Effects of Vitamin E on the immune system and tumor growth during radiotherapy.

PURPOSE: The purpose of this study is to evaluate the effects of Vitamin E (VE) on the immune system and tumor growth during radiotherapy (RT) in mice model. METHODS: C57BL/6NCrSlc mice were randomly distributed in four groups (control, VE alone, RT alone, and VE + RT). In the VE and VE + RT groups, VE was administered in the diet at 500 mg/kg. Radiation was delivered at 2 Gy in a single fraction on the whole body or at 6 Gy in three fractions locally in the RT and VE + RT groups. Changes in leukocytes and T lymphocytes were counted and compared between the four groups. To evaluate the effects on tumor growth, Ehrlich carcinoma cells were injected into the thighs of mice, and tumor volumes and growth inhibition rates were compared. RESULTS: The number of leukocytes was increased in the VE group compared with that in the control group. The magnitude of leukocyte recovery after RT was also increased by VE. This change was affected largely by alterations in lymphocytes and monocytes rather than that in granulocytes. Both CD4+ and CD8+ T lymphocytes were positively affected by VE. The tumor growth was inhibited not only by RT but also by VE alone. If RT was delivered with VE, tumor growth was markedly inhibited. CONCLUSION: VE could increase the number of leukocytes, primarily lymphocytes, even after RT was delivered. VE also inhibited the tumor growth in addition to RT. Thus, VE may be a useful radioprotective supplement in radiotherapy without inducing tumor growth.

Plant Enzymes Decrease Prostate Cancer Cell Numbers and Increase TNF-α In Vivo: A Possible Role in Immunostimulatory Activity.

Increased caloric intake and Westernized dietary choices may be contributing toward a recent rising trend of incidences of chronic lifestyle-related diseases. In this study, we evaluated the anticancer properties of Plant Enzyme Validux (PEV) using a mouse model. Five-week-old male C3H mice were randomly distributed into four experimental groups: Control, PEV only, 6Gy irradiation only, and PEV + 6Gy. PEV was orally administered daily at 500 mg/kg for 14 days prior to three rounds of 2Gy irradiation. We focused on the anticancer action and immunostimulatory effects of PEV with and without irradiation. Oncogene suppression was observed after PEV treatment as was an increase in TNF-α, suggesting an antitumor effect. PEV administration also appeared to reduce oxidative stress as evidenced by a decrease in lipid peroxidation. In addition, PEV confirmed radioprotective effect by radical blocking ability by radiation irradiation. Immunological responses to PEV administration were evidenced by an increase in number of total white blood cells and T lymphocytes. Immunotherapy is drawing more and more attention as a treatment for prostate cancer, suggesting that there will be a need for the identification of specific targets for prostate cancer and for more basic research on the genetic aspects of immunotherapy. Thus, PEV may be of use as a radioprotective supplement during radiotherapy for tumor treatment.

Cellular and Molecular Level Mechanisms against Electrochemical Cancer Therapy.

Electrochemical treatment (ECT) is a promising new way to induce tumor regression by flowing direct current into the cancer tissue. ECT was applied to different kinds of tumors in clinical studies and showed good results. In addition, basic research has almost not been done in the field of evaluation of efficacy, dose-response, and cytotoxicity. Therefore, the objective is to study the cellular mechanism in the antitumor effect of ECT and to contribute data of basic research of ECT. In the cell-level study, tumor cells (Sarcoma-180, Scc-7, Ehrlich Carcinoma) were studied using ICR mice and C3H mice. In the study group, pH values of control, 10mA × 150secs, 10mA × 300secs, and 10mA × 600secs groups were measured five times each. In histological level studies, ECT was performed on tumors inoculated on the upper part of the right foot of C3H mice. In each group, mice were sacrificed by cervical dislocation 6, 12, and 24 hrs after ECT treatment, and tumors were removed. The excised tumor was fixed in tissue with 10% formalin, and HE staining and apoptosis antibody staining were carried out from the obtained tissue section and observation. In the study at the cellular level, statistically significant differences were observed in all ECT groups in Sarcoma in the tumor growth measurement study compared with the control group. Statistically significant differences were also observed in Scc-7 in all ECT groups compared to the control group. In the intratumoral pH measurement study, there was a statistically significant difference between the anode and the cathode in each group compared to the control group. In the examination at the histological level, microscopic observation of a slide stained with apoptosis antibody with a magnification of 400 times showed that 6hrs after ECT it was stronger and then decreased. By performing ECT, a weak current flows in the living body. As a result, changes in tissue pH, generation of gas, etc. occur. In this study, it was also confirmed that the intratumor pH value becomes strongly acidic on the anode side and strongly alkaline on the cathode side. In addition, this study confirmed the occurrence of gas during treatment of ECT. Changes in the pH and the like cause changes in the environment in the cell, denaturation of proteins, apoptosis, and necrosis. In this study, a significant increase in apoptosis was confirmed in each ECT group compared to the control group. Treatment effects by ECT were also observed in tumor growth measurement studies and tumor weight measurement studies. From these research results, ECT is considered to be effective as a tumor treatment method.

Subchronic Oral Dose Toxicity Study of Enterococcus Faecalis 2001 (EF 2001) in Mice.

As a part of general toxicity studies of Enterococcus Faecalis 2001 (EF 2001) prepared using heat-treatment bacillus mort body EF 2001 in mice, this study examined the toxicity of EF 2001 in single and repeated administrations following the previous report in order to apply this product to preventive medicine. The safety of oral ingestion of EF 2001 was examined in 6-week-old male and female ICR mice with 1,000 mg/kg, 3,000 mg/kg and 5,000 mg/kg body weight/day administrated by gavage of the maximum acceptable dose of EF 2001. The study was conducted using distilled water as a control following the methods for general toxicity studies described in the "Guidelines for Non-clinical Studies of Pharmaceutical Products 2002". As a control, 1) observation of general conditions, 2) measurement of body weight, 3) determination of food consumption, 4) determination of water consumption, 5) blood test and urinalysis and 6) pathological examination were performed for the administration of EF 2001. Mice received EF 2001 for 13 weeks and results were compared with those of the control group that received distilled water. The results of the above examinations revealed no significant differences between control and EF 2001 groups for both males and females. Thus, no notable toxicity was confirmed with single and repeated oral administrations of EF 2001. Oral administration in the above doses did not result in abnormal symptoms or death during the observation period. No abnormalities in blood cell count or organ weights were seen. Without any evidence of toxicity to cells and organs, EF 2001 is speculated to not adversely affect living organisms. The 50% lethal dose of EF 2001 with oral administration in mice is estimated to be greater than 5,000 mg/kg body weight/day for both male and female mice. Therefore, LD50 value for animals was 5,000 mg/kg or more.

Pharmaceutical Production of Anti-tumor and Immune-potentiating Enterococcus faecalis-2001 ß-glucans: Enhanced Activity of Macrophage and Lymphocytes in Tumor-implanted Mice.

BACKGROUND: Enterococcus faecalis 2001 is a probiotic lactic acid bacterium and has been used as a biological response modifier (BRM). From physiological limitation of bacterial preservation in storage and safety, the live E. faecalis 2001 has been heat-treated and the BRM components containing high level of ß-glucan, named EF-2001, were prepared. METHOD: The heat-treated EF-2001 has been examined for the antioxidative potential for radical scavenging and anti-tumor activities as well as immune-enhancing response in mice. Lymphocyte versus polymorphonuclear leukocyte ratio was increased in mice upon treatment with EF-2001. The number of lymphocytes was increased in the EF-2001-treated group. In the mice bearing two different Ehrlich solid and Sarcoma-180 carcinomas, the treatment with EF-2001 resulted in anti-tumor action. Tumor-suppressive capacity upon treatment with EF-2001 was significantly increased compared to normal controls. RESULTS: During the time interval administration of 5 weeks between the priming and secondary administration of EF-2001, the expression and production levels of TNF-α were also observed in the EF- 2001-administered mice. Additionally, anti-tumor activity examined with the intravenous administration of EF 2001 with a 34 times interval was also observed, as the growth of Sarcoma180 cells was clearly inhibited by the EF-2001. CONCLUSION: From the results, it was suggested that the immune response is enhanced due to antioxidative activity caused by the EF-2001 and anti-tumor activity by NK cells and TNF-α.

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.

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.

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.

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-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.

Water soluble vitamin E (TMG) as a radioprotector.

Tocopherol monoglucoside (TMG), a water soluble derivative of vitamin E offers protection against deleterious effects of ionizing radiation, both under in vivo and in vitro conditions, to biological systems. TMG was found to be a potent antioxidant and an effective free radical scavenger. It forms a phenoxyl radical similar to trolox upon reaction with various one-electron oxidants. TMG protected DNA from radiation-induced strand breaks. It also protected thymine glycol formation induced by gamma-radiation. Gamma-radiation-induced loss of viability of EL-tumor cells and peroxidation of lipids in microsomal and mitochondrial membranes were prevented by TMG. TMG was nontoxic to mice when administered orally up to 7.0 g/kg body weight. The LD50 dose of TMG for ip administration in mice was 1.15 g/kg body wt. In rats, following oral and ip administration of TMG, the absorption (distribution) half lives were 5.8 and 3.0 min respectively and elimination half lives were 6.7 and 3.1 min respectively. Embryonic mortality resulting from exposure of pregnant mice to ionizing radiation (2 Gy) was reduced by 75% by ip administration of TMG (0.6 g/kg, body wt) prior to irradiation. TMG offered protection to mice against whole body gamma-radiation-induced lethality and weight loss. The LD50(30) of mice increased from 6 to 6.72 Gy upon post irradiation administration of a single dose of TMG (0.6 g/kg, body wt) by ip.

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.