Rostyslav R. Panchuk

Structure-Related Mode-of-Action Differences of Anticancer Organoruthenium Complexes with β-Diketonates.

A series of organoruthenium(II) chlorido complexes with fluorinated O,O-ligands [(η(6)-p-cymene)Ru(F3C-acac-Ar)Cl] (1a-6a) and their respective 1,3,5-triaza-7-phosphaadamantane (pta) derivatives [(η(6)-p-cymene)Ru(F3C-acac-Ar)pta]PF6 (1b-6b) were synthesized and fully characterized in both solution and solid state. All complexes were inactive against nonmalignant keratinocytes but displayed variable activity against cancer cell models (ovarian, osteosarcoma). Compounds with a ligand containing the 4-chlorophenyl substituent (6a and 6b) exhibited the strongest anticancer effects. Despite a marginally lower cellular Ru accumulation compared to the chlorido complexes, pta analogues showed higher activity especially in the osteosarcoma model. Reduction of glutathione levels by buthionine sulfoximine (BSO) significantly enhanced the activity of all compounds with the most pronounced effects being observed for the pta series resulting in IC50 values down to the nanomolar range. While all chlorido complexes potently induce reactive oxygen species, DNA damage, and apoptosis, the respective pta compounds widely lacked ROS production but blocked cell cycle progression in G0/G1 phase.

Complex of C60 Fullerene with Doxorubicin as a Promising Agent in Antitumor Therapy

The main aim of this work was to evaluate the effect of doxorubicin in complex with C60 fullerene (C60 + Dox) on the growth and metastasis of Lewis lung carcinoma in mice and to perform a primary screening of the potential mechanisms of C60 + Dox complex action. We found that volume of tumor from mice treated with the C60 + Dox complex was 1.4 times less than that in control untreated animals. The number of metastatic foci in lungs of animals treated with C60 + Dox complex was two times less than that in control untreated animals. Western blot analysis of tumor lysates revealed a significant decrease in the level of heat-shock protein 70 in animals treated with C60 + Dox complex. Moreover, the treatment of tumor-bearing mice was accompanied by the increase of cytotoxic activity of immune cells. Thus, the potential mechanisms of antitumor effect of C60 + Dox complex include both its direct action on tumor cells by inducing cell death and increasing of stress sensitivity and an immunomodulating effect. The obtained results provide a scientific basis for further application of C60 + Dox nanocomplexes as treatment agents in cancer chemotherapy.

Specific antioxidant compounds differentially modulate cytotoxic activity of doxorubicin and cisplatin: in vitro and in vivo study

Aim To use the antioxidant compounds (sodium selenite, selenomethionine, D-pantethine) for modulation of cytotoxic effect of doxorubicin and cisplatin toward wild type and drug-resistant mutants of several human tumor cells. Similar treatments were applied in vivo toward adult male Wistar rats. Methods Human tumor cells of different lines (HCT-116, Jurkat and HL-60) with various mechanisms of drug-resistance were treated with doxorubicin or cisplatin, alone or in combination with sodium selenite, selenomethionine, or D-pantethine. Cell viability, induction of apoptosis, and production of O2- radicals were measured. Activity of redox potential modulating enzymes was measured in the liver and blood plasma of adult male Wistar rats subjected to similar treatments. Results All antioxidants used in physiologically harmless concentration inhibited cytotoxic action of doxorubicin toward tumor cells sensitive to chemotherapy treatment by 15%-30%, and slightly enhanced cytotoxic effect of this medicine toward drug-resistant malignant cells. At the same time, there was no significant effect of these antioxidants on cisplatin action. Such effects were accompanied by a complete inhibition of production of superoxide radicals induced by doxorubicin. The results of in vivo study in adult male Wistar rats were in agreement with the results of in vitro study of human tumor cells. Conclusion Protective effect of specific antioxidant agents during cytotoxic action of doxorubicin was demonstrated in vitro in drug-sensitive human tumor cells and in adult male Wistar rats, while there was no protective effect in drug-resistant sub-lines of these tumor cells during action of doxorubicin and cisplatin.

C60 fullerene enhances cisplatin anticancer activity and overcomes tumor cell drug resistance

We formulated and analyzed a novel nanoformulation of the anticancer drug cisplatin (Cis) with C60 fullerene (C60+Cis complex) and showed its higher toxicity toward tumor cell lines in vitro when compared to Cis alone. The highest toxicity of the complex was observed in HL-60/adr and HL-60/vinc chemotherapy-resistant human leukemia cell sublines (resistant to Adriamycin and Vinculin, respectively). We discovered that the action of the C60+Cis complex is associated with overcoming the drug resistance of the tumor cell lines through observing an increased number of apoptotic cells in the Annexin V/PI assay. Moreover, in vivo assays with Lewis lung carcinoma (LLC) C57BL/6J male mice showed that the C60+Cis complex increases tumor growth inhibition, when compared to Cis or C60 fullerenes alone. Simultaneously, we conducted a molecular docking study and performed an Ames test. Molecular docking specifies the capability of a C60 fullerene to form van der Waals interactions with potential binding sites on P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP-1), and multidrug resistance protein 2 (MRP-2) molecules. The observed phenomenon revealed a possible mechanism to bypass tumor cell drug resistance by the C60+Cis complex. Additionally, the results of the Ames test show that the formation of such a complex diminishes the Cis mutagenic activity and may reduce the probability of secondary neoplasm formation. In conclusion, the C60+Cis complex effectively induced tumor cell death in vitro and inhibited tumor growth in vivo, overcoming drug resistance likely by the potential of the C60 fullerene to interact with P-gp, MRP-1, and MRP-2 molecules. Thus, the C60+Cis complex might be a potential novel chemotherapy modification.

Changes in signaling pathways of cell proliferation and apoptosis during NK/Ly lymphoma aging.

Expression of specific proteins involved in regulation of cell proliferation and apoptosis was studied at the initial (7–8 days after tumor inoculation), median (13–14 days), and terminal (20–21 days) stages of murine NK/Ly lymphoma development. Western‐blot analysis using antibodies to MEK—ERK signaling pathway, E2F‐1/2 and c‐Myc, pSTAT1, pSTAT3, pSTAT5, anti‐apoptotic Bcl‐XL and pro‐apoptotic p53 and Rb proteins, as well as active cleaved forms of caspases‐3, −6, −7, was carried out to investigate the growth and survival status of NK/Ly cells. There was a marked increase in the expression of E2F‐1/2 transcription factors, MAPK signaling cascade and c‐Myc, which suggests intensive proliferation of lymphoma cells at terminal stage of tumor development. However, cytomorphological investigation and electrophoretic study of DNA fragmentation have shown degeneration of NK/Ly lymphoma cells and increase in their death. No expression of p53 protein or cleaved forms of caspases‐3, −6, −7 was found, which suggests a caspase‐independent type of apoptosis in these cells. Ascitic fluid collected at a terminal stage of NK/Ly lymphoma development was significantly weaker in supporting tumor cell growth than ascitic fluid collected at the initial stage of tumor development. It is suggested that uncontrolled cell proliferation at terminal stage of the NK/Ly lymphoma development causes nutrient deprivation and deficiency of specific growth factors in the ascitic fluid, due to overexpression of MEK—ERK, E2F and c‐Myc, thereby leading to the induction of apoptosis.

Rapid generation of hydrogen peroxide contributes to the complex cell death induction by the angucycline antibiotic landomycin E

Abstract Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance‐mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T‐cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE‐induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell‐derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24 h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC‐1 staining, was weakly enhanced already at 3 h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase‐9 was not detectable before 12 h exposure. In contrast, cleavage of the down‐stream caspase substrate PARP‐1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase‐7 was induced at this early time points paralleling the LE‐induced oxidative burst. Accordingly, this massive cleavage of caspase‐7 at early time points was inhibitable by the radical scavenger N‐acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE‐induced apoptosis. Specific scavengers of both H2O2 and OH• effectively decreased LE‐induced ROS production, but only partially inhibited LE‐induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main cardiotoxic mechanism of Dx, LE might represent a better tolerable drug candidate for further (pre)clinical development. HighlightsRole of ROS in cell death induction by landomycin E as compared to doxorubicin.Rapid H2O2 production and effector caspase‐7 activation by landomycin E but not doxorubicin.Landomycin E in contrast to doxorubicin does not induce superoxide radical generation.

Tissue-protective activity of selenomethionine and D-panthetine in B16 melanoma-bearing mice under doxorubicin treatment is not connected with their ROS scavenging potential

Aim To evaluate molecular mechanisms of tissue-protective effects of antioxidants selenomethionine (SeMet) and D-pantethine (D-Pt) applied in combination with doxorubicin (Dx) in B16 melanoma-bearing-mice. Methods Impact of the chemotherapy scheme on a survival of tumor-bearing animals, general nephro- and hepatotoxicity, blood cell profile in vivo, and ROS content in B16 melanoma cells in vitro was compared with the action of Dx applied alone. Nephrotoxicity of the drugs was evaluated by measuring creatinine indicator assay, hepatotoxicity was studied by measuring the activity of ALT/AST enzymes, and myelotoxicity was assessed by light microscopic analysis of blood smears. Changes in ROS content in B16 melanoma cells under Dx, SeMet, and D-Pt action in vitro were measured by incubation with fluorescent dyes dihydrodichlorofluoresceindiacetate (DCFDA, H2O2-specific) and dihydroethidium (DHE, O2--specific), and further analysis at FL1 (DCFDA) or FL2 channels (DHE) of FACScan flow cytometer. The impact of aforementioned compounds on functional status of mitochondria was measured by Rhodamine 123 assay and further analysis at FL1 channel of FACScan flow cytometer. Results Selenomethionine (1200 µg/kg) and D-pantethine (500 mg/kg) in combination with Dx (10 mg/kg) significantly reduced tumor-induced neutrophilia, lymphocytopenia, and leukocytosis in comparison to Dx treatment alone. Moreover, SeMet and D-Pt decreased several side effects of Dx, namely an elevated creatinine level in blood and monocytosis, thus normalizing health conditions of B16 melanoma-bearing animals. Conclusions Our results showed that antioxidants selenomethionine and D-pantethine possess significant nephroprotective and myeloprotective activity toward Dx action on murine B16 melanoma in vivo, but fail to boost a survival of B16 melanoma-bearing animals. The observed cytoprotective effects of studied antioxidants are not directly connected with their ROS scavenging.

Antioxidants selenomethionine and D-pantethine decrease the negative side effects of doxorubicin in NL/Ly lymphoma-bearing mice

Aim To investigate the potential tissue-protective effects of antioxidants selenomethionine and D-pantethine applied together with doxorubicin (Dx) on NK/Ly lymphoma-bearing mice. The impact of this chemotherapy scheme on animal survival, blood cell profile, hepatotoxicity, glutathione level, and activity of glutathione-converting enzymes in the liver was compared with the action of Dx applied alone. Methods The hematological profile of animals was studied by the analysis of blood smears under light microscopy. Hepatotoxicity of studied drugs was evaluated measuring the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, De Ritis ratio, and coenzyme A fractions by McDougal assay. Glutathione level in animal tissues was measured with Ellman reagent, and the activity of glutathione reductase, transferase, and peroxidase was measured using standard biochemical assays. Results D-pantethine (500 mg/kg) and, to a lower extent, selenomethionine (600 µg/kg) partially reduced the negative side effects (leukocytopenia and erythropenia) of Dx (5 mg/kg) in NK/Ly lymphoma bearing animals on the 14th day of their treatment. This increased animal survival time from 47-48 to 60+ days and improved the quality of their life. This ability of D-pantethine and selenomethionine was realized via hepatoprotective and immunomodulating activities. D-pantethine also restored the levels of acid-soluble and free CoA in the liver of tumor-bearing animals, while selenomethionine caused the recovery of glutathione peroxidase levels in the liver, which was significantly diminished under Dx treatment. Both compounds decreased glutathione level in the liver, which was considerably induced by Dx. Conclusions Antioxidants selenomethionine and D-pantethine partially reversed the negative side effects of Dx in NK/Ly lymphoma-bearing mice and significantly increased the therapeutic efficiency of this drug in tumor treatment.

Putative anticancer potential of novel 4-thiazolidinone derivatives: cytotoxicity toward rat C6 glioma in vitro and correlation of general toxicity with the balance of free radical oxidation in rats

Aim To evaluate the cytotoxic action of 4-thiazolidinone derivatives (ID 3288, ID 3882, and ID 3833) toward rat glioma C6 cells and to compare the effects of these compounds and doxorubicin on the balance of free radical oxidation (FRO) and antioxidant activity (AOA) in the serum of rats. Methods Glioma cells were treated with ID 3882, ID 3288, ID 3833, and doxorubicin, and their cytotoxicity was studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Trypan blue exclusion test, light and fluorescent microscopy, and flow cytometric study of cell cycling and apoptosis, including measuring of Annexin V-positive cells. The contents of superoxide radical, hydrogen peroxide, hydroxyl radical, malonic dialdehyde, and hydrogen sulfide were measured in the serum of rats. Enzymatic activity of superoxide dismutase (SOD), catalase (Cat), and glutathione peroxydase (GPO) was determined. Results Among novel 4-thiazolidinone derivatives, ID 3288 was most toxic toward rat glioma C6 cells, even compared with doxorubicin. All applied derivatives were less active than doxorubicin in inducing reactive oxygen species-related indicators in the serum of rats. A similar effect was observed when enzymatic indicators of AOA processes were measured. While doxorubicin inhibited the activity of SOD, GPO, and Cat, the effects of 4-thiazolidinone derivatives were less prominent. Conclusion Novel 4-thiazolidinone derivatives differ in their antineoplastic action toward rat glioma C6 cells, and ID 3288 possesses the highest activity compared to doxorubicin. Measurement of indicators of FRO and AOA in the serum of rats treated with these compounds showed their lower general toxicity compared with doxorubicin’s toxicity.

Differential pro-apoptotic effects of synthetic 4-thiazolidinone derivative Les-3288, doxorubicin and temozolomide in human glioma U251 cells.

Aim To compare various pro-apoptotic effects of synthetic 4-thiazolidinone derivative (Les-3288), doxorubicin (Dox) and temozolomide (TMZ) in the treatment of human glioma U251 cells to improve treatment outcomes of glioblastoma and avoid anticancer drug resistance. Methods The cytotoxic effects of drugs used in human glioma U251 cells were measured by cell viability and proliferation assay (MTT), Trypan blue exclusion test, and Western-blot analysis of the apoptosis-related proteins. In addition, flow cytometry study of reactive oxygen species (ROS) level in glioma cells was carried out. Cytomorphological changes in treated cells were monitored by fluorescent microscopy after cell staining with Hoechst 33342 and ethydium bromide. Results Half-maximal inhibitory concentration (IC50) of Les-3288, Dox, and TMZ was calculated for human glioblastoma U251 cells. The rating of the values of this indicator of cellular vitality was assessed. The results of MTT assay proved the superiority of Les-3288 vs Les-3288>Dox>TMZ, which is in agreement with the results of Trypan blue testing showing Les-3288 ≈ Dox>TMZ. In general, such ranking corresponded to a scale of pro-apoptotic impairments in the morphology of glioma U251 cells and the results of Western-blot analysis of cleaved Caspase 3. Contrary to Dox, Les-3288 and TMZ did not affect significantly ROS levels in the treated cells. Conclusion The effect of the synthetic 4-thiazolidinone derivative Les-3288 is realized via apoptosis mechanisms and does not involve ROS. In comparison with Dox and TMZ, it is more effective in destroying human glioblastoma U251 cells. Les-3288 compound has a potential as an anticancer drug for glioblastoma. Nevertheless, further preclinical studies of the blood-brain barrier are needed.