Studies On The Mechanism Of Oxidative Stress And Bcl-2 In HEK293 Cells Apoptosis Induced By Cadmium

Cadmium can induce apoptosis in many kinds of cells. Studying the apoptosis induced by cadmium is necessary for us to cure lots of diseases caused by cadmium. And it is also very important for the molecular toxicological study of cadmium. This paper studied some aspects of the mechanism of apoptosis induced by cadmium:1 The oxidative stress effect on the apotosis of HEK293 cells induced by cadmiumUnder the condition of 37℃and 5% CO₂, HEK293 cells was incubated with different concentrations of cadmium or treated by a certain density cadmium for different hours, then the apoptosis were measured by flow cytometry , lactate dehydrogenase (LDH) activity in cultured medium were detected in spectro photometric assay and the cytosolic reactive oxygen species ( ROS) content were measured respectively by flow cytometry and confocal laser microscopy . The results showed that the apoptosis increased with cadmium concentrations increased in definite concentration extent. 6 hours after 60μmol/L CdCl₂ treatment, the apoptosis was peak , the cytosolic ROS content decreased significantly . We observed the dose and time-dependent increase of LDH activity. It is suggested that cadmium might cause apoptosis with enhancive ROS.2 The function of reactive oxygen species (ROS) in the apoptosis of HEK293 cells induced by cadmiumThe HEK293 cells homogenates were prepared to detect the levels of malondialdehyde (MDA) and reduced glutathione hormone (GSH) in spectro photometric assay . Western blot was used to analyze the expressions of Bcl-2 and Caspase-3 proenzyme . The results showed that the dose and time-dependent increase of MDA content, the dose and time-dependent decrease of GSH content and GSH-PX activity. 6 hours after 60μmol/L CdCl₂ treatment , the cytosolic ROS content was peak, MDA content increased, GSH content and GSH-PX activity decreased significantly (P<0.01) . Furthermore , the expression of Bcl-2 was lower than the expression of Bcl-2 3h after 60μmol/L CdCl₂ treatment. The expression of Caspase-3 proenzyme was decreased , compared with the control. It is suggested that ROS caused oxidative injury , the expression of Bcl-2 down regulation and the activation of Casepase-3 in HEK293 cells .Theses had the relation with the apoptosis of HEK293 induced by cadmium.3 The study on the anti-apoptotic function of Bcl-2 in the apoptosis of HEK293 cells induced by cadmiumHEK293 cells were transfected by pcDNA3 /Bcl-2 and pcDNA3/Bcl-2ΔC. Then the growth inhibition was detected by MTT and the apoptosis was detected by using acridine orange / ethidium bromide double fluorescent dye staining. 6 hours after 60μmol/L CdCl₂ treatment , the cytosolic reactive oxygen species ( ROS) content were measured respectively by flow cytometry. The results showed that, comparing to HEK293 cells transfected by pcDNA3, the vitality of HEK293 cells transfected by pcDNA3 / Bcl - 2 was increased significantly (P<0.01), the apoptosis and the cytosolic ROS content was lower. But the HEK293 cells transfected by pcDNA3/Bcl-2ΔC had no significant differences. It is suggested that Bcl-2 could evidently lower prevent the cytosolic ROS content induced by cadmium. The C terminal hydrophobicity amino acids part of the Bcl-2 has an important function to its function.