Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway.

Identification of unique features of cancer cells is important for defining specific and efficient therapeutic targets. Mutant p53 is present in nearly half of all cancer cases, forming a promising target for pharmacological reactivation. In addition to being defective for the tumor-suppressor function, mutant p53 contributes to malignancy by blocking a p53 family member p73. Here, we describe a small-molecule RETRA that activates a set of p53-regulated genes and specifically suppresses mutant p53-bearing tumor cells in vitro and in mouse xenografts. Although the effect is strictly limited to the cells expressing mutant p53, it is abrogated by inhibition with RNAi to p73. Treatment of mutant p53-expressing cancer cells with RETRA results in a substantial increase in the expression level of p73, and a release of p73 from the blocking complex with mutant p53, which produces tumor-suppressor effects similar to the functional reactivation of p53. RETRA is active against tumor cells expressing a variety of p53 mutants and does not affect normal cells. The results validate the mutant p53-p73 complex as a promising and highly specific potential target for cancer therapy.

[Transcriptional inhibition of human papilloma virus in cervical carcinoma cells reactivates functions of the tumor suppressor p53].

Inactivation of tumor suppressor p53 accompanies the majority of malignant diseases in humans. Restoration of p53 functions in tumor results in death of cancer cells, which can be used in cancer therapy. In cervical cancer a product of E6 gene of the human papilloma virus promotes accelerated degradation of p53 in proteasome system. Therefore, one of the approaches to reactivation of p53 in cervical carcinoma cells could be the use of small molecules that inhibit functions of viral proteins. By using as a test system human cervical carcinoma cells (HeLa cell line bearing human papilloma virus type 18, HPV-18) with introduced reporter construct that expresses beta-galactosidase under control of a p53-dependent promoter we carried out screening of a library of small molecules to select small molecules capable of reactivating transcriptional activity of p53. We then characterized the effects of two most active compounds in cell lines that differ in the status of p53-dependent signaling pathway. Both of the compounds caused specific activation of p53 in the cell lines expressing HPV-18, to a lesser extent--HPV-16, and do not cause any effect in control p53 negative cells, or in the cells with undisrupted p53 pathway. Activation of p53 in cervical carcinoma cells was accompanied by the induction of the p53-dependent gene CDKN1 (p21), by inhibition of proliferation, and by the induction of apoptosis. Both of the compounds were capable of deep inhibition of transcription from the HPV genome, which apparently was the cause for p53 reactivation in response to decreased expression of the E6 protein. The observed low toxicity for normal cells allows considering these chemical compounds as prototypes for future anticancer drugs.

Small molecules that dramatically alter multidrug resistance phenotype by modulating the substrate specificity of P-glycoprotein.

By screening a chemical library for the compounds protecting cells from adriamycin (Adr), a series of small molecules was isolated that interfered with the accumulation of Adr in mouse fibroblasts by enhancing efflux of the drug. Isolated compounds also stimulated efflux of Rhodamine 123 (Rho-123), another substrate of multidrug transporters. Stimulation of drug efflux was detectable in the cells expressing P-glycoprotein (P-gp), but not in their P-gp-negative variants, and was completely reversible by the P-gp inhibitors. A dramatic stimulation of P-gp activity against Adr and Rho-123 by the identified compounds was accompanied by suppression of P-gp-mediated efflux of other substrates, such as Taxol (paclitaxel) or Hoechst 33342, indicating that they act as modulators of substrate specificity of P-gp. Consistently, P-gp modulators dramatically altered the pattern of cross-resistance of P-gp-expressing cells to different P-gp substrates: an increase in resistance to Adr, daunorubicin, and etoposide was accompanied by cell sensitization to Vinca alkaloids, gramicidin D, and Taxol with no effect on cell sensitivity to colchicine, actinomycin D, puromycin, and colcemid, as well as to several non-P-gp substrates. The relative effect of P-gp modulators against different substrates varied among the isolated compounds that can be used as fine tools for analyzing mechanisms of drug selectivity of P-gp. These results raise the possibility of a rational control over cell sensitivity to drugs and toxins through modulation of P-gp activity by small molecules.

p53 Involvement in the control of murine hair follicle regression.

p53 is a transcription factor mediating a variety of biological responses including apoptotic cell death. p53 was recently shown to control apoptosis in the hair follicle induced by ionizing radiation and chemotherapy, but its role in the apoptosis-driven physiological hair follicle regression (catagen) remains to be elucidated. Here, we show that p53 protein is strongly expressed and co-localized with apoptotic markers in the regressing hair follicle compartments during catagen. In contrast to wild-type mice, p53 knockout mice show significant retardation of catagen accompanied by significant decrease in the number of apoptotic cells in the hair matrix. Furthermore, p53 null hair follicles are characterized by alterations in the expression of markers that are encoded by p53 target genes and are implicated in the control of catagen (Bax, Bcl-2, insulin-like growth factor binding protein-3). These data suggest that p53 is involved in the control of apoptosis in the hair follicle during physiological regression and imply that p53 antagonists may be useful for the management of hair growth disorders characterized by premature entry into catagen, such as androgenetic alopecia, alopecia areata, and telogen effluvium.

p53 is essential for chemotherapy-induced hair loss.

Anticancer drugs stimulate apoptosis in the hair follicles (HF) and cause hair loss, the most common side effect of chemotherapy. In a mouse model for chemotherapy-induced hair loss, we demonstrate that p53 is essential for this process: in contrast to wild-type mice, p53-deficient mice show neither hair loss nor apoptosis in the HF keratinocytes that maintained active proliferation after cyclophosphamide treatment. HF in p53 mutants are characterized by down-regulation of Fas and insulin-like growth factor-binding protein 3 and by increased expression of Bcl-2. These observations indicate that local pharmacological inhibition of p53 may be useful to prevent chemotherapy-associated hair loss.

A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-alpha, protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction.

Activation of the LRP (lung resistance-related protein) gene by short-term exposure of human leukemia cells to phorbol ester and cytarabine.

Treatment-induced secondary drug resistance of tumor cells is a major cause of relapsed disease and therapeutic failure in cancer patients. It has been shown that the expression of the multidrug resistance MDR1/P-glycoprotein gene could be induced by short-term in vitro exposure of cells to protein kinase C (PKC) agonists or different chemotherapeutic drugs. We studied whether other genes involved in drug resistance are regulated by similar signaling pathways. Transient (up to 24 h) treatment of HL-60 or K562 leukemia cells with phorbol 12-myristate 13-acetate (TPA) resulted in increased steady-state level of LRP (lung resistance-related protein) mRNA and protein. Among conventional chemotherapeutic drugs tested, only cytarabine (Ara C) induced the LRP mRNA expression though no increase in LRP protein was detected. LRP gene activation was not detectable in either H9 T-cell leukemia or in solid carcinoma cell lines (BT-20, ZR-75-1, and SW 1573). None of the agents influenced the levels of MRP (multidrug resistance-associated protein) mRNA in any cell line tested. In HL-60 cells, the LRP activation by TPA or Ara C was sustained for at least 23 days after withdrawal of inducing agents. bis-Indolylmaleimide I, a potent PKC inhibitor, attenuated TPA-induced LRP activation. In contrast, the inhibitor had no effect on the LRP induction by Ara C. These data indicate that the LRP gene can be activated by different mechanisms, some of which involve PKC.

Inhibition of cytarabine-induced MDR1 (P-glycoprotein) gene activation in human tumor cells by fatty acid-polyethylene glycol-fatty acid diesters, novel inhibitors of P-glycoprotein function.

Fatty acid ester surfactants Cremophor EL and Solutol HS 15 were described earlier as modulators of multidrug resistance mediated by MDR1 P-glycoprotein (Pgp). We have shown that the most active components of these polydisperse surfactants are fatty acid-polyethylene glycol-fatty acid diesters (FA-PEG-FA). A new generation of Pgp-surfactant inhibitors of defined structure was therefore synthesized. In the present study we show that these compounds are also able to inhibit up-regulation of MDR1 gene expression caused by cytarabine (ARA-C) and doxorubicin in human tumor cell lines H9 and KB 3-1, which express minimal levels of MDR1 mRNA. The surfactant inhibitors, however, had no effect on the induction of MDR1 gene expression by protein kinase C agonists. Using a set of FA-PEG-FA diesters with various fatty acids and different lengths of the PEG domain, we demonstrated that the activity of diester preparations as inhibitors of drug-induced MDR1 activation was in proportion to their activity as inhibitors of Pgp function. Oleic and stearic acid diesters with PEG 900 (20 ethylene oxide units) were the most potent. The poloxamer analogs of these diesters demonstrated similar effects. In contrast, the well-known, structurally unrelated inhibitors of Pgp activity, verapamil, cyclosporin A and PSC 833, had no inhibitory effect on drug-induced MDR1 activation. The ability of FA-PEG-FA diesters to inhibit both Pgp function and drug-induced MDR1 activation suggests that these chemomodulators may be uniquely useful for the prophylaxis of Pgp-mediated multidrug resistance in drug-treated tumors.

[Changes in EEG and higher nervous activity of rats during cerebral anti-ischemic protection by acelysin]. / Izmenenie EEG i VID krys pri ispol'zovanii dlia protivoishemicheskoi zashchity golovnogo mozga atselizina.

The water-soluble aspirin (acelysin) has been used as an anti-ischaemic protector when injected in the dose of 150 mg/kg 30 min before ischaemia. The EEG has been registered during the whole period of experiment and the total EEG power index has been calculated. The higher nervous activity has been evaluated during analysis of rat's abilities for elaboration of conditional reflex of an active escape reaction in Y-labyrinth. The results have demonstrated the complete rehabilitation and restoration of brain functional activity 8 days after endurance of brain ischaemia under protection of acelisin.

[Anti-ischemic protection of the brain using water-soluble form of aspirin-acelisin]. / Protivoishemicheskaia zashchita golovnogo mozga s pomoshch'iu vodorastvorimoi formy aspirina-atselizina.

The domestic water-soluble aspirin (acelisin) has been used as an anti-ischemic brain protector. The total brain ischemia has been implemented in accordance with an original technique for 17 to 35 min. The doses of acelisin from 25 to 250 mg/kg have been tested during experiments. The infusion of solutions has been carried out before ischemia, 15 min before reperfusion and just after the beginning of reperfusion. The functional status and survival of rats have been evaluated during a week. The best result has been reached with 150 mg/kg acelisin injected 30 min before ischaemia. A positive effect was reported when acelisin was used in early postischaemic period.

[Anti-ischemic action of a 1-hydroxy-2,2,6,6-tetramethylpiperidine derivative from a series of nitroxyl bioantioxidants]. / Protivoishemicheskoe deistvie 1-gidroksi-proizvodnogo 2,2,6,6-tetrametilpiperidina iz riada nitroksil'nykh bioantioksidantov.

The experiments have been performed on 216 Wistar rats to examine anti-ischaemic action of the 1-hydroxy-2,2,6,6-tetramethyl-piperidine derivative (I), whose antioxidant properties were, earlier shown for model systems. Introduction of I (10(-4) M) into the perfusion medium and the subsequent storage (37 degrees C) of isolated liver was shown to decrease the accumulation of lipid peroxidation products (MDA). Compound I administrated (5 x 10(-6)-10(-5) M) in perfuse medium of isolated (Langendorff method) and ischemized (30 min, 37 degrees C) heart improves contractile function (Pmax) and decreases end-diastolic pressure at postischaemic period. In vivo injection of I increases (12 mg/kg, i.p.) the number of rats survival after sublethal time (2.5 h) of liver total ischaemia, increase (35 mg/kg, i.p.) the number of rats survival and improves parameters of heart function after ischaemic shock (6 h ischaemia and reperfusion of limbs). The analog of I, corresponding amine, possessing no antioxidant properties also fails to exhibit any anti-ischaemic effect.

Sterically-hindered hydroxylamines as bioactive spin labels.

The use of sterically hindered hydroxylamines for the regulation of free radical reactions in biological systems and for the pharmacological correction of pathological conditions is described. They are shown to possess a number of advantages over nitroxyl radicals. They are more soluble in water, are less toxic and are easily oxidized to nitroxyl radicals in aqueous solutions. Hindered hydroxylamines can be also used for the study of pharmacokinetics of bioactive spin labels by the EPR technique. Pharmacokinetic parameters for spin-labeled analogues of tetronal are evaluated. Sulfur-containing hindered hydroxylamines are effective bioantioxidants, inhibiting efficiently the LPO of the microsomal fraction of liver and ADP- and thrombin induced plasma platelet aggregation. They increase also the survival of animals under ischemic shock. A cyclic mechanism for its antioxidative action is suggested. Recent data on the influence of the nitroxyl in equilibrium hydroxylamine moiety on bio-activity are summarized.

[Disorders of cardiac contractile function in ischemic shock; the protective effect of antioxidants and liposomes made from egg phospholipids]. / Narusheniia sokratitel'noi funktsii serdtsa pri ishemicheskom shoke; zashchitnyi éffekt antioksidantov i liposom iz iaichnykh fosfolipidov.

Experiments were made on Wistar rats with 6h tourniqueting of the hind limbs to study animal survival rate, myocardial contractile function and protective action of antioxidants and egg phospholipid liposomes during ischemic shock. It has been shown that reperfusion of the limbs leads to a high animal lethality, make lower myocardial contractile function and coronary flow of the hearts isolated from rats following a 6h reperfusion of the limbs. Well-known antioxidant butylated hydroxytoluene and a new antioxidant tetramethylpiperidine derivative bring animal lethality down and improve coronary flow and contractile function of the isolated heart. Phospholipid liposomes increase survival rate moderately but have no any effect on the heart contractile function. It has been deduced that lipid peroxidation takes part in the disturbance of heart contractile function and genesis of the death within ischemic shock.

[Effect of ischemia and reperfusion of the rat brain on lipid peroxidation and the protective effect of antioxidants]. / Vliianie ishemii i reperfuzii golognogo mozga krys na protsessy perekisnogo okisleniia lipidov i zashchitnyi éffekt antioksidantov.

The experiments have been performed on 179 Wistar rats to examine the changes in the brain level of lipid peroxidation products upon 5-, 15-, 30- and 60-min ischemia and 5-, 20- and 60-min reperfusion and to study the protective effect of antioxidants. It has been found that ischemia is accompanied by the accumulation of lipid peroxidation products. The content increases by an average of 138-213% of the initial level. Brain reperfusion after 30-min ischemia was accompanied by an increase or maintenance of a high level of lipid peroxidation products. Ionol injection was accompanied by an increase in the survival of rats and prevented the accumulation of lipid peroxidation products after brain ischemia and reperfusion.

[Disordered functioning of the superoxide radical-superoxide dismutase system in rat liver ischemia]. / Narushenie v funktsionirovanii sistemy superoksidnyi radikal-superoksiddismutaza pri ishemii pecheni krys.

The rate of O2 radical generation in microsomal membranes (VO2), the activity of cytosol superoxide dismutase (Cu, ZnSOD) and mitochondrial superoxide dismutase (MnSOD), and the activity of xanthine oxidizing system (XO) after a two-hour ischemia following a 24-hour reoxygenation of the rat liver were investigated. The high value of VO2, as compared to Cu, ZnSOD activity, may result in regulation disorders in O2-SOD system during ischemia. During reoxygenation, xanthine oxidizing system in combination with lowered Cu, ZnSOD activity may substantially contribute to the disturbance.

[Assessment of the effectiveness of the action of chemical compounds on the enzymatic peroxidation of lipids]. / Otsenka éffektivnosti deístviia khimicheskikh soedinenii na fermentativnoe perekisnoe okislenie lipidov.

Inhibitory effects of 3-hydroxypyridines and of some drugs on NADPH-dependent lipid peroxidation was studied in liver microsomes of rats using a spectrophotometric technique with continuous monitoring of accumulation of peroxidation products. At the same time, inhibition of fatty acyl peroxidation catalyzed by the NADPH-dependent system and lipoxygenase from soybean was studied. The data obtained suggest that peroxidation, catalyzed by enzymes of two different types, may be inhibited to different degree by the same chemical compounds.