Study On The Apoptosis In HL-7702 Cells Induced By Cadmium Chloride And Its Mechanisms

Cadmium (Cd) is a toxic heavy metal drawing continuous occupationaland environmental concerns due to its wide variety of adverse effects. It caneasily accumulate in the body especially in the liver, which is considered one ofthe most important target organs, in acute cadmium poisoning. Therefore, thestudy of cadmium hepatotoxicity becomes more and more important. Thisstudy is designed to learn the detailed mechanism of apoptosis induced bycadmium in liver cells, especially in normal tissues. We choose the normalhuman liver cell line, HL-7702 cell line, as the model to study the process ofapoptosis induced by cadmium chloride, observe in both cellular and molecularbiology levels. Results showed that cadmium chloride had toxic effects onHL-7702 cells and the possible mechanisms were related to S phase blocking,oxidase damage and the induction of apoptosis by upregulating ordownregulating apoptosis-related genes and proteins.1. Inhibitory effects of cadmium chloride on HL-7702 cells1.1 Changes of growth curve of HL-7702 cells after exposure to cadmiumchlorideHL-7702 cells were exposed to cadmium chloride at differentconcentrations (5, 10, 20, 30, and 40μmol/L). The inhibition of the cells during8 days was determined by cell counting and then the cell growth curve wasmade. The results showed that the cell amount in control group was more thanthat of experimental groups during the course. It suggested that cadmiumchloride at different concentrations could inhibit the growth and proliferationof HL-7702 cells in a dose-dependent manner with the cell growth curvechanged accordingly.1.2 Toxic effects of cadmium chloride on HL-7702 cellsHL-7702 cells were exposed to cadmium chloride at differentconcentrations (5, 10, 20, 30, and 40μmol/L). MTT assay was applied toobserve the toxic effects of cadmium chloride, and the results were describedby inhibitory rate. The results showed that the inhibitory rate of HL-7702 cellsat 24h increased significantly with the concentrations of 30 and 40μmol/Lcompared with NC group (P<0.05, P<0.01, respectively). The inhibitory rate ofHL-7702 cells at 48h increased significantly with all concentrations comparedwith NC group (P<0.05-P<0.01). And the inhibitory rate of HL-7702 cells at72h also increased significantly with concentration of 40μmol/L compared withNC groups (P<0.05). It indicated that cadmium chloride had toxic effects onHL-7702 cells.2. Effects of cadmium chloride on HL-7702 cell cycleFCM assay was performed to study the changes in cell cycle of HL-7702cells in five concentrations (5, 10, 20, 30, and 40μmol/L) at 24, 48 and 72h.The results showed that the percentage of HL-7702 cells in G0/G1 phasedecreased significantly 24h after treated by cadmium chloride withconcentration of 40μmol/L compared with NC group (P<0.01). And 48h aftertreatment, the percentage of cells in G0/G1 phase decreased significantly in40μmol/L group compared with NC group (P<0.01), the percentage of cells inS phase increased significantly with concentrations of 20, 30 and 40μmol/Lcompared with NC group (P<0.05-P<0.01), the percentage of cells in G2+Mphase decreased significantly with concentrations of 30 and 40μmol/Lcompared with NC group (P<0.05). And 72h after treatment, the percentage ofcells in G0/G1 phase decreased significantly with concentrations of 10, 20, 30and 40μmol/L compared with NC group (P<0.05-P<0.01), the percentage ofcells in S phase increased significantly with all concentrations compared withNC group (P<0.05-P<0.01), the percentage of cells in G2+M phase decreasedsignificantly with all concentrations compared with NC group (P<0.01). Itindicated that S phase blocking could be induced by cadmium chloride inHL-7702 cells.3. Induction of apoptosis in HL-7702 cells with cadmium chloride3.1 Effects of cadmium chloride on apoptosis of HL-7702 cells determinedby AnnexinV-FITC/PI double fluorescent staining FCMFCM assay was performed to study the changes in apoptosis of HL-7702cells in four concentrations (5, 10, 20 and 40μmol/L) at 12, 24, 48 and 72h.The results showed that the percentage of apoptotic cells changed slightly 12hafter treatment while after 24h, the percentage of apoptotic cells increasedsignificantly with concentrations of 20 and 40μmol/L compared with NC group(P<0.05). 48h after treatment, the percentage increased significantly withconcentration of 40μmol/L compared with NC group (P<0.01). And after 72hof treatment, the percentage increased dramatically with all concentrationscompared with NC group (P<0.05-P<0.01). It indicated that cadmium chloridecould induce apoptosis in HL-7702 cells.3.2 Effects of cadmium chloride on apoptosis of HL-7702 cells determinedby AO/EB double fluorescent stainingFour concentrations (5, 10, 20 and 40μmol/L) of cadmium chloride wereused to study the changes in apoptosis of HL-7702 cells by AO/EB doublefluorescent staining assay 24h after treatment by cadmium chloride. The resultsshowed that the percentage of apoptotic cells increased significantly with allconcentrations when compared with NC group (P<0.05-P<0.01). It suggestedthat cadmium chloride could induce apoptosis of HL-7702 cells.4. Effects of cadmium chloride on oxidase damage of HL-7702 cellsFour concentrations (5, 10, 20 and 40μmol/L) of cadmium chloride wereused to study the changes of activities of SOD, content of MDA, and activitiesof GSH-Px in HL-7702 cells 24h after treatment by cadmium chloride and allwere measured by kit.4.1 Effects of cadmium chloride on activities of SOD in HL-7702 cellsResults showed that the activities of SOD were decreased significantlywith concentration of 40μmol/L compared with NC group (P<0.05). Itindicated that the activities of SOD in HL-7702 cells could be decreased bycadmium chloride.4.2 Effects of cadmium chloride on content of MDA in HL-7702 cellsResults showed that the content of MDA increased significantly withconcentrations of 20 and 40μmol/L compared with NC group (P<0.05, P<0.01,respectively). It indicated that the content of MDA in HL-7702 cells could beincreased by cadmium chloride.4.3 Effects of cadmium chloride on activities of GSH-Px in HL-7702 cellsResults showed that the activities of GSH-Px changed slightly in allconcentrations. But there was a decreasing trend of activities with theincreasing of cadmium chloride. It indicated that the activities of GSH-Px inHL-7702 cells probably could be decreased by cadmium chloride.5. Changes in mRNA level of apoptosis-related genes in HL-7702 cells afterexposure to cadmium chlorideHL-7702 cells were treated by cadmium chloride for 6-48h withconcentrations of 20μmol/L, and five genes were studied, caspase-3, caspase-8,Bax, bcl-2 and surviving. RT-PCR assay was employed for the measurement ofmRNA level.5.1 Changes in caspase-3 mRNA levelResults showed that caspase-3 mRNA level increased at 12 and 24h, andslightly decreased at 6 and 48h.5.2 Changes in caspase-8 mRNA levelResults showed that caspase-8 mRNA level increased at 6-48h, and it gotto the highest at 24h.5.3 Changes in Bax mRNA levelResults showed that Bax mRNA level also increased at 6-48h, and it got tothe highest at 24h.5.4 Changes in bcl-2 mRNA levelResults showed that bcl-2 mRNA level decreased at 6h, and increased at12-48h.5.5 Changes in survivin mRNA levelResults showed that survivin mRNA level slightly increased at 6h, anddecreased at 12-48h.6. Changes of protein expression of apoptosis-related genes in HL-7702cells after exposure to cadmium chloride6.1 Changes of caspase-3 protein expressionFour concentrations (5, 10, 20 and 40μmol/L) of cadmium chloride wereused to study the caspase-3 protein expression, 24h after treatment by cadmiumchloride.6.1.1 Changes of caspase-3 protein expression detected by FCMResults showed that the caspase-3 protein expression increased slightlyalong with the elevating of concentrations, but there was no statistic differencebetween experimental groups and NC group.6.1.2 Changes of caspase-3 protein expression detected by Western BlotResults showed that all groups could express 32kDa procaspase-3.Besides, a unique cleaved protein fragment at 20kDa could be detected in allexperiment groups, and the color of this fragment increased with the elevatingof concentrations gradually.6.2 Changes of Fas protein expressionImmunocytochemistry assay was applied to observe the changes of Fasprotein expression induced by cadmium chloride at 24h in four concentrations(5, 10, 20 and 40μmol/L). Results showed that the Fas protein expression inHL-7702 cells increased significantly with all concentrations compared withNC group (P<0.05-P<0.01).6.3 Changes of FasL protein expressionImmunocytochemistry assay was applied to observe the changes of FasLprotein expression induced by cadmium chloride at 24h in four concentrations(5, 10, 20 and 40μmol/L). Results showed that the FasL protein expression inHL-7702 cells increased significantly with concentrations of 10, 20 and40μmol/L compared with NC group (P<0.01).