RalA regulates ROS and ATP production in cancer cells by association with cavelolin-1 / 医用生物力学

Objective By analyzing mitochondrial function,reactive oxygen species (ROS) and adenosine triphosphate (ATP) production under different levels of RalA and caveolin-1 (Cav-1) expression,to investigate the regulation role of RalA played in cancer metabolism and explore the possibility of its regulation role involved in Cav-1 and caveolae motility.Methods Firstly,RalA and Cav-1 expression were inhibited by siRNA in breast cancer cell line MDA-MB-231,and then the changes of mitochondrial membrane potential (MMP),ROS produc tion,ATP generation and L-lactate level before and after inhibition were assessed by Western blotting,confocal microscope and fluorescence quantification.Results (1) The decreased RalA and Cav-1 expression led to a significant reduction of MMP directly.(2) Low RalA and Cav-1 expression resulted in an inhibition of ATP production and an increase of H2O2 generation.With the reduction of MMP,mitochondrial malfunction was observed.(3) With mitochondrial function suppression,an elevated level of glycolysis metabolite L-lactate was also detected in RalA and Cav-1 deprived cells.Conclusions RalA and Cav-1 mediate cellular metabolic switch by inhibiting mitochondrial function and simultaneously boosting glycolysis.This regulation role of RalA depends on its association with Cav-1,and possibly is related to the endocytosis and motility of caveolae.The research findings enrich the cancer metabolic studies,and provide a novel approach for cancer therapeutic strategy targeted to cellular metabolism.

RalA regulates ROS and ATP production in cancer cells by association with cavelolin-1 / 医用生物力学

Objective By analyzing mitochondrial function, reactive oxygen species (ROS) and adenosine triphosphate (ATP) production under different levels of RalA and caveolin-1 (Cav-1) expression, to investigate the regulation role of RalA played in cancer metabolism and explore the possibility of its regulation role involved in Cav-1 and caveolae motility. Methods Firstly, RalA and Cav-1 expression were inhibited by siRNA in breast cancer cell line MDA-MB-231, and then the changes of mitochondrial membrane potential (MMP), ROS production, ATP generation and L-lactate level before and after inhibition were assessed by Western blotting, confocal microscope and fluorescence quantification. Results (1) The decreased RalA and Cav-1 expression led to a significant reduction of MMP directly. (2) Low RalA and Cav-1 expression resulted in an inhibition of ATP production and an increase of H2O2 generation. With the reduction of MMP, mitochondrial malfunction was observed. (3) With mitochondrial function suppression, an elevated level of glycolysis metabolite L-lactate was also detected in RalA and Cav-1 deprived cells. Conclusions RalA and Cav-1 mediate cellular metabolic switch by inhibiting mitochondrial function and simultaneously boosting glycolysis. This regulation role of RalA depends on its association with Cav-1, and possibly is related to the endocytosis and motility of caveolae. The research findings enrich the cancer metabolic studies, and provide a novel approach for cancer therapeutic strategy targeted to cellular metabolism.

Regulation of GTP-binding state in RalA through Ca2+ and calmodulin

RalA GTPase, a member of Ras superfamily proteins, shows alternative forms between the active GTP-binding and the inactive GDP-binding states. Ral-specific guanine nucleotide exchange factor such as RalGDS interacts with activated Ras and cooperates with Ras indicating that Ral can be activated through Ras signaling pathway. Another activation path for Ral are through Ca2+-dependent but Ras-independent manner. In this study, studies were carried out to examine possible effects of Ca2+ and calmodulin, Ca2+-binding protein, directly on the GTP/GDP-binding state to recombinant unprenylated GST-RalA proteins. The results showed that Ca2+ stimulated the binding of GTP to RalA, whereas it reduced the binding of GDP to RalA. However, it does not involve a high affinity association of Ca2+ with RalA. Ca2+/calmodulin stimulated the GTPase activity of RalA. These results indicate that Ca2+ alone activates RalA by stimulating GTP-binding to RalA and Ca2+/calmodulin inactivates RalA by increasing the activity of RalGTPase.