Anti-tumor Activity And Mechanism Of Apoptosis Of Tumor Cells Induced By Ruthenium Complexes

The Chapter 1 mainly illustrates the relationship between cancer treatment and apoptosis, introduces the basic concept of apoptosis; morphological characteristics of apoptosis; several major signaling pathways of apoptosis and proteins of Bcl-2 family which is closely related to mitochondria death pathway. Review the progress of mechanism of tumor cell apoptosis induced by ruthenium complexes.In chapter 2, the cytotoxicity of Ru(MeIm)4(pip) 1 and Ru(MeIm)4(4-mopip) 2 were tested by MTT assay and IC50 values was calculated, both of ruthenium complexes 1 and 2 showed growth inhibition on selected cell lines. Changes in cell morphology were observed by inverted microscope. Typical apoptosis-specific morphological and biochemical characteristics were observed by fluorescence microscopy with Hoechst 33342 staining. Nuclear morphology solid shrinkage, chromatin condensation and margination, nuclear fragmentation, apoptotic bodies and other characteristics were seen. DNA ladder was showed on DNA agarose gel electrophoresis. Flow cytometry analysis showed that with the increasing of concentrations, the rates of apoptosis were also increasing gradually. These results showed that ruthenium complexes induced tumor cell apoptosis to different extent. Flow cytometry results showed that the ruthenium complex can arrest the cell cycle of tumor cell at S phase; they also can lead to theΔΨm of tumor cell decreased, indicating that the occurrence of intracellular mitochondrial membrane depolarization; In addition, the ruthenium complexes can induce the levels of intracellular ROS increased, confirmed that drugs can destroy the defense system of tumor cell and induce oxidative damage to cells. By Western Blot analysis, we found that:while the expression of Bax protein increased, the expression of Bcl-2 and Bcl-xl protein decreased. Indicated that ruthenium complexes can block the cell cycle of tumor cell and the effect of ruthenium complex 2 on inducing-apoptosis of A549 cells may be related to chondrosome. In chapter 3, the interaction of ruthenium complex 3 with Pu39,mutPu39 was investigated by UV spectral titration, luminescence spectral titration, CD spectral experiments, FRET and PCR stop assay. These results clearly showed that the ruthenium complex 3 was capable of inducing the formation of the G-quadruplex and interacting with Pu39,mutPu39 G-quadruplex. These results indicated that Bcl-2 is a possible anti-tumor target of complex 3. MTT assay illustrated that Ru(phen)2 (5-idip) 3 and Ru(bpy)2(5-idip) 4 showed growth inhibition on selected cell lines. Changes in cell morphology were observed by inverted microscope. Typical apoptosis-specific morphological and biochemical characteristics were observed by fluorescence microscopy with Hoechst 33342 staining. DNA ladder was showed on DNA agarose gel electrophoresis. Flow cytometry analysis showed that with the increasing of concentrations, the rates of apoptosis were also increasing gradually. These results showed that ruthenium complexes induced apoptosis of HeLa cells. Flow cytometry results showed that the ruthenium complex can arrest the cell cycle of tumor cell at S phase; they also can lead to theΔΨm of tumor cell decreased, indicating that the occurrence of intracellular mitochondrial membrane depolarization. By Western Blot analysis, we found that:while the expression of Bax protein increased, the expression of Bcl-2 and Bcl-xl protein decreased. Indicated that ruthenium complexes can block the cell cycle of tumor cell and the effect of ruthenium complex 3 on inducing-apoptosis of HeLa cells may be related to the pathway of chondrosome, Bcl-2 may be the target of complex 3. Complex 3 interacted with the G-rich sequences of bcl-2 promoter and induced the formation G-quadruplex, prevented the transcription and translation of Bcl-2 to decrease the expression of Bcl-2 protein, and ultimately induced the apoptosis of HeLa cells through mitochondrial pathway.