Influence of exogenous overexpression of p21WAF1/Cip1 gene and various chemotherapeutics on proliferation of colon cancer cells

Protein p21 je primarno definiran kao inhibitor ciklin ovisnih kinaza te faktor koji u slučaju staničnog stresa (oštećenje DNA, oksidativni stres, kemoterapeutici i sl.) uzrokuje kočenje staničnog ciklusa. Noviji literaturni podaci ukazuju na važnu ulogu tog proteina u regulaciji replikacije DNA te vrlo kompleksnu ulogu u indukciji ili inibiciji stanične smrti, koja ovisi o tipu stanica, staničnom kontekstu i vrsti tretmana koji se koristi na tumorskim stanicama. Zbog mnogoznačne i često kontradiktorne uloge proteina p21, izuzetno je važno proučiti mehanizme njegovog djelovanja nakon oštećenja DNA različitim kemoterapeuticima kako bi se odredio njegov utjecaj na kemosenzitivnost tumorskih stanica, što bi moglo dovesti do usmjerenijih oblika liječenja tumora. U ovom smo radu, pratili utjecaj pet poznatih kemoterapeutika te povećane ekspresije gena p21 na rast tumorskih stanica debelog crijeva HCT116, SW480, SW620 i CaCo-2. Povećanu ekspresiju proteina p21WAF1/Cip1 postigli smo zaražavanjem stanica replikacijski nesposobnim adenovirusnim vektorima s ugrađenim genom p21WAF1/Cip1 (Ad5CMV-p21), dok je kao kontrola korišten vektor s ugrađenim genom za β-galaktozidazu. Kako bismo ispitali utjecaj egzogenog proteina p21 na učinak kemoterapeutika, stanice smo nakon infekcije adenovirusnim vektorom tretirali kemoterapeuticima kamptotecinom, doksorubicinom i cisplatinom u različitom rasponu koncentracija. Preživljenje stanica pratili smo MTT testom, a ekspresiju proteina p21 metodom Western blot. Svi su spojevi usporavali rast tumorskih stanica, pri čemu je 5-flurouracil pokazao najslabije, a kamptotecin najjače djelovanje. Pet dana nakon infekcije stanica vektorom Ad5CMV-p21 došlo je do značajnije inhibicije rasta staničnih linija HCT116 i SW620 u koncentracijama virusa višim od 60MOI. Povećana ekspresija gena p21WAF1/Cip1 izrazito je pojačala učinak cisplatine i kamptotecina te vrlo slabo doksorubicina u stanicama HCT116 i SW620, dok je sličan, ali slabije izražen učinak pokazan u stanicama CaCo-2. S druge strane, iako su se stanice SW480 pokazale najosjetljvijima na kemoterapeutike, njihov učinak nije pojačan u kombinaciji s pojačanom ekspresijom proteina p21 u toj staničnoj liniji.p21WAF1/Cip1 protein is primarily defined as a cyclin dependent kinase inhibitor which induces cell cycle arrest under stress conditions (DNA damage, oxidative stress, anticancer agents...). Recent studies point out an important role of p21 protein in regulation of DNA replication as well as very complex part in induction or inhibition of cell death, which depends on tretment type, cell type and genetic context. Since p21Waf1/Cip1 revealed equivocal and somewhat controversial role, it is a major issue to elucidate mechanism of it`s action after DNA damage caused by common chemotherapeutics in order to establish the influence of p21 to cell chemosensitivity , which coluld give rise to more targeted tumor therapy. In our expreiments we investigated the effects of five common chemotherapeutics and overexpression of exogenous p21WAF1/Cip1 on the growth of colon cancer cell lines HCT116, SW480, SW620, CaCo-2. Enhanced expression of protein p21WAF1/Cip1 was accomplished by infection with replication-defective adenoviral recombinant vector Ad5CMVp21, whereas Ad5CMVβ-gal was used as a control vector. In order to evaluate the effect of exogenous protein p21WAF1/Cip1 on the sensitivity to chemotheraputics, we treated cells with chemotherapeutics camptothecin, doxorubcin and cisplatin at different concetration range after infection wtih adenoviral vectors. Cells’ viability was monitored by MTT assay, and the expression of p21 protein by Western blot method. All of the tested chemotherapeutics inhibited growth of the tumor cells whereby 5-fluorouracil revealed least and camptothecin strongest effect. We detected significant growth inhibition of HCT116 and SW620 cells five days after infection with Ad5CMV-p21 at MOI 60. Furthermore, p21WAF1/Cip1 gene overexpression in HCT116 and SW620 cells distinctly enhanced cisplatin and camptotecin influence, whereas it had faint effect on doxorubicin influence. We detected similar but weaker effect of p21 on CaCo-2 cells. On the other hand, even though SW480 appeared to be the most sensitive to chemotherapeutics, their effect wasn`t enhanced when combined with p21WAF1/Cip1 overexpression in this cell line

Influence of exogenous overexpression of p21WAF1/Cip1 gene and various chemotherapeutics on proliferation of colon cancer cells

Protein p21 je primarno definiran kao inhibitor ciklin ovisnih kinaza te faktor koji u slučaju staničnog stresa (oštećenje DNA, oksidativni stres, kemoterapeutici i sl.) uzrokuje kočenje staničnog ciklusa. Noviji literaturni podaci ukazuju na važnu ulogu tog proteina u regulaciji replikacije DNA te vrlo kompleksnu ulogu u indukciji ili inibiciji stanične smrti, koja ovisi o tipu stanica, staničnom kontekstu i vrsti tretmana koji se koristi na tumorskim stanicama. Zbog mnogoznačne i često kontradiktorne uloge proteina p21, izuzetno je važno proučiti mehanizme njegovog djelovanja nakon oštećenja DNA različitim kemoterapeuticima kako bi se odredio njegov utjecaj na kemosenzitivnost tumorskih stanica, što bi moglo dovesti do usmjerenijih oblika liječenja tumora. U ovom smo radu, pratili utjecaj pet poznatih kemoterapeutika te povećane ekspresije gena p21 na rast tumorskih stanica debelog crijeva HCT116, SW480, SW620 i CaCo-2. Povećanu ekspresiju proteina p21WAF1/Cip1 postigli smo zaražavanjem stanica replikacijski nesposobnim adenovirusnim vektorima s ugrađenim genom p21WAF1/Cip1 (Ad5CMV-p21), dok je kao kontrola korišten vektor s ugrađenim genom za β-galaktozidazu. Kako bismo ispitali utjecaj egzogenog proteina p21 na učinak kemoterapeutika, stanice smo nakon infekcije adenovirusnim vektorom tretirali kemoterapeuticima kamptotecinom, doksorubicinom i cisplatinom u različitom rasponu koncentracija. Preživljenje stanica pratili smo MTT testom, a ekspresiju proteina p21 metodom Western blot. Svi su spojevi usporavali rast tumorskih stanica, pri čemu je 5-flurouracil pokazao najslabije, a kamptotecin najjače djelovanje. Pet dana nakon infekcije stanica vektorom Ad5CMV-p21 došlo je do značajnije inhibicije rasta staničnih linija HCT116 i SW620 u koncentracijama virusa višim od 60MOI. Povećana ekspresija gena p21WAF1/Cip1 izrazito je pojačala učinak cisplatine i kamptotecina te vrlo slabo doksorubicina u stanicama HCT116 i SW620, dok je sličan, ali slabije izražen učinak pokazan u stanicama CaCo-2. S druge strane, iako su se stanice SW480 pokazale najosjetljvijima na kemoterapeutike, njihov učinak nije pojačan u kombinaciji s pojačanom ekspresijom proteina p21 u toj staničnoj liniji.p21WAF1/Cip1 protein is primarily defined as a cyclin dependent kinase inhibitor which induces cell cycle arrest under stress conditions (DNA damage, oxidative stress, anticancer agents...). Recent studies point out an important role of p21 protein in regulation of DNA replication as well as very complex part in induction or inhibition of cell death, which depends on tretment type, cell type and genetic context. Since p21Waf1/Cip1 revealed equivocal and somewhat controversial role, it is a major issue to elucidate mechanism of it`s action after DNA damage caused by common chemotherapeutics in order to establish the influence of p21 to cell chemosensitivity , which coluld give rise to more targeted tumor therapy. In our expreiments we investigated the effects of five common chemotherapeutics and overexpression of exogenous p21WAF1/Cip1 on the growth of colon cancer cell lines HCT116, SW480, SW620, CaCo-2. Enhanced expression of protein p21WAF1/Cip1 was accomplished by infection with replication-defective adenoviral recombinant vector Ad5CMVp21, whereas Ad5CMVβ-gal was used as a control vector. In order to evaluate the effect of exogenous protein p21WAF1/Cip1 on the sensitivity to chemotheraputics, we treated cells with chemotherapeutics camptothecin, doxorubcin and cisplatin at different concetration range after infection wtih adenoviral vectors. Cells’ viability was monitored by MTT assay, and the expression of p21 protein by Western blot method. All of the tested chemotherapeutics inhibited growth of the tumor cells whereby 5-fluorouracil revealed least and camptothecin strongest effect. We detected significant growth inhibition of HCT116 and SW620 cells five days after infection with Ad5CMV-p21 at MOI 60. Furthermore, p21WAF1/Cip1 gene overexpression in HCT116 and SW620 cells distinctly enhanced cisplatin and camptotecin influence, whereas it had faint effect on doxorubicin influence. We detected similar but weaker effect of p21 on CaCo-2 cells. On the other hand, even though SW480 appeared to be the most sensitive to chemotherapeutics, their effect wasn`t enhanced when combined with p21WAF1/Cip1 overexpression in this cell line

Influence of exogenous overexpression of p21WAF1/Cip1 gene and various chemotherapeutics on proliferation of colon cancer cells

Protein p21 je primarno definiran kao inhibitor ciklin ovisnih kinaza te faktor koji u slučaju staničnog stresa (oštećenje DNA, oksidativni stres, kemoterapeutici i sl.) uzrokuje kočenje staničnog ciklusa. Noviji literaturni podaci ukazuju na važnu ulogu tog proteina u regulaciji replikacije DNA te vrlo kompleksnu ulogu u indukciji ili inibiciji stanične smrti, koja ovisi o tipu stanica, staničnom kontekstu i vrsti tretmana koji se koristi na tumorskim stanicama. Zbog mnogoznačne i često kontradiktorne uloge proteina p21, izuzetno je važno proučiti mehanizme njegovog djelovanja nakon oštećenja DNA različitim kemoterapeuticima kako bi se odredio njegov utjecaj na kemosenzitivnost tumorskih stanica, što bi moglo dovesti do usmjerenijih oblika liječenja tumora. U ovom smo radu, pratili utjecaj pet poznatih kemoterapeutika te povećane ekspresije gena p21 na rast tumorskih stanica debelog crijeva HCT116, SW480, SW620 i CaCo-2. Povećanu ekspresiju proteina p21WAF1/Cip1 postigli smo zaražavanjem stanica replikacijski nesposobnim adenovirusnim vektorima s ugrađenim genom p21WAF1/Cip1 (Ad5CMV-p21), dok je kao kontrola korišten vektor s ugrađenim genom za β-galaktozidazu. Kako bismo ispitali utjecaj egzogenog proteina p21 na učinak kemoterapeutika, stanice smo nakon infekcije adenovirusnim vektorom tretirali kemoterapeuticima kamptotecinom, doksorubicinom i cisplatinom u različitom rasponu koncentracija. Preživljenje stanica pratili smo MTT testom, a ekspresiju proteina p21 metodom Western blot. Svi su spojevi usporavali rast tumorskih stanica, pri čemu je 5-flurouracil pokazao najslabije, a kamptotecin najjače djelovanje. Pet dana nakon infekcije stanica vektorom Ad5CMV-p21 došlo je do značajnije inhibicije rasta staničnih linija HCT116 i SW620 u koncentracijama virusa višim od 60MOI. Povećana ekspresija gena p21WAF1/Cip1 izrazito je pojačala učinak cisplatine i kamptotecina te vrlo slabo doksorubicina u stanicama HCT116 i SW620, dok je sličan, ali slabije izražen učinak pokazan u stanicama CaCo-2. S druge strane, iako su se stanice SW480 pokazale najosjetljvijima na kemoterapeutike, njihov učinak nije pojačan u kombinaciji s pojačanom ekspresijom proteina p21 u toj staničnoj liniji.p21WAF1/Cip1 protein is primarily defined as a cyclin dependent kinase inhibitor which induces cell cycle arrest under stress conditions (DNA damage, oxidative stress, anticancer agents...). Recent studies point out an important role of p21 protein in regulation of DNA replication as well as very complex part in induction or inhibition of cell death, which depends on tretment type, cell type and genetic context. Since p21Waf1/Cip1 revealed equivocal and somewhat controversial role, it is a major issue to elucidate mechanism of it`s action after DNA damage caused by common chemotherapeutics in order to establish the influence of p21 to cell chemosensitivity , which coluld give rise to more targeted tumor therapy. In our expreiments we investigated the effects of five common chemotherapeutics and overexpression of exogenous p21WAF1/Cip1 on the growth of colon cancer cell lines HCT116, SW480, SW620, CaCo-2. Enhanced expression of protein p21WAF1/Cip1 was accomplished by infection with replication-defective adenoviral recombinant vector Ad5CMVp21, whereas Ad5CMVβ-gal was used as a control vector. In order to evaluate the effect of exogenous protein p21WAF1/Cip1 on the sensitivity to chemotheraputics, we treated cells with chemotherapeutics camptothecin, doxorubcin and cisplatin at different concetration range after infection wtih adenoviral vectors. Cells’ viability was monitored by MTT assay, and the expression of p21 protein by Western blot method. All of the tested chemotherapeutics inhibited growth of the tumor cells whereby 5-fluorouracil revealed least and camptothecin strongest effect. We detected significant growth inhibition of HCT116 and SW620 cells five days after infection with Ad5CMV-p21 at MOI 60. Furthermore, p21WAF1/Cip1 gene overexpression in HCT116 and SW620 cells distinctly enhanced cisplatin and camptotecin influence, whereas it had faint effect on doxorubicin influence. We detected similar but weaker effect of p21 on CaCo-2 cells. On the other hand, even though SW480 appeared to be the most sensitive to chemotherapeutics, their effect wasn`t enhanced when combined with p21WAF1/Cip1 overexpression in this cell line