BIX-01294 inhibits the proliferation of esophageal squamous cell carcinoma cells by inducing DNA damage and activating the mitochondrial apoptosis pathway / 中国胸心血管外科临床杂志

@#Objective    To explore the effects and molecular mechanisms of histone methylase G9a inhibitor BIX-01294 on apoptosis in esophageal squamous cell carcinoma (ESCC). Methods    MTT assay and Colony-forming Units were adopted to determine the effects of BIX-01294 on the growth and proliferation of ESCC cell lines EC109 and KYSE150. Flow cytometry was used to analyze the apoptosis status of ESCC cells after the treatment of BIX-01294. The effects of BIX-01294 treatment on the expressions of G9a catalytic product H3K9me2, DNA double-strand break (DSB) markers, and apoptosis-related proteins were detected by Western blotting. Results    BIX-01294 inhibited the growth of EC109 and KYSE150 cells in a dose-dependent manner (P<0.05), and BIX-01294 with the inhibitory concentration 50%(IC50) significantly inhibited the formation of colony (P<0.05). After 24 hours treatment of BIX-01294 (IC50), the apoptosis rate of EC109 cells increased from 11.5%±2.1% to 42.5%±5.4%, and KYSE150 cells from 7.5%±0.9% to 49.2%±5.2%(P<0.05). The expression level of the G9a catalytic product, H3K9me2, significantly decreased (P<0.05); while the expression of the DSB marker γH2AX was dramatically enhanced (P<0.05). We also found that the mitochondrial apoptosis pathway was activated and the expression levels of cleaved caspase3 and cleaved PARP were significantly elevated (P<0.05). Conclusion    BIX-01294, the inhibitor of methyltransferase G9a, prompted apoptosis in ESCC cells by inducing DSB damage and activating mitochondrial apoptosis pathway.

Nicotinamide activates latent HIV-1 ex vivo in ART suppressed individuals, revealing higher potency than the association of two methyltransferase inhibitors, chaetocin and BIX01294

ABSTRACT Background: Latent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n = 17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n = 25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals. Results: NAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log10 RNA copies/mL, respectively (p = 0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p = 0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n = 4). Conclusion: NAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use.

Inhibition of G9a attenuates cell proliferation via the mitochondrial apoptosis pathway in lung adenocarcinoma / 中华肿瘤杂志

Objective@#The aim of this study is to investigate the effect of G9a inhibitor BIX-01294 on attenuating cell proliferation in human lung adenocarcinoma A549 cell line and the underlying molecular mechanism.@*Methods@#Treated with BIX-01294, the growth and proliferation of A549 cells were detected by MTT assay and colony formation assay, and its impact on cell apoptosis was analyzed using flow cytometry. By Western blot, we explored the alterations in the expression of apoptosis-related proteins and the G9a catalysate, H3K9me and H3K9me2. In addition, in the pretreatment with caspase inhibitor Z-VAD-FMK, we detected the apoptotic dependence of BIX-01294 attenuating impact on A549 cell proliferation.@*Results@#Compared with the control group, the histone methyltransferase G9a inhibitor BIX-01294 attenuated cell proliferation in A549 cells in a dose- and time-dependent manner. There were 42.5±8.7 colonies after BIX-01294 (10 μmol/L) treatment for 7 days, while 172.7±23.0 colonies in the control group, with a statistical significance (P<0.05). After treatment with BIX-01294 (10 μmol/L) for 24 hours, the cell apoptotic rate was(47.6±8.4)%, with a significant difference in comparison with the control group [(7.2±3.6)%, P<0.05]. The expression of G9a catalysate, H3K9me and H3K9me2 was downregulated, the same with anti-apoptotic protein Bcl-2, while the proteins in mitochondrial apoptosis pathway, Bax, Bak and cleaved caspase-9, were upregulated, so was the expression of cleaved caspase-3 and cleaved PARP, and there was no alteration in the expression of cleaved caspase-8, which is a protein related with death receptor apoptosis pathway. Furthermore, after Z-VAD-FMK pretreatment, the cell apoptotic rate was decreased significantly, and the expression of apoptosis-related proteins were downregulated.@*Conclusions@#Our results indicate that BIX-01294 can attenuate cell proliferation in lung adenocarcinoma, and it can be considered as one of the underlying mechanisms, the apoptosis may be induced by activating mitochondrial pathway.

Effect of BIX-01294 on cell proliferation, apoptosis and histone methylation in Molt-4 line / 中国药理学通报

Aim To investigate the effect of the specif-ic inhibitor BIX-01294 of G9a on the proliferation, ap-optosis,DNA methylation and histone modulation of a-cute leukemia cell line, Molt-4. Methods Cells were cultured with different concentrations of BIX-01294 . Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of Caspase-3, Bcl-2, Bax, P15, acetylated H3, H3 K9 me1 , H3 K9 me2 , H3 K9 me3 and H3 K27 me1 , H3K27me2 methylation were detected by Western blot. Results BIX-01294 downregulated bcl-2 , and upreg-ulated Bax, Caspase-3 and induced apoptosis in (5. 54 ± 1. 35)%, (10. 24 ± 2. 26)%, (32. 28 ± 3. 26)%, (47. 52 ± 4. 37 )% after 24 hours exposure to BIX-01294 in 0 , 1 , 2 , 4 μmol · L-1 . The difference be-tween them was statistically significant ( P <0. 05 ) . BIX-01294 inhibited DMNT1 and promoted P15 , which resulted in cell proliferation inhibition. Further studies showed that BIX-01294 decreased H3 K9 me1 , H3 K9 me2 , and H3 K27 me1 , H3 K27 me2 , and didn′t change protein expression of acetylated H3 and H3 K9 me3. Conclusion BIX-01294 inhibits G9a, resulting in downregulation of methylation of H3 K9 me1 , H3 K9 me2 , H3 K27 me1 and H3 K27 me2 and DMNT1 and P15 denovo. It inhibits cell proliferation and in-duces cell apoptosis.