Pharmacological Strategies for Overcoming Multidrug Resistance to Chemotherapy.

Actual mechanisms of multidrug resistance (MDR) to chemotherapy in oncology are considered. ABC-transporters such as P-glycoprotein, BCRP protein, and MRP proteins take part in the development of resistance. The review presents the main classes of chemosensitizers, i.e., inhibitors of ABC transporters of the 1st-4th generations. Plant polyphenols, i.e., flavonoids, are commonly referred to as the last (4th) generation of MDR inhibitors. Chemosensitizers of different classes should be chosen with allowance for the patient mutation-expression profile and the receptor status of a particular tumor. The appropriate dosage of the chemosensitizer and the administration schedule can enhance the process of counteracting MDR.

[The mechanisms of anti-inflammatory action of enisamium iodide].

AIM: The role of cyclooxygenases (COX-1 and/or COX-2), transcription nuclear factor NF-κB, anti-inflammatory cytokines - TGF1b, IL-4, IL-10 and pro-inflammatory cytokines IL-1, IL-6 were studied to substantiate the expediency of antiviral agent enisamium iodide (Nobazit) using to regulate key inflammatory components in acute respiratory infections, IL-8, TNF-alpha in the realization of the pharmacological activity of this drug. MATERIALS AND METHODS: Gene expression was determined by real-time RT-PCR, the concentration of interleukins was determined by ELISA, and the viability of peripheral blood mononuclear cells (PBMC) was assessed by the MTT spectrophotometric method. The chemiluminescence method was used to assess PBMC oxidant activity. RESULTS: Enisamium iodide (10 µM) reduced mRNA levels of COX-1, COX-2, NF-κB, TGF1b, IL-1, IL-6 in stimulated PBMC of healthy donors by an average of 48% (p≤0.05). At 5 times higher concentration, 50 µM, enisamium iodide suppressed the expression of these genes by an average of 43% (p≤0.05). At a concentration of 100 µM, enisamium iodide reduced the expression of COX-2, TGF1b, IL-1, IL-6 by an average of 47% (p≤0.05). At a concentration of 10 µM, enisamium iodide stimulated the secretion of IL-10 by mononuclear cells by 1.2 times, p≤0.05. The tested drug at a concentration of 50 µM did not affected on the concentration of IL-1, IL-4, IL-8 and TNF-alpha, but significantly stimulated the production of IL-10 by 1.5 times, p≤0.05. The chemiluminescence method revealed that enisamium iodide in the entire concentration range (10-100 µM) does not reduce the viability of macrophages, but inhibits their oxidative activity (maximum value of CL intensity) by an average of 55% (p≤0.05). CONCLUSION: The anti-inflammatory effect of enisamium iodide at a concentration of 10 µM may be associated with inhibition of the expression of COX-1, 2, NF-κB, IL-1, IL-6, TGF1b and an increase in the expression and production of IL-10. An additional contribution to the anti-inflammatory activity of enisamium iodide is made by its antioxidant and antiradical activity. The absence of the effect of enisamium iodide (10-100 µM) on the viability of PBMC indicates its safety for the cells of the immune system and the expediency of using it to suppress inflammatory reactions in acute respiratory infections, restore the quality of life of patients and the possibility of using Nobazit as an effective agent for treatment of these infections of various etiologies.

Clinical Use of Progestins and Their Mechanisms of Action: Present and Future (Review).

This review summarizes the current opinions on the mechanisms of action of nuclear, mitochondrial, and membrane progesterone receptors. The main aspects of the pharmacological action of progestins have been studied. Data on the clinical use of gestagens by nosological groups are presented. Particular attention is paid to progesterone, megestrol acetate, medroxyprogesterone acetate due to broadening of their spectrum of action. The possibilities of using gestagens as neuroprotectors, immunomodulators, and chemosensitizers are considered.

Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.

[INFLUENCE OF PROGESTERONE DERIVATIVES ON THE VIABILITY AND EXPRESSION OF ESTROGEN RECEPTOR-ALPHA MRNA IN HELA CELLS.]

We have studied the effect of new ligands of progesterone receptors, including pregna-D'-pentaran 6-methoxyimino-16a,17a-cyclohexanopregn-4-en-3,20-dio-ne (K1047), 17a-acetoxy-3b-butanoyloxy-6-methylpregna-4,6-dien-20-one (buterol), progesterone (P4), and medroxyprogesterone acetate on the viability of HeLa cells and expression of estrogen receptor-alpha (Era) mRNA gene in these cells. K1I047 and buterol exhibited high cytostatic activity, which exceeded the activity of reference compounds on the average by 15% (p < 0.05). Both buterol and K-1047 (at 10(-6)M) effectively suppressed ERa mRNA gene expression in HeLa cell culture by 83.4 - 9 8.6%.

[ANTIOXIDANT AND CYTOPROTECTOR PROPERTIES OF GENISTEIN - ISOFLAVONE WITH ESTROGENIC ACTIVITY.]

Phytoestrogen genistein can exhibit cytoprotective and antioxidant properties, providing chemopreventive action, and produce cytotoxic effects on some tu- mors. In this work, the cytotoxic, cytoprotective, and antioxidant properties of genistein have been studied on model tumor cells (human cervical cancer HeLa cells) and normal cells (rat dermal fibroblasts, RDF). For assessing the cytotoxic effect of genistein (spectrophotometric MTT assay), the reference drug was cis-diaminodichloroplatinum (cisplatin); for evaluating antioxidant action, beta-estradiol was the reference drug. It is established that genistein produces a cyto- toxic effect only at high concentrations, IC50 = 20 mM and 14 mM for RDF and HeLa cells, respectively, which is 30 and 10 times higher than IC50 for cisplatin on these cells. Genistein like estradiol, but unlike cisplatin, had no effect on the mitochondrial pore induction from rat liver mitochondria. Thus, genistein at physiological concentrations (up to 200 n) acts as a cytoprotective agent. High antioxidant activity of genistein also suggests the possibility of its use as a chemopreventive drug.

[Cytoprotective Effects of Phytoestrogen Genistein against Cancer Cells].

In this paper we study the effect of synthetic isoflavonoid genistein against cancer HeLa cells, which contain estrogen receptors alpha but not beta, with the aim to determine the cytotoxic or cytoprotective effect of genistein. It is shown that the half maximal inhibitory concentration (IC50) value of genistein (0.2 mM) for the growth inhibition of HeLa cells is at least ten times higher than that one of tamoxifen and cisplatin--drugs, used in cervical cancer treatment. In micromolar concentrations (0.1-10 µM) genistein decreased the cytotoxic effects of cisplatin and tamoxifen. The decreased Bax mRNA expression and increased Bcl-2 mRNA expression after incubation .of the cells with genistein also demonstrate the cytoprotective, anti-apoptotic effect of genistein. Genistein, even in high concentrations, had no effect on membrane potential and calcium capacity of isolated mitochondria, without activating the opening of Ca(2+)-induced mitochondrial pore. Thus, these data demonstrate a cytoprotective effect of isoflavonoid genistein against this type of cancer cells.

[Antitumor and antiproliferative action of the steroidal cytostatic antiestrogen cytestrol acetate on hormone-dependent tumor models].

Cytestrole acetate (CA), in the structure of which the steroidal antiestrogen component is associated with bis-ß-cloroethylamino group, exhibits a strong cytotoxic activity against hormone-dependent cancer cell lines (CaOV, HeLa, MCF-7). In doxorubicin-resistant MCF-7 cells, CA potentiates the cytotoxic effect of etoposide and doxorubicin, and the IC50 for CA in these cells is 40 times lower than that for tamoxifen (TAM). In transplantable mice breast adenocarcinoma Ca-755, the therapeutic CA dose is 25 mg/kg when administered subcutaneously in oil solution for 5 days. On the DMBA-induced mammary tumors in rats, CA injected subcutaneously led to partial regressions 4 weeks after treatment in 75% of test rats, whereas TAM produced this effect in 43% of rats. Among various drug forms of CA, the most active were oil solution of CA in gelatin capsules for oral use and liposomal emulsion for intravenous administration, since these forms exhibited the highest values of Ca-755 tumor growth inhibition index (TGI = 97 - 98%).

[Redox-dependent ferric oxide nanoparticles loaded with doxorubicin and their influence on the functions of mitochondria].

The spectral, fluorescent and functional properties of ferric oxide and ferric hydroxide nanoparticles loaded with doxorubicin and stabilized with citric acid or lysine were studied in comparison with free doxorubicin. Their effect on the opening of calcium-induced mitochondrial pore and the possibility of the controlled release of doxorubicin under the influence of redox stimuli were investigated. The data show that the effect of nanoparticles on mitochondria depends on the type of a stabilizer. The spectral and fluorescence methods used allow us to estimate the presence or absence of free doxorubicin in solution of nanoparticles and the:concentration of bound doxorubicin. It is shown that the dithiotreitol and glutathione increase the amplitude of absorption and fluorescence of doxorubicin during incubation with nanoparticles. It is assumed that this effect may be associated with the reduction of the oxidized iron by thiols with subsequent release of doxorubicin.

[Effect of steroid hormones on production of reactive oxygen species in mitochondria].

Among the targets of the steroid hormones are mitochondria, which as the main source of reactive oxygen species (ROS) in the cell play a central role in the development of various pathologies. We studied the effect of progesterone and its synthetic analogues on mitochondrial ROS production. It was found that progesterone activates the formation of superoxide anion and hydrogen peroxide in mitochondria during oxidation of complex I substrates of the respiratory chain and exerts no influence on production of ROS during oxidation of succinate, complex II substrate of the respiratory chain. Synthetic analogues of progesterone - medroxyprogesterone acetate, buterol, acetomepregenol, megestrol acetate, have different effects on ROS production, depending on their chemical structure. By the effectiveness of impact on ROS production in mitochondria all the steroids tested can be classified in the descending order as follows: progesterone > buterol > or = atsetomepregenol > medroxyprogesterone acetate > megestrol acetate. Activation of ROS production by progesterone and buterol has different mechanisms: progesterone acts as an inhibitor of NAD-dependent respiration, while buterol and acetomepregenol form noncovalently associated complexes by hydrogen bonds between the ester carbonyl at C3 and SH-groups of the respective targets.

[Molecular mechanisms of cytostatic and chemosensitizing action of gestagens].

In this study cytostatic and chemosensitizing actions of gestagens are described. Cytostatic mechanisms can be divided into estrogen-mediated and independent ones. The former include down-regulation of estrogen receptors, induction of 17?-hydroxysteroid dehydrogenase and estron sulfotransferase. The latter are MAP-kinase inhibition, induction of cell differentiation and apoptosis. Chemosensitization by gestagens is due to inhibition of P-glycoprotein, GST and MRP. Another putative chemosensitization mechanisms involves mitochondrial transition pore (MTP), at least for such gestagens as buterol and acetomepregenol. Elucidation of common mechanisms of MTP and MDR regulation and the possible role of MTP in chemosensitization is a new direction of investigations into the action of steroid hormones.

[Modern approaches to human cognitive activity enhancement].

Recent literature on human cognitive activity enhancement is reviewed and summarized. Two classes of pharmacological approaches are picked out, i.e. modern aspects of traditional substance utilization and application of specially developed drugs. Among non-pharmacological approaches a number of psychological, physiological, behavioral and biophysical methods to improve human cognition, memory and learning are analyzed. The most attention is paid to non-drug approaches that utilize bioelectric processes of the individual including characteristics of its brain electrical activity--electroencephalogram (EEG) to enhance different aspects of cognitive functioning. Some promising lines of these investigations are delineated.

[Estrogen receptors in the diagnosis and treatment of hormone-dependent tumors].

The paper presents the current data available on the etiology of tumor diseases, among other things, shows the role of hormones, namely: estrogens and their receptors, in the mechanism responsible for the occurrence of these diseases. Each tumor was shown to have its receptor status and to be unresponsive to this or that therapy differently. The efficiency of treatment and its prognosis may be determined if the receptor composition of an involved organ is estimated correctly. The authors summarize the data available in the literature, as well as the results of their own investigations explaining the selective effect of estradiol receptor agonists and antagonists in various tissues. The mRNA varieties that potentially encoding for the synthesis of different subtypes of estrogen receptors (ER) - ER-α and ERß, which are also coded by various genes located in different chromosomes. Pituitary adenomas as hormone-dependent masses are described in detail. The authors data on the concentration of nuclear estrogen receptors in different types of adenomas are given.

[A new Russian gestagen with anticancer activity].

The authors present results obtained in a complex study of 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-on (ABMP). ABMP was shown to differ from existing analogues by high gestagen activity and prolonged action. The substance does not possess androgenic or mineralocorticoid activity, is not toxic when used in high doses, and possesses significant cytostatic and chemiosensitizing activity. These properties of ABMP demonstrate that the substance should be studied in clinical setting as a gestagen with anticancer and chemiosensitizing activity for further application to therapy of hyperplastic processes in the female genital system and tumors that are sensitive to female sex hormones.

Combined effect of bis-beta-chloroethylamine estrogen derivatives and doxorubicin on proliferation of sensitive and resistant MCF-7 cells.

The sensitivity of normal (MCF-7/WT) and doxorubicin-resistant (MCF-7/R) breast cancer cells to the antiproliferative effect of ethynylestradiol 11alpha-derivatives with the cytostatic residue in the 3-position of the steroid ring (antiestrogen cytostatics) was studied by evaluating cell viability using methylthiazole tetrazolium staining. The antiproliferative effects of these agents on cell lines in the presence of doxorubicin were compared. Antiestrogen cytostatics produced weaker cytostatic effect on MCF-7/WT cells, but more potent cytostatic effect on MCF-7/WT cells compared to those of doxorubicin. Moreover, administration of these agents in combination with doxorubicin more significantly suppressed proliferation of tumor cells. Accumulation and efflux of cytostatic doxorubicin in MCF-7/R cells were studied in the presence and absence of antiestrogen cytostatic Po716. Confocal laser microscopy showed that doxorubicin accumulation in MCF-7/R cells in the absence of Po716 took 20 min, while in the presence of antiestrogen cytostatic this process took 5 min. The rate of doxorubicin transport from tumor cells was much lower in the presence of the test antiestrogen cytostatic. Our results suggest that antiestrogen cytostatics increase the sensitivity of resistant MCF-7/R cells to doxorubicin by modulating the mechanisms of multidrug resistance of tumor cells.

[Antitumor activity of the new gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-one]. / Izuchenie protivoopukholevoi aktivnosti novogo gestagena 17 al'fa-atsetoksi-3 beta-butanoiloksi-6-metil-pregna-4,6-dien-20-ona.

Antitumor activity of a new highly active promising gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-one (butagest) was studied in mice with model cervical carcinoma (RShM-5). The reference drug was medroxyprogesteron acetate (MPA, Depo Provera) used in clinics. The new preparation introduced perorally in a dose of 1 mg per mice inhibited the model tumor growth by 73%, which was 18% (p < 0.01) more effective than the action of the reference drug MPA. The effect of the new gestagen was also studied in vitro with respect to human breast carcinoma of the MCF-7 line and human cervical carcinoma HeLa. The viability of the tumor cells was studied during a 6-day incubation with the drug at a concentration of 10(-7)-10(-5) M (MTT test). The reference compounds were progesterone and MPA. These drugs suppressed the growth of both MCF-7 and, in higher concentrations, of HeLa. Butagest inhibited the growth of HeLa in all concentrations. Thus, the new gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-one is capable of suppressing the viability of human breast carcinoma and human cervical carcinoma, being comparable or even more effective than the reference drugs.

Combined action of doxorubicin and gestagens on doxorubicin-sensitive and doxorubicin-resistant MCF-7 cells.

The combined cytostatic effect of doxorubicin and gestagens progesterone, medroxyprogesterone acetate, mecigestone, and butagest on doxorubicin-resistant and doxorubicin-sensitive human breast cancer MCF-7 cells was studied by the MTT assay. On the 6th day of incubation progesterone, medroxyprogesterone acetate, mecigestone, and butagest in high concentrations (10(-5) M) potentiated the cytostatic action of doxorubicin in sensitive and resistant cells by 30-50%. Potentiation of the cytostatic effect produced by doxorubicin in sensitive cells is related to intrinsic cytotoxic activity of gestagens. In resistant cells these changes are associated with potentiation of the effect of doxorubicin.

6alpha-Methyl-16alpha,17alpha-cyclohexane progesterone and progesterone inhibit growth of doxorubicin-sensitive MCF-7 and HeLa tumor cells.

Temporal and concentration dependencies of the effects of gestagens (6alpha-methylpentarane and progesterone) and cytostatic doxorubicin on proliferation of MCF-7 and HeLa tumor cells was studied using(3)H-thymidine test. Gestagens produced the maximum inhibitory effect of on cell proliferation in a concentration of 10(-5)M; the effect developed on day 6 of incubation. 6 alpha-Methylpentarane in a concentration of 10(-8)inhibited proliferation of HeLa cells more effectively than progesterone (p<0.05). In experiments with combined treatment of doxorubicin-sensitive MCF-7 and HeLa cells, progesterone in a concentration of 10(-7)M attenuated the cytostatic effect of doxorubicin (p<0.05), while 6alpha-methylpentarane in the studied concentrations did not modulate it.