The Role Of Telomere And Telomerase In Apoptosis Of Malignant Cells Induced By Bortezomib And Suppression Of Gastric Cancer By ARID1A

BackgroundTelomeres are complexes located at the ends of eukaryotic chromosomes, consisting of short DNA repeats "TTAGGG" and associated proteins. The main role of telomeres is to protect chromosomes through preventing them from degradation by nuclease and end fusion. Besides, telomere length can predict the potential of cell division and they are therefore called "cell division clock". With each cell division, telomeric DNA losts 50-200bp until it reaches a certain limit. Then chromosome DNA can not achieve successful replication and cell division will be arrest. Telomerase is an RNA-dependent DNA polymerase, which comprises telomerase reverse transcriptase (hTERT), telomerase RNA (hTER) and associated proteins. The most important contribution of telomerase is to elongate telomeres. In most normal cells, telomerase is silent. However, in pathological state, such as malignant cells, it is strongly activated to ensure unlimited proliferation of cells. The maintainance of telomere length preserved by telomerase is essential for tumor progression.Bortezomib is an anti-cancer agent which is already used clinically for the treatment of multiple myeloma(MM). As a proteasome inhibitor, its well characterized mechanism is to inhibit the activity of NF-κB, which is an outstanding transcription factor. With NF-κB inhibition, the anti-apoptotic bcl-2 expression is down-regulated and caspase 3 is activated, which contribute to cell apoptosis. In order to resolve drug resistance and acquire more reasonable clinical applications, we have to search novel mechanisms and targets of bortezomib.Tumor suppressor aridla is a major component of SWI/SNF complexes, which is responsible for chromatin remodeling. Along with another element of this complex BRG1 or BRM which hydrolyzes ATP to generate energy, aridla achieves its chromatin remodeling function. SWI/SNF complexes remodel chromatin conformation to expose or hide transcriptional start sites(TSS) of target genes through removing or altering the location of histone core. Thus, the expression of target genes are regulated at the transcriptional level. Recent sequencing results showed ARID1A was mutated or lost in a series of cancers, such as ovary clear cell carcinoma, gastric cancer, renal carcinoma and pancreatic cancer. Thus, aridla is considered to be a tumor suppressor. However, little has been known about its anti-cancer mechanisms.Aims1. To explore the role of telomere and telomerase in apoptosis of malignant cells induced by bortezomib and the role of hTERT in the resistance of malignant cell to bortezomib.2. To determine the negative regulation of hTERT by aridla and associated mechanisms, which will provide evidence for the anti-cancer effect of aridla.Mothods and Results1. Bortezomib-mediated down-regulation of telomerase and disruption of telomere homeostasis contributes to apoptosis of malignant cells.(1) Bortezomib down regulated the activity of telomerase and disrupted telomere homeostasis.Before and after bortezomib treatment, we detected the telomerase activity by PCR-ELISA kit, the mRNA expression of hTERT, hTER and telomere binding proteins TRF1, TRF2, POT1, RAP1, TPP1 and TIN2 by qRT-PCR, the protein expression of TRF1, TRF2, POT1 by western blotting, the telomere length by flow-FISH and qPCR and the telomeric DNA damage by immuno-FISH in HEL and BGC-823 cells. The results showed that bortezomib decreased the telomerase activity, down regulated the mRNA and(or) protein expressions of hTERT, hTER and telomere binding proteins, shortened the telomere and induced telomere dysfunction in HEL and BGC-823 cells.(2) hTERT was capable of preventing telomere dysfunction induced by bortezomib.We treated hTERT-overexpressed HEL and BGC-823 cells with bortezomib for 24 hours, then detected their telomere length by flow-FISH and qPCR, the telomeric DNA damage by immuno-FISH, the mRNA expression of telomere binding proteins TRF1, TRF2, POT1, RAP1, TPP1 and TIN2 by qRT-PCR and the protein expression ofTRF1, TRF2, POT1 by western blotting. There was no significant difference in telomere length before and after bortezomib treatment in HEL-hTERT and BGC-823-hTERT cells. Telomere dysfunction and dysregulation of telomere binding proteins induced by bortezomib were attenuated by hTERT overexpression.(3) Bortezomib-induced cell apoptosis was attenuated by over-expression of hTERT.We treated control-and hTERT-expressed HEL and BGC-823 cells with bortezomib, then determined cell viability by trypan blue staining and detected apoptotic cells by Annexin-V staining and flow cytometry. The viable cells in HEL-hTERT and BGC-823-hTERT cells were more than those of control cells and the fraction of apoptotic cells in hTERT overexpressed cells were smaller than those of control cells.(4) hTERT over-expression protected malignant cells from bortezomib-killing effect, likely by up-regulating bcl2 expression and attenuating bortezomib-induced DNA damage.Control- and hTERT-expressed HEL and BGC-823 cells were treated with bortezomib for 24 hours, then we detecteed their DNA damage by immunofluorescence and the expression of bcl-2 by western blotting. The results showed over expression of hTERT attenuated DNA damage induced by bortezomib and upregulated bcl-2 expression. The residual bcl-2 protein in HEL-hTERT cells after bortezomib treatment was more than that in HEL-control cells.2. Chromatin remodeling factor aridla suppressed the development of gastric cancer by targeting hTERT.(1) Arid la downregulated hTERT expression.Gastric cancer cells AGS and SGC were transfected with PcDNA6.0 or arid la-over-expression plasmids. Then we detected the mRNA and protein expression of hTERT by qRT-PCR and western blotting. We found hTERT expression was decreased after arid la over expression,(2) Arid1a decreased the activity of hTERT promoter.Both control and arid la-over-expressed AGS and SGC cells were transfected with hTERT promoter reporter plasmids. Then detected the hTERT promoter activity with dual luciferase reporter assay. The results showed hTERT promoter activities in arid1a-over-expressed AGS and SGC cells were lower than that of control ones.(3) Arid la blocked the binding of c-myc at the promoter of hTERT, but did not affected the expression of c-myc.AGS and SGC cells were transfected with PcDNA6.0 or arid la-over-expression plasmids. Then detected the mRNA expression of c-myc by qRT-PCR. We found c-myc mRNA expression was not affecte by arid1a. Both control and arid1a-over-expressed AGS and SGC cells were transfected with hTERT promoter reporter plasmids in which the binding site of c-myc was mutated. Then detected the hTERT promoter activity with dual luciferase reporter assay. The results showed that the activity of mutated hTERT promoter was not affected by arid1a.Conclusions(1) Bortezomib induced the apoptosis of HEL and BGC-823 cells through downregulating telomerase activity and disrupting telomere homeostasis. hTERT attenuated the effect of bortezomib on telomere funcion/homeostasis to mediate cell resistance to bortezomib.(2) Aridla downregulated the expression of hTERT through blocking the binding of c-myc at the promoter of hTERT to suppress the development of gastric cancer.