Design Of Folate-Linked Liposomal Doxorubicin To Its Antitumor Effect In Mice
Maitani Y, Yamada A, Taniguchi Y, Kawano K, Hattori Y
Hoshi University, Tokyo, Japan
Background: Tumor cell targeting is a promising strategy for enhancing the therapeutic potential of chemotherapy agents. Polyethylene glycol (PEG)-coated (sterically stabilized) liposomes show enhanced accumulation on the surface of tumors, but steric hindrance by PEGylation reduces the association of the liposome-bound ligand with its receptor. To increase folate receptor (FR)-targeting, we optimized the concentration and PEG spacer length of folate-PEG-lipid in liposomes.
Methods: Three types of folate-linked liposomal doxorubicin (DXR) were designed and prepared by optimizing the concentration and PEG-spacer length of folate-PEG-lipid in PEGylated or non-PEGylated liposomes, and by masking folate-linked liposomes where the folate ligand is “masked” by adjacent PEG spacers. The liposome targeting efficacy was evaluated in vitro and in vivo.
Results: The effects of PEG-spacer length and ligand density on folate receptor-targeted liposomes were evaluated. In human oral carcinoma KB cells, which overexpress FR, modification with sufficiently long PEG spacer and a high concentration of folate ligand to non-PEGylated liposomes increased the FR-mediated association and cytotoxicity more than with PEGylated and masked folate-linked liposomes. On the contrary, in mice bearing murine lung carcinoma M109, modification with the folate ligand in PEGylated and masked folate-linked liposomes showed significantly higher antitumor effect than with non-PEGylated liposomes irrespective of the length of time in the circulation after i.v. injection.
Conclusions: 1) Folate ligands with 0.25 mol% and sufficiently long PEG spacers (F-PEG5000-DSPE) of the folate ligand of liposomes without PEG-coating increased the folate receptor association and cytotoxicity compared with those with PEG-coating and masking folate-linked liposomes in vitro. 2) On the contrary, folate-linked and masking folate-linked liposomes showed a higher tumor killing effect than folate-linked liposomes without PEG-coating in vivo. The results of this study will be beneficial for the design and preparation of ligand-targeting carriers for cancer treatment.
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Competitive Interactions In Escherichia Coli Populations: The Role Of Bacteriocins
MAJEED H1, RILEY MA2 and GILLOR O1
1Department of Environmental Hydrology & Microbiology Zuckerberg Institute. Ben-Gurion University, Sede Boker, Israel; 2Department of Biology, University of Massachusetts, Amherst, USA
Introduction: Bacteriocins are highly toxic, naturally occurring antimicrobials found in all lineages of bacteria. Hundreds of these toxins have been identified each active only against its close relatives, thus they were proposed for drug development enabling the creation of pathogen-specific designer drugs. Bacteriocins producers were shown to competitively exclude non-producing strains, yet, the dynamics between bacteriocin producers are largely unknown. Here we studied the competitive interactions between them employing in vitro and in vivo models.
Methods: Two Escherichia coli strains were generated, each carrying a unique bacteriocin (E2 & E7). The bacteriocins encoding gene promoters were amplified, fused to reporter vectors and transferred to bacterial hosts. The reporter strains were used to monitor bacteriocin mediated induction. The bacteriocin producers were first competed against each other in a static culture environment and then employed in a murine model. Each strain was established in 7 mice and 3 mixed (E2/E7) and 4 control cages (E2/E2 & E7/E7) were generated. Cell density and killing phenotypes were monitored by fecal sampling.
Results: Using reporter gene assays we found that each bacteriocin is not only lethal to its opponent but at lower doses can also induce its expression. In the static culture the ratios of the distribution of the bacteriocinogenic populations did not change over time. In the mixed cages, no strain replacement occurred between mice harboring the experimental cages.Bacterial density fluctuated over time in the control cages while, the mixed cages showed reduced yet stable cell concentration over the sampling period.
Conclusions: We have demonstrated that due to mutual induction each population excluded its competitor by inhibiting invasion. Both models imply that bacteriocin-mediated bacteriocin induction enable producers to successfully compete and defend their niche against challenging invaders. We thus suggest that bacteriocin producing strains can potentially be used as bio-protective agents controlling the invasion of un-desired bacterial species.
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The Thyroid Gland Function Assessment In Women After Mastectomy And Chemotherapy During Breast Cancer Therapy.
MAJKOWSKA-MŁYNARCZYK A1,3, KINALSKI M², ZACZEK-KUCHARSKA E³
1Endocrinological Clinic of District Hospital, Kielce, 2Department of Gynecology of District Hospital, Białystok, ³Department of Health Natural –Humanistic Univwersity, Kielce, Poland
Background: For many years much attention has been focused on an interaction between the breast disease and the thyroid gland function in the literature. In those studies the question whether disease changes in the thyroid gland can induces the breast disease was addressed. On the other hand there are a few works concerning the inverted question whether the breast cancer therapy, in particular after mastectomy and chemotherapy, can disturb the thyroid gland function. So, the aim of the study is to investigate the influence of the mastectomy and chemotherapy on the thyroid gland function in women after breast cancer therapy.
Materials and methods: 173 patients aged 30-80 (average 56) were included in this study. The studied group comprised 97 women after breast cancer therapy (average age 60 ).The control group consisted of 76 patients (average age 55). 75 patients after mastectomy of the studied group were additionally treated with chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil;FAC: 5-fluorouracil, adriamecine, cyclophosphamide). The following methods were used to carry out the research: the USG method was applied to evaluate thyroid morphological condition in women after mastectomy and chemotherapy; the Color-Doppler technique was used for dynamic presentation and fine- needle aspiration biopsy: examination of the thyroid functional state by measuring the TSH, fT3, fT4 hormone concentration and the level of antithyroid antibodies.
Results: the average concentration of antithyroid antibodies: anty-TPO and anty-Tg was found significantly higher in the studied group of women after chemotherapy, comparing with the control group. The level of fT3 hormone concentration was comparable in all investigated groups. Nevertheless, the average concentration of TSH was found higher in women after mastectomy and chemotherapy and as a consequence leading to hypothyroidism.
Conclusion: Taking into consideration the high level of the concentration of antithyroid antibodies: (anty-TPO and anty-Tg), which lead to destruction of the thyroid gland tissue, the thyroid gland function of the women after mastectomy and chemotherapy should be monitored morphologically as well as functionally.
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Pulmonary Drug Delivery System For Efficient Treatment Of Tuberculosis
MAKINO K1-3, TERADA H2
1Faculty of Pharmaceutical Sciences, 2Center for Drug Delivery Research, 3Institute of Colloid and Interface Science, Tokyo University of Science, Noda, Japan
Background: Inhalable aerosol particles containing tubaculostatic agents are useful for the treatment of initial infection with Mycobacterium tuberculosis in the lung so that multiple drug resistant strains do not occur. Mycobacteria, which are resistant to biological digestion in macrophages, continuously grow in the phagosomes of alveolar macrophages using macrophages as incubators. Hence, the biocompatible and biodegradable microspheres containing anti-tuberculosis agents will be efficient for therapy of tuberculosis, if they are taken up well by alveolar macrophages through phagocytosis.
Methods: We have prepared PLGA microspheres containing the anti-tuberculosis agent rifampicin (RFP), by spray drying method, and they were administered to NR8383 cells derived from rat alveolar macrophages. RFP-loaded PLGA microspheres were administered by inhalation to rats which were infected with mycobacterium tuberculosis every day for 2 weeks and their killing effects against tubercle bacilli were evaluated.
Results: (1) NR8383 cells phagocytosed most efficiently the PLGA microspheres with a diameter of about 3 m, and phagocytosis attained the maximal level after 4 hrs, (2) the phagocytosis of PLGA microspheres stimulated phagocytic activity of macrophages, (3) the amount of RFP taken up by macrophages in a form of particles was about 20 times greater than that administered in a form of solution, and (4) RFP in macrophages efficiently killed BCG, used as a model of Mycobacterium tuberculosis, taken up by phagocytosis. Also we have shown that the RFP-loaded PLGA microspheres can reach deep in lung by inhalation and have shown the therapeutic effects in vivo.
Conclusions: It is possible that tuberculosis can be overcome efficiently by administration of anti-tuberculosis agents in a form of inhalable PLGA microspheres to alveolar macrophages.
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A New Application Of Granulocyte Colony-Stimulating Factor (G-CSF) In The Female Reproductive Medicine - For Prevention Of Luteinized Unruptured Follicle (LUF) -
MAKINODA S, TOMIZAWA H, FUJITA S, WASEDA T, FUJII R
Kanazawa Med. Univ., Uchinada, Japan
Background: G-CSF is detected in follicles and it shows a peak concentration in serum on a few days before ovulation. The expression level of G-CSF mRNA in the follicular wall at similar phase is 10-fold greater than other phases. These findings indicate that G-CSF plays an important role in the mechanism of follicle rupture, ovulation. LUF is an ovulation disorder that shows follicular growth and luteinization but lacks follicle rupture. We have conducted a clinical trial to induce ovulation by G-CSF for the patients of LUF.
Methods: Patients who received Clomiphene (CC) - human Chorionic Gonadotropin (hCG) treatment for ovulation induction and showed LUF at the last induction cycle participated in this study with informed consent. In addition to CC - hCG, G-CSF 100 μg was administered at 24 - 48 hours before hCG administration in 62 cycles of 56 patients, considering the natural cyclic changes of serum G-CSF. Ovulation was then confirmed by ultrasonography.
Results: The total numbers of LUF cycles before G-CSF treatment were 59 of 130 cycles (45.4%). Ovulation was successfully induced in 54 cycles (87.1%) with G-CSF, which is significantly higher than the cycles without G-CSF (P<0.0001). Pregnancy was confirmed in 4 cases of G-CSF treated cycles.
Conclusions: G-CSF administration during CC - hCG treatment is very effective to prevent LUF. Since there are no other treatments at present, G-CSF must be used for LUF patients as the first choice.
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Argyrin A A New Type Of Proteasome Inhibitor With Potent Anti-Proliferative And Anti-Angiogenic Activities
NICKELEIT I, MALEK NP
Institute for Molecular Biology, Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School
The cyclin kinase inhibitor p27kip1 acts as an important tumor suppressor protein in a variety of human cancers. Its expression levels closely correlate with the prognosis of the affected patient and also predict the outcome of various treatment modalities. While many tumors express low levels of p27 its genomic locus is rarely mutated in cancer cells. We previously showed that ubiquitin dependent turnover of p27 is part of the progression sequence of intestinal cancers. Using a mouse system in which p27 turnover is partly inhibited we were able to demonstrate that interference with p27 degradation results in a block to tumor cell de-differentiation in the intestine. Based on these results we decided to conduct a small molecule screen to identify substances which block p27 degradation. We identified Argyrin A, a natural compound derived from myxobacteria as a potent inducer of p27 expression in a variety of cancer cells. Treatment with Argyrin A induces apoptosis in human colon carcinoma cells in vitro and in xenotransplant tumors derived from these cell lines in vivo. Moreover Argyrin A also leads to a dramatic reduction of tumor vasculature in xenotransplants and an inhibition of HUVEC tube formation in vitro. All of these effects are dependent on p27 expression as cells derived from p27 knockout mice or cells in which p27 expression was lowered by siRNA treatment did not respond to Argyrin A. Interestingly cells in which the activity of the proteasome was lowered using siRNA specific for the beta1,2,5 subunits also displayed cellular phenotypes only when p27 was expressed in these cells.
Our results point towards a central role of p27 in controlling the cellular responses connected with proteasome inhibition. They also point towards the existence of signalling events downstream of the proteasome which use distinct proteins to mediate specific cellular phenotypes. The high efficiency with which Argyrin A targets cancer cells and tumor vasculature combined with its very low toxicity in vivo makes a p27 directed therapy using Argyrin A worthy of further clinical development.
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The Natural Estrogens And Mycoestrogens: From Simple Cell Proliferation To Transcriptional Effects On Cytochrome P450 1A1 And 1B1 Expression In Human Breast Cancer Cells
MALEKINEJAD H1, 2, FINK-GREMMELS J2
1Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Urmia University, Iran; 2Department of Veterinary Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
Background: In last decades much focused on compounds which acting as endocrine disrupters and among others mycoestrogens attracted much scientific attention.
Aims: 1- to study the mycoestrogen Zearalenone's (ZEA) toxicokinetics (TK) in animals and human in order to uncover potential target receptors other than estrogen receptors; 2- to evaluate any effect of estrogens and mycoestrogens at transcriptional level, to find possible approach for therapeutic purposes.
Methods: To study the biotransformation of ZEA, incubation of ZEA with hepatic sub-cellular fractions of animals and human was conducted. Real-time PCR analysis was also performed to determine the enzyme(s) which are involved in biotransformation of ZEA. Finally to determine the effect of the E2 and mycoestrogens on Cytochrome P450 1A1 and 1B1, qPCR analyses were performed.
Results: the biotransformation studies revealed that ZEA is converted predominantly into -Zearalenol as potent estrogenic metabolite in pig and human and into -Zearalenol as the weakest estrogenic metabolite in chicken and rat. We demonstrated that 3-HSD and 3-HSD are expressed in tissues that are exposed to ZEA and consequently ZEA metabolites are formed.
Exposing MCF-7 cells against E2 and ZEA reduced EROD activities significantly. Subsequent qPCR studies showed that the treatment of MCF-7 cells with E2 and mycoestrogens resulted to down-regulation of CYP 1A1 and up-regulation of CYP 1B1 in mRNA level.
Conclusions: 1) as -ZOL is the major metabolite of ZEA in human and pig, thus the reason of being sensitive to ZEA could be justified and equally as enzymes which are involved in this processes were identified, therefore any inhibitor of mentioned enzymes could be potential therapeutic agent in ZEA intoxication; 2) as CYP 1A1 and 1B1 are oxidative enzymes in steroids metabolism and any alteration in expression of these two could disturb the balance of the catechol estrogens formation, therefore prevention of any potential effect of ZEA and its metabolites on transcriptional level could be count as a novel approach in prevention of carcinogenesis.
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Potential Of JAK/STAT Inhibitors For Treating RA
MALEMUD CJ
Department of Medicine, Division of Rheumatic Diseases, Case Western Reserve University, Cleveland, Ohio (USA)
Background: IL-1, IL-6, IL-2, IL-7, IL-12, IL-15, TNF-, and the interferons are proinflammatory cytokines that contribute to cartilage destruction in RA, in part, by maintaining synoviocyte survival. These cytokines may also induce chondrocyte apoptosis. Whereas IL-1 and TNF- bind to IL-1R and TNFR1/TNFR2, respectively, where they activate the SAP/MAPK pathway, IL-6, IL-2, IL-7, IL-12, IL-15 and interferons are known to bind to Type I and Type II cytokine receptors and activate the JAK/STAT pathway. In this study, cultured human chondrocytes were used to determine which SAP/MAPKs and/or STAT proteins were specifically activated by human recombinant TNF-.
Methods: Chondrocytes were liberated from human osteoarthritic (OA) cartilage by enzymatic digestion. After growth in monolayer culture, chondrocytes were grown in high-density microcultures which were previously shown to maintain the chondrogenic phenotype (Malemud CJ et al. Cells Tissues Organs 2003; 174: 34-48) and treated with TNF- (10 ng/ml) for 24 hr after which cell lysates were collected at 1, 5, 30 and 60 min. SDS/PAGE combined with Western blotting determined the extent to which TNF- altered STAT, p38 kinase (p38), JNK1(p46), JNK2 (p54) phosphorylation and protein.
Results: Within 1 min TNF- caused specific phosphorylation of STAT3. STAT3 was maintained in the activated state for up to 30 min after which phosphorylated STAT3 was no longer detectable. TNF- also gradually activated p38 where maximal phosphorylated p38 was detected at 30 min. TNF- preferentially activated JNK2 within 5 min which was maintained for up to 60 min, whereas JNK1 phosphorylation increased for up to 5 min but was no longer detectable after that time. STAT3, p38 or JNK protein levels were not altered.
Conclusions: These studies showed that TNF- specifically activated chondrocyte STAT3 as well as p38 and JNK1, 2. Since it was previously shown that STAT3 was critical in order for RA or OA synoviocytes to survive (Krause A et al. J Immunol 2002; 169: 6610-6) the results of these in vitro studies suggested that a JAK2 inhibitor such as AG-490 may be useful for determining if STAT3 inhibition alone will result in NF-κB activation and induce chondrocyte apoptosis or whether additional specific p38 and JNK small molecule inhibitors can counteract TNF- induced chondrocyte STAT3 activation which may be critical for limiting chondrocyte apoptosis in RA.
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