Preliminary Study On The Inhibitory Effect, The Gene Expression Profile And Molecular Mechanisms Of Arsenic Trioxide-induced Apoptosis In Human Adenoid Cystic Carcinoma ACC-2Cells In Vitro

ObjectivesThis study aims to (1)observe the inhibitory effect of arsenic trioxide (As2O3) on adenoid cystic carcinoma (ACC) cell line ACC-2, and to investigate the cellular and molecular mechanisms of As2O3-induced apoptosis in ACC-2cells;(2)detect the changes of gene expression profile of ACC-2cell line before and after AsO3's induction, and to screen the apoptosis-related genes;(3)investigate the role of apoptosis-related genes bcl-2, c-myc in the ACC-2cell apoptosis induced by As2O3, and to verify the gene expression profiling test results of microarray, explore the molecular mechanisms of As2O3-induced apoptosis in human adenoid cystic carcinoma ACC-2cells, provide a theoretical basis for the clinical application of As2O3drugs;(4)explore the role of hTERT in the ACC-2cell apoptosis induced by As2O3;(5)investigate the role of mitochondrial membrane potential (Δψm) and Caspase3in the ACC-2cell apoptosis induced by As2O3.Methods(1)ACC-2cells were cultured. The different drug concentration gradient (0,1.0,2.0,4.0,8.0umol/L) of As2O3were applied to ACC-2cells respectively. The morphological changes of ACC-2cells before and after As2O3's induction were observed under inverted phase contrast microscope, and photographic records were taken. MTT assay was performed after As2O3application24h,48h,72h, and the absorbance OD values were measured by TECAN Safire Ⅱ multifunctional microplate reader to get the cell growth inhibition rate. Hoechst33342/PI double staining by immunofluorescence staining was performed. Annexin V-PI double staining was performed by flow cytometry to analyze the apoptosis rate of cells. PI staining was performed by flow cytometry to detect DNA content and cell cycle. The ultrastructural changes of ACC-2cells before and after As2O3's induction were observed by transmission electron microscopy.(2) The gene expression profile of ACC-2cells was detected before and after As2O3's induction with application of gene chip. Bioinformatics analysis was used to explore the functional networks of the differentially expressed genes in order to get the significantly differentially expressed candidate genes.(3) The mRNA levels and protein levels expression of the apoptosis related genes bcl-2, c-myc of ACC-2cells before and after As2O3's induction were detected by RT-PCR and Western Blot.(4) The mRNA expression of hTERT of ACC-2cells before and after As2O3's induction was detected by RT-PCR.(5) The mitochondrial membrane potential (Δψm) of ACC-2cells before and after As2O3's induction (8.0μmol/L for24h) was detected by flow cytometry with Rh123staining. Caspase3activity was detected by the multifunctional microplate reader.Results(1) AS2O3could inhibit the adenoid cystic carcinoma ACC-2cells growth, and the inhibition presented a time-dose-dependent relationship. With the increased AS2O3drug concentration and action time, nuclei of apoptosis cells stained strong blue became more and more by immunofluorescence staining. Ultrastructure observation under the transmission electron microscope showed that some of ACC-2cells presenting apoptotic morphology, such as cell shrinkage, nucleus concentrated shrinkage, transparent area in the nucleus, nucleolus disappearing, nuclear vacuolization, chromatin condensation, gathering in the nuclear membrane, or integrating clumps and attaching to the nuclear membrane, or chromatin margination forming of crescent-shaped substances, organelles decreased, cytoplasm concentration, or cracking into the plasma membrane surrounding the chromatin fragments (apoptotoc bodies). Flow cytometry results showed that ACC-2cells apoptosis rate increased gradually with the increase of the concentration of AS2O3. Except for the low concentration group (1.0μmol/L), the others'(2.0,4.0,8.0μmol/L) apoptosis rate were higher, compared with that of the control group, the difference being significant (P<0.05). Sub-G1apoptosis peak was detected in As2O3action groups, among which, sub-G1apoptosis peak was not obvious in the low concentration groups (1.0,2.0umol/L), while it was obvious in the high concentration group (4.0,8.0μmol/L). After4.0.8.0μmol/L As2O3action to ACC-2cells for48h, G2-M phase cells significantly increased. Compared with that of the control group, it was with an increase of30.23%and33.43%, respectively.(2)After As2O3action to ACC-2cells, its gene expression profile changed greatly, with1160genes expression significantly up-regulated, and1084genes significantly downregulated.(3)RT-PCR results showed that bcl-2, c-myc mRNA expression were significantly higher in the control group, while after As2O3action to ACC-2cells for24h and48h, with the increase of As2O3concentration (1.0μmol/L,2.0μmol/L,4.0μmol/L,8.0μmol/L), bcl-2, c-myc mRNA expression decreased gradually. Western Blot results showed that bcl-2, c-myc protein expression were significantly higher in the control group, while after As2O3action to ACC-2cells for24h and48h, with the increase of As2O3concentration (1.0μmol/L,2.0μmol/L,4.0μmol/L,8.0μmol/L), bcl-2, c-myc protein expression decreased gradually. The trend was consistent with mRNA expression.(4)RT-PCR results showed that hTERT mRNA expression was significantly higher in the control group, while after As2O3action to ACC-2cell for24h and48h. with the increase of As2O3concentration (1.0μmol/L,2.0μmol/L,4.0μmol/L,8.0μmol/L), hTERT mRNA expression decreased gradually.(5)Rh123fluorescence intensity in ACC-2cells was the strongest in the control group, while it weakened in ACC-2cells in8.0μmol/L As2O3treatment group. The difference was significant (P<0.05). With the increase of As2O3concentration (0μmol/L,1.0μmol/L,2.0μmol/L,4.Oμmol/L,8.0μmol/L), Caspase3enzyme activity unit in ACC-2cells gradually increased.Conclusions(1) As2O3can inhibit the adenoid cystic carcinoma ACC-2cells growth, mainly through the induction of apoptosis. After As2O3treatment to ACC-2cells, the cells show typical morphological changes of apoptosis. Sub-Gi apoptotic peak can be detected and cells are in G2/M phase arrest. The apoptosis rate increased gradually with the increase of As2O3concentration.(2)After As2O3treatment to ACC-2cells, its gene expression profile changed greatly, with the significant changes of apoptosis-related genes.(3) As2O3can induce apoptosis of ACC-2cells by downregulating bcl-2, c-myc, hTERT mRNA transcript expression and bcl-2, c-myc protein expression.(4) As2O3can induce apoptosis of ACC-2cells by reducing the mitochondrial membrane potential. With As2O3concentration increased, Caspase3enzyme activity unit of ACC-2cells gradually increases, which results in activation of the expression of Caspase3, and the cells immediately enter the irreversible apoptosis process.(5)On the whole, As2O3can induce the apoptosis of adenoid cystic carcinoma ACC-2cells, at least through the early downregulating bcl-2, c-myc mRNA transcript expression and protein expression, and positively regulating hTERT mRNA expression downregulation, bcl-2protein expression inhibition, the mitochondrial membrane potential decline or even dissipation, which can cause increased permeability of the mitochondrial outer membrane, MPTP opening up, promoting the release of cytochrome C, activating Caspase3, which occurs in apoptosis, cells arrest at the G2/M phase. As2O3-induced ACC-2cells apoptosis achieves through the complex process in which multi-step, multiple fagtors of oncogenes, tumor suppressor genes, protease are all involved, and through the interchange network of signal transduction pathways.