Effect of combination usage of TSA and CYA on pancreatic cancer PANC-1 cell apoptosis
- Author:
He-Yi YOU
1
Author Information
- Publication Type:Journal Article
- Keywords: Apoptosis; Cyclopamine; Hedgehog; Pancreatic cancer; Trichostatin A
- From: Chinese Pharmaceutical Journal 2014;49(11):971-977
- CountryChina
- Language:Chinese
- Abstract: OBJECTIVE: To evaluate the apoptosis effect of HDAC inhibitor trichostatin A (TSA) combination with Hedgehog (Hh) signaling inhibitor cyclopamine (CYA) on the PANC-1 cells line. METHODS: Hochest 33258 staining was used to measure apoptosis of PANC-1 cells treated with TSA and CYA respectively or cooperatively. The 24 h IC50 of PANC-1 cells line responding to TSA and CYA were detected by CCK8. The mRNA expression of the gene relative with apoptosis and Hh signaling were evaluated by SYBR GREEN based FQ-PCR (fluorescent quantization polymerase chain reaction). The proteins associated with apoptosis (Bcl-2 and activated caspase-3, activated caspase-8, activated caspase-9) and Hh signalling pathway (Gli1, Smo and Ptc-1) were measured by Western blotting (WB). Gli1 was also detected by cell immunofluorescence. RESULTS: TSA and CYA could induce apoptosis of PANC-1 cells by Hochest 33258 stain, respectively or combinatively. The 24 h IC50 of PANC-1 cells line treated with chemical were (0.51±0.07) μmol·L-1 (TSA) and (33.6±2.3) μmol·L-1 (CYA), respectively; the combination index (CI) of TSA and CYA on the PANC-1 is 0.835. Combination usage of TSA and CYA caused more significantly decreased expression of Bcl-2 mRNA and increased Bax mRNA as compared with the TSA or CYA treatment. TSA (0.5 μmol·L-1) inhibited the expression of Gli1 mRNA, TSA promoted the inhibition effect of CYA on Hh pathway, down-regulation of Gli1, Smo mRNA and up-regulation of Gli3 mRNA. Western blotting showed that combination usage of TSA and CYA induced more significant activation of activated caspase-3, activated caspase-8, activated caspase-9 and reduction of Bcl-2, Gli1, Smo and Ptc-1 expression than TSA or CYA treatment. Decreased Gli 1 also confirmed by immunofluorescence. CONCLUSION: Combination usage of TSA and CYA could induce PANC-1 cells line apoptosis and synergistically inhibit Hh pathway.
