O Anthony Holland, καθηγητής στο Skidmore College στην Αμερική, είναι ένας πρωτοπόρος ερευνητής που έχει δείξει με διάφορες παρουσιάσεις του ότι τα καρκινικά κύτταρα, όπως και τα διάφορα μικρόβια μπορούν να εξοντωθούν με απλό και αποτελεσματικό τρόπο και μάλιστα χωρίς παρενέργειες με την επίδραση παλμικών ηλεκτρομαγνητικών πεδίων-συχνοτήτων!
Η εταιρεία Novobiotronics Inc. που έχει ιδρύσει είναι μια μη κερδοσκοπική επιστημονική και εκπαιδευτική ερευνητική εταιρεία αφιερωμένη στον εργαστηριακό πειραματισμό σχετικά με τις επιδράσεις των παλμικών ηλεκτρομαγνητικών πεδίων (OPEF) που σχετίζονται με συχνότητες σε καρκινικά κύτταρα και παθογόνους μικροοργανισμούς. Αυτή η τεχνολογία προσφέρει ένα μη τοξικό, μη επεμβατικό και φθηνό μέσο για τη «θραύση» των καρκινικών κυττάρων και την απενεργοποίηση της ανθεκτικότητας στα αντιβιοτικά διαφόρων μικροβίων, όπως η Ψευδομονάδα, ή και άλλων επικίνδυνων παθογόνων μικροβίων που αποτελούν ένα μείζον πρόβλημα για την ιατρική επιστήμη σήμερα.
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CELL FRAGMENTATION AND INHIBITION OF PROLIFERATION OF HUMAN
LEUKEMIA CELLS IN VITRO BY FREQUENCY SPECIFIC AMPLITUDE
MODULATED RF PULSED PLASMAS
Anthony G. Holland, Skidmore College; Novobiotronics Inc.; Saratoga Springs, NY, USA.
Summary of Abstract.
Frequency-specific oscillating pulsed electric fields (OPEF)
Frequency-specific oscillating pulsed electric fields (OPEF)
generated by an amplitude modulated (AM) radio frequency (RF) transmitter utilizing an
enclosed gas plasma antenna have demonstrated an ability in vitro to fragment cancer cells
and inhibit proliferation in two different human leukemia cell lines.
Objectives.
Tumor-Treating (electric) Fields (TTF) technology has been approved in
several countries for clinical treatment of certain types of cancer, but the current apparatus
depends upon external attachment of specialized insulated electrodes placed at
complementary angles to a specific tumor location [1]. Cancers which have no one specific
tumor location, such as leukemia, require a new approach. Our use of an enclosed gas
plasma antenna to project OPEF signals of sufficient strength to significantly negatively
impact cancer cells from a distance of over 60 cm. offers a potential solution.
Methods. OPEF was tested against two cancer cells lines: chronic myelogenous leukemia
K562, [Coriell Institute, catalog No. GM05372] and acute lymphocytic leukemia (ALL)
[Coriell Institute, catalog No. GM03671]. Cell lines were maintained in IMDM media with
10% FBS(K562) or RPMI1640 media with 10% FBS (ALL) in a humidified CO2 incubator
at 37 deg. C.. 1.5 mL of cells in suspension were plated in multiple wells of a 12 well plate
and exposed to OPEF while sitting on the stage of a phase contrast inverted tissue
microscope. OPEF treatment durations varied: 10.5 hours nonstop, 24 hours nonstop, 21
hours (10.5 hours/day for two days), 31.5 hours (10.5 hours/day for 3 days) and 42 hours
(10.5 hours/day for 4 days). Cells which were treated for 10.5 hours/day over multiple days
were returned to the CO2 incubator between treatments. The plasma antenna distance from
the cells varied: 6 inches, 12 inches, 18 inches and 24 inches. Time lapse photomicrographs
were taken every 3 minutes and later combined into time-lapse video sequences. Cells were
assayed before and after OPEF treatments using a hemocytometer and trypan blue exclusion
method. Parallel control experiments were also run for comparison to OPEF treated cells.
Digitally synthesized square waves, duty cycle 70%, were used as control input for the
amplitude modulation (AM) of a 27 MHz carrier signal of an RF transmitter (OM2 from
Plasmasonics Ltd.) where the index of modulation was higher than 1. This ‘over-
modulation’ of the RF carrier results in a pulsed RF output. These pulses are then used to
stimulate an argon/helium/neon gas mixture inside an enclosed glass tube into a plasma
state. Input control frequencies and resultant RF and electric field pulse rates ranged from
A. Holland: BioEm2015, Inhibition of Human Leukemia Cells in Vitrο.
100kHz-212kHz. Electric field strength at 197kHz from a distance of 18 inches (45.72cm)
was measured to be 33V/cm [2].
Results.
OPEF broadcast by an enclosed gas plasma antenna demonstrated an ability at all
distances from the plasma tube to dramatically fragment a significant number of cells of
both types of human leukemia cell lines and to inhibit their proliferation. The greatest effect
was seen after four consecutive days of treatment, 10.5 Hrs/day, with the plasma tube 24
inches away (60.96cm.), where the percent of viable cells was decreased by 61%. Time
lapse microphotography reveals a dramatic cancer cell fragmentation process that begins
within the first few hours of an OPEF treatment that is within an appropriate “frequency and
amplitude window”.
Conclusions & Discussion.
Our experiments confirm earlier work done in 2009-10
(unpublished) with leukemia cell line K562, that OPEF can fragment and inhibit the
proliferation of human leukemia cells in vitro. The effects were seen in two different
leukemic cell lines with the plasma antenna at all distances from the cells (6, 12, 18, 24
inches) but with the greatest effect seen at 24 inches [60.96cm], suggesting an ‘Adey-like’
window for both frequency and amplitude. The most effective frequencies were a sweep
from 196.5kHz-197.5kHz in 50Hz. steps, each frequency for 30 minutes, with the most
effective treatment duration being 10 ½ hours/day over four consecutive days for a total of
42 hours of OPEF treatment.
The use of Tumor Treating (electric) Fields [TTF] has been demonstrated both in vitro and
in vivo to cause an inhibition of proliferation of many types of cancer cells[3][4][5], but
current TTF technology requires a specific tumor location so that TTF electrodes may be
attached at complementary angles to the tumor[1]. Existing TTF technology applied
through electrodes is non-pulsed and has been shown to heat biological tissues[6] raising
concerns about heat shock proteins and other deliterious thermal damage. Our new
approach of OPEF broadcast by an enclosed gas plasma antenna seems to offer several
advantages over currently approved TTF technology: (1) the OPEF pulsed output induces a
nonthermal biological effect [7], (2) current data seem to indicate that OPEF appears to be
more effective in inhibiting the proliferation of cancer cells, (3) OPEF causes cancer cell
fragmentation for a significant number of cells, (4) OPEF may provide a new approach for
cancers which do not have specific tumor locations (are systemic in nature), (5) OPEF has
shown to be effective at distances in excess of 60cm., indicating a potential upward
scalability for whole body clinical treatments, (6) OPEF appears to generate and broadcast
an electric field an order of magnitude greater in amplitude than current TTF technologies
[1][2], thus easily exceeding the minimum field strength needed to negatively impact cancer
cells from a greater distance [8], (7) there is a physiological limit to the amount of power
that may be applied through electrodes/transducers without causing tissue damage at the
point of application. This both limits the depth of penetration and the affected area by the
A. Holland: BioEm2015, Inhibition of Human Leukemia Cells in Vitro
electrode/transducer array [9], (8) electrode/transducer arrays must be placed in specific
locations and the electric field direction regularly alternated at angles perpendicular to the
tumor so as to maximize the number of cell mitotic spindles which can be brought into
alignment with the directional electric field and therefore which can be affected by the
electrode broadcast electric fields [10], but when the pulsed electric field (OPEF) is
broadcast by an enclosed gas plasma antenna, the field is isotropic in nature. This means that
all dividing cancer cells have their mitotic spindles in optimal alignment with the field at all
times. The field broadcast by an enclosed gas plasma antenna is not a compromise as found
with the use of electrodes/transducers [8].
OPEF assays with a hemocytometer offer new challenges because many cells are broken up
into fragments and dispersed while the trypan blue exclusion method counts only whole in-
tact viable and nonviable cells, therefore it seems likely that the number of nonviable cells
caused by OPEF is larger than our current data indicate. Future assay approaches are needed
which can account for cell fragmentation caused by OPEF.
Acknowledgements. Dr. James Bare, Plasma Sonics Ltd.;
Prof. Gérard DuBost, Pr. Emerite Institut d’Electronique et de Télécommunications
de Rennes, Université de Rennes
References
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Steinberg-Shapira S, Gurvich Z, Schneiderman R, Wasserman Y, Salzberg M, Ryffel B,
Goldsher D, Dekel E, Palti Y. “Alternating electric fields arrest cell proliferation in
animal tumor models and human brain tumors”. Proc Natl Acad Sci USA.
2007;104:10152–10157. doi: 10.1073/pnas.0702916104
[2] Dubost G, Bare J, Holland A, Bellossi F.
“Destructive Effects of Pulsed Electric Fields on Cancer Cells: The Microtubules
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European BioElectromagnetics Association BioEM2013 Abstract Collection - Complete
Collection June 10, 2013 - June 14, 2013 Thessaloniki, Greece
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[6] Giladi M, Porat Y, Blatt A, Shmueli E, Wasserman Y, Kirson E, Palti Y.
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[7] Dubost G, Bellossi A. “Thermal and non thermal effects of electromagnetic
fields on bio-systems” January 2012, 221 pages, ISBN 987-1-4710-5249-1
[8] Dubost G, Holland A, Bare J, Bellosi F. “Morphological transformations of
human cancer cells and microtubules caused by frequency specific pulsed electric
fields broadcast by an enclosed gas plasma antenna”. Proceedings-7th International
Workshop on Biological Effects of EMF – October 2012 (Malta) ISBN: 978-99957-
0-361-5
[9] Palti Y. “Apparatus for destroying dividing cells”. United States Patent No. US
7,136,699 B2. Nov. 14, 2006
[10] Kirson E, Gurvich Z, Schneiderman R, Dekel E, Itzhaki A, Wasserman Y,
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