Ponatinib ve VS-5584 terapötik ajanlarının kronik miyeloid lösemi lökomogenezine etkileri

Kronik miyeloid lösemi (KML), sürekli tirozin kinaz aktivitesi sergileyen BCR-ABL1 füzyon proteinine sahip hücrelerle karakterize miyeloproliferatif bir hastalıktır. Bu nedenle hastalığın tedavisinde tirozin kinaz inhibitörleri kullanılmaktadır. Ponatinib, T315I dahil olmak üzere birçok BCR-ABL1 otofosforilasyonunu inhibe edebilen ATP-yarışmalı tirozin kinaz inhibitörüdür. Lösemi kök hücreleri, normal hematopoietik kök hücrelerle benzer özelliklere sahip malignant hücrelerdir. Tirozin kinaz inhibitörleri, primitif lösemi kök hücreleri üzerinde etkili olmadıkları için tedavinin etkinliği düşük olabilmektedir. Ayrıca, ciddi yan etkilerin önlenmesi için kullanılan düşük tirozin kinaz inhibitörü dozları, KML hücrelerinde aktif halde bulunan alternatif sağkalım yolaklarının baskılanmasını sağlayamamaktadır. Tirozin kinaz inhibitörü tedavisinin başarısının düşük olduğu KML olgularında ponatinibin tek ilaç olarak kullanımından ziyade çoklu hedefli tedavi yaklaşımlarının geliştirilmesine ihtiyaç olduğu açıktır. KML'de aktif olduğu bilinen PI3K/Akt/mTOR yolağının inhibitörlerinden VS-5584, tercihen kanser kök hücrelerini öldüren, potent, seçici bir dual inhibitördür. Bu tez çalışmasında KML tedavisindeki eksikliklerin üstesinden gelebilecek in vitro kombinasyon terapisinin belirlenebilmesi hedeflenmiştir. Bu amaçla VS-5584 ile ponatinibin kombinasyonlarıyla BCR-ABL1 ve PI3K/Akt/mTOR yolaklarının eş zamanlı inhibe edilmesinin KML ve lösemi kök hücrelerinin eliminasyonundaki etkisi ve rolü moleküler düzeyde araştırılmıştır. KML hücre modeli K562, lösemi kök hücre modeli LSC ve normal hematopoietik kök hücre modeli HSC hücre hatlarında, VS-5584'ün, ponatinibin ve kombinasyonlarının sitotoksik etkileri WST-8 testi ile belirlendi. Kombinasyon indeksleri CalcuSyn izobologram analiziyle değerlendirildi. Belirlenen IC50, IC75, ED50 ve ED75 dozlarının apoptotik etkileri Anneksin V ve aktif kaspaz-3 analizleri ile hücre döngüsüne etkileri PI analizi ile akım sitometride ve siklin D1, p27 antikorlarıyla western blotma ile değerlendirildi. PI3K/Akt/mTOR yolağındaki proteinlerin aktivitelerindeki değişimler, antikor array ile fosforile protein seviyelerinin kantitasyonu yapılarak, ölçüldü. Transkripsiyon faktörlerinin aktivelerindeki değişimler uygun vektörlerin transfeksiyonu sonrasında dual lusiferaz aktivitesi ile kantite edildi. Aktivitesi değişen transkripsiyon faktörlerinin hedef genlerinin mRNA seviyelerindeki değişimleri saptamak için qRT-PCR gerçekleştirildi. Ponatinib ve VS-5584'ün K562 ve LSC hücrelerinde doza ve zamana bağlı artan sitotoksik etkiye sahip olduğu saptandı. Ponatinibden farklı olarak, VS-5584'ün sağlıklı HSC hücre hattında belirgin şekilde daha az sitotoksik olduğu gösterildi. Kombinasyon dozlarından ED50 additif, ED75 ve ED90 sinerjistik etkili olarak belirlendi. Ponatinibin indüklediği apoptoza ek olarak, VS-5584'ün hücre döngüsünde belirgin G0/G1 duraklamasına neden olduğu gösterildi. Özellikle LSC hücre hattında, ponatinib Akt yolağını baskılamada yetersiz kalırken, ponatinibin VS-5584 ile kombinasyonunun başta S6, S6K, BAD olmak üzere birçok proteinin fosforilasyonu inhibe ederek PI3K/Akt/mTOR yolağını baskıladığı belirlendi. Elk-1/SRF, AP-1 ve NFkB transkripsiyon faktörlerinin lösemi hücre ve lösemi kök hücre modellerinde belirgin şekilde yüksek aktivite sergiledikleri saptandı. Kombinasyon uygulaması sonrasında K562 ve LSC hücrelerinde, seçici bir müdahale noktası olabilecek bu transkripsiyon faktörlerine ek olarak Myc/Max, CREB, STAT3 ve E2F/DP1 transkripsiyon faktörlerinin aktivitelerinde de baskılanma gözlendi. K562 hücrelerinde C/EBP ve FOXO1 transkripsiyon faktörlerinin, LSC hücrelerinde ise C/EBP ve p53 transkripsiyon faktörlerinin aktivitelerinde kombinasyon uygulamasını takiben artış olduğu gösterildi. Ekspresyonları bu transkripsiyon faktörleri tarafından kontrol edilen birçok genin mRNA seviyelerinde değişiklik meydana geldiği belirlendi. Sonuçlar, ponatinib ve VS-5584 ile sağkalım yolaklarında görevli transkripsiyon faktörlerinin regülasyonlarının anti-proliferatif ve apoptotik süreçlere katkı sağladığını gösterdi. Ponatinib ile VS-5584'ün kombinasyonunun, KML blastlarının ve kök hücrelerinin kontrolsüz proliferasyonunu selektif olarak önleyebilecek ve ponatinibin bilinen ciddi yan etkilerini azaltabilecek umut verici bir tedavi stratejisi olabileceğini düşünmekteyiz.Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by cells with the BCR-ABL1 fusion protein, which exhibits continuous tyrosine kinase activity. Therefore, tyrosine kinase inhibitors are used in the treatment of CML. Ponatinib is an ATP-competitive tyrosine kinase inhibitor capable of inhibiting BCR-ABL1 autophosphorylation, including T315I-mutant. Leukemia stem cells are malignant cells with similar properties to normal hematopoietic stem cells. Since tyrosine kinase inhibitors are not effective on primitive leukemia stem cells, the efficacy of treatment may be low. In addition, low tyrosine kinase inhibitor doses that use to prevent severe side effects, do not suppress alternative survival pathways that are active in CML cells. It is clear that there is a need to develop multi-targeted treatment approaches instead of single drug ponatinib therapies for patients with CML who exhibit sub-optimal response to tyrosine kinase inhibitor treatment. VS-5584, an inhibitor of the PI3K/Akt/mTOR pathway known to be active in CML, is a potent, selective dual inhibitor that preferably kills cancer stem cells. In this thesis study, it was aimed to determine an in vitro combination therapy which could overcome the main causes of failure of CML therapy. For this purpose, the effects and roles of simultaneous inhibition of BCR-ABL1 and PI3K/Akt/mTOR pathways by combination of ponatinib and VS-5584 on elimination of CML and leukemic stem cells has been investigated at the molecular level. Cytotoxic effects of ponatinib, VS-5584, and their combinations on CML cell model K562, leukemia stem cell model LSC, and normal hematopoietic stem cell model HSC were measured with WST-8 test. Combination indices were evaluated by CalcuSyn isobologram analysis. Apoptotic effects of IC50, IC75, ED50, ED75 doses were evaluated with Annexin V and active caspase-3 assays by using flow cytometry, effects on cell cycle were evaluated by using PI assay with flow cytometry and by cyclinD1, p27 antibodies with western blot. Changes in the activities of the PI3K/Akt/mTOR pathway proteins were measured by quantitation of the phosphoprotein levels with the antibody array. Changes in the activity of transcription factors were quantified by dual luciferase activity after transfection of appropriate vectors. qRT-PCR was performed to detect changes in mRNA levels of the target genes of the altered transcription factors. Ponatinib and VS-5584 were found cytotoxic for K562 and LSC cells in time and dose dependent manner. Unlike ponatinib, VS-5584 was shown to be significantly less cytotoxic in the healthy HSC cell line. ED50 doses were additive, ED75 and ED90 showed synergistic effect. In addition to ponatinib-induced apoptosis, VS-5584 was shown to cause significant G0/G1 arrest. Especially in LSC cell line it was shown that while ponatinib was insufficient to suppress Akt pathway, combination of ponatinib and VS-5584 inhibited PI3K/Akt/mTOR pathway through inhibiting the phosphorylation of several proteins, particularly S6, S6K, BAD. It was found that the Elk-1/SRF, AP-1 and NFkB transcription factors displayed markedly elevated activity in leukemia and leukemia stem cell models. In addition to these transcription factors which may be a selective intervention point, suppression of Myc/Max, CREB, STAT3 and E2F/DP1 transcription factors was also observed in K562 and LSC cells after the combination treatment. It was determined that the activities of C/EBP and FOXO1 transcription factors in K562 cells and of C/EBP and p53 transcription factors in LSC cells increase following the application of the combination. Many genes whose expressions are regulated by these transcription factors have also been shown altered mRNA levels. The results showed that regulation of transcription factors involved in survival pathway with ponatinib and VS-5584 contributed to anti-proliferative and apoptotic processes. We believe that the combination of ponatinib and VS-5584 can be a promising treatment strategy that can selectively inhibit the uncontrolled proliferation of CML blasts and stem cells and reduce the known serious side effects of ponatinib

The lncRNA expression profile signature of leukemia stem cells is altered upon PI3K/mTOR inhibition: an in vitro and in silico study

Genetic and/or epigenetic alterations in hematopoietic stem cells (HSCs) contribute to leukemia stem cell (LSC) formation. We aimed to identify alterations in the lncRNA expression profile signature of LSCs upon inhibition of PI3K/Akt/mTOR signaling, which provides selective advantages to LSCs. We also aimed to elucidate the potential interaction networks and functions of differentially expressed lncRNAs (DELs). We suppressed PI3K/Akt/mTOR signaling in LSC and HSC cell-lines by specific PI3K/mTOR dual-inhibitor (VS-5584) and confirmed the inhibition by antibody-array. We defined DELs by qRT-PCR. Increased SRA, ZEB2-AS1, antiPeg11, DLX6-AS, SNHG4, and decreased H19, PCGEM1, CAR-Intergenic-10, L1PA16, IGF2AS, and SNHG5 levels (|log2fold-change|>5) were strictly associated with PI3K/Akt/mTOR pathway inhibition in LSC. We performed in silico analyses for DELs. ZEB2-AS1 was found to be specifically expressed in normal bone marrow and predominantly lower in leukemic cell-lines. Three sub-clusters were identified for DELs and they were associated with abnormality of multiple cell lineages in the bone marrow. DELs were most highly enriched for glucuronidation Reactome pathway and ascorbate and aldarate metabolism and inositol phosphate metabolism KEGG pathways. Transcription factors, MBD4, NANOG, PAX6, RELA, CEBPB, and CEBPA were predicted to be associated with the DEL profile. SRA was predicted to interact with CREB1, RARA, and PPARA. The possible DELs' targets were predicted to form six ontological groups, be highly enriched for phosphoprotein, and be involved in PPAR signaling pathway and ChREBP regulation by carbohydrates and cAMP. These results will help to elucidate the roles of lncRNAs in the mechanisms that provide selective advantages to leukemia stem cells

Effect of CCT137690 on long non-coding RNA expression profiles in MCF-7 and MDA-MB-231 cell lines

Long non-coding RNAs (lncRNAs) are involved in a range of biological processes, such as cellular differentiation, migration, apoptosis, invasion, proliferation, and transcriptional regulation. The aberrant expression of lncRNAs plays a significant role in several cancer types. Aurora kinases are increasingly expressed in various malignancies; accordingly, the inhibition of these enzymes may represent a novel approach for the treatment of various cancers. CCT137690, an Aurora kinase inhibitor, displays an anti-proliferative activity in human cancer cell lines. The aim of the present study was to investigate the anti-proliferative and cytotoxic effects of CCT137690 on estrogen receptor (ER)-positive human breast cancer cell line (MCF-7) and ER-negative human breast cancer cell line (MDA-MB-231). In addition, this study was targeted toward determining the changes induced in lncRNA expression levels following the initiation of Aurora kinase inhibitor treatment. The cytotoxic effects of CCT137690 were determined by means of the xCELLigence system. Furthermore, the anti-proliferative role of CCT137690 in breast cancer was investigated by checking the changes in lncRNA expression profiles using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The half-maximal inhibitory concentrations (IC50) of CCT137690 were determined as 4.5 μM (MCF-7) and 7.27 μM (MDA-MB-231). Several oncogenic lncRNAs (e.g., PRINS, HOXA1AS, and NCRMS) were downregulated in both ER-negative and ER-positive cell lines. On the other hand, tumor suppressor lncRNAs (e.g., DGCR5 and IGF2AS) were upregulated in the ER-positive cell line. After CCT137690 treatment, HOXA11AS and PCAT-14 lncRNAs were downregulated in the ER-positive cell lines. In addition, MER11C, SCA8, BC200, HOTAIR, PCAT-1, UCA1, SOX2OT, and HULC lncRNAs were downregulated in the ER-negative cell lines. The results of the present study indicated that Aurora kinase inhibitor CCT137690 could be a potential anti-cancer agent for breast cancer treatment

Design, synthesis, cytotoxic activity, and apoptosis inducing effects of 4- and N-substituted benzoyltaurinamide derivatives

In this study, a group of 4-substituted benzoyltaurinamide derivatives were designed, synthesized, and investigated for their anticancer activity against three cancer cell lines and one nontumorigenic cell line by MTT assay. Among the final compounds, methoxyphenyl derivatives 14, 15, 16 were found to be effective against all the tested cancerous cell lines with promising selectivity. The most active compounds were further evaluated to determine the molecular mechanism of their anticancer activity by using western blot assay and the Annexin V-FITC/PI test. Compound 14 (in SH-SY5Y and MDA-MB-231 cell lines) and 15 (in SH-SY5Y cell line) were found to induce intrinsic apoptotic pathway by upregulating BAX, caspase-3, and caspase-9, while downregulating Bcl-2 and Bcl-xL expression levels. According to mechanistic studies, compounds displayed their anticancer activity via three different mechanisms: a. caspase-dependent, b. caspase-independent, and c. caspase-dependent pathway that excluded caspase-9 activation. As a result, this study provides interesting data which can be used to design new taurine-based anticancer derivatives

Genistein-Induced Apoptosis Affects Human Telomerase Reverse Transcriptase Activity in Acute Promyelocytic Leukemia

BACKGROUND/AIMS The purpose of this study was to research the effects of genistein on telomerase activity and apoptosis in an acute promyelocytic leukemia cell line (HL-60). MATERIAL and METHODS In HL-60 cells, the cytotoxic effect of commercially available genistein was evaluated by the XTT method. The XTT method is a Cell Proliferation Assay. The Annexin V-EGFP method was used to determine apoptosis. The human telomerase reverse transcriptase (hTERT) is a marker of telomerase activity. hTERT gene expression analysis was performed by LightCycler real-time RT-PCR. RESULTS In HL-60 cells, the IC50 of genistein was found to be 50 ?M at 72 hours. It was observed that the induction of apoptosis was 4.25-fold higher compared to the genistein untreated cells used as the control group. Compared to the control group, hTERT activity was found to decrease by 5.16, 3.81 and 5.04-fold at 24, 48 and 72 hours, respectively. CONCLUSION Induced apoptosis of HL-60 cells by the reduction of human telomerase reverse transcriptase activity may be a beneficial parameter in leukemia patient

Cytotoxic effects of silver nanoparticles synthesized using Asphodelus aestivus Brot. aqueous extract

The aim of the study is to examine the cytotoxic, apoptotic effects and gene expressions of Asphodelus aestivus water extract (ASP) and silver nanoparticles (AgNPS) on decided cancer lines. Breast cancer cell lines MCF-7 and MDA-231; melanoma cancer lines MEWO and CHL-1; fibroblast cancer lines WI-38 and HEL 299 were selected for biological activities. xCELLigence system was used for cytotoxicity. Annexin V-EG FP Apoptosis detection kit was used for apoptosis and gene expressions were assessed by real time online RT-PCR by using cancer cell lines Qiagen kits. AgNPS showed significant cytotoxicity in all cell lines. The most prominent apoptosis was determined in MCF-7 cell line for AgNPS. It has been observed that the most important progress in gene expression, the suppression capacity of MUC1-C, a breast cancer-associated oncoprotein, is greatly increased by nanoparticle formation. In addition, cells that were not affected at all in MDA-MB-231 breast cancer oncoprotein started to suppress with nanoparticles. In conclusion, it was determined that silver nanoparticles increased the effect especially in breast cancer cell lines and could be considered for use.This research was supported by TUBITAK (project number SBAG-114S730) . We are thankful to FABAL (Ege University, Faculty of Pharmacy Pharmaceutical Sciences Research Centre) and Izmir Institute of Technology, Center for Materials Research.TUBITAK [SBAG-114S730]; FABAL (Ege University, Faculty of Pharmacy Pharmaceutical Sciences Research Centre); Izmir Institute of Technology, Center for Materials Researc