CHARACTERISTICS OF TUMORS THAT DEVELOPED IN MICE AFTER TREATMENT WITH IRRADIATED SYNGENEIC MESENCHYMAL STEM CELLS OF BONE MARROW.

Mesenchymal stem cells (MSCs) are present in almost all organs and tissues of the organism. It is believed, that MSCs could be transformed into cancer stem cells spontaneously or under influence of genotoxic factors and trigger the growth of tumors. The aim of this work was to study the possibility of malignant transformation of cultured MSCs from murine bone marrow (MSCs-BM) after g-irradiation in vitro and characterize of biochemical and histological features of the tumors that developed after transplantation of MSCs-BM into syngeneic mice. Tumors were observed in 3­4 months after MSCs-BM transplantation. After administration of MSCs-BM irradiated at a dose of 1 Gy, tumors were seen in 2 of 5 mice. After transplantation of MSCs-BM irradiated at a dose of 6 Gy, tumors were found in all 5 of 5 mice. In the case of control MSCs-BM, only one tumor appeared in 6 months after transplantation. The telomerase activity was two times higher in the tumor developed from 6 Gy irradiated MSCs-BM than from 1 Gy irradiated MSCs-BM. The tumors developed from control and irradiated MSCs-BM were classified as multicomponent mesenchymomas («mixture of sarcomas¼). Histological examination showed that tumors contained tissue areas of different histogenesis. Thus, MSCs-BM g-irradiated at doses of 1 and 6 Gy and, much less frequently, control MSCs-BM can transform into tumor cells and induce development of multicomponent mesenchymomas.

[Effects of Low and Sublethal Doses of γ-Radiation on Mesenchymal and Neural Stem Cells from Mouse Brain].

Mesenchymal stem cells (MSC) exist in the brain in addition to the neural stem cells (NSC). The aim of this work was to investigate the sensitivity of mouse brain MSC (MSC(BR)) to sublethal doses of γ-radiation in comparison with the sensitivity of bone marrow MSC (MSC(BM)) and NSC and to study the effects of γ-irradiation at low doses on these cells. Cells were exposed to γ-radiation (137Cs) at the doses of 10 to 200 mGy at a dose rate of 10 mGy/min; higher doses were achieved at the dose rates of 200 and 500 mGy/min (60Co). The survival of cells was assessed by counting living cells after staining with trypan blue in the Goryaev's chamber or using the MTT test for NSC growing as neurospheres. SP fraction was measured using flow cytometry after incubation with rhodamine-123. Exposure to the doses in the range of 10 to 500 mGy stimulated cell proliferation. The maximum decrease in the cells number was seen on the seventh day after irradiation and it was practically the same for the MSC(BR) and MSC(BM). NCS were more radiosensitive than MSC. Exposure to the doses of 100 to 500 mGy stimulated cells proliferation of all SCs except of MSC(BM). It was shown that the size of SP fraction of MSC(BR) was diminished after γ-irradiation at low doses. Thus, the stimulation of cell proliferation after γ-irradiation at low doses is accompanied by the redistribution of distinct cell subpopulations: the decrease in the SP fraction and the increase in the general population of cells were observed.

[NEURONAL DIFFERENTIATION OF PC12 CELL LINE AND MURINE NEURAL STEM CELLS ON THE CARBON NANOTUBES FILMS].

The study of the interaction of nerve cells with specially designed substrates (scaffolds) with different surface characteristics at the nanoscale is a necessary step in the development of methods of stimulation of regeneration of nervous tissues, as well as to create next generation of bioelectronic devices. A promising material for such scaffolds may be carbon nanotubes (CNT) that are flexible films of graphene rolled into nano-sized cylindrical tubes. CNT were produced by chemical deposition from the gas phase. The analysis of the PC12 cells cultivated on quartz glass coated by carbon nanotubes films using electron and light microscopy has shown that CNT stimulate the proliferation and do not inhibit neuronal differentiation of PC12 cells. We have found that it is possible to obtain differentiated neurons from murine neural stem cells on the quartz glasses covered with CNT films. The data obtained indicate that the CNT films produced by chemical deposition from the gas phase onto quartz glass may be used as the electro conductive scaffold to obtain and study the functions of neural cells and possibly of mature neurons.

[The selective toxic effect of dialdehyde derivatives of the pyrimidine nucleosides on human tumor cells].

The impact of a number of synthetic nucleoside derivatives on the growth and survival of cultured human ovarian tumor cells (line SKOV-3) and normal human lung fibroblasts was investigated. It was shown that the dialdehyde derivatives of uridine, 1-ß-D-eritrofuranozyl uracil and 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine, in contrast to their unoxidized counterparts, exert marked toxic effect on SKOV-3 cells. Cultured human fibroblasts were less susceptible to the damaging effect of the dialdehyde nucleosides. The dialdehyde derivative of 1-ß-D-eritrofuranozyl uracil demonstrated greatest differences in the cytotoxic effect on these cultures: inhibition of tumor SKOV-3 cells growth on 50% or more was achieved at the concentrations of this compound ten times lower than in the case of normal fibroblasts.

Targeted delivery of doxorubicin: drug delivery system based on PAMAM dendrimers.

Polyamidoamine (PAMAM) dendrimers of the second generation (G2) are branched polymers containing 16 surface amino groups that allow them to be used as universal carriers on creating systems for drug delivery. G2 labeled with fluorescein isothiocyanate (FITC) efficiently bound with the surface of tumor cells at 4°C and was absorbed by the cells at 37°C. The covalent binding to G2-FITC of a vector protein, a recombinant fragment of the human alpha-fetoprotein receptor-binding domain (rAFP3D), increased the binding and endocytosis efficiency more than threefold. Covalent conjugates of G2 with doxorubicin (Dox) obtained by acid-labile linking of cis-aconitic anhydride (CAA) without the vector protein (G2-Dox) and with the vector protein rAFP3D (rAFP3D-G2-Dox) were accumulated by the tumor cells with high efficiency. However, a selective effect was observed only in rAFP3D-G2-Dox, which also demonstrated high cytotoxic activity against the human ovarian adenocarcinoma SKOV3 cells and low cytotoxicity against human peripheral blood lymphocytes. Based on these results, rAFP3D-G2 conjugate is promising for selective delivery of antitumor drugs.

A new system for targeted delivery of doxorubicin into tumor cells.

The use of vector molecules for the targeted delivery of antitumor drugs provides their selectivity for cancer cells. The recombinant receptor-binding fragment of alpha-fetoprotein (rAFP3D) was used as a vector molecule. The specific receptor of alpha-fetoprotein is a universal tumor marker, being expressed on the surface of many tumor cells, but not in normal human tissues. And rAFP3D includes the receptor binding cite of AFP. A three-component delivery system including vector protein rAFP3D, PAMAM G2 dendrimer and antitumor antibiotic doxorubicin (Dox) was synthesized. The attachment of two dendrimer molecules to the vector protein did not affect the effectiveness of rAFP3D binding to AFP receptor on the surface of tumor cells nor the effectiveness of receptor-mediated endocytosis. Dox was conjugated with G2 via cis-aconitic anhydride (acid labile linker). The in vitro Dox release study showed that the conjugate was stable at neutral pH but was labile at pH<6. The Dox release was correlated with the intracellular distribution of conjugate in tumor cells. The rAFP3D-G2-Dox conjugate demonstrated a high cytotoxic activity against human ovarian adenocarcinoma cell lines: Dox-sensitive SKOV3 cells and Dox-resistant SKVLB cells and was low-toxic against human peripheral blood lymphocytes. Based on our findings, we may conclude that it is possible to significantly increase the effectiveness of Dox delivery to tumor cells by using the targeted delivery system comprising the recombinant third domain rAFP3D as a vector molecule.

[Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters.

[New protein vectors based on an alpha-fetoprotein fragment for targeted DNA delivery into cancer cells].

A human alpha-fetoprotein fragment (AFP) modified with oligocationic homologs of nuclear localization signal was used to construct new target cell-selective DNA-carrier proteins. The new recombinant vectors containing C- or N-terminal polynucleotide-binding domains are able to form stable complexes with single- or double-stranded oligonucleotides and plasmid DNA. Using flow cytometry and fluorescent microscopy, it was shown that such nucleoprotein complexes can be selectively internalized in target cells receptors superexpressing AFP receptors. The results obtained are important both for understanding mechanisms of formation of DNA-protein complexes and for studying their interaction with intracellular molecular targets. The new proteins can be used as a tool for the development of highly selective and efficacious gene-selective antitumour drugs.

Targeted delivery of anti-cancer growth inhibitory peptides derived from human alpha-fetoprotein: review of an International Multi-Center Collaborative Study.

The alpha-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide ((125)I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX-GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

[Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain].

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.

Localization of Mullerian inhibiting substance receptors in various human cancer cell lines.

Recombinant human MIS (rhMIS) produced in transfected Chinese hamster ovary cells has been purified by immunoaffinity chromatography. In the absence of reducing agents, 140 kD homodimer and several oligomers with molecular masses from 280 to 1000 kD are present. Homodimer, tetramer, and higher-molecular-weight rhMIS fractions reduced survival of tumor cells. For these experiments, FITC-labeled rhMIS was used for binding and endocytosis studies by flow cytometry. Flow cytometry performed on MIS-sensitive cancer cell lines demonstrated specific binding of rhMIS. The majority of rhMIS receptors have cytosolic localization. Thus, the level of MIS receptors on the cell membrane was proportional to the content of MIS-binding proteins in the whole cell and defines a level of receptor-mediated endocytosis. The immunopurified rhMIS caused significant growth inhibition of ovarian and prostate adenocarcinoma and melanoma human cell lines in inhibition assays.

[Telomeric oligonucleotide complexes with PGEk protein vector: internalization by target cells and antiproliferative properties].

The recombinant protein PGEk, containing residual of the human epidermal factor (hEGF) bearing DNA binding sequence, retains ability of hEGF to bind with hEGF receptor and to induce cell proliferation was shown. On an example of PGEk complexes with telomeric mimic-oligodeoxyribonucleotide d(TTAGGG)4 and with its thio-analogue we had found such systems can be effectively and selectively internalized by hEGF receptors super expressing cells. The association of this process with a protein/oligonucleotide ratio in complexes was investigated. The intracellular localization of oligonucleotides was explored. We had shown that PGEk not only promotes intensive delivery of oligonucleotides, but also protects them from degradation by nucleases. The oligonucleotides in composition of complexes have considerably more expressed cytotoxic activity in comparison with free oligonucleotides.

Antitumor activity of alpha-fetoprotein conjugate with doxorubicin in vitro and in vivo.

alpha-Fetoprotein (AFP) was conjugated with doxorubicin (DR) using glutaraldehyde as a cross-linking agent. The protein/DR molar ratio in the conjugate is 1 : 2. Cytotoxic activities (CTA) of the AFP-DR conjugate and of the free DR were compared using human mammary gland carcinoma cells, both DR-sensitive (MCF-7Wt) and DR-resistant (MCF-7AdrR). The CTA of the AFP-DR conjugate was fivefold higher than the CTA of the free DR for sensitive cells of the MCF-7Wt line and sevenfold higher for resistant cells of the MCF-7AdrR line. The CTA of the AFP-DR conjugate was also studied in vitro using the proliferating endothelium taken for a model of endothelial cell lining of blood vessels that supply the tumor. The AFP-DR conjugate was shown to have a high CTA for the endothelial cells (IC50 = 2.5 nM); thus, the conjugate is suggested to manifest an anti-angiogenic effect in vivo. The antitumor activity of the AFP-DR conjugate was studied using mice with inoculated melanoma B16 tumors. The treatment of animals significantly inhibited the tumor growth (>97%) and increased by 60% the mean life span of the animals compared to the control. The high antitumor efficiency of the AFP-DR conjugate and the possibility to significantly decrease the tumor cell resistance to DR make this conjugate a promising chemotherapeutic agent.

Antitumor activity of alpha fetoprotein and epidermal growth factor conjugates in vitro and in vivo.

Conjugates of carminomycin (Cm) with alpha-fetoprotein (AFP) and epidermal growth factor (EGF) were prepared and their cytotoxic activities were studied in vitro. Both conjugates showed cytotoxic activity which exceeded that of free Cm in tumor cell cultures of MCF-7, SKOV3, QOS, P388 and B16 cells. The antitumor effects of the conjugates were studied in vivo in mice with subcutaneous tumors of B16 and P388 cells. The Cm-AFP and Cm-EGF conjugates inhibited tumor growth and noticeably increased the mean life span in experimental animals. Our results suggest that the therapeutic activity of Cm can be significantly enhanced by conjugation to AFP or EGF.

Targeting phthalocyanines to tumor cells using epidermal growth factor conjugates.

Epidermal growth factor (EGF) was used as a vector for targeted delivery of phthalocyanines to tumor cells. The conjugates of EGF with disulfochloride aluminum phthalocyanine [Pc(Al)] and disulfochloride cobalt phthalocyanine [Pc(Co)] were synthesized. The cytotoxic activity of the conjugates against the human breast carcinoma cell line MCF-7 was determined. The cytotoxic activity of the EGF-Pc(Co) conjugate was 4.5 times higher than that of the EGF-Pc(Al) conjugate. The antitumor activity of the EGF-Pc(Co) conjugate was also studied in vivo in murine melanoma B16. Compared to free Pc(Co), intravenous injections of Pc(Co) conjugated with EGF inhibited tumor development and increased mean life span and mean survival time of experimental animals.

Water-soluble 2,3,7,8-tetrachlorodibenzo-p-dioxin complex with human alpha-fetoprotein: properties, toxicity in vivo and antitumor activity in vitro.

The conditions for the formation of a non-covalent complex between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the human transport fetal protein, alpha-fetoprotein (AFP), have been studied. TCDD has been shown to form a stable complex with AFP in a 2:1 (TCDD:AFP) ratio. The apparent solubility of TCDD in water increases 10(5)-fold after complex formation. The toxicity of the TCDD:AFP complex injected into mice by the intravenous route is comparable with that of free TCDD administered in oil solution per os. The complex manifests very much higher toxicity (200-1400 times) against human tumor cells (CEM, MCF-7, HepG2) in vitro and surpasses TCDD in selectivity. AFP may facilitate TCDD transport in embryonic tissues and enhance its embryotoxic and teratogenic effects.

Antitumor activity of conjugates of the oncofetal protein alpha-fetoprotein and phthalocyanines in vitro.

The several conjugates of aluminium and cobalt complexes of phthalocyanines with human alpha-fetoprotein have been synthesized. Their cytotoxic activity against tumor cells and human peripheral blood lymphocytes was studied. The experimental data demonstrate that the cytotoxic activity of alpha-fetoprotein-phthalocyanine conjugates against three types of tumor cells of various origin is much higher (for aluminium and cobalt complexes more than 1000 and 50 times, respectively) in comparison with phthalocyanines themselves. The application of phthalocyanines as conjugates with alpha-fetoprotein makes it possible to markedly enhance the selective toxicity of phthalocyanines against human tumor cells.