Andrey A. Karelin

Proteolytic degradation of hemoglobin in erythrocytes leads to biologically active peptides

A number of hemoglobin-derived homogeneous peptides were isolated from erythrocyte lysate. The amino acid sequences of nine peptides were determined. Seven out of nine peptides were relatively long, 30-32 membered peptides covering the N- or C-terminal sequences of globin chains. The remaining two were the pentapeptide neo-kyotorphin and its tetrapeptide derivative.

Antitumor effect of valorphin in vitro and in vivo: combined action with cytostatic drugs.

The action of the cytostatic drugs (epirubicin and vincristine) in combination with the endogenous antiproliferative £]-hemoglobin fragment (33-39), valorphin, was studied in tumor (L929 and A549) cell cultures, primary culture of murine bone marrow cells and in murine model of breast carcinoma in vivo. Simultaneous application of 1 µM valorphin and 1 µM epirubicin, in vitro, did not result in an additive suppressive effect on cell culture growth. Additive effects were achieved with alternating applications of the peptide and the drugs, namely, 0.5 µM (but not 1 µM) epirubicin added 24 h prior to 1 µM valorphin; 1 µM valorphin added 48 h prior to 0.1 µM epirubicin, or 0.1 µM vincristine, or 0.05 µM vincristine, which resulted in 100% cell death in the both series with vincristine and up to 78% cell biomass reduction in the experiments with epirubicin. In the in vivo model (female BLRB mice with subcutaneously inoculated syngeneic mammary carcinoma), simultaneous treatment with 25 mg/m2 epirubicin and 1 mg/kg valorphin resulted in 42% of tumor growth inhibition, as compared with the negative control group and 22% inhibition as compared with the epirubcin-treated group (at 20th day of treatment). Survival was significantly improved (69% compared to 39% in the group treated with epirubicin only) at day 26 after the treatment beginning.

Tumor Cell Cytolysis Mediated by Valorphin, an Opioid-Like Fragment of Hemoglobin β-Chain

Valorphin, an endogenous opioid-like hemoglobin fragment, is cytotoxic for L929 and K562 tumor cells in 10(-7)-10(-13) M concentration range. Because cytolytic effects induced by valorphin in K562 cells are inhibited by naloxone, opioid receptors should be involved in induction of valorphin-mediated tumor cell death. Three distinct cytolytic processes, differing in the onset time and the development time, take place with K562 cells within 10-18 h of incubation with valorphin. All three processes are not associated with apoptotic mechanism of cell death.

Stimulation of fibroblast proliferation by neokyotorphin requires Ca2+ influx and activation of PKA, CaMK II and MAPK/ERK

Neokyotorphin [TSKYR, hemoglobin α‐chain fragment (137–141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8‐Br‐cAMP, but not the PKC activator 4β‐phorbol 12‐myristate, 13‐acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+L‐type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester, kinase inhibitors H‐89 (PKA), KN‐62 (Ca2+/calmodulin‐dependent kinase II) and PD98059 (mitogen‐activated protein kinase). The proliferative effect of 8‐Br‐cAMP was also suppressed by KN‐62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP‐like action for neokyotorphin.

Met-enkephalin induces cytolytic processes of apoptotic type in K562 human erythroid leukemia cells

Cytolytic activity of Met‐enkephalin, an endogenous opioid peptide, was studied within the 10−7−10−17 M concentration range in K562 human erythroid leukemia cells. Cytolytic activity was determined by the trypan blue inclusion method after 13, 15 and 18 h of Met‐enkephalin co‐incubation with target cells. Discrete maxima of cytolytic activity were detected at concentrations of 10−9−10−10, 10−13 and 10−15 M. Cytolysis was accompanied by internucleosomal DNA fragmentation which is indicative of the mechanism of cell death being apoptosis.

Cytolytic processes induced by TNF in L929 and K562 differ in DNA fragmentation mechanisms

OBJECTIVE Cytolytic activity of TNF was analysed at L929 and K562 tumor cell lines. METHODS TNF-mediated cytotoxicity was studied within 10(-6)-10(-17) M concentration range after 18 h of incubation with target cells. RESULTS TNF caused reliable cytotoxicity values in both cell lines, while L929 cells were more sensitive to cytolytic action of the protein than K562 cells. Three cytotoxicity maxima were detected at each cell line: at concentrations of 10(-6) M, 10(-17) M and 10(-15) M in K562 cells and at concentrations of 10(-7) M, 10(-11) M, 10(-14), 10(-16) M in L929 cells. CONCLUSIONS DNA fragmentation analysis demonstrated that all cytolytic processes induced by TNF in L929 cells are associated with apoptotic mechanism of cell death, while cytolytic process induced in K562 cells differed in DNA fragmentation patterns: cytolytic processes induced by 10(-6) M of TNF was of apoptotic type, while the other processes were not associated with internucleosomal DNA cleavage.

Both neurotoxin II from venom of Naja naja oxiana and its endogeneous analogue induce apoptosis in tumor cells

Both neurotoxin II (NT II from venom of Naja naja oxiana) and 20–30 kDa proteins partially purified from pig brain (NTlm) cross‐reacting with antibodies to NT II were cytotoxic for L929 and K562 tumor cells at concentrations of 10−6–10−8 M. Modification of Cys residues significantly reduced cytotoxicity of NT II. Both NT2 and NTlm induced apoptosis in L929 and K562 cells.

Alteration of Intraerythrocyte Proteolytic Degradation of Hemoglobin During Hodgkin's Disease

The erythrocyte lysate samples obtained from 10 healthy donors (aged of 23 +/- 12 years) and 16 patients with Hodgkins disease (aged of 39 +/- 25 years) with the following histological types: 12 mixed cellularity and 4 nodular sclerosis, were studied. Patients with Hodgkins disease (HD) were randomly selected before, during or after the completion of combined chemotherapy. A comparative analysis of peptide components of erythrocyte lysate samples of HD patients and healthy donors was carried out. The amino acid sequences of 4 peptides, corresponding to fragments 1-33, 1-32, 1-31 and 1-30 of human hemoglobin (Hb) alpha-chain were determined. Increase of the content of two fragments corresponding to 1-31 and 1-32 amino acid residues of alpha-globin were detected for HD patients. The link between the normal process of proteolytic degradation and those occurring during HD is proposed. The possibility of using the identified alterations recorded during HD diagnosis is discussed.

Tubocurarin induces the wide spectrum of cytolytic effects in tumor cells

Tubocurarin was shown to mediate a wide spectrum of cytolytic processes in tumor cells while peritoneal murine macrophages were resistant to its action. Group I of these processes was due to rapid (within 1-3 h) and nonspecific lysis mediated by relatively high (> 10(-5) M) concentrations of tubocurarin. Membrane damage with subsequent osmotic lysis was associated with these events. Processes of groups II and III were highly specific, cytolysis of group II having developed within 4-5 h and characterized by saturation at 5 x 10(-7) M. It was proposed that two types of necrosis contributed to the processes of group II. These pathways were induced by different concentrations of tubocurarin and also differed in their sensitivity to lysosomal inhibitor NH4Cl. Cytolysis of group III was associated with apoptosis and developed within 8-24 h. The significant acceleration of DNA fragmentation and subsequent cell death was achieved by increase of tubocurarin concentration from 5 x 10(-8) to 10(-6) M.

Cytotoxic activity of acetylcholine receptor ligands

Acetylcholine receptor ligands were studied for cytotoxicity in K562 human erythroid leukemia tumor cells. Cytotoxicity of carbachol, an agonist of acetylcholine receptors, atropine, an antagonist of muscarinic acetylcholine receptor, neurotoxin 11 (NT II) from Naja naja oxiana cobra venom and tubocurarine, antagonists of acetylcholine receptor of nicotinic type was exhibited in the 10‐7‐10‐5 M concentration range. Several cytolytic processes, two for carbachol and three for other ligands, corresponding to different concentrations of each ligand were detected. All acetylcholine receptor ligands induced internucleosomal DNA fragmentation.