In vivo drug screening method of radiosensitizers using tumor-bearing chick embryo.

In radiotherapy, tumor hypoxia is the main factor responsible for treatment resistance, and the development of radiosensitizers that can overcome this is imperative. However, many drugs that are effective in vitro and in vivo fail in clinical trials, and thus it is necessary to develop an animal model that can be used for the correct evaluation of pharmacokinetics and activity. Developing chicken eggs are commonly used in various research fields such as anticancer drug sensitivity tests and cardiotoxicity tests. We examined whether the radiosensitizing activity of etanidazole, as a hypoxic cell radiosensitizer, could be evaluated using tumor-bearing chick embryo. Following the transplantation of mouse mammary carcinoma EMT6 cells on day 11, a solid tumor was formed on day 15 and an evaluation of the time-course of the tumor revealed that the tumor weight was the highest on day 18. The maximum dose of etanidazole that did not affect tumor growth and fetal survival was 1.0mg and the maximum X-ray dose was 8Gy. Etanidazole was intravenously administered 10min prior to single dose X-ray irradiation. A significant tumor growth inhibitory effect was confirmed with 1.0mg of etanidazole in combination with 8Gy X-ray. In the case of mouse colon cancer colon26 cells, the combination of 3.0mg of etanidazole and 2Gy X-ray showed 2.79 times higher radiosensitizing activity than that observed for the control group. These results demonstrate that it is possible to evaluate the activity of radiosensitizers using tumor-bearing chick embryo.

Case Report: A Non-small Cell Lung Cancer Patient Treated with GcMAF, Sonodynamic Therapy and Tumor Treating Fields.

BACKGROUND/AIM: Macrophage activating factor (MAF)-based immunotherapy has a wide application for use in treating many diseases via macrophage activation. Sonodynamic therapy (SDT) using low-intensity ultrasound and tumor treating field (TTF) therapy are novel therapeutic modalities. SDT is usually combined with ozone therapy to improve local hypoxia within the tumor environment. CASE REPORT: We treated a 77-year-old male diagnosed with non-small cell lung cancer ((NSCLC) stage 3B) using second-generation serum GcMAF and oral colostrum MAF-based immunotherapy combined with SDT, TTF and ozone therapies. RESULTS: This case report demonstrates that GcMAF, oral colostrum MAF, SDT, TTF and ozone therapy can be used for NSCLC without adverse effects. CONCLUSION: This case report suggests a new concept of cancer treatment using local destruction of cancer tissue, in this case conducted with SDT and TTF therapy, to be used in combination with serum GcMAF and colostrum MAF immunotherapy as a systemic treatment.

Degalactosylated/Desialylated Bovine Colostrum Induces Macrophage Phagocytic Activity Independently of Inflammatory Cytokine Production.

BACKGROUND/AIM: Colostrum contains antibodies, such as immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM), and, therefore, has potent immunomodulating activity. In particular, IgA has an O-linked sugar chain similar to that in the group-specific component (Gc) protein, a precursor of the Gc protein-derived macrophage-activating factor (GcMAF). In the present study, we investigated the macrophage-activating effects of degalactosylated/desialylated bovine colostrum. RESULTS: We detected the positive band in degalactosylated/ desialylated bovine colostrum by western blotting using Helix pomatia agglutinin lectin. We also found that degalactosylated/ desialylated bovine colostrum could significantly enhance the phagocytic activity of mouse peritoneal macrophages in vitro and of intestinal macrophages in vivo. Besides, degalactosylated/desialylated bovine colostrum did not mediate the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). CONCLUSION: Similar to the use of GcMAF, degalactosylated/desialylated bovine colostrum can be used as a potential macrophage activator for various immunotherapies.

Oral Colostrum Macrophage-activating Factor for Serious Infection and Chronic Fatigue Syndrome: Three Case Reports.

BACKGROUND: Gc protein-derived macrophage-activating factor (GcMAF) immunotherapy has been steadily advancing over the last two decades. Oral colostrum macrophage-activating factor (MAF) produced from bovine colostrum has shown high macrophage phagocytic activity. GcMAF-based immunotherapy has a wide application for use in treating many diseases via macrophage activation or for use as supportive therapy. RESULTS: Three case studies demonstrate that oral colostrum MAF can be used for serious infection and chronic fatigue syndrome (CFS) without adverse effects. CONCLUSION: We demonstrate that colostrum MAF shows promising clinical results in patients with infectious diseases and for symptoms of fatigue, which is common in many chronic diseases.

Clinical experience of integrative cancer immunotherapy with GcMAF.

BACKGROUND: Immunotherapy has become an attractive new strategy in the treatment of cancer. The laboratory and clinical study of cancer immunotherapy is rapidly advancing. However, in the clinical setting, the results of cancer immunotherapy are mixed. We therefore contend that cancer immunotherapy should be customized to each patient individually based on their immune status and propose an integrative immunotherapy approach with second-generation group-specific component macrophage activating factor (GcMAF)-containing human serum. PATIENTS AND METHODS: The standard protocol of our integrative cancer immunotherapy is as follows: i) 0.5 ml GcMAF-containing human serum is administered intramuscularly or subcutaneously once or twice per week for the duration of cancer therapy until all cancer cells are eradicated; ii) hyper T/natural killer (NK) cell therapy is given once per week for six weeks; iii) high-dose vitamin C is administered intravenously twice per week; iv) alpha lipoic acid (600 mg) is administered orally daily; v) vitamin D3 (5,000-10,000 IU) is administered orally daily. RESULTS: By March 2013, Saisei Mirai have treated over 345 patients with GcMAF. Among them we here present the cases of three patients for whom our integrative immunotherapy was remarkably effective. CONCLUSION: The results of our integrative immunotherapy seem hopeful. We also plan to conduct a comparative clinical study.>

Design, synthesis and destructive dynamic effects of BODIPY-containing and curcuminoid boron tracedrugs for neutron dynamic therapy.

BACKGROUND: We previously designed the boron tracedrugs UTX-42, UTX-43, and UTX-44, which possess antioxidant potency. In order to explore their destructive dynamic effects when bombarded by weak thermal neutrons, we performed thermal neutron irradiation of bovine serum albumin (BSA) treated with the boron tracedrugs. MATERIALS AND METHODS: Boron tracedrugs, including the boron dipyrromethene (BODIPY)-containing compounds UTX-42, UTX-44, and UTX-47 and the curcuminoid compounds UTX-50 and UTX-51, were designed for neutron dynamic therapy based on their molecular orbital calculation. Newly designed UTX-47, UTX-50, and UTX-51 were synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect decomposition by thermal neutron irradiation of BSA treated with these boron tracedrugs. RESULTS: The combination of 1.0 µM BSA with 100 µM of each of the boron tracedrugs showed a decrease in band intensity after irradiation. CONCLUSION: All boron tracedrugs tested caused destructive dynamic damage of BSA during thermal neutron irradiation, suggesting that boron tracedrugs could be used as dynamic drugs for neutron dynamic therapy.

Effect of the Gc-derived macrophage-activating factor precursor (preGcMAF) on phagocytic activation of mouse peritoneal macrophages.

BACKGROUND: The 1f1f subtype of the Gc protein (Gc(1f1f) protein) was converted into Gc-derived macrophage-activating factor (GcMAF) by enzymatic processing in the presence of ß-galactosidase of an activated B-cell and sialidase of a T-cell. We hypothesized that preGc(1f1f)MAF, the only Gc(1f1f) protein lacking galactose, can be converted to GcMAF in vivo because sialic acid is cleaved by residual sialidase. Hence, we investigated the effect of preGc(1f1f)MAF on the phagocytic activation of mouse peritoneal macrophages. RESULTS: We examined the sugar moiety of preGc(1f1f)MAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We also found that preGc(1f1f)MAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; the level of phagocytic activity was the same as that observed for GcMAF. CONCLUSION: PreGc(1f1f)MAF can be used as an effective macrophage activator in vivo.

Artepillin C (ARC) in Brazilian green propolis selectively blocks oncogenic PAK1 signaling and suppresses the growth of NF tumors in mice.

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.

The usefulness of mild temperature hyperthermia combined with a newly developed hypoxia-oriented 10B conjugate compound, TX-2100, for boron neutron capture therapy.

PURPOSE: To evaluate the usefulness of a new 10B-compound (TX-2100) as a 10B-carrier in boron neutron capture therapy (BNCT), compared with the simultaneous use of its component drugs, sodium borocapate-10B (BSH) and 3-amino-2-quinoxalinecarbonitrile 1,4-dioxide (TX-402). Further, the usefulness of mild temperature hyperthermia (MTH, 40 degrees Celsius, 30 min) combined with TX-2100 was also examined compared with MTH combined with the concurrent administration with its component drugs. MATERIALS AND METHODS: TX-2100 is a hybrid compound that has both a hypoxic cytotoxin unit (TX-402) and a thermal neutron-sensitizing unit (BSH). TX-2100 or both TX-402 plus BSH in combination with MTH or not was administered to SCC VII tumour-bearing mice intra-peritoneally. Then, the 10B concentrations in the tumours and normal tissues were measured by gamma-ray spectrometry. Meanwhile, SCC VII tumour-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumours, then treated with TX-2100, TX-402 plus BSH or BSH only, in the same manner as in the biodistribution experiments, either with or without MTH. Right after thermal neutron irradiation during which intra-tumour 10B concentrations remained at similar levels, the tumours were excised, minced and trypsinized. The tumour cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker) and the micronucleus (MN) frequency in cells without BrdU labelling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in the total (P + Q) tumour cell population was determined from the tumours that were not pre-treated with BrdU. The clonogenic cell survival was also determined in mice given no BrdU. RESULTS: 10B biodistribution analyses in tumours, brain, skin, muscles, blood and liver indicated that the administration of TX-2100 plus MTH is most favourable for concentrating a sufficient amount of 10B in tumours and maintaining a high enough 10B concentration during irradiation. In addition, MTH had a stronger sensitizing effect when combined with TX-2100 than with the concurrent administration of its components TX-402 and BSH on both the total and Q cell populations in solid tumours. CONCLUSION: MTH was very effective in combination with the newly-developed TX-2100. The sensitizing effect in combination with MTH should be examined when new 10B-carriers are designed.

Reevaluation of the radiosensitizing effects of sanazole and nimorazole in vitro and in vivo.

Sanazole (AK-2123, 3-nitrotriazole derivative, N1-(3-methoxypropyl)-2-(3-nitro-1 H-1,2,4-triazol-1-yl)acetamide) and nimorazole (5-nitroimidazole derivative, 4-(2-(5-nitro-1H-1-imidazolyl)ethyl)morpholine) have been tested clinically as hypoxic cell radiosensitizers, mainly outside Japan. To determine if these sensitizers deserve clinical investigation in Japan, we reevaluated the radiosensitizing effects of these compounds in vitro and in vivo, in comparison with a fluorinated 2-nitroimidazole derivative KU-2285 (N1-(2-hydroxyethyl)-1,2-difluoro-3-(2-nitro-1 H-1-midazolyl)propanamide). KU-2285 is a known and established radiosensitizer, but is not suitable for clinical studies because of the high cost of synthesis. In vitro, the radiosensitizing effects of the three compounds on SCCVII (squamous cell carcinoma line in C3H mice) tumor cells were examined at 0.5 and 1 mM under aerobic or hypoxic conditions, using a colony assay. In vivo, SCCVII tumors grown subcutaneously in the hind legs of C3H/HeN mice were irradiated with or without prior intraperitoneal administration of 100, 200 or 400 mg/kg of the drugs. Thereafter, tumor growth delay was measured. In vitro, no sensitizing effect was observed under aerobic conditions at 1 mM. Under hypoxic conditions, the sensitizer enhancement ratio (SER) determined at 1% cell survival level for sanazole, nimorazole and KU-2285 was 1.55, 1.45 and 1.95, respectively, at 1 mM, and 1.40, 1.40 and 1.75, respectively, at 0.5 mM. In vivo, all three compounds had significant radiosensitizing effects; their effects appeared to decrease in the order of KU-2285, sanazole, and nimorazole. It was suggested that sanazole may be more suitable for clinical trials than nimorazole.

Usefulness of combined treatment with mild temperature hyperthermia and/or tirapazamine in the treatment of solid tumors: its independence of p53 status.

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received tirapazamine (TPZ) with or without mild temperature hyperthermia (40 degrees C, 60 min) (MTH), gamma-ray irradiation with or without MTH and/or TPZ, cisplatin (CDDP) with or without MTH and/or TPZ, or paclitaxel (TXL) with or without MTH and/or TPZ. After each treatment, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling (i.e., quiescent (Q) cells) was determined by using immunofluorescence staining for BrdU. Meanwhile, 6 h after gamma-ray irradiation or 24 h after other cytotoxic treatments, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN frequency and apoptosis frequency in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. On the whole, gamma-ray irradiation and CDDP injection induced a higher frequency of apoptosis and lower frequency of MN in SAS/neo cells than SAS/mp53 cells. There were no apparent differences in the induced frequency of apoptosis and MN between SAS/neo and SAS/mp53 cells after TPZ or TXL treatment. MTH sensitized cells to TPZ-inducing cytotoxicity more markedly in SAS/mp53 and Q cells than in SAS/neo cells and total cells, respectively. In gamma-ray irradiation and CDDP treatment, the enhancement in combination with MTH and/or TPZ was more remarkable in SAS/mp53 cells and Q cells than in SAS/neo and total tumor cells, respectively. Also in the case of TXL treatment, the combination with MTH and/or TPZ induced a slightly greater enhancement effect in SAS/mp53 cells and Q cells. In view of the difficulty in controlling mutated p53 status tumors and intratumor Q cells, combination treatment with MTH and/or TPZ as a cooperative modality in cancer therapy is considered to have potential for controlling solid tumors as a whole.

Potential of alpha-amino alcohol p-boronophenylalaninol as a boron carrier in boron neutron capture therapy, regarding its enantiomers.

PURPOSE: We evaluated the potential of a newly developed (10)B-containing alpha-amino alcohol of p-boronophenylalanine-(10)B (BPA), p-boronophenylalaninol (BPAol), as a boron carrier in boron neutron capture therapy. METHODS: C57BL mice bearing EL4 tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously via implanted mini-osmotic pumps to label all proliferating (P) cells. After oral administration of L-BPA or D-BPA, or intraperitoneal injection of L-BPAol or D-BPAol, the tumors were irradiated with reactor thermal neutron beams. Some of the tumors were heated at 40 degrees C for 30 min (mild temperature hyperthermia (MTH)) right before neutron exposure, and/or tirapazamine (TPZ) was intraperitoneally injected 30 min before irradiation. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling [ =quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The apoptosis and MN frequency in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. RESULTS: Without TPZ or MTH, L- and D-BPAol increased both frequencies markedly, especially for total cells. Although not significantly larger, L-BPA and D-BPAol increased both frequencies slightly more than D-BPA and L-BPAol, respectively. Combination with both MTH and TPZ markedly reduced the sensitivity difference between total and Q cells. CONCLUSION: Both L- and D-BPAol have potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.

An unusual sesquiterpene derivative from Ferula kuhistanica.

An unusual new sesquiterpene derivative, kuhistaferone (1), was isolated from the fruits of the Uzbekistan medicinal plant Ferula kuhistanica. The structure of 1 was established on the basis of spectroscopic evidence. Compound 1 showed moderate cytotoxicity against the human colon tumor cell line HCT116.