Naoto Tatewaki

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC50: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy.

Squalene as Novel Food Factor

Currently, health beneficial roles of natural products attract much attention and diverse functional ingredients have been extensively studied their preventive effect in many diseases such as cardiovascular diseases and cancer. Squalene is one of those examples. It distributes in nature from plant to animal but extraordinarily concentrated in the liver of certain species of shark (Squalidae family) as it was first identified as a healing substance in the shark liver oil. It is now well-known that squalene is the physiological substance functioning in animal as the precursor of cholesterol biosynthesis. On the other hand, it has long history of using as an attractive resource for functional food, supplement or even pharmaceutics because it has unique physical property and wide variety of physiological functions such as anticancer and anti-hyper cholesterolemia. The antioxidant and oxygen carrying properties of squalene predicts its potential in preventing cardiovascular disease. We reviewed recent progress in functional studies of squalene both in vitro and in vivo models.

Synergistic inhibition of cancer cell proliferation with a combination of δ-tocotrienol and ferulic acid

Rice bran consists of many functional compounds and thus much attention has been focused on the health benefits of its components. Here, we investigated the synergistic inhibitory effects of its components, particularly δ-tocotrienol (δ-T3) and ferulic acid (FA), against the proliferation of an array of cancer cells, including DU-145 (prostate cancer), MCF-7 (breast cancer), and PANC-1 (pancreatic cancer) cells. The combination of δ-T3 and FA markedly reduced cell proliferation relative to δ-T3 alone, and FA had no effect when used alone. Although δ-T3 induced G1 arrest by up-regulating p21 in PANC-1 cells, more cells accumulated in G1 phase with the combination of δ-T3 and FA. This synergistic effect was attributed to an increase in the cellular concentration of δ-T3 by FA. Our results suggest that the combination of δ-T3 and FA may present a new strategy for cancer prevention and therapy.

Non-thermal DNA damage of cancer cells using near-infrared irradiation.

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)

Objective assessment of skin tightening in Asians using a water-filtered near-infrared (1,000–1,800 nm) device with contact-cooling and freezer-stored gel

Background Near-infrared has been shown to penetrate deeper than optical light sources independent of skin color, allowing safer treatment for the Asian skin type. Many studies have indicated the efficacy of various types of devices, but have not included a sufficiently objective evaluation. In this study, we used three-dimensional imaging for objective evaluation of facial skin tightening using a water-filtered near-infrared device. Methods Twenty Japanese patients were treated with the water-filtered near-infrared (1,000–1,800 nm) device using a contact-cooling and nonfreezing gel stored in a freezer. Three-dimensional imaging was performed, and quantitative volume measurements were taken to evaluate the change in post-treatment volume. The patients then provided their subjective assessments. Results Objective assessments of the treated cheek volume evaluated by a three-dimensional color schematic representation with quantitative volume measurements showed significant improvement 3 months after treatment. The mean volume reduction at the last post-treatment visit was 2.554 ± 0.999 mL. The post-treatment volume was significantly reduced compared with the pretreatment volume in all patients (P < 0.0001). Eighty-five percent of patients reported satisfaction with the improvement of skin laxity, and 80% of patients reported satisfaction with improvement of rhytids, such as the nasolabial folds. Side effects, such as epidermal burns and scar formation, were not observed throughout the study. Conclusion The advantages of this water-filtered near-infrared treatment are its high efficacy for skin tightening, associated with a minimal level of discomfort and minimal side effects. Together, these characteristics facilitate our ability to administer repeated treatments and provide alternative or adjunctive treatment for patients, with improved results. This study provides a qualitative and quantitative volumetric assessment, establishing the ability of this technology to reduce volume through noninvasive skin tightening.

Synergistic Anticancer Effect of Tocotrienol Combined with Chemotherapeutic Agents or Dietary Components: A Review

Tocotrienol (T3), unsaturated vitamin E, is gaining a lot of attention owing to its potent anticancer effect, since its efficacy is much greater than that of tocopherol (Toc). Various factors are known to be involved in such antitumor action, including cell cycle arrest, apoptosis induction, antiangiogenesis, anti-metastasis, nuclear factor-κB suppression, and telomerase inhibition. Owing to a difference in the affinity of T3 and Toc for the α-tocopherol transfer protein, the bioavailability of orally ingested T3 is lower than that of Toc. Furthermore, cellular uptake of T3 is interrupted by coadministration of α-Toc in vitro and in vivo. Based on this, several studies are in progress to screen for molecules that can synergize with T3 in order to augment its potency. Combinations of T3 with chemotherapeutic drugs (e.g., statins, celecoxib, and gefitinib) or dietary components (e.g., polyphenols, sesamin, and ferulic acid) exhibit synergistic actions on cancer cell growth and signaling pathways. In this review, we summarize the current status of synergistic effects of T3 and an array of agents on cancer cells, and discuss their molecular mechanisms of action. These combination strategies would encourage further investigation and application in cancer prevention and therapy.

Persimmon Leaf Extract Inhibits the ATM Activity during DNA Damage Response Induced by Doxorubicin in A549 Lung Adenocarcinoma Cells

Persimmon leaf (PL) has been commonly recognized for its wide variety of health benefits. A previous study has reported that persimmon leaf extract (PLE) contained flavonols with the 2″-galloly moiety (PLEg). Galloylated homologues generically show stronger activity in their biological function, so enhanced functions can be expected for PLEg. We investigated in this present study the effect of PLEg on the cellular DNA damage checkpoint signaling to sensitize cancer chemotherapy. Treatment with PLE and PLEg significantly increased the cytotoxicity of doxorubicin (DOX) in A549 adenocarcinoma cells. PLE and PLEg reduced the phosphorylation of checkpoint proteins such as structural maintenance of chromosomes 1 (SMC1), checkpoint kinase 1 (Chk1), and p53 in DOX-treated cells. Moreover, PLE decreased the phosphorylation of ATM (ataxia telangiectasia mutated) in a dose-dependent manner. PLE, and especially PLEg, abrogated the G2/M checkpoint during DOX-induced DNA damage. These results suggest that PLEg specifically inhibited ATM-dependent checkpoint activation by DOX, and that PLEg might be a useful sensitizer in cancer chemotherapy.

Amadori-glycated phosphatidylethanolamine up-regulates telomerase activity in PANC-1 human pancreatic carcinoma cells

Several lines of experimental data have highlighted a key role of Amadori‐glycated phosphatidylethanolamine (Amadori‐PE) in the development of diabetic complications. Recent epidemiological studies suggest that diabetes mellitus could be a risk factor for some cancers. A characteristic of cancer cells is their immortal phenotype, and the enzyme telomerase contributes to the infinite replicative potential of cancer cells. The purpose of this study was to obtain new information about the effect of Amadori‐PE on the regulation of telomerase in PANC‐1 human pancreatic carcinoma cells. Amadori‐PE enhanced cellular telomerase in a time‐ and dose‐dependent manner by up‐regulating hTERT expression through induction of c‐myc. These results provide experimental evidence for a novel role of Amadori‐PE in linking diabetes and cancer.

Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

Metabolism and cytotoxic effects of phosphatidylcholine hydroperoxide in human hepatoma HepG2 cells.

In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis.