Research Article |
Open Access |
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Non-thermal Effects of Far-Infrared Ray (FIR) on Human
Hepatocellular Carcinoma Cells HepG2 and their Tumors |
Tatsuo Ishikawa 1, Jun Ishibashi 2, Kikuji Yamashita 1*, Shine-Od Dalkhsuren 1,
Kaori Sumida 1, Takahumi Masui 1, Seiichiro Kitamura 1 |
1Department of Oral and Maxillofacial Anatomy, Medical Science for Oral and Maxillofacial Regeneration,
Graduate School of Health Biosciences, University of Tokushima, 3-18-15 Kuramoto, Tokushima, 770-8504 Japan |
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| *Corresponding author: |
Dr . Kikuji Yamashita, Department of Oral and
Maxillofacial Anatomy,
Medical Science for Oral and
Maxillofacial Regeneration,
Graduate School of Health Biosciences,
University of Tokushima, 3-18-15 Kuramoto,
Tokushima, 770-8504 Japan,
Tel : +81-88-6339120,
Fax : +81-88-6337320,
E-mail : kikuji@dent.tokushima-u.ac.jp |
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| Received November 03, 2009; Accepted December 29, 2009; Published
December 29, 2009 |
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Citation: Ishikawa T, Ishibashi J, Yamashita K, Dalkhsuren SO, Sumida
K, et al. (2009) Non-thermal Effects of Far-Infrared Ray (FIR) on Human
Hepatocellular Carcinoma Cells HepG2 and their Tumors. J Cancer Sci Ther 1: 078-082. doi:10.4172/1948-5956.1000012 |
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Copyright:© 2009 Ishikawa T, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution
License,which permits unrestricted use, distribution, and reproduction
in any medium, provided the original author and source are credited. |
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| We detected an efficacy of Far-Infrared Ray (FIR) on
HepG2 cell lines and their implanted cancer tumors by
using an incubator and a murine raising rack which irradiate
FIR at the wavelength of it was 4-20μm that we developed.
In this study we used hepatocytometer, WST-1 assay
and Brd-U assay in order to detect cell proliferation
and HE staining, DAPI staining, TUNEL staining, histology
and microarray assay were performed to examine both
cells and tumors under the non-thermal conditions of
25.0±5°C and 37.0±5°C degrees. Consequently cancer cell
proliferation was inhibited and tumors by implanted
HepG2 cells into the dorsal subcutaneous layer of SCID
mice disappeared or reduced. We found out dehydrogenase
was activated by FIR and mRNA of Vascular Endothelial
Growth Factor (VEGF) decreased. Thus these results
suggest that electron transfer system of HepG2 might
be related to inhibition of cancer cell growth and inhibition
of vascular growth of cancer tumors. |
Keywords: |
| Far-Infrared Radiation (FIR); FIR incubator; Human
cancer cell |
Introduction |
| Infrared Radiation (IR) is an electromagnetic wave which
was found out by W. Hershel (UK) in 1800. He found out this
new invisible ray with surprise while measuring spectrointensity
distribution in sunlight by using a mercury thermometer and a
trigonal prism. When he happened to measure the degree of
the thermometer of an area beyond the red light limit at the
wavelength of 0.78μm he saw temperature upshift. So IR was
called ‘dark heat’ or ‘invisible rays’ in the earliest keeps. In
1895 Rubens (Germany) could measure up to, but nowadays
researchers can measure reliable spectrum of several millimeters
(Mitsuishi, 2007). And, one example, it is divided into two
regions-Near Infrared Ray and Far Infrared Ray. The former
(0.78~3μm, it looks like visible light) is used as CCD camera,
IR camera, heater and vein authentication system. The latter
(3~1000μm, looks like electromagnetic wave) is used as thermography
and heater. |
Recently, adding to the heat effect, biological effects of FIR
have been reported. For instance, at 39~41°C, mammary tumor
growth in mice was inhibited (Udagawa et al., 1999;
Udagawa and Nagasawa, 2000; Udagawa et al., 2000). Furthermore
even at normal temperature, at 25.5°C, it was also
inhibited (Nagasawa et al., 1999). |
As we had had FIR incubator and animal raiser made, we
had carried out the experimental study as former reported
(Hosokawa et al., 2005; Hosokawa et al., 2005; Yamashita et
al., 2005) about HSC3 (human tongue cancer cell), Sa3 (human
gum cancer cell), A549 (human lung cancer cell), A431
(human vulva cancer cell) and MCF7 (human breast cancer
cell) (Udagawa et al., 2000). |
Exceedingly, in this article, we’ve performed experimental
study of effects of FIR on cultured hepatocellular cartinoma
cell HepG2 to detect another cancer responsible for its anticancer
effects. |
Materials and Methods |
Instruments |
| We fabricated an FIR radiant-panelled cell incubator and a
murine keeping rack by coating ceramics consist of carbon/
silica/aluminium oxide/titanium oxide( radiating efficiency >
97% compared with an ideal blackbody of 100%) using a polycarbonate
printing technique ( Bloodissue Co. Tokushima Japan).
They have a stably irradiate system with FIR at wavelength
between 4 and 20μm ( maximum at 7-12μm) under conditions
of 100% humidity, at 37.0 ± 0.5°C degrees and 5% CO2
in air. |
Cell line and cell culture |
| Human hepatoma HepG2 cell line was purchased from Riken
Cell Bank (Tsukuba, Japan). HepG2 cells were cultured in
Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient mixture
(Sigma, St. Louis, MO, USA). The medium was supplemented
with 10% heat-inactivated fetal bovine serum (FBS),
100μg/mL penicillin G and 100μg/mL streptomycin. Cells were
maintained in a humidified atmosphere of 5% CO2 in 95% air at 37°C for 24 hours. The medium were replaced every 3 days. |
Measurement of cell number and growth |
| Cells(5×104) were plated in triplicate in 24-well plates (Nunc,
Roskilde, Denmark). The attached cell populations were measured
on day 0, 2, 4, 6, 8, 10, 12, 14 using 3% Trypan blue
(Wako Junyaku, Osaka) and a homocytometer (SLGC, Tokyo).
And also WST-1 assay was performed. Cells (1×103 cells/well)
were scattered into 96-well plates( Nunc, Roskilde, Denmark).
Having confirmed the cells in the non-confluent stage, we
administerd 10mL WST-1 (Roche, Mannheim, Germany) into
100 mL medium. After 4 hours reaction at 37°C, Absorbance
values (at 405nm) were measured by an ImmunoMini NJ-2300
plate reader (System Instrument, Tokyo, Japan). |
Morphological observation of HepG2 cells in vitro |
| Cells were incubated on coverslips of 22×22mm (Matsunami
Glass Inc. Osaka) on 35mm dishes (Nunc). After 4 days of
incubation irradiated by FIR, they were rinsed with PBS (--),
fixed by 4% formalin in PBS on ice for 30 minutes. Then Hematoxylin
and Eosin staining was performed. Living cells were
photographed by CK40 upside down Microscope (Olympus,
Tokyo) and PM-BP35 photo instrument (Olympus). |
A study of effects of FIR on tumors of mice |
| 2×106 HepG2 cells in PBS containing OD matrigel were implanted
in dorsal panniculus layer against 8-10 week CB17/
Icr-Prkdc/CrjCrj SCID mice. Immediately after infusion they
were divided into with and without FIR treatment groups. And
raised in FIR-irradiating animal raising rack for 50 days. For
the duration of the experiment we measured their body weight
and shortest and longest axis of tumors every 5 days and calculated
the tumor volume by the next equation. |
tumor volume =0.5×(longest axis)×(shortest axis)2 |
Mice were feeded with solid food irradiated by sterilizing
radiation (Oriental Kobo Industrial Inc., Tokyo, Japan) and sterilized
water. After 50 days they were euthanized subjected to
the university codes. Tumor tissues were excised and mRNA
of them were extracted. During these experiments temperature
condition of with and without FIR were set precisely the same. |
Histological investigation of tumor tissue in vivo |
| After 50 days mice with and without FIR treatment were
euthanized and only tumors were excised and fixed with 4%
paraformaldehyde wi th PBS at 4°C for 4 hours. Then
dehydrized by ethanol and embedded in paraffin, produced tissue
sections at 4-μm-thick tissue sections and Hematoxylin and
Eosin staining was performed. |
cDNA microarray assay and data analysis |
| cDNA microarray analysis was performed in order to elucidate
the influence of FIR in vitro and in vivo on the expression
of genes. Total RNA was extracted from the 4 day incubated
cells of HepG2 with and without FIR and the tumors from 50
day raised tumor-bearing mice with and without FIR according
to the protocol of Qiagen Rne mini kit (Qiagen, Valencia,
CA, USA). After ascertained A260/A280 rate was over 1.8 with
UV spectrophotometer DC530 (Beckman Coulter, Fullerton,
CA, USA), we ascertained the no fragmentation of RNA with
Agilent 2100 bioanalyzer( Agilent Technologies, Palo Alto, CA,
USA). Next amplification of each RNA and labellation of Cy3
(control group) and Cy5 (FIR group) was carried out using
Low RNA Input Fluorescent Linear Amplification Kit (Agilent
Technologies). Hybridization of these RNA was performed
according to the protocol of Agilent human 1A ver.2 microarray
slides (Agilent Technologies) which carries 19,000 typical human
genes. After rinsing and drying, fluorescent light of Cy3
and that of Cy5 was scanned by 670 and 770 nm by Agilent
Technologies Microarray Scanner (Agilent Technologies). |
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Figure 1: Cell proliferation. |
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GeneSpring 7.0 software (Silicon Genetics, CA, USA) was used
for data analysis and extracted the genes whose expression was
significant. And we made a list of FIR-sensitive genes by per
spot and per chip intensity-dependent (LOWESS) normalization
by using raw data. Then we extracted the data whose value
was at once p-value < 0.01 against the control group and expression
rate ≥ ± 1.5 against the control groups, and then except
even one condition as follows genes whose fluorescence
were filled, genes whose fluorescent value were differences
with background fluorescence were within+2.6×SD. We classified
these FIR-sensitive genes according to GO term information
(http://www.godatabase.org). |
Detection of tumor cell apoptosis by TUNEL staining |
| Isolated tumor tissue from euthanized mice was fixed in 4%
paraformaldehyde in PBS at 4°C for 4 hours. Dehydrated by
ethanol, embedded in paraffin, we produced 4-μm-thick tissue
sections. Then In Situ Detection Kit (Takara Shuzo, Osaka)
was carried out. After deparaffinization, treated by proteinase
K for 15 minutes at room temperature, rinsed with PBS(--). Labeling reaction was performed with TdT enzyme, and labeling
FITC, dUTP at 37°C for 90 minutes. After rinsing with
PBS (--), observation of apoptosis was performed with BX51
fluorescent microscope( OLYMPUS). |
Statistical analysis |
| Data are means ± SE of replicate samples in single experiments
or replicate experiments as described in the figure legends.
Student’s t-test was used for comparisons between two
groups-treated groups and control groups. And significant
threshould was set to P < 0.05. Multiple group comparisons
were performed by one-way ANOVA, followed by the Tukey-
Kramer multiple group comparisons test. All statistical analysis
were carried out using Statcel 2 software (OMS publishing,
Saitama, Japan). |
Results |
Inhibition of HepG2 cell growth by FIR |
| After 14 day incubation FIR inhibited cell growth of HepG2
by 30% by cell count method by hepatocytometer (Figure 1A). And by BrdU assay( Quantity of DNA) absorbance was
downregurated after 2 days by 53%, 3days by 20% and 4days
28% (Figure 1C). By WST-1 assay, however, absorbance of
FIR treated cells decreased only 0.0, 5.4, 3.3% after 2, 4.6
days (Figure 1B). |
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Figure 2: Tumor-bearing mice. |
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Figure 3: Histological images of HepG2 tumor tissue (x 40). |
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Figure 4: histological observation. |
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Morphological analysis of HepG2 cells |
| After 4 days of exposure to FIR we examined the morphology.
But they showed no apparent change in morphology. |
Effects of FIR on the expression of HepG2 genes
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| mRNA of albumin( function marker of hepatocell), alphafetoprotein(
AFP) and asialoglycoprotein receptor 2(ASGR2)
decreased by 36.1%,36.0% and 30.7%. VEGF decreased
36.1%. |
Effects of FIR on implanted HepG2 tumor |
| Tumor volume of HepG2 of FIR groups decreased significantly
after 30 days. On the other band body weight decreased
slightly compared to control groups (Figure 2A, B). |
By histological analysis, Cell swellings were observed in control
groups (Figure 4A). Disruption of microtubule cytoskeleton
of control groups were also observed (Figure 4B). But we
could not find the difference of apoptosis between treated and
non-treated groups (Figure 4C). |
Discussion |
| There have been few reports about HepG2 and mice (Eguchi
et al., 1997). In the present study we have carried out these
experiments and got some findings. At first we have investgated
the effect of FIR on the proliferation of HepG2 cells. As a result,
though cell number and DNA quantity (by BrdU assay)
decreased 32.5% at its peak (Figure 1A, C), dehydrogenase
activity (by WST-1 assay) decreased only 3.3% at its peak (Figure
1B). This fact means that activity of dehydrogenase increased
comparatively. As WST-1 assay measures Formazan
(dark red) which was resolved from water-soluble tetrazolium
WST-1 (blight red) by the function of dehydrogenase using
spectrophotometer (Isiyama et al., 1993), it showed the strength
of the activity of dehydrogenase. This means dehydrogenase
released H+ by FIR by co-enzyme NADH oxidation. This result
is similar to other reports that FIR (2-25μm at its wavelength)
from ceramics (silicon nitride/ silicon carbon) and Glucose-
6-phosphate dehydrogenase at 37°C was used (Kohashi et al., 1996; Kohashi et al., 1993) and that electromagnetic
waves of 875 MHz (343mm at its wavelength) and NADH oxidation
and plasma membrane were used (Friedman et al., 2007).
Then ROS (reactive oxygen species) andMMP (matrix
metalloproteinase) may become active (Friedman et al., 2007).
Our histological findings and other report (Eguchi et al., 1997)
are of one accord in many venule and their disruption. Tumors
of our SCID mice (2 out of 10 mice, 5.2×6.2, 5.2×5.7μm after
20 days disappeared after 25days) disappeared. We suppose
that MMP might affect this distruction. In tumors there were
much mass of erythrocyte and they are. |
| Table 1: microarry assay of HepG2 in vivo. |
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Acknowledgements |
| This research was supported in part by following aids and
grants: Ministry of Economy, Trade and Industry, 41104503;
Toseki Matera f19008001; Y. Tsukada |
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