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Histone acetyltransferase inhibitor II induces apoptosis in glioma cell lines via the p53 signaling pathway.

Xu LX, Li ZH, Tao YF, Li RH, Fang F, Zhao H, Li G, Li YH, Wang J, Feng X, Pan J - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: Here, we examined the effects and mechanism of action of HATi II in glioma cell lines.Upregulation of p53 signaling pathway-related genes in HATi II-treated cells was confirmed by quantitative RT-PCR and Western blotting.HATi II deserves further investigation as a novel treatment for glioma.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pediatric Research, Children's Hospital affiliated to Soochow University, Suzhou, 215006, China. xulixiao2013@hotmail.com.

ABSTRACT

Background: Histone acetyltransferase (HAT) inhibitors can inhibit proliferation and induce apoptosis in cancer cell lines. The novel cell-permeable p300/CREB-binding protein (CBP)-selective HAT inhibitor HATi II can reduce histone H3 acetylation and induce chromatin condensation in HeLa cells. Here, we examined the effects and mechanism of action of HATi II in glioma cell lines.

Methods: Cell viability was assessed using the CCK-8 assay. Cell cycle analysis was performed using flow cytometry. Apoptosis was evaluated using Annexin V staining and flow cytometry, Hoechst 33342 staining and the TUNEL assay. Expression and cleavage of caspase-3, caspase-9 and poly ADP-ribose polymerase (PARP) were assessed by Western blotting. Statistical analysis was performed using two-tailed Student's t-tests. The gene expression profiles of U251 glioma cells treated with HATi II or DMSO were analyzed using the Arraystar Human 8 x 60 K LncRNA/mRNA expression array; data was analyzed using MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profiles (≥2-fold) derived from the cluster analyses were subjected to gene ontology and pathway analysis.

Results: HATi II inhibited the proliferation of U251, U87, HS683 and SHG44 cells in a dose-dependent manner. HATi II induced cell cycle arrest at the G2/M phase, and induced significant levels of apoptosis, apoptotic body formation and DNA fragmentation in HATi II-treated U251 and SHG44 cells. HATi II induced cleavage of caspase-3, caspase-9 and PARP in U251 and SHG44 cells. In HATi II-treated U251 cells, 965 genes were upregulated, 984 genes were downregulated and 3492/33327 lncRNAs were differentially expressed. GO analysis showed the differentially expressed genes with known functions are involved in a variety of processes; alcoholism, p53 signaling pathway, cytokine-cytokine receptor interaction and transcriptional mis-regulation in cancer were the four most significant pathways. Upregulation of p53 signaling pathway-related genes in HATi II-treated cells was confirmed by quantitative RT-PCR and Western blotting.

Conclusions: HATi II inhibits proliferation and induces apoptosis via the caspase-dependent pathway in human glioma cell lines, possibly by activating the p53 signaling pathway. HATi II deserves further investigation as a novel treatment for glioma.

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Bioinformatic analysis of the differentially expressed genes in HATi II-treated U251 cells. Gene ontology enrichment analysis provides a controlled vocabulary to describe the attributes of differentially expressed transcripts in all organisms (p-values ≤ 0.05 are recommended). (A) Upregulated genes and (B) downregulated genes in HATi II-treated U251 cells. Pathway analysis is a functional analysis that maps genes to KEGG pathways. The top five upregulated pathways (C) and top five downregulated pathways (D) in HATi II-treated U251 cells are listed. The “p53 signaling pathway”, which is associated with apoptosis, was activated in HATi II-treated U251 cells. (E) Confirmation of the microarray results. The expression of p53 signaling pathway-related genes in HATi II-treated U251, HS683, U87 and SHG44 cells was validated by quantitative real-time PCR. The columns represent the log-transformed median fold changes in expression relative to DMSO-treated cells.
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Fig5: Bioinformatic analysis of the differentially expressed genes in HATi II-treated U251 cells. Gene ontology enrichment analysis provides a controlled vocabulary to describe the attributes of differentially expressed transcripts in all organisms (p-values ≤ 0.05 are recommended). (A) Upregulated genes and (B) downregulated genes in HATi II-treated U251 cells. Pathway analysis is a functional analysis that maps genes to KEGG pathways. The top five upregulated pathways (C) and top five downregulated pathways (D) in HATi II-treated U251 cells are listed. The “p53 signaling pathway”, which is associated with apoptosis, was activated in HATi II-treated U251 cells. (E) Confirmation of the microarray results. The expression of p53 signaling pathway-related genes in HATi II-treated U251, HS683, U87 and SHG44 cells was validated by quantitative real-time PCR. The columns represent the log-transformed median fold changes in expression relative to DMSO-treated cells.

Mentions: We found that the most enriched GOs targeted by the upregulated and downregulated transcripts were involved in a variety of functions including cellular processes, biological regulation, cell cycle, response to stimulus, immune and defense response, signal transduction, transcriptional regulation and metabolism (Figure 5A and 5B). The genes associated with the most enriched GO terms in HATi II-treated U251 cells were linked to DNA ligation, regulation of protein processing, cell cycle and mitosis, and oxygen transporter activity, and included HBMG2, CRCC4, TOP2A, RAD51, A2M, C3, F12, KLKB1, GTSE1, CETN2, PLK4, KLF11, KLF3B and HBM (Additional file 9: Table S7). In contrast, the genes associated with the most enriched GO terms in DMSO-treated U251 control cells included factors involved in response to stimulus, immune system and defense processes, and included S100A7, MMP3, BCL2, HMOX1, APCS, IGF1, SP3, FOS, CXCL1, TIMP3, DUSP6, MAPK10, CLEC7A and TLR8 (Additional file 10: Table S8).Figure 5


Histone acetyltransferase inhibitor II induces apoptosis in glioma cell lines via the p53 signaling pathway.

Xu LX, Li ZH, Tao YF, Li RH, Fang F, Zhao H, Li G, Li YH, Wang J, Feng X, Pan J - J. Exp. Clin. Cancer Res. (2014)

Bioinformatic analysis of the differentially expressed genes in HATi II-treated U251 cells. Gene ontology enrichment analysis provides a controlled vocabulary to describe the attributes of differentially expressed transcripts in all organisms (p-values ≤ 0.05 are recommended). (A) Upregulated genes and (B) downregulated genes in HATi II-treated U251 cells. Pathway analysis is a functional analysis that maps genes to KEGG pathways. The top five upregulated pathways (C) and top five downregulated pathways (D) in HATi II-treated U251 cells are listed. The “p53 signaling pathway”, which is associated with apoptosis, was activated in HATi II-treated U251 cells. (E) Confirmation of the microarray results. The expression of p53 signaling pathway-related genes in HATi II-treated U251, HS683, U87 and SHG44 cells was validated by quantitative real-time PCR. The columns represent the log-transformed median fold changes in expression relative to DMSO-treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4321714&req=5

Fig5: Bioinformatic analysis of the differentially expressed genes in HATi II-treated U251 cells. Gene ontology enrichment analysis provides a controlled vocabulary to describe the attributes of differentially expressed transcripts in all organisms (p-values ≤ 0.05 are recommended). (A) Upregulated genes and (B) downregulated genes in HATi II-treated U251 cells. Pathway analysis is a functional analysis that maps genes to KEGG pathways. The top five upregulated pathways (C) and top five downregulated pathways (D) in HATi II-treated U251 cells are listed. The “p53 signaling pathway”, which is associated with apoptosis, was activated in HATi II-treated U251 cells. (E) Confirmation of the microarray results. The expression of p53 signaling pathway-related genes in HATi II-treated U251, HS683, U87 and SHG44 cells was validated by quantitative real-time PCR. The columns represent the log-transformed median fold changes in expression relative to DMSO-treated cells.
Mentions: We found that the most enriched GOs targeted by the upregulated and downregulated transcripts were involved in a variety of functions including cellular processes, biological regulation, cell cycle, response to stimulus, immune and defense response, signal transduction, transcriptional regulation and metabolism (Figure 5A and 5B). The genes associated with the most enriched GO terms in HATi II-treated U251 cells were linked to DNA ligation, regulation of protein processing, cell cycle and mitosis, and oxygen transporter activity, and included HBMG2, CRCC4, TOP2A, RAD51, A2M, C3, F12, KLKB1, GTSE1, CETN2, PLK4, KLF11, KLF3B and HBM (Additional file 9: Table S7). In contrast, the genes associated with the most enriched GO terms in DMSO-treated U251 control cells included factors involved in response to stimulus, immune system and defense processes, and included S100A7, MMP3, BCL2, HMOX1, APCS, IGF1, SP3, FOS, CXCL1, TIMP3, DUSP6, MAPK10, CLEC7A and TLR8 (Additional file 10: Table S8).Figure 5

Bottom Line: Here, we examined the effects and mechanism of action of HATi II in glioma cell lines.Upregulation of p53 signaling pathway-related genes in HATi II-treated cells was confirmed by quantitative RT-PCR and Western blotting.HATi II deserves further investigation as a novel treatment for glioma.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pediatric Research, Children's Hospital affiliated to Soochow University, Suzhou, 215006, China. xulixiao2013@hotmail.com.

ABSTRACT

Background: Histone acetyltransferase (HAT) inhibitors can inhibit proliferation and induce apoptosis in cancer cell lines. The novel cell-permeable p300/CREB-binding protein (CBP)-selective HAT inhibitor HATi II can reduce histone H3 acetylation and induce chromatin condensation in HeLa cells. Here, we examined the effects and mechanism of action of HATi II in glioma cell lines.

Methods: Cell viability was assessed using the CCK-8 assay. Cell cycle analysis was performed using flow cytometry. Apoptosis was evaluated using Annexin V staining and flow cytometry, Hoechst 33342 staining and the TUNEL assay. Expression and cleavage of caspase-3, caspase-9 and poly ADP-ribose polymerase (PARP) were assessed by Western blotting. Statistical analysis was performed using two-tailed Student's t-tests. The gene expression profiles of U251 glioma cells treated with HATi II or DMSO were analyzed using the Arraystar Human 8 x 60 K LncRNA/mRNA expression array; data was analyzed using MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profiles (≥2-fold) derived from the cluster analyses were subjected to gene ontology and pathway analysis.

Results: HATi II inhibited the proliferation of U251, U87, HS683 and SHG44 cells in a dose-dependent manner. HATi II induced cell cycle arrest at the G2/M phase, and induced significant levels of apoptosis, apoptotic body formation and DNA fragmentation in HATi II-treated U251 and SHG44 cells. HATi II induced cleavage of caspase-3, caspase-9 and PARP in U251 and SHG44 cells. In HATi II-treated U251 cells, 965 genes were upregulated, 984 genes were downregulated and 3492/33327 lncRNAs were differentially expressed. GO analysis showed the differentially expressed genes with known functions are involved in a variety of processes; alcoholism, p53 signaling pathway, cytokine-cytokine receptor interaction and transcriptional mis-regulation in cancer were the four most significant pathways. Upregulation of p53 signaling pathway-related genes in HATi II-treated cells was confirmed by quantitative RT-PCR and Western blotting.

Conclusions: HATi II inhibits proliferation and induces apoptosis via the caspase-dependent pathway in human glioma cell lines, possibly by activating the p53 signaling pathway. HATi II deserves further investigation as a novel treatment for glioma.

Show MeSH
Related in: MedlinePlus