Maintenance of genome stability: the unifying role of interconnections between the DNA damage response and RNA-processing pathways.

Endogenous and exogenous factors can severely affect the integrity of genetic information by inducing DNA damage and impairing genome stability. The protection of genome integrity is ensured by the so-called "DNA damage response" (DDR), a set of evolutionary-conserved events that, triggered upon DNA damage detection, arrests the cell cycle, and attempts DNA repair. Here, we review the role of the DDR proteins as post-transcriptional regulators of gene expression, in addition to their roles in DNA damage recognition, signaling, and repair. At the same time, we discuss recent insights into how pre-mRNA splicing factors go beyond their splicing activities and play direct functions in detecting, signaling, and repairing DNA damage. The importance of extensive two-way crosstalk and interaction between the RNA processing and the DDR stems from growing evidence that the defects of their communication lead to genomic instability.

Novel nonclassical antifolate, 2-[N-(2´-Hydroxyethyl)amino]methyl-3H-quinazolin-4-one, with a potent antineoplastic activity toward leukemia cells.

This study was aimed to investigate the therapeutic potential of novel nonclassical antifolate, 2-[N-(2´-Hydroxyethyl)ami-no]methyl-3H-quinazolin-4-one (HEAMQ), toward human promonocytic U937 and murine lymphoblastic L1210 cell lines. The antiproliferative activity of HEAMQ was determined by MTT assay and its effects on cell cycle progression and apoptosis were studied by flow cytometry, and by immunoblots, respectively. In addition, combination chemotherapy of HEAMQ with cisplatin and temozolomide under in vitro and in vivo conditions was tested. HEAMQ showed concentration- and time-dependent cytotoxicity toward U937 and L1210 cells. It induced G2/M arrest that in U937 cells was associated with a marked decrease in the protein expressions of cyclin A, cyclin B, and cyclin-dependent kinase Cdk1. HEAMQ-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and down-regulation of the protein expression of Bcl-2, Mcl-1, XIAP, and survivin, resulting in cytochrome c release and activation of caspases. Inhibitors of JNK (SP600125) and p38 MAPK (SB203580) suppressed HEAMQ-induced apoptosis and G2/M phase arrest, attenuated the activation of Bax, and blocked down-regulation of Bcl-2, XIAP and survivin in HEAMQ-treated U937 cells. In addition, combinations of HEAMQ with cisplatin and temozolomide resulted in synergistic inhibition of cell growth, producing long-term survivors of L1210 tumor-bearing mice. In conclusion, these results indicate that HEAMQ antineoplastic activity toward leukemia cells is associated with cell cycle arrest and apoptosis. The in vivo studies further confirmed the antitumor activity of HEAMQ and highlighted that this agent could be used to further increase therapeutic efficacies of traditional chemotherapeutic agents.

PARP-1 inhibitors: a novel genetically specific agents for cancer therapy.

The nuclear poly(ADP-ribose) polymerase-1 (PARP-1) represents an important novel target in cancer therapy. The enzyme is essential for single strand DNA breaks repair via base excision repair pathway. Inhibition of PARP-1 exerts "synthetic lethality" effect towards the tumors with defects in DNA repair by homologous recombination, specifically the tumors with mutations in the breast cancer associated BRCA1 and BRCA2 genes. Recent clinical data confirmed the early in vitro studies and suggest that PARP-1 inhibitors could be used not only as chemosensitizers but as well as single agents to selective kill tumors with defective DNA repair by homologous recombination. Such concept of "synthetic lethality" for tumors which have lost one DNA repair pathway by targeting a second DNA repair pathway, represents groundbreaking therapeutic strategy. The review highlights our current knowledge and ongoing clinical development/trials of PARP-1 inhibitors.

3-(5-Nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline induces ROS-mitochondrial mediated death signaling and activation of p38 MAPK in murine L1210 leukemia cells.

Quinazoline derivatives are multitarget agents with a broad spectrum of biological activity. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline (NTCHMTQ) is a new synthetically prepared derivative, which in our previous studies showed antiproliferative and apoptosis inducing activities towards murine L1210 leukemia cells. The aim of this study was to provide the insight into the molecular mechanism regulating NTCHMTQ-induced apoptosis in L1210 cells. The activity of caspases 3, 8 and 9, generation of reactive oxygen species (ROS), mitochondrial membrane potential changes, release of cytochrome c, degradation of PARP and activation of c-Jun N-terminal kinase 1/2 (JNK1/2), p38 MAPK and extracellular-regulated kinase 1/2 (ERK1/2) were investigated. NTCHMTQ induced production of ROS, activation of caspases 3 and 9, cytochrome c release, PARP cleavage and activation of p38 MAPK, with no activation of JNK1/2 and ERK1/2. Our resuls clearly demonstrate that NTCHMTQ induces apoptosis of L1210 leukemia cells through ROS-mitochondrial mediated death signaling and activation of p38 MAPK.

Apoptosis induced by 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile in human leukemia cells involves ROS-mitochondrial mediated death signaling and activation of p38 MAPK.

Benzothiazoles are multitarget agents with broad spectrum of biological activity. 2-Acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile (AMBAN) is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects towards tumor cells. The aim of the present study was to examine the antiproliferative and apoptosis inducing activities of AMBAN towards human leukemia HL60 and U937 cells. Further, the molecular mechanism involved in AMBAN-induced apoptosis was investigated. Benzothiazole inhibited the growth and induced programmed cell death of HL60 and U937 cells. In addition, AMBAN elevated the level of reactive oxygen species, decreased the mitochondrial membrane potential, activated caspases 9 and 3, induced the cytochrome c release and PARP cleavage and led to intranucleosomal DNA fragmentation. Further, p38 MAPK was associated with the apoptotic activity of AMBAN. It can be concluded that AMBAN-induced apoptosis in HL60 and U937 cells through mitochondrial/caspase 9/caspase 3-dependent pathway.

Antiproliferative activity and apoptosis induced by 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline on cells of leukemia lines.

UNLABELLED: Quinazolines are known to be multitarget agents with broad spectrum of biological activity. AIM: To investigate anticancer activity of newly prepared 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline (BMAQ) towards L1210, HL-60 and U-937 leukemia cells. MATERIALS AND METHODS: Growth inhibition of BMAQ-treated cells was determined by cell counting using trypan blue staining technique. Apoptosis and cell cycle profile changes were analysed using internucleosomal DNA fragmentation assay, fluorescence microscopy and flow cytometry. Activity of caspase-3 was determined using colorimetric method. RESULTS: Cell proliferation assay showed that BMAQ caused significant decrease of cell number in a dose-dependent manner. BMAQ induced cell death by apoptosis, based on results from DNA fragmentation, fluorescence microscopy and caspase-3 assays. CONCLUSION: Presented results clearly demonstrate that BMAQ is a promising anticancer agent with significant antiproliferative and apoptotic activities towards leukemia cells in vitro.

4-Amino-3-acetylquinoline-induced apoptosis of murine L1210 leukemia cells involves ROS-mitochondrial-mediated death signaling and activation of p38 MAPK.

Quinolines are known to be multitarget agents with a broad spectrum of biological activity. In a previous study, we showed that newly prepared 4-amino-3-acetylquinoline (AAQ) possesses strong anticancer activities. In this study, we investigated whether AAQ has cytotoxicity in murine L1210 leukemia cells. Results from cell proliferation assays showed that AAQ caused significant decrease in cell number in a dose-dependent manner. The cell death induced by AAQ appeared to involve apoptosis, based on evidence from apoptotic DNA fragmentation, flow cytometry, fluorescence microscopy, and Western blot analyses. We found that AAQ-treated cells had activated p38 MAPK and that apoptosis was processed through a reactive oxygen species (ROS)-dependent mitochondrial pathway. In summary, our results suggest that AAQ can induce apoptosis, at least in part, through the activation of the p38 MAPK pathway in L1210 leukemia cells.

Cytotoxicity of hydroxyapatite, fluorapatite and fluor-hydroxyapatite: a comparative in vitro study.

The purpose of this study was to evaluate the cytotoxicity of two formulations of hydroxyapatite (HA), namely fluorapatite (FA) and fluor-hydroxyapatite (FHA). HA is used as carrier material for antibiotics or anticancer drugs during treatment of bone metastasis. Negative control, represented by HA, was included for comparative purposes. Leukemia cells were used as a model cell line, and the effect of eluates of tested biomaterials on cell proliferation/viability and mechanism of antiproliferative activity were assessed. Study design attempted to reveal the toxicity of tested biomaterials with an emphasis to decide if tested biomaterials have promise for further studies in vivo. Results showed that eluates of FA and FHA inhibit the growth of leukemia cells and induce programmed cell death through mitochondrial/caspase-9/caspase-3-dependent pathway. Due to these differences compare to HA, it is concluded that FA and FHA have promise for evaluation of their behaviour in vivo.

Antigenotoxic effect of extract from Cynara cardunculus L.

The extract of artichoke Cynara cardunculus L. (CCE) was investigated for its potential antigenotoxic and antioxidant effects using four experimental model systems. In the Saccharomyces cerevisiae mutagenicity/antimutagenicity assay, CCE significantly reduced the frequency of 4-nitroquinoline-N-oxide-induced revertants at the ilv1 locus and mitotic gene convertants at the trp5 locus in the diploid Saccharomyces cerevisiae tester strain D7. In the simultaneous toxicity and clastogenicity/anticlastogenicity assay, it exerted an anticlastogenic effect against N-nitroso-N'-methylurea-induced clastogenicity in the plant species Vicia sativa L. On the contrary, despite CCE not being mutagenic itself, in the preincubation Ames assay with metabolic activation, it significantly increased the mutagenic effect of 2-aminofluorene in the bacterial strain Salmonella typhimurium TA98. In the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, CCE exhibited considerable antioxidant activity. The SC50 value representing 0.0054% CCE corresponds to an antioxidant activity of 216.8 microm ascorbic acid which was used as a reference compound. Although the mechanism of CCE action still remains to be elucidated, different possible mechanisms are probably involved in the CCE antigenotoxic effects. It could be concluded that CCE is of particular interest as a suitable candidate for an effective chemopreventive agent.

Growth inhibitory effect of ethyl acetate-soluble fraction of Cynara cardunculus L. in leukemia cells involves cell cycle arrest, cytochrome c release and activation of caspases.

An extract of artichoke Cynara cardunculus L. (CCE) has been shown to exhibit antioxidant and antigenotoxic properties. In this study, the ability of CCE to inhibit the growth of L1210 and HL-60 leukemia cells was studied. Treatment of leukemia cells with a variety of concentrations of CCE (500-2500 microg/microL) for 24 h resulted in dose-dependent inhibition of leukemia cell growth. The cell growth inhibition was accompanied by G(0)/G(1) cell cycle arrest and by a loss of cells in S phase. Futhermore, apoptosis detected as a sub-G(0) cell population and apoptotic DNA fragmentation was observed. More detailed analyses of apoptosis induced by CCE in HL-60 cells revealed that apoptosis progressed through the caspase-9/-3 pathway, as release of cytochrome c, caspase-9/-3 activations and specific proteolytic cleavage of poly(ADP-ribose) polymerase. Taken together, the results suggest that CCE exerts an antiproliferative activity on leukemia cells and induces apoptosis of these cells through a mitochondrial/caspase dependent pathway.

Growth inhibition and apoptosis induced by 2-phenoxymethyl-3H-quinazolin-4-one in HL-60 leukemia cells.

AIM: The aim of the study was to investigate anticancer activity of newly synthesized 2-phenoxymethyl-3H-quinazolin-4-one (PMQ). MATERIALS AND METHODS: Anticancer activity of PMQ was studied towards human HL-60 leukemia cells. Antiproliferative activity of PMQ was determined by direct counting of cells using trypan blue staining technique. Apoptosis and cell cycle profile changes were analysed using internucleosomal DNA fragmentation assay and flow cytometry. Activation of caspases and changes in glutathione level were monitored using colorimetric or luminiscent methods. RESULTS: PMQ induced concentration-dependent cytotoxicity in leukemia cells, with IC(50) of 10.8 +/- 0.9 microM. DNA flow cytometry analysis and DNA ladder formation assay indicated that PMQ actively induced apoptosis of cells accompanied by a block of cells in G(2)/M phase and a marked loss of cells in G(0)/G(1) and S phases. Additionally, the activities of caspase-3 and caspase-9 were increased significantly and a markedly increased level of oxidized glutahione was observed. Inhibition of glutahione synthesis using buthionine sulfoximine sensitized leukemia cells to PMQ, confirming the involvement of ROS in PMQ-induced apoptosis. CONCLUSION: The results of this study clearly demonstrate that PMQ is a promising anticancer drug showing cytostatic and apoptotic effects toward HL-60 leukemia cells mainly through mitochondrial/caspase-9 dependent pathway.

Flavonoids potentiate the efficacy of cytarabine through modulation of drug-induced apoptosis.

Recent studies have provided strong evidence for potential beneficial effects of flavonoids in chemoprevention or in combination with chemotherapeutics in tumor cells treatment. The aim of this work was to compare the antioxidant properties of four flavonoids with emphasis on association of these antioxidant properties with their effects on the therapeutic efficacy of cytarabine (AraC) using L1210 leukemia cells. The results of antiproliferative studies showed that antiproliferative potential of flavonoids tested decreased in the order: isorhamnetin > kaempferol > myricetin > rutin, while their antioxidant properties decreased in the order: rutin > myricetin > kaempferol > isorhamnetin. Combinational treatment of isorhamnetin, kaempferol and myricetin with AraC led to synergism in their antiproliferative activities (CIs < 1). Rutin exhibited antagonism with AraC (CIs > 1). Apoptotic DNA fragmentation and flow cytometry analyses revealed that synergism in antiproliferative activities of compounds tested might be due to potentiation of AraC-induced apoptosis. In conclusion, our results clearly indicate that isorhamnetin, kaempferol and myricetin despite their antioxidant properties might be used to increase the sensitivity of leukemia cells to AraC treatment.

New [1,2,4]triazolo[4,3-c]quinazoline enhances cisplatin- and temozolomide-induced growth inhibition and apoptosis in HL-60 cells.

The purpose of this study was to investigate the therapeutic potential of a newly synthesized [1,2,4]triazolo[4,3-c]quinazoline (NTCHMTQ) alone and in combination with two anticancer drugs (cisplatin and temozolomide) against HL-60 leukemia cell line. The IC50 value of NTCHMTQ toward HL-60 cells was 19.7 microM. No apoptosis and cell cycle changes were observed in cells treated with 5 microM NTCHMTQ alone. Combination of non-toxic concentrations of NTCHMTQ (1-5 microM) with cisplatin or temozolomide sensitized HL-60 cells to these two drugs and significantly enhanced their efficacies, that is illustrated by combination indexes, sub-G0 cell fraction, apoptotic DNA fragmentation and caspase-3 activity. The results suggest that combined therapy of non-toxic concentrations of NTCHMTQ with chemotherapeutics may provide synergistic regimen for treatment of leukemia. However, further in vitro and in vivo experimental drug-cell and drug-drug studies are warranted.

Photochemical and phototoxic activity of berberine on murine fibroblast NIH-3T3 and Ehrlich ascites carcinoma cells.

The present study demonstrates photoinduced generation of superoxide anion radical and singlet oxygen upon UVA irradiation of berberine chloride, and its cytotoxic/phototoxic effects on murine fibroblast non-cancer NIH-3T3 and Ehrlich ascites carcinoma (EAC) cells. The EPR spectra monitored upon photoexcitation of aerated solutions of berberine evidenced the efficient activation of molecular oxygen via Type I and II mechanisms, as the generation of superoxide anion radical and singlet oxygen was observed. The EAC cell line was more sensitive to the effect of non-photoactivated and photoactivated berberine than the NIH-3T3 cell line. UVA irradiation increased the sensitivity of EAC cells to berberine, while the sensitivity of NIH-3T3 cells to photoactivated berberine was not changed. Berberine significantly induced direct DNA strand breaks in tested cells, oxidative lesions were not detected, and the effect of irradiation of cells after berberine treatment did not affect the increase of DNA damage in EAC and NIH-3T3 cells. The DNA damage generated by a combination of berberine with UVA irradiation induced a significant blockage of EAC cells in the S and G(2)/M phases and the stopping/decrease of cell proliferation after 24h of influence. On the other hand, after 36h or 48h of berberine treatment, the DNA damage induced necrotic or apoptotic death of EAC cells. Whether these divergences are caused by differences in the properties of two non-isogenic cell lines or by different berberine uptake and cell localization will be analyzed in our further investigations.

Berberine-antiproliferative activity in vitro and induction of apoptosis/necrosis of the U937 and B16 cells.

Berberine, an isoquinoline plant alkaloid, is widely distributed in plants used in the traditional Chinese medicine. It displays a wide range of biological activities and the mechanism of action. Our previous studies of the anticancer activity of berberine against the cancer cell lines HeLa and L1210 were extended to the human tumour U937 cell line and the murine melanoma B16 cell line growing in vitro. Cytotoxicity was measured by the growth inhibition assay and by the cell morphology monitoring. The in vitro cytotoxic studies were complemented by the cell cycle analysis and determination of apoptotic DNA fragmentation. Berberine acted cytotoxically on both tumour cell lines. The melanoma B16 cells were much more sensitive to berberine treatment than the U937 cells. The value of IC(100) was below 100 microg/ml for the U937 cells and below 1 microg/ml for the B16 cells. As for both cell lines under the long-term influence the values of IC(50) were found to be less than 4 microg/ml. No effect of berberine on the cell cycle profile of the U937 and B16 cells was detected, however, berberine induced apoptosis of the U937 cells. On the other hand, cell lysis/necrosis of the berberine-treated B16 cells was observed as the result of the integrity damage of the cytoplasmic membrane.

Potentiation of doxorubicin-induced apoptosis and differentiation by indomethacin in K-562 leukemia cells.

In this study we have examined the antitumor effect of combined administrations of indomethacin (IND) with doxorubicin (DOX) on growth of K-562 leukemia cells. Although, as single drug treatment, only high concentrations of IND reduced growth (>200 microM) and induced apoptosis (>800 microM) of the K-562 cells, a synergistic effects on DOX-induced cell growth inhibition, apoptosis and differentiation were observed during the co-administration of DOX with 10 microM IND. Cells treated with this combination had elevated GSHt level compare to DOX-treated cells. Modulation of GSHt level of DOX-treated cells with Cd2+ ions or BSO confirmed its important role in processes of DOX-induced differentiation. Results of this study showed that IND has a positive effect on therapeutic efficacy of DOX, and could be a perspective modulator in cancer chemotherapy.

Effects of flavonoids on glutathione and glutathione-related enzymes in cisplatin-treated L1210 leukemia cells.

Connections between the ability of quercetin (Qu)and galangin (Ga) to differentially modulate cis-Pt-induced apoptosis and their effects on glutathione system of murine L1210 leukemia cells were studied. The results showed that total glutathione (GSHt) level is increased significantly (cca 123% of control level), both in cells treated with 10 microM Qu and in cells treated with 4 microM cis-Pt and 10 microM Qu in combination. 10 microM Ga had no effect on GSHt content. Activities of glutathione S-transferase (GST) and glutathione reductase (GR) were not changed significantly when 10 microM flavonoids were used. Significant inhibition of GR activity was observed when flavonoids were used in concentrations higher than 25 microM. The presented data indicate that Qu change the redox state of the cells that is implicated in regulation of apoptosis, due to its ability to increase the GSHt level, and thus may potentiate cis-Pt-induced apoptosis of L1210 cells.

Effect of berberine on proliferation, cell cycle and apoptosis in HeLa and L1210 cells.

Previous studies on the anticancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human tumour HeLa and murine leukemia L1210 cell lines. An attempt was also made to investigate the relationship between the cytotoxic activity of berberine and its molecular mechanism of action. Cytotoxicity was measured in-vitro using a primary biochemical screening according to Oyama and Eagle, and the growth inhibition assay. The in-vitro cytotoxic techniques were complemented by cell cycle analysis and determination of apoptotic DNA fragmentation in L1210 cells. Berberine acted cytotoxically on both tumour cell lines. The sensitivity of leukemia L1210 cells to the berberine was higher than that of HeLa cells. The IC(100) was below 100 microg mL(-1) for HeLa cells and approached a 10 microg mL(-1) limit for the leukemia L1210 cells. For both cell lines the IC(50) was found to be less than 4 microg mL(-1), a limit put forward by the National Cancer Institute (NCI) for classification of the compound as a potential anticancer drug. In L1210 cells treated with 10-50 microg mL(-1) berberine, G(0)/G(1) cell cycle arrest was observed. Furthermore, a concentration-dependent decrease of cells in S phase and increase in G(2)/M phase was detected. In addition, apoptosis detected as sub-G(0) cell population in cell cycle measurement was proved in 25-100 microg mL(-1) berberine-treated cells by monitoring the apoptotic DNA fragmentation (DNA ladder) using agarose gel electrophoresis.

Induction of cytotoxicity and ssDNA breaks by 9-bromo-5-morpholino-tetrazolo[1,5-c]quinazoline in tumor cells cultured in vitro.

9-Bromo-5-morpholino-tetrazolo[1,5-c]quinazoline (BMTQ) acted cytotoxically on murine leukemia cell line L1210 and human colon carcinoma cells Caco-2. We found the two highest concentrations of BMTQ (149.2 and 74.6 microM) induced an acute cytotoxic effect, however other tested concentrations (<74.6 microM) manifested a concentration/dependent and time/dependent cytotoxic effect. The sensitivity of murine leukemia cells L1210 and human colon carcinoma cells Caco-2 was expressed in the same order. The cytotoxicity of BMTQ was not accompanied by changes of the cell cycle profile. Following the cytotoxicity-related effects of BMTQ we observed the induction of ssDNA breaks after BMTQ treatment. All the concentrations of BMTQ increased the level of ssDNA breaks 1.3-2.9 times (after 2 h of treatment) and 1.6-2.8 times (after 4 h of treatment) in Caco-2 cells compared to the control. No apoptotic DNA fragmentation induced by BMTQ in Caco-2 cells was recorded.

Comparative DNA protectivity and antimutagenicity studies using DNA-topology and Ames assays.

Two experimental techniques, the DNA-topology assay and the Ames assay, were proved to be suitable for monitoring compounds with a genotoxic potential and/or with an antimutagenic effect. Both procedures were used in assaying the acid-mine water (AMW) containing toxic metals and sulfoethyl chitin-glucan (SE-Ch-G), a derivative of chitin-glucan, in which bioprotective activities were detected earlier. It was shown that after toxic metal concentrations were decreased due to AMW dilution to the limits that correspond with those set by the Slovak Technical Norm (STN) for drinking water, AMW was not genotoxic in the Ames assay. As it is possible to detect any single-strand DNA (ssDNA) break in the DNA-topology assay, the SE-Ch-G protective effect against the ssDNA breaks induced by Fe(2+) in the DNA-topology assay was recorded. SE-Ch-G exhibited the antimutagenic potential after its application simultaneously with diagnostic mutagens in the Ames assay. These results demonstrate the complementarity of both experimental systems.