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Previously, we reported that near-infrared irradiation that simulates solar near-infrared irradiation with pre- and parallel-irradiational cooling can non-thermally induce cytocidal effects in cancer cells. To explore these effects, we assessed cell viability, DNA damage response pathways, and the percentage of mitotic cancer cells after near-infrared treatment. Further, we evaluated the anti-cancer effects of near-infrared irradiation compared with doxorubicin in xenografts in nude mice by measuring tumor volume and assessing protein phosphorylation by immunoblot analysis. The cell viability of A549 lung adenocarcinoma cells was significantly decreased after three rounds of near-infrared irradiation at 20 J ⁄ cm2. Apoptotic cells were observed in near-infrared treated cells. Moreover, near-infrared treatment increased the phosphorylation of ataxia-telangiectasia mutated (ATM) at Ser1981, H2AX at Ser139, Chk1 at Ser317, structural maintenance of chromosome (SMC) 1 at Ser966, and p53 at Ser15 in A549 cells compared with control. Notably, near-infrared treatment induced the formation of nucleic foci of γH2AX. The percentage of mitotic A549 cells, as measured by histone H3 phosphorylation, decreased significantly after three rounds of near-infrared irradiation at 20 J ⁄cm2. Both near-infrared and doxorubicin inhibited the tumor growth of MDA-MB435 melanoma cell xenografts in nude mice and increased the phosphorylation of p53 at Ser15, Chk1 at Ser317, SMC1 at Ser966, and H2AX at Ser139 compared with control mice. These results indicate that near-infrared irradiation can non-thermally induce cytocidal effects in cancer cells as a result of activation of the DNA damage response pathway. The near-infrared irradiation schedule used here reduces discomfort and side effects. Therefore, this strategy may have potential application in the treatment of cancer. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02310.x, 2012)
Near-infrared (NIR) is electromagnetic radiation that simultaneously exhibits both wave and particle properties and is known to be strongly absorbed by water, hemoglobin, and myoglobin. Previously, we reported that NIR irradiation that simulates solar NIR with pre- and parallel-irradiational cooling can penetrate the skin and non-thermally affect the dermis,[1-4] subdermal blood plexus, superficial skeletal muscles,[5, 6] and other tissues.[7-10] Furthermore, NIR induces apoptotic changes in both smooth muscle fibers of the subdermal blood plexus and skeletal muscle fibers of the panniculus carnosus in rats, which results in long-lasting vasodilation and muscle thinning.[5, 6]
At a wavelength of 904 nm, NIR has been shown to have antitumor activity and to increase cytomorphological changes by inducing apoptosis in neoplastic cells. In addition, actively proliferating cells exhibit increased sensitivity to red and NIR wavelengths.[12, 13] It appears that NIR irradiation induces DNA strand breaks and cell death by apoptosis, and can elicit photodisruptive destruction of tumor tissue. However, in-depth studies to date have not explored the optimal NIR wavelength that is most effective for treating cancer.
In a previous study, we found that the NIR spectrum between 1100 and 1800 nm, with water filtering to remove wavelengths between 1400 and 1500 nm, significantly suppresses the proliferation of various cancer cell lines and significantly inhibits the growth of MCF7 breast cancer cells transplanted into SCID mice and MDA-MB435 melanoma cells transplanted into nude mice. We hypothesized that this specialized NIR irradiation may induce DNA damage in cancer cells and therefore provide a potentially effective approach for cancer treatment.
It is well known that DNA damage checkpoint proteins, including ataxia-telangiectasia mutated (ATM), play a pivotal role in the maintenance of chromosomes.[16, 17] To avoid the carryover of damaged DNA to the next generation of cells, checkpoint signals provide a safeguard in several critical phases of the cell cycle. The requirement of ATM and downstream molecules for checkpoint activation and cell survival following DNA damage, γ-irradiation, or treatment with genotoxic agents has been well-documented.[18-20] Ataxia-telangiectasia mutated phosphorylates various substrates, including p53, Chk1, and structural maintenance of chromosome (SMC) 1 , and it is therefore important to define that exact biological mechanism taking place in cells treated with NIR irradiation.
To explore the biological effects of NIR irradiation for cancer treatment, we performed both ex vivo and in vivo testing. The MTT assay was used to investigate the cell viability of cultured cancer cells after they had been treated with NIR irradiation. We also evaluated the percentage of mitotic cancer cells by performing a G2/M checkpoint analysis and assessed the DNA damage response by immunoblot analysis. Finally, we performed in vivo studies to examine the anticancer effects of NIR irradiation compared with doxorubicin by measuring tumor volume and performing immunoblot analysis of samples from xenografts.
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The present study has demonstrated that a specific wavelength of NIR irradiation, which simulates solar NIR with pre- and parallel-irradiational cooling, can non-thermally induce cytocidal effects in cancer cells by inducing DNA damage. Near-infrared is an electromagnetic wave that simultaneously exhibits both wave and particle properties and is absorbed by sweat on the skin surface, water in the dermis, hemoglobin in dilated vessels,[2, 9] myoglobin in superficial muscles,[5, 6] and bone cortical mass. Near-infrared can non-thermally induce the degeneration of myoglobin, which results in apoptosis of vascular smooth muscle cells and marked long-lasting vasodilation. Near-infrared penetrates superficial layers and is absorbed by chromophores, such as hemoglobin and myoglobin,[21, 22] which are oxygen-carrying proteins that have many α-helices.[10, 23, 24] The α-helices have strong amide bands in the infrared spectra that have characteristic frequencies and intensities and are thought to be involved in the resonance by NIR. Therefore, NIR may induce the resonance of α-helices in these oxygen-carrying proteins, resulting in protein degeneration, damage to oxygen storage and transport, and apoptosis.
Previously, NIR was been reported to induce cell death by apoptosis. Actively proliferating cells also show increased sensitivity to red and NIR radiation.[12, 13]
The NIR spectrum of biological materials is a result of the overtones and combination of O-H, C-H, and N-H group bond stretching vibrations. It is hypothesized that NIR mainly resonates helical structures, α-helices, and DNA. Moreover, it is predicted that NIR induces DNA damage, and this could be one of the mechanisms underlying apoptosis. However, to date there is no evidence demonstrating the effects of NIR on cellular DNA damage checkpoint signals.
In the present study, we used an NIR device emitting a spectrum of NIR irradiation from 1100 to 1800 nm with a water filter that excluded wavelengths between 1400 and 1500 nm, which are strongly absorbed by water and hemoglobin. By filtering out wavelengths below 1100 nm, around 1450 nm, and above 1850 nm, NIR irradiation can reach deeper tissues. However, an NIR device increases skin surface temperature and induces perspiration and blood vessel dilation, even with a water filter, which mediates the absorption of NIR radiation by water and hemoglobin. To counter this effect, in the present study we used contact cooling through a temperature-controlled sapphire window to reduce the skin surface temperature and thereby reduce perspiration and blood vessel dilation. These specific wavelengths and the cooling system enabled NIR irradiation to penetrate the skin surface without pain or epidermal burns,[27, 28] which was evident by the ability to treat mice without anesthesia. Moreover, contact burns or other adverse events were not observed.
In our previous study, we demonstrated that A549 lung adenocarcinoma cells responded well to DNA damage induced by ultraviolet and γ-irradiation. In addition, several DNA damage checkpoint signals have been discovered using A549 cells.[30, 31] Therefore, the A549 cell line provides a good in vitro model for understanding how NIR modulates cellular signal transduction related to the DNA damage response pathway. In the present study, A549 cell proliferation was significantly inhibited by NIR irradiation and this was due to apoptosis, as expected. Total NIR output appeared to correlate with cell survival. In addition, the correlation with efficacy seemed to be highest with the total amount of energy delivered and not the per pulse fluence, because multiple rounds of irradiation with a lower output appeared equally effective as higher fluence irradiation. Ten exposures at 20 J/cm2 achieved a comparable significant reduction in cell count as that of three exposures at 40 J/cm2. In addition, three exposures at 20 J/cm2 appeared to be close to an in vitro threshold energy dosage in the present study, as well as in our previous studies.[7, 10]
To determine whether NIR induces a DNA damage response, including the checkpoint signaling pathway, the phosphorylation of H2AX at Ser139 was measured as an index of the DNA damage of cells. After treatment with NIR, phosphorylation of H2AX occurred after two rounds of NIR at 20 J⁄cm2. The formation of γH2AX foci was clearly observed in NIR irradiation-treated cells. Ataxia-telangiectasia mutated is a key molecule in the DNA damage response and was clearly phosphorylated at Ser1981 after NIR irradiation. We also confirmed the dose-dependent phosphorylation of several DNA damage checkpoint molecules, including Ser15 of p53, Ser966 of SMC1, and Ser317 of Chk1 by NIR irradiation. Notably, KU55933, an ATM-specific inhibitor, markedly decreased the phosphorylation of checkpoint molecules. These results indicate that ATM is activated by NIR irradiation and transduces the downstream activation of other factors in the pathway. Therefore, our findings indicate that NIR irradiation exerts checkpoint signals by damaging cellular DNA.
We further examined the mitotic transition of cells using flow cytometry to assess the effect of the cellular DNA damage response by NIR. The percentage of mitotic A549 cells was evaluated by histone H3 phosphorylation at Ser10, and was found to be significantly decreased after more than three rounds of NIR irradiation at 20 J ⁄cm2. These data indicate that the G2/M checkpoint is activated by NIR-induced DNA damage to avoid mitotic error.
Near-infrared irradiation suppresses the proliferation of various kinds of malignant cells and damages highly proliferative cells, such as bone marrow cells. In our in vivo studies, we used MDA-MB435 cells because they develop highly proliferative skin neoplasms that appear to be susceptible to solar and artificial NIR irradiation. Near-infrared irradiation induced marked cytocidal tumor shrinkage compared with control after seven rounds of NIR treatment at 40 J/cm2, whereas DOX only statically inhibited MDA-MB435 tumor growth in nude mice. We further examined whether NIR irradiation treatment also activates the DNA damage response in tumors and found that NIR irradiation increased the phosphorylation of p53 at Ser15, SMC1 at Ser966, and Chk1 at Ser317 in the tumor samples. Similarly, DOX increased the phosphorylation of p53, SMC1, and Chk1 at these sites as well. These data indicate that each checkpoint protein was activated by NIR irradiation treatment via ATM activation.
The ATM protein kinases act as master controllers in DNA damage checkpoint signaling. In a previous study, ATM-deficient cells, derived from human ataxia telangiectasia (AT) patients, were found to exhibit chromosomal instability, telomere shortening, and defects in cellular responses to DNA double-strand breaks following exposure to infra-red and radiomimetic chemicals, including DOX. The results of the present study indicate that NIR irradiation causes cytocidal effects in cancer cells by inducing double-strand DNA breaks in an in vivo tumor model.
In our previous in vivo studies, histological findings showed tumor shrinkage and cell death in the center of the tumor mass, which supports the hypothesis that NIR electromagnetic properties non-thermally induce the biological effects observed.[7, 10] Near-infrared irradiation penetrates the skin and reaches the subcutaneous tissues without a significant increase in skin temperature, and the effects of NIR irradiation are independent of the generation of heat. If the cytocidal effect of NIR irradiation was induced thermally, the histology would have shown a gradient cytocidal effect from the superficial layer to the center of the tumor, and the thermal effect would be reduced by the contact cooling (20°C) of the NIR device. Due to surface cooling, NIR irradiation can penetrate deeper tissue and induce a marked non-thermal cytocidal effect in the center of the tumor mass. Furthermore, NIR irradiation treatment with very low output and fewer exposures (10 exposures of NIR at 20 and 40 J/cm2) also inhibited tumor growth. This output was so low that, on human skin, the sensation of heat would not be felt because of contact cooling. The NIR irradiation induced no pain and the mice did not withdraw from the treatment even though NIR treatment was performed without anesthesia. In addition, side effects, such as epidermal burns, were not observed and the mice appeared to be healthy throughout the study. Further studies are necessary to determine whether more output, increased frequency of treatments, or longer periods of irradiation may be even more effective in suppressing tumor growth.
There are several advantages of the NIR irradiation schedule determined in the present study, including a reduction in discomfort, limited side effects, and low cost. These characteristics were facilitated by repeated rounds of irradiation, which, if proven beneficial for cancer cell reduction in humans, may provide an alternative or adjunct treatment for transient mass reduction before surgery and could provide improved results and quality of life for patients. Near-infrared irradiation is frequently administered at a level of 40 J/cm2 for other indications and has a very high safety record with no significant complications. It should be noted that the present study was a preliminary study based on experiments in a limited variety of cancer cell lines. Additional studies are warranted in larger numbers and various types of cancer cell types and with longer post-treatment periods to evaluate the variations in treatment parameters and correlations with other antitumor therapies. Importantly, however, these studies hold promise in the design of more efficacious cancer treatments.