Changes in the Morphological and Immunological Characteristics of Mel Ibr Melanoma Cells in Response to Chicken Embryo Extract.

Exposure of Mel Ibr human melanoma cells to chicken embryo extract resulted in the appearance of a subclone with morphological and growth characteristics similar to those of embryonic stem cells. The subclone differed from the parental line cells by a sharply reduced percentage of HLA-DR(+) and CD54(+) cells, a significantly elevated percentage of CD63(+) cells, and appearance of CD133(+) and Oct-4A(+) cells. Hence, the subclone cells were characterized by the same features as stem tumor cells and could be responsible for further progress of tumor growth.

Optimized thermosensitive liposomes for selective doxorubicin delivery: formulation development, quality analysis and bioactivity proof.

In our study we examined thermosensitive liposomal formulations (TL) from the perspective to minimize the general toxicity drawbacks of chemotherapy. The TL become active in response to local hyperthermia (LH), and remain inactive at physiological conditions. Here, we formulated novel doxorubicin loaded thermoliposomes (Dox-TL) with optimized characteristics and tested their biological activity in vitro. The liposomal membrane composition of Dox-TL and their preparation technology were adjusted for high drug loading and extended formulation stability. The 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol(Chol):1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-2000) in molar ratio 9:1:0.2:0.02:0.2 and drug/lipid weight ratio 0.13-0.20/1 composition has demonstrated best results. The freshly-prepared vesicles contained 94% doxorubicin. The Dox-TL, freeze-dried with 4% sucrose, maintained high level of encapsulated drug, remained stable in serum and prevented premature drug leakage. The Dox-TL proved to be significantly less toxic at 37°C than free Dox. In combination with local hyperthermia of 42.5°C Dox-TL were as effective as free Dox in cell survival, and even outperformed free Dox in proliferation activity suppression, colony proliferation rate, and cellular uptake. These findings represent a solid basis for a safer and more effective antitumor therapy.

Integrins LFA-1 and Mac-1 and cytokines IL-6 and IL-10 in high-cancer mice under the influence of phytoadaptogen.

The expression of leukocyte integrins LFA-1 and Mac-1 and cytokines IL-6 and IL-10 was studied in mice predisposed to spontaneous hepatocarcinomas. The efficacy of a phytoadaptogen in correcting these parameters was evaluated. The role of adhesive interactions between immune cells and target cells in the recovery of antitumor regulatory mechanisms was estimated.

Oncolytic adenoviruses: A thorny path to glioma cure.

Glioblastoma Multiforme (GBM) is a rapidly progressing brain tumor. Despite the relatively low percentage of cancer patients with glioma diagnoses, recent statistics indicate that the number of glioma patients may have increased over the past decade. Current therapeutic options for glioma patients include tumor resection, chemotherapy, and concomitant radiation therapy with an average survival of approximately 16 months. The rapid progression of gliomas has spurred the development of novel treatment options, such as cancer gene therapy and oncolytic virotherapy. Preclinical testing of oncolytic adenoviruses using glioma models revealed both positive and negative sides of the virotherapy approach. Here we present a detailed overview of the glioma virotherapy field and discuss auxiliary therapeutic strategies with the potential for augmenting clinical efficacy of GBM virotherapy treatment.

[Optimization of policy aimed at forming a permanent access based on ultrasonographic duplex scanning for programmed dialysis].

The study was aimed at optimizing the policy of forming a permanent vascular access (PVA) by means of preoperative colour duplex scanning (CDS) of vessels in patients on programmed haemodialysis. The study included 420 patients undergoing from September 2003 to September 2011 a total of 595 PVAs. The Study Group (Group I) patients (351 PVAs) were subjected to preoperative PVA of vessels of limbs accompanied by assessing velocity parameters of the venous blood flow. The comparison group (Group II) patients (244 PVAs) underwent clinical examination only. The end point of the study was early diagnosis of incompetence of the PVA. Using preoperative PVA of vessels improved the outcomes of forming the PVA, significantly increasing the number of native arteriovenous fistulas (AVF) as a whole: (88.0% for Group I and 65.6% for Group II; p<0.01), their variants: radial-cephalic (I - 57.5%, II - 32.2%; p<0.01) and secondary radial-cephalic variants (I - 8.3%, II - 3.7%; p<0.01), leading to decreased frequency of using synthetic prostheses both totally (I - 12.0%, II - 34.4%; p<0.01) and in all positions (p<0.01) taken apart. Also decreased the frequency of the development of incompetence of all PVAs (I - 10.8%, II - 29.9%; p<0.01), AVFs as a whole (I - 7.4%, II - 18.0%; p<0.01) and accesses with a synthetic prosthesis both as a whole (I - 1.7%, II - 11.9%; p<0.01) and in all positions separately (p<0.01). Determining the velocity parameters of venous blood flow made it possible to exclude the development of significant proximal venous obstruction and to refuse phlebography. We consider it obligatory to perform CDS of vessels prior to forming a PVA.

[DR3/LARD mRNA spliced variants' frequency at colorectal cancer].

There are a lot of "death receptor" DR3/LARD mRNA variants that are formed during the alternative splicing. Membrane and soluble forms of the receptor are encoded with various types of the DR3/LARD mRNA and perform different functions. Using RT-PCR, the DR3/LARD mRNA spliced variants' frequency was measured in colon cancer patients' samples and cancer cell lines. In samples under investigation, four forms of the DR3/LARD mRNA were found with various frequencies. Two of them encoded the membrane receptors (LARD la mRNA and DR3beta mRNA) and other two expressed the soluble molecules (LARD 3 mRNA and soluble DR3beta mRNA). In blood of healthy volunteers 11 combinations (spectrums) of the DR3/LARD mRNA forms were revealed, and the "full" spectrum including all four variants of DR3/LARD mRNA dominated. In blood of colon cancer patients and tumour tissue samples, 6 DR3/LARD mRNA spectrums were found. The diversity of the DR3/LARD mRNA spectrums was decreased in colon cancer patients because of the frequency reduction of soluble DR3beta mRNA. Reduction of a variety of spectrums in cells of the patients was caused by decrease in occurrence of mRNA of the soluble DR3beta form. In samples of the tumor centers the spectrum with absence only mRNA of the soluble DR3beta form dominated. In blood of patients two spectrums prevailed: "full" range and presented mRNA LARD la and mRNA LARD 3. Only these two spectrums of mRNA DR3/LARD were also found in the tumor cell lines. Distinctions in occurrence of spectrums of DR3/LARD mRNA at healthy volunteers and colon cancer patients can define a different susceptibility of immunocompetent and tumor cells for apoptosis signals.

[Heterogeneous expression of CD38 gene in tumor tissue in patients with colorectal cancer].

The CD38 gene codes a membrane protein which takes part in cell adhesion and catalyzes the formation of cyclic ADP-ribose. Using RT-PCR method we tested the presence of full-size and alternative forms of mRNA CD38 in samples of tumor tissue of patients with colorectal cancer and in tumor cell lines. It was shown that there are the cells in the tumor tissue which expressed CD38 gene. In tumor tissue of patients the alternative form of mRNA CD38 was detected less frequently than full-size form. Cells of lines Colo-205, T-84, HCT15 and HCT116 contained mRNA CD38, in cells of lines Caco-2 and SW-620 mRNA CD38 was absent. In cells of tumor tissue on the first stage of colorectal cancer CD38 gene was expressed in 100% of cases. On the second, third and fourth stages of the disease gene expression was observed less often. The frequency of mRNA CD38 detection not depend on tumor localization, tumor grade and presence of metastases. Using method of restriction analysis CpG methylation was detected in binding sites of transcription factor Sp1 and receptor of retinoic acid (RARE) in all tested samples of tumor tissue independently of the presence or absence of mRNA CD38. The obtained data suggest that in the tumor cells of patients with colorectal cancer the expression of the CD38 gene is heterogeneous.

[Nanostructured liposomal systems as transport agents for anticancer drugs].

Liposomes quite recently have turned from a model of biological membranes into an object of extensive research and practical use. The versatile traits of liposomal formulation allow its' universal implementation, especially in cancer chemotherapy. The advantages of liposomal use as a carrier of an anticancer drug for its targeted selective accumulation are discussed in this article. This article contains description of new types of liposomes, differing in contents and use, such as: simple, sterically stabilized, targeted (immunoliposomes),cationic, sensitive to physical and chemical stimuli. The characteristics of liposomal systems of anticancer drug delivery designed at Blokhin Russian Oncological Scientific Centre is given in the article.

[pAkt expression in cervical intraepithelial neoplasia (CIN) and in microinvasive cervical cancer].

The study objective was an immunohistochemical evaluation of pAkt expression in 81 CIN and microinvasive cervical cancer tissue samples and 10 samples of relatively "normal" cervical epithelium of HPV-infected women. PAkt expression showed significant up-regulation in CIN2, CIN3 and microinvasive cancer in compare to CIN1 and "normal" epithelium. The rate of pAkt- positive cells increased progressively by cervical neoplasia grade advancement reaching 7 +/- 5% in CIN2, 15 +/- 13% in CIN3 and 17 +/- 15% in microinvasive cancer. The rate of pAkt-positive cases in general was 1,7-fold higher in CIN3 (41%) than in CIN2 (24%). pAkt expression in conjunction with other markers may be used in immunohistochemical studies for individual CIN outcome prognosis and prospectively in immunocytochemical tests for CIN grade diagnostics improvement before using invasive methods. To elaborate multicomponent system of markers with their indexation there is a need for further investigations with greater number of cases.

[Ki-67 expression, thymidine phosphorylase and PTEN in intraepithelial cervical carcinoma].

Expression of Ki-67, thymidine phosphorylase (TP) and PTEN were assessed in various grades of cervical intraepithelial neoplasia (CIN) in order to evaluate their potentials of predicting the gravity of possible damage to the epithelium as well as pro- or regression of CIN. Ki-67 and TP levels were shown to correlate directly with CIN grade. It was suggested that a small number of cases of Ki-67 and TP expression absence (15%), exclusively in CIN3 samples, be due to imminent progression to invasive cancer. Both separately and in combination, Ki-67 and TP expression indices should be regarded as having a potential as markers for cervical carcinoma diagnosis, grade and clinical course.

Prospects of using phytoadaptogen in the treatment of diffuse stomach cancer.

The efficiency of treatment of diffuse stomach cancer can be improved by using a complex phytoadaptogen. In groups receiving phytoadaptogen, the level of tumor marker CA 19-9 decreased and the mean life span of patients increased by 2.5 times. The drug exhibited immunomodulating (including interferonogenic and adhesiogenic), antioxidant, and hormone-modulating effects.

[Composite phytoadaptogens in oncology and gerontology].

The article describes results of research devoted to Phytomix-40, a mixture of plant adaptogens. It focuses on immunobiological criteria for its formulation, chemical composition and manufacture procedures, biological standartization tests, in vitro and in vivo preclinical studies, clinical trials in patients with non-malignant tumours (benign prostatic hyperplasia), precancer (oral leukoplakia), advanced cancer (malignant gastric cancer), and age-related neurodegenerative disease (parkinsonism). Prospects for the development of other plant preparations for non-toxic prevention and treatment of cancer and prolongation of life span of the affected subjects are discussed.

[Pharmacodynamic characteristics of velcade efficacy in resistant and recurrent multiple myeloma: determination of free light chains of blood serum immunoglobulins].

AIM: To ascertain individual sensitivity to velkeid in patients with resistant and recurrent multiple myeloma (MM) by determination of free light chains (FLC) of serum immunoglobulins. MATERIAL AND METHODS: Fourteen patients with MM stage III (Gcappa-5, Glambda-2, Acappa-3, Alambda-1, BJcappa-3) aged 52-75 years with documented resistance to treatment or recurrence received second-line monotherapy with velkeid. The drug was injected intravenously (jet) in a dose 1.3 mg/m2 on the treatment day 1, 4, 8 and 11. Free light chains concentration was examined with antibodies to their latent determinants (Binding Site, UK) on nephelometer (Hitathi-911, Japan) on velkeid treatment day 1, 2, 3, 5, 9 and 12. RESULTS: Nine patients showed lowering of FLC concentration and responded to treatment by Durie criteria. CONCLUSION: Dynamic follow-up of FLC concentration of the tumor clone in resistant and recurrent MM evaluates pharmacodynamics of the drug. The method provides prognosis of late clinical results.

[Parameters of specific antibody interaction with P-gp in T-cell leukemia Jurkat cells].

UNLABELLED: Special features of Pgp expression evaluation by flow cytometry were investigated. Indexes of interaction of FITC-conjugated Becton Dickinson Pharmingen monoclonal antibodies to external Pgp epitope (clone 17F9) were analyzed depending on the cell concentration (400000 to 3000000 cells/ml) and the specific antibody concentration (5, 10 and 20 microl of the market product solution per 300 microl of the cell suspension). RESULTS: 1. Optimal condition of incubation with the antibodies was revealed--after the cell fixation in 4% formaldehyde. 2. Character of the increase of the cell fluorescence average intensity in the suspension totally according to the concentration of the Pgp-specific antibodies did not depend on the number of the cells. 3. Both the absolute value of the average intensity of the cell specific fluorescence as well as cell number out of the isotypic control fluorescence region depended on the ratio of the cell number to monoclonal antibody concentration. CONCLUSION: 1. It was shown that Pgp was practically expressed in all Jurkat cells. 2. By the Pgp expression level, the Jurkat cell culture was sufficiently homogeneous and stable in various passages. 3. Jurkat cells could be used as test culture in estimation of the market antibody activity. 4. For immunofluorescent assay of the Pgp expression in human tumor biopsy specimens, it is necessary to use not less than three concentrations of the specific antibodies, not less than three concentrations of the cells in the suspension as well as concurrent assay of the cell culture characterized previously. In particular, for investigated Pgp monoclonal antibodies, it is possible to use Jurkat cell culture. It allows revealing not only the fact of the Pgp expression but the level of the expression as well, i.e. to estimate severity of multidrug resistance phenotype.

[Detection rate of MAGE-A1-A6 mRNA in the peripheral blood of cancer patients].

The mRNA expression of 6 MAGE-A (MAGE-A1-A6) antigens by the tumor focal cells and cancer cells circulating in blood was studied in solid malignant and benign neoplasms. MAGE- A1-A6 mRNA expression was detected in the tumor focal cells in more than 90% of cases of cancer of the breast, lung, and stomach and melanoma cell lines. The detection rate of peripheral blood MAGE-A1-A6-positive tumor cells was 95% in lung cancer, 53% in corpus uteri cancer, 67% in gastric cancer, 63% in breast cancer, 33% in melanoma, and 42% in uterine myoma. MAGE-A3 and MAGE-A4 mRNA expression was more commonly observed.

[The serum levels of a soluble form of HLA I molecules in patients with lung cancer].

The paper presents the results of studying the content of soluble HLA I (sHLA-I) molecules in the blood samples from 56 patients first admitted to hospital for histologically verified lung cancer and 23 healthy donors matched by the patients' sex and age. The findings suggest that the development of Stages IB, IIA, and IIIA lung cancer is accompanied by a significant reduction in the serum levels of sHLA-I molecules. The serum concentration of sHLA-I molecules is decreased when the involvement foci are located in the lymph nodes of the lung root or in the mediastinum, but significantly increased when distant metastases emerge. The different morphological variants of the structure of a tumor do not differ in the serum content of sHLA molecules in the patients.

[High-dose gamma-irradiation enhance expression of transgene under control of immediate-early CMV promoter in stably transfected tumor cells].

Gamma-irradiation is a usual method to inactivate whole-cellular anticancer vaccines consisting viable tumor cells. To evaluate the effect of gamma-irradiation to transgene expression in tumor cells we constructed several stably transfected clones of human and mouse cell lines expressing transgenic GM-CSF or GFP under control of IE-CMV promoter. Irradiation of those cells with different doses (ranged from 20 to 100 Gr) of gamma-radiation caused loss of proliferation capacity with survival of the cells population clearly depended on irradiation dose. Cell-cycle staining reveals accumulation of the cells with G2/M DNA content and almost loss of cells in S-phase. Substantial proportion of irradiated cells shows beta-galactosidase activity and morphological changes associated with cell senescence. An irradiated cell shows no changes in the level of mitochondrial dehydrogenase activity regardless irradiation dose exposed. Irradiated cells retain their ability to express transgene. Moreover, amount of the secreted GM-CSF as well as MFI in GFP-expressing cells significantly increases after gamma-irradiation up to 10 fold for cells exposed with 100 Gr. Enhancing of the transgene expression in both human and mouse cells positively correlates with total dose of gamma-irradiation gained by the cells and demonstrates gradual nature. Overall, our results supports using of 100 Gr of gamma-irradiation as the optimal dose for whole-cell anticancer vaccine inactivation.

[Efficacy of various methods of a reporter gene transfer to chicken embryonic cells].

The methods of transfection ofa plasmid with a reporter gene involving DNA injection into chicken embryonic cells were studied. The parameters of the efficient transfection of chicken blastodermal cells with a foreign gene have been determined (20-24 and up to 40% in culture and embryos, respectively). A high efficiency of transfection of primordial germ cells isolated from the gonads has been obtained after DNA injection into the dorsal aorta of 2.5-day-old chicken embryos.

[Multiple medication resistance of apoptosis-resistant tumoral cells].

A4 clone cells, received by CD95-mediated selection from the parental line of Jurkat T-lymphoblast human leukosis, lost their ability of apoptosis as a result of programmed cell death mechanism breakdown. The complex of their acquired phenotypic properties meets tumor progression criteria: oxidative stress resistance, active immune suppression, and low requirement for growth factors. The loss of A4 cell ability of apoptosis is accompanied by acquisition of the phenotype of multiple medication resistance to a wide spectrum of antineoplastic chemotherapeutic drugs and cytotoxins.