Masaharu Hazawa

Suppressive effects of novel derivatives prepared from Aconitum alkaloids on tumor growth

SummaryLittle information has so far been reported regarding the antiproliferative properties of Aconitum alkaloids against human tumor cells despite of their intense toxicities. In the present study, the antitumor properties and radiation sensitizing effects were investigated by various types of novel derivatives prepared from Aconitum alkaloids. The antitumor properties were investigated against human tumor cell lines, A172, A549, HeLa and Raji, respectively, by a cell growth, a clonogenic assay, cell cycle distribution, cell cycle related molecules and γH2AX expression. The novel compounds derived from C20-diterupenoid alkaloids showed a significantly suppressive effect in all cell lines. In contrast, natural C19-norditerpenoid alkaloids and their derivatives showed either no effect or only a slight effect. One of the compounds also showed radiosensitizing properties on A549 cells. These effects are not related to either the cell cycle distribution, the enhancement of apoptosis or the γH2AX expression. Novel derivatives prepared from Aconitum alkaloids, not but natural alkaloids, clearly showed anti-proliferative activity in human tumor cell lines.

Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation.

Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome-cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

Regulation of DNA damage response and cell cycle in radiation-resistant HL60 myeloid leukemia cells

The acquisition of resistance to radiation has been a matter of concern in clinical cases. However, mechanisms underlying the acquisition of radiation resistance are yet to be elucidated. We established a radiation-resistant strain (Res-HL60 cells) from HL60 leukemic cells and investigated their response to radiation. Res-HL60 cells not only proliferated on the fifth day after radiation but also had a high survival rate in a clonogenic assay. Although Chk1 was activated in HL60 cells after irradiation, the expression levels of Chk1 in Res-HL60 cells decreased. There were few differences between the two cell lines with regard to Chk2 activity. Res-HL60 cells show prolonged G2/M arrest and an early decrease in the extent of DNA damage. However, inhibitors against ATM/ATR and DNA-dependent protein kinase (DNA-PK) both abrogated the radiation resistance capacity of the cells. These results reveal that radiation-resistant strains have a high repair capacity, and inhibition of the repair system at an early stage is an effective strategy in the second round of radiation therapy.

Functional properties of synthetic N-acyl-L-homoserine lactone analogs of quorum-sensing gram-negative bacteria on the growth of human oral squamous carcinoma cells

SummaryQuorum sensing is defined as the ability of microorganisms to sense their population density via the release of signaling molecules composed of acyl-homoserine lactone (AHL), which is a type of autoinducer (AI). Previous structure-activity relationship (SAR) studies demonstrated that the 3-oxo group, homoserine lactone of L-form, and long acyl side chain have crucial roles in apoptosis induction. Various types of synthetic AI analogs of Pseudomonas aeruginosa were prepared, and SAR study was conducted to determine their effects against human oral squamous carcinoma cells derived from gingival carcinoma Ca9-22 cells and tongue cancer SAS cells. Not only the antiproliferative potential but also the radiation-sensitizing effects against these cells were examined. It was found that antiproliferative activity partly depended on HSL structure and acyl side chain length. Moreover, a few compounds, compound 5 and 87, showed antiproliferative effects against both Ca9-22 and SAS cells, and also induced radiation-sensitizing effects against Ca9-22 cells. Compound 5 alone induced apoptotic cell death accompanied by sub-G1 phase accumulation in cell cycle and caspase-3 activation, and radiation-sensitizing effects of compound 5 could be attributed to enhanced apoptosis induction. In contrast, there were no remarkable alterations in cell cycle distribution in Ca9-22 treated with compound 87 alone or in combination. However, both compounds lack 3-oxo and their acyl side chain lengths are not necessarily long. This SAR study demonstrated that HSL analogs, which lacked the recommended characteristics for apoptosis induction clearly showed antiproliferative and radiation-sensitizing activity in Ca9-22 cells.

Mitigative Effects of a Combination of Multiple Pharmaceutical Drugs on the Survival of Mice Exposed to Lethal Ionizing Radiation

It is important to establish an easy-to-use therapeutic protocol for the emergency medical care of patients involved in radiation accidents to reduce the radiation-related casualties. The present study aimed to establish an optimum therapeutic protocol using currently approved pharmaceutical drugs to increase the survival of victims exposed to lethal radiation. Different combinations of four drugs-recombinant human erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), c-mpl receptor agonist romiplostim (RP) and nandrolone decanoate (ND)-were administered to mice within 2 h after exposure to a lethal 7 Gy dose of γ-irradiation. On day 30 after irradiation, the condition of the mice was analyzed using various hematological parameters, such as the number of peripheral blood cells, bone marrow cells, hematopoietic progenitor cells and the expression of cell surface antigens. Approximately 10% of the untreated irradiated control mice survived for 21 days, but all of the control mice died by day 30. The combined administration of G-CSF, EPO and RP for five days immediately after irradiation led to a complete survival of the irradiated mice until day 30. However, the treatment with G-CSF, EPO and RP with ND led to only 75% survival at day 30. The hematological analyses showed that the numbers of almost all of hematopoietic cells in the surviving mice treated with effective medications recovered to the levels of non-irradiated mice. The present findings show that the combination of G-CSF, EPO and RP may be a useful countermeasure for victims exposed to accidental lethal irradiation.

Sulfation of keratan sulfate proteoglycan reduces radiation-induced apoptosis in human Burkitt's lymphoma cell lines.

This study focuses on clarifying the contribution of sulfation to radiation‐induced apoptosis in human Burkitts lymphoma cell lines, using 3′‐phosphoadenosine 5′‐phosphosulfate transporters (PAPSTs). Overexpression of PAPST1 or PAPST2 reduced radiation‐induced apoptosis in Namalwa cells, whereas the repression of PAPST1 expression enhanced apoptosis. Inhibition of PAPST slightly decreased keratan sulfate (KS) expression, so that depletion of KS significantly increased radiation‐induced apoptosis. In addition, the repression of all three N‐acetylglucosamine‐6‐O‐sulfotransferases (CHST2, CHST6, and CHST7) increased apoptosis. In contrast, PAPST1 expression promoted the phosphorylation of p38 MAPK and Akt in irradiated Namalwa cells. These findings suggest that 6‐O‐sulfation of GlcNAc residues in KS reduces radiation‐induced apoptosis of human Burkitts lymphoma cells.

Caspase-independent apoptosis induction of quorum-sensing autoinducer analogs against chronic myeloid leukemia K562

SummaryQuorum sensing is defined as the ability of microorganisms to sense their population density via the release of signaling molecules called autoinducers (AIs). Various types of AI analogs were prepared and their antitumor properties against chronic myeloid leukemia (CML) K562 cells were investigated. Two AI analogs induced progressive apoptosis with JNK activation and p21 induction. In addition, this induction of apoptosis is not related to bcr-abl kinase, which sustains CML proliferation. However, the progression of　apoptosis was not inhibited by a caspase family inhibitor. These results suggested that AI analogs could induce caspase-independent apoptosis in CML K562.

(-)-Epigallocatechin-3-O-gallate induces nonapoptotic cell death in leukemia cells independent of the 67 kDa laminin receptor.

The 67 kDa laminin receptor (67 LR) mediates (-)-epigallocatechin-3-O-gallate (1; EGCG)-67 LR direct action only at physiological concentrations. The relevancy of biological effects of 1 at physiological concentrations to 67 LR was investigated in myeloid and lymphoid leukemia cells using flow cytometric analysis. It was shown that physiological concentrations of 1 suppressed the cell growth of HL60 myeloid leukemia cells and Raji lymphoid leukemic cells independent of 67 LR expression. Moreover, there was no discernible change in the levels of intracellular reactive oxygen species, characteristics of apoptosis such as phosphatidylserine translocation and activated caspase-3. The activity of 1 at physiological concentrations does not depend on direct 67 LR-mediated actions, and this compound induces necrosis-like death of promyelocytic leukemia and non-Hodgkins lymphoma cells.

Suppressive effects of liquid crystal compounds on the growth of the A549 human lung cancer cell line

SummaryThe aim of this study was to evaluate the biological activity and pharmacological activity of several amphiphilic liquid-crystalline compounds (LCs), i.e. phenylpyrimidine derivatives possessing D-glucamine and cyanobiphenyl derivatives with a terminal hydroxyl unit, to explore novel anti-cancer functions of the LCs. The anti-cancer properties of the LCs were investigated in A549 human lung cancer cells by assessing cell growth, cell cycle distribution, and cell signaling pathways using a flow cytometer and a Western blot analysis. In addition, the effect of LCs on the growth of WI-38 normal fibroblasts was examined. Consequently, the phenylpyrimidine derivatives and cyanobiphenyl derivatives showed cytostatic effects, causing the suppression of cell growth through G1 phase arrest in A549 cells. Further analyses using phenylpyrimidine derivatives and precursors of a cyanobiphenyl compound demonstrated the structure-activity relationships. One of the phenylpyrimidine derivatives inhibited A549 growth without any toxicity to normal fibroblasts. As a result, a novel pharmacological function was hypothesized to be inherent in the structure of the LCs themselves, and the dependence of the tumor-specific activity on the hydrophobic group of phenylpyrimidine derivatives therefore remains an interesting issue. Our results suggest the possibility that the LCs themselves may act as a novel type of chemotherapeutic agent.

Estimation of secondary measles transmission from a healthcare worker in a hospital setting

Measles among healthcare workers (HCWs) is associated with a significant risk of nosocomial transmission to susceptible patients. When a measles case occurs in the healthcare setting, most guidelines recommend exhaustive measures. To evaluate the effects of measures against measles transmission in the healthcare setting precisely, it is essential to determine whether secondary transmission generally occurs. This study describes, for the first time, the actual secondary transmission rate for a measles-infected HCW in a ward with no special air ventilation capacity. The routine treatment of a number of immunocompromised patients occurs in this ward, and thus patients as well as HCWs have a thorough understanding and practice of standard and extended precautions. Our paired serum sample study revealed that none of the people in the ward exposed to the HCW at the catarrhal stage over a period of 4 days exhibited elevated levels of antibodies against measles. We suggest that strict adherence to standard and expanded precautions among patients and HCWs may be effective for preventing the transmission of a highly airborne disease, such as measles.