Apoptosis induction in human leukemia cells by novel 2-amino-5-benzylthiazole derivatives

Derivatives of 2-amino-5-benzylthiazole are heterocyclic pharmacophores that exhibit different pharmacological activities including anticancer action. The mechanisms of such action of these compounds are not clear. The aim of the present study was to investigate apoptosis induction, particularly DNA damage in human leukemia cells, by the novel synthesized thiazole derivatives ‒ 2,8-dimethyl-7-(3-trifluoromethyl-benzyl)pyrazolo[4,3-e]thiazolo[3,2-a]pyrimidin-4(2H)-one (compound 1) and 7-benzyl-8-methyl-2-propylpyrazolo[4,3-e]thiazolo[3,2-a]pyrimidin-4(2H)-one (compound 2). Western-blot analysis, DNA comet assay in alkaline conditions, diphenylamine DNA fragmentation assay, agarose gel retardation, and methyl green DNA intercalation assays were used to study the effects of the studied compounds in human leukemia cells. These compounds induced PARP1 and caspase 3 cleavage in the leukemia cells, also increased the level of pro-apoptotic Bim protein and the mitochondrion-specific EndoG nuclease, and decreased the level of the anti-apoptotic Bcl-2 protein. They caused DNA single-strand breaks and DNA fragmentation in the leukemia cells without direct DNA binding or DNA intercalation. Thus, novel 2-amino-5-benzylthiazole derivatives may be promising agents for apoptosis induction in the targeted human leukemia cells

Acute and Long-Term Effects of Hyperthermia in B16-F10 Melanoma Cells

OBJECTIVE: Hyperthermia uses exogenous heat induction as a cancer therapy. This work addresses the acute and long-term effects of hyperthermia in the highly metastatic melanoma cell line B16-F10. MATERIALS AND METHODS: Melanoma cells were submitted to one heat treatment, 45°C for 30 min, and thereafter were kept at 37°C for an additional period of 14 days. Cultures maintained at 37°C were used as control. Cultures were assessed for the heat shock reaction. RESULTS: Immediately after the heat shock, cells began a process of fast degradation, and, in the first 24 h, cultures showed decreased viability, alterations in cell morphology and F-actin cytoskeleton organization, significant reduction in the number of adherent cells, most of them in a process of late apoptosis, and an altered gene expression profile. A follow-up of two weeks after heat exposure showed that viability and number of adherent cells remained very low, with a high percentage of early apoptotic cells. Still, heat-treated cultures maintained a low but relatively constant population of cells in S and G(2)/M phases for a long period after heat exposure, evidencing the presence of metabolically active cells. CONCLUSION: The melanoma cell line B16-F10 is susceptible to one hyperthermia treatment at 45°C, with significant induced acute and long-term effects. However, a low but apparently stable percentage of metabolically active cells survived long after heat exposure

Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production

Apparent diffusion coefficient of water in evaluation of treatment response in animal body tumors

The review summarizes the author’s results and literature data on the evaluation of diffusion-weighted magnetic resonance imaging (DWI) as a cancer biomarkers that reflects structural, cellular, apoptotic, and necrotic changes in tumor tissue. Diffusion measurements reflect the effective displacement of water molecules allowed to migrate for a given time. It was demonstrated that apparent diffusion coefficient (ADC) of water estimated from 1H DWI is important tool for the detection and characterization of neoplastic transformation as well as monitoring response to therapy. The possible mechanisms of pre- and post-therapy changes in water ADC in animal body tumors are discussed

Contribution of perfusion in piffusion-weighted 1H-MRI of intrahepatic and subcutaneous hepatocellular carcinoma in rat

Hepatocellular carcinoma (HCC) and liver metastases are an increasing problem worldwide. Non-invasive methods for understanding of HCC growth mechanisms are highly desirable. A biexponential model for analysis of non-invasive diffusion-weighted 1H magnetic resonance imaging (MRI) provides important information about neoplastic transformation in capillary liver tissue perfusion and water molecular diffusion. Fast and slow components of water apparent diffusion coefficient (ADC) were separated in the normal rat liver, intrahepatic, and subcutaneous HCCs. MRI was acquired with a Varian 9.4 T horizontal bore system. The fast component of ADC (ADCfast), which contributes 38% to total signal in the intrahepatic HCC, was significantly lower compared to normal liver value, while the slow component of ADC did not differ in liver, intrahepatic, and subcutaneous HCCs. A decrease in ADCfast may be caused by restricted perfusion in abnormal tumor microvessels. Thus, a reported earlier decrease in ADC in HCC compared to normal liver was mostly due to a decreased in tumor perfusion rather than a decrease in water diffusion. Subcutaneous HCC showed a very limited vasculature development, which makes the tumor perfusion extremely poor and hypoxic. Simultaneous monitoring of water ADC changes in orthotopic and subcutaneous HCCs may be useful, but a possibility of location-based physiological and metabolic differences must be recognized

Comparative characteristics of respiration and oxidative phosphorylation in mitochondria of cells of mouse liver and lymphoma NK/Ly

Parameters of respiration and oxidative phosphorylation in mouse liver cell mitochondria and experimental lymphoma NK/Ly was investigated. The suspensions of mitochondria were obtained by the differential centrifugation. Oxygen uptake rate was measured using platinum polarographic Clark electrode. α-Ketoglutarate and succinate were used as substrates. It was established that the rate of oxygen uptake in mitochondria of mouse liver cells is higher in all investigated metabolic states compared to the rate of oxygen uptake in mitochondria of lymphoma NK/Ly cells when both α-ketoglu­tarate and succinate were used as substrates. Also we found lower rates of respiratory control and longer time of phosphorylation in tumor cell mitochondria compared to liver cell mitochondria for the oxidation of α-ketoglutarate, while the ratio of ADP/O was lower in mitochondria of lymphoma cells for the oxidation of succinate

Processes of lipopero­xidation and respiration of mitochondria in rat liver under the action of thiazoles derivatives in vitro

One of the main problems of chemotherapy is development of negative side effects, when anti-tumor drugs damage healthy cells, in particular hepatocytes. Liver is the main detoxifying organ in human and animals. This organ plays an important role in the excretion of drugs from the body. Changes in free radical oxidation processes and respiratory function of mitochondria in liver cells following the effects of newly synthesized antitumor agents may indicate adverse side effects that often occur after taking such substances. It was shown that thiazole derivatives passess anti-neoplastic activity against cancer cells in vitro. The influence in vitro of newly synthesized derivatives of thiazoles (N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide and 8-methyl-2-Me-7-[tri­fluoro­methyl-phenylmethyl]-pyrazolo-[4,3-e]-[1,3]-thiazolo-[3,2-a]-pyrimidin-4(2H)-one) in concentarion of 1, 10 and 50 mM on lipid peroxidation processes in hepatocyte membranes, respiration and oxidative phosphorylation in rat liver mitochondria was studied. The effects of these substances did not reveal changes in the products of the primary peroxide lipid oxidation, and the content of secondary products was significantly reduced. Such results may indicate that the studied substances might to interact with the active forms of Oxygen, while the antioxidant defense system was not changed. These results may also indirectly indicate that the thiazole derivatives not only do not activate, but also decrease the formation of peroxide oxidation products. The processes of respiration and oxidative phosphorylation in liver mitochondria practically did not change due to the influence of the studied thiazole derivatives. The only exceptions when energy changes were observed at using high doses (50 mM) of substances with nonselective effects. Since the studied thiazole derivatives, as shown earlier, exhibit high cytotoxicity to cancer cells, these substances can be applied as antitumor drugs with minimal negative side effects

Prooxidant and antioxidant processes in lymphoma cells under the action of pyrazolopyrimidine derivative

Background. The influence in vitro of thiazole derivative 8-methyl-2-Me-7-[trifluoro­methyl-phenylmethyl]-pyrazolo-[4,3-e]-[1,3]-thiazolo-[3,2-a]-pyrimidin-4(2H)-one (PP2) on the level of lipid peroxidation products, superoxide anion radical and antioxidant system activity in lymphoma cells was studied. A pronounced cytotoxic action of the thiazole derivative on the tumor cells in vitro was reported earlier, however, no cytotoxicity of this substance was detected toward non-cancerous cells. In addition, it was shown that the sca­vengers of active forms of Oxygen significantly reduced the cytotoxic effect of the studied compound. The purpose of this work was to investigate the effect of 8-methyl-2-Me-7-[trifluoromethyl-phenylmethyl]-pyrazolo-[4,3-e]-[1,3]-thiazolo-[3,2-a]-pyrimidin-4(2H)-one on the content of lipid peroxidation products, superoxide radical and the activity of enzymes of antioxidant defense in the lymphoma cells. Materials and Methods. Experiments were conducted on white wild-type male mice with grafted NK/Ly lymphoma. Ascites tumor cells were passaged by the intreperitoneal inoculation to mice. Abdominal drainage with ascites was performed with a sterile syringe under ether anesthesia. PP2 was dissolved in dimethylsulfoxide. The product content and enzymatic activity were determined spectrophotometrically. Statistical analysis of obtained results was carried out using MS Excel-2013 program. Results. The influence of the pyrazolopyrimidine derivative on the content of lipid peroxidation products and superoxide radical in lymphoma cells was investigated. It was found that the studied compound did not change the amount of the primary lipid peroxidation products, but reduced the amount of secondary products. A decrease in the MDA content under the action of the studied derivative indicates probable interaction of the substance with the reactive Oxygen species. Pyrazolopyrimidine derivative did not change the level of the superoxide radical. The effect of the thiazole derivative on the activity of key enzymes of the antioxidant system in lymphoma cells was investigated. The studied compound at the concentration of 10 µM activated superoxide dismutase. Pyrazolopyrimidine derivative decreased the activity of catalase and glutathione peroxidase. Such changes in the activity of enzymes can cause the growth of hydrogen peroxide in the cell, which is toxic in large quantities. Conclusions. The obtained results may indicate that the studied pyrazolopyrimidine derivative can realize its cytotoxic effect on lymphoma cells though the action on the pro­ducts of lipid peroxidation and antioxidant system activity. These data can be used to understand the mechanism of action of the studied compounds and for further improvement of their antitumor effect

Effects of new derivatives of 2-amino-5-benzylthiazole of genotoxicity and acute toxicity in Allium bioassays

We have found that new derivatives of 2-amino-5-benzylthiazole possess cytotoxic action towards human tumor cells (Finiuk et al., Biopolym. Cell, 2017; Finiuk et al., Ukr. Biochem. J., 2018). A release of the chemotherapeutic drugs into the environment may cause adverse effects towards ecosystems. To promote further these derivatives as potential anticancer agents, it was necessary to evaluate their genotoxicity and acute toxi­city, namely in plants that have an important trophic level in ecosystems. To do that, we used towards plant bioassays for new derivatives of N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide (compound 1) and 2,8-dimethyl-7-(3-trifluoromethyl-benzyl)pyrazolo[4,3-e]thiazolo[3,2-a]pyrimidin-4-one (compound 2). Allium cepa ana-telophase assay was applied to monitor the genotoxicity of the studied compounds. Besides, the acute toxic effects such as inhibition of cell division, seed germination and growth of Allium roots were estimated. The compound 1 (10 mM) in concentration equal to the IC50 for tumor cells, and compound 2 in 1 mM (1´ concentration, equal to the IC50 for tumor cells) and 10 mM (10´ concentration) did not possess acute toxicity towards Allium cepa. A significant inhibition of root growth and seed germination effects were detected at using the compound 1 only in dose that is 10 times higher than the IC50 for tumor cells. The ana-telophase assay did not reveal the genotoxic effect of the compounds 1 (10 mM) and 2 (1 and 10 mM). The compounds 1 (10 mM) and 2 (1 and 10 mM) did not affect the mitotic and phase (prophase, metaphase, anaphase, telophase) indices. A commercial anticancer drug Doxorubicin (0.1 and 1 mM) possessed a significant inhibitory effect on root growth and seed germination, mitotic index and enhanced a level of chromosomal aberrations in Allium cepa. The compound 1 at 10 mM and compound 2 at 1 mM and 10 mM did not possess a significant acute toxicity (inhibition of cell division, seed germination and growth of Allium roots), did not demonstrated the genotoxic effects (induction of chromosomal aberrations) in Allium bioassay. These results give primary evidence about a possibility of using the synthetic 2-amino-5-benzylthiazole derivatives – compounds 1 and 2 – as novel antineoplastic agents that will have no negative side effects in the treated plant organism. Additional experiments should be performed in order to evaluate the adverse effects of new derivatives of 2-amino-5-benzylthiazole in a vide spectrum of the concentrations for the prediction of environmental toxicity and genotoxicity of chemicals