Sudden death occurs as a result of a fast-flowing latent or clinically pronounced painful condition. As medical practice shows, sudden death in adults often occurs due to acute coronary insufficiency, congenital or acquired cardiac and vascular pathologies. Find out what symptoms may indirectly indicate hidden threat.

What is sudden death

According to international medical recommendations, a person’s death within 6 hours after the appearance of the first symptoms of a pathological condition is considered sudden. Instant death, or translated into English sudden death, occurs without a known cause. In addition, there are no morphological signs on the basis of which an appropriate diagnosis of the patient’s sudden death can be made at autopsy.

However, during a post-mortem examination of a person, a pathologist, having compared all available data, can make a logical conclusion about the instantaneous or violent death of the person. In most cases, instant death is supported by changes in organs in which continuation of life for the shortest period of time is impossible.

Causes of sudden death

Statistics show that the main cause of most deaths is heart disease: ischemic pathology, the onset of ventricular fibrillation. At the same time, when answering what causes instant death, experts often name chronic illnesses that occur in a latent form for a long time, after which they suddenly worsen and lead to the unexpected death of a person. One of these is deadly dangerous diseases is cancer.

In most cases, oncology develops asymptomatically and makes itself felt when the patient is often considered hopeless. Thus, malignant liver disease is the main cause of unexpected deaths in China. Another insidious disease that can lead to sudden death is AIDS, which claims millions of lives in Africa every year. In addition, it is worth mentioning separately about Mexico. This is the only country in which cirrhosis of the liver occurs main reason high mortality rate of the population.

In young age

Today, boys and girls are exposed every day negative influence modern lifestyle. From TV screens and the covers of fashion magazines, the cult of a slender (often dystrophic) body, accessibility and promiscuity is imposed on young people. Therefore, it is quite understandable that the mortality rate of people just beginning their life journey will increase over time. The main causes of instant death among boys and girls under 25 years of age are considered to be:

• alcohol;
• smoking;
• promiscuity;
• drug addiction;
• poor nutrition;
• psychological sensitivity;
• hereditary diseases;
• severe congenital pathologies.

In a dream

Unexpected death in this condition occurs due to the loss of special cells responsible for the contractility of the lungs. Thus, scientists from the USA were able to prove that people die in their sleep in most cases due to central sleep apnea. In this case, a person may even wake up, but still leave this mortal world due to oxygen starvation caused by a stroke or cardiac arrest. As a rule, elderly people are susceptible to this syndrome. There are no specific treatments for central sleep apnea.

Sudden infant death

This syndrome was first described in the early 60s of the last century, although cases of instant death of infants were recorded earlier, but they were not subjected to such a thorough analysis. Young children have very high adaptive abilities and incredible resistance to a variety of negative factors, because death infant is considered to be an exceptional situation. However, there are a number of external and internal reasons that can lead to sudden child death:

• elongation QT interval;
• apnea (the phenomenon of periodic breathing);
• deficiency of serotonin receptors;
• overheat.

Risk factors

Due to the fact that the main cardiogenic cause of instant death is ischemic disease, it is quite logical to assume that the syndromes accompanying this heart pathology can be fully attributed to conditions that can increase the likelihood of sudden death. With all this, it has been scientifically proven that this connection is mediated through the underlying disease. Clinical risk factors for the development of clinical death among patients with ischemic syndrome are:

• acute myocardial infarction;
• post-infarction macrofocal sclerosis;
• unstable angina;
• heart rhythm disturbance due to ischemic changes (rigid, sinus);
• ventricular asystole;
• myocardial damage;
• episodes of loss of consciousness;
• damage to the coronary (heart) arteries;
• diabetes;
• electrolyte imbalance (eg, hyperkalemia);
• arterial hypertension;
• smoking.

How does sudden death occur?

This syndrome develops in a matter of minutes (less often hours) without any warning in the midst of complete well-being. In most cases, instant death affects young men aged 35 to 43 years. Moreover, often during the pathological examination of the deceased, vascular causes of sudden death are discovered. Thus, studying the increasing cases of instant death, experts came to the conclusion that the main provoking factor in the occurrence of this syndrome is a violation of coronary blood flow.

For heart failure

In 85% of cases, immediate death is recorded in individuals with structural abnormalities of the organ that pumps blood into the vessels. In this case, sudden cardiac death looks like a lightning-fast clinical variant of coronary disease. Medical practice shows that in a quarter of people who die instantly, bradycardia and episodes of asystole are observed before the onset of primary symptoms. Death from cardiac arrest occurs due to the launch of the following pathogenetic mechanisms:

• Reducing left ventricular fractional ejection by 25-30%. This syndrome greatly increases the risk of sudden coronary death.
• Ectopic focus of automatism in the ventricle (more than 10 ventricular extrasystoles per hour or unstable ventricular tachycardia), arising as a consequence of ventricular arrhythmias. The latter mostly develop against the background of acute transient myocardial ischemia. An ectopic focus of automatism is usually classified as a risk factor for sudden arrhythmic death.
• The process of spasm of the blood vessels of the heart, which leads to ischemia and contributes to the deterioration of the restoration of blood flow to damaged areas.

It is worth noting that tachyarrhythmia is a particularly significant electrophysiological mechanism resulting in sudden coronary death in a person with heart failure. At the same time, timely treatment of this condition using a defibrillator with a modified pulse configuration significantly reduces the number of deaths among patients who have suffered sudden cardiac arrest.

From a heart attack

Blood enters the heart through the coronary arteries. If their lumen closes, the formation of primary foci of necrosis and ischemia in the heart occurs. Acute manifestation of cardiac pathology begins with damage to the vascular wall with further thrombosis and spasm of the arteries. As a result, the load on the heart increases, the myocardium begins to experience oxygen starvation, which affects its electrical activity.

As a result of a sudden coronary spasm, ventricular fibrillation occurs, a few seconds after which a complete cessation of blood circulation to the brain occurs. At the next stage, the patient experiences respiratory arrest, atony, and absence of corneal and pupillary reflexes. After 4 minutes from the onset of ventricular fibrillation and complete cessation of blood circulation in the body, irreversible changes occur in the brain cells. In general, death from a heart attack can occur in 3-5 minutes.

From a blood clot

In the venous bed, these pathological formations arise due to the uncoordinated work of the coagulation and anticoagulation systems. Thus, the onset of the appearance of a clot is caused by damage to the vascular wall and its inflammation against the background of thrombophlebitis. Perceiving the appropriate chemical signal, the coagulation system comes into action. As a result, fibrin threads form near the pathological area, in which blood cells become entangled, creating all the conditions for the blood clot to break off.

In arteries, the formation of clots occurs due to narrowing of the vascular lumen. Thus, cholesterol plaques block the path of free blood flow, resulting in the formation of a lump of platelets and fibrin threads. It is important to note that in medicine a distinction is made between floating and mural thrombi. Compared to the first type, the latter has a slight chance of breaking off and causing a blockage (embolism) of the vessel. In most cases, the causes of sudden cardiac arrest from a blood clot are due to the movement of a floating thrombus.

One of the serious consequences of the separation of such a clot is blockage of the pulmonary artery, which is expressed in a strong cough and bluish skin. Often there is respiratory failure followed by cessation of cardiac activity. An equally serious consequence of the detachment of a blood clot is a violation of cerebral circulation due to embolism of the main vessels of the head.

Diagnosis of sudden death

A timely physical examination is the key to the success of further cardiopulmonary resuscitation (CPR) measures. Diagnosis of instant death is based on symptoms characteristic of the patient's natural death. Thus, absence of consciousness is determined if no external stimuli cause reactions on the part of the person being resuscitated.

Diagnosis of breathing disorders is noted when within 10-20 s. observation fails to detect coordinated movements of the sternum and the noise of the air exhaled by the patient. In this case, agonal breaths do not provide adequate ventilation of the lungs and cannot be interpreted as spontaneous breathing. During ECG monitoring, pathological changes characteristic of clinical death are detected:

• ventricular fibrillation or flutter;
• cardiac asystole;
• electromechanical dissociation.

Clinical manifestations

In 25% of cases, sudden death occurs instantly without any warning signs. Some patients, a week before clinical death, complain of various prodromal manifestations: increased pain in the sternum, general weakness, shortness of breath. It is important to note that today there are already methods for preventing heart attacks based on early diagnosis of the warning symptoms of this condition. Immediately before the onset of sudden death, half of the patients experience an anginal attack. Clinical signs of a patient’s imminent death include:

• loss of consciousness;
• absence of pulse in the carotid arteries;
• dilated pupils;
• lack of breathing or the appearance of agonal breaths;
• change in skin color from normal to gray with a bluish tint.

Medical care for sudden death

Typically, most cases of unexpected cardiac arrest occur outside the hospital. For this reason, it is extremely important to master the technique of providing emergency care in case of sudden clinical death. This is especially true for subjects of society who, due to their job responsibilities come into contact with a large number of people. Remember, competent resuscitation actions immediately in the first minutes after the onset of symptoms of cardiac arrest will help gain time before arrival medical workers.

Urgent Care

The main problem that arises in unconscious persons is obstruction of the airways by the root of the tongue and the epiglottis due to muscle atony. It must be said that this condition develops in any position of the body, and when the head is tilted forward, it develops in 100% of cases. Therefore, the first thing that needs to be done is to ensure proper airway patency. For this purpose, you need to use P. Safar’s triple technique, consisting of the following sequential actions:

1. Throwing back the head;
2. Moving the lower jaw forward;
3. Opening the mouth.

Once airway patency is ensured, you should proceed to artificial pulmonary ventilation (ALV). When providing first aid, this activity is carried out using the mouth-to-mouth method. So, one hand is placed on the victim’s forehead, while the other pinches his nose. Then the resuscitator fixes his own lips around the mouth of the person being revived and blows air, while controlling the excursion of the patient's chest. When it is visible, you need to release the victim’s mouth, giving him a chance to exhale passively.

At the next stage, artificial maintenance of blood circulation is carried out, to ensure which an algorithm for performing indirect cardiac massage or chest compression is used. For this purpose, you need to correctly lay the person being resuscitated on a flat surface. Next, you should determine the compression points: by palpating the xiphoid process and moving away from it 2 transverse fingers upward.

The hand must be placed on the border of the middle and lower part of the sternum so that the fingers are parallel to the ribs. Pushes are performed with the limbs straightened at the elbows. Chest compression is performed at a frequency of 100 compressions per minute with a break for artificial ventilation. The depth of the shocks is about 4-5 cm. Measures to restore cardiac activity should be stopped if:

1. A pulse appeared in the main arteries.
2. The actions taken do not have the desired effect within 30 minutes. The exception is the following conditions that require prolongation of resuscitation:

• hypothermia;
• drowning;
• overdose medicines;
• electrical injury.

Resuscitation measures

Today, the concept of CPR is based on strict rules that ensure complete safety of the activities carried out for human life. In addition, an algorithm for the resuscitator’s actions in case of sudden cardiac arrest or sudden loss of respiratory function in the injured person is presented and scientifically substantiated. When these conditions develop main role Time plays: only a few minutes separate a person from death. The algorithm for performing cardiopulmonary resuscitation involves performing next steps:

1. Determining the condition of the victim, on the basis of which the range of measures necessary for revival is selected;
2. Early initiation of CPR, which involves performing two manipulations: chest compressions and artificial ventilation.
3. If the second stage is ineffective, they proceed to defibrillation. The procedure involves applying an electrical impulse to the heart muscle. In this case, direct current discharges should be applied only if the electrodes are correctly positioned and have good contact with the victim’s skin.
4. At this stage, as a rule, the victim is provided with specialized medical care, including the following early treatment measures:

• artificial ventilation with tracheal intubation;
• drug support, involving the use of:
• catecholamines (adrenaline, atropine);
• antidiuretic hormones (Vasopressin);
• antiarrhythmic drugs (Cordarone, Lidocaine);
• fibrinolytic agents (Streptokinase).
• intravenous drip administration of electrolyte or buffer solutions (for example, sodium bicarbonate is administered for acidosis)

Video

Soviet fighter pilot Valentin Bondarenko could become the first man in space. In any case, he had a chance of this... But he simply did not live to see his first flight: he died by a stupid accident during testing.

First squad

Valentin Vasilyevich Bondarenko was born on February 16, 1937 in Kharkov. His father, the head of a workshop at the Kharkov fur factory, went to the front in the first days of the war. Together with his mother and older brother, Valentin survived the German occupation. As a high school student, he began studying at the Kharkov flying club. In 1954, after graduating from school, he entered the Voroshilovgrad Military Aviation School, after its disbandment he transferred to Grozny, and then to the Armavir School, from which he graduated with honors in 1957. Bondarenko served in the aviation units of the Air Force of the Baltic Military District.

On April 28, 1960, Valentin’s cherished dream came true: after careful selection, he was enrolled in the first squad of Soviet cosmonauts. Out of several thousand applicants, only 29 people were selected.

Initially, Valentin was not one of the six candidates for space flight on the Vostok spacecraft. But for various reasons, several selected future cosmonauts dropped out of action, and Bondarenko was brought in for training.

Ridiculous death

The tests included a ten-day stay in a pressure chamber, the purpose of which was to test the reaction to the absence external stimuli. It was believed that the conditions in the chamber were close to the conditions inside spacecraft. It was located at the Air Force Research Institute-7 (now the Institute of Aviation and Space Medicine).

At the end of the experiment, Valentin was informed that he could remove the medical sensors attached to his body. There were red marks left at the attachment points, which Bondarenko wiped with a cotton swab soaked in alcohol. After that, the young man, without looking, threw the cotton wool towards the trash can. But by an unfortunate accident, it landed on the spiral of a hot electric stove and immediately burst into flames... Since the chamber was filled with almost pure oxygen, the flame quickly spread. Bondarenko's woolen training suit caught fire.

It was not possible to open the chamber quickly due to the large pressure drop. When it was finally opened, the cadet was still alive. Doctors at the Botkin Hospital fought for his life for 8 hours. He died on March 23, 1961, just 19 days before Gagarin’s flight, who, together with his squad comrades, spent several hours in his hospital... The cause of death was burn shock.

Secret Hero

The death of Senior Lieutenant Valentin Bondarenko was not reported anywhere: in those days, everything related to space was strictly classified. However, on June 17, 1961, by decree of the Presidium Supreme Council USSR Bondarenko “for the successful completion of the government’s assignment” was posthumously awarded the Order of the Red Star.

At Bondarenko’s grave, located in Kharkov at the Filippovsky cemetery, an obelisk was erected with the inscription: “In blessed memory from fellow pilots.” Only in the 80s did the postscript appear: “-cosmonauts of the USSR.”

Bondarenko left behind his wife Anna and son Alexander. For some time they continued to live in Star City, where Anna worked at the Cosmonaut Training Center, then they left for Kharkov, where they had relatives. Until Sasha’s 16th birthday, he was paid a pension of 100 rubles for his father - quite decent money at that time. Subsequently, Alexander Bondarenko followed in his father’s footsteps and became a military pilot.

Only in 1980 did the Western press begin to write about the death of Valentin Bondarenko. In the USSR, an article about him was first published in 1986 in Izvestia. In 1991, one of the lunar craters was named after Bondarenko, and in July 2013, the name of the cosmonaut was given to school No. 93 in the city of Kharkov, where he once studied.

It would seem that the death of Valentin Bondarenko has nothing to do with the space flights themselves - it is simply a tragic accident. However, without such mistakes and tragedies, astronautics could not develop. By the way, this story forced engineers and scientists to reconsider the design of the test pressure chamber, in particular, changing the composition and pressure of the atmosphere, which was taken into account in the further development of manned spacecraft.

Today in the Museum of the First Flight, located in the city of Gagarin, Smolensk region, on small homeland the first person in the world to go into space, you can see the deaf chamber, which is an exact copy of the one in which Valentin Bondarenko died.

The life and death of the wonderful Russian actor Yuri Demich date back to ancient Soviet and post-Soviet times. He was born three years after the end of the Great Patriotic War and the main peak of his work came in the 70-80s, when public interest and respect for acting were well deserved and natural. The cause of death of Yuri Demich was blood loss incompatible with life.

Yuri Demich is known to moviegoers for his many film roles, but his amazing theatrical work could only be appreciated by theater lovers of the northern capital who had the opportunity to visit the Leningrad Bolshoi Drama Theater. He was an actor from God - talented, fantastically able-bodied, versatile and unique.

Demich chose his profession following the example of his father, who became a victim Stalin's repressions and spent 20 years in exile in Magadan, where Demich Jr. was born. Following the example of his father, who freely did somersaults at the age of 60, the young actor became interested in sports and for a long time kept himself in excellent physical shape.

Since childhood, he began to appear on stage with his father’s troupe of the Kuibyshev Drama Theater. M. Gorky, graduated from his studio, and then entered GITIS. In 1973, the role of Hamlet, masterfully played in Kuibyshev, became for Demich a ticket to the stage of the Leningrad Bolshoi Drama Theater. He captivated the audience, his colleagues, and the director A. G. Tovstonogov, who led the team, with his performance in the plays “Last Summer in Chulimsk” based on the play by Vampilov and “Three Bags of Weedy Wheat” by the master himself.

Having become one of the leading actors of this theater, Demich brilliantly played about 40 roles over 7 theater seasons and every time he went on stage, he was ready to play as if for the first time. In cinema, he was also often offered big roles, and in total there were almost 50 of them in 20 years. Such a powerful waste of energy, despite an excess of health, temperament and strength, could be disastrous. The actor began to drink a little and one day fell under the hot hand of Tovstonogov, an implacable opponent of drunkenness on stage.

Few know whether Demich was actually to blame, as the director was told, but he had to leave his native theater. This tragedy was not easy for the actor: having moved to the capital and joined the troupe of the Moscow Ermolov Theater, he began to lose control over his weakness. His theatrical repertoire shrank, although he was still invited to films. But he started drinking openly.

When Yuri Alexandrovich, having pulled himself together, began to establish a sober life, economic problems began in the country and it turned out to be difficult for him to find work. Demich earned money by traveling around Russia giving performances.

The nomadic life completely undermined his health. In December 1990, the actor began to experience severe bleeding, which doctors could not stop. The cause of Yuri Demich's death at the age of 42 was a rupture of the veins of the esophagus, which led to enormous blood loss.

He is buried at the Vagankovskoye cemetery in Moscow.

In medicine, sudden death from heart failure is regarded as a fatal outcome that occurs naturally. This happens both to people who have had heart disease for a long time, and to people who have never used the services of a cardiologist. A pathology that develops quickly, sometimes even instantly, is called sudden cardiac death.

Often there are no signs of a threat to life, and death occurs within a few minutes. The pathology can progress slowly, starting with pain in the heart area and rapid pulse. The duration of the development period is up to 6 hours.

Cardiac death is distinguished between quick and instantaneous. The fulminant variant of coronary heart disease causes death in 80-90% of incidents. Also among the main causes are myocardial infarction, arrhythmia, and heart failure.

Read more about the reasons. Most of them are associated with changes in blood vessels and the heart (arterial spasms, hypertrophy of the heart muscle, atherosclerosis, etc.). Among the common prerequisites are the following:

• ischemia, arrhythmia, tachycardia, impaired blood flow;
• weakening of the myocardium, ventricular failure;
• free fluid in the pericardium;
• signs of heart and vascular diseases;
• heart injuries;
• atherosclerotic changes;
• intoxication;
• congenital defects of valves, coronary arteries;
• obesity, as a result of poor nutrition and metabolic disorders;
  unhealthy lifestyle, bad habits;
• physical overload.

More often, the occurrence of sudden cardiac death is provoked by a combination of several factors simultaneously. The risk of coronary death increases in people who:

• there are congenital cardiovascular diseases, ischemic heart disease, ventricular tachycardia;
• there was a previous case of resuscitation after a diagnosed cardiac arrest;
• a previous heart attack was diagnosed;
• there are pathologies of the valve apparatus, chronic insufficiency, ischemia;
• facts of loss of consciousness were recorded;
• there is a reduction in blood ejection from the left ventricle by less than 40%;
• A diagnosis of cardiac hypertrophy was made.

Secondary significant conditions for increasing the risk of death are considered to be: tachycardia, hypertension, myocardial hypertrophy, changes in fat metabolism, diabetes. Bad influence are caused by smoking, weak or excessive physical activity

Signs of heart failure before death

Cardiac arrest is often a complication after cardiac arrest. vascular disease. Because of this, the heart can suddenly stop its activity. After the first signs appear, death can occur within 1.5 hours.

Previous dangerous symptoms:

• shortness of breath (up to 40 movements per minute);
• pressing pain in the heart area;
• the skin becomes gray or bluish and becomes colder;
• convulsions due to hypoxia of brain tissue;
• separation of foam from the oral cavity;
• feeling of fear.

Many people experience symptoms of exacerbation of the disease within 5-15 days. Heart pain, lethargy, shortness of breath, weakness, malaise, arrhythmia. Shortly before death, most people experience fear. You should immediately contact a cardiologist.

Signs during an attack:

• weakness, fainting due to the high rate of ventricular contraction;
• involuntary muscle contraction;
• facial redness;
• pale skin (it becomes cold, bluish or gray);
• inability to determine pulse, heartbeat;
• lack of reflexes of the pupils, which have become wide;
• irregularity, convulsive breathing, sweating;
• loss of consciousness is possible, and after a few minutes breathing cessation.

In case of death, against the background of seemingly good health, symptoms could be present, just not clearly manifested.

Mechanism of disease development

As a result of a study of people who died due to acute heart failure, it was found that most of them had atherosclerotic changes that affected the coronary arteries. As a result, myocardial circulation was disrupted and it was damaged.

Patients experience enlargement of the liver and neck veins, and sometimes pulmonary edema. Coronary circulatory arrest is diagnosed; after half an hour, abnormalities in the myocardial cells are observed. The whole process lasts up to 2 hours. After cardiac activity stops, irreversible changes occur in brain cells within 3-5 minutes.

Often cases of sudden cardiac death occur during sleep after breathing has stopped. In a dream, the chances of salvation are practically absent.

Statistics of mortality from heart failure and age characteristics

One in five people will experience this during their lifetime. Instant death occurs in a quarter of victims. The mortality rate from this diagnosis exceeds the mortality rate from myocardial infarction by approximately 10 times. Up to 600 thousand deaths are reported annually due to this reason. According to statistics, after treatment for heart failure, 30% of patients die within a year.

More often, coronary death occurs in persons 40-70 years old with diagnosed vascular and cardiac disorders. Men are susceptible to it more often: at a young age 4 times, in the elderly – 7 times, by the age of 70 – 2 times. A quarter of patients do not reach the age of 60 years. The risk group includes not only elderly people, but also very young people. The cause of sudden cardiac death at a young age can be vascular spasms, myocardial hypertrophy caused by drug use, as well as excessive stress and hypothermia.

Diagnostic measures

90% of sudden cardiac death episodes occur outside of hospitals. It’s good if the ambulance arrives quickly and the doctors carry out a quick diagnosis.

Emergency doctors note the absence of consciousness, pulse, breathing (or its rare presence), and the absence of pupillary response to light. To continue diagnostic measures, resuscitation actions are first required (indirect cardiac massage, artificial ventilation of the lungs, intravenous administration of medications).

After this, an ECG is performed. In case of a cardiogram in the form of a straight line (cardiac arrest), the administration of adrenaline, atropine, and other drugs is recommended. If resuscitation is successful, further laboratory examinations, ECG monitoring, and cardiac ultrasound are carried out. Based on the results, surgical intervention, implantation of a pacemaker, or conservative treatment with medications is possible.

Urgent Care

With symptoms of sudden death from heart failure, doctors have only 3 minutes to help and save the patient. Irreversible changes occurring in brain cells after this time period lead to death. Timely first aid can save lives.

The development of symptoms of heart failure is facilitated by a state of panic and fear. The patient must calm down, relieving emotional stress. Call ambulance(team of cardiologists). Sit comfortably, lower your legs down. Take nitroglycerin (2-3 tablets) under the tongue.

Cardiac arrest often occurs in crowded places. Those around you need to urgently call an ambulance. While waiting for her arrival, you need to provide the victim with an influx of fresh air, if necessary, perform artificial respiration, and perform a cardiac massage.

Prevention

To reduce mortality, preventive measures are important:

• regular consultations with a cardiologist, preventive procedures and prescriptions (special attention
• patients with hypertension, ischemia, weak left ventricle);
• refusal from provoking bad habits, ensuring proper nutrition;
• control of blood pressure;
• systematic ECG (pay attention to non-standard indicators);
• prevention of atherosclerosis ( early diagnosis, treatment);
• implantation methods in risk groups.

Sudden cardiac death is a severe pathology that occurs instantly or in a short period of time. The coronary nature of the pathology is confirmed by the absence of injuries and the sudden and rapid cardiac arrest. A quarter of cases of sudden cardiac death are lightning fast, and without the presence of visible precursors.

You may also be interested in:

How to recognize and treat acute heart failure
Signs of coronary heart disease in men: diagnostic methods

EXPERIMENTAL ARTICLES

UDC 577.15:576.367

Acadesine causes non-apoptotic death of tumor cells

V. A. Glazunova1*, K. V. Lobanov2, R. S. Shakulov2, A. S. Mironov2, A. A. Shtil1 "N.N. Blokhin Russian Oncology Research Center of the Russian Academy of Medical Sciences, 115478, Moscow, Kashirskoe sh. ., 24

■State Research Institute of Genetics and Selection of Industrial Microorganisms, 117545, Moscow, Dorozhny pr-d, 1 *E-mail: [email protected] Received by the editor December 27, 2012

ABSTRACT The effect of acadesine (5-aminoimidazole-4-carboxamide-1-0-O-ribofuranoside) on tumor and non-tumor cells of various species and tissue origin was studied. It has been established that acadesine causes non-apoptotic death of tumor cells; The sensitivity of non-tumor cells to the action of this compound is significantly lower. Acadesine causes the death of tumor cells with a drug resistance phenotype due to the expression of the P-glycoprotein transporter and inactivation of the proapoptotic protein p53. The activity of adenosine transporters is a necessary condition for cell death, whereas the function of AMP-activated protein kinase is not required. The predominant death of tumor cells under the influence of acadesine and the peculiarities of the mechanism of its cytotoxicity make this compound promising as an antitumor agent. keywords acadesine, cell death, tumor cells.

introduction

Acadesine (5-aminoimidazole-4-carboxamide-1-P-O-ribofuranoside, AICAR) is being tested in clinical trials as a drug for the treatment of chronic lymphocytic leukemia. An important property of acadesine is its primary toxicity to tumor cells with less pronounced damage to non-tumor cells. Previously, it was shown that acadesine is able to stimulate AMP-activated protein kinase (AMPK), an important regulator energy balance cells, controlling the oxidation of fatty acids, glucose metabolism, synthesis of proteins, fatty acids and cholesterol. The mechanism of action of acadesine is due to its phosphorylation by adenosine kinase to form ZMP (5-amino-4-imidazolecarboxamide ribotide), an intermediate in the de novo synthesis of purine bases. ZMP, by mimicking the metabolic effects of AMP, is able to activate AMPK. The antitumor effect of acadesine is associated with the induction of apoptosis. At the same time, there is evidence of non-apoptotic cell death and an AMPK-independent mechanism of action of acadesine on tumor cells.

In this work, the effect of acadesine on mammalian cells was studied. It has been shown that acadesine causes the death of tumor cells of various tissues.

of new origin, including cells resistant to a number of antitumor agents. The mechanisms of cell death are different from apoptosis; their important feature it turns out that adenosine transport is necessary. Non-tumor cells are less sensitive to the action of acadesine. The selectivity of the cytotoxic effect and the specific mechanisms of tumor cell death may be important factors determining the prospects of using acadesine in tumor therapy.

experimental part

The following human cell lines were used in the experiments: HCT116 (colon adenocarcinoma), HCT116p53KO (isogenic subline in which p53 does not function), K562 (promyelocytic leukemia), K562/4 (subline obtained after selection for survival in the presence of doxorubicin; expressed multidrug resistance protein (MDR) P-glycoprotein; Pgp), MCF-7 (breast adenocarcinoma), MCF-7Dox (subline after selection for survival in the presence of doxorubicin; Pgp-mediated MDR phenotype), fibroblast culture PFC-2, blood lymphocytes from healthy donors, as well as mouse cells: P388 (lymphocytic leukemia) and Sp2/0 (myeloma). Reagents were purchased from PanEco, Russia (except where otherwise specified). Cells were cultured in mo-

Dulbecco's modified Eagle's medium with the addition of 5% fetal bovine serum (Bio-Whittaker, Austria), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C, 5% CO2 in a humidified atmosphere. In the experiments, cultures in the logarithmic growth phase were used. Lymphocytes were isolated from the peripheral blood of donors by centrifugation in a Ficoll-urografin density gradient (d = 1.077 g/cm3).

Acadesine was obtained at GosNIIGenetika using a microbiological method using an original recombinant strain. In addition, the cytotoxicity of acadesine from Sigma was assessed. The same company purchased dipyridamole, an adenosine receptor inhibitor, 5-iodotubercidine, an adenosine kinase inhibitor that prevents the conversion of acadesine to ZMP, and zVAD-fmk (carbobenzoxyvalylalanyl-aspartyl-fluoromethylketone), a pan-caspase inhibitor. All compounds were dissolved in dimethyl sulfoxide or water (10-20 mM) and stored at -20°C. On the day of the experiment, dilutions of the drug were prepared in a culture medium. To assess the cytotoxicity of acadesine, we used the MTT test, staining of cells with propidium iodide and annexin V conjugated with fluorescein isothiocyanate (FITC), determination

cell cycle in flow cytometry and electrophoretic analysis of genomic DNA integrity. In some experiments, the reference drug was the alkyl cationic glycerolipid gas-P-(4-[(2-ethoxy-3-octadecyloxy)prop-1-yloxycarbonyl]butyl)-N-methylimidazolium iodide, an apoptosis inducer.

RESULTS AND DISCUSSION

Preferential sensitivity of tumor cells to acadesine

In preliminary experiments, we established that the microbiologically obtained acadesine preparation and commercial acadesine are identical in physicochemical properties, purity, storage stability and cytotoxicity (data not shown). For further studies, we used acadesine obtained by the author's method. In table Figure 1 shows the cytotoxicity of acadesine for transformed and non-transformed cells (cultured or freshly isolated) of various species and tissue origin.

From the data presented in table. 1, it follows that the most sensitive to the action of acadesine are

Table 1. Cytotoxicity of acadesine for mammalian cells

Acadesine chains, mM

G G.125 G.25 G.5 1.G 2.G

K562 1GG* 1GG 70 46 9 G

P388 1GG 36 30 20 9 G

Sp2/0 1GG 34 29 14 G G

K562/4 1GG 1GG 72 42 8 G

MCF-7 1GG 1GG 82 50 15 2

MCF-7Dox 1GG 1GG 86 48 17 1

HCT116 1GG 1GG 50 36 23 G

HCT116p53KO 1GG 1GG 54 34 25 G

HPF-2, proliferating 1GG 1GG 1GG 96 96 86

HPF-2, non-proliferating** 1GG 1GG 1GG 1GG 95 92

Donor lymphocytes 1GG 1GG 1GG 98 94 90

Note. The results of the MTT test after 72-hour incubation of cells are presented. "The survival rate of cells incubated without acadesine was taken as 100%. Each value is the average of five independent experiments, standard deviation < 0%. ""Пролиферацию фибробластов останавливали культивированием клеток до монослоя (контактное торможение деления клеток).

P388 cells (mouse leukemia) and Sp2/0 (mouse myeloma): at an acadesine concentration of 0.125 mM, ~1/3 of the cell population survives. Other transformed cell lines studied also die when exposed to submillimolar concentrations of acadesine. It is important that the cytotoxicity of acadesine is almost the same in the case of the leukemia line K562 and its subline with Pgp-mediated MDR (K562/4). The same is true for the MCF-7 breast adenocarcinoma line and the MDR subline (Table 1). A comparison of the cytotoxicity of acadesine against the HCT116 line and the HCT116p53KO subline (resistant to a number of DNA-damaging antitumor compounds) showed that inactivation of the proapoptotic protein p53 does not lead to an increase in cell survival in the presence of acadesine.

Equally important is the significantly higher survival of non-tumor cells in the presence of acadesine: the death of donor lymphocytes and non-transformed fibroblasts was practically absent even when exposed to acadesine in millimolar concentrations for 72 hours of continuous exposure (Table 1). Thus, acadesine causes preferential death of transformed cells (suspension and epithelial), including sublines resistant to other antitumor compounds. Non-tumor cells are damaged by acadesine to a much lesser extent. These features make it promising to use acadesine as an antitumor agent. However, for this it is important to establish the mechanisms of acadesine toxicity for tumor cells.

Acadesine induces non-apoptotic cell death

The effect of acadesine on the distribution of ploidy of the colon adenocarcinoma cell line HCT116 was studied by flow cytometry. 24 hours after the addition of acadesine (0.25 mM), the accumulation of cells in the S phase was determined, and after 48 hours (Fig. 1) - mass death cells (region to the left of the G1 peak; hypodiploid nuclei).

The accumulation of fragmented DNA may be a sign of apoptotic cell death if DNA cleavage occurs in the internucleosomal spaces, as evidenced by the formation of a set of fragments 140-170 bp long. with electrophoresis. To test this possibility, DNA integrity was determined in HCT116 cells treated with acadesine. It turned out that acadesine, unlike the reference drug - an alkyl cationic glycerolipid, does not lead to the appearance of a “ladder” of DNA fragments characteristic of apoptosis (Fig. 2).

Fluorescence

Rice. Fig. 1. Distribution of HCT116 cell line across phases of the cycle under the influence of 0.4 mM acadesine. A - intact cells; B - accumulation in the S phase after 24 hours; B - accumulation in the sub^1 area after 48 hours

An argument in favor of a non-apoptotic mechanism of death of HCT116 cells under the influence of acadesine is the results of cell staining with annexin U-FITC and propidium iodide (Fig. 3). Annexin U binds phosphatidylserine at the plasma membrane (translocation of phosphatidylserine from the inner lipid layer of the membrane

Rice. 2. DNA integrity in HCT116 cells.

1 - Intact cells;

2 - acadesine, 0.4 mM, 24 h;

3 - alkyl cationic glycerolipid, 6 µM, 24 h (method control)

Rice. 3. Staining of HCT116 cells with annexin V-FITC and propidium iodide. Pseudocolors: red - intact cells; purple - acadesine (0.4 mM, 24 h); blue - alkyl cationic glycerolipid (method control; see caption to Fig. 2)

externally is considered a sign of apoptosis). Propidium iodide is able to penetrate cells undergoing necrosis (violation of the integrity of the plasma membrane). HCT116 cells treated with acadesine (0.4 mM, 24 h) were not stained with annexin V-FITC; in contrast, the cells accumulated propidium iodide (Fig. 3), suggesting a necrotic component of the death mechanism. Similar results were obtained when recording necrotic cells using trypan blue (data not shown). It is likely that disruption of the integrity of the plasma membrane is a late event in acadesine-induced cell death. The comparison drug, an alkyl cationic glycerolipid, caused an increase in annexin V-positive cells characteristic of apoptosis (Fig. 3).

Since apoptotic cell death suggests an active role for caspases, the effect of the pan-caspase inhibitor zVAD-fmk on the cytotoxicity of acadesine was studied. HCT116 cells were incubated with 200 μM zVAD-fmk for 30 min, after which acadesine was added to the cultures and incubation continued for 24 hours. The presence of zVAD-fmk did not reduce cell death, which confirms the conclusion about the non-apoptotic mechanism of acadesine cytotoxicity.

Interaction with adenosine receptors is necessary for the death of tumor cells under the influence of acadesine

The transfer of acadesine from the extracellular environment into cells can be carried out by adenosine transporters. We studied the effect of dipyridamole, an inhibitor of these transporters, on the cytotoxicity of acadesine in the P388 cell line. It turned out that in the presence of dipyridamole, cells are insensitive even to relatively high (up to 0.8 mM) concentrations of acadesine (Table 2).

To clarify the role of the acadesine-MP-AMPK metabolic pathway in the cytotoxicity of acadesine

Table 2. Cytotoxicity of acadesine in combinations with dipyridamole or 5-iodotubercidin

Exposure to Acadesine, mM

0 0.08 0.1 0.2 0.4 0.8

Acadezine 100* 79 З8 ЗЗ 20 18

Acadesine + dipyridamole, 5 µM 100 100 99 99 100 101

Acadesine + 5-iodotubercidin, 0.05 µM 100 76 39 31 22 16

*Survival (%) of P388 leukemia cells according to the MTT test after incubation for 72 hours.

(its phosphorylation by adenosine kinase to form ZMP and activate AMPK), cells were incubated with acadesine and the adenosine kinase inhibitor 5-iodotubercidin. The inhibitor did not affect the cytotoxicity of acadesine (Table 2). It follows from this that cell death in response to acadesine is not due to the formation of ZMP and activation of AMPK.

Thus, the study of the mechanisms of cytotoxicity of acadesine revealed a number of features indicating the non-trivial nature of the pharmacological effects of this compound. Acadesine causes the death of cultured tumor cells with a significantly less pronounced effect on non-tumor cells. Acadesine is toxic to cells with molecular determinants of drug resistance - Pgp expression and non-functioning p53. It is important to emphasize the non-apoptotic nature of tumor cell death under the influence of acadesine. These results allow us to regard acadesine as a unique reagent for studying the mechanisms of tumor cell death and a promising drug candidate.

The question remains open about the intracellular target of acadesine, the interaction with which causes the death of tumor cells. We have shown that the condition for cell death is the functioning of adenosine transporters, while activation of AMPK is not required. It is reasonable to assume that tumors expressing these adenosine transporters and receptors will be most sensitive to acadesine. The role of purine base transport in cell death is not well understood; analysis of differential expression of adenosine transporters and receptors in tumors is required different types. It is likely that increased expression of these molecules will be a new molecular marker of tumor sensitivity to acadesine and a criterion for selecting patients for appropriate therapy.

The work was supported by the Ministry of Education and Science Russian Federation(State contract No. 16.N08.12.1010), and was also partially supported by the Dynasty Non-profit Programs Foundation.

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