What is Far infrared Ray?

Far infrared ray is part of the sunlight spectrum that is invisible to the human eye. The complete spectrum of sunlight consists of visible and invisible rays. The visible rays are red, orange,yellow, green, indigo, blue, and violet in color. The invisible rays are Ultra Violet, X-rays, Gamma, Cosmic, Microwave, Long Wave, Electrical Wave, and Infrared.Electromagnetic waves between visible light and the microwave are called infrared waves. The wavelength of infrared waves range from 0.76 micron to 1,000 microns. This range is further subdividedinto the ranges of Near, Medium, and Far-Infrared Rays with following wavelength: 0.76 to 1.5microns; 1.5 to 4 microns; and 4 to 1,000 microns respectively.Far infrared waves are thermal. In other words, we experience this type of infrared radiation every day in the form of heat. Unlike ultraviolet light, FIR is harmless to the body.

How Can A Fiber Emit Far Infrared Ray?

How can the bio-ceramic emit far infrared? Will there be any side effects? Actually, the principle is very simple. It is a well-known fact that human body constantly emits thermo energy. When the bio-ceramic is stimulated by the thermo energy of human body, it converts it into the far infrared rays and reflects it back into the human body. So the far infrared ray emitted by the far infrared fiber is really powered by our own body and there are no known side effects.

What do FIR do?

The FIRs whose wavelength falls between 4 to 50 microns are also commonly called Biogenetic rays. Biogenetic rays have been shown to promote the healing and growth of living cells especially inplants, animals and human beings. All of the FIR products we make emit FIR with wavelength concentrated between 8 to 15 microns

How can FIR heal human body?

One of the reasons that FIR has beneficial results in a variety of illnesses is because FIR can deeply penetrate skin and underlying tissues. When FIR penetrates the skin, they come into contactwith protein, collagen and fats. By stimulating micro-vibrations, FIR can elevate tissue temperatures. The human body then can be revitalized because of the improved micro circulation.Another reason is that FIR has the ability to remove toxins in the body, which are often at the core of many health problems. The build up of toxins in a health body could block the normal bloodcirculation and impair the cellular energy. When FIR waves are applied, the water molecules that encapsulate the toxins get heat up, and start to vibrate. This vibration reduces the ion bonds of theatoms that are holding together the molecules of water. As the breakdown of the water molecules occurs, encapsulated gases and other toxic materials are released and the body gets rejuvenated.In addition, FIR also improves human health through following functions:

Rejuvenates the skin and muscle tone.

Increases oxygen in the blood cell

Promotes regeneration and fast healing

Improves the autonomic functions of the nervous system

Reduces fatty acids in the tissues

Increase metabolism between blood and tissue.

What are the symptoms that FIR products are effective for?

FIR products have been shown to have therapeutic effect to many chronic healthy symptoms. Enumerating all of them here might be difficult. However, following list contains some of the symptomsthat have been shown to respond very well to FIR products:ArthritisMuscle pain and spasmShoulder or joint stiffnessRaymaud’s syndromeWith long sleeve shirts and long john bottoms, FIR products have also been shown to speed up weight loss, improve immune system and the overall health.

What is microcirculation?

Microcirculation refers to the flow of blood through the vascular network lying between the arterioles and venules; includes capillaries, metarterioles and arteriovenous anastomoses. The mainfunction of microcirculation is carry oxygen and nutrition matter and ship out carbon dioxide, lactic acid and metabolic production.

How long does it take to feel the effects?

Time taken to feel the effect varies depends on the symptom one has, the products one uses and personal health condition. One may start feeling the pain relieving impacts of a knee bandimmediately, while others wearing our garments may take several weeks to notice the profound whole body results.Is it possible that one may not feel anything using these products?Depends on the individual’s health needs, some time, the effects of our products are more subtle. If you do not feel anything by using our FIR products, your body may be healthier thanothers. In such case, using FIR garments will provide the benefits of long term preventative therapy.

How often can I use FIR products?

Our FIR health products can be conveniently used as often as you like, and as long as you like. Our products are very comfortable to wear and many people use them for 8 - 10 hours a day.

Are there any side effects of FIR products?

There are no side-effects from using FIR products. As the body gets rid of old toxins in the body, some individuals may experience some initial detoxification symptoms. This is usually a good signthat healing effects of the FIR garment is working, and may be expected. If you continue experiencing prolonged symptoms, discontinue using the product and contact your physician.

What are your FIR products made from?

Our FIR products are made from man-made fibers impregnated with micro-particles of bio-ceramic powder. All of our FIR products are made from fibers with thisadvanced technology.

What is the “bio-ceramics”?

The bio-ceramics used in the FIR production are a special kind of natural lead free stones and minerals (like Silica Oxide (SiO2).

What is the FIR wavelength of your product?

The recommended length of the Far Infrared wave length is 8-15 Microns. This is the wave length that can easily be absorbed by the body.

Are FIR product washable?

Yes, FIR health products are washable: Hand wash or gentle cycle in cold water. Detergent or mild soap can be used. Natural dry in room temperature.

Avoid heat and direct sunlight. dry clean, iron or bleach

Is there any age limit for using this product?

There is no age limit for using this product. In fact, FIR is needed from the day you were born until old age.

In Vivo. 2000 Mar-Apr;14(2):321-6.
Effects of far-infrared ray on reproduction, growth, behaviour and some physiological parameters in mice.
Udagawa Y, Nagasawa H.
Source

Experimental Animal Research Laboratory, Meiji University, Kanagawa, Japan.
Abstract

The effects of chronic exposure to far-infrared ray (FIR) on reproduction, growth, behaviour, survival time and some related parameters were examined in SHN mice. The reproductive parameters differed slightly between the females on the normal racks and those on the FIR racks, which emitted FIR from the ceiling. The age and body weight on the day of vaginal opening was lower in the experimental mice born and maintained on the FIR rack than in the control on the normal rack. In both sexes, the levels of urinary components in the experimental group was significantly higher than the control at 6-7 months of age. Spontaneous motor activity of females during the light and dark phases were higher and lower, respectively, in the experimental group than the control. The survival rate was significantly higher in the experimental group than the control. These findings suggest that FIR has 'normalization effects' on the organisms.

Frequently Asked Questions about

DRY

The Feel good Technology  is a functional-finishing for all kinds of textiles. They stay three times dry.

Dry on the inside: Moisture caused by perspiration is quickly absorbed, transported away from the body and can evaporate faster.

Dry on the outside: Moisture is reliably repelled.

Dry in a flash: Textiles with this finishing dry significantly faster than without it.

How does  work?

The finishing combines two technologies on just one textile: From the outside to the middle of the textile it is water repellent, while from the inside to the middle it is treated to absorb water. The optical, hand and air permeability features are not influenced by the treatment.

What effect does  have?

ensures a feel good effect during all activities:

Diminishes perspiration marks

Just the textile-inside absorbs moisture. That’s why perspiration marks are as good as invisible from the outside.

Generates a cooling effect

With perspiration evaporates close to the body. This supports the natural cooling function of the body and thus ensures more energy during active phases.

Improved dirt and water repellence

Textiles with -finishing are water repellent and significantly less susceptible to dirt.  

What makes  different from competitor products?

Is the only technology that displays hydrophilic and hydrophobic properties on a single layered textile. The combination of water repellence and moisture-management is unique in finishing procedures and is patented .

Is particularly suitable for textiles that are directly worn on the skin (next-to-skin). For instance, tee shirts, pullovers, blouses, shirts, pants, or underwear. It makes sense for those who find themselves time after time in intensive, hectic, or hot environments – e.g. sport, daily work, outdoor activities or while traveling.

Is applicable on all textiles (cotton, synthetics, etc.). Through the finishing, all natural fiber textiles (cotton or wool) become functional, while textiles made of synthetic fibers are even more functional.

Will the air permeability or hand properties of the material be affected by DRY?

DRYneither has an influence on the air permeability nor the hand properties of a material.

How long does the finishing stay intact?

The wash permanence  is circa 50 washing cycles. We recommend that textiles treated with ironed or tumble dried . No fabric softener should be used during the wash.

The feelgood effect  has got weaker – what to do?

In order to guarantee the optimal function over a longer period of time, the product should be ironed or tumble dried after washing. Thereby the finishing can almost invariably often be regenerated.

Is there a certain washing detergent that I can’t use?

No fabric softener should be used that is water repellent or has hydrophilic properties, or both functions would be impaired.

cold Advantages

Optimal protection from heating up due to sunlight
Because textiles finished with cold heat up less in all colors compared with material without a cold finish.

Textiles stay cool to the touch
When applied to clothing this means that the wearer perspires less, feels better and is capable of greater performance.

Reliable protection from UV rays (minimum UPF 30)
Protection from UV-A und UV-B rays. UV-A rays accelerate the aging of skin. UV-B rays cause a pigment change, resulting in long term browning. Without effective UPF protection (Ultraviolet Protection Factor), aggressive oxygen molecules are released which cause sunburn or which can lead to the formation of melanoma.

FIR AND CANCER

Jun Ishibashi,¹ Kikuji Yamashita,¹ Tatsuo Ishikawa,¹ Hiroyoshi Hosokawa,² Kaori Sumida,¹ Masaru Nagayama,² and Seiichiro Kitamura¹

Corresponding author.

This article has been cited by other articles in PMC.

• Other Sections▼

Materials and methods

FIR incubator

As previously reported [11], we fabricated an FIR radiant-panel incubator by coating a carbon/silica/aluminum oxide/titanium oxide ceramic (radiation efficiency > 97%) using a polycarbonate printing technique (Bloodissue Co. Ltd. Tokushima, Japan). The incubator has a stably irradiate system with FIR at wavelengths between 4 and 20 μm (maximum at 7–12 μm) under conditions of 100% humidity, 37.0 ± 0.5°C, and 5% CO₂ in air.

Calculation of FIR absorbed per 10-cm tissue culture dish in the FIR incubator

Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed that the ceramics coating inside the CO₂ incubator emits FIR at 4 W m⁻¹ str⁻¹ μm⁻¹ at wavelengths between 4.486 and 20.256 μm, with a maximal emission of 11.6 W m⁻¹ str⁻¹ μm⁻¹ at 9 μm, which is >95% of the emission rate of an ideal black body. Since the ceramic coating was maintained at 40°C, the total generating energy, integrated over the entire range of wavelengths, was calculated to be 130.225 Wm⁻² str⁻¹. The total area of the FIR-emitting ceramic surface was 1.2385 (m²). Therefore, the total energy emitted into the incubator was 161.28366 W/str. Assuming that FIR is emitted in all directions, the total emission was 2026.7502 J/sec. Given that the volume of the CO₂ incubator was 0.1257 m³ and the volume of culture medium was 6 ml, the amount of energy absorbed by each 10-cm culture dish was 0.09674 J/sec. The surface area of each 10-cm culture dish was 78.5 cm², so that the energy reaching the base of the dish was 0.001232 J/sec/cm². Thus, over a 1-h period 4.4352 J/h cm² was absorbed by each 10-cm culture dish.

Cell lines and cell culture

A431 human epithelial vulva carcinoma cells and Sa3 human gingival squamous carcinoma cells were purchased from RIKEN Cell Bank (Tsukuba, Japan). A HSC3 human tongue squamous carcinoma cells, A549 human lung carcinoma cells, and MCF7 human breast carcinoma cells were purchased from Health Science Research Resources Bank (Sennan, Japan). A431, A549, and MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient mixture (Sigma, St. Louis, MO, USA). HSC3 and Sa3 cells were cultured in Eagle’s basal medium (Sigma). All culture medium was supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/ml penicillin G, 100 μg/ml streptomycin sulfate, and 250 ng/ml amphotericin B (Invitrogen, Carlsbad, CA, USA). Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air. The medium were replaced every 2 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 8 using 0.2% Trypan blue and a hemocytometer. Incorporation of 5-bromo-2′-deoxyuridine (BrdU) was used to determine the amount of DNA synthesis. DNA synthesis by proliferating cells was assessed using a BrdU labeling and detection kit III (Roche, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, cells (5 × 10³ per well) were seeded in 96-well tissue culture plates (Nunc) and then placed in the FIR incubator for 4 days, and BrdU incorporation was measured during the logarithmic growth phase (i.e., before the cells were confluent) by treating the cells for 4 h at 37°C with 10 μM BrdU. BrdU incorporation was quantified by measuring the absorbance of the substrate reaction (405 nm) and the absorbance at the reference wavelength (590 nm) using an ImmunoMini NJ-2300 (System Instruments, Tokyo, Japan). Absorbance values directly correlated with the amount of DNA synthesis and therefore the number of proliferating cells.

Histochemistry

Cells were grown on 22-mm² glass coverslips in 6-well culture dishes (Nunc). After 4 days of FIR irradiation, the cells were observed with a CK40 phase contrast microscope (Olympus, Tokyo, Japan), fixed, and stained with hematoxylin and eosin. For immunofluorescent staining of heat shock protein (HSP) 70, cells were washed in Phosphate Buffered Saline (PBS), fixed for 20 min in 4% paraformaldehyde in PBS, washed three times for 5 min each in PBS, and blocked for 1 h at room temperature with 5% goat serum. Cells were incubated at 4°C overnight in 1:200 mouse monoclonal antibody to HSP70 (Stressgen, Victoria, Canada) in PBS containing 1 mg/ml bovine serum albumin. After washing, the cells were incubated with 1:400 FITC (Fluorescein isothiocyanate)-labeled goat anti mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The localization of intracellular HSP70 protein was identified using a BX51 confocal microscope (Olympus) and a Cool SNAP CF digital camera (Roper Scientific, Trenton, NJ, USA) and calibrated using RS Image Express software (Roper Scientific).

Microarray studies and data analysis

Four days after FIR irradiation, two control and two FIR-irradiated samples were prepared for microarray hybridization. Total RNA was extracted using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Agilent human 1A ver.2 microarray slides (Agilent Technologies, Palo Alto, CA, USA) were used for the hybridization. The quality of RNA samples was monitored using an Agilent 2100 bioanalyzer (200 ng each). To produce labeled cRNA (complementary RNA), high-quality RNA was amplified and labeled with Cy5-and Cy3-CTP (Amersham Biosciences, Buckinghamshire, UK) using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent) according to the manufacturer’s protocol. After the amplification and labeling, the dye incorporation ratio was determined using a Nanodrop spectrophotometer, and the ratios were within 10–20 pmol per μg cRNA, which is the range suggested by the manufacturer for hybridization. For hybridization, an Agilent 60-mer oligo microarray (Rev. 7, SSC Wash/6-screw hybridization chamber) was used according to the manufacturer’s protocol. Briefly, 750 ng Cy3-labeled control and 750 ng Cy5-labeled MPP+-treated sample were mixed and incubated for 17 h with an SSC-washed microarray slide from an Agilent In situ Hybridization Kit. Sample pairs were dye-swapped and processed at the same time. The washed slides were immediately dried under a stream of ultrapure N₂ in an ozone-free atmosphere. After drying, the slides were scanned using an Agilent Technologies Microarray Scanner with the PMT setting at 770 for Cy5 and 670 for Cy3, and the raw data were normalized and analyzed using GeneSpring 7.0 software (Silicon Genetics, Santa Clara, CA, USA). For normalization, per spot and per chip intensity-dependent (LOWESS) normalization was used to correct for the intensity-dependent ratio bias¹². In addition, the following filters were applied to improve the quality of the data: eliminate saturated signal, eliminate non uniformity of background, eliminate non uniformity of feature, Feature Population Outlier, eliminate low-signal feature of background signal + 2.6 × SD, and eliminate P-value < 0.01. Genes were further classified for process and function according to their GO term information (http://www.godatabase.org).

Stable transfection of HSP70A

The HSP70A expression vector pcDNA3-HSP70A containing the cDNA for full-length human HSP70A was a generous gift from Dr. Hector R. Wong (Children’s Hospital Medical Center, Cincinnati, OH, USA). HSP70A cDNA was subcloned into the Xba I and Bam HI sites of pcDNA3.1(−) (Invitrogen). Cells grown on 60-mm dishes were transfected with 8 μg of pcDNA3-HSP70A, or pcDNA3.1 (Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The transfected cells were selected with 400 μg/ml G418 (Sigma), and clones formed were collected and maintained separately in medium supplemented with 400 μg/ml G418.

Reverse transcription (RT)-polymerase chain reaction (PCR)

Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer’s instructions. The concentration and purity of the RNA were determined spectrophotometrically. One microgram of total RNA was reverse-transcribed into first-strand cDNA. Next, oligo(dT_12–18) primer (Invitrogen), 10 mM dNTP mix (Invitrogen), 25 mM MgCl₂ (Promega, Madison WI, USA), and 0.1 M dithiothreitol (Invitrogen) were added, and the mixture was incubated for 2 min at 42°C. Next, the RT reaction was performed by adding SuperScript II RT (Invitrogen) and incubating at 42°C for 50 min, followed by 70°C for 15 min. The reaction was terminated by rapid cooling on ice, after which RNA in the sample was degraded by treatment with RNase H (Invitrogen) at 37°C for 20 min. Polymerase chain reaction (PCR) was carried out using a 1-μl sample of the RT reaction and Ready Mix PCR Master Mix (AB gene, Epsom, Surrey, UK). The analyzed genes and the respective primer sequences were as follows: HSP70A, 5′-TGTTCCGTTTCCAGCCCCCAA-3′ (sense) and 5′-GGGCTTGTCTCCGTCGTTGAT-3′ (antisense); HSP70B, 5′-CTCCAGCATCCGACAAGAAGC-3′ (sense) and 5′-ACGGTGTTGTGGGGGTTCAGG-3′ (antisense); HSP70C, 5′-TTGAGGAGGTGGATTAGGGGC-3′ (sense) and 5′-AGCCTTTGTAGTGTTTTCGCC-3′ (antisense); glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense). PCR was carried out using cycles (30 for HSPs and 23 for G3PDH) of 94°C for 45 s, 58°C for 30 s (all HSPs) or 52°C (G3PDH), and 72°C for 90 s. Finally, primer extension was performed for 10 min at 72°C. A 10-μl sample of each PCR product was separated by electrophoresis on a 1.5% polyacrylamide gel in Tris borate/EDTA buffer and stained with ethidium bromide.

Quantitative real-time RT-PCR data analysis

To determine the level of HSP70A mRNA, quantitative real-time RT-PCR was carried out using a LightCycler and the Fast Start DNA Master SYBR Green I Kit (Roche). The reaction contained 50 ng of cDNA and 100 pmol of each primer in a final volume of 10 μl. The gene-specific primers were as follows: HSP70A, 5′-TGTTCCGTTTCCAGCCCCCAA-3′ (sense) and 5′-GGGCTTGTCTCCGTCGTTGAT-3′ (antisense); and α-actin, 5′-ATAGCACAGCCTGGATAGCAACGTAC-3′ (sense) and 5′-CACCTTCTACAATGAGCTGCGTGTG-3′ (antisense). The concentration of Mg²⁺ was 3 mM. In all cases, a first phase of denaturation was performed at 95°C for 10 min. Amplification was carried out for cycles of denaturation at 95°C for 10 s, hybridization for 10 s (58°C for HSP70A or 60°C for α-actin), and elongation at 72°C (20 s for HSP70A or 10 s for α-actin). Product specificity was evaluated by melting curve analysis. Fluorescence data were analyzed using LightCycler Software Ver. 3.5 (Roche). Crossing points were established using the second derivative method. The relative amount of target transcript in the sample was calculated by dividing the amount of target by the amount of internal standard (α-actin). Results were expressed as the target/internal standard concentration ratio calculated from the calibration curve. Since the target and internal standard genes had different sequence and amplicon lengths, it was expected that they would show different PCR efficiencies. Therefore, the PCR efficiency (10^−1/m, where m is the slope from the calibration curve) was first established for each pair of primers. All reactions were performed in triplicate.

HSP70 Enzyme-Linked Immuno Sorbent Assay (ELISA)

HSP70 protein was quantified in cell lysates using a commercially available ELISA kit for human HSP70 (Stressgen). Cells (10⁵ per well) were seeded in 6-well plates (Nunc). After 4 days, the cells were lysed, and all samples were assayed at optimal dilutions according to the manufacturer’s instructions.

Protein extraction and Western blotting

Cells (1 × 10⁶) were grown in 60-mm tissue culture dishes (Nunc). After removing the cell culture medium from the culture dishes (Nunc) and washing the cells twice with cold PBS(−), the cells were lysed in lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1 μg/mL leupeptin). Protein levels were measured by the Lowry method [13] using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Cell lysate containing 15 μg of protein for HSP70 was subjected to Sodium Dodesyl Sulfate (SDS)-polyacrylamide gel electrophoresis. Separated proteins were then transferred from the gel to a polyvinylidene difluoride membrane. After blocking with 5% skim milk in PBS-Tween, the membrane was incubated for 1 h at room temperature with primary antibody in PBS-T containing 5% skim milk, followed by three 10-min washes with PBS-T. Next, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-labeled secondary antibody and washed three times for 10 min with PBS-T. Immunoreactive protein was detected using an ECL plus kit (Amersham Biosciences) and visualization by exposure to Hyperfilm (Amersham Biosciences). The primary antibodies used were rabbit anti-HSP70 (Stressgen), anti α-actin (Sigma), and the secondary antibody was horseradish peroxidase-conjugated anti-mouse (Zymed Laboratories, South San Francisco, CA, USA) or anti-rabbit IgG (Amersham Biosciences).

Small interfering RNA (siRNA)

We designed 21-nucleotide siRNAs targeting human HSP70A and HSP70C according to the manufacturer’s instructions (Dharmacon, Lafayette, CO, USA) and corresponding to the sequence 5′-AAGAACCAGGUGGCGCUGAAC-3′. It was not possible to design a siRNA specific to HSP70A because its mRNA was highly homologous to the mRNA for HSP70C (see Table 1). We used siCONTROL Non-Targeting siRNA (Dharmacon; 5′-UAGCGACUAAACACAUCAAUU-3′) as a negative control because it does not match the sequence of any known human or mouse genes. Cells (1 × 10⁴/well) were plated in 96-well culture dishes (Nunc) and cultured for 24 h at 37°C in a 5% CO₂ atmosphere. When the cells reached 70–90% confluence, they were transfected with 100 nM siRNAs to HSP70A and HSP70C or control siRNAs complexed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. The growth medium was removed after 6 h, and the culture dish was transferred to the FIR incubator. After incubation for 48 h at 37°C in a 5% CO₂ atmosphere, cell proliferation was assessed by BrdU incorporation.

Table 1 Characterization of Hsp70 (A, B, and C)

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. Multiple group comparisons were performed by one-way ANOVA, followed by the Tukey–Kramer multiple group comparisons test. All statistical analyses were performed using Statcel 2 software (OMS publishing, Saitama, Japan).

• Other Sections▼

Results

FIR irradiation selectively inhibits the growth of specific cancer cell lines

To clarify the effect of FIR irradiation on the proliferation of cancer cell lines, we irradiated five cancer cell lines (A431, HSC3, Sa3, A549, and MCF7) with FIR and measured the number of live cells by Trypan blue dye exclusion. Although the proliferation of HSC3, Sa3, and A549 cells were significantly suppressed on day 8 of culture (45.75%, 74.63%, and 65.79%, respectively), FIR irradiation had little effect on the growth of A431, or MCF7 cells (Fig. 1).

Fig. 1 Effect of FIR irradiation on cell growth of five cancer cell lines. Cells (1 × 105) were plated in 24-well dishes and cultured for 8 days. Cell numbers were counted every other day. Although proliferation of HSC3, Sa3, (more ...)

Microarray analysis and extraction of candidate gene for FIR control

Several gene families showed high correlations between endogenous expression (signal in the microarray) and the growth rate, proteins involved in cell proliferation, cytoskeletal proteins, cell cycle components, and protein kinases. Especially, in the analysis of stress factor, some genes encoding the HSPs which were well known that they participate in the cellular resistance to stress were focused. Of the 35 HSP genes on the microarray, HSP70 showed the highest correlation with the growth rate (Fig. 2a, b). We examined this further by real time RT-PCR, and we found that HSP70 is most highly expressed in A431 cells at all stages of the cell cycle. Lower levels of expression were found in HSC3 and Sa3 cells (Fig. 2c). We did not find a statistically significant difference between the expression of this gene between control (unirradiated) and FIR-irradiated A431, HSC3, or Sa3 cells. An ELISA for HSP70 in untreated cells showed similar results, specifically, that the expression of HSP70A was higher in A431 cells than in the HSC3 or Sa3 cells (Fig. 2d).

Fig. 2 Relationship between the expression of HSP70 and the suppression of proliferation by FIR irradiation. (a) Correlation between the signal from the microarray for the HSP family members in the control group and the correlation coefficient. (b) Correlation (more ...)

Increased expression of HSP70A improves the survival of HSC3 cells after a limited exposure to FIR

To directly determine whether HSP70 can protect cells from FIR-induced cell death, we developed A431 and HSC3 cell lines stably expressing human HSP70A (A431-HSP70A and HSC3-HSP70A cells, respectively). Control cells were transfected with empty pcDNA3.1 (A431-Neo and HSC3-Neo; Fig. 3a, b). In our initial experiments, we found that exposure of HSC3 and Sa3 cells but not A431 cells to limited FIR causes G2/M arrest and induces partial hypertrophy to necrosis (data not shown). To determine whether increased expression of HSP70A confers protection against FIR, cell survival was examined in FIR-irradiated A431-HSP70, A431-Neo, A431-wt, and HSC3-HSP70, HSC3-Neo, and HSC3-wt cells. We found that over expression of HSP70A increased cell proliferation in A431 and HSC3 cells. Furthermore, the proliferation of FIR-irradiated and control (unirradiated) A431-HSP70A cells was similar (Fig. 3c). The survival rate after 6 days of FIR irradiation was significantly higher in HSC3-HSP70A cells than in HSC3-Neo or HSC3-wt cells. In addition, the proliferation of FIR-treated HSC3-HSP70A cells was similar to that of control HSC3-HSP70A cells. BrdU incorporation was significantly higher in FIR-irradiated or control A431-HSP70A cells than in A431-Neo or A431-wt cells (Fig. 3d). Although BrdU incorporation of FIR-irradiated HSC3-wt and HSC3-Neo cells was lower than in unirradiated HSC3-wt and HSC3-Neo cells, it was similar in FIR-irradiated and unirradiated HSC3-HSP70A cells. Surprisingly, BrdU incorporation by HSC3-HSP70A cells was significantly higher in both FIR-irradiated and unirradiated cells. These data demonstrate that over expression of HSP70A in HSC3 and A431 cells did not affect their proliferation, and also their morphology, even when they were irradiated with FIR (Fig. 3e).

Fig. 3 Over expression of HSP70 prevents the suppression of cell growth and induction of cell hypertrophy by FIR. (a) Real-time RT-PCR of HSP70 expression. Cells overexpressing HSP70 were established. (b) Representative Western blots demonstrating increased (more ...)

Knocking down HSP70A by using siRNA improves the deth of HSC3 cells after a limited exposure to FIR

We next examined the effect of knocking down HSP70A and HSP70C mRNA and HSP70 protein expression using siRNA. Transfection with HSP70A/C siRNA effectively decreased HSP70A and HSP70C mRNA (Fig. 4a) and protein levels (Fig. 4b) in both A431 and HSC3 cells without affecting the level of HSP70B mRNA or protein. HSP70A/C siRNA did not suppress BrdU incorporation in unirradiated A431 cells, but it suppressed BrdU incorporation in cells irradiated with FIR (Fig. 4c). Similarly, the HSP70A/C siRNA enhanced the suppression of BrdU incorporation by FIR irradiation. FIR irradiation also significantly suppressed BrdU incorporation in HSC3 cells transfected with the negative control siRNA (Fig. 4c). These results indicate that a decrease in HSP70 protein mediates the ability of limited FIR to inhibit the proliferation of A431 and HSC3 cells.

Fig. 4 Inhibition of HSP70 expression and BrdU incorporation in A431 and HSC3 cells after 48 h of transfection with 100 nM HSP70 siRNA. (a) RT-PCR of HSP70A, HSP70B, and HSP70C in A431 and HSC3 cells after treatment with siRNAs. C, wild-type (more ...)