Material with a very optimistic subtitle “Genetically engineered drug for all types and stages of malignant tumors patients can get it in three to four years.”
However, any person who knows at least some about the treatment of oncological diseases will, at best, raise his eyebrows in surprise, and at worst, be indignant at the sight of such a prognosis. We tell you what’s wrong with the latest “scientific sensation.”
What's happened?
The development of the drug, which was described in Izvestia, is being carried out at the State Research Institute of Highly Pure Drugs of the Federal Medical and Biological Agency (FMBA) of Russia. Deputy Director for scientific work Institute, Corresponding Member of the Russian Academy of Sciences and Doctor medical sciences, Professor Andrei Simbirtsev, in this article entitled “In Russia they created a cure for cancer and tested it in space,” told an Izvestia correspondent about the “heat shock protein,” which was crystallized in zero gravity on the ISS, and is now undergoing preclinical testing.
Currently, the research is being carried out with a grant from the Ministry of Education and Science, and scientists plan to find 100 million rubles for clinical trials with the help of private investors and a 50% state co-financing program. To attract him, the developers are going to “knock on all doors, because the drug is unique. We are on the verge of discovering a completely new cancer treatment. It will help people with incurable tumors.”
“We are already producing the drug using production sites Research Institute,” Andrei Simbirtsev tells inspired journalists, adding that tests are currently underway on mice, and it will reach patients in just three to four years.
What's the catch?
All this sounds very inspiring, but heat shock proteins have actually been known for a long time, but for some reason people still have not made them a panacea for all types of cancer. This is a fairly large family of proteins that are activated in response to stress when temperature increases (and sometimes decreases). They help the cell fight the consequences of degradation of the structure of other proteins. The most famous example of such a change is the coagulation of the main component of egg white, albumin, during frying or boiling, when it turns from clear to white. So, heat shock proteins eliminate the consequences of these changes: they “repair” or finally utilize degraded structures. Many heat shock proteins are also chaperones that help other proteins fold correctly.
Reference:
Chaperones are a class of proteins whose main function is to restore the tertiary or quaternary structure of proteins; they are also involved in the formation and dissociation of protein complexes.
Heat shock proteins are found in all cells. However, in different cells (especially tumor cells, which differ greatly in different types cancer both from each other and from normal cells in the body), these proteins behave differently. For example, in some types of cancer, the expression of the HSP-70 protein can be either increased (in malignant melanoma) or decreased (in kidney cancer).
To understand what kind of protein we are talking about and whether it is really used in cancer therapy and can help with all types of cancer, we talked with Doctor of Biological Sciences Alexander Sapozhnikov. This scientist is the head of the laboratory of cellular interactions at the Institute of Bioorganic Chemistry named after M.M. Shemyakin and Yu.A. Ovchinnikov RAS, which has been working on one of the most promising heat shock proteins for development in this direction for many years. He commented on this article:
“I won’t say that this is nonsense, but this is absolutely incorrect information. The author of the idea of using heat shock proteins with a molecular weight of 70 kilodaltons (the so-called HSP-70, in English HSP70) is my friend and colleague Boris Margulis. He works at the Institute of Cytology in St. Petersburg.
He and his wife Irina Guzhova have been working on this protein all their lives (I have also been working on it for many years, but not with research related to cancer therapy). Formally, the head of the laboratory is Irina, who studies how proteins are associated with neurodegenerative diseases, and Boris is the head of the department. He is the first person in the world to propose the use of a “naked” protein, not loaded with any tumor-associated antigens.
I did not believe in his ideas about this use of this protein (in fact, it has not yet been proven that it will be effective). If we “dance from the stove,” there is a certain Hindu, Pramod Srivastava, who was born in India, but studied, lives and works in America. A long time ago, he not only made a “vaccine” against a tumor using HSP-70, but also opened a clinic and treats cancer patients with it. Srivastava isolates this protein directly from the tumor: he takes biopsies from patients, isolates it from pieces of tissue (there are special ways to obtain a very high fraction of this protein).
However, the protein, which is obtained from the tissues of cancer patients, is in strong contact with tumor-associated peptides - those hallmarks of a tumor that are recognized by the immune system. Therefore, when this complex is administered to patients, a large number of patients develop an immune response, and a positive effect is obtained for the patient.
In fact, according to statistics, this effect does not exceed the effect of chemotherapy. But still, chemotherapy “poisons” the body, but such “vaccination” does not “poison” the body. This is a very long history; this approach has been used in the clinic for a long time.
Alexander Sapozhnikov. Doctor of Biological Sciences, Professor
As for Boris Margulis, he (in particular, based on my laboratory) showed (and published the results of his work) that if pure protein, without any tumor load, is added to tumor cells, then this exogenous protein causes tumor cells to expose the same tumor-associated peptides that are normally located inside these cells, in the cytoplasm. Then the immune system will recognize them, and the body will reject these cells on its own and fight the tumor.
This has been shown in culture in vitro, that is, not in the body, but in a test tube. In addition, Boris Margulis claimed only for childhood leukemia, since he is connected with clinicians in St. Petersburg. What Simbirtsev said in his interview is an expansion of this method of using naked, pure protein.
The mechanism of action of this pure protein is to force the tumor to pull to the surface (as Margulis himself called it, “squeeze out”) these peptides with their endogenous protein. This protein is present in all cells, and there is not a single cell in the world that does not have this protein. This is a very ancient, very conservative protein, everyone has it (I’m not talking about viruses now).
Margulis himself would not have carried out preclinical research; he received (five years ago) a grant together with the Institute of Highly Pure Drugs. Apparently, this institute is where this Simbirtsev works, I have heard his name many times, but since this is the Federal Medical-Biological Agency, which includes the Institute of Immunology on Kashirka, where I worked for many years, then most likely this is the Institute highly pure drugs, with which he received a grant for preclinical research. IN Soviet years it was the Third Directorate of the Ministry of Health. It was with this institute that a grant for the preclinic was received from the Ministry of Education for 30 million for three years, which ended two years ago.
The Institute of Highly Pure Preparations did all the paperwork, they reported on their grant, as for the next stage, promotion of the drug, money is also needed there. This is the first stage clinical trials. Here Boris Margulis, as far as I understand, has already moved away from development, leaving it to the Institute of Highly Pure Preparations.
They make this protein, they made biotechnology, I even have it in the refrigerator, Boris gave it to me for testing. They make it in large quantities and store it in lyophilized form (dry) in sterile ampoules. Actually, this drug should be used, perhaps with some additives, in clinical trials. But this requires money.
Having accidentally seen the news with Simbirtsev’s interview, I read it, sent it to Margulis, and asked if he had read it. Boris answered me that Andrey (whom he knows well) did something stupid, he didn’t even refer to the authors. The author of this idea (to use pure protein as an antitumor drug in oncology), I repeat, is Boris Margulis. But, as far as I’ve heard from him lately, he has moved away from this issue.
I'm working on this protein, but as an immunomodulator, like my laboratory. We worked a little with antitumor properties, in mouse models. It really worked out there good results. I mean "naked" protein, it simply has immunostimulating properties. By the way, the big question is what is the reason for its immunostimulating properties: the protein itself or some small impurities, for example lipopolysaccharides. This protein is obtained in bacterial culture (in E.coli), this is the most common technique for producing recombinant proteins. Lipopolysaccharides (LPS) - component cell wall bacteria, and it is very difficult to completely purify the culture from this impurity. Of course, they clean it, but some tiny concentrations remain. These LPS impurities also have immunostimulating properties simply because the immune system has evolved to develop its own defenses against bacteria. As soon as the “smell” of bacteria appears in the body, the immune system is activated. Therefore, many authors now believe that the immunostimulating properties of this protein, which also modulate the antitumor response, are caused not by HSP as such, but by its admixture. But this question is scientific, debatable and has nothing to do with practice.
Now, I repeat, Boris Margulis is moving away from this topic, from oncology, and is working on small molecules that can regulate the production of this protein. He contacted chemists who know how to make inhibitors - such specific kinases, some enzymes inside cells that stop their work. Inhibitors can tell some enzyme: “No, you have no right to work.”
This is done very simply: all enzymes have a substrate binding center, and if you take some small molecule that is built into this substrate binding center, it will no longer be able to process this substrate. Boris is now working on molecules that inhibit the intracellular synthesis of this HSP-70. And, indeed, such molecules are very relevant, and not only for fundamental biology, but also for practice and clinical medicine.”
Alexander Sapozhnikov does not agree with this theoretical basis mechanism of action of the drug. According to him, HSP70 may work in a different way, which remains to be studied, but the fact remains that in cell cultures and a number of tumors in two lines of rats in which “human” tumor cells were inoculated, the protein actually shows activity.
According to the authors of the work, the temperature at which they work with HSP70 in cell cultures is 43°C, and it is too high for living organisms, however, other mechanisms appear to be involved, which also remain to be understood. This also applies to the action of exogenous cellular protein heat shock inside the body. “Each of us has enough high level HSP70 - up to 900 nanograms per milliliter. We injected it into the animal and tried to see what happened to the protein next. Within 40 minutes we saw traces of HSP70 in the blood, and then it disappeared. There is an opinion that the protein breaks down, but we don’t think so.”
Impressive results awaiting verification
Irina Guzhova also spoke about further testing of the drug: “We tested this mechanism on mouse melanoma B16, which grows subcutaneously, and used it in the form of a gel applied to the surface of the skin. The result was impressive: the survival rate of mice was much higher than that of the control group, which was treated with gel without active substance or not treated at all. The difference was about ten days. For mice and of this type tumors are a very good delay. Similar results were shown in rat C6 glioma (this is a tumor that grows directly in the brain).
Animals treated with a single injection into the brain were given an extra ten days to live, while animals given the protein continuously for three days via a pump had this duration extended by an additional ten days as the tumor grew more slowly. We showed that if you deplete the population of T cells from a mouse that had a tumor, and remove the already “learned” NK cells or CD8-positive lymphocytes, they will not recognize the tumor as well. It can be concluded that the main function of HSP70 in this process is activation specific immunity».
These data prompted scientists to conduct a limited study at the Polenov Clinic (Research Institute of Neurosurgery in St. Petersburg). “At this time, our team included neurosurgeon Maxim Shevtsov, who, simultaneously with Boris Alexandrovich’s postgraduate studies (Margulis, - website note) completed his residency at this research institute. He convinced his supervisor, Professor Khachaturian, to test this drug. According to the legislation of that time, the decision of the scientific council and the informed consent of the patients were sufficient, and we were allocated 25 patients. They all had various brain tumors, and they all received what they were entitled to under insurance, but plus after surgical removal Maxim injected HSP70 solution into the operating bed.
The problem is that brain tumors are difficult to completely remove. There are always small pieces left that are dangerous to remove, because along with them the personality can be removed, and these pieces give rise to relapses. But the results turned out to be absolutely amazing: after the operation, the number of specific immune cells in patients increased, the number of pro-tumor (“switched to the tumor side”) T-lymphocytes decreased, and the amount of interleukin-10 (an information molecule of the immune system) decreased.
The study was only a pilot, not randomized, there was no control group, and it was conducted in 2011. That same year, a law was passed prohibiting such tests, and they had to be stopped as soon as they began. We have 12 operated patients left. Anyone familiar with the clinical part of the research has an idea of how difficult it is to track the fate of patients after each of them leaves the clinic. Therefore, we only know of eight who remained contactable, and all of them are still alive. At the beginning of autumn last year, they were quite healthy, and those who continued to study went to school in the fall, although the average prognosis for life expectancy with a detected glioma is 14 months.”
Now, according to the speakers, preclinical trials are coming to an end, and the drug requires multi-stage testing on patients, which will take several years (that’s why the Izvestia article included such an incredibly short period of time before the drug enters the market - 3-4 years).
Alexander Sapozhnikov also emphasized the importance of clinical trials: “A tumor grafted into mice and a human tumor are heaven and earth. The drug may work on this tumor, but be ineffective on either a normal mouse tumor or a human one. Reassure your colleagues, there is no cure for all diseases at once.”
The researchers themselves think so. “At these stages, everything works (and very well), but, of course, this is not the medicine that raises Lazarus,” says Irina Guzhova, “however, it is quite effective and worthy of undergoing clinical trials. And we hope that this will happen."
Simply space
The reader may have a reasonable question: where did space come from? Irina Guzhova explains: “The fact is that the tests took place on the basis of the Institute of Highly Pure Preparations, whose employees have good experience in registering patents and writing papers, so we gave this matter to them. At the same time, they began producing this protein, and we did experiments on animals. But in the process, a representative of Roscosmos approached them and asked if we had some kind of uncrystallized protein that could be crystallized in space, in orbit. And they were given HSP70, they tried to grow crystals in orbit, but nothing worked.”
The problem turned out to be in the structure of the protein. A very mobile part in the structure of the protein interfered with crystallization, so they began to try to crystallize it in pieces, to bind the moving part with a special molecule so that it would “hold” it. They are still trying. “This is where this story about cells that grow in space and cure everyone from cancer arose,” comments Irina Guzhova.
She also said that for testing in space and on mice, the protein was subjected to a very high degree of purification - about 99%. As for doubts that it is not the chaperone that activates the immune system, but lipopolysaccharide (LPS) - a component of the cell wall of bacteria in which this protein is produced - such a probability is small. Although LPS “sticks” to HSP very strongly, and it is quite difficult to purify the protein from its most minute impurities. Scientists set up additional controls to show that it is not he, but the chaperone, that is the cause of the drug’s effect. For example, the drug can be boiled, which does not affect LPS, but destroys the protein structure. Then its HSP properties are lost and the drug stops working, which would not happen if it was mainly bacterial LPS that acted in it.
In addition, the researchers compared the effect of introducing bacterial cell wall components with the effect of HSP70, and these comparisons clearly favored the latter.
“We didn’t say anything stupid. And what? “Zero emotions!”
Irina reports that scientists have not yet discovered any adverse reactions during the tests, but they may be delayed. “I believe that a researcher should first of all try everything on herself, and I completed two courses of chaperone therapy. There were no side effects; on the contrary, it seemed that minor sores were going away and wings were growing behind my back.”
“On the other hand, everything that was in the media was a real disgrace,” the researcher notes. - But, as they say, there would be no happiness, but misfortune would help: the Institute of Highly Pure Preparations is already receiving calls with offers to help with clinical trials. We spoke at conferences and in various more modest media, talking about the same thing, but checking our words and not saying anything stupid. And what? - Zero emotions! And then this kind of dregs flashed across the screens, and please! Such an interesting society, such an interesting country.”
However, according to sources on the site, Simbirtsev was forced to give the interview that started it all. offered to give an interview to stimulate interest in the problems of the Institute and attract additional funding for clinical trials. In addition, there are rumors about the possible loss of a legal entity by the institute due to mergers of scientific organizations occurring throughout the country. Apparently, the scientist was not ready to tell the newspaper in detail and popularly about what was happening. “This time, everything that could have been misunderstood was misunderstood,” notes the source.
As a result, the situation is becoming more and more like a well-known fable, when Roscosmos and government agencies distributing grants are rushing into the clouds, expecting immediate results from fundamental science, cancer is moving backwards, journalists are spilling structured water... And Russian science once again finds itself in an unenviable position, forced to justify herself for crimes she did not commit.
Heat shock heat shock- heat shock.
Stressful state of the body after exposure elevated temperature, in particular, T.sh. used to induce polyploidy<induced polyploidy> mainly for animals that reproduce in water (fish, shellfish): the water temperature is increased to 29-33 o C for 2-20 minutes. ( normal temperature incubation is usually 15-20 o C) after 3-10 minutes. (induction of triploidy) or after 20-40 minutes. (induction of tetraploidy) after fertilization; also able T.sh. analyze the activity of specific heat shock proteins<heat-shock proteins>, pouf activity<puffing> in fruit flies (in this case T.sh. at 41-43 o C).
(Source: "English-Russian Dictionary genetic terms." Arefiev V.A., Lisovenko L.A., Moscow: Publishing House VNIRO, 1995)
See what “heat shock” is in other dictionaries:
Heat shock- * ceplav shock * heat shock is a stressful state of the body due to exposure to elevated temperature. T. sh. used: a) to induce polyploidy (see) in fish, mollusks, incubation of individuals after fertilization at tо = 29-33 °C (instead of ... ... Genetics. encyclopedic Dictionary
heat shock- Stressful state of the body after exposure to elevated temperature, in particular, T.sh. used to induce polyploidy mainly in water-reproducing animals (fish, shellfish): the water temperature is increased to 29-33 oC for 2-20 minutes... ... Technical Translator's Guide
Thermal shock- Syn: Thermal exhaustion. Occurs when overheating due to insufficient response of the heart vessels to extreme high temperature, develops especially often in older people taking diuretics. Shows weakness... Encyclopedic Dictionary of Psychology and Pedagogy
OVERHEATING AND HEAT STROKE- honey Overheating (heat syncope, heat prostration, heat collapse) and heat stroke (hyperpyrexia, sunstroke, overheating of the body) are pathological reactions of the body to high temperature environment, Related… … Directory of diseases
- (English HSP, Heat shock proteins) is a class of functionally similar proteins, the expression of which increases with increasing temperature or under other conditions that stress the cell. Increased expression of genes encoding thermal proteins... ... Wikipedia
A tetramer consisting of four identical p53 protein molecules. They are interconnected by domains responsible for oligomerization (see text). p53 (p53 protein) is a transcription factor that regulates the cell cycle. In a non-mutated state... ... Wikipedia
In 1962 in Italy, a young geneticist Ferruccio Ritossa discovered swelling (puffing) of some chromosome regions Drosophila with a random increase in temperature in the thermostat. This turned out to be a manifestation of gene activation and was called " heat shock response (link ),
and the inducible proteins were named heat shock proteins, HSP (heat shock proteins , HSP).
This class of proteins was later found in all cells of all living organisms - from bacteria to humans. It is known that such a response manifests itself, in addition to heat, under various biological (infection, inflammation), physical (radiation, hypoxia), chemical (alcohols, metals) and others stress influences. That's why Heat shock proteins are also called stress proteins.
Increased expression of HSP proteins protects the cell by stabilizing denatured or misfolded peptides. Accumulating under various harmful influences, Heat shock proteins help the cell maintain homeostasis under stress (cm). HSP proteins respond not only to external stressful situations, they appear in many diseases, such as neurodegeneration, metabolic disorders, ischemic injury and cancer, which determines the increased interest in these proteins and the search for therapeutic tools that regulate their reactions ( 2006
,
2007
,
2007a ).
Heat shock proteins serve biological markers of an unfavorable state of the body.
Cellular response to stress regulated primarily at the level transcriptions(DNA to RNA) using heat shock factors (heat shock factor, HSF) (). The HSF family contains 4 species, of which HSF1, HSF2 and HSF4 are expressed in mammals and humans, with HSF1 being a universal stress-responsive activator, while HSF2 is more associated with differentiation processes. In the absence of stress, these factors are located in the nucleus and cytoplasm in monomeric form and are not able to bind to DNA. In response to stress HSF form trimers(possible HSF1 homotrimers or HSF1-HSF2 heterotrimers) (cm .) and move into the nucleus, where do they contact heat shock elements (HSE) - specific DNA sequences in promoters of heat shock genes.
Subsequent phosphorylation of HSF trimers accompanied by activation of heat shock gene transcription and increased HSP levels, leading to formation of HSF-HSP complexes. When the stress stops, the trimeric forms of HSF separate from the DNA, turning back into inactive monomers, and the cell returns to normal protein synthesis (link).
It is assumed that heat shock proteins themselves can regulate the expression of their genes through "autoregulation loop". According to this hypothesis, an increase in the concentration of misfolded proteins resulting from stress leads to the binding of specific HSPs and activation of HSF.
Heat shock proteins as molecular chaperones
Further study of the HSP class showed that these proteins are not only induced by stress, but many of them function constitutively asmolecular chaperones, participating in the stabilization and movement of immature peptides during normal growth. For example, proteins Hsp70, Hsp90 are present in high concentrations in non-stressed cells, accounting for 1-1.5% of total cellular protein, which indicates the constant need of the cell to maintain the conformational homeostasis of its proteins. These proteins are found in the cytosol, mitochondria, endoplasmic reticulum and nucleus. The molecular masses of HSPs range from 15-110 kDa. The most studied proteins in mammals are the 60, 70, 90, and 110 kDa HSP proteins, which play important roles in fundamental intracellular processes, from antiapoptotic effects to protein unfolding and intracellular trafficking.
Functions of HSPs as chaperones can be reduced to the following:
1. Clotting immature polypeptide chains;
2. Relief movements proteins through different cellular compartments;
3. Modulation of protein activity through stabilization and/or maturation to a functionally competent conformation;
4. Support the formation/cleavage of multiproteins complexes;
5. Correction incorrectly folded proteins;
6. Protecting proteins from aggregation;
7. Direction completely damaged proteins for breakdown;
8. Organization units from destroyed proteins;
9. Solubilization of protein aggregates for further degradation.
Co-chaperones
The activity of heat shock proteins is regulated by other proteins - co-chaperones, which contribute to the basic functions of the HSP. Although many co-chaperones are soluble cytosolic proteins, some are localized within cell membranes ah or elements of the cytoskeleton. These specialized co-chaperones include auxilin, Tom70, UNC-45, Bag-1 homologs. Co-chaperones may be involved in the ATP-dependent activities of HSP70 and HSP90, including functions such as secretion, protein transport, and protein complex formation/cleavage (link).
Co-chaperones Hip, Hop, Hup, CHIP modulate nucleotide exchange and substrate binding HSP70 proteins, coordinating the folding of newly synthesized proteins, correct the incorrect folding of damaged and denatured proteins, direct the transport of proteins across cell membranes, inhibit protein aggregation and carry out degradation along the proteasomal pathway ().
Functions of some co-chaperones
HSP70 proteins together with co-sharepons carry out at least 2 alternative activities: prevent aggregation of non-native proteins when binding to hydrophobic regions of substrate molecules, protecting them from intermolecular interactions (“security”, “holder” activity), and also contribute to the folding of non-native intermediates to the native state (“folding”, “folder” activity).
HSP and the ATPase cycle
Heat shock proteins in mammals are represented by 6 families depending on molecular weight: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and small Hsps (15 to 30 kDa), including Hsp27. High molecular weight HSPs are ATP dependent, while the activity of small HSPs is independent of ATP.
Genetic and biochemical data have shown that ATP hydrolysis is an essential element of HSP70 chaperone activity. Proteins of this family bind to intermediate peptides through cycles of ATP binding and hydrolysis, and subsequent ADP/ATP exchange is accompanied by the release of peptides. HSP70 molecules contain two conserved regions - N-terminal ATP-binding(45 kDa) and C-terminal (15 kDa), binding hydrophobic peptides. In between is the more variable alpha helical cap region. ATP-bound HSP70 (the “lid” is open) freely interacts with immature or misfolded peptides, causing conformational changes that lead to ATPase activation and enhances association with the co-chaperone HSP40, which promotes the transition to ADP-bound (the “lid” closed) form. For the effective coupling of ATP hydrolysis with the binding and subsequent release of peptide substrates, co-chaperones of the JDP family (J-domain proteins) ( ; ).
Heat shock proteins during ischemia
The cytoprotective properties of HSP70 class proteins have been demonstrated in various models of ischemic disorders in vitro and in vivo ( , , , , , ). Initially, this protection was explained by the action of HSPs as chaperones (maintaining the correct folding of proteins and preventing their aggregation), but then it turned out that HSP70 can directly react with cell death pathways - apoptosis and necrosis.
As can be seen from the figure, cerebral ischemia induces apoptosis different ways, and HSP70 reduces the effect of all of them. The “intrinsic” pathway of apoptosis consists of the release of pro-apoptotic mitochondrial substances, the opening of the mitochondrial pore and the activation of caspases (see). Another ("external") pathway is associated with receptor activation plasma membrane(Fas and TNFR) inducing apoptosis through caspase-8 using the TRAF factor. In addition, mechanisms of caspase-independent apoptosis are known (see).
HSP70 proteins can inhibit the release of cytochrome c (cyt c) from mitochondria and the translocation of the apoptosis-inducing factor AIF into the nucleus, reducing ischemic brain damage (see), as well as inhibit the release of the pro-apoptotic protein Smac/DIABLO from the mitochondria of myocytes.
Expression of HSP72 in astrocytes leads to decreased production of reactive oxygen species (ROS) and maintenance of mitochondrial membrane potential, as well as glutathione levels and increased superoxide dismutase activity during ischemic disorders in cardiocytes.
Increased expression of HSP72 is able to reduce apoptosis directly through increasing Bcl-2 levels and by inhibiting the translocation of the proapoptotic factor Bax.
Proteins of the HSP70 class have been shown to inhibit the dephosphorylation of JNK kinase (c-Jun N-terminal kinase), which plays a significant role in neuronal apoptosis and is one of the targets for stroke therapy.
In addition, Hsp proteins interact with topoisomerase 1 (a regulator of apoptosis) and are effectors of the important antiapoptotic kinase Akt/PKB (see). Significant activation of glutathione peroxidase and glutathione reductase by heat shock proteins is an essential element in the mechanism of the cytoprotective action of HSP during ischemia ().
Anti-inflammatory effect of heat shock proteins
Heat shock proteins have a strong anti-inflammatory effect by preventing cell responses to inflammatory cytokines such as TNF and IL-1.
Inflammation is known to produce ROS through activation of inducible NO synthase (iNOS) and NADPH oxidase, with iNOS occurring in response to cytokine release. Nitrous oxide (NO) synthesized by iNOS reacts with superoxide to form the highly toxic oxidizing agent peroxynitrite: -O2− + -NO → ONOO−
and HSP72 inhibits iNOS expression by reducing NFkappaB activation (link). In addition, heat shock proteins reduce NADPH oxidase activity in neutrophils and activate superoxide dismutase in phagocytes, and also regulate the activity of matrix metalloproteinases in astrocytes.
A significant part of the intracellular effects of HSP proteins during inflammation is associated with their regulation of the nuclear factor NFκB pathway, since transcription factors of this family are key participants in triggering the inflammatory response. Translocation of the dimers that make up NFkB into the nucleus, where they induce the expression of many inflammatory genes, is inhibited by heat shock proteins through direct interaction or through influence on NFkB signaling pathways.
It has also been shown that Hsp72 interacts with the IKK kinase complex, which is necessary for the release of NFkB and its transition to the nucleus.
Thus, HSP70 class proteins use many pathways to prevent inflammatory processes in the body (review).
Extracellular action of heat shock proteins
HSP proteins have long been considered cytoplasmic, with functions limited to the intracellular compartment. However, recently there has been an increasing number of observations that these proteins can be released into the extracellular environment and have an effect on other cells. This was first shown in the glial cells of the squid giant axon, from which the HSP70 proteins released moved into the axon. Work from several laboratories has investigated the effects of astrocyte- or Schwann-cell-derived HSP72 on neighboring neurons and axons. Extracellular effects of HSP have also been obtained on epithelial cells, rat embryonic cells, B lymphocytes, dendritic and tumor cells.
It has been shown that extracellular HSP72 can induce the release of cytokines (TNF, IL-6, IL-1beta) from monocytes, which is provided by TLR2, TLR4 receptors and activation of NFkB.
Extracellular HSPs can interact with cell membrane lipids and insert into membranes, forming ATP-gated cation channels (see). In addition, HSP72, by interacting with phosphatidylserine on the surface of apoptotic cells, accelerates the death of these cells.
There is a significant correlation between increased level serum HSP70 and a decrease in the development of atherosclerosis, determined by the thickness of the intima of the carotid artery ().
The fact that patients with coronary insufficiency have inverse correlation between serum HSP70 levels and the risk of this disease, shown by a coronary artery angiogram (see).
The role of heat shock proteins in immune responses
HSP and anticancer therapy
Heat shock proteins are highly expressed in many types of human cancer and are involved in the proliferation, differentiation, metastasis and recognition of tumor cells by the immune system. They are useful biomarkers of carcinogenesis in some tissues and signal the degree of differentiation and aggressiveness of certain types of cancer. In addition, levels of circulating HSP and anti-HSP antibodies may be useful for diagnosing cancer. Increased HSP expression may also sometimes predict the answer to anticancer treatment
. For example, HSP27 and HSP70 have been implicated in chemotherapy resistance in breast cancer, and elevated HSP27 levels predict poor response to chemotherapy in leukemia. At the same time, HSP70 expression suggests good chemotherapeutic effects in osteosarcomas ( see review).
Their role in the development of anticancer therapy with the participation of HSP dual function in the body: On the one side - intracellular cytoprotective/anti-apoptotic, and on the other - extracellular/immunogenic.
This allowed us to develop 2 main strategies in anticancer therapy:
1) Pharmacological modification of HSP expression and their activity as molecular chaperones;
2) Use of HSPs in cancer vaccines based on their ability to act as immunological adjuvants.
Most promising as an anticancer pharmacological target turned out to be the HSP90 protein. Its level is 1-2% of the total protein content in the absence of stress, and its amount client proteins exceeds 100, many of which are associated with tumorigenesis. Increased expression of HSP90 has been found in breast tumors, lung cancer, leukemia, Hodgkin's disease, lymphomas and other cancers. Therefore, inhibition of HSP90 may simultaneously destroy a large number of oncogenic signaling pathways. Many laboratories are engaged in the development of HSP90 inhibitors (, , 2007a, 2007b, etc.).
Natural HSP90 Inhibitors - geldanamycin (GA) and 17-allylamino-17-demethoxygeldanamycin (17-AAG)- interact with the ATP-binding site of the HSP90 molecule with higher affinity than natural nucleotides and interfere with ATP-ADP protein transitions, disrupting the activity of HSP90 as a chaperone, and its client proteins are degraded by the proteasome. It is essential that HSP90 inhibitors, while removing client proteins in cancer cells, do not affect the same proteins in normal tissues, since their affinity for HSP90 isolated from tumors is 20-200 times higher (see).
Learn more about natural and artificial heat shock protein inhibitors and their mechanisms of action can be read in reviews , .
The ability of heat shock proteins to bind antigen peptides formed the basis immunotherapeutic approach to the treatment of cancer. Peptide complexes Hsp70 and Grp96 isolated from tumors of cancer patients are used as anti-cancer vaccines for the treatment and prevention of cancer. Heat shock proteins, in addition to exhibiting chaperone activity towards tumor peptide antigens, facilitate the entry of HSP-peptide complexes into cells due to receptor endocytosis. This made it possible to quickly transfer HSP-based vaccines from study in animal models to treatment. cancer diseases in the clinic. Improved forms of HSP vaccines are obtained by isolating HSP70-peptide complexes from dendritic cells fused with tumor cells.
Pramod K. Srivastava ( Pramod K. Srivastava, a professor of medicine and director of the Center for Immunotherapy of Cancer and Infectious Diseases at the University of Connecticut School of Medicine)- one of the first researchers to study the role of heat shock proteins in immune system. With his participation, the company Antigenics was created, which successfully develops anti-cancer vaccines based on HSPs isolated from individual patient tumors.
These drugs, based on various heat shock proteins, are currently in clinical trials.
Heat shock proteins during aging
As organisms age, they lose the ability to adequately respond to external stress and maintain homeostasis. Old cells are more susceptible to disorders and diseases, so susceptibility to these factors increases with age.
During the life of a stable protein, various post-translational changes occur. Protein stability is impaired due to numerous harmful effects - oxidation of side chains, glycation, deamination of asparaginyl and glutaminyl residues, which leads to the formation of isopeptide bonds. Sensitivity to proteotoxic damage increases due to errors in transcription and translation and manifests itself as defects in protein folding. Aging is characterized by an increase in protein modifications associated with coagulation homeostasis ( cm. ) . The functions of chaperones are disrupted, the need for protein degradation increases, but the activity of the main proteolytic apparatus, the proteasome, also decreases with age, leading to the danger of glycation. Aggregation is also accompanied by proteasome inhibition and stopping cell cycle. With age lysosomal protein degradation is also impaired(possibly due to suppression by lipofuscin). Accumulation of misfolded proteins and weakening defense mechanisms leads to
Alexander Sapozhnikov does not agree with this theoretical justification for the mechanism of action of the drug. According to him, HSP70 may work in a different way, which remains to be studied, but the fact remains that in cell cultures and a number of tumors in two lines of rats in which “human” tumor cells were inoculated, the protein actually shows activity.
According to the authors of the work, the temperature at which they work with HSP70 in cell cultures is 43°C, and it is too high for living organisms, however, other mechanisms appear to be involved, which also remain to be understood. This also applies to the action of exogenous non-cellular heat shock protein inside the body. “Each of us has fairly high levels of HSP70 in our bloodstream - up to 900 nanograms per milliliter. We injected it into the animal and tried to see what happened to the protein next. Within 40 minutes we saw traces of HSP70 in the blood, and then it disappeared. There is an opinion that the protein breaks down, but we don’t think so.”
Impressive results awaiting verification
Irina Guzhova also spoke about further testing of the drug: “We tested this mechanism on mouse melanoma B16, which grows subcutaneously, and used it in the form of a gel applied to the surface of the skin. The result was impressive: the survival rate of the mice was much higher than that of the control group, which was treated with a gel without the active substance or not treated at all. The difference was about ten days. For mice and this type of tumor, this is a very good delay. Similar results were shown in rat C6 glioma (this is a tumor that grows directly in the brain).
Animals treated with a single injection into the brain were given an extra ten days to live, while animals given the protein continuously for three days via a pump had this duration extended by an additional ten days as the tumor grew more slowly. We showed that if you deplete the population of T cells from a mouse that had a tumor, and remove the already “learned” NK cells or CD8-positive lymphocytes, they will not recognize the tumor as well. We can conclude that the main function of HSP70 in this process is the activation of specific immunity."
These data prompted scientists to conduct a limited study at the Polenov Clinic (Research Institute of Neurosurgery in St. Petersburg). “At this time, our team included neurosurgeon Maxim Shevtsov, who, simultaneously with Boris Alexandrovich’s postgraduate studies (Margulis, - website note) completed his residency at this research institute. He convinced his supervisor, Professor Khachaturian, to test this drug. According to the legislation of that time, the decision of the scientific council and the informed consent of the patients were sufficient, and we were allocated 25 patients. They all had various brain tumors, and they all received what they were entitled to under insurance, but plus, after surgical removal of the tumor, Maxim injected an HSP70 solution into the operating bed.
The problem is that brain tumors are difficult to completely remove. There are always small pieces left that are dangerous to remove, because along with them the personality can be removed, and these pieces give rise to relapses. But the results turned out to be absolutely amazing: after the operation, the number of specific immune cells in patients increased, the number of pro-tumor (“switched to the tumor side”) T-lymphocytes decreased, and the amount of interleukin-10 (an information molecule of the immune system) decreased.
The study was only a pilot, not randomized, there was no control group, and it was conducted in 2011. That same year, a law was passed prohibiting such tests, and they had to be stopped as soon as they began. We have 12 operated patients left. Anyone familiar with the clinical part of the research has an idea of how difficult it is to track the fate of patients after each of them leaves the clinic. Therefore, we only know of eight who remained contactable, and all of them are still alive. At the beginning of autumn last year, they were quite healthy, and those who continued to study went to school in the fall, although the average prognosis for life expectancy with a detected glioma is 14 months.”
Now, according to the speakers, preclinical trials are coming to an end, and the drug requires multi-stage testing on patients, which will take several years (that’s why the Izvestia article included such an incredibly short period of time before the drug enters the market - 3-4 years).
Alexander Sapozhnikov also emphasized the importance of clinical trials: “A tumor grafted into mice and a human tumor are heaven and earth. The drug may work on this tumor, but be ineffective on either a normal mouse tumor or a human one. Reassure your colleagues, there is no cure for all diseases at once.”
The researchers themselves think so. “At these stages, everything works (and very well), but, of course, this is not the medicine that raises Lazarus,” says Irina Guzhova, “however, it is quite effective and worthy of undergoing clinical trials. And we hope that this will happen."
Simply space
The reader may have a reasonable question: where did space come from? Irina Guzhova explains: “The fact is that the tests took place on the basis of the Institute of Highly Pure Preparations, whose employees have good experience in registering patents and writing papers, so we gave this matter to them. At the same time, they began producing this protein, and we did experiments on animals. But in the process, a representative of Roscosmos approached them and asked if we had some kind of uncrystallized protein that could be crystallized in space, in orbit. And they were given HSP70, they tried to grow crystals in orbit, but nothing worked.”
The problem turned out to be in the structure of the protein. A very mobile part in the structure of the protein interfered with crystallization, so they began to try to crystallize it in pieces, to bind the moving part with a special molecule so that it would “hold” it. They are still trying. “This is where this story about cells that grow in space and cure everyone from cancer arose,” comments Irina Guzhova.
She also said that for testing in space and on mice, the protein was subjected to a very high degree of purification - about 99%. As for doubts that it is not the chaperone that activates the immune system, but lipopolysaccharide (LPS) - a component of the cell wall of bacteria in which this protein is produced - such a probability is small. Although LPS “sticks” to HSP very strongly, and it is quite difficult to purify the protein from its most minute impurities. Scientists set up additional controls to show that it is not he, but the chaperone, that is the cause of the drug’s effect. For example, the drug can be boiled, which does not affect LPS, but destroys the protein structure. Then its HSP properties are lost and the drug stops working, which would not happen if it was mainly bacterial LPS that acted in it.
In addition, the researchers compared the effect of introducing bacterial cell wall components with the effect of HSP70, and these comparisons clearly favored the latter.
“We didn’t say anything stupid. And what? “Zero emotions!”
Irina reports that scientists have not yet discovered any adverse reactions during the tests, but they may be delayed. “I believe that a researcher should first of all try everything on herself, and I completed two courses of chaperone therapy. There were no side effects; on the contrary, it seemed that minor sores were going away and wings were growing behind my back.”
“On the other hand, everything that was in the media was a real disgrace,” the researcher notes. - But, as they say, there would be no happiness, but misfortune would help: the Institute of Highly Pure Preparations is already receiving calls with offers to help with clinical trials. We spoke at conferences and in various more modest media, talking about the same thing, but checking our words and not saying anything stupid. And what? - Zero emotions! And then this kind of dregs flashed across the screens, and please! Such an interesting society, such an interesting country.”
However, according to sources on the site, Simbirtsev was forced to give the interview that started it all. offered to give an interview to stimulate interest in the problems of the Institute and attract additional funding for clinical trials. In addition, there are rumors about the possible loss of a legal entity by the institute due to mergers of scientific organizations occurring throughout the country. Apparently, the scientist was not ready to tell the newspaper in detail and popularly about what was happening. “This time, everything that could have been misunderstood was misunderstood,” notes the source.
As a result, the situation is becoming more and more like a well-known fable, when Roscosmos and government agencies distributing grants are rushing into the clouds, expecting immediate results from fundamental science, cancer is moving backwards, journalists are spilling structured water... And Russian science once again finds itself in an unenviable position, forced to justify herself for crimes she did not commit.
