The decreasing of sensitivity or resistance to tamoxifen occured after long-term treatment in breast cancer. One of the major factor in tamoxifen resistance is over expression of efflux transporter ...P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). Curcumin has known as inhibitor of P-gp and BCRP. The addition of curcumin to the tamoxifen resistant cells is expected to increase the sensitivity of breast cancer cells to tamoxifen. This study aim to know the effect of curcumin in increasing the cell sensitivity to tamoxifen through inhibition of P-gp and BCRP transporter efflux. MCF-7 breast cancer cell line was induced with tamoxifen 1 µM for 10 passage (MCF-7(T)), then cell viability and mRNA expression of P-gp and BCRP were analyzed. To the MCF-7(T) cells, curcumin was given at of 5/10/20 µM with or without tamoxifen for 5 days and cell viability and mRNA expression of P-gp and BCRP were analyzed on day 5th. As positive control, verapamil 50 µM was used as P-gp inhibitor, ritonavir 15 µM and nelfinavir 15 µM were used as BCRP inhibitor. The results showed that MCF-7(T) cells sensitivity to tamoxifen decreased with 11.8 times, the cell viability increased 10.82 fold and mRNA expression of P-gp and BCRP increased 4.04 fold. Then after administration of curcumin with or without tamoxifen for 5 days, the cell viability and the mRNA expression of P-gp and BCRP decreased. As conclusion, curcumin increased the sensitivity of MCF-7(T) to tamoxifen characterized by the decreasing of cell viability and mRNA expression of P-gp and BCRP. However, the administration of combination of curcumin with tamoxifen was more potent than just curcumin. The increased sensitivity was estimated at least partly through the inhibition of P-gp and BCRP mRNA expression by curcumin
Crohn's disease and ulcerative colitis are both chronic inflammatory disorders of the gastrointestinal tract, the main causes of which remain unknown. Crohn's disease and ulcerative colitis are ...characterized by cell-mediated immune response against the luminal bacteria. It is suggested that expression levels and function of P-glycoprotein, encoded by the MDR1 gene, are important for protection of the gut against xenobiotics and bacterial toxins. Therefore, the mutations of the MDR1 gene are thought to be related with the pathogenesis of inflammatory bowel disease. The aim of this study was to investigate the G2677T/A polymorphism in the MDR1 gene in Turkish patients with inflammatory bowel disease and a healthy control group.
In our study, the genotypes of endoscopically or histopathologically diagnosed Crohn's disease (n: 35; 14 F, 21 M) and ulcerative colitis (n: 82; 36 F, 46 M) patients and of 70 healthy individuals (39 F, 31 M) were compared. In the patient and control groups, polymerase chain reaction restriction fragment length polymorphism analysis was performed for two polymorphisms (G2677T and G2677A) of the MDR1 gene.
In this study, the frequency of alleles at position 2677 of the MDR1 gene, which has a triallelic polymorphism, was not found to be significantly different between the patient and the healthy control groups. Moreover, the 2677A allele was not detected in either the patient group or the healthy control group.
In this study, the G2677T/A polymorphism observed in the MDR1 gene was not found to be a risk factor for Crohn's disease or ulcerative colitis.
Genus Euphorbia, with more than 2,000 species of annual, biennial or perennial flowering plants belonging to the family Euphorbiaceae, is one of the largest and most diverse genera not only in the ...spurge family but in the entire plant kingdom. Plants of this genus are used in traditional medicine since ancient times. Phytochemical studies up to now have shown that the species of the genus Euphorbia produce a variety of secondary metabolites, including various terpenes (sesquiterpenes, diterpenes and triterpenes), steroids, cerebrosides, glycerols and phenolic compounds (phloracetophenones, flavonoids, tannins, coumarins). These compounds perform many different activities, including modulability of multidrug resistance, microtubuleinteracting activity, antiproliferative, cytotoxic, antiviral, antimicrobial, anticancer, antiinflammatory, tumor promoting and proinflammatory effects. Genus Euphorbia is a rich source of jatrophanes, macrocyclic diterpenes with basic trans-bicyclo10.3.0pentadecane structure, which are characterized by the existence of a flexible twelve membered ring. These are very functionalized compounds with various oxygenation stages; in plants are usually found in the form of polyhydroxylated diterpenes esterified with various acids (acetyl, propanoil, n-butanoyl, isobutanoyl, isovaleryl, angeloyl, tigloyl, benzoyl, nicotinoyl derivatives, etc...), and are sometimes called jatrophane polyesters. In this dissertation, jatrophane diterpenes from Euphorbia dendroides L. originating from Montenegro were investigated. The study comprises investigation of the aerial parts and of the milky latex. From the aerial parts of E. dendroides six new jatrophanes, namely euphodendrophanes A - F (1 - 5, and 16), were isolated and characterized. From the milky latex of E. dendroides, in addition to 1, 2, and 16, the constituents of the aerial parts, thirteen new jatrophanes, euphodendrophanes G - S (6 - 15, and 17 - 19), were isolated and characterized. All isolated jatrophanes have very similar structures. Jatrophanes 1 15 are differing only in the type, number and position of substituents, while jatrophanes 16 19 differ from them in that they contain endocyclic (Δ5) instead of exocyclic (Δ6(17)) double bond. Compounds 1 14 are penta- or hexaesterified jatrophane-type polyols containing a keto carbonyl group at C-14, an endocyclic 11 Eand an exocyclic C- 6(17) double bond. Compound 15 is triesterified jatrophane-type polyol containing two keto carbonyl groups (at C-9 and C-14), an endocyclic 11 Eand an exocyclic C-6(17) double bond. Compounds 16 19 are heptaesterified jatrophane-type polyols containing a ketone carbonyl group at C-14 and two endocyclic double bonds (Δ5 and Δ11). The biological activities of the representative set of isolated jatrophanes have been assessed. Compounds 1 – 7, 9, 10, and 16 – 19 were evaluated for their capacity to inhibit in vitro growth of six human cancer cell lines: non-small cell lung carcinoma (NCIH460 and NCI-H460/R), colorectal carcinoma (DLD-1 and DLD-TxR), and glioblastoma (U-87 and U87-TxR) using a sulforhodamine B (SRB) assay. Paclitaxel was used as a positive control. Compounds 2, 6, 9, 10, and 17 19 more efficient than the others inhibited the growth of both NCI-H460 and NCI-H460/R cells (IC50 4.7 16.2 and 8.3 26.8 μM, respectively). Only the sensitivity to 16 was decreased considerably against the NCI-H460/R cell line. Compounds 10 and 18 the most efficient of the tested jatrophanes inhibited the growth of U-87 cells (IC50 10.0 and 8.6 μM, respectively) and quite effectively inhibited the growth of U87-TxR cells (IC50 57.1 and 28.5 μM, respectively). Jatrophane diterpenoids 1, 2, 6 – 10, and 15 – 19 were tested for their P-gp inhibiting activity on three different human MDR cancer cell lines: NCI-H460/R, DLD1-TxR and U87-TxR. Standard functional assay with Rhodamine (Rho) 123 as a fluorescent substrate for P-gp was assessed by flow-cytometry. Among the investigated jatrophanes, compounds 6 i 9 were found to be the most powerful inhibitors of P-gp in NCI-H460/R and DLD-TxR cells. Their efficacy was significantly higher than R(+)- verapamil’s taken as a positive control. Tested compounds (1, 2, 6 – 10, and 16 – 19), along with positive controls (verapamil and tariquidar), were moderately active against P-gp pump in glioblastoma U87-TxR cell line. Only compound 15 was ineffective. The interaction and reversal potential of compounds 1, 2, 6, 8, 18 and 19 in simultaneous treatment with paclitaxel, and effects of 1 and 2 in simultaneous combination with doxorubicin were examined. All jatrophanes induced the increase in paclitaxel or doxorubicin sensitivity in resistant NCI-H460/R cell line. These results point to the potential of tested jatrophanes to reverse paclitaxel and doxorubicin resistance in the MDR cancer cell line used. Preliminary structure–activity relationship showed no obvious difference in the activity of jatrophanes with 6(17) exo- (1, 2, 6 – 10, 15), and those with 5 endo double bond (16 – 19), what suggests that double bond position make little change in activity, rather the oxygenation pattern governs the activity. The biological evaluation of two groups of exo jatrophanes with identical structures except for the substitution on C-8, namely 1, 6 – 8 and 2, 9 and 10, have shown dramatic increase in the activity upon substitution of either acetyloxy, isobutanoiloxy or nicotinoiloxy with benzoyloxy group at C-8. Within the set of jatrophanes with 6(17) exo double bond ((1, 2, 6 – 10, 15)), the compounds 6 and 9 with benzoyloxy group at C-8 were the most effective inhibitors of P-gp activity. Among tested jatrophanes with 5 endo double bond (16 – 19) the most powerful inhibitor of P-gp was 19 with benzoyloxy group at C-9. Substitution of propanoyloxy by isobutanoyloxy group at C-3 do not influence on inhibitory activities of compounds 1 i 2 or 6 i 9. However substitution of acetyloxy by propanoyloxy group at C-3 completely abolished inhibitory effect of 17 compared with 16. Poor activity of compound 15 confirms the relevance of the substitution pattern at C-7, C-8 and C-9. The lack of substitution at these positions dramatically decreased the MDR-modulatory activity. Within the set of jatrophanes isolated and tested in this work were closely related compounds based on structurally homogeneous skeleton with main differences in the substitution pattern on medium sized ring. Thus it can be stressed that the activity is strongly affected by the benzoyloxy group at the positions C-8 and C-9 for jatrophanes with exo- and endo double bond, respectively.
Rod Euphorbia, sa više od 2.000 vrsta jednogodišnjih, dvogodišnjih ili višegodišnjih cvetnica koje su članovi familije Euphorbiaceae, jedan je od najvećih i najraznovrsnijih rodova ne samo u svojoj familiji, već u celom biljnom carstvu. Biljke ovog roda koriste se u tradicionalnoj medicini još od antičkih vremena. Dosadašnja fitohemijska proučavanja pokazala su da vrste roda Euphorbia proizvode veoma raznovrsne sekundarne metabolite, uključujući različite terpene (seskviterpeni, diterpeni i triterpeni), steroide, cerebrozide, glicerole i fenolna jedinjenja (floracetofenoni, flavonoidi, tanini, kumarini). Mnoga od ovih jedinjenja pokazuju biološke aktivnosti, a najvažnije su reverzija višestruke rezistencije na lekove, antimitotsko, antiproliferativno, citotoksično, antivirusno, antimikrobno, antitumorsko, antiinflamatorno, tumor promotorsko i proinflamatorno dejstvo. Vrste iz roda Euphorbia su bogat izvor jatrofana, makrocikličnih diterpena osnovne trans-biciklo10.3.0pentadekanske strukture, za koje je karakteristično postojanje veoma fleksibilnog dvanaestočlanog prstena. To su veoma funkcionalizovana, različito oksigenovana jedinjenja; u biljkama se obično nalaze u obliku polihidroksilovanih diterpena esterifikovanih različitim kiselinama (acetil, propanoil, nbutanoil, izobutanoil, izovaleril, angeloil, tigloil, benzoil, nikotinoil derivati, itd.), zbog čega su poznati i pod nazivom jatrofanski poliestri. U okviru ove disertacije ispitivani su jatrofanski diterpeni iz vrste Euphorbia dendroides L. poreklom iz Crne Gore koja do sada nije fitohemijski proučavana. Proučavanje je obuhvatalo dve celine: nadzemni deo i mlečni lateks. Iz nadzemnog dela E. dendroides izolovano je i okarakterisano šest novih jatrofana, koji su nazvani eufodendrofani A – F (1 – 5, 16), a iz mlečnog lateksa E. dendroides izolovano je i okarakterisano trinaest novih jatrofana, eufodendrofani G – S (6 – 15, 17 – 19), kao i tri jatrofana izolovana iz nadzemnih delova (1, 2, 16). Izolovani jatrofani imaju vrlo slične strukture. Jatrofani 1 – 15 se međusobno razlikuju samo po vrsti, broju i položaju supstituenata, dok se jatrofani 16 – 19 od njih razlikuju po tome što sadrže endocikličnu (Δ5) umesto egzociklične (Δ6(17)) dvostruke veze. Jedinjenja 1 – 14 su penta- ili heksaesterifikovani jatrofanski polioli koji sadrže keto grupu u položaju C-14, jednu endocikličnu 11 E– i jednu egzocikličnu C-6(17) dvostruku vezu. Jedinjenje 15 je triesterifikovani jatrofanski poliol koji sadrži dve keto grupe (u položajima C-9 i C-14), jednu endocikličnu 11 E– i jednu egzocikličnu C- 6(17) dvostruku vezu. Jedinjenja 16 – 19 su heptaesterifikovani jatrofanski polioli koji sadrže keto grupu u položaju C-14 i dve endociklične dvostruke veze (Δ5 i Δ11). Neka od izolovanih jedinjenja su podvrgnuta testovima za ispitivanje bioloških aktivnosti. Sposobnost jedinjenja 1 – 7, 9, 10 i 16 – 19 da in vitro inhibiraju rast senzitivih i rezistentnih ćelijskih linija humanog karcinoma pluća (NCI-H460 i NCI-H460/R), karcinoma debelog creva (DLD1 i DLD-TxR) i glioblastoma (U87 i U87-TxR) procenjivana je pomoću sulforodamin B (SRB) testa. Kao kontrolna supstanca upotrebljen je paklitaksel. Od ukupno trinaest analiziranih jatrofana, jedinjenja 2, 6, 9, 10, 17 – 19 efikasnije od ostalih inhibiraju rast kako NCI-H460 tako i NCI-H460/R ćelija (IC50 4,7 16,2 μ
Multi-drug resistance (MDR) is a major obstacle to successful cancer treatment. The efficacy of paclitaxel (PTX) is often limited by appearance of drug resistance. The aim of this study was to ...explore molecular and phenotypic alterations during development of MDR induced by PTX in human colon carcinoma (DLD1) and glioblastoma (U87) cell lines. We also tested the usefulness of developed MDR models in the evaluation of four anti-cancer agents. Continuous treatment with PTX led to the development of MDR in both tested cancer cell lines that became resistant to structurally and functionally unrelated chemotherapeutics. After confirmation of the cross-resistance in newly established DLD1-TxR and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased. Over-expression of P-glycoprotein (P-gp), coded by mdr1, was observed in both MDR cancer cell lines. Flow cytometry analyzes showed that the accumulation of P-gp substrates (rhodamine 123 and doxorubicin) in DLD1-TxR and U87-TxR was significantly lower compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression and glutathione (GSH) concentration was observed in U87-TxR. Vascular Endothelial Growth Factor (VEGF) secretion was inhibited by single PTX treatment of colon cancer and in continuous treatment of glioblastoma cell lines. The analysis of cell cycle kinetics revealed extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX treatment, while glioblastoma cells died through interphase (G1, S or G2). The MDR cancer cell lines acquired novel structural or numerical chromosomal aberrations. Polyploidy reduction was observed after development of MDR in U87-TxR. Losses of 6q in both resistant cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also revealed. We evaluated the anti-cancer activities and MDR reversal potential of the Akt inhibitor (GSK690693), the Ras inhibitor (Tipifarnib) and two P-gp inhibitors (jatrophane diterpenoids Euphodendrophane H-Euph H and Euphodendrophane S -Euph S). Their effects vary due to the cell-type differences, existence of MDR phenotype or tumor suppressors’ alterations. Tipifarnib, Euph H and S, significantly sensitized MDR cancer cells to PTX. In conclusion, continuous PTX treatment caused the over-expression of P-gp and acquisition of MDR in colon cancer and glioblastoma cell lines. MDR cancer cells obtained new molecular and cytogenetic characteristics, while some mechanisms of MDR and tumor progression such as GSH detoxification system and VEGF secretion were suppressed. The results implicate the PTX treatment as an important clinical tool for colon carcinoma and glioblastoma treatment even in the presence of MDR. Despite limitations discussed in literature due to the usefulness of MDR in vitro models, further examinations of changes provoked by artificially developed MDR are still emerging. Therefore, our MDR cancer cell lines present valuable tool for pre-clinical evaluation of new anti-cancer agents.
Glavni uzrok neuspeha hemioterapije u lečenju kancera je pojava višestruke (engl. „multi-drug“) rezistencije (MDR). Efikasnost paklitaksela (PTX) je često ograničena pojavom rezistencije. Cilj ove doktorske disertacije je bio ispitivanje molekularnih i fenotipskih promena u toku razvoja MDR-a indukovanih PTX-om kod ćelijskih linija humanog karcinoma debelog creva (DLD1) i glioblastoma (U87). Takođe je testirana upotrebljivost dobijenih MDR modela u evaluaciji četiri anti-kancer agensa. Kontinuirani tretman PTX-om doveo je do razvoja MDR-a kod obe ispitivane ćelijske linije koje su postale rezistentne na strukturno i funkcionalno različite hemioterapeutike. Nakon potvrde prisustva ukrštene rezistencije kod novouspostavljenih ćelijskih linija DLD1-TxR i U87-TxR, analizirana je ekspresija membranskih trasportera uključenih u razvoj MDR-a na nivou iRNK. Ćelije su imale povišen nivo ekspresije mdr1 gena i smanjen nivo ekspresije mrp1 gena. Prekomerna ekspresija P-glikoproteina (P-gp), koji kodira mdr1 gen, je uočena kod obe MDR ćelijske linije. Analiza na protočnom citofluorimetru je pokazala da je akumulacija Pgp supstrata (rodamina 123 i doksorubicina) kod DLD1-TxR i U87-TxR ćelija značajno manja u poređenju sa odgovarajućim parentalnim ćelijama, DLD1 i U87. Značajno smanjenje ekspresije gst-π gena i koncentracije glutationa (GSH) je uočeno kod U87- TxR ćelija. Sekrecija vaskularnog endotelijalnog faktora rasta (VEGF) je inhibirana u jednokratnom tretmanu kod ćelijskih linija karcinoma debelog creva i u kontinuiranom tretmanu kod ćelijske linije glioblastoma. Analiza ćelijskog ciklusa je pokazala da je jednokratni tretman PTX-om kod ćelijskih linija humanog karcinoma debelog creva praćen porastom subG0 faze, odnosno povećanjem procenta mrtvih ćelija, dok je kod ćelija glioblastoma došlo do umiranja tokom interfaze (G1, S ili G2). MDR tumorske ćelijske linije su stekle nove strukturne i numerićke hromozomske aberacije. Sticanje MDR fenotipa kod U87-TxR ćelija je praćeno smanjenem jednog nivoa ploidije. Takođe je uočen gubitak hromozomskog regiona 6q kod obe rezistentne ćelijske linije, kao i inaktivacija p53 tumor spresor gena kod U87-TxR ćelija i PTEN tumor supresor gena kod DLD1-TxR ćelija. Dalje je analizirana anti-kancer aktivnost i potencijal za reverziju MDR-a Akt inhibitora (GSK690693), Ras inhibitora (Tipifarnib) i dva inhibitora P-gp-a (jatrofanski diterpenoidi Eufodendrofana H-Euph H i Eufodendrofane S -Euph S). Njihova efikasnost varira u zavisnosti od razlika u tipu ćelija, postojanja MDR fenotipa ili promena u tumor supresorima. Tipifarnib, Euph H i S su značajno povećali senzitivnost MDR ćelijskih linija na PTX. Kontinuirani tretman PTX-om dovodi do prekomerne ekspresije P-gp-a i sticanja MDR fenotoipa kod ćelijskih linija humanog karcinoma debelog creva i glioblastoma. MDR ćelije su stekle nove molekularne i citogenetske karakteristike, dok su suprimirani neki mehanizmi MDR-a i progresije tumora kao što su GSH detoksifikacioni sistem i sekrecija VEGF-a. Ovi rezultati ukazuju da tretman PTX-om može imati veliki značaj u terapiji karcinoma debelog creva i glioblastoma, čak i u prisustvu MDR-a. Uprkos ograničenjima o kojima se govori u literaturi u vezi upotrebljivosti MDR in vitro modela, ispitivanja promena izazvanih veštačkim razvojem MDR-a su i dalje neophodna i aktuelana. Zbog toga, ove MDR tumorske ćelijske linije predstavljaju značajan eksperimentalni model za testiranje novih antikancer agenasa.
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