parameters (10 KV/cm, 150 ns, 80 pulses). 7 V 1.67 Hz 3.33 Hz 6.67 Hz Hz 600 ms 300 ms 150 ms 75 ms 10 V 3.33 Hz 6.67 Hz Hz Hz 300 ms 150 ms 75 ms 37.5 ms 14 V 6.67 Hz Hz Hz Hz 150 ms 75 ms 37.5 ms ms 20 V Hz Hz Hz Hz 75 ms 37.5 ms ms 9.38 ms Table 2. LVEF parameters equivalent to mild IRE voltages parameters (100 V/cm, 24 ms, 8 pulses). 7 V 1.04 Hz 2.08 Hz 4.17 Hz 8.34 Hz 960 ms 480 ms 240 ms 120 ms 10 V 2.08 Hz 4.17 Hz 8.33 Hz Hz 480 ms 240 ms 120 ms 60 ms 14 V 4.17 Hz 8.33 Hz Hz Hz 240 ms 120 ms 60 ms 30 ms 20 V 8.33 Hz Hz Hz Hz 120 ms 60 ms 30 ms 15 ms

3 DOI: / Low electric field parameters required to induce death of cancer cells 161 Table 3. LVEF parameters equivalent to moderate high IRE voltages parameters (575 V/cm, 100 ms, 80 pulses). 7 V 0.75 Hz 1.49 Hz 2.99 Hz 6 Hz 1340 ms 670 ms 335 ms ms 10 V 1.49 Hz 2.99 Hz 5.97 Hz Hz 670 ms 335 ms ms ms 14 V 2.99 Hz 5.97 Hz Hz Hz 335 ms ms ms ms 20 V 5.97 Hz Hz Hz 47.9 Hz ms ms ms ms Table 4. LVEF parameters equivalent to high IRE voltages parameters (1000 V/cm, 50 ms, 80 pulses). 7 V 0.5 Hz 1 Hz 2 Hz 4 Hz 2000 ms 1000 ms 500 ms 250 ms 10 V 1 Hz 2 Hz 4 Hz 8 Hz 1000 ms 500 ms 250 ms 125 ms 14 V 2 Hz 4 Hz 8 Hz 16 Hz 500 ms 250 ms 125 ms 62.5 ms 20 V 4 Hz 8 Hz 16 Hz 32 Hz 250 ms 125 ms 62.5 ms ms Materials and methods Cell culture Hela cell line were revived from frozen storage and cultured at 37 C under 7% CO 2 in low glucose medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin/ glutamate. After 2 days incubation of Hela cell line in 2 T-75 flasks, cells were trypsinized, then counted and viability was measured using trypan blue. The total cells count was 5x10 6 and the viability was 98%. The whole pellet was resuspended in 50 ml phosphate buffer saline, then divided into 50 plastic tubes (Rahbari et al., 2009). Exposure of cells to LVEF The exposure was performed in a plastic tube by placing the cell suspension between two rings Ag/AgCl electrodes of radius 6 mm. Unipolar square pulses were applied to the cells through digital function generator (CALTEK, CA1640P-02 function generator/counter, serial number:06mg0676, made in USA). The distance between the electrodes was kept at 1 cm. Each LVEF protocol mentioned in the Theory and Calculation section (48 electric field parameter combinations; 16 in each protocol) was applied. Viability test After protocol application, cell viability was determined by mixing 10 ml of cells from the tubes with 90 ml of Trypan Blue solution. The mixture was placed in a hemocytometer under a contrast light microscope. Trypan Blue can only penetrate dead cells, so cells that were dyed blue are dead. Viability percent is calculated by using the following formula (Masters, 2000): Figure 1. Example Control 1 (Live cells in hemocytometer under light microscope, X ¼ 150). Figure 2. Example Control 2 (Live cells in hemocytometer under light microscope, X ¼ 150). Some cells are adjacent in couples or triplets. % viability ¼ ðlive cell count=total cell countþ100 ð8þ Temperature measurement The temperature for each of the examined exposure protocol was measured in separate pilot study before and immediately after the exposure using digital thermometer (made in China). The measurements were repeated three times for each sample. Averages and standard deviations were calculated for each sample. Paired t-test was used to determine the difference significance; a value of p50.05 was considered significant. Results Hundred percent cell death was obtained using all the previously mentioned electric field exposures. Figures 1 and 2 show examples of control cells which are live and maintain their normal shape. Figures 3 5 show examples of the exposed cells; complete lysis and destruction with loosing the normal shape of all the cells are achieved.

4 162 M. M. Shawki and A. Farid Electromagn Biol Med, 2014; 33(2): There was no significant temperature difference in the cell suspensions before and after exposure to different electric field protocols. Figure 3. Example cells exposed to electric field protocol 1 (Destroyed Figure 4. Example cells exposed to electric field protocol 2 (Destroyed Figure 5. Example cells exposed to electric field protocol 3 (Destroyed Discussion Nowadays the science tries to reach drug-free techniques and for that IRE using high-voltage, very short duration pulses is being used. Besides the advantages of high voltage electrochemotherapy, there are also some disadvantages of it. The most important unpleasant sensation of electrochemotherapy is muscle contraction as well as slight edema or erythema. Edema results from high local current density (Mir et al., 2006; Pucihar et al., 2002; Sersa et al., 2008). Pain can be avoided by lifting the treated tumor nodule while applying electric pulses, by increasing the repetition frequency of electric pulses above the tetanic frequency, or by using low-voltage pulses. In this article, IRE technique has been developed in order to use low-voltage pulse amplitude. For the purpose of using LVEF to induce cell apoptosis, many electric parameters were examined that the used LVEF pulses of 7 20 V, with pulse number from 40 to 320 pulses, with frequency from 0.5 to Hz and pulse width from 9.38 to 2000 ms. These parameters were calculated to produce electrical energy equivalent to the popular used IRE parameters. This technique can be used after some development to have a treatment for cancer without the need of high technology methods. This method is also cheap, drug-free, and has minimum occupational risk. Antov et al. (2005) state that the linear decline of viability of cells exposed to LEF probably reflects cytotoxic effects of electrolysis products. The amount of these products is expected to be proportional to the total current (charge) passing through the sample. Viability of COS 5 7 cells (fibroblast-like cells derived from African green monkey kidney), exposed to LEF of 20 V/cm decreased linearly down to 85% upon lengthening pulse duration to 250 ms. At the same time, viability of cells decreased down to 73% when electric field strength increased to 43 V/cm. Shankayi and Firoozabadi (2011) investigated the effect of using a high repetition frequency electric pulses (5 khz) with different low amplitudes of electrochemotherapy (70, 100 and 150 V/cm) in the treatment of invasive ductal carcinoma tumors using bleomycin as a cytotoxic drug and to examine the possibility of using this suggested protocol compared to currently used standard and clinical protocols (1000 V/cm, a 1 Hz electric pulse repetition frequency and 5 khz electric pulse repetition frequency). Their results showed that the equivalent protocol is successful. Matsuki et al. demonstrated that apoptosis increased in accordance with the duration and number of LVEF in B16 cells. They showed that LVEF-induced apoptosis was achieved through caspase-8 and caspase-9 activation and subsequent caspase-3 activation. Caspase-3 is a key enzyme in the caspase induced apoptotic pathway, it is the main downstream effecter caspase that is activated by the initiator caspases, caspase-8 of the cell death receptor pathway and caspase-9 of the mitochondrial pathway, leading to the cleavage of a number of cellular substrates leading to apoptosis (Matsuki et al., 2008, 2010).

5 DOI: / Low electric field parameters required to induce death of cancer cells 163 Temperature change DT was determined from the assumption that electrical energy is transformed into heat completely (Pucihar et al., 2011) as in the following equation: DT ¼ VItN=vc ð9þ Here, V is the amplitude of the pulse, I is the current through cell suspension, t is the pulse duration, N is the number of pulses, is the specific density of the medium ( ¼ 1000 kg/m 3 ), v is the volume of the medium (v ¼ 1 ml) and c is the specific heat capacity of the medium. Equation (9) can be rewritten as follows: DT ¼ V 2 tn=vcr ð10þ Comparing DT induced through IRE through high voltage (DT h ) with that induced through low voltage (DT l ), we can find that: V 2 h t hn h =vcr ¼ V 2 l t ln l =vcr ð11þ So, V 2 h t hn h ¼ V 2 l t ln l ð12þ We can see that Equations (12) and (5) are the same, so DT will remain constant and no heat induction can be expected when using LVEF energy equivalent to IRE protocol. Practically we find no significant increase in the cell suspension temperature which agrees with the theoretical calculation. Conclusions Using low electric field (pulses with low volt amplitude and wider pulse width) gives the same lethal biological effect obtained when using pulses with high-voltage exposure with short exposure time. The method of using the equivalent energy will facilitate the using of electric field in many biomedical applications without high technology need. More studies in this area can lead to great advances in low technology drug-free cancer treatment. Fractionation of the total energy dose used in this article will be our next target to know the LD50 (lethal dose of the electric field which can lead to the death of 50% of the cells). Taking the sight toward the killing effect of low electric field which is nonionizing energy will have great effect in the field of biology and medicine. Declaration of interest The authors report no conflicts of interest. 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