Kanser tedavisinde kullanılan anti-mikrotübül ilaçlardan vinca alkaloidler ve paklitaksel’in DNA bağlanma aktiviteleri

Microtubules are an essential part of the intracellular cytoskeletal structure and possess unique polymerization dynamics that are critical for many cellular functions, including cell division. Anti-microtubule drugs that interfere with microtubule formation are important chemotherapeutic agents for the treatment of various cancer. These drugs that block mitosis seem to work by a common mechanism, which suppresses the dynamics of microtubules, slows cells, induces apoptosis and subsequently kills tumor cells. Vinca alkaloids (vinblastine, vincristine and vinorelbine) and Taxanes (paclitaxel) are two different classes of anti-microtubule drugs that cause microtubule dysfunction and inhibit cancer cell proliferation. The main activity of vinca alkaloids and taxanes result from their binding interactions with tubulin proteins. However, studies on DNA interactions of these anti-microtubule drugs are not sufficient. In this study, it was aimed to investigate the DNA binding activities of the vinca alkaloids (vinblastine, vincristine, vinorelbine) and paclitaxel.MATERIAL AND METHODS: The interactions of the drugs with DNA were analyzed by agarose gel electrophoresis assay. Three types of DNA were used in each experiment, including 100bp marker DNA, pUC19 plasmid DNA (2686 bp), and pBR322 plasmid DNA (4361 bp). After the DNAs were incubated with different concentrations of the drugs under certain conditions, agarose gel electrophoresis was performed. DNA band distributions were analyzed with a gel analysis system so that the drugs-DNA interactions could be interpreted.RESULTS: According to our results, it was found that among the vinca alkaloids, especially vinorelbine binds to DNA with higher activity than vincristine and vinblastine. The Vinca alkaloids have structural properties required for DNA binding activity and there is a similarity in their DNA binding models. However, the results showed that paclitaxel, which is from the taxane group, did not have DNA binding activity. This may be because the chemical structure of paclitaxel is not suitable for binding to DNA.CONCLUSIONS: The interaction of drugs with DNA play an important role in determining the pathways of drugs action and their ability to cause DNA damage. Consequently, the findings of our study will contribute to elucidating the effect mechanisms and the genotoxic potentials of these drugs, which are microtubule inhibitors.Mikrotübüller, hücre içi hücre iskeleti yapısının önemli bir parçasıdır ve hücre bölünmesi dahil birçok hücresel işlev için kritik olan benzersiz polimerizasyon dinamiklerine sahiptirler. Mikrotübüllere müdahale eden anti-mikrotübül ilaçlar, çeşitli kanserlerin tedavisi için çok önemli kemoterapötik ajanlardır. Mitozu bloke eden bu ilaçlar, mikrotübüllerin dinamiğini baskılayan, hücreleri yavaşlatan, apoptozu indükleyen ve ardından tümör hücrelerini öldüren ortak bir mekanizma ile çalışmaktadır. Vinka alkaloidleri (vinblastin, vincristin ve vinorelbin) ve Taksanlar (paklitaksel), mikrotübüllerin işlev bozukluğuna neden olan ve kanser hücresi proliferasyonunu inhibe eden iki farklı anti-mikrotübül ilaç sınıfıdır. Vinka alkaloidlerinin ve taksanların ana aktivitesi, tübülin proteinleri ile bağlanma etkileşimlerinden kaynaklanmaktadır. Ancak, bu anti-mikrotübül ilaçların DNA etkileşimleri ile ilgili çalışmalar yeterli değildir. Bu çalışmada, vinka alkaloidleri (vinblastin, vincristin, vinorelbin) ve paklitakselin DNA bağlanma aktivitelerinin araştırılması amaçlanmıştır.GEREÇ VE YÖNTEM: İlaçların DNA ile etkileşimleri agaroz jel elektroforez deneyi ile analiz edildi. Her deneyde, 100 bp marker DNA, pUC19 plazmid DNA (2686 bp) ve pBR322 plazmid DNA (4361 bp) dahil olmak üzere üç tip DNA kullanıldı. DNA'lar belirli koşullar altında farklı ilaç konsantrasyonları ile inkübe edildikten sonra agaroz jel elektroforezi yapıldı. İlaç-DNA etkileşimlerinin yorumlanabilmesi için DNA bant dağılımları jel analiz sistemi ile analiz edildi.BULGULAR: Sonuçlarımıza göre vinka alkaloidlerinden özellikle vinorelbinin, vinkristin ve vinblastine göre daha yüksek aktiviteyle DNA'ya bağlandığı bulunmuştur. Vinca alkaloidleri, DNA bağlanma aktivitesi için gerekli yapısal özelliklere sahiptir ve DNA bağlanma motiflerinde benzerlik vardır. Ancak sonuçlar, taksan grubundan olan paklitakselin DNA bağlama aktivitesine sahip olmadığını gösterdi. Bunun nedeni, paklitakselin kimyasal yapısının DNA'ya bağlanmaya uygun olmaması olabilir.SONUÇ: İlaçların DNA ile interaksiyonu, ilaçların etki yollarını ve DNA hasarına neden olma yeteneklerini belirlemede önemli bir rol oynar. Sonuç olarak, çalışmamızın bulguları mikrotübül inhibitörü olan bu ilaçların etki mekanizmalarını ve genotoksik potansiyellerini aydınlatmaya katkı sağlayacaktır

Efficacy of multi-drug resistance transporters and glutathione s-transferase P-1 at developing bortezomib resistance in multiple myeloma cell lines

Multiple myeloma (MM) is a hematologic cancer characterized by the accumulation of malignant plasma cells. Bortezomib is the most effective chemotherapeutic drug used in treatment. However, drug resistance hinders efficient chemotherapy. One of the factors causing resistance is overexpression of multidrug-resistance genes. In this study, the expression of P-gp, MRP-1, MRP-2, MRP-3, MRP-6, MRP-7 and GSTP1 genes were investigated in MM cells. MTT assay was performed to determine bortezomib cytotoxicity in multiple myeloma KMS20 (bortezomib-resistant) and KMS28 (bortezomib-sensitive) cell lines. RNA isolation and cDNA synthesis were realized and the expressions of genes were analyzed by Realtime-qPCR. The results of the study suggest that P-gp is the main factor responsible for bortezomib resistance. MRP-1 expression appeared after P-gp expression reached a certain limit. This study is the first report indicating that MRP-7 is associated with bortezomib resistance in MM. Furthermore, the results demonstrated that bortezomib may be excreted from the cells by MRP-transporters after binding to glutathione with GSTP1. These findings will allow to develop new treatment strategies to prevent bortezomib resistance in MM

Inhibition of apoptosis may lead to the development of bortezomib resistance in multiple myeloma cancer cells

Background: Multiple myeloma (MM), a malignancy of plasma cells, is the second most prevalent hematological cancer. Bortezomib is the most effective chemotherapeutic drug used in treatment. However, drug-resistance prevents success of chemotherapy. One of the factors causing drug-resistance is dysfunction of apoptotic-pathways. This study aimed to evaluate the relationship between expression levels of Bcl-2, Bax, caspase-3 and p-53 genes involved in apoptosis and the development of bortezomib-resistance in MM cell lines. Materials and methods: Multiple myeloma KMS20 (bortezomib-resistant) and KMS28 (bortezomib-sensitive) cell lines were used. 3-[4,5-Dimethylthiazol-2-yl] 1-2,5-diphenylte-trazolium bromide (MTT) assay was performed to determine IC50 values of bortezomib. RNAs were isolated from bortezomib-treated cell lines, followed by cDNA synthesis. Expression levels of the genes were analyzed by using q-Realtime-PCR. Results: As a result, Bcl-2/Bax ratio was higher in KMS20 (resistant) cells than in KMS28 (sensitive) cells. Expression of caspase-3 decreased in KMS20-cells, whereas increased in KMS28-cells. The results indicate that apoptosis was suppressed in resistant cells. Conclusion: These findings will enable us to understand the molecular mechanisms leading to drug-resistance in MM cells and to develop new methods to prevent the resistance. Consequently, preventing the development of bortezomib resistance by eliminating the factors which suppress apoptosis may be a new hope for MM treatment

Patterns of the expression of cyclin genes in bortezomib-sensitive and resistant cells of multiple myeloma

Multiple myeloma (MM) is a blood cancer defined by the accumulation of abnormal plasma cells in the bone marrow. Bortezomib is still one of the most effectual anti-cancer drugs used for the treatment of MM. However, drug-resistance prevents the efficacy of chemotherapy. One of the mechanisms causing drug-resistance is changes in regulatory factors involved cell-cycle-checkpoints. In this study, the expression levels of cyclin-D1, cyclin-D2, cyclin-A1, cyclin-E1 and cyclin-B1 genes in bortezomib-sensitive (KMS-28)/resistant (KMS-20) multiple myeloma cell lines and their effects on resistance were investigated. MTT-assay was performed to determine bortezomib cytotoxicity in both cell lines. RNA isolation and cDNA synthesis were done from bortezomib treated cells. Expressions of the genes were analyzed by Real-Time-qPCR. The results of the study showed that cyclin-A1 was not expressed in bortezomib-sensitive cells, but overexpressed in bortezomib-resistant cells. This study was the first to demonstrate a direct role of cyclin-A1 at the development of bortezomib-resistance in MM. It was observed that cyclin-D1 expression increased in KMS-28 and decreased in KMS-20 cells. The results of the study suggest that suppression of cyclin-D1 in KMS-20 cells prevents apoptosis formation and therefore causes resistance. However, we found no evidence of an association of the cyclin-D2, cyclin-E1 and cyclin-B1 genes with the development of resistance. Consequently, the findings of the study will enable us to better understand MM disease at the molecular level and to develop new treatment strategies to prevent bortezomib resistance

The combined effects of proteasome inhibitor bortezomib with topoisomerase I and II inhibitors on topoisomerase enzymes

Background/aim: DNA topoisomerases are ubiquitous enzymes that regulate conformational changes in DNA topology during essential cellular processes, and, for this reason, have been characterized as the cellular targets of a number of anticancer drugs. Bortezomib is a powerful proteasome inhibitor used in the treatment of hematological malignancies. In this study, we investigated the inhibitory effects of bortezomib on human topoisomerase I and II enzymes both alone and in combination modes with camptothecin and etoposide. Materials and methods: The interactions of these drugs with topoisomerase enzymes were evaluated by relaxation assay in cell-free systems. IC50 values of the drugs on topoisomerase enzymes were calculated using the S probit analysis program. Results: Bortezomib showed a very weak inhibition effect on topoisomerase I (IC50 = 87.11 mM). On the other hand, it had a strong inhibitory effect on topoisomerase II (IC50 = 1.41 mM). Our results indicated that bortezomib is effective not only on proteasome but also on topoisomerase II. In addition, bortezomib possesses an increased synergistic effect when used in combination with camptothecin and etoposide than when used alone. Conclusion: The results of this study point out that these data may build a framework for combination studies with bortezomib, camptothecin, and etoposide in the treatment of cancer...

Antiproliferative and genotoxic activities in L929 and HeLa cell lines, mutagenic effects in Salmonella strains of novel benzoxazole derivatives

Some novel fused heterocyclic compounds of 2,5-disubstituted-benzoxazole derivatives, which were previously synthesized by our group, were investigated for their mutagenic properties on Salmonella typhimurium TA 98 and TA 100 strains, cytotoxic activity in L929 and HeLa cell lines by Sulforhodamine B (SRB) cytotoxicity test, and genotoxic potentials in the comet assay. By using Ames/Salmonella assay in the presence of S9 fraction, B22 (5-nitro-2-(p-nitrobenzyl)benzoxazole) was found to be mutagenic in both S. typhimurium TA98 and TA100 strains at all tested doses. IC50 values which were evaluated by SRB cytotoxicity assay revealed that B11 (2-(p-nitrobenzyl)benzoxazole) (IC50 = 99.16 µM) was the most anti-proliferative compound on HeLa cancer cells. Compounds were also tested for their genotoxicity by using comet assay, and it was found that all the compounds had DNA-damaging genotoxic activity on HeLa cells. The comet assay results showed that B11 produced DNA damage at lower concentrations than the other compounds tested on HeLa cancer cells. The results obtained from all the tests suggest that B11 could be a good candidate as a new anticancer agent. © 2016, Colegio de Farmaceuticos de la Provincia de Buenos Aires. All rights reserved

A study on antiproliferative and genotoxic potentials in L929 and HeLa cell lines - the mutagenic activities in Salmonella strains of novel 2,5-disubstituted-benzoxazole derivatives

41st FEBS Congress on Molecular and Systems Biology for a Better Life -- SEP 03-08, 2016 -- Kusadasi, TURKEYWOS: 000383616900256FEB

Antitumor activity against human promyelocytic leukemia and in silico studies of some benzoxazines

Cancer is one of the deadliest diseases in the world today, and the incidence of cancer is increasing. Leukemia is a type of blood cancer defined as the uncontrolled proliferation of abnormal leukocytes in the blood and bone marrow. The HL-60 (human promyelocytic leukemia) cell line, derived from a single patient with acute promyelocytic leukemia, provides a unique in vitro model system for studying the cellular and molecular events involved in the proliferation and differentiation of leukemic cells. In this study, antitumor activities on the HL-60 of some of the resynthesized benzoxazine derivatives (BXN-01 and BXN-02) were investigated. The results of in vitro studies obtained were compared a standard drug, etoposide. In vitro results showed that BXN-01 and BXN-02 were found to be extremely effective compared to etoposide (IC50 value: 10 µM) with IC50 values of 5 nM and 25 nM, respectively. Furthermore, molecular docking studies were carried out for preliminary prediction of possible interaction modes between compounds and the active site of the target macromolecules, hTopo IIα, HDAC2, and RXRA. Then, in silico ADME/Tox studies were performed to predict drug-likeness and pharmacokinetic properties of BXN-01 and BXN-02. Communicated by Ramaswamy H. Sarma

Antitumor activities on HL-60 human leukemia cell line, molecular docking, and quantum-chemical calculations of some sulfonamide-benzoxazoles

WOS: 000413975300017PubMed: 27829297We previously synthesized some novel benzoxazole derivatives-containing sulfonamide. In this study, the compounds were investigated for their antitumor activities against the HL-60 human leukemia cells, using the MTT assay. Moreover, quantum chemical calculations using the DFT methods were applied for understanding the difference in antitumor activity. Additionally, molecular docking into active site of the DNA Topo II enzyme was performed on 3QX3. PDB file in order to find out possible mechanism of antitumor effect. According to all obtained results showed that compounds 1b, 1c, and 1d could be potential drug candidates as new antitumor agents, and are promising for cancer therapy.Scientific Research Project of Aksaray University [2014-022]; Scientific Research Project of Ankara University [16H0237002]This study was supported by the Scientific Research Project of Aksaray University [grant number: 2014-022] and the Scientific Research Project of Ankara University [grant number: 16H0237002]

Economic burden of gastric cancer in Turkey

WOS: 000413599900160Human papillomavirus (HPV) infection is a pre-requisite for vaginal and vulval cancers. The incidence of vaginal cancer in the UK has remained relatively stable over the past 25 years; however the age-standardised incidence of vulval cancer has been increasing. Whilst a body of literature exists for the epidemiology and aetiology of vaginal and vulval cancer, a dearth of evidence exists regarding the economic burden. The objective of the present study was to quantify the economic burden these cancers place on the NHS in England