Prohydrojasmon Promotes the Accumulation of Phenolic Compounds in Red Leaf Lettuce.

Prohydrojasmon (PDJ) is a synthetic jasmonate derivative that is primarily used as a growth regulator, but its mechanism of action is unclear. In this study, we elucidated the effects of PDJ on phytochemical production in red leaf lettuce. The PDJ treatments promoted the accumulation of phenolic compounds in aerial plant parts. An LC-MS analysis revealed that these accumulated compounds were identified as cyanidin-3-O-glucoside, cyanidin-3-O-(6″-O-malonyl)-glucoside and cyanidin-3-O-(6″-O-malonyl)-glucoside methyl ester. The abundance of these compounds in lettuce extracts increased significantly in response to the PDJ treatment. Additionally, the LC-MS analysis also identified the accumulated phenolic compounds in the extracts of PDJ-treated lettuce, including caffeoyltartaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, and dicaffeoylquinic acid. Gene expression analyses indicated the PDJ treatments upregulated the expression of PAL, F3H, and ANS genes in lettuce. These results suggest that PDJ treatments enhance the expression of genes involved in the synthesis of anthocyanins and phenolic compounds, resulting in an increase in the quantities of these compounds, which reportedly have various functions affecting human physiology.

Effect of Prohydrojasmon on the Growth of Eggplant and Komatsuna.

Prohydrojasmon (PDJ) can improve the polyphenol and anthocyanin content and antioxidant activity of some crop plants, but it also shows a suppressive effect on the plant growth. This study aimed to investigate the inhibitory effect of PDJ on the growth of two crop plants: komatsuna (Brassica rapa var. periviridis) and eggplant (Solanum melongena). We applied various concentrations of PDJ drip-wise or by spraying to eggplant and komatsuna seedlings and made detailed observations of growth. In general, no significant suppressive effect of PDJ was observed in the aerial parts in both plants. However, a significant inhibitory effect was found in roots treated with PDJ at concentrations of 600 and 1000 ppm. Interestingly, komatsuna treated with PDJ at a concentration of 200 ppm in both approaches resulted in a significant increase in root weight up to 37%. At a concentration range of 200-400 ppm, PDJ showed no inhibitory effects, and in some cases slightly promoted root growth. Therefore, this could be the recommended concentration range. We conclude that application of PDJ can still be beneficial to the vegetable crops without causing serious inhibition or suppression effects on the growth, as long as it is kept at rather low concentrations.

Effect of prohydrojasmon on total phenolic content, anthocyanin accumulation and antioxidant activity in komatsuna and lettuce.

Prohydrojasmon has been reported to improve the quality of crops. However, most previous studies have investigated its application on fruits. Here, we evaluated the effect of prohydrojasmon on the growth and total phenolic content, anthocyanin content, and antioxidant activity in komatsuna (Brassica rapa var. periviridis) and lettuce (Lactuca sativa L.). Prohydrojasmon did not show any serious inhibitory effect. Prohydrojasmon applied to komatsuna at a concentration of 0.5 µM significantly increased the total phenolic content and anthocyanin content, and a concentration of 1 µM increased the antioxidant activity. In lettuce, prohydrojasmon at a concentration of 400 µM significantly increased the total phenolic content and anthocyanin content, while a concentration of 0.5 µM significantly increased the antioxidant activity. These results suggest that prohydrojasmon positively affects the phenolic compound and anthocyanin accumulation and antioxidant activity in komatsuna and lettuce without adversely affecting growth.

Relative biological effectiveness in canine osteosarcoma cells irradiated with accelerated charged particles.

Heavy ions, characterized by high linear energy transfer (LET) radiation, have advantages compared with low LET protons and photons in their biological effects. The application of heavy ions within veterinary clinics requires additional background information to determine heavy ion efficacy. In the present study, comparison of the cell-killing effects of photons, protons and heavy ions was investigated in canine osteosarcoma (OSA) cells in vitro. A total of four canine OSA cell lines with various radiosensitivities were irradiated with 137Cs gamma-rays, monoenergetic proton beams, 50 keV/µm carbon ion spread out Bragg peak beams and 200 keV/µm iron ion monoenergetic beams. Clonogenic survival was examined using colony-forming as says, and relative biological effectiveness (RBE) values were calculated relative to gamma-rays using the D10 value, which is determined as the dose (Gy) resulting in 10% survival. For proton irradiation, the RBE values for all four cell lines were 1.0-1.1. For all four cell lines, exposure to carbon ions yielded a decreased cell survival compared with gamma-rays, with the RBE values ranging from 1.56-2.10. Iron ions yielded the lowest cell survival among tested radiation types, with RBE values ranging from 3.51-3.69 observed in the three radioresistant cell lines. The radiosensitive cell line investigated demonstrated similar cell survival for carbon and iron ion irradiation. The results of the present study suggest that heavy ions are more effective for killing radioresistant canine OSA cells when compared with gamma-rays and protons. This markedly increased efficiency of cell killing is an attractive reason for utilizing heavy ions for radioresistant canine OSA.

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges.

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of 15O, 13N, and 11C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.

VE-821, an ATR inhibitor, causes radiosensitization in human tumor cells irradiated with high LET radiation.

BACKGROUND: High linear energy transfer (LET) radiation such as carbon ion particles is successfully used for treatment of solid tumors. The reason why high LET radiation accomplishes greater tumor-killing than X-rays is still not completely understood. One factor would be the clustered or complex-type DNA damages. We previously reported that complex DNA double-strand breaks produced by high LET radiation enhanced DNA end resection, and this could lead to higher kinase activity of ATR protein recruited to RPA-coated single-stranded DNA. Although the effect of ATR inhibition on cells exposed to low LET gamma-rays has recently been reported, little is known regarding the effect of ATR inhibitor on cells treated with high LET radiation. The purpose of this study is to investigate the effects of the ATR inhibitor VE-821 in human tumor and normal cells irradiated with high LET carbon ions. FINDINGS: HeLa, U2OS, and 1BR-hTERT (normal) cells were pre-treated with 1 µM VE-821 for 1 hour and irradiated with either high LET carbon ions or X-rays. Cell survival, cell cycle distribution, cell growth, and micronuclei formation were evaluated. VE-821 caused abrogation of G2/M checkpoint and forced irradiated cells to divide into daughter cells. We also found that carbon ions caused a higher number of multiple micronuclei than X-rays, leading to decreased cell survival in tumor cells when treated with VE-821, while the survival of irradiated normal cells were not significantly affected by this inhibitor. CONCLUSIONS: ATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation.

Novel characteristics of CtIP at damage-induced foci following the initiation of DNA end resection.

Homologous recombination (HR) is a major repair pathway for DNA double strand breaks (DSBs), and end resection, which generates a 3'-single strand DNA tail at the DSB, is an early step in the process. Resection is initiated by the Mre11 nuclease together with CtIP. Here, we describe novel characteristics of CtIP at DSBs. At early times following exposure of human cells to ionizing radiation, CtIP localized to the DSB, became hyperphosphorylated and formed foci in an ATM-dependent manner. At later times, when the initiation of resection had occurred, CtIP foci persist but CtIP is maintained in a hypophosphorylated state, which is dependent on ATM and ATR. Exposure to cycloheximide revealed that CtIP turns over at DSB sites downstream of resection. Our findings provide strong evidence that CtIP is continuously recruited to DSBs downstream of both the initiation and extension step of resection, strongly suggesting that CtIP has functions in addition to promoting the initiation of resection during HR.

The combination of Hsp90 inhibitor 17AAG and heavy-ion irradiation provides effective tumor control in human lung cancer cells.

Hsp90 inhibitors have become well-studied antitumor agents for their selective property against tumors versus normal cells. The combined treatment of Hsp90 inhibitor and conventional photon radiation also showed more effective tumor growth delay than radiation alone. However, little is known regarding the combined treatment of Hsp90 inhibitor and heavy-ion irradiation. In this study, SQ5 human lung tumor cells were used in vitro for clonogenic cell survival and in vivo for tumor growth delay measurement using a mouse xenograft model after 17-allylamino-17-demethoxygeldanamycin (17AAG) pretreatment and carbon ion irradiation. Repair of DNA double strand breaks (DSBs) was also assessed along with expressions of DSB repair-related proteins. Cell cycle analysis after the combined treatment was also performed. The combined treatment of 17AAG and carbon ions revealed a promising treatment option in both in vitro and in vivo studies. One likely cause of this effectiveness was shown to be the inhibition of homologous recombination repair by 17AAG. The more intensified G2 cell cycle delay was also associated with the combined treatment when compared with carbon ion treatment alone. Our findings indicate that the combination of Hsp90 inhibition and heavy-ion irradiation provides a new effective therapeutic alternative for treatment of solid tumors.

Hyperthermia-induced radiosensitization in CHO wild-type, NHEJ repair mutant and HR repair mutant following proton and carbon-ion exposure.

The DNA repair mechanisms involved in hyperthermia-induced radiosensitization with proton and carbon ion radiation exposure were investigated in the present study. In a previous study, Chinese hamster ovary (CHO) cells were exposed to low linear energy transfer (LET) photon radiation. These cells can be sensitized by hyperthermia as a result of inhibition of homologous recombination (HR) repair. The present study used wild-type, non-homologous end joining (NHEJ) and HR repair-deficient CHO cells to define the contributions of each repair pathway to cellular lethality following hyperthermia-induced hadron radiation sensitization. The cells were exposed to ionizing radiation, followed by hyperthermia treatment (42.5°C for 1 h). Hyperthermia-induced radiosensitization was determined by the colony formation assay and thermal enhancement ratio. HR repair-deficient cells exhibited no hyper-sensitization to X-rays, protons, or low and high LET carbon ions when combined with hyperthermia. Wild-type and NHEJ repair-deficient cells exhibited significant hyperthermia-induced sensitization to low LET photon and hadron radiation. Hyperthermia-induced sensitization to high LET carbon-ion radiation was less than at low LET radiation. Relative biological effectiveness (RBE) between radiation alone and radiation combined with hyperthermia cell groups was not significantly different in any of the cell lines, with the exception of wild-type cells exposed to high LET radiation, which exhibited a lower RBE in the combined group. The present study investigated additional cell lines to confirm the lower RBE observed in DNA repair-deficient cell lines. These findings suggested that hyperthermia-induced hyper-sensitization to hadron radiation is also dependent on inhibition of HR repair, as was observed with photon radiation in a previous study.

Monoglucosyl-rutin as a potential radioprotector in mammalian cells.

In the present study, the role of monoglucosyl­rutin as a potential radioprotector was investigated using mammalian cell culture models. Cell survival and DNA damage were assessed using colony formation, sister chromatid exchange and γH2AX assays. It was demonstrated that monoglucosyl­rutin was able to increase cell survival when exposed to ionizing radiation, possibly by decreasing the amount of base damage experienced by the cell. However, the present study also demonstrated that, despite monoglucosyl­rutin exhibiting radioprotective effects at low doses, high doses of monoglucosyl­rutin led to a decrease in plating efficiency and an increased doubling time. This effect may be due to double­strand breaks caused by high concentrations of monoglucosyl­rutin.

The complexity of DNA double strand breaks is a critical factor enhancing end-resection.

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) are deleterious damages. Two major pathways repair DSBs in human cells, DNA non-homologous end-joining (NHEJ) and homologous recombination (HR). It has been suggested that the balance between the two repair pathways varies depending on the chromatin structure surrounding the damage site and/or the complexity of damage at the DNA break ends. Heavy ion radiation is known to induce complex-type DSBs, and the efficiency of NHEJ in repairing these DSBs was shown to be diminished. Taking advantage of the ability of high linear energy transfer (LET) radiation to produce complex DSBs effectively, we investigated how the complexity of DSB end structure influences DNA damage responses. An early step in HR is the generation of 3'-single strand DNA (SSD) via a process of DNA end resection that requires CtIP. To assess this process, we analyzed the level of phosphorylated CtIP, as well as RPA phosphorylation and focus formation, which occur on the exposed SSD. We show that complex DSBs efficiently activate DNA end resection. After heavy ion beam irradiation, resection signals appear both in the vicinity of heterochromatic areas, which is also observed after X-irradiation, and additionally in euchromatic areas. Consequently, ~85% of complex DSBs are subjected to resection in heavy ion particle tracks. Furthermore, around 20-40% of G1 cells exhibit resection signals. Taken together, our observations reveal that the complexity of DSB ends is a critical factor regulating the choice of DSB repair pathway and drastically alters the balance toward resection-mediated rejoining. As demonstrated here, studies on DNA damage responses induced by heavy ion radiation provide an important tool to shed light on mechanisms regulating DNA end resection.

Comparison of human chordoma cell-kill for 290 MeV/n carbon ions versus 70 MeV protons in vitro.

BACKGROUND: While the pace of commissioning of new charged particle radiation therapy facilities is accelerating worldwide, biological data pertaining to chordomas, theoretically and clinically optimally suited targets for particle radiotherapy, are still lacking. In spite of the numerous clinical reports of successful treatment of these malignancies with this modality, the characterization of this malignancy remains hampered by its characteristic slow cell growth, particularly in vitro. METHODS: Cellular lethality of U-CH1-N cells in response to different qualities of radiation was compared with immediate plating after radiation or as previously reported using the multilayered OptiCell™ system. The OptiCell™ system was used to evaluate cellular lethality over a broad dose-depth deposition range of particle radiation to anatomically mimic the clinical setting. Cells were irradiated with either 290 MeV/n accelerated carbon ions or 70 MeV accelerated protons and photons and evaluated through colony formation assays at a single position or at each depth, depending on the system. RESULTS: There was a cell killing of approximately 20-40% for all radiation qualities in the OptiCell™ system in which chordoma cells are herein described as more radiation sensitive than regular colony formation assay. The relative biological effectiveness values were, however, similar in both in vitro systems for any given radiation quality. Relative biological effectiveness values of proton was 0.89, of 13-20 keV/µm carbon ions was 0.85, of 20-30 keV/µm carbon ions was 1.27, and >30 keV/µm carbon ions was 1.69. Carbon-ions killed cells depending on both the dose and the LET, while protons depended on the dose alone in the condition of our study. This is the first report and characterization of a direct comparison between the effects of charged particle carbon ions versus protons for a chordoma cell line in vitro. Our results support a potentially superior therapeutic value of carbon particle irradiation in chordoma patients. CONCLUSION: Carbon ion therapy may have an advantage for chordoma radiotherapy because of higher cell-killing effect with high LET doses from biological observation in this study.

Comparison of cellular lethality in DNA repair-proficient or -deficient cell lines resulting from exposure to 70 MeV/n protons or 290 MeV/n carbon ions.

Charged particle therapy utilizing protons or carbon ions has been rapidly intensifying over recent years. The present study was designed to jointly investigate these two charged particle treatment modalities with respect to modeled anatomical depth-dependent dose and linear energy transfer (LET) deliveries to cells with either normal or compromised DNA repair phenotypes. We compared cellular lethality in response to dose, LET and Bragg peak location for accelerated protons and carbon ions at 70 and 290 MeV/n, respectively. A novel experimental live cell irradiation OptiCell™ in vitro culture system using three different Chinese hamster ovary (CHO) cells as a mammalian model was conducted. A wild-type DNA repair-competent CHO cell line (CHO 10B2) was compared to two other CHO cell lines (51D1 and xrs5), each genetically deficient with respect to one of the two major DNA repair pathways (homologous recombination and non-homologous end joining pathways, respectively) following genotoxic insults. We found that wild-type and homologous recombination-deficient (Rad51D) cellular lethality was dependent on both the dose and LET of the carbon ions, whereas it was only dependent on dose for protons. The non-homologous end joining deficient cell line (Ku80 mutant) showed nearly identical dose-response profiles for both carbon ions and protons. Our results show that the increasingly used modality of carbon ions as charged particle therapy is advantageous to protons in a radiotherapeutic context, primarily for tumor cells proficient in non-homologous end joining DNA repair where cellular lethality is dependent not only on the dose as in the case of more common photon therapeutic modalities, but more importantly on the carbon ion LETs. Genetic characterization of patient tumors would be key to individualize and optimize the selection of radiation modality, clinical outcome and treatment cost.

Genomic instability and telomere fusion of canine osteosarcoma cells.

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.

Induction of matrix metalloproteinase gene transcription by nitric oxide and mechanisms of MMP-1 gene induction in human melanoma cell lines.

Expression of 12 matrix metalloproteinases (MMPs) after exposure of human melanoma cell lines C32TG and Mewo to nitric oxide (NO) was investigated by the reverse transcription-polymerase chain reaction. Expression of the mRNA of MMP-1, -3, -10 and -13 in C32TG cells was transcriptionally enhanced in a dose-dependent manner by exposure to an NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) and mRNA expression of MMP-1 and -10 was similarly enhanced in Mewo cells. Exposure of C32TG cells to NO increased the MMP-1 protein concentration in the culture medium. Testing with the luciferase gene fused to the 1.5 Kbp 5'-flanking region of the human MMP-1 gene showed that exposure to NO upregulated MMP-1 promoter activity in C32TG cells. Endogenous NO production after introduction of inducible NO synthase cDNA also enhanced MMP-1 promoter activity in C32TG cells. Deletion and mutational analysis identified a critical AP-1 binding site required for NO regulation of MMP-1. A neighboring Ets motif from the AP-1 site in the promoter region acted as an accessory to enhance MMP-1 expression. Electromobility shift analysis using the AP-1 binding site showed that NO enhanced the AP-1 binding ability of nuclear factors in C32TG cells. PD98059, a selective MEK inhibitor and SB202190, a p38 MAPK inhibitor, attenuated the MMP-1 mRNA expression enhanced by NO. Thus, MMP-1 was transcriptionally enhanced by NO via MAPK (ERK and p38) pathways. The results of our study suggest that the increased expression of MMPs in response to NO may be associated with tumor progression under inflammation.

Interferon-beta from melanoma cells suppresses the proliferations of melanoma cells in an autocrine manner.

Interferon (IFN)-alpha and IFN-beta have been utilized in the treatment of melanoma as a form of cytokine therapy. While previous studies have demonstrated that melanocytes and melanoma cells produce a number of cytokines, it remains unclear whether or not melanocytes and melanoma cells per se produce IFN-alpha or IFN-beta. In the present study, we investigated the expression of IFN-alpha or IFN-beta in human melanocytes and five melanoma cell lines: G-361, C32TG, MMAc, MEWO and VMRC-MELG at both mRNA and protein levels. Both IFN-alpha and IFN-beta mRNA were detected in normal human melanocytes. Likewise, IFN-alpha mRNA was detected in all five melanoma cell lines. However, IFN-beta mRNA was only detected in one melanoma cell line, VMRC-MELG. When melanocytes and melanoma cells were treated with a potent IFN inducer, polyinosinic:polycytidylic acid (poly I:C), the mRNA expression of both IFN-alpha and IFN-beta was significantly upregulated. Poly I:C was not able to induce melanocytes or melanoma cells to produce detectable amounts of IFN-alpha protein, but able to induce a significant amount of IFN-beta in melanocytes and two of the melanoma cell lines: MMAc and VMRC-MELG. Moreover, similar to exogenous IFN-alpha and IFN-beta, poly I:C significantly inhibited the proliferation of all five melanoma cell lines. This suppressive effect was partially blocked by anti-IFN-beta antibody treatment in the IFN-beta-producing melanoma cell lines: MMAc and VMRC-MELG, but not in the non-IFN-beta-producing cell lines: G-361, C32TG and MEWO. Collectively, these studies have demonstrated for the first time that human melanocytes and melanoma cells produce IFN-beta. Furthermore, melanoma cells are capable of suppressing their own proliferation via secretion of endogenous IFN-beta. This finding may have important implications for melanoma therapy.