Effects of microenvironment on apoptotic pathway in adult and children cancers therapy models, in in vitro environment.

Aim and Hypothesis: One of the main targets of the studies which aim to optimize appliedchemotherapy strategies more effectively and to develop new therapy strategies is the tumormicroenvironment and natural anti tumor systems. It is thought that many interactionsbetween tumor cell and its microenvironment play an effective role in the survival of thetumor cell. It is known that a microenvironment including reactive oxygen and nitrogenproducts, may affect distinct pathways by producing distinct metabolites in cellular processes.Effects of the RNS and ROS based microenvironment components on therapy processes;apoptotic pathways are new study areas which are aimed to develop therapy strategies andchemotherapy therapy in more appropriate compositions. The aim of this study is to showhow a reactive oxygen and nitrogen based microenvironment may affect the apoptoticpathway and therapy processes by forming an in vitro cancer therapy model.Method: An in vitro therapy model was formed in SHSY5Y, Kelly, HeLa and HEp -2 celllines. In experimental design of ROS and RNS based microenvironment L -Arg, GSNO, SIN -1, H2O2, and L -NAME, SOD, Ebselen, Aminoguanidine, Bicarbonate (as inhibitors) wereused. BHV -1 was used as a viral infection model. Cytotoxic effects on cells were determinedby MTT and XTT methods. For apoptotic cellular death; Hoechst 33342 / Propidium iodidenuclear staining, in situ TUNEL technique (TdT-mediated dUTP nick end labeling technique),and for early determination of the apoptosis; Annexin V labeling were used. Nitric oxideresponses were determined as total nitrite by Griess Reagent.Results and Conclusion: In experiments which the responses against microenvironmentchanges in cervical carcinoma cell line are searched, L -Arg (endogen NO donor) stimulatedthe apoptotic pathway in a dose-response manner. Cellular death increased synergistically1when NO was stimulated in conditions which superoxide was in the microenvironment.Endogen NO stimulated cellular death type was apoptotic, but dominant cellular death typewas necrotic when superoxide and NO were together. These results showed that NO played anessential role in mediating the cellular death according to the dominant radical in themicroenvironment.The proteins expressed during the transition from ?E? stage to ?L? stage either in normal or incancer microenvironment in BHV -1 infection; also inhibit the staurosporin stimulatedapoptosis. On the contrary, apoptosis was stimulated at most in a normal microenvironment(MDBK cells) when BHV -1 ?E? genes are expressed whereas when ?IE? genes wereexpressed in a cancer (HEp -2 cells) microenvironment. It was shown that BHV -1 stimulatedapoptosis was through caspase-8 pathway (means through ?outer membrane receptor?).In DOXO, CIS and ETOP therapy protocols in SHSY5Y cells, cell death was increased.When exogenous NO (GSNO) and peroxynitrite (SIN -1) were dominant inmicroenvironment, In CARBO + ETOP therapy protocols in SHSY5Y cells; cell death wasincreased when peroxynitrite and superoxide were dominant in microenvironment.And in CIS+ETOP therapy protocols in SHSY5Y cells, cell death was increased whenexogenous NO and superoxide were dominant.In DOXO, CIS, ETOP therapy protocols in Kelly cells, cell death was increased when ROSand RNS were dominant in microenvironment. In CARBO application, peroxynitrite andsuperoxide radicals; in CIS+ETOP application, exogenous NO; in CARBO+ETOPapplication both exogenous NO and superoxide increased the cellular death.Apoptotic cellular death in neuroblastoma cells during therapy process was increased 3,45times when ROS was dominant; 2,18 times when exogenous NO was dominant; and 2,74times when peroxynitrite was dominant in the microenvironment.These results support that therapy processes are affected by the type of free radical in themicroenvironment.KEY WORDSNeuroblastoma; tumor microenvironment; in vitro therapy model; ROS; RNS; apoptoticpathway2