Radiation is an effective cancer treatment, but cancer cells can acquire radioresistance, which is associated with increased DNA damage response and enhanced proliferative capacity, and therefore, it ...is important to understand the intracellular biochemical responses to γ-irradiation. The transient receptor potential melastatin 8 (TRPM8) channel plays roles in the development and progression of tumors, but it is unclear whether it is involved in the DNA damage response induced by γ-irradiation. Here, we show that a TRPM8 channel inhibitor suppresses the DNA damage response (phosphorylated histone variant H2AX-p53-binding protein 1 (γH2AX-53BP1) focus formation) and colony formation of B16 melanoma cells. Furthermore, the TRPM8 channel-specific agonist WS-12 enhanced the DNA damage response and increased the survival fraction after γ-irradiation. We found that the TRPM8 channel inhibitor enhanced G2/M phase arrest after γ-irradiation. Phosphorylation of ataxia telangiectasia mutated and p53, which both contribute to the DNA damage response was also suppressed after γ-irradiation. In addition, the TRPM8 channel inhibitor enhanced the γ-irradiation-induced suppression of tumor growth in vivo. We conclude that the TRPM8 channel is involved in radiation-induced DNA damage repair and contributes to the radioresistance of B16 melanoma cells. TRPM8 channel inhibitors might be clinically useful as radiosensitizers to enhance radiation therapy of melanoma.
It is therapeutically important to elucidate the factors involved in the radiation resistance of tumors. We previously showed that ATP is released from mouse melanoma B16 cells in response to ...γ-irradiation, but the role of adenosine, a metabolite of ATP, is still unclear. Here, we show that the adenosine A2B receptor is involved in DNA damage repair and radioresistance in mouse melanoma B16 cells. The DNA damage response after γ-irradiation was attenuated by pretreatment with A2B receptor antagonists, such as PSB603, while it was enhanced by pretreatment with A2B receptor agonists, such as BAY60-6583. γ-Irradiation decreased the cell survival rate, and pretreatment with PSB603 further reduced the survival rate. On the other hand, pretreatment with BAY60-6583 increased the cell survival rate after irradiation. The DNA damage response and the cell survival rate after γ-irradiation were both decreased in A2B-knockdown cells. In vivo experiments in mice confirmed that tumor growth was suppressed and delayed in the irradiated group pretreated with PSB603, compared with the irradiation-alone group. Our results indicate that adenosine A2B receptor contributes to radioresistance, and could be a new target for the development of agents to increase the efficacy of radiotherapy.
Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer ...cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software “Cancer Cell Finder” that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.
Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and ...pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.
Background: EGFR signaling maintains aerobic glycolysis, but the molecular mechanism is still undefined.
Results: Drug inhibition studies reveal that downstream signaling via the PI3K pathway is critical for glucose transport and metabolism.
Conclusion: The PI3K signaling regulates key metabolic activities in EGFR-mutant lung adenocarcinoma.
Significance: These data may guide the development of chemotherapeutic options, including targeting of the PI3K pathway and glucose transporter machinery.
Herein, we propose a spherical shell robot that can roll and move on its legs, and develop a prototype of the robot. Recently, there has been a growing demand for robots that can move freely and ...gather information on rough terrains, such as disaster sites, which are not accessible to humans. The robot developed here has two types of mobilities: rolling movement using a spherical shape and walking movement using its legs. Because the morphological transformation does not require recombination of parts, it can be reversibly performed via remote control. Therefore, the robot can select the movement method according to the environment, and reach the target point reliably even on uneven terrains, such as a disaster site. We designed a mechanism that enabled the transformation of the form and devised an operation method. Accordingly, a prototype was developed and tested. A rolling test on flat ground confirmed that the robot can roll over 5.0 m and its speed could be controlled using a gyro sensor. The leg locomotion test confirmed that the robot can turn and move straight ahead without turning over. In addition, we also conducted experiments, such as sudden stops and remote morphological deformation, to confirm the operation of the robot during rolling.
Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in ...the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.
Astaxanthin, a natural antioxidant, exists in non-esterified and esterified forms. Although it is known that astaxanthin can improve exercise endurance and cause metabolic improvement in skeletal ...muscle, the effects of the two different forms are unclear. We investigated the effects of the different forms of astaxanthin on endurance in mice. Eight-week-old ICR mice were divided into four groups: control; astaxanthin extracted from Haematococcus pluvialis in an esterified form; astaxanthin extracted from Phaffia rhodozyma in a non-esterified form; and astaxanthin synthesized chemically in a non-esterified form. After 5 weeks of treatment, each group was divided into sedentary and exercise groups. In the group fed astaxanthin from Haematococcus, the running time to exhaustion was longest, and the plasma and tissue concentrations of astaxanthin were significantly higher than those in the other groups. Astaxanthin from Haematococcus increased 5'-adenosine monophosphate-activated protein kinase levels in the skeletal muscle. Although the mice in the Haematococcus group ran for longer, hexanoyl lysine adduct levels in the skeletal muscle mitochondria were similar in the control and Haematococcus groups. Our results suggested that esterified astaxanthin promoted energy production and protected tissues from oxidative damage during exercise owing to its favorable absorption properties, leading to a longer running time.
Cationic amphiphilic peptides have the potential to function as agents for the treatment of microbial infections and cancer therapy. The cationic and hydrophobic parts of these molecules allow them ...to associate strongly with negatively charged bacterial or cancer cell membranes, thus exerting antimicrobial and anticancer activities through membrane disruption. Meanwhile, cyclometalated iridium(III) complexes such as fac-Ir(ppy)3 (ppy = 2-phenylpyridine) and fac-Ir(tpy)3 (tpy = 2-(4′-tolyl)pyridine) possess C 3-symmetric structures and excellent photophysical properties as phosphorescence materials, which make them important candidates for use in biological applications such as chemosensors, biolabeling, living cell staining, in vivo tumor imaging, and anticancer agents. We recently reported on some regioselective substitution reactions of Ir(tpy)3 and Ir(ppy)3 at the 5′-position (p-position with respect to the C–Ir bond) on the 2-phenylpyridine ligands and their subsequent conversions to a variety of functional groups. We report here on the design and synthesis of amphiphilic and luminescent tris-cyclometalated Ir complexes in which cationic peptides are attached through alkyl chain linkers that work as inducers and detectors of cell death. Ir complexes containing cationic peptides such as a KKGG sequence and alkyl chain linkers of adequate length (C6 and C8) exhibit considerable cytotoxicity against cancer cells such as Jurkat, Molt-4, HeLa-S3, and A549 cells, and that dead cells are well stained with these Ir complexes. Furthermore, an Ir complex in which the KKGG peptide is attached through a C6 linker displayed lower cytotoxicity against normal mouse lymphocytes. Mechanistic studies suggest that Ir complexes containing the KKGG peptide interact with anionic molecules on the cell surface and/or membrane receptors to trigger the Ca2+ dependent pathway and intracellular Ca2+ response, resulting in necrosis accompanied by membrane disruption.
Platypnea-orthodeoxia syndrome (POS) is a rare disease characterized by dyspnea and hypoxemia in orthostatism that improves in the recumbent position. We herein report an 81-year-old woman with ...dyspnea in the upright position following thoracic vertebral compression fractures. After the patient's daughter brought a recording showing decreasing SpO2 (peripheral capillary oxygen saturation) in the upright position as measured by a portable pulse oximeter outside the hospital, a small atrial septal defect (ASD) was detected. A contrast echocardiogram and four-dimensional flow magnetic resonance imaging demonstrated a right-to-left shunt. The patient's symptoms dramatically improved after percutaneous ASD closure. In conclusion, such new technologies are useful for diagnosing POS.
Suppression of T-cell growth is an important mechanism for establishment of self-tolerance and prevention of unwanted prolonged immune responses that may cause tissue damage. Although negative ...selection of potentially self-reactive T cells in the thymus as well as in peripheral tissues has been extensively investigated and well documented, regulatory mechanisms to dampen proliferation of antigen-specific effector T cells in response to antigen stimulation remain largely unknown. Thus, in this work, we focus on the identification of growth suppression mechanisms of antigen-specific effector T cells. In order to address this issue, we investigated the cellular and molecular events in growth suppression of an ovalbumin (OVA)-specific T-cell clone after stimulation with a wide range of OVA-peptide concentrations. We observed that while an optimal dose of peptide leads to cell cycle progression and proliferation, higher doses of peptide reduced cell growth, a phenomenon that was previously termed high-dose suppression. Our analysis of this phenomenon indicated that high-dose suppression is a consequence of cell cycle arrest, but not Fas-Fas ligand-dependent apoptosis or T-cell anergy, and that this growth arrest occurs in S phase, accompanied by reduced expression of CDK2 and cyclin A. Importantly, inhibition of MEK/ERK activation eliminated this growth suppression and cell cycle arrest, while it reduced the proliferative response to optimal antigenic stimulation. These results suggest that cell cycle arrest is the major mechanism regulating antigen-specific effector T-cell expansion, and that the MEK/ERK signaling pathway has both positive and negative effects, depending on the strength of antigenic stimulation.
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