Rat C6 glioma model as a tool for discovering new therapeutic strategies: characterization of the resistant phenotype in vitro and in vivo

Uspešnost terapije glioblastoma, najĉešćeg i najagresivnijeg malignog tumora centralnog nervnog sistema, je osujećena usled visokog stepena invazivnosti, kao i pojave rezistencije na hemioterapiju. Cilj ove doktorske disertacije je bio da se uspostavi i okarakteriše rezistentni gliomski model koji bi imao primenu u ispitivanju novih terapeutskih strategija u leĉenju ove teške bolesti. Kao poĉetni materijal korišćena je pacovska C6 ćelijska linija glioma, fenotipski sliĉna humanim glioblastomima. Za uspostavljanje rezistencije korišćen je prvi odobreni antigliomski lek 1,3-bis(2-hloroetil)-1-nitrozoureom (BCNU, karmustin), alkilirajući agens koji se koristi u terapiji primarnih i rekurentnih glioblastoma. Kontinuirana primena BCNU je dovela do razvoja rezistencije C6 ćelija ne samo na ovaj hemioterapeutik, već i na druge DNK oštećujuće agense, cisplatin (CPt) i temozolomid (TMZ). Novouspostavljena rezistentna ćelijska linija je oznaĉena kao RC6. Ispitivanje mehanizama rezistencije je obuhvatilo analizu promena na nivou apoptotske mašinerije i oštećenja DNK lanaca, kao i analizu promena u antioksidativnom kapacitetu izmeĊu C6 i RC6 ćelija. Rezistentna RC6 linija pokazuje smanjenu osetljivost na indukciju ćelijske smrti koja je praćena smanjenjem ekspresije antiapoptotskog faktora Bcl-2, proapoptotskog faktora Bad i efektorske kaspaze 3 na nivou iRNK i proteina. TakoĊe je pokazano smanjenje ekspresije iRNK antiapoptoskog Bcl-Xl i proapoptotskog Bax-a. Razvoj rezistencije je doveo i do znaĉajnog povećanja produkcije reaktivnih kiseoniĉnih vrsta (ROS) i smanjenja odnosa koncentracije oksidovanog i redukovanog glutationa. Zapaţeno je povećanje ekspresije iRNK komponenti antioksidativnog sistema (MnSOD, iNOS, GPx i MRP1) i smanjenje ekspresije iRNK za HIF-1α kod RC6 ćelija u odnosu na C6 ćelije.RC6 ćelije pokazuju povećanu osetljivost na delovanje doksorubicina (DOX). UtvrĊeno je da RC6 ćelije akumuliraju više DOX-a od C6 ćelija ĉemu doprinosi znaĉajno smanjenje ekspresije iRNK za ABCB1 membranski transporter, kao i smanjenje unutarćelijskog pH.Pored toga, RC6 ćelije proliferišu znatno sporije u odnosu na C6 ćelije što je karakteristiĉno za rezistentne ćelije, ali i za ćelije sa većim invazivnim sposobnostima. Rezultati potvrĊuju da invazivnosti RC6 ćelija doprinosi povišena ekspresija iRNK za metaloproteinazu MMP9...The most common and aggressive malignant tumor of the central nervous system is glioblastoma which successful treatment is compromised due to its high invasiveness, and resistance to chemotherapy. This study aimed to establish and characterize resistant glioma model for testing new therapeutic strategies for treatment of this serious disease. Rat C6 glioma cell line, which is phenotypically similar to human glioblastoma, was chosen as starting material. The first approved antiglioma drug, 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU, carmustine), an alkylating agent for treatment of primary and recurrent glioblastoma was used for establishing the resistant phenotype. Continuous treatment with BCNU led to the development of resistance not only to this chemotherapeutic, but also to other DNA damaging agents, cisplatin (CPt) and temozolomide (TMZ). The newly established drug resistant cell line was labeled as RC6. Resistance mechanisms analysis adressed the changes in apoptotic machinery and DNA damage, as well as the changes in antioxidant capacity between C6 and RC6 cells. Resistant RC6 line showed reduced sensitivity to cell death induction which was accompanied by a significant decrease in mRNA and protein expression of antiapoptotic factor Bcl-2, proapoptotic factor Bad and effector caspase 3. The mRNA expression of antiapoptotic factor Bcl-Xl and proapoptotic factor Bax was also decreased. The development of resistance was accompanied by significantly increased reactive oxygen species (ROS) production and decreased oxidized to reduced glutathione ratio. mRNA expression of antioxidant system components (MnSOD, iNOS, GPx and MRP1) was significantly increased in RC6 compared to C6 cells, while HIF-1α mRNA expression was decreased.RC6 cells exibited increased sensitivity to doxorubicin (DOX). It was determined that RC6 cells accumulated more DOX than C6 cells as a result of decreased mRNA expression of ABCB1 membrane transporter, as well as lower intracellular pH.In addition, RC6 cells proliferated significantly slower compared to C6 cells which is characteristic for drug resistant cells and cells with greater invasive capacity. The results confirmed that increased mRNA expression of metalloprotease MMP9 contributed to RC6 cells invasiveness..

Rat C6 glioma model as a tool for discovering new therapeutic strategies: characterization of the resistant phenotype in vitro and in vivo

Uspešnost terapije glioblastoma, najĉešćeg i najagresivnijeg malignog tumora centralnog nervnog sistema, je osujećena usled visokog stepena invazivnosti, kao i pojave rezistencije na hemioterapiju. Cilj ove doktorske disertacije je bio da se uspostavi i okarakteriše rezistentni gliomski model koji bi imao primenu u ispitivanju novih terapeutskih strategija u leĉenju ove teške bolesti. Kao poĉetni materijal korišćena je pacovska C6 ćelijska linija glioma, fenotipski sliĉna humanim glioblastomima. Za uspostavljanje rezistencije korišćen je prvi odobreni antigliomski lek 1,3-bis(2-hloroetil)-1-nitrozoureom (BCNU, karmustin), alkilirajući agens koji se koristi u terapiji primarnih i rekurentnih glioblastoma. Kontinuirana primena BCNU je dovela do razvoja rezistencije C6 ćelija ne samo na ovaj hemioterapeutik, već i na druge DNK oštećujuće agense, cisplatin (CPt) i temozolomid (TMZ). Novouspostavljena rezistentna ćelijska linija je oznaĉena kao RC6. Ispitivanje mehanizama rezistencije je obuhvatilo analizu promena na nivou apoptotske mašinerije i oštećenja DNK lanaca, kao i analizu promena u antioksidativnom kapacitetu izmeĊu C6 i RC6 ćelija. Rezistentna RC6 linija pokazuje smanjenu osetljivost na indukciju ćelijske smrti koja je praćena smanjenjem ekspresije antiapoptotskog faktora Bcl-2, proapoptotskog faktora Bad i efektorske kaspaze 3 na nivou iRNK i proteina. TakoĊe je pokazano smanjenje ekspresije iRNK antiapoptoskog Bcl-Xl i proapoptotskog Bax-a. Razvoj rezistencije je doveo i do znaĉajnog povećanja produkcije reaktivnih kiseoniĉnih vrsta (ROS) i smanjenja odnosa koncentracije oksidovanog i redukovanog glutationa. Zapaţeno je povećanje ekspresije iRNK komponenti antioksidativnog sistema (MnSOD, iNOS, GPx i MRP1) i smanjenje ekspresije iRNK za HIF-1α kod RC6 ćelija u odnosu na C6 ćelije.RC6 ćelije pokazuju povećanu osetljivost na delovanje doksorubicina (DOX). UtvrĊeno je da RC6 ćelije akumuliraju više DOX-a od C6 ćelija ĉemu doprinosi znaĉajno smanjenje ekspresije iRNK za ABCB1 membranski transporter, kao i smanjenje unutarćelijskog pH.Pored toga, RC6 ćelije proliferišu znatno sporije u odnosu na C6 ćelije što je karakteristiĉno za rezistentne ćelije, ali i za ćelije sa većim invazivnim sposobnostima. Rezultati potvrĊuju da invazivnosti RC6 ćelija doprinosi povišena ekspresija iRNK za metaloproteinazu MMP9...The most common and aggressive malignant tumor of the central nervous system is glioblastoma which successful treatment is compromised due to its high invasiveness, and resistance to chemotherapy. This study aimed to establish and characterize resistant glioma model for testing new therapeutic strategies for treatment of this serious disease. Rat C6 glioma cell line, which is phenotypically similar to human glioblastoma, was chosen as starting material. The first approved antiglioma drug, 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU, carmustine), an alkylating agent for treatment of primary and recurrent glioblastoma was used for establishing the resistant phenotype. Continuous treatment with BCNU led to the development of resistance not only to this chemotherapeutic, but also to other DNA damaging agents, cisplatin (CPt) and temozolomide (TMZ). The newly established drug resistant cell line was labeled as RC6. Resistance mechanisms analysis adressed the changes in apoptotic machinery and DNA damage, as well as the changes in antioxidant capacity between C6 and RC6 cells. Resistant RC6 line showed reduced sensitivity to cell death induction which was accompanied by a significant decrease in mRNA and protein expression of antiapoptotic factor Bcl-2, proapoptotic factor Bad and effector caspase 3. The mRNA expression of antiapoptotic factor Bcl-Xl and proapoptotic factor Bax was also decreased. The development of resistance was accompanied by significantly increased reactive oxygen species (ROS) production and decreased oxidized to reduced glutathione ratio. mRNA expression of antioxidant system components (MnSOD, iNOS, GPx and MRP1) was significantly increased in RC6 compared to C6 cells, while HIF-1α mRNA expression was decreased.RC6 cells exibited increased sensitivity to doxorubicin (DOX). It was determined that RC6 cells accumulated more DOX than C6 cells as a result of decreased mRNA expression of ABCB1 membrane transporter, as well as lower intracellular pH.In addition, RC6 cells proliferated significantly slower compared to C6 cells which is characteristic for drug resistant cells and cells with greater invasive capacity. The results confirmed that increased mRNA expression of metalloprotease MMP9 contributed to RC6 cells invasiveness..

Decreased TSPAN14 Expression Contributes to NSCLC Progression

Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients' samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression

Association of Overexpressed MYC Gene with Altered PHACTR3 and E2F4 Genes Contributes to Non-Small Cell Lung Carcinoma Pathogenesis

Background: C-Myc is one of the major cellular oncogenes overexpressed in non-small cell lung carcinoma (NSCLC). Its deregulated expression is necessary but not sufficient for malignant transformation. We evaluated expression of MYC gene in NSCLC patients and its association with alterations in the genes previously identified to be related to NSCLC pathogenesis, PHACTR3 and E2F4. Methods: We analyzed MYC gene expression by qRT-PCR in 30 NSCLC patients' samples and paired normal lung tissue. MYC expression was further statistically evaluated in relation to histopathological parameters, PHACTR3 and E2F4 gene alterations and survival. Alterations in aforementioned genes were previously detected and identified based on AP-PCR profiles of paired normal and tumor DNA samples, selection of DNA bands with altered mobility in tumor samples and their characterization by the reamplification, cloning and sequencing. Results: MYC expression was significantly increased in NSCLC samples and its overexpression significantly associated with squamous cell carcinoma subtype. Most importantly, MYC overexpression significantly coincided with mutations in PHACTR3 and E2F4 genes, in group of all patients and in squamous cell carcinoma subtype. Moreover, patients with jointly overexpressed MYC and altered PHACTR3 or E2F4 showed trend of shorter survival. Conclusions: Overall, MYC is frequently overexpressed in NSCLC and it is associated with mutated PHACTR3 gene, as well as mutated E2F4 gene. These joint gene alterations could be considered as potential molecular markers of NSCLC and its specific subtypes

Modulation of Antioxidant Potential with Coenzyme Q10 Suppressed Invasion of Temozolomide-Resistant Rat Glioma In Vitro and In Vivo.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity

Rat C6 glioma model as a tool for discovering new ttherapeutic strategies: characterization of the resistant phenotype in vitro and in vivo

Uspešnost terapije glioblastoma, najĉešćeg i najagresivnijeg malignog tumora centralnog nervnog sistema, je osujećena usled visokog stepena invazivnosti, kao i pojave rezistencije na hemioterapiju. Cilj ove doktorske disertacije je bio da se uspostavi i okarakteriše rezistentni gliomski model koji bi imao primenu u ispitivanju novih terapeutskih strategija u leĉenju ove teške bolesti. Kao poĉetni materijal korišćena je pacovska C6 ćelijska linija glioma, fenotipski sliĉna humanim glioblastomima. Za uspostavljanje rezistencije korišćen je prvi odobreni antigliomski lek 1,3-bis(2-hloroetil)-1-nitrozoureom (BCNU, karmustin), alkilirajući agens koji se koristi u terapiji primarnih i rekurentnih glioblastoma. Kontinuirana primena BCNU je dovela do razvoja rezistencije C6 ćelija ne samo na ovaj hemioterapeutik, već i na druge DNK oštećujuće agense, cisplatin (CPt) i temozolomid (TMZ). Novouspostavljena rezistentna ćelijska linija je oznaĉena kao RC6. Ispitivanje mehanizama rezistencije je obuhvatilo analizu promena na nivou apoptotske mašinerije i oštećenja DNK lanaca, kao i analizu promena u antioksidativnom kapacitetu izmeĊu C6 i RC6 ćelija. Rezistentna RC6 linija pokazuje smanjenu osetljivost na indukciju ćelijske smrti koja je praćena smanjenjem ekspresije antiapoptotskog faktora Bcl-2, proapoptotskog faktora Bad i efektorske kaspaze 3 na nivou iRNK i proteina. TakoĊe je pokazano smanjenje ekspresije iRNK antiapoptoskog Bcl-Xl i proapoptotskog Bax-a. Razvoj rezistencije je doveo i do znaĉajnog povećanja produkcije reaktivnih kiseoniĉnih vrsta (ROS) i smanjenja odnosa koncentracije oksidovanog i redukovanog glutationa. Zapaţeno je povećanje ekspresije iRNK komponenti antioksidativnog sistema (MnSOD, iNOS, GPx i MRP1) i smanjenje ekspresije iRNK za HIF-1α kod RC6 ćelija u odnosu na C6 ćelije. RC6 ćelije pokazuju povećanu osetljivost na delovanje doksorubicina (DOX). UtvrĊeno je da RC6 ćelije akumuliraju više DOX-a od C6 ćelija ĉemu doprinosi znaĉajno smanjenje ekspresije iRNK za ABCB1 membranski transporter, kao i smanjenje unutarćelijskog pH. Pored toga, RC6 ćelije proliferišu znatno sporije u odnosu na C6 ćelije što je karakteristiĉno za rezistentne ćelije, ali i za ćelije sa većim invazivnim sposobnostima. Rezultati potvrĊuju da invazivnosti RC6 ćelija doprinosi povišena ekspresija iRNK za metaloproteinazu MMP9...The most common and aggressive malignant tumor of the central nervous system is glioblastoma which successful treatment is compromised due to its high invasiveness, and resistance to chemotherapy. This study aimed to establish and characterize resistant glioma model for testing new therapeutic strategies for treatment of this serious disease. Rat C6 glioma cell line, which is phenotypically similar to human glioblastoma, was chosen as starting material. The first approved antiglioma drug, 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU, carmustine), an alkylating agent for treatment of primary and recurrent glioblastoma was used for establishing the resistant phenotype. Continuous treatment with BCNU led to the development of resistance not only to this chemotherapeutic, but also to other DNA damaging agents, cisplatin (CPt) and temozolomide (TMZ). The newly established drug resistant cell line was labeled as RC6. Resistance mechanisms analysis adressed the changes in apoptotic machinery and DNA damage, as well as the changes in antioxidant capacity between C6 and RC6 cells. Resistant RC6 line showed reduced sensitivity to cell death induction which was accompanied by a significant decrease in mRNA and protein expression of antiapoptotic factor Bcl-2, proapoptotic factor Bad and effector caspase 3. The mRNA expression of antiapoptotic factor Bcl-Xl and proapoptotic factor Bax was also decreased. The development of resistance was accompanied by significantly increased reactive oxygen species (ROS) production and decreased oxidized to reduced glutathione ratio. mRNA expression of antioxidant system components (MnSOD, iNOS, GPx and MRP1) was significantly increased in RC6 compared to C6 cells, while HIF-1α mRNA expression was decreased. RC6 cells exibited increased sensitivity to doxorubicin (DOX). It was determined that RC6 cells accumulated more DOX than C6 cells as a result of decreased mRNA expression of ABCB1 membrane transporter, as well as lower intracellular pH. In addition, RC6 cells proliferated significantly slower compared to C6 cells which is characteristic for drug resistant cells and cells with greater invasive capacity. The results confirmed that increased mRNA expression of metalloprotease MMP9 contributed to RC6 cells invasiveness..