Destructive Effects of Pulsed Electric Fields on Cancer Cells: The Microtubules Mechanical Resonance Clue

G. Dubost1; E.J. Bare2; A. Holland3; F. Bellossi4

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1Emeritus Professor University of Rennes I France; 2DC Albuquerque NM USA; 3Associate Professor Skidmore College NY USA; 4ESE Engineer Bordeaux France

Introduction
The anti-proliferative effect of amplitude-modulated frequencies appears to be probably due to the perturbation of the microtubule bundle and mitotic spindle during cell division. Because the axis of cell division is randomly
oriented, only a fraction of the dividing cells is subject to optimal treatment by polarized electric field. Two perpendicular fields are to be 20% more effective than a single directional [1]. Our special confined plasma antenna,
called “phanotron”, coupled to an electronic device, overcomes this limitation since the plasma antenna acts as an isotropic and randomly polarised antenna.

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Frequency specific, specially tuned, amplitude modulated radio frequency fields utilising the phanotron, showed dramatic disruptive effects on the morphology of human cancer cells in vitro, following the Pulsed Emission
Field Treatment (PEFT) protocol defined in a recent book ([2] chapter 4.2.1, p. 81).
Based on the phanotron model and on the results of the PEFT and Tumor Treating Fields (TTF) protocols, the present paper explains the destruction of the cancer cells by non thermal pulsed electric fields.

Material

The confined plasma antenna: Characterization of the electric and magnetic fields generated by In vitro experimental set-up to expose cancer cells to Pulsed

Square wave
generator
f
the confined plasma antenna (phanotron)

Transmitter
(27.12 MHz)
13
Plasma antenna
Phanotron f (kHz) 100 120 140 150 170 200
Emission Field treatment (PEFT) protocol

Power Antenna tuner P (dBm) -67 -66 -66 -66 -65 -64
amplifier with balun

The confined plasma antenna (phanotron), filled with

NO
B (T)

Pd (mW/cm2)
2.4 10-6 2.2 10-6 1.9 10-6 1.8 10-6 1.7 10-6 1.7 10-6

1.8 10-7 2.2 10-7 2.2 10-7 2.2 10-7 2.8 10-7 3.5 10-7
helium gas, is fed by a 27.12 MHz carrier signal over- Table 1: P is the power measured with the spectrum analyzer at the
modulated with square pulses in the frequency range distance from the phanotron sphere center r = 5 cm; B is the measured
of 100 to 200 kHz. induction magnetic field radiated by the phanotron and Pd is the power
density at r = 5 cm. B and Pd vary following the law 1/r2.
VO
f (kHz) 100 120 140 150 170 200

EP (V/m) 9.2 103 7.2 103 5.3 103 4.6 103 4.0 103 3.3 103 The plasma tube is located near the stage of an inverted research
microscope with a high-resolution video camera.
Table 2: The deduced pulsed electric field EP in terms of the frequency
radiated in air medium at r = 45 cm. It varies following the law 1/r3. 96-well plates of human cancer cells are set on the microscope stage,
exposed to the PEFT and the results are photographed and videotaped
in real time. The distance from the phanotron electrodes to the cancer
Measurement of the phanotron radiated power cells is 45 cm for all experiments.
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Method – Theoretical model Results
A microtubule is a hollow cylinder of about 24 nm in diameter and 1 to 50 µm in length, polarized, and is The polar microtubules emanate from the two centrosomes during the metaphase and overlay to each
compared to a cylindrical rod fixed at both ends. Mechanical resonance of the microtubule is other in the centre, pushing against each other, forcing the centrosomes toward the poles of the cell.
considered together with the energy accumulation over exposure time. They can then reach a size similar to the cell size. Considering the in vitro results obtained according to
the PEFT protocol (§ 4.2.1, p.82 of [2]), the destruction of different cancer cells by a pulsed electric field
Microtubule polarization: A microtubule is composed of 13 protofilaments made of tubulin proteins.
of a few V/cm is obtained with modulation frequency fr between 100 and 200 kHz.
Each protein is equivalent to a dipole with an electric charge of 36 elementary charges:
QP = ± 36e = ±5.8 10-18 Coulombs. The 13 protofilaments of the microtubule are equivalent to a dipole From equation (5) and taking into account a Young Modulus EY = 6 107 Pa at 20 °C [3], we can deduce
supporting 468 elementary charges i.e. Q = 7.5 10 -17 C (1). that the transverse vibration has destructive effect on polar microtubules being 8.6 to 6.1 µm long.

The static electric field ES along the microtubule is equal to (2), the opposed longitudinal static forces FS The static electric field ES (2) is then comprised between 9.1 and 18 V/cm. The static pressure PS (4)
TR
applied at its ends is given in (3) and the static pressure PS in (4): varies between 136 and 270 Pa and the static strength FS (3) between 6.8 10-14 and 1.3 10-13 N. The
pulsed electric field EP is comprised between 92 and 33 V/cm (Table 2).
ES = 6.75 10-8 / L2 (2) FS = 5 10-24 / L2 (3) PS = 10-8 / L2 (4) in SI units.
With a mean value Pd = 3 10-8 W/m² between 100 and 200 kHz extracted from Table 1 for r = 45 cm,
Microtubule resonance frequency: The transverse vibration of the polar microtubule firmly fixed at its gt =0.42 10-6 s and EY = 6 107 Pa (20° C), we deduce from (12):
both ends has a resonance frequency equal to: fr  9.5 10 10 EY L2 (5 ) Wa  6 10 28 i  fr  Dt i (13 ) in SI units.
where EY is the Young modulus of the microtubule as measured in [3].

Microtubule absorbed energy:

Cancer Resonance Pulsed Electric Exposure time Microtubule Aborbed Energy
The microtubule absorption area sM is given in ([2] § 6.3.3): sM = sP. NP = 1.75 L (6) in SI units, 10-11
ON

cells frequency (fr)i Field EP Dti length L Wa (computed)

where sP is the protein absorption and Np the number of proteins in the microtubule of length L with: (computed) (9) (13)
[4], [5], [6] (measured) (measured–Table 2) (measured)
s P  8QP2  t P  Z0 MP (7) and NP = 13 L / DP (8)
114 kHz 80 V/cm 1.8 104 s 8.0 µm
with tP being the protein relaxation time, MP the mass of the protein, DP the diameter of the protein, and Z0 Ovarian 3.6 10-21 J
150 kHz 46 V/cm 1.1 103 s 7.0 µm
the vacuum medium impedance.
150 kHz 46 V/cm 7.2 103 s 7.0 µm
The energy absorbed by the microtubule during one phanotron pulse is equal to: wa = sM .gt .Pd (9), Pancreatic 1.7 10-21 J
158 kHz 43 V/cm 2.9 104 s 6.8 µm
gt is the sum of the rise and fall times of one pulse and Pd the incident power density.
Leukemia 197 kHz 22 V/cm 3.6 104 s 6.1 µm 9.6 10-21 J
From (5) and (6) we deduce the microtubule absorption area sM as a function of the transverse resonance
Table 3: PEFT protocol in vitro results at 20°C
frequency: s M  5.4 10 16 EY1/ 4 fr1/ 2 (10) in SI units.
The accumulated energy is close to the thermal energy kT equal to 4.04 10-21 J, at 20°C. The
IC

With (9) and (10), we determine the energy absorbed by the microtubule at the frequency resonance (fr)i
during a time (Δt)i:  
Wa i  w a  fr  Dt i  5.4 10 16 g t  Pd  EY1/ 4  fr1/ 2  Dt i (11)
microtubule dimensions are similar to the dimensions of the cells. As an example cells radiuses of 9 and
5 µm correspond approximately to those of melanoma and glioma respectively at frequencies of 100
The whole energy accumulated by the microtubule during the therapy PETF is: and 200 kHz [7].

Wa  5.4 10 16 g t  Pd  EY1/ 4  i fr1/ 2  Dt  i (12)

Conclusion
SI

The hypothesis related to the in vitro destruction of different cancer cells caused by energy accumulation in their polar microtubules illuminated by an electric field of a few V/cm at frequencies between 100 and 200 kHz
equal to their transverse resonance frequencies is in line with the experimental results. These frequencies depend on the length of the microtubule which depends on the cell size and on the Young Modulus of
the microtubule which varies with the temperature. Hence in vivo frequencies at 37°C are lower than in vitro frequencies (20°C). Centrosomes are frequently altered in many types of human tumours cancer cells. These
alterations can modify the transversal resonance frequencies of polar microtubules which depend on the setting quality upon the centrosomes. The adequacy between the theory and the experimental results opens a
promising way to a better understanding of the potential destructive effects of low intensity electric fields on cancer cells.
Action of lower frequencies (100 Hz to 21 kHz), experimented through the decrease of the growth of hepatocellular carcinoma [8], could complement the protocol: due to their short length (1 µm < L < 4 µm), astral
microtubules undergo longitudinal resonance and depolarization at frequencies 117 Hz < fr < 7.5 kHz according to the model developed in ([2], §6.3.2), together with a high static pressure (625 Pa < PS < 104 Pa).
More detailed consideration on specificities of cancer cells, to be supported by further tests, will enable refining the model and optimising the efficiency of the PEFT which has the unique benefit of the phanotron acting as
NC

an isotropic and randomly polarised antenna.

Acknowledgements The authors thank the Professor Jean Dupuy for the continuous interest for our research and the encouragements he bestowed for many years.

References
[1] Eilon D. Kirson, Vladimir Dbaly et al. “Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors”. Proc Natl Acad Sci USA 2007, 104(24): 10152-10157.
[2] G. Dubost, A. Bellossi. “Thermal and non thermal effect of electromagnetic fields on bio- systems”. January 2012, 221 pages, ISBN 987-1-4710-5249-1, available on Amazon.fr
[3] A. Kis, S. Kasas et al “Nanomechanics of microtubules” Physical Revue Letters, Vo. 89 number 24, 9 Dec.2002.
[4] Ovarian 081210_9D copy2 http://novobiotronics.com/CancerResults9.htm
[5] March 3_2010 PancreaticClose1E copy2 http://novobiotronics.com/CancerResults11.html
.

[6] March 10_2010 LeukemiaCloseup 2Dcopy http://novobiotronics.com/CancerResults8.html

[7] “Relation of the Optimal TTField Frequency to Cell Size”. Link: http://www.pnas.org/content/suppl/2007/05/30/0702916104.DC1#A1
[8] JW Zimmerman, MJ Pennison et al. “Cancer cell proliferation is inhibited by specific modulation frequencies”. British Journal of Cancer (2012) 106, 307 - 313