Open access peer-reviewed chapter
Abstract
The proline-rich peptide (PRP-1) isolated from neurosecretory granules of the bovine neurohypophysis, produced by N.supraopticus and N.paraventricularis, has many potentially beneficial biological effects. PRP-1 has been shown to have the opposite effects on cell death in neurodegenerative and cancer diseases. It significantly reduces staurosporine-induced apoptosis of postnatal hippocampal cells, as well as doxorubicin-induced apoptosis of bone marrow monocytes and granulocytes, in both time- and dose-dependent manner. PRP-1 also exerts the opposite effect on the proliferation of bone marrow stromal cells obtained from normal humans and on the stromal cells isolated from human giant-cell tumor. PRP-1 cytostatically inhibits chondrosarcoma bulk tumor but exerts drastic cytotoxic effect on sarcomas cancer stem cells. The same peptide caused cell death through apoptosis in rats with Ehrlich Ascites Carcinoma model.
Keywords
- proline-rich peptide (PRP-1)
- neurodegeneration
- cancer diseases
- ehrlich ascites carcinoma (EAC)
- chondrosarcoma
1. Introduction
Our long-term scientific work has been aimed at studying the protective effects of certain physiologically active compounds, including proline-rich peptide (PRP-1, comprised of 15 amino acids residues, AGAPEPAEPAQPGVY, with an apparent molecular mass of 1475.25 Da) on brain plasticity in rats with different neurodegenerative models. While displaying neuroprotective role in those models, in cancer related studies, on the other hand, PRP-1 displayed its beneficial antiproliferative effect in cellular context dependent manner by triggering cell death leading to drastic decrease of cancer stem cell population responsible for disease relapse and drug resistance.
2. Effect of PRP-1 on cell death in the neurodegenerative diseases
The protection of neurons from damage and death in neurodegenerative disorders, such as Alzheimer disease (AD), ischemic insults, Parkinson disease (PD), is a major challenge for neuroscientists in the twenty first century. Much attention is focused on the discovery of novel biomarkers for diagnosis and therapy of neurodegenerative diseases.
The new family of peptide neurohormones consisting of 10–15 amino acid residues isolated by prof. Galoyan and coworkers from bovine and human neurohypophysis neurosecretory granules are synthesized in the form of a common precursor protein (neurophysin-vasopressin associated glycoprotein) [1, 2]. These five peptides contain a high proportion of proline residues and, therefore, were designated as proline rich peptides (PRPs). The most studied is the bovine PRP-1 (also known as galarmin). The polypeptide is not species-specific; hence it is active in mice, humans, and rats. It has been shown that PRP-1, as well as its synthetic analogue, has many beneficial biological effects.
Neuronal injuries have been suggested to promote PRP-1 synthesis and its release as a messenger, modulating the signaling cascades and, therefore, contributing to protection, regeneration, and repair of the neurons.
The neuroprotective [3, 4, 5, 6, 7] and immunoregulatory [8, 9, 10] effects of PRP-1 through its involvement in the neuro-immuno-hematopoietic interaction have been demonstrated in the following models of central nervous system damage: spinal cord hemisection; βA-peptide injection (model of Alzheimer disease); vestibular nuclei damage through vibration and unilateral labyrinthectomy; immobilization stress (IMO stress).
Our previous report on the effects of PRP-1 on SC-injured rats indicated the possibility of PRP-1 involvement in the mechanisms of neuronal repair [3]. Immunohistochemical study demonstrated that treatment with PRP-1 resulted in the recovery and growth of nerve fibers, glia proliferation, and motoneuron survival. Therefore, PRP-1 has been found to be a highly active neurotrophic-like substance (Figure 1).
Figure 1.
PRP-1-Ir Spinal Cord (SC) nerve structures in SC-hemisectioned rats with and without PRP-1 injection. Degenerative and strongly immunoreactive for PRP-1 motoneurons (MNs) with no processes are demonstrated in the spinal cord anterior horn, situated in the lightened pericellular area, perhaps, brain edema. After daily administration of PRP-1 to trauma-injured animals for 3 weeks, regeneration of strongly immunostained MNs and their processes was observed suggested the possibility of PRP-1 involvement in the mechanisms of neuronal repair: growth of nerve fibers and motoneurons survival. ABC immunohistochemical method.
PRP-1 participation in the regeneration of the nerve structures was also immunohistochemically demonstrated in the trauma-injured rats with SC hemisection treated by the administration of Naja Naja Oxiana (NOX) snake venom. NOX venom prevented the scar formation, well observed 2 months after the SC injury in the control rats, and resulted in the regeneration of the nerve fibers growing through the trauma region. It was suggested to exert the neuroprotective effect by involving the endogenous PRP-1 in the underlying mechanism of the neuronal recovery – based on the data regarding the survival of the immunoreactive to PRP-1 (PRP-1-Ir) motoneurons, the increased number of PRP-1-Ir nerve fibers in the SC lesion region, and appearance in the white matter of the PRP-1-Ir astrocytes migrating towards the injured side [7] (Figures 2 and 3).
Figure 2.
SC nerve fibers in the SC hemisectioned rats regularly treated with NOX venom. Naja Naja Oxiana (NOX) snake venom prevented the scar formation, well observed two months after SC injury in the control rats (A) and resulted in the regeneration of nerve fibers growing through the trauma region (B, C). Histochemical method on detection of Ca2+-dependent acidic phosphatase activity.
Figure 3.
PRP-1-Ir structures in SC of injured rats treated with NOX venom. NOX increased the number of PRP-1-Ir nerve fibers (A) and astrocytes (B) in the SC lesion region and promoted the survival of the PRP-1-Ir motoneurons (C). In the boxed area the PRP-1-Ir astrocytes are seen migrating toward the injured side. ABC immunohistochemical method.
Different forms of injury and types of stress induce different morphological responses. For example, 5-hour IMO stress induces deeper neurodegenerative changes, which is manifested by the histochemical method on detection of Ca2+-dependent acid phosphatase activity. The final product – phosphate precipitate of various sizes and shapes was localized in both the neurons and the extracellular region (Figure 4).
Figure 4.
Degenerative nerve structures in the hypothalamic SON and PVN of rats exposed to 5 h IMO stress. In the hypothalamic SON and PVN, the central chromatolysis and ectopied negative nuclei are revealed in the hypertrophied cells with no processes. High phosphatase activity is seen like a thich ring under the cellular membrane and extracellular area. Histochemical method on detection of Ca2+-dependent acidic phosphatase activity.
In the next neurodegenerative model, the vestibular nuclei injury caused by vibration and unilateral labyrinthectomy, in various brain regions of rats, including hypothalamus, hippocampus, brain stem (locus coureleus, nucleus hyppoglosus), cerebellum, the neurodegeneration was revealed by the presence of the hypertrophied cells situated in the tissue edema [11]. Single PRP-1 administration triggered regeneration and survival of neurons in the same brain regions. Interestingly, obvious regeneration of the structures is revealed in labyrinthectomized rats exposed to the 2 h daily vibration for 2 weeks.
Apart from this, in the distinct stress-related brain regions of labyrinthectomized rats, neurons, demonstrating PRP-1-immunoreactivity, (PRP-1-IR) were revealed in the cell nuclei, as opposed to the PRP-1-IR in the cytoplasm of the intact cells (Figure 5).
Figure 5.
PRP-1-Ir neural structures in the (A, B) supraoptic nucleus (SON), (C, D) locus coureleus (LC) and (E, F) nucleus Hyppoglosus (n.Hyp.) of intact and labyrintectomized rats brain. In the distinct stress-related brain regions of labyrintectomized rats, the stress-induced activation of neurons was found out by detection of the increased number of cell nuclei demonstrating PRP-1-IR and immediated early gene c-fos-IR. ABC immunohistochemical method.
According to the literature, the nuclear localization of the proteins such as c-Fos, c-Jun, as well as the so-called Heat Shock factors with a molecular weight of 1–70 kD was detected in rats under stressful conditions (exposure to radiation, temperature, chemicals, etc.) [12, 13].
Using the immediate early gene c-fos-antibody, the stress-induced activation of neurons was immunohistochemically demonstrated by detection of the c-fos-immunoreactive (c-fos-Ir) nuclei in the distinct stress-related brain regions of labyrinthectomized rats. We assume that PRP-1 can control the DNA transcription and can function as a transcription factor similar to c-fos [14].
In the same stress-related brain regions of rats, high phosphatase (APh) activity was also detected in cellular nuclei in 15 minutes after stress, earlier than gene c-fos, which can be explained by the activation of cellular activators like c-fos through their phosphorylation (Figure 6).
Figure 6.
PRP-1-IR, transcription factor early gene c-fos-IR (c-fos-IR) and Acid Phospatase (APh) activity in the cortex of labyrintectomized rats. Nuclei of pyramidal cells in the brain cortex of labyrintectomized rats, demonstrated (A) PRP-1- and (B) c-fos-IR, as well as (C) high APh activity. Detection of APh activity in the injured cells nuclei can be explained by activating of cellular activators like c-fos through their phosphorylation. ABC immunohistochemical method and histochemical method on detection of Ca2+-dependent acidic phosphatase activity.
Our results obtained in the mentioned models of the central nervous system injury indicate that PRP-1 therapy may protect against the neurodegeneration by enhancing the survival of the damaged neurons.
3. PRP-1 participation in the generating of new neurons
Adult brain cell regeneration, also known as neurogenesis, demonstrated in many species, including rodents, is the process of generating new neurons, [15].
Today, there is scientific evidence of the stem cells presence in many more tissues and organs. One of their characteristics is ability to self-renew and to differentiate, to secure primary steady state functioning of a cell, called homeostasis, and, with limitations, to replace cells that die because of injury or disease [16, 17]. The list of adult tissues reported to contain stem cells is growing and includes bone marrow, peripheral blood, brain, spinal cord, muscle, and other tissues. Multipotent adult progenitor cells (MAPCs), derived from pluripotent mesenchymal stem cells, purified and isolated by Jiang et al. [18], could differentiate both into mesenchymal and neural cells.
Our recent histochemical and immunohistochemical studies in newborn rats exposed to the acute prenatal immobilization (psychogenic) stress brought new information about PRP-1 participation in the brain recovery process through generating new neurons [19].
To differentiate PRP-1-immunoreactive (PRP-1-Ir) glial cells and small undifferentiated cells found in the injured brain, we used antibodies against the astrocyte marker GFAP, neuroepithelial stem cells marker gene nestin [20, 21], and mouse stem cells. Considering the obtained results regarding the detection of GFAP-, nestin-, and PRP-1-immunoreactive radial astrocytes and cell structures of different sizes and forms, we suggest that they could be the intermediate neural progenitor cells, and that PRP-1 could participate in the generation of new functional neurons following the injury (Figure 7).
Figure 7.
GFAP-, Nestin- and PRP-1-Immunoreactive structures in the brain of 45-days aged rats exposed to prenatal IMO stress. Using markers against the neural progenitor cells, GFAP- and nestin-Ir radial astrocytes (A, B, D), nestin- and PRP-1-Ir cell structures of different sizes and forms (C, F), as well as PRP-1-Ir varicose nerve fibers and varicosities (E) were detected in the distinct stress-related brain regions. Taking into account the results obtained, we suggest that they could be the intermediate neural progenitor cells and that PRP-1 could participate in the generation of new functional neurons following the injury. ABC immunohistochemical method.
We succeeded also in detecting the cellular structures resembling the mesenchymal cells in both bone marrow and different stress-related brain regions of the immobilized rats some of which expressed immunoreactivity against MSCs, nestin and PRP-1 (Figure 8).
Figure 8.
Cellular structures resembling mesenchymal cells in the bone marrow (BM) and brain of the immobilized rats. (A-C) numerous round in shape and fusiform small cells with short axon-like extensions, being in the various proliferative stages (arrows), are visible in the BM stroma (B). (D) small cells (arrow heads) and (E, F) dark-colored fusiform cells with processes are detected in the cerebellum (G-I). Using antibodies against the mouse stem cells (MSCs), nestin and PRP-1, in the spinal cord of the injured rats, (G) MSCs-Ir, (H) PRP-1-Ir and (I) nestin-Ir cellular structures are revealed. Histochemical method on detection of Ca+2-dependant acid phosphatase activity (A-E) and ABC Immunohistochemical method (G-I).
In addition to the established functions of PRP-1 for cell survival and neurogenesis, using the PRP-1-antiserum, as well as antiserum against the synaptophysin (presynaptic vesicle protein, marker for the functional synapsis), we also suggested that PRP-1 could mediate higher brain activity through the formation of new synapses, thus, increasing the number of connections between the neurons (Figure 9).
Figure 9.
Synaptophysine-Ir (Syn-Ir) and PRP-1-Ir varicose nerve fibers and varicosities in the brain of 75-days aged rats exposed to prenatal IMO stress and injected with PRP-1. Syn-Ir (A-C) and PRP-1-Ir (D-F) nerve fibers and varicosities, possibly synapses, scattered in the brain, are well demonstrated. ABC immunohistochemical method.
We suggest that the process of generating new neurons occurs in injured brain, most probably proceeding from BM-derived cells that migrate into the brain and express neuronal marker genes, or from the neural stem/progenitor cells (NPCs) in non-neurogenic regions giving rise to the neurons mediated by the local astrocyte populations.
4. Protective effect of PRP-1 on the immune system cells death
Histochemical and immunohistochemical studies were also carried out to investigate the morpho-functional states of bone marrow (BM) structures of intact rats, rats injected with PRP-1, and rats exposed to immobilization stress and to bilateral electro-stimulation of the hypothalamic paraventricular nucleus [11].
The increased number of PRP-1-Ir blood-forming cells were observed in BM stroma and sinusoidal capillaries after PRP-1-antiserum was applied (Figure 10).
Figure 10.
Hematopoiesis in the bone marrow of rats injected with PRP-1. (A) sinusoidal capillary in the intact rat BM appeared to be empty, in general (black asterisk). Increased number of immune system cells both in sinusoid (asterisk) (B) and stroma (B-D) are well seen in the BM of injected with PRP-1 rats. (B) in the sinusoid, a megakaryocyte (arrow) with the homogenously and densely stained nuclei is demonstrated in the stage of platelets release. Histochemical method on detection of Ca2+-dependent acidic phosphatase activity.
In addition, PRP-1-Ir varicose nerve fibers and islands of PRP-1-Ir immune system cells were detected in the surrounding areas of sinusoids (Figure 11).
Figure 11.
PRP-1-Ir structures in bone marrow of the immobilized rats. The increased number of PRP-1-Ir blood-formed cells is detected in BM sinusoidal capillaries and stroma. Among these cells, islands of PRP-1-Ir immune system cells (white asterisks) were found inside and around the sinusoids (black asterisks). A PRP-1-Ir capillary (arrow) (A) and a few single PRP-1-Ir varicose nerve fibers (arrows) (C) are seen. (D): fragment of 10C. ABC immunohistochemical method.
The possibility that the PRP-1 synthesis takes place in the immune cells exists due to the evidence of distinct neuropeptides biosynthesis in the lymphocytes. Based on this, we conducted in vitro experiments in intact lymphocytes isolated from the rat bone marrow. By using PRP-1-antiserum and the flow cytofluorimetric analysis, about 4–5% PRP-1 was detected in the lymphocytes in the presence of some activators, such as phytohemagglutinin (FGA), phorbol miristil acetate (FMA), and concanavaline A (ConA), compared to near 0% in the intact immune cells. Data obtained indicate the possible synthesis of PRP-1 in the immune system cells in vivo.
Thus, adult stem cells plasticity in response to the immobilization stress is assumed in some of the studied brain regions. However, whether these cells are indeed bone marrow-derived stem cells circulating in the blood is a question to be answered. In regards to the PRP-1-Ir migrating cells from the SC central canal, we assumed that they could be the SC stem cell-derived structures.
5. Effect of PRP-1 on inflammation
The inflammation, a physiological response to a variety of tissue damages, is made up of multiple related chains of cellular and chemical reactions. This cascade of responses activates the localized production of cytokines implicated in the cell recruitment and differentiation through specific gene expression. Though the processes behind the acute neuroinflammation following trauma or stroke may worsen the initial lesion through the increased neuronal loss, they stimulate the subsequent functional recovery through promoting neuronal plasticity. However, the consequences of chronic neuroinflammation are suspected to include neuronal loss in pathological conditions including neurodegenerative and autoimmune disorders and diseases [22].
Inflammation of the brain is linked to the biosynthesis and secretion of several neuroactive molecules, such as oxygen and nitrogen free radicals, cytokines, excitatory amino acids, proteases, complement proteins, and others, by the activated glial cells [23]. Though chronic, unregulated, and ongoing inflammation is highly prejudicial, inflammation is generally a beneficial process for the organisms due to its role in containing and curbing the survival and the spread of pathogens, as well as energy conservation and tissue recovery [24].
Beta Amyloid peptide (Aβ) (1–42) by its nature is neurotoxic and is linked to dysregulation of the brain function during Alzheimer’s disease (AD) and, therefore, is strongly associated with the brain function loss through the course of AD. The accumulation of Beta Amyloid triggers the induction of neuronal cytotoxic pathways, involving microglia induced activation of pro-inflammatory cytokines IL-β and TNF-α and formation of free radicals [25, 26].
TNF-α can promote tumorigenesis leading to prostate and other cancers, and initiate apoptotic cell death [27]. Inflammatory processes are correlated with the neuronal apoptosis found in neurodegenerative diseases. Whether apoptosis plays an overall beneficial or detrimental role in neuroinflammation is unclear and topic remains controversial.
PRP-1 has roles as a caspases-2 and -6 activator [28], immunocompetent cells (Т and В lymphocytes and macrophages) stimulator [8, 9], and pro-apoptotic caspases-3 and -9 inhibitor. Furthermore, it is a tumor necrosis factor alfa (TNFα) and interleukins (IL-1, IL-6) inductor in lymphocytes, astrocytes, macrophages, and fibroblasts.
PRP-1 exhibits regulatory effect on myelo- and lymphopoiesis [29, 30, 31] and neuroprotection, countering multiple toxic endogen agents [32, 33], as well as demonstrates strong neurotrophic effect on glial fibrillary acidic protein (GFAP) biosynthesis in astrocytes [34].
PRP-1’s influence on staurosporine-promoted apoptosis of postnatal hippocampal cells as well as on doxorubicin-induced bone marrow mono/granulocyte apoptosis was investigated [35]. We characterized PRP-1’s activity on the neuron survival rate (in a myelopoiesis context) by demonstrating the significant reduction of staurosporine-induced apoptosis of postnatal hippocampal cells from PRP-1 treatment. PRP-1’s protective function against apoptosis was shown to be both dose- and time- dependent. Prolonged PRP-1 treatments showed more pronounced neuroprotection against staurosporine-induced apoptosis. A similar significant reduction was seen in bone marrow monocyte and granulocyte apoptosis by doxorubicin. The neuroprotective effect lasted for 2–4 hours and was no longer effective at 24 h when doxorubicin and PRP-1 were simultaneously added. In conclusion, the endogenous peptide PRP-1 has the primary functions of regulating myelopoiesis and neuroprotection.
Multiple experiments were carried out to understand the effect of PRP-1 on the proliferation and the colony formation of multipotent mesenchymal stromal cells (MMSCs). The dose response effect of PRP administration to rats was observed with the increased number of MMSCs in bone marrow and spleen. In ex vivo condition, the addition of PRP into the culture medium led to up to 2.5-fold increase by stimulation.
On the contrary, the proliferation was inhibited 1.5 to 2-fold in the cultures of giant-cell tumor (GCT) stromal cells, when the same PRP concentrations and cultivation periods were used. PRP-1 demonstrated also the opposite effects on the proliferation of the human bone marrow stromal cells obtained from normal humans, and the stromal cells isolated from human GCT [36].
6. Effect of PRP-1 on cell death in the cancer diseases
Other results demonstrating antitumor activity of PRP1 followed, opening up new perspective of PRP-1 antitumorigenic activity. PRP-1 induced the decay of tumor cells L929 and decreased the mitotic activity of transformed mouse fibroblast cells [37, 38, 39, 40]. The peptide caused shrinkage of tumor in sarcoma C45 after subcutaneous injection [39].
These experimental results served as predecessors of intensive studies on other musculoskeletal malignancies, particularly chondrosarcoma. Chondrosarcoma is the second most common bone malignancy, which primarily affects the cartilage cells of the femur (thighbone), arm, pelvis, knee, and spine and even larynx, head, and neck. Chondrosarcoma is a rare disease and it does not appear to respond to either chemotherapy or radiation.
Surgical resection is the only option for the treatment, although metastatic spread to lungs occurs, eventually leading to dismal prognosis as these tumors are highly aggressive. Therefore, the search for new therapies is extremely important and urgent.
To date, only a few drugs have been identified that have been successfully shown to have clinical efficacy through the inactivation of a specific oncogene phenotype [41]. Inactivation of a single oncogene can induce cancer cells to differentiate into cells with a normal phenotype, or to undergo cellular senescence and/or apoptosis [42]. Upon MYC inactivation [43], tumors variously undergo proliferative arrest, cellular differentiation, and apoptosis.
In our most early publications, we provided experimental data indicating that PRP-1 caused inactivation of cMyc oncogene in human chondrosarcoma cells, prompting us to further investigate this peptide antitumorigenic potential [44].
The cytostatic effect of PRP-1 in human chondrosarcoma JJ012 cell line was demonstrated as 80% inhibition of cell proliferation on PRP-1 treatment in comparison with the nontreated cells. Interestingly, PRP-1 did not have any effect on immortalized chondrocytes culture, which spoke to the fact that PRP-1 selectively targeted malignant sarcoma cells and not the benign cells. Caspase-3 activity was not affected and no apoptosis was detected, thus the inhibition was due to cytostatic and not cytotoxic effect [45].
The mammalian target of rapamycin (mTORC) is an intracellular serine/threonine protein kinase, which is linked to cell growth because of its important role in nutrient signaling processes. PRP-1 was revealed as mTORC1 inhibitor, as it was able to inhibit this kinase activity in statistically significant manner [46].
The fact that the concentration lower than 10 μg/ml peptide with cytostatic effect did not inhibit mTORC1 but inhibited its target cMyc prompted us to assume the possibility of PRP-1 binding to two different receptors, facilitating the antiproliferative effect.
The cytostatic, antiproliferative action of PRP-1 was also demonstrated in the triple negative breast carcinoma MDA MB 231 cell lines [47] and the cell cycle experiments pointed on obvious stall in S phase, delaying the progression to the next stage of cell cycle upon PRP-1 treatment.
Through our study, we sought to ascertain the condition of JJ012 human malignant chondrosarcoma cells’ expression of intercellular junction proteins, as well as determine the effect of the antitumorigenic cytokine PRP-1 on their expression. The experimental data suggested that tumor suppressor desmosomal proteins expression in JJ012 chondrosarcoma cells is restored and H3K9 demethylase activity comprised of a pool of JMJD1 and JMJD2 is inhibited by PRP-1, suppressing the tumorigenic potential of chondrosarcoma cells [48].
Identifying the PRP-1’s receptor was very important to discern the mechanism behind its action. G protein coupled receptors (GPCR) and nuclear pathway receptor assays determined that PRP-1 receptors do not belong to nuclear or orphan receptor families, and neither were they G protein coupled. We have demonstrated in our study that the interacting partners of PRP-1 binding belong to the gel forming secreted mucin MUC5B, as well as to the innate immunity pattern recognition toll-like receptors TLR1/2 and TLR6. The experimental data indicated that the aforementioned receptors had tumor suppressive function in this cellular context [49].
When examined, the microRNA expression profiles specific to tumors showed that, throughout diverse cancers, there was widespread deregulation of these molecules. MicroRNAs have been reported to have the potential to function in disease diagnostics and therapy as novel biomarkers, as well as a novel class of tumor suppressor and oncogenes. Tumor suppressors, such as miR20a, miR125b, and miR192, were significantly upregulated by mTORC-1 inhibitor PRP-1 while onco-miRNAs, miR509-3p, miR589, miR490-3p, and miR550 were downregulated in the human chondrosarcoma JJ012 cell line [50]. The fact that PRP-1 manifests itself as a powerful epigenetic regulator was confirmed with the experiments demonstrating inhibition of BAFF Chromatin remodeling complexes [51].
Experimental results indicated that among the miRNA significantly downregulated by PRP-1 treatment was miRNA 302c. miRNA 302c is a part of the embryonic human stem cell stemness regulator cluster miR302367. miR302367 is expressed in embryonic stem cells, as well as in certain tumors, but its expression is not found in normal tissue or in adult HMSCs [52]. PRP-1 had a strong effect on chondrosarcoma and multilineage induced multipotent adult cells (embryonic primitive cell type) viability by inhibiting their proliferation. However, PRP-1 did not have any cell proliferation inhibitory action on glioblastoma, because the miR-302-367 cluster in glioblastoma exhibits an opposite effect and its expression is enough to inhibit the stemness inducing properties. The antiproliferative activity of PRP-1 and its action on downregulation of miR302c has an observed correlation that explains the peptide’s opposite effects on the downregulation of miR302c targets, the stemness markers Nanog, c-Myc, and polycomb protein Bmi-1 [52].
We concluded that the inhibition of H3K9 demethylase activity by PRP-1 leads to downregulation of miR302c and its targets, defining the antiproliferative role of PRP-1.
Effects of PRP-1 on a 3D chondrosarcoma tumor model in vitro (known as spheroids) and on the cancer stem cells (CSCs) that form the spheroids, was evaluated in another study [53]. Spheroid formation and colony formation assays of cell fractions (including CSCs) were used in comparing PRP-1 treated groups with the controls. The CSCs were assessed with a modified Annexin V/propidium iodide assay for early apoptosis and cell death. Western blotting confirmed mesenchymal marker expression, and the spheroid self-renewal assay demonstrated the presence of the self-renewing CSCs. The study’s results determined that PRP-1 eliminates spheroid formation and independent CSC growth, indicating the PRP-1 potential to inhibit tumor formation in a murine model. Another indication of an advantageous decline in tumor stromal cells is the decrease in non-CSC bulk tumor cells. These findings lead us to conclude that PRP-1 inhibits CSC proliferation in 3D tumor models that mimic the behavior of in vivo chondrosarcoma.
Although the cytostatic effect of PRP-1 has been demonstrated in various tumors we studied, the potential of PRP-1-related apoptosis in other types of cancer has not been ruled out.
PRP-1 action is cellular and disease context dependent. Morpho-functional study on the effect of PRP-1 on a mouse Ehrlich ascites carcinoma (EAC) model was conducted [54]. The number of viable cells in the suspension was determined by the histological method of exclusion with trypan blue (diazo live dye). The percentage of dead and alive cells was calculated after 24 h of incubation in the control samples and in those treated with PRP-1 at 0.1 and 1 μg/ml concentrations. The effect of PRP-1 on the number of tumor cells incubated for 24 h and their viability led to a 44% reduction in the number of viable cells on day 11 post-inoculation, vs. the 22% inhibition of viable cells after PRP-1 treatment (0.1 μg/ml) on day 7 post-inoculation (Figures 12 and 13).
Figure 12.
Effect of the hypothalamic PRP-1 on the growth and viability of mouse isolated EAC cells on the 7th day of tumor growth. By the histological method with Tr-Bl staining, viable EAC cells were revealed in the control samples before their culture (control). Few number of dead Tr-Bl-positive tumor cells were detected among the viable cells in the non-treated control samples 24 h after culture (control 24 h). An increased number of Tr-Bl-positive dead cells was evident 24 h after 0.1 and 1 μg/ml PRP-1 administration. The PRP-1 (0.1 and 1 μg/ml) inhibitory effect on the number of (B) total and (C) viable tumor cells treated for 24 h was statistically (***P<0.001) significant, difference compared to the control at 24 h. Histological method with Tr-Bl staining.
Figure 13.
Effect of the hypothalamic PRP-1 on the growth and viability of mouse isolated EAC cells on the 11th day of tumor growth. (A) Histological method with Tr-Bl staining detected viable EAC cells before their culture (control). EAC control cells after 24 h of incubation were mainly viable, although several dead Tr-Bl-positive cells were present (control 24 h). In samples treated with 0.1 and 1 μg/ml PRP-1, an increased number of Tr-Bl-positive non-viable cells was detected; along with various viable cells (arrows), apoptotic cells with fragmented nuclei (double arrows), as well as various Tr-Bl-positive cells surrounded by apoptotic bodies (arrow heads) were observed. The PRP-1 (0.1 and 1 μg/ml) inhibitory effect on the number of (B) total and (C) viable tumor cells treated for 24 h was statistically (***P<0.001) significant, difference compared to the control at 24 h. Histological method with TR-Bl staining.
Based on the PRP-1-induced morphological features of EAC cells, the apoptotic nature of PRP-1 was confirmed histologically as manifested by cell shrinkage, membrane blebbing, chromosome condensation (pyknosis), and nuclear fragmentation (karyorrhexis) (Figures 14 and 15).
Figure 14.
Histological evaluation of the hypothalamic PRP-1 effect on mouse-isolated EAC cells on day 7 of tumor growth. Morphological changes of tumor cells (A) 24 and (B) 72 h after culture. (A) In control samples, numerous EAC cells linked with each other were detected at 24 h, whereas a decreased number of cells was observed with both doses of PRP-1. In the experimental samples, PRP-1-induced morphological changes were similar for both the two time-points of culture. Apoptotic membrane blebbing and apoptotic bodies (arrowheads), smaller and round-shaped cells with eosinophilic cytoplasm and condensed nuclei (pyknosis), and loss of reticular extensions and contacts with adjacent cells were observed. (B) Necrotic EAC cells containing no nuclei (karyolysis) or cells with lost membrane integrity (arrows) were mainly presented in the control samples after 72 h of culture, whereas few necrotic cells with lost plasma membrane integrity and released cell death products (arrows) were detected in the samples treated with PRP-1 for 72 h. Statistical data regarding the PRP-1 (0.1 and 1 μg/ml) effect on the apoptosis and necrosis in tumor cells treated for (C) 24 h and (D) 72 h were presented according to the H&E exclusion test in comparison with the findings in the untreated control cells. Data are presented as the mean ± standard deviation (n = 3), and represent ≥3 independent experiments. **P<0.01; ***P<0.001, significant difference compared to (C) the control at 24 h and (D) the control at 72 h. Histological method with H&E staining.
Figure 15.
Histological evaluation of the hypothalamic PRP-1 effect on mouse-isolated EAC cells on the 11th day of tumor growth by H&E staining. Morphological changes of tumor cells at (A) 24 and (B) 72 h after culture. In the untreated control samples, tumor cells (arrows) with typical morphology were observed after both culture time-points. In comparison to cells in the control group, cells exposed to 0.1 and 1 μg/ml PRP-1 were smaller in size and exhibited a round shape. EAC cells with the apoptotic bodies (arrowheads) were observed having no contact to adjacent cells. Statistical data regarding the PRP-1 (0.1 and 1 μg/ml) effect on the apoptosis and necrosis in tumor cells treated for (C) 24 h and (D) 72 h was presented according to the H&E exclusion test in comparison with the findings in untreated control cells. Data are presented as the mean ± standard deviation (n=3), and represent ≥3 independent experiments. **P<0.01; ***P<0.001, significant difference compared to (C) the control at 24 h and (D) the control at 72 h. Histological method with H&E staining.
To verify this observation, a series of experiments were performed, which were focused on the determination of apoptosis in cultured tumor cells using an Annexin V-Cy3 apoptosis detection kit and fluorescence microscopy. The analysis of the apoptosis on the 7-day inoculated mice EAC-cultured cells exposed to 0.1 μg/ml PRP-1 for 24 h revealed a significant increase in the number of apoptotic cells, reaching 50.33%, compared to 8.33% in the control sample on day 7. Besides, early apoptotic cells, as well as late apoptotic cells, containing and surrounded by fragments of necrotic nuclei were also detected, in contrast to the numerous viable tumor cells detected in the untreated control samples.
In late apoptotic cells, the apoptotic bodies undergo secondary necrotic changes and turn to detritus, known as a secondary form of necrosis mainly
Figure 16.
Analysis of apoptosis/necrosis in cultured mouse EAC cells exposed to the hypothalamic PRP-1 on the 7th (A, B) and 11th days (C, D) of tumor growth according to fluorescence detection with Annexin V-cyanine 3. (A, C) viable EAC cultured cells (green) were detected 24 h after growing in the control untreated samples. In contrast to control samples, on the 7th day of tumor growth (B) an increased number of early apoptotic cells (orange) was revealed 24 h after treatment with 0.1 μg/ml PRP-1. Fragments of necrotic nuclei (red) were clearly detected in late apoptotic cells. (D) on the 11th day of tumor growth after treatment with 0.1 μg/ml PRP 1, the plasma membrane and certain weakly stained intracellular components could be indicative of early-stage apoptosis. Fluorescent method with Annexin V-Cy3 staining.
In addition, a series of experiments aimed at elucidating the possible participation of PRP-1 in antitumorigenic processes was carried out by detecting the immunohistochemical localization of PRP-1 in the control and experimental EAC cells (Figures 17 and 18).
Figure 17.
Immunohistochemical localization of the hypothalamic PRP-1 in cultured mouse EAC cells on the 7th day of tumor growth. (A) All microimages demonstrated no PRP-1-IR in EAC cells before culture (control). PRP-1-IR in tumor cells (B) at 24 h and (C) 72 h after culture. (B) No intracellular PRP-1-IR was detected in control EAC cells after 24 h of culture, whereas the plasma membrane exhibited weak PRP-1-IR in the form of a narrow ring (arrows). In experimental samples, the sub-membrane cytoplasm with dense PRP-1-IR was detected in the tumor cells (arrows) exposed to 0.1 μg/ml PRP-1. (C) After 72 h of culture, nuclear localization of PRP-1 was detected in certain control (arrows) and PRP-1 treated (not shown) tumor cells. PRP-1-Ir cytoplasm was released from necrotic control cells with lost membrane integrity (double arrows). The strong PRP-1-Ir cytoplasm was revealed both in control (not demonstrated) and exposed to PRP-1 EAC cells. Notably, the apoptotic cells with the apoptotic bodies (arrowheads) also demonstrated strong PRP-1-Ir cytoplasm. ABC immunohistochemical method.
Figure 18.
Immunohistochemical localization of the hypothalamic PRP-1 in cultured mouse EAC cells on the 11th day of tumor growth according to the ABC immunohisto-chemical method. (A) EAC control cells before culture, where PRP-1 was localized in the cell membrane, cytoplasm (arrows) and nucleoli (double arrows) of certain tumor cells. PRP-1-IR in the tumor cells (B) 24 h and (C) 72 h after their culture. (B) In the untreated control samples at 24 h, weak PRP-1-IR was mainly observed in the perinuclear zone of cell cytoplasm. In the experimental samples exposed to 0.1 μg/ml PRP-1 for 24 h, PRP-1-IR was observed in the cell nucleoli (double arrows). (C) At 72 h after EAC cell culture, dense cytoplasmatic IR for PRP-1 was detected in tumor cells both in the control and PRP-1 treated samples. Morphological changes of the cells undergoing death-related processes (apoptosis and necrosis) were clearly observed, including release of PRP-1-Ir intracellular contents from necrotic cells into the extracellular space, which was detected predominantly in the control samples (arrows), while PRP-1-Ir plasma blebs and apoptotic bodies (arrowheads) were revealed mainly in the experimental samples. ABC immunohistochemical method.
In the mice of control non-cultured EAC cells, PRP-1-IR was not detected on the seventh day of tumor growth. No intracellular PRP-1-IR was detected in the untreated control EAC cells cultured for 24 h however the presence of PRP-1-Ir cell membrane (21%) in the shape of narrow ring was registered. In the samples with 0.1 μg/ml PRP-1 treatment, strong PRP-1-IR was observed in the cytoplasm (46.5%) and nucleoli (10%).
However, the number of EAC cells with cytoplasmic PRP-1-IR of inoculated for 11 days mice and cultured for 72 h in the presence of PRP-1, constituted 73.4%, in contrast to 32.6% of the control cells with cytoplasmic PRP-1-IR. Notably, dense PRP-1-IR was noticed in 25% of apoptotic cells with membrane blebbing, in contrast to 4% of the untreated control cells.
Fluorescent nuclear staining of DAPI nevertheless proved statistically insignificant nuclear localization of PRP-1 in both control and PRP-1-treated samples after 72 h of incubation (4% of the total number of tumor cells).
Thus, the findings provide evidence that the effect of PRP-1 is cellular context dependent in EAC cells, with PRP-1 acting as a cytotoxic agent by inducing programmed cell death type I apoptosis. With regard to the detection of PRP-1-IR in the nucleus and cytoplasm of apoptotic EAC cells cultured for 72 h in both control (untreated) and experimental (PRP-1 treated) samples, the possible biosynthesis of the endogenous PRP-1 in the studied cancer cells should be taken into account.
7. PRP-1 as a circulating biomarker
Today, much attention is paid to the discovery of circulating biomarkers in the blood serum of patients with different disorders.
Previously, we succeeded in the detection and quantification of PRP-1 in rat blood serum by an enzyme linked immunosorbent assay (ELISA) developed for PRP-1 [56]. The minimum detectable concentration of PRP-1 in the intact rat blood serum has been shown to be approximately 1.78 ng/ml. Furthermore, the effect of the exogenous PRP-1 on the endogenous PRP-1 concentration was identified in the blood after 5 h and 2 days of its administration.
The significant increase of PRP-1 concentration observed in the blood samples in 5 h after the PRP-1 intraperitoneal injection was decreased in the 2-day post-injection period to approximately the control level.
Based on the recent data [57] pointing to PRP-1 being a new natural substrate for the multifunctional dipeptidyl protease (DPP-IV) that hydrolyses the peptide bonds formed by the proline residues, the decrease in the peptide concentration could be explained by the proteolytic processing of PRP-1 by DPP-IV.
The results serve as a basis for suggesting the involvement of different factors (neuropeptides, enzymes, neurotransmitters, etc.) in the mechanism of the PRP-1 action, and justify the need for additional studies for demonstrating the potential role of PRP-1 in the stress-induced disorders obtained on the animal models and in the pathogenesis of various human diseases.
References
- 1.
Galoyan A. New polypeptides of hypothalamus: Isolation and primary structure. Proceeding NAS RA. 1996; 96 :555-570. DOI: 10.54503/0321-1339 - 2.
Galoyan AA. Biochemistry of Novel Cardioactive Hormones and Immunomodulators of the Functional System Neurosecretory Hypothalamus–Endocrine Heart. Design, Nauka Publishers: Moscow; 1997. p. 240 ISBN: 5-02-022509-6 - 3.
Galoyan A, Sarkissian J, Kipriyan T, Sarkissian E, Sulkhanyan R, Grigorian Y, et al. Comparison of the protection against neuronal injury by hypothalamic peptides and by dexamethasone. Neurochemical Research. 2000; 25 (12):1567-1578. DOI: 10.1023/A:1026662318816 - 4.
Galoyan A, Sarkissian J, Kipriyan T, Sarkissian E, Chavushyan E, Sulkhanyan R, et al. Protective effects of proline-rich peptides against cobra venom and trauma induced neuronal injury. Neurochemical Research. 2001; 26 :1023-1038. DOI: 10.1023/A:1012353005489 - 5.
Abrahamyan S, Meliksetyan I, Galoyan A. Immunohistochemical analysis of the brain immunomodulators (proline rich polypeptide and immunophilin) in the normal and pathological conditions. In: Proceedings in the International Conference “Biochemical and Mollecular-Biological Aspects of the Brain Immune System”; Yerevan. Tsakhkadzor. RA. September 15-19 2001. pp. 62-73 - 6.
Abrahamyan S, Sarkissian J, Meliksetyan I, Galoyan A. Survival of trauma-injured neurons in rat brain by treatment with proline-rich peptide (PRP-1): An immunohistochemical study. Neurochemical Research. 2004; 29 (4):695-708. DOI: 10.1023/B:NERE.0000018840.19073.0b - 7.
Abrahamyan S, Meliksetyan I, Chavushyan V, Aloyan M, Sarkissian J. Protective action of snake venom Naja Naja Oxiana at spinal cord hemisection. Clinical Neuroscience. 2007; 60 (3-4):148-153 - 8.
Aprikyan V, Galoyan A. Antibacterial activity of a new hypothalamic peptide. Proceeding NAS RA. 1999; 99 :367-371 - 9.
Galoyan A, Aprikyan V. A new hypothalamic polypeptide is a regulator of myelopoiesis. Neurochemical Research. 2002; 27 (4):305-312. DOI: 10.1023/A:1014959212651 - 10.
Abrahamyan S, Sahakyan I, Meliksetyan I, Tumasyan N, Badalyan B, Galoyan A. Histochemical and immunohistochemical study of morpho-functional state of brain and bone marrow cell structures of rats under stress. The New Armenian Medical Journal. 2011; 5 (2):60-68 - 11.
Tumasyan N, Sahakyan I, Kocharyan N, Abrahamyan S. Effect of PRP-1 on the morpho-functional state of brain cellular structures of rats with various neurodegeneration. Journal “Asymmetria”. 2018; 12 (4):509-518 (In russian) - 12.
Palkovits M, Deli M, Gallatz K, Tóth Z, Buzás E, Falus A. Highly activated c-fos expression in specific brain regions (ependyma, circumventricular organs, choroid plexus) of histidine decarboxylase deficient mice in response to formalin-induced acute pain. Neuropharmacology. 2007; 53 (1):101-112. DOI: 10.1016/j.neuropharm.2007.04.001 - 13.
Zhou H, Gao J, Lu Z, Lu L, Dai W, Xu M. Role of c-Fos/JunD in protecting stress-induced cell death. Cell Proliferation. 2007; 40 (3):431-444. DOI: 10.1111/j.1365-2184.2007.00444.x - 14.
Abrahamyan S, Sahakyan I, Meliksetyan I, Tumasyan N, Badalyan B, Galoyan A. Histochemical study of the brain plasticity in the condition of the vestibular system neuronal disorder and the effect of hypothalamic proline-rich-polypeptide (PRP-1). Materials of the Russian Federation Conference with International Participation dedicated to “Functional interhemispheric asymmetry and brain plasticity”; Moscow. December 13-14 2012. pp. 223-227. (In russian) - 15.
Aimone JB, Jessberger S, Gage FH. Adult neurogenesis. Journal of Scholarpedia. 2007; 2 (2):2100 - 16.
Holtzer H. Cell lineages, stem cells and the ‘quantal’ cell cycle concept. In: Stem Cells and Tissue Homeostasis. Lord BI, Potten CS, Cole RJ. editor. Cambridge, New York: Cambridge University Press; 1978. pp. 1-28. ISBN: 9780521217996 - 17.
Leblond C. Classification of cell populations on the basis of their proliferative behavior. National Cancer Institute Monograph. 1964; 14 :119-150. PMID: 14147128 - 18.
Jiang Y, Jahagirdar B, Reinhardt R, Schwartz R, Keene C, Ortiz-Gonzalez X, et al. Pluripotency of mesenchimal stem cells derived from adult marrow. Nature. 2008; 418 :41-49. DOI: 10.1038/nature00870 - 19.
Abrahamyan S, Meliksetyan I, Sahakyan I, Tumasyan N, Badalyan B, Galoyan A. Brain plasticity of exposed to prenatal immobilization stress rats. Biopolymers and Cell. 2011; 27 (5):339-343 - 20.
Lu P, Blesch A, Tuszynsky M. Induction of bone marrow stromal cells to neurons: Differentiation, transdifferentiation, or artefact? Journal of Neuroscience Research. 2004; 77 :174-191. DOI: 10.1002/jnr.20148 - 21.
Vogel W, Grunebach F, Messam C, Kanz L, Bruger W, Buhring H. Heterogeneity among human bone-marrow-derived mesenchymal stem cells and neural progenitor cells. Haematologica. 2003; 88 :126-133 PMID: 12604402 - 22.
Chauveau F, Boutin H, Van Camp N, Dollé F, Tavitian B. Nuclear imaging of neuroinflammation: A comprehensive review of [11C] PK11195 challengers. European Journal of Nuclear Medicine and Molecular Imaging. 2008; 35 :2304-2319. DOI: 10.1007/s00259-008-0908-9 - 23.
Gahtan E, Overmier B. Inflammatory pathogenesis in Alzheimer’s disease: Biological mechanisms and cognitive sequel. Neuroscience & Biobehavioral Reviews. 1999; 23 :615-633. DOI: 10.1016/s0149-7634(98)00058-x - 24.
Allan S, Rothwell N. Inflammation in central nervous system injury. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 2003; 358 :1669-1677. DOI: 10.1098/rstb.2003.1358 - 25.
Monsonego A, Imitola J, Zota V, Oida T, Weiner LL. Microglia-mediated nitric oxide cytotoxicity of T cells following amyloid β-peptide presentation to Th1 cells. Journal of Immunology. 2003; 171 :2216-2224. DOI: 10.4049/jimmunol.171.5.2216 - 26.
Royuela M, Rodriguete-Berriguete G, Fraile B, Paniagua R. TNF-α/IL-1/NF-κB transduction pathway in human cancer prostate. Histology and Histopathology. 2008; 23 :1279-1290. DOI: 10.14670/HH-23.1279 - 27.
Abdi A, Sadraie H, Dargahi L, Khalaj L, Ahmadiani A. Apoptosis inhibition can be threatening in Aβ-induced neuroinflammation, through promoting cell proliferation. Neurochemical Research. 2011; 36 :39-48. DOI: 10.1007/s11064-010-0259-3 - 28.
Galoyan A, Terio T, Berg M, Marks N. Effects of prolin-rich peptide derived from Neurophysin-II on caspases of murine neuroblastoma: Evidences for caspase-2 and -6 activation. Neurochemistry (RAS and NAS RA). 2000; 17 :185-188. DOI: 10.1007/s11064-005-2686-0 - 29.
Galoian K, Luo S, Qureshi A, Patel P, Price R, Morse A, et al. Proline rich polypeptide-1 and inflammatory pathway signaling in chondrosarcoma. Molecular and Clinical Oncology. 2016; 5 :618-624. DOI: 10.3892/mco.2016.1010 - 30.
Durgaryan A, Matevosyan M, Seferyan T, Sargsyan M, Grigoryan S, Galoian K, et al. The protective and immunomodulatory effects of hypothalamic proline-rich polypeptide galarmin against methicillin-resistant Staphylococcus aureus infection in mice. European Journal of Clinical Microbiology & Infectious Diseases. 2012; 3 :2153-2165. DOI: 10.1007/s10096-012-1550-9 - 31.
Galoyan A, Korochkin L, Rybalkina E, Pavlova G, Saburina I, Zaraiski E, et al. Hypothalamic proline-rich polypeptide enhances bone marrow colony-forming cell proliferation and stromal progenitor cell differentiation. Cell Transplantation. 2008; 17 :1061-1066. DOI: 10.3727/096368908786991579 - 32.
Galoyan K, Scully S, Galoyan A. Myc-oncogene inactivating effect by proline rich polypeptide (PRP-1) in chondrosarcoma. Neurochemical Research. 2009; 34 :379-385. DOI: 10.1007/s11064-008-9794-6 - 33.
Galoian K, Temple T, Galoyan A. Cytostatic effect of the hypothalamic cytokine PRP-1 is mediated by its inhibition of mTOR and cMyc in high grade metastatic chondrosarcoma. Neurochemical Research. 2011; 36 :812-818. DOI: 10.1007/s11064-011-0406-5 - 34.
Chekhonin V, Gurina O, Ruabukin I, Savchenko E, Galoyan A. Immunochemical study of the effect of neurotrophins on the GFAP synthesis in astrocyte culture. 2001. In: Proceed. of the Intern. Conf. on Bioch. and Molec. Biol. Aspects of the Brain Immune System; Yerevan-Tsakhkadzor. 15-19 September. 2001. pp. 140-145 - 35.
Galoyan A, Josef K, Klumpp S, Danielian K, Galoian K, Kremer W, et al. Effect of hypothalamic proline-rich peptide (PRP-1) on neuronal and bone marrow cell apoptosis. Neurochemical Research. 2007; 32 :898-1905. DOI: 10.1007/s11064-007-9379-9 - 36.
Chailakhyan R, Gerasimov Y, Chailakhyan M. Proline-rich hypothalamic polypeptide has opposite effects on the proliferation of human normal bone marrow stromal cells and human giant-cell tumour stromal cells. Neurochemical Research. 2010; 35 :934-939. DOI: 10.1007/s11064-009-0113-7 - 37.
Galoyan A, Shakhlamov V, Kondakova L, Altukhova V, Polyakova G. The influence of proline-rich polypeptide on the morphology and mitotic activity of neurinoma Hassel node of tumor cells at rats (electron microscopic investigations). PNAS (Russian Academy of Sciences). 2001; 101 (2):279-286 - 38.
Galoyan A, Shakhlamov V, Malaytsev V. Changes in tumor cells L929 under PRP effect in vitro. Medical Science of Armenia. 2001; 41 (1):25-29 - 39.
Simonyan G, Nersesyan A, Simonyan R, Babayan M, Simonyan M, Galoyan A. Antitumor and antistressor effects of hypothalamic proline rich peptide (PRP-1) in sarcoma 45; possible biochemical mechanisms. Neurochemistry (RAS and NAS RA). 2005; 22 (2):125-130 - 40.
Galoian K, Scully S, Galoyan A. Myc-oncogene inactivating effect by proline rich polypeptide (PRP-1) in chondrosarcoma JJ012 Cells. Neurochemical Research. 2009; 34 (2):379-385. DOI: 10.1007/s11064-008-9794-6 - 41.
Driker B, Talpaz M, Resta D, Peng B, Buchdunger E, Ford J, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. The New England Journal of Medicine. 2001; 344 :1031-1037. DOI: 10.1056/NEJM200104053441401 - 42.
Felsher D. Oncogene addiction versus oncogene amnesia: Perhaps more than just a bad habit? Cancer Research. 2008; 68 (9):3081-3086. DOI: 10.1158/0008-5472.CAN-07-5832 - 43.
Shachaf C, Felsher D. Tumor dormancy and Myc inactivation: Pushing cancer to the brink of normalcy. Cancer Research. 2005; 65 :4471-4474. DOI: 10.1158/0008-5472.CAN-05-1172 - 44.
Galoian K, Scully S, Galoyan A. Myc-oncogene inactivating effect of proline rich polypeptides (PRP-1) in the human chondrosarcoma JJ012 cells. Neurochemical Research. 2009; 34 (2):379-385. DOI: 10.1007/s11064-008-9794-6 - 45.
Galoian K, Scully S, Galoyan A. Antitumorigenic effect of brain proline rich polypeptide-1 in human chondrosarcoma. Neurochemical Research. 2009; 34 (12):2117-2121. DOI: 10.1007/s11064-009-0009-6 - 46.
Galoian K, Temple T, Galoyan A. Cytostatic effect of the hypothalamic cytokine PRP-1 is mediated by mTOR and cMyc inhibition in high grade chondrosarcoma. Neurochemical Research. 2011; 36 (5):812-818. DOI: 10.1007/s11064-011-0406-5 - 47.
Galoian K, Temple T, Galoyan A. Cytostatic effect of novel mTOR inhibitor, PRP-1 (galarmin) in MDA 231 (ER-) breast carcinoma cell line. PRP-1 inhibits mesenchymal tumors. Tumour Biology. 2011; 32 :745-751. DOI: 10.1007/s13277-011-0176-3 - 48.
Galoian K, Qureshi A, Wideroff G, Temple H. Restoration of desmosomal junction protein expression and inhibition of H3K9-specific histone demethylase activity by cytostatic proline-rich polypeptide-1 leads to suppression of tumorigenic potential in human chondrosarcoma cells. Molecular and Clinical Oncology. 2015; 3 (1):171-178. DOI: 10.3892/mco.2014.445 - 49.
Galoian K, Abrahamyan S, Chailyan G, Qureshi A, Patel P, Metzer G, et al. Toll like receptors TLR1/2, TLR 6 and MUC5b as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma. International Journal of Oncology. 2018; 52 (1):139-154. DOI: 10.3892/ijo.2017.4199 - 50.
Galoian K, Guettouche T, Issac B, Qureshi A, Temple H. Regulation of onco and tumor suppressor miRNAs by mTORC1 inhibitor PRP-1 in human chondrosarcoma. Tumour Biology. 2014; 35 (3):2335-2341. DOI: 10.1007/s13277-013-1309-7 - 51.
Moran A, Hoyt A, Sedani A, Granger C, Blonska M, Zhao-Jun L, et al. Proline-Rich-Polypeptide (PRP-1) drastically decreases cancer stem cell (CSC) population targeting BAFF chromatin-remodeling-complexes in human chondrosarcoma JJ012-cells. Oncology Reports. 2020; 44 :393-403. DOI: 10.3892/or.2020.7612 - 52.
Galoian K, Qureshi A, Dippolito G, Schiller P, Molinari M, Johnstone A, et al. Epigenetic regulation of embryonic stem cell marker miR302C in human chondrosarcoma as determinant of antiproliferative activity of proline rich polypeptide 1. International Journal of Oncology. 2015; 47 :465-472. DOI: 10.3892/ijo.2015.3054 - 53.
Granger C, hoyt A, Moran A, Becker B, Sedani A, Saigh S, et al. Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for proline rich polypeptide-1. Molecular Medicine Reports. 2020;22(5):3747-3758. DOI: 10.3892/mmr.2020.11480 - 54.
Abrahamyan S, Sahakyan I, Tumasyan N, Kocharyan N, Simonyan A, Aroutiounian R, et al. Morpho-functional study of the hypothalamic proline-rich polypeptide apoptotic activity against mouse Ehrlich ascites carcinoma. Oncology Reports. 2020; 44 :196-212. DOI: 10.3892/or.2020.7604 - 55.
Shacter E, Williams JA, Hinson RM, Senturker S, Lee Y. Oxidative stress interferes with cancer chemotherapy: Inhibition of lymphoma cell apoptosis and phagocytosis. Blood. 2020; 96 :307-313 - 56.
Abrahamyan S, Davtyan T, Khachatryan A, Tumasyan N, Sahakyan I, Harutyunyan H, et al. Quantification of the hypothalamic proline rich polypeptide-1 in rat blood serum. Neurochemical Journal. 2014; 36 (1):38-43. DOI: 10.1134/S1819712414010024 - 57.
Antonyan A, Sharoyan S, Mardanyan S, Galoyan A. Proline-rich cytokine from neurosecretory granules: A new natural substrate for dipeptidyl peptidase IV. Neurochemical Research. 2011; 36 (1):34-38. DOI: 10.1007/s11064-010-0258-4