vivek

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Effect of pulse field electromagnetism in enhancing the effect of
chemotherapeutic drugs Methotrexate and 5’-Fluorouracil on
THP-1 cell line
Abstract:
Pulsating electromagnetic field (PEMF) has become an indispensable tool that has a wide
variety of usage. One of its uses is to stimulate the cells to uptake the drugs, in the present
study low frequency PEMF (40Hz at 1.5mT magnetic flux) was used to stimulate growth of
THP-1 cells with and without anti-metabolite drugs like Methotrexate and 5’-Fuorouracil. It
was proved that the HEPES under PEMF stimulation significantly (p = 0.0001 and 0.0012)
decreased the THP-1 cell viability under both 24hrs exposure and the 2hr alternate pulses of
electromagnetic field. However under the normal conditions of PEMF treatment prolonged
i.e. 24hrs exposure significantly induced growth signals in the THP-1 cells compared to the
2hr alternate pulse exposed THP-1 cells (p = 0.19). Constant exposure of THP-1 cells to
PEMF in the presence of 5 & 20µg/ml Methotrexate (p =1.42E-07 & p = 1.78E-07) and 5’-
Fluorouracil (p = 1.9E-08 & p = 1.13E-06) drastically killed or reduced the protein levels.
NaOH and NH4OH used for dissolving Methotrexate and 5’Fluorouracil had a mere effect on
THP-1 cell cytotoxicity. Finally 5’Fluorouracil was shown to be more cytotoxic than
Methotrexate.
Keywords: PEMF, Magnetic Flux, HEPES, THP-1, 5’-Fluorouracil and Methotrexate

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1.0 Introduction:
There was an exponential change in the field of diagnostics and the type of diseases that
being diagnosed by the use of latest or current 21st century technology. One of the major
diseases that kill nearly 7.9 million people around the globe is cancer (CRUK-2009). It is the
second largest causes of death in the western world after the heart disease. It is estimated that
by 2030 there will be a staggering 12million people dying out of cancer. A lot of money is
invested in the cancer research field for the sake of drug development and cancer eradication.
Cancer can be simply defined as an abnormal growth of cells which tend to proliferate in an
uncontrolled way and metastasize to other parts or organs of the body. Cancer is not one
disease. It is a group of more than 100 different and distinctive diseases. Cancer can involve
any tissue of the body and have many different forms in each body area. Most cancers are
named for the type of cell or organ in which they start. If a cancer spreads (metastasizes), the
new tumour bears the same name as the original (primary) tumour. Cancer is a highly
heterogeneous disease; each cell in a tumour lump is distinct and has its own genetic and
expression profile. The exact reason for cancer occurrence in unknown it’s simply a disease
caused due to multiple factors (age, familial, environment, sex etc...). There are many drugs
for treating cancer but none of them can permanently diffuse the tumour to a base level. The
major treatment strategies followed by the physicians are Chemotherapy, Surgery and
radiotherapy. Each strategy will be used basing on the stage, location and type of cancer.
Nowadays combinational therapy has become a trend to treat cancer. Besides these
approaches there were many advanced combinational approaches that are used to treat
cancers. One of them is Pulsating electromagnetic field (PEMF) usage to enhance the drug
diffusion capability in to the cancer cells. Therapeutic effects of electromagnetic fields at a
broad range of frequencies were used to activate different metabolic processes depending on
the tissue type. Initially differential frequency EMF is used to treat endocrine related
disorders (Bersani et al., 1997). Later on the optimisation of EMF frequencies and their
characterisation lead to their extensive usage in the field of medical therapeutics. Low
frequency EMFs are used for heart etc... The mode of PEMF interaction might occur outside
the cell membrane, but it may involve interactions with cell membrane or trans-membrane
proteins (Berton et al., 1993). This phenomenon was recently demonstrated in the NIH3T3
cells at 50Hz PEMF signal for greater than 2hrs increased the clustering of trans-membrane
proteins (Luben et al., 1982). Due this clustering a chain of intracellular signals are evoked by
G proteins leading to the enhancement of the growth signal. This amplification of

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intracellular signals aids TGF-b signalling to induce growth responsive stimulus (Lohmann et
al., 2003). This was also demonstrated invivo in wounded patients. Other in vitro studies
have shown that PEMF exerts a reproducible osteogenic effect on rat osteoblasts and induces
transcription of bone morphogenetic protein (BMP)-2 and BMP-4 mRNA (Bodamyali et al.,
1998). Combined treatment of BMP-2 and PEMF had additive effects on osteoblastic cell
proliferation and differentiation (Selvamurugan et al., 2007). Very recently, an ample review
appeared regarding microvasculature and SMF, PEMF and EMF (McKay et al., 2007).
Sontag and Dertinger (1998) investigated the liberation of PGE2 (prostaglandin E2) of human
granulocytes (HL – 60 g) during application of EMF of different frequencies: they found
“windows” at 6 and 16 Hz where PGE was 200% above 0 Hz baseline and beneath these
“windows” (e.g. at 10 Hz) PGE was only slightly above the baseline. PEMF can influence the
movement of whole organisms during development. Komazaki and Takano (2007) examined
the influence of EMF on early development of the amphibian embryo. When embryos
developed under the influence of a low-frequency PEMF (50 Hz, 5–30 mT) the rate of early
development was accelerated. EMF effects were preferentially exerted at the gastrula stage,
as the period of gastrulation was shortened. Histological observations showed that EMF
promoted morphogenetic cell movements during gastrulation. PEMF specifically increased
the intracellular calcium concentration of gastrula cells, thereby accelerating the rate of
morphogenetic cell movements. PEMF also acts directly on cell differentiation when coupled
to Ca signalling. Lisi et al. (2006) found that exposure to a 50 Hz ELF EMF (magnetic flux
density of 2 mT) promotes differentiation of pituitary corticotrope-derived cells from an
AtT20 D16V cell line that responds to nerve growth factor (NGF) by extending neurite-like
processes and differentiating into neurosecretory-like cells. During exposure, intracellular
calcium ([Ca2+]i) significantly increased and intracellular pH decreased. PEMF is also able
to stimulate neurogenesis in the sub ventricular zone of adult rats (Arias-Carrion et al., 2004).
This leads to studies of PEMF on stem cells. Nikolova et al. (2005) found that PEMF affect
transcript levels of apoptosis-related genes in mouse ES cell-derived neural progenitors. ES
cells were exposed to ELF PEMF simulating power line MF (PLMF) at 50 Hz. This
significantly affected transcript levels of the apoptosis-related bcl-2, bax and cell cycle
regulatory growth arrest DNA damage inducible (GADD45) genes, whereas mRNA levels of
neural-specific genes were not affected. No effects were found on mitochondrial function,
nuclear apoptosis, cell proliferation, or chromosomal alterations. Czyz et al. (2004)
investigated the non-thermal effects of PLMF as (50 Hz) on gene expression levels of
pluripotent ES cells and the role of the tumour suppressor p53. In the model of pluripotent

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mouse ES cells, they found that 5 min ON/30 min OFF intermittent PLMF (50 Hz) exposure
is capable of evoking non-thermal responses in ES cells, dependent on the cellular p53
function. The current trend in medical research related to PEMF is to study different
pathways related to cell physiology, proliferation, toxicity of chemicals, gene expression, etc.
Activated by PEMF, (Miyagi, Sato et al. 2000; Laque-Ruperez, Ruiz-Gomez et al. 2003). The
resistance of tumour cells to different antineoplastic agent is an obstacle for cancer
chemotherapy. The main mechanism in drug resistance is the multidrug resistance (MDR)
phenomenon, which constitutes the reduction of intracellular drug level due to the P-
glycoprotein pump function (Murthy 1999). Animal studies have shown that the use of EMF
can enhance drug delivery across biological barriers (rat abdominal skin), using benzoic acid
as the drug candidate. It has been reported by various authors that PEMF can produce
alterations in antineoplastic drugs cytotoxicity (Omote, Hosokawa et al. 1990; Hannan, Liang
et al. 1994; Miyagi, Sato et al. 2000). In addition, the use of unpulsed magnetic fields has
been found to produce alterations in MCF-7 cells. In this way, Harland and Liburdy (Harland
and Liburdy 1997) have observed that 1.2 μT, 60 Hz EMF partially blocked tamoxifen's
inhibitory action on the growth of this human mammary tumour cells in vitro. Leukaemia
THP 1 cancer cell line was used to detect the possible effects in pulse electromagnetic field.
The motive of this study is to investigate the effect of pulse electromagnetic fields on the
growth and activity of THP 1 cell lines and also to find whether pulse electromagnetic field
can enhance the effects of various chemotherapy drugs. So the two hypotheses in this project
were, to stimulate the growth of leukaemia THP 1 cells in Pulse Electromagnetic Field and
PEMF will enhance the effect of chemotherapy drug. To achieve our hypothesis the project
was classified in to two parts. The first part was to investigate the growth rate of THP 1 cells
in PEMF, which was carried out in two different experimental conditions. The second part
was the investigation of the growth rate of THP 1 cells in the PEMF with the addition of
chemotherapy drugs.

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2. OBJECTIVE:
2.1 Aim: Investigate the effect of pulse electromagnetic field on the growth and activity of
THP-1 cell line and further explore the combined growth inhibitory effect of pulse
electromagnetic field (PEMF) and Methotrexate and 5’-Fluorouracil chemotherapeutic drugs
on Thp-1 suspension cell line. THP 1 cells under pulse electromagnetic field (PEMF)
enhances the effect or efficacy of chemotherapeutic drugs (Methotrexate and 5’-Fluorouracil)
by modulating the cell permeability and by activating the expression of various pathways.
Decrease in the protein concentration is inversely proportional to cell viability or simply is an
index for decreased cell viability.
3. MATERIALS AND METHODS:
3.1 Materials:
3.1.1 THP 1 cells: Human Acute Monocytic Leukemia Cell Line. These were derived from
the peripheral blood of a one year old male boy with Acute Monocytic Leukemia, which are
large, round and single cells. They are non adherent cells and they grow in suspension
cultures.
Figure.1 schematic of thp-1 cell line in suspension (copied from
www.chenlab.clemson.edu/chenlabimg/thp1.jpg )
3.1.2 CO2 Incubator: Heraeus® HERA™ (Germany) Cell culture Incubator was used. It was
CO2 cell culture incubator. Inside of the Incubator comes with stainless steel interior. The
Hera cell 150 provides a stable, safe and precisely-controlled environment with convenience
and proven contamination control.

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3.1.3 G306 Magnetic Stimulator M KIII (20x5mm): G306 magnetic stimulator was used as
a Pulse Electromagnetic Field system and up to 10-50Hz frequency range.
3.1.4 HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was a zwitterionic
organic chemical buffering agent. It was better in maintaining pH despite changes in CO2.
Hence it was widely used in cell culture. For maintaining enzyme structure and functioning at
low temperatures made it as effective buffering agent (Baicu et al,. 1987).
3.1.5 Trypan Blue: This was the dye used for the staining of cells. The reactivity of trypan
blue depends on the fact that the chromophore is negatively charged and does not interact
with the cell unless damaged (Fresheney et al., 2002).
3.1.6 Haemocytometer: It was invented by Louis-Charles Malassez. It consists of a thick
microscopic slide which consists of a rectangular indentation chamber which engraved with a
laser-etched grid.
Number of cells/ml = Average cell number in A, B, C and D X 10,000
3.1.7 Cell Media: At Roswell Park Memorial Institute, RPMI 1640 was developed by Moore
and his group in 1966. To this media, 10% FBS and 500 micro liters of penicillin
streptomycin solution was added. This media was used for both suspension culture and
adherent cell culture.
Chemicals Used:
 Methotrexate: anti metabolite stops cells from dividing; (Lederle-Cyaramid Ibérica,
Madrid)
 5’-Fluoro Uracil: anti-metabolite;
 Trypan blue: for counting viable cells

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 NaOH- Sodioum hydroxide: Aids in dissolving Methotrexate
 NH4OH- Ammonium hydroxide: Aids in dissolving 5’-Fluoro Uracil
3.2 Methods:
3.2.1 Cell Culture:
Thp-1 cells were cultured RPMI 1640 (Lonza, MD, USA) medium with10% FBS (Lonza,
MD, USA). The cell density was maintained in between 3-9 x 105cells/ml and grown under
constant 5% CO2 at 37oC in an incubator. For every 3 days the media was changed on the
cells. Thp-1 cells are non adherent cells and the cells were passaged/sub-cultured over the
time period when they reach 70 to 80% confluence.
3.2.2 Counting cells: Trypan blue method
0.1 ml of cells was placed in a screw cap test tube and to them 0.1 ml of trypan blue was
added and mixed thoroughly. The mix was incubated for 5min at room temperature. 0.01ml
of the incubated mix was added to the Haemocytometer for counting the cells. All the viable
blue stained cells were counted from the four grids ABCD (fig.). The average of all the grids
was taken and multiplied by 10,000 to give the total number of cells per ml of dissolved
medium. Initially if the cells were dissolved in 5mls of RPMI, the average was multiplied by
a factor of 50,000 (dilution factor-5 x 10,000).
3.2.3 Biuret Assay:
The biuret assay was used to determine the protein concentration in control and test plates.
The following chemicals and composition was used to prepare biuret reagent.
1. NaOH, sodium hydroxide - 8gm/ml
2. NaKT, sodium potassium tartrate - 9gm
3. CuSO4.5H2O - 3gm
4. Potassium Iodide - 5gm
The two flasks, one in PEMF and other in incubator are taken out after the completion of
exposure time. For the Thp-1 suspension culture, cells were centrifuged at 1200rpm for
5minutes at room temperature, supernatant was taken out and the proteins were extracted
from the pelleted cells by using the lysis buffer (50 mM Tris-HCl pH 7.5, 10% SDS) (200μL/
well of 6 well plates) and incubated for 4minutes at RT. The lysed cells were centrifuged at

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13000rpm for 5minutes to remove cell debri in the form of pellet. The super was collected
and to 1ml of every lysate, 2 ml of biuret reagent was added and allowed to incubate for
10minutes at RT. Then spectrophotometer readings were observed at 550nM.
3.2.4 Pulse electromagnetic field exposure: (PEMF)
3.2.4.1 Effect of HEPES and PEMF on Thp-1 cell viability:
100,000 Thp-1 cells were aliquoted in to each well of a 6well plate and 2ml of medium was
added to each well. Thp-1 cells were subjected to PEMF for 24hrs and the control group
cells/ plate was incubated in CO2 incubator. The PEMF subjected cells were allowed to
recover for another 24hrs without PEMF. As a second set a time dependent experiment was
done by exposing the Thp-1 cells to PEMF at regular intervals of 2hrs ON/OFF module for a
total time of 24hrs and then the cells were allowed to recover for 24hrs without PEMF while
the control group was treated the same as the first set.
The lysates were extracted from these samples and the protein concentration was determined.
Later 0.6 ml of HEPES was added to 5ml of cell media and subjected to PEMF The flask was
introduced in to this exposure system by two different exposure intervals as the first one with
24hr ON and 24hr OFF and second one was ON for two hour and OFF for two hour up to
eight times or for a total period of 24hrs. This stimulator was incorporated in to CO2
incubator and it was controlled with specific interval by an automatic timer. Protein lysates
from these samples were extracted and their concentrations were determined. Below is the
tabulated layout of 6 well plates for studying the PEMF effects on Thp-1 cells with HEPES
respectively:
Control plate:
THP-1 cells THP-1 cells THP-1 cells
THP-1 cells THP-1 cells THP-1 cells
HEPES test plate:
THP-1 + HEPES THP-1 + HEPES THP-1 + HEPES
THP-1 + HEPES THP-1 + HEPES THP-1 + HEPES

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3.2.4.2 Effect of Methotrexate and 5’-Fluorouracil anti-metabolites on THP-1 cell lines
under PEMF:
The THP-1 cells were constantly exposed to PEMF followed by 24hrs recovery with and
without drugs at 5µg/ml and 20µg/ml of Methotrexate and 5’-Fluorouracil.
Protein lysates were extracted from the treated and control groups and the viability of THP-1
cells was assessed basing on the protein concentrations. The experimental setup was done in
the below mentioned fashion.
Control-THP-1-
Normal medium
Control-THP-1-with
NaOH/NH4OH
THP-1 cells with 5µg/ml of
Methotrexate/5’Fluorouracil
Control-THP-1-
Normal medium
Control-THP-1-with
NaOH /NH4OH
Methotrexate/5’-Fluorouracil
Control-THP-1-
Normal medium
Control-THP-1-with
NaOH /NH4OH
Table: 2 Layout of 12 well plates used for studying the effects of PEMF on increasing the penetrance of
Methotrexate and 5’-Fluorouracil drugs. Every sample is done in triplicates.
3.2.5: Statistical analysis: All the data was presented as mean and standard error of mean.
One sample t-Test was used to compare the data consistency and significance at 99.95%
confidence interval i.e. (p=< 0.05).

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4.0 Results:
4.1 Protein standard curve with BSA (Biuret’s assay):
Table3: Standard protein concentrations and Standard graph and the equation for the best fit line with the values
from table-3. Y (protein concentration) = 12.607X (OD550) + 0.3643
Absorbance (550nM) Std BSA concentration µg/µl
0 0
0.004 0.15
0.012 0.3
0.027 0.6
0.055 1.25
0.096 2.5
0.348 5
0.815 10
1.54 20
BSA statndard curve with Biuret's assay
y = 12.607x + 0.3643
0
5
10
15
20
25
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
OD (550nM)
Concentrationinug/ul

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4.2 PEMF Enhances the growth of THP-1 cells on the other hand HEPES reduces the
THP-1 cell viability when exposedto PEMF:
Thp-1 cells when exposed to PEMF showed a significant (p = 0.000163) increased growth
response compared to control plate; however the same cells significantly (p = 0.00013)
reduced their growth in the presence of HEPES buffer and PEMF. PEMF treated 2hr interval
sample showed no significant (p = 0.198) difference in the growth profile compared with
controls. Interestingly the HEPES and 2hr interval PEMF exposure sample showed a
significant (p = 0.0012) growth difference when compared to the controls sample. The
variance among the samples seems to be very less as the concentrations in each well was
almost equal between the wells.
Table: 3 List of protein concentrations with PEMF and HEPES exposure in 24hrs ON/OFF mode and 2hr
interval mode. The statistical tests were conducted with respect to control plates; p<0.05 were considered
significant
Sno/ well no Control plate
(μg/μl)
PEMF treated
plate 24hrs
(μg/μl)
PEMF treated
at 2hrs
intervals
(μg/μl)
PEMF+HEPES
24hrs (μg/μl)
PEMF+HEPES
2hr regular
intervals (μg/μl)
1
1.12 1.33 1.1 0.86 0.997
2
0.99 1.24 1.06 0.754 0.974
3
1.034 1.37 1.09 0.754 0.843
4
1.122 1.34 1.07 0.891 0.965
5
1.102 1.214 1 0.914 0.989
6
1.099 1.201 0.995 0.679 0.919
Mean
1.077833333 1.2825 1.0525 0.808666667 0.947833333
SE
0.021906493 0.029642031 0.018337121 0.037990935 0.023743654
T-test wrt
control plate;
99.95%CI 0.00
0.000163711 0.198329666 0.000139278 0.001227089

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4.3 Effect of PEMF on THP-1 cell viability: PEMF enhances the cell growth at low
frequencies and HEPES significantly decreases the cell viability of THP-1 cells upon PEMF
exposure (Fig: 2).
Figure: 2 PEMF enhances THP-1 cell growth and HEPES suppresses the THP-1 cell viability: (A) The
protein concentration scatter plot showing the highest range of protein per well in PEMF treated THP-1 cells
compared to other treatments. HEPES treated THP-1 cells under PEMF influence decreased the protein
concentration reflecting the lower growth rate. (B) THP-1 cells under normal conditions grow significantly
slower than cells exposed to constant PEMF (p = 0.000163). Differential kinetic PEMF treatment for every 2hrs
ON/OFF protocol significantly (p = 0.19) didn’t show any change in the growth pattern of THP-1 cells,
intriguingly HEPES treated cells significantly (p = 0.000139278; p = 0.00122) reduced proliferation under both
constant and the differential PEMF treatments respectively.
*
*
*
* p < 0.05 significant
B
A

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4.4 Combined effect of Methotrexate and PEMF on THP-1 cells: Simultaneous exposure
to PEMF and Methotrexate treatment significantly decreased the THP-1 cell viabilty
compared to the normal controls. The reduced cell survival increases with the concnetration
of the drug (fig.3). Sodium hydroxide (NaOH) is nearly significant (0.041) in reducing the
THP-1 cell growth Table-4).
Table: 4 Protein concentrations of THP-1 cells when exposed to Methotrexate and PEMF at low frequency
20Hz under 24 hr constant exposures with a 24hrs recovery period. NaoH is merely significant (p = 0.041) in
reducing the THP-1 cell viability.
Each lysate was duplicated during quantification to get maximum inter sample consistency.
The variance was so low and the samples showed no drastic change in the sampling.
Sno control+PEMF PEMF+NaoH PEMF+Methotrexate(5µg/ml) PEMF+Methotrexate(20µg/ml)
1 1.39 1.22 0.56 0.13
1 1.34 1.29 0.44 0.201
1 1.27 1.134 0.49 0.11
2 1.44 1.182 0.391 0.109
2 1.314 1.192 0.414 0.12
2 1.101 1.199 0.379 0.09
Mean 1.309166667 1.202833333 0.445666667 0.126666667
SE 0.048129282 0.020973661 0.027991268 0.015827543
t-Test 0 0.04173282 1.42114E-07 1.78729E-07

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Figure: 3 Coupled Methotrexate and PEMF exposure for 24hrs drastically reduces the THP-1 cell survival in a
concentration gradient fashion: (A) THP-1 cells protein concentrations duplicated from single well showed no
significant change in the variability and the notably the controls displayed high protein concentrations compared to
Methotrexate treated sample wells at different concentrations. Intriguingly NaOH treated cells showed a slight
reduction in the protein levels. (B) THP-1 cells under increased concentrations (5-20µg/µl) of Methotrexate with
coupled PEMF exposure significantly induced cell death or viability respectively. NaOH showed mere significance (p
= 0.041) in reducing the cell viability but compared to Methotrexate its highly insignificant (p = 1.2E-08).
A
B

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Combined effect of 5’-Fluorouracil and PEMF on THP-1 cell viability: Simultaneous
exposure to PEMF and 5’-Fluorouracil treatment significantly decreased the THP-1 cell
viability compared to the normal controls. The reduced cell survival increased with the
increase in concentration of the 5’-Fluorouracil (fig.5). Ammonium hydroxide (NH4OH) is
nearly significant (0.0541) in reducing the THP-1 cell growth Table-5).
Table: 5 Protein concentrations of THP-1 cells when exposed to 5’-Fluorouracil and PEMF at low frequency
20Hz under 24 hr constant exposures followed by a 24hrs recovery period. NH4OH is merely significant ( p =
0.0541) in reducing the THP-1 cell viability.
Sno Control+PEMF PEMF+NH4OH PEMF+5'-Fluorouracil
(5µg/ml)
PEMF+5'-Fluorouracil
(20µg/ml)
1 1.34 1.32 0.23 0.087
1 1.24 1.19 0.143 0.067
1 1.37 1.134 0.254 0.0652
2 1.54 1.142 0.35 0.0689
2 1.214 1.162 0.164 0.071
2 1.201 1.29 0.11 0.0599
Mean 1.3175 1.206333333 0.2085 0.069833333
SE 0.052617963 0.032415703 0.035836434 0.003764277
t-Test 0.0 0.054165402 1.95512E-08 1.13836E-06

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Figure: 5 Coupled 5’-Fluorouracil and PEMF exposure for 24hrs drastically reduces the THP-1 cell survival
in a concentration gradient fashion and is potent than Methotrexate in reducing the cell viability: (A) THP-1
cells protein concentrations duplicated from single well showed no significant change in the variability and the
notably the controls displayed high protein concentrations compared to 5’-Fluorouracil treated sample wells at
different concentrations. Intriguingly NaoH treated cells showed a slight reduction in the protein levels. (B) THP-1
cells under increased concentrations (5-20µg/µl) of 5’-Fluorouracil with coupled PEMF exposure significantly
induced THP-1 cell death or viability (p = 1.9 E-08; p = 1.31E-06). NH4OH showed mere significance (p = 0.0541)
in reducing the cell viability, but compared to5’-Fluorouracil its highly insignificant (p = 1.7E-09).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control+PEMF PEMF+NH4OH PEMF+5'-Fluorouracil
(5µg/ml)
(20µg/ml)
Proteinconcentrationinµg/µl
Effect of 5'-Fluorouracil on Thp-1 cells viability
***
***
* p < 0.05 significant
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.5 1 1.5 2 2.5
Control+PEMF
PEMF+NH4OH
(5µg/ml)
(20µg/ml)
Proteinconcentrationinµg/µl
Duplicates

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5.0 Discussion:
Cancer is the second leading cause of death after the heart disease. There are many methods
and drugs to treat cancer but none of them could solve the problem on the whole. During this
process the patient has to undergo many hurdles moth physically and mentally. Due to the
recent advancements in the technology Pulsative electromagnetic field is used to boost the
chemotherapy and induce cancer cell specific death. In the present research Initial 24hrs
constant exposure of THP-1 cell lines to PEMF showed a significant (p = 0.00016) increase
in the cell growth similar to osteocyte growth in the presence of weak PEMF (Ottani,
Raspanti et al. 2002). Interestingly HEPES under PEMF influence reduced the THP-1 cell
viability, which seems to be a novel finding where most of the reports showed HEPES as
standard buffer that could induce the endothelial cell growth (Bowman, Berger et al. 1985).
From the past none of the of the researchers have proved the HEPES toxicity to cells (Lepe-
Zuniga, Zigler et al. 1987). Pulsed exposure of PEMF and HEPES also seems to be very
effective. On the other hand the pulsed treatment of PEMF didn’t show any significant
changes in the growth of THP-1 cells. This indirectly suggests that the constant exposure of
cells to PEMF seems to be more effective than the pulsed or interval exposure. The use of
low frequency up to 40Hz at 1.55mT was optimised and previous studies have also shown
that the use of high frequency electromagnetic waves could induces drastic changes in the
cell behaviour and induces apoptosis or necrosis (Yang, Huo et al. 2007; Wei, Xiaolin et al.
2008). This scenario cannot used when working on animal models (Cadossi, Hentz et al.
1989). Studies on similar PEMF stimulations on normal cells enhanced the proliferative
capability in (Satake 1990). Many of the previous studies have proved that the use of PEMF
as an aid to increase the drug efficacy. In the present study Methotrexate and 5’Fluorouracil
were used at two different concentrations (5 & 20µg/µl) where both of them induced a drastic
growth repression in THP-1 cells. This decrease in cell viability was dependent upon the
concentrations and PEMF exposure. Intriguingly the NaOH and NH4OH treated THP-1 cells
also reduced the cell viability but it’s merely significant. The previous study by (Laque-
Ruperez, Ruiz-Gomez et al. 2003) have shown in MCF-7s increased use of Methotrexate
under low PEMF didn’t alter the cell viability or cytotoxicity. But the present result
contradicts to Ruperez et als findings. Maybe they should have increased their frequency to
40 instead of 25Hz. Another interesting observation which supports the data from the
literature is 5’-Fluorouracil was potently cytotoxic than Methotrexate. This may be due the
fact that the 5’-Fluorouracil is directly involved in the DNA and RNA synthesis so it has the

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immediate effect. On the other hand Methotrexate was shown to be one of the potent drugs
but 24hrs may not be enough for Methotrexate to show enough cytotoxicity on THP-1 cells.
Other findings from the fellow mates with MCF-7 have shown the same effect, but the HeLA
cells responded differently to THP-1 cells under PEMF stimulation and drug treatments. This
effect may be a dynamic and heterogeneous effect. So each cell line should be examined
because of this heterogeneous behaviour. Overall the HEPES induced cell death seems to be
strange, a further extension would benefit or support the present finding by repeating the
similar stimulation experiments with other cancer cell lines. Protein measurement as an index
of cell viability seems to be diluted concept a further apoptotic marker profiling or annexin V
staining would give more clues or insights of which molecular mechanism or signalling
pathway is activated or down regulated.
6.0 Conclusions:
 Growth of THP-1 cells is stimulated by a 24hr constant exposure under PEMF.
 Differential PEMF exposure at 2hr ON/OFF didn’t induce a significant growth
response inTHP-1 cells.
 HEPES treatment combined with PEMF stimulation repressed THP-1 cell viability or
proliferation in both modes of exposure.
 Methotrexate and 5’-Fluorouracil decrease the THP-1 cell proliferation under PEMF
stimulation and the viability of THP-1 cells are dependent the concentration of both
the drugs increased concentration decrease the cell viability.
 5’-Fluorouracil has shown the maximum cytotoxicity than Methotrexate at 24hr
stimulation by PEMF.
 Low frequency (40Hz frequency at 1.5mT magnetic flux) PEMF was highly effective
in inducing the THP-1 growth stimulus.

19
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22
CONTENTS
1. Introduction…………………………………………………………………….2-4
2. Objective……………………………………………………………………..……5
3. Materials and Methods…………………………………………………..5-9
4. Results…………………………………………………………………………10-16
5. Discussion………………………………………………………………………17-18
6. Conclusions………………………………………………………………………18
7. References…………………………………………………………………….19-21

23
Acknowledgment
I am heartily thankful to my supervisor, Dr. George
Worthington, whose encouragement, supervision and support
from the preliminary to the concluding level enabled me to
develop an understanding of the subject. Lastly, I offer my
regards and blessings to all of those who supported me in any
respect during the completion of the project.

vivek

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