The Mechanisms of Regulation of Aerobic Glycolysis (Warburg Effect) by Oncoproteins in Carcinogenesis.

According to modern concepts, tumor formation is associated with impairments in the structure of protooncogenes and/or deactivation of suppressor genes, regardless of the nature of carcinogenic factor. As a consequence, unregulated oncoproteins activate extracellular proteases, resulting in the destruction of the extracellular matrix, which facilitates cell invasion, deterioration of the cell-cell contacts, and metastasis. Tumor development requires activation of certain transcription factors; however, many oncoproteins are not transcription factors. It can be assumed that these oncoproteins are not the ultimate effectors of tumor development, but rather transmitters of the carcinogenic signal to the transcription factors promoting tumorigenesis. Here, we describe the mechanisms of carcinogenesis caused by various oncogenes/oncoproteins. We conclude that the common feature of these mechanisms is stimulation of aerobic glycolysis (Warburg effect) regulated, as a rule, through the activation of the HIFα transcription factor. The role of aerobic glycolysis at the early stages of carcinogenesis is discussed.

Role of Proton Pumps in Tumorigenesis.

One of the differences between normal and cancer cells is lower pH of the extracellular space in tumors. Low pH in the extracellular space activates proteases and stimulates tumor invasion and metastasis. Tumor cells display higher level of the HIF1α transcription factor that promotes cell switch from mitochondrial respiration to glycolysis. The terminal product of glycolysis is lactate. Lactate formation from pyruvate is catalyzed by the specific HIF1α-dependent isoform of lactate dehydrogenase A. Because lactate accumulation is deleterious for the cell, it is actively exported by monocarboxylate transporters. Lactate is cotransported with proton, which acidifies the extracellular space. Another protein that contributes to proton concentration increase in the extracellular space is tumor-specific HIF1α-dependent carbonic anhydrase IX, which generates a proton in the reaction between carbon dioxide and water. The activity of Na+/H+ exchanger (another protein pump) is stimulated by stress factors (e.g. osmotic shock) and proliferation stimuli. This review describes the mechanisms of proton pump activation and reviews results of studies on effects of various proton pump inhibitors on tumor functioning and growth in cell culture and in vivo. The prospects of combined application of proton pump inhibitors and cytostatics in cancer therapy are discussed.

HYPOXIA AND GLYCOLYSIS AS FACTORS DETERMINING THE MALIGNANT PHENOTYPE.

The review deals with the role of hypoxia and glycolysis in the development of cancer. Experimental results demonstrate that the function of glycolysis in tumour cells is not limited to providing energy. Glycolysis stimulates the activity of transcription factor HIF1a. HIF1a in complex with protein ARNT stimulates expression of numerous genes. There are genes encoding proteins of glycolysis, telomerase, P-glycoproteins, antiapoptotic proteins belonging to bcl2 family, inhibitor of pyruvate dehydrogenase­pyruvate dehydrogenase kynase, dedifferentiation genes and others. Inhibition of mitochondria respiratory chain by inhibiting of pyruvate dehydrogenase stimulates accumulation of pyruvate in the cell. Lactate dehydrogenase transforms pyruvate on lactate. Accumulation of lactate in tumour cells activates monocarboxylate transporter. As a result, lactate and proton are displayed in the intercellular space. There is a drop in the pH in tumour tissue. The low pH promotes the activity of various proteases that degrade intercellular matrix. The enhancement of invasion is observed in tumours area with low pH level. The restoration of normal pH level in tumour tissue inhibits invasion and metastasis. Thus, it is possible to conclude that hypoxia is a physiological state of cells that stimulates and mantaines tumour process. Aerobic glycolisis (Warburg effect) stimulates tumour growth even in the case of sufficient oxygenation of the cells. Modern views on the mechanism of the Warburg effect is given. The possibility of using inhibitors of different stages of glycolysis as mono anticancer agents or in combination with conventional anticancer compounds is discussed.

[Effect of cell microenvironment on cell functions associated with tumour promotion and progression].

To study the tumour-promotion activity of cell environment the transformed embryonic rat fibroblasts (clone CL-1-1) were transfect to immunodeficient mice then the cells of the formed tumour were cultivated (clone CL-1-1). The cells before and after transplantation were compared by morphology, proliferation activity and gap junction intracellular communications. The clone CL-1 cells proliferated much faster than clone CL-1 cells. The CL-1-1 cells had changed morphology structure and unlike CL-1 the contract inhibition was absent in CL-1-1. The number of CL-1 cells in phase G1 was significantly greater than that of CL-1-1 cells, while the number of CL-1-1 cells in G2/M phases was much more the number of CL-1 cells. The activity of gap junction intercellular communications in both cell types was near the same. It was concluded that cell microenvironment act as a tumour-promoter and tumour progression factor in the case of cell transplantation to immunodeficient mice.

[Inhibitory effect of human lactoferrin (neolactoferrin) on the growth of transplantable tumor in the uterine cervix of mice].

There was studied effect of recombinant form of human breast milk component-lactoferrin, received from milk of goats-producers (neolactoferrin), on growth of transplantable tumor of the cervix in mice (TTC-5). Neolactoferrin in dose of 100 mg/kg and 200 mg/kg of animals' mass inhibited the rate of tumor growth. The most effective was the dose of 200 mg/kg, which was entered a week before transplantation. In contrast to the control group, in groups where neolactoferrin was entered it was fixed resorption of TTC-5 in 6 mice. Repeated transplantation TTC-5 to these mice led to reducing of the rate of tumor growth and increasing of duration of their lives. To investigate if tumor-braking effect neolactoferrin connected with direct effect on the tumor or due to the general effect of the organism, TTC-5 cells were transformed in culture and they were exposed by neolactoferrin in dose of 10 and 100 mkg/ml. In investigated doses neolactoferrin did not influence on tumor cells growth. There is discussed possible mechanism of anti-tumor effect of neolactoferrin.

[Activation of transcription factor NF-kappaB by carcinogenic polycyclic aromatic hydricarbons].

Effect of carcinogenic polycyclic aromatic hydrocarbons (PAH) benzo(a)pyrene (BP) and 3-methylcholanthrene (MC) on transcription factor NF-kappaB activation was studied. The determination of NF-kappaB activity was performed by two different methods: determination of mRNA expression of NF-kappaB-dependent I-kappaB gene, and determination of transcription activity of co-transfected with the plasmid containing the luciferase reporter gene under the NF-kappaB-sensitive promoter. As a subject of inquiry the hepatoma cell cultures HepG2 expressed Ah receptor and G27 not expressed Ah receptor were used. BP and MC weekly enhanced NF-kappaB activity in proliferating HepG2 cells. The enhance of NF-kappaB activity was significantly higher in resting cells. NF-kappaB activation by BP and MC in hepatoma G27 cells was significantly higher in hepatima G27 cells than in HepG2 cells both in proliferating and resting cells. The role of Ah receptor in PAH action on NF-kappaB activation is discussed.

Mechanisms of tumor promotion by reactive oxygen species.

This review analyzes the available information concerning mechanisms of non-genotoxic action of reactive oxygen species (ROS) during tumor promotion and pathways of their generation under the influence of chemical compounds. Special attention is given to the ability of ROS to induce pseudohypoxia through inhibition of prolyl oxidase, which is an oxygen sensor in the cell. Functions of HIF-1alpha as a main contributor to the ROS-induced promotion are analyzed. Data suggest that an unregulated high level of HIF-1alpha in the cell could induce the development of tumors. Hypothetical possibilities of ROS production under the influence of different environmental pollutants, which are promoters of tumorigenesis, include functioning of cytochrome P450 during oxidation of substrates, functioning of the mitochondrial respiratory chain, and action of peroxisome proliferators.

Appearance of differentiation characteristics (induction of Ah-receptor-dependent genes) during cultivation of transformed cell clone K8 from embryonic rat fibroblasts.

The differentiation status of fibroblasts can be characterized by their ability to induce Ah-receptor-dependent genes. The ability to induce Ah-receptor-dependent genes encoding cytochrome P450 isoforms, Ah-receptor repressor, and NADPH-quinine oxidoreductase were studied in the transformed cell clone K8 obtained from immortalized embryonic rat fibroblasts by treatment with benzo(a)pyrene and in the parental clone F27. Treatment with benz(a)anthracene did not induce the genes in the transformed clone K8 on passages 4-14, but the induction was recorded in the transformed clone beginning from the 16th passage and later, whereas in F27 cells the induction was observed throughout the experiment. Induction levels of mRNA of the induction-regulating genes encoding the Ah-receptor and Ah receptor nuclear translocator were similar in F27 cells and in the transformed cell clone K8 in both early and late passages. Electrophoretic mobility shift assay showed that in clone K8 transmission of the induction signal was disturbed in the early passages before interaction of the activated Ah-receptor with the recognizing region of DNA. Possible mechanisms responsible for the absence of induction in the early passages in the transformed cells are discussed.

[Ah receptor-independent inhibition of gap junction intercellular communications in hepatoma cell culture 27 by polycyclic aromatic hydrocarbons].

One of the systems that regulate tissue homeostatis is gap junction intercellular communications (GJIC). Inhibition of GJIC is widely used in experiments as a characteristic of tumor promotion. It is accepted that the down-regulation of GJIC is tightly related with the tumor-promoting properties of carcinogens. In this study, the effect of some carcinogenic polycyclic aromatic hydrocarbons on GJIC in cell cultures of hepatoma 27 lacking cytochrome P450 and Ah receptor was investigated. It was shown that inter 6 compounds studied only benzo/a/pyren and 3-methylcholanthrene were able to inhibit GJIC. We have concluded that an unknown factor is present in hepatoma cells and its interaction with some polycyclic aromatic hydrocarbons results in GJIC inhibition. The investigation of mutual effect of benzo/a/pyrene and non carcinogenic benzo/e/pyrene with similar structure has shown that GJIC inhibition by benzo/a/pyrene is at least double stepped.

[A comparative study of induction regulation in cytochromes family 1 P450 in cell cultures at different stages of tumor transformation].

Lipophilic xenobiotics, including some carcinogenic agents and cytostatics, are metabolized by cytochrome P450 isoforms (CYP). In tumours expression of CYP genes and their inducibility are lower than in a homologous normal tissue. This phenomenon determines the known higher cytostatic stability of tumour cells. To clarify, at which particular stage of tumour transformation the level of family 1 CYP may change, we compared mRNA expression of CYP1A1, CYP1B1 and also of proteins regulated CYP expression: Ah receptor, ARNT and AHRR. For this aim we studied embryonic and fibroblast-like cells, in addition to cells of the same types but immortalized by the Rausher virus, or spontaneously after crisis. Besides, we investigated transformed clones obtained by means of benzo/a/pyrene action on Rausher virus-immortalized cells. Constitutive expression of genes studied in all cell cultures was shown. Benzo/a/anthracene induction increases the mRNA expression of all inducible genes (CYP1A1, CYP1B1, AHRR) in the original embryonic cells, in Rausher virus-immortalized cells, and in transformed clone K2. In both spontaneously immortalized cells and transformed clone K1 only CYP1B1 was induced. In transformed clone K8 no inducible gene was induced. In summary, we have shown that: (1) the ability of immortalized cells to CYP induction is determined not only by their capacity for a non-limited persistence, but also by the nature of immortalization; (2) despite their common genesis, the transformed clones differ in their ability to induce CYP. In addition to Ah receptor and ARNT, some other, yet unknown factors may also take part in CYP induction.

[Specificity of cytochrome P-450 isoforms contained in endoplasmic reticulum membranes and nuclear membranes in relation to benzo(a)pyrene]. / Spetsifichnost' izoform tsitokhroma P-450, soderzhashchikhsia v membranakh éndoplazmaticheskogo retikuluma i iadernykh membranakh, po otnosheniiu k benz(a)pirenu.

The metabolic activation of benz(a)pyrene (BP) catalyzed by rat liver microsomes and isolated liver nuclei in the presence of certain inhibitors of the monooxygenase system was studied. Several forms of cytochrome P-450 are supposed to participate in metabolic conversion of BP. The specificity of cytochrome P-450 isoforms contained in the endoplasmic reticulum and in nuclear membranes is discussed.

[The changes in the sphingenine/sphinganine ratio in sphingolipids of transplantable rat tumors depends on a transplantation organ]. / Izmenenie otnosheniia sfingenin/sfinganin v sfingolipidakh perevivaemykh opukholei krys v zavisimosti ot organa transplantatsii.

The content of sphingenine (sphingosine) and sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.

[Effect of some polycyclic aromatic hydrocarbons on gap junction intercellular communication in hepatoma Hep G2 cell culture]. / Deistvie riada politsiklicheskikh aromaticheskikh uglevodorodov na mezhkletochnye shchelevye kontakty v kul'ture kletok gepatomy Hep G2

Systems regulating tissue homeostasis are gap junction intercellular communications (GJIC). It is accepted that the down-regulation of GJIP has been due to tumor promoting properties of carcinogens. In this study, effects of some carcinogenic and noncarcinogenic polycyclic aromatic hydrocarbons (PAH) on GJIC were investigated. Noncarcinogenic PAHs do not influence GJIC function. In dose 5 microg/ml carcinogenic PAHs down-regulated GJIC by 70-100% after a 24 h treatment. Dependent on the structure of PAHs, down-regulation was observed after a 1 h treatment. The methyl group in PAH structure decreased down-regulation of GJIC in 1 h experiments, whereas after a 24 h treatment the down-regulation caused by methyl group either contained or not contained PAH was nearly the same. To clarify the role of Ah-receptor in PAH action on GJIC, the effect of 2,3,7,8-tetrachlorodibezdioxin, a specific ligand of Ah-receptor was studied, which appeared to be insignificant. Benzo/a/pyrene does not influence the functioning of gramicidine channels formed in the phospholipid membrane. This result indicates that PAH action on GJIC is not associated with non-specific destruction of the membrane. Thus, two steps are there in PAH action on GJIC: one is fast and caused by specific interaction of unchanged PAH molecule, the other develops in time and is presumably associated with the formation of active metabolites.

[Effect of uncoupling agents and benz (a) pyrene metabolites on respiration in L cells and normal mouse embryonal fibroblasts]. / Distvie razobshchaiushchikh agentov i metabolitov benz(a)pirena na dykhanie kletok L i normal'nykh émbrional'nykh fibroblastov myshi

A method is proposed for a polarografic study of the respiration of cell suspensions obtained from monolayer cultures of L-cells and from normal embryonic fibroblasts of mice (C3HA line). 6-hydroxybenzo(a)pyrene (6-OHBP). The metabolite of the carcinogenic hydrocarbon benzo(a)pyrene, was shown to be a strong uncoupler of of oxidation and phosphorylation of cell suspensions. Its effect was influenced by the presence of calf serum in the incubation media. Possible relationships between the toxic effect of 6-OHBP on monolayer cultures of normal and tumor cells, and its effect on cell energetics are discussed.

[Phenols as toxic metabolites of benz(a)pyrene]. / Fenoly kak toksicheskie metabolity benz(a)pirena.

The effect of benzo(a)pyrene and two its phenolic metabolites 3-hydroxybenzo(a)-pyrene and 6-hydroxybenzo(a)pyrene--on the cultures of normal and transformed fibroblasts has been studied. In was shown that unlike the parent carcinogen its phenolic metabolites exerted only toxic (but not transforming) effect on cultured cells, and this effect has been developed at a faster rate than that produced by benzo(a)pyrene. 6-hydroxybenzo(a)pyrene was more toxic than 3-hydroxybenzo(a) pyrene. It was concluded that both metabolites produced their effects without preliminary activation by microsomal enzymes.

[Respiration of the cells and isolated mitochondria of the rat liver in the early stages of hepatocarcinogenesis]. / Dykhanie kletok i izolirovannykh mitokhondrii pecheni krys na rannikh stadiiakh gepatokantserogeneza.

Under study was the respiration of mitochondria and cell aggregates (slices) of rat liver during the process of chemical carcinogenesis, induced by 3'-Me-DAB and AAP. The study of the hepatic mitochondria respiration indicated no impairment of the mitochondria functions investigated at early stages of carcinogenesis. The examination of hepatic slices respiration has demonstrated that during the process of chemical carcinogenesis the stimulation of endogenic respiration affected by DNP is decreased, whereas such effect is not observed while using succinate as a substrate. During the carcinogens metabolism compounds are assumed to be formed, those are able to affect oxidative phosphorylation without forming any stable link with the respiratory chain components. Which of the carcinogens metabolites may render similar effects is the question to be discussed.

[Microsomal oxygenase enzyme system during chronic administration of 2-acetylaminofluorene]. / Izuchenie sostoianiia fermentnoi sistemy mikrosomal'nykh oksigenaz pri khronicheskom vvedenii 2-atsetilaminofluorena.

Alteration in content of enzymatic components was studied in microsomal oxygenases system of rats administered with 2-acetyl aminofluorene. Content of cytochrome R-450 was decreased beginning from the fourth week of the experiment. Activity of NADP cytochrome c reductase was higher in animals, treated with the drug as compared with controls. Inductors of the I and II type were capable to induce the enzymatic system up to the end of the experiment - within 16 weeks as far as liver tumors developed.

[State of the monooxygenase enzymatic system during chronic diethylnitrosamine administration]. / Sostoianie monooksigenaznoi fermentnoi sistemy pri khronicheskom vvedenii diétilnitrozamina.

Content of components and functional activity of monooxygenase enzyme system were studied in rat liver tissue during hepatocarcinogenesis produced by diethyl nitrosamine treatment. Content of cytochromes P-450 and b5, NADPH- NADH-cytochrome c reductase and demethylase activities as well as capacity to induce the monooxygenase enzyme system after administration of its inductors were not altered in chronic treatment with carcinogenic doses of diethyl nitrosamine. The higher doses of the amine were shown to reduce the content of the cytochromes in liver tissue.

[New models of experimental chemotherapy of tumors]. / Novye modeli dlia éksperimental'noi khimioterapii opukholei.

New neoplastic models such as orthotopic solitary hepatic tumor (cholangiocellular cancer PC-1) as well as different strains of malignant pleuritis (hemoblastosis and ascitic tumors) have been evolved by transplanting tumor cell suspension to rat liver or murine pleural cavity. Intrahepatic cancer PC-1 has a solid mucosa-excreting structure and is characterized by low activity of detoxication enzymes of such xenobiotics as glutathione-S-transferase, NAD(p)N-chinonoxyreductase and aldehyde dehydrogenase. Orthotopic hepatic tumor PC-1 may be used for evaluating systemic and regional therapy. Models of tumorous pleuritis described with respect to their response to chemotherapy, may have an application in screening and preclinical examination of newly-developed antitumor drugs.