Study Of Cancer Cells' Autophagy Induced By Arsenic Trioxide And Its Mechanism In Leukemia

Autophagy is evolutionarily conserved and occurs in all eukaryotic cells,from yeast to mammals.Autophagy is activated in response to nutrient starvation,differentiation,and developmental triggers.It is an adaptive process responding to metabolic stresses that results in degradation of intracellular proteins and organelles.During autophagy, portions of the cytoplasm are encapsulated in a doubl membrane structure referred to as an autophagosome.Autophagosomes then fuse with lysosomes where the contents are delivered,resulting in their degradation by lysosomal hydrolases.Under normal physiological conditions,autophagy occurs at basal levels in most tissues,contributing to the routine turnover of cytoplasmic components.It can promote cell adaptation and survival during stresses such as starvation,but under some conditions cells undergo death by excessive autophagy.Though the phenomenon of apoptosis had been described for almost a century,in 1972,Kerr,Wyllie,and Currie first coined the term "apoptosis" in order to differentiate naturally occurring developmental cell death from necrotic cell death that results from acute tissue injury.They also noted that apoptosis was responsible for maintaining tissue homeostasis by mediating the equilibrium between cell proliferation and death.Morphologic characteristics of apoptosis include cell membrane blebbing,cell shrinkage,chromatin condensation,and nucleosomal fragmentation.Under normal circumstances,cells undergoing apoptosis are recognized by macrophages,or neighboring cells that consume the cells' fractionated carcasses.Apoptosis has been considered a major mechanism of chemotherapy-induced cell death,and pathways regulating apoptosis are the focus of many preclinical drug discovery investigations.In yeast,a cassette of autophagy-related genes (referredto as ATG) have been identified that regulate autophagy induction,autophagosome formation and expansion, fusion with lysosomes,and the recycling of autophagosome contents.Some of the mammalian orthologs to these genes have been identified.Studies involving Beclin 1,the mammalian ortholog of yeast Atg6,gave the first indications linking dysfunctional autophagy with tumorigenesis.Beclin 1 is required for autophagosome formation and has been suggested to be a haploinsufficient tumor suppressor gene.Beclin 1 +/-mice suffer from a high incidence of spontaneous tumors,and Beclin 1 is monoallelically deleted in a high percentage of sporadic human breast,ovarian,and prostate carcinomas.Several lines of evidence have found that a cross-talk exists between autophagic and apoptotic pathways.Beclin1 was originally identified through its interaction with Bcl-2.Recent findings have shown that Bcl-2 and Bcl-xL expression can sensitize cells to autophagic death induced by etoposide,and that Bcl-2 inhibits Beclin 1-mediated autophagy in response to starvation. These contradictory findings suggest that the outcome of the autophagic response may vary depending on the type of insult or cellular stress. The role of autophagy in cancer and in the response to chemotherapeutic agents is slowly gaining attention.A number of studies have reported that autophagy,or autophagic cell death,is activated in cancer cells derived from a variety of tissues in response to various anticancer therapies.For example tamoxifen induces autophagic cell death in cultured breast cancer cells,in part,through a down-regulation of Akt. Autophagy also plays a role in protecting some cells from chemotherapy-induced death.Bafilomycin A1 can inhibit autophagy by preventing the fusion of autophagosomes and lysosomes.It is a V-type ATPase inhibitor that prevents acidification of lysosomal-based compartments including autophagosomes.Bafilomycin A1 inhibitedγ-irradiation-induced autophagy in cancer cell lines derived from different tissues,and increased the amount of IR-induced death.As₂O₃-induced apoptosis was a general mechanism found in most of clinical applications for cancer chemotherapy. As₂O₃-induced apoptosis has been observed in different types of human cancer cells and in brain tissue cultures.In these models, increased intracellular oxidative stress was demonstrated to be one of the important mechanisms that lead to As₂O₃-induced cell death.Previous studies indicated that autophagic cell death and apoptosis are pathways to the same end;a functional connection between both forms of cell death is likely to be operative.Both forms of cell death can act as backup mechanisms of each other,under conditions where cell death is imperative.As an anticancer drug,the main mechanism of Arsenic trioxide(As₂O₃) is to induce tumor cell apoptosis.There have been few reports about whether As₂O₃ can induce tumor cells to have non-apoptotic death(such as autophagy,etc.).Through the observation of different levels of autophagy in HepG-2 cells after induction with different concentrations of As₂O₃,we preliminarily investigate its mechanism.The results are reported as follows.Objective:To assess the level of autophagy in HepG-2 cells,HL60 cells and k562 cells after induction with arsenic trioxide, and to investigate its mechanism.Methods:1.Cell culture and grouping The experimental cells were inoculated into the culture bottles with 2×10⁵/ml and the culture fluid was 10%fetal calf serum RPMI1640.They were placed into the incubator at 37℃with saturated humidity and 5%CO₂ for cell crawling growth.After the cells being adhered, different concentrations of As₂O₃ were added for treatment. According to the concentrations of As₂O₃,the cells were divided into five groups:0μM /L(control group),1.0μM/L,2.0μM/L,4.0μM/L and 8μM/L group;Each group was trypsinized with 0.25%trypsin 24 h after the treatment to harvest cells or lamella of crawling cell for observation.2. The Observation of general morphology Useing inverted microscope to observe cell general morphology before and after the addition of the drugs and the corresponding photos were taken.3.Detection of autophagy with MDC fluorescent staining Staining of the autophagicvacuoles(AV):the lamella of crawling cells were taken out and rinsed with PBS twice.The substrates containing serum were removed.Then they were incubated with 0.05 mmol/L MDC at 37℃for 60 min and fixed with 4%paraformaldehyde for 15 min.Following that,they were again rinsed with PBS twice.After the airing,the fluorescent microscope(Olympus) along with 356 nm emission filter and 545 nm barrier filter was used to observe the cells and take their photos.4.The flow cytometry(FCM)-based quantitative analysis for autophagy According to the above method of MDC staining,the Elite flow cytometry(COULTER company,USA) was used to determine the fluorescence intensity of the cells stained with MDC at 488 nm excitation wavelength.The light scattering and fluorescent data were input into the computer for analysis.5.Detection of Bcl-2 mRNA expression with RT-PCR Cells were collected after they were cultured in the incubator at 37℃with saturated humidity and 5%CO₂ after 24 h and then were rinsed with PBS for twice.The guanidinium thiocyanate one-step method was used to extract RNA and the concentration and purity were determined at the lenth of 260/280nm wave; RT-PCR processes were completed according to the notes on reverse transcriptase reagent kit.The sequences of primers for PCR amplification were as follows:Bcl-2:Sense 5'-TGTGGCCTTCTTTGAGTTCG-3',Antisense 5'-TCACTTGTGGCTCAGATAGG-3',fragment 280 bp;β-action:Sense 5'-GGGTCAGAAGGATTCCTGTG-3',Antisense 5'-GGTCTCAAACATGATC TGGG-3',fragment 317 bp.20μl of each PCR product was taken for electrophoresis in 10g /L agarose gel and stained with ethidium bromide.Then the photos were developed under ultraviolet lamps.6.Detection of positive rate of Bcl-2 protein with immunochemical method APAAP method was adopted for staining.The concrete staining procedures referred to the notes on reagent kit.The positive results presented as red particles in cytoplasm,100 cells were counted under the oil lens and the percentage of positive cells was calculated.The experiment of each group was repeated three times and the average value of the results was considered as the final outcome. 7.Statistical process The data were presented as x±s;The comparison of average values between the two samples was tested with t-test.Pearson linear correlation was used for correlation analysis(α=0.05) and SPSS10.0 software package was adopted for statistical analysis.RESULTS:1.The effects of As₂O₃ on the growth of three kinds cells lines There were more cells with good stretching abilities and even and transparent cytoplasm in control group when they were observed under the inverted microscope.24 h after the induction with As₂O₃,the number of cells in all experimental groups significantly decreased compared with that in the control group.In addition,the adhering abilities of some cells decreased and grew in a floating state.The cell shapes turned round gradually and began to become smaller.2. The change of HepG-2 autophagy level MDC could be assimilated by HepG-2 cells and selectively gathered in autophagy vesicles.When the AVs stained with MDC fluorescence were observed under microscope,they presented as dark green or yellow-green spotty structure and scattered around the nucleus.In the control group,only very few MDC-positive cells could be seen with each cell containing a small number of AVs. In the As₂O₃ treatment groups,the number of MDC positive cells increased significantly with every cell containing more AVs. The percentage of MDC-positive cells by FCM quantitative determination increased with the enhanced concentration of As₂O₃,which had a significant difference compared with that in the control group(P＜0.05).3.The effect of As₂O₃ on Bcl-2 gene transcription level and Bcl-2 protein expression 24h after the induction with different concentrations of As₂O₃,Bcl-2 gene expression in each group significantly weakened than that in the control group,and was correlated with As₂O₃ concentration. Bcl-2 protein expression could be detected in both control group and all treatment groups.The positive results presented as red particles within the cytoplasm.The average number of Bcl-2 protein positive cells in all treatment groups would increase with the decrease of As₂O₃ concentration and had a significant difference compared with that in the control group (P＜0.05).4.The correlation of cell autophagy and Bcl-2 gene expression The correlation showed that the level of cell autophagy was negatively correlated with Bcl-2 protein positive cells(r=-0.47,P＜0.05).Conclusion:1.Arsenic trioxide can induce the autophagy of the three kinds cells whose mechanism might he associated with the downregulation of Bcl-2 gene expression.2.The percentage of MDC-positive cells by FCM quantitative determination increased with the enhanced concentration of As₂O₃.3 Autophagy and apoptosis may have some associations.Autophagy occurred prior to apoptosis.4.Autophagy delayed the apoptosis.