Cytoprotective Effects Of Selenium On Cadmium-induced Growth Inhibition And Apoptosis In LLC-PK₁ Cells

Cadmium is a widespread environmental and industrial pollutant,which is classified by IARC as Groupâ… carcinogen to humans. It has beenwell established that chronic exposure to cadmium causes irreversiblekidney damage and renal tubular dysfunction. During the last decade, anumber of studies have shown that Cd induces apoptosis of the proximaltubular cells. At present, oxidative stress has been considered animportant possible mechanism of cadmium toxicity. Nevertheless, theexact mechanism in Cd-induced apoptosis is still unclear. The c-JunN-terminal kinase (JNK), also known as stress-activated protein kinase(SAPK), belongs to the mitogen-activated protein kinases (MAPK)superfamily, which regulates cell differentiation, proliferation, cellsurvival and cell death in response to stress signals. Based on substantialevidence, JNK signaling is also sensitive to a decreased or increasedoxidative environment.Selenium is an essential micronutrient for organisms. Recently, thestudy show that selenium may have antagonistic joint action in exposureto cadmium, but the mechanism is still not clear. In this study, in order togain further insights into the toxic mechanism response to cadmium, weinvestigated oxidative stress involvement on JNK pathway activation.The objectives of this study was to study the protective mechanism of selenium involved in cadmium-induced apoptosis in LLC-PK1 cell lines.Section 1 Effects of selenium on cadmium-induced cell growthinhibition and apoptosis in LLC-PK₁ cells.Objective: To determine the effect of selenium oncadmium-induced cell growth inhibition and apoptosis in LLC-PK1 cells.Methods: MTT assay was used to observe the effects on growthinhibition induced by cadmium in LLC-PK1 cells. The effect of seleniumwas observed in cadmium-induced apoptosis with the fluoromi croscopedyed by AO/EB and Hoechst 33258, Annexin V-FITC/PI was used toanalyze apoptosis by flow cytometry. Results: Our study clearly revealedthe protective effects of selenium were observed to inhibitcadmium-induced LLC-PK₁ cells growth in a dose-dependent manner,and cadmium treatment caused apoptosis in LLC-PK₁ cells, which waspartially suppressed by pretreatment with selenium, an antioxidantnutrient. Conclutions: Selenium plays a protective role oncadmium-induced cell growth inhibition and apoptosis in LLC-PK1 cells.Section 2 Cytoprotective effctes of selenium on cadmium-inducedLLC-PK₁ cells apoptosis by activating JNK pathwayObjective: The purpose of this study was to determine therelationships between the activation of p-JNK and cadmium-inducedapoptosis, and assess the possible cytoprotective mechanism of selenium.Methods: The levels of P-JNK and JNK, caspase-3, bcl-2,p53 proteinswere detected by western blot. Results: The studies found thephosphorylation of JNK kinase increased with exposure to cadmium for 3h, even remained elevated at 9 h in the time course study, and theactivation of phosphorylated JNK was detected in a dose-dependentmanner. In addition, a concomitant time-dependent increase in caspase-3 activities was observed by cadmium treatment. During the process,selenium played the same role as N-acetyl-L-cysteine (NAC), a freeradical scavenger. Pretreatment of cells with selenium partiallysuppressed of the phosphorylation of JNK, coupled with caspase-3activation involved in cadmium-induced apoptosis. Furthermore, Onexposure to 40μM cadmium for 12h, the expression levels of p53 proteinswere upregulated. At the same time, there was a downregulation of bcl-2protein in LLC-PK₁ cells. The selenium pretreatment abrogated of thecadmiumd-induced p53 proteins upregulation, however, did not affect thebcl-2 expression levels, when compared with those of the treated cellswith cadmium alone. Conclutions: Our studies provided a molecularlinkage between the phosphorylation of JNK and cadmium-inducedLLC-PK₁ cells apoptosis, and demonstrated selenium also contributed apotentially protection to prevent cadmium-cytotoxicity.