| BackgroundZinc finger protein which regulates apoptosis and cell cycle arrest (ZAC) is a transcription factor with transcription activating and DNA binding function, and can induce apoptosis and cell cycle G1arrest. P300is a transcription activating factor with a wide range functions, its histone acetyltransferase (HAT) domain is the core domain by which P300acetylates substrate protein and activates transcription. ZAC acts as a transcriptional cofactor cooperated with HAT and/or histone deacetylases (HDAC) to regulate gene expression. Our previous study showed that somatostatin (SST) analogue, otreotide (OCT), could induce ZAC gene expression in a time-and concentration-dependant manner in gastric cancer cell, and ZAC play an important role in the pathway of OCT inhibiting proliferation of gastric cancer cell. The inhibitor of HDAC, trichostatin A (TSA), could upregulate ZAC gene expression in gastric cancer cell.ObjectTo observe the effects of OCT on P300-HAT activity in gastric cancer cell SGC7901and explore its mechanism.Materials and methods1. Experimental group Gastric cancer cell SGC7901was randomly divided into four groups:the experimental groups1-3were cultured with medieum containing OCT at a final concentration of10nmol/L for12h,24h and48h, respectively; the control group was cultured with medieum containing no OCT. The cells of the four groups were collected, and the nuclear proteins were extracted, respectively.2. Effects of OCT on P300-HAT activity in gastric cancer cell SGC7901The P300-HAT activity in the nucleoprotein of four groups was detected by activity spectrophotometry to observe the effects of OCT on P300-HAT in gastric cancer cell SGC7901.3. Effects of OCT on P300gene expression in gastric cancer cell SGC7901The amount of P300protein in the four groups was detected by using Western blot to observe the effects of OCT on P300gene expression in gastric cancer cell SGC7901.4. Interaction between ZAC and P300in gastric cancer cell SGC7901ZAC-P300complex was extracted by co-immunoprecipitation assay and the amount of ZAC-P300complex of four groups was detected by using Western blot to observe to the Interaction between ZAC and P300in gastric cancer cell SGC7901.5. Statistical analysis All data were showed as mean±standard deviation (x±s) and analyzed with SPSS20.0statistical software. P<0.05was considered as statistically significant.Results1. Effects of OCT on P300-HAT activity in gastric cancer cell SGC7901The P300-HAT activity in control,12h,24h and48h group of gastric cancer cell SGC7901was0.00068627433±0.000254713082,0.00163398700±0.00012336330,0.00269607833±0.000084904553and0.00388888867±0.000141507396, respectively, and a significant difference occurred between the the P300-HAT activity of control group and those of experimental groups (P<0.05).10nmol/L OCT enhanced P300-HAT activity in gastric cancer cells SGC7901in a time-dependent manner.2. Effects of OCT on P300gene expression in gastric cancer cell SGC7901The P300/β-Tubulin value in control,12h,24h and48h group of gastric cancer cell SGC7901was0.211±0.002,0.210±0.007,0.208±0.014and0.216±0.004, respectively. There was no significant difference between P300gene expression in control group and those in three experimental groups (P>0.05).3. Interaction between ZAC and P300in gastric cancer cell SGC7901ZAC could interact with P300protein in gastric cancer cell SGC7901. The relative amount of ZAC-P300complex in control,12h,24h and48h group of gastric cancer cell SGC7901was5812.568±933.548,5869.995±1067.049,5810.128±975.091and3563.223±382.192, respectively. There was no significant difference between the amount of ZAC-P300complex in12h and24h groups and that in control group (P>0.05), however, that in48h group was significantly decreased (P <0.05).Conclusion:OCT may ehance P300-HAT activity in gastric cancer cell SGC7901through the interaction between ZAC and P300. |
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