Dimethylsulfoxide extracts of Chilean berries Myrteola nummularia, ‘Murtilla-like’ vs. well known ‘Murtilla’, Chilean and Polish blueberries, and Chilean raspberries were investigated for their ...antioxidant, quenching and antiproliferative activities. The significantly highest levels of polyphenols, flavonoids, flavanols and their antioxidant activities were estimated in ‘Murtilla’ (MT) berries (P<0.05), than in other investigated samples. DPPH kinetic measurements were calculated to compare, distinguish and discriminate the antiradical activity among berry extracts by multivariate analysis. The lowest IC50 values, 751 and 858μg/ml, were obtained for MT extract on colon cancer cell lines HT-29 and SW48. HT-29 cells treated with MT extract showed a decrease in G1 phase cells from 77% to 56% (P<0.05). At the highest concentration of 2000μg/ml MT extract caused 90–100% cell growth inhibition. Percentage of death cells treated with MT extract was 80.1% and 72.5% for SW48 and HT-29 cells, respectively. The inhibition of cancer cell proliferation highly correlated with the levels of polyphenols, flavonoids and their antioxidant activities. The interaction between drugs and serum albumin plays an important role in the distribution and metabolism of drugs, therefore the complexation reaction between flavonoids, and berries extracts, and bovine serum albumin (BSA) was investigated by 3-D fluorescence and FTIR spectroscopy. The results indicated that flavonoids and polyphenol extracts have strong ability to quench the intrinsic fluorescence of BSA by forming complexes. A shift in the maximum of amides FTIR-bands appeared. In conclusion, these findings suggest that the intake of a new kind of berry, as a source of natural antioxidants, may reduce colon cancer risk.
DNA methylation is an epigenetic phenomenon known to play an important role in the development and progression of human cancer. Enzyme responsible for this process is DNA methyltransferase 1 (DNMT1) ...that maintains an altered methylation pattern by copying it from parent to daughter DNA strands after replication. Aberrant methylation of the promoter regions of genes critical for normal cellular functions is potentially reversible. Therefore, inactivation of DNMT1 seems to be a valuable target for the development of cancer therapies. Currently, the most popular DNMT inhibitors (DNMTi) are cytidine analogues like 5-azacytidine, 5-aza-2'-deoxycytidine (decitabine) and pyrimidin-2-one ribonucleoside (zebularine). In colorectal cancer, epigenetic modifications play an essential role at each step of carcinogenesis. Therefore, we have addressed the hypothesis that DNA methyltransferase inhibitors may potentiate inhibitory effects of classical chemotherapeutic agents, such as oxaliplatin and 5-fluorouracil (5-FU), commonly used in colorectal cancer therapy. Here, our report shows that DNMTi can have positive interactions with standard chemotherapeutics in colorectal cancer treatment. Using pharmacological models for the drug-drug interaction analysis, we have revealed that the combination of decitabine with 5-FU or oxaliplatin shows the most attractive interaction (synergism), whereas the effect of zebularine in combinations with chemotherapeutics is moderate and may be depended on genetic/epigenetic background of a cell line or secondary drug used in combination. Our results suggest that DNMTi administered in combination with standard chemotherapeutics might improve the treatment of patients with colorectal cancers.
Abstract 520
Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of BCR-ABL1 kinase-positive chronic myeloid leukemia in chronic phase (CML-CP). ...Unfortunately, 15–25% of patients initially responding favorably to imatinib will develop acquired drug resistance, which in 40–90% of cases is caused by genomic instability resulting in the appearance of clones expressing TKI resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species (ROS) resulting in excessive oxidative DNA damage such as oxidized DNA bases (8-oxoguanine and 5-hydroxycytosine→uracil) (Nieborowska-Skorska et al., Blood, 2012). Unfaithful and/or inefficient repair of these lesions generates TKI resistant point mutations in BCR-ABL1 kinase. Oxidative DNA lesions may be removed by base excision repair (BER) or, if not removed, will create mismatches, which are repaired by mismatch repair (MMR). Since we found that MMR is inhibited in CML-CP (Stoklosa et al., Cancer Res., 2008), the activity of BER is critical to prevent the accumulation of point mutations. Using an array of specific substrates and inhibitors/blocking antibodies we found that two major glycosylases, uracil-DNA glycosylase UNG2 and 8-oxoguanine glycosylase (OGG1) responsible for the excision of uracil (product of oxidation of cytosine) and 8-oxoguanine (8-oxoG) from DNA, respectively, were inhibited in BCR-ABL1 –transformed cell lines and CD34+ CML cells. The inhibitory effect was even more pronounced in CML blast phase (CML-BP) in comparison to CML-CP, it depended on BCR-ABL1 kinase activity and was not accompanied by deregulation of nuclear expression and/or chromatin association of these glycosylases. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases such as TEL-ABL1, TEL-PDGFbetaR and NPM-ALK did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI we found that UNG2 activity diminished the number of oxidized DNA bases detected by modified comet assay and prevented accumulation of point mutations in reporter gene Na+/K+ATPase, which encode resistance to ouabain. In conclusion, we hypothesize that inhibition of UNG2 and OGG1, accompanied by reduced MMR activity is responsible for accumulation of TKI-resistant BCR-ABL1 kinase point mutations and perhaps also other point mutations facilitating malignant progression of CML.
No relevant conflicts of interest to declare.
Abstract 909
Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in the appearance of clones carrying BCR-ABL1 kinase mutations encoding resistance to ...tyrosine kinase inhibitors (TKIs) and/or those harboring additional chromosomal aberrations, eventually leading to disease relapse and/or malignant progression to blast phase (CML-BP) Skorski, T., Leukemia and Lymphoma, 2011. We found that Lin−CD34+CD38− human leukemia stem cells (huLSCs), including the quiescent sub-population, and Lin−CD34+CD38+ human leukemia progenitor cells (huLPCs) accumulate high levels of reactive oxygen species (ROS) resulting in numerous oxidative DNA lesions such as 8-oxoguanine (8-oxoG) and DNA double-strand breaks (DSBs) Nieborowska-Skorska, Blood, 2012. huLSCs and huLPCs treated with TKIs continue to exhibit ROS-induced oxidative DNA damage suggesting the persistence of genomic instability in TKI-treated patients. Furthermore, genomic instability in TKI-refractory huLSCs and TKI-sensitive huLPCs may have a varying impact on disease progression and determining novel treatment modalities. To determine if TKI-refractory huLSCs are a source of genomic instability we employed a tetracycline-inducible murine model of CML-CP: SCLtTA/p210BCR-ABL1. Mice exhibiting CML-CP -like disease demonstrated splenomegaly, leukocytosis, and expansion of mature Gr1+/CD11b+ cells. ROS were elevated in Lin−c-Kit+Sca-1+ cells (muLSCs), but not Lin−c-Kit+Sca-1− cells (muLPCs), which was associated with higher mRNA expression of BCR-ABL1 in muLSCs. In addition, ROS levels were directly proportional to BCR-ABL1 kinase expression in transduced CD34+ human hematopoietic cells, thus confirming the “dosage-dependent” effect of BCR-ABL1 on ROS. Among the Lin−c-Kit+Sca-1+ cells, enhanced ROS were detected in TKI-refractory quiescent muLSCs, in CD34−Flt3− long-term and CD34+Flt3− short-term muLSCs, and also in CD34+Flt3+ multipotent progenitors. High levels of ROS in muLSCs were accompanied by aberrant expression of genes regulating ROS metabolism (mitochondrial electron transport, oxidative phosphorylation, hydrogen peroxide synthesis, and detoxification). In addition, muLSCs, including the quiescent sub-population, displayed high levels of oxidative DNA lesions (8-oxoG, and DSBs). ROS-induced oxidative DNA damage in muLSCs was accompanied by genomic instability in CML-CP –like mice, which accumulated a broad range of genetic aberrations recapitulating the heterogeneity of sporadic mutations detected in TKI-naive CML-CP patients. These aberrations include TKI-resistant BCR-ABL1 kinase mutations, deletions in Ikzf1 and Trp53 and additions in Zfp423 and Idh1 genes, which have been associated with CML-CP relapse and progression to CML-BP. Imatinib caused only modest inhibition of ROS and oxidative DNA damage in TKI-refractory muLSCs. In concordance, CML-CP –like mice treated with imatinib continued to accumulate genomic aberrations. Since BCR-ABL1(K1172R) kinase-dead mutant expressed in CD34+ human hematopoietic cells did not enhance ROS, it suggests that BCR-ABL1 kinase-independent mechanisms contribute to genomic instability. In summary, we postulate that ROS-induced oxidative DNA damage resulting in genetic instability may originate in the most primitive TKI-refractory huLSCs in TKI-naive and TKI-treated patients.
Lange:Novartis: Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees.
Abstract 2736
BCR-ABL1 –positive chronic myeloid leukemia in chronic phase (CML-CP) is a leukemia stem cell (LSC)-derived but leukemia progenitor cell (LPC)-driven disease, which may eventually ...develop resistance to the tyrosine kinase inhibitors (TKIs) and progress to fatal CML blast phase (CML-BP). In CML-CP, LSCs and LPCs reside in the CD34+CD38- and CD34+CD38+ populations, respectively. In addition, majority of LSCs and LPCs belong to quiescent (CFSEmax) and proliferative (CFSElow) populations, respectively. Quiescent LSCs are intrinsically insensitive to TKIs, and LPCs can acquire resistance to TKIs. In the TKI era, these cells may eventually initiate the disease relapse and progression to CML-BP, which is associated with genomic instability manifested by accumulation of a new or additional TKI-resistant BCR-ABL1 kinase mutations and chromosomal aberrations. We reported that BCR-ABL1 –positive leukemia cells contain high levels of the reactive oxygen species (ROS)-induced oxidative DNA damage resulting in genomic instability (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). These studies highlighted the importance of identification of the origin of leukemia cell lineage accumulating genomic instability and the mechanisms responsible for generation of ROS-mediated oxidative DNA damage.
Here we show that LSC-enriched CD34+CD38- cells and quiescent LSCs, and also LPC-enriched CD34+CD38+ and proliferating CML-CP cells contain higher levels of ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and oxidative DNA lesions (8-oxoG and DNA double-strand breaks) than corresponding cells from healthy donors. Surprisingly, the most primitive quiescent LSCs accumulated the highest levels of ROS and oxidative DNA damage. On the basis of these observations and the studies in murine hematopoietic 32Dcl3 cells expressing TKI-resistant BCR-ABL1 kinase variants (Y253F, T315I, H396P), we would predict that primitive CML-CP cells carrying these mutations would also contain high levels of ROS and oxidative DNA damage. Moreover, inhibition of BCR-ABL1 kinase with imatinib exerted only modest, if any, effect on ROS and oxidative DNA damage in LSCs/LPCs in the presence of growth factors (GFs).
Among numerous signaling proteins activated in CML cells, Rac GTPases were potential candidates to regulate production of ROS. Importantly, Rac was stimulated in leukemia cells expressing non-mutated BCR-ABL1 and TKI-resistant kinase mutants and it remained active in CML-CP cells treated with imatinib in the presence of GFs. We used Rac dominant-negative mutant (RacT17N), Rac specific inhibitors (NSC23766 and EHT1864) and Rac1, Rac2 and Rac3 knockout cells to document that Rac2 GTPase is responsible for elevation of ROS and oxidative DNA damage in LSC-enriched CD34+CD38- cells, quiescent LSCs, and also in LPCs. Active Rac2 reduced mitochondrial membrane potential (ΔΨm) and slowed the electron flow between mitochondrial respiratory chain (MRC) complexes I-II and I-III leading to overproduction of ROS. Using cells depleted of functional mitochondria (Rho0 cells), applying specific probes to measure mitochondrial ROS (MitosoxRed and mitochondria matrix-targeted circularly permuted yellow fluorescence protein = mt-cpYFP) and employing a specific inhibitor of mitochondrial ROS (MitoQ) we determined that mitochondria are the main source of ROS causing oxidative DNA damage in CD34+CD38- and quiescent LSCs and in LPCs. Furthermore, using selective inhibitors of various MRC complexes we pinpointed complex III as major producer of ROS in LSCs and LPCs. This conclusion is supported by the observation that BCR-ABL1 –positive cells with genetically inactivated complex III, but not complex I, displayed diminished capability to generate ROS. Targeting Rac2 GTPase by RacT17N and reduction of mitochondrial ROS by mitochondrial-targeted catalase and by mitochondrial-targeted ROS-scavenging peptide aptamers prevented genomic instability.
Altogether, Rac2 - MRC-cIII pathway is a major source of ROS-mediated oxidative DNA damage resulting in genomic instability in LSCs and LPCs, which could be targeted to prevent the relapse and malignant progression of CML. We also postulate that similar mechanisms cause genomic instability in FLT3(ITD)-positive acute myeloid leukemia cells and in JAK2(V617F)-positive polycythemia vera cells.
Holyoake:Novartis: Consultancy, Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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