Non-Thermal Effects of Far-Infrared Ray (FIR) on Human Hepatocellular Carcinoma Cells HepG2 and their Tumors

Background: We developed a cell culture CO2 incubator and a mice rack that can continuously irradiate cells or murine with FIR. Our goal is to make clear the non-thermal effect of FIR on HepG2 with these instruments morphologically. Methods: By using them, in vitro , we examined the proliferation of cultured HepG2 cells with hematocytometer, BrdU assay, WST-1 assay, HE staining, Toluidine blue staining and microarray studies. And in vivo, we measured the tumors, observed the sections by IHC, DAPI staining with light microscopes and performed microarray studies. Results: Proliferation of HepG2 cells were suppressed (e.g., cell count declined by 34% after 10 days of FIR irradiation), tumor volumes reduced by 86% after 30 days of FIR irradiation, mRNA of Vascular Endothelial Growth Factor (VEGF) decreased by 48%, vascular area in cross sections from the tumors decreased 60% compared with the control. More frequent properties in apoptosis were observed by TUNEL and DAPI staining in FIR-treated groups. Body weight of mice increased compared with the control. Oxydation and Reduction (Redox) reactions by H+ (proton and electron)/O2(a kind of Reactive Oxygen Species (ROS)) were induced by FIR. Conclusions: These results clarified that FIR inhibited the proliferation of HepG2 at non-thermal circumstances (at 25±0.5, 37±0.5°C). FIR will serve as a tool against diseases induced by HepG2. Non-Thermal Effects of Far-Infrared Ray (FIR) on Human Hepatocellular Carcinoma Cells HepG2 and their Tumors Tatsuo Ishikawa1, Jun Ishibashi2, Kikuji Yamashita1*, Shine-Od Dalkhsuren1, Kaori Sumida1, Takahumi Masui1 and Seiichiro Kitamura1 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 2Blanka Dental Office Journal of Cancer Science & Therapy Open Access JCST/Vol.1 Issue 2


Introduction
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 and Nagasawa, 2000;Udagawa, 2000). Furthermore, even at normal temperature, at 25.5°C, it was also inhibited . As we had FIR incubator and animal raiser made, we had carried out the experimental study as former reported (Hosokawa et al., 2005;Yamashita et al., 2005;Ishibashi et al., 2008) 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,200). Exceedingly, in this article we have performed experimental study of effects of FIR on cultured hepatocellular carcinoma cells HepG2 to detect another cancer responsible for its anticancer effects.

Instruments
We fabricated an FIR radiant-paneled cell incubator and a murine keeping rack by coating ceramics consist of carbon/ silica/aluminum 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 20ìm (maximum at 7-12ìm) under conditions of 100% humidity, at 37.0±0.5ºC degrees and 5% CO 2 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% CO 2 in 95% air at 37ºC for 24 hours. The medium were replaced every 3 days.

Morphological observation of HepG2 cells in vitro
Cells were incubated on cover slips 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 2×10 6 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. Their cages were made of plastics such as to penetrate FIR. And raised in FIR-irradiating mice rack for 50 days. For the duration of the experiment we measured their body weight, 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 fed 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 Tokushima University animal care and use guidelines. Tumor tissues were excised and mRNA of them was 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 with PBS at 4ºC for 4 hours. Then dehydrized by ethanol and embedded in paraffin, produced tissue sections at 4-ìm-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 of Cy5 was scanned by 670 and 770 nm by Agilent Technologies Microarray Scanner (Agilent Technologies). 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 Figure 1A: Cell number were counted using a hemocytometer. Cell count were significantly lower compared with controls on 8, 10, 12, 14 days.   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 FIRsensitive 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 4ºC for 4 hours. Dehydrated by ethanol, embedded in paraffin, we produced 4-ìm-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 37ºC 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 groupstreated groups and control groups. And significant threshold 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 was carried out using Statcel 2 software (OMS publishing, Saitama, Japan).

Effects of FIR on HepG2 Cell Proliferation
To evaluate the effects of FIR on HepG2 cell proliferation, we performed assays as described in the Material and Methods. Figure 1A shows viable cell counts at days 2, 4, 6, 8, 10, 12 respectively after FIR exposure using hematocytometer. Cell counts of FIR-treated group decreased 36, 34, 26, 21% at days 8, 10, 12, 14 respectively compared with control one significantly. Similar result was obtained by BrdU assay. Synthesis of DNA by incorporated BrdU of FIR-treated group decreased by 53, 20, 28% at days 2, 3, 4 compared with control one significantly ( Figure 1B). WST-1 assay, however, exhibited similar levels. Absorbance of FIR-treated cells decreased only 0.0, 5.4, 3.3% after 2, 4, 6 days ( Figure 1C). This means FIR Water Soluble Tetrazolium salt was cleaved into formazan by Journal of Cancer Science & Therapy -Open Access JCST/Vol.1 Issue 2 oxidation-reduction (redox) reaction or disproportionation reaction by O 2 -in the FIR-treated tumor compared to the control.

Morphological analysis of HepG2 cells
We were not able to find any difference between experimental and control groups.

Effects of FIR on implanted HepG2 tumor
Cell count of FIR-treated group decreased 18.4% compared to the control. We measured HepG2 cell-size (n=1000) with and without FIR (Figure 2A). Longest axis and volume of cells decreased but the volume of nucleus increased by 5 '16% with FIR group. Therefore nucleus to cell (N/C) ratio of FIR group increased 13 '14%. Disruption of microtubule cytoskeleton of control groups was also observed ( Figure 2B). And we could observe cell apoptosis more frequently in FIR-treated groups ( Figure 2C). 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 3A, Table 1: microarry assay of HepG2 in vivo. VEGF (Vescular Endothelial Growth Factor) mRNA decreased by 40%. ALB (Albumin) mRNA decreased by 36%. AFP (Alpha-Fetoprotein, a meter of fiver cancer usually produced only in fetallife) mRNA decreased by 36%. ASGR1 (Asialoglycoprotein) mRNA decreased by 8%, ASGR2 by 31%.

Figure 2:
Upper row is shown control group, lower row is shown FIR group; A. HE staining, Cell size is relatively smaller in FIR group than control; B.Disruption of microtubule cytoskeleton in control group and shrinkage of microtubule in FIR group were observed; C.We observed cell apoptosis more frequently in FIR-treated group.     Figure 3B). Vessel areas also decreased with FIR-treated groups ( Figure 4A, Figure 4B).  (Kohashi, 1996;Kohashi, 1993;Friedman, 2007). But we newly found that without cells they are produced by FIR irradiation.

Discussion
3) This facts are pretty commonly observed. I think this is not only a cause but also a result of cell death. Swelling of nucleus means disruption of nuclear lamina. This was induced by FIR and affect next results.