The Role Of Energy Metabolic Balance In The Regulation Of Cell Growth And Transformation

ObjectivesIt has been suggested that micro-environment redox status in tumor cells is different from that in normal cells.Cell energy metabolism transition from normal to abnormal exists in the process of cell transformation.If the metabolic stress is too high,cell growth and transformation will be blocked.Since the biological characteristics and energy metabolism mechanism of tumor cells are quite different from normal cells,its special energy metabolism may play an important role in the maintenance of the redox status.This study was based on the preliminary work concerning fatty acid synthesis and tumor suppressor Rb.The aim of this study is to explore the regulation of energy metabolism balance tothe cell growth and transformation from culture cells and soft agar levels,and to clarify the role of fatty acid synthesis pathway and tumor suppressor Rb in the control of energy metabolism balance via cell biology and molecular biology techniques.Methods1.The expressions of endogenous FAS and CPT-1 protein in breast cancer cells were detected by Western Blot method.2.The method to measure the rate of growth is to count the number of cells at several time points and plot a growth curve.To determine cell viability,the MTT assay was used.3.The intracellular ROS levels were determined using DCFH-DA probe and DHE probe.The intracellular NADPH/NADP+ proportion was detected by NADPH/NADP+ assay kit.By intracellular expression of Grx1-roGFP2 protein that is highly sensitive to redox,the changes of the proportion of-SH/-SS were measured by the ratio of GFP400/GFP480.4.The lentiviral vector system was used to establish stable cell lines by expressing over-expression plasmids,knockdown plasmids,restore expression plasmids and empty vector plasmids.The expression efficiency was determined by Western Blot method.5.The numbers of colonies were detected by soft agar colony formation assay.6.The mRNA expressions of mitochondrial membrane proteins were detected by RT-PCR method.Results1.No significant correlation between the protein expressionsof FAS and CPT-1 with the proliferation rate in four types of breast cancer cells.Only in cancer cells,the key enzymes of fatty acid synthesis and oxidation were highly expressed at the same time.2.The specific inhibitors of fatty acid metabolism enzymes,including cerulenin,orlistat,TOFA,etomoxir and triacsin C,exhibited killing effect with varying degrees on MCF-7,MDA-MB-231,BT474 and SKBR3 breast cancer cells.After the treatment of cerulenin on BT474 cells,intracellular ROS levels and NADPH/NADP+ ratio were increased rapidly.Antioxidative agent NAC can protect breast cancer cells from apoptosis induced by cerulenin,indicating that cell apoptosis induced by inhibition of fatty acid synthesis was mainly mediated by ROS.3.Oxidative stress and redox imbalance were induced by Rb loss.The intracellular ROS levels in 3T3Rb-/-cells were significantly higher than that in the control cells.Moreover,the-S-S-/SH ratio in the 3T3Rb-/-cells was higher than 3T3/wt cells.4.The mitochondrial membrance potential was decreased by Rb loss.Rb was involved in modulating the expression of mitochondrial membrance protein.The intracellular ROS levels were reduced by re-expression of Rb or over-epression of Bcl-XL in 3T3Rb-/-cells.The data showed that ROS and redox imbalance induced by Rb loss was mainly derived from mitochondrial.5.NAC treatment inhibited the production of ROS in and rescued the mitochondrial membrance potential.Moreover,Rb reexpression in 3T3Rb-/-cells inhibited the production of ROS in and rescued the mitochondrial membrance potential.6.Cell fate controlled by redox balance was determined by Rb status.Antioxidative agent NAC promoted the apoptosis of 3T3/wt cells,yet 3T3Rb-/-cells was sensitive to oxidative agent H2O2.7.Rb loss prevented the transformation of 3T3/Ras cells.Precise control of redox balance promoted the transformation of 3T3/Rb-/-cells.8.When Gpxl and GSR was overexpressed in 3T3/wt and 3T3Rb-/-cells,colony formation was observed in 3T3/Gpxl and 3T3Rb-/-/GSR cells,yet no colony formation was observed in 3T3/GSR and 3T3Rb-/-/Gpxl cells.These data indicated that was redox homeostasis is involved in the significant mechanism that Rb regulates energy metabolism.9.Oxidative stress was induced by Rb knockdown.The ROS levels in MCF-10A/shRb cells were higher than that in the control cells.The results of soft agar colony formation experiments showed that the numbers of colonies of MCF-10A/shRb/Ras cells were significantly less than the control cells,and NAC may promote the colony formation of MCF-10A/shRb/Ras cells.10.The growth and transformation ability was markedly damaged by GSR silence,indicating GSH redox buffer system plays an important role in regulating the growth and transformation of cancer cells.11.Rb loss had no effect on the transformation of Dul 45 cells.Instead,NAC can promote the colony formation of Du145/Ras cells.When Rb was silenced in MCF-7,the cell growth rate did not change significantly,yet the ability of colony formation was reduced markedly.Conclusions1.Comparing the relationship between the protein expressionsof FAS and CPT-1 with cell proliferation speed in four types of breast cancer cells,we found that the fatty acid metabolism,in addition to provide nutrition and energy for the rapid proliferation of tumor cells,but also have other biological functions.We found that the killing effect of cerulenin on BT474 cells was closely related to the imbalance of redox status,confirming that fatty acid metabolism is extremely important for the maintenance of intracellular redox status.2.Rb definitely regulates the balance of cell metabolism,and modulate cell transformation by controlling the metabolic balance.GSH system may regulate this function of Rb.Moreover,these data indicated that Rb loss can promote cell transformation by inducing cell cycle deregulation,and Rb loss also may inhibit cell transformation by causing metabolic imbalance.3.Our study clarifies the key role of energy metabolic balance in cell growth and transformation from the above two different angles.It is expected to provide theoretical basis and new ideas for exploring effective tumor interventions.