Biochemical and Molecular Mechanisms of Platelet-Rich Plasma in Ameliorating Liver Fibrosis Induced by Dimethylnitrosurea.

BACKGROUND/AIMS: Hepatic fibrosis is a wound-healing process in the chronically injured liver. Clinical application of platelet-rich plasma (PRP) is of considerable interest for wound healing and regeneration. In view of the regeneration effect of PRP, we designed this study to explore the hypothesis that PRP could play a role in improving the biochemical and molecular changes that occur in liver fibrosis induced by dimethylnitrosamine (DMN) in rats. METHODS: Four groups were studied: control, PRP control, DMN (liver fibrosis), and DMN+PRP groups. Serum liver enzymes (alanine amino transferase ALT, aspartate amino transferase AST, gamma glutamyl transferase GGT, and lactate dehydrogenase LDH), and liver hydroxyproline content were measured colorimetrically.Interleukin-8 (IL-8) and B-cell lymphoma (Bcl2) were determined by enzyme-linked immunosorbent assay. And the expression levels of alpha-smooth muscle actin (α-SMA) ,transforming growth factor (TGF-ß), and nuclear factor kappa B1(NF-қB1) were evaluated by quantitative real-time polymerase chain reaction. RESULTS: Our results showed that PRP markedly improved the DMN-induced changes in liver enzymes accompanied by a significant decrease in liver hydroxyproline content and IL-8 level induced by DMN, and an increase in the anti-apoptotic marker Bcl-2. PRP also showed significant down-regulation of fibrosis-related genes α-SMA and TGF-ß and a significant decrease in the inflammatory marker NF-қB1. CONCLUSION: Based on these encouraging results, we consider that PRP could be a promising new agent for liver regeneration and alleviation of fibrosis.

Inhibitor of vasculogenic mimicry restores sensitivity of resistant melanoma cells to DNA-damaging agents.

The increasing incidence of melanoma makes this cancer an important public health problem. Therapeutic resistance is still a major obstacle to the therapy of patients with metastatic melanomas. The aim of this study was to develop the melanoma cell line resistant to DNA-alkylating agents and to elucidate the mechanisms involved in acquired drug resistance. We established a unique melanoma subline Mel MeR resistant to DNA-alkylating drug aranoza by continuous stepwise selection of the Mel Me/WT cell line with increasing concentrations of this drug. Mel MeR cells were also cross-resistant to streptozotocin or cisplatin. Here, we show that aranoza-resistant melanoma cells modulate the ABC transporter activity, upregulate the expression of PRAME, adopt a vascular-related phenotype and engage in vasculogenic mimicry. LCS1269, a vasculogenic mimicry low-molecular-weight inhibitor, reverses the sensitivity of resistant melanoma cells to DNA-damaging agents. In this study, we provide experimental evidence that LCS1269 might be considered as a new potential anticancer agent capable of overcoming multidrug resistance for DNA-damaging agents in melanoma.

Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells.

Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.

[Hematological toxicity of some combined chemotherapy schemes involving aranoza].

Hematological toxicity of four combined chemotherapy schemes--TAr, TPAr, EPAr, and IPAr--involving aranose (Ar), cisplatin (P), etoposide (E), irinotecan (I), and topotecan (T) in therapeutic regimes has been studied on healthy mice in comparison to the treatment with Ar in a high therapeutic dose. It is established that Ar alone induces early leucopenia, mostly as a result of lymphocytopenia followed by the absolute and relative neutrophilia; the TAr treatment leads to a moderate neutropenia; whereas the ternary combinations cause a moderate lymphocytopenia. A relatively high hematological toxicity among the ternary combinations was observed for TPAr. The total number of erythrocytes and thrombocytes in the peripheral blood of mice under the action of Ar and all combinations remains on the control level. The inclusion of Ar into the indicated schemes neither modifies the spectrum of hematological toxicity nor increases its level. The observed changes in the peripheral blood were reversible and were not accompanied by any impairment in the state of mice. The obtained results allow the combinations involving P, E, I, T, and Ar to be considered as promising for further investigation.

[Establishment of an animal model with thymic lymphoma in mice].

The objective of study was to establish an animal model with thymic lymphoma in mice induced by intraperitoneal injection of DNA alkylating agent N-methyl-N-nitrosourea (MNU). Male and female mice of the C57BL/6 strain were injected by the intraperitoneal route with MNU solution in a dosage of 50 mg/kg body weight. The injection was repeated at week 8. Following injection of MNU, the general status of mice was observed. All mice were sacrificed for autopsy at the 22nd experimental week. Complete gross examination was performed for detection of tumor masses. The results showed that at the 22nd week, the incidence of thymic lymphoma in MNU-treated animals was 67.5% (27/40). No significant sex difference in the incidence of thymic lymphoma was observed. In conclusions, an animal model with thymic lymphoma in mice can be established by twice intraperitoneal administration of MNU. The biological behavior of the induced tumors resembles to those of human thymic lymphoma derived from thymic T-cells.

[Pre-clinical study of combined aranosa, cisplatin and irinotecan in the treatment of experimental lung cancer].

Data are presented on evaluation of use of combinations of aranosa (Ar), irinotecan (I) and cisplatin (C) in treatment of Lewis lung carcinoma. The drugs were administered i.p. simultaneously, in single doses: irinotecan 20-100 mg/kg, single dose, cisplatin 2.5 3.0 mg/kg and aranosa 100-200 mg/kg, three doses. High synergism (IP, PAr, IPAr) and sufficient tolerance were reported. Efficiency of IPAr regimen was significantly higher while hematotoxity prognosis - comparable to those of IP.

[Combination chemotherapy with newly-developed cytostatics for disseminated small cell lung cancer].

The paper discusses the results of phase II clinical trials of chemotherapy regimens using newly-developed cytostatics for disseminated small cell lung cancer. Taxotere (docetaxel)/cisplatin and campto(irinotecan)/cisplatin were investigated as first-line treatment. Doxorubicin and vincristine in combinations with a novel antitumor cytostatic aranoza were studied for application as second-line treatment. Safety and immediate- and end results were reviewed. Taxotere (docetaxel)/cisplatin and campto(irinotecan)/cisplatin regimens were compared.

[Chemoimmunotherapy with dacarbazine and aranose combined with interferon-alpha in disseminated cutaneous melanoma]. / Khimioimmunoterapiia dakarbazinom i aranozoi s interferonom-al'fa disseminirovannoi melanomy kozhi (DMK).

Aranoza and dacarbazine (DTIC) monotherapy and in combinations with aranoza and interferon-alpha (IPHN-alpha) as well as DTIC and IPHN-alpha was given to 175 patients (89 males and 86 females, aged 23-78) with disseminated skin melanoma. The effectiveness of aranoza and DTIC monotherapy was practically identical. Total response to DTIC used in combination with IPHN-alpha increased insignificantly: 25.6%--in combination vs. 19.5% DTIC alone (p > 0.05). IPHN-alpha was most effective when used with aranoza (total objective response--31.7% vs. aranoza alone--16%). The best results were reported in cases heretofore untreated with cytostatic drugs. Total response (complete or partial remission) was recorded in 63% vs. 4.5% (p < 0.05) of patients who had received chemotherapy. In cases of aranoza+ IPHN-alpha treatment, complete and partial remission was reported in metastasis of the skin, subcutaneous fat, peripheral, retroperitoneal and abdominal (mesenteric) lymph nodes and lungs. There was no relapse of metastasis of the liver, intestinal tract, bones, breast or ovary.

Apoptosis and mutation in the murine small intestine: loss of Mlh1- and Pms2-dependent apoptosis leads to increased mutation in vivo.

The mismatch repair (MMR) protein Msh2 has been shown to function in the apoptotic response to alkylating agents in vivo. Here, we extend these studies to the MutL homologues (MLH) Mlh1 and Pms2 by analysing the apoptotic response within the small intestine of gene targeted strains. We demonstrate significant differences between Msh2, Mlh1 and Pms2 mutations in influencing apoptotic signalling following 50mg/kg N-methyl-nitrosourea (NMNU), with no obvious reliance upon either Mlh1 or Pms2. However, following exposure to 100mg/kg temozolomide or lower levels of NMNU (10mg/kg) both Mlh1- and Pms2-dependent apoptosis was observed, indicating that the apoptotic response at these levels of DNA damage is dependent on the MutL homologues. Given our ability to observe a MutLalpha dependence of the apoptotic response, we tested whether perturbations of this response directly translate into increases in mutation frequency in vivo. We show that treatment with temozolomide or 10mg/kg NMNU significantly increases mutation in both the Mlh1 and Pms2 mutant mice. At higher levels of NMNU, where the apoptotic response is independent of Mlh1 and Pms2, no gene dependent increase in mutation frequency was observed. These results argue that the MutSalpha and MutLalpha are not equally important in their ability to signal apoptosis. However, when MMR does mediate apoptosis, perturbation of this response leads to long-term persistence of mutant cells in vivo.

Subcutaneous undifferentiated sarcoma induced by N'-ethyl-N'-nitrosourea in rat: radiology, histopathology and mutagenesis.

The aim of the present study was to investigate high dose and long-term effects of a common industrial agent, N'-ethyl-N'-nitrosourea (ENU), on soft tissues in a rat model. ENU, which was dissolved in polyethyleneglycol (PEG) was injected intra-peritoneally once a week (300 mg/kg) in the first experimental group. The second group received only PEG. The control group was free of any agent administration. Only rats treated with ENU for a period of 45 weeks developed large subcutaneous tumours (approximately 5-9 cm in size). Tumoral tissues were examined radiologically, histopathologically and immunohistochemically. There was no bone destruction beneath the soft tumoral tissues in direct X radiograms. Computed tomographic (CT) images showed heterogeneous soft tissue masses with a density ranging from 50 to 65 HU. Macroscopically, all tumors were circumscribed with a gray-white surface in the cross-sections. The histopathological and immunohistochemical examination of the subcutaneous tumoral tissues showed a spindle cell type of sarcoma. Lymphatic and skeletal muscle invasion, atypical mitoses and necroses were determined in all tumoral tissues in the experimental group. A somatic point mutation was detected in exon 2 of KRAS oncogene in sarcoma tissues using the single strand conformational polymorphism (SSCP) analysis. In conclusion, the activated KRAS oncogene might contribute to the progression of subcutaneous sarcoma in experimentally ENU induced rats due to point mutation.

Semi-automated method of quantifying vasculature of 1-methyl-1-nitrosourea-induced rat mammary carcinomas using immunohistochemical detection.

Studies of the vascularization of autochthonous rodent mammary tumors are limited in number, and the majority have used Factor VIII staining for blood vessel detection. Moreover, little effort has been directed at measuring the vascularization of tissue immediately adjacent to a tumor despite its central importance in the process of angiogenesis. Thirty-six chemically-induced mammary carcinomas and tissue immediately adjacent to these carcinomas were used to develop a census counting method for quantitative assessment of intra- and extra-tumor vascularization. Blood vessels were identified using antiserum directed against either CD31 or Factor VIII. Techniques used to create digitized images of all tumors and the semi-automated methods for circumscribing the extra-tumoral region are described. For Factor VIII, CD31 allowed greater discrimination of blood vessels with areas <25 microm(2) and demonstrated crisp staining of blood vessels, with minimal background and excellent preservation of tissue architecture. Census counting data support the use of CD31 for quantifying both intra- and extra-tumoral vascularization. This method provides a basis for standardizing the approach to evaluation of experimentally induced premalignant and malignant mammary lesions in rodent model systems used to investigate potential anti-angiogenic cancer preventive agents.

[Aranoza -- a new Russian antineoplastic drug]. / Aranoza--novyi otechestvennyi protivoopukholevyi preparat.

Myelosuppression is a toxicity-related limitation for aranoza dosage. The drug proved effective in the treatment of uterine sarcoma, cancer of the head and neck, breast, Hodgkin's disease and lymphosarcoma during stage II of clinical studies. Complete regression was reported in the treatment of melanoma (ca. 12%). Good results of chemoimmunotherapy should be expected in untreated patients as well as intraarterial infusions for local lesions of the extremities. Clinical trials of aranoza used in combined modalities of therapy in various sites continue.

[Sensitivity of human melanoma xenografts to nitrosoalkylurea antineoplastic agents]. / Chuvstvitel'nost' ksenograftov melanomy cheloveka k protivoopukholevym preparatam klassa nitrozoalkilmochevin.

We studied the sensitivity of human melanoma (Bro strain) xenografts to drugs of the nitrosoalkylurea (NAU) class: nitrosomethylurea (NMM), karmustin (BCNU), nimustin (ACNU), nitrulin, and ADEKO. High antitumor activity of NAM was shown when the drugs were applied not only at the early, but also at the late stages of tumor progression (tumor mass 400 and 1200 mg, respectively). The therapeutic effect of the drugs was estimated with the use of criteria characterizing the kinetics of tumor regression, increased life span, and survival of treated animals. After early administration of the drugs (Day 4 after tumor transplantation), 67% and 50% of animals survive under the influence of nitrulin and ACNU, respectively, while the rate of tumor regression increased in the sequence nitrulin < karmustin < NMM < ACNU. After late administration (11 days after tumor transplantation), NMM was most effective at increasing survival (35% of survived animals by 35 days of observation), while the rate of tumor regression increased in the sequence ADEKO < NMM < karmustin < nitrulin < ACNU.

Studies of initiation and promotion of carcinogenesis by N-nitroso compounds.

Complete carcinogens must possess both initiating and promoting properties. Most N-nitroso compounds are mutagens and are considered to be initiators, but some are not mutagenic and yet are complete carcinogens. To investigate the two activities, brief treatments of male F344 rats with each of three mutagens, nitrosodimethylurea, nitrosodiethylurea and nitrosobis-(2-oxopropyl)-amine, were followed by chronic treatment (40 weeks) with one of four non-mutagens, nitrosomethyl-3-carboxypropylamine, nitrosodiethanolamine, nitrosomethyl-2-hydroxypropylamine or phenobarbital, the last being a well-known promotor of liver tumors in rats. Each treatment group consisted of 18 animals and there were control groups of 15 animals without initiation and 15 animals without promotion. All surviving rats were sacrificed at week 78. There were almost no tumors in untreated controls or in groups treated with the promotors, other than bladder tumors in one group. Certain tumors were numerous in the initiated groups, but there were only a few instances of increased incidences after treatment with the promotors. The action of the initiators appeared to be the dominant factor and there was scant indication in this experiment of the induction of tumors by the promotors of promotion of initiated cells in most organs (e.g. the liver). This indicates that it is unlikely that non-genotoxic carcinogens induce tumors by promotion of already-initiated cells, but that some other mechanism prevails.

[Mutagenic effect of nitrosodimethylurea in male mice. Induction of dominant lethal mutations and chromosome aberrations in germ cells; genotype influence on the clastogenic effect in bone marrow cells]. / Mutagennyi effekt nitrozodimetilmocheviny u samtsov myshei. Induktsiia dominantnykh letal'nykh mutatsii i khromosomnykh narushenii v polovykh kletkakh, vliianie genotipa na klastogennyi éffekt v kletkakh kostnogo mozga.

The ability of dimethylnitrosourea (DMNU) to induce dominant lethal mutations in germ cells and chromosome aberrations in bone marrow cells of male CBAB6F1 mice was studied. At a mutagen dose of 200 mg/kg, mortality was 19% in the second week after treatment (being 5% in the control) and the frequency of bone marrow cells containing chromosome aberrations was 19.6% 24 h after treatment. DMNU induces dominant lethals in postmeiotic cells and in spermatogonia; the effects in spermatozoids and spermatogonia are equal. Chromosome aberrations in spermatocytes induced by DMNU are not realized as dominant lethals. The sensitivity of mouse strains to the clastogenic effect of DMNU ranged in an order similar to that observed in experiments with thioTEPA. The most sensitive was the TPS strain (29.2 +/- 4.6% of cells with chromosome aberrations), the most resistant-the CBA/Lac strain (9.5 +/- 2.9%). DMNU exhibited a relatively poor clastogenic activity; the effect in bone marrow cells was higher than that in male germ cells.

[A comparative study of the action of 1-methyl-1-nitrosourea and 1,3-dimethyl-1-nitrosourea on tumor cell DNA in vitro and in vivo by the alkaline elution method]. / Sravnitel'noe izuchenie deistviia 1-metil-1-nitrozomocheviny i 1,3-dimetil-1-nitrozomocheviny na DNK opukholevykh kletok in vitro i in vivo metodom shchelochnoi éliutsii.

DNA damage of the tumor cells was studied by the method of alkali elution from filters after introduction of 1-methyl-1-nitrosourea (MNU) and 1,3-dimethyl-1-nitrosourea (DMNU) to mice with Ehrlich ascites carcinoma or after treatment of the cultivated cells with these drugs. DNA was essay fluorometrically using DAPI. The degree of DNA damage was characterized by the constant of the alkali elution rate (Kae), which was estimated according to the anamorphism of the kinetic curves of elution. It was shown that in the case of MNU application the tumor cell DNA was damaged to a greater extent than in the case of DMNU application. Kae increased with the concentration of drugs. A correlation was established between the antitumor activity of the drug (kappa), K(ae), and the number of chromosome defects per cell (gaps, deletions, microfragments, ring chromosomes, and translocations). This suggests that kappa is due both to DNA damage and chromosome defects.

[A trial of the efficacy of using the synthetic antioxidant dibunol in the chemotherapy of experimental tumors]. / Opyt éffektivnosti primeneniia sinteticheskogo antioksidanta dibunola pri khimioterapii éksperimental'nykh opukholei.

We studied the therapeutic efficiency of the synthetic antioxidant Dibunol alone and in combination with cytostatic drugs on animals with experimental tumors after simultaneous and successive administration. Introduction of Dibunol to animals (tumor carriers) soon after the transplantation of tumor cells had adverse consequences, stimulated tumor growth and reduced the life span of animals. Dibunol exerted a therapeutic effect only in cases of developed tumors. Simultaneous introduction of cytostatic drugs (cyclophosphamide, dimethylnitrosourea, ADEKO) and Dibunol to tumor carriers was efficient and increased the mean life span of animals. The antitumor effect of the cyclophosphamide-Dibunol combination did not, in practice, depend on the interval between introductions of these drugs. However, the mean life span and survival of animals were somewhat higher the intervals between introductions or simultaneous application of the drugs were short.

Unexpected genetic toxicity to rodents of the N',N'-dimethyl analogues of MNU and ENU.

Lijinsky and his colleagues have reported that the N',N'-dimethyl analogues of ENU and MNU [N',N'-dimethyl-N-ethyl-N-nitrosourea (DMENU) and trimethylnitrosourea (TMNU), respectively] are carcinogenic to rats despite their extreme hydrolytic stability which would reduce or preclude generation of alkylating species analogous to those formed upon hydrolysis of ENU and MNU. Lijinsky and his colleagues were unable to rationalize those activities of DMENU and TMNU despite extensive experimentation. We therefore decided to study this problem further. Whichever mode is accepted for the generation of electrophilic/mutagenic/carcinogenic reactive species from ENU and MNU, blocking of the free-NH2 group with methyl groups (-NMe2) should ablate or abolish activity. Consistent with this DMENU and TMNU gave negative results in the NBP alkylation test while the parent compounds gave an instantaneous deep blue coloration. Studies of the rate of hydrolysis of these four compounds revealed ENU and MNU to have half-lives of 8 min, while the alkylated analogues (DMENU and TMNU) had half-lives of 25 and 41 days, respectively. Hydrolysis of ENU and MNU, to yield the alkylating species, proceeds either via proton abstraction from the -NH2 group or by attack by water on the carbon of the carbonyl group. Methylation will inhibit both of these pathways, the first absolutely (no -NH2 protons) and the second partially, via steric inhibition. The slow hydrolysis observed for DMENU and TMNU suggests that the latter route of hydrolysis is applicable. Studies with strain TA1535 of Salmonella typhimurium (without S9 mix) confirmed the potent mutagenic activity for ENU and MNU (approximately 300-fold increase in revertants at 2,000 micrograms/plate and approximately 180-fold increase in revertants at 150 micrograms/plate respectively). In contrast, the methylated analogues showed only weak mutagenic activity (approximately 3-fold) at approximately 100-fold higher dose-levels. Addition of S9 mix did not affect the mutagenicity of DMENU or TMNU. To this point, hypothesis and data coincide. ENU and MNU are potent micronucleus-inducing agents to the mouse bone marrow, and given the above data, it was expected that DMENU and TMNU would show weak or no activity in that assay. In fact, the methylated analogues were as effective as ENU and MNU as clastogens to the mouse bone marrow. Four possible reasons for this conflict of theory and data are explored. The speculative explanation we favour for these effects is that the net alkylation of bone marrow DNA is the same for all four chemicals. With ENU and MNU, most of the alkylating activity is dissipated by rapid hydrolysis. Thus, only a small fraction of the administered dose survives to alkylate the bone marrow. Due to the enhanced stability of the methyl analogues most of the delivered dose will reach the bone marrow. However, because of their lower intrinsic reactivity, only a small fraction of the target dose will alkylate the bone marrow DNA during the time window of the experiment. If these opposing influences happen to balance out, the essentially identical bone marrow genetic toxicity for the four chemicals could be explained.

Alterations in DNA and cell membranes in an initiation process of carcinogenesis by N-nitrosourea.

We examined DNA alkylation of rat organs (liver, stomach, kidney, lung, and brain) treated with a tissue specific carcinogen, N-methyl-N'-(beta-naphthyl)-N-nitrosourea (MNaNU) (200 mg/kg wt.). There is no correlation between the level of O6-methylguanine (O6-MeG) and formation of tumors with these organs. Fourier-transform infrared (FT-IR) spectra of the treated tissues were compared with those obtained form the non-treated organs in the region between 1000 and 1350 cm-1. The infrared spectra of the treated stomach which is a target organ for induction of tumors showed the formation of hydrogen bonding in carbohydrates, disturbance of nucleic acid structures, and presence of disordered states in the membranes. Further, the stomachs were divided into the forestomachs (target tissue) and glandular stomachs, and their FT-IR spectra were also examined. Using 1134 cm-1 band due to the C-H plane bending of the naphthyl ring of MNaNU, we estimated approximately 0.82 mg of MNaNU would persist in the forestomach of one rat at 6h after administration. The present results demonstrate importance of pharmacokinetics and receptor sites in cell membrane in addition to DNA alkylation for carcinogenesis by directly acting N-nitroso compounds.