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.

Caffeic acid phenyl ester in propolis is a strong inhibitor of matrix metalloproteinase-9 and invasion inhibitor: isolation and identification.

BACKGROUND: Propolis has been used as a folk medicine and has several proven biological activities. Herbal remedies recommended for cancer therapies in Korea. METHODS: Matrix metalloproteinase (MMP)-9-inhibitory activity of propolis has been assessed. CAPE as an acting compound was isolated and molecular structure was determined. Anti-invasion activity of CAPE was assayed using hepatocarcinoma cells. RESULTS: Propolis ethanol extracts showed a strong inhibitory effect of MMP-9 activity, which is known to be involved in tumor cell invasion and metastasis in a concentration-dependent manner on zymography. Assay guided fractionation led to the isolation of a caffeic acid phenyl ester (CAPE) as the compound responsible for the anti-MMP-9 activity. CAPE was obtained by reversed-phase HPLC, and its structure was elucidated by fast atom bombardment mass spectrometry and tandem mass spectrometry. The purified CAPE inhibited MMP-9 activity with the IC(50) of 1.0-2.0 nmol/l. CONCLUSIONS: CAPE possesses selective antiproliferative activity toward hepatocaricoma cell line Hep3B, but not primary cultured mouse hepatocytes.

Acanthopanax senticosus Harms as a prophylactic for MPTP-induced Parkinson's disease in rats.

The aim of this study was to determine whether Acanthopanax senticosus Harms (ASH) offers protection against Parkinson's disease (PD) and its related depressive behaviors in rats administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We examined how ASH affected the MPTP-induced loss of tyrosine hydroxylase (TH)-positive neurons in the midbrain of rats. Extract from the stem bark of ASH prepared with hot water was dissolved in distilled water. Rats were then orally administered ASH (250 mg/kg) once a day for 2 weeks before ASH administration plus an intraperitoneal injection of MPTP (20 mg/kg). The pole test and catalepsy test were used to evaluate the effects of ASH administration on bradykinesia and depressive behaviors in the PD model of rats given MPTP for 2 weeks. Treatment with ASH for 2 weeks resulted in prophylactic effects on MPTP-induced Parkinsonian bradykinesia and catalepsy. Immunohistochemistical analysis using TH antibody showed that ASH provided cytoprotective effects against MPTP-induced loss of dopamine (DA) cells. The present results suggest that it may be possible to use ASH for the prevention of nigral degenerative disorders, e.g., PD with depression, caused by exposure to toxic substances.

Enhancement of radioprotection and anti-tumor immunity by yeast-derived beta-glucan in mice.

Intraperitoneal injection of beta-glucan was shown to greatly delay mortality in mice exposed to whole-body X-ray radiation and tumor growth in tumor-bearing mice. Since the leukocyte and lymphocyte numbers were increased by a single dose of beta-glucan, the radioprotective effect of beta-glucan is probably mediated, at least in part, by a hemopoietic action in irradiated mice. In addition, both natural killer (NK) and lymphokine-activated killer (LAK) activities were significantly increased by repeated doses of beta-glucan. Augmented immunological activity as seen in increased NK and LAK activity by beta-glucan seems to play a role in preventing secondary infections associated with irradiation, and probably contributes to the attenuated tumor growth in tumor-bearing mice through enhanced anti-tumor immunity. These results suggest that beta-glucan may be a promising adjunct treatment for cancer patients receiving radiotherapy.

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.

Prolactin prevents acute stress-induced hypocalcemia and ulcerogenesis by acting in the brain of rat.

Stress causes hypocalcemia and ulcerogenesis in rats. In rats under stressful conditions, a rapid and transient increase in circulating prolactin (PRL) is observed, and this enhanced PRL induces PRL receptors (PRLR) in the choroid plexus of rat brain. In this study we used restraint stress in water to elucidate the mechanism by which PRLR in the rat brain mediate the protective effect of PRL against stress-induced hypocalcemia and ulcerogenesis. We show that rat PRL acts through the long form of PRLR in the hypothalamus. This is followed by an increase in the long form of PRLR mRNA expression in the choroid plexus of the brain, which provides protection against restraint stress in water-induced hypocalcemia and gastric erosions. We also show that PRL induces the expression of PRLR protein and corticotropin-releasing factor mRNA in the paraventricular nucleus. These results suggest that the PRL levels increase in response to stress, and it moves from the circulation to the cerebrospinal fluid to act on the central nervous system and thereby plays an important role in helping to protect against acute stress-induced hypocalcemia and gastric erosions.

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.

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.

Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells.

Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 microg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2 alpha and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells.

Effect of sesamin in Acanthopanax senticosus HARMS on behavioral dysfunction in rotenone-induced parkinsonian rats.

The aim of this study was to determine whether sesamin, a component from Acanthopanax senticosus HARMS (ASH) pharmacologically offers protection against Parkinson's disease (PD) and its related depressive behavior in rats administered rotenone. We also examined how sesamin affected the rotenone-induced loss of tyrosine hydroxylase (TH) or glial cell line-derived neurotrophic factor (GDNF)-positive neurons in the midbrain of rats. Rats were orally administered sesamin (3, 30 mg/kg) once a day for 2 weeks before an intraperitoneal injection of rotenone (2.5 mg/kg). The pole test and catalepsy test were used to evaluate the effects of sesamin administration on bradykinesia and depressive behaviors in the PD model of rats given rotenone for 5 weeks. Those effects were compared with the ASH administrated group (250 mg/kg). Treatment with sesamin for seven weeks resulted in prophylactic effects on rotenone-induced parkinsonian bradykinesia and catalepsy, and the effects were equivalent to ASH effects. Immunohistochemistical analysis using TH or GDNF antibody showed that sesamin provided cytoprotective effects against rotenone-induced loss of DA cells. The results suggest that it may be possible to use the ASH and sesamin for the prevention of nigral degenerative disorders, e.g., PD with depression, caused by exposure to pesticide or environmental neurotoxins in general.

Orally administered beta-1,6-D-polyglucose extracted from Agaricus blazei results in tumor regression in tumor-bearing mice.

There is an increasing demand from both patients and practicing oncologists for orally formulated chemotherapy. The present study focused on the oral formulation for natural products that may be effectively used in oncologic treatment regimens. Tumor-bearing mice treated with intratumoral administration of aqueous ammonium oxalate-soluble and ethanol-insoluble derivatives of Agaricus blazei showed marked tumor regression at doses ranging from 0.1 to 2.5 mg (p < 0.05 vs. saline control; n = 7). However, oral administration of this same fraction, either prior to, simultaneously with, or after, tumor cell inoculation did not result in tumor regression (p > 0.05 vs. control). When this fraction was treated with hydrochloric acid (acid-treated fraction; ATF), intratumoral administration resulted in a marked regression of tumor growth comparable to that of the acid-untreated fraction. More importantly, parenteral administration of ATF resulted in a significantly greater regression of tumor growth than that produced by the untreated fraction (p < 0.05 vs. untreated; n = 7). When a total of 4.5 mg of ATF was given orally at varying schedules prior to, simultaneously with, or after, tumor inoculation, a significant regression was seen using a schedule starting 4 days prior to inoculation (p < 0.05 vs. all other treatments; n = 7). NMR and molecular analyses showed that the ATF fraction had a molecular weight of approximately 10 kDa and consisted mainly of only (1,6)-beta- D-polyglucose. These results suggest that the oral administration of simple acid-treated ATF results in a remarkable tumor regression. Thus, simple acid hydrolysis of natural products may not only bring measurable benefits in oncological practice, but may also be a useful general formulation for natural products for oral chemotherapy.

Antidiabetic activity of Lyophyllum decastes in genetically type 2 diabetic mice.

The antidiabetic activity of Lyophyllum decastes (Tricholomataceae) was investigated in KK-Ay mice, an animal model of genetically type 2 diabetes with hyperinsulinemia. The water extract of Lyophyllum decastes (LD) (500 mg/kg body weight) reduced the blood glucose of KK-Ay mice 7 h after a single oral administration (p<0.05) when compared with control. LD reduced the blood glucose of KK-Ay mice 3 weeks after repeated administration (p<0.05), and also significantly lowered the serum insulin of KK-Ay mice under similar conditions (p<0.01). However, LD did not affect the blood glucose in normal mice. LD tended to decrease of the blood glucose in an insulin tolerance test. In addition, the muscle content of facilitative glucose transporter isoform 4 (GLUT4) protein content in the plasma membrane fraction from muscle significantly increased in the orally LD-treated KK-Ay mice when compared to that of the controls (p<0.01). These results suggest that the antidiabetic activity of LD is derived, at least in part, from a decrease in insulin resistance, due to the increase of GLUT4 protein content in the plasma membrane of the muscle.

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

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) 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-1beta 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-1beta 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-1beta on days 1 and 2. These results demonstrate that the potency of induction of MMP-2 by IL-1beta and TNF-alpha 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-1alpha and IL-1beta.