Effect of Lysine Acridone Acetate on the Level of Apoptosis and Expression of Apoptosis-Associated Proteins during Antioncogenic Therapy in Colorectal Cancer in Mice.

We studied the mechanism of action of cytostatics with the addition of lysine acridone acetate to evaluate the possibility of its use for improving the effectiveness of antioncogenic therapy in colorectal cancer. In Nude mouse model, the level of apoptosis (TUNEL) and expression of proteins CD95, p53, Bcl-2, histone H3, and Ki-67 (immunohistochemistry) were assessed in primary tumor biopsy specimens. It has been shown that cytostatic treatment led to stimulation of p53-mediated apoptosis and suppression of proliferation (Ki-67 expression) of tumor cells, and apoptosis level was increased in groups receiving lysine acridone acetate. H3 expression in the experimental groups was changed.

[FAS- and TNF-dependent ways participation in apoptosis mechanisms in hypotalumus in physiological and pathological aging.]

The cell resistance to apoptosis can be related to the activity of cytokine-dependent signaling. So, the aim of the work is to investigate the mechanisms of cytokine-dependent FAS/TNF-mediated regulation of apoptosis of neurosecretory cells in the physiological and pathological (overexpression of the oncogene HER-2/Neu) aging. HER2/Neu transgenic accelerated aged mice of different ages and wild type FVB/N were examined. The apoptosis level of neurons in hypothalamic sections (supraoptic and paraventricular nuclei) (TUNEL) and expression of caspase-8, CD178 (FASL), FAS, FADD, TRADD (Western blotting) was determined. Participation of the proinflammatory component in the aging process is shown. FAS, adapter proteins associated with the death domain (FADD and TRADD), caspase-8 expression is activated in hypothalamus in FVB/N mice (wild type) during aging, and it correlates with an increase in the apoptosis level. HER-2/Neu expression leads to the extrinsic apoptotic pathway suppression. In this case, the reception of an apoptotic signal (FAS-receptor expression) and its further transmission (expression of FADD and TRADD) is suppressed. However, in young transgenic mice, increased expression of TRADD can activate one of the survival ways - NF-κB, ERK or PI3K-AKT cascade. Thus, the HER-2/Neu tyrosine kinase receptor plays a role in the mechanism of cell resistance to age-dependent apoptosis, and the FAS/TNF-signaling pathway is one of the targets of HER-2/Neu.

[Ultradian rhythms and oxidative stress in the tissue of the lymph nodes in ontogenesis.]

We studied the age-related features of the ultradian rhythms of levels of total protein and albumin in the lymph nodes under normal condition and chronic toxic oxidative stress condition using the chronobiological approach and the biochemical methods. During the experiment, we compared the rhythmic activity of levels of total protein and albumin in the rat lymph nodes in ontogenesis and during chronic exposure to sulfur-containing natural gas. The revealed fluctuations of the levels of total protein and albumin can be characterized as around-hourly (ultradian) rhythms, which period is approximately 20-40 to 60 minutes. The obtained experimental data indicate a decrease in the adaptive resistance of lymph node with age and the depletion of the antioxidant system of rats in aging. These results could have a practical value for the development of correction methods of negative effects of toxic oxidative stress in ontogenesis.

[Cytoflavin effect on locomotor and psychoemotional status in physiological and pathological aging.]

Currently, the actual problem is the correction of motor, cognitive and psychoemotional disorders in physiological aging, as well as in various pathological processes that accompany aging and accelerate it. In this regard, it became necessary to search for drugs that can restore age-related disorders of the brain. The aim of the study was to evaluate the possibility of Cytoflavin as a pharmacological corrector of age-dependent disorders of the functions of the cerebral cortex during physiological and pathological, accelerated aging. The mouse sensorimotor cortex of the brain was the material for study. The transgenic male mice with HER2/neu overexpression at the age of 2 and 10 months were used as an experimental model, male wild-type FBV/N mice at the age of 2 and 18 months served as a control. We studied locomotor activity, orientational research behavior and the psychoemotional status of animals using the «open field¼ test and the Suok test. It was found that in old FBV/N mice, after the cytoflavin treatment, recovery of locomotor functions and orientational-research behavior is observed. Under conditions of HER2/neu overexpression after the Cytoflavin treatment, an improvement in motor functions occurs. It was also shown that the studied drug has an anxiolytic effect on both wild-type FBV/N mice and transgenic HER2/neu mice during aging. Thus, the positive effect of Cytoflavin on the dynamics of the behavior of experimental mice during physiological and pathological accelerated aging allow to suggest that in the late stages of ontogenesis, Cytoflavin restores the cerebral cortex functions and prevents neurodegeneration.

Functional Imbalance of Glutamate- and GABAergic Neuronal Systems in the Pathogenesis of Focal Drug-Resistant Epilepsy in Humans.

The mechanisms of the formation of pharmacological resistance in temporal focal epilepsy remain poorly understood, and effective treatment strategies that can suppress epileptogenesis do not currently exist. We studied the imbalance between the glutamatergic (stimulating) and GABAergic (inhibitory) neuronal systems, as well as the role of apoptotic processes in the pathogenesis of drug-resistant epilepsy. To this end, the expression of Gad65, Vglut2, NR2B, Bcl-2, and caspase-8 proteins was analyzed in the gray and white matter of the temporal cortex of human brain. It was shown that pathological processes in the glutamatergic and GABAergic systems related to drug-resistant epilepsy are accompanied by changes in the content of apoptotic proteins, which can be the cause of neuronal death.

[Pharmacological correction of alterations of apoptosis of the neurons of the hypothalamus suprachiasmatic nucleus and pinealocytes during aging and stress.]

As is known, the pineal gland plays an important role in adaptogenesis, and the hypothalamus is one of the main links of the stress-reactive system and is involved in the regulation of the involution of the whole organism. So, the study of changes in these organs during stress and aging is very interesting. The aim of the work is to study the mechanisms of apoptosis of pinealocytes and neurosecretory cells of the suprachiasmatic nucleus of the hypothalamus during aging, stress, and under the conditions of pharmacological correction of involutional processes and stress response (antioxidant alpha-tocopherol acetate, immunomodulator cycloferon). We used Wistar rats as model, young (2-4 months) and old (30 months). Age-related features of the apoptosis dynamics of pinealocytes and neurosecretory cells of the hypothalamic suprachiasmatic nucleus were studied using TUNEL and immunohistochemistry, and the possibilities of pharmacological correction of apoptotic processes are determined. An age-dependent increase of apoptosis level of cells of suprachiasmatic nucleus and epiphysis in rats was revealed. The stress effect (immobilization) led to the intensification of cell death, more significant in older animals. The pineal gland and suprachiasmatic nucleus, traditionally regarded as regulators of circadian rhythms, are at the same time actively involved in general adaptation processes. The studied drugs (α-tocopherol-acetate, cycloferon, and their combination) have a pronounced anti-apoptotic, cytoprotective effect under physiological conditions during aging, as well as during non-specific emotional stress (immobilization) in young and old animals. The regulatory effect is accomplished by activating the expression of the anti-apoptotic protein Bcl-2 in the neurosecretory cells of the suprachiasmatic nucleus and pinealocytes.

Analysis of HER-2/neu Receptor Expression and the Level of Neuronal Apoptosis in HER-2/neu Transgenic Mice during Aging.

We studied neuronal death in the sensorimotor cortex, hippocampus, and supraoptic and paraventricular nuclei of the hypothalamus and dynamics of HER-2/neu expression in late ontogenesis in young and old transgenic HER-2/neu mice. Wild-type FVB/N mice served as the control. The intensity of apoptosis (TUNEL) and HER-2/neu expression (Western blotting) in the same brain regions were measured. HER-2/neu was detected in the cortex, hippocampus, and hypothalamus of transgenic and wild-type mice, and its expression increased with age. The effect of HER-2/neu on the intensity of cell death in various brain regions depended on the stage of ontogenesis and animal genotype. Enhanced expression of HER-2/neu determines low rate of cell death in the studied brain regions during pathological ageing.

[Effects of cytoflavin on neuronal apoptotic processes in the murine cerebral cortex on a model of physiologicaland pathological aging]. / Vliianie tsitoflavina na protsessy apoptoza neironov kory golovnogo mozga u myshei na modeli fiziologicheskogo i patologicheskogo stareniia.

Involutional changes in the cerebral cortex substantially affect the activity of the cortex itself and the function of target organs. This necessitates pharmacological correction of age-related diseases, primarily a high level of cell death. OBJECTIVE: To investigate the role of cytoflavin in mechanisms for the apoptotic regulation of cerebral cortical cells during physiological and pathological aging (in the presence of HER-2/neu overexpression). MATERIAL AND METHODS: HER-2/neu transgenic mice were used; wild-type FVB/N mice served as controls. The levels of apoptosis (TUNEL) and the expression of its associated proteins (p53, CD95, Mcl-1, p-AKT, and p-ERK) (Western blotting) were estimated in the sensorimotor cortex. RESULTS: Activation of fundamental AKT and ERK survival pathways promotes a low level of cell death in young FVB/N mice; the extrinsic receptor mechanism of apoptosis is observed to be initiated by aging. The high p-AKT levels in the cortical cells provide suppressed cell death in transgenic mice regardless of their age. After cytoflavin administration, the old wild-type mice show a lower level of apoptosis in the cortical neurons apparently due to the increased expression of the anti-apoptotic protein Mcl-1, while the old transgenic mice exhibited suppression of the AKT and ERK survival pathways and, accordingly, activation of the extrinsic receptor and p53-dependent apoptosis pathways. CONCLUSION: Thus, cytoflavin exerts a pronounced neuroprotective effect during physiological and accelerated aging, while its effect on the level of neuronal apoptosis is ambiguous and depends on the genetic line of animals. So, this is a moderate stimulation of apoptosis when its level is low in HER-2/neu mice with a high level of carcinogenesis, as well as a decrease in the high level of apoptosis in old wild-type animals, which prevents neurodegeneration.

The role of STAT transcription factors in apoptosis regulation of hypothalamic neurons in aging in HER-2/neu transgenic mice and wild-type FVB/N mice.

For the firsts time, the involvement of the STAT pathway in the regulation of neuronal apoptosis in physiological aging and in old mice overexpressing the HER-2/neu oncogene was studied. We showed that suppression of STAT3, STAT5, and STAT6 and overexpression of the proapoptotic factor STAT1, which provides p53-mediated apoptosis, are the causes for increasing the number of apoptotic neurons in physiological aging. HER-2 tyrosine kinase receptor overexpression promotes neuronal survival through activation of STAT-signaling pathway with simultaneous suppression of the proapoptotic factor STAT1.

[PHARMACOLOGICAL CORRECTION OF APOPTOSIS LEVEL OF CORTICAL NEURONS IN AGED HER2/NEU TRANSGENIC MICE].

Neurodegenerative changes and neuronal death are the basis for development of the nervous system aging. We investigated the mechanism of apoptosis of the sensorimotor cortex neurons of transgenic mice HER2/neu during aging, changes in the cortex function and the participation of exogenous neurometabolites (cytoflavin, piracetam) in regulation of neuronal death and locomotor and psycho-emotional status of mice. The level of apoptosis and expression of apoptosis markers (TUNEL, immunohistochemistry, Western blotting) in HER2/neu transgenic mice as compared to wild type mice (FBV line) were determined. In aging FBV mice the basal activity was shown to decrease and anxiety to increase correlating with the high level of neuronal apoptosis. We identified behavioral characteristics of transgenic HER2/neu mice and found that their low basal activity does not change with aging. Previously we have shown that in this strain of mice the apoptosis level is low, without any age-related changes, due to the suppression, first of all, of the p53-dependent pathway by HER2 (tyrosine kinase receptor) overexpression. Cytoflavin and piracetam were revealed to possess a marked neuroprotective effect, preserving and restoring functions of the nervous system (improving locomotion and psychological status) in both strains of mice. The effect of neurometabolites studied on neuronal apoptosis is ambiguous. In case of its low level it is a moderate stumulation of apoptosis via the external p53-dependent pathways with activation of caspase-3 in transgenic HER2/neu mice with high carcinogenesis level that can possibly prevent tumor development. On the contrary, in old wild-type animals we observed a significant decrease of age-dependent apoptosis level (by stimulating expression of the anti-apoptotic protein Mcl-1), which prevents neurodegeneration.

Pathways of apoptosis regulation in hepatocytes induced by first-line antitubercular drugs.

We studied pathways of apoptosis regulation during experimental hepatopathy caused by treatment with antitubercular drugs and involvement of some hepatoprotectors and immunomodulators in the regulation of hepatocyte apoptosis induced by antitubercular drugs. The intensity of apoptosis and expression of apoptosis-associated molecules were evaluated. It was shown that antitubercular drugs induce apoptosis in hepatocytes by triggering external signaling pathway and p53-dependent signaling pathway and simultaneously reducing the level of anti-apoptotic Bcl-2 protein. Runihol, remaxol, and cycloferon reduced degenerative effects in the liver, though the level of apoptosis remained high. Ademetionine in tablets and reamberin improved the microstructure of the liver by inhibiting both apoptotic pathways induced by the antitubercular drugs; in other words, they have distinct hepatoprotective and apoptosis-protective effects, which is especially important at the late stages of ontogeny.

[Comparative experimental study of the influence of drugs that improve brain metabolism (angiogen, cytoflavin) on neuronal apoptosis and function of cerebral cortex during aging].

The safety of cortical neurons and their functional activity is essential for organism at all stages of ontogenesis. However, aging changes leading to an increase in apoptosis level may cause considerable damage to cerebral cortex function, including sensorimotor. We have studied the role of exogenous neurometabolites (angiogen, cytoflavin) in apoptosis regulation and correction of age-related motor and behavioral disturbances. To study the regulation of neuronal morphofunctional activity, we used accelerate-senescent transgenic HER2 mice in comparison to wild type FBV mice. Functional changes in cerebral cortex were studied by the Suok test and open field test, the level of neuronal apoptosis was assessed by TUNEL method, the expression of apoptosis-modulating proteins was detected by immunohistochemistry and Western blotting. We have revealed differences in psycho-emotional and locomotor activity of these strains of mice. In addition, results of our study showed morphological differences: increase in the apoptosis level of cortical neurons in aged FBV type mice, but no changes in aged HER2 mice. The investigated drugs induce cell death of cortical neurons in transgenic mice of both ages and in young wild-type mice by p53-dependent pathway. Increased apoptosis in the cortex of old transgenic mice has important clinical implications, because reduced apoptosis during aging is one of the causes of cancer. The treatment of old wild-type animals reduces elevated neuronal apoptosis, which decreases risk of age neurodegeneration. Thus, revealed morphological changes in the cerebral cortex are the basis for involutional disabilities (including reduced locomotor activity and increased anxiety level). The use of angiogen and cytoflavin treatment improves functional activity of the cortex and protects normal structure of nervous tissue.

[Role of hepatoprotectors and immunomodulators in regulation of hepatocyte apoptosis induced by antituberculosis treatment].

UNLABELLED: It was currently shown that hepatopathy due to drug toxicity is associated with increased apoptosis of hepatocytes. Therefore, development of drugs which regulate cell death is of great importance. AIM: To involve some hepatoprotectors (ademethionine, reamberin, remaxol) and immunomodulators (cycloferon) into regulation of apoptosis in experimental models of liver first-line antituberculousis drugs (isoniazid, rifampicin, pyraztinamide). MATERIALS AND METHODS: Levels of apoptoasis (TUNEL), expression of CD95 (receptor of tumor necrosis factor - by immunohistochemistry), expression of caspase-8, caspase-3 and pS3 (Western-blotting) were measured. RESULTS: Exposition offirst-line antituberculousis drugs leads to dysthrophia of liver parenchyma cells with increased apoptosis of hepatocytes and activation of CD95, caspase-8 (external way) and overexpression of p53 and caspase-3. It was found that reamberin, cycloferon and remaxol have hepatoprotective effect improving liver histology; ademethionine administered by intraperitoneal injection showed no positive effects. Reamberin demonstrated apoptosis-inhibiting effect in the experiment whereas other drugs were found to be apoptosis inductors for hepatocytes in toxic hepatopathy. CONCLUSIONS: Legulation of apoptosis by cycloferon and remaxol mediated by external and p53-dependent pathway is confirmed by increased expression of CD95 and p53 protein. Ademethionine might induce apoptosis by the intrinsic pathway.

[Mechanism of hepatocyte apoptosis in experimental toxic liver injury].

One of the causes of drug hepatopathy is hepatocyte apoptosis, the mechanisms of which are still unclear. The experiments were performed in 24 Wistar rats to study the role of hepatoprotectors in the regulation of hepatocyte apoptosis in liver damage induced by administration of antituberculosis drugs (ATD). The level of apoptosis (TUNEL) was evaluated, and the expression of apoptosis-associated molecules was detected by immunohistochemistry and Western blotting. It was shown that a signaling cascade induced by ATD involved the activation of cell surface receptors (CD95) and caspase-8, i.e. apoptosis was mediated by extrinsic pathway. In addition,ATD induced p53 oncosuppressor synthesis with further activation of caspase-3 effector. Runihol administration during ATD treatment administration improved the condition of the liver, despite some apoptosis stimulating effect, mediated by an intrinsic pathway. It was found that runihol blocked both FAS- and p53-dependent pathways. Ademethionine during drug intoxication acts as a hepatoprotector, blocking extrinsic and p53-dependent pathways.

[Cycloferon: mechanism of action, functions and application].

In recent years, both interferons and inductors of endogenous interferon production find increasing use in clinical practice. The latter agents are characterized by high antiviral and immunomodulatory activity in the absence of serious side effects, which makes it possible to prescribe long courses if necessary. One of the most frequently used interferon inductors is cycloferon. Diverse effects of cycloferon on biochemical and cellular cascades (including induction of alpha- and beta-interferon, inhibition ofproapoptotic factors such as tumor necrosis factor and interleukin 1-beta) suggest that it also takes active part in the regulation of apoptosis, one of the most important processes of cell activity that opens up new prospects for the therapeutic use of cycloferon.

[Role of cycloferon and interferon-alphain apoptosis regulation in neuroendocrinal system on aging].

A detailed analysis of the literature data gives contradictory information about the role of interferon-alpha in the regulation of apoptosis, while there are almost no data on the participation of cycloferon in this process. Results of original experiments in recent years showed that exogenous interferon-alpha is not apoptosis protector in hypothalamic neurons on aging. The treatment with interferon-alpha activates dystrophic processes in neurosecretory cells of aged mice. However, endogenous interferon induced by cycloferon leads to a decrease in the apoptosis of hypothalamic neurons in both young and old animals. Antiapoptotic activity of interferon-alpha and cycloferon has been found in aged animals under stress condition. Thus, the role of immunomodulators in apoptosis regulation in hypothalamic neurons depends on the age and the type of immunomodulators. This fact opens new prospects for the clinical use of interferon-alpha and cycloferon.

[Mechanisms of apoptosis in drug-resistant epilepsy]. / Mekhanizmy apoptoza pri farmakorezistentnoi epilepsii.

Epilepsy is a chronic neurological disease with regular spontaneous seizures associated with neuroinflammatory, autoimmune and neurodegenerative processes. Approximately 40% of patients suffer from drug-resistant epilepsy, which leads to an increased risk of premature death, injury, irreversible brain damage, psychosocial dysfunction, and reduced quality of life. Apoptosis of neurons and glial cells of the brain is of great importance in the pathogenesis of epilepsy, especially drug-resistant epilepsy. Investigation of the mechanisms of apoptosis is necessary for the creation of a new generation of neuroprotective and anticonvulsant drugs, effective, in particular, in the case of drug-resistant epilepsy. The aim of the study was to analyze the mechanisms and role of apoptosis in epileptogenesis and the development of resistance. The review considers current data on the main mechanisms of apoptosis in epilepsy, especially its drug-resistant forms.

[Influence the water deprivation and alpha-tocopheroli acetates on the expression of apoptosis regulator proteins].

We revealed ontogenetic features in apoptosis level, apoptosis signal proteins expression, antioxidant (alpha-tocoferoli acetate) effects in neurons of magnocellular hypothalamic centers of BALB-c mice. It was obtained that water deprivation stress leads to apoptosis initiation of neurons in both age groups. Stress-protected action of alpha-tocoferoli acetate was more significantly in young mice compared to old ones. In our subsequent work (immunocytochemical reactions) we obtained further regular occurrences. Dehydration leads to increase of proapoptotic protein Bax synthesis in hypothalamic neurosecretory cells in young mice and in age-independent manner, this stress leads to decrease of antiapoptotic proteins Bcl-2 and Mcl-1 synthesis. So, the apoptosis level increases. More significant antiapoptotic action of alpha-tocoferoli acetate in stress condition in young mice is obviously connected with quick reaction of compensatory mechanisms (low expression of proapoptotic proteins p53, Bax and high expression of antiapoptotic protein Bcl-2).

[Ontogenetic peculiarities of regulation of apoptosis in hypothalamic neurosecretory cells in thf-knockout mice].

Tumor necrosis factor (TNF) participates in regulation of many processes including carcinogenesis and apoptosis. However, at present, there are practically absent the works on peculiarities of regulation of apoptosis in tnf-knockout (tnf-/-) mice. These mice develop without morphological abnormalities, but they seem to have disturbances of many biological processes, such as inflammation, programmed cell death, etc. Therefore, the goal of our work was to study possible pathways of regulation of apoptosis in the absence of TNF in neurosecretory cells (NSC) of young and old mice. For this purpose, we determined immunohistochemically expression of apoptosis markers caspase-8, -9. Bax, Bcl2, Mcl1, neuropeptide vasopressin, and the apoptosis level in hypothalamus in tnf-knockout mice of different ages as compared with mice with unchanged level of TNF synthesis. It was shown that the apoptosis activation observed during aging did not depend on the tnf gene, and apoptosis at aging was caspase-dependent. It has been revealed that at aging in mouse NSC the external cell death pathway with participation of caspase-8 is activated. The pathways mediating cell death in different neurosecretory centers at aging are different. Thus, in supraoptic nucleus (SON), in all studied animal groups, animal groups, an important cause of the NSC apoptosis is Bax. In paraventricular nucleus (PVN), of the greater importance is a decrease of the antiapoptotic protection. Hence, misbalance of synthesis of proteins of the Bcl-2 family plays an important role in development of senescent apoptosis.

Aging-related changes in the expression of apoptosis-associated molecules in neurosecretory cells of the mouse hypothalamus.

The aim of the present work was to identify the characteristics of the control of apoptosis in neurosecretory centers of aged (18 months) mice on exposure to the immunomodulator interferon-alpha (IA) in comparison with young (two months) mice. Age-related activation of apoptosis in the supraoptic (SON) and paraventricular (PVN) nuclei was found to be mediated by different pathways and could result from changes in the ratio of synthesis of pro-(Bax) and antiapoptotic (Mcl-1, Bcl-2) molecules. In addition, the apoptosis signal cascade in young mice treated with IA was identical in both nuclei, while in aged mice there were differences between the SON and the PVN.