| Hepatocellular carcinoma (HCC) belongs to one of the common malignant tumors.Therefore, it affects human health in the world, not only greatly reduces the survival timeof patients, but also reduces their quality of life. Its incidence ranks fifth in the worldamong malignant tumors, the third mortality rates in the world, and there are one millionnew cases each year. HCC is proven to be invasive tumors that growth rapidly，resistanceto chemotherapy, high recurrence rate to postoperative. Recent years, despite the newchemotherapy drugs and treatment strategies are used widely, however, the treatment ofHCC is still not satisfactory. Therefore, how to increase survival and improve the qualityof life of patients and how to more effectively treat HCC, has become very urgent andnecessary.Arsenic trioxide (As2O3) is the effective components of traditional Chinese medicineof arsenic. Early in the1970s, As2O3has became the clinical medicine for the treatment ofacute promyelocytic leukemia (APL). In recent years, numerous studies have confirmedthe role of As2O3in solid tumor cells in vitro and in vivo, especially in liver cancer cells, further confirmed that it can inhibit proliferation of hepatoma cells. Related studies showthat the apoptosis of liver cancer cells is associated with mitochondrial pathways. Thestudy showed that when As2O3induced apoptosis of hepatoma cells, there will be a lot ofreactive oxygen species(ROS) generation, however, when added antioxidantN-acetylcysteine (NAC), it can suppress As2O3-induced apoptosis of hepatoma cells. Themechanism of As2O3-induced apoptosis of hepatoma cells through activating themitochondrial pathway, it can inhibit the expression of Bcl-2proteins and increaseexpression of Bax, while the release of cytochrome c into the cytoplasm, therebyactivating cysteine aspartate-specific enzyme (caspase), and then cascade can eventuallylead to apoptosis. Smac and cyt c are both of mitochondrial protein, they inducedapoptosis by suppressing Inhibitors of apoptosis protein (Inhibitors of apoptosis protein,IAP), to eliminate the inhibition of caspase and caspase thereby into the cytoplasm, play arole of resulting in apoptosis. In order to further investigate the mitochondrial pathway ofAs2O3induced liver cancer cells apoptosis, as well as the expression of related proteins,we select two common HCC cell lines, respectively, to further explore whether As2O3playa role in inducing apoptosis of hepatoma cells by regulating ROS-mediated mitochondrialpathway, and whether As2O3induced apoptosis of hepatoma cells by regulating theexpression of mitochondrial related proteins.【Objective】1. Cultured liver cancer cells, HepG2hepatocellular carcinoma cell line andhepatocellular carcinoma cell line SMMC–7721.2. To investigate whether As2O3can inhibit proliferation and induced apoptosis ofhepatoma cells.3. To investigate whether As2O3through ROS-mediated mitochondrial pathway plays arole in inducing apoptosis of hepatoma cells.4. To explore whether As2O3play a role in inducing apoptosis of hepatoma cells byregulating the expression of mitochondrial proteins. 【Methods】1. Cell culture and grouping.(1) SMMC-7721human liver cells were placed into1640medium containing10%fetal bovine serum which was supplemented with100U/ml penicillin and100mg/mlstreptomycin, in a humidified atmosphere of5%COo2at37C. The cells weresubsequently treated with0,5,10,20μM As2O3for24h.Thus were to become thecontrol group, low-dose group, middle dose group and high-dose groups.(2) HepG2cells were grown in Dulbecco’s Modified Eagle Medium containing10%fetal bovine serum which was supplemented with100U/ml penicillin and100mg/mlstreptomycin, in a humidified atmosphere of5%COo2at37C. The cells weresubsequently treated with0,2,5,15μM As2O3for24h with or without pretreatmentwith10mM N-acetylcysteine (NAC) for1h, respectively. Thus were to become thecontrol group, low-dose group, middle dose group, high-dose group and NACblockers.2. MTT reduction method (MTT method) to observe the effect of As2O3on liver cancercell activity.3. Through Hoechst33258staining to observe morphological changes of apoptoticnuclei.4. The percentage of apoptotic cells was measured by flow cytometry using an annexinV-FITC/PI kit.5. DCFH-DA reactive oxygen test kit to measure the amount of intracellular ROS.6. Rhodamine123detects test in mitochondrial membrane potential.7. Detection of apoptotic protein expression levels of protein immunoblot (Western Blot).【Results】1. MTT assay showed:(1) With the increasing concentrations of As2O3and prolonging of the treatment time, HCC cell’s vitality showed a decreasing trend. Concentration of5μmol/L of As2O3effected in hepatoma cells after48h, cell vitality decreased(94.23±1.52%，P<0.05).Concentration of10μmol/L of As2O3effected in hepatoma cells after72h, cell vitalitydecreased(78.21±1.73%，P<0.01). When the concentration of20μmol/L of As2O3effected in hepatoma cells after72h, cell vitality was still significantlydecreased(49.65±0.83%，P<0.01).(2) With the increasing concentrations of As2O3and prolonging of the treatment time,HCC cell vitality showed a decreasing trend. Concentration of5μmol/L of As2O3effected in hepatoma cells after48h, cell vitality decreased(78.12±1.51%，P<0.01).Concentration of15μmol/L As2O3effected in HepG2cells after72h, cell vitalitydecreased(59.13±1.72%，P<0.01). However, when given the antioxidant NAC, adownward trend in cell vitality were suppressed, after72h, compared with the higherdose group, the cell vitality increased(84.23±2.12%，P<0.01).2. Through Hoechst33258staining to observe morphological changes of apoptotic nucleishowed:(1) In hepatocellular carcinoma SMMC-7721cells, with increasing concentrations ofAs2O3, each group appeared increasingly obvious signs of apoptosis. After giving5,10,20μmol/L As2O3to SMMC-7721cells, nuclear condensed, the number ofchromatin condensed cells were increased respectively to (11.23±1.52%, P<0.01),(23.22±2.29%, P<0.0),(42.45±3.17%, P<0.01).(2) In hepatocellular carcinoma HepG2cells, with the increasing concentrations ofAs2O3, each group appeared increasingly obvious signs of apoptosis. After giving2,5,15μmol/L As2O3to HepG2cells, nuclear condensed, the number of chromatincondensed cells were increased respectively to (17.73±0.91%, P<0.01),(24.23±2.21%,P<0.0),(47.54±2.92%, P<0.01). However, when given antioxidant NAC, apoptoticcells were decreased. Compared with the high-dose group, nuclear condensed, thenumber of chromatin condensed cells decreased to (19.77±2.12%, P<0.01).3. Apoptosis detected by flow cytometry Showed: (1) In hepatocellular carcinoma SMMC-7721cells, with increasing doses of As2O3,eachgroup’s apoptotic and necrotic rate were gradually increased. Meanwhile, the livingrate of cells was decreased. After giving5,10,20μmol/L As2O3to liver cancer cells,the proportion of apoptotic cells increased from1.94±0.58%(As2O3unused) to(23.67±2.19%, P<0.01),(39.22±1.95%, P<0.01),(54.23±2.46%, P<0.01).(2) In hepatocellular carcinoma HepG2cells, with increasing doses of As2O3,eachgroup’s apoptotic and necrotic rate were gradually increased. Meanwhile, the livingrate of cells was decreased, showing a dose-dependent relationship.. After giving2,5,15μmol/L As2O3to liver cancer cells, the proportion of apoptotic cells increasedfrom2.82±0.55%(As2O3unused) to (18.91±2.77%, P<0.01),(38.75±1.92%, P<0.01),(59.18±1.79, P<0.01). However, when given the antioxidant NAC, apoptotic cellswere reduced, compared with the high-dose group, the number of apoptotic cellsreduced to (13.1±2.13%, P<0.01).4. Measuring the amount of intracellular ROS:(1) With an increasing amount of As2O3, the amount of intracellular ROS was alsoincreased, showing a dose-dependent relationship. After giving5,10,20μmol/LAs2O3to SMMC-7721cells，the amount of intracellular ROS detected wererespectively1.2times（P<0.05）,2.1times （P<0.01）and3.2times（P<0.01）of thecontrol groups.(2) With an increasing amount of As2O3, the amount of intracellular ROS was alsoincreased, showing a dose-dependent relationship. After giving2,5,15μmol/L As2O3to HepG2cells, the amount of intracellular ROS detected were respectively1.1times（P<0.05）,1.8time（sP<0.01）and3.2time（sP<0.01）of the control groups. However,when given the antioxidant NAC，the amount of ROS produced in cells was reduced.In contrast, the amount of ROS reduced to0.33times of the high-dose groups（P<0.01）.5. Rhodamine123detects changes in mitochondrial membrane potential：(1) With increasing dosage of As2O3, mitochondrial membrane potential showed a downward trend, with a dose-dependent manner. After giving5,10,20μmol/L As2O3to SMMC-7721cells, the decreasing amount of mitochondrial membrane potentialwere respectively0.91times (P<0.05),0.73times (P<0.01) and0.52times(P<0.01)of the control groups.(2) With increasing dosage of As2O3, detected mitochondrial membrane potential showeda downward trend, with a dose-dependent relationship. After giving2,5,15μmol/LAs2O3to HepG2cells, the decreasing amount of mitochondrial membrane potentialwere respectively0.95times(P<0.05),0.74times(P<0.01) and0.53times(P<0.01) ofthe control groups. However, when given antioxidant NAC，the downward trend ofmitochondrial membrane potential was suppressed. The increase of mitochondrialmembrane potential was1.64times of the higher dose group (P<0.01).6. Using Western Blot assay to detect the apoptosis protein level showed:(1) In SMMC-7721cells, intracellular Bax/Bcl-2ratio increased with the increasingconcentration of As2O3.Specifically, the expression of Bax protein increased, and theexpression of Bcl-2protein decreased. Similarly, with the increasing concentration ofAs2O3,the expression of cyt c, Smac, caspase-3, caspase-6and caspase-9proteinincreased, with a dose-dependent manner, the differences were statisticallysignificant.(2) In HepG2hepatoma cell lines, detected the expression of caspase-3, caspase-6andcaspase-9protein, finding an upward trend in the three proteins, with adose-dependent manner. However, when given the antioxidant NAC, the increasingexpression of three proteins were suppressed. Using Western Blot assay to detect, theexpression of cyt c and Smac increased, so as to the ratio of Bax/Bcl-2. Theexpression of Bax protein increased, and the Bcl-2protein decreased, with adose-dependent manner. However, when adding antioxidant NAC, the increasingexpression of cyt c, Smac, Bax/Bcl-2were suppressed. The differences werestatistically significant.(p <0.05).【Conclusion】1. As2O3can inhibit the proliferation of liver cancer cells and induce apoptosis of hepatoma cells.2. As2O3induced apoptosis of hepatoma cells is mediated by mitochondrial ROSpathway.3. As2O3induced apoptosis of liver cancer cells by regulating the expression of relatedproteins. |
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