Ikukatsu Suzuki

Propolis induces cell cycle arrest and apoptosis in human leukemic U937 cells through Bcl-2/Bax regulation

We investigated mechanism(s) where propolis induces apoptosis in human leukemic U937 cells. Propolis inhibited the proliferation of U937 cells in a dose-dependent manner by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Western blot analysis showed that propolis increases the expression of p21 and p27 proteins, and decreases the levels of cyclin B1, cyclin A, Cdk2 and Cdc2, thereby contributing to cell cycle arrest. DAPI staining assay revealed typical morphology features of apoptotic cells. Propolis-induced apoptosis was also confirmed by assays with annexin V-FITC, PI-labeling and DNA fragmentation assay. The increase in apoptosis level induced by propolis was associated with down-regulation of Bcl-2 and activation of caspase-3, but not with Bax. These results suggests that propolis-induced apoptosis is related to the selective activation of caspase-3 and induction of Bcl-2/Bax regulation.

Antitumor and anticytopenic effects of aqueous extracts of propolis in combination with chemotherapeutic agents.

Using an ICR mouse model bearing a syngeneic Ehrlich ascitis carcinoma, the present study was undertaken to examine the effects of crude, water-soluble propolis (CWSP) on tumor progression, chemotherapeutic efficacy, and hematopoiesis in the peripheral blood. It was demonstrated that CWSP, administered subcutaneously, resulted in marked regression of tumor growth in mice, at the early phase after tumor inoculation (CWSP, p < 0.05 vs. saline control). Molecular analysis indicated that the CWSP is composed of 8.4% protein, 4.2% quercetin plus a variety of saccharides with a molecular weight of 29 kDa. Orally administered CWSP did not produce any regression for the observation period (oral CWSP, p > 0.05 vs. saline control). Peritoneal injection of CWSP into neonatal mice resulted in an increased lymphocyte/polymorphonuclear leukocyte ratio activity, indicating the potential activation of lymphoid cell lineages. These observations suggest that subcutaneously injected CWSP could regulate the development of tumors by possibly stimulating multicellular immunity. In addition, oral administration of CWSP concurrently with 5-fluorouracil (5-FU) or mitomycin C (MMC), significantly increased tumor regression as compared with the respective chemotherapy alone, illustrating the adjuvant effect of orally administered CWSP for tumor regression when combined with chemotherapeutic agents. To examine further the potential usefulness of CWSP for chemotherapeutic regimens, which induce profound multilineage hematopoietic suppression, mice that received CWSP orally in addition to a 5-FU or MMC were followed for absolute numbers of platelets and white and red blood cells. The oral administration of CWSP significantly ameliorated the cytopenia induced by 5-FU, resulting in recovery of white as well as red blood cell counts (5-FU plus CWSP, p < 0.05 vs. 5-FU alone or water control; white blood cells on day 15, red blood cells on day 25), but no marked effects on platelet counts was observed (5-FU plus CWSP, p > 0.05 vs. 5-FU alone or water control). On the other hand, CWSP significantly reduced all three MMC-induced cytopenias, especially at the later stage of the chemotherapeutic course (after day 30), suggesting repetitive requirements of oral administration of CWSP. In summary, subcutaneous administration of an aqueous CWSP resulted in marked regression of transplanted tumors. Orally administered CWSP combined with chemotherapeutic agents significantly increased tumor regression and ameliorated the cytopenia induced by the chemotherapeutic agents alone. These results suggest the benefits of potential clinical trials using CWSP combined with chemotherapeutic agents in order to maximize enhanced immunity while potentially minimizing postchemotherapeutic deteriorated reactions.

Immune Activation and Radioprotection by Propolis

In this study, we focused on immune stimulation by Propolis, and examined changes in the effect of irradiation after Propolis administration. We also examined the radioprotective effect of Propolis by observing its effect on the immune system. The effect of immune activation by Propolis was investigated by measuring the total immunoglobulin (Ig) G and IgM. The radioprotective effect of immune activation by Propolis was investigated by measuring the T-lymphocyte subsets in the peripheral blood of mice following whole body irradiation. Compared with the control group, the IgG was significantly reduced in the Propolis group, indicating that Propolis suppressed IgG production. ELISA revealed that the amount of IgM in mouse serum was significantly higher in the Propolis group as compared with the control group, indicating that Propolis increased IgM production. The number of CD4-positive cells was increased only in the Propolis group. Likewise, the number of CD4-positive cells increased by 81% in the Propolis with irradiation group compared with the irradiation group alone. Compared with the control group, the Propolis group increased CD8-positive cells. Compared with the irradiation alone group, CD8-positive cells were decreased by Propolis with irradiation group. Propolis activated macrophages to stimulate interferon (IFN)-gamma production in association with the secondary activation of T-lymphocytes, resulting in a decrease in IgG and IgM production. Cytokines released from macrophages in mouse peripheral blood after Propolis administration activated helper T-cells to proliferate. In addition, activated macrophages in association with the secondary T-lymphocyte activation increased IFN-gamma production and stimulated proliferation of cytotoxic T-cells and suppressor T-cells, indicating the activation of cell-mediated immune responses.

Interleukin-1 and tumor necrosis factor-α induce collagenolysis and bone resorption by regulation of matrix metalloproteinase-2 in mouse calvarial bone cells

Abstract Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. We examined the effects of the two cytokines on the collagenolysis and bone resorption by induction of matrix metalloproteinases (MMPs). The cells were analyzed using zymographic analysis. It was shown that the mouse calvarial osteoblasts constitutively synthesize progelatinase‐A (MMP‐2). Interleukin‐1β markedly enhanced the messenger RNAs (mRNAs) expression of MMP‐2 (gelatinase A), but slightly MMP‐9 (gelatinase B), which associated with increases in bone matrix degradation. Both pro‐ and active‐forms of MMP‐2 were detected in the conditioned medium collected from calvarial cultures, and IL‐1β markedly stimulated both pro‐ and active‐forms of the MMP‐2. The expression of MMP‐2 mRNAs could be detected, and they were markedly enhanced by IL‐1β on days 1 and 2. These results demonstrate that the potency of induction of MMP‐2 by IL‐1β and TNF‐α is closely linked to the respective bone‐resorbing activity, suggesting that MMP‐2‐dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines. On the other hand, when the mouse osteoblasts were stimulated with parathyroid hormone, 1,25(OH)2D3, mononuclear cell conditioned medium (MCM) and IL‐1 as bone resorption agents, collagenolysis was increased by producing the active gelatinase. Interleukin‐1 in stimulating bone resorption was examined using fetal mouse long bone organ culture. Interleukin‐1 stimulated bone resorption and produced marked resorption when present simultaneously. Furthermore, treatment of indomethacin and dexamethasone clearly abolished the responses of IL‐1α and IL‐1β.

Combined effects of radiation and ultrasound on ICR mice in the preimplantation stage

Embryos of ICR mice at the preimplantation stage of development were used to examine the single and combined effects of ultrasound (US) and radiation. Pregnant mice were exposed to a single dose of whole body gamma radiation and/or US at 2 hpc (hours postconception) or 3 hpc. The exposure duration was 10 min with 1-MHz continuous wave US at 1 or 2 W/cm(2) by I(SPTA) (intensities of spatial peak temporal average). Gamma irradiation of pregnant mice (2 hpc) was at 0.5 Gy at a dosage rate of 0.2 Gy/min. The rate for ultrasonic irradiation was a dose of 1 or 2 W/cm(2), considered by some to be too high in therapy, such as for arthritis in the rehabilitation area of the medical application. Various malformations were recognized, and the incidence of malformations in the 0.5-Gy exposure and the control groups were compared. A greater number of malformations were observed in the 0.5-Gy exposure group relative to the 0.5-Gy plus 1.0 W/cm(2) US exposure group. Therefore, the synergistic effects of radiation relate to external malformations, the number of implantations, and the rate of embryonic death due to US. It appears that US may act to repair DNA damaged by ionizing radiation. The cell cycle of the fertilized egg is delayed, which may be the mechanism by which lesions induced by ionizing radiation are healed by ultrasonic irradiation.

Inactivated Actinobacillus suis in Synergy with Interferon-α Protects Mice against Herpes Simplex Virus-2 Infection

A single intraperitoneal administration of heat-killed and lyophilized cells ofActinobacillus suis ATCC 15557 (AS preparation) into 5-week-old female BALB/c mice led to potent, but not statistically significant, nonspecific resistance to intravenous challenge with a lethal dose of herpes simplex virus, type 2 (HSV-2). A combination treatment with the intraperitoneal AS preparation and intravenous interferon (IFN)-α markedly elevated the survival rates of the mice. The production of endogenous IFN-γ was also enhanced by the combination treatment but the production of other endogenous cytokines such as tumor necrosis factor α and interleukin-12 was minimal. The focus of in vitro formation of HSV-2 was markedly inhibited by the peritoneal exudate cells induced by AS preparation in combination with IFN-α. The antiviral activity of peritoneal exudate cells induced by the combined agents was transferrable to syngeneic recipient mice. These results suggest the possibility that the AS preparation in combination with IFN-α may play an important role in controlling intracellular HSV-2 infection through the activation of immunocompetent cells and the production of endogenous IFN-γ in BALB/c mice.