Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria.

Cadmium (Cd) is a well-known toxic compound for the kidney in vivo and in vitro. It has been demonstrated to induce nephrotoxicity via in part by apoptotic cell death, but the precise mechanism is still unclear. Therefore, we have studied the effects of Cd on HEK 293 cells and investigated the mechanisms of Cd-induced apoptosis. Studies of morphology and oligonucleosomal DNA fragmentation demonstrated that 30-60 microM Cd induced apoptosis as early as 6-9h with strong effects on MTT activity, whereas 120 microM Cd revealed mainly necrosis, and the result of flow cytometry confirmed it. A concomitant time-dependent decrease of mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 expression was observed, subsequently, release of cytochrome c (Cyt c) and activation of caspase-3 were detected, suggesting a caspase-dependent pathway. Meanwhile, mitochondrial AIF was released to cytoplasm and nucleus, suggesting a caspase-independent pathway. Furthermore, when cells were transfected with pcDNA3/Bcl-2 before exposed to CdCl(2), alleviated apoptosis was assessed by part of the apoptotic features in this study. Taken together, our results showed that CdCl(2) caused time- and dose-dependent apoptosis or even necrosis in HEK 293 cells depending on the exposure conditions. The apoptotic events may involve mitochondrial disruption including both caspase-dependent and -independent pathways.

[Cadmium induced apoptosis of HEK293 cells and its mitochondrial apoptosis pathway].

HEK293 cell was chose to study the kidney damage of cadmium and to explore the significance of caspase 3,Bcl-2 and AIF (apoptosis inducing factor) in the apoptosis of cells induced by cadmium. Inhibition of the cell proliferation was measured by MTT assay. The structure of apoptotic cells was observed by light microscopy and electron microscopy; moreover, apoptotic cells were detected by DNA electrophoresis, flow cytometry and confocal laser microscopy. Furthermore,the expressions of Pro-caspase-3, Bcl-2 and the location of AIF in cells (mitochondria,cytoplasm or nuclei) were tested by western blot and immunofluorescence assay. CdCl2 exhibited anti-proliferative activity in dosage and time-dependent manner. DNA ladders of HEK293 cells were showed on agarose gel electrophoresis and the fragments of DNA were integral of 180-200 bp. 6-9 hours after 30 micromol/L CdCl2 treatment,DNA ladders were distinct. However, mistiness DNA ladder or smear was found when HEK293 cells were treated with CdCl2 on higher concentration or treated longer. It suggests that necrosis may happen, and flow cytometry results confirmed it. Morphological examination showed cell shrinkage, chromosomal condensation, karyotheca margination, nucleus cracking, vacuoles formed in cytoplasm and the presence of apoptotic bodies. At the same time,mitochondrial membrane potential (MMP) decreased, and the expression of Pro-caspase-3, Bcl-2 were decreased in time-dependent manner. Furthermore, AIF was released from mitochondria,and then traveled to nuclei. It suggests that CdCl2 may induce the apoptosis of HEK293 cells involving mitochondrial disruption including AIF migration and Cyt c release through both caspase-independent and -dependent pathways, and Bcl-2 and Caspase-3 are important factors which participate in the processes.