In vitro ispitivanje uloge enzima ciklooksigenaze-2 u etiopatogenezi i terapiji adenokarcinoma pluća i adenokarcinoma kolona

Abstract

Uvod: Ciklooksigenaza-2 (COX-2) predstavlja inducibilan COX izoenzim, čija je ekspresija u većini tkiva veoma niska, ali se povećava u stanjima akutne i hronične inflamacije, pod dejstvom brojnih fizičkih, hemijskih i bioloških faktora, uključujući i karcinogene. Povećana ekspresija enzima COX-2 i njegovog najvažnijeg produkta prostaglandina E2 (PGE2) detektovana je u malignim tumorima. Smatra se da metilacija DNK, epigenetski događaj posredovan enzimima DNK metiltransferazama (DNMT), ima važnu ulogu u transkripcionom „utišavanju” tumor-supresor gena koji su povezani sa COX-2 signalnim putevima. Cilj: In vitro utvrđivanje citotoksične aktivnosti celekoksiba, selektivnog COX-2 inhibitora, kao i 5-aza-2'-deoksicitidina (decitabina), demetilacionog agensa, na humanim ćelijskim linijama adenokarcinoma pluća (A549), adenokarcinoma kolona (HT-29) i fetalnih plućnih fibroblasta (MRC-5). Cilj jeste i ispitivanje ekspresije gena, koji su uključeni u proces kancerogeneze, u A549, HT-29 i MRC-5 ćelijama koje su tretirane celekoksibom, odnosno decitabinom, kao i njihovim kombinacijama sa prirodnim supstancama – silibininom i žučnim kiselinama (henodeoksiholnom (HDH) i ursodeoksiholnom kiselinom (UDH)). Metode: Citotoksičnost ispitivanih supstanci na A549, HT-29 i MRC-5 ćelijama evaluirana je MTT testom. Kvantifikacija genske ekspresije izvedena je metodom qRT-PCR, a analizirana je komparativnom ΔΔCt metodom, uz ACTB kao referentni gen. Statistička obrada rezultata je urađena jednofaktorskom analizom varijanse (ANOVA) sa Takijevim post-hoc testom. Rezultati: Celekoksib i decitabin ispoljavaju koncentracijski-zavisnu citotoksičnost, odnosno inhibiciju vijabilnosti i proliferacije malignih i normalnih ćelija. U A549 i HT-29 ćelijama, u poređenju sa rezultatima dobijenim u netretiranoj, kontrolnoj grupi ćelija, uočava se da je celekoksib smanjio ekspresiju gena koji su uključeni u sintezu COX-2 i PGE2 (PTGS2 i PTGES), a povećao ekspresiju gena zaduženog za katabolizam PGE2 (HPGD), koji kodira sintezu enzima 15 hidroksiprostaglandin dehidrogenaze (15-PGDH). Silibinin je pokazao sinergističko antiinflamatorno dejstvo sa celekoksibom u pogledu povećanja ekspresije HPGD gena u A549 i HT-29, ali i smanjenja ekspresije PTGS2 gena u HT-29 ćelijama. UDH je ispoljila potpuno sinergističko antiinflamatorno dejstvo sa celekoksibom u A549, dok se u HT-29 ćelijama taj sinergizam zapaža u pogledu smanjenja ekspresije PTGS2 i povećanja ekspresije HPGD gena. U obe maligne ćelijske linije celekoksib je smanjio nivo mRNK za proinflamatorni transkripcioni faktor NFκB, uz uočeno aditivno dejstvo kotretmana sa silibininom. Ispitivane žučne kiseline su to sinergističko dejstvo ispoljile samo u HT-29 ćelijama. Ispitivanje ekspresije tumor-supresor gena pokazalo je da je celekoksib povećao ekspresiju TP53 u A549 ćelijama, a u A549 i HT-29 PTEN ekspresiju. U obe ćelijske linije dodatak silibinina i UDH celekoksibu povećao je količine TP53 i PTEN. U A549 ćelijama i HDH je delovala sinergistički sa celekoksibom na povećanje nivoa ovih tumor-supresora. Celekoksib je izazvao povećanje količine mRNK za proapoptotski BAX gen, a smanjio količine mRNK za antiapoptotski BCL2 gen u A549 i HT-29 ćelijama. Silibinin i žučne kiseline su različito delovali na gene uključene u regulaciju procesa apoptoze, ali je dodatak ovih prirodnih supstanci celekoksibu neminovno usmeravao maligne ćelije ka apoptozi, posmatrajući odnos mRNK BAX/BCL2. Rezultati pokazuju da je tretman malignih ćelija celekoksibom povećao ekspresiju CDH1 gena, koji kodira sintezu tumor-supresorskog epitelnog proteina E-kadherina, a smanjio nivo gena zaduženih za sintezu važnih matriksnih metaloproteinaza (MMP2 i MMP9) koje imaju ulogu u tumorskoj invaziji i metastaziranju. Silibinin i UDH su pokazali povoljna dejstva na smanjenje metastatskog potencijala A549 i HT-29 ćelija, s obzirom na sinergistički uticaj sa celekoksibom u pogledu modulacije ekspresije navedenih gena. Decitabin je negativno delovao na količine mRNK za sva tri enzima DNMT (DNMT1, DNMT3A i DNMT3B) u A549 i HT-29 ćelijama. Rezultati pokazuju da je tretman A549 ćelija decitabinom, smanjujući ekspresiju transkripcionog represora ZEB1, doveo do aktivacije transkripcije CDH1 i HPGD tumor-supresorskih gena. Potpuno sinergističko dejstvo sa decitabinom u A549 ispoljili su silibinin i UDH. Zaključak: S obzirom na dobijene IC50 vrednosti za celekoksib, zaključuje se da su maligne ćelije osetljivije na selektivnu COX-2 inhibiciju, u poređenju sa normalnim ćelijama, što ukazuje na važnu ulogu enzima COX-2 u preživljavanju i proliferaciji ćelija adenokarcinoma pluća i kolona. Zaključuje se i da postoji mogućnost modulacije ekspresije gena uključenih u kancerogenezu primenom selektivnog COX-2 inhibitora. Pokazano je da demetilacioni agens ima ulogu u reaktivaciji epigenetski „utišanih” tumor-supresorskih gena koji kodiraju sintezu E-kadherina i 15-PGDH u ćelijama adenokarcinoma pluća. Takođe, potvrđena su sinergistička antineoplastična dejstva silibinina i žučnih kiselina sa celekoksibom i decitabinom, što otvara mogućnosti primene novih kombinovanih farmakoloških strategija u terapiji adenokarcinoma pluća i kolona.

Introduction: Cyclooxygenase-2 (COX-2) is an inducible COX isoenzyme, whose expression is very low in most tissues, but increases in states of acute and chronic inflammation, under the influence of numerous physical, chemical and biological factors, including carcinogens. Increased expression of the enzyme COX-2 and its most important product prostaglandin E2 (PGE2) has been detected in malignant tumors. DNA methylation, an epigenetic event mediated by DNA methyltransferases (DNMT) enzymes, is thought to play an important role in the transcriptional "silencing" of tumor-suppressor genes associated with COX-2 signaling pathways. Objective: In vitro determination of the cytotoxic activity of celecoxib, a selective COX-2 inhibitor, as well as 5-aza-2'-deoxycytidine (decitabine), a demethylating agent, on human cell lines of lung adenocarcinoma (A549), colon adenocarcinoma (HT-29) and fetal lung fibroblasts (MRC-5). The aim is also to examine the expression of genes involved in the process of carcinogenesis in A549, HT-29 and MRC-5 cells treated with celecoxib and decitabine, as well as with their combinations with natural substances – silibinin and bile acids (chenodeoxycholic (CDCA) and ursodeoxycholic acid (UDCA)). Methods: The cytotoxicity of the tested substances on A549, HT-29 and MRC-5 cells was evaluated by the MTT test. Quantification of gene expression was performed using the qRT-PCR method, and was analyzed using the comparative ΔΔCt method, with ACTB as a reference gene. Statistical processing of the results was done by one-factor analysis of variance (ANOVA) with Taki's post-hoc test. Results: Celecoxib and decitabine exhibit concentration-dependent cytotoxicity, i.e. inhibition of viability and proliferation of malignant and normal cells. In A549 and HT-29 cells, compared to the results obtained in the untreated, control group of cells, it was observed that celecoxib decreased the expression of the genes involved in the synthesis of COX-2 and PGE2 (PTGS2 and PTGES), and increased the expression of the gene responsible for the catabolism of PGE2 (HPGD), which encodes the synthesis of the enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Silibinin showed a synergistic anti-inflammatory effect with celecoxib in terms of increasing HPGD gene expression in A549 and HT-29, but also decreasing PTGS2 gene expression in HT-29 cells. UDCA exhibited a fully synergistic anti-inflammatory effect with celecoxib in A549, while in HT-29 cells this synergism was observed in terms of decreasing PTGS2 expression and increasing HPGD gene expression. In both malignant cell lines, celecoxib reduced the mRNA level for the pro-inflammatory transcription factor NFκB, with observed the additive effect of co-treatment with silibinin. The tested bile acids exhibited this synergistic effect only in HT-29 cells. Examining the expression of tumor-suppressor genes showed that celecoxib increased the expression of TP53 in A549 cells, and PTEN expression in A549 and HT-29. In both cell lines addition of silibinin and UDCA to celecoxib increased the amounts of TP53 and PTEN. In A549 cells CDCA also acted synergistically with celecoxib to increase the levels of these tumor-suppressors. Celecoxib caused an increase in the amount of mRNA for the proapoptotic BAX gene, and decreased the amount of mRNA for the antiapoptotic BCL2 gene in A549 and HT-29 cells. Silibinin and bile acids acted differently on the genes involved in the regulation of the apoptosis, but the addition of these natural substances to celecoxib inevitably directed the malignant cells towards apoptosis, observing the BAX/BCL2 mRNA ratio. The results show that the treatment of malignant cells with celecoxib increased the expression of the CDH1 gene, which encodes the synthesis of the tumor-suppressor epithelial protein E-cadherin, and decreased the level of the genes responsible for the synthesis of important matrix metalloproteinases (MMP2 and MMP9) that play a role in tumor invasion and metastasis. Silibinin and UDCA showed beneficial effects on reducing the metastatic potential of A549 and HT-29 cells, considering the synergistic effect with celecoxib regarding the modulation of the expression of the mentioned genes. Decitabine negatively affected mRNA levels for all three DNMT enzymes (DNMT1, DNMT3A, and DNMT3B) in A549 and HT-29 cells. The results show that the treatment of A549 cells with decitabine, by reducing the expression of the transcriptional repressor ZEB1, led to the transcriptional activation of CDH1 and HPGD tumor-suppressor genes. A complete synergistic effect with decitabine in A549 was exhibited by silibinin and UDCA. Conclusion: Taking into account the obtained IC50 values for celecoxib, it is concluded that malignant cells are more sensitive to selective COX-2 inhibition, compared to normal cells, which indicates an important role of the COX-2 enzyme in the survival and proliferation of lung and colon adenocarcinoma cells. It is also concluded that there is a possibility of modulating the expression of genes involved in carcinogenesis using the selective COX-2 inhibitor. It was shown that the demethylating agent has a role in the reactivation of epigenetically "silenced" tumor-suppressor genes that encode the synthesis of E-cadherin and 15-PGDH in lung adenocarcinoma cells. Also, the synergistic antineoplastic effects of silibinin and bile acids with celecoxib and decitabine were confirmed, which opens the possibility of applying new combined pharmacological strategies in the therapy of lung and colon adenocarcinoma.